"PROCEEDINGS OF THE POSTER COMMUNICATIONS Distribution ofa"
42P PROCEEDINGS OF THE POSTER COMMUNICATIONS Distribution of a-adrenergic, muscarinic and opiate receptors within lumbar spinal cord of cat BY M. R. DASHWOOD*, S. M. FLEETWOOD-WALKERt, M. P. GILBEY*, R. MITCHELLt and K. M. SPYER*. * Department of Physiology, Royal Free Hospital School of Medicine, Rowland Hill Street, London, t Department of Physiology, Royal (Dick) School of Veterinary Studies, Summerhall, Edinburgh, t MRC Brain Metabolism Unit, 1 George Square, Edinburgh The selective antinociceptive effect of ionophoretically applied noradrenaline (NA), on the responses of cat dorsal horn neurones, is mediated by a2 adrenoreceptors (Fleetwood-Walker, Hope, Iggo, Mitchell & Molony, 1983; Fleetwood-Walker, Mitchell, Hope, Molony & Iggo, 1984; Davies, Johnston, Miller, Quinlan & Sheardown, 1984). In some cases a, receptors appeared to mediate non-selective depression of neuronal activity. The present experiments investigate the distribution of a, and a2 receptors within the lumbar dorsal horn of cat spinal cord. In vitro autoradiographic techniques (Dashwood, 1984; Young & Kuhar, 1980) were used to detect these sites, together with muscarinic and naloxone-sensitive opiate receptors for comparison. Histologically prepared sections of lumbar cord (L6/L7) from 6 cats were incubated at 4 0C in appropriate buffers with low concentrations of [3H]prazosin, [3H]rauwolscine, [3H]quinuclidinyl benzilate and [3H]naloxone. Sections were covered by cover-slips coated with tritium-sensitive emulsion or [3H]Ultrofilm and exposed at 4 0C for 4-6 weeks. Development revealed dense accumulations of silver grains over particular areas of grey matter, which were absent when incubated with excess unlabelled ligand/displacer. Specific binding of [3H]prazosin (al) was present at very low levels, reaching only 2-3 times non-specific. Most of the sites were in ventral horn and the deeper laminae of dorsal horn. Alpha-2 receptors, however (labelled by [3H]rauwolscine) were present at high levels, concentrated in laminae I/II and the intermediomedial zone, with moderate density in the rest of the grey matter. The opiate receptors were highly concentrated in laminae I/II, as were muscarinic receptors, which also showed moderate levels throughout most of the grey matter. The results accord with the brief report of Young & Kuhar (1980) in rat cervical cord and are consistent with a2- but not al-mediated effects of NA on the processing of nociceptive input in the superficial laminae of dorsal horn. This work was supported in part by the British Heart Foundation. REFERENCES DASHWOOD, M. R. (1984). J. Physiol. 350, 66P. DAVIES, J., JOHNSTON, S. E., MILLER, A. J., QUINLAN, J. E. & SHEARDOWN, M. (1984). J. Physiol. 350, 24P. FLEETWOOD-WALKER, S. M., HOPE, P. J., IGGo, A., MITCHELL, R. & MOLONY, V. (1983). J. Physiol. 343, 67-68P. FLEETWOOD-WALKER, S. M., MITCHELL, R., HOPE, P. J., MOLONY, V. & IGoo, A. (1984). Pain Supply. 2, 417P. YOUNG, W. S. III & KUHAR, M. J Physiol (jp.physoc.orgAcad. Sci. 77(3), 1696-1700. Downloaded from J. (1980). Proc. natn. ) by on September 19, 2009 PHYSIOLOGICAL SOCIETY, JANUARY 1985 43P GABA-related anion channels, labelled by [35S]t-butylbicyclophosphoro- thionate (I35SJTBPS), in rat pituitary BY R. A. ANDERSON, R. H. MCALLISTER-WILLIAMS and R. MITCHELL. MRC Brain Metabolism Unit, 1 George Square, Edinburgh EH8 9JZ Multiple receptors for y-aminobutyric acid (GABA) are present in both anterior and neurointermediate pituitary gland (Anderson & Mitchell, 1983 a), where they may be involved in controlling the secretion of hormones such as prolactin (Anderson & Mitchell, 1984) and a-MSH (Tomiko, Tareskevich & Douglas, 1983). Recognition sites for benzodiazepines that represent a component of some GABAA-type receptor complexes are also present (Anderson & Mitchell, 1983 b). The experiments described here use a specific ligand ([35S]TBPS) to label the picrotoxinin/barbiturate-sensitive site of the anion channel component in GABAA receptor complexes (Squires, Casida, Richardson & Saederup, 1983). Pituitary glands were removed from adult male Wistar rats and dissected into anterior (AP) and neurointermediate (NI) lobes. Tissue was homogenized and centrifuged in 100 volumes of 5 mM-Tris HOl buffer, containing 1 mM-EDTA, pH 7.5 and washed three further times in fresh buffer. Binding of [35S]TBPS was studied by the method of Squires et al. (1983), as described by McAllister-Williams & Mitchell (1984). Specific binding of 2 nM-[35S]TBPS (defined by 100 /LM-picrotoxinin) was detected in both AP and NI at 278 + 26 and 133 + 13 fmol/g tissue respectively (n = 5). This relative concentration of sites in AP matches that of GABAA receptors (Anderson & Mitchell, 1983 a) but contrasts with the distribution of benzodiazepine recognition sites (Anderson & Mitchell, 1983b). Displacement of [35S]TBPS binding to AP membranes by muscimol (2-2000 nM) revealed two components, similar to observa- tions in certain CNS areas such as cerebellum, where only some of the complexes are regulated by benzodiazepines (McAllister-Williams & Mitchell, 1984). These results demonstrate the presence of an anion channel component linked to GABAA receptors in the pituitary gland, as in the CNS, and are consistent with the existence of both benzodiazepine-regulated and benzodiazepine-independent GABAA receptor complexes in AP. R. A. A. is a Houldsworth Scholar of Edinburgh University. R. H. McA-W. was supported in part by the S.H.H.D. REFERENCES ANDERSON, R. A. & MITCHELL, R. (1983a). J. Physiol. 346, 126P. ANDERSON, R. A. & MITCHELL, R. (1983b). Brit. J. Pharmacol. 79, 290P. ANDERSON, R. A. & MITCHELL, R. (1984). J. Physiol. 353, 100P. MCALLISTER-WILLIAMS, R. H. & MITCHELL, R. (1984). Proceeding of the British Pharmacological Society, December 1984. SQUIRES, R. F., CASIDA, J. E., RICHARDSON, M. & SAEDERUP, E. (1983). Mol. Pharmac. 23,326-336. TOMIKO, S. A., TARASKEVICH, P. S. & DOUGLAS, W. W. (1983). Nature, Lond. 301, 706-707. Downloaded from J Physiol (jp.physoc.org) by on September 19, 2009 44P PROCEEDINGS OF THE Semliki Forest Virus induced demyelination in mice: some pathological observations and a growth study BY T. G. J. ALLEN and E. M. TANSEY. Vision Research Unit of Sherrington School, The Rayne Institute, St Thomas's Hospital, London SEJ 7EH Inoculation of Semliki Forest Virus (SFV) into mice induces patchy demyelination throughout the CNS (e.g. Chew-Lim, Suckling & Webb, 1977). Daily observations of a large population of control and SFV-infected mice have enabled us to quantify other pathological changes such as weight loss, hind limb paralysis and conjunctivitis both before and during the demyelinating period (which is at a peak from post-infection day (PID) 14 to PID 20). Control Swiss/A2G mice received i.P. inoculations of 04 ml BAPS (bovine albumin phosphate saline) at 3-4 weeks of age, whilst litter-mate animals received i.p. inoculations of 041 ml SFV in BAPS at the same age, and all were observed daily up to PID 30. Less than 0 3 % (N = 704) of the control mice died, compared with 4 % (N = 800) of the SFV mice. Paralysis of one or both hind limbs was seen in over 14 % (N = 508) of the SFV mice and nearly 5 % (N = 508) developed inflammation of one or both eyes. In contrast only one control animal developed slight lameness (0 3 %, N = 464) and only three developed conjunctivitis (0 7 %, N = 