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Chylomicron Components Mediate Lipid-Induced Inhibition Of Gastric Motility In Rats. Jorg Glatzle, UC Davis, Dept Anat Physiol Cell Big, Sch of Vet Med, Davis, CA; Theodore J. Kalogeris, Dept Surg, LSU Sch of Medicine, Shreveport, I_A; Tilman T. Zittel, Dept of Surg, Univ Tuebingen, Tubingen Germany; Stephania Guerrini, CURE, UCLA, Sch of Medicine, Los Angeles, CA; Patrick Tso, Dept Pathology, Univ of Cincinnati, Cincinnati, OH; Helen E. Raybould, Dept Anat, Physiol, Cell Big, Sch of Vet Meal, UC Davis, Davis, CA

Lipid, particularly long chain trigylcedde (LCT), initiates feedback inhibition of gastric secreto- motor function and food intake; these effects are mediated by a CCK A receptor and vagal afferent capsaicin-sensitive pathway. The mechanism by which luminal lipid activates this pathway is not clear. Aim: To determine the role of chylomicrons and chylomicron components, in particular apolipoprotein AN (apoAIV), in feedback inhibition of gastric motility. Methods: Mesenteric lymph was obtained from awake rats fitted with lymph fistulas during intestinal perfusion with lipid (Intralipid, 170/Lmol/h; chylous lymph) or a maintenance solution (dextrose + electrolytes; control lymph). Lymph was collected after perfusion with maintenance solution, lipid, lipid + L81 (a surfactant that inhibits chylomicron formation and apolipoproteinAIV secretion) or lipid + L63, (a non-inhibitory surfactant). Inhibition of gastric motility was measured manometrically in urethane-anesthetized recipient rats in response to intra-arterial (ia) injection of lymph, lipid or apoAIV close to the upper GI tract. Results: Injection of chylous lymph significantly inhibited gastric motility; this inhibition was significantly greater than that obtained in response to control lymph (decrease intragastric pressure (IGP) in cmH20; 0.75 + 0.10 vs 0.44 + 0.09, n = 15, p<O.01). Injection of lipid emulsion with a similar fat content as chylous lymph had no significant effect on gastric motility. Inhibition of gastric motility was significantly reduced following injection of lymph harvested during infusion of lipid + L81, but not lipid + L63 (decrease IGP in cmH20; 0.45 + 0.06 vs 0.73 + 0.14, n=lO, p<O.05). Injection of apoAIV (200 p.g ia) significantly inhibited gastric motility compared to vehicle injections (0.51 + 0.10 vs 0.13 + 0.09 cm H20, n=lO, p<O.01). Conclusions: Chylous lymph significantly inhibited gastric motility; this effect was not dependent on lipid content but was dependent on the presence of chylomicrons. These data suggest that a component of chylous lymph associated with chylomicrons, possibly apoAIV, is involved in activation of the CCK A receptor and vagal afferent pathway mediating lipid-induced feedback inhibition of gastric motor function. Supported by DK 41004 (HER), DK 56910 & DK 54504 (PT), and DFG GL 311/1-1 (JG).

299

Characterization Of The Populations Of Mesenteric Afferenl Nerves Inhibited By Octreotide In Anesthetized Rats. Charlotte E. Booth, Anthony J. Kirkup, Univ of Sheffield, Sheffield United Kingdom; Gareth A. Hicks, Patrick P A Humphrey, Glaxo Institute of Applied Pharmacology, Cambridge United Kingdom; David Grundy, Univ of Sheffield, Sheffield United Kingdom

