Catecholamine secretion by isolated adrenal cells

168

Biochimica et Biophysica Acta, 421 (1976) 168--175

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 27792

CATECHOLAMINE SECRETION BY ISOLATED A D R E N A L CELLS

JUDITH HOCHMAN and ROBERT L. PERLMAN Department o f Physiology, Harvard Medical School Boston, Mass. 02115 (U.S.A.)

(Received July 7th, 1975)

Summary Isolated adrenal cells were prepared by collagenase digestion of guinea pig adrenal glands. A~etylcholine stimulates the secretion of catecholamines by these isolated adrenal cells. Acetylcholine-stimulated catecholamine secretion is inhibited by cholinergic blocking agents {atropine and h e x a m e t h o n i u m ) and by local anaesthetics (tetracaine), and is d e p e n d e n t upon the concentration of Ca :÷ in the incubation medium. In the presence of Ca :÷, catecholamine secretion is also stimulated by two divalent cation ionophores, A23187 and X-537A. Cyclic nucleotides and 5'-nucleotides cause a small, non-specific stimulation of catecholamine secretion. These results indicate that isolated adrenal cells are a useful system in which to study catecholamine secretion, and support the hypothesis that increased Ca :÷ entry into chromaffin cells is a sufficient stimulus for catecholamine secretion.

Introduction Chromaffin cells in the adrenal medulla synthesize and store the catecholamine hormones, epinephrine and norepinephrine, and secrete these hormones in response to stimulation by a variety of secretagogues. Acetylcholine, released from preganglionic sympathetic nerve terminals within the adrenal gland, is the physiological stimulus for catecholamine secretion [1]. Studies with perfused adrenal glands have shown that catecholamine secretion occurs by exocytosis [ 2 ] , and have demonstrated that extracellular Ca 2÷ plays a critical role in this process [3]. Acetylcholine increases the uptake of 4 s Ca2÷ into the adrenal gland [ 4 ] , and Ca 2÷ is required for the stimulation of catecholamine secretion by acetylcholine [5] and by ot her secretagogues [6]. Douglas has proposed that the entry of Ca 2÷ into chromaffin cells is itself a sufficient stimulus for catecholamine secretion, and that acetylcholine and other secretagogues act merely to increase the permeability of chromaffin cells to Ca 2÷

169 [ 3 ] . The intracellular events which are stimulated by Ca ~÷, and which lead to exocytosis, are still unknow n. We have developed m e t h o d s for studying catecholamine secretion by isolated adrenal cells. This r e p o r t describes the preparation of isolated adrenal cells f r o m guinea pig adrenal glands, and some aspects of catecholamine secretion by these cells. Materials and Methods Guinea pigs were killed by decapitation, and their adrenal glands (weighing ab o u t 50 mg each) were dissected free of surrounding tissue, rinsed in ice-cold Ca2÷-free Krebs-Ringer bicarbonate glucose buffer (Buffer I), and sliced with a razor blade into 1.5 m m slices (about 5 slices per gland). In most experiments, adrenal glands from three or four guinea pigs were used. The pooled adrenal slices were incubated at 37°C in 5 ml of Ca2÷-free Buffer I containing 0.5--2.0 mg collagenase/ml, under 95% O2/5% CO2, in a shaking water bath at 125 oscillations per min. After 30 min incubation, the tissue was dispersed by repeated (20--30 times) gentle pipetting through a wide bore (5 mm diameter) pipette, the supernatant was removed with a Pasteur pipette, and the undigested tissue was incubated with fresh collagenase solution. Collagenase digestion was cont i nued for four or five 30-min periods, by which time the adrenal medullas were almost completely dispersed. The supernatant obtained after the first collagenase digestion contained m a n y red blood cells and few adrenal cells, and so was discarded. Subsequent supernatants, which contained isolated adrenal cells, were centrifuged at 0°C for 7 min at 200 × g, and the cell pellets were washed once with 5 ml of Buffe~" I containing 2.2 mM Ca ~÷ and 5 mg/ml bovine serum albumin (Buffer II), resuspended in this buffer, and incubated at 37°C until the collagenase digestion was terminated. The pooled cells were c o u n t e d with a h e m o c y t o m e t e r , and were then centrifuged, washed once with ice-cold Buffer II, and resuspended in cold Buffer II. Aliquots of the cells were washed in Buffer I to remove the albumin, and their protein c o n t e n t was d eter min ed by the m e t h o d of Lowry et al. [ 7]. For the measurement of catecholamine secretion, about 3--4 • 10 s adrenal cells (containing a bout 200 pg cell protein) were incubated with the test substances in Buffer II, in a final volume of 1.5 ml. Unless otherwise indicated, samples were incubated for 10 min at 37°C in 95% O2/5% CO2, and were then rapidly chilled in an ice bath. Cells were removed by centrifugation, and the catecholamine c o n t e n t of the supernatant solutions was measured by the fluorimetric m e t h o d of Shore and Olin [ 8 ] . Oxidation was carried out at pH 5, in order to measure bot h epinephrine and norepinephrine. Catecholamine secretion was estimated after subtraction of the catecholamine c o n t e n t found in samples maintained at 0°C; this small background catecholamine c o n t e n t is presumably due to the release of catecholamines from dead or broken cells. Catecholamine secretion is expressed in terms of nm ol / m g cell protein. Each e x p er imen t was repeated several times, with similar results. However , because o f the quantitative variation in catecholamine secretion in different experiments, we have presented the results of representative individual experiments. Weanling Hartley-strain guinea pigs (150--200 gm) were obtained from the

