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The effect of substance P on cyclic AMP and cyclic GMP levels in actively secreting pancreatic lobules BY JANET ALBANO, K. D. BHOOLA and R. F. HARVEY. Departments of Pharmacology and Medicine, The Medical School, University of Bristol, Bristol BS8 1TD Although the endogenous occurrence of substance P was reported more than 40 years ago, its physiological function is as yet not clearly defined. Recently this peptide has been shown to increase adenylate cyclase activity in brain membrane preparations (Duffy & Powell, 1975), but to stimulate amylase secretion from parotid gland slices without altering cyclic nucleotide levels (Rudich & Butcher, 1976). We have previously shown that secretion of proteases from pancreatic slices in reponse to activation of muscarinic receptors by acetylcholine and hormonal activation by cholecystokinin (C0K) is preceded by a rapid but transient rise in intracellular cyclic GMP (Albano, Bhoola & Harvey, 1976). The experiments reported in this study were designed to determine the effect of synthetic substance P (gift from CibaGeigy, Basle) on protease secretion and on intracellular levels of cyclic GMP and cyclic AMP using isolated guinea-pig pancreatic lobules. Guinea-pigs were killed by cervical dislocation and pancreatic lobules were prepared with the aid of a dissecting microscope and flasks containing 6-8 lobules, weighing 30-40 mg, incubated at 37 00 in Krebs-Ringer bicarbonate buffer containing 01°% bovine serum albumin and 50 ,ugfml. bacitracin. Flasks were gassed throughout the incubation period with 95 %0 2-5 % C02. Cyclic GMP was measured by radioimmunoassay, cyclic AMP by a binding protein method and the serine proteases by hydrolysis of a synthetic substrate after activation of the proenzymes by enterokinase. Substance P (2.5 x 10-6 M) increased intracellular cyclic GMP concentrations eight- to tenfold within 30 sec returning to near control levels by 5 min. Release of proteases into the incubation medium increased 30-40 % above controls at 30 min. The cyclic GMP increase showed dose dependency with the threshold between 10-8 and 10-7 M; no changes in intracellular cyclic AMP levels were observed with substance P concentrations up to 2-5 x 10-5 M. Atropine did not affect the cyclic GMP increase elicited by substance P, under conditions in which the acetylcholine response was abolished. In the presence of the phosphodiesterase inhibitor 3isobutyl-1-methyl xanthine (IBMX, 0.1 mm), basal levels of cyclic AMP were increased about fourfold, but no further increase was observed with substance P. Basal levels of cyclic GMP were increased two- to threefold by IBMX and the application of substance P (2.5 x 10-6 M) produced an additive effect on cyclic GMP levels when compared with lobules incubated in the absence of the methylxanthine. These results suggest a possible physiological role for substance P as a secretogogue in the exocrine pancreas and that its action may be mediated through activation of guanylate cyclase. Furthermore, the fundamental difference in the finding that substance P stimulates adenylate cyclase in brain membrane fragments but increases cyclic GMP in pancreatic lobules may reflect two separate receptor populations. The financial support of the Wellcome Trust, Medical Research Council and the South West

Regional Health Authority is gratefully acknowledged.

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REFERENCES

ALBANO, J., BHOOLA, K. D. & HARVEY, R. F. (1976). Nature, Lond. 262, 404-406. DUFFY, M. J. & POWELL, D. (1975). Biochim. biophys. Acta 385, 275-280. RuIDICH, L. & BuITcHEI, F. R. (1976). Biochim. biophy8. Acta 444, 704-711.

Incorporation of acetylmonoethylcholine into the transmitter pool at the mammalian neuromuscular junction BY W. A. LARGE and H. P. RANG. Department of Pharmacology, St George's Hospital Medical School, Cranmer Terrace, London SW17 ORE Nerve stimulation in the presence of monoethylcholine (MECh) causes acetylcholine (ACh) to be replaced by acetylmonoethylcholine (AMECh) as the transmitter at the neuromuscular junction of the rat diaphragm, and this is associated with a characteristic shortening of the time constant of decay (r) of the miniature end-plate currents (m.e.p.c.s) recorded with a voltage-clamp technique (Colquhoun, Large & Rang, 1977). In this work we have investigated whether, at intermediate stages of the replacement of ACh by AMECh, it is possible to detect two separate populations of quanta, corresponding to newly synthesized AMECh quanta and pre-existing ACh quanta, or whether the quanta released represent a homogeneous pool of intermediate composition. Stimulation of the phrenic nerve at 3 Hz for 15-25 min in the presence of 0.1 mM MECh caused the mean r to decrease from 1-57 + 0-07 msec to 1-29 + 0-08 msec (results from seven muscles). Stimulation for a further 30-60 min reduced T to 0-89 + 0 05 msec, which represents complete replacement of ACh by AMECh. Calculating directly from the reduction in mean r indicated that the transmitter reponsible for the m.e.p.c.s occurring after 15-25 min stimulation comprised 44 + 5 % AMECh (mean of six muscles). Under normal conditions the coefficient of variation (CV) of T for a single cell was 0 10 + 0 004 (sixty cells). This was unaffected by stimulation in the absence of MECh. After 15-25 min stimulation in the presence of MECh, the mean CV for m.e.p.c.s recorded during the succeeding 40 min period increased to 0.16+0-012 (thirty-five cells). Histograms revealed the increase in scatter of the r values in many cells, but in no case was the distribution clearly bimodal. It can be calculated that, had the quanta represented two distinct populations, the CV should have been increased to 0X28. The measured increase in CV averaged only about 30 % of the predicted effect, and in no case exceeded 50 %. The small degree of heterogeneity found agrees with the result of Elmqvist & Quastel (1965), who found that hemicholinium caused a gradual reduction in quantum size, suggesting that the available transmitter remains evenly distributed throughout the population of quanta. Various explanations of our result are possible. The mixing of AMECh and ACh in individual quanta could result from admixture of choline and MECh prior to transmitter synthesis, which would imply that the tissue can draw on a substantial pool of choline, even when MECh is present. Alternatively, the admixture might be occurring after the synthesis of transmitter, implying either that the bulk of the 21

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PROCEEDINGS OF THE 62P stored transmitter is in the form of a miscible cytoplasmic pool or that rapid exchange of transmitter between vesicles can take place. We are grateful to Dr D. Colquhoun for useful advice and discussion. REFERENCES COLQUHOUN, D., LARGE, W. A. & RANGE, H. P. (1977). J. Phy8iol. 266, 361-395. ELMQVIST, D. & QUASTEL, D. M. J. (1965). J. Physiol. 177, 463-482.

Temperature dependence of the cation affinities of the sodium pump in red cells from hibernators and non-hibernators BY J. C. ELLORY* and J. S. WILLIS. Department of Physiology and Biophysics, University of Illinois, Urbana, Illinois Red cells from thirteen-lined ground squirrels, a hibernating species, have been shown to give greater sodium pump activity at 5 0C than cells from non-hibernating species (Kimzey & Willis, 1971). This cold resistance could directly reflect an adaptive difference in the properties of the sodium pump, or could result secondarily from changes in substrate availability, e.g. internal Na, ATP. We have estimated the change with temperature in external affinity for K and internal affinity for Na of the pump, by using ouabain-sensitive K influx into guinea-pig and ground-squirrel red cells with high internal ATP. Red cell cation composition was altered by the Nystatin method (Cass & Dalmark, 1973). Ouabain-sensitive K influx was measured at high internal Na and ATP, at varying [K]0 in a high Na medium. The data were fitted linearly as S/4v vs. S. The KO.5 (KO) was 3-83 + 0-10 (3) mm for ground squirrel at 37 'C, falling to 075 + 004 at 5 0C; guinea-pig cells gave values of 2-24 + 0 07 (3) and 070 + 0 05 mm respectively. The ratio of apparent Vmax values for pump K influx at 5 00 vs. 37 00 was 1-7 % for ground squirrel and 0X3 % for guinea-pig. This fivefold difference between the ratio for a hibernator and non-hibernator confirms Kimzey & Willis (1971) under more rigorous conditions. Similar experiments in which internal Na was varied with internal K fixed at 100 mM were fitted as (83VV) vS. S (cf. Garay & Garrahan, 1973). Ouabain-sensitive K influx in guinea-pig cells gave Ko.5 (Na1) 9.9 + 2-2 (4) mM-Na at 37 0C, which fell to 1-5 + 0 3 (3) mM-Na at 5 0C. In contrast, ground squirrel cells show a high apparent Na affinity even at 37 0C, KO.5 (Nal) < 2 mM-Na, which did not decrease dramatically on cooling. Thus the affinity of the sodium pump for both external K and internal Na increases on cooling. However, neither of these kinetic changes can account for the difference in cold sensitivity between the hibernator and non-hibernator, even though there are clearly kinetic differences between the two species. This work is supported by NIH Grant GM 11494.