Background: We have previously demonstrated a dose-dependent inhibitory action of octrootide upon rat jejunal afferent discharge and upon the neuronal excitations produced by high threshold distension (Booth et al, 99. Neurogastroenterol. Motil., 11,251) and bradykinin (BK) (Booth eta/, 00. GasfroenteroL 118;A380). To identify the populations of afferent fibers sensitive to octreotide, we investigated its effects on these parameters in vagotomized animals. Methods: Extracallular recordings of jejunal afferent nerve discharge were obtained from pentobarbitone-anesthetized (60mg/kg, i.p.) male Wistar rats (330-450g) as previously described (Kirkup etal, '99 J. PhysioL520, 277). 10 animals underwent chronic sub-diaphrag- matic vagotomy 5-10 days prior to experimentation. Ramp distensions (to a maximum of 60mmHg) were performed by infusion of saline (lml/min). Data are the mean_+S.E.M from 6-10 animals, analyzed using the rank-sum test. Results: In vagotomized animals the mean baseline afferent discharge rate was 30+5 spikes/s. BK (0.1, 0.3 & 1/.,,g.kg ~, i.a.) evoked dose- dependent increases in afferent discharge by 58-+4%, 161 _+23% and 387-+92% respectively (P<O.05) above baseline. Furthermore, high threshold distension evoked increases in afferent discharge by 65_+17-135-+24 spikes/s, over a pressure range of 25-55mmHg. Octreotide (lO0/.~/kg, i.v.) had no effect on baseline discharge, but inhibited the afferent responses to BK by 39_+10%, 43_+18% and 63_+6% respectively, and attenuated the high threshold response to distension by 74_+11%-81_+6% (over 25-55mmHg range). Conclusions: The inhibitory effects of octreotide upon BK and high threshold distension responses observed here in vagotomized animals were not significantly different to those in vagus-intact animals previously reported. However, the inhibition of baseline discharge was not observed in the vagotomized population. The inhibition produced by octreotide on baseline discharge is thus likely to represent an effect on vagally projecting mesentedc afferents. In contrast, afferent responses to BK and high threshold distension are most likely conveyed by spinaUy projecting fibers, which are sensitive to octreotide. These latter effects may contribute to the ability of octreotide to modulate visceral perception in humans (Hasler et al, '93 GastroenteroL 104, 1390-7).

300

Experimental Ulcers Alter Voltage-Dependent Sodium Currents in Rat Gastric Sensory Neurons Klaas Bieleteldt, Noriyuki Ozaki, Gerald F. Gebhart, Univ of Iowa, Iowa City, IA

Voltage-dependent Na t currents are important determinants of neural excitability. We hypothe- sized that gastric inflammation may alter Na + current properties in primary sensory neurons, thereby potentially contributing to the development of dyspeptic symptoms. METHODS: The stomach was exposed in male Sprague Dawley rats through a midline incision and the retrograde tracer Dil was injected into the gastric wall. Control animals received additional injections of normal saline, while in the ulcer group, 20 % acetic acid was injected into the gastric wall. Seven days after surgery, the nndose ganglia were harvested, neurons were dissociated and cultured fro 4 - 24 h. Whole cell Na ÷ currents were recorded with the patch clamp technique. RESULTS: While none of the control animals developed mucosal lesions, 3_+0.5 ulcers were counted in the stomach of acetic acid-treated rats. Gastric sensory neurons

expressed voltage-dependent Na ÷ currents that could be differentiated based on their sensitivity to tetrodotoxin (TrX). The peak Na ÷ current increased from -2474 -+ 290 pA in control cell (n =64) to -4197 -+ 580 pA in cells from animals with gastric ulcers (n ~48; p-<O.01). While the peak amplitude of the l-fX-sensitive current did not differ (-2032 _+ 276 pA vs. -2414 _+ 384pA), there was an increase in the TTX-resistant current (-1199 _ 269 pA vs. -2525 -+ 482 pA; p-<O.05). Na + currents recovered more rapidly from inactivation in cells harvested from animals with gastric ulcers compared to controls (recovery 10 ms after repolarization: 71 -+ 4 % vs. 62 -+ 4 %; p-~O.05). This was due to the increase in the relative contribution of Tl'X-rosistant current, which recovers faster from inactivation. In addition, the properties of the TrX-sensitive current changed with a more rapid recovery from inactivation compared to control cells (recovery 10 ms after repoladzation: 52 -+ 5 % vs. 33 -+ 4 %; p<-O.05). CONCLUSION: Induction of gastric ulcers increases the peak Na + current and accelerates Na + current recovery from inactivation in gastric vagal sensory neurons. This may enhance excitability, thereby contributing to peripheral sensitization and the development of dyspep- tic symptoms.

