ROYAL A C A D E M Y OF MEDICINE IN IRELAND

SECTION OF BIOMEDICAL SCIENCES

Proceedings of Winter Meeting held in Trinity College Dublin, 1996.

PROTECTION OF G6PD DEFICIENT ERYTHROCYTES BY LAZAROIDS

S Sharma*, S. C. Sharma. *Department of Surgery, St. Vlncent's Hospital, Elm Park,

Dublin 4 and Department of Pharmacology and Therapeutics, Trinity College, Dubhn 2.

Lazarolds (21-ammosteroids) are novel antioxidants which can protect normal human erythrocytes against haemolysls mduced by hypotomc sahne or hydrogen peroxide ~ In the present study we have investigated if lazarolds can also protect erythrocytes which are deficient in glucose -6 phosphate dehydrogenase (G6PD) enzyme Samples of antecubltal vein blood were collected from two male subjects of Mediterranean origin who were known to have G6PD deficmncy Erythrocytic deficiency of the enzyme however was confirmed using Sigma dye decoloration method, Two lazaroids (U74500A and U 74389F) were selected for the study

Erytbroeyte suspensions were prepared and incubated for 20 minutes at 37~ with varying concentrations of the lazaroid before exposing them to a haemolytie injury This was caused by adding sulphantlamtde (l raM), qulmne (0.031xM) and H202 (25pM) to erythrocyte suspensions in saline phosphate buffer. In some experiments the ability of lazaroids to protect against haemolysls was also compared with vitamin E (~-tocopherol) Stock solutions of lazarolds and vitamin E were prepared in 95 per cent ethanol and added slowly in 25 [tl volume to erythrocyte suspension which was steadily mixed Final concentration of ethanol in experimental and control tubes was kept at 1.75 per cent Mean (+ SEM) IC~0 and Cma ' (concentrations which produced 50 per cent inhibition and almost complete inhibition of erythrolysls respectively) for various haemolytm agents are shown in the table

Lazaroid (~Mol/I) Sulphamlamlde Quinine H2O 2

U 74500A ICs0 1.35+0.11 1,62_+0,06 0.80_+0.09 Cm~ ~ 273_+028 2 9 7 + 0 1 5 225+013

U 74389F IC5~ 22 95 + 2 49 35 21 + 1.49 18.75 -+ 1.50 C.~ 6784+391 6270-+250 5760-+386

The results show that lazaroids can effectively protect G6 PD deficient erythrocytes against haemolysis reduced by various agents Out of the two lazaroids studied compound U74500A was several times more effective than U74389F in providing protectmn to erythrocytes,

While the two compounds were able to provide protection over a wide range of concentrations their high concentrations (several fold higher than Cm,~) were pro-haemolytic Vitamin E added in physiological concentrations however failed to provide any slgmflcant protection to G6 PD erythrocytes It was also pro-haemolytic in high concentrations

Acknowledgements . The lazarolds were kindly prowded by Upjohn.

Reference 1 Sharma, S C Protection of human erythrocyte~ by lazarolde~ Br J Pharmacol 1992, 106 l14p

ASSESSMENT OF A NOVEL APPROACH TO SPINAL ANAESTHESIA

P D. Byrne j, J B Lyons z, J F Boylan 2, A. J. MeShane z. ~Faculty of Medicine, University College, Dublin,

2Department of Anaesthesia and Intensive Care, St. Vincent's Hospital, Elm Park, Dubhn

When using 0 5 per cent hyperbaric bupivacaine for spinal blockade, the segmental spread and cardiovascular effects of the block have been shown to be dependent on the volume of local anaesthetic injected 1.

Following hospital ethical committee approval, the effects of reducing the volume of hyperbaric buplvacame by giving half the volume as isobaric bupivacalne were assessed in a prospective, randomlsed, double-blind trial, in 40 patients scheduled for elective surgery. Group 1 (20 patients) received their local anaesthetic as 2 equal aliquots of 0 5 per cent hyperbaric bupivacaine and 0 5 per cent isobaric buplvacaine respectively; Group 2 (20 pahents) received their local anaesthetic as 2 equal ahquots of 0.5 per cent hyperbaric buplvacame Statistical analysis was carried out using Student's 't ' test to compare parametrm data (age, weight, and height), Analysis of Variance (ANOVA) to compare blood pressure, Wllcoxon signed-rank test to compare segmental spread of anaesthesia, and chl squared analysis was used to compare categorical data such as sex, needle usage, vasopressor usage, and patient posltlomng There was no sigmficant difference found between the 2 groups wlth regard to maximal height of block achieved (group 1, mean (range), T7 (7), group 2, T8 (6)), rate of onset of blockade, or time to maximal blockade (group 1, mean (SEM), 23 55 (2 41) min, group 2:20.89 (2.95) rain The trial group had a higher eephalad spread between times 25 min and 60 min, however, this difference was not statistically significant, There was no difference found between each group m either cardiovascular stablhty or vasopressor usage,

Conclusion' The admimstratlon of a mixture of 0 5 per cent isobaric bupivacalne and 0 5 per cent hyperbaric hupivacaine, confers no advantages over administration ot the same volume of 0.5 per cent hyperbaric buplvacaine alone

The authors acknowledge grant support from the Health Research Board

Reference 1 Chambers, W A, LIttlewood, D G, Edstrom, H H, Scott. D B

Spinal anaesthesia with hyperbaric buptvacame Effects of concentratmn and volume admlmstered Br J Anaesth 1982, 54 75-80

AN INVESTIGATION OF THE RADIOPROTECTIVE ACTION OF DIETARY ANTIOXIDANTS ON P388

MURINE LYMPHOBLASTIC CELLS

E S Galhgan, S F Sweetman, S, R. McKeown, J J Strain School of Biomedical Scmnces, University of Ulster,

Jordanstown. N Ireland.

Low energy iomsing radlahon such as X- and T-rays reduces the majority of cellular damage indirectly through the production

304

Vol 165 No 4

of free radicals Antioxldants both endogenous and those derived from the diet are thought to protect the cell from free radical induced DNA damage Green et al. (1994) ~ have shown that dietary supplementation with vitamin C can reduce DNA damage to human lymphocytes exposed to ",/-rays in wtro To further examine this hypothesis we have exposed P388 munne lymphoblast]c cells to X-irradiation (0-5 Gray (Gy)) The ceils were previously exposed for 24 h to a range of antlox]dants: B- carotene (2p.m), vitamin C (60~tm), vitamin E (90ktm). The Comet assay z was used to measure DNA damage immediately following exposure to the X-irradiation: cells m which the DNA contamed single and double strand breaks showed streaming of the DNA from the nucleus on electrophoresis under alkaline conditions. (Cells were wsuahsed using ethldmm bromide staming and analysed using image analysis.)

All of the antioxidants showed significant protection of the cells (p<0 05) at the lower radlatmn doses (I, 2 and 3 Gy). In addition B-carotene and vitamin E showed protection of the cells at 5 Gy, with tall moment (TM) values of 2.25_+0.02 and 1.82_+0 14 respectively in comparison to non-supplemented ceils (TM=3 92_+0 47) A protective effect was not afforded with wtamm C at this radiation dose (TM=2.62_+0 11) This showed that supplementatlon of cells In wtro with dietary antioxldants cali reduce the extent of radiation damage at low doses.

References l Green, M H L, Lowe, 1 E, Waugh, A P W, Aldndge, K E, Cole, J Arlett, C F Effect of diet and vitamin C on DNA strand breakage in freshlyqqolated human white blood cell, Mutat Res 1994; 316 91- 102 2 McKelvey-Martm, V J, Green. M H L, Sehmezer, P et al The single cell gel electrophoresls assay (comet assay) A European Review Murat Re~ 1993, 288 47-63

INVESTIGATION OF THE EFFECTS OF LOW LEVEL LASER IRRADIATION UPON PERIPHERAL BLOOD

FLOW IN THE HUMAN FOREARM IPSILATERALAND CONTRALATERAL EFFECTS

B A Clements, A S Lowc, K J M, McCormaek, D. M. Walsh, G D Baxter, J M Allen

Rehabilitation Sciences Research Group, Schools of Health Sciences and Biomedical Sciences, University of Ulster,

Jordanstown, BT37 OQB, N, Ireland.