REFERENCES CASS, A. & D

M. (1973). Nature, New Biol. 244, 47. GARAY, R. P. & GARRmiAw, P. J. (1973). J. Phy8iol. 231, 297-335. KIMZEY, S. L. & WILLIs, J. S. (1971). J. gen. Phy8iol. 58, 620-633. * Permanent address: The Physiological Laboratory, Cambridge University.

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Shortening of tetanized skeletal muscle causes a fall of intracellular calcium concentration BY D. G. ALLEN. Department of Physiology, University College London, Gower Street, London WC1E 6BT Single twitch muscle fibres were dissected from frogs and microinjected with the calcium-sensitive photoprotein aequorin (Blinks, Prendergast & Allen, 1976). Tension, length and light emission (a function of intracellular calcium concentration) were measured and stretches and releases imposed on the fibre during tetanic contractions. Fig. 1 shows a tetanic contraction in which there was rapid shortening from a

[ 60,000 counts/s

[ 200 mN/mm2

L 25 pim

2\22Bm

2 sec

Fig. 1. Single fibre from semitendinosus muscle of Rana temporaria, 10 0C. Record shows from above downward: light emission, tension, length and stimuli.

sarcomere spacing of 2-5 to 2-15 #tm. After full tetanic tension had been developed, light emission rose steadily but fell sharply during the period when the fibre was shortening and redeveloping tension. When the fibre was stretched from 2-15 to 2-5 /m, there was no detectable effect on light emission. The magnitude and time course of the fall in light emission was found to depend on the size and speed of the shortening step. When the same length change was made from different starting lengths, the fall in light emission was absent in fibres whose sarcomere spacing was greater than 3-6 #um, but at very short sarcomere spacings the light emission rose during shortening. This work was done during the tenure of a British-American Research Fellowship of the American Heart Association and British Heart Foundation. Supported by U.S. Public Health Service Grant HL 12186. REFERENCE

BLINKS, J. R., PRENDERGAST, F. G. & ALLEN, D. G. (1976). Pharmacy. Rev. 28, 1-93.

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On the use of nitrous oxide/oxygen mixtures for anaesthesia in cats BY P. HAMMOND. Department of Commmunication and Neuroscience, University of Keele, Keele, Staffordshire STh 5BG Claims that nitrous oxide obtains adequate anaesthesia in cats in restricted situations (Venes, Collins & Taub, 1971; Blakemore, Donaghy, Maffei, Movshon, Rose & van Sluyters, 1974) have been contested. Even 80 %:20 % N20:02 neither induces anaesthesia in cats (Russell, 1973) nor satisfactorily maintains anaesthesia induced with short-acting barbiturates (Richards & Webb, 1975). Our own early (Hammond, 1971) and more recent experience with N20:02 mixtures consistently matches the latter observations, even with atraumatic head restraint and after local anaesthesia of wound margins. Despite this, nitrous oxide continues to be widely used for 'anaesthesia' in paralysed cats, where controls may be inadequate. We have therefore been at pains to develop an alternative, stable, lightly anaesthetized preparation with adequate controls. Following induction and surgical preparation under halothane, cats were ventilated on N20:02 mixtures ranging from 80 %:20% to 70 %:30 %, with careful monitoring of e.e.g., e.c.g. and/or blood pressure, end-tidal CO2 (3.8-4.0%) and rectal temperature (38&5 'C). Reflex state and e.e.g., or e.e.g. in cats paralysed with gallamine triethiodide (Flaxedil, May and Baker) for visual recording, together with other monitored parameters, indicated that N20:02 alone was never satisfactory for long-term recording. After a period during which a continuously spindling e.e.g. gave way progressively to episodes of spindling interspersed with small-amplitude fast activity, the e.e.g. finally became indistinguishable from desynchronized activity typical of arousal in conscious controls. At this point supplementary anaesthetic was immediately administered. We have established that 72-5 %: 27-5 % N20 :02 supplemented with even small doses of pentobarbitone (Nembutal, Abbott; averaging 1 mg kg-' hr-', compared with some 3 mg kg-' hr-4 for the conventional barbiturate-anaesthetized preparation) leads to a periodically synchronized e.e.g., and (in non-paralysed preparations) loss of muscle tone, absence of withdrawal reflex and cessation of any spontaneous movements consistent with satisfactory anaesthesia (see also Hammond & MacKay, 1978). This preparation has proved suitable for short- or long-term visual cortical recording: units are sharply tuned, responses are brisk and most complex cells possess a pronounced resting discharge. REFERENCES

BLAKEMOBE, C., DONAGHY, M. J., MAFFEI, L., MOVSHON, J. A., ROSE, D. & vAN SLUTYERS, R. C. (1974). J. PI&yiol. 237, 39-41P. HAMMOND, P. (1971). J. Physiol. 213, 475-494. HAMMOND, P. & MACKAY, D. M. (1978). Expl Brain. Re8. (In the Press.) RICHARDS, C. D. & WEBB, A. C. (1975). J. Phy8iol. 245, 72-73P. RUSSELL, W. J. (1973). J. Phy8iol. 231, 20-21P. VENES, J. L., CoLLiNs, W. P. & TAUB, A. (1971). Am. J. Phy8iol. 220, 2028-2031.

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The effect upon the masseteric electromyogram of double mechanical stimuli applied to teeth BY J. M. REYNOLDS and A. J. THEXTON. Department of Physiology, Royal Dental Hospital School of Dental Surgery, Jenner Wing, St George's Hospital Medical School, London S.W. 17 A period of silence can be induced in the electromyogram (e.m.g.) of the human masseter either by applying mechanical stimuli to individual teeth or to mucosa or by voluntarily tapping the teeth together. When the teeth are tapped together, the silent period duration is usually about 15 msec; it is suggested that longer periods are indicative of the myo-facial pain dysfunction syndrome (McNamara, 1976). In this study we have applied a servo-controlled stimulus consisting of either two triangular force profiles or two ramp force profiles in the form of a double step, the interval between the rising phases being precisely controlled (McGarrick Reynolds & Thexton 1977). The stimulus was applied axially to an upper incisor tooth whilst the subject bit on a rubber bung between the molar teeth; the force of closure was equivalent to 30 % of the maximum bite as judged by the r.m.s. value of the surface e.m.g. of the ipsilateral masseter. The threshold for eliciting e.m.g. silence varied from subject to subject but the silent period could always be obtained if a force of 3 N was applied at 200 N sec-' or more. The latency of the inhibitory period was 15-20 msec and its duration 9-15 msec. Separate silent periods of the same duration were obtained if the rising phases of the stimulus were more than 23 msec apart. A small increase of e.m.g. activity (E1) often preceded the silent period and another period of increased activity (E2) followed it. When the interval between the rising phases was adjusted to try and make E2 of the first response coincide with El of the second response there was either an apparent increase in e.m.g. activity at that instant or both E1 and E2 disappeared so that the silent periods fused. The former response was critically dependent upon the interval between the stimulus slopes. Using the double triangle stimulus, a fused inhibitory period with a duration of 160210 % of the response to a single stimulus could be obtained in all subjects. Using the stepped stimulus the silent periods could merge to give an increased duration of 220-300 % over the single silent period. Since multiple asynchronous contacts are obtained when unevenly occluding teeth are voluntarily tapped together it is probable that mechano-receptors receive stimuli similar to those used in this study. Therefore the production of prolonged silent periods by tapping the teeth together may be due as much to the interaction of the responses to multiple mechanical transients as to any underlying functional disorder. REFERENCES McGAluucK, J., REYNOLDS, J. M. & THExTON, A. J. (1977). 275, 9-10 P. McNABARA, D. C. (1976). Arch. oral Biol. 21, 325-328.

