15P PHYSIOLOGICAL SOCIETY, MAY 1978 that the lag is followed by a single exponential (first-order kinetic) process. For the sodium currents, again close fits can be obtained assuming heterogeneity and having the kinetics follow a simple sequential form: A -- C -> D with C conducting. All resulting kinetic variables vary monotonically with voltage. AO is a function of the number of channels, their conductivity, and the electro-chemical potential. A simple physical explanation of the lag may be that it is a time needed to make or break a number of hydrogen bonds. This could explain the pronounced effect on the time course when replacing H20 by D20. Even though the appearance of the conducting form, C, is heterogeneous, its disappearance in response to a step repolarization can be simple exponential; for instance if C is not stabilized in the same way as A. REFERENCE

HODGKIN, A. L. & Huxixy, A. F. (1952). J. Phyaiol. 117, 500-544.

COMMUNICATIONS Effect of sodium removal on tension and membrane potential after inhibition of the sodium pump in dog Purkinje fibres BY E. CORABOEUF*, P. GAUTIERt and P. GUIRAUDOUt. t Centre de Recherches ClinMidy, 34082 Montpellier Cedex, and * Laboratoire de Physiologie compare, 91405 Orsay Cedex, France In cardiac tissues low-Na media increase Na efflux, inducing a fall in [Na]i (Ellis, 1977) while contracture develops as a consequence of the Na-Ca exchange. In nerve this exchange is suppressed by Mn at high concentrations (Baker, 1972) and acid media (Baker & McNaughton, 1977). We measured changes in mechanical tension and membrane potential during perfusion with low Na (13.7 mM) or Na-free media in dog Purkinje fibres in which the sodium pump had been previously inhibited by K-free media or ouabain 10-4 M. In K-free media replacement of Na with Tris, Li, choline or sucrose produced a contracture (C) and a hyperpolarization (AEm). Both C and AEm show a maximum after 1-2 min. Fig. 1. A, Potassium currents of squid under voltage clamp. Holding potential -60 mV. Temperature 6 'C. Depolarization is by a step change to the potential in mV shown at the , the best fit with a function which is right of each curve. 0O experimental points; the same as for the sodium currents but with k2 set at zero. Data courtesy of Dr Y. Pichon. B, Sodium currents from squid, 1'5 'C. Data courtesy of Dr J. Kimura. Holding potential - 100 mV with a step depolarization to the voltage shown near each curve. The equation used to fit the currents is

1(t) =

(27ro02)-i[k1/(k2-kl)] .A0.

{exp [-k (t-u- At)]

-exp -k2 (t- U-At)]}. exp [- U2/2o-2] du.

This is simply the equation for C in the sequential reaction A k, C with a Gaussian function having its centre shifted to a time At.

k) D convoluted

16P PROCEEDINGS OF THE When Tris was used, AEm reached 32 mV (Em increased from -40+3 mV to -72 + 10 mV, n = 39). Verapamil (5-20 mg/I.), Mn (4 mM), Cs (20 mM in the presence of ouabain 10-4 M), TEA (5-20 mM) and ouabain 10-6 M did not alter C, Em or AEm. Tetrodotoxin (1.2 x 10-5 M) increased Em by about 8 mV without altering O and AEm. 4-aminopyridine (5 mM) did not alter AEm. By contrast, in the presence of Mn at a high concentration (20 mm), Na-free solutions no longer induced C and markedly decreased AEm: only a small hyperpolarization (6-8 mV) remained which did not occur when Mn and TTX were both present. Ca-free solutions containing EGTA 10-4 M gave results similar to those obtained with Mn 20 mM. At low pH (after 45-60 min at pH 6-0, HEPES buffer) C and AEm were reduced by 75 % or more. In the presence of ouabain 1o-4 M ([K]o = 5-4 mm), Em generally reached a stable value of -40 mV. Under this condition Na-free media induced C and AEm similar to those previously described. The fact that Na-free media induce a contracture and a hyperpolarization which are insensitive to inhibitors of 9Ng, YCa-Na and qE and suppressed by Mn 20 mm and acid media, suggests that both phenomena are due to Na-Ca exchange. As hyperpolarization can hardly be related to changes in passive conductance, it may result from an electrogenic Na-Ca exchange as previously suggested in frog atrium (Horackova & Vassort, 1976). REFERENCES BAKER, P. F. (1972). Prog. Biophy8. molec. Biol. 24, 177-223. BAKER, P. F. & McNAUGHTON, P. A. (1977). J. Phy8iol. 269, 78-79P.

ErLIs, D. (1977). J. Phy8iol. 273, 2 11-239. HORACKOVA, M. & VASSORT, G. (1976). J. Phy8iol. 258, 77-78P.

Effect of pH on the uptake and efflux of calcium from cardiac sarcoplasmic reticulum vesicles BY JANE DUNNETT and WINIFRED G. NAYLER. Cardiothoracic Institute, London University, 2 Beaumont Street, London WiN 2DX It has repeatedly been shown that myocardial function is impaired when acidotic conditions prevail (see, for example, Williamson, Safer, Rich, Schaffer & Kobayashi, 1975; Cingolani, Maas, Zimmerman & Meijler, 1975). Because it seems probable that the contraction and relaxation of the myofilaments is controlled by variations in the intracellular availability of ionized calcium produced by the uptake and release of calcium by the sarcoplasmic reticulum, the following series of experiments were carried out to ascertain whether pH affects the affinity of the sarcoplasmic reticulum for calcium. Cardiac microsomal fractions rich in sarcoplasmic reticulum were prepared from guinea-pig ventricular muscle by homogenization and differential centrifugation. Net calcium uptake and release in the presence of 2-5 mM-K oxalate, and unidirectional calcium efflux rates were measured as described in a previous communication to this Society (Dunnett & Nayler, 1977) at pH 6-4, 6-8, 7-2 and at varying concentrations of free calcium in the incubation medium [Ca]0. It was found that at pH 6-4 the amount of calcium taken up by the microsomal

PH YSIOLOGICAL SOCIETY, MAY 1978 17P vesicles over the first 15 min was less than at higher pH, but the uptake of calcium continued for 50 min. No net release of calcium was observed. At pH 6*8 net release of calcium occurred after 30 min incubation at high [Ca].. At pH 7-2 the amount of calcium taken up over the first 15 min was greater than at pH 6*4 or 6-8, but uptake was followed by the early onset of a relatively rapid net release of calcium at all levels of [Ca]o studied. These results may explain earlier observations in which the pH optimum for calcium uptake was shown to shift towards acidity with increasing incubation time (Streter, 1969). Unidirectional calcium efflux rates were depressed at pH 6-4 relative to rates at pH 6-8 and 7 2. At pH 7 2 an increase in calcium efflux rate at low levels of [Ca]0 was observed. These results show that the ability of cardiac sarcoplasmic reticulum initially to take up calcium, and to release calcium, is impaired at low pH, and suggest that an effect of pH at the excitation-contraction coupling level may contribute to the negative inotropic effect of an acidosis on the myocardium. These investigations were supported by a programme grant from the Medical Research Council.

