J. Phyeiol. (1975), 252, pp. 363-378 With 4 text-ft gures Printed in Great Britain
363
CALCIUM AND STIMULUS-SECRETION COUPLING IN THE ADRENAL MEDULLA: CONTRASTING STIMULATING EFFECTS OF THE IONOPHORES X-537A AND A23187 ON CATECHOLAMINE OUTPUT
BY D. E. COCHRANE,* W. W. DOUGLAS, T. MOURI AND Y. NAKAZATO From the Department of Pharmacology, Yale University of Medicine, New Haven, Connecticut 06510, U.S.A.
(Received 3 February 1975) SUMMARY
1. The ionophores X-537A and A23187, which are known to transfer calcium across cell membranes, stimulated catecholamine release from perfused cat adrenal glands. 2. These stimulant effects persisted in the presence of hexamethonium and atropine and are therefore attributable to direct actions of the ionophores on the adrenal chromaffin cells. 3. Perfusion with calcium-free Locke abolished responses to A23187 and reduced those to X-537A. 4. Responses to X-537A were consistently large and comparable with those produced by 56 mm potassium. By contrast, responses to A23177, over the wide range of concentrations tested, were variable and much smaller. 5. That the two ionophores can stimulate through calcium-dependent mechanisms is considered fresh support for the calcium hypothesis of stimulus-secretion coupling. That they differ in effectiveness may mean that factors besides calcium are important. The greater potency of the less specific ionophore, X-537A, may be attributable to its ability to depolarize as well as carry calcium, while the relatively small effects of A23187, a generally more effective ionophore for calcium, may indicate that inward movement of calcium without a background of membrane perturbation such as may be produced by depolarization, is insufficient to elicit strong secretary responses. *
NIH Postdoctoral
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364
D. E. COCHRANE AND OTHERS INTRODUCTION
Experiments on many different secretary cells have revealed a common requirement for calcium in stimulus-secretion coupling and it has been suggested that a rise in the concentration of free calcium ions at some critical site within the cell (possibly at the inner surface of the plasma membrane) in response to the physiological stimulus, may in each instance set in motion a common process, exocytosis (Douglas, 1968, 1974a; Rubin, 1971, 1974). Two recent sets of observations are in harmony with this scheme. First, the intracellular injection of calcium by microiontophoresis has been found to release neural transmitter at the squid giant synapse (Miledi, 1973) and also to cause the expulsion of secretary granules from mesenteric mast cells (Kanno, Cochrane & Douglas, 1973). Secondly, the ionophores A23187 and X-537A, which can act as mobile carriers to transfer calcium across cell membranes (Reed & Lardy, 1972 a, b; Pressman, 1972, 1973), have been shown to elicit secretion in various cells where exocytosis has either been demonstrated, or is suspected, to operate. Thus, A23187 releases histamine from mast cells (Foreman, Mongar & Gomperts, 1973) and this effect is accompanied by light microscopic (Cochrane & Douglas, 1974) and electron microscopic (Kagayama & Douglas, 1974) evidence of exocytosis. The same ionophore has also been found to elicit release of amylase from pancreatic slices (Eimerl, Savion, Heichal & Selinger, 1974; Williams & Lee, 1974) and perfused intact pancreas (W. W. Douglas, T. Mouri & Y. Nakazato, unpublished observation). It will be recalled that secretion of pancreatic enzymes is the archetypical example of exocytosis (Palade, 1958). However, in this laboratory, A23187 has been found to be a relatively feeble stimulus for the secretion of vasopressin from isolated neurohypophyses (Nakazato & Douglas, 1974), yet here too secretion normally involves exocytosis (Douglas, Nagasawa & Schulz, 1971; Douglas, 1973, 1974b). Differences in the behaviour of various tissues are also evident in response to the ionophore X-537A which, in contrast to A23187, strongly stimulates vasopressin secretion from neurohypophysial terminals (Nakazato & Douglas, 1974) and has a corresponding effect on release of acetylcholine from motor nerve endings (Kita & van der Kloot, 1974). Yet, X-537A, unlike A23187, has little or no stimulant effect on amylase output from the perfused pancreas when indirect effects attributable to acetylcholine release are blocked by atropine (W. W. Douglas, T. Mouri & Y. Nakazato,
unpublished observation). It seemed to us possible that this diversity in the responses to the two ionophores might provide a fresh clue to the still uncertain events involved in stimulus-secretion coupling. Clearly, evidence from a wider
IONOPHORES AND CHROMAFFIN CELL SECRETION 365 spectrum of secretary cells was required. This led us to study the effects of A23187 and X-537A on the adrenal medulla. METHODS
Adult cats were anaesthetized by i.P. injection of sodium pentobarbitone (35 mg/kg) and their adrenals, acutely denervated, were perfused in situ or in vitro with Locke of the following composition (mm): NaCl, 154; KCl, 5-6; CaCl2, 2-2; Na2HPO4-NaH2PO4 buffer (pH 7 0), 3; glucose, 10. The solution was equilibrated with pure oxygen and perfusion was carried out at room temperature (about 220 C). The perfusion methods and techniques for fluorometric assay of catecholamines were as described by Douglas & Rubin (1961, 1963) except that perfusion was by peristaltic pump adjusted to yield an adrenal effluent of 1 ml./min. Perfusion of the adrenal glands in situ was by a cannula inserted into the lower aorta below the renal artery. The aorta was tied immediately above the coeliac axis and all other vessels were tied except those directly supplying the adrenals. The perfusate was collected through a cannula inserted into the vena cava at the same level as the arterial cannula. For perfusion of the isolated adrenal glands, a fine cannula, connected to the perfusion system, was inserted into the adrenolumbar vein lateral to the gland and pointing toward it. The adrenolumbar vein was then tied close to the vena cava and perfusion begun. The adrenal gland was then cut free from the animal as rapidly as possible and transferred to a filter funnel for collection of the perfusate. A thorough description of the procedures used to perfuse the isolated glands and the glands in situ is given in Douglas & Rubin (1961). In Ca-free Locke, CaCl2 was omitted and, where indicated, EGTA (Ethylenebis (oxyethylene-nitrilo) tetra-acetic acid), 1 or 5 mm, was present. High potassium Locke contained 56 mMKCl with NaCl reduced to maintain isotonicity (Douglas & Rubin. 1963). Except where indicated, the perfusion fluids contained hexamethonium chloride (C6), 5 x 10-4 g/ml., and atropine sulphate, 1O-- g/ml., to block possible indirect effects that might arise from the release of acetylcholine from splanchnic nerve endings and consequent activation of either nicotinic or muscarinic receptors on the chromaffin cells (Douglas & Poisner, 1965; Douglas, Kanno, & Sampson, 1967a). Perfusion fluids containing the ionophores X-537A or A23187 were prepared using stock solutions of the drugs in dimethylsulphoxide (DMSO). The final concentration of the solvent never exceeded 0-5 % which was without effect on catecholamine output (Fig. 3a). Samples of adrenal effluent were generally collected for successive 5 min periods. In a few experiments with A23187, successive samples were collected at 1 min intervals to determine whether any brief stimulant effects occurred. However, as any transient effects were small, the average 5 min value has been plotted. To determine the presence of ionophore activity, perfusion fluids to which the adrenals were exposed were sometimes examined for their ability to stimulate mast cell secretion, measured by degranulation or histamine release. Mast cells were isolated from the rat peritoneum, suspended in Locke, and degranulation observed by phase contrast microscopy as previously described (Cochrane & Douglas, 1974). Histamine release was determined by conventional methods as follows: suspensions of the mast cells were incubated with samples of perfusion fluid at room temperature. After 10 min, several volumes of ice-cold Locke were added and the samples centrifuged to sediment the cells. Cellular histamine was extracted by boiling for 5 min in 2% perchloric acid. Cells and supernatant fractions were then deproteinized and histamine determined fluorometrically as described by Kremzner & Wilson (1961). Differences between sample means were tested for significance by Student's t test.
