Use of isolated chromaffin cells to study basic release mechanisms

An account is given of the authors' work with isolated adrenal chromaffin cells to study the synthesis, storage and release of catecholamines and of a number of neuropeptides endogenous to the adrenal medulla. A review of other studies in the literature with the isolated chromaffin cell system is included. It is seen that the isolated chromaffin cells are a convenient in vitro system well-suited to studies of basic release mechanisms. The isolated adrenal chromaffin cells maintain high levels of catecholamines and opiates and release them by exocytosis. The cells have both nicotinic and muscarinic receptors but only the nicotinic are involved in the agonist-evoked release of catecholamines (EC50 nicotine 5 X 10(-6) M: ACh 5 X 10(-5) M). The cells can synthesize AChE and selectively release the 10S molecular form by a mechanism different from exocytosis. Substance P (SP) modulates the secretion of catecholamines and ATP evoked by ACh or nicotine but not that evoked by K+ or veratridine. SP appears to interact with the nicotinic receptor-ionophore complex to regulate Na+ entry. SP receptors on the chromaffin cells show similar structural requirements to SP receptors in other SP responsive tissues. Binding studies on isolated chromaffin cell membranes with [4-3H-Phe]SP have shown specific binding in the nM range. In addition, at high concentrations of ACh, SP protects against nicotinic receptor desensitization. Since SP is contained in the splanchnic nerve terminals that innervate the medulla, the demonstration of SP action and SP receptors on the chromaffin cells suggests a physiological role for SP in the regulation of secretion from the adrenal medulla. Somatostatin (SS) and a number of SS analogues also inhibit release, but are approximately 15-fold less potent than SP. Leu- and Met-enkephalin, which are co-stored with adrenaline in the bovine adrenal medullary cells produce a non-specific inhibition of the nicotine-evoked release of CA, but enhance the basal release of endogenous catecholamines by a mechanism that is Ca2+-dependent, stereospecific and reversible by naloxone and naltrexone. The implication of these peptide-amine interactions for physiological processes regulating homeostasis in the adrenal are discussed.     

Vasoactive intestinal peptide elevates cyclic AMP levels and potentiates secretion in bovine adrenal chromaffin cells

Vasoactive intestinal peptide (VIP) elevates the levels of cyclic AMP in primary cultures of highly purified chromaffin cells isolated from the bovine adrenal medulla, an effect that is potentiated by inhibitors of cyclic nucleotide phosphodiesterases. This elevation occurs within minutes and elevated levels of cyclic AMP are observed for as long as 24 hr in culture. The half-maximally effective concentration of VIP was approximately 1 microM. The VIP-induced elevation in chromaffin cell cyclic AMP levels was potentiated by simultaneous addition of secretagogues which alone also elevated cellular cyclic AMP levels. VIP alone failed to evoke catecholamine secretion from chromaffin cells, but potentiated potassium-, veratridine-, and nicotine-evoked secretion. Because VIP is found in nerve terminals innervating the adrenal medulla, this neuropeptide appears to be a modulator of chromaffin cell function, possibly via regulation of cyclic AMP levels.     

Substance P in the adrenal gland: Origin and species distribution

Substance P-like immunoreactivity (I-SP) was found in adrenal glands of cat, rabbit, guinea-pig, rat, cattle and man, as well as in a pheochromocytoma. I-SP was reduced in chronically denervated adrenal glands of rats and in adrenal glands of guinea-pigs pretreated with capsaicin, suggesting innervation of the adrenal glands by I-SP containing nerves, possibly of sensory origin. I-SP could partially be recovered from the chromaffin granule fraction of bovine adrenal medulla and a pheochromocytoma tumor. HPLC analysis of bovine chromaffin granules and a pheochromocytoma extract showed that the measured immunoreactivity corresponded to authentic substance P. Therefore, a dual localization of I-SP in nerve fibers and chromaffin cells can be assumed. The presence of somatostatin-like immunoreactivity in human adrenal medullae and in a pheochromocytoma is also reported. No neurotensin could be detected in the pheochromocytoma.     

