Ninth Annual Meeting

Nebraska Physiological Society A Chapter of the American Physiological Society

Program and Abstract Book

May 12, 2006 University of Nebraska Medical Center

Durham Research Center

Nebraska Physiological Society Ninth Annual Meeting

University of Nebraska Medical Center Durham Research Center Room 1002

May 12, 2006

8:00 a.m. Check in at Registration Table - Lobby of Durham Research Center Set up Posters - 5th floor of Durham Research Center 9:00 a.m. Welcome and Introductory Remarks NPS President – William G. Mayhan, Ph.D. 9:15 a.m. Educational Seminar Robert G. Carroll, Ph.D. East Carolina University “The Role of the Teacher in Physiology Education” 10:15-10:30 Break 10:30 a.m. Student and Fellow Presentations 11:45 a.m. Break – View Exhibits 12:00 noon Lunch APS Sponsored - Keynote Address Michael S. Wolin, Ph.D. New York Medical College “Oxidant Redox Signaling Mechanisms in Vascular Regulation and Dysfunction” 1:15 p.m. NPS Business Meeting 1. State of American Physiological Society - Martin Frank, Ph.D. 2. NPS Business Meeting – William G. Mayhan, Ph.D. 3. LOTS Activities – Janet Steele, Ph.D. 4. Student Awards 1:45 -2:00 p.m. Break - View Exhibits 2:00 – 4:00 p.m. View posters - 5th Floor of Durham Research Center 4:00 p.m. Adjourn

Robert G. Carroll, Ph.D. Professor, East Carolina University

Robert G. Carroll earned his Ph.D. under the direction of Dr. David F. Opdyke from the Department of Physiology of the Graduate School of Biomedical Sciences of the University of Medicine and Dentistry of New Jersey-Newark in 1981. He completed a 3 year post-doc at University of Mississippi Medical Center in Jackson, MS under the sponsorship of Drs. Thomas E. Lohmeier and Arthur C. Guyton. In 1984, he became an Assistant Professor of Physiology at East Carolina University. He is currently Professor of Physiology at the Brody School of Medicine at East Carolina University, and holds adjunct appointments in the Departments of Emergency Medicine and in the Department of Biology. Back when he did the actual experiments, Rob’s dissertation examined blood pressure regulation in sharks. He now sits around and spends his time in front of a computer, and has an addiction to “free cell”. For the past 20 years, the real work in the laboratory was accomplished by his technician, one doctoral student, 2 masters’ students, and numerous medical students, residents and fellows, resulting in 47 research manuscripts. He uses travel as another mechanism to avoid lab work, presenting seminars and workshops in Hungary, Indonesia, Jamaica, Mexico, New Zealand, Pakistan, Russia, and Scotland; and hosting visiting faculty members from Indonesia, Sudan, and Jamaica. Apart from his bench research, Rob has published 11 peer reviewed education manuscripts, edited one book, is a section editor for a Medical-Surgical Nursing textbook, and is writing a review book for medical students preparing for their licensing examination. Rob currently chairs the Education Committee for the American Physiological Society, and is a member of the Education Committee of the International Union of Physiological Sciences. He is an associate editor of the journal “Advances in Physiology Education”. In the past, he served on the USMLE Step I Physiology Test Material Development Committee of the National Board of Medical Examiners, and as Secretary for the International Association of Medical Science Educators. In 2002, he was recognized in the inaugural class of Master Educators at the Brody School of Medicine, and received the Arthur C. Guyton Physiology Educator of the Year from the American Physiological Society in 2004. He received the Outstanding Alumni Award from the University of Medicine and Dentistry of New Jersey in 2005. His three teenage children, however, still question his sanity and judgment.

Michael S. Wolin, Ph.D. Professor, New York Medical College

American Physiological Society Sponsored Keynote Speaker

The research of Michael S. Wolin has focused on the elucidation of multiple fundamental concepts of vascular oxidant signaling mechanisms. Dr. Wolin earned his Ph.D. in Chemistry from Yale University in 1981 and his thesis work focused on understanding how adenylate cyclase made cAMP. His subsequent postdoctoral work with Louis J. Ignarro in the Department of Pharmacology of Tulane University investigated how the soluble form of guanylate cyclase was regulated by nitric oxide, porphyrins and free radicals. Dr. Wolin joined the Department of Physiology of New York Medical College as an Assistant Professor in 1983 and subsequently rose to the rank of Professor in 1995. Dr. Wolin was an Established Investigator of the AHA and a recipient of a Merit Award from the NHLBI. He was a member of the Research and Scientific Sessions Program Committees of the AHA, a past chair of the AHA Cardiopulmonary, Perioperative and Critical Care Council and the recipient of this council’s 2005 AHA Distinguished Achievement Award. Dr. Wolin served as a member of the NIH VCMB study section and as an Associate Editor for the Journal Microcirculation, and he is currently an Associate Editor for the American Journal of Physiology. His research has focused on how reactive oxygen species and their interactions with nitric oxide control vascular signaling mechanisms associated with endothelial function, mitochondrial respiration and guanylate cyclase. Through collaborations at NYMC he has been able to investigate how vascular oxidant-NO signaling mechanisms control microcirculatory function and are altered by complications of hypertension, diabetes, heart failure and aging. More recent interests include understanding the regulatory role of cytosolic NAD(P)H redox and how these systems control of differences in oxygen sensing mechanisms between the coronary and pulmonary vasculature.

Martin Frank, Ph.D.

Executive Director, American Physiological Society

Dr. Frank received his Ph.D. at the University of Illinois, Urbana-Champaign in 1973. Dr. Frank began his career as all other newly minted PhDs did and soon accepted a postdoctoral position at the Michigan Cancer Foundation. This was followed by another position at Michigan State University, East Lansing. After completing two postdoctoral positions, Dr. Frank accepted a position as an Assistant Professor in the Department of Physiology at the George Washington University Medical School in Washington, DC. He looked forward to a life as an “absent-minded professor” who rode his bike to his laboratory each day and initiated scores of eager students into the mysteries of physiology.

However, life had other plans for Dr. Frank. As an Assistant Professor, Dr. Frank found himself interested in science policy. As he likes to tell people, he was bit by “Potomac Fever.” After three years at GWU, he moved on to NIH where he served as the Executive Secretary of the Physiology Study Section, Division of Research Grants. There he played the role of arbitrator and champion of the investigator. In his continual quest to expand his knowledge base, he applied for and was accepted into the Senior Executive Service Candidate Program in the Department of Health and Human Services. Participation in that program allowed him to work with a variety of people in different capacities within DHHS. That knowledge would serve him well in future years.

It was in 1985 that Dr. Frank accepted a new challenge – Executive Director of The American Physiological Society. As Executive Director of the oldest biomedical sciences research society in America, he is responsible for managing the Society, a non-profit association established to promote the physiological sciences. Under his leadership APS has grown exponentially to become a $16 million business with over 70 employees. He works directly with the elected Presidents and Council members and the appointed committee chairs to ensure the publication of the Society’s 14 top-rated journals, the management of the scientific meetings highlighting the latest in physiological research, the development and implementation of educational programs to meet the broad and varied needs of the scientific community, and the ongoing vigilance needed for continued increased funding for the discipline. He also works closely with the Council to ensure the continued availability of research animals for their humane use in experimental studies.

Physiology is the basis of medicine and provides us with an understanding of how our bodies function. Dr. Frank expounds physiology as an exciting field that will help to answer the questions raised by the various genome projects. The genome projects have provided scientists with the A, C, T, and G of the alphabet. It is the physiologists who will be called upon to put those letters into words, sentences, and paragraphs, helping to define the function of the genes and how they relate to the physiology of the organism.

For students working toward a career in physiology Dr. Frank would encourage them to develop not only an understanding of the molecular and genomic aspects of our science, but also an understanding of how to measure function in the organism, both the intact organism and its various organ systems. In so doing, a student will be able to participate in the excitement of translating the genome. The student will also be able to translate their discoveries at the bench into possible treatments at the bedside.

POSTER 1 Exercise Training Normalizes Enhanced NMDA-Mediated Changes in Renal Sympathetic Nerve Activity and NR1 Expression Within the PVN in Heart Failure Rats Allison Kleiber, Hong Zheng, Xuefei Liu, and Kaushik P. Patel

POSTER 2 Exercise Training Blunt RyR2 Dysfunction in Hearts of Streptozotocin-Induced Diabetic Rats Chun-Hong Shao, George J. Rozanski, Kaushik P. Patel and Keshore R. Bidasee

POSTER 3

Heart Rate Variability and Central Angiotensin II Receptors in Heart Failure: Role of Exercise Training Tarek M Mousa, Kurtis G. Cornish, and Irving H. Zucker

POSTER 4 Exercise Training Alters Muscle Pressor Reflex in Rats With Heart Failure Yan-Xia Pan, Mohammad Fahim, Lie Gao, Weizhong Wang, Kurtis G. Cornish, Irving H. Zucker, and Wei Wang

POSTER 5

Effect of Fluid Replacement During Distance Running on Gastrointestinal Barrier Function G.P. Lambert, J.A. Lang, A.J. Bull, P.C. Pfeifer, J.M. Eckerson, and G.A. Moore

POSTER 6

Effect of Exercise Intensity on Active and Passive Carbohydrate Absorption James A. Lang, Carl V. Gisolfi, and G. Patrick Lambert

POSTER 7 Neuronal AT1 Receptor Upregulation in Heart Failure: Activation of AP-1 and JNK Dongmei Liu, Lie Gao, Shyamal K. Roy, Kurtis G. Cornish, and Irving H. Zucker

POSTER 8 Cardiac Sympathetic Afferent Stimulation Inhibits the Arterial Baroreflex in Heart Failure at the Level of the Nucleus of the Solitary Tract W- Z Wang, Y-X Pan, L. Gao, I.H. Zucker, and W. Wang

POSTER 9

Disruption of the Cardiovascular Circadian Rhythm in Mice Post Myocardial Infarction: Relationship to Central Angiotensin II Receptor Expression Tarek M. Mousa, and Irving H. Zucker.

POSTER 10 Sympatho-Excitation in Chronic Heart Failure: Ang II Induced Inhibition of Voltage-Gated K+ Channel – an In Vivo and In Vitro Study Lie Gao, Wei Wang, Ethan Mann, Marcus Finch, Yulong Li, Dongmei Liu, Harold D. Schultz, and Irving H. Zucker.

POSTER 11

Angiotensin II-Mediated Sympathoexcitation in Diabetes: Role of Superoxide Hong Zheng, Xuefei Liu, Allison Kleiber, Robin L. Davisson, and Kaushik P. Patel

POSTER 12

Influence of Diabetes Mellitus and Exercise Training on Oxidative Stress Repair Mechanisms in Cardiac Tissue Mary Connealy, Cory Ciccone, Karynn Kucera, and Janet Steele

POSTER 13

Influence of Diabetes Mellitus and Exercise Training on Gene Expression in Cardiac Muscle Tissue Cory Ciccone, Mary Connealy, Karynn Kucera, and Janet Steele

POSTER 14

Testosterone does not Increase Kidney Angiotensin II in Experimental Diabetes Pascale H. Lane, William J. Langer, Kay Devish, and Jianhong Sun.

POSTER 15

Kidney Gene Expression in Diabetes Pre- and Post-Puberty Pascale H. Lane and William J. Langer

POSTER 16 Role of Cyp2E1 in Impaired Nitric Oxide Synthase Nos-Dependent Cerebral Vasodilation During Chronic Alcohol Consumption Hong Sun, Denise M. Arrick, and William G. Mayhan

POSTER 17

Role for Poly (Adp-Ribose) Polymerase (Parp) Activation in Impaired Responses of Cerebral Arterioles in Type 1 Diabetes Mellitus (T1D) Denise M. Arrick, Hong Sun, Glenda Sharpe and William G. Mayhan

POSTER 18

Functional and Immunohistochemical Characterization of Hyperpolarization Activated Current in Rat Suprachiasmatic Nucleus Nermien Waly and Richard Hallworth

POSTER 19

Downregulation of Carbon Monoxide as well as Nitric Oxide Contributes to Peripheral Chemoreflex Hypersensitivity in Heart Failure Rabbits YanFeng Ding, Yu-Long Li, and Harold D. Schultz

POSTER 20

Endogenous Neuronal Nitric Oxide Systhase and AT1 Receptor do not Correlate the Enhanced Peripheral Chemoreflex Function in Early Stage of Chronic Heart Failure Rabbits Yu-Long Li, Chad Agnew, and Harold D. Schultz

POSTER 21 Enhancement of Cholinergic Activity Elicited Cardiac Protection and Anti-Inflammatory Effect Yi-Fan Li, Jessica Freeling, Kristina Wattier, and Carly LaCroix

POSTER 22 Aging-Related Cardiac Dysfunction in Mice – The Role of ErbB Tyrosine Kinase Receptors Viswanathan Rajagopalan, Irving H. Zucker, Tarek M. Mousa, and Ying J. Ma

POSTER 23 Protein Kinase C-Dependent Superoxide Production by the Renal Medullary Thick Ascending Limb in Normal and High Glucose Environments Pamela K. Carmines, William G. Mayhan, and Jennifer S. Pollock

POSTER 24

In Vitro Differentiation of Bone Marrow Mesenchymal Stem Cells into Renal Tubular Epitheliallineage Kurinji Singaravelu and Babu J. Padanilam.

POSTER 25 The Localization of the Beta-1, -2, and -4 Subunits to the Large Conductance, Ca2+- Activated K+ Channel (Bk) within the Mouse Kidney P. Richard Grimm, Ruth M. Foutz, Peilin Wei, Jennifer L. Pluznick and Steven C. Sansom

POSTER 26

The Role of VASP in No/CGMP-Mediated Inhibition of SOC in Human Mesangial Cells Xiaoxia Wang, Jennifer L. Pluznick, P. Richard Grimm, Peilin Wei, and Steven C. Sansom

POSTER 27

Effects of Warm Acclimation and Papaverine on Serum Osmolality in Antarctic Fish H.A. Hudson, P.R. Brauer, M.A. Scofield, and D.H. Petzel

POSTER 28

Warm Acclimation Affects Serum Osmolality and Na+/K+Atpase Activity in the Gilland Gut of Trematomus Bernacchii K. Smith, F. Dowd, P. Brauer, J. Morrison, M. Scofield, and D. Petzel

POSTER 29

GnRH Signaling During Early Embryonic Development William E. Pohlmeier, Marcelo M. Montagner, Rebecca A. Cederberg, and Brett R. White

POSTER 30

Glucocorticoid Responsiveness of the Porcine GnRH Receptor (GnRHR) Gene C. Lee, R. A. Cederberg, and B. R. White.