Whale the use of low power lasers (low level laser therapy / LLLT) has recently been promoted as an effective treatment for a varmty of wounds, the precise mechanism and the optimal conditions under which LLLT produces such blost]mulatory effects are still under question. In particular, a lack of research evidence from properly controlled studies limits acceptance of its use in some clinical situations

Alter ethical approval was obtained this double-bhnded, controlled study was carried out to assess the effects of LLLT (830nm, continuous wave; 1.5 and 9.0 Jcm-'-, CB Medico, Vaeloese, Denmark) upon peripheral blood flow in forearms ol resting, healthy, human, caucasian volunteers (n=15) An index of blood flow was recorded bilaterally using 2 laser doppler flowmeters SubJects were randomly and equally allocated to 1 of 2 treatment groups or a control group After artificially raising blood flow by immersion of the forearms in water baths (45~ I 0 min), red cell flux (tissue perfusmn units, TPU) was measured at 2 min Intervals, concomitant with mmutorlng of skin surface temperature, from probes positioned over the mid-points of the

Sec t ion o f B i o m e d i c a l Sc i ences 305

forearms along the courses of the median nerves Irradiation at the parameters specified was applied, after 10 rain, to 10 equidistant points over the course of the median nerve on the right (dominant) forearm

Analysis of results (ANOVA) showed a dramatm and statistically slgnlheant reduction in right arm red cell flux paral leled with a notable reductmn In superfmlal skin temperature at a radiant exposure of 1 5 Jcm -2 when compared to controls (p<0 05, mean reduction 10 3+1 18 TPU treatment group 1 5 Jcm 2, 2.5_+0 81 TPU control group) Right arm flux and temperature values from the 9 0 Jcm 2 radiant group showed minimal variation from controls (mean reduction 2,7_+1 34 TPU) In contrast, no significant findings were reported from the left arm flux or temperature values indicating that the observed laser- mediated reduction in blood flow (and skin temperature) was not systemically affected

While the precise mechanism underlying the results reported, and their relevance to the therapeutic effects of this modahty are unclear, laser Irradiation at the parameters indicated has been demonstrated to have a significant effect upon physiological function,

TUMOUR NECROSIS FACTOR-~t STIMULATES PROTEIN KINASE C ACTIVITY IN PULMONARY VASCULAR

ENDOTHELIAL CELLS

S Kelleher, A K Keenan Department of Pharmacology, University College Dubhn,

Belfleld. Dublin 4

This study investigated the hypothesis that TNF-ct can In the short term stimulate vascular endothehal cell protein kmase C (PKC), an enzyme whose acuvation has previously been hnked to aspects of endothelial dysfunction j

Bovine pulmonary artery endothchal cells (BPAEC) were maintained and subcultured as described prevmusly 2 and were used between passage numbers 13-16 Tutal cellular mositol phosphate lIP) accumulation was measured by incubating [~H]mosltol-loaded cells (2-4 BC1/ml for 24 h) +_TNF-tr at 37~ in the presence of 20 mM Li* PKC activity was measured by a method based on the transfer of the "t-phosphate of ['y~:P]ATP to a PKC-specifm pcpttde (l) In cell lysates or (u) in cytosohc and plasma membrane fractlon~ prepared from lysates Data presented are mean values + s e m of n observations and significant differences were determined by ANOVA, followed by Dunnett's or Bonferronl's multiple comparisons post test as appropriate

lncubahon of BPAEC with 100 ng/ml TNF-o~ marginally increased IP accumulation at 10 mm (by I 20 + t/07 fold, n=6, p<0 05) In cell lysates 9 7 p.M phorbol myristyl acetate (PMA) + phosphatidyl serme (PS/PMA) and T N F - a (40 ng/ml) stimulated PKC activity at 10 rain (from 35 8 + 5 7 (basal) to 132.8 + 14-.4 and 118 4 _+ 8 6 pmol phosphate transterred/mm/ mg respectively, n=3, p<0 05 m each case) While plasma membrane fractions lacked stlmulatable PKC activity, m cytosol significant fold mt, reases in activity over basal were again stimulated by PS/PMA (p<0 01L TNF-tr (p<0 001). or a combmatmn of the two (p<0 001 ) where acnwty increased to a greater extent than had been the case with rather agent alone

We conclude that TNF-tr increases PKC achvRy in BPAEC by a mechanism which may include a marginal activation of cellular phosphohpase C

Supported by Forbalrt

306 Royal Academy of Medtctne in Ireland

References 1 Moloney, L,, Armstrong, L Cytoskeletal reorgantzanons in human umbthcal veto endothehal cells as a result of cytokme exposu,~e Exp Cell Res 1991, 196 40-48 2 Rcdmond, E M , Cahlll, P C, Keenan, A K Atrial natrluretlc factor recogrllze~ two receptor subtype~ in endothehal cells cultured from bnwne pulmonary artery FEBS Lett 1990, 269. 157-162

THE RADIOSENSITIZER N1COTINAMIDE ATTENUATES RAT TAIL ARTERY CON STRICTIONS EXAMINATION OF ITS EFFECTS ON VASCULAR SMOOTH MUSCLE

INTRACELLULAR SIGNALLING

D M Burns ~, J. C W Brown t, M.W Ruddock ~,G D Kennovin 2, E L. Dykes'-, E W Fhtney 2, D. G. HtrsP. tSchool of Biomedical Sciences, Umverstty of Ulster,

Newtownabbey, Co Antrim, N Ireland ~Caacer Biology Research Group, University of St. Andrews. St Andrews,

Fife. Scotland.

Extensive pre-clinical studies have demonstrated the efficacy oI nlcotlnamide (NA) as a radlosenslttzer for cancer treatment. To date efforts to develop improved agents have been unsuccessful because the mechanism of action of NA is unknown, Previously, we have shown that NA (8.2 mM) inhibits phenylephrme (PE) or high [K § induced-constriction of rat tail artery in ex vivo perfuslon experiments t. Thus NA blocks constriction induced by ealetum influx due to opening of either voltage dependent or receptor dependent calcium channels Here we report the results of further work examming the effect of NA on intracellular signalling m vascular smooth muscle. Isolated pieces of rat tail artery were perfused externally and internally with Krebs solution at constant flow rate. Constriction was induced by bolus lnjechons of vasoconstrictors (10 ~tM) with any increased perfuslon pressure measured by pressure transducers Dose responses to these agents were performed in the presence and absence of NA and other mhibttors which were continuously perfused NA (8 2 mM) markedly inhibited vasopressln (VP) lnduccd-transtent constrictions, which ls believed to act via activation of phospholipase C and generation of inositol trisphosphate (IP~) and diacylglycerol NA mediated decrease m smooth muscle permeability to calcium ts unlikely as NA also markedly attenuates PE and VP induced-transient constrictions m calcium-free Krebs Recent reports found that PE and calcium influx generate another second messenger, cychc-ADP-ribose (C-ADP-R) in vascular smooth muscle, The generation of this agent also produces NA. however NA does not appear to exert its vasorelaxant effect via blockage of this signalling pathway In our system continuous perfusion of ryanodme (1 laM) which binds to the C-ADP-R receptor on the sarcoplasmic retlculum blocking its activity, el'so reduced PE induced-transient constriction, and in combination with NA (8.2 raM) greatly potentiated NA lnhibttory activity. Similarly, the phosphod]esterase inhibitor 3-tsobutyl-l-methylxanthane (IBMX) (10 ktM) did not prevent NA mediated relaxation, indmcatlng that NA did not impair reactivation of cychc-AMP, which causes vasorelaxatton In conclusion, NA does not appear to exert Its principal mode of action in smooth muscle via the pathways exammed above

This work is supported by the Cancer Research Campaign.

Reference 1 Htr~t, D G , Kennovm, G D, Fhmey, F W The radlosensmzer meonnamtde mh,blts arterial vasoconstncnon Br J Radiol 1994, 67 59799

IJMS October, November, December, 1996

POTASSIUM CHANNEL ACTIVATION BY CALCIUM- MEDIATED SECRETAGOGUES IN HUMAN COLON

D. C Winter J. Cuffe G C O'Sullivan*, B. J Harvey. Wellcome Trust Cellular Physiology Research Unit,

Department of Physiology and *Department of Surgery. Mercy Hospital, University College Cork

Charge balance during transcellular chloride (CI) transport is maintained via potassium (K*) exit across the basolateral membrane of secretory tissues The mechanisms of cross-talk between the apical CI- and basolateral K § channels during CI" secretion are ill-defined We investigated the effect of the C1- secretagogues histamine (H) and carbachol (C) on basolateral K* channels in the human colon

Electrogenlc ion transport across sheets of normal mucosa from colectomy specimens was measured with short circuit techniques in Usslng chambers. Permeabllisation of the apical membrane with an ionophore (nystatm) permitted examination of basolateral K+ flux Modification of the mucosal bathing solutton in order to clamp the free intracel lular calcium concentration ([CaZ*]l) at 10nM was effected using EGTA and gluconate Increases in short circuit current (Alse) are expressed as micro-amperes per cm z and all values represent mean+s e m. at 100ktM doses.

Scrosal application of carbachol or histamine to intact- membrane samples induced a transient C1- secretion that was inhibited by pro-treatment of the basolateral membrane with 1001aM tetrapentylammonium (TPA) but not tolbutamide (results not shown), This suggests K* re-cycling through calcium activated (KCa2+), but not adenosine trlphosphate-dependent (KATP) channels

Experiments on permeablhsed tissue demonstrated an augmentation of basolateral K ~ flux by H (22,24_+0 71; n=16) or C (31 67+1 86, n=16) The increases m K* conductance were inhibited by TPA (H=0 53_+0.43' C=0 77_+0.85; n=16) but not by tolbutamide (n=12) Neither secretagogue evoked changes in K* current when [Ca:*]i 'was clamped (n=18).