REFERENCES CI`NOLAWI, H. E., MAAS, A. J. H., ZIMMERMAN, A. N. E. & MEIJLER, F. L. (1975). Eur. J. Cardiol. 3/4, 329-336. DUNNETT, J. & NAYLER, W. G. (1977). J. Physiol. 266, 79-80P. STRETER, F. A. (1969). Archs Biochem. Biophy8. 134, 25-33. WILLIAMSON, J. R., SAFER, B., RICE, T., SCHAFFER, S. & KOBAYASHI, K. (1975). Acta med.

8cand. suppl. 587, 95-111.

Actin in squid retinal photoreceptors BY HELEN SAIBIL. The M.R.C. Cell Biophysics Unit, University of London, King's College, 26-29 Drury Lane, London, W.C. 2 The rhabdomes of the distal segments of squid retinula cells consist of 300 ,um long stacks of 60 nm microvilli, whose membranes contain the visual pigment rhodopsin. In order to investigate the major proteins present in these photoreceptors, isolated rhabdomes (Sperling & Hubbard, 1975) have been analysed by sodium dodecyl sulphate acrylamide gel electrophoresis. The strongest feature observed on the gels is the complex rhodopsin band (Hagins, 1973) containing four components. These have been separated by two-dimensional gel electrophoresis. At a slightly lower molecular weight (42,000), a less intense band is observed and identified as actin. Four other significant bands, somewhat less intense than the actin, were also observed, of molecular weights approximately 170,000, 140,000, 90,000 and 35,000. Actin was positively identified by the gel peptide mapping technique of Cleveland, Fischer, Kirschner & Laemmli (1977), in which the band was cut from a lightly stained gel, placed in the sample well of a second gel, incubated with papain, and its digestion products electrophoresed alongside those of known actin samples. This gave a digest pattern of seven peptide fragments identical to those derived from vertebrate muscle actin.

PROCEEDINGS OF THE 18P The exact cellular location and the function of this actin are as yet unknown. Transmission electron microscopy demonstrates microtubules concentrated around the microvillar borders of the photoreceptors, and a weak tubulin fraction can be separated from the diffuse rhodopsin band on gels by iso-electric focusing. However, no obvious concentrations of microfilaments have been seen by electron microscopy, though individual microvilli do contain an indistinct core filament. This study demonstrates that actin is a major protein of squid photoreceptor distal segments. It exists there in a form easily sedimented from dilute preparative media, and thus may be associated with the photoreceptor membranes. I am grateful to Drs D. Gilbert and J. Scholes for valuable help and discussion and to the staff of the Laboratory of the Marine Biological Association, Plymouth, for the supply of squid and use of their facilities. I am supported by National Research Council of Canada Research Fellowship.

REFERENCES CLEVELAND, D. W., FISCHER, S. G., KRsCHNER, M. W. & LAEMMLI, U. K. (1977). J. biol. Chem. 252, 1102-1106. HAGINS, F. (1973). J. biol. Chem. 248, 32-39. SPERLING, L. & HulBBARD, R. (1975). J. gen. Phy8iol. 65, 235-251.

Veratridine-induced secretion in medullary cells isolated from the bovine adrenal gland BY D. E. KNIGHT and M. J. WHITAKER. Department of Physiology, King's College, Strand, London WC2R 2LS The alkaloid veratridine depolarizes many excitable cells as a result of alteration of the kinetics of the sodium channel, and in the case of neurosecretory cells evokes the release of transmitter. Catecholamine release and 45Ca tracer uptake resulting from veratridine application have been studied in cell suspensions obtained by protease digestion of thin medullary slices. The suspensions are responsive to both high-K solutions and to carbamyl choline (550 /M). Unstimulated cells in Locke solution (Hepes-buffered pH 7-4 and saturated with 02) containing 3-6 mM-Ca have a resting secretion of 0-1 n-mole catecholamine per min. per 106 cells and a linear tracer calcium uptake equivalent to 0-01 n-mole Ca per min. per 106 cells. Maximal rates of catecholamine secretion and tracer uptake occur immediately after addition of the alkaloid (100 jug/ml.) and are 2 and 0-2 n-mole per min per 106 cells respectively. Equimolar substitution of choline for sodium in the incubation medium has little effect on resting rates but abolishes both catecholamine release and tracer uptake. Similar results are obtained with TTX (Ki = 25 nM). Substitution of Mg for Ca also prevents a secretary response, as does the inclusion of cobalt (5 mM) or methoxyverapamil, D 600 (Ki = 1 ,uM). Cells depolarized by suspension in Locke solution containing 70 mM-K and 66 mM-Na are insensitive to veratridine. Preliminary experiments with the carbocyanine dye DiS-C3(5), whose partitioning between cells and suspending medium has been shown to depend on membrane potential (Hoffman & Laris, 1974), suggest that little depolarization results from application of 170 /UM of the drug. A concentration of 860 SUM applied to the cells gives a very similar magnitude of both tracer uptake and secretary response and in addition produces

PHYYSIOLOGICAL SOCIETY, MAY 1978 19P a significant change in the partition of the dye, consistent with a depolarization. The changed partition is reduced in the presence of TTX but persists in the presence of D 600. These results suggest that the primary site of action of veratridine in the bovine adrenal medullary cell is the sodium channel. Interaction with this channel initiates secretion by promoting Ca entry via a route which closely resembles the 'late Ca channel' (Baker, Hodgkin & Ridgway, 1971). The small effect of the lower concentration of alkaloid on dye partition and the relatively slow inactivation of the secretory response perhaps imply that the extra entry of calcium results from an increased action potential frequency rather than gross depolarization, an explanation suggested by analogy with the action of acetylcholine on rat medullary cells (Brandt, Hagiwara, Kidokoro & Miyazaki (1976). We wish to thank Dr B. Salzburg for the gift of the dye DiS-C3(5) and the S.R.C. and M.R.C. for support. REFERENCES

BAKR , P. F., HODGKIN, A. L. & RIDGWAY, E. B. (1971). J. Phy8iol. 218, 709-755. BRANDT, B. L., HIAGIWARA, S., KIDOKORO, Y. & MIYAZAK, S. (1976). J. Phy8iol. 263, 417-439. HOFFMAN, J. F. & LARIs, P. C. (1974). J. Phy8iol. 239, 519-522.