POSTER 31

Evidence Supporting a Role for GSK3β/Β-Catenin in Hormone-Stimulated Progesterone Production in the Bovine Corpus Luteum Lynn Roy, Xiaoying Hou, Edward Arvisais, Chao Jiang, and John S. Davis

POSTER 32 The Identification of Critical Time Periods for Atypical Reproductive and Immune Development due to Genistein Exposure in Male Rats Evan Ball, Alicia Fisher, Stacie Holmgren, Stacy Knight, Raime Robinson, and Amy Wisniewski

POSTER 33 Expression of MRNA for Hyaluronan Metabolic Enzymes in the Embryonic and Perinatal Rat Gonads Natalie C. Hart, Rebecca C. Bott, Robin A. Ten Broeck, Debra T. Clopton, Michelle M. Baltes and Andrea S. Cupp.

POSTER 34 Vascular Endothelial Growth Factor (VEGF) 164 Increases Vascular Density during Testis Morphogenesis and may be Regulated by the Inhibitory Isoform VEGF164b M.M. Baltes, R.A. Ten Broeck, R.C. Bott, D.T. Clopton, and A.S. Cupp

POSTER 35 Localization of Period 1 in the Ruminant Oocyte and Investigations of its Role in Ovarian Function R.A. Cushman, M.F. Allan, S.A. Jones, G.P. Rupp, and S.E. Echternkamp

POSTER 36

Inhibition of Vascular Endothelial Growth Factor Signaling Blocks Primordial Follicle Activation in Bovine Ovarian Cortical Cultures D.T. Clopton, A.S. Cupp, M.F. Allan, S.E. Echternkamp and R.A. Cushman

POSTER 37

Vascular Endothelial Growth Factor (VEGF) 164 Increases Vascular Development in the Perinatal Rat Ovary while Inhibition of VEGF Receptor 2 (VEGFR2) does not Alter Vascular Development or Follicle Progression R.A. Ten Broeck, D.T. Clopton, M.M. Baltes, and A.S. Cupp

POSTER 38

Liver Receptor Homologue-1 (LRH-1) Regulates Progesterone Synthesis in the Bovine Corpus Luteum X. Hou, C, Jiang, E.W. Arvisais, and J. S. Davis

POSTER 39

Vascular Endothelial Growth Factor (VEGF) 120 but not VEGF164 Expression is Increased in Granulosa Cells of Bovine Dominant Follicles due to Nutrition Jeremy L. Martin, Rick N. Funston, Debra T. Clopton, Heidi L. Harris, and Andrea S. Cupp

POSTER 40

Engaging Middle School Students through Distance Learning from Antarctica Yvette K. McCulley, Philip R. Brauer, Hilary A. Hudson, Margaret A. Scofield, Kimberly A. Smith Hailaeos Troy, and David H. Petzel

POSTER 1 Exercise Training Normalizes Enhanced NMDA-Mediated Changes in Renal Sympathetic Nerve Activity and NR1 Expression within the PVN in Heart Failure Rats Allison Kleiber, Hong Zheng, Xuefei Liu, and Kaushik P. Patel Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, NE 68198-5850

Exercise training (ExT) normalizes increased sympathetic outflow in heart failure (HF), but the mechanism(s) is/are not known. We hypothesized ExT would improve glutamatergic mechanisms mediated by paraventricular nucleus (PVN) NMDA receptors. Four groups of rats were used: 1) Sham Sedentary (Sed); 2) Sham ExT; 3) HF Sed; and 4) HF ExT. HF was induced by left coronary artery ligation, and ExT consisted of 3 weeks of treadmill running. In a-chloralose-urethane-anesthetized rats, the dose-dependent increase in renal sympathetic nerve activity (RSNA) in response to NMDA injected into the PVN was potentiated in HF Sed compared to Sham Sed. At the highest dose of NMDA (200 pmol), this response in HF Sed was 101% +18% (n=4) compared to 52%+9% (n=4) in Sham Sed. In HF ExT the response (41%+3%, n=4) was not different from Sham Sed or ExT (33% ±9%, n=6). Relative NMDA receptor subunitNR 1expression was increased in HFSed (1.70+0.30 relativeunits) compared to Sham Sed (1.0), but in HF ExT was significantly attenuated (0.68 +0.18 relative units) compared to HF Sed. NR1 protein expression increased 61% in HF Sed compared to Sham Sed, but was significantly attenuated in HF ExT. Thus, ExT normalizes PVN NMDA-mediated changes in RSNA, which may be due to normalization of PVN NR1 expression, and this may be a mechanism by which ExT alleviates the increased sympathetic outflow associated with HF.

POSTER 2

Exercise Training Blunt RyR2 Dysfunction in Hearts of Streptozotocin Induced Diabetic Rats

Chun-Hong Shao1, George J.Rozanski2, Kaushik P. Patel2 and Keshore R. Bidasee1

1Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198-5800 2Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, NE 68198-5850

The synchronous release of Ca2+ from the sarcoplasmic reticulum (SR) via type 2 ryanodine receptors (RyR2) is an integral step in the transduction cascade leading to cardiac muscle contraction. We and others found that this process becomes defective during type 1 diabetes (T1D) in part from a dysfunction of RyR2. Exercise training (ExT) minimizes myocardial contractility loss during T1D. However, molecular mechanisms responsible for its beneficial effect remain incompletely understood. The present study was undertaken to determine whether ExT prevents RyR2 dysregulation and normalize Ca2+ release from the SR during T1D. After 7-8 weeks of streptozotocin (STZ)-induced diabetes, expression of RyR2 remained unchanged in hearts of male Sprague-Dawley rats. However, the number of functional RyR2 decreased by 42% and their sensitivity to Ca2+ activation increased 2.5 fold. When field stimulated, intracellular Ca2+ release in diabetic ventricular myocytes was dyssynchronous and time to peak Ca2+ increased 3.6 fold. Diabetic myocytes also exhibited diastolic Ca2+ release and a 2.2-fold increase in Ca2+ spark frequency. Four weeks of ExT training, initiated after 3-4 weeks of diabetes minimized RyR2 dysregulation, blunted dyssynchronous and diastolic Ca2+ releases and normalize Ca2+ spark frequency. ExT also blunted phosphorylation of RyR2 at Ser2809. These data suggest that ExT is minimizing myocardial contractility loss during T1D in part by preventing dysregulation of RyR2.

POSTER 3 Heart Rate Variability and Central Angiotensin II Receptors in Heart Failure: Role of Exercise Training

Tarek M. Mousa, Kurtis G. Cornish, Irving H. Zucker Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, Nebraska 68198-5850

Exercise training (EX) has become an important modality enhancing survival of patients with chronic heart failure (CHF). Heart rate variability (HRV) is a non-invasive method assessing sympathovagal balance and predicting mortality in CHF. Enhanced HRV occurs in CHF following EX. We showed a down-regulation of brain angiotensin II receptors (AT1), a reduction in plasma Ang II and normalization of baseline renal sympathetic nerve activity (SNA) and an enhanced HRV for total power and standard deviation of R-R interval (SDRR) with EX in a rapid pacing model of CHF in New Zealand white rabbits. We hypothesized that prevention of Ang II normalization in CHF-EX rabbits would prevent the beneficial effects of EX on AT1 expression, SNA and HRV. The table shows HRV parameters, baseline SNA and AT1 expression using Western-blot and RT-PCR in the rostral portion of the ventrolateral medulla. When plasma Ang II levels were “clamped” and prevented from normalization with EX, down-regulation of AT1 expression was abolished and the total power was depressed through a reduction in the low frequency (LF) component of the total power (TP). The enhanced SDRR with EX was preserved despite Ang II infusion with EX. These data suggest that the reduction in plasma Ang II and/or AT1 expression may be responsible for lowering SNA and that Ang II influences on HRV are reflected by the LF component of the TP and this influence does not affect SDRR, perhaps due to maintenance of vagal tone following EX in CHF. CHF (n=9) CHF-EX (n=9) CHF-EX-Ang II (n=10)

TP(ms2) 57.2±4.4 90.9±12.4* 65.4±9.2 LF (ms2) 11.0±2.3 15.9±4.3* 6.2±1.2 HF (ms2) 11.1±2.4* 23.2±2.6 19.3±3.4

SDRR (ms) 6.6±1.2* 11.7±1.2 10.2±0.9 SNA (%) 49.4±2.3 35.5±1.8* 53.6±3.6 Western 1.5±0.2 0.8±0.1* 1.4±0.2

*p<0.05 compared to either groups

POSTER 4 Exercise Training Alters Muscle Pressor Reflex in Rats with Heart Failure Yan-Xia Pan, Mohammad Fahim, Lie Gao, Weizhong Wang, Kurtis G. Cornish, Irving H. Zucker, Wei Wang Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, NE 68198-5850.

Muscle pressor reflex (EPR) consists of mechanoreflex and metaboreflex. In chronic heart failure (CHF), there is exaggerated mechanoreflex and attenuated metaboreflex during exercise, which limits tolerance to physical activity. The mechanisms underlying these abnormalities are poor understood. Exercise training (EX) has been shown to improve vasodilatation of skeletal muscle via nitric oxide (NO) dependent mechanism. In the present study, we hypothesized that EX restored abnormal EPR in CHF rats via increasing NO synthase (NOS) expression in skeletal muscle. To test this hypothesis, rats were divided into four groups; sham, CHF, sham+EX, CHF+EX. The mechanoreflex was elicited by electrical stimulation of L4/L5 spinal ventral roots and the metaboreflex was evoked by intra-arterial injection of capsaicin. NOS protein expression in the skeletal muscle was measured by Western blot. This result showed that EX significantly decreased MAP and HR responses to muscle contraction of hindlimb in CHF rats (MAP, 15.3 ± 2.3mmHg in CHF+Ex vs. 29.5 ± 3.0 mmHg in CHF; HR, 14.7 ± 1.8 bpm in CHF+Ex vs. 20.5 ± 1.0 bpm in CHF, P<0.05). EX also abolished exaggerated RSNA response to capsaicin and restored attenuated blood pressure response to capsaicin In CHF rats. In addition, eNOS and nNOS protein in the soleus of CHF rats was decreased by 35% and 69% compared with sham rats (eNOS, 0.68 ± 0.05 in CHF vs. 0.44 ± 0.04 in sham; nNOS, 1.08 ±0.05 in CHF vs. 0.34±0.04 in sham, respectively), accompanying by decreased citrate synthase activity (CS) in the soleus. EX significantly increased in CS of the soleus and up-regulated protein expression of eNOS and nNOS in the soleus in both CHF and sham rats. These results suggest that adaptation to EX increased eNOS and nNOS protein expression and decreased oxidative stress of skeletal muscle which might be responsible for improvement in cardiovascular function in CHF.

POSTER 5 Effect of Fluid Replacement During Distance Running on Gastrointestinal Barrier Function G.P. Lambert, J.A. Lang, A.J. Bull, P.C. Pfeifer, J.M. Eckerson, and G.A. Moore Department of Exercise Science and Athletic Training, Creighton University, Omaha NE.

The purpose of this study was to determine if moderately intense distance running causes gastrointestinal (GI) barrier dysfunction and whether this is affected by fluid replacement. Twenty trained runners (11 males, 9 females; age = 22 ± 1 (SE) yrs; mean VO2 max = 55.7 ± 1.8 ml/kg/min) completed four experiments separated by one week. These were: 1) rest, 2) running with no fluid ingestion (NF), 3) running with ingestion of a 4% glucose solution (GLU), and 4) running with ingestion of a water placebo (PLA). The running experiments consisted of 60 min of treadmill exercise at 70% VO2 max in a relatively thermoneutral environment (24.4 ± 04 °C, 32.7 ± 0.55 %RH). Before and after each experiment, measures of nude body weight (BW) and rectal temperature were obtained. During the running experiments with fluid ingestion, subjects drank 3 ml/kg BW every 10 min. Immediately prior to each experiment, subjects ingested a solution (150 ml) containing 5 g sucrose (S), 5 g lactulose (L), and 2 g rhamnose (R). Urinary excretion of S is an indicator of gastric permeability and the excretion ratio of L to R (L/R) is an index of small intestinal permeability. Increased permeability above resting conditions indicates GI barrier dysfunction. There were no differences among the running trials for final rectal temperature (mean = 38.5 ±0.1 °C), final heart rate (mean = 172 ± 2 bpm) or sweat rate (mean = 1.1 ± 0.1 L/h), although all of the running experiments significantly (P < 0.05) differed from rest for these variables. BW loss was significantly greater for NF (1.5 ± 0.1%) compared to the other trials (mean = 0.05 ± 0.08%) with GLU and PLA not differing from each other or rest. Compared to rest, NF significantly increased both gastric permeability (S excretion) and intestinal permeability (L/R). There were no other differences among trials for either S excretion or L/R. These data indicate that running for 60 min at 70% VO2 max without fluid replacement impairs the GI barrier and suggests that fluid replacement may be protective in this regard. Supported by the Gatorade Sports Science Institute

POSTER 6 Effect of Exercise Intensity on Active and Passive Carbohydrate Absorption James A. Lang1, Carl V. Gisolfi2, and G. Patrick Lambert1 1Department of Exercise Science and Athletic Training, Creighton University, Omaha, NE 2Department of Exercise Science, University of Iowa, Iowa City, IA

The purpose of this study was to determine the effects of exercise intensity on active and passive intestinal carbohydrate (CHO) absorption. Eight trained runners (age = 23 ± 2 (SE) yr; VO2max = 62.1 ± 5.8 ml/kg/min) performed a 1-hr resting experiment and three 1-hr treadmill experiments at 30, 50, or 70% VO2max in a thermoneutral environment (22.0 ± 2.0 oC, 47.4 ± 4.2 %RH). Immediately prior to each experiment, euhydrated (specific gravity < 1.020) subjects initiated a 5-hr urine collection period after ingesting a solution (100 ml) containing the non-metabolizable sugars 3-O-methyl-D-glucose (5 g; 3MG; actively absorbed) and D-xylose (5 g; passively absorbed). Immediately post-exercise, subjects ingested water equal to their sweat loss. High pressure liquid chromatography was used to quantify the amount of these CHO’s in the urine. Intestinal absorption was assessed as the urinary excretion of each CHO (i.e., percentage excreted of the administered dose). There were no significant differences in final urine volume (1.35 + 0.1 L) or in final urine specific gravity (1.07 + 0.01) among trials indicating renal function was not affected by exercise intensity. For 3MG, subjects excreted 45.4 ± 5.1%, 51.2 ± 2.3%, 43.9 ± 3.0%, and 35.7 ± 3.8% during the rest, 30%, 50%, and 70% VO2max trials, respectively. For D-xylose, subjects excreted 32.2 ± 3.8%, 34.0 ± 2.0%, 28.9 ± 1.7%, and 21.1 ± 2.4%, respectively, during the same trials. A significant (P < 0.05) reduction in urinary excretion of each CHO was observed at 70% VO2max compared to the other intensities suggesting that both active and passive intestinal absorption of CHO may be reduced during prolonged running at this intensity. Supported by the Gatorade Sports Science Institute

POSTER 7

Neuronal AT1 Receptor Upregulation in Heart Failure: Activation of AP-1 and JNK Dongmei Liu, Lie Gao, Shyamal K. Roy, Kurtis G. Cornish, Irving H. Zucker The University of Nebraska Medical Center, Department of Cellular and Integrative Physiology, Omaha, NE 68198-5850

Angiotensin II (Ang II) plays an important role in the development and progression of chronic heart failure (CHF). This study investigated the Ang II type I receptor (AT1R) in the rostral ventrolateral medulla (RVLM) of rabbits with CHF, its downstream pathway and gene regulation of the transcription factor AP1. The data (Table) show that AT1R mRNA and protein expressions, plasma Ang II, and AP-1 DNA binding activity were significantly higher in CHF than in Sham. The analysis of the SAPK/JNK pathway showed phosphorylated c-Jun proteins, phosphorylated JNK proteins and JNK activity increased significantly in CHF compared to sham. Importantly, intracerebroventricular (ICV) injection of Losartan in CHF rabbits attenuated these increases. ICV injection of Ang II in normal rabbits simulated the molecular changes seen in CHF. This effect was blocked by ICV losartan. We also observed Ang II-induced-AT1R expression was blocked by losartan and Jnk Inhibitor II in neuronal cell culture. These data suggest that central Ang II may activate the AT1R, SAPK/JNK pathway. AP1 may further regulate gene expressions in sympathetic neurons in the CHF state.