366
D. E. COCHRANE AND OTHERS RESULTS
Responses to X-537A When adrenal glands perfused with Locke were exposed to X-537A in a concentration of 10 jug/ml. for 10 min, the output of catecholamines increased greatly (Fig. 1A). Similar responses to the drug were obtained from each of nine glands perfused with Locke containing hexamethonium (5 x 10-4 g/ml.) and atropine (10-5 g/ml.) to prevent indirect effects 8 7
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mediated by acetylcholine release from the splanchnic terminals (Douglas & Poisner, 1965; Douglas et al. 1967a). The result shown in Fig. 1B is typical: catecholamine output rose to very high levels during perfusion with X-537A and remained elevated long after the drug had been withdrawn. The mean rate of catecholamine release in these experiments during the 10 min when X-537A was present was more than a hundred-
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D. E. COCHRANE AND OTHERS 368 fold greater than the mean basal rate immediately before introducing the drug. In the 10 min following exposure to X-537A, secretion was still about 60 times the basal value, and even 1 hr after withdrawing the drug, secretion was far above the normal basal level. These large increases in catecholamine output in response to X-537A are comparable with those produced by acetylcholine in high concentration or by strongly depolarizing concentrations of potassium (Douglas, 1975). Such responses to acetylcholine and potassium are known to be critically dependent on calcium and cannot be elicited when the adrenals 1-2 ug/min A
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Fig. 2. Catecholamine outputs in response to X-537A in the presence (A) and absence (B) of calcium. X-537A (0-5 tg/ml.) was present for the periods indicated by the horizontal bars. In A an adrenal gland was perfused with Locke. In B a second adrenal gland was perfused for the period indicated and for 15 min before, with calcium-free Locke with the addition of EGTA (1 mM). The large response obtained in the last 5 min was the ,result of switching the perfusion to Locke and thus introducing calcium. Both experiments were conducted in the presence of C6 (5 x 104 g/ml.) and atropine (10-5 g/ml.).
are perfused for several minutes with Ca-free Locke (Douglas & Rubin,. 1961, 1963). By contrast, it was found that the responses to X-537A were less sensitive to calcium deprivation. However, a substantial reduction was achieved by perfusing glands for 90 min with calcium-free Locke containing 5 mM-EGTA. Under these conditions, catecholamine output during exposure to X-537A, although still many times higher
IONOPHORES AND CHROMAFFIN CELL SECRETION 369 than the basal rate, was only about 12 % of that obtained with this same concentration (10 ,g/ml.) of X-537A given in the conventional, calciumcontaining medium (Table 1). From results yielded by mast cells (Foreman et al. 1973; Cochrane & Douglas, 1974; Kagayama & Douglas, 1974) and neurohypophyses (Nakazato & Douglas, 1974) it appears that secretary responses to X537A show a greater dependence on extracellular calcium when lower concentrations of the drug are used. This prompted us to examine the effect of calcium-deprivation on responses to X-537A in a lower concentration (0.5 4ag/ml.) given for a shorter time (5 min). In the control glands perfused with calcium-containing Locke, this treatment with X-537A elicited responses that were smaller than those obtained with the drug in twentyfold higher concentration but still many times above resting level. Catecholamine output always reached a maximum some time after exposure to the drug-containing solution and remained elevated long after such exposure had ceased (Fig. 2A). By contrast, in glands perfused with calcium-free Locke containing 1 mM-EGTA for 30 min beforehand, X-537A, in this low concentration, produced only feeble responses (Fig. 2B). The results of seven such experiments are presented in the lower part of Table 1. Responses to A23187 A23187 was a much less effective stimulus for catecholamine release than X-537A and its effects were more variable in magnitude and time course. The variability was such that we have been unable to present either a typical response or a meaningful summary. For this reason we have chosen to present, in Fig. 3, all the results we have obtained. A23187 was given in concentrations ranging from 2 to 50 /ug/ml. and for periods of 10 to 30 min. Although it always stimulated, the outputs of catecholamines were not obviously related to the concentrations of the ionophore. In all but two experiments (Fig. 3F and G) the maximum rate of catecholamine output following exposure to A23187 was less than 5 times the resting level. As with X-537A, the response outlasted exposure to the ionophore-containing Locke and in several experiments it became progressively larger. A23187 had no effect in glands perfused for 45 min with calcium-free Locke (Fig. 3M), and its stimulant effect in Locke was reversed by perfusing with calcium-free Locke containing 5 mM-EGTA (Fig. 3N). In five experiments, X-537A was introduced after exposure to A23187 and in each instance had its usual powerful stimulant effect on catecholamine output. In three other experiments, exposure to A23187 was followed by treatment with high potassium Locke and this, too, caused
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IONOPHORES AND CHROMAFFIN CELL SECRETION 371 a massive outpouring of catecholamines. The contrasting effects of A23187 on the one hand and X-537A or high potassium on the other are illustrated in Fig. 3C, G-J and Fig. 3F, K and L respectively. In several other studies, A23187-containing media have been prepared from stock solutions of the ionophore in ethanol or methanol (e.g. Eimerl et al. 1974; Prince, Rasmussen & Berridge, 1973). We adopted this procedure in several additional experiments but were still unable to elicit larger outputs of catecholamines with A23187. Unlike DMSO, which was without effect on catecholamine output (Fig. 3A), both ethanol and methanol in a final concentration of 1 % stimulated catecholamine output somewhat and were thus less suitable than DMS0 for the present study. Since the responses of the chromaffin cells to A23187 were very much less than those of rat peritoneal mast cells (Foreman et al. 1973; Cochrane & Douglas, 1974; Kagayama & Douglas, 1974) it was thought possible that there might have been some loss of the drug in the perfusion system. Although this did not seem likely, as perfusion with X-537A was very effective, we tested the adrenal perfusion medium to which we had added A23187 for its ability to stimulate mast cell secretion measured by histamine release or degranulation. In each instance, the perfusion fluid, sampled at the point it entered the tissue (and after passage through the pump circuit), elicited widespread degranulation and the release of most of the histamine. The effects were indistinguishable from those elicited by samples of A23187-containing Locke obtained before passage through the perfusion system and were comparable to the effects of A23187 on mast cells reported previously (Foreman et al. 1973; Cochrane & Douglas, 1974; Kagayama & Douglas, 1974). Moreover, the effluent collected from Fig. 3. Effects of the ionophore A23187 (A) on the output of catecholamines from perfused cat adrenal glands. The thick horizontal bars show the duration of exposure to A23187 and the number in brackets below indicates the concentration (,zg/ml.). A, control experiments (mean+ S.E., n = 5) with the solvent DMSO (open bar, 05 %). B-L show the generally small and variable responses to A23187 which contrast with the large responses to X-537A, 10 ig/ml., (X) or high potassium Locke (K). M, responses (mean S.E., n = 2) to A23187 in the absence of extra-cellular calcium. Perfusion was with calcium-free Locke for the period indicated and for 45 min before. N, effect of perfusing with calcium-free Locke containing 5 mm-EGTA on the response to A23187. In Al and N the final 5 min of perfusion was with Locke (calcium). In records B, D and I perfusion was orthograde; in the others it was retrograde. Where the records are discontinuous, the period (min) omitted is indicated by the number below the interruption. Perfusion in all experiments was with Locke except where shown and hexamethonium (5 x 10-4 g/ml.) and atropine (10-5 g/ml.) were present throughout. Numbers beside the interrupted vertical columns indicate the catecholamine output (,ug/ml.) during these 5 min periods.
