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Cyclic AMP inhibits secretion from bovine adrenal chromaffin cells evoked by carbamylcholine but not by high K+
Biochirnica et Biophysica Acta 846 (1985) 388-393 Elsevier
388
BBA 11552
Cyclic AMP
i n h i b i t s s e c r e t i o n f r o m b o v i n e a d r e n a l c h r o m a f f i n c e l l s e v o k e d by c a r b a m y l c h o l i n e b u t n o t by h i g h K ÷
Elizabeth M. Baker, Timothy
R. C h e e k a n d R o b e r t D . B u r g o y n e
*
M. R.C. Secreto~' Control Group, The Physiological Laboratory, University of Liverpool, P.O. Box 147, Liverpool, L69 3BX (U.K.) (Received April 25th, 1985)
Key words: Cyclic AMP; Carbamylchotine; K+: Catecholamine secretion; (Chromaffin cell)
The role of cAMP in the control of secretion from bovine adrenal chromaffin cells was examined using the adenylate cyclase activator, forskolin. Treatment of chromaffin cells with forskolin resulted in a rise in cAMP levels. Forskolin inhibited catecholamine release elicited by carbamylcholine or nicotine but had no effect on secretion evoked by 55 mM K +. Inhibition of carbamylcholine-stimulated release by forskolin was half-maximal at 10/~M forskolin. The inhibition by forskolin of secretion evoked by carbamylcholine was at a step distal to the rise in intracellular free calcium concentration ([Ca2+li), since this rise was not inhibited by forskolin, which itself produced a small rise in [Ca2+]i. The results suggest that secretion evoked by carbamylcholine is due to the activation of an additional second messenger pathway acting with the rise in [Ca2+] i. This additional pathway may be the target for cAMP action.
Introduction Calcium plays a major role in stimulus-secretion coupling in the adrenal chromaffin cell [1,2]. Nicotinic stimulation of these cells results in a rise in the intracellular free calcium concentration ([Ca2+]~) detectable using the calcium indicator, quirt2 [3,4]. We have suggested that calcium may not be the only signal generated by nicotine cholinergic agonists in chromaffin cells. High K + and the calcium ionophore A23187 raise [Ca2+]i to a level similar to or higher than that due to cholinergic agonists. However, high K + and A23187 result in a secretory response that is both smaller and shorter-lived than that elicited by nicotinic agonists * To whom correspondence should be addressed. Abbreviations: [Ca2+]i, the intracellular concentration of ionised calcium; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulphonic acid.
[3,4]. Furthermore, the calmodulin antagonist calmidazolium is more potent in inhibiting secretion due to carbamylcholine than that due to high K + or A23187 [5]. These data are consistent with the requirement for a signal, other than a rise in [Ca2+]~, for the generation of a full secretory response. The identity of the alternative signalling pathway is unknown, but both the breakdown of phosphoinositides [5-7] and a rise in cyclic G M P [8] can be ruled out, since these occur in response to muscarinic but not nicotinic agonists. A candidate for an additional second messenger in chromaffin cells is cyclic AMP. Nicotinic stimulation results in an elevation of cAMP levels in chromaffin cells [9,10] which leads to an increase in biosynthesis of secretory products [10,11]. The role of cAMP in secretion is unclear due to the contradictory nature of reports that cAMP analogues in some cases have a marginal stimulatory effect [12,13] and in others [11,14] have no effect on secretion.
0167-4889/85/$03.30 '~' 1985 Elsevier Science Publishers B.V. (Biomedical Division)
389
In order to determine the role of c A M P in secretion from chromaffin cells we have used the adenylate cyclase activator forskolin [15], which has been reported to elevate intracellular c A M P reliably over a wide range of cell types [16]. The results show that elevation of cAMP in chromaffin cells inhibits secretion elicited by carbamylcholine but has no effect on secretion elicited by high K ÷. Materials and Methods
Freshly excised bovine adrenal glands were transported from the local abattoir on ice. Chromaffin cells were dissociated from the isolated medullase by enzymatic digestion as previously described [17] in a Ca2+-free Krebs-Ringer buffer comprising 145 m M NaC1/5 m M KCI/1.3 mM MgCI2/1.2 mM N a H 2 P O 4 / 1 0 mM glucose/20 m M Hepes (pH 7.4), (buffer A). The cells were then washed twice by centrifugation in buffer A containing 3 mM CaCI 2 and 0.5% bovine serum albumin, (buffer B), examined using Trypan blue exclusion and phase-contrast microscopy and counted using a haemocytometer. All buffers were equilibrated with 95% 0 2 / 5 % CO 2. For determination of [Ca2+]i cells were loaded by incubation at room temperature with 10 ~M quin2 acetoxymethyl ester in buffer B for 30 min. The cells were washed and resuspended in buffer B and incubated for a further 40 min at. room temperature. After this time, the cells were washed by centrifugation and resuspended in buffer B without bovine serum albumin. The [Ca2+]i was continuously monitored at 20°C in a PerkinElkmer LS-5 luminescence spectrometer (excitation 339 nm; emission 492 nm) fitted with a magnetic stirrer. Calibration of fluorescence and calculation o f [Ca2+]i was as previously described [18], following lysis with 50 ~M digitonin and then addition of 40 m M EGTA. The results of all quin2 experiments were confirmed on more than one batch of cells. For catecholamine release, cells were incubated in buffer B for 1 h at room temperature, washed by centrifugation and resuspended in buffer B. Aliquots were added to centrifuge tubes containing appropriate concentrations of secretagogue. Incubation of the cells at room temperature for a specified time was followed by termination of the
reaction by the addition of an equal volume of ice-cold buffer A containing 20 m M EGTA. The cells were centrifuged at 14000 × g for 3 min in an MSE microcentaur and aliquots of the supernatants assayed for total catecholamine content (adrenaline and noradrenaline) by a previously described fluorimetric assay [19] using a PerkinElmer LS-5 luminescence spectrometer. Aliquots of the original cell suspension were assayed to determine total catecholamine content of the cells and released catecholamine was expressed as a percentage of this total value. For the assay of cAMP levels, cells were incubated with or without forskolin for 10 min at room temperature. After pelleting by centrifugation, the cells were lysed by resuspension in 4 mM E D T A / 5 mM Tris-HC1 (pH 7.5), boiled, sonicated, recentrifuged and the supernatant was taken for assay of cAMP using a cyclic A M P assay kit (Amersham International, Bucks, U.K.) as described in the manufactures' instructions. Results
Treatment of chromaffin cells with forskolin resulted in a rise in intacellular cAMP levels
3e o
~
32
28
i
24
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~ 2o
× ,
8r
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J
I
6
I
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s
4
Fig. 1. Effect of forskolin on intracellular levels of cAMP in bovine chromaffin cells. Aliquots of cells were incubated without or with varying concentrations of forskolin for 10 min. The cells were pelleted by centrifugation, lysed and assayed for cAMP. The data shown are means of duplicate determinations.