*p<0.05, **p<0.01. EF, Ejection Fraction; a: ratio to control group; b: gray level density; c: ratio to GAPDH house keeping protein. d: ratio to total protein. Supported by NIH grant PO-1 HL 062222.

EF, % 75.7±4.2 36.3±4.2* 41.6±3.7* 79.6±6.2 74.2±3.3 Ang II (pg/ml)

15.6±2.4 43.8±5.7* 18.9±11.2 13.5±9.9 21.2+11.6

aAT1R mRNA

1.08±0.33 2.46±0.67** 1.40±0.44 2.28±0.4 1.22±0.52

cAT1R Protein

0.45±0.12 0.93±0.26* 0.51±0.15 0.88±0.18 0.42±0.11

bAP-1 DNA Binding

21150±508 43872±3481** 27224±7711 39086±3001 25061±4600

ac-Jun mRNA

1.06±0.41 2.08±0.58* 1.2±0.29 1.98±0.21 1.13±0.16

dp-c-Jun Protein

0.27±0.10 0.71±0.13** 0.38±0.07 0.68±0.11 0.23±0.06

dp-Jnk protein

0.36±0.05 0.77±0.1** 0.40±0.06 0.67±0.08 0.42±0.04

bJnk-activity 38875±2859 52076±3917** 40315±4180 50876±4830 42315±5291

POSTER 8

Cardiac Sympathetic Afferent Stimulation Inhibits the Arterial Baroreflex in Heart Failure at the Level of the Nucleus of the Solitary Tract W-Z Wang, Y-X Pan, L. Gao, I.H. Zucker, W. Wang Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, NE 68198 -5850

The enhanced cardiac sympathetic afferent reflex (CSAR) has been demonstrated to contribute to increased sympathetic outflow and blunted arterial baroreflex function in chronic heart failure (CHF). Recently, it was demonstrated that central angiotensin II (Ang II) is involved in the interaction between the arterial baroreflex and the CSAR in CHF. The nucleus of the solitary tract (NTS) modulates these cardiovascular reflexes. The present study is designed to determine the roles of the NTS -Ang II mechanism in the interaction between the baroreflex and the CSAR using single-unit extracellular recording techniques in anesthetized and paralyzed rats with CHF. CHF was produced by coronary artery ligation. Of 48 spontaneously discharging NTS cells tested for baroreceptive sensitivity by elevation of arterial pressure (35 ± 6 mmHg), 26 (resting activity: 4.7 ± 0.9 spikes/sec) were recorded in CHF rats and 10 (resting activity: 6.2 ± 1.1 spikes/sec) were tested in sham rats. Activation of CSAR by epicardial application of capsaicin (0.4 µg) powerfully (P<0.01) attenuated the barosensitivity of 12 NTS baroreceptive neurons by 86.2 ±9.3%, and attenuation of the magnitude of barosensitivity by CSAR activation was significantly greater in CHF rats than in sham rats (65.7 ± 6.3%, n=10, P<0.01). In 8 cells from CHF rats, tonic inhibition of cardiac sympathetic afferent input by epicardial lidocaine (6 µ l) effectively (P<0.01) sensitized (improved) the barosensitivity of NTS neurons by 42.8 ± 5.9%. In the remaining 8 cells from CHF rats, intravenous injection of the Ang II AT1 receptor antagonist losartan (2 mg/kg) not only markedly sensitized the neuronal barosensitivity by 83.1±7.2% but also effectively reversed the inhibitory effect of CSAR activation on neuronal barosensitivity (-84.7 ± 9.6 vs. -12.5 ± 4.8 %, P<0.01). In conclusion, the present results suggest that the NTS plays an important role in mediating the blunting of the arterial baroreflex by activation of the CSAR via an AT1 receptor dependent mechanism in CHF.

POSTER 9 Disruption of the Cardiovascular Circadian Rhythm in Mice Post Myocardial Infarction: Relationship to Central Angiotensin II Receptor Expression Tarek M. Mousa and Irving H. Zucker Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, Nebraska 68198-5850

Angiotensin II (Ang II) is well known to participate in the abnormal autonomic cardiovascular control that occurs during the development of chronic heart failure (CHF) following acute myocardial infarction (MI). Disrupted cardiovascular circadian rhythm in CHF is also well known; however, the mechanism/s underlying and the proposed role of central Ang II type 1 receptors (AT1) and oxidative stress in mediating such changes is not clear. In a post MI CHF C57BL/6 mouse model we investigated the circadian rhythm for blood pressure (BP), heart rate (HR), and baroreflex sensitivity (BRS) following MI. Data were acquired using radiotelemetry in the conscious unrestrained state. BRS was assessed using the spontaneous baroreflex sequence technique. We measured central AT1 expression and the gp91phox subunit of NAD(P)H oxidase as a marker of oxidative stress. Results are shown in the table. The cardiovascular parameters represent the middle 6 hour averages during daytime (6:00-18:00) and nighttime (18:00-6:00). Baseline HR positively correlated with the severity of CHF reaching its maximum by 12 wks post MI; loss of circadian HR & BRS rhythms were observed as early as 4 wks post MI in conjunction with a significant blunting in the BRS and an up-regulation in the AT1 and gp91phox proteins. Loss of BP circadian rhythm was observed 8 wks post MI. The loss of circadian rhythm for BP was due primarily to post MI mice becoming non dippers during the daytime hours. The disruption of circadian rhythm for HR, BP and BRS along with an early up regulation of AT1 and gp91phox suggests a possible role for central oxidative stress as a mediator of circadian cardiovascular parameters in the post MI & CHF states.

Sham 4 wks MI 8 wks MI 12 wks MI 16 wks MI

Day Night Day Night Day Night Day Night Day Night

MAP (mmHg) 99.4±1 118.2±2* 106.5±1 114.2±1* 117.2±2 122.9±2 113.0±2 112.2±2 116.4±4 116.8±2HR (bpm) 590±7 624.6±7* 654.8±6 668.2±6 705.6±8 723.3±5 731.4±11 732.6±4 694.6±9 704.7±12

BR S(bpm/mmHg) 2.74±0.3 2.54±0.3* 1.36±0.1† 1.25±0.1† 1.1±0.2† 1.12±0.1† 1.1±0.2† 1.14±0.1† 1.14±0.3† 1.12±0.3†

AT1 protein 0.3±0.03 0.51±0.08 0.55±0.02 0.55±0.11 0.51±0.07 gp91 protein 0.35±0.01 0.59±0.06 0.59±0.03 0.59±0.03 0.58±0.01

Data are mean ± SEM * p<0.05 compared to daytime within the same group (paired t test). † p< 0.05 compared to respective sham group

POSTER 10 Sympatho-Excitation in Chronic Heart Failure: Ang II Induced Inhibition of Voltage-Gated K+ Channel – an In Vivo and In Vitro Study Lie Gao, Wei Wang, Ethan Mann, Marcus Finch, Yulong Li, Dongmei Liu, Harold D. Schultz, and Irving H. Zucker Department of Cellular and Integrative Physiology, University of Nebraska, Omaha, Nebraska 68198-5850

We have reported that elevated central Ang II plays a critical role in sympathoexcitation of chronic heart failure (CHF) by stimulating up-regulated AT1 receptors in rostral ventrolateral medulla (RVLM). However, the link between enhanced Ang II signaling and electrophysiological characteristics of neurons in RVLM remains unclear. In the present experiments, we first screened for potentially altered genes in the brainstem of rats with CHF that relate membrane conductance in neurons using the Rat Genome 230 2.0 Array GeneChip. We found that Kv 4.3 gene was significantly down-regulated in the CHF rats (-2.1 ± 0.2 times, P < 0.05, n = 3). Second, we used real-time PCR and Western blot further to confirm this down regulation of Kv 4.3 in the RVLM of CHF rats (mRNA: 1.3 ± 0.1 to 0.7 ± 0.1, P < 0.05; protein: 2.0 ± 0.1 to 0.9 ± 0.1, P < 0.05). Finally, we used a neuronal cell line (CATH.a neurons) to explore the effect of Ang II on Kv 4.3. We found that Ang II treatment (1) down-regulated the mRNA (0.4 ± 0.1 to 0.2 ± 0.1, P < 0.05) and protein (1.5 ± 0.2 to 0.6 ± 0.1, P < 0.05) of Kv 4.3; (2) decreased the K+ peak current (Ik: 289.6 ± 31.5 to 143.3 ± 8.9 pA, P < 0.05); and (3) up-regulated the mRNA (0.7 ± 0.1 to 1.8 ± 0.2, P < 0.05) and protein (0.8 ± 0.1 to 1.5 ± 0.2, P < 0.05) of the AT1 receptor. We conclude that the sympathoexcitation of CHF may be due to, in part, the effect of Ang II down-regulating the Kv 4.3 expression and decreasing Ik, and thereby increasing the excitability of neurons in the RVLM, via the enhanced AT1 receptor expression. Supported by NIH grant HL PO-1 62222.

POSTER 11 Angiotensin II-Mediated Sympathoexcitation in Diabetes: Role of Superoxide Hong Zheng*, Xuefei Liu*, Allison Kleiber*, Robin L. Davisson** & Kaushik P. Patel* Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, Nebraska 68198-5850. **Department of Anatomy and Cell Biology, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, Iowa 52242

Our previous study has demonstrated an enhanced angiotensin II (Ang II) mediated sympathoexcitation in diabetes. Recent studies have shown that a superoxide mechanism is involved in Ang II signaling in the central nervous system. We hypothesized that Ang II activates sympathetic outflow by stimulation of superoxide within the paraventricular nucleus (PVN) of diabetic rats. In a-chloralose and urethane anesthetized rats, microinjection of AngII into the PVN (50, 100 and 200pmol) produced dose-dependent increases in renal sympathetic nerve activity (RSNA), arterial pressure (AP) and heart rate (HR) in control rats and streptozotocin (STZ)-induced diabetic rats. There was a potentiation of the increase in RSNA (27.7±2.1% vs. 19.1±1.1%, P<0.05), AP and HR due to Ang II AT1 receptor activation in diabetic rats compared to control rats. In diabetic rats, significant increases in mRNA and protein expression of NAD(P)H oxidase subunits p22phox, p47phox and p67phox in the PVN were observed. p47phox mRNA expression increased 36.0±4.0% and p47phox protein level increased 31.1±1.7% (n=6, P<0.05). The increased responses to Ang II were attenuated by pretreatment with adenoviral vector-mediated over-expression of human mitochondrial superoxide dismutase (AdMnSOD) within the PVN of the diabetic rats (RSNA 20.4±0.7% vs. 27.7±2.1%, n=6, P<0.05). These data support the conclusion that superoxide contributes to enhanced Ang II-mediated signaling in the PVN involved with the exaggerated sympathoexcitation in diabetes.

POSTER 12 Influence of Diabetes Mellitus and Exercise Training on Oxidative Stress Repair Mechanisms in Cardiac Tissue Mary Connealy, Cory Ciccone, Karynn Kucera, and Janet Steele Department of Biology, University of Nebraska at Kearney, Kearney, NE 68849

Diabetes mellitus and exercise training both induce oxidative stress and reactive oxygen species formation (ROS). Recent work has demonstrated that intracellular glutathione (GSH), an endogenous antioxidant, plays an important role in preventing electrochemical changes in the failing heart, particularly those changes which affect K+ channels. Specifically, augmentation of GSH has been shown to upregulate the density of transient outward K+ currents. The goal of this study was to examine antioxidant formation and enzyme efficiency in pathways limiting the damage caused by ROS in cardiac tissue. Fifty female Sprague-Dawley rats served as subjects. Half the rats exercise-trained by swimming up to 2 hr per day for 10 weeks while the others served as non-trained controls. Half the rats in each group were made diabetic via i.p. injection of streptozotocin. Rats were euthanized via overdose of sodium pentobarbital and hearts removed. Total GSH and the activity of glutathione reductase (GR) and aconitase (ACON) were analyzed in cardiac tissue. Results suggest that diabetes mellitus, exercise training, or a combination of the two, did not significantly alter the formation of GSH or the activity of GR or ACON. This work was supported by NIH grant 1 P20 RR16469 BRIN Program of the National Center for Research Resources.

POSTER 13 Influence of Diabetes Mellitus and Exercise Training on Gene Expression in Cardiac Muscle Tissue Cory Ciccone, Mary Connealy, Karynn Kucera, and Janet Steele Department of Biology, University of Nebraska at Kearney, Kearney, NE 68849

Cardiac gene expression distinguishes the heart with physiological left ventricular hypertrophy (LVH) from the heart with pathological LVH. The failing heart undergoes a remodeling process that is accompanied by electrical changes in K+ channels, and oxidative stress has been identified as a key factor in this process. The goal of this study was to examine changes in gene expression caused by oxidative stress brought on by diabetes mellitus and exercise training. Fifty female Sprague-Dawley rats served as subjects. Half the rats exercise-trained by swimming up to 2 hr per day for 10 weeks while the others served as sedentary controls. Half the rats in each group were made diabetic via i.p. injection of streptozotocin while the remaining rats were injected with citrate vehicle. Rats were euthanized via overdose of sodium pentobarbital and hearts removed. RNA was isolated from cardiac tissue and microarray analysis performed. Preliminary results suggest that 1) diabetes mellitus upregulates genes for glutathione synthase, 2) diabetes mellitus upregulates genes for enzymes such as acyl-CoA dehydrogenase and thiolase, which are involved in lipid metabolism, and 3) exercise training upregulates genes for voltage-gated K+ channels. This work was supported by NIH grant 1 P20 RR16469 BRIN Program of the National Center for Research Resources.