Histamme and carbachol stimulate TPA-sensitive basolateral K* channels via a mechanism mediated by [Ca'-+]l. Activation of these KCa'-* channels es tabl ishes a membrane hyperpolarisatlon that would drive apical CI- flux

ABSORPTION OF CARBON DIOXIDE AND ANAESTHETIC GASES BY ZEOLITE 5A

G H Thompson, J M. Murray, J P H Fee Department of Anaesthetics, The Queen's University, Belfast.

Zeolites have been shown to be capable of removmg carbon dioxide in a simulated low-flow anaesthetic circuit t Advantages over currently used soda hme are the possibility of regeneration for reuse and avoidance of potentially toxic degeneration products of volatile agents.

The following gasses were passed through a canister containing 62g zeolite 5A at flow rate of 1 l/mm. (a) carbon dioxide 4 per cent in oxygen, (b) nitrous oxide 50 per cent in oxygen, (c) isoflurane 1.2% m oxygen. Gas was supplied continuously distal to the camster. Carbon dioxide was completely absorbed by the zolite for 34.8 mm, thereafter absorption decreased gradually. Nitrous oxide was completely absorbed for 5.3 min, then absorption rapidly dechned lsoflurane was completely absorbed for 2 8 mln, then absorption rapidly declined

Gas mixtures with 3 8 per cent carbon dioxlde at a flow rate

Vol 165 No 4

of 21/rain were passed through 62g zeolite 5A as follows: (a) carbon dioxide 3.8 per cent in oxygen with fresh zeolite, (b) carbon dioxide 3.8 per cent, nitrous oxide 50 per cent in oxygen with zeolite previously exposed to nitrous oxide, (c) carbon dioxide 3.8 per cent, isoflurane 1 2 per cent in oxygen with zeolite previously exposed to isoflurane Carbon dioxide was completely absorbed by the fresh zeolite for 138 min, and thereafter absorption gradually decreased. With the zeolite previously exposed to nitrous oxide, carbon dioxide was completely absorbed only transiently and absorption continued to decline rapidly. With the zeolite previously exposed to isof lurane, complete absorption of carbon dioxide was maintained for 10 7 rain, thereafter absorption declined gradually.

It is concluded that zeolite 5A absorbs carbon dioxide effectively when delivered in dry oxygen. The zeohte also extracts anaesthetic agents but these saturate quickly. The capacity of zeolite 5A to absorb carbon dioxide is markedly impaired by nitrous oxide but not greatly affected by isoflurane Therefore zeolite 5A could be used as a carbon dioxide absorbent in an anaesthetic breathing system but not when nitrous oxide is used in the gas mixture Reference 1 Fee, J P H,, Murray, J M, Luney, S R Molecular sieves an altemanve method of carbon dioxide removal which does not generate compound A during ~lmulated low flow sevoflurane anaesthesia Anaesthesia 1995, 50' 841-845

AN INVESTIGATION OF THE EFFECTS OF 8-HYDROXY- 2-(DI-N-PROPYLAMINO)-TETRAL1N (8-OH-DPAT), ON

BEHAVIOURAL PARAMETERS IN RATS TREATED ACUTELY AND CHRONICALLY WITH METYRAPONE

F Brehony, J P Kelly, B E. Leonard Department of Pharmacology, University College, Galway.

Both abnormal glucocorttcold secretion and dysfunction of central serotonergic neurotransmission have been implicated in the pathogenesis of affective disorders ~ Metyrapone, a potent Inhibitor of cort isol production and 8-hydroxy-2-(di-n- propylamino)- tetrahn (X-OH-DPAT), a selective 5-HT~, x receptor agomst, are utlhsed in this study to examine the relationship between glucocortlcoid regulation and serotonergic activity. The obJective of this study was to compare the effects of acute and chronic administration of metyrapone to rats subsequently challenged with 8-hydroxy- 2-(di-n-propylamino)- tetrahn (8-OH-DPAT), on activity and temperature responses, Metyrapone (50mg/kg i p.) was administered daily to male Sprague Dawley rats (260-280g) After one administration of metyrapone and after 17 days of daily treatment, 8-OH-DPAT (0 1 and 1 0mg/kg s.c.) was admimstered 30 mm later. Chrome, but not acute metyrapone treatment potentiated the hyperactive response (p<0 05 Mann Whitney) of 8-OH-DPAT as recorded by the home cage activity monitor. In addition a hyperactive response was mduced by chronic metyrapone administration alone (p<0 05 Mann Whitney). The 8-OH-DPAT induced hypothermlc response was attenuated following chronic but not acute metyrapone treatment. These results suggest that the function of 5-HT1A receptors are altered after chronic metyrapone administration and imphcate a role for the 5-HTjA receptor in glucocorttcoid regulation

Reference l Dman, T G Glueocortleotds and the genesis of depressive dlness Apsychoblologtcal model Br J Pharmacol 1994, 164 365-371

Section of Biomedical Sctences 307

ENHANCEMENT BY REACTIVE BLUE 2 OF OUTWARD CURRENT IN ISOLATED SMOOTH MUSCLE CELLS OF

THE SHEEP BLADDER

K D. Cotton M A Hollywood, N G McHale, K. D Thornbury

School of Biomedical Science, Queen's Untverslty, 97 Lisburn Road, Belfast, BT9 7BL

Cibacron Blue and Bastlen Blue (Isomers of Reactive Blue 2) are widely used as selective blockers of P2> purmergic receptors but recent results suggest that their action may not be due solely to specific receptor antagonism ~ In the present study we examined the effect of both isomers on outward current in freshly dispersed cells from the sheep bladder using the whole- cell patch clamp technique Noisy outward currents were evoked by stepping from a holding potential of -60 mV to +40 mV Cibacron Blue (10-4 M) increased peak outward current from a mean of 1747 + 509 to 4151 + 805, S.E M., n=7. The effects were concentration-dependent with a half maximal response at 10 -~ M and a maximal response at 10 4 M. Very similar effects were seen with Basden Blue In contrast , suramin in concentrations up to l0 -3 M, had no such effect The Clbacrnn Blue response was greatly reduced in Ca2*-free physiological salt solution suggesting that it was Ca 2§ dependent Similarly the Cibacron Blue-sensitive current was significantly attenuated by charybotoxm. In 4 experiments Ctbacron Blue increased the peak outward current from a mean of 1759 + 288 pA to 3577 + 656 in the absence of toxin and from 437 _+ 58 pA to 627 -+ 85 in the presence of 10 -7 M ChTX (p<0.05, Student's paired t-test) Clbacron Blue did not modulate inward current nor were its effects modified by caffeine or hepann suggesting that its effect on outward current was not secondary to an increase in mtracellular Ca 2. These results suggest that the isomers of Reactive Blue 2 are potent activators of a Ca 2+ dependent outward current in bladder smooth muscle ceils in addition to their actions as purinerglc blockers Reference Bndgewater, M, Cunaane, T C. Bradlng, A F Characteristic features of mhtbltoly]uncOon potentials evoked by single sttmuh in the guinea- pig isolated taema cacti J Physlol 1995, 485 145-155,

MODULATION BY C1BACRON BLUE OF Ca 2§ DEPENDENT K § CURRENT IN ISOLATED SMOOTH

MUSCLE CELLS FROM THE SHEEP BLADDER

M.A. Hollywood, K D. Cotton, K D. Thornbury, N G McHalc

School of Biomedmal Science, Queen's University, 97 Ltsburn Road, Belfast, BT9 7BL.

The inside/out patch clamp techmque was used to study modulatlon of K + current by Cibacron Blue. The dominant channel in inside-out patches had a large conductance and showed both voltage and Ca z+ sensitivity Channel conductance was studied by measuring the amplitudes of unitary currents at holding potentials from 0 - 100 mV Data were fitted by hnear regression and the average slope conductance in symmetrmal 140 mM K § at 37~ was calculated to be 298 _+ 8.3 pS (SEM) and the reversal potential was 0 8 + 2 5 mV (n=7). There was typically a 90 mV decrease in the voltage of half maximal activation when Ca 2" concentration was increased from 10 -7 to 104 M. These properties are typical of the maxi K + channel found ubiquitously in smooth muscle cells. The voltage profile of channel activation was studied using ramp protocols Activation

308 Royal Academy of Medicine zn Ireland

curves were calculated by averagmg current responses to 15 potenDal ramps and dividing each data pomt of the averaged current by the smgle-channel amplitude at that holding potential after leakage current correction Under control conditions (5x10 7 M Ca 2.) significant activation occurred only in the positive range When these data were fitted with a Boltzmann function the voltage of half maximal activation was 98 mV Addition of 10 ~ M Cibacron Blue caused the channels to activate at much more negative potentials (V~r z = -21 mV) In 6 such experiments Clbacron consistently shifted the activation curve to the left with a mean shift in V~a of 102 _+ 20 mV (p<0 005, Student's paired t-test).