390
TABLE 1
T A B L E II
EFFECT OF FORSKOLIN AND CARBAMYLCHOLINE ON cAMP LEVELS IN BOVINE CHROMAFFIN CELLS
EFFECT OF FORSKOLIN ON CATECHOLAMINE RELEASE DUE TO CARBAMYLCHOLINE A N D 55 m M K +
A l i q u o t s of cells w e r e i n c u b a t e d with 30 ,aM f o r s k o l i n a n d 2 , 1 0 4 M c a r b a m y l c h o l i n e as i n d i c a t e d for 10 min. Cyclic A M P levels a r e s h o w n as m e a n s ± S.E, of three d e t e r m i n a t i o n s .
C h r o m a f f i n cells w e r e c h a l l e n g e d for 10 m i n with 2 . 1 0 4 M c a r b a m y l c h o l i n e or 55 m M K + in the p r e s e n c e or a b s e n c e of 30 ,aM forskolin. C a t e c h o l a m i n e release w a s e x p r e s s e d as a p e r c e n t a g e of total c a t e c h o l a m i n e a n d s h o w n as release a b o v e b a s a l levels. T h e d a t a s h o w n are m e a n s i S . E , of three determinations.
Carbamylcholine
Forskolin
C o n c e n t r a t i o n of cAMP (pmol/106 cells)
+ +
Forskolin
7.0±0.7 6.7±0.8 41.4±2.5 47.6+3.6
-
+ +
(Fig. 1). At the highest concentration tested (60 ~M), forskolin resulted in a 6.3-fold rise in cAMP concentration from a mean control level of 5.4 ± 0.15 p m o l / 1 0 6 cells. Similar levels of cAMP were attained when cells were challenged with forskolin in the presence of 2 - 1 0 -.4 M carbamylcholine (Table I). Carbamylcholine alone did not raise cAMP over the 10 min time-period used in the latter experiments (Table I). However, treatment of cells with 2 . 1 0 4 M carbamylcholine for 30 rain, by which time secretion had terminated (Fig. 2), resulted in a 2.2-fold rise in cAMP concentration from 6.6to14.5 p m o l / 1 0 6 cells (means of two determinations). The effect of 30/~M forskolin on catecholamine release is compared to the response to 2 . 1 0 4 M
+ + +
Carbamylcholine
+ + -
55 m M K +
~ ~
Catecholamine release (%)
Inhibition (%)
0 ±0.3 6.7±0.2 1.7±0.1 3.7±0.3 4.3±0.2
75 16
carbamylcholine in Fig. 2. Forskolin did not elicit secretion, nor did it have any effect on basal levels of release at any concentration tested in the range 0.1-60 ttM. Table II shows the effects on secretion of challenging chromaffin cells simultaneously with 30 /xM forskolin and either carbamylcholine or 55 m M K +. Forskolin almost completely abolished secretion due to carbamylcholine but had no effect on secretion elicited by 55 mM K ÷. The possibility existed that the faster secretory response due to 55
10
T A B L E 1II J
J
8
EFFECT OF FORSKOLIN ON CATECHOLAMINE RELEASE DUE TO CARBAMYLCHOLINE AND NICOTINE
,.0, (/)
C h r o m a f f i n cells w e r e c h a l l e n g e d for 10 m i n w i t h 2 . 1 0 4 M c a r b a m y l c h o l i n e o r 10 5 M n i c o t i n e in the p r e s e n c e or a b s e n c e of 30 ,aM forskolin. C a t e c h o l a m i n e release w a s e x p r e s s e d as a p e r c e n t a g e o f total c a t e c h o l a m i n e a n d s h o w n as release a b o v e b a s a l levels. T h e d a t a s h o w n are m e a n s i S . E , of three determinations.
¢[
.=
8 ~2 .- r 2
A 4
-
I 6
~ 8
I 10
I 12
1~4
116
I 18
Forskolin
Carbamylcholine
+ +
+ + -
1
20
Nicotine
22
TIME (min$)
Fig. 2. T i m e - c o u r s e of c a t e c h o l a m i n e release f r o m c h r o m a f f i n cells in r e s p o n s e to 2 . 1 0 4 M c a r b a m y i c h o l i n e ( e ) or 30 ,aM f o r s k o l i n (B). R e l e a s e d c a t e c h o l a m i n e w a s e x p r e s s e d as a perc e n t a g e of total c a t e c h o l a m i n e a n d is s h o w n as release a b o v e b a s a l levels.