301

Pancreatic Polypeptides Inhibit Excitatory But Not Inhibitory Synaptic Transmission To Gastrointestinal-Projecting Neurons Of The Rat Dorsal Motor Nucleus Of The Vagus. Kirsteen N. Browning, R Alberto Travagli, Univ of Michigan, Ann Arbor, MI

Whole cell recordings were made from identified gastrointestinal- projecting neurons of the rat dorsal motor nucleus of the vagus (DMV). Paired pulse electrical stimulation of the medialis subnucleus of the nucleus of the tractus solitarius (medNTS) evoked excitatory or inhibitory synaptic currents (EPSCs and (PSCs, respectively). In 15 of 22 gastrointestinal proiecting neurons, NPY (1-300nM) inhibited the peak amplitude of evoked EPSCs in a concentration- dependant manner. This effect was prevented by the Yl-receptor selective antagonist, BIBP3226 (lp.M, 3 of 3 neurons). An increase in the paired pulse ratio indicated that such an inhibition occurred due to actions at a presynaptic site. This presynaptic inhibitory action was mimicked by the Y1 receptor selective agonist, Leu31Pro~NPY (lO0-300nM; 8 of 11 neurons), an action also prevented by BIBP3226 (1/~M, 2 of 2 neurons). The amplitude of evoked EPSCs was also inhibited by PYY in a concentration-dependent manner(1-3OOnM) in 7 of 11 neurons and by the Y2-receptor selective agonist, NPY3-36 (lO0-300nM; 6 of 9 neurons). Again, an increase in the paired pulse ratio indicated a presynaptic site of action. The inhibitory effect of NPY3-36 was unaffected by the Y1 receptor selective antagonist, BIBP3226 (1/~M; n = 1 ). In contrast, the amplitude of evoked I PSCs was not affected by either NPY (lOOnM, 7 of 7 neurons) or by PYY (lOOnM, 5 of 5 neurons). These results suggest that within the dorsal vagal complex, both Y1 and Y2 receptors are present on presynaptic news terminals of excitatory but not inhibitory inputs to gastrointestinal motoneurons, and may imply that the central inhibitory actions of pancreatic polypeptides targets selected parasympathetic pathways. Supported by NIH grant DK-55530 to AT.

302

The Role of Na+/Ca 2÷ Exchanger in Opossum Esophageal Smooth Muscle Contractility. Praveen K. Roy, Yong Zhang, Sandra L. Lourenssen, Micheal G. Blennerhasett, William G. Paterson, Queen's Univ, Kingston Canada

The Na+/Ca2+ exchanger (NCX), an ion transport protein, is an important mediator of Ca2 + homeostasis. In cardiac and vascular smooth muscle increased expression occurs in cardiac failure and hypertrophic states, while hypoxia and ischemia tend to decreased expres- sion. However, its role in esophageal smooth muscle contractility is unknown. Its importance in smooth muscle contraction suggests that an understanding of its role in esophageal contractility may lead to a better understanding of motility disorders of esophagus, including abnormal motility associated with GERD. Therefore, the role of NCX in the opossum lower esophageal sphincter (LES) and esophageal longitudinal smooth muscle (LM) was investigated. Isometric tension recordings were performed on LES and LM strips from the opossum esophagus. Data were standardized to maximal contraction induced by bath application of 60 rnM KCI. Amiloride (lmM) and its analogue benzamil (500/.~I), were used as inhibitors of NCX. NCX expression was investigated by Western immunoblot analysis, with an antibody raised against the canine cardiac sacrolemmal NCX protein (SWant, Switzerland) and using rat heart muscle as a positive control, For cellular Iocaization of the protein, immunohistochem- istry (IHC) was done on isolated LES and LM cells. IHC and immunoblot were also done on cells obtained from animals infused with acid over 4 consecutive days. In the LES, low Na solution induced a contraction of 168-+16% of KCi response. Amiloride and benzamil caused a 22.4_+10% and 8-+14 % reduction in the basal tone of the LES respectively. Both agents also significantly inhibited the response to low Na. Similarly, in LM the low Na response was inhibited by amilodde and benzamil. In both LES and LM, western blot revealed a 85-kDa band which is comparable to NCX protein reported from the cardiac sacmlemma. Expression was localized to the plasma membrane on IHC. Preliminary data from both IHC and immunoblot showed strongly decreased expression of NCX in cells isolated from animals with acid induced esophagitis as compared to controls. In summary, NCX is present and functional in the esophageal smooth muscle, and may be a target of inflammation in acid induced esophageal injury. Pharmacological manipulation of this exchanger may have therapeutic potential in primary or GERD-related motor disorders. (Supported by CIHR).

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