Guinea-pig Chromaffin Cells Research Articles

Muscarinic receptors in adrenal chromaffin cells: physiological role and regulation of ion channels

Adrenal medullary chromaffin cells in mammals are innervated by sympathetic preganglionic nerve fibers, as are sympathetic ganglion neurons. Acetylcholine in the ganglion neurons is well established as mediating fast and slow excitatory postsynaptic potentials through nicotinic and muscarinic acetylcholine receptors (AChRs), respectively. The role of muscarinic AChRs during neuronal transmission in chromaffin cells varies among different mammals. Furthermore, the ion channel mechanisms associated with the muscarinic AChR-mediated increase in excitability of chromaffin cells are complicated and different from the excitation of ganglion neurons, which has been ascribed to the inhibition of M-type K+ channels. In this review, we focus on muscarinic receptor-mediated excitation in rodent and guinea pig chromaffin cells, in particular, on the role of muscarinic receptors in neuronal transmission, the muscarinic receptor subtypes involved in excitation and secretion, and the muscarinic regulation of ion channels including TWIK-related acid-sensitive K+ channels. Finally, we discuss prospectively the future of muscarinic receptor research in adrenal chromaffin cells.

GABA Signaling and Neuroactive Steroids in Adrenal Medullary Chromaffin Cells.

Gamma-aminobutyric acid (GABA) is produced not only in the brain, but also in endocrine cells by the two isoforms of glutamic acid decarboxylase (GAD), GAD65 and GAD67. In rat adrenal medullary chromaffin cells only GAD67 is expressed, and GABA is stored in large dense core vesicles (LDCVs), but not synaptic-like microvesicles (SLMVs). The α3β2/3γ2 complex represents the majority of GABAA receptors expressed in rat and guinea pig chromaffin cells, whereas PC12 cells, an immortalized rat chromaffin cell line, express the α1 subunit as well as the α3. The expression of α3, but not α1, in PC12 cells is enhanced by glucocorticoid activity, which may be mediated by both the mineralocorticoid receptor (MR) and the glucocorticoid receptor (GR). GABA has two actions mediated by GABAA receptors in chromaffin cells: it induces catecholamine secretion by itself and produces an inhibition of synaptically evoked secretion by a shunt effect. Allopregnanolone, a neuroactive steroid which is secreted from the adrenal cortex, produces a marked facilitation of GABAA receptor channel activity. Since there are no GABAergic nerve fibers in the adrenal medulla, GABA may function as a para/autocrine factor in the chromaffin cells. This function of GABA may be facilitated by expression of the immature isoforms of GAD and GABAA receptors and the lack of expression of plasma membrane GABA transporters (GATs). In this review, we will consider how the para/autocrine function of GABA is achieved, focusing on the structural and molecular mechanisms for GABA signaling.

Catecholamine secretion by chemical hypoxia in guinea-pig, but not rat, adrenal medullary cells: Differences in mitochondria

The effects of mitochondrial inhibitors (CN−, a complex IV inhibitor and CCCP, protonophore) on catecholamine (CA) secretion and mitochondrial function were explored functionally and biochemically in rat and guinea-pig adrenal chromaffin cells. Guinea-pig chromaffin cells conspicuously secreted CA in response to CN− or CCCP, but rat cells showed a little, if any, secretory response to either of them. The resting metabolic rates in rat adrenal medullae did not differ from those in guinea-pig adrenal medullae. On the other hand, the time course of depolarization of the mitochondrial membrane potential (ΔΨm) in guinea-pig chromaffin cells in response to CN− was slower than that in rat chromaffin cells, and this difference was abolished by oligomycin, an F1F0-ATPase inhibitor. The extent of CCCP-induced decrease in cellular ATP in guinea-pig chromaffin cells, which was indirectly measured using a Mg2+ indicator, was smaller than that in rat chromaffin cells. Relative expression levels of F1F0-ATPase inhibitor factor in guinea-pig adrenal medullae were smaller than in rat adrenal medullae, and the opposite was true for F1F0-ATPase α subunit. The present results indicate that guinea-pig chromaffin cells secrete more CA in response to a mitochondrial inhibitor than rat chromaffin cells and this higher susceptibility in the former is accounted for by a larger extent of reversed operation of F1F0-ATPase with the consequent decrease in ATP under conditions where ΔΨm is depolarized.

