THE RELEASE OF ADRENALINE AND NORADRENALINE FROM THE ADRENAL GLAND OF THE CAT BY ACETYLCHOLINE

Transcription

1 Brit. J. Pharmacol. (1957), 12, 422. THE RELEASE OF ADRENALNE AND NORADRENALNE FROM THE ADRENAL GLAND OF THE CAT BY ACETYLCHOLNE BY K. R. BUTTERWORTH* AND MONCA MANN From the Department of Pharmacology, School of Pharmacy, Bloomsbury Square, London (RECEVED APRL 1, 1957) The adrenal glands of atropinized cats were depleted of their content of adrenaline and noradrenaline by repeated intravenous doses of acetylcholine. n each experiment there was a similar percentage loss of both amines. This was irrespective of the degree of depletion, which ranged from 7.1% to 86.5%.. n some experiments, adrenal blood was collected and it was found that the percentage of noradrenaline in the adrenal venous effluent (mean 62.%) was similar to that in the amine lost from the depleted gland (mean 63.7%). n some experiments, the adrenaline and noradrenaline lost from the gland could be recovered quantitatively in the adrenal blood. The results indicate that, under these experimental conditions, synthesis is not taking place concurrently with the liberation of amines or that it is taking place so slowly as not to be detected. One of the first workers to study the liberation of catechol amines from the adrenal gland was Elliott (1912). He studied the effects of both chemical stimuli (for example, morphine, tetrahydro-fl-naphthylamine, gaseous anaesthetics) and physical stimuli (such as splanchnic nerve stimulation, pressure on the glands) on the loss of adrenaline from the adrenal glands of cats. He found that the chemical means was considerably more effective in causing a depletion than the physical means. Elliott and other early workers in this field only assayed the adrenal glands for adrenaline, and many years elapsed before it was shown that the adrenal medulla of most species contain two catechol amines, adrenaline and noradrenaline. Euler (195) was the first to suggest that these two hormones can be liberated independently, and support of this hypothesis has come from many workers. Burn, Hutcheon, and Parker (195) and Hokfelt (1951) have shown that there is a greater loss of adrenaline than of noradrenaline from the adrenal gland of the rat, following the administration of insulin. Outschoorn (1952), using the same species, found that insulin, morphine, and tetrahydro-,8-naphthylamine caused a significant loss of adrenaline but not of noradrenaline. n the cat he found that stimulation of the glands by acetylcholine, *Present address: Department of Pharmacology, St. Mary's Hospital Medical School. London, W.2. potassium chloride, or electrical stimulation of the splanchnic nerve caused an increase in the adrenaline and noradrenaline content of the adrenal venous effluent. n the acetylcholine experiments the adrenal glands were not assayed, but with splanchnic stimulation there was a similar % depletion of both amines. Dundr (1954) found a marked increase in the secretion of adrenaline into the venous effluent from the adrenal gland of the cat in response to insulin hypoglycaemia, while there was only a slight increase in the noradrenaline output. Workers such as Briicke, Kaindl, and Mayer (1952), Euler and Folkow (1953), and Redgate and Gellhorn (1953) have studied the effect of electrical stimulation of the hypothalamus on the release of catechol amines from the adrenal gland. They found in the cat that the proportion of the two amines in the adrenal venous effluent varied considerably according to the area that was stimulated. Thus it was concluded that some of the adrenal medullary cells produce adrenaline and others noradrenaline, and that these are innervated by separate fibres with a different hypothalamic representation. Since adrenaline and noradrenaline can be liberated from the adrenal gland independently, it was thought of interest to study the effect of the natural mediator of splanchnic nerve transmission, namely acetylcholine, on the liberation

2 RELEASE OF AMNES FROM THE ADRENAL GLAND 423 of the adrenal medullary amines in the cat. The degree of depletion of the glands, the relative amounts of adrenaline and noradrenaline lost from the depleted gland, the proportion of adrenaline to noradrenaline in. the adrenal venous effluent, and the correlation between the amines lost from the gland and those found in the plasma have been studied. METHOD Healthy, adult cats of both sexes were used. Series 1. Depletion Experiments.-The animal was given 6 mg. of atropine sulphate/kg. of body weight intraperitoneally and anaesthetized 1 min. later, in an anaesthetic chamber, with ether followed by 6 mg./kg. of chloralose intravenously. One adrenal gland was removed and an extract prepared (see below). The other gland was depleted of its catechol amines by repeated intravenous doses of acetylcholine. The dose and the number of doses varied in each experiment so that differing degrees of depletion could be obtained. The dose range was.2 to 3. mg./kg. of body weight. The approximate amount of amine liberated was determined by comparing the acetylcholine-induced pressor responses with the rises in blood pressure produced by doses of adrenaline and of noradrenaline. Following the last dose of acetylcholine the second gland was removed and an extract prepared as for the first gland. Series 2. Depletion Experiments with Collection of Adrenal Venous Effluent.