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Measurements of extracellular potassium and calcium concentration during passage of current across the surface of the brain BY A. R. GARDNER-MEDWIN* and C. NICHOLSON. Department of Physiology and Biophysics, New York University Medical Center, New York 10016, and Department of Physiology, University College London, London WC1E 6BT* When an electric current is passed across the surface of the brain there is a flux of K+ between the tissue and the fluid bathing it which is greater than should be expected if K+ moves through the brain primarily along the intercellular clefts (Gardner-Medwin, 1977). This suggests that much of the K+ movement may be transcellular, possibly involving glial cells. We have now used ion selective micro-electrodes (Nicholson, ten Bruggencate, Steinberg & Stbckle, 1977) to investigate the effects of current passage on the extracellular [K+] and [Ca2+] at various depths in the neocortex and cerebellar cortex of rats anaesthetized with urethane. Currents of approximately 20,uA/mm2 at the surface of the brain were passed between an electrode in a cup containing artificial c.s.f. and an indifferent electrode elsewhere on the animal's head. The cup fluid was continuously replenished to maintain a constant composition (NaCl 140, KCl 3, CaCl2 1D6, Na2HPO4 4, NaH2PO4 2 mmole/l.; pH 7). C currents from the cup into the brain resulted in a gradual lowering of [K+] in the superficial 1 mm of tissue. The largest changes were seen within 100 to 300 pum of the surface, where the concentration reached values 08-P15 mmole/l. below the normal ambient level (3-3.5 mmole/l.), after 400 see of current passage. The opposite current for the same time produced increases in [K+] of 1-5-2-5 mmole/l. Similar results were obtained with both the cerebellum and neocortex, though the [K+] base-line levels in the cerebellum were more stable. Experiments on the cerebellum using Ca2+-sensitive electrodes under identical conditions showed either no detectable [Ca2+] changes, or changes which were much smaller (< 0-25 mmole/l.). Changes in neural activity seem unlikely to have been the cause of the [K+] changes, since these were still seen with tetrodotoxin (10-4 M) in the cup. Under the normal experimental conditions the Purkinje-cell firing rates were changed in either direction, mostly so as to increase during passage of current into the brain. The results show that extracellular K+ can be depleted or augmented in the superficial layers of the brain when currents are passed across the interface between the tissue and the fluid. Calcium levels, on the other hand, are almost unaffected. These results are to be expected if the brain possesses a specific mechanism for migration of K+ with a higher transport number than c.s.f., such as a mechanism involving movement across cell membranes (Gardner-Medwin, 1977). Potassium would be removed from or added to the extracellular environment at the superficial sites in the tissue where there is net passage of current into or out of the cells. The results thus support the conclusion that transcellular migration of K+ may contribute significantly to the movement and redistribution of K+ through brain tissue. W. Simon, Zurich, provided the Ca2+ exchanger. The work was supported by USPHS grant NS-13740-01, and a Royal Society Travel Grant to A.R.G.-M.

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REFERENCES GARDNER-MEDWIN, A. R. (1977). J. Phy8iol. 269, 32-33P. NiCHOLSON, C., TEN BRUGGENCATE, G., STEINBERG, R. & STOCKLE, H. (1977). Proc. natn. Acad. Sci. U.S.A. 74, 1287-1290.

Phase lags between e.m.g. and flexion during voluntary oscillatory motion of the elbow BY MARILYN LORD. Department of Mechanical Engineering, University College London, London WC1E 6BT The elbow flexion system exhibits an oscillatory mechanical response to sudden load disturbances, at frequencies between 4 and 6 Hz (Robson, 1962), and can be induced to sustained tremor in the range 2-12 Hz when appropriately loaded (Joyce & Rack, 1974). Consideration of the possible servo origins of these oscillations requires knowledge of the delays between muscle activation and limb motion. These have been measured during voluntary oscillations of the forearm. The subject 'stiffened' the elbow by contraction of biceps and triceps whilst oscillating the forearm at a target frequency (1-5 Hz) and amplitude (110 peak to peak), using visual and auditory feed-back. The surface e.m.g. from biceps and the elbow angle were recorded. Cross-correlation of e.m.g. and angle enabled an estimate of the phase lag and correlation coefficient, p, to be made at each test frequency. Phase lags ranged from 180 to 2050 at 1 Hz (p = 0 2-0 35) and 245 to 2550 at 5 Hz (p = 0.70-0.90). In comparison to control correlations taken between pure sinusoids and e.m.g. recorded during a steady contraction (p = 0.10-0.15), the test correlation coefficients are significant. The phase lags compare with those given by Neilson (1974) in a similar test on athetotic patients (180-2700 at 1-5-2 Hz). In these subjects there is an involuntary co-contraction during voluntary movement. REFERENCES JOYCE, G. C. & RACK, P. M. (1974). J. Phy8iol. 240, 375-396. NEILSON, P. D. (1974). J. Neurol. Neuro8urg. Psychiat. 37, 162-170. RoBsoN, J. G. (1962). Ph.D. Thesis. University of Cambridge.

The role of periaqueductal grey matter and of spinal serotonergic pathways in morphine analgesia By J. F. W. DEAEIN,* J. 0. DoSTROVSKYt and A. LONGDEN. National Institute for Medical Research, Mill Hill, London NW7 1AA, and Clinical Research Centre, Watford Road, Harrow, Middlesex HAl 3UJ Several lines of evidence suggest that the periaqueductal grey matter (PAG) is a central site of action of morphine in producing analgesia. Morphine microinjection M.R.C. Training Fellow. t Fellow of the Canadian M.R.C. Present address: Physiology Department, University of Toronto, Toronto, Ontario M5S 1A8, Canada. *

68P 68PtPROCEEDINGS OF THE into and electrical stimulation of this area produces analgesia (see Mayer & Price, 1976) and it is rich in opiate receptors and endogenous morphine-like substances. Recently, it has been shown that electrolytic lesions of the PAG markedly reduced the analgesic effects of morphine (Dostrovsky & Deakin, 1977). This effect could have been due to damage to the dorsal raphe nucleus or the periventricular system of catecholamine neurones. Further experiments to test these two possibilities are now reported. The role of the descending serotonergic innervation of the spinal cord has been reinvestigated since previous lesion studies implicating this system either lacked biochemical lesion confirmation (Proudfit & Anderson, 1975) or used unusually low doses of morphine (Vogt, 1974). Electrolytic destruction of the PAG in rats reduced the analgesic effects of 10 and 20 mg/kg morphine i.P. in tail-flick and hot-plate tests as in the previous study. Histologically, the lesions damaged the dorsal raph6 nucleus to variable extent which was correlated with reductions in striatal 5-hydroxytryptamine (5-HT) and 5hydroxyindole acetic acid (5-HIAA) concentrations. These measures were not related to lesion effects on morphine analgesia. Furthermore, a similar 25 % reduction in striatal 5-HIAA achieved by dorsal raph6 microinjections of the specific 5-HT neurotoxin 5,6-dihydroxytryptamine, were without effect on morphine analgesia. Selective 6-hydroxydopamine induced destruction of catecholamine neurones in the PAG resulted in an equivocal reduction of morphine analgesia which was not related to reduction in amygdala noradrenaline concentrations. Selective destruction of spinal serotonergic neurones by intraspinal microinjection of 5,7-dihydroxytryptamine resulted in substantial reductions of morphine analgesia. The lesions were biochemically confirmed (spinal 5-HT depleted by 70 %) and a large dose of morphine (10 mg/kg) was used. The results suggest that systemic morphine produces analgesia by an action on opiate receptors in the PAG. In addition, a role for the descending spinal serotonin innervation has been demonstrated. It remains to be demonstrated that the spinal serotonergic system is the effector pathway for opiate actions in the PAG. REFERENCES & DOSTROVSKY, J. 0. DEAKN, J. F. W. (1977). Neurosci. Lett. 4, 99-103. MAYER, D. J. & PRICE, D. 0. (1976). Pain 2, 379-404. PROUDFIT, H. K. & ANDERSON, E. G. (1975). Brain Res. 98, 612-618. VOGT, M. (1974). J. Physiol. 236, 483-498.