170 Harvard Medical School colony or from Charles River Laboratories. A23187 was obtained from Eli Lilly Co., and X-537A was a gift of Hoffmann-La Roche. These compounds were both dissolved in methanol (1 mg/ml) before use. Collagenase was Type I, 150--250 units/mg, from Worthington Biochemicals Corp.; bovine serum albumin was Fraction V, from Sigma Chemical Co. All other chemicals were reagent grade. Glass distilled water was used throughout. Plastic flasks, centrifuge tubes and pipettes were used whenever possible. All glassware with which the adrenal cells came in contact was siliconized with Siliclad (Clay Adams). Buffer I contained 118 mM NaC1, 4.7 mM KC1, 1.2 mM KH2 PO~, 1.2 mM MgSO4,25 mM NaHCO3,10 mM glucose, and, unless otherwise indicated, 2.2 mM CaC12. Buffer II contained, in addition to the above ingredients, 5 mg/ml bovine serum albumin. These buffers were equilibrated with 95% O2/5% CO2 at room temperature before use. Results

When adrenal slices are incubated with collagenase and subjected to gentle mechanical agitation, the medulla is disrupted more rapidly than is the cortex. After several hours of incubation, a small hole can be seen in the center of the adrenal slices, while the periphery of the slices remains largly intact. By careful inspection of the slices during collagenase treatment, it is possible to prepare a chromaffin-rich suspension of isolated adrenal cells. The enrichment of adrenal cell suspensions for chromaffin cells was assessed by measuring the catecholamine content of these suspensions. Guinea pig adrenal glands contain about 10 nmoles of catecholamine per mg protein. In ten preparations, the catecholamine content of adrenal cell suspensions has ranged between 14 and 150 n m o l / mg protein, with a mean values of 52 nmol/mg protein. Epinephrine accounts for more than 95% of the total catecholamine content in intact guinea pig adrenal glands and in the isolated adrenal cell suspensions. In typical preparations, approximately 106 cells (about 0.7 mg of protein) are obtained per

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adrenal gland. The isolated cells contain about 15--20% of the total catecholamines in the adrenal glands. The yield of chromaffin cells has not yet been determined. When isolated adrenal cells are incubated at 37°C, they release a small a m o u n t of catecholamine into the incubation medium. The addition of acetylcholine to these cells produces a rapid burst of catecholamine secretion (Fig. 1). Although there is some quantitative variation in catecholamine secretion in different preparations, the stimulation of ca~echolamine secretion by acetylcholine is reproducible. In nine separate experiments, 10 -4 M acetylcholine stimulated catecholamine secretion between six and t w e n t y fold; in most experiments, acetylcholine increased secretion by more than ten fold. The absolute q u an tity of catecholamine secreted in these experiments varied between 5 and 67 nm ol / m g protein. The stimulation of catecholamine secretion by acetylcholine is dose-dependent. Fig. 2 shows a typical dose-response curve for this action o f acetylcholine. The stimulation of catecholamine secretion by acetylcholine was evident at 10 .6 M, was half-maximal at about 10 -4 M, and was maximal at 10 .3 M. In the experiments presented in Figs 1 and 2, prostigmine (10 -5 M) was added to all incubation flasks in order to prevent hydrolysis of acetylcholine by acetylcholinesterase. Control experiments showed that this concent rat i on of prostigmine by itself p r o d u c e d a negligible stimulation of catecholamine secretion, and that 10 -4 M acetylcholine stimulated catecholamine secretion equally well in the presence and absence of prostigmine. The response of isolated adrenal cells to acetylcholine was also characterized in terms of its susceptibility to pharmacological inhibition. The results o f these studies are summarized in Table I. The cholinergic blocking agents, atropine and h e x a m e t h o n i u m , both inhibit this action of acetylc,holine; at a c o n c e n t r a t i o n o f 10 -4 M, atropine is a more p o t e n t inhibitor than is hexam e t h o n iu m. In four separate experiments, the stimulation of cateeholamine