372 D. E. COCHRANE AND OTHERS the gland during perfusion with Locke to which the ionophore had been added also stimulated histamine release and degranulation although it appeared to be less potent in this regard suggesting that a significant uptake by the tissue had occurred. A typical experiment is shown in Fig. 4, in which it may be seen that even after passage through the adrenal the A23187-containing medium that had increased catecholamine output only slightly caused a massive release of histamine even when diluted 1:5. To emphasize the difference in behaviour of the two secretary systems, *j C
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Fig. 4. Effects of A23 187 (50 ,sg/ml. for the period indicated by the horizontal bar) on catecholamine output from a perfused adrenal gland (upper record) and on the histamine-releasing activity of the adrenal effluent (lower record). The latter is considered attributable to the presence of A23187. Effluent was collected during successive 5 min periods and four samples (vertical arrows), obtained before, during, and after perfusion with A23187, were diluted 1:5 and tested for their ability to release histamine from rat peritoneal mast cells. Note that the samples of adrenal effluent obtained during perfusion with A23187 (short vertical arrows) had potent histamine-releasing activity whereas those obtained before or after (long vertical arrows) had little or no such activity (the values for histamine release with the latter are comparable with controls obtained in the absence of perfusate). To illustrate the contrasting intensity of the secretory responses of chromaffin and mast cells the release of catecholamine and of histamine is expressed as a percentage of the amount in the tissue. Mast cell histamine was experimentally determined. Medullary catecholamine was estimated at 300 lig (Butterworth & Mann, 1957).
IONOPHORES AND CHROMAFFIN CELL SECRETION 373 we have plotted release of catecholamines and histamine as percentages of the totals present in the chromaffin cells and mast cells respectively. The adrenal effluent collected before or after perfusion with A23187 did not stimulate histamine release: the values shown are similar to the basal rate of histamine release. Control experiments showed that adrenaline (5 x 10-7 g/ml.) and noradrenaline (5 x 10-7 g/ml.) had no stimulant effect on histamine release. DISCUSSION
Our experiments show that the ionophores, X-537A and A23187, stimulate catecholamine output from the adrenal gland by actions exerted directly on the chromaffin cells. Because these ionophores have been shown to transport calcium ions across cell membranes and because their stimulant effects on catecholamine output were greatly weakened by perfusion with calcium-free media the experiments may be considered additional support for the calcium-entry hypothesis of stimulus-secretion coupling (Douglas & Rubin, 1961, 1963; Douglas, 1968). The results also raise several questions. Why, for example, did catecholamine output remain at high levels long after perfusion with the ionophore-containing Locke had stopped? Why did X-537A have a considerable residual effect after removal of extracellular calcium? And why were responses relatively small and variable with A23187 which is a more specific and effective ionophore for calcium than is X-537A (Pressman, 1972, 1973; Reed & Lardy, 1972a, b; Pfeiffer, Reed & Lardy, 1974)? That responses to the ionophores long outlasted the period of perfusion may have a simple explanation, namely that once taken up by the tissue, these drugs, because of their lipophilic nature, will not pass easily back into the aqueous perfusion medium but will continue to exert their actions. The residual responses to X-537A in calcium-free media, which were about 12 % of those obtained in the presence of extracellular calcium, may be due to mobilization of cellular calcium. X-537A releases calcium from sarcoplasmic reticulum (Entman, Gillette, Wallick, Pressman & Schwartz, 1972; Pressman, 1972; Scarpa & Inesi, 1972; Scarpa, Baldassare & Inesi, 1972; Levy, Cohen & Inesi, 1973) and from mitochondria (Lin & Kun, 1973). Alternatively, these residual responses may be due to the ability of X-537A to complex amines (Pressman, 1973). By such an action, coupled with an ability to traverse biological membranes, X-537A might transfer catecholamines from the chromaffin granules to the extracellular environment directly without activation of the normal secretary process, exocytosis. There is some evidence for this from mast cells (Foreman et al. 1973) and adrenergic neurones (Thoa, Costa, Moss & Kopin, 1974). Two other possibilities that might contribute to prolonged responses and
374 D. E. COCHRANE AND OTHERS residual effects in the absence of extracellular calcium are: (1) inhibitory effects on the metabolism of the chromaffin cells (Rubin, 1970) by the ionophores (Reed & Lardy, 1972a; Lin & Kun, 1973; Wong, Wilkinson, Hamill & Horng, 1973; Andreo & Vallejos, 1974); and (2) ill-defined actions resulting in cell damage (Chambers, Pressman & Rose, 1974; Gerrard, White & Rao, 1974). The remaining question, which concerns the remarkable difference in potency of the two ionophores as medullary secretagogues, has the most immediate relevance to the problem of stimulus-secretion coupling. Whereas the hundredfold increases in catecholamine output observed with the higher doses of X-537A equalled or exceeded maximal outputs obtainable by splanchnic nerve stimulation or perfusion with acetylcholine or excess potassium (Douglas, 1975), the responses to all concentrations of A23187 were, by comparison, small and involved only a several-fold increase over resting levels. These relatively small responses to A23187 are, on first view, surprising since A23187 is generally believed to be a good ionophore for calcium (Reed & Lardy, 1972 a, b; Pfeiffer et al. 1974) and is, for example, sixtyfold more potent than X-537A in transporting calcium ions across the membranes of sarcoplasmic reticulum (Pressman, 1972). On the calcium hypothesis therefore, A23187 might have been expected to be more active in releasing catecholamines. One explanation for its relatively modest activity may be that within the membrane of the chromaffin cell the behaviour of A23187 may not conform to the pattern observed in other systems, and that, in this cell, A23187 is less effective in transporting calcium than is X-537A. An alternative possibility, with interesting physiological implications, is that the difference in efficacy of the two ionophores as medullary secretagogues may be explainable not by differences in ability to transport calcium ions but by differences in ability to transport monovalent cations. A23187 is relatively specific for divalent cations, particularly at the pH we have used (Pfeiffer et al. 1974), but X-537A can transport monovalent cations in addition to divalent cations (Pressman, 1972, 1973). The ability to transport sodium and potassium ions might allow X-537A to depolarize chromaffin cells and thereby stimulate secretion quite independently of effects arising from its action as a calcium ionophore. It is known that potassium, in concentrations that depolarize chromaffin cells (Douglas et al. 1967b), provides a strong stimulus for catecholamine secretion that requires extracellular calcium (Douglas & Rubin, 1961, 1963) and is accompanied by accumulation of 45Ca in the medulla (Poisner & Douglas, 1962). We have not examined the effect of the ionophores on the membrane potential of chromaffin cells but have found that X-537A promptly depolarizes frog skeletal muscle fibres in calcium-containing Ringer