Studies on the mediation of the atropine-resistant response to neurostimulation of the guinea-pig distal colon BY C. C. EAGLESOM and I. J. ZEITLIN. Department of Physiology and Pharmacology, University of Strathelyde, Glasgow G1 1XW Transmural neurostimulation of isolated guinea-pig distal colon produces an atropine-resistant motility response unaffected by adrenergic antagonists and consisting of relaxation and, on ending stimulation, a contraction. Burnstock (1972) has proposed that this response is purine-mediated. Kinins have recently been implicated in the mediation of a non-cholinergic, non-adrenergic response in the cat colon

PHYSIOLOGICAL SOCIETY, NOVEMBER 1977 69P (Fasth, Hulten, Johnson & Zeitlin, 1976). We are therefore re-examining the pharmacology of this response in the guinea-pig colon. Guinea-pig distal colons, suspended in 20 ml. Tyrode solution (37 TC, air), were stimulated transmurally using coaxial electrodes (Paton, 1975). The tissue load was 0.5 g. Contractions were detected using an isotonic transducer. Tissues were stimulated (5 Hz, maximal voltage, 1 ms pulse width, 10 see trains) at 2 min intervals. Neurostimulation produced a biphasic response unaltered by atropine (106 M), phentolamine (106 M) and propranolol (10-6 M), methysergide (2 x 1O-5 M) or mepyramine (10-5 M) in concentrations which blocked the responses to acetylcholine, noradrenaline, 5-hydroxytryptamine and histamine respectively. None of these agonists produced the typical biphasic response. The response was also unaltered by guanethidine (3 x 106 M) or pretreatment with reserpine (1 mg . kg-1, 24 hr previously). The response was blocked by hexamethonium (2-7 x 104 M) indicating a preganglionic site of stimulation. The biphasic response was mimicked closely by ATP (10-7 M- 4 X 10- M) and bradykinin (2 x 10-9 to 9-6 x 10-7 M). Indomethacin (2.3 x 104 M) completely blocked the after-contraction produced by neurostimulation, by ATP (3.2 x 10- M) and by bradykinin (1.9 x 10-7 M) but had no effect on the initial relaxation. Prostaglandin E2 (9 x 10-9 to 7.3 x 10-7 M) produced a contraction which was much slower (2 min to peak) than the after-contraction to neurostimulation (7 see to peak). In addition to inhibiting prostaglandin synthesis, indomethacin depletes intestinal tissues of calcium (Northover, 1972). The calcium-chelating agent, EDTA, at a concentration (1.7 x 103 M) which almost completely inhibited the neurostimulation response, had little effect on the responses to either ATP or bradykinin and certainly had no selective action on the after-contraction. These findings support the suggestion (Burnstock, Cocks, Paddle & StaszewskaBarczak, 1975) that the response to neurostimulation may be partially mediated by prostaglandins. The results obtained so far do not distinguish between ATP and bradykinin as possible mediators of this response and indicate that their actions may also be partially mediated by prostaglandins. This work was supported by an M.R.C. project grant to I.J.Z. We thank Sandoz Ltd and Upjohn Ltd for gifts of bradykinin and prostaglandin. REFERENCES BtTRNSTOCK, G. (1972). Pharmac. Rev. 24, 509-558. BURNSTOCK, G., CocKs, T., PADDLE, B., STAsZEWSKA-BARcZAI, J. (1975). Eur. J. Pharmac. 31,

360-362. FASTH, S., HuLTEN, L., JoHNsoN, B. J. & ZEITLIN, I. J. (1977). J. Physiol. 265, 56-57P. NORTHOVER, B. J. (1972). Br. J. Pharmac. 45, 651-659. PATON, W. D. M. (1975). In Methods in Pharmacology, vol. 3, ed. DANEL, E. E., pp. 313-320. New York: Plenum.

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Imprinting and the incorporation of uracil in the chick brain: a radioautographic study BY P. P. G. BATESON, G. HORN and B. J. MCCABE. Sub-Department of Animal Behavior, University of Cambridge, and Department of Anatomy, University of Bristol The social preferences of day-old chicks can be restricted to a visually conspicuous object (e.g. a hen or a flashing light) by exposing the chicks to that object (see Sluckin, 1972). A series of experiments (see Horn, Rose & Bateson, 1973) has suggested that net incorporation of radioactive uracil into acid-insoluble substances in the 'roof' of the forebrain is intimately associated with this learning process ('imprinting'). The present study was undertaken to specify more precisely the sites of such biochemical changes in order to analyse the cellular activities which these changes reflect. Domestic chicks were exposed to a rotating flashing light for either 45 or 180 min on the first day after hatching (60 or 240 min in the case of one pair): on the second day all birds were exposed for a further 63 min. This experimental design presupposes that birds exposed to the stimulus for the shorter period on day 1 (undertrained chicks) will have more to learn about this stimulus on day 2 than birds exposed for the longer period on day 1 (overtrained chicks): accordingly the net incorporation of [3H]uracil in the forebrain roof was predicted and found to be greater in the undertrained chicks than in the overtrained chicks (Bateson, Rose & Horn, 1973). Twenty birds were matched in pairs: each chick received an intracardiac injection of 50 jtc[14C]uracil before exposure on day 2. The chicks were decapitated 150 min after injection and serial coronal sections of their brains cut: alternate pairs of sections were prepared for radioautography (Horn & McCabe, 1978). The optical density for a number of major anatomical regions was measured. The measurements for each region were averaged over 0-5 mm 'slabs' of brain and expressed as a percentage of the mean of all measurements for that brain. Differences were found in two adjacent 0 5 mm slabs, lying just caudal to the midpoint between the anterior and the posterior poles of the forebrain. For this 1 mm of brain the standardized mean optical density, d, of the medial part of hyperstriatum ventrale was significantly (P < 0.002) greater in the undertrained chicks (d = 119-9 %) than in the overtrained chicks (d = 114-1 %). No other regional differences between the two groups of chicks were found, even though two of the regions, the ectostriatum and the hyperstriatum intercalatus, are visual projection areas. We thank the S.R.C. for financial support. REFERENCES BATESON, P. P. G., ROSE, S. P. R. & HORN, G. (1973). Science, N.Y. 181, 576-578. HORN, G. & MCCABE, B. J. (1978). J. Phy8iol. 275, 2-3P. HORN, G., ROSE, S. P. R. & BATESON, P. P. G. (1973). Science, N.Y. 181, 506-514.

SLUCKN, W. (1972). Imprinting and Early Learning. London: Methuen.

PHYSIOLOGICAL SOCIETY, NOVEMBER 1977

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The properties of substantia gelatinosa units BY P. D. WALL. Anatomy Department, University College London, WC1E 6BT We have examined the properties of 308 units located either within the substantia gelatinosa (SG) or in the lateral Lissauer tract (LLT) to which SG cells project. The units were recorded in the L4-S1 segments of decerebrate cat spinal cord with platinum-plated tungsten micro-electrodes; unit locations were marked. Seventeen units failed to respond to peripheral stimulation, 171 were excited by brush and touch, 61 by brush, touch and pressure and 59 required pressure in their receptive field (RF). Eighty-six of the RF's were strikingly small (< 1.5 cm2), 167 RF's were small (a fraction of the upper leg, lower leg, foot or toes) and 40 had large receptive fields extending over two or three parts of the leg. Electrical stimulation of the RF excited all the tested units with response latencies which showed that they were excited by myelinated afferents. None were peripheral afferent axons since they failed to respond to peripheral nerve stimulation with a fixed latency or at frequencies above 100 Hz. They were not marginal cells because the optimal recording sites were clearly in laminae 2 and 3. They were not axons of long descending systems since they failed to follow high-frequency stimulation of the Lissauer tract or the dorso-lateral fasciculus (DLF) if the stimulus was applied more than 2 mm rostral to the recording site. The spikes were not caused by dendritic activity or field spread from the large cells of lamina 4 since their receptive field properties differed from lamina 4 cells, none of them projected axons into the DLF, and, as shown previously (Merrill & Wall, 1975) using two independent electrodes, their firing did not synchronize with the action potentials in deep cells. Furthermore, using micro-electrode stimulation of the LLT (Merrill & Yaksh, 1978), a representative sample of 56 of the 308 cells were shown to project axons into the LLT and not to DLF, dorsal columns or peripheral nerve. Two highly unusual response properties were observed. Forty-three cells completely habituated after one, two or three stimuli to their receptive field and failed to return to their former responsiveness for 30 see or more. Fifty-six cells showed a very prolonged after discharge lasting 10 see to more than 2 min after a single brief natural stimulus to their RF. We show here that it is possible to record from single units within SG and that their afferent drive is not monopolized by unmyelinated afferents. Some of them show unusual properties not previously recorded in other dorsal horn cells. REFERENCES

MERRILL, E. G. & WALL, P. D. (1975). J. Phy8iol. 245, 82-83P. MERRILL, E. G. & YAxsH, T. L. (1978). J. Phy8iol. 275, 71-72P.