+
+ +
Catecholamine release (%) 0.5±0.4 5.1±0.4 1.3±0.3 4.9±0.3 1.6±0.1
Inhibition
83 76
391
lO
i-Ca2+3i nM
9 8
300--
~
7
lOO-
~wl
6
Ca~'b
2O0-
E gl
z
5
Z ~ O
Carb + Forsk
4
b
Z
o
~
300-200--
3
U
1OO--
2 1
I-----4
I 5
I 4
I 3
lmin Forsk
r
1
-LOG ~ARBAMYLCHOLINE~ M
Fig. 3. Effect of forskolin on catecholamine release stimulated by carbamylcholine. Chromaffin cells were challenged with a range of concentrations of carbamylcholine in the presence (11) or absence (e) of 30 ,aM forskolin. Catecholamine released within 10 min was expressed as a percentage of total cellular catecholamine. Data are means + S.E. of these determinations.
mM K + [4] was already completed before cAMP was raised sufficiently to inhibit secretion. However, almost identical results were obtained in experiments in which chromaffin cells were preincubated with 30 ~ M forskolin for 5 min prior to the addition of carbamylcholine or 55 mM K +.
c
200--
100-
5. Effect of carbamylcholine (a), carbamylcholine plus forskolin (b), or forskolin (c) on [Ca 2÷ ]i- Concentration of carbamylcholine (Carb.) 2-10 4 M; concentration of forskolin (Forsk.), 30 ,aM. Fig.
The inhibitory effect of forskolin was due to inhibition of secretion elicited by nicotinic receptor activation, since nicotine-stimulated catecholamine release was inhibited by forskolin to the same 14
8C-
C~ -r ~4 4C U 20
-LOG Q~ARBAMYLCHOLINE3(MI 7
6
5
I
4
Fig. 4. Concentration dependence of inhibition of carbamylcholine-stimulated release by forskolin. Chromaffin cells were challenged with 2-10 4 M carbamylcholine in the presence of a range of concentrations of forskolin for 10 min. Data are means of two or three determinations.
Fig. 6. Effect of a low dose of forskolin on catecholamine release due to carbamylcholine. Chromaffin cells were challenged with a range of concentrations of carbamylcholine in the presence (D) or absence (B) of 1 ,aM forskolin. Catecholamine released within 20 min was expressed as a percentage of total cellular catecholamine. Data are m e a n s + S . E , of three determinations. In some cases S.E. is not shown as this was smaller than the symbol used.
392 extent as that elicited by carbamylcholine (Table III). The inhibition of carbamylcholine-stimulated catecholamine release by forskolin was not overcome by increasing the concentration of carbamylcholine (Fig. 3). The concentration dependence of inhibition of carbamylcholine-stimulated release by forskolin is shown in Fig. 4. Inhibition was half-maximal at a forskolin concentration of approx. 10 /~M. By comparison with Fig. 1 it is apparent that halfmaximal inhibition of carbamylcholine-stimulated secretion occurs at a forskolin concentration that produces a 2.7-fold increase in the level of intracellular cAMP over the 10 rain time-period studied. Forskolin over the concentration range 0.1 60 ~M had no effect on catecholamine release elicited by 55 mM K +. The possibility that the inhibitory effect of forskolin on carbamylcholine-stimulated catecholamine release was due to inhibition of Ca 2+ influx and the subsequent rise in [Ca2+]i was examined using the fluorescent calcium indicator quin2 (Fig. 5). The rise in [Ca2+]i due to carbamylcholine was not inhibited by forskolin. However, the timecourse of the changes in [Ca2+]i due to carbamylcholine was apparently modified by forskolin. Carbamylcholine challenge resulted in a rise in [Ca2+]i from a basal level of 145 +_ 5 nM (n = 13) to 313 +_ 17 nM (n = 4 ) with the peak level of [Ca2+]i reached within 15 s (Fig. 5A). Simultaneous challenge with carbamylcholine and 30 ~M forskolin resulted in a rise in [Ca2+]~ to 373 + 23 nM (n = 4) with the peak level of [Ca2+]i reached within 60-90 s. The longer-lasting rise in [Ca2+]i to a higher level could be accounted for by a rise in [Ca2+]i due to forskolin itself (Fig. 5C). The forskolin-mediated rise in [Ca2+]i occurred after a lag of 20-30 s, peaked within 60-90 s resulting in an increase in [Ca2+]i to 42 _+ 5 nM (n = 5) above the resting level and w a s not affected by the removal of external Ca 2+ (not shown). The timecourse shown in Fig. 5B can be attributed to a combination of a fast rise in [Ca2+]i due to carbamylcholine and a slow long-lasting rise due to forskolin. The small additional rise in [Ca2+]i due to forskolin is unlikely to be related to the inhibition of carbamylcholine-stimulated catecholamine release, since this rise was also elicited by 1 /~M
forskolin (not shown), a concentration that had no effect on secretion due to carbamylcholine. In PC12 cells [20], forskolin has biphasic effects on secretion and therefore the possibility that a low level of forskolin, that raised [Ca2+]i and cAMP, could potentiate carbamylcholine-stimulated catecholamine release was tested by challenging cells with 1 ~M forskolin and submaximal doses of carbamylcholine (Fig. 6). Under these conditions forskolin had no effect on the level of catecholamine released. Discussion Since nicotinic cholinergic agonists elevate c A M P in adrenal chromaffin cells [9,10] we examined the possible involvement of cAMP in a signalling pathway leading to secretion, by using the adenylate cyclase activator forskolin. Surprisingly, the present results indicate that cAMP may be involved in an inhibitory pathway in bovine adrenal chromaffin cells. The inhibitory effect of elevated cAMP seems to have as its target those intracellular events associated with secretion elicited by cholinergic nicotinic agonists but not those associated with secretion due to high K +. It has been commonly supposed [17] that the intracellular mechanisms by which nicotinic agonists and high K + elicit secretion from chromaffin cells are identical. The only difference between the two situations being that depolarisation by high K + bypasses receptor-linked membrane depolarisation and directly depolarises the membrane to open voltage-dependent calcium channels. In each case the subsequent rise in [Ca2+], would bring about the secretory response. The present results do not support such a simple interpretation. The inhibition of carbamylcholine-stimulated secretion by forskolin appears to be due to an effect on a step distal to Ca 2+ entry, since forskolin did not inhibit the rise in [Ca2+]~ due to carbamylcholine. If secretion elicited by both carbamylcholine and high K + was simply a consequence of the rise in [Ca2+],, then secretion stimulated by both agents should be equally sensitive to inhibitory factors acting distal to the rise in [Ca2+],. It should be noted that the present results are consistent with the observation that cAMP did