IONOPHORES AND CHROMAFFIN CELL SECRETION 375 whereas A23187 has only a feeble, and delayed, depolarizing effect (Cochrane & Douglas, 1975). It is possible then, that the vigorous responses to X-537A reflect a combination of depolarization and calcium entry while the relatively feeble responses to A23187 may reflect the effect of inward movement of calcium unaccompanied by substantial depolarization. If the inward transport of calcium achieved by A23187 is similar to that achieved by X-537A (or by the other strong secretagogues, acetylcholine and 56 mM potassium) then cne reasonable inference would be that a rise in intracellular calcium alone is insufficient to elicit maximal secretary rates in the chromaffin cell. The problem whether a rise in intracellular calcium alone will induce secretion in various cells or is effective only when the plasma membranes are perturbed, as by depolarization or exposure to secretagogue, has been raised on several occasions (e.g. Douglas, 1968; Woodin & Wienecke, 1970; Poste & Allison, 1973). Although the secretory responses that have been observed upon microiontophoretic injection of calcium ions into squid nerve terminals (Miledi, 1973) and mast cells (Kanno et al. 1973) suggest that calcium alone may be effective, the ionejecting currents used may have perturbed the cell membranes and contributed an additional co-operative effect. The question is still unsettled. In tissues developmentally related to chromaffin cells it is noteworthy that X-537A is also a relatively potent stimulus for release of secretary product. It causes a discharge of acetylcholine from motor neurones (Kita & van der Kloot, 1974), noradrenaline from adrenergic neurones (Pressman, 1973; Schwartz, Lewis, Hanley, Munson, Dial & Ray, 1974; Thoa et al. 1974), and vasopressin from neurohypophysial terminals (Nakazato & Douglas, 1974). On the other hand, A23187 has not been reported to stimulate acetylcholine release, has a relatively feeble action on adrenergic terminals (Schwartz et al. 1974; Thoa et al. 1974), and has little or no stimulant effect on the output of vasopressin from isolated neurohypophyses in calcium-containing medium (Nakazato & Douglas, 1974). Interestingly, neurohypophyses exposed to A23187 in a calcium-free medium for a prolonged period have been observed to release vasopressin when calcium is reintroduced (Russell, Hansen & Thorn, 1974). This again may indicate a co-operative action between calcium and depolarization for we have observed, on skeletal muscle fibres, that calcium deprivation potentiates the depolarizing action of A23187 (Cochrane & Douglas, 1975). It may be significant that those cells that have responded to A23187 with a vigorous extrusion of preformed secretary product, namely mast cells (Foreman et al. 1973; Cochrane & Douglas, 1974) and cells of the exocrine pancreas (Eimerl et al. 1974; Williams & Lee, 1974) differ from
D. E. COCHRANE AND OTHERS chromaffin cells, neurones, and neurosecretory fibres in that they do not secrete when depolarized by excess potassium (Guschin, Orlov & Tsyu, 1973; Matthews, Petersen & Williams, 1973; Nishiyama & Petersen, 1974). Further comparative studies of the effects of ionophores on diverse secretary systems may help to define better the events in stimulussecretion coupling. 376
We thank Dr M. W. Osborne of Hoffman-La Roche, Inc. and Dr R. L. Hamill of Eli Lilly and Co. for generous gifts of X-537A and A23187 respectively. This work was supported by USPHS grants NS 08546 and NS 09137. REFERENCES ANDREO, C. S. & VALLEJOS, R. H. (1974). Uncoupling of photophosphorylation in spinach chloroplasts by the ionophorous antibiotic A23187. FEBS Lett. 46, 343-346. BAKER, P. F. (1972). Transport and metabolism of calcium ions in nerve. Prog. Biophys. molec. Biol. 24, 177-223. BUTTERWORTH, K. R. & MANN, M. (1957). A quantitative comparison of the sympathomimetic amine content of the left and right adrenal glands of the cat. J. Physiol. 136, 294-299. CHAMBERS, E. L., PRESSMAN, B. C. & ROSE, B. (1974). The activation of sea urchin eggs by the divalent ionophores A23187 and X-537A. Biochem. biophys. Res. Commun. 60, 126-132. COCHRANE, D. E. & DOUGLAS, W. W. (1974). Calcium-induced extrusion of secretary granules (exocytosis) in mast cells exposed to 48/80 or the ionophores A-23187 and X-537A. Proc. natn Acad. Sci. U.S.A. 71, 408-412. COCHRANE, D. E. & DOUGLAS, W. W. (1975). Depolarizing effects of the ionophores X-537A and A23187 and their relevance to secretion. Br. J. Pharmac. (in the Press). DOUGLAS, W. W. (1968). Stimulus-secretion coupling: The concept and clues from chromaffin and other cells. The First Gaddum Memorial Lecture, Cambridge, 1967, Br. J. Pharmac. 34, 451-474. DOUGLAS, W. W. (1973). How do neurones secrete peptides? Exocytosis and its consequences, including 'synaptic vesicle' formation, in the hypothalamo-neurohypophyseal system. In Drug effects on Neuroendocrine Regulation, ed. ZIMMERMAN, E., GISPEN, W. H., MARKS, B. H. & DE WIED, D. Progress in Brain Research vol. 31, pp. 21-39. Amsterdam: Elsevier. DOUGLAS, W. W. (1974a). Involvement of calcium in exocytosis and the exocytosisvesiculation sequence. In Calcium and Cell Regulation, ed. SMELLIE, R. M. S. Biochem. Soc. Symp. vol. 39, pp. 1-28. London: The Biochemical Society. DOUGLAS, W. W. (1974b). Mechanism of release of neurohypophysial hormones: stimulus-secretion coupling. In Handbook of Physiology, section 7: Endocrinology. vol. iv, The Pituitary Gland and its Neuroendocrine Control, part 1, ed. KNOBIL, E. & SAWYER, W. H., pp. 191-224. Washington, D.C.: Am. Physiol. Soc. DOUGLAS, W. W. (1975). Secretomotor control of adrenal medullary secretion: synaptic, membrane and ionic events in stimulus-secretion coupling. In Handbook of Physiology, section 7: Endocrinology. vol. vi, The Adrenal Gland, ed. BLASCHKO, H., SAYERS, G. & SMITH, A. D., pp. 367-388. Washington, D.C.: Am. Physiol. Soc. DOUGLAS, W. W., KANNO, T. & SAMPSON, S. R. (1967a). Effects of acetylcholine and other medullary secretagogues and antagonists on the membrane potential of adrenal chromaffin cells: an analysis employing techniques of tissue culture. J. Physiol. 188, 107-120.
IONOPHORES AND CHROJIAFFIN CELL SECRETION 377 DOUGLAS, W. XV., KANNO, T. & SAMPSON, S. R. (1967b). Influence of the ionic environment on the membrane potential of adrenal chromaffin cells and on the depolarizing effect of acetylcholine. J. Physiol. 191, 107-121. DOUGLAS, W. XV., NAGASAWA, J. & SCHULZ, R. A. (1971). Electron microscopic studies on the mechanism of secretion of posterior pituitary hormones and significance of microvesicles ('synaptic vesicles'):' Evidence of secretion by exocytosis and formation of microvesicles as a by-product of this process. Mem. Soc. Endocr. 19, 353-378. DOUGLAS, W. W. & POISNER, A. M. (1965). Preferential release of adrenaline from the adrenal medulla by muscarine and pilocarpine. Nature, Lond. 208, 1102-1103. DOUGLAS, WV. W. & RUBIN, R. P. (1961). The role of calcium in the secretary response of the adrenal medulla to acetylcholine. J. Physiol. 159, 40-57. DOUGLAS, W. W. & RUBIN, R. P. (1963). The mechanism of catecholamine release from the adrenal medulla and the role of calcium in stimulus-secretion coupling. J. Physiol. 167, 288-310. EIMERL, S., SAVION, N., HEICHAL, 0. & SELINGER, Z. (1974). Induction of enzyme secretion in rat pancreatic slices using the ionophore A-23187 and calcium. J. biol. Chem. 249, 3991-3993. ENTMAN, M. L., GILLETTE, P. C., WALLICK, E. T., PRESSMAN, B. C. & SCHWARTZ, A. (1972). A study of calcium binding and uptake by isolated cardiac sarco-plasmic reticulum: the use of a new ionophore (X537A). Biochem. biophys. Res. Commun. 48, 847-853. FOREMAN, J. C., MONGAR, J. L. & GOMPERTS, B. D. (1973). Calcium ionophores and movement of calcium ions following the physiological stimulus to a secretary process. Nature, Lond. 245, 249-251. GERRARD, J. M., WHITE, J. G. & RAO, G. H. R. (1974). Effects of the ionophore A23187 on blood platelets. II. Influence on Ultrastructure. Am.J. Path. 77, 151-166. GUSCHIN, I. S., ORLOV, S. M. & TsYu, N. L. (1973). Membrane potential of mast cells and histamine release from them. Bill. eskp. Biol. Med. 76, 20-23. KAGAYAMA, M. & DOUGLAS, WV. W. (1974). Electron microscopic evidence of calciuminduced exocytosis in mast cells treated with 48/80 or the ionophores A-23187 and X-537A. J. cell Biol. 62, 519-526. KANNO, T., COCHRANE, D. E. & DOUGLAS, W. W. (1973). Exocytosis secretaryy granule extrusion) induced by injection of calcium into mast cells. Can. J. Physiol. Pharmacol. 51, 1001-1004. KITA, H. & VAN DER KLOOT, W. (1974). Calcium ionophore X-537A increases spontaneous and phasic quantal release of acetylcholine at frog neuromuscular junction. Nature, Lond. 250, 658-660. KREMZNER, L. T. & XVILSON, I. B. (1961). A procedure for the determination of histamine. Biochim. biophys. Acta 50, 364-367. LEVY, J. V., COHEN, J. A. & INESI, G. (1973). Contractile effects of a calcium ionophore. Nature, Lond. 242, 461-463. LIN, D. C. & KUN, E. (1973). Mode of action of the antibiotic X-537A on mitochondrial glutamate oxidation. Biochem. biophys. Res. Commun. 50, 820-825. MATTHEWS, E. K., PETERSEN, 0. H. & WILLIAMS, J. A. (1973). Pancreatic acinar cells: acetylcholine-induced membrane depolarization, calcium efflux and amylase release. J. Physiol. 234, 689-701. MILEDI, R. (1973). Transmitter release induced by injection of calcium ions into nerve terminals. Proc. R. Soc. B 183, 421-425. NAKAZATO, Y. & DOUGLAS, W. W. ( 1974). Vasopressin release from the isolated neurohypophysis induced by a calcium ionophore, X-537A. Nature, Lond. 249, 479-481. NISHIYAMA, A. & PETERSEN, 0. H. (1974). Pancreatic acinar cells: membrane potential and resistance change evoked by acetylcholine. J. Physiol. 238, 145-158.