EFFECT OF MYOSIN AND TROPONIN-I MIMETIC PEPTIDES ON RIGOR IN ISOLATED RAT

CARDIOMYOCYTES

M T Stapleton, A P Allshire Department of Pharmacology & Therapeutics, University

College Cork (Introduced by A K Xeenan)

Following contractile failure in ischaemic myocardlum diastolic pressure increases (independent of cytosohc free calcmm), reflecting rigor activation of contractile elements at low ATP concentratmns

Isolated cardtomyocytes subjected to hypoxla or metabolic mhlbltlou undergo analogous calcium-independent shortening accompanied by abrupt depletion of residual ATP 1, probably through rigor-activation of myosin ATPase, followed by loss of calcium homeostasis that primes cells for injury at reoxygenatmn Preventing rigor may therefore attenuate the deterioration of ischacmlc myocardium

In thus study isolated rat vent r lcu lar myocytes were permeablhzed with digitonm, then rigor (cell shortening) and myosin ATPase activity were measured in parallel. For these measurements the cells were placed m calcium-free lntracellular- type solutions (pH 7 l), comprising KCI/EGTA/MOPS with MgCl 2 and ATP added to give defined [MgATP] Non-myosin ATPase mhlbitors ouabam, cyclopiazonlc acid and ollgomycln were included so that inorganic phosphate measurements would reflect prednmmantly myosin ATPase activity

Interactions between contractile and regulatory proteins m the sarcomere represent potential targets for rigor inhibition Recent structural data show that regions 403-414 and 529-544 of the myosin head are revolved in actin binding 2 However we find that peptide mimetlcs of these two domains affect neither rigor mduced at low ATP concentrations nor myosin ATPase activity By contrast a mimetic pepttde of the troponin-I region (Tn-ll]7.t48) that brads to both actln and troponln-C shows dose dependent abolition of rigor and myosin ATPasc activity. At 56p.M MgATP and 1141.tM peptide rigor is mhlbltecl by 95 per cent + 7 5 per cent (mean 4- S.D, n=4) and myosin ATPase by 88 per cent 4- 4,65 per cent (mean + S D , n=7) lnhlbitmn of rigor is sequence-specific rather than charge dependent since control pephdes with scrambled sequences have no effect on rigor Scrambled pepDdes only partially lnhxhtt myosin ATPase suggestmg both sequence and charge effects

Refcrence~ 1 Bowers. K , Allqhtre, A , Cobbold, P Blolumlne~cem measurement in gmgle cardiomyoeytes of sudden eytosohc ATP depleoon coincident with rigor J Mol Cell Cardlol 1992 24, 213-218 2 Rayment. 1, Rypmewskl, W, SchmJdt-Base. K et al Three- dimensional structure of myosin subfragment-1 a molecular motor Science 1993; 261 50-58

IJMS October, November, December, 1996

EVALUATION OF CARDIOVASCULAR RISK-FACTORS IN ANGINA AND M1 PATIENTS

J. C Freeburn I, J M. W. Wallace ~, D. Sinnamon 2, B. Craig 2, J- J Strain t, W. S. Gilmore t.

School of ~Biomedical Sciences, University of Ulster, Coleraine BT52 ISA, ~auseway Health & SOCLal Services

Trust, Coleraine BT52 IJA, Northern Ireland

The pathology of atherosclerosts has been the subject of much speculation over the past 50 yr Currently the disease may be regarded as a consequence of the body's normal response to injury, an hypothesis originally proposed by Ross ~

We evaluated a number of established and novel putative cardiovascular disease risk factors within serum hplds and clotting factors The study was approved by the research ethical committee of the University of Ulster. All subjects were male aged 37-72 yr Blood fractious were obtained from stable angina patients (n=26), post myocardml infarction (MI) patients (n=29) and apparently healthy controls (n=27) Statistical analysts was performed using analysis of variance (ANOVA) with least significant difference (LSD).

Table I shows the concentration of various clotting factors and serum hplds. Values are means 4- sere fZ denotes significant differences between control and angina groups; A denotes significant differences between control and post MI groups; u denotes significant differences between angina and post MI groups

Control (n=27) Angina (n=26) Post M[ (n=29)

Fibrmogen (g/l) 2 58-+0 11 3 69~0 26 fl** 3 49-+0 20 A** Factor V (%) 8767+352 112 32+5 58 fl** 119 04+7 52A*** Factor VI1 (%) 10456-+383 10072+437 lfil 50+--~ 82 Factor VIII (%) 96 25-+4 66 104 68-+9 62 118 83-+10 37 Total cholesterol (mmolll) 8 6-+0 36 7 07_+051 fZ* 6 94-+0 46A** HDL-cholesterol (mmol/I) 1 08+007 1 04-40.04 0 77-+0 IA***Y** LDL-cholesterol (retool/l) 7 55-+0 38 6 03-+0 50[2* 6 23-+0 49A* Trlglyeende~ (retool/l) 2 53+0 27 2 29x~0 18 2 78-+0 33

*P_<0 05, **P-<0 01, ***P-<0 fill

These findings suggest that serum hptds and coagulation parameters may enhance and/or mediate atherothrombotJc ischaemlc events

References 1 Ross, R, Atherosclerosls J Cardlovasc Pharmacol 1984, 6, $7t4- $719

THE EFFECTS OF FASTING AND ANAESTHESIA ON ANTIOXIDANT STATUS IN HUMANS

J D M Lyons ~, J Murray j,2, T Trlnick 3, J P H. Fee z Departments of ~Anaesthesxa, Ulster Hospital, Dundonald and 2The Queens University of Belfast and ~Blochemlstry, Ulster

Hospital, Dundonald

Free radicals are highly reactive and a potentially toxic species A number of exogenous substances exert their toxic effects on the body through the secondary generation of reactive oxidants by their metabohsm Antioxldants arc important defence mechanisms against such free radical mediated injury A new method has recently been developed for measuring the total antloxtdant capacity of body fluids and drug solutions based on the absorption of the ABTS (2 ,2 ' -az luobls - (3- ethylbenzothiazoline-6-st~lphonlc acid) radical cation (serum trolox)l The response to anaesthesia and surgery appears to be quahtatively mdtstlngmshable from other injury, including bums. Blebuyck and colleagues have demonstrated that nutrition

Vol t65 No 4

can have a protective effect against the metabolic change 2. The aim of this study was to es tabl ish the effects o f halothane, lSOflurane and propofol anaesthesia on antioxidant status and hepatic function of fasted and non-fasted patients

Fol lowing local ethics commit tee approval and informed consent, 53 unpremedieated patients scheduled to undergo body surface surgery were studied. They were randomly allocated to receive e i ther halothane (group 1), tsoflurane (group II), or propofol (group III). Each of these groups was further stratified to receive either n saline 11 or Vitrimix 11 (a proprietary IV feed). Forearm venous blood samples were drawn before IV therapy and before inductaon. Further blood samples were drawn at the end o f surgery and at 24 and 48 h after surgery. Bi l i rubin , albumin, urate, and trolox concentrattons were estimated. Data were analysed using parametr ic and non-parametric tests where appropr ia te A value o f p<0 05 was considered s ignif icant . Patients were comparable for age, gender and pre-operat lve phys ica l state. Serum trolox concent ra t ions dec l ined af ter operation, and gradually returned towards normal over the study period. At 48 h the groups receiving Vi tnmix had signif icantly better recovery of serum trolox than the fasted groups (p<0.05) (Table). The variance of serum trolox concentrations between anaesthetic agents did not reach significance

Table - Serum Trolox (mmol I -t) f~ted and non-f&~ted patients (mean (SD))

Pre- Pre- End of 24h post- 48h post- infusion induction operation operative operative

N Saline 1 43(009) 1 37(0.11) l 21(0.16)* 1 32(0 16) 1.27(0 14)**

Vnnmix 1 47(0 19) 1 44(0 15) l 25(0 19)* 1 39(0 21) 1 38(0.17)**

* p<0 05 within group variation from pre-lnfuslon and pre-laduclaon ** p<0.05 between group difference at 48 h

Conclusion. These data show that the recovery of serum trolox after body surface surgery is accelerated by the preoperattve infusion of IV Vltr imix. The authors acknowledge grant support from DHSS (NI). References l Miller, N J , Rice-Evans, C , Davies, M. J , Gopmathan, V. Mflner, A A novel method for measuring antloxtdant capacity and its apphcanon to monitoring the antloxldant status In premature neonates Chn Scl 1972, 84 407-412 2. Blebuyck, J. F., Albera, K G M M The effects ~f anaesthetic agents on carbohydrate metabohsm m the rat Chn SCL 1972, 42 4-5

AEROBIC FITNESS CORRELATES INVERSELY WITH B O D Y MASS C H A N G E IN JOCKEYS

L Hennessy ~, A. W. S. Watson 2, W. L. Halley 3 tDepartment of Exercise Physiology, Blackrock Clinic,

Blackrock, Co. Dublin; ZSports Injuries Research Centre, Universi ty of Limerick, Limerick; 3Turf Club Medical Officer,

Haddmgton Road, Ballsbrtdge, Dublin 4

Body fat content is inverse ly re la ted to many physical performance character is t ics 'Z. It is, however, unclear i f this relationship exists in a homogenous group of leading jockeys T h e r e f o r e , the purpose o f this study was to e x a m m e the r e l a t i onsh ips among an th ropome t r l c and phys ica l f i tness variables and body mass change before a race meeting Statistical analyses were carr ied out using SPSS Release 6.0.