POSTER 14 Testosterone does not Increase Kidney Angiotensin II in Experimental Diabetes Pascale H. Lane, William J. Langer, Kay Devish, and Jianhong Sun Department of Pediatrics, University of Nebraska Medical Center, Omaha, NE.

Kidney diseases, including diabetes mellitus (DM), accelerate during puberty. Rats with experimental DM induced before puberty show less renal and glomerular growth over 6 weeks than those with adult onset. Androgens, which rise at puberty, are reported to promote kidney growth and stimulate the intrarenal renin-angiotensin system (RAS). To test the interrelationship of these factors, we examined effects of early testosterone treatment in juvenile or castration in adult rats. Juveniles included intact animals with or without testosterone treatment given streptozocin or vehicle at 4 weeks of age. Adults received streptozocin or vehicle at 14 weeks of age; groups included intact animals, animals castrated during the second week of life, and castrated animals given testosterone. Insulin palmitate pellets were placed to maintain glucose levels of 300-400 mg/dl. After 6 weeks of study rats underwent conscious decapitation. Trunk blood and kidney were immediately chilled and processed for measurement of angiotensin II (AngII). Results were compared with two-factor ANOVA; factors were metabolic state and hormonal state. Post-hoc comparisons were made to the reference group, intact adults matched for metabolic state, via Holms-Sidak method.

Plasma AngII (left graph) was lower with DM, and castration also reduced it. In nondiabetic juvenile animals, testosterone raised AngII substantially. DM also lowered kidney AngII content, particularly in the juvenile groups (right graph). Testosterone treatment in nondiabetic juveniles significantly reduced renal AngII. These data do not support a detrimental role for androgens via the intrarenal RAS in DM kidney growth.

POSTER 15 Kidney Gene Expression in Diabetes Pre- and Post-Puberty Pascale H. Lane and William J. Langer Department of Pediatrics, University of Nebraska Medical Center, Omaha, NE

Many kidney diseases accelerate during puberty, including diabetes mellitus (DM). Rats with experimental DM induced prior to puberty show less renal and glomerular hypertrophy over 6 weeks than those with adult onset. Transforming growth factor β appears to be differentially regulated; however, it is likely that other genes are as well. Male littermates had one member of each litter assigned to each of these groups: Young Control, Young DM, Adult Control, and Adult DM. Young groups received streptozocin, 65 mg/kg IV, or vehicle at 4 weeks of age. Adult groups received similar treatment at 14 weeks of age. Insulin pellets were placed 3 days after injection to maintain blood glucose 300-400 mg/dl for 6 weeks. Under isoflurane anesthesia the kidneys were removed and snap-frozen in liquid nitrogen. Once all animals had completed the protocol, RNA was isolated and analyzed with a commercial array including more than 28,000 genes. Data were analyzed by two-factor ANOVA. Differences; 1.7-fold with p<0.05 were considered significant; 4,278 genes met these criteria. Known profibrotic growth factors were differentially regulated, including connective tissue growth factor (CTGF) and components of the transforming growth factor &beta; signaling pathway. Primers were designed and real time RT-PCR used to confirm age-specific upregulation of CTGF in DM, and CTGF protein was assessed via immunoblotting.

Many biological processes are differentially regulated in response to DM pre-and post-puberty in the male rat, including CTGF. Further study will likely result in new directions for experimentation at the level of the gene, protein, and integrated function.

POSTER 16 Role of Cyp2E1 in Impaired Nitric Oxide Synthase Nos-Dependent Cerebral Vasodilation during Chronic Alcohol Consumption Hong Sun, Denise M. Arrick, and William G. Mayhan Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, NE 68198-5850

Oxidative stress is associated with alcohol-induced impairment of NOS-dependent cerebral vasodilation. We determined whether CYP2E1 and xanthine oxidase contribute to impaired NOS-dependent dilation of cerebral arterioles during alcohol consumption. Using a cranial window, we measured effects of topical application of a CYP2E1 inhibitor, diallyl sulfide, and a xanthine oxidase inhibitor, allopurinol, on responses of parietal pial arterioles to eNOS-dependent agonists (Ach and ADP), a nNOS-dependent agonist (NMDA), and a NOS-independent agonist (nitroglycerin) in nonalcohol-fed and 2-3 months alcohol-fed rats. Protein expression of CYP2E1 in parietal cortex and pial arterioles was measured by Western blot analysis. Response of pial arterioles to nitroglycerin was similar in nonalcohol-fed and alcohol-fed rats. Compared to nonalcohol-fed rats, however, vasodilation to Ach (1 and 10 mM), ADP (10 and 100 mM), and NMDA (0.1 and 0.3 mM) was significantly less in alcohol-fed rats. Topical application of diallyl sulfide (1 mM) did not alter vasodilation in nonalcohol-fed rats, but significantly improved vasodilation to Ach, ADP, and NMDA in alcohol-fed rats. The expression of CYP2E1 was not altered in parietal cortex and pial arterioles of alcohol-fed rats. In addition, topical application of allopurinol (0.3 mM) had no effect on responses of cerebral arterioles in nonalcohol-fed and alcohol-fed rats. Our findings suggest that CYP2E1, but not xanthine oxidase, may play an important role in impaired NOS-dependent dilatation of cerebral arterioles during alcohol consumption.

POSTER 17 Role for Poly (Adp-Ribose) Polymerase (Parp) Activation in Impaired Responses of Cerebral Arterioles in Type 1 Diabetes Mellitus (T1D) Denise M. Arrick, Hong Sun, Glenda Sharpe and William G. Mayhan Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, NE 68198 -5850.

An important component of T1D-induced endothelial dysfunction is the formation of oxygen derived free radicals. Since PARP may contribute to the formation of oxygen radicals, the goal of the current study was to investigate the role of PARP in T1D-induced cerebral endothelial dysfunction. Craniotomies were performed on nondiabetic and streptozotocin-induced diabetic rats in order to measure changes in diameter of parietal pial arterioles to a NOS-independent agonist (nitroglycerin) and eNOS-dependent agonists (Ach and ADP). Comparison of nondiabetic and diabetic rats revealed that cerebral dilation was impaired to ADP and Ach, but not to nitroglycerin. Arteriolar response was then measured in the presence of PJ34 (1 µM), a phenanthridinone based PARP inhibitor. PARP inhibition did not alter reactivity of pial arterioles to nitroglycerin, ADP or Ach in nondiabetic rats, but restored vasodilation to ADP and Ach in diabetic rats. In contrast, PARP inhibition did not alter dilation of pial arterioles to nitroglycerin in diabetic rats. These findings suggest that PARP may play an important role in the pathogenesis of endothelial dysfunction of cerebral resistance arterioles during T1D. Supported by NIH grant HL 079587.

POSTER 18 Functional and Immunohistochemical Characterization of Hyperpolarization Activated Current in Rat Suprachiasmatic Nucleus Nermien Waly 1 and Richard Hallworth 2

1 Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, NE 68198-5850. 2 Biomedical Sciences Department, School of Medicine, Creighton University, Omaha, NE, 68178.

The hyperpolarization-activated current (Ih) is a mixed Na+/K+ inward current that is believed to regulate a wide variety of physiological function in both the central nervous system (CNS) and in the heart. It plays an important role in suprachiasmatic nucleus (SCN), which is the circadian pacemaker, where it regulates neuronal excitability by limiting the duration of hyperpolarizing events. The mammalian Ih is encoded by four members of the Hyperpolarization-activated and Cyclic-Nucleotide gated non selective cation channel (HCN) gene family (HCN1-4). There was a conflict in previous reports regarding the fraction of SCN neurons that exhibited Ih. So, the hypothesis that whether or not HCN channel subunits are found within the SCN was tested. Using immunohistochemistry the distribution of HCN1, HCN2, and HCN4 in the SCN was studied. Ih was also recorded from SCN neurons using whole cell voltage clamp. The results show that Ih is functionally and anatomically well expressed in the SCN neurons. Both HCN1 and HCN2 subunits are present in the SCN but with different patterns of localization. HCN4 was not detected in the SCN. 84% of SCN neurons exhibited Ih. Ih recorded had the activation constant (τ) of 236±2 ms and amplitude of 25±1 pA at -40 mV step. At -60 mV step, τ was significantly reduced to 167±3 ms (p<0.5) while the current amplitude was significantly increased to 34±2 pA (p<0.01). In conclusion Ih channel subunits abundant in the SCN; the kinetic properties of the recorded Ih resemble those of HCN2 homomers. These results support the hypothesis that Ih may play a major role in the SCN excitability. This research was completed at the Biomedical Sciences Department of Creighton University

POSTER 19 Downregulation of Carbon Monoxide as well as Nitric Oxide Contributes to Peripheral Chemoreflex Hypersensitivity in Heart Failure Rabbits YanFeng Ding, Yu-Long Li, Harold D. Schultz Department of Integrative and Cellular Physiology, University of Nebraska Medical Center, Omaha NE 68198-5850

Peripheral chemoreflex sensitivity (PCS) is potentiated in both clinical and experimental chronic heart failure (CHF). Downregulation of carotid body (CB) nitric oxide (NO) synthase contributes to this effect. However, it remains poorly understood whether carbon monoxide (CO) also contributes to the altered PCS in CHF. This work highlights the effect of NO and CO on renal sympathetic nerve activity (RSNA) in response to graded hypoxia in conscious rabbits. RSNA responses to graded hypoxia were enhanced in CHF rabbits compared with that in sham rabbits. The NO donor (S-nitroso-N-acetylpenicillamine, SNAP, 1.2 µg/kg/min) or CO donor (tricarbonyldichlororuthenium (II) dimmer, [Ru(CO)3Cl2]2, 3.0 µg/kg/min) alone attenuated hypoxia-induced increases of RSNA in CHF rabbits (P<0.05) but no additive effect was found in SNAP plus [Ru(CO)3Cl2]2. Conversely, NO synthase (NOS) inhibitor (Nω-nitro-L-arginine methyl ester, LNAME, 30mg/kg) or the heme oxygenases (HO) blocker (Cr (III) mesoporphyrin IX chloride, CrMP, 0.5 mg/kg) alone augmented RSNA response to hypoxia in sham rabbits but LNAME plus CrMP didn’t induce an additive effect. In addition, protein expressions of nNOS (0.18±0.08) and HO-2 (0.11±0.03) in carotid bodies from CHF are lower than those from sham rabbits (0.64±0.06 and 0.28±0.02, respectively). These results suggest that a deficiency of both NO and CO in the carotid body augments PCS to hypoxia in CHF.

POSTER 20 Endogenous Neuronal Nitric Oxide Systhase and AT1 Receptor do not Correlate the Enhanced Peripheral Chemoreflex Function in Early Stage of Chronic Heart Failure Rabbits Yu-Long Li, Chad Agnew, Harold D. Schultz Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, NE 68198-5850

Our previous study has shown that decreased endogenous nNOS activity and increased expression of AT1 receptor in the carotid body (CB) play an important role in the enhanced peripheral chemoreflex function in chronic heart failure (CHF, 3-4 week pacing). Here we will investigate the relationship between the peripheral chemoreflex function and these two endogenous modulators in the early stage of CHF (one-week pacing). Renal sympathetic nerve activity (RSNA) in response to graded hypoxia was measured in sham and one-week pacing rabbits. One week pacing enhanced the response of RSNA to graded levels of isocapnic hypoxia, compared with that in sham rabbits (33.8 ± 5.1 % versus 40.3 ± 4.2 % at 60.5 ±3.9 mm Hg and 51.3 ± 4.9 % versus 62.6 ± 3.6 % at 40.3 ± 3.3 mm Hg, p<0.05). We also measured the protein expression of nNOS and AT1 receptor in CB using immunofluorescent and western blot analyses. The nNOS and AT1 receptor protein expressions were not different between sham and one-week pacing rabbits. These results suggest that endogenous nNOS and AT1 receptor in CB were not involved in the enhancement of RSNA in the early stage of CHF.