Functional relation between caffeine- and muscarine-sensitive Ca 2+ stores and no Ca 2+ releasing action of cyclic adenosine diphosphate-ribose in guinea-pig adrenal chromaffin cells

In voltage-clamped guinea-pig chromaffin cells, muscarine (50 μM) or caffeine (30 mM) produced a transient intracellular Ca 2+ concentration ([Ca 2+] i) increase, catecholamine release and an outward K + current mediated through Ca 2+ released from internal Ca 2+ stores at a holding potential of −40 mV. Caffeine followed by muscarine failed to evoke these responses, while muscarine followed by caffeine was effective in producing about 30% of [Ca 2+] i increase and catecholamine secretion. In cells dialyzed with inositol 1,4,5-trisphosphate (IP 3), caffeine failed to produce the [Ca 2+] i increase. Intracellular application of cyclic adenosine 5′-diphosphate-ribose (cADP-ribose) or 8-bromo cADP-ribose exerted no effect on the resting [Ca 2+] i and the caffeine-induced [Ca 2+] i increase. These results suggest that IP 3-sensitive stores are functionally divided into two subpopulations, sensitive and insensitive to caffeine, and it is unlikely that cADP-ribose plays a role as a Ca 2+ releaser in guinea-pig adrenal chromaffin cells.

Ca2+-dependent K+ currents induced by muscarinic receptor activation in guinea pig adrenal chromaffin cells.

The characteristics of the outward current (I(out)) induced by muscarine were examined by using the whole-cell patch-clamp technique with a K+-containing pipette solution in combination with fura-2 microfluorometry in guinea pig chromaffin cells. Muscarine caused a transient increase in the cytosolic Ca2+ concentration ([Ca2+]i) and activated an I(out) with a reversal potential close to a K+ equilibrium potential. Under symmetric K+ conditions, muscarine produced a transient inward current and an increase in [Ca2+]i at -60 mV. At -15 mV, apamin and charybdotoxin, respective SK and BK channel blockers, decreased the I(out) but scarcely affected the [Ca2+]i response to muscarine. Muscarine produced an I(out) and an increase in [Ca2+]i even after a removal of external Ca2+ and in the presence of Co2+, indicating that these responses are mediated by Ca2+ release from intracellular stores. The I(out) evoked by the Ca2+ release was much smaller than that evoked by the voltage-dependent Ca2+ influx, even when similar [Ca2+]i changes assessed by fura-2 microfluorometry occurred. Inositol 1,4,5-trisphosphate (InsP3) applied intracellularly and the photolysis of caged InsP3 each evoked current changes similar to those induced by muscarine. These results indicate that the I(out) evoked by muscarinic stimulation is mediated by Ca2+-dependent K+ channels (probably BK and SK channels), which are activated by Ca2+ released from intracellular Ca2+ stores in guinea pig chromaffin cells.

Muscarinic receptor activation in guinea-pig chromaffin cells causes decreased membrane conductance and depolarization

Membrane potentials were recorded with conventional intracellular microelectrodes from chromaffin cells in isolated, bisected adrenal glands from guinea-pigs. The local pressure ejection of muscarinic agonists, acetylcholine (in the presence of hexamethonium) or bethanecol, caused a transient depolarization that was relatively slow (1–2 s) in onset compared with the depolarization associated with the activation of nicotinic receptors. Muscarinic receptor-induced depolarization was associated with an increase in input resistance and the firing of action potentials. Repetitive stimulation of splanchnic nerve fibers within the gland, in the presence of hexamethonium, caused a maintained depolarization that was slow in both onset and decay and in many cells caused the repetitive firing of action potentials. It is suggested that, in this species, the exocytosis of catecholamines caused by the activation of muscarinic receptors, described by others, may be due to the initiation of tetrodotoxin-sensitive action potentials and consequent opening of voltage sensitive Ca 2+ channels.

Cyanide suppression of inwardly rectifying K+ channels in guinea pig chromaffin cells involves dephosphorylation.