-The animal was prepared as in Series 1 and the right adrenal gland was removed as a control. The left lumbar vein was then cannulated towards the adrenal gland, after tying off the surrounding blood vessels, care being taken to leave the arterial supply to the gland intact. Heparin (1, units/kg.) was given intravenously. When the adrenal vein was temporarily occluded, blood flowed into the cannulated lumbar vein. mmediately after each dose of acetylcholine had been given, blood was collected into ice-cooled centrifuge tubes. Collection was for a little longer than the duration of the rise in blood pressure, as determined by giving acetylcholine without occlusion of tle adrenal vein. Half of each blood sample was retained (see below). The other half of each sample was returned to the cat to ensure that the acetylcholine was exerting a pressor effect and also to minimize the loss of blood from the animal. n addition, suitable control blood samples were collected. n each experiment about 3 doses of acetylcholine were given. The dose range was similar to that of Series 1, although here the main factor was that the dose should be sufficient to cause a high concentration of amines in the adrenal venous effluent. Following depletion, the right adrenal gland was removed. Preparation of the Extracts of the Adrenal Glands. -mmediately the adrenal gland was removed, it was freed of connective tissue, weighed on a microtorsion balance and a 5 mg./ml. extract prepared by grinding the gland in.1 N hydrochloric acid with 3 mg. of acid-washed sand/1 mg. of gland. After centrifugation at 5, rev./min. for 2 min., the supernatant fluid was withdrawn and stored in an airtight container at 4'. Preparation of the Plasma Samples.-Each half sample of blood was centrifuged at 5, rev./min. for 3 min. immediately after collection. The plasma was withdrawn into a graduated measuring cylinder containing 2 to 3 drops of concentrated hydrochloric acid. The remaining cells were re-centrifuged and any further plasma obtained was added to the first sample. All the samples from one experiment were pooled, measured, and then stored at 4. Assay of the Adrenal Gland Extracts and the Plasma Samples.-The adrenaline and noradrenaline contents of the adrenal gland extracts and the plasma samples were determined by differential biological assays. The assay was first performed on the acutely denervated nictitating membrane of the cat. The animal was then given hexamethonium bromide and the samples were assayed on the blood pressure. At least two assays of each extract or plasma sample were performed on each preparation. The adrenaline and noradrenaline contents were calculated, as the laevo isomers of the bases, by the formula of Bulbring (1949). The amine contents of the adrenal glands are expressed in jug./gland in view of the findings of Butterworth and Mann (1957a). n calculating the adrenaline and noradrenaline content of the adrenal venous effluent it was necessary to determine the distribution of amines between the cell and the plasma fractions. n each experiment a correction factor was applied for the small amount (approximately 1%) of adrenaline and noradrenaline which was present in the cell fraction. n addition to the biological assays, a paper chromatographic analysis was made by the method of Shepherd and West (1951), which employs n-butanol-acetic acid-water as the solvent and potassium iodate to locate the catechol amines. RESULTS Series 1. Depletion Experiments.-A marked rise in blood pressure was obtained in response to each dose of acetylcholine. nitially repeated doses caused rises in blood pressure of a similar magnitude (Fig. 1), but gradually, as the gland became depleted, the responses became smaller. f the dose of acetylcholine was then increased, the response returned to its initial magnitude. rl some experiments the doses of acetylcholine were increased both in size and in number until no further pressor response was elicited (Fig. 2). Comparing the amine contents of the depleted and the control glands, it was found that there was a similar % depletion of adrenaline and noradrenaline in every experiment (Fig. 3). This similar degree of depletion of both amines was independent of the degree of depletion of the