POSTER 21 Enhancement of Cholinergic Activity Elicited Cardiac Protection and Anti-Inflammatory Effect Yi-Fan Li, Jessica Freeling, Kristina Wattier, Carly LaCroix Division of Basic Biomedical Sciences, University of South Dakota, Vermillion, SD 57069

Enhancement of parasympathetic nerve activity exerted beneficial effect on congestive heart failure (CHF). However, the underlying mechanisms remain to be determined. The present study was to investigate if this beneficial effect involves regulation of pro- and anti-inflammatory cytokines in the heart. Transverse aortic constriction (TAC)-induced cardiac hypertrophy and dysfunction in rats was used in the present study. To augment cholinergic activity, a low dose of a peripheral cholinesterase inhibitor, neostigmine (Neo, 0.01 mg/kg per day) was injected subcutaneously for two weeks in TAC and sham rats. There was a slight decrease in heart rate in Neo treated group (318±25 bpm), compared with saline group (341±23 bpm), indicating an increase in parasympathetic control of the heart. The ratios of heart weight vs. body weight were 3.46±0.10 in TAC-Neo group and 4.47±0.45 in TAC-saline group, suggesting a significant reduction in TAC-induced cardiac hypertrophy by Neo. The treatment with Neo also preserved left ventricular function in TAC. Moreover, the protein level of pro-inflammatory cytokine TNFα was decreased in heart tissues in the TAC rats treated with Neo compared with saline control. In contrast, the level of anti-inflammatory cytokine IL-10 was increased in the TAC rats treated with Neo. Furthermore, in vitro study showed that co-incubation with acetylcholine (5 uM) attenuated phenylephrine-induced TNFα release in primary cultured neonatal cardiomyocytes. These data suggest that augmentation of parasympathetic activity may exert cardiac protection at least in part via regulation of the productions of pro- and anti-inflammatory cytokines in the heart. Supported by American Heart Association Beginning Grant-in-Aid 0460063Z, NIH INBRE Grant No. 2 P20 RR016479, and NIH COBRE grant No. 5 P20 RR017662

POSTER 22 Aging-Related Cardiac Dysfunction in Mice – The Role of ErbB Tyrosine Kinase Receptors Viswanathan Rajagopalan, Irving H. Zucker, Tarek M. Mousa, Ying J. Ma Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, Nebraska 68198-5850

Background: Aging is an important independent risk factor for cardiovascular disorders. Multiple factors are known to be involved in age-related cardiac dysfunction. Evidence from the literature indicates that conditional ErbB tyrosine kinase receptor gene knock-outs and ErbB antibody (e.g. Trastuzumab-Herceptin) used for cancer therapy demonstrate cardiac dysfunction. Unpublished data from our lab shows that a conditional ErbB mutation leads to cardiac dysfunction in young adult mice. We thus investigated the cardiac physiological and molecular (ErbB receptors) changes in aged mice. Methods: Mice were selected in three different age groups; Young Adult (YA) 4-7 months, Middle Aged (MA) 9-12 months and Aged Adult (AA) mice 18-22 months (n = 4-5 per group). Mice underwent echocardiography using the VEVO 770 (Visual Sonics, Inc.) to assess cardiac function. We also measured LV pressures and dP/dt values (measures of cardiac contractility) in conscious mice using Radiotelemetry (Data Sciences Intl). Heart rates during echocardiography were maintained within normal limits. Standard procedures of RT-PCR and Western Blotting were employed to study the expression profile of ErbB receptors. One-way ANOVA and Newman-Keuls Multiple Comparison Test were used for statistical analyses and results are tabulated as mean±sem. Results: Hemodynamic data are shown in the table. Echocardiography showed that the left ventricular (LV) mass, LV Posterior Wall dimension (indicating hypertrophy), and aortic diameter increased significantly and progressively with increase in age. Ejection Fraction (EF) and Fractional Shortening (FS) were decreased in the aged mice, although they did not reach statistical significance. LV End Diastolic Pressure (LVEDP) was significantly increased in aged mice (AA>MA>YA) while, LV dP/dt was significantly decreased in aged mice (AA<MA<YA). ErbB-1 and ErbB-4 receptor expression of mRNA, protein and phospho-protein were found to be significantly diminished in AA as compared to YA. Conclusion: These observations demonstrate the age-related physiological and molecular cardiac dysfunction in mice. Future experiments will focus on dissecting the role of ErbB receptors in cardiac aging and age-related cardiac dysfunction. Parameters YA MA AA P-value

YA vs AA P-value

YA vs MA P-value

MA vs AA LVEDP (mmHg)

5.4±0.5 12.3±0.7 22.8±0.8 <0.001 <0.001 <0.001

LV dP/dt (mmHg/s)

7831±561.3 5995±483.9

3420±494.2 <0.001 <0.05 <0.001

LVMass (mg/g bw)

3.2 ±0.3 3.7±0.2 4.5±0.2 <0.01 ns

<0.05

LVPWd (mm) 0.8±0.1 0.99±0.1 1.1±0.1 <0.05 ns ns Aortic Diameter (mm)

1.3±0.1 1.6±0.1 1.7±0.03 <0.01 <0.05 ns

Ejection Fraction (%)

66.6±5 59.7±5 56.2±2 ns ns ns

Fractional Shortening (%)

36.7±4 31.5±3 28.9±1 ns ns ns

ns - not significant

POSTER 23 Protein Kinase C-dependent Superoxide Production by the Renal Medullary Thick Ascending Limb in Normal and High Glucose Environments Pamela K. Carmines1, William G. Mayhan1, Jennifer S. Pollock2 1Univ. of Nebraska College of Medicine, Department of Cellular and Integrative Physiology Omaha, NE, 68198-5850 2Medical College of Georgia, Vascular Biology Center, Augusta, GA, 39012

Experiments were performed to determine the enzymatic source of glucose-induced superoxide anion (O2

•–) production by the medullary thick ascending limb (mTAL) and the involvement of protein kinase C (PKC) in this phenomenon. mTAL suspensions prepared from normal rat kidney were incubated 30 min in normal (5.5 mM; NG) or high (20 mM; HG) glucose media. O2

•– production (lucigenin chemiluminescence) by the mTALs averaged 484 ± 107 RLU/sec/mg protein (n = 9) in NG media and was significantly greater in HG media (1146 ± 297 RLU/sec/mg; n = 8). Pre-incubation in either 100 μM apocynin (APO; NAD(P)H oxidase inhibitor) or 250 μM L-NAME (NO synthase inhibitor) did not alter O2

•– production in NG media, but prevented the stimulatory impact of HG (HG + APO, 204 ± 92 RLU/sec/mg; HG + L-NAME, 490 ± 168 RLU/sec/mg; both P > 0.05 vs NG). PKC inhibition (30 μM chelerythrine) virtually abolished O2

•– production in both NG and HG media. PKC activation (1 μM PMA in NG media; 30 min) markedly increased O2

•– production (4712 ± 808 RLU/sec/mg), a response that was ~50% abated by pretreatment with APO, L-NAME, or APO + L-NAME (2389 ± 333, 2392 ± 302, and 2806 ± 467 RLU/sec/mg, respectively). We conclude that both NAD(P)H oxidase and NO synthase contribute to glucose-stimulated O2

•– production by the mTAL. PKC is essential for O2

•– production under basal and glucose-stimulated conditions. Moreover, PKC-stimulated O2•–

production by the mTAL involves, at least in part, NAD(P)H oxidase and NO synthase acting in a sequential manner. Supported by NIH DK63416

POSTER 24 In Vitro Differentiation of Bone Marrow Mesenchymal Stem Cells into Renal Tubular Epitheliallineage Kurinji Singaravelu and Babu J. Padanilam Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, Nebraska 68198-5850

Bone marrow stem cells (BMSCs) have been shown to differentiate in to a number of renal lineages including renal tubular epithelium, mesangial cells, podocytes, interstitial cells and renal vascular endothelium in different in vivo renal injury models. Nevertheless, the in vitro differentiation of bone marrow cells in to a renal lineage has not been investigated. We hypothesize that factors secreted by injured renal tubular cells may facilitate the differentiation of pluripotent BMSCs to differentiate into renal lineages. In this study, we investigated the potential of bone marrow mesenchymal stem cells for in vitro renal lineage differentiation by co-culturing injured renal tubular cells with mesenchymal stem cells (MSCs). Mouse MSCs were isolated from bone marrow and cultured for 2 to 3 weeks. to semiconfluence. Mouse cortical tubular epithelial cell line (MCTs) was cultured in the Transwell insert and injured using H2O2. The injured MCT cells were then co-cultured with MSCs in the Transwell system to facilitate the interactions between MCTs and MSCs without cell fusion. MSCs cultured alone served as control. The co-culturing system was incubated for up to 7 days and MSCs were examined for the expression of the renal specific epithelial marker, kidney-specific cadherin (ksp-cadherin) and the renal proximal tubular cell marker, aquaporin 1 (AQP1). The expression of ksp-cadherin and AQP1 on MSCs was detected as early as day 3 by immunocytochemistry followed by light microscopy or confocal microscopy. There was widespread expression of ksp-cadherin and AQP1 at day 5 and 7 compared to that at day 3 in co-cultured MSCs. No ksp-cadherin or AQP1 immunostaining was detected on MSCs that were cultured alone. MCT cells were used as a positive control for the expression of ksp-cadherin and AQP1. We conclude that bone marrow-derived mesenchymal stem cells might differentiate into renal cells independent of cell fusion when co-cultured with injured renal cells. The use of an in vitro system to facilitate differentiation of MSCs to renal cells offers new opportunities for using the model to investigate early development, drug discovery and generating cells for replacement therapy.

POSTER 25 The Localization of the Beta-1, -2, and -4 Subunits to the Large Conductance, Ca2+- Activated K+ Channel (BK) within the Mouse Kidney P. Richard Grimm, Ruth M. Foutz, Peilin Wei, Jennifer L. Pluznick and Steven C. Sansom Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, Nebraska 68198 -5850

The BK channel is comprised of pore-forming alpha subunits and one of four accessory beta subunits which confer variable properties to the channel. The alpha subunit is expressed in many segments of the nephron; however, less is known about the location of the various beta subunits. We recently showed the presence of the beta-1 subunit in the apical membrane of the CNT and that it was essential for flow-mediated K+ secretion. We propose the expression of the different beta subunits throughout the nephron help to explain BK's function within that section of the tubule. Although the CNT is comprised of a heterogeneous mixture of three cell types, many studies have concluded that principal cells (PC) of the distal nephron (including the connecting-tubule cells of the CNT) are ideally suited to respond to mediators of K+ secretion. In immunohistochemical experiments, we found that peanut lectin (an intercalated cell (IC) marker) and a beta-1 subunit antibody labeled separate cells in the same tubule. To confirm these results, we used a beta-1 subunit antibody along with an antibody for the vacuolar hydrogen ATPase (V-ATPase), which labels intercalated cells (IC). Once again, we found that the beta-1 subunit and V-ATPase antibodies labeled separate cells. Using an antibody to the beta-4 subunit, we were able to localize it to the IC cells of the CNT and cortical collecting duct (CCD), as well as, to the proximal tubule and thick ascending limb (TAL). Lastly, we have been able to determine that within the mouse kidney the beta-2 subunit is expressed in specific regions of cortex and prominently in the outer medulla, although the exact tubule location has not been determined yet. We conclude that the beta-1 subunit is located exclusively within the apical membrane of the connecting-tubule cells of the CNT were it serves in flow-mediated K+ secretion, while the beta-4 subunit in the IC cells most likely plays a role in potassium recycling along with hydrogen/potassium exchanger (H /K+ exchanger). Supported by RO1-DK49561 and AHA fellowship.

POSTER 26 The Role of VASP in No/CGMP-Mediated Inhibition of SOC in Human Mesangial Cells Xiaoxia Wang, Jennifer L. Pluznick, P. Richard Grimm, Peilin Wei, Steven C. Sansom Department of Cellular and Integrative Physiology, University of Nebraska Medical Center Omaha, NE, 68198-5850

In several cell types, local nitric oxide (NO) production reduces proliferation, migration, matrix deposition and dedifferentiation by signaling mechanisms which lead to reductions in store-operated or receptor-operated Ca2+ entry. In mesangial cells, nitric oxide reduces cell contraction, expansion and collagen deposition; however, the effects of NO on mesangial Ca2+

entry pathways have not been determined. We used the fura–2 ratiometric method for measuring intracellular Ca2+ concentration [Ca2+]i to determine whether NO and the cGMP-activated protein kinase (PKG) signaling pathway governs [Ca2+]i by modulating store-operated Ca2+ channel (SOC) activity and if so, whether phosphorylation of S-239 site of vasodilator-stimulated phosphoprotein (VASP) was involved in this process. SOC, at least partially comprised of the TRPC4 molecules, was measured as the elevation in intracellular Ca2+ when increasing extracellular [Ca2+] from <0.01 nM to 1 mM in the continued presence of thapsigargin (thaps). In the presence of 100 µM SNP, an NO donor, we found that the SOC-TRPC4 response, which was 110 ±10 nM in control, was significantly attenuated to 55±10 nM. Similarly, application of 8-Br-cGMP (100 µM), a membrane permeable second messenger of NO, reduced the SOC-TRPC4 response significantly to 58 ±15 nM. Addition of DT-3 (0.25 µM), a specific PKG Iα inhibitor, completely reversed the SOC in response to 8-Br-cGMP. Application of 100 µM cAMP decreased the SOC-TRPC4 response slightly, but not significantly. RT-PCR revealed PKG Iα mRNA and immunocytochemical analysis revealed protein expression in the plasma membrane and cytoplasm of MC. We found no direct phosphorylation of TRPC4 by 8-Br-cGMP as detected by immunoprecipitation. However, VASP, a substrate for PKG at site S239, was shown to regulate migration and matrix deposition in vascular smooth muscle cells. We therefore hypothesized that VASP, abundantly expressed in HMC, played a role in the cGMP dependent protein kinase-mediated inhibition of TRPC4-SOC. Using Western blot analysis, we found that 8-Br-cGMP enhanced the phosphorylation of VASP at Ser239. When HMC were incubated with 8-Br-cGMP for 10 minutes, we found by co-immunoprecipitation that S239-VASP associated with SOC-TRPC4. Therefore, VASP may be an intermediary for the inhibition of SOC-TRPC4 by the NO-PKG pathway.

POSTER 27 Effects of Warm Acclimation and Papaverine on Serum Osmolality in Antarctic Fish H.A. Hudson¹, P.R. Brauer¹, M.A. Scofield², and D.H. Petzel¹ ¹Biomedical Sciences, ²Pharmacology, Creighton University School of Medicine, 2500 California Plaza, Omaha, NE, 68178

Antarctic fish have some of the highest serum osmolalities of any teleost which colligatively lower the freezing point of their blood. In response to warm acclimation, the Antarctic fish,Trematomus bernacchii, lowers its serum osmolality. The renin-angiotensin system (RAS), which regulates blood pressure, drinking rate, and serum ion composition, may be responsible for this change in serum osmolality. Few studies have been done regarding the renin-angiotensin system in Antarctic fish, however renin and angiotensin converting enzyme activity have been reported T. bernacchii. To determine if a functional renin-angiotensin system is operating in T. bernacchii, fish were acclimated to warmer temperatures for different periods of time and were subjected to 20% blood volume hemorrhage, 25% blood volume hypervolemia, or 10 mg/kg papaverine injection (a hypotensive agent). Serum was collected for serum osmolality and future renin measurements. After 2 days, fish acclimated at +4°C showed a significant decrease in osmolality (499 ± 1 mOsm/kg) when compared to the environmental control temperature of -1.5°C fish (547 ± 4 mOsm/kg; p<0.001), whereas fish acclimated at +1°C did not show a significant decrease in osmolality until 14 days (511 ± 14 mOsm/kg; p<0.05). Upon changing the blood volume and possibly blood pressure, the only significant change (p<0.05) in osmolality occurred in papaverine treated fish 180 min and 6 hr after injection (increasing from 550 ± 5 mOsm/kg to 585 ± 9 mOsm/kg and from 565 ± 8 mOsm/kg to 607 ± 12 mOsm/kg, respectively). These results showed serum osmolality was not affected by acute changes in blood volume, and that papaverine, a drug known to cause hypotension and renin release, changes osmolality within 3 hr. Thus, changes in RAS activity could be responsible for the relatively sudden decrease in serum osmolality observed in warm acclimated fish. These results suggest T. bernacchii may have an functional RAS. Supported by NSF #OPP-0229462.