Treatment of chromaffin cells with cyanide induced a gradual decrease in an inwardly rectifying K+ current (IIR), and washout of the mitochondrial inhibitor resulted in a rapid recovery of IIR. This diminution of IIR was reversed in a time-dependent manner by infusion of ATP or UTP, but not by that of GTP, ITP, or CTP. The restoration by ATP was not altered by addition to the pipette solution of 50 microM fluorescein 5-isothiocyanate, an inhibitor of various ATPases. A similar recovery of IIR occurred with injection of adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), but not of 5'-adenylylimidodiphosphate or alpha,beta-methyleneadenosine 5'-triphosphate. The ATP gamma S effect was biphasic, resulting in first a run-up of the current in ATP-depleted cells followed by a rundown of the current. This rundown was almost abolished by addition of guanosine 5'-O-(2-thiodiphosphate) to the ATP gamma S solution, suggesting the involvement of a G protein. Bath application of the protein kinase inhibitor 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine at 100 microM, but not N-(2-[methylamino]-ethyl)-5-isoquinolinesulfonamide, induced a reversible inhibition of IIR in the presence of pipette ATP, and the inhibition was diminished by 1 microM calyculin A, a phosphatase inhibitor. Bath application of 1 microM phorbol 12,13-dibutyrate did not affect IIR. It is concluded that cyanide suppresses inward rectifier K+ channel activity via dephosphorylation and that protein kinase C, adenosine 3',5'-cyclic monophosphate-dependent kinase, or guanosine 3',5'-cyclic monophosphate-dependent kinase is not involved in modulation of the channel.

Fluctuation analysis of nonselective cation currents induced by A1F complex in guinea-pig chromaffin cells

Properties of aluminium fluoride (A1F) complex-activated nonselective cation (NS) channels in guinea-pig chromaffin cells were investigated using the patch clamp technique. As the membrane potential was hyperpolarized from the holding potential of −55 mV, the A1F-induced nonselective cation current (INS) diminished progressively. With hyperpolarizations to −100 MV or more negative potentials, the A1F INS almost instantaneously disappeared. The apparent unit conductance of A1F INS was estimated to be 3 pS by fluctuation analysis. The open state probability of A1F-activated NS channels became large with a decrease in concentration of free Mg2+ ions inside the cell and was less than 0.5 at 12 μM Mg2+. It is concluded that NS channels in the chromaffin cell apparently differ from those in smooth muscle cells.

Role and source of ATP for activation of nonselective cation channels by AlF complex in guinea pig chromaffin cells.

Intracellular dialysis with the solution containing the G protein activator, AlF complex, induced an inward nonselective cation current (INS) at -55 mV in chromaffin cells. Amplitudes of INS induced by dialysis with ATP-free AlF solutions progressively diminished as cells were pretreated with cyanide, a mitochondrial inhibitor. After a 10-min pretreatment, generation of INS by the AlF complex depended on exogenous ATP delivered from pipette solution. The relationship between amplitudes of INS and concentrations of MgATP was well expressed by a rectangular hyperbola with an EC50 of 0.265 mM. This result suggests that the cyanide treatment almost depleted ATP near the plasma membrane. On the other hand, a similar cyanide treatment of adrenal medullary preparations did not induce a marked decrease in cellular ATP content. GTP, ITP, or UTP could not substitute for ATP in generation of INS by the AIF complex. Similarly, the substitution of ATP with non- or poorly hydrolyzable ATP analogues did not aid in generating INS. Bath application of the kinase inhibitor, H-7 (100 microM), suppressed AlF-induced INS in a manner depending on intracellular Mg2+. We conclude that ATP is a prerequisite for generation of INS as a phosphoryl donor and that mitochondria is the main source of ATP.