Nicotinic stimulation of catecholamine synthesis and tyrosine hydroxylase phosphorylation in cervine adrenal medullary chromaffin cells

The synthesis and secretion of catecholamines by the adrenal medulla is of major importance in the stress response. Tyrosine hydroxylase, the rate-limiting enzyme for catecholamine biosynthesis, has been extensively studied in adrenal medullary chromaffin cells from a number of species. Cervine chromaffin cells are of interest because the deer is known to be a relatively stress-prone reactive species. We report the first characterisation of tyrosine hydroxylase regulation in cervine chromaffin cells. Nicotinic receptor activation resulted in a time- and concentration-dependent increase in catecholamine synthesis, which was significantly reduced by the extracellular signal-regulated kinase (ERK)1/2 signalling pathway inhibitor PD98059 and the calcium/calmodulin protein kinase II inhibitor KN-93, but not by H89 or bisindolylmaleimide I, inhibitors of protein kinase A and C, respectively. Nicotinic stimulation also increased the phosphorylation of ERK1/2 and tyrosine hydroxylase. This latter response occurred on serine residues 19, 31 and 40 of the enzyme. The nicotinic-induced phosphorylation of ERK1/2 and serine 31 of tyrosine hydroxylase was suppressed by PD98059 but not bisindolylmaleimide I. These data indicate that nicotinic stimulation of tyrosine hydroxylase involves the phosphorylation of serine 31 via an ERK1/2-dependent, protein kinase C-independent pathway. Protein kinase C activation by phorbol 12-myristate 13-acetate also caused an ERK1/2-dependent increase in the serine 31 phosphorylation of tyrosine hydroxylase but, in contrast to the nicotinic response, was not accompanied by an increase in enzyme activity. Thus, ERK1/2-mediated serine 31 phosphorylation of tyrosine hydroxylase appears necessary but not sufficient for nicotinic activation of catecholamine synthesis in cervine chromaffin cells. These data present potentially important similarities and differences between the regulation of catecholamine synthesis in cervine and the more widely studied bovine adrenal medulla.     

Regulation of acetylcholinesterase secretion from perfused bovine adrenal gland and isolated bovine chromaffin cells

The secretion of acetylcholinesterase (AChE) was studied in an isolated perfused bovine adrenal gland preparation and in cultured bovine adrenal medullary chromaffin cells. Electrical field stimulation (10 Hz) of splanchnic nerve terminals in the isolated perfused gland resulted in a two-fold increase in AChE secretion from the gland. Perfusion with the cholinergic receptor antagonists mecamylamine (5 μ M) and atropine (1 μ M) inhibited 70% of the stimulated secretion of AChE, demonstrating that most of the stimulated secretion was derived from chromaffin cells. The effect of nicotine stimulation on the secretion of AChE from isolated bovine chromaffin cells was compared with that produced by other compounds (histamine, angiotensin II) which are known to stimulate secretion of catecholamines. Incubation with nicotine (1–25 μM) stimulated the secretion of catecholamines and AChE. Histamine (1 nM–10 μ M) and angiotensin II (10 pM–10 μ M) did not stimulate AChE secretion. Time-course studies of AChE resynthesis after irreversible inhibition with the esterase inhibitor diisopropylfluorophosphate (DFP) demonstrated that AChE is stored within chromaffin cells for at least 11 h before being secreted. AChE secretion was inhibited within 2–3 h by 10 μg/ml brefeldin A (BFA), a compound known to block protein translocation from the endoplasmic reticulum (ER) to the Golgi apparatus (GA). The results suggest that AChE may reside for 8–9 h within the lumen of the ER before being actively secreted by processing through the GA.     

The substance P receptor subtype modulating catecholamine release from adrenal chromaffin cells