378
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PALADE, G. (1958). Functional changes in the structure of cell components. In Subcellular Particles, ed. HAYASHI, T., pp. 64-80. New York: Ronald Press. PFEIFFER, D. R., REED, P. W. & LARDY, H. A. (1974). Ultraviolet and fluorescent spectral properties of the divalent cation ionophore A23187 and its metal ion complexes. Biochemistry 13, 4007-4014. POISNER, A. M. & DOUGLAS, W. W. (1962). Enhanced uptake of Ca45 in the adrenal medulla in response to acetylcholine or potassium. Fedn Proc. 21, no. 2, 193. POSTE, G. & ALLISON, A. C. (1973). Membrane Fusion. Biochim. biophys. Acta 300, 421-465. PRESSMAN, B. C. (1972). Carboxylic ionophores as mobile carriers for divalent ions. In The Role of Membranes in Metabolic Regulation, ed. MEHLMAN, M. A. & HANSON, R. W., pp. 149-164. New York: Academic Press. PRESSMAN-, B. C. (1973). Properties of ionophores with broad range cation selectivity. Fedn Proc. 32, 1698-1703. PRINCE, W. T., RASMUSSEN, H. & BERRIDGE, M. J. (1973). The role of calcium in fly salivary gland secretion analysed with the ionophore A-23187. Biochim. biophys. Acta 329, 98-107. REED, P. W. & LARDY, H. A. (1972a). A23187: A divalent cation ionophore. J. biol. Chem. 247, 6970-6977. REED, P. W. & LARDY, H. A. (1972b). Antibiotic A23187 as a probe for the study of calcium and magnesium function in biological systems. In The Role of Membranes in Metabolic Regulation, ed. MEHLMAN, M. A. & HANSON, R. W., pp. 111- 131. New York: Academic Press. RUBIN, R. P. (1970). The role of energy metabolism in calcium-evoked secretion from the adrenal medulla. J. Physiol. 206, 181-192. RUBIN, R. P. ( 1971). The role of calcium in the release of neurotransmitter substances and hormones. Pharmac. Rev. 22, 389-428. RUBIN, R. P. (1974). Calcium and the Secretory Process. New York: Plenum Press. RUSSELL, J. T., HANSEN, E. L. & THORN, N. A. (1974). Calcium and stimulus-secretion coupling in the neurohypophysis. IV. Ca2+ ionophore (A23187)-induced release of vasopressin from isolated rat neurohypophyses. Acta endocr., Copnh. 77, 443-450. SCARPA, A., BALDASSARE, J. & INESI, G. (1972). The effect of calcium ionophores on fragmented sarcoplasmic reticulum. J. gen. Physiol. 60, 735-749. SCARPA, A. & INESI, G. (1972). Ionophore mediated equilibration of calcium ion gradients in fragmented sarcoplasmic reticulum. FEBS Lett. 22, 273-276. SCHWARTZ, A., LEWIS, R. M., HANLEY, H. G., MUJNSON, R. G., DIAL, F. D. & RAY, M. V. (1974). Hemodynamic and biochemical effects of a new positive inotropic agent. Circulation Res. 34, 102-111. THOA, N. B., COSTA, J. L., Moss, J. & KOPIN, I. J. (1974). Mechanism of release of norepinephrine from peripheral adrenergic neurones by the calcium ionophores X-537A and A23187. Life Sci. Oxford 14, 1705-1719. WILLIAMS, 'J. A. & LEE, M. (1974). Pancreatic acinar cells: use of a Ca++ ionophore to separate enzyme release from the earlier steps in stimulus-secretion coupling. Biochem. biophys. Res. Commun. 60, 542-548. WONG, D. T., WILKINSON, J. R., HAMILL, R. L. & HORNG, J.-S. (1973). Effects of antibiotic ionophore, A23187, on oxidative phosphorylation and calcium transport of liver mitochondria. Archs. Biochem. 156, 578-585. WOODIN, A. M. & WIENEKE, A. A. (1970). Site of protein secretion and calcium accumulation in the polymorphonuclear leucocyte treated with leucocidin. In Calcium and Cellular Function, ed. CUTHBERT, A. W., pp. 183-197. New York: St Martin's Press.
363
CALCIUM AND STIMULUS-SECRETION COUPLING IN THE ADRENAL MEDULLA: CONTRASTING STIMULATING EFFECTS OF THE IONOPHORES X-537A AND A23187 ON CATECHOLAMINE OUTPUT
BY D. E. COCHRANE,* W. W. DOUGLAS, T. MOURI AND Y. NAKAZATO From the Department of Pharmacology, Yale University of Medicine, New Haven, Connecticut 06510, U.S.A.
(Received 3 February 1975) SUMMARY
1. The ionophores X-537A and A23187, which are known to transfer calcium across cell membranes, stimulated catecholamine release from perfused cat adrenal glands. 2. These stimulant effects persisted in the presence of hexamethonium and atropine and are therefore attributable to direct actions of the ionophores on the adrenal chromaffin cells. 3. Perfusion with calcium-free Locke abolished responses to A23187 and reduced those to X-537A. 4. Responses to X-537A were consistently large and comparable with those produced by 56 mm potassium. By contrast, responses to A23177, over the wide range of concentrations tested, were variable and much smaller. 5. That the two ionophores can stimulate through calcium-dependent mechanisms is considered fresh support for the calcium hypothesis of stimulus-secretion coupling. That they differ in effectiveness may mean that factors besides calcium are important. The greater potency of the less specific ionophore, X-537A, may be attributable to its ability to depolarize as well as carry calcium, while the relatively small effects of A23187, a generally more effective ionophore for calcium, may indicate that inward movement of calcium without a background of membrane perturbation such as may be produced by depolarization, is insufficient to elicit strong secretary responses. *
NIH Postdoctoral
Fellow.