Glucose transport in squid axons BY A. CARRUTHERS. Department of Physiology, King's College, Strand, London WC2R 2LS The squid axon should be very useful for studying sugar transport; but little work has been reported (Hoskin & Rosenberg, 1966). Glucose influx into isolated axons of Loligoforbesi immersed in sea water containing 20

@

-

4+

6

15 ~~~~ 10 ~~

~

~

~

~

~

~

4

C

05

0

10

20

30

40

50

External glucose concentration (mM)

Fig. 1. Dependence of a.D[U.14Cjglucose influx on external glucose concentration in choline (0O) and Na (@*) sea waters. Exposure to isotope was 20 miin and the number of experiments is shown by each point. The curve is a rectangular hyperbola with K,,, = 3e61 mM;VEa., = 11x16 pmol.cm-2.sec-1. Temperature = 21 °C.

PROCEEDINGS OF THE 20P 1 mM glucose, the concentration in squid blood, is roughly linear for 60 min and averages 1 84 + 0-14 pmol. cm-2. sec-. Fig. 1 shows that in choline sea water, Dglucose influx becomes saturated at higher external glucose concentrations. A second component of influx can be detected in media containing Na at glucose concentrations greater than 15 mm. In Na media D-glucose influx is unaffected by L-glucose (10 mm), methyl-fi-D-glucopyranoside (10 mM) and phloridzin (0.1 mM); but is reduced by methyl-a-D-glucopyranoside which is itself transported into the axon (apparent Km = 28-6 mM; Vmax. = 3-15 pmol. cm-2. sec-'). Once inside the axon methyl-cz-D-glucopyranoside seems not to be metabolized and has a longitudinal diffusion coefficient of 4 x 106 cm2. sec-'. Glucose uptake is reduced to 33 / (n = 8) in cyanide-poisoned axons. Efflux of the non-metabolized analogue methyl-a-D-glucopyranoside into Na-sea water averages 0-0179+0-0003pmol.cm-2.sec-1 at an internal concentration of 1 mm. The efflux into Na sea water is reversibly reduced by cyanide and dinitrophenol and is increased transiently by external glucose. This work was supported by an M.R.C. Research Studentship REFERENCE

Hosxnq, F. C. G. & ROSENBERG, P. (1966). J. gen. Phyeiol. 49, 47-56.

Effects of vibration on blood viscosity in vitro and in vivo BY J. E. RAMCHARAN, H. S. SHOJA and D. E. M. TAYLOR. Department of Applied Physiology and Surgical Sciences, Royal College of Surgeons of England, Lincoln's Inn Fields, London WC2A 3PN Acute exposure to vibration of 30-80 Hz produces vasodilatation (Ryan & Salter, 1976). Investigations were carried out to see whether vibration also produced changes in viscosity in vitro and in vivo. TABLE 1. Effect of in vitro and in vivo vibration on anomalous viscosity and yield stress of blood. (Mean ± s.E.) Initial Post-vibration A

A

slope (mN. m-2/

Low-shear Casson slope (mnN. m-2/

8-l)1

8-l)4

High-shear

Casson

In vitro

1-68 +0-11

In vivo Difference (In vivoIn vitro) Paired t test

2-22 +0-08

Yield stress mN.M-2

0-548 +0-013

High-shear Casson slope mN. m-2/ 8-l)*

1-69 +0-08

Low-shear Casson slope

(mi.N m-2/ 8-*

2-15 +0-14

Yield stress mN.m-2

0-451 +0-039

1-66

2-24

0-571

1-69

2-49

0-354

+0-09

+0-08

+0-014

+0-12

+0-17

+0-032

0-0223 ±0-0778

-0-0288 +0-0482

-0-0227

0-0008 +0-0790

-0-3416

-0-0967 +0-0215

N.S.

N.5.

+0-0128

N.5.

N.5.

±0-1150 0-02>P>0-01

P P> 0.001) (Chi-square contingency table analysis). A correlation has been reported between responsiveness of spinal neurones to substance P and peripherally applied noxious heat (Henry, 1976). Thermal injury has been demonstrated to cause the release of kinins (Rocha e Silva & Rosenthal, 1961) which may activate perivascular pain receptors (Juan & Lembeck, 1974). These present studies demonstrate a correlation between spinal neurones responsive to ionophoretic application of a substance P analogue and bradykinin I.A. This may suggest that substance P primary afferents are activated peripherally by kinins, possibly released during inflammation. They might therefore be involved with the central transmission of pain associated with such conditions. REFERENCES

HENRY, J. L. (1976). Brain Re8 114, 439-451. JuAN, H. & LEMBECK, F. (1974). Naunyn-Schmiedeberg'8 Arch. exp. path. Pharmak. 283, 151-164. OTSUKA, M., KoNmsmi, S. & TAxAHAsHi, T. (1972). Proc. Jap. Acad. 48, 342-346. RocHA E SILVA, M. & ROSENTHAL, S. R. (1961). J. Pharmac. exp. Ther. 132, 110-116.

Habituation of the auditory startle reflex in decerebrate rats BY J. E. Fox. Department of Physiology, The Medical School, Birmingham B15 2TJ Groves & Lynch (1972) proposed that short-term habituation of the auditory startle reflex is mediated by the brainstem reticular formation. If this hypothesis is correct, it should be possible to demonstrate habituation of the reflex in decerebrate animals. Davis & Gendelman (1977), however, reported that the reflex was not habituated in the acute decerebrate rat. The present experiments were carried out to investigate further this hypothesis. 0)c'

1 00'

vE\

C-

0-)

Eo (0 =

z,~0 0>5 50, )0)

Groups of 5 stimuli

Fig. 1. Mean size of response evoked by successive groups of five auditory stimuli. Stimulus rate

=

I per 20 see. Bar shows 8.E., n

=

5.

35P PH YSIOLOGICAL SOCIETY, MAY 1978 Rats were decerebrated at the level of the superior colliculus under Avertin anaesthesia. They were then left for 24 hr. Integrated e.m.g. recordings were made from posterior neck muscles and auditory stimuli (110 dB, 1 kHz, sine wave, 20 msec duration) were presented to the animals at rates between 1 per 5 sec and 1 per 100 sec. The initial presentation of the auditory stimulus gave a recordable e.m.g. response (mean latency = 13 msec; mean duration 70 msec). With stimulus repetition, the response was habituated (see Fig. 1). After an interval of a few mins, the response recovered and was again habituated when a further series of stimuli was presented. Higher rate stimulation caused more rapid habituation and noxious cutaneous electrical stimulation brought about dishabituation. It therefore appears that the neuronal apparatus necessary for short-term habituation of startle exists entirely within the brain stem.

REFERENCES DAvIs, M. & GENDELMAN, P. M. (1977). J. comp. phy8iol. Psychol. 91, 549-563. GROVES, P. M. & LYrNCH, G. S. (1972). Paychol. Rev. 79, 237-244.