Substance P (SP) has two distinct actions on catecholamine (CA) release from cultured bovine adrenal chromaffin cells: SP inhibits acetylcholine (ACh)- or nicotine-induced [3H]norepinephrine ([3H]NE) release; and SP protects against desensitization of ACh- or nicotine-induced [3H]NE release. In order to characterize the SP receptor subtypes modulating these two actions, we have tested several SP-related tachykinins and SP analogues for their ability to act as agonists of SP for these actions. The naturally occurring tachykinins, physalaemin, eledoisin and kassinin, were about equipotent and all much less potent than SP in inhibiting nicotine-induced [3H]NE release. Physalaemin and eledoisin were also approximately equipotent and much less potent than SP in protecting against desensitization of nicotine-induced [3H]NE release; kassinin slightly enhanced nicotinic desensitization. These results indicate that neither a kassinin-like nor a physalaemin-like peptide are likely to modulate adrenal CA release. The 'SP-P' receptor-selective analogue, SP-methyl ester, was an agonist, equipotent with SP, for both of SP's actions on [3H]NE release. [D-Pro2,D-Trp7,9]-SP, a potent antagonist of SP's actions in many SP-responsive tissues, was inactive as an antagonist of both of SP's actions on [3H]NE release from adrenal chromaffin cells; however, this analogue was a potent SP agonist in chromaffin cells. The rank order of potency of these and other SP-related peptides for inhibition of [3H]NE release was similar to their order of potency for protection against desensitization. Thus the chromaffin cell SP receptors mediating inhibition of nicotine-induced [3H]NE release appear to be very similar to those mediating protection against desensitization of nicotine-induced [3H]NE release. The results indicate that the SP receptors modulating adrenal CA release are different from either the 'SP-P' or 'SP-E' receptor subtypes; bovine adrenal chromaffin cells appear to possess a third subtype of SP receptor which shares similar characteristics with the SP receptors mediating histamine release from rat mast cells.     

Noradrenergic,but not Adrenergic Chromaffin Cells in the Adrenal Gland Express Neuromodulin (GAP-43)

Neuroendocrine chromaffin cells of the adrenal gland express certain molecular markers either transiently during development or permanently. In the present study, the expression of neuromodulin (GAP-43), a neuronal protein often associated with neurite outgrowth, was examined in adult adrenals. Neuromodulin was detected by Western blot analysis in extracts of both rat adrenals and cultured bovine chromaffin cells, and was localized in situ in a subpopulation of chromaffin cells, as well as in nerve fibres and Schwann cells. The use of anti-tyrosine hydroxylase or anti-phenylethanolamine N -methyltransferase antibodies in combination with anti-neuromodulin antibodies in double immunofluorescent labelling of cryostat sections of rat glands demonstrated that neuromodulin is expressed by noradrenergic, and not by adrenergic chromaffin cells. The results provide further evidence that neuromodulin is not limited to neurons; it is also expressed in a subpopulation of neuroendocrine chromaffin cells. Neuromodulin may play a role in the development of the adrenal medulla or in the specific regulation of noradrenalin secretion from chromaffin cells.     

Bovine adrenal chromaffin cells: high-yield purification and viability in suspension culture

A method for purifying chromaffin cells from adult, bovine, adrenal medullae and the techniques for maintaining the cells in suspension culture for at least 14 days are presented. Perfusion of medullae with a collagenase-containing medium produced a cell fraction that contained, in addition to chromaffin cells, a significant percentage of non-chromaffin cells. These cells were found to attach more rapidly than chromaffin cells to glass and tissue-culture plasticware. Using this property, we devised a selective plating procedure that yielded ～ 1–2 × 10⁸ chromaffin cells per adrenal medulla at a purity of 95% or higher.On the basis of catecholamine levels and enzyme activities, suspension (as opposed to monolayer) cultures were chosen to further investigate their potential as a model system for the regulation of adrenergic function. In contrast to chromaffin cells cultured in monolayer, chromaffin cells in suspension had a more rounded appearance and formed multicellular aggregates with time in culture. Very few neurite-like structures, commonly observed in monolayer cultures, were present in the suspension cultures. Also, inhibitors of mitosis were not necessary to prevent overgrowth by non-chromaffin cells as there was little or no cell division in the suspension cultures. Catecholamine levels were relatively stable for at least 2 weeks, although a gradual decline in epinephrine occurred after day 5. Unlike other enzymes involved in catecholamine metabolism, phenylethanolamine N-methyl transferase activity declined significantly with time in culture in parallel to the gradual loss of epinephrine. In addition, both oxygen consumption and amino acid incorporation into proteins were relatively stable. Thus, the primary suspension cultures of adult, bovine chromaffin cells seem to offer several advantages for studying long-term regulation of chromaffin cell function and provide a stable source of adrenergic cells for examining short-term regulatory processes.     