364
D. E. COCHRANE AND OTHERS INTRODUCTION
Experiments on many different secretary cells have revealed a common requirement for calcium in stimulus-secretion coupling and it has been suggested that a rise in the concentration of free calcium ions at some critical site within the cell (possibly at the inner surface of the plasma membrane) in response to the physiological stimulus, may in each instance set in motion a common process, exocytosis (Douglas, 1968, 1974a; Rubin, 1971, 1974). Two recent sets of observations are in harmony with this scheme. First, the intracellular injection of calcium by microiontophoresis has been found to release neural transmitter at the squid giant synapse (Miledi, 1973) and also to cause the expulsion of secretary granules from mesenteric mast cells (Kanno, Cochrane & Douglas, 1973). Secondly, the ionophores A23187 and X-537A, which can act as mobile carriers to transfer calcium across cell membranes (Reed & Lardy, 1972 a, b; Pressman, 1972, 1973), have been shown to elicit secretion in various cells where exocytosis has either been demonstrated, or is suspected, to operate. Thus, A23187 releases histamine from mast cells (Foreman, Mongar & Gomperts, 1973) and this effect is accompanied by light microscopic (Cochrane & Douglas, 1974) and electron microscopic (Kagayama & Douglas, 1974) evidence of exocytosis. The same ionophore has also been found to elicit release of amylase from pancreatic slices (Eimerl, Savion, Heichal & Selinger, 1974; Williams & Lee, 1974) and perfused intact pancreas (W. W. Douglas, T. Mouri & Y. Nakazato, unpublished observation). It will be recalled that secretion of pancreatic enzymes is the archetypical example of exocytosis (Palade, 1958). However, in this laboratory, A23187 has been found to be a relatively feeble stimulus for the secretion of vasopressin from isolated neurohypophyses (Nakazato & Douglas, 1974), yet here too secretion normally involves exocytosis (Douglas, Nagasawa & Schulz, 1971; Douglas, 1973, 1974b). Differences in the behaviour of various tissues are also evident in response to the ionophore X-537A which, in contrast to A23187, strongly stimulates vasopressin secretion from neurohypophysial terminals (Nakazato & Douglas, 1974) and has a corresponding effect on release of acetylcholine from motor nerve endings (Kita & van der Kloot, 1974). Yet, X-537A, unlike A23187, has little or no stimulant effect on amylase output from the perfused pancreas when indirect effects attributable to acetylcholine release are blocked by atropine (W. W. Douglas, T. Mouri & Y. Nakazato,
unpublished observation). It seemed to us possible that this diversity in the responses to the two ionophores might provide a fresh clue to the still uncertain events involved in stimulus-secretion coupling. Clearly, evidence from a wider
IONOPHORES AND CHROMAFFIN CELL SECRETION 365 spectrum of secretary cells was required. This led us to study the effects of A23187 and X-537A on the adrenal medulla. METHODS
Adult cats were anaesthetized by i.P. injection of sodium pentobarbitone (35 mg/kg) and their adrenals, acutely denervated, were perfused in situ or in vitro with Locke of the following composition (mm): NaCl, 154; KCl, 5-6; CaCl2, 2-2; Na2HPO4-NaH2PO4 buffer (pH 7 0), 3; glucose, 10. The solution was equilibrated with pure oxygen and perfusion was carried out at room temperature (about 220 C). The perfusion methods and techniques for fluorometric assay of catecholamines were as described by Douglas & Rubin (1961, 1963) except that perfusion was by peristaltic pump adjusted to yield an adrenal effluent of 1 ml./min. Perfusion of the adrenal glands in situ was by a cannula inserted into the lower aorta below the renal artery. The aorta was tied immediately above the coeliac axis and all other vessels were tied except those directly supplying the adrenals. The perfusate was collected through a cannula inserted into the vena cava at the same level as the arterial cannula. For perfusion of the isolated adrenal glands, a fine cannula, connected to the perfusion system, was inserted into the adrenolumbar vein lateral to the gland and pointing toward it. The adrenolumbar vein was then tied close to the vena cava and perfusion begun. The adrenal gland was then cut free from the animal as rapidly as possible and transferred to a filter funnel for collection of the perfusate. A thorough description of the procedures used to perfuse the isolated glands and the glands in situ is given in Douglas & Rubin (1961). In Ca-free Locke, CaCl2 was omitted and, where indicated, EGTA (Ethylenebis (oxyethylene-nitrilo) tetra-acetic acid), 1 or 5 mm, was present. High potassium Locke contained 56 mMKCl with NaCl reduced to maintain isotonicity (Douglas & Rubin. 1963). Except where indicated, the perfusion fluids contained hexamethonium chloride (C6), 5 x 10-4 g/ml., and atropine sulphate, 1O-- g/ml., to block possible indirect effects that might arise from the release of acetylcholine from splanchnic nerve endings and consequent activation of either nicotinic or muscarinic receptors on the chromaffin cells (Douglas & Poisner, 1965; Douglas, Kanno, & Sampson, 1967a). Perfusion fluids containing the ionophores X-537A or A23187 were prepared using stock solutions of the drugs in dimethylsulphoxide (DMSO). The final concentration of the solvent never exceeded 0-5 % which was without effect on catecholamine output (Fig. 3a). Samples of adrenal effluent were generally collected for successive 5 min periods. In a few experiments with A23187, successive samples were collected at 1 min intervals to determine whether any brief stimulant effects occurred. However, as any transient effects were small, the average 5 min value has been plotted. To determine the presence of ionophore activity, perfusion fluids to which the adrenals were exposed were sometimes examined for their ability to stimulate mast cell secretion, measured by degranulation or histamine release. Mast cells were isolated from the rat peritoneum, suspended in Locke, and degranulation observed by phase contrast microscopy as previously described (Cochrane & Douglas, 1974). Histamine release was determined by conventional methods as follows: suspensions of the mast cells were incubated with samples of perfusion fluid at room temperature. After 10 min, several volumes of ice-cold Locke were added and the samples centrifuged to sediment the cells. Cellular histamine was extracted by boiling for 5 min in 2% perchloric acid. Cells and supernatant fractions were then deproteinized and histamine determined fluorometrically as described by Kremzner & Wilson (1961). Differences between sample means were tested for significance by Student's t test.