393
not affect secretion triggered by raising intracellular Ca 2+ in leaky chromaffin cells [21]. The site of action of cAMP in the secretory pathway is unknown. In both platelets [22] and neutrophils [23] elevating cAMP inhibits secretory responses independent of effects on [Ca2]i. The action of cAMP in these cell types may be due to inhibition of phosphatydylinositol turnover [24]. In the case of bovine chromaffin cells such an explanation for the inhibitory effects of cAMP can be excluded; phosphatidylinositol turnover in these cells is linked to muscarinic but not nicotinic receptors [25] and does not appear to play any significant role in the secretory response [6]. Does the cAMP-dependent inhibitory pathway function during the normal secretory events in chromaffin cells? Forskolin inhibits carbamylcholine-induced secretion half-maximally at a concentration that produces a 2.7-fold rise in cAMP. Elevation of cAMP to this extent does occur in chromaffin cells challenged with nicotinic agonists [9A0,26]. However, as we have shown, the rise in c A M P is slower than secretion and thus could play a role in the termination of the secretory response. Elevation of c A M P in chromaffin cells by forskolin has a second effect which appears to be independent of its effect on secretion. Forskolin alone or in combination with carbamylcholine produces a small long-lasting rise in [Ca2+]i . This rise occurs with a similar lag to that of the rise in [Ca2+]i due to release of Ca 2+ from internal stores by muscarinic agonists [6]. The rise in [CaZ+]i produced by forskolin is unaffected by the removal of external Ca 2+ and is probably a result of the release of Ca 2+ from internal stores. The present results add further support to the notion that nicotinic cholinergic agonists do not trigger secretion simply by raising [Ca2+]i but use an additional unknown signalling pathway. This additional pathway may be the target for the inhibitory action of cAMP. Acknowledgements We thank Mrs. K.M. Norman for technical assistance. T.C. was in receipt of an M.R.C. Re-
search Studentship. This work was supported by a project grant from the M.R.C. References 1 Burgoyne, R.D. (1984) Biochim. Biophys. Acta 779,201-216 2 Baker, P.F. and Knight, D.E. (1982) Philos. Trans. R. Soc. Lond. B. 296, 83 103 3 Burgoyne, R.D. (1984) Biosci. Rep. 4. 605-611 4 Burgoyne, R.D. and Cheek, T.R. (1985) FEBS Lett. 182, 115-118 5 Burgoyne, R.D. and Norman. K.M. (1984) Biochim. Biophys Acta 805, 37-43 6 Cheek, T.R. and Burgoyne, R.D. (1985) Biochim. Biophys Acta 846, 167 173 7 Swilem, A.-M.F. and Hawthorne, J.N. (1985) Biochem. Soc. Trans. 13, 184-185 8 Derome, G., Tseng, R., Mercier, P., Lemaire, J. and Lemaire, S. (1981) Biochem. Pharmacol. 30, 855-860 9 Guidotti, A. and Costa, E. (1974) J. Pharmacol. Exp. Ther. 189, 655-675 10 Eiden. L.E., Giraud, P., Dave, J.R., Hotchkiss, A.J. and Affolter, H.-U. (1984) Nature 312, 661-663 11 Kumakura, K., Guidotti, A. and Costa, E. (1979) Mol. Pharmacol. 16, 865-876 12 Greenberg, A. and Zinder, O. (1982) Cell Tiss. Res. 226, 655-665. 13 Michener, M.J. and Peach, M.J. (1984) Biochem. Pharmacol. 33, 1819-1823 14 Hochman, J. and Perlman, R.L. (1976) Biochim. Biophys. Acta 421, 168-175 15 Seamon, K.B., Padgett, N. and Daly, J.W. (1981) Proc. Natl, Acad. Sci. USA 78, 3363-3367 16 Seamon, K.B. and Daly, J.W. (1983) Trends. Pharmacol. Sci. 4, 120-123 17 Knight, D.E. and Baker, P.F. (1983) Q. J. Exp. Physiol. 68, 123-143 18 Tsien, R.Y., Pozzan, T. and Rink, T.J. (1982) J. Cell Biol. 94, 325-334 19 Von Euler, U.S. and Floding, 1. (1955) Acta Physiol. Scand. Suppl. 118, 45-56 20 Rabe, C.S.. Schneider, J. and McGee (1982) J. Cyclic Nucleotide Res. 8, 371-384 21 Niggli, V., Knight, D.E., Baker, P.F., Vigny, A. and Henry, J.P. (1984). J. Neurochem. 43, 646-658 22 Rink, T.J. and Sanchez, A. (1984) Biochem. J. 222, 833-836 23 De Togni, P., Cabrini, G. and Di Virgillo, F. (1984) Biochem. J. 224, 629-635 24 Knight, D.E. and Scrutton. M.C. (1984) Nature 309, 66-68 25 Fisher, S.K., Holz, R.W. and Agranoff, B.W. (1981) J. Neurochem. 37, 491-497 26 Schneider, A.S., Cline, H.T. and Lemaire, S. (1979) Life Sci. 24, 1389-1394
388
BBA 11552
Cyclic AMP
i n h i b i t s s e c r e t i o n f r o m b o v i n e a d r e n a l c h r o m a f f i n c e l l s e v o k e d by c a r b a m y l c h o l i n e b u t n o t by h i g h K ÷
Elizabeth M. Baker, Timothy
R. C h e e k a n d R o b e r t D . B u r g o y n e
*
M. R.C. Secreto~' Control Group, The Physiological Laboratory, University of Liverpool, P.O. Box 147, Liverpool, L69 3BX (U.K.) (Received April 25th, 1985)
Key words: Cyclic AMP; Carbamylchotine; K+: Catecholamine secretion; (Chromaffin cell)
The role of cAMP in the control of secretion from bovine adrenal chromaffin cells was examined using the adenylate cyclase activator, forskolin. Treatment of chromaffin cells with forskolin resulted in a rise in cAMP levels. Forskolin inhibited catecholamine release elicited by carbamylcholine or nicotine but had no effect on secretion evoked by 55 mM K +. Inhibition of carbamylcholine-stimulated release by forskolin was half-maximal at 10/~M forskolin. The inhibition by forskolin of secretion evoked by carbamylcholine was at a step distal to the rise in intracellular free calcium concentration ([Ca2+li), since this rise was not inhibited by forskolin, which itself produced a small rise in [Ca2+]i. The results suggest that secretion evoked by carbamylcholine is due to the activation of an additional second messenger pathway acting with the rise in [Ca2+] i. This additional pathway may be the target for cAMP action.