3 424 mm. Hg 2a 1 K. R. BUTTERWORTH and MONCA MANN Fo..-Cat (3.4 kg.), 6 mg./kg. of atropine sulphate intraperitoneally followed by ether and 6 mg./kg. of chloralose intravenously. The right adrenal gland was removed. The blood pressure tracing shows thesimilarity of successive responses to 2 mg. doses of acetylcholine given intravenously. Time, 3 sec. mm. Hg Doses of acetylcholine FG.2.-Cat(3.9kg.),6mg./kg. of atropine sulphate intraperitoneally followed by ether and 6 mg./kg. of chloralose intravenously. The left adrenal gland was removed. The blood pressure tracing shows the ultimate diminution in the response to repeatedintravenous doses of acetylcholine. Time, 3 sec 'a Q C FG. 3-Results of Series 1, showing the similarity in the percentage loss ofadrenaline (U) and of noradrenaline (U) when the adrenal gland of the atropinized cat is depleted by repeated doses of acetylcholine. glands (7.1% to 86.5%) and of the actual % of noradrenaline present in the glands (33.4% to 63.6%). The total absolute loss of amines ranged from 11.6 rug. to 1,245,ug. From these 18 results the mean % depletion of adrenaline was 53.3 and of noradrenaline was 55.5, there being no significant difference (.8>P>.7) between the adrenaline depletion values and those of the noradrenaline. n three cats a constant infusion of acetylcholine was given in an attempt to produce a continuous liberation of amine from the adrenal medulla and hence a greater depletion of the glands in a shorter period of time. At first a maintained rise in blood pressure was produced, but a marked tachyphylaxis soon developed and the blood pressure returned to normal while the acetylcholine infusion was still being given. To determine whether the vasopressor effect was due solely to the amines liberated from the adrenal medulla, further doses of acetylcholine were given in some cats after removing the second gland. No rise in blood pressure was produced with the dose of acetylcholine which caused a marked vasopressor effect before the bilateral adrenalectomy, but in a few cases, on increasing the dose 1 to 2 times, a very small rise in blood pressure was produced. Series 2. Depletion Experiments with Collection of Adrenal Venous Effluent.-As in Series 1, there was always a similar % depletion of adrenaline (mean 57.5%) and of noradrenaline (mean 56.3%) in each cat (Fig. 4). Again this similar % depletion of the two amines in each experiment was irrespective of the degree of depletion of the glands (35.3% to 78.5%) and of the actual

4 RELEASE OF AMNES FROM THE ADRENAL GLAND 425 c._ Fo. 4.-Results of Series 2, showing the similarity in the percentage loss of adrenaline (U) and of noradrenaline (U) when the adrenal gland of the atropinized cat is depleted by repeated doses of acetylcholine. % of noradrenaline present in the glands (34.5% to 9.7%). There was no significant difference (.9>P>.8) between the depletion of adrenaline and of noradrenaline. n every experiment there was a similar % of noradrenaline in the control adrenal gland, the depleted gland and the plasma obtained from the adrenal venous effluent (Table ) and therefore between the % of noradrenaline TABLE SERES 2. DEPLETON OF CAT ADRENAL GLANDS BY ACETYLCHOLNE, WTH COLLECTON OF ADRENAL VENOUS EFFLUENT Y. of Total Total % of Noradrenaline Total Amines AJUineCs._ CtAmines Lost in Ls G from Dfrpolte AdErentl Control Depleted Adrenal from Depleted Gland Effluent Gland Gland Effluent Depleted Gland Lu(g.) (pg.) Gland S * * * * S * * Mean= *7 in the adrenal venous effluent and in the loss from the gland (Fig. 5). The total amount of amines in the plasma samples was equal to or less than the total amount of amines lost from the depleted gland (Table ). n three experiments (Table, cat Nos. 23, 26, and 27), the total amine content of the adrenal venous effluent was only approximately 5% of the total amine lost from the gland. This poor recovery is ascribed to the fact that these were the first three experiments in which adrenal blood was collected and there was a "leak" into the general circulation, due to incomplete ligation of the small blood vessels surrounding the gland. This " leak " was detected 1 8 O6 z Fio. 5.-Results of Series 2, showing the relationship between the percentage of noradrenaline in the adrenal venous effluent (U) and in the loss from the depleted adrenal gland ((D). by the rise in blood pressure that occurred when each dose of acetylcholine was given. n those experiments where there was no such " leak " the recovery of amines was satisfactory. n no case did the control plasma samples possess any sympathomimetic activity. By means of the chromatographic analysis the presence of adrenaline and noradrenaline in all the adrenal glands was confirmed. No other catechol amines were detected. DscussoN Acetylcholine always caused a similar % loss of adrenaline and noradrenaline, irrespective of whether the degree of depletion was small or large. This absence of preferential loss of either amine may be of some significance in connexion with the normal physiological functioning of the gland. A maximum depletion to 13.5% of the initial amount was obtained. However, it might be possible to deplete the glands to an even greater extent if the doses of acetylcholine were continued when there was no longer a release of amine sufficient to produce a vasopressor effect. As might be expected, the rises in blood pressure produced by the acetylcholine became smaller as the doses were repeated. This was due to the liberation of less amine by each dose of acetylcholine and not to a reduction in the sensitivity to adrenaline or noradrenaline. After the response to the same dose of acetylcholine had gradually decreased it could be increased temporarily by giving a larger dose of acetylcholine. However, when the gland was nearly exhausted this could not be done. There was no precise relationship between the dose of acetylcholine, the number of doses given, and the degree of depletion obtained. Obviously there cannot be, since