Anthropornetric and physical fitness characterist ics of 10 leading Irish jockeys were assessed. A reduction of 1.25 _+ 1.28 kg (mean + standard deviation) in body mass (body mass change, BMC) during the week before a race meeting was recorded. Relationships between the variables were investigated by means of a zero order correlation matrix (Table I).

Section of Btomedical Sciences 3 0 9

Table l Zero order correlation matrix for selected variables

BMC 5SF %Fat VO2max VAT Body mass change (BMC) I 0 0 49 0 37 -0 9 l* -0 83* Sum of 5 skmfolds (5SF) l 0 097* -067* -087* % Fat 1 0 -0 58 -0 79* VO 2 max l 0 0 92* Ventilatory anaerobic threshold (VAT) 1 0

* Indicates that correlation coefficient is slgmficantly different from zero (p < 0 05)

Maximum oxygen uptake and the sum of 5 sklnfolds were found to have an inverse re la t ionship , conf i rming previous findings ~ Significant inverse relationships were found between maximum oxygen uptake, ventilatory anaerobic threshold and body mass change, Further, l inear regression analysis indicated that maximum oxygen uptake accounted for 83 per cent of the var iance m body mass change that occurred pr ior to a race meeting. These findings indicate that jockeys with a high level of aerobic fitness tend to have lower levels of body fat. and that they also demonstrate lower pre-race changes in body weight

T h i s s tudy was k i nd l y sponso red by The lrlsh Fteld Newspaper.

References 1 Watson, A W S Quantification of the influence of body fat content on selected physical performance variables m adolescent boys lr J Ivied Sol 1988, 157 383-384 2 Mayhew, J L , Schwegler, T M , Piper, F C Relationship of acceleration momentum to anaerobic power measurements. J Sports Med Phys Fit 1986, 2(3) 209-213

A N T H R O P O M E T R I C AND PHYSI CAL FITNESS CHARACTERISTICS OF LEADING IRISH JOCKEYS

L. Hennessy t, A. W, S. Watson 2, W. L. Halley 3. IDepartment of Exercise Physiology, Blackrock Chine,

Blackrock, Co Dublin, 2Sports Injuries Research Centre, Universi ty of Limerick, Limerick, 3Turf Club Medical Officer,

Haddington Road, Bal lsbridge, Dublin 4.

Li t t le publ ished informat ion is ava i l ab le descr ib ing the anthropometric and physical fitness characterist ics of jockeys. The purpose of this study was to provide such information. Ten leading jockeys (mean age 28,7 _+ 4.6 yr) volunteered for the study and gave their informed consent. All tests employed were found to have acceptable test-retest rehabdi t ies (see Table I)

Table 1 Anthropometrle, physical fitness and pulmonary function data (mean • standard devlanon) for 10 leading Irish iockey~

Test-Retest 95% Confidence Variable correlation L~mlts of Jockeys Reference Man

Measurement (Mean• (Mean)

Height (ram) 1680!--50 1740 Body mass (kg) 58,5+4 9 70 0 Sum of 5 sklnfolds (mm) 0 90 5 3 20 2-+6 9 %Fat 095 3 4 73-+24 150 VO~max (ml/kg/mm) 0 98 3 1 62 0+74 40-45 Ventllatory Anaerobic 0 95 6 0 52 ff2_8 3 Threshold (ml/kg/mm) Forced Vital Capacity 0 90 7 2 5 2 _+0 6 5 33 (FVC) (hires) Forced Expiratory Volume 0 91 8 1 44 -+0 5 440 m 1 second (FEVI) (hires) Peak Expiratory Flow Rate 0 89 8 5 10 64-2 1 9 48 (PEFR) (htres/second)

All jockeys were shorter in stature and displayed lower than ave rage body mass as compared to r e fe rence man t. Body

310 Royal Academy of MedlcinP in Ireland

composition was similar to levels reported foi" elite endurance ~.thletes arid subjects had ~levels of aerobic fitness comparable to 'many el i te sporting groups z The FVC, FEV1, PEFR of the subjects were 106 per cent, 110 per cent and 117 per cent of the age and height corrected norms for males These results indicate that Irish jockeys have umque authropometrlc characteristics and high levels o f the physiological variables that are associated with aerobic fitness This study was kindly sponsored by the Irish Fteld newspaper. Permission for the study was granted by the Stewards of the Irish Turf Club.

References 1 Kateh, F 1, McArdle. W D In Nutntton. Weight Control, and Exerclse(3rdeditmn) 134 Lea&Fehlger, Phdadetphta, 1988 2 Watson, A W S Physical Funess and Athletic Performance (2nd edmon) Longrnans Scientific and Technical, London. 1996

AN EMPIRICAL EXAMINATION OF SOME COMMON HYPOTHESES CONCERNING THE CAUSES OF

O V E R U S E IN'JURIES IN ADOLESCENTS

M. A. Weir, A W. S.,Watson. Umvers i ty of Limerick.

Overuse injuries are common in adolescents involved m a substantial amount of athletic activity. It has been suggested that certain physical character is t ics , such as above average height and anaerobic power, may predispose to overuse injury 1 The purpose o f the present study w~as to test.this hypothesis on Irish adolescents and to determine whether simple tests,-such as might be carr ied out in schools, could be used to identify adolescents at high risk of overuse injury The subjects of the study were 149 male and 117 female adolescents between the ages o f 12 and 16 yr. All subjects and their parents gave wri t ten in fo rmed consent to pa r t i c ipa te in the study The fol lowing measurements were taken by standard methods,- height, wmght, shuttle run, standing long jump, sit and reach test, smt-ups and 12 rain run-walk z, With the ~xception o f flexibili ty the 95 per cent confidence hmits of all measures was within +- 7 71 per cent of the mean. The results of the study are shown in the fol lowing table.

SubJects who SubJeCtS who SubJeCts who Un-mjured had prevJously had prewously had prewously SubJects suffered from suffered from suffered from any overuse knee overuse thigh overuse injury injury injury

(Group A) (Group K) (Group T) (Group U) Mean• Dev Mean• Dev Mean• Dev Mean• Dev

Height (cm) 160 53+-8,31 163 25-+7 31' 157 77+-8 34 159 76+-8 11 Weight (kg) 53 73-+12 34 56,26-+12 65 51.88-+13 9l 52 92-+g,3 ShuttleSrun (s) 19 17-+1 61' 19 01~_1 37* 18 90s 1" 19 95-+1 82 Standing long

jump (mt 1 65-+0 24* 1 69-+0 26* 1 67+-0 19 1 58-+0 25 Sltandreach(cm) 1493:i:796 1242-+91 I* 1568-+572 1608-+596 flexlblhty Sit-ups Ino l 22 14+-5 43 21 96-+5 7 23 05-+5 92 21 19+4 2 12 mm run-walk (km) 2 65+049' 270:[:042* 271:s 2 4Ok0 43

*Significant difference from Group U (p<0135)

Wi th in the l imi t s o f the e x p e r i m e n t a l e r ro r of the m e a s u r e m e n t s i t was found tha t t he re were s i g n i f i c a n t

I I M S October, November, December, 1996

differences in the shuttle run, standing broad jump and 12 min ' run -wa lk scores of adolescents who suffered f rom overuse injuries compared to un-injured subjects. Though scores on the 12 rain run-walk test were not expected to be associated with overuse injury the explanation for this relationship probably lies in the fact that subjects with better scores on tlus test were those who did more training This greater amount of training provided far more opportunity for them to incur an overuse injury thereby creating an artificial relationship between scores on the 12 mm run-walk test and incidence of overuse inJUry. Although the above results suggest that certain physiological factors are associated with the incidence of overuse injury further research is required in order to establish causality.

References 1 Lysens, R , O~tyn M S, Vanden A Y "The accident-prone and overuse-prone profiles of the young athlete" Am, J Sport Med, 1989, 17 612-619 2. Campbell, W R, Tucker, N M, An Introduction To Tests And Measurements In Physical Edueanon Bell, London, 1967

INTERACTION OF T H E NOVEL B I O R E D U C T I V E DRUG AQ4N WITH RADIATION IN BDF MICE I M P L A N T E D

WITH T H E T50/80 T U M O U R

O. E Frlery 1, M. V HejmadP, I. A Mclntyre ~, J J A McALeer 2, L H~ Patterson 3, S. R McKeown I (Introduced by

J M. Allen.) ~B omedlcal Sc Umversi ty of Ulster, Jordanstown N.

, Ire.land; ~Oncology, Queen's University Belfast ~Pharrnaceutical SCl, De Monttort University, Leicester, U K.