Introduction Calcium plays a major role in stimulus-secretion coupling in the adrenal chromaffin cell [1,2]. Nicotinic stimulation of these cells results in a rise in the intracellular free calcium concentration ([Ca2+]~) detectable using the calcium indicator, quirt2 [3,4]. We have suggested that calcium may not be the only signal generated by nicotine cholinergic agonists in chromaffin cells. High K + and the calcium ionophore A23187 raise [Ca2+]i to a level similar to or higher than that due to cholinergic agonists. However, high K + and A23187 result in a secretory response that is both smaller and shorter-lived than that elicited by nicotinic agonists * To whom correspondence should be addressed. Abbreviations: [Ca2+]i, the intracellular concentration of ionised calcium; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulphonic acid.
[3,4]. Furthermore, the calmodulin antagonist calmidazolium is more potent in inhibiting secretion due to carbamylcholine than that due to high K + or A23187 [5]. These data are consistent with the requirement for a signal, other than a rise in [Ca2+]~, for the generation of a full secretory response. The identity of the alternative signalling pathway is unknown, but both the breakdown of phosphoinositides [5-7] and a rise in cyclic G M P [8] can be ruled out, since these occur in response to muscarinic but not nicotinic agonists. A candidate for an additional second messenger in chromaffin cells is cyclic AMP. Nicotinic stimulation results in an elevation of cAMP levels in chromaffin cells [9,10] which leads to an increase in biosynthesis of secretory products [10,11]. The role of cAMP in secretion is unclear due to the contradictory nature of reports that cAMP analogues in some cases have a marginal stimulatory effect [12,13] and in others [11,14] have no effect on secretion.
0167-4889/85/$03.30 '~' 1985 Elsevier Science Publishers B.V. (Biomedical Division)
389
In order to determine the role of c A M P in secretion from chromaffin cells we have used the adenylate cyclase activator forskolin [15], which has been reported to elevate intracellular c A M P reliably over a wide range of cell types [16]. The results show that elevation of cAMP in chromaffin cells inhibits secretion elicited by carbamylcholine but has no effect on secretion elicited by high K ÷. Materials and Methods
Freshly excised bovine adrenal glands were transported from the local abattoir on ice. Chromaffin cells were dissociated from the isolated medullase by enzymatic digestion as previously described [17] in a Ca2+-free Krebs-Ringer buffer comprising 145 m M NaC1/5 m M KCI/1.3 mM MgCI2/1.2 mM N a H 2 P O 4 / 1 0 mM glucose/20 m M Hepes (pH 7.4), (buffer A). The cells were then washed twice by centrifugation in buffer A containing 3 mM CaCI 2 and 0.5% bovine serum albumin, (buffer B), examined using Trypan blue exclusion and phase-contrast microscopy and counted using a haemocytometer. All buffers were equilibrated with 95% 0 2 / 5 % CO 2. For determination of [Ca2+]i cells were loaded by incubation at room temperature with 10 ~M quin2 acetoxymethyl ester in buffer B for 30 min. The cells were washed and resuspended in buffer B and incubated for a further 40 min at. room temperature. After this time, the cells were washed by centrifugation and resuspended in buffer B without bovine serum albumin. The [Ca2+]i was continuously monitored at 20°C in a PerkinElkmer LS-5 luminescence spectrometer (excitation 339 nm; emission 492 nm) fitted with a magnetic stirrer. Calibration of fluorescence and calculation o f [Ca2+]i was as previously described [18], following lysis with 50 ~M digitonin and then addition of 40 m M EGTA. The results of all quin2 experiments were confirmed on more than one batch of cells. For catecholamine release, cells were incubated in buffer B for 1 h at room temperature, washed by centrifugation and resuspended in buffer B. Aliquots were added to centrifuge tubes containing appropriate concentrations of secretagogue. Incubation of the cells at room temperature for a specified time was followed by termination of the
reaction by the addition of an equal volume of ice-cold buffer A containing 20 m M EGTA. The cells were centrifuged at 14000 × g for 3 min in an MSE microcentaur and aliquots of the supernatants assayed for total catecholamine content (adrenaline and noradrenaline) by a previously described fluorimetric assay [19] using a PerkinElmer LS-5 luminescence spectrometer. Aliquots of the original cell suspension were assayed to determine total catecholamine content of the cells and released catecholamine was expressed as a percentage of this total value. For the assay of cAMP levels, cells were incubated with or without forskolin for 10 min at room temperature. After pelleting by centrifugation, the cells were lysed by resuspension in 4 mM E D T A / 5 mM Tris-HC1 (pH 7.5), boiled, sonicated, recentrifuged and the supernatant was taken for assay of cAMP using a cyclic A M P assay kit (Amersham International, Bucks, U.K.) as described in the manufactures' instructions. Results
Treatment of chromaffin cells with forskolin resulted in a rise in intacellular cAMP levels
3e o
~
32
28
i
24
T
~ 2o
× ,
8r
1
J
I
6
I
I
s
4
Fig. 1. Effect of forskolin on intracellular levels of cAMP in bovine chromaffin cells. Aliquots of cells were incubated without or with varying concentrations of forskolin for 10 min. The cells were pelleted by centrifugation, lysed and assayed for cAMP. The data shown are means of duplicate determinations.