464). A growth study of 354 mice (168 controls and 186 SFV) from inoculation to PID 30 revealed that by PID 4 the mean weight of the SFV group was significantly lower than that of controls, and it remained so for the rest of the observation period. A further 32 mice (16 controls and 16 SFV) were caged individually and weight, food, water and waste monitored daily. Again, the weight of the SFV group was significantly lower than the control group by PID 4 and at the same time food and water consumption and waste production dropped in the SFV group and did not return to control levels until PID 13-14, although they never compensated for their weight loss. Paralysis and weight loss have been reported also for an autoimmune model of demyelination, experimental allergic encephalomyelitis (e.g. Pender & Sears, 1984). Whilst the relationship of such changes to the subsequent demyelination remains to be elucidated in both models, they emphasize the complexity of the pathophysiology of demyelination. This work was supported by grants from the Multiple Sclerosis Society and the Special Trustees of St Thomas's Hospital to Professor Hisako Ikeda and we are grateful to her for encouragement and critical comments. We also thank Dr Hugh Webb for the gift of SFV and Mrs Gill Blake and Mrs Janet Turner for assistance. REFERENCES CHEW-LIM, M., SUCKLING, A. J. & WEBB, H. E. (1977). Vet. Pathol. 14, 67-72. PENDER, M. P. & SEARS, T. A. (1984). Brain 197, 699-726. Downloaded from J Physiol (jp.physoc.org) by on September 19, 2009 PHYSIOLOGICAL SOCIETY, JANUARY 1985 45P Patch-clamp of rat cerebellar neurones in tissue culture BY S. G. CULL-CANDY, P. DILGER, D. C. OGDEN and S. TEMPLE*. MRC Receptor Mechanisms Group, Department of Pharmacology, University College, London WC1E 6BT Small explants of cerebellum from newborn rats were tissue cultured in Dulbecco's minimum essential medium, 10 % foetal calf serum (and 100 units per ml penicillin- /streptomycin) on glass cover-slips for periods up to one month. Neurones were identified with neurofilament-selective monoclonal antibody and rhodamine- conjugated goat anti-mouse immunoglobin; glial cells were also identified with indirect immunofluorescence (fluorescein) after staining with antibodies to glial fibrillary acid protein (a known marker for glial cells). This allowed confirmation that neurones could be visually identified with phase or Nomarski interference optics for patch-clamp recording (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). Fig. 1. Photomicrographs of a single field of cerebellar explant. A, cerebellar explant viewed wihphase contrast; B, fluorescence micrograph of neurones labelled with rhodamine; C, glial cells labelled with fluorescein. Arrows in A, B indicate examples of identified neurones; their corresponding positions are indicated in C. We have recorded voltage-activated inward Na+ and outward K+ currents. The cells also possessed GABA-activated anion channels and glutamate-activated cation channels (Nowak, Bregestovski, Ascher, Herbert & Prochiantz, 1984; Cull-Candy & Ogden, 1985) the properties of which have been examined by means of noise analysis from whole-cell recordings and single-channel recording. We have obtained evidence for more than one type of glutamate-activated receptor channel in these cells. This work is supported by the Wellcome Trust and the MRC.