The effects of temperature on the local anaesthetic action of primary alcohols BY J. C. METCALFE and C. D. RICHARDS. Department of Biochemistry, Tennis Court Road, Cambridge CB2 1QW and The National Institute for Medical Research, Mill Hill, London NW7 1AA Several theories postulate that anaesthesia is primarily caused by the disorganizing influence of anaesthetic molecules on the lipid moiety of nerve cell membranes (Ashcroft, Coster & Smith, 1977; Lee, 1976; Mullins, 1954; Trudell, 1977). All of these theories require that the effectiveness of an anaesthetic depends upon its concentration in the membrane lipid. The solubility of benzyl alcohol in both natural and artificial membranes increases with temperature (Colley, 1971) and that of butanol may be expected on theoretical grounds to show similar behaviour. Therefore, these theories predict that the ability of both alcohols to block nerve conduction should decrease with cooling. However, as cooling a nerve impairs normal impulse propagation, it is possible that cooling may make a nerve more susceptible to the action of alcohols and balance out the effect of the decrease in membrane concentration. Nevertheless, any compensatory changes should affect the potency of both alcohols equally. These predictions have been tested using in vitro preparations of the lateral olfactory tract (l.o.t.) of the guinea-pig which was supramaximally stimulated at 1 Hz. Its response was recorded monopolarly with an extracellular glass micropipette (see Richards, 1973). When the l.o.t. was maintained at 37 'C and the concentration of benzyl alcohol was raised from 5 to 15 mm the l.o.t. compound action potential became progressively attenuated. This effect was accompanied by a broadening of the action potential, a decrease in conduction velocity and an increase in threshold, and was indistinguishable from that caused by procaine. At lower temperatures the effects of benzyl alcohol were potentiated. Thus, in one preparation 10 mm benzyl alcohol reduced the

PROCEEDINGS OF THE 36P l.o.t. action potential by 40 % at 37 0, by 50 % at 30 00 and by 90 % at 21 00, whereas the potency of 50 mM butanol over the temperature range 37-20 00 decreased by 8 %. This suggests that the nerve had not become more sensitive to block by benzyl alcohol simply as a result of cooling. Our experimental results are not easily reconciled with those theories of anaesthesia which postulate that membrane lipids are the primary site of action of anaesthetics, but can be accommodated by assuming that anaesthetics produce their characteristic effects by direct interactions with functional membrane proteins (Richards, 1978). REFERENCES

ASHCROFT, R.G., COSTER, H. G. L. & SMITH, J. R. (1977). Nature, Lond. 269, 819-820. COLLEY, C. M. (1971). Ph.D. Thesis. University of Cambridge. LEE, A. G. (1976). Nature, Lond. 262, 545-548. MULLINS, L. J. (1954). Chem. Rev. 54, 289- 323. RICHARDS, C. D. (1973). J. Phy8iol. 233, 439-456. RiCUaMDS, C. D. (1978). Ch. 2 in MTP Rev. Biochem., ser. II ed. METCALFE, J. C., Baltimore, Md.: University Park Press. (In the press.) TRUDELL, J. R. (1977). Ane8the8iology 46, 5-10.

Gastric acid secretion studies in response to pentagastrin in duodenal reflux and bile diversion BY W. E. G. THoMAs*. Department of Applied Physiology and Surgical Sciences, Royal College of Surgeons of England, Lincoln's Inn Fields, London WC2A 3PN Reflux of duodenal contents is known to cause significant changes in the gastric mucosa, but its effect on acid secretion has been little studied. This trial was designed to assess and compare the effects of duodenal contents and bile alone on acid secretion in the dog. Adult beagles were used to achieve uniformity of weight, size and temperament, and randomized into four groups, each of five animals. (1) A control group. (2) Bile diversion by cholecysto-gastrostomy and ligation of the common bile duct. (3) Duodenal reflux by dividing the duodenum distal to the entry of the common bile duct and pancreatic duct and anastomosing this to the upper lesser curve, and restoring continuity with a gastro-jejunostomy. (4) Duodenal reflux, as before, with truncal vagotomy just below the diaphragm. Surgery was performed using Sagatal as anaesthetic, and each animal was equipped with a Gregory-type gastrostomy tube. One month after surgery dose-response curves to an intravenous infusion of pentagastrin were constructed, using doses of 0 5, 1-0, 2-0, 4 0 and 8-0 ,sg/kg. hr and acid output in m-moles/30 min, calculated by titration of 15 min aliquots against 0.1 m-NaOH, using screened methyl orange as an indicator. Two further tests on each dog were subsequently performed at submaximal doses of 1@0 ,ug/kg . hr and maximal doses of 8-0 jug/kg . hr. In all groups basal acid output was virtually nil. In the duodenal reflux group maximal acid output showed an increase of 62-7 % (P < 0.01) over the control group with an increase in the gradient of the curve. There was no shift of the curve to the *

Bernard Sunley Research Fellow.

PH YSIOLOGICAL SOCIETY, MAY 1978 37P left or the right and submaximal stimulation with l0 #ug/kg. hr pentagastrin showed no significant difference from that of the controls. Vagotomy completely abolished this hypersecretion caused by duodenal reflux and showed a marked decrease of acid secretion at both doses of pentagastrin, reducing the response to 8 jug/kg. hr by 60-8 % over the controls and 75*9 % over the reflux group (P < 0-01). Fasting serum gastrin levels showed no significant difference between all four groups, and their means all fell well within normal limits of 50 + 25 pg/ml. Therefore, the hypersecretion due to duodenal reflux does not appear to be simply due to a trophic effect of raised circulating serum gastrin caused by alkalinization of the antrum, nor does the response show the characteristics of supersensitivity. It therefore seems possible that the effect is due to depression of some inhibitory

mechanism. Integration of metabolic and physical control into a model of feeding behaviour in ruminants BY J. M. FORBES. Department of Animal Physiology and Nutrition, University of Leeds LS2 9JT Ruminants are thought to use levels of absorbed nutrients to control their meal size unless the physical capacity of the reticulo-rumen intervenes (Baile & Forbes, 1974). In this model it has been assumed that feeding is switched off when absorption of energy exceeds energy requirements for maintenance, growth, pregnancy and lactation plus an upper threshold (Anil & Forbes, 1977), and that feeding recommences when the rate of absorption falls below rate of utilization minus a lower threshold. A meal is also terminated if the physical capacity of the gut is reached; this capacity is reduced by increasing deposition of abdominal fat which occurs when rate of absorption exceeds rate of utilization for purposes other than fattening. Body fat is used to supply energy when absorption is insufficient. The rates of digestion and passage of the residues of each previous meal are calculated each minute and compared with the thresholds and with gut capacity. Simulation with a moderately thin sheep offered a feed of 65 % digestibility results in meals which are initiated by a fall in energy absorption below the lower threshold, but which are terminated either by a rise in absorption above the upper threshold or by reaching the physical limit. Thus it is possible that metabolic and physical control of intake occur in the same animal at different times of day, as well as with changes in physiological condition, as previously discussed (Forbes, 1977). Predicted meal patterns and total daily intakes are similar to those observed, and wide variations in the thresholds, a quantitatively weak part of the model, result in little change in predicted feeding behaviour or weight eaten. The following changes have been simulated with realistic changes in food intake (in many cases observations on feeding behaviour with which to compare the predictions are lacking): (1) fattening (by reducing gut contents); (2) lactation (by increasing energy requirements); (3) growth (by increasing energy requirements and gut capacity); (4) pregnancy (by increasing energy requirements and decreasing gut capacity); (5) grazing (by decreasing rate of eating and increasing energy requirements); (6) restricting food by time of access or weight offered; (7) grinding of food (by increasing

38P PROCEEDINGS OF THE rate of passage); (8) different feeds (by changing energy content and rates of passage and absorption). REFERENCES

ANiL, H. & FORBES, J. M. (1977). Proc. Nutr. Soc. 36, 68A. BAILE, C. A. & FORBES, J. M. (1974). Physiol. Rev. 54, 160-214. FORBES, J. M. (1977). Anim. Prod. 24, 91-101.