390
TABLE 1
T A B L E II
EFFECT OF FORSKOLIN AND CARBAMYLCHOLINE ON cAMP LEVELS IN BOVINE CHROMAFFIN CELLS
EFFECT OF FORSKOLIN ON CATECHOLAMINE RELEASE DUE TO CARBAMYLCHOLINE A N D 55 m M K +
A l i q u o t s of cells w e r e i n c u b a t e d with 30 ,aM f o r s k o l i n a n d 2 , 1 0 4 M c a r b a m y l c h o l i n e as i n d i c a t e d for 10 min. Cyclic A M P levels a r e s h o w n as m e a n s ± S.E, of three d e t e r m i n a t i o n s .
C h r o m a f f i n cells w e r e c h a l l e n g e d for 10 m i n with 2 . 1 0 4 M c a r b a m y l c h o l i n e or 55 m M K + in the p r e s e n c e or a b s e n c e of 30 ,aM forskolin. C a t e c h o l a m i n e release w a s e x p r e s s e d as a p e r c e n t a g e of total c a t e c h o l a m i n e a n d s h o w n as release a b o v e b a s a l levels. T h e d a t a s h o w n are m e a n s i S . E , of three determinations.
Carbamylcholine
Forskolin
C o n c e n t r a t i o n of cAMP (pmol/106 cells)
+ +
Forskolin
7.0±0.7 6.7±0.8 41.4±2.5 47.6+3.6
-
+ +
(Fig. 1). At the highest concentration tested (60 ~M), forskolin resulted in a 6.3-fold rise in cAMP concentration from a mean control level of 5.4 ± 0.15 p m o l / 1 0 6 cells. Similar levels of cAMP were attained when cells were challenged with forskolin in the presence of 2 - 1 0 -.4 M carbamylcholine (Table I). Carbamylcholine alone did not raise cAMP over the 10 min time-period used in the latter experiments (Table I). However, treatment of cells with 2 . 1 0 4 M carbamylcholine for 30 rain, by which time secretion had terminated (Fig. 2), resulted in a 2.2-fold rise in cAMP concentration from 6.6to14.5 p m o l / 1 0 6 cells (means of two determinations). The effect of 30/~M forskolin on catecholamine release is compared to the response to 2 . 1 0 4 M
+ + +
Carbamylcholine
+ + -
55 m M K +
~ ~
Catecholamine release (%)
Inhibition (%)
0 ±0.3 6.7±0.2 1.7±0.1 3.7±0.3 4.3±0.2
75 16
carbamylcholine in Fig. 2. Forskolin did not elicit secretion, nor did it have any effect on basal levels of release at any concentration tested in the range 0.1-60 ttM. Table II shows the effects on secretion of challenging chromaffin cells simultaneously with 30 /xM forskolin and either carbamylcholine or 55 m M K +. Forskolin almost completely abolished secretion due to carbamylcholine but had no effect on secretion elicited by 55 mM K ÷. The possibility existed that the faster secretory response due to 55
10
T A B L E 1II J
J
8
EFFECT OF FORSKOLIN ON CATECHOLAMINE RELEASE DUE TO CARBAMYLCHOLINE AND NICOTINE
,.0, (/)
C h r o m a f f i n cells w e r e c h a l l e n g e d for 10 m i n w i t h 2 . 1 0 4 M c a r b a m y l c h o l i n e o r 10 5 M n i c o t i n e in the p r e s e n c e or a b s e n c e of 30 ,aM forskolin. C a t e c h o l a m i n e release w a s e x p r e s s e d as a p e r c e n t a g e o f total c a t e c h o l a m i n e a n d s h o w n as release a b o v e b a s a l levels. T h e d a t a s h o w n are m e a n s i S . E , of three determinations.
¢[
.=
8 ~2 .- r 2
A 4
-
I 6
~ 8
I 10
I 12
1~4
116
I 18
Forskolin
Carbamylcholine
+ +
+ + -
1
20
Nicotine
22
TIME (min$)
Fig. 2. T i m e - c o u r s e of c a t e c h o l a m i n e release f r o m c h r o m a f f i n cells in r e s p o n s e to 2 . 1 0 4 M c a r b a m y i c h o l i n e ( e ) or 30 ,aM f o r s k o l i n (B). R e l e a s e d c a t e c h o l a m i n e w a s e x p r e s s e d as a perc e n t a g e of total c a t e c h o l a m i n e a n d is s h o w n as release a b o v e b a s a l levels.