- S. G. C. -C. is a Wellcome Trust Senior Lecturer. REFERENCES CULL-CANDY, S. G. & OGDEN, D. C. (1985). Proc. R. Soc. Lond. B (in the Press). HAMILL, 0. P., MARTY, A., NEHER, E., SAKMANN, B. & SIGwoRTH, F. J. (1981). Pfluiger8 Arch. 391, 85-100. NOwAK, L., BREGESTOVSKI, P., ASCHER, P., HERBERT, A. & PROCHIANTZ, A. (1984). Nature, Lond. 307, 462-465. * Department of Zoology, University College London. Downloaded from J Physiol (jp.physoc.org) by on September 19, 2009 46P 46P PROCEEDINGS OF THE Single Ca2+-activated K+ channels recorded from cultured rat sympathetic neurones BY T. G. SMART. MRC Neuropharmacology Research Group, Department of Pharma- cology, School of Pharmacy, 29/39 Brunswick Square, London WC1N lAX The macroscopic Ca2+-activated K+ current (I,) recorded from mammalian and amphibian neurones has been well documented (Adams, Constanti, Brown & Clark, 1982; Galvan & Sedlmeier, 1984) but the corresponding single-channel currents have not yet been fully characterized. Patch-clamp recordings were made from dissociated rat superior cervical ganglionic neurones (obtained from 20-day-old embryos) at 20-23 'C. The most predominant single-channel currents observed at the resting potential exhibited a large unitary conductance with multiple closures. These channels were markedly modulated by membrane voltage, with the open-state probability increasing with depolarization and decreasing with hyperpolarization, becoming virtually inactive at membrane potentials more negative than -100 mV. In cell free membrane patches (inside-out configuration) the channel open-state probability and frequency was increased by raising the Ca2+ concentration at the cytoplasmic face of the membrane. The channels exhibited no inactivation on prolonged exposure to high Ca2+ (3 /LM). Virtually no channel activity was observed at [Ca2+] = 0.01 jFM. The reversal potential for these currents was -60 mV patch potential (n 0 mV membrane potential) with 150 mM-KCl patch electrode solution, indicating a channel selective for K+ over Na+ or Cl-. The current-voltage relationship displayed linearity over a range of -50 to + 50 mV, producing a slope conductance of 200 pS. In cell - free membrane patches, channel current amplitudes were reversibly reduced by tetraethylammonium (0-125-5 mm outside, 5-10 mm inside) and also by Cs+ (5 mM outside). 1 mm external 4-aminopyridine did not affect channel activity whereas 1 mM-Ba2+ reduced the open state probability but did not affect the amplitude. These preliminary properties of the neuronal I, suggest many similarities with I, single channels on other preparations (Marty, 1981; Barrett, Magleby & Pallotta, 1982). This work was supported by the M.R.C. REFERENCES ADAMS, P. R., CONSTANTI, A., BROWN, D. A. & CLARK, R. B. (1982). Nature, Lond. 296, 746-749. BARRETT, J. N., MAGLEBY, K. L. & PALLOTTA, B. S. (1982). J. Phy8iol. 331, 211-230. GALVAN, M. & SEDLMEIER, C. (1984). J. Physiol. 356, 115-133. MARTY, A. (1981). Nature, Lond. 291, 497-500. Downloaded from J Physiol (jp.physoc.org) by on September 19, 2009 PHYSIOLOGICAL SOCIETY, JANUARY 1985 47P Interaction of verapamil with tubocurarine at the chick neuromuscular junction BY F. A. WALL. Anaesthetics Unit, The London Hospital Medical College, Whitechapel, London El 1BB Calcium channel blockers, e.g. verapamil, are increasingly used in the treatment of cardiovascular disorders. Since calcium ions are essential for neuromuscular transmission and muscle contraction, it is possible that calcium antagonists will inhibit neuromuscular transmission and will increase the neuromuscular blockade produced by muscle relaxants, e.g. tubocurarine. In the present investigation, the interaction of verapamil with tubocurarine and cholinergic drugs was studied in the isolated chick biventer cervicis skeletal muscle (Ginsborg & Warriner, 1960). The preparation was set up in an organ bath (20 ml) containing Krebs-bicarbonate solution maintained at 38 + 2 0C and bubbled with 5 % CO2 in oxygen. The motor nerve was stimulated at a repetitive rate of 0-2 Hz with