POSTER 28 Warm Acclimation Affects Serum Osmolality and Na+/K+Atpase Activity in the Gilland Gut of Trematomus Bernacchii K. Smith1 , F. Dowd2 , P. Brauer1 , J. Morrison1 , M. Scofield 2, and D. Petzel1. Creighton University School of Medicine, Departments of 1Biomedical Sciences and 2Pharmacology, Omaha, NE, 68178

The purpose of this experiment was to determine the effects of warm acclimation from -1.5°C to +4°C on serum osmolality and Na+K+ATPase activity in both the gill and gut of the Antarctic teleost,Trematomus bernacchii. Blood for serum osmolality was sampled from six fish from the experimental temperature groups every three days for 12 days. Serum osmolality was determined with a vapor pressure osmometer. Tissue for the Na+/K+-ATPase assay was collected from fish acclimated to -1.5°C and +4°C. Na+/K+-ATPase was defined as the amount oubain inhibitable Pi released by enzymatic activity. Graphing and statistical analysis was performed using Microsoft Excel and GraphPad Prism. Warm acclimation of T. bernachii from -1.8°C reduced the serum osmolality via an increase in gill Na+/K+-ATPase enzymatic activity (expressed as micromoles of Pi/mg protein/hour).Upon warm acclimation to +1°C for 12 days, T. bernacchii significantly lowered their serum osmolalities to 513.2 ± 3.9 mOsm/kg (p<0.01). Acclimation to +4°C also resulted in a significant lowering of osmolalitiesto 491.8 ± 2.8 mOsm/kg (p<0.01) after 12 days. Acclimation to +4°C significantly increased (p<0.05) gill Na+/K+-ATPase activity from 2.5 ± 1.1 µmol Pi/mg/hr. to 15.6 ± 5.2 µmol Pi/mg/hr. The gill Na+/K+-ATPase activity of fish acclimated to +4°C was 15.6 ± 5.2 µmol Pi/mg/hr., which was significantly higher (p<0.05) than gut activity of 3.726 ± 2.1µmol Pi/mg/hr. In conclusion, there was a significant change in osmolality after six days of warm acclimation at +4°C and +1°C. There was a significant change in gill Na+/K+-ATPase activity upon warm acclimation with a significant difference between -1.5°C gill Na+/K+-ATPase activity and +4°C gill Na+/K+-ATPase activity. After warm acclimation there was also a significant difference between Na+/K+-ATPase activity in the gill and gut however there was no difference between the tow gut samples. These results indicate that upon warm acclimation, T.bernachii generate significant changes in Na+/K+-ATPase activity in both the gill and gut, and subsequently serum osmolality as early as 6 days.

POSTER 29 GnRH Signaling During Early Embryonic Developement William E. Pohlmeier, Marcelo M. Montagner, Rebecca A. Cederberg, Brett R. White Department of Animal Science, University of Nebraska-Lincoln, Lincoln, NE

In addition to the classical production of gonadotropin-releasing hormone (GnRH) from the hypothalamus, GnRH has been detected in a number of extra-hypothalamic tissues. Importantly, GnRH has been observed in mouse and human embryos, potentially representing a critical developmental process. Consistent with this, our laboratory established that the addition of a GnRH antagonist, SB-75, to the in vitro culture medium results in reduced developmental rates of mouse embryos. Further, SB-75 inhibition of embryonic development can be rescued by the addition of a GnRH agonist, histrelin, confirming a receptor-mediated mechanism. We examined the mechanisms underlying GnRH regulation of pre-implantation embryonic development, focusing on cell signaling pathways that are activated following GnRH binding to its receptor in early embryos. Two pathways of primary interest were apoptosis and cell cycle control. We have previously shown, via Western blotting procedures, that SB-75 had little effect on cleaved caspase-3 protein levels in pre-implantation embryos. In this study, embryos were cultured in 50 &mu;L microdrops of KSOM under mineral oil at 37°C in a humidified 5% CO2 in air environment and media was changed every 24 h. Embryos in the treatment groups were cultured with 10 µM of the GnRH antagonist, SB-75. Following in vitro culture for either 72 or 96 h, embryos were stained to determine live/dead cells using a fluorescein diacetate (FDA) and ethidium bromide (EtBr). The staining solution consisted of 0.05 mg/ml EtBr and 0.005 mg/ml FDA in 10 ml of Dulbecco&rsquo;s PBS. Each treatment group was placed into a 50 µl drop of staining solution on individual glass slides. Slides were then incubated on a slide warmer at 37°C for 3 min in the dark. Next, embryos were viewed under UV epiluminesence using a fluorescent microscope. Following SB-75 treatment, none of the embryos developed past the 2-cell stage. This differs from the 96.8% of control embryos that developed into compact morulae. In addition, embryos treated with SB-75 were able to survive without further development for up to 48 hours. Thus, we suggest that GnRH signaling during early embryonic development may activate cell-cycle pathways. This research was funded through a UCARE -Undergraduate Creative Activity Research Experience fellowship from the Pepsi Foundation.

POSTER 30 Glucocorticoid Responsiveness of the Porcine GnRH Receptor (GnRHR) Gene C. Lee, R. A. Cederberg, B. R. White Department of Animal Science, University of Nebraska-Lincoln, Lincoln, NE.

The binding of GnRH to its receptor results in the synthesis and secretion of the gonadotropins as well as stimulation of the gene encoding its own receptor. Thus, the interaction between GnRH and GnRHR represents a central point for regulation of reproductive function. Glucocorticoids can alter reproduction by reducing GnRH responsiveness of gonadotropes within the anterior pituitary gland, potentially via transcriptional regulation of the GnRHR gene. Consistent with this, transcription of the murine GnRHR gene is stimulated by glucocorticoids. To determine the effect of glucocorticoids on porcine GnRHR gene expression, we isolated approximately 5000 bp of 5' flanking sequence for the porcine GnRHR gene and produced reporter constructs containing the GnRHR promoter fused to the cDNA encoding luciferase (-5000LUC). Gonadotrope-derived αT3-1 cells were transiently transfected with -5000LUC for 12 hr and treated with increasing concentrations of the glucocorticoid agonist, dexamethasone (1, 10, 100 and 1,000 nM) for an additional 12 hr prior to harvest. A dose-dependent increase in luciferase activity was observed with maximal induction noted at 100 nM dexamethasone (2-fold over untreated controls; P < 0.05). The dexamethasone induction of the -5000 promoter was blocked by the glucocorticoid antagonist, mifepristone (100 pM). To determine the location of the glucocorticoid response element(s) within the GnRHR promoter, we performed transient transfection assays with luciferase reporter constructs containing progressively less 5' flanking region for the porcine GnRHR gene. Dexamethasone stimulated luciferase activity was maintained following reduction of the full length GnRHR promoter to 300 bp upstream of the translational start site. However, further deletion to 150 bp of proximal promoter eliminated glucocorticoid responsiveness, suggesting the presence of a glucocorticoid response element(s) within this region. Sequence analysis of this region has revealed a number of putative elements including a consensus activator protein-1 element and binding sites for cyclic AMP response element-binding protein, GATA-1 and glucocorticoid receptor. In summary, glucocorticoid responsiveness of the porcine GnRHR gene is conferred by an element(s) located between 150 and 300 bp of proximal promoter.

POSTER 31 Evidence Supporting a Role for GSK3β/Β-Catenin in Hormone-Stimulated Progesterone Production in the Bovine Corpus Luteum Lynn Roy1, Xiaoying Hou1, Edward Arvisais1, Chao Jiang1, John S.Davis1,2 1Olson Center for Women’s Health, Departments of Ob/Gyn and Pharmacology, University of Nebraska Medical Center, Omaha, NE 68198, 2VA Medical Center, Omaha, NE 68105.

The secretion of progesterone by the steroidogenic cells of the corpus luteum (CL) is essential for the maintenance of pregnancy. In the CL, the rate limiting step in the synthesis of progesterone is the transport of cholesterol to the inner mitochondrial membrane. This process is mediated by steroidogenic acute regulatory protein (StAR), a gene that is under the transcriptional regulation of luteinizing hormone (LH) and prostaglandin E2 (PGE2). However, the exact mechanism of this regulation is unknown. Our working hypothesis is that LH and PGE2 increase progesterone via a cAMP/protein kinase A (PKA) dependent pathway requiring the inhibition of the Wnt signaling pathway intermediate glycogen synthase kinase-3 (GSK3). Phosphorylation of GSK3β at N-terminal Ser9 results in its inactivation and, as a result, levels of phosphorylated β-catenin decrease, which reduces its degradation and allows for its nuclear accumulation. β-catenin acts as a co-transcription factor by binding to members of the TCF/LEF family that regulate growth and development. It may also interact with the transcription factors that promote expression of proteins necessary for progesterone synthesis. In this project, we have examined a role for GSK in progesterone synthesis in steroidogenic cells isolated from the bovine corpus luteum. Utilizing western blot analysis, we observed that low doses of LH (1-100 ng/mL) stimulate the rapid phosphorylation of GSK3β (peak at 5 min). PGE2 (1 µM) mimics this response showing a dramatic increase in phospho-GSK3β (peak 5 min). The protein levels of β-catenin were also increased after stimulation with LH, PGE2, or the GSK inhibitors LiCl (30 mM) and GSK inhibitor IX (1 µM). In addition, treatment with LH, PGE2, LiCL, and GSK inhibitor IX increased in progesterone as detected using a progesterone RIA kit. We have also used adenoviral mediated overexpression of mutant GSK3β proteins and wild-type β-catenin to evaluate whether the modulation of GSK activity affects steroidogenesis in luteal cells. By itself, overexpression of constitutively active GSK3β mutant (Ad.GSK-S9A), kinase dead GSK3β mutant (Ad.GSK-Km), or wild-type β-catenin (Ad.βcat) had no effect on the production of progesterone. However, luteal cells transfected with either Ad.GSK-Km mutant or Ad.βcat showed augmented progesterone production when treated with PGE2, when compared to cells transfected with a control adenovirus. In contrast, Ad.GSK-S9A transfected cells did not show an augmented progesterone response when treated with PGE2. The data suggest that GSK3β-catenin signaling may contribute to the steroidogenic response to LH and PGE2 in luteal cells. Supported by the VA, NIH, and Olson Center for Women’s Health.

POSTER 32

The Identification of Critical Time Periods for Atypical Reproductive and Immune Development Due to Genistein Exposure in Male Rats Evan Ball2 , Alicia Fisher1 , Stacie Holmgren1,2 , Stacy Knight.1,3, Raime Robinson1 , Amy Wisniewski1

Drake University Des Moines, IA 50311 Departments of: Biology,1 Pharmaceutical Sciences,2

Environmental Sciences3

Exposure to environmental estrogens, also known as endocrine disrupting compounds, disrupts the action of endogenous estrogens. Synthetic chemicals have been the focus of most previous studies of endocrine disruption. Due to the lack of information on long-term effects of exposure to phytoestrogens (plant-derived estrogenic substances), it is important to study the potential impact of genistein, a phytoestrogen found in soy products, on development. The popularity of soy based products has increased in recent years, due in part to its protein content and its promotion as a health food. The present research examines the effects of early exposure to genistein on male development. Specifically, the goal of the present study is to identify a critical period during which exposure to genistein disrupts reproductive and immune development in male rats. We hypothesize that genistein exposure during a specific time interval (gestation and/or lactation) results in demasculinization of reproductive and immune function. Four groups (n = 6 dams per group) of rat pups were exposed to genistein or control diet during gestation and/or lactation. At weaning all the pups were placed on control food. Reproductive measures were obtained from birth through adulthood and immune measures were taken from reproductively mature males only. Males exposed to genistein during both gestation and lactation exhibited the most demasculinized external genitalia (F(3, 20) = 4.45, p<0.05). Exposure to genistein during gestation or lactation resulted in the earlier onset of puberty ( F(3, 20) = 5.28, p<0.05) and smaller immune organs compared to other males. Exposure to genistein during different critical time periods (gestation and lactations versus gestation or lactation) results in demasculinzation of different reproductive and immune outcomes.

POSTER 33 Expression of mRNA for Hyaluronan Metabolic Enzymes in the Embryonic and Perinatal Rat Gonads Natalie C. Hart, Rebecca C. Bott, Robin A. Ten Broeck, Debra T. Clopton, Michelle M. Baltes and Andrea S. Cupp Department of Animal Science, University of Nebraska- Lincoln, Lincoln, NE

A multifunctional carbohydrate polymer, hyaluronan resides primarily in extracellular matrices and may be necessary in many morphogenetic processes. The biosynthesis and catabolism of hyaluronan is regulated by hyaluronan synthases (HAS 1,2,3) and hyaluronidases (Hyal 1,2,3,4,5). Hyaluronan in its large, native form is antiangiogenic while digestion through Hyal enzymes to smaller oligosaccharides results in more angiogenic molecules. Our laboratory has determined that the angiogenic growth factor Vascular Endothelial Growth Factor (VEGF) is important for normal vascular development during both testis and ovarian morphogenesis. Therefore, the long-term objective of the current project was to determine the role of hyaluronan synthesis and degradation products in vascular formation during gonadal differentiation. For the current study, the objective was to investigate the presence of mRNA for hyaluronan synthase and hyaluronidase enzymes during gonadal development. Male and female gonads were collected from embryonic day 13 (E13; E0 = plug date) E14, E16, E18, postnatal day 0 (P0), P3 and P5 rats. Conventional RT-PCR was conducted to determine presences of mRNA for HAS 2, HAS 3, and Hyal 1 at these developmental time points. HAS2 mRNA was expressed in the E13, E16, E18, P0, and P5 ovaries. HAS3 mRNA was expressed in the P0 and P5 ovaries and in the P0, P3, and P5 testes. Hyal 1 expression was detected in the E16, P0, P3, and P5 ovaries. From P0 to P3 oogonial clusters are rearranging to form primordial follicles. In P3 ovaries there was an absence of mRNA expression for HAS2 and HAS3 and expression of Hyal1. We speculate that the rearrangement and development of primordial follicles may necessitate breakdown of large native forms of hyaluronan to allow for cell migration and vascular remodeling. Further experiments are planned to determine the potential role of hyaluronan metabolic enzymes in sex-specific morphogenesis within the developing gonads. This research was funded through a UCARE -Undergraduate Creative Activity Research Experience fellowship from the Pepsi Foundation.