The in vtvo anti- tumour eff icacy of A Q 4 N ( l~4-b ls - [ [2- ( d i m e t h y l a m i n o - N - o x i d e ) - e t h y l ] a m r u o } 5 , 8 - d i h ~ d r o x y - anthracene-9,10-dione) has been studied in combinat ion wl~h radiat ion (smgle and mult iple ' fractions). Experlm'ents were carr ied out in B 6 D 2 F 1 mice bear ing the T 5 0 / 8 0 maminary carcinoma. Treatment was initiated when the turnout diameter reached 6 5-7 5ram (geomet r i c mean o f three o r thogoua l diameters). Tumour size was measured 3 times weekly The time taken for the tumour to reach double its treatment volume (VDT) was used as a measure o f anti-turnout efficacy A Q 4 N (50- 200mg/kg) was admimstered as a single intraperttoneal mjechon 30 min prior to a single dose of radiation (12Gy). The interaction was at least additive result ing in a tumour growth delay (TGD) of 16,84+_0.86 (50mg/kg) to 23.32+-1 98 days (200mg/kg) A Q 4 N reduced the r ad ia t ion dose by 50 per cen t for an eqmvalent anti-turnout effect, this maximal effect ~vas observed w h e n the drug was a d m i n i s t e r e d f rom 4 days b e f o r e (TGD=I8 55+_1 41) to 6 h (after radiation (TGD=22 18+-1 00) ~ AQ4N (150mg/kg) provided an increase in turnout growth delay m combinat ion with al l rad ia t ion doses tested (10-Z~0Gy) Increased antl-tumour efficacy was also found when radiation was given in fractlonated regimens (5 X 3Gy; 10 X 2Gy) with single and multi-dose AQ4N In conclusion, AQ4N can be used to significantly reduce the radiatmn dose required to achieve an equivalent anti-tumour effect

Reference 1 McKeown, S ~ R , Hejmadi, M V, MeIntyre, 1 A , McAleer, J, J A , Patterson, L H AQ4N an alkylammoanthraqumohe N-oxide showing bloreductlve potef~tial and pOSltlVe interaction with radiation in vlvo Br J Cancer 1995.7~" 76-8.1

Vol 165 No 4

URINARY POLYAMINES AS A MARKER OF DISEASE RECURRENCE IN TRANSITIONAL CELL CARCINOMA

(TCC)

J L Leckey, G B Nevin, S. R. McKeown, D. S. Soppttt. (Introduced by J M Allen)

School of Biomedical Sciences, University of Ulster, Jordanstown, N l,reland.

Transitional cell carcinoma (TCC) of the bladder has a high recurrence rate and a slgfllficant proportion progress to a more malignant disease At present this requires a lifetime follow-up by cystoscopy; an invasive procedure, often requiring a general anaesthetic Suitable urinary markers would make the need for regular cystoscopy redundant Polyamtnes are positively charged, basic cell components, found in most body flmds Their exact function is still unclear but they are believed to influence processes involved in the formation of DNA, RNA and protein molecules. Studies on urinary polyamine levels in cancer patients have shown confltctmg results Some authors report that they act as a marker for disease recurrence I while others have found that they are of hmlted use z,

Polyamine analysis was carried out on 28 TCC urines, and 22 age and sex matched control urines using HPLC. Creatinine concentrations were determined spectrophotometrically and were used to standardise polyamme levels.

The polyamines putresclne, cadavarlne, spermidlne and spermlne were individually invesUgated using Students t-tests. The mean urinary putrescine levels were 0 688ng/mg creatlnme for controls and 0.175ng/mg creatlnine for TCC patients, showing a statistically significant difference (p<0.0014). No differences were found between the two populations for cadavartne, spermidine and spermine The mean spermidine/ spermlne ratios were 3.584 for controls and 0 619 for TCC patients , showing a s ta t t s t ica l ly s igni f icant di f ference (p<0.0079) Results obtained to date, suggest that putresclne levels and the spermldme/spermine ratio may act as a suitable marker for TCC recurrence.

References I Lmsalata, M, Ru~o, F, Cavalhnl. A, Berloco, P, Dt Leo, A Polyammes, dlamlne oxldase, and ornlthme decarboxylase activity in colureetal cancer and in normal surrounding mucosa Diseases of the Colon and Rectum 1993, 36 662-667 2 Becctolim, A , Porclam, S , Lanlnl, A , Sontoln, R , CtOll]-nl, L Urinary polyamlnes in patients with advanced cervical cancer or pelvic recurrence during and after radiotherapy Acta Oncologwa 1992, 31 (3) 327-331

AN INVESTIGATION OF THE EFFECTS OF 8-HYDROXY-2- (DI-N-PROPYLAMINO)-TETRLIN (8-OH-DPAT), ON BEHAVIOURAL PARAMETERS IN RATS TREATED

ACUTELY AND CHRONICALLY WITH METYRAPONE

F Brehony, J, P Kelly, B E Leonard. Department o~ Pharmacology, Umversity College, Galway.

Both abnormal glueocorticold secretion and dysfunction of central serotoncrgic neurotranstmssLon have been implicated in the pathogenesls of affecttvc disorders ~. Metyrapone, a potent inhibi tor of cortlsot production and 8-hydroxy-2-(dl-n- propylamino)-tetral ln (8-OH-DPAT), a selective 5-HTIA receptor agonlst, are utillsed in this study to examine the relationship between glucocortlcoid regulation and serotonergic activity The objective of this study was to compare the effects

Section of Biomedtcat Science~ 311

of acute and chronic administration of metyrapone to rats subsequently challenged with 8-hydroxy-2-(di-n-propylammo)- tetralin (8-OH-DPAT), on achvlty and temperature responses Metyrapone (50mg/kg i p ) was administered daily to male Sprague Dawley rats (260-280g) After one administration of metyrapone and after 17 days of daily treatment, 8-OH-DPAT (0 1 and 1 0mg/kg s c ) was admimstered 30 mm later Chrome, but not acute metyrapone treatment potentiated the hyperacnve response (p<0.05 Mann Whitney) of 8-OH-DPAT as recorded by the home cage activity monitor In addition a hyperactive response was induced by chromc metyrapone admintstrauon alone (p<0.05 Mann Whitney), The 8-OH-DPAT induced hypothermlc response was attenuated following chronic but not acute metyrapone treatment These results suggest that the function of 5-HTtA receptors are altered after chronic metyraponc administration and implicate a role for the 5-HT~A receptor in glucocorticold regulation

Reference I Dinah, T G Glueocorticolds and the genesis of depressive illness Apsychoblologmal model Br J Pharmacol 1994, 164 365 371

THE COMPARATIVE EFFECTS OF LONG-TERM ADMINISTRATION OF SERTRALINE, PAROXETINE,

CITALOPRAM, FLUOXETINE AND AMITR1PTYLINE ON MONOAMINE CONTENT IN RAT BRAIN

A, J. Harkm, J P Kelly, B E Leonard Department of Pharmacology, University College, Galway

The objective of this study was to compare the effects of long-term administration of sertrahne, paroxetlne, cltalopram, fluoxetlne and amitriptyhne on monoamme content in rat braJ n Sertrahne (5mg/kg), paroxetine (5mg/kg), citalopram (5mg/kg), f luoxetine (5mg/kg) and ami t r ip ty l ine (10mg/kg) were administered once daiIy p.o, to male Sprague Dawley rats (250- 270g) Twenty-four'hours after the last treatment, animals were sacrificed and discrete brain regions (brain stem, hlppoeampus and frontal cortex) were analysed for monoammc content by a standard HPLC method Fluoxehne induced a persistent depletion in 5-hydroxytryptamlne (5-HT) and 5-hydroxymdole acetic acid (5-HIAA) in discrete areas of rat brain 5-HT ctmtent was reduced to approximately 85 per cent of control levels (P< .05 Student's t-test) 5-HIAA content was reduced to 70 per ceut of control levels (P<0 01 ) The other selective seotonin reuptake inhibilors and amttrlptyhne failed to reduce this effect However fluoxetlne, sertraline and paroxetine reduced the turnover of 5- HT in the hippocampus (P<0 05) Regional differences m the effects of repeated administration of the drugs on the changes in 5-HT and 5-HIAA were evident The hippocampus was particularly sensitive to drag-induced changes m 5-tIT turnover The depletion of 5-HT and 5-HIAA, a result unique to fluoxetme at the pharmacological dose employed, may be a refleehon ot a combined effect of fluoxetlne and its active metnbolite norfluoxetlne I 2

Referel~ees 1 Cacela, S, Fracasso, C, Galattml, S, Saran, S Effect~ of short- term and long-term admlnlslratlon of fluoxetme on monoamme content m rat bram Neuropharmacology 1992 3 I, 342-347 2 Gobbl, M , Frlttolh, E, Mennml, T, GarattmL S Releasing actJwttes of d-fenfluramme and fluoxetme on rat hlppocampal synapto~omes preloaded with [1HJ-serotonln Naunyn Schmledeberg's Archlve~ of Pharmacology [992,345 1-6 ,

312 Royal Academy of Medicine in Ireland

AN INVESTIGATION OF THE BEHAVIOURAL AND PHYSIOLOGICAL EFFECTS OF ACUTE AND CHRONIC ADMINISTRATION OF METYRAPONE, CHALLENGED

WITH (-+) 3,4-METHYLENE-DIOXY- METHAMPHETAMINE

F Brehony, J P Kelly, B. E ',,Leonard. Department of Pharmacology, Universit'y, College, Galway.