366
D. E. COCHRANE AND OTHERS RESULTS
Responses to X-537A When adrenal glands perfused with Locke were exposed to X-537A in a concentration of 10 jug/ml. for 10 min, the output of catecholamines increased greatly (Fig. 1A). Similar responses to the drug were obtained from each of nine glands perfused with Locke containing hexamethonium (5 x 10-4 g/ml.) and atropine (10-5 g/ml.) to prevent indirect effects 8 7
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mediated by acetylcholine release from the splanchnic terminals (Douglas & Poisner, 1965; Douglas et al. 1967a). The result shown in Fig. 1B is typical: catecholamine output rose to very high levels during perfusion with X-537A and remained elevated long after the drug had been withdrawn. The mean rate of catecholamine release in these experiments during the 10 min when X-537A was present was more than a hundred-
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D. E. COCHRANE AND OTHERS 368 fold greater than the mean basal rate immediately before introducing the drug. In the 10 min following exposure to X-537A, secretion was still about 60 times the basal value, and even 1 hr after withdrawing the drug, secretion was far above the normal basal level. These large increases in catecholamine output in response to X-537A are comparable with those produced by acetylcholine in high concentration or by strongly depolarizing concentrations of potassium (Douglas, 1975). Such responses to acetylcholine and potassium are known to be critically dependent on calcium and cannot be elicited when the adrenals 1-2 ug/min A
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are perfused for several minutes with Ca-free Locke (Douglas & Rubin,. 1961, 1963). By contrast, it was found that the responses to X-537A were less sensitive to calcium deprivation. However, a substantial reduction was achieved by perfusing glands for 90 min with calcium-free Locke containing 5 mM-EGTA. Under these conditions, catecholamine output during exposure to X-537A, although still many times higher
IONOPHORES AND CHROMAFFIN CELL SECRETION 369 than the basal rate, was only about 12 % of that obtained with this same concentration (10 ,g/ml.) of X-537A given in the conventional, calciumcontaining medium (Table 1). From results yielded by mast cells (Foreman et al. 1973; Cochrane & Douglas, 1974; Kagayama & Douglas, 1974) and neurohypophyses (Nakazato & Douglas, 1974) it appears that secretary responses to X537A show a greater dependence on extracellular calcium when lower concentrations of the drug are used. This prompted us to examine the effect of calcium-deprivation on responses to X-537A in a lower concentration (0.5 4ag/ml.) given for a shorter time (5 min). In the control glands perfused with calcium-containing Locke, this treatment with X-537A elicited responses that were smaller than those obtained with the drug in twentyfold higher concentration but still many times above resting level. Catecholamine output always reached a maximum some time after exposure to the drug-containing solution and remained elevated long after such exposure had ceased (Fig. 2A). By contrast, in glands perfused with calcium-free Locke containing 1 mM-EGTA for 30 min beforehand, X-537A, in this low concentration, produced only feeble responses (Fig. 2B). The results of seven such experiments are presented in the lower part of Table 1. Responses to A23187 A23187 was a much less effective stimulus for catecholamine release than X-537A and its effects were more variable in magnitude and time course. The variability was such that we have been unable to present either a typical response or a meaningful summary. For this reason we have chosen to present, in Fig. 3, all the results we have obtained. A23187 was given in concentrations ranging from 2 to 50 /ug/ml. and for periods of 10 to 30 min. Although it always stimulated, the outputs of catecholamines were not obviously related to the concentrations of the ionophore. In all but two experiments (Fig. 3F and G) the maximum rate of catecholamine output following exposure to A23187 was less than 5 times the resting level. As with X-537A, the response outlasted exposure to the ionophore-containing Locke and in several experiments it became progressively larger. A23187 had no effect in glands perfused for 45 min with calcium-free Locke (Fig. 3M), and its stimulant effect in Locke was reversed by perfusing with calcium-free Locke containing 5 mM-EGTA (Fig. 3N). In five experiments, X-537A was introduced after exposure to A23187 and in each instance had its usual powerful stimulant effect on catecholamine output. In three other experiments, exposure to A23187 was followed by treatment with high potassium Locke and this, too, caused
D. E. COCHRANE AND OTHERS
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IONOPHORES AND CHROMAFFIN CELL SECRETION 371 a massive outpouring of catecholamines. The contrasting effects of A23187 on the one hand and X-537A or high potassium on the other are illustrated in Fig. 3C, G-J and Fig. 3F, K and L respectively. In several other studies, A23187-containing media have been prepared from stock solutions of the ionophore in ethanol or methanol (e.g. Eimerl et al. 1974; Prince, Rasmussen & Berridge, 1973). We adopted this procedure in several additional experiments but were still unable to elicit larger outputs of catecholamines with A23187. Unlike DMSO, which was without effect on catecholamine output (Fig. 3A), both ethanol and methanol in a final concentration of 1 % stimulated catecholamine output somewhat and were thus less suitable than DMS0 for the present study. Since the responses of the chromaffin cells to A23187 were very much less than those of rat peritoneal mast cells (Foreman et al. 1973; Cochrane & Douglas, 1974; Kagayama & Douglas, 1974) it was thought possible that there might have been some loss of the drug in the perfusion system. Although this did not seem likely, as perfusion with X-537A was very effective, we tested the adrenal perfusion medium to which we had added A23187 for its ability to stimulate mast cell secretion measured by histamine release or degranulation. In each instance, the perfusion fluid, sampled at the point it entered the tissue (and after passage through the pump circuit), elicited widespread degranulation and the release of most of the histamine. The effects were indistinguishable from those elicited by samples of A23187-containing Locke obtained before passage through the perfusion system and were comparable to the effects of A23187 on mast cells reported previously (Foreman et al. 1973; Cochrane & Douglas, 1974; Kagayama & Douglas, 1974). Moreover, the effluent collected from Fig. 3. Effects of the ionophore A23187 (A) on the output of catecholamines from perfused cat adrenal glands. The thick horizontal bars show the duration of exposure to A23187 and the number in brackets below indicates the concentration (,zg/ml.). A, control experiments (mean+ S.E., n = 5) with the solvent DMSO (open bar, 05 %). B-L show the generally small and variable responses to A23187 which contrast with the large responses to X-537A, 10 ig/ml., (X) or high potassium Locke (K). M, responses (mean S.E., n = 2) to A23187 in the absence of extra-cellular calcium. Perfusion was with calcium-free Locke for the period indicated and for 45 min before. N, effect of perfusing with calcium-free Locke containing 5 mm-EGTA on the response to A23187. In Al and N the final 5 min of perfusion was with Locke (calcium). In records B, D and I perfusion was orthograde; in the others it was retrograde. Where the records are discontinuous, the period (min) omitted is indicated by the number below the interruption. Perfusion in all experiments was with Locke except where shown and hexamethonium (5 x 10-4 g/ml.) and atropine (10-5 g/ml.) were present throughout. Numbers beside the interrupted vertical columns indicate the catecholamine output (,ug/ml.) during these 5 min periods.
372 D. E. COCHRANE AND OTHERS the gland during perfusion with Locke to which the ionophore had been added also stimulated histamine release and degranulation although it appeared to be less potent in this regard suggesting that a significant uptake by the tissue had occurred. A typical experiment is shown in Fig. 4, in which it may be seen that even after passage through the adrenal the A23187-containing medium that had increased catecholamine output only slightly caused a massive release of histamine even when diluted 1:5. To emphasize the difference in behaviour of the two secretary systems, *j C
A23187
0050
40
0-2 ~~t C
record and onthhistmine-eleasngacivityoft 100
I
minsamples
I
80 -
~~60
~
0
20 0
Fig. 4. Effects of A23 187 (50 ,sg/ml. for the period indicated by the horizontal bar) on catecholamine output from a perfused adrenal gland (upper record) and on the histamine-releasing activity of the adrenal effluent (lower record). The latter is considered attributable to the presence of A23187. Effluent was collected during successive 5 min periods and four samples (vertical arrows), obtained before, during, and after perfusion with A23187, were diluted 1:5 and tested for their ability to release histamine from rat peritoneal mast cells. Note that the samples of adrenal effluent obtained during perfusion with A23187 (short vertical arrows) had potent histamine-releasing activity whereas those obtained before or after (long vertical arrows) had little or no such activity (the values for histamine release with the latter are comparable with controls obtained in the absence of perfusate). To illustrate the contrasting intensity of the secretory responses of chromaffin and mast cells the release of catecholamine and of histamine is expressed as a percentage of the amount in the tissue. Mast cell histamine was experimentally determined. Medullary catecholamine was estimated at 300 lig (Butterworth & Mann, 1957).