Metorphamide, a novel endogenous adrenal opioid peptide, inhibits nicotine-induced secretion from bovine adrenal chromaffin cells

Opioid peptides are found in high concentrations in the adrenal medulla. Recently, a novel opioid octapeptide, metorphamide, possessing an amidated C-terminal, was characterized and also found to be present in adrenal tissue. We have studied the ability of this novel peptide to modify nicotine-induced secretion from isolated bovine adrenal chromaffin cells. Exocytosis was monitored by measuring adenosine triphosphate (ATP) release on-line by the luciferin-luciferase bioluminescence method, or by measuring endogenous catecholamine release by high-performance liquid chromatography (HPLC) with electrochemical detection. Metorphamide inhibited 5 microM nicotine-induced ATP release from fresh chromaffin cells by almost 50% at 5 microM. Metorphamide at concentrations less than 1 microM had no effect on 5 microM nicotine-induced adrenaline and noradrenaline release from cultured cells, but at higher concentrations inhibited their release equally, with an IC50 of approximately 10 microM. By contrast, Met5-enkephalin inhibited the release of both catecholamines equally with an IC50 of greater than 1 mM, making metorphamide greater than 100-fold more potent than Met5-enkephalin in this system. Naloxone (10 microM) and diprenorphine (1 microM) failed to antagonise the inhibitory action of metorphamide on nicotine-induced catecholamine release. Metorphamide inhibited the nicotinic response in a non-competitive manner, and failed to affect either adrenaline or noradrenaline release induced by elevated potassium ion concentrations. The results suggest metorphamide acts on naloxone- and diprenorphine-resistant receptors to inhibit chromaffin cell nicotinic secretion and that the novel amidated C-terminal of the peptide is important for this action.     

Effects of decrease of extracellular sodium in carbachol-evoked catecholamine secretion in isolated adrenal medullae of rats

The effect of extracellular Na(+) deprivation on the carbachol-evoked catecholamine secretion was evaluated in chromaffin cells. Isolated adrenal medullae of male Wistar rats were incubated in solutions with different sodium concentrations (144,0; 75,0; 25,0 and psi mM). Catecholamine secretions inversely increased as a response to fall of extracellular concentration of sodium. The magnitude of response to cholinergic stimulus (carbachol 100 microM) was decreased in low extracellular sodium concentration. Atropine (100 microM) inhibited secretion of catecholamine induced by carbachol in the presence and in the absence of extracellular sodium. Results suggest that in isolated adrenal medullae of rats (1) decrease in concentration of extracellular sodium increases secretion of catecholamines, perhaps by a greater influx of calcium from the extracellular environment through reversal of Na(+) /Ca(2+) exchanger; (2) intensity of catecholamine secretion induced by cholinergic stimulus seems to depend on extracellular sodium.     

Porcine chromaffin cells, culture, and transplant for antinociceptive effects in rodents and primates

It has been shown that xenografts and allografts of spinally transplanted adrenal chromaffin cells produce antinociception in animals and pain relief in patients with cancer pain. As there is a very limited availability of human adrenal tissue to serve as allografts, the clinical need for xenogeneic chromaffin cells as transplants is obvious. Bovine adrenal glands as a steady source of chromaffin cells have been extensively studied. There is however concern about the possible infection in humans with retrovirus following transplantation. The purpose of this study is to use the pig as a preferred donor animal species for xenotransplantation into rat and monkey. As pigs have been cloned, this opens the door to gene-targeted technologies and allows for genetic modifications, which possibly could improve the efficacy and safety of chromaffin cell transplantation. Porcine chromaffin cells were isolated from adrenal glands of 6-8-month-old pigs. After culturing cells for 1 week in a medium containing serum, the release of met-enkephalin and norepinephrine from the cells was detected by high-performance liquid chromatography and radioimmunoassay with nicotine stimulation, lasting approximately 3 weeks. Transplantation of these cells into the subarachnoid space of rats produced antinociceptive effects on Adelta and C fiber-mediated responses lasting 2-3 weeks. Similar findings were observed in studies with macaque monkeys. Compared with the same number of bovine chromaffin cells, porcine chromaffin cells showed a more robust and longer antinociceptive effect, and could be a better source of cells for human transplantation.     