supramaximal stimuli (5-10 V) and 0-2 ms pulse duration. The contractile responses produced either by motor nerve stimulation or by drug application were recorded isometrically using a force displacement transducer and a Washington pen recorder. Verapamil (2-04-204 /UM) produced concentration-dependent reductions in the amplitude of the indirectly elicited twitch contractions in the chick muscle. The mean (± S.E.M.) IC 50s (concentration to produce 50 % inhibition) for the verapamil-induced inhibition oftwitch tension was 85 + 4-6 #sM (n = 6). High concentrations of verapamil (> 200 /LM) produced a marked contracture in the chick skeletal muscle (1 0 + 0 1 g, n = 6). In concentrations that had little effect (< 15 %) on twitch tension, verapamil (20 /LM) increased the neuromuscular blockade produced by tubocurarine (1-27-127 ,UM). The mean IC 50s for the tubocurarine-induced inhibition of twitch tension in the control Krebs solution and in Krebs containing verapamil were 40 + 3-2 and 25 + 1-6 ,UM respectively (n = 6, P < 0 001). Verapamil (20 ,tM) also reduced the contractures produced by acetylcholine (05-1 1 mM) and tetraethylammonium (24-12 mM) in the chick muscle (reduced by 40-50 % and 50-60 %, n = 6, respectively). The interaction of verapamil with other neuromuscular blocking agents, e.g. pancuronium, vecuronium and atracurium, have been studied by other workers (Bikhazi, Leung & Foldes, 1982; Carpenter & Mulroy, 1983), who concluded that verapamil augmented twitch depression. It is not clear whether verapamil augments twitch blockade by inhibition of ACH release due to calcium entry blockade. Since the blockade by verapamil was not completely reversible upon washing out the preparation, it is possible that it may accumulate at the neuromuscular site, thereby increasing the effects of muscle relaxants. REFERENCES BIKHAZI, G. B., LEUNG, I. & FOLDES, F. F. (1982). Anesthesiology 57, A268. CARPENTER, R. L. & MuLRoY, M. F. (1983). Anesthesiology 59, A272. GINSBORG, B. L. & WARRINER, J. (1960). Br. J. Pharmac. 15, 410-411. 17 7 PHY 362 Downloaded from J Physiol (jp.physoc.org) by on September 19, 2009 48P PROCEEDINGS OF THE Plasma volumes, electrolytes and osmolalities in rats lacking hypothalamic vasopressin (Brattleboro strain): effects of water deprivation BY T. BENNETT and S. M. GARDINER. Department of Physiology and Pharmacology, Medical School, Queen's Medical Centre, Clifton Boulevard, Nottingham NG7 2UH Dehydration-induced natriuresis, which could serve to buffer increases in plasma [Na+ ] and osmolality, may involve a vasopressin-dependent process (Luke, 1973). In this study we have measured plasma osmolalities and electrolyte concentrations in normal (Long Evans; LE) rats and in rats lacking hypothalamic vasopressin (Brattleboro strain) when plasma volumes were reduced to a similar degree by water deprivation. Male, age-matched animals were used. The water-deprived groups had their drinking water (but not food) removed either 14 h (Brattleboro, n = 8) or 53 h (LE, n = 7) before the experiment; the water-replete groups (Brattleboro, n = 8; LE, n = 9) had free access to water and food until the time of study. The animals were anaesthetized with sodium pentobarbital (60 mg/kg I.P.); plasma volumes were measured by dye dilution (Belcher & Harris, 1957) and blood samples were taken by cardiac puncture. In the water-replete state, plasma volumes were higher (P < 0 02) in Brattleboro rats (3-25 + 0-15 ml/I00 g; mean+S.E. of mean) than in LE rats (2-79+0-08 ml/100 g); since our schedule for water deprivation produced similar proportional reductions in plasma volumes in the two strains, the difference persisted in the water-deprived groups (Brattleboro = 2 