5 426 K. R. BUTTERWORTH and MONCA MANN the amount of amine liberated by each dose varies from animal to animal and also as the experiment proceeds. n three experiments the acetylcholine was injected into the central stump of the left brachial artery. t was hoped that by this route a smaller dose of acetylcholine would release as much amine as a larger dose given intravenously. A further possible advantage was that it might have avoided any fall in blood pressure, which occurred in a few experiments, due to incomplete atropinization of the animal However, the intraarterial route was no better than the intravenous route and it also had the disadvantage of causing a pronounced contracture of the muscles of the chest and left fore-limb. After double adrenalectomy, the dose of acetylcholine had to be increased many times to produce even a small rise in blood pressure. This indicated that the doses that were used to cause depletion of the glands were exerting their effect mainly on the adrenal medulla. The small rise occasionally seen after bilateral adrenalectomy, when a large dose of acetylcholine was given, was probably due to an effect on the sympathetic chain or. on accessory chromaffin tissue. That there was very little or no loss of amine from the gland during its removal was shown by the absence of any rise in blood pressure during this time. n Series 1 either the right or the left gland was used as a control, but in Series 2 the right gland was always the control, due to the difficulty of collecting blood from this gland. Obviously it would have been preferable to have taken control glands from either side in the latter series of experiments. However, Butterworth and Mann (1957a) have shown that the left and right glands of each cat contain the same total amount of amine and the same proportion of adrenaline and noradrenaline, irrespective of any difference in the weights of the two glands. Hence, any bias in these experiments was not considered to be a disadvantage. From the recorded rises in blood pressure in Series 1 it was obvious that much amine was liberated during the course of each experiment. Although assay of the glands showed a depletion of catechol amines it was possible that some synthesis was taking place while the amines were being liberated. This was investigated in Series 2, where the amounts of amines released in the blood stream were compared with the loss from the gland. n these results the total amount of catechol amines was equal to or less than that lost from the gland. Therefore it was inferred that no synthesis of adrenaline or noradrenaline was taking place while there was a liberation of amines. An alternative interpretation of the results of this series is that synthesis was occurring concurrently with the depletion and that this was masked by only a % of the amines being recovered in the adrenal venous effluent. This opinion is not held by the authors, since when there was a "leak" of amines into the general circulation, as in cat Nos. 23, 26, and 27, the amine content of the adrenal blood was appreciably less than the loss from the gland. Also, if such a synthesis was occurring under the conditions of these experiments, it must have taken place at such a rate as to replace similar % of adrenaline and noradrenaline. This is unlikely in view of the subsequent observations of Butterworth and Mann (1957b). Nevertheless, it may well be that under certain conditions-such as stimulation of the splanchnic nerve, as shown by Holland and Schumann (1956)-the rate of formation is considerably increased. These workers suggest that "the chemical processes in the adrenal gland which underlie the formation of noradrenaline can be greatly accelerated by the stimulation of the splanchnic nerves, and that statements concerning rates of formation in conditions in which there is no stimulation may be inapplicable during splanchnic activity." The authors wish to express their sincere thanks to Professor G. A. H. Buttle and Dr. G. B. West for their constant advice and criticism; to Professor J. H. Bum for his helpful discussion of the manuscript; and to Mr. J. W. Conway and Mr. J. D. Caisey for technical assistance. REFERENCES Brucke, F., Kaindl, F., and Mayer, H. (1952). Arch. int. Pharmacodyn., 88, 47. Bilbring, E. (1949). Brit. J. Pharmacol., 4, 234. Bum, J. H., Hutcheon, D. E., and Parker, R. H.. (195). bid., 5, 417. Butterworth, K. R., and Mann, M. (1957a). J. Physiol., 136, (1957b). Brit. J. Pharmacol., 12, 415. Dun6r, H. (1954). Acta physiol. scand., 32, 63. Elliott, T. R. (1912). J. 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