+
+ +
Catecholamine release (%) 0.5±0.4 5.1±0.4 1.3±0.3 4.9±0.3 1.6±0.1
Inhibition
83 76
391
lO
i-Ca2+3i nM
9 8
300--
~
7
lOO-
~wl
6
Ca~'b
2O0-
E gl
z
5
Z ~ O
Carb + Forsk
4
b
Z
o
~
300-200--
3
U
1OO--
2 1
I-----4
I 5
I 4
I 3
lmin Forsk
r
1
-LOG ~ARBAMYLCHOLINE~ M
Fig. 3. Effect of forskolin on catecholamine release stimulated by carbamylcholine. Chromaffin cells were challenged with a range of concentrations of carbamylcholine in the presence (11) or absence (e) of 30 ,aM forskolin. Catecholamine released within 10 min was expressed as a percentage of total cellular catecholamine. Data are means + S.E. of these determinations.
mM K + [4] was already completed before cAMP was raised sufficiently to inhibit secretion. However, almost identical results were obtained in experiments in which chromaffin cells were preincubated with 30 ~ M forskolin for 5 min prior to the addition of carbamylcholine or 55 mM K +.
c
200--
100-
5. Effect of carbamylcholine (a), carbamylcholine plus forskolin (b), or forskolin (c) on [Ca 2÷ ]i- Concentration of carbamylcholine (Carb.) 2-10 4 M; concentration of forskolin (Forsk.), 30 ,aM. Fig.
The inhibitory effect of forskolin was due to inhibition of secretion elicited by nicotinic receptor activation, since nicotine-stimulated catecholamine release was inhibited by forskolin to the same 14
8C-
C~ -r ~4 4C U 20
-LOG Q~ARBAMYLCHOLINE3(MI 7
6
5
I
4
Fig. 4. Concentration dependence of inhibition of carbamylcholine-stimulated release by forskolin. Chromaffin cells were challenged with 2-10 4 M carbamylcholine in the presence of a range of concentrations of forskolin for 10 min. Data are means of two or three determinations.
Fig. 6. Effect of a low dose of forskolin on catecholamine release due to carbamylcholine. Chromaffin cells were challenged with a range of concentrations of carbamylcholine in the presence (D) or absence (B) of 1 ,aM forskolin. Catecholamine released within 20 min was expressed as a percentage of total cellular catecholamine. Data are m e a n s + S . E , of three determinations. In some cases S.E. is not shown as this was smaller than the symbol used.
392 extent as that elicited by carbamylcholine (Table III). The inhibition of carbamylcholine-stimulated catecholamine release by forskolin was not overcome by increasing the concentration of carbamylcholine (Fig. 3). The concentration dependence of inhibition of carbamylcholine-stimulated release by forskolin is shown in Fig. 4. Inhibition was half-maximal at a forskolin concentration of approx. 10 /~M. By comparison with Fig. 1 it is apparent that halfmaximal inhibition of carbamylcholine-stimulated secretion occurs at a forskolin concentration that produces a 2.7-fold increase in the level of intracellular cAMP over the 10 rain time-period studied. Forskolin over the concentration range 0.1 60 ~M had no effect on catecholamine release elicited by 55 mM K +. The possibility that the inhibitory effect of forskolin on carbamylcholine-stimulated catecholamine release was due to inhibition of Ca 2+ influx and the subsequent rise in [Ca2+]i was examined using the fluorescent calcium indicator quin2 (Fig. 5). The rise in [Ca2+]i due to carbamylcholine was not inhibited by forskolin. However, the timecourse of the changes in [Ca2+]i due to carbamylcholine was apparently modified by forskolin. Carbamylcholine challenge resulted in a rise in [Ca2+]i from a basal level of 145 +_ 5 nM (n = 13) to 313 +_ 17 nM (n = 4 ) with the peak level of [Ca2+]i reached within 15 s (Fig. 5A). Simultaneous challenge with carbamylcholine and 30 ~M forskolin resulted in a rise in [Ca2+]~ to 373 + 23 nM (n = 4) with the peak level of [Ca2+]i reached within 60-90 s. The longer-lasting rise in [Ca2+]i to a higher level could be accounted for by a rise in [Ca2+]i due to forskolin itself (Fig. 5C). The forskolin-mediated rise in [Ca2+]i occurred after a lag of 20-30 s, peaked within 60-90 s resulting in an increase in [Ca2+]i to 42 _+ 5 nM (n = 5) above the resting level and w a s not affected by the removal of external Ca 2+ (not shown). The timecourse shown in Fig. 5B can be attributed to a combination of a fast rise in [Ca2+]i due to carbamylcholine and a slow long-lasting rise due to forskolin. The small additional rise in [Ca2+]i due to forskolin is unlikely to be related to the inhibition of carbamylcholine-stimulated catecholamine release, since this rise was also elicited by 1 /~M
forskolin (not shown), a concentration that had no effect on secretion due to carbamylcholine. In PC12 cells [20], forskolin has biphasic effects on secretion and therefore the possibility that a low level of forskolin, that raised [Ca2+]i and cAMP, could potentiate carbamylcholine-stimulated catecholamine release was tested by challenging cells with 1 ~M forskolin and submaximal doses of carbamylcholine (Fig. 6). Under these conditions forskolin had no effect on the level of catecholamine released. Discussion Since nicotinic cholinergic agonists elevate c A M P in adrenal chromaffin cells [9,10] we examined the possible involvement of cAMP in a signalling pathway leading to secretion, by using the adenylate cyclase activator forskolin. Surprisingly, the present results indicate that cAMP may be involved in an inhibitory pathway in bovine adrenal chromaffin cells. The inhibitory effect of elevated cAMP seems to have as its target those