Properties of cerebellum parallel fibres and the Lissauer tract of the cat BY E. G. MERRILL and T. L. YAxSH. Anatomy Department, University College London, WC1E 6BT Microstimulation and single fibre recording techniques, using platinum-plated tungsten micro-electrodes, have been developed for use in the Lissauer tract (LT) and substantia gelktinosa. Because of the similarity of the parallel fibres (PF) to the

PROCEEDINGS OF THE 72P LT (i.e. both consist of densely packed unmyelinated fibres running longitudinally along the surface of the c.N.s.), we have also evaluated these techniques in the cerebellum. Surface microstimulation (05-5 ,uA, 50 ,sec duration) produced excitation of a narrow beam of fibres in both cases, up to 2-5 mm (1.5 mm for LT) long by 150,um wide by less than 100,um deep. The PF beam was as described previously. Fibre conduction velocities were 0S3-0 5 m/sec (occasionally as high as 2-0 m/sec). The absolute refractory periods for single fibres were between 0 7 and 1 0 msec, followed by a supranormal period lasting approximately 25 msec, during which time threshold was reduced by as much as 25 % and conduction velocity increased by as much as 18% (PF, 7-18%, LT, 3-10%), as shown by Gardner-Medwin (1972) for the PF compound action potential. No subnormal period was observed after the supranormal period. Individual PF and LT fibres followed stimuli at rates in excess of 100 Hz. Chronaxies for PF and LT single fibres varied with conduction velocity, from 180 ,#sec (2 m/sec conduction velocity) to 360 ,usec (0.4 m/sec), in both PF and LT. The PF compound action potential chronaxie was 155 ,usec (0 70 m/sec). Koslow, Bak & Li (1973), report a chronaxie of approximately 400,sec for the saphenous nerve C fibre population. When a subthreshold conditioning pulse was used in the assessment of excitability, a surprisingly long-lasting reduction in threshold (up to 40 % initially) and increase in conduction velocity (up to 18 %) could be shown. This increase in excitability declined gradually over the 20 msec following the conditioning stimulus. Two mechanisms may be involved - a passive one governed by the membrane time constant (estimated to be about 400 tsec from the chronaxie) and a longer-lasting one due to active processes either in the tested axon or in its neighbours. An estimate of the space constant for PF and LT fibres was attempted using two stimulating electrodes, yielding measurements of the order of 100 jtm. Thus the data obtained with our stimulation and recording methods are completely consistent with the known anatomy and physiology of the PF and LT, but differ in a few details from what is known about peripheral C fibres. REFERENCES GARDNER-MEDWIN, A. R. (1972). J. Phyiol. 222, 357-371. KOSLOW, M., BAx, A. & Li, C. L. (1973). Expl Neurol. 41, 745-753.

A precentral macropotential with short latency as a possible indicator of response reafferent activity in man BY D. PAPAKOSTOPOULOS. Burden Neurological Institute, Bristol Brain macropotentials were recorded with scalp electrodes from sixteen normal subjects, and with chronically implanted cortical electrodes from two patients (Crow, Cooper & Phillips, 1961; Crow, 1973) executing self-paced thumb movements. The movement involved pressing a button which initiated the sweep of an oscilloscope beam in each of the following four conditions: left-hand press, right-hand press, pressing with both hands simultaneously, and performing a skilled performance

PHYSIOLOGICAL SOCIETY, NOVEMBER 1977 73P task in which the sweep was initiated by a left-hand press and terminated within 40-60 msec by a right-hand press. Electromyograms were recorded from flexor and extensor muscles of each forearm. Scalp macropotentials were recorded from the bridge of the nose and the vertex and bilaterally from pre- and post-central locations, each electrode being referred to linked mastoids. During corticographic studies the pre- and post-central gyri were identified by the reversal of the polarity of the early component of the somatosensory evoked potential (Papakostopoulos, Cooper & Crow, 1975; Papakostopoulos & Crow, 1978). On-line averaging and other data processing was carried out by a PDP-12 computer. A negative potential with maximum amplitude over precentral cortex, the motor cortex potential, was recorded from all normal subjects in all four conditions. Its mean peak latency was 32 msec after the peak e.m.g. of the initiating press. The corticographic data of the two patients were similar to the scalp data both in spatial and latency characteristics. On the basis of its spatial and temporal characteristics it is suggested that the motor cortex potential is an indicator of response reafferent sensory input to precentral cortex amenable to study with non-invasive techniques in man. As such it may be seen as a neurophysiological index of the transcortical reflex loop, the evidence for which has hitherto come only from neurophysiological experiments in animals and human behavioural experiments (Marsden, Merton & Morton, 1973). REFERENCES

CROW, H. J., COOPER, R. & PmILLPS, D. G. (1961). J. Neurol. Neuroaurg. Psychiat. 24, 353-360. CROW, H. J. (1973). P.ychiat. Neurol. Neurochir. Am8t. 76, 365-381. MARSDEN, C. D., MERTON, P. A. & MORTON, H. B. (1973). J. Phygiol. 230, 58-59P. PAIA'OSTOPOUILOS, D. & CROW, H. J. (1978). Progre88 in Clinical Neurophysiol, ed. DESMEDT, J. E. S. Basel: Karger. (In the Press.)

PAPAKOSTOPOULOS, D., COOPER, R. & CROW, H. J. (1975). Nature, Lond. 258, 321-324. Abnormal reinnervation of muscles in dystrophic mice BY C. R. SLATER and A. F. R. WOLFE. Muscular Dystrophy Group Research Laboratories, Regional Neurological Centre, Newcastle General Hospital, Newcastle upon Tyne After a crush injury of motor axons, muscle reinnervation occurs primarily at the sites of the original nerve-muscle junctions (cf. Bennett & Pettigrew, 1976). It seems likely that some form of interaction between the muscle fibre and the ingrowing axon at these sites promotes the differentiation of the presynaptic terminal. J. B. Harris & M. W. Marshall (personal communication) have recently shown that after a crush, functional reinnervation in dystrophic mice of the strain Bar Harbor ReJ 129 dy/dy occurs after a greater delay than in normal animals. We have studied nerve terminal morphology in this situation to learn more about the process of reinnervation in dystrophic mice. Reinnervation was studied in the soleus muscle of eight-week-old normal and dystrophic mice after crushing the right sciatic nerve in the thigh. At various times after the operation animals were killed and the degree of functional reinnervation

74P 7PPROCEEDINGS OF THE was determined, in isolated nerve-muscle preparations at room temperature. Following nerve crush, junctional transmission fails and the nerve terminals degenerate within 12-24 hr. In control animals spontaneous miniature end-plate potentials and indirectly evoked muscle action potentials were first seen 6-8 days after the crush and all muscle fibres had become reinnervated by 12 days. In dystrophic mice, functional reinnervation was delayed about 2 days. To study nerve terminal morphology the preparations were then stained by the ZnI-0s04 method (Akert & Sandri, 1968), which stains cholinergic nerve terminals. In control animals, easily recognized motor axon terminals were seen as soon as transmission could be detected. The average length of these terminals, as measured along the longitudinal axis of the muscle, was around 62,um some 40 % greater than in the unoperated contralateral limbs, and remained so for at least 3 weeks. The soleus nerve terminals in unoperated limbs of dystrophic animals were slightly longer than in control animals (about 25 %). Following reinnervation, however, the terminals in dystrophic mice quickly grew to around 250 % of their normal length and remained at that size for at least 7 weeks. Initial studies of the distribution of persisting post-synaptic features of the original nerve-muscle junction cholinesterase (Karnovsky & Roots, 1964) and acetylcholine receptor, as defined by labelling with fluorescent conjugated a-bungarotoxin (Anderson & Cohen, 1974), confirm that following reinnervation of soleus muscle in dystrophic mice the differentiated axon terminals extend far beyond the limits of the original end-plate. REFERENCES

AKDERT, K. & SANDRI, C. (1968). Brain Re&. 7, 286-295. ANDERSON, M. J. & COHEN, M. W. (1974). J. Physiol. 237, 385-400. BENNETT, M. R. & PETrIGREW, A. G. (1976). Cold Spring Harb. Symp. quaint. Biol. 40, 409-424. KARNOVsKY, M. J. & RooTs, L. (1964). J. Histochem. Cytochem. 12, 219-221.