POSTER 34 Vascular Endothelial Growth Factor (VEGF) 164 Increases Vascular Density During Testis Morphogenesis and may be Regulated by the Inhibitory Isoform VEGF164b M.M. Baltes, R.A. Ten Broeck, R.C. Bott, D.T. Clopton, and A.S. Cupp Department of Animal Science, University Of Nebraska- Lincoln, Lincoln, NE 68583-0908

Vascular Endothelial Growth Factor (VEGF) is a potent regulator of angiogenesis in ovary during follicle development and corpus luteum function but little is known about its role in testis morphogenesis. Alternative splicing of the VEGF gene produces both angiogenic and inhibitory (anti-angiogenic) isoforms. Angiogenic VEGF isoforms include 120, 164, 188, and 205. One human inhibitory VEGF isoform has been identified, VEGF165b, but the current hypothesis is that for every angiogenic isoform there is an inhibitory isoform. Previous studies from our lab have shown that VEGF164 is present during seminiferous cord formation (embryonic day 13, E13; plug date = E0) in the rat testis. Additionally, inhibition of VEGF arrests both cord formation and vascular development during testis morphogenesis. Therefore, the objectives of this study were to: 1) determine the effects of 50 ng/ml of recombinant VEGF164 on testis morphogenesis and 2) determine if inhibitory VEGF isoforms are present during testis development. Rat E13 testis organ culture pairs (n=20) were treated with either 50ng/ml of recombinant VEGF164 or vehicle control. After organ culture, organs were imaged and processed for whole mount immunohistochemistry with PECAM antibody to determine vascular density. Area and depth of the VEGF164 treated organs were not different from controls. Sliced organ vascular density tended to be greater in the treatments compared to controls (213.24% increase; P < 0.079). Total organ vascular density of VEGF164 treated organs were greater (157.15% increase; P < 0.05) than vehicle controls. To determine if inhibitory VEGF isoforms were present during testis development, conventional RT-PCR was conducted and bands were subcloned and sequenced to confirm inhibitory products. There was little to no expression of VEGF164b rat isoform expression at E13 during endothelial cell migration in the indifferent testis. However, expression of VEGF164b was present after cord formation (E14) and was maintained through postnatal day 0 (P0). The results of these experiments support our previous data that VEGF164 is involved in testis vascular development. The dynamic expression of VEGF164b during testis development suggests that this isoform regulates vascular patterns during testis morphogenesis.

:

POSTER 35 Localization of Period 1 in the Ruminant Oocyte and Investigations of its Role in Ovarian Function

R.A. Cushman, M.F. Allan, S.A. Jones, G.P. Rupp, S.E. Echternkamp U.S. Meat Animal Research Center and Great Plains Veterinary Educational Center-University of Nebraska, Clay Center, NE 68933

The clock gene Period 1 (Per1) may be a prolificacy gene, because it localized to the mouse oocyte and Per1-null drosophila shed fewer eggs. Because Per1 mapped to a region of mouse chromosome 11 homologous to bovine chromosome 19 where a QTL for ovulation rate existed, we hypothesized that Per1 influenced folliculogenesis and ovulation rate in ruminants. Ovarian cortex was collected at slaughter on Days 5, 12, 15, 17, and 20 after estrus for real-time RT-PCR evaluation of Per1 mRNA expression in Dorset (n = 18); Romanov (n = 10); Romanov/Dorset (n = 21); and Composite (n = 22) ewes. Ovarian cortex was also collected from cows selected for increased ovulation rate (n = 37) or unselected controls (n = 28) on days 4, 5, and 6 of the estrous cycle for in situ hybridization and real-time RT-PCR. To examine the role of Per1 in early follicular development, ovarian cortex from neonatal calves (n = 5) was cultured for ten days and Per1 mRNA levels were measured on Day 0 and on Day 10 of culture. The primers generated a 483 bp amplicon with 100% homology to bovine RIGUI-like protein (Per1) which is 20 cM from the QTL. Per1 mRNA expression was unaffected by prolificacy, day of the cycle, or pregnancy status in ewes or cows. The riboprobe hybridized to oocytes of bovine preantral and antral follicles. In bovine ovarian cortical cultures on Day 0, the tissue contained mostly primordial follicles (5.6 ± 0.6 follicles/section); however, after 10 days in culture, the number of primordial follicles per section decreased (0.5 follicles/section) and the number of primary follicles increased as follicles activated (Day 0 = 0.5 ± 0.6 vs. Day 10 = 10.4 ± 0.6 primary follicles/ section; P < 0.001). Per1 mRNA did not change over time in culture. We conclude that Per1 mRNA is expressed by ruminant oocytes in preantral and antral follicles; however, its physiological role in mammalian ovarian function remains to be elucidated.

POSTER 36 Inhibition of Vascular Endothelial Growth Factor Signaling Blocks Primordial Follicle Activation in Bovine Ovarian Cortical Cultures D.T. Clopton, A.S. Cupp, M.F. Allan, S.E. Echternkamp and R.A. Cushman Department of Animal Science, University of Nebraska- Lincoln, Lincoln, NE and U.S. Meat Animal Research Center Clay Center, NE.

Depletion of the ovarian reserve is associated with the end of reproductive life in mammals, and is associated with activation of primordial follicles for further development. Therefore, understanding the mechanisms regulating follicle activation could lead to methods to increase lifetime productivity of female domestic livestock and enhance reproductive outcomes in women. In previous studies, inhibition of Vascular Endothelial Growth Factor (VEGF) signaling through the VEGF receptor decreased primordial follicle activation in neonatal rat ovary cultures. Therefore, we hypothesized that inhibition of the VEGF receptor signal cascade would also inhibit primordial follicle activation in bovine ovarian cortical cultures. Ovaries were collected by mid-line laparotomy from neonatal calves (n = 5) at 44 ± 1.2 days of age, and the ovarian cortex was dissected from the medulla. Ovarian cortical pieces (2 pieces/well) were cultured in serum-free medium, with 0, 2, 4 or 8 mM of VEGF receptor tyrosine kinase inhibitor (VEGF-TKI) or DMSO (vehicle control) for 10 days in duplicate wells. VEGF-TKI was replenished every day and medium was changed every other day. On Day 0 or Day 10, cortical pieces (4 pieces/calf/day/treatment) were collected and embedded in London Resin White resin for morphometric analysis. In control and DMSO-treated cultures, the number of primordial follicles per section decreased (Day 0 = 4.6 ± 0.7 vs. Day 10 = 0.7 ± 0.3; P < 0.001) and the number of primary follicles per section increased (Day 0 = 1.3 ± 0.3 vs. Day 10 = 5.4 ± 0.5; P < 0.001). Primordial follicle activation was not inhibited by 2 or 4 mM of VEGF-TKI; however, after 10 days in the presence of 8 mM VEGF-TKI, the number of primordial follicles per section was not different from Day 0 controls (3.4 ± 0.4 follicles/section). Primordial follicles increased in diameter after 10 days in control and DMSO-treated cultures (Day 0 = 16.0 ± 2.1 mm vs. Day 10 = 26.0 ± 2.1 mm; P < 0.05), and 8 mM VEGF-TKI did not inhibit the growth of primordial follicles after 10 days (30.3 ± 2.1 mM). Thus, VEGF-TKI (8 mM) inhibited primordial follicle activation in bovine ovarian cortical cultures. The current study supports previous results in the perinatal rat and indicates that VEGF may be a regulator of primordial follicle activation.

POSTER 37 Vascular Endothelial Growth Factor (VEGF) 164 Increases Vascular Development in the Perinatal Rat Ovary while Inhibition of VEGF Receptor 2 (VEGFR2) does not Alter Vascular Development or Follicle Progression R.A. Ten Broeck, D.T. Clopton, M.M. Baltes, A.S. Cupp Department of Animal Science, University of Nebraska- Lincoln, Lincoln, NE 68583-0908

Folliculogenesis is a fundamental process in female reproduction that is necessary to prepare the primordial follicle for ovulation. The regulatory mechanisms involved in primordial follicle development, follicle progression, and depletion of atretic follicles are still not clear. Our laboratory has demonstrated that inhibition of VEGF signaling through both receptors arrests primordial follicle progression and vascular density in the perinatal rat ovary. Thus, our objectives were to determine if inhibition of signal transduction through VEGFR2 or treatment with the predominant VEGF isoform, VEGF164, would affect vascular development and follicle progression. Postnatal day 3 (P3) and P4 rat ovary pairs were cultured for 14 days and treated with one of two doses of a VEGFR2 inhibitor, V1 (15 µM, n =27; or 30 µM, n = 16), VEGF164 (50 ng/ml; n= 12), or vehicle control. Organs were processed for histology or whole mount immunohistochemistry using PECAM antibody to determine vascular density. Histological sections of cultured ovaries were examined to determine follicle stage (0= primordial, 1=early primary, 2=primary, 3=transitional). There was no effect on vascular density with either dose of V1; however, 15 µM V1 decreased organ area by 18% and 30 µM V1 by 15 % when compared to controls (P < 0.001). Stage 1 follicle number was decreased in 30 µM V1 treated organs (by 20 %; P < 0.02) and tended to increase stage 2 follicle number (P < 0.07). VEGF164 treated organs had a 100 % increase in vascular density per organ slice compared to controls (P < 0.05). Area and depth of organs treated with VEGF164 increased by 10 % and 14 % (P < 0.01), respectively. There was no effect on follicle stage as measured per average field of view with VEGF164 treatment; however, area and depth of organs was increased suggesting a total increase in follicle numbers. Therefore, VEGF164 treatment increased total ovarian size and vascular density and may be the result of greater follicle activation. Inhibition of VEGFR2 signal transduction does not inhibit vascular density but decreases ovarian size resulting in a reduction in stage 1 follicle number and an increase in number of stage 2 follicles. Therefore, we support our previous data and conclude that VEGF is involved in vascular development and follicle activation in the perinatal rat ovary.

POSTER 38 Liver Receptor Homologue-1 (LRH-1) Regulates Progesterone Synthesis in the Bovine Corpus Luteum X. Hou, C, Jiang, E.W. Arvisais, and J. S. Davis Olson Center for Women’s Health, Departments of Ob/Gyn and Pharmacology, University of Nebraska Medical Center, and VA Medical Center, Omaha, NE

Progesterone secretion is a primary function of the corpus luteum and a prerequisite for normal maintenance of pregnancy in all mammals. Luteinizing hormone (LH), irrespective of species, is the single most important regulatory factor involved in progesterone secretion. It has been shown recently that LRH-1, an orphan nuclear receptor (NRXXX), plays an important regulatory role in steroid hormone biosynthesis in ovarian follicles. However, the molecular mechanism of LRH-1 on progesterone synthesis in the corpus luteum has not been demonstrated. We investigated the functional role of LRH-1 on progesterone secretion in primary bovine luteal cells. Ovaries were obtained from a local abattoir and granulosa and luteal cells were placed into primary culture. Western blot analysis demonstrated that LRH-1 was predominantly expressed in luteal cells as compared to the granulosa cells from the same ovary. Transduction of luteal cells with an adenovirus that expresses LRH-1 (provided by Dr. Zeleznik) resulted in overexpression of LRH-1 protein, as well as an elevation of progesterone secretion measured by radioimmunoassay. Furthermore, LRH-1 overexpression significantly (p < 0.05) enhanced progesterone synthesis in response to either LH (100 ng/ml, 6 hours) or 8-Br-cAMP (1 mM, 6 hours). Adenovirus-mediated overexpression of LRH-1 in ovarian cells (SKOV3) transfected with a full-length human StAR promoter-luciferase reporter increased the StAR promoter activity by 8 fold. Treatment with 8-Br-cAMP further increased the StAR promoter activity by 14 fold under similar conditions. Additionally, using a chromatin-immunoprecipitation (ChIP) assay we found that LH treatment of bovine luteal cells (100 ng/ml, 30 min) induced LRH-1 binding to the StAR promoter. Taken together, these findings indicate that LRH-1 mediates LH-induced progesterone synthesis in the corpus luteum by activating the transcription of StAR. Supported by VA, NIH, and the Olson Center for Women’s Health.

POSTER 39 Vascular Endothelial Growth Factor (VEGF) 120 but not VEGF64 Expression is Increased in Granulosa Cells of Bovine Dominant Follicles due to Nutrition Jeremy L Martin 1, Rick N Funston 1,2, Debra T Clopton1, Heidi L Harris1, Andrea S. Cupp1

1Department of Animal Science, University of Nebraska-Lincoln, Lincoln, NE 2West Central Research and Extension Center, University of Nebraska-Lincoln

Vascular endothelial growth factor (VEGF) is involved in bovine follicle maturation, ovulation and corpus luteum development. Alternative splicing of the VEGF gene produces both angiogenic and inhibitory isoforms of the protein. The predominant angiogenic isoforms are VEGF164 and VEGF120. The objective of this study was to determine whether heifer nutrition influences VEGF expression in dominant follicles. Crossbred yearling beef heifers were assigned randomly to receive 1.65 kg soybeans ground with 0.40 kg corn (SOY; n = 12) or an isonitrogenous, isocaloric control diet (CON; n = 10) for 157 days during the development phase. The SOY diet contained more lipid and three phytoestrogens were detected in the soybeans. Heifers were synchronized with two shots of prostaglandin F2α (PGF) administered 14 days apart, and dominant follicles were measured and aspirated 60 hours following the second PGF injection using transvaginal ultrasonography. Quantitative RT_PCR was conducted on granulosa cell aspirates to quantify VEGF isoform expression relative to GAPDH expression. Only samples from follicles with an estrogen to progesterone ratio > 1 in the follicular fluid were included in the analysis. Dominant follicle diameter was greater (P = 0.09) for SOY than CON heifers at the time of aspiration (11.10 ± 0.47 vs. 12.23 ± 0.43 mm, respectively). However, follicular fluid estrogen and progesterone concentrations, and the ratio of estrogen to progesterone, were similar (P > 0.05). Expression of VEGF120 was greater (P = 0.05; 4.74 ± 0.96 vs 2.04 ± 0.82) in granulosa cell extracts from CON heifers than SOY heifers. No differences (P > 0.05) were detected in VEGF164 expression. Simple correlation analysis failed to demonstrate (P > 0.05) a correlation between VEGF120 expression and follicle diameter, follicular fluid estrogen concentration, or follicular fluid progesterone concentration, suggesting that VEGF120 expression is altered independently of these parameters. These data demonstrate that nutrition can modulate VEGF120 but not VEGF164 expression in bovine dominant follicles.

POSTER 40 Engaging Middle School Students through Distance Learning from Antarctica Yvette K. McCulley1 , Philip R. Brauer 2, Hilary A. Hudson2 , Margaret A. Scofield2 ,Kimberly A. Smith2 , Hailaeos Troy3 , David H. Petzel2 1King Science and Technology Magnet Center, Omaha, NE, 2Creighton University School of Medicine, Omaha, NE, 3Raytheon Polar Services, Centennial, CO

Long distance learning from remote areas such as Antarctica are challenging for educating middle school students. Interactive methods must be used to maintain student interest. The objective of this project was to determine the interactive communication methods available at McMurdo Station, Antarctica , a site having very limited bandwidth. These methods facilitate student understanding of the scope of scientific research conduct in Antarctica and enable students to exchange of ideas with Antarctic researchers (National Science Standards 02 and 01, respectively). Students from King Science and Technology Magnet Center remained in Omaha while their science magnet coordinator joined the Creighton University science team in Antarctica to develop a student website and organize real-time video conferences with students in Omaha . Video conference technology included Yahoo Instant Messenger with an iSight camera connected to Apple computers and an analog phone line. A wireless connection, using Cisco 1200 access points, allowed real-time roaming video recording of remote areas of McMurdo station, and a second stationary computer allowed scientists to see and describe the video that students observed. Students were also assigned questions about Antarctica to discuss during the follow week&rsquo;s conference. This use of roaming video conferencing in Antarctica opens new doors for the Antarctica science community to reach out and facilitate scientific inquiry for students around the world. Supported by NSF #OPP-0229462.