The objective of this study was to compare the effects of acute and chrome administration of metyrapone to rats subsequently challenged with (-+) 3,,1-methylene- dioxymethamphetamine (MDMA). Metyrapone (50 mg/kg) was administered daily i.p to male Sprague Dawley rats (260-280g), After l administration of metyrapone and after 19 days of daily treatment, MDMA (10mg/kg) was admimstered i.p, 30 min later. Activity and temperature profiles were recorded using a home cage activity monitor and a colonic thermometer respectively. Six hours after MDMA challenge, animals were sacrificed and discrete brain regions (amygdala, frontal cortex and hippocampus) were analysed for monoamine content by a standard HPLC method and y-aminobutync aetd concentration by a photometric analysis. Adrenal ascorbic acid concentrations, plasma cortieosterone concentrations and white blood cell counts were also determined. Acute and cbrootc metyrapone treatment elicited no significant alterations in the biochemical parameters examined Chronic metyrapone treatment did not affect the significant reductions m 5-HT and 5-HIAA (p<0.001 Student's t-test) concentrations following MDMA administration in the amygdala, frontal cortex and hippocampus, Acute and chronic metyrapone treatment potentiated the hyperactive response (p<0.05 Mann Whitney) of MDMA as recorded by the home cage activity monitor. This effect was more apparent after chronic treatment and this may be due to an upregulation of the 5-HTIB receptorL Colonic temperature profiles demonstrated that chronic, but not acute, administration of metyrapone disrupted a biphasic temperature response of 10mg/kg MDMA, possibly due to alterations m 5-HTjA receptors I. Thus chronic, but not acute, metyrapone treatment alters the behavloural, but not the neurochemical profile of MDMA in the rat.

Reference 1 McNamara, M G, Kelly, J, P., Leonard. B. E The effect of acute MDMA administration on body temperature, serum corhcosterone and neurotransmltter concentrations In male and female rats Human Psychopharmacol. 1995, 10. 373-383

A COMPARATIVE BEHAVIOURAL AND HISTOLOGICAL STUDY OF INTRA-PERITONEAL AND INTRA-

AMYGDALOID LESIONS INDUCED BY KAINIC ACID

A S. Wrynn, B Earley, B. E. Leonard Department of Pharmacology College Galway,

Kalnic acid (KA), an agontst at ionotropic non-NMDA glutamate receptors and a potent neurotoxin, induces neuronal damage by both direct excitotoxic action and by induction of continuous seizure activity It is a neuron-specific, axon sparing lesioning tool with particular sensitivity towards amygdaloid neurons'

A comparahve study was carried out to assess the role of the anterior cortical nucleus (ACe) of the amygdala on memory following intra-pehtoneal (I P.) and intra-amygdaloid (1.A,) lesions reduced by KA. The I A. lesions of the ACe (0.2p_g/ 2btl) were performed on male Sprague Dawley rats under 2-2-2 tribromoethanol anaesthesia. In a separate experiment KA was

IJMS October, November, December, 1996

also given I P (8 mg/kg). Following 2 weeks recovery from surgery passive avoidance behaviour and 8 arm radial maze tests were undertaken No deficits in any group were seen in the ra&al maze. However, the I.A. group alone exhibited deficits in passive avoidance learning (p<0.01) His tological analysis of hlppocampal CA3 and CA4 regions indicated severe damage with almost complete cell loss in the I.A. group wlth less damage occurring in the I.P. group.

These results indicate that, similar to lesions of other amygdaloid auele~ z, the ACe. of the amygdala may play a role in fear-motivated, but not spatial learning. However the hippocampal damage also indicates that the ACe alone is not responsible for such deficits and indeed suggests such distant damage as a feature of KA-induced neurotoxicity

References 1. Berger. M L. La~sman, H , Hornyklewicz, O Llmble seizures wnhoat brain damage after rejection of low doses of kainic acid rote the amygdala of free moving tats Brain Res 1989, 489 261-272 2 Sarter, M,, Markowitsch, H J. Involvement of the amygdala m learmng and memory A critical review, with emphasis on anatomical relanons Bvhav Neurosclence 1992, 99 342-380

MAPPING THE Xp22 1 - Xp22.3 REGION OF THE HUMAN X CHROMOSOME BY FLUORESCENT IN SITU

HYBRIDIZATION

T. M Dineen, R Powell, J. A. Houghton Cytogenetics Unit, Department of Microbiology, University

College, Galway.

The most common inherited defect of phosphate transport is X-linked hypophosphataemic (vitamin D resistant) rickets with an incidence of 1 in 225,000 hve births. The defect is caused by a failure of the kidneys to reabsorb phosphate from the glomerular fi l trate, resul t ing in a phosphate leak and hypophosphataemia.

The production of accurate gene maps is dependent on the ability to order genes and markers Methods include linkage studies and analysis of lnterspeelfiC somatic cell hybrids and pulsed field gel electrophoresls. Such genetic linkage analyses have loealised the hypophosphataemia rickets gene to Xp22.1.

Fluorescent m situ hybridization (FISH) has made a major contribution to physical gene mapping and ordering FISH permits putative single-locus sequences to be assigned to metaphase chromosome bands at a resolution of about 3-20 Mb, as well as determining distance and ordering sequences in the mterphase nucleus that are separated by about 50-850 Kb.

In this study, FISH has been applied to the development of a physteal map of the distal section of the short (p) arms of the human X chromosome in the region Xp22.1 to Xp22 3. A series of cosmid contigs which have already been assembled for this region, were labelled using the hapten digoxigenin These were hybridized to the X chromosome and detected using an anti- dJgoxigenin antibody that hacl FITC conjugated to it. FISH signals of metaphase chromosomes provided immediate coarse data on the chromosomal location of the cosmids on the X chromosome and gave, an approximate regional mapping. Cosmid maps will now be produced with increasingly finer resolution For example, multtcolour FISH in which the location of 2 or mum probes can be distinguished. Using probes of known relative order and distance will improve the resolution of the mapping

The authors acknowledge financial support from Forbairt and the EU under the Biomed Programme: Human Genuine Analysis.

Vol 165 No 4

EFFECTS OF CERVICAL SYMPATHETIC TRUNK SECTION ON VENTILATION IN CONSCIOUS RATS

D Cantillon, I McDaid, K, D. O'Halloran, A. Bradford Department of Physiology, Royal College of Surgeons in

Ireland, St Stephen's Green, Dublin 2.

The carotid body receives a rich sympathetic mnervation from the cervical sympathetic trunk which can excite or inhibit chemoreceptor atferent activity Hypoxia and hypercapma stimulate these sympathetic efferents ~ but very little is known about their role in ventilatory control

Ventilation was measured by barometric plethysmography in 12 adult, male, cousclous, unrestrained Wlstar rats breathing air and hypoxic and hypercapmc gas mixtures Animals were anaesthetized with ether and sham-operated (n=6,) or both cervical sympathetic nerve trunks were cut (n=6,) and allowed to recover Bilateral ptosm was present in all sympathectomlzed animals Values for minute ventilation in ml/min/100g body weight (rutact vs. sympathectomlzed) were 45 8 • 4 8 vs 24 8 + 8 1 (mean + SD) for air, 65 9 + 25.2 vs. 37.4 _+ 22.3 for 10 per cent 02 , 52.3 _+ 12 2 vs. 38.5 + 11.4 for 3 per cent CO2, 81 7 • 2 1 3 v s 4 5 0 + 1 6 1 f o r 6 p e r c e n t C O z a n d l 1 0 0 + 2 7 5 v s 519 • 11 8 for 9 per cent CO 2. Sympathectomy significantly (ANOVA, p < 0 05) reduced ventilation for all inspired gases but did not affect venti latory responses to hypoxla and hypercapnia except for a reduced 9 per cent CO 2 response These results show that the sympathetic nerves to the carotid body exert an important influence on ventilatory control, possibly by stimulating chemoreceptor afferent activity. Supported by the Health Research Board and Royal College of Surgeons in Ireland.

Reference I Matsumoto, S, Mokashl, A, Lahm, S Cerw~,al preganghonic sympathetic nerve activity and chemoreflexes m the cat J Appl Physlol 1987, 62 1713-1720

SUPERIOR LARYNGEAL NERVE SECTION ALTERS THE VENTILATORY RESPONSE TO ALM1TRINE IN

CONSCIOUS RATS

K. D O'Halloran, A K Curran, A. Bradford Department of Physiology, Royal College of Surgeons m

Ireland, St Stephen's Green, Dublin 2

Almltrlne is an arterial chemoreceptor stimulant whose ventilatory effects have been well described The superior laryngeal nerves (SLN) contain paragangha morphologically and hiochemically similar to chemoreceptor tissue but the function of these paragangha is unknown The present lnvesttgatlon examines the effects of SLN section on the ventllatory response to almltrlne

Ventilation was measured by barometric plethysmography in 27 conscious, unrestrained, tracheostomlsed (under ether inhalation), male, Wlstar rats (400-500g body weight) during normoxia, hypoxm and hypercapma before (control) and after administering almltrme (5 mg/kg i p ) or vehicle (n=9) In 1 group (SLN Intact, n=18), the SLN were carefully identified but remained intact In a second group (SLN cut, n=9), both SLN were Cut.