The kinetics of active, electrogenic hexose transport in the proximal and distal rat small intestine following overnight fasting BY E. S. DEBNAM. Department of Physiology, Royal Free Hospital School of Medicine, London WC1N 1BP Overnight fasting is a common procedure in the preparation of animals intended for intestinal absorption experiments. To investigate possible transport effects of this treatment, the kinetics of glucose and galactose absorption have been determined in vito using proximal and distal rat small intestinal segments before and after overnight fasting. The apparent Km for galactose absorption in both proximal and distal TABLE 1. 'Apparent Kin' and V... for active, electrogenic glucose and galactose absorption across proximal and distal rat small intestine of anaesthetised rats. The techniques used have been described previously (Debnam & Levin, 1975). Mean values + s.E.M., number in parentheses. Fed Overnight fasting A

A

Distal 124 + 12

Glucose Apparent Km (mM)

Proximal 19-6 + 10

Vmax. (/zmoles/10 cm/15 min)

67-8 + 4.8

19-8 + 0 9

(7)

(6)

Galactose Apparent Km (mm)

43.5+ 2.3

23X3+1X9

(7)

(7)

VM.. (#moles/10 cm/15 min)

(7)

(6)

Proximal 217 ± + 12

(7)

Distal 121 + 10

(6)

60-6 + 3.2

14*4 + 1.0*

34*0 + 2.9**

14-4 + 1-3*

(7)

(6)

(6)

(6)

99.4+ 8.7

26-3 + 3.4

86.9 + 9.3

29*1 + 5.3

(7)

(7)

(6)

(6)

*P 0 03 >P

>0*02

intestine was reduced by fasting (Table 1), but the apparent Kms for glucose absorption were unchanged. The maximum transport capacity (nVmax.) for glucose absorption in the distal segment was reduced by fasting while that for galactose was unaffected. The Vmax.s for glucose and galactose absorption in the proximal intestine were reduced by 10x6 and 12x6 % respectively after fasting. These complex, dietary-induced kinetic changes, which were independent of alterations in unstirred water layer thickness, mucosal wet or dry weights and villus heights, illustrate the danger of regarding as normal rats which have been fasted overnight, and also further support the concept of multiple transport mechanisms for hexoses across the rat small intestine. M.R.C. support is gratefully acknowledged.

PH YSIOLOGICAL SOCIETY, MAY 1978

39P

REFERENCES

DEBNAM, E. S. & LEvN, R. J. (1975). J. Phy8iol. 246, 181-196.

Electrolyte changes in human gastric juice during steady-state secretion BY M. HOBSLEY and P. F. WHITFIELD. The Department of Surgical Studies, The Middlesex Hospital and Medical School, London, W. 1 On the assumption that the primary secretion of the stomach is pure hydrochloric acid, Teorell (1947) advanced the concept of back-diffusion to account for the presence of sodium ion. His findings suggested that H+ was lost from the lumen twice as fast as Na+ was gained from the gastric mucosa. The findings of Davenport (Davenport, Warner & Code, 1964) suggest a ratio of 0-85:1 for Na/H exchange. During gastric secretary plateau consecutive timed samples can differ in volume because of the sequestration of fluid between folds of gastric mucosa (Hobsley & Silen, 1969). The sample aspirated after the release of sequestration (desequestrated sample) contains a portion of gastric juice which has been exposed to back-diffusion for double the length of time of the aspirated portion of the preceding sequestrated sample. In 215 separate gastric secretion studies on patients and volunteers, secretary plateaux were established with histamine acid phosphate intravenous infusion (0.13 molee kg-' hr-1, or 0-03 molee kg-1 hr-1). Forty-six sequestration/ desequestration sample pairs were identified by changes in recovery of a marker (phenolsulphonphthalein) (Hobsley & Silen, 1969), and the electrolyte changes between the first and second sample of each pair studied. The observed ratio of changes in [H+] and [Na+] were different (P < 0-00001) from the postulated ratios of Teorell and Davenport. Moreover, there was in general a decrease in [Na+], instead of the increase expected with back-diffusion. However, if the electrolyte changes were attributed to the alternative concept of varying amounts of reflux from the duodenum, the observed changes in electrolyte composition were found to be compatible with the known constitution of duodenal contents under these circumstances (Na+ 150 m-mole/l., HCO3- 70 m-mole/l., Cl- 93 m-mole/i.) (Tankel, Lester, Richman & Hollander, 1957). If back-diffusion cannot be detected in such favourable conditions, then perhaps it does not occur in the actively secreting stomach. In that case, the composition of pure gastric juice, as reported by Hobsley & Whitfield (1977), would also be the composition of primary secretion of the human stomach. REFERENCES

DAVENPORT, H. W., WARNER, H. A. & CODE, C. F. (1964). Gastroenterology 47, 142-152. HOBSLEY, M. & SILEN, W. (1969). Gut 10, 787-795. HOBSLEY, M. & WMITFIELD, P. F. (1977). J. Phy8iol. 271, 57-58P. TANKEL, M. I., LESTER, L. J., RICHMAN, A. & HOLLANDER, I. F. (1957). Gastroenterology 32, 642-650. TEORELL, T. (1947). Gastroenterology 9, 425-443.