39 + 0-05; LE = 2-16 + 009 ml/ 100 g; P < 0 05). Plasma [Na+] was higher (P < 0 002) in the water-replete Brattleboro rats (148 1 + 0-8 m-mole/l) than in the LE rats (141 9 + 0-2 m-mole/l) but the difference was greater after water deprivation (Brattleboro = 156-8 + 0-5; LE = 144'2 + 0-2 m- mole/l; P < 0 002). Likewise, in the water-replete state, plasma osmolality was higher (P < 0-002) in the Brattleboro rats (318 + 1 m-osmole/kg) than in the LE rats (305+1 m-osmole/kg) but the difference was greater after water deprivation (Brattleboro = 333 + 2 m-osmole/kg; LE = 310+1 m-osmole/kg; P < 0 002). Plasma [K+] was not different in the two groups of water-replete rats (Brattleboro = 5-06 + 0-15; LE = 5-05 + 002 m-mole/l) but after water deprivation plasma [K+] was lower (P < 0-002) in the LE rats (4-71 ±0-12 m-mole/l) than in the Brattleboro rats (5 90 + 0-35 m-mole/l). These results show that a similar degree of plasma volume reduction during water deprivation is associated with markedly different changes in plasma electrolyte concentrations and osmolalities in Brattleboro and LE rats. We are presently unable to comment on the relative contributions of changes in electrolyte intake, output or distribution to these findings. We wish to thank the British Heart Foundation for financial support. REFERENCES BELCHER, E. H. & HARRIS, E. B. (1957). J. Physiol. 139, 64-78. LUKE, R. G. (1973). Am. J. Physiol. 224, 13-20. Downloaded from J Physiol (jp.physoc.org) by on September 19, 2009 PHYSIOLOGICAL SOCIETY, JANUARY 1985 49P Time-dependent respiratory effects of injected phenyldiguanide on vagoto- mized rabbits BY A. DAVIES and H. JONES. Department of Physiology, Massey University, Palmerston North, New Zealand. Intravenous injections of phenyldiguanide (PDG) have been used to provoke respiratory reflexes attributed to the activity of receptors associated with unmyel- inated vagal fibres from the gut, heart and lungs. Section of the vagi below the diaphragm and injection of local anaesthetic into the pericardial sac (Anand & Paintal, 1980) have been used to abolish afferent activity from the gut and heart respectively. Stimulation by PDG of nerve endings in the carotid region can be abolished by stripping the carotid bifurcations (Dawes, Mott & Widdicombe, 1951). It would be expected that cervical vagal section would almost totally abolish all respiratory reflexes if they only originated from the sites outlined above. We and other workers have demonstrated this in rabbits (Dawes, Mott & Widdicombe, 1951; Karczewski & Widdicombe, 1969; Guz & Trenchard, 1971; Davies, Dixon, Callanan, Huszczuk, Widdicombe & Wise, 1978). On the other hand Miserocchi, Trippenbach, Mazzarelli, Jasper & Hazucha (1978) found little diminution ofthe effect of PDG after vagotomy. Our present study was intended to resolve this difference and demonstrate the sites at which PDG acts in rabbits. We measured inspiratory duration (t1) and expiratory duration (tE) in 19 New Zealand White rabbits, anaesthetized with sodium pentobarbitone (40 mg/kg), while 30 pg/kg PDG was given (via an intravenous catheter whose tip lay close to the right atrium) to the intact rabbit; after injecting 1 ml 2 % xylocaine into the pericardial sac; immediately after bilateral cervical vagotomy; 15 min after vagotomy and after cutting the glossopharangeal nerves near the base of the skull. The respiratory reflex after injection of xylocaine, 15 min after vagotomy and after cutting the glossopharangeal nerves, was as pronouned as in the intact state, and consisted of an increase in frequency almost totally due to a reduction in tE. With injections given up to 3 min after bilateral vagotomy the respiratory response was greatly attenuated and variable. We suggest this question of timing may contribute to the differences seen by different groups of workers. REFERENCES ANAND, A. & PAITAL, A. S. (1980). J. Physiol. 