An immunological model of the sweet-taste receptor BY J. A. EDWARDSON and C. A. M. HOUGH. Department of Physiology, St George's Hospital Medical School, London SW17 ORE, and Tate and Lyle Ltd, Philip Lyle Memorial Research Laboratories, P.O. Box 68, Reading, Berks. Thaumatins are sweet-tasting proteins produced in the aril of fruit of the West African plant Thaumatococcus danielli (Bentham). They are approximately 2 x 105 times sweeter than sucrose, compared on a molar basis, and thus have potential as low-calorie sweeteners (Van der Wel & Loeve, 1972). A radioimmunoassay has been developed to measure low, palatable concentrations of thaumatin in solution. Antibodies raised against the protein show marked cross-reaction with other non-protein sweet substances, suggesting that the major antigenic determinant is also the active centre which reacts with the sweet receptor in vivo. Antisera were raised in New Zealand White rabbits which received multiple intradermal injections of thaumatin in Freund's complete adjuvant. A solid-phase radioimmunoassay was developed using 'MI-labelled thaumatin and antibodycoated tubes, a method first described for the assay of peptide hormones (Catt & Tregear, 1967). Binding of the labelled protein to tubes was readily displaced with

75P PHYSIOLOGICAL SOCIETY, NOVEMBER 1977 unlabelled thaumatin and non-specific binding to tubes without antibody was small. When 5-5 x 104M sodium saccharin was added in the absence of unlabelled sweet protein a significant displacement of the labelled thaumatin from antibody was observed. A variety of sweet substances were similarly tested in the assay, including aspartame (1-aspartyl-1-phenylalanyl methyl ester), calcium cyclamate, several chlorinated sucrose derivatives, neohesperidin dihydrochalcone, and monellin, which is another sweet-tasting plant protein (Morris & Cagan, 1972). In each case the sweet substances partially displaced labelled thaumatin and their immunoreactivity was proportional to their reported values for sweetness relative to sucrose. When immunoreactivity and sweetness for the seven compounds tested were ranked, a correlation coefficient of 0 9606 was obtained (P < 0 001). None of these sweet substances produced non-specific interference in two radioimmunoassays for peptide hormones, and direct evidence for specific binding to the anti-thaumatin antibodies was obtained with 125I-labelled monellin. This remarkable correlation between the recognition of sweet substances in vitro by antibodies raised against thaumatin and by sweet-taste receptors in vivo, points to some common molecular features of both recognition sites. The phenomenon may provide a useful model for studying the physico-chemical properties which confer sweetness on certain molecules. REFERENCES

CAWr, K. & TREGEAR, G. W. (1967). Science, N.Y. 158, 1570-1571. MoRRIs, J. A. & CAGAN, R. H. (1972). Biochim. biophy8. Acta 261, 114-122. VAN DER WEL, H. & LOEVE, K. (1972). Eur. J. Biochem. 31, 221-225.

Ouabain-induced contractures in mammalian heart muscle BY WINIFRED G. NAYLER, P. A. POOLE-WILsoN and A. WiurMs. Cardiothoracic Institute, London Univer8ity, 2 Beaumont Street, London WIN 2DX Ouabain-induced contractures in mammalian heart muscle are usually explained in terms of a raised tissue Ca2+ (Langer & Serena, 1970). Our experiments were designed to study the temporal relation between the ouabain-induced rise in resting tension, the gain in tissue Ca2 , the depletion of high-energy phosphates, and the ability of the sarcoplasmic reticulum (SR) to accumulate Ca2+. Isolated rabbit hearts were perfused by the Langendorff technique at 37 00 and a mean perfusion pressure of 80 mmHg. The perfusate contained, in m-mole/l., NaCl 1 15-0, NaHCO3 25-0, KCl 4 0, KH2PO4 0 9, Mg2SO4 1 1, CaCl2 1'5 and glucose 110, and was gassed with 95 % 02+ 5 % C02. Tension was measured with a transducer (Grass FTO3) attached via a nylon ligature to the ventricular apex. Control hearts were perfused for 90 min. After 30 min, ouabain was added to provide a final concentration of 5 x 104 M. After varying periods of perfusion the hearts were assayed for Ca2+ (by atomic absorption spectrometry), adenosine triphosphate and creatine phosphate (CP) (Nayler, Grau & Slade, 1976), mitochondrial respiratory activity (Nayler, Fassold & Yepez, 1977) - expressed in terms of Qo2 and RCl, and the Ca2+-accumulating activity of the SR - measured by the Millipore filtration technique (Nayler, Dunnett & Berry, 1975). After 15 min perfusion in the presence of 5 x 10-6 M ouabain, resting tension was

76P 7PPROCEEDINGS OF THE elevated (P < 0 01). At this time no change could be detected in tissue Ca2+, ATP, OP, mitochondrial Qo2 and RCl or the ability of the SR to accumulate Ca2 . Extending the duration of the ouabain perfusion to 60 min resulted in a raised (P < 0'001) tissue Ca2+ and a diminished tissue ATP (P < 0-001), CP (P < 0.001), mitochondrial RCl (P < 0 001) and Q02 (P < 0.01). The Ca2+-accumulating activity of the SR was normal. Removing Ca2+ from the extracellular phase early but not late in the development of the contracture response abolished the contracture. These results show that the raised tissue Ca2+ caused by high concentrations of ouabain occurs as a late response at a time when the high-energy phosphate reserves are diminished. The onset of the contracture requires the presence of COa+ in the extracellular phase. The contracture is not attributable to irreversible damage of the SR or the mitochondria. This work was supported by the Medical Research Council. REFERENCES

LANGER, G. A. & SERENA, S. D. (1970). J. mol. cell Cardiol. 1, 65-90. NAYLER, W. G., DUNNETT, J. & BERRY, D. (1975). J. mol. cell. Cardiol. 7, 275-288. NAYLER, W. G., GRAu, A. & SLADE, A. (1976). Cardiovasc. Res. 10, 650-662. NAYLER, W. G., FASSOLD, E. & YEPEZ, C. (1977). Cardiovaec. Re8. (In the Press.)

Properties of plasma plasminogen activators BY B. BENNETT, M. MACKiE and D. OGSTON. Departments of Medicine and Physiology, University of Aberdeen, Aberdeen Circulating blood contains small quantities of plasminogen activator activity; the level can be markedly enhanced by such stimuli as exercise, venous occlusion and the injection of vasoactive agents. The number and origin of activators of plasminogen in circulating human blood is not established. The vascular wall is believed to be an important source while an activator formed by a pathway dependent on the activation of factor XII and involving the kallikrein-kinin system may be physiologically relevant. In this study some properties of a circulating activator isolated from blood after venous occlusion by a previously described technique (Ogston, Bennett & Mackie, 1976) has been compared with vascular activator prepared from cadaveric veins, an activator derived from factor XII activation, and urokinase, the activator present in urine. The circulating activator, vascular activator and the factor XII-dependent activator all had an elution volume on gel filtration suggesting a molecular size similar to that of albumin, while urokinase consistently eluted behind the albumin position. In addition similar patterns of inhibition were noted when circulating, vascular and factor XII-dependent activators were exposed to iodoacetamide, phenylmethylsulphonyl fluoride, tosyl-L-lysine chloromethylketone and tosyl-phenylalanine chloromethylketone. Urokinase differed in its inhibition by iodoacetamide. An antibody, raised in rabbits, to the cadaveric vascular wall activator was tested on double diffusion against the four activators: circulating and factor XII-dependent activator appeared to share a common antigen, but no precipitin line appeared with

PHYSIOLOGICAL SOCIETY, NOVEMBER 1977 77P urokinase. In a functional assay the antibody inhibited the activity of vascular activator. REFERENCE QOSTON, D., BENNETT, B. & MACKIE, M. (1976). Thromb. Re8. 8, 275-284.