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Hypoxic pulmonary vasoconstriction in the ferret BY GWENDA BARER, F. MOHAMMED, A. SUGGETT and C. TWELVES. Experimental Medicine, Academic Division of Medicine, University of Sheffield, New Medical School, Beech Hill Road, Sheffield S10 2RX The stimulus-response curve may provide a clue to the mechanism underlying hypoxic pulmonary vasoconstriction. It was approximately sigmoidal in cats and dogs (Barer, Howard & Shaw, 1970; Benumof & Wahrenbrock, 1975) but linear in coati mundi (Grant, Davies, Jones & Hughes, 1976). In the ferret (urethane anaesthetized) which gives a large response, it was curvilinear with maximum slope in the physiological range of oxygen tensions. Fig. 1 A shows the relationship, in six ferrets, between alveolar Po2 (PA,O,) and blood flow measured (using an electromagnetic flowmeter) in a circuit introduced between the left lower lobe pulmonary vein and the B

-

0 0

3: ~010

1(

co 0 0

1:

20

40

60

80

100

120

140

PA, 0 Fig. 1. Relationship between

20

--

40

60

80

100

120

140

torr

Pa0O. and, A, blood flow; B, Pp.; ten ferrets.

left atrium. The lobe was ventilated with decreasing 02 concentrations. Pulmonary artery pressure (Ppa) varied insignificantly. They resemble O2-dissociation curves but show dilatation at low PA, °s. Fig. lB shows the relationship between PAO. and PPa in ferrets in which the blood from the right atrium was pumped into the left lower lobe artery at constant flow. For both circuits the mean PA,02 at 50 % maximum response was 44 9 + 9-2 torr S.D.

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REFERENCES BARER, G., HOWARD, P. & SHAW, J. (1970). J. Physiol. 211, 139-155. BENUMOF, J. & WAHRENBROCK, E. (1975). J. apple. Physiol. 38, 846-850. GRANT, B., DAVIES, E., JONES, H. & HUGHES, J. (1976). J. apple. Physiol. 40, 216-228.

The half-life and cardiovascular effects of angiotensin II in rats on high, normal or low sodium diets BY D. P. BROOKS, B. J. CHAPMAN, C. F. CHEN and K. A. MUNDAY. Department of Physiology and Pharmacology, School of Biochemical and Physiological Sciences, University of Southampton, Southampton S09 3TU It has been postulated that the half-life of angiotensin II in the circulation may be altered by variations in sodium balance (Peach, 1977), but most evidence to date is indirect. For example, the studies of Leary & Leadingham (1970) indicate that the capacity of the kidney (but not liver) to inactivate angiotensin II is reduced markedly by sodium loading and enhanced by sodium depletion. We have studied this further, and have also tested the sensitivity of the cardiovascular system to angiotensin II in rats on different salt diets. Three groups of rats were maintained for at least 8 weeks on diets differing only in their NaCl content. Groups (1) and (2) received diets containing 0 or 1 % NaCl. Group (3) received a diet containing 3 % NaCl and in addition their drinking water was replaced by 0 9 % saline. Food intakes and body weight increments were similar in the three groups. The rats were anaesthetized with pentobarbitone, and the femoral vein and artery cannulated. The half-life of angk-oVnsin II was then determined using the method of Brooks, Chapman & Munday (1977), which evaluates the half-life from the decay of the pressor response elicited by injected angiotensin II. In other work, using this method, we have been able to show that the half-life of angiotensin II may be altered by various experimental procedures. The measured half-lives of angiotensin II in the high, normal and low NaCl dietary groups were 19-5 + 2-6 sec. 21-8 + 3-2 see and 18-9 + 3-2 see (six rats in each group); the small differences between groups were not statistically significant when evaluated by analysis of variance. This suggests that differences in sodium balance have no effect on the disappearance of angiotensin II from the circulation. Cardiac output was measured by impedance cardiography using methods described by Chapman, Chen & Munday (1977). The basal cardiac outputs in all three groups were similar: group (1) 194 + 15 ml. min-. kg body wt-1, group (2) 199 + 12, group (3) 187 + 22; but the ability of angiotensin II to cause decreases in cardiac output was diminished in the group on the high sodium diet. We are indebted to Professor T. G. Taylor who designed the diets employed in this study. REFERENCES B. J. & D. MUNDAY, K. A. (1977). J. Physiol. 265, 35-36P. BROOKS, P., CHAPMAN, CHAPMAN, B. J., CxEN, C. F. & MUNDAY, K. A. (1977). J. Physiol. 270, 3-5P. LEARY, W. P. P. & LEADINGHAM, J. G. G. (1970). Clin Sci. 38, 573-582. PEACH, M. (1977). Physiol. Rev. 57, 313-370.

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Modulation of slow-reacting substance of anaphylaxis and histamine release by prostacyclin and thromboxanes BY DINAZ M. ENGINEER, P. J. JOSE, PRISCILLA J. PIPER and J. R. TIPPINS. Department of Pharmacology, Royal College of Surgeons, London WC2A 3PN Treatment of sensitized guinea-pig lungs with indomethacin potentiates substance of anaphylaxis (SRS-A) and histamine release fivefold (Engineer, Niederhauser, Piper& Sirois, 1978), suggesting that some product(s) of arachidonic acid metabolism by cyclo-oxygenase (prostaglandins, thromboxanes and prostacyclin) depresses the anaphylactic release of SRS-A and histamine. We investigated the effects of indomethacin, inhibition of thromboxane synthesis by imidazole (Moncada, Bunting, Mullane, Thorogood & Vane, 1977) and of prostacyclin by 15-hydroperoxy arachidonic acid (15-HPAA) (Gryglewski, Bunting, Moncada, Flower & Vane, 1976) on mediator release from perfused lungs using the methods of Engineer et al. (1978). Mediator release from chopped lung was measured by incubating aliquots (500 mg) in Tyrode solution (3 ml.) with ovalbumin (50 jug/ml.) for 15 min and assaying the supernatant. The effects of thromboxane B2 and prostacyclin were investigated by administration with antigen. In perfused lungs indomethacin (1 jug/ml.) increased the peak concentration of SRS-A and duration of the enhanced release. Total release of histamine was increased but the duration of output unchanged; prostaglandin production was negligible. Imidazole (1 mg/ml.) increased the release of SRS-A, histamine and prostaglandins E2 and F2a and reduced histamine output from challenged lungs. Thromboxane B2 (1 jug/ml.) caused a reduction (50 %) in SRS-A release and a small decrease in histamine output from perfused lungs and 1-500ng/ml. caused a dose-related inhibition of SRS-A release (maximum 55 %) in chopped lung. 15-HPAA (1 ,sg/ml.) increased release of SRS-A, histamine and prostaglandins E2 and F2a from perfused lungs. Prostacyclin (1-300 ng/ml.) depressed the anaphylactic release of SRS-A in chopped lung. However, the largest increase in mediator release from perfused lungs was produced by a combination of 15-HPAA (1 ug/ml.) and imidazole (500 jug/ml.): increases were SRS-A eight, histamine seven and prostaglandins twelve times. These results show that both prostacyclin and thromboxane(s) depress the release of SRS-A and histamine from guinea-pig lung and suggest that prostaglandins E2 and F2x are relatively unimportant. We wish to thank the M.R.C., Asthma Research Council and Fisons Ltd for grants. REFERENCES

ENGINEER, D. M., NIEDERHAUSER, U., PIPER, P. J. & Smois, P. (1978). Br. J. Pharmac. 62, 61-66. GRYGLEWSKI, R., BUNTING, S., MONCADA, S., FLOWER, R. J. & VANE, J. R. (1976). Pro8taglandin8 12, 685-713. MONCADA, S., BUNTING, S., MULLANE, K., TEOROGOOD, P. & VANE, J. R. (1977). Pro8taglandins 13, 611-618.