Find out more about the

Nebraska Physiological Society

Slate of Candidates

Thomas E. Pisarri, Ph.D. Nominated for President-Elect

Thomas Pisarri is Assistant Professor of Biomedical Sciences at Creighton University School of Medicine in Omaha, Nebraska, where he has served since his appointment as in 1993. Born in Albany, New York, he attended the State University of New York at Buffalo, earning the BA degree in Biology in 1973. He began his graduate study in the Department of Physiology at the University of Wisconsin in 1973, and received his M.S. degree in 1975. He interrupted his graduate education to spend four years as a United Nations Volunteer on the faculty of the National Teacher Training College in Maseru, Lesotho, in southern Africa. On his return to the U.S, he spent a year teaching high school science and math before returning to the University of Wisconsin, where he received the Ph.D. degree in 1983 under the direction of John "Ed" Kendrick. His dissertation research focused on the interaction of the arterial baroreflex and arterial chemoreceptors in control of cardiovascular function. Later that year, he began his post-doctoral training under the direction of Drs. John and Hazel Coleridge at the Cardiovascular Research Institute at the University of California, San Francisco. His work there focused on stimuli to and reflexes evoked by afferent fibers from the lung and airways. He was appointed Assistant Research Physiologist in 1985 and subsequently Assistant Adjunct Professor of Physiology in 1992, a position he held until moving to Creighton in 1993. His current research interests focus on the role of pulmonary afferents in regulation of the bronchial circulation. He is also the Director of the second year Medical Curriculum in the Creighton School of Medicine. He has been a member of the Nebraska Physiological Society since its founding in 1997, served as a Councilor (1998- 2001), and has been a member of the Local Outreach Team since 2001.

Janet E. Steele, Ph.D. Nominated for President-Elect

Janet E. Steele is a Professor of Biology at the University of Nebraska at Kearney, where she has served since her appointment as Assistant Professor in 1993. Born in Charleston, Illinois, she attended Texas A&M University in College Station, Texas, and earned the BS degree in Bioenvironmental Sciences in1984. She began graduate study at Eastern Illinois University in Charleston, Illinois, and completed the MS degree in Environmental Biology in 1987. She continued her graduate education at Miami University in Oxford, Ohio, under the direction of Ronald L. Wiley, earning the Ph.D. in 1991. Her dissertation research focused on the influence of exercise training on autonomic control of arterial blood pressure in borderline hypertensive rats. In the fall of that year she began post-doctoral training under the direction of Steven L. Britton and Patricia J. Metting at the Medical College of Ohio in Toledo, Ohio. There she worked on the influence of high and low salt diets on the expression of spontaneous pressure diuresis in rats. Her current research interests focus on the influence of exercise training on enzyme repair mechanisms and gene expression in diabetic rats and is a part of the Nebraska INBRE team. She has served the Nebraska Physiological Society s Councilor (1999-2001) and Secretary (2001-2003), and she heads the Nebraska Local Outreach Team, a branch of the American Physiological Society’s Frontiers in Physiology program

Robert A. Cushman, Ph.D. Nominated for Councilor

Bob Cushman is a research physiologist with the RLH US Meat Animal Research Center (USMARC), Clay Center, NE. He received his B.S. degree in Animal Science from the University of Maine in 1988 and worked for 2 years as a research technician at the Worcester Foundation for Experimental Biology. In 1990, he entered graduate school at the University of Connecticut and obtained his M.S. degree in Animal Science with a concentration in Reproductive Physiology in 1992. Later that year, he entered the Physiology program at North Carolina State University, and in 1998 received a Ph.D. with a minor in Biotechnology. From 1998 to 2003 he was a post-doctoral fellow at Cornell University where he was supported by a National Research Service Award from the NIH. In May 2003 he joined USDA-ARS at MARC. Dr. Cushman’s research has focused on mammalian ovarian physiology, specifically the mechanisms controlling oocyte development, follicle growth, ovulation rate, and luteal function.

G. Patrick Lambert, Ph.D. Nominated for Councilor

G. Patrick Lambert is Assistant Professor of Exercise Science and Athletic Training at Creighton University. He also holds a secondary appointment in Biomedical Sciences at Creighton. Born in Michigan, he received his B.S. degree in Exercise and Health Science from Alma College (1988), his M.S. degree in Exercise Physiology from Ball State University (1990), and his Ph.D. degree in Exercise Physiology from the University of Iowa (2001). At Ball State, he worked under Dr. David Costill. At the University of Iowa he was mentored by the late Dr. Carl Gisolfi and by Dr. Kevin Kregel. He conducted research for 8 years as Dr. Gisolfi’s lab technician prior to finishing his Ph.D. His dissertation examined the effects of heat stress on intestinal barrier function. Following his Ph.D. work, he moved to Omaha to conduct postdoctoral research in the Pulmonary and Critical Care Division of Internal Medicine at the University of Nebraska Medical Center. While at UNMC he studied the effects of endotoxin on cytokine and matrix metalloproteinase release under the direction of Drs. Todd Wyatt, Debra Romberger, and Susanna Von Essen. His current research interests include GI barrier dysfunction and endotoxemia during exercise-heat stress and gastric emptying and intestinal absorption of fluids during exercise.

NEBRASKA PHYSIOLOGICAL SOCIETY BALLOT

PRESIDENT-ELECT - (Vote for One)

� Thomas E. Pisarri, Ph.D. Department of Biomedical Sciences Creighton University

� Janet K. Steele, Ph.D. Department of Biology University of Nebraska at Kearney

� _______________________________ Write-in (must agree to serve) COUNCILOR - (Vote for One) � Robert Cushman, Ph.D. United States Department of Agriculture Meat Animal Research Center

� G. Patrick Lambert, Ph.D. Exercise Science and Athletic Training Department of Biomedical Sciences Creighton University

� ________________________________ Write in (must agree to serve)

Nebraska Physiological Society

Oral Presentation Reviewer Form Poster 31

Title of Abstract: Evidence Supporting a Role for GSK3β/Β-Catenin in Hormone- Stimulated Progesterone Production in the Bovine Corpus Luteum

Presenter: Lynn Roy Grading Scale: 1 (Poor) – 5 (Excellent)

Reviewer’s Evaluation: (Please circle one score in every category.) Content Hypothesis / Question Is the hypothesis or question stated 1 2 3 4 5 clearly and succinctly? Introduction Does the introduction provide 1 2 3 4 5 sufficient background to understand problem or hypothesis under study? Methods / Procedures Are procedures or protocols 1 2 3 4 5 described clearly? Results Are results described clearly? Have 1 2 3 4 5 controls been used effectively? Conclusion / Discussion Are conclusions supported by the data? 1 2 3 4 5 Presentation Organization Is the presentation organized logically? 1 2 3 4 5 Speech Skills Is the overall style effective? 1 2 3 4 5 Figures and Tables Are figures and tables easy to 1 2 3 4 5 read and interpret? Response to Questions / Knowledge Knowledge Does the presenter’s style, format, 1 2 3 4 5 etc. foster communication of the investigator’s work? Response to Questions Are responses direct and insightful? 1 2 3 4 5 Understanding of Work Does the presenter understand the 1 2 3 4 5 technical and conceptual aspects of the work? Discussion Can the presenter discuss ideas freely? 1 2 3 4 5

Total

Comments: (Faculty reviewer’s comments are greatly appreciated and very important.)

Nebraska Physiological Society

Oral Presentation Reviewer Form Poster 34

Title of Abstract: Vascular Endothelial Growth Factor (VEGF) 164 Increases Vascular Density During Testis Morphogenesis and may be Regulated by the Inhibitory

Isoform VEGF164b. Presenter: Michelle Baltes Grading Scale: 1 (Poor) – 5 (Excellent)

Reviewer’s Evaluation: (Please circle one score in every category.) Content Hypothesis / Question Is the hypothesis or question stated 1 2 3 4 5 clearly and succinctly? Introduction Does the introduction provide 1 2 3 4 5 sufficient background to understand problem or hypothesis under study? Methods / Procedures Are procedures or protocols 1 2 3 4 5 described clearly? Results Are results described clearly? Have 1 2 3 4 5 controls been used effectively? Conclusion / Discussion Are conclusions supported by the data? 1 2 3 4 5 Presentation Organization Is the presentation organized logically? 1 2 3 4 5 Speech Skills Is the overall style effective? 1 2 3 4 5 Figures and Tables Are figures and tables easy to 1 2 3 4 5 read and interpret? Response to Questions / Knowledge Knowledge Does the presenter’s style, format, 1 2 3 4 5 etc. foster communication of the investigator’s work? Response to Questions Are responses direct and insightful? 1 2 3 4 5 Understanding of Work Does the presenter understand the 1 2 3 4 5 technical and conceptual aspects of the work? Discussion Can the presenter discuss ideas freely? 1 2 3 4 5

Total

Comments: (Faculty reviewer’s comments are greatly appreciated and very important.)

Nebraska Physiological Society

Oral Presentation Reviewer Form Poster 4

Title of Abstract: Exercise Training Alters Muscle Pressor Reflex in Rats with Heart Failure

Presenter: Yan-Xia Pan Grading Scale: 1 (Poor) – 5 (Excellent)

Reviewer’s Evaluation: (Please circle one score in every category.) Content Hypothesis / Question Is the hypothesis or question stated 1 2 3 4 5 clearly and succinctly? Introduction Does the introduction provide 1 2 3 4 5 sufficient background to understand problem or hypothesis under study? Methods / Procedures Are procedures or protocols 1 2 3 4 5 described clearly? Results Are results described clearly? Have 1 2 3 4 5 controls been used effectively? Conclusion / Discussion Are conclusions supported by the data? 1 2 3 4 5 Presentation Organization Is the presentation organized logically? 1 2 3 4 5 Speech Skills Is the overall style effective? 1 2 3 4 5 Figures and Tables Are figures and tables easy to 1 2 3 4 5 read and interpret? Response to Questions / Knowledge Knowledge Does the presenter’s style, format, 1 2 3 4 5 etc. foster communication of the investigator’s work? Response to Questions Are responses direct and insightful? 1 2 3 4 5 Understanding of Work Does the presenter understand the 1 2 3 4 5 technical and conceptual aspects of the work? Discussion Can the presenter discuss ideas freely? 1 2 3 4 5

Total

Comments: (Faculty reviewer’s comments are greatly appreciated and very important.)

Nebraska Physiological Society

Oral Presentation Reviewer Form Poster 3

Title of Abstract: Heart Rate Variability and Central Angiotensin II Receptors in Heart Failure: Role of Exercise Training

Presenter: Tarek Mousa Grading Scale: 1 (Poor) – 5 (Excellent)

Reviewer’s Evaluation: (Please circle one score in every category.) Content Hypothesis / Question Is the hypothesis or question stated 1 2 3 4 5 clearly and succinctly? Introduction Does the introduction provide 1 2 3 4 5 sufficient background to understand problem or hypothesis under study? Methods / Procedures Are procedures or protocols 1 2 3 4 5 described clearly? Results Are results described clearly? Have 1 2 3 4 5 controls been used effectively? Conclusion / Discussion Are conclusions supported by the data? 1 2 3 4 5 Presentation Organization Is the presentation organized logically? 1 2 3 4 5 Speech Skills Is the overall style effective? 1 2 3 4 5 Figures and Tables Are figures and tables easy to 1 2 3 4 5 read and interpret? Response to Questions / Knowledge Knowledge Does the presenter’s style, format, 1 2 3 4 5 etc. foster communication of the investigator’s work? Response to Questions Are responses direct and insightful? 1 2 3 4 5 Understanding of Work Does the presenter understand the 1 2 3 4 5 technical and conceptual aspects of the work? Discussion Can the presenter discuss ideas freely? 1 2 3 4 5

Total

Comments: (Faculty reviewer’s comments are greatly appreciated and very important.)

Evaluation: Nebraska Physiological Society Meeting

May 12, 2006 Please complete and return evaluation at the conclusion of the program. Disagree Agree

1 2 3 4 5

Poster Set-up and Registration University of Nebraska Medical Center Durham Research Center

• Area and amount of time given adequate • Help available if you needed it • Registration table was well prepared

1 1 1

2 2 2

3 3 3

4 4 4

5 5 5

Educational Seminar Robert G. Carroll, Ph.D.

• Material presented was clear and well organized • Information presented is relevant and useful to me • Audio-visual aids added to the presentation

1 1 1

2 2 2

3 3 3

4 4 4

5 5 5

Student Presentations

• Material presented was clear and well organized • Information presented is relevant and useful to me • Audio-visual aids added to the presentation

111

2 2 2

3 3 3

4 4 4

5 5 5

APS Keynote Address Michael S. Wolin, Ph.D.

• Material presented was clear and well organized • Information presented is relevant and useful to me • Audio-visual aids added to the presentation

1 1 1

2 2 2

3 3 3

4 4 4

5 5 5

Lunch

• State of American Physiological Society – Dr. Frank • Area and amount of time given adequate for lunch • Visited the exhibits • Help available if you needed it

1 1 1 1

2 2 2 2

3 3 3 3

4 4 4 4

5 5 5 5

Business Meeting William G. Mayhan, Ph.D.

• Material presented was clear and well organized • Information presented is relevant and useful to me

1 1

2 2

3 3

4 4

5 5

Poster Session Durham Research Center – 5th Floor

• Area and amount of time given adequate 1

2

3

4

5

Would you be willing to travel to Nebraska City to attend a joint meeting with Iowa Physiological Society? _____Yes _____No Do you have any suggestion regarding the electronic abstract submission process?

Acknowledgments

We would like to acknowledge and thank our sponsors who have provided support to the Nebraska Physiological Society meeting for 2006.

We would especially like to thank Dorothy Burgin, Pearl Sorensen, Linda Tegeder from the

Department of Cellular and Integrative Physiology at the University of Nebraska Medical Center for their help in organizing the Nebraska Physiological Society meeting.

For additional information on the NPS sponsors, please visit www.unmc.edu/physiology/nps/sponsors.