IONOPHORES AND CHROMAFFIN CELL SECRETION 373 we have plotted release of catecholamines and histamine as percentages of the totals present in the chromaffin cells and mast cells respectively. The adrenal effluent collected before or after perfusion with A23187 did not stimulate histamine release: the values shown are similar to the basal rate of histamine release. Control experiments showed that adrenaline (5 x 10-7 g/ml.) and noradrenaline (5 x 10-7 g/ml.) had no stimulant effect on histamine release. DISCUSSION
Our experiments show that the ionophores, X-537A and A23187, stimulate catecholamine output from the adrenal gland by actions exerted directly on the chromaffin cells. Because these ionophores have been shown to transport calcium ions across cell membranes and because their stimulant effects on catecholamine output were greatly weakened by perfusion with calcium-free media the experiments may be considered additional support for the calcium-entry hypothesis of stimulus-secretion coupling (Douglas & Rubin, 1961, 1963; Douglas, 1968). The results also raise several questions. Why, for example, did catecholamine output remain at high levels long after perfusion with the ionophore-containing Locke had stopped? Why did X-537A have a considerable residual effect after removal of extracellular calcium? And why were responses relatively small and variable with A23187 which is a more specific and effective ionophore for calcium than is X-537A (Pressman, 1972, 1973; Reed & Lardy, 1972a, b; Pfeiffer, Reed & Lardy, 1974)? That responses to the ionophores long outlasted the period of perfusion may have a simple explanation, namely that once taken up by the tissue, these drugs, because of their lipophilic nature, will not pass easily back into the aqueous perfusion medium but will continue to exert their actions. The residual responses to X-537A in calcium-free media, which were about 12 % of those obtained in the presence of extracellular calcium, may be due to mobilization of cellular calcium. X-537A releases calcium from sarcoplasmic reticulum (Entman, Gillette, Wallick, Pressman & Schwartz, 1972; Pressman, 1972; Scarpa & Inesi, 1972; Scarpa, Baldassare & Inesi, 1972; Levy, Cohen & Inesi, 1973) and from mitochondria (Lin & Kun, 1973). Alternatively, these residual responses may be due to the ability of X-537A to complex amines (Pressman, 1973). By such an action, coupled with an ability to traverse biological membranes, X-537A might transfer catecholamines from the chromaffin granules to the extracellular environment directly without activation of the normal secretary process, exocytosis. There is some evidence for this from mast cells (Foreman et al. 1973) and adrenergic neurones (Thoa, Costa, Moss & Kopin, 1974). Two other possibilities that might contribute to prolonged responses and
374 D. E. COCHRANE AND OTHERS residual effects in the absence of extracellular calcium are: (1) inhibitory effects on the metabolism of the chromaffin cells (Rubin, 1970) by the ionophores (Reed & Lardy, 1972a; Lin & Kun, 1973; Wong, Wilkinson, Hamill & Horng, 1973; Andreo & Vallejos, 1974); and (2) ill-defined actions resulting in cell damage (Chambers, Pressman & Rose, 1974; Gerrard, White & Rao, 1974). The remaining question, which concerns the remarkable difference in potency of the two ionophores as medullary secretagogues, has the most immediate relevance to the problem of stimulus-secretion coupling. Whereas the hundredfold increases in catecholamine output observed with the higher doses of X-537A equalled or exceeded maximal outputs obtainable by splanchnic nerve stimulation or perfusion with acetylcholine or excess potassium (Douglas, 1975), the responses to all concentrations of A23187 were, by comparison, small and involved only a several-fold increase over resting levels. These relatively small responses to A23187 are, on first view, surprising since A23187 is generally believed to be a good ionophore for calcium (Reed & Lardy, 1972 a, b; Pfeiffer et al. 1974) and is, for example, sixtyfold more potent than X-537A in transporting calcium ions across the membranes of sarcoplasmic reticulum (Pressman, 1972). On the calcium hypothesis therefore, A23187 might have been expected to be more active in releasing catecholamines. One explanation for its relatively modest activity may be that within the membrane of the chromaffin cell the behaviour of A23187 may not conform to the pattern observed in other systems, and that, in this cell, A23187 is less effective in transporting calcium than is X-537A. An alternative possibility, with interesting physiological implications, is that the difference in efficacy of the two ionophores as medullary secretagogues may be explainable not by differences in ability to transport calcium ions but by differences in ability to transport monovalent cations. A23187 is relatively specific for divalent cations, particularly at the pH we have used (Pfeiffer et al. 1974), but X-537A can transport monovalent cations in addition to divalent cations (Pressman, 1972, 1973). The ability to transport sodium and potassium ions might allow X-537A to depolarize chromaffin cells and thereby stimulate secretion quite independently of effects arising from its action as a calcium ionophore. It is known that potassium, in concentrations that depolarize chromaffin cells (Douglas et al. 1967b), provides a strong stimulus for catecholamine secretion that requires extracellular calcium (Douglas & Rubin, 1961, 1963) and is accompanied by accumulation of 45Ca in the medulla (Poisner & Douglas, 1962). We have not examined the effect of the ionophores on the membrane potential of chromaffin cells but have found that X-537A promptly depolarizes frog skeletal muscle fibres in calcium-containing Ringer
IONOPHORES AND CHROMAFFIN CELL SECRETION 375 whereas A23187 has only a feeble, and delayed, depolarizing effect (Cochrane & Douglas, 1975). It is possible then, that the vigorous responses to X-537A reflect a combination of depolarization and calcium entry while the relatively feeble responses to A23187 may reflect the effect of inward movement of calcium unaccompanied by substantial depolarization. If the inward transport of calcium achieved by A23187 is similar to that achieved by X-537A (or by the other strong secretagogues, acetylcholine and 56 mM potassium) then cne reasonable inference would be that a rise in intracellular calcium alone is insufficient to elicit maximal secretary rates in the chromaffin cell. The problem whether a rise in intracellular calcium alone will induce secretion in various cells or is effective only when the plasma membranes are perturbed, as by depolarization or exposure to secretagogue, has been raised on several occasions (e.g. Douglas, 1968; Woodin & Wienecke, 1970; Poste & Allison, 1973). Although the secretory responses that have been observed upon microiontophoretic injection of calcium ions into squid nerve terminals (Miledi, 1973) and mast cells (Kanno et al. 1973) suggest that calcium alone may be effective, the ionejecting currents used may have perturbed the cell membranes and contributed an additional co-operative effect. The question is still unsettled. In tissues developmentally related to chromaffin cells it is noteworthy that X-537A is also a relatively potent stimulus for release of secretary product. It causes a discharge of acetylcholine from motor neurones (Kita & van der Kloot, 1974), noradrenaline from adrenergic neurones (Pressman, 1973; Schwartz, Lewis, Hanley, Munson, Dial & Ray, 1974; Thoa et al. 1974), and vasopressin from neurohypophysial terminals (Nakazato & Douglas, 1974). On the other hand, A23187 has not been reported to stimulate acetylcholine release, has a relatively feeble action on adrenergic terminals (Schwartz et al. 1974; Thoa et al. 1974), and has little or no stimulant effect on the output of vasopressin from isolated neurohypophyses in calcium-containing medium (Nakazato & Douglas, 1974). Interestingly, neurohypophyses exposed to A23187 in a calcium-free medium for a prolonged period have been observed to release vasopressin when calcium is reintroduced (Russell, Hansen & Thorn, 1974). This again may indicate a co-operative action between calcium and depolarization for we have observed, on skeletal muscle fibres, that calcium deprivation potentiates the depolarizing action of A23187 (Cochrane & Douglas, 1975). It may be significant that those cells that have responded to A23187 with a vigorous extrusion of preformed secretary product, namely mast cells (Foreman et al. 1973; Cochrane & Douglas, 1974) and cells of the exocrine pancreas (Eimerl et al. 1974; Williams & Lee, 1974) differ from
D. E. COCHRANE AND OTHERS chromaffin cells, neurones, and neurosecretory fibres in that they do not secrete when depolarized by excess potassium (Guschin, Orlov & Tsyu, 1973; Matthews, Petersen & Williams, 1973; Nishiyama & Petersen, 1974). Further comparative studies of the effects of ionophores on diverse secretary systems may help to define better the events in stimulussecretion coupling. 376
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