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A vagal reflex contributes to the hypotensive effect of prostacyclin in anaesthetized dogs By D. J. CHAPPLE, G. J. DUSTING, R. HUGHES and J. R. VANE. Wellcome Research Laboratories, Beckenham, Kent. Prostacyclin (PGI2), biosynthesized from arachidonic acid by blood vessels (Moncada & Vane, 1977), causes vasodilatation in -rats, rabbits (Armstrong, Lattimer, Moncada & Vane, 1978) and dogs (Armstrong, Chapple, Dusting, Hughes, Moncada & Vane, 1977; G. J. Dusting, D. J. Chapple, R. Hughes, S. Moncada & J. R. Vane, submitted). In dogs, prostacyclin, unlike prostaglandin E2, often induced bradycardia despite hypotension (Armstrong et al., 1977; Dusting et al. 1978). We have now shown that this bradycardia is reflex in origin. Anaesthesia was induced with thiopentone (20-25 mg kg-', i.v.), maintained with chloralose (50 mg kg-', i.v.), with supplements (5 mg kg-' i.v.) as required. Dogs were mechanically ventilated, arterial Po, being maintained above 100 mmHg and PCO2 at 28-40 mmHg. An electromagnetic flow probe was placed on the ascending aorta and cardiovascular variables were recorded as described previously (Hughes, 1971). Prostacyclin infused (0-05-1P0 ,tg kg-' min-') or injected (0-025-10 ,ug kg-') intravenously or into the left atrium caused dose-dependent reductions in blood pressure and peripheral resistance but inconsistent changes in myocardial contractility, stroke volume and cardiac output. Whereas intra-atrially administered prostacyclin produced bradyeardia in four out of five dogs, intravenous administration produced tachycardia in three of these dogs. Bradycardia induced by prostacyclin given intraatrially or intravenously was abolished or reversed to tachycardia after vagotomy (six dogs). Furthermore, the effects of prostacyclin on blood pressure and peripheral resistance were significantly attenuated by vagotomy. In one dog with intact vagi, atropine (0 04 mg kg-') reversed the bradycardia induced by prostacyclin but did not alter the hypotensive response. Thus, bradyeardia after systemic prostacyclin is of reflex origin, the afferent (and probably efferent) arcs being carried in the vagus. Furthermore, the hypotension induced by prostacyclin has at least two components: direct relaxation of vascular smooth muscle and reflex vasodilatation. Colleridge, Colleridge, Ginzel, Baker, Banzett & Morrison (1976) found that prostaglandin F2. stimulates afferent C-fibres in the lungs, but clearly the reflex effects of prostacyclin arise in other parts of the circulation, perhaps the left atrium. We are grateful to Mark Hunt for expert technical assistance.

REFERENCES ARMSTRONG, J. M., CHAPPLE, D. J., DuSTING, G. J., HUGHES, R., MONCADA, S. & VANE, J. R. (1977). Br. J. Pharmac. 61, 136P. ARMSTRONG, J. M., LATTIMER, N., MONCADA, S. & VANE, J. R. (1978). Br. J. Pharmac. 62, 125130. COLLERIDGE, H. M., COLLERIDGE, J. C. G., GINZEL, K. H., BAKER, D. G., BANZETT, R. B. & MORRISON, M. A. (1976). Nature, Lond. 264, 451-453. HUGHES, R. (1971). Med. Biol. Engng. 9, 603-610.

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MONCADA, S. & VANE, J. R. (1977). In Biochemical Aspects of Prostaglandin8 and Thromboxanes, ed. KARAScO, N. & FlRED, J., pp. 155-177. New York, San Francisco, London: Academic Press.

A proposed mediator of increased vascular permeability in acute inflammation in the rabbit BY T. J. WILLIAMS. Department of Pharmacology, Royal College of Surgeons, London WC2A 3PN Intradermal (I.D.) injection of Bordetella pertussis kills organisms or zymosan in the unsensitized rabbit, resulting in plasma exudation with a peak at 60-80 min and a duration of 120-180 min (Williams & Peck, 1977). It has been proposed that the exudation is produced by the combined action of a vascular permeability-increasing mediator and a vasodilator mediator (Williams & Peck, 1977). The vasodilator mediator, probably an E-type prostaglandin (PGE) or PGJ2, markedly potentiates exudation. Little exudation is observed when the production of the vasodilator is specifically inhibited by the prostaglandin synthetase inhibitor, indomethacin. Until now, the source and nature of the permeability-increasing mediator has remained undetermined since its action is not antagonized by the antihistamine mepyramine, and its production is not suppressed by the inhibitors of kinin formation, soya bean trypsin inhibitor (SBTI) and Trasylol. This paper considers the possibility that the permeability mediator is derived from blood plasma. Plasma exudation was measured for 30 min following I.D. injection (0.1 ml. samples) into dorsal skin (6 replicate doses per rabbit), using the accumulation of intravenously-injected [1311]albumin. Zymosan (1 mg/ml.) was added to fresh rabbit plasma containing heparin (10 u./ml.) and incubated at 37 0C for 30 min. The zymosan was then removed by centrifugation. To reproduce conditions in the inflammatory reaction, and to facilitate exudation measurements, PGE1 (100 ng/0 1 ml.) was routinely added to samples before I.D. injection. Without PGE1, activated plasma samples (P*) produced little exudation, 5*7 + 3*1 1d. (n = 6 animals). Addition of PGE1 produced a large potentiated response, 72 8 + 7 9 p1. (n = 11 animals), whereas control plasma incubated without zymosan when PGE1 was added gave responses of 11.1 + 25 1ul. (n = 6 animals). PGE1 alone at the same dose produced little exudation, 3 6 + 0 9 Al. (n = 4 animals). Responses comparable with those produced by P*+PGE1 were produced by histamine (2.5 ,tg/0.1 ml.) +PGE, (100 ng/0 1 ml.). However, although addition of mepyramine (1 jug/0 1 ml.) reduced these responses by 87-0 + 3.30/ (n = 5), those to P*+PGE1 were reduced only by 20-8+3-1% (n = 11). This indicates the presence of a histamine-independent plasma activation permeability agent (PAPA), probably a complement component. PAPA was also produced when antigen-antibody complexes were used to activate plasma. The generation of PAPA was unaffected when SBTI (100 ,ug/ml.) or Trasylol (100 ,ug/ml.) were added to plasma before activation, but pre-incubation of plasma at 56 0C for 30 min abolished activity. It is suggested that generation of PAPA in extracellular fluid initially, and in

PHYSIOLOGICAL SOCIETY, MAY 1978 45P plasma exudate subsequently, could account for the increased permeability observed in acute (non-specific and Arthus-type) inflammation in the rabbit. REFERENCE WmrARMs, T. J. & PECK, M. J. (1977). Nature, Lond. 270, 530-532.