Echo cardiographic left ventricular dimensions in the supine and upright positions and on upright exercise in normal subjects By D. BENNETT, R. GOLDSTEIN and G. LEACH. Department of Medicine I, St George's Hospital Medical School, Cranmer Terrace, London SW17 ORE An Ekoline ultrasonic cardiogram has been used to measure left ventricular dimensions in five normal male subjects. Measurements of left ventricular enddiastolic and end-systolic dimensions have been made at rest in the supine and upright positions, and on upright exercise at increasing work loads (40, 80, 120 and 160 W). On assuming the upright position, four out of five subjects showed a mean fall of 5 mm (10%) in left ventricular end diastolic dimension. One subject showed no change. All five subjects reduced their left ventricular end systolic dimension by a mean of 4*2 mm (14 %). The mean percentage left ventricular shortening (i.e. the difference between end systolic and end diastolic dimension) increased by 3 % following the change in posture. Exercise at 40 W produced a uniform rise of 8 7 % in end-diastolic dimension to a level similar to supine rest. Increasing levels of exercise produced no further systematic change in end diastolic dimension. In contrast, end-systolic dimension decreased so that at maximum exercise it was 25 % below upright resting levels. These measurements, therefore, confirm the well-established fact that end-diastolic dimension decreases on assuming the upright posture. However, they imply that low levels of exercise rapidly restore the heart to its supine dimension, and that no further increase in heart size occurs with higher levels of exercise. The decreasing systolic dimension with exercise strongly implies that the heart empties to a greater degree as the exercise level increases. Although ultrasonic measurements of left ventricular dimensions should not be considered true measurements of left ventricular volume, they are probably closely related to volume, particularly in the normal heart. If this is so, it is concluded that the heart on exercise increases its output, first in increasing its rate, and secondly by decreasing its systolic volume, not by an increase in end-diastolic volume. The structure of the augmented breath By M. ROUMY. Department of Physiology, St George's Hospital Medical School, Cranmer Terrace, Tooting, London SW17 ORE In a previous communication (Davies, Roumy, Widdicombe & Wise, 1977) it was reported that short pulses of inflation and deflation given during inspiration produce an inspiratory augmented response. These breaths showed a biphasic tidal volume and phrenic integral. The inspiratory time was 1-8 times that of a normal breath,

78P PROCEEDINGS OF THE and expiratory time was decreased. In anaesthetized rabbits, spontaneous augmented breaths occur with quite a regular frequency, and their characteristics are the same as those of the triggered augmented breaths. In addition, both natural and triggered augmented breaths are followed by a refractory period of up to 2 min. Both depend on a vagal reflex, thought to be initiated by lung irritant receptors. We were therefore satisfied that the triggered augmented breaths could be used to investigate the mechanisms underlying the spontaneous ones. Recordings of lung irritant receptor activity during an augmented breath showed an excitation not only during the pulse but also in the second part of the breath. There is therefore a possibility that this excitation is partly responsible for the augmented breath by a positive feed-back. To test this hypothesis, pulses of inflation and deflation (which stimulate lung irritant receptors) were delivered to anaesthetized (40 mg/kg sodium pentobarbitone), paralysed and artificially ventilated rabbits. Under these conditions phrenic discharge and tidal volume were out of phase. When pulses were given during phrenic inspiration, inspiratory activity was lengthened. The paralysed animals differed in at least two respects from the nonparalysed ones: (1) responses were obtained when the pulse was given at the end of inspiratory activity, and (2) they showed no refractoriness. By giving pulses during phrenic expiration, it became apparent that the pulses were producing a burst of phrenic activity of constant duration which, when produced at the end of a normal inspiration, gave the appearance of an augmented breath. When a pulse of deflation was given during phrenic expiration and at FRC (functional residual capacity), stimulating strongly irritant receptors, the burst of phrenic activity was produced without latency. When a pulse of inflation was given in the same conditions, producing a weaker stimulation of irritant receptors, the burst of phrenic activity was phase-locked with tidal volume inspiration. The latter case suggests that the natural discharge of irritant receptors and the activity produced by the pulse are summed to trigger the phrenic response. The relevance of this summation to the mechanisms involved in the spontaneous augmented breath was discussed. REFERENCE

DAVIES, A., Roumy, M., WIDDICOMBE, J. G. & WISE, J. C. M. (1977). In Proc. 27th Int. Cong. Physiological Sciences, Paris, vol. 13, p. 163.

Experiments on the origin of vasomotor tone BY P. G. GUERTZENSTEIN, S. M. HILTON, J. M. MARSHALL and R. J. TIMMS. Department of Physiology, Medical School, Birmingham B15 2TJ Bilateral application of glycine to the ventral surface of the cat's medulla causes a pronounced and prolonged fall of arterial blood pressure: the site of action is restricted and near the surface (Guertzenstein & Silver, 1974). We have tested the hypothesis that the drug thus applied acts by blocking synapses in the efferent pathway for the autonomic components of the defence reaction, as this pathway is known to descend in this region of the ventral medulla (Abrahams, Hilton & Zbroiyna, 1960; Schramm & Bignall, 1971). In cats anaesthetized by continuous infusion of Althesin (Glaxo), electrodes were

PHYSIOLOGICAL SOCIETY, NOVEMBER 1977 79P implanted stereotactically in the regions of the amygdala or hypothalamus from which defence reactions are elicited. The ventral surface of the medulla was then exposed. With bilateral application of glycine restricted to the sensitive area, respiration ceased and, with the animal now artificially ventilated, arterial blood pressure fell to a low level over some 10-15 min, due mainly to a pronounced vasodilatation in the mesenteric bed. Vascular conductance changed little, if at all, in skeletal muscle, and there was slight bradycardia. After 5-10 min, when mean arterial pressure had fallen to around 70 mmHg, the circulatory response elicited from the amygdala or hypothalamus was substantially reduced, particularly the rise in arterial pressure and mesenteric vasoconstriction. The increase in muscle conductance was smaller, but the tachycardia persisted almost unchanged. Pupillary dilatation, retraction of the nictitating membranes and piloerection were all reduced. Localized electrical stimulation in the glycine-sensitive area of the ventral medulla elicited all the autonomic components of the defence reaction, including muscle vasodilatation and mesenteric vasoconstriction, together with increased rate and depth of respiration. Further mapping showed that this pattern could be elicited from a narrow longitudinal strip in the medulla, 3-4 mm each side of the mid line. This strip becomes superficial within the glycine-sensitive area, where it runs less than 500 #sm from the surface. After a unilateral lesion within the glycine-sensitive area, restricted to this strip, the whole response to ipsilateral defence area stimulation was much reduced. Glycine then applied to the contralateral medullary area alone produced fully the changes which, without the lesion, would only be seen after bilateral application. We conclude that, in so far as the 'resting' blood pressure level is of central nervous origin, it results from continuing activity in the brainstem defence areas, relayed through the ventral medulla. Supported by grants from the Medical Research Council and Wellcome Trust. REFERENCES ABRAHAMS, V. C., HILTON, S. M. & ZBROiYNA, A. W. (1960). J. Physiol. 154, 491-513. GUERTZENSTEIN, P. G. & SILVER, A. (1974). J. Phygiol. 242, 489-503. ScHRAMM, L. P. & BIGNALL, K. E. (1971). Am. J. Phy8iol. 221, 754-767.