In SLN-intact animals, the minute ventilation responses (control vs almltrrue) were 43,2 5:41 7 vs. 52 1 5:70 6 for 10 per cent O z ('per cent change from air + SD, n = 9), 40 4 5:33 8

Sectton ofBiomedtcalSciences 313

vs. 38.5 + 19 2 for 3 per cent COz, 63 3 _+ 58 1 vs, 92 2 + 63,6 for6 per centCO zand 80 6 +52 5 vs 85 8 +58 2 for9 per cent CO 2 The hypoxlc ventdatory response was unaffected but the response to hypercapnia was increased by almttrlne (ANOVA, p< 0 05) Vehicle had no effects, In SLN-denervated ammals, the minute ventilation responses (control vs almitrme) were 15 4 +36 .4vs 3 9 0 + 6 5 l for l 0 per cent O z, 22 2 _+ 29 5 vs 57,75: 92.5 for 3 per cent CO2, 44 9 5:32 5 vs 58 5 + 56 1 for 6 per cent CO 2 a n d 5 8 4 _ + 5 1 5 vs 8 6 2 + 5 7 5 for 9 p e r c e n t C O 2, There was a reduction in the ventllatory response to almitrme breathing air (vs SLN-rutact) but the ventdatory responses to hypoxla and hypercapnia were unaffected.

In conclusion, almitrine exerts part of its ventllatory effects through SLN afferents suggesting a functional chemosensltlvtty of SLN paragangha

Supported by the Health Research Board, Royal College of Surgeons in Ireland and Welleome Trust,

EFFECTS OF HYPERCAPNIA ON DEPOLARIZATION INDUCED TENSION IN ISOLATED RINGS OF THE RAT

PULMONARY ARTERY

D Harmon. M Sweeney, P. McLoughhn Department of Human Anatomy and Physiology, University

College, Earlsfort Tee, Dublin 2

Hypercapnla causes vasodilatlon through endothelium- dependent and independent mechanisms in systemic vascular beds ~. In this study we have examined the modltmatrun by hypcrcapnia (mean PCO z + SEM = 59 9 • 4 2 mmHg, at constant extracellular pH) of isometric tension development, in isolated, submaxlmally contracted (30 mM KCI) rings of rat pulmonary artery. Rats were killed by stunning and cervical dislocation Their lungs were then removed and rings of pulmonary artery were dissected free and mounted in physiologmal sahne (PSS) at 37~ for recording of isometric tension development All tensions were expressed as per cent T max in response to 80 mM KCI exposure. The presence of intact endothelium was confirmed by demonstra t ing re laxat ion in response to acetylchohne (10"SM) In some experiments endothehum removal was demonstrated by the absence of relaxation m response to ACh. In endothehum intact rings (n = 6) following a switch from control to control soluuon (PCO z = 34.8 • 2 2 mmHg) a mean increase in tension of 12 per cent (4-- 3 4) was produced over 45 mln In endothelium-intact rings (n = 9) a switch to hypercapnic solution produced a mean increase in tension of 22 per cent (5: 4.5). The difference between the two groups was not statistically significant (p = 0 08, ANOVA) Following removal of the endothehum, hypercapma (n = 5) caused a reducuon in mean tension of -3.0 per cent (_+ 5 3), statistically szgnlficantly different from the response to hypercapma with endothehum intact (p < 0 01) and control (p < 0 05) switches The absence of a vasoddator response m these pulmonary arterial rings is in contrast to those from systemic vascular beds ~. The response of pulmonary arterial rings to hypercapma is significantly modified by the cndothellum.

Reference 1 Carr, P, Graves, J E , Ponstan, L Carbon dioxide induced vasorelaxatlon in rat me,enteric small arteries precontracted wnh noradranahne ts endothehum dependent and medmted by nurlc oxide Pflugers Arch 1993, 423 343 345

314 Royal Academy o] Medtcme m Ireland

MAP4 DOES NOT ANTAGONISE THE ATTENUATING EFFECT OF LAP4 ON EARLY PAIRED PULSE

DEPRESSION 1N THE RAT HIPPOCAMPUS IN VITRO

D O'Leary, J J, O'Connor. Department of Human Anatomy & Physiology, Umverslty College, Earlsfort Terrace, Dublin 2 (Introduced by M J

Rowan).

Field potentials evoked in the medial perforant path of the hlppocampus exhibit paired-pulse depression (PPD), the extent ol which is partially dependent on the Inter stlmulu~ interval (1SI)I We have prewously demonstrated to the Society 2 that metabotroplc glutamate receptors (mGluRs) can attenuate the PPD observed upon medial pathway stimulation in the rat dentate gyrus in vitro In the present study we have investigated the role of a mGluR antagomst on early (_<50ms) and late (_<500ms) phase PPD

Transverse hippocampal slices were prepared from male Wlstar rats (50-150g) by standard methods Field excitatory post- synaptic potentials were evoked in the medial perforant pathway every 20s. Paired pulses were applied at ISis of 10, 100 and 500ms.

In control slices the second synaptic response (S 2) was consistently smaller than the first at all 1Sis tested (S z reduced by 37.2_+5.6 per cent, 2 0-2_1 5 per cent and 14 5+1 5 per cent at I 0ms, 100ms and 500ms ISI respecttvely; n=5; mean_+sem) The mGluR agonist, L-2-amino-4-phosphonobutyric acJd (LAP4; 20p.M) sigmflcantly attenuated this PPD (e.g. in the presence of LAP4 S z reduced by 25 1_+4 2 per cent and 9 0-+3 75 per cent at 10ms and 500ms ISI respectively; p<0.01; n=4 compared to controls). The putative and selective presynaptlc mGluR antagonist o~-methyl-L-AP4 (MAP4) at 2001,tM did not block the LAP4-1nduced reduction in PPD at 10ms ISI (p>0 05, n=4). However MAP4 significantly reduced the LAP4 effect on PPD at both lOOms and at 500ms (n=4 for both concentrations; p<0,05)

These results show disparities m the action of MAP4 on early and late phase PPD in the medial perforant pathway. The authors acknowledge grant support from Forbalrt

References 1 Kahle, J S , Cotman, C W L-2-amJno-4-phoqphonobutanolc acid and IS,3R-I-ammocyelopentane-I ,3-dicarboxyhc acid reduce paired pulse depression recorded from the medial perforant path in the dentate gyrus of rat hlppocampal shces J Pharmacol Exp, Ther 1993, 266 207-215 2 Cassldy, E M , O'Connor, J J The effects of presynaptlC metahotroplc glutamate receptors on paired pulse depression in the rat hlppo~,ampus in wtro lr J Med Scl 1996, m pre~s

IJMS October, November. De~ember. 1996

JUN K1NASE ACTIVITY IN THE PREGNANT AND INVOLUTING MOUSE MAMMARY GLAND

A. M. Broderlck j, N Keon:, F Martin 2. ~Our Lady's Hospital for Sick Children, Crumlin, Dubhn and

2Pharmacology Department and Biotechnology Centre, University College Dublin, Dublin 4

The post-laetational involuting mouse mammary gland undergoes a significant and rapid loss of its epithelial component by a programmed cell death In carher studies, we, and others, have shown that this programmed cell death is accompanied by new gene expression and that this IS most likely modulated by the actlvahon of transcription factors such as AP-I (Fos/Jun). It has been proposed that a key trigger of this programmed cell death ts a loss of contact between the epithelial cells and their underlying extracellular matrix. It is possible that the loss of this contact triggers a new pattern of signal transductlon cascades within the epithelial cells and that this results In the activation of specific tran~crlption factors. Recent studies have shown that both Fos and Jun are activated by second messenger cascades which mvolve specific kmases which phosphorylate a very limited number of substrates. In particular, Jun has been shown to be phosphorylated by the SAP/JNK family of klnases.

In this study, analyhcal methods for the measurement of the Jun kmases, In particular JNK I and JNK 11, were established These were A) a solid state assay which used the bacterial fusion protein GST-JUN(1-223) linked to the matrix GSH-agarose as both a specific affinity binder and substrate for JNK 1 and 11. GST-JUN(I-223mut) m which serine63 and serine73, the two JNK target amino acids were mutated to alamne was used as negative control; and B) an in-gel klnase assay in which GST-JUN or 14-mer synthetic pepttdes spanning serme63 and serlneT~ in eJun were incorporated into polyacrylamide matrices and acted as In sttu substrates for .INK 1 and II These assays were used to measure Jan klnase acUvlty m pregnant, lactating and involuting mouse mammary gland and in normal and mvolatlng rat ventral prostate

Using the solid state assay we detected a kmase activity which phosphorylated serlne63 and serineT~ of cJuo whose activity transiently increased in the early involuting mouse mammary gland Using the m-gel kinase assays with either GST-JUN (1- 223) or the serine63 or serlneT~ peptides as substratea we detected JNK 1 (44kd) and JNK II (55kd) activities With both assay systems we found JNK 1 and JNK II actlwty to be highest in the lactating mammary gland and normal prostate and to decrease in revolution. Our results suggest that increased JNK 1 and JNK I1 activity probably do not contribute to activation of cJun and increased AP 1 activity in mammary gland and prostatic involution However, a separate kinase activity detectable, using the solid state activity, may Increase the levels of Jun phosphorylatlon in early lnvolutmn and contribute to increased AP 1 activity.

This work was funded by grants from Forbalrt, Ireland and The Health Research Board, Ireland