172 TABLE

I

STIMULATION

OF CATECHOLAMINE

SECRETION

BY ACETYLCHOLINE

Stimulation of catecholamine secretion by acetylcholine. Cells were incubated for 10 min at 37~C in Buffer II, unless otherwise indicated. In this and in subsequent tables, acetylcholine, atropine, and hexamethonium were added to a concentration of 0.1 mM, and tetracaine was used at a concentration of 0.5 mM. The results of two separate experiments are shown. Additions

Catecholamine Experiment

None Acetylcholine Acetylcholine Acetylcholine Acetylcholine

(0.1 mM) + atropine (0.1 mM) + hexamethonium (0.1 mM) + atropine (0.1 raM) + hexamethonium (0.1 mM) Acetylcholine + tetracaine (0.5 mM) Acetylcholine, no Ca + Acetylcholine, 0 ° C

secretion (nmol/mg)

1

Experiment

0.4 7.3 1.0 2.6

2.1 16.5 3.8 5.2

0.4 2.4 0.8 0.1

2.0 5.6 3.4 1.0

2

secretion produced by 10 -4 M acetylcholine was inhibited by more than 75% by 10 -4 M atropine. The response to acetylcholine can be completely inhibited by a combination of atropine and hexamethonium. The action of acetylcholine is also inhibited by the local anaesthetic, tetracaine. Furthermore, the stimulation of catecholamine secretion by acetylcholine is temperature dependent, and is abolished at 0 ° C. Finally, this response of chromaffin cells to acetylcholine is dependent upon extracellular Ca2÷; in the absence of added Ca 2÷, the response to acetylcholine is inhibited by about 90%. The dependence of acetylcholine-

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173 TABLE

II

STIMULATION Stimulation II m o d i f i e d shown. Incubation

OF CATECHOLAMINE

SECRETION

BY 56 mM K +

o f c a t e c h o l a m i n e s e c r e t i o n b y 5 6 m M K +. C e l l s w e r e i n c u b a t e d f o r 1 0 m i n a t 3 7 ° C i n B u f f e r t o c o n t a i n 5 6 m M K +, u n l e s s o t h e r w i s e i n d i c a t e d . T h e r e s u l t s o f t w o s e p a r a t e e x p e r i m e n t s a r e

conditions

Catecholamine Experiment

Buffer II 56 mM K + 56 mM K + + atropine (0.1 mM) + hexamethonium (0.1 raM) 56 mM K + + tetracaine (0.5 mM) 56 mM K + + no Ca 2+ 5 6 m M K +, 0 ° C

1

secretion (nmol/mg) Experiment

2.9 11.7

1.5 8.1

10.8 13.1 1.2 0.6

8.1 8.9 1.8 1.1

2

stimulated catecholamine secretion on the extracellular Ca 2÷ concent rat i on is illustrated in Fig. 3. Catecholamine secretion increases with increasing Ca 2÷ up to a c o n c e n t r a t i o n of 8.8 mM. Catecholamine secretion by isolated adrenal cells is also stimulated by 56 mM K÷ (Table II). The effect of 56 mM K ÷ is not inhibited by atropine and h e x a m e t h o n i u m , or by tetracaine, but, like the effect of acetylcholine, is almost co mp letely abolished in the absence of extracellular Ca 2÷. The role of Ca 2÷ in catecholamine secretion was studied further by use of the divalent cation ionophores, A23187 and X-537A. As shown in Fig. 4, A23187 produces a Ca~÷-dependent stimulation of catecholamine secretion. Catecholamine secretion in response to this i onophore is linear for at least 20 min, and is d e p e n d e n t on the i o n o p h o r e c o n c e n t r a t i o n (data n o t shown). In seven separate experiments, 10 pM A23187 has p r o d u c e d a greater than fivefold increase in catecholamine secretion. Some aspects of the effect of A23187 are characterized in Table III. The action of the i onophore is t e m p e r a t u r e dependent, but is n o t significantly inhibited by atropine and h e x a m e t h o n i u m , TABLE

III

STIMULATION

OF CATECHOLAMINE

SECRETION

BY IONOPHORES

Stimulation of catecholamine secretion by ionophores. Cells were incubated for 10 rain at 37°C in Buffer II, unless otherwise indicated. The ionophores A23187 and X-537A were used at a concentration of 0.01 mM. Additions

None Ionophore (0.01 mM) Ionophore + atropine (0.1 mM) + hexamethonium (0.1 mM) Ionophore + tetracaine (0.5 mM) Ionophore, no Ca 2+ Ionophore, 0°C