intracellular events associated with secretion elicited by cholinergic nicotinic agonists but not those associated with secretion due to high K +. It has been commonly supposed [17] that the intracellular mechanisms by which nicotinic agonists and high K + elicit secretion from chromaffin cells are identical. The only difference between the two situations being that depolarisation by high K + bypasses receptor-linked membrane depolarisation and directly depolarises the membrane to open voltage-dependent calcium channels. In each case the subsequent rise in [Ca2+], would bring about the secretory response. The present results do not support such a simple interpretation. The inhibition of carbamylcholine-stimulated secretion by forskolin appears to be due to an effect on a step distal to Ca 2+ entry, since forskolin did not inhibit the rise in [Ca2+]~ due to carbamylcholine. If secretion elicited by both carbamylcholine and high K + was simply a consequence of the rise in [Ca2+],, then secretion stimulated by both agents should be equally sensitive to inhibitory factors acting distal to the rise in [Ca2+],. It should be noted that the present results are consistent with the observation that cAMP did
393
not affect secretion triggered by raising intracellular Ca 2+ in leaky chromaffin cells [21]. The site of action of cAMP in the secretory pathway is unknown. In both platelets [22] and neutrophils [23] elevating cAMP inhibits secretory responses independent of effects on [Ca2]i. The action of cAMP in these cell types may be due to inhibition of phosphatydylinositol turnover [24]. In the case of bovine chromaffin cells such an explanation for the inhibitory effects of cAMP can be excluded; phosphatidylinositol turnover in these cells is linked to muscarinic but not nicotinic receptors [25] and does not appear to play any significant role in the secretory response [6]. Does the cAMP-dependent inhibitory pathway function during the normal secretory events in chromaffin cells? Forskolin inhibits carbamylcholine-induced secretion half-maximally at a concentration that produces a 2.7-fold rise in cAMP. Elevation of cAMP to this extent does occur in chromaffin cells challenged with nicotinic agonists [9A0,26]. However, as we have shown, the rise in c A M P is slower than secretion and thus could play a role in the termination of the secretory response. Elevation of c A M P in chromaffin cells by forskolin has a second effect which appears to be independent of its effect on secretion. Forskolin alone or in combination with carbamylcholine produces a small long-lasting rise in [Ca2+]i . This rise occurs with a similar lag to that of the rise in [Ca2+]i due to release of Ca 2+ from internal stores by muscarinic agonists [6]. The rise in [CaZ+]i produced by forskolin is unaffected by the removal of external Ca 2+ and is probably a result of the release of Ca 2+ from internal stores. The present results add further support to the notion that nicotinic cholinergic agonists do not trigger secretion simply by raising [Ca2+]i but use an additional unknown signalling pathway. This additional pathway may be the target for the inhibitory action of cAMP. Acknowledgements We thank Mrs. K.M. Norman for technical assistance. T.C. was in receipt of an M.R.C. Re-
search Studentship. This work was supported by a project grant from the M.R.C. References 1 Burgoyne, R.D. (1984) Biochim. Biophys. Acta 779,201-216 2 Baker, P.F. and Knight, D.E. (1982) Philos. Trans. R. Soc. Lond. B. 296, 83 103 3 Burgoyne, R.D. (1984) Biosci. Rep. 4. 605-611 4 Burgoyne, R.D. and Cheek, T.R. (1985) FEBS Lett. 182, 115-118 5 Burgoyne, R.D. and Norman. K.M. (1984) Biochim. Biophys Acta 805, 37-43 6 Cheek, T.R. and Burgoyne, R.D. (1985) Biochim. Biophys Acta 846, 167 173 7 Swilem, A.-M.F. and Hawthorne, J.N. (1985) Biochem. Soc. Trans. 13, 184-185 8 Derome, G., Tseng, R., Mercier, P., Lemaire, J. and Lemaire, S. (1981) Biochem. Pharmacol. 30, 855-860 9 Guidotti, A. and Costa, E. (1974) J. Pharmacol. Exp. Ther. 189, 655-675 10 Eiden. L.E., Giraud, P., Dave, J.R., Hotchkiss, A.J. and Affolter, H.-U. (1984) Nature 312, 661-663 11 Kumakura, K., Guidotti, A. and Costa, E. (1979) Mol. Pharmacol. 16, 865-876 12 Greenberg, A. and Zinder, O. (1982) Cell Tiss. Res. 226, 655-665. 13 Michener, M.J. and Peach, M.J. (1984) Biochem. Pharmacol. 33, 1819-1823 14 Hochman, J. and Perlman, R.L. (1976) Biochim. Biophys. Acta 421, 168-175 15 Seamon, K.B., Padgett, N. and Daly, J.W. (1981) Proc. Natl, Acad. Sci. USA 78, 3363-3367 16 Seamon, K.B. and Daly, J.W. (1983) Trends. Pharmacol. Sci. 4, 120-123 17 Knight, D.E. and Baker, P.F. (1983) Q. J. Exp. Physiol. 68, 123-143 18 Tsien, R.Y., Pozzan, T. and Rink, T.J. (1982) J. Cell Biol. 94, 325-334 19 Von Euler, U.S. and Floding, 1. (1955) Acta Physiol. Scand. Suppl. 118, 45-56 20 Rabe, C.S.. Schneider, J. and McGee (1982) J. Cyclic Nucleotide Res. 8, 371-384 21 Niggli, V., Knight, D.E., Baker, P.F., Vigny, A. and Henry, J.P. (1984). J. Neurochem. 43, 646-658 22 Rink, T.J. and Sanchez, A. (1984) Biochem. J. 222, 833-836 23 De Togni, P., Cabrini, G. and Di Virgillo, F. (1984) Biochem. J. 224, 629-635 24 Knight, D.E. and Scrutton. M.C. (1984) Nature 309, 66-68 25 Fisher, S.K., Holz, R.W. and Agranoff, B.W. (1981) J. Neurochem. 37, 491-497 26 Schneider, A.S., Cline, H.T. and Lemaire, S. (1979) Life Sci. 24, 1389-1394