Actions of a proposed endogenous inhibitor of prostaglandin synthetase from plasma on cyclo-oxygenase activity in platelets and gastric mucosa BY B. J. R. WHrrTLE. Department of Prostaglandin R6search, Wellcome Research Laboratories, Beckenham, Kent BR3 3BS The occurrence of a potential endogenous inhibitor of prostaglandin synthetase in plasma fractions has recently been reported (Saeed, McDonald-Gibson, Cuthbert, Copas, Schneider, Gardiner, Butt & Collier, 1977). The present experiments investigate its actions in vitro in an intact cell system using platelets, and in vivo on the formation of prostacyclin by the rat gastric mucosa. Platelet aggregation induced by arachidonic acid (100-500 ,zg/ml.) in human citrated platelet-rich plasma (PRP) can readily be inhibited (Vargaftig & Zirinis, 1973) by known cyclo-oxygenase inhibitors such as indomethacin or aspirin (Vane, 1971) and thus can be a rapid and sensitive test for cyclo-oxygenase inhibition in an intact-cell system. Aliquots of PRP were incubated at 37 'C for up to 15 min with human Cohn fraction IV-4 (Miles Labs. Inc.; 1-10 mg/ml.), reported to be the most active plasma fraction. In experiments using PRP from four different donors, no inhibition of arachidonate-induced aggregation was observed, whereas indomethacin (1 jug/ml.) caused complete inhibition of aggregation. Furthermore, incubation of PRP from three other donors with Cohn IV-4 (1-10 mg/ml.) at room temperature, for 1 5, 3 and 6 hr did not lead to the inhibition of arachidonate-induced aggregation. Prostacyclin generation by the rat gastric mucosa in vitro can be markedly inhibited by administration of indomethacin (5 mg/kg s.c.) or aspirin (100 mg/kg s.c.) to rats 1-3 hr prior to removal and incubation of the mucosa (Moncada, Salmon, Vane & Whittle, 1978). Thus, cyclo-oxygenase inhibition in vivo prevents subsequent prostacyclin generation in vitro. In the present study, Cohn IV-4 (50 mg/kg) was administered by intravenous or intraperitoneal injection in a dose used in the original paper (Saeed et al. 1977). The prostacyclin-like activity, generated by strips of rat gastric mucosa incubated for 1 min at room temperature in 50 mM Tris buffer, was assayed in terms of authentic prostacyclin via inhibition of platelet aggregation (Moncada et al. 1978). In three replicate experiments, using three rats in each group, there was no significant inhibition (P> 0 05) of prostacyclin generation by gastric mucosa from rats pretreated for 3 hr with Cohn fraction IV-4 compared to the controls (94 + 8 ng/g tissue; n = 9). Thus, the inhibition of the cyclo-oxygenase by plasma fraction Cohn IV-4, demonstrated by Saeed et at. (1977) using bovine seminal vesicle homogenates, does not occur in vivo in the gastric mucosa or in vitro in an intact cell system using platelets. It may not be possible, therefore, to extrapolate the original findings with Cohn INV-4 fraction to all other systems or to use it as a means to study prostaglandin biosynthesis.

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MONCADA, S., SALMON, J. A., VANE, J. R. & WITrrLE, B. J. R. (1978). J. Phy8iol. 275, 4-AP. SAEED, S. A., MCDONALD-GIBsoN, W. J., CUT BERT, J., COPAS, J. L., SCHNEIDER, C., GARDINER, P. J., BUTT, N. M. & COLLIER, H. 0. J. (1977). Nature, Lond. 270, 32-36. VANE, J. R. (1971). Nature, New Biol. 231, 232-239. VARGAFTIG, B. B. & Zmns, P. (1973). Nature, New Biol. 244, 114-116.

Renal excretion of solutes and water in conscious rats after inhibition of prostaglandin synthetase by indomethacin BY J. HAYLOR and C. J. LOTE. Department of Physiology, Medical School, Birmingham B15 2TJ The role of endogenous prostaglandins in determining renal solute and water excretion is uncertain because, following prostaglandin synthetase inhibition, solute and water excretion may be increased (Kirschenbaum & Stein, 1976), decreased (Leyssac, Christensen, Hill & Skinner, 1975) or unaffected (Berl, Raz, Wald, Horrowitz & Czaczkes, 1977). TArLE 1. Effect of indomethacin on renal excretion in conscious rats. Values are Mean + S.E.M. (control series, n = 9; experimental series, n = 10). P values refer to paired t-test within series, and unpaired t-test between series. Indomethacin to experimental series Time from start of saline infusion (hr) 6-8 4-6 Urine flow (/zl./min) Control 85-7+4-5 N.S. 97-7±5-3 Experimental 92-1+4-7 59-5+4-2 P < 0-001 P < 0-001 N.S. Osmolal output Control 42-0 + 2-6 43-0 + 1-8 N.s. Experimental 29-0+ 2-0 44-2+1-6 P < 0-001 (/Z-osmole/min) P < 0-001 N.S. Sodium output + Control 13-0 0-8 13-7 + 0-7 N.S. (Im/min) Experimental 8-9 + 0-7 14-4+0-7 P < 0-001 N.S. P < 0-001

We have determined urine flow and composition from conscious rats undergoing minimal (< 1-5 % body wt.) volume expansion before and after administration of indomethacin. Sodium chloride (0.15 M; 5-2 ml./hr) was infused into a tail vein for 8 hr. Six hours after starting the infusion, either indomethacin (1 mg/t00 g body wt.) in buffered saline (0.05 ml./100 g body wt.) or buffered saline alone (control) was administered via the tail vein. The results (Table 1) indicate that in conscious rats, prostaglandin synthetase inhibition decreases urine flow, osmolal output and sodium output. These findings are unlike those obtained in conscious dogs, where prostaglandin synthetase inhibition produced a natriuresis (Kirschenbaum & Stein, 1976). However, our results are similar to those seen when prostaglandin synthetase inhibitors are administered to conscious human subjects (e.g. Haylor, 1977). We thank the National Kidney Research Fund for financial support.

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REFERENCES

BERL, T., RAz, A., WALD, H., HORROWITZ, J. & CZACZEEs, W. (1977). Am. J. Phy8iol. 232, F529-537. HAYLOR, J. (1977). Ph.D. Thesis, University of Birmingham. KIRSCEHNBAUM, M. A. & STEIN, J. H. (1976). J. clin. Inve8t. 57, 517-521. LEYSSAC, P. P., CHRISTENSEN, P., HILu, R. & SKINNER, S. L. (1975). Acta phy8iol. 8cand. 94, 484-496.