The effects of acetylcholine and histamine on total lung resistance, dynamic lung compliance and lung irritant receptor discharge in the anaesthetized dog By M. DIXON, D. M. JACKSON, I. M. RICHARDS and K. VENDY. Department of Pharmacology, Fisons Ltd., R. & D. Laboratories, Loughborough, Leics. Histamine, which may be an important mediator in asthma, can excite lung irritant receptors and could therefore initiate a reflex bronchoconstriction. Mills, Sellick & Widdicombe (1969) attribute this stimulation by histamine to contraction of underlying bronchial smooth muscle, whereas Sampson (1977) reports that histamine can stimulate the receptors directly. We have assessed the effects of acetylcholine and histamine on total lung resistance (RL), dynamic lung compliance (Cdyn) and lung irritant receptor discharge in anaesthetized dogs.

APAROCEEDINGS OF THE 80P Beagle dogs (9-12 kg) were sedated with thiopentone (5-10 mg.kg-', i.v.) and anaesthetized with chloralose (80 mg.kg-, i.v. followed by 10-15 mg.kg-' every 15 min). Blood pressure and respiratory mechanics were measured (Richards & Jackson, 1976). Thoracotomy was routinely performed. In one group of dogs both cervical vagi were cut and action potentials were recorded from single fibres teased from the left vagus nerve and originating from irritant receptors. Two criteria were used to characterize irritant receptors. First, the receptor was stimulated by, and adapted rapidly to, hyperinflation and deflation of the lungs, and secondly, the receptor was located within the lung. Acetylcholine (5-40 jug. kg-') and histamine (5-20 jg. kg-') were given i.v. and the changes in receptor discharge and in RL and Cdyn were measured. In a second group of dogs the cervical vagi were placed on silver thermodes which could be cooled to 0. 5 C0. Acetylcholine (40 jug. kg-1) and histamine (10 jug.kg-') were given ix. and the changes in RL and CdYn were measured with the vagi at body temperature and when cooled. When doses of agonist were chosen which gave equal changes in RL of approximately 0 4 kPa 1-1 see (40,ug. kg-' acetylcholine, 10 jg. kg-' histamine), the change in irritant receptor discharge was significantly (P < 0.05) higher for histamine than for acetylcholine (268 + 30 and 132 + 51 impulses/15 sec respectively). The falls in CdYn were 64 + 5 ml. kPa-1 for acetylcholine and 46 + 6 ml. kPa-1 for histamine (all values are means + S.E., n = 10). Mechanical distortion of the airways, measured by changes in RL and CdyD, apparently cannot entirely account for the increase in discharge of lung irritant receptors caused by histamine. Histamine can probably stimulate or sensitize the receptors directly. The second group of experiments enforces this suggestion. Histamine (10 jg. kg-', i.v.) increased RL, but significantly less when the vagi were cooled (0.63 + 0.07 kPa l-1 see reduced to 0 29 + 0.05 kPa 1-1 sec, n = 5). Acetylcholine (40 ug. kg-', i.v.) increased RL, but not significantly less during vagal cooling (0.42 + 0-02, kPa 1-1 see reduced to 0 40 + 0.02 kPa l-1 sec, n = 4). REFERENCES

MMLLS, J. E., SELLICK, H. & WIDDICOMBE, J. G. (1969). J. Physiol. 203, 337-357. RICHARDS, I. M. & JACKSON, D. M. (1976). Br. J. Pharmac. 57, 436P. SAMPSON, S. R. (1977). Am. Rev. Rest. Di8., 115, 107-115.

The effects of calcium and calcium ionophore A23187 on mucin secretion and potential difference in the isolated chicken trachea BY KIM BALFRE. Department of Physiology, St George's Hospital Medical School, London SW17 ORE Calcium is known to be intimately involved in many secretary systems (see Rubin, 1974). There have been no previous reports on the effects of calcium on tracheal mucin production. The aim of these experiments was to investigate the effects of calcium concentration and calcium ionophore A23187 on tracheal mucin production and on the spontaneous electrical potential difference found to exist across the chicken trachea in vitro.

PHYSIOLOGICAL SOCIETY, NOVEMBER 1977 81P A modified version of the method used by Phipps & Richardson (1975) for mucus collection in vivo has been used. Atropine (1 jug/ml.) was present throughout the experiment. The trachea was suspended in avian Ringer in an organ bath. The mucus-secreting cells took up [3H]glucose given into the lumen at the start of the experiment and incorporated the tritium into mucins. The output of radioactivity bound to these macromolecules was monitored during the rest of the experiment. Increasing calcium concentration from the control value of 1-8-18 mm on both sides of the trachea resulted in an increase in mucin secretion (A = + 44-4 + 6*10% (mean and s.E.), n = 9, P < 0.001). It also resulted in an increase in potential difference across the tracheal wall - that is, the lumen became more negative with respect to the submucosal surface. (Control potential = 4 4 + 0 3 mV, test potential= 6-7 + 0-4 mV, mean increase = + 2-4 + 0 3 mV, n = 9, P < 0 001). Introduction of calcium-free solutions for 15 min on to both luminal and submucosal surfaces of the trachea resulted in large increases in mucin output. (A = 158 + 45.5 %, n = 13, P < 0 01). In a further eight experiments 1 mm EGTA (a chelating agent) was added to the Ca-free Ringer, and again mucin output increased (A = + 352 + 44 %, n = 7, P < 0 001; in the eighth experiment a 40-fold percentage increase in mucin output occurred). In those experiments in which potential difference was also recorded, this increased (mean increase = + 3 1 + 0-7 mV, n = 11, P < 0 01). It is surprising that both increased and decreased calcium concentrations augmented secretion. However, preliminary results suggest that calcium-free solutions work predominantly on the luminal surface of the trachea whereas calcium-enriched solutions act mainly on the submucosal side. Introduction of calcium ionophore A23187 (1.9 x 106 M) into the lumen of chicken tracheas always resulted in increases in mucin production (A = + 51 + 12*3 %, n = 5, P < 0 01). Potential difference also increased (mean increase = + 3 0 + 0 9 mV, n = 5, P < 0.05). Subsequent administration of the same dose of ionophore into the same trachea only produced small increases in mucin output. None of the above effects of calcium or calcium ionophore on mucin production or potential difference were blocked by atropine. These results indicate that calcium may play a role in release of mucins from hen trachea. Changing calcium concentration or calcium ionophore administration increases both mucin secretion and potential difference across the tracheal wall. REFERENCES

PHIPPS, R. J. & RIcHARDsoN, P. S. (1975). J. Phy8iol. 247, 27-28P. RUBIN, R. P. (1974). Calcium and the Secretory Process. New York: Plenum Press.

Impedance related to local blood flow in cerebral cortex BY N. M. BRANSTON, A. J. STRONG and L. SYMON. Institute of Neurology, The National Ho8pital, Queen Square, London WC1 3B1 Total cerebral ischaemia produces a large increase in cortical impedance (Van Harreveld, 1966). In twenty-two baboons anaesthetized with alpha-chloralose, we measured impedance (Fig. 1A), tissue blood flow (hydrogen clearance) and somatosensory-evoked responses (Branston, Strong & Symon, 1977) in the same cortical region.

82P PROCEEDINGS OF THE Specific impedance of normally perfused cortex was 437 + 16 Qcm (mean + s.E.). To reduce flow in controlled stages, the middle cerebral artery was occluded, with exsanguination as necessary (Branston et al. 1977). Impedance and flow were correlated (r = - 0 95, P < 0 005) from 0-10 ml./ 100 g. min (Fig. I B, left), as were evoked response and flow (r = 0 95, P < 0 02) from 13-18 ml./ 100 g.min (Fig. IB, right). The regression lines (dotted) intercepted the flow axis at 9-6 and 13-3 ml./ 100 g. min, respectively; 95 % confidence bands are indicated. Hydrogen clearance system

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The effect of substance P on cyclic AMP and cyclic GMP levels in actively secreting pancreatic lobules [proceedings].

60P PPROCEEDINGS OF THE The effect of substance P on cyclic AMP and cyclic GMP levels in actively secreting pancreatic lobules BY JANET ALBANO, K. D...
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