Immunological identification of a nicotinic acetylcholine receptor on bovine chromaffin cells

Two probes previously shown to distinguish the nicotinic ACh receptor of chick ciliary ganglion neurons also recognize a component on the surface of bovine chromaffin cells in culture that displays the properties expected for the chromaffin nicotinic ACh receptor. The first probe is a monoclonal antibody, mAb 35, raised against ACh receptor from Electrophorus electric organ, and the second is an alpha-neurotoxin, Bgt 3.1, purified from B. multicinctus venom. mAb 35 binds specifically to a single class of high-affinity sites on the chromaffin cells in culture. Scatchard analysis indicates a KD of 2.1 +/- 0.2 nM for the binding and a Bmax of 1.6 +/- 0.1 X 10(4) mAb 35 sites per cell. The number of sites on the cells can be reduced through modulation by exposure of the cells to Bgt 3.1. The modulation can be blocked by the cholinergic ligands d-tubocurarine and carbamylcholine. Long-term exposure to the agonist carbamylcholine alone also reduces the number of mAb 35 binding sites. Bgt 3.1 inhibits nicotine-induced catecholamine release from the cells to the same extent and with the same concentration dependence that it modulates the number of mAb 35 sites on the cells. In addition, mAb 35 treatment of the cells causes a specific and almost complete blockade of nicotine-induced catecholamine release, apparently through a modulation of the receptor. These results indicate that the bovine chromaffin component recognized by mAb 35 and Bgt 3.1 is likely to be the nicotinic ACh receptor on the cells and that it has many similarities to ACh receptors on chick autonomic neurons.     

Prolactin inhibits the activity of tyrosine hydroxylase in cultured bovine adrenal chromaffin cells in a dose-dependent manner

Prolactin can modulate the adrenal medulla function, but it has not yet been established whether its action is directly exerted on the adrenal medulla cells. In this work, we have studied the effect of several concentrations of prolactin on the synthesis, storage and release of norepinephrine and epinephrine using cultured bovine adrenal chromaffin cells. In these cells, prolactin inhibited the activity of tyrosine hydroxylase, the rate-limiting enzyme in the catecholamine synthesis, in a dose-dependent manner, from a concentration above 50 ng/ml of prolactin in the incubation medium. Surprisingly, this dose-dependent decrease was not accompanied by changes in the catecholamine release, since the secretion of both norepinephrine and epinephrine as well as the total catecholamine secretion were not significantly altered by the different prolactin concentrations. Moreover, the cellular content of both catecholamines was not altered by prolactin. In summary, these observations allow us to conclude that prolactin exerts a direct inhibitory effect on the tyrosine hydroxylase activity in cultured adrenal chromaffin cells without altering catecholamine release.     

Simultaneous release of neurotensin, somatostatin, enkephalins and catecholamines from perfused cat adrenal glands

Acid extracts of cat adrenal medullae were found to contain neurotensin-like (0.71 +/- 0.21nmol/gm), met-enkephalin-like (11.71 +/- 2.87nmol/gm), leu-enkephalin-like (1.95 +/- 0.11nmol/gm) and somatostatin-like (1.57 +/- 0.63pmol/gm) immunoreactivities. Using isolated retrogradely perfused cat adrenal glands the secretory responses to acetylcholine (ACh), nicotine and potassium ions (K+) were studied. Spontaneous secretion of catecholamines, neurotensin-like, met-enkephalin-like, leu-enkephalin-like and somatostatin-like immunoreactivities was negligible. However, ACh (5.5 X 10(-5)M), nicotine (2.2 X 10(-5)M) and 55mM K+ all evoked the simultaneous release of noradrenaline, adrenaline and the four neuropeptide immunoreactivities. The ACh stimulated secretion of the four neuropeptides could be prevented by perfusion with hexamethonium (C6, 2.8 X 10(-4)M). The concomitant release of these neuropeptides with catecholamines suggests that they may have a role in the modulation of stress responses.     

Transplantation of microencapsulated bovine chromaffin cells reduces lesion-induced rotational asymmetry in rats

Surrounding bovine chromaffin cells by a semipermeable membrane may protect the transplanted cells from a host immune response and shield them from the inflammatory process resulting from the surgical trauma. Encapsulation of the chromaffin cells was achieved by inter-facial adsorption of a polycation on a polyanionic colloid matrix in which the chromaffin cells were entrapped. Basal and potassium-evoked release of catecholamines from encapsulated bovine chromaffin cells was analyzed over a 4-week period in vitro. Norepinephrine and dopamine release remained constant over time whereas epinephrine release significantly decreased. The chromaffin cells also retained the capacity for depolarization-elicited catecholamine release 4 weeks following the encapsulation procedure. Morphological analysis revealed the presence of intact chromaffin cells with well-preserved secretory granules. Striatial implantation of chromaffin cell-loaded capsules significantly reduced apomorphine-induced rotation compared to empty polymer capsules in animals lesioned with 6-hydroxydopamne frr at least 4 weeks. Intact chromaffin cells expressing tyrosine hydroxylase and dopamine-β-hydroxylase were observed in all capsules implanted in the striatum for 4 weeks. The assessment of the clinical potential of transplanting encapsulated adrenal chromaffin cells of either allo- or xenogeneic origin for Parkinson's disease will require long-term behavioral studies. The present study suggests, however, that the polymer encapsulation procedure may offer an alternative to adrenal autografts as a source of dopaminergic tissue.     