299, 553-572. DAVIES, A., DIXON, M., CALLANAN, D., HuSZCZUK, A., WIDDICOMBE, J. G. & WISE, J. C. M. (1978). Respir. Physiol. 34, 83-101. DAWES, G. S., MoTT, J. C. & WIDDICOMBE, J. G. (1951). J. Physiol. 115, 258-291. Guz, A. & TRENCHARD, D. W. (1971). J. Physiol. 213, 345-371. KARaZEWSKI, W. & WIDDICOMBE, J. G. (1969). J. Physiol. 201, 271-291. MISEROCCHI, G., TRIPPENBACH, T., MAZZARELLI, M., JASPAR, N. & HAZUCHA, M. (1978). Respir. Physiol. 32, 141-153. 17-2 Downloaded from J Physiol (jp.physoc.org) by on September 19, 2009 50P PPROCEEDINGS OF THE The effect of aspartocholate on bile flow in the rat BY E. R. L. O'MAILLE and T. PETTERSON. Department of Physiology, University of Liverpool, Liverpool L69 3BX The effect on bile flow of the artificial bile salt, aspartocholate, has been compared to that of the natural bile salt, taurocholate, in the anaesthetized rat to assess the influence of bile salt structure on bile formation. The increase in bile flow which follows the administration of a bile salt such as taurocholate is believed to be the osmotic consequence of the secretion of the bile salt anion and its counter-ion (chiefly Na+) into bile. Disodium aspartocholate, when fully ionized in monomeric solution, dissociates into three particles (a divalent anion and two Na+ ions), whereas sodium taurocholate in similar circumstances dissociates into only two particles (a monovalent anion and a Na+ ion). (Since the higher pKa of aspartocholate's two acid groups is 5-2 (Lack & Weiner, 1967) and the pH of rat bile is about 7.5 (Zouboulis-Vafiadis, Dumont & Erlinger, 1982), aspartocholate would exist predominantly in the dibasic form in these experiments; taurocholate (pKa about 2) would be fully ionized.) In addition, the critical micellar concentration (well exceeded in bile for both bile salts) of aspartocholate is greater than that of taurocholate (Roda, Hofmann & Mysels, 1983). Both of these factors would lead to the prediction that the choleretic potency of aspartocholate would be greater than that of taurocholate. To test this prediction, aspartocholate or taurocholate was infused via a femoral vein at rates of, in the majority of experiments, 2-7 ,tmol/min per kg body wt in rats anaesthetized with pentobarbitone (50 mg/kg i.P.) in which the common bile duct was cannulated. Both bile salts were quantitatively secreted into bile in unchanged form. The increase in bile flow per unit increase in bile salt secretion for aspartocholate (1 1-8 + 1 1 (S.D.) ,ul/,umol, n = 7 rats) was significantly greater than that for taurocholate (7-7 + 0-92 (S.D.) ,u/,umol, n = 8 rats) (P < 0001). The use of disodium aspartocholate, in which the extra Na+ ion secreted with each molecule bestows the major extra osmotic force (compared to taurocholate), thus provides a novel means of demonstrating the operation of the osmotic theory for the bile-salt-dependent bile flow. The aspartocholate used in these experiments was a gift from Professor A. F. Hofmann, Department of Medicine, University of California at San Diego, U.S.A. This work formed part of a project dissertation presented by T. Petterson for one section of the B.Sc. Honours degree at the University of Liverpool in June, 1983. REFERENCES LACK, L. & WEINER, I. M. (1967). Biochim. Biophys. Acta 135, 1065-1068. RODA, A., HOFMANN, A. F. & MYSELS, K. J. (1983). J. biol. Chem. 258, 6362-6370. ZOUBOULIS-VAFIADIS, I., DUMONT, M. & ERLINGER, S. (1982). Am. J. Phy8iol. 243, G208-G213. Downloaded from J Physiol (jp.physoc.org) by on September 19, 2009