Catecholamine

secretion (nmol/mg)

A23187

X-537A

0.7 11.4

0.7 14.7

9.8 12.0 2.2 1.8

14.7 17.3 3.8 0.9

174 or by tetracalne. The ionophore X-537A also causes a Ca~÷-dependent stimulation of catecholamine secretion (Table III). The action of X-537A is similar to that of A23187. Because of the interest in the possible role of cyclic nucleotides in the regulation of catecholamine secretion, we also studied the effects of a number of nucleotides in isolated adrenal cells. A variety of nucleotides, including AMP, cyclic AMP, dibutyryl cyclic AMP, GMP, cyclic GMP, dibutyryl cyclic GMP, and 8-bromo cyclic GMP, were all found to stimulate catecholamine secretion (data not shown). However, the effects of these nucleotides is small in comparison to the effect of acetylcholine. In an experiment in which acetylcholine caused a twelve-fold increase in catecholamine release, the nucleotides stimulated catecholamine release approximately two fold. Furthermore, there is no specificity to this action of the nucleotides. Adenine nucleotides are as potent as guanine nucleotides, and 5'-nucleotides are as active as cyclic nucleotides. The effect of these nucleotides on catecholamine secretion has not been further characterized. Discussion

Isolated adrenal cell suspensions have been prepared by a number of investigators; our m e t h o d is a modification of that used by Kloppenborg et al. [9]. These isolated adrenal cell suspensions have been used for studies of adrenal cortical functions; isolated adrenal cells secrete glucocorticoids in response to adrenocorticotropin [9] and to various cyclic nucleotides [10]. Douglas et al. have performed electrophysiological studies on isolated adrenal cells in culture [11]. To our knowledge, the presence of functional chromaffin cells in isolated adrenal cell suspensions has not previously been reported. It is not possible to compare the properties of isolated adrenal cells with those of intact adrenal glands in great detail, because of differences in species and in incubation conditions. In general, however, catecholamine secretion by isolated adrenal cells is similar to secretion by intact, perfused adrenal glands. In both experimental systems, catecholamine secretion is stimulated by acetylcholine and by 56 mM K÷; in both systems, the effect of acetylcholine is inhibited by atropine and hexamethonium, and by tetracaine, and is dependent upon extracellular Ca 2÷. Isolated adrenal cells appear to be a useful system in which to study catecholamine secretion. The study of catecholamine secretion by the sequential stimulation of perfused adrenal glands is complicated by the fact that these glands may become depleted of catecholamines (or undergo other metabolic changes) during the course of the experiment. The measurement of catecholamine secretion during the simultaneous incubation of aliquots of chromaffin cell suspensions avoids this problem. Isolated cell suspensions lack vascular permeability barriers that might prevent the response of perfused adrenal glands to secretory stimuli. Finally, isolated chromaffin cells can be maintained in tissue culture. Cells prepared by this m e t h o d adhere to culture dishes, and continue to secrete catecholamines in response to acetylcholine and to 56 mM K ÷ (unpublished observations). The studies of Douglas have documented the important role of Ca 2÷ in the regulation of catecholamine secretion by perfused adrenal glands [3]. Recent-

175

l y , divalent cation ionophores have becom e available as pharmacological tools for studying the role of Ca 2÷ in biological systems [ 1 2 ] . We have f o u n d that t w o of these ionophores mimic the effect of acetylcholine in stimulating catecholamine secretion by adrenal cells. The fact that two different ionophores both stimulate catecholamine secretion, and the fact t hat the stimulation of catecholamine secretion by these c o m p o u n d s is d e p e n d e n t u p o n extracellular Ca 2÷, suggest that the effects of these c o m p o u n d s on catecholamine secretion are indeed due to their f unct i on as Ca ~÷ ionophores, and not to some unrelated biological activity. The action of these ionophores supports the hypothesis that the increased entry of Ca 2÷ into chromaffin cells is a sufficient stimulus to initiate the events that result in catecholamine secretion by these cells. Our findings do n o t provide evidence for an i m p o r t a n t role of cyclic nucleotides in the regulation of catecholamine secretion. Acknowledgments We thank Ms. Michele Carvotta for excellent technical assistance. We are grateful to Mr. Peter MacLeish for m any helpful discussions. This research was s u p p o r ted in part by research grant AM15135 from the National Institutes of Health. This wo r k was done during the tenure of a research grant-in-aid award from the American Heart Association, Greater Boston Massachusetts Chapter No. 1256. R.L.P. is the recipient of a research career d e v e l o p m e n t award, AM70648, from the National Institutes of Health. References 1 2 3 4 5 6 7 8 9

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