Altered reactivity of the rat adrenal medulla

Previous studies have suggested that experimental alterations in adrenomedullary reactivity, i.e., changes in catecholamine release in response to a standard dose of acetylcholine, may be partially accounted for by changes occurring at the level of the adrenal medulla itself, independent of both the central nervous system and the innervation of the adrenal gland. The present study was designed to investigate the morphology of adrenal chromaffin cells in rats subjected to chronic hypoglycemia induced by long acting insulin, and to assess this morphology in terms of associated changes in catecholamine content and release. Surgically isolated, perfused adrenal gland preparations were utilized to characterize the functional release of catecholamines from the adrenal medulla. Pretreatment with long acting insulin resulted in a selective depletion of epinephrine stores and acetylcholine-mediated epinephrine release, but did not appear to significantly affect either the levels or the release of norepinephrine. The biochemical effects of long acting insulin persisted for several days after termination of the treatment, exhibiting a gradual recovery over a period of approximately 5 days. Electron microscopic examination of the adrenal chromaffin cells revealed a progressive degranulation and vacuolization of numerous chromaffin cells followed by a compensatory biosynthetic response and a gradual recovery toward the morphology of control cells. The functional release of catecholamines from adrenal chromaffin cells was further examined in preparations of perfused adrenal slices. Acetylcholine-mediated catecholamine release was significantly decreased in slices of adrenal glands prepared from insulin treated rats when compared with that of control animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Progesterone regulation of catecholamine secretion from chromaffin cells

Stress stimulates the adrenal medulla to rapidly secrete catecholamines (CAs), and the adrenal cortex to release progesterone (PROG), which may locally regulate stress-induced CA release. We used bovine chromaffin cells to investigate the effects of PROG on CA secretion. PROG dose-dependently inhibited CA secretion induced by nicotinic acetylcholine receptor (nAChR) agonist 1,1-dimethyl-4-phenlypiperazinium iodide (DMPP) up to 77%. Pre-incubation with PROG up to 1 h increased this inhibition. 3alpha,5alpha-Tetrahydroprogesterone (3alpha,5alpha-THP) and dexamethasone were less potent inhibitors. Patch-clamp techniques revealed that PROG co-applied with DMPP inhibited peak DMPP-induced current up to 68% and with 3 min pre-incubation inhibited both peak and integrated current up to approximately 95%. Monitoring of FURA-2 showed that PROG similarly inhibited parallel changes in intracellular-free Ca(++) concentration. PROG also inhibited CA secretion elicited by elevated K(+) (38%), and, in single cells, suppressed Ca(++) current evoked by step depolarization, inhibiting amplitude by 15%, and reducing the time constant of current decay during depolarization by 57%. In contrast to the immediate inhibition of nicotinic current, inhibition of Ca(++) current became statistically significant only after 1 min exposure to PROG. PROG did not inhibit secretion stimulated by high Ca(++) perfusion of permeabilized cells. These data suggest that PROG inhibits CA secretion from chromaffin cells predominantly by rapidly inhibiting nAChRs, and by gradually enhancing the inactivation of voltage-dependent Ca(++) channels (VDCCs), but not by affecting secretory processes downstream of Ca(++) influx. This study supports a role for adrenocortical PROG in the regulation of CA secretion during stress.     

Two GTP-binding Proteins Control Calcium-dependent Exocytosis in Chromaffin Cells

The effect of guanosine triphosphate analogues on catecholamine secretion from permeabilized bovine chromaffin cells was examined. Guanosine 5'-[γ-thio]triphosphate was demonstrated to produce a dual effect on calcium-evoked secretion, enhancing the release through a mechanism involving protein kinase C and inhibiting secretion by a protein kinase C-independent pathway. We propose that two functionally distinct G-proteins control the stimulus - secretion coupling in chromaffin cells.