ADP indirectly supports activation of non-selective cation channels by AIF complex in guinea-pig chromaffin cells

Infusion of the G protein activator, AIF complex, into guinea-pig adrenal chromaffin cells produced an inward non-selective cation current ( I NS) at −55 mV. Under metabolically suppressed conditions, this current was abolished by removal of ATP from the AIF-containing pipette solution. ADP could substitute for ATP in generation of the current. This supporting action of ADP was eliminated by addition to the AIF solution of the ATP-consuming system (hexokinase and 2-deoxyglucose). A similar generation of INS occurred when AMP and phosphocreatine were simultaneously added to AIF solution, but not when either of the agents was added separately. These results suggest that under conditions of whole-cell current recordings, the machinery for ATP production is preserved and that ADP indirectly supports generation of INS by the AIF complex.

Mg 2+-dependent phosphatase as an inhibitory mediator of the nonselective cation current induced by aluminum fluoride in guinea-pig chromaffin cells

Internal administration of the G protein activator, guanosine-5′- o-(3-thiotriphosphate) (GTPγS) or aluminum fluoride (AlF) complex, produce an inward nonselective cation current ( I NS) at −55 mV. This current was rapidly diminished under conditions of high intracellular Mg 2+ ([Mg 2+] = 979 μM), the half decay time ( T 1 2 ) being 80 to 100 s. As [Mg 2+] in AlF solutions decreased from 400 to 12 μM, the maximum amplitude of AlF-induced I NS became larger and the current was diminished more slowly. The AlF I NS in the presence of 12 μM Mg 2+ reversed polarity at about + 9 mV, irrespective of the extent of decline. Bath application of muscarine produced a sustained I NS was rapidly diminished. Addition of vanadate (0.5 mM) to 979 μM Mg 2+-containing AlF solution mimicked the effects of low Mg 2+ solution. Inversely, addition of alkaline phosphatase (40 units/ml) to 12 μM Mg 2+ AlF solution reproduced the effects of high Mg 2+ solution. It is suggested that AlF complex deactivates I NS through facilitating an apparent activity of Mg 2+-dependent phosphatase.

ATP-evoked membrane current in guinea pig adrenal chromaffin cells

Adenosine 5′-triphosphate (ATP) evoked an inward current in whole-cell voltage-clamped adrenal chromaffin cells of the guinea pig. The reversal potential ( E rev) of ATP-evoked current was about 0 mV in normal external solution and was shifted towards negative potentials by substituting Tris + or sucrose, but not Ca 2+, for the extracellular Na +. This current was mediated by the activation of non-selective cation channels and had some different properties from nicotinic current. It is suggested that these channels may function as a part of the ATP-induced Ca 2+ influx pathway in guinea pig chromaffin cells.

Phosphatase is responsible for run down, and probably G protein-mediated inhibition of inwardly rectifying K+ currents in guinea pig chromaffin cells.

The mechanism of G protein-mediated inhibition of an inwardly rectifying K+ current (IIR) in adrenal chromaffin cells was investigated using the whole-cell version of the patch clamp technique. In case of recording with use of ATP-containing patch solution, the IIR was well maintained; otherwise, it ran down within 15 min. This run down was not prevented by replacement with adenylyl-imidodiphosphate, a nonhydrolysable analogue of ATP, but was markedly reduced by the addition to the ATP-free solution of 1 microM calyculin A, a specific inhibitor of serine/threonine phosphatase 1 (PP1) and 2A (PP2A). The addition of alkaline phosphatase to the ATP-containing solution facilitated run down of the current, and application of 100 microM H-7, a general kinase inhibitor, reversibly suppressed IIR. These results taken together suggest that inwardly rectifying K+ channels are under the influence of kinase and phosphatase without external signals. Infusion of nonhydrolysable analogues of GTP, guanosine-5'-O-(3-thiophosphate) (GTP gamma S) or guanylyl-imidodiphosphate, through the pipette produced little inward current at -55 mV, but completely inhibited IIR within approximately 5 or 6 min in all cells tested in the presence of 12 microM Mg2+ inside the cell. In contrast, infusion of aluminum fluoride (AlF) complex, another GTP binding (G) protein activator, consistently produced large inward currents, but did not alter IIR noticeably for 15 min in 17% of the cells tested. In the other cells, the inhibition of IIR developed slowly after long latent periods. This inhibitory potency of AlF was not enhanced by an increase in Mg2+ concentrations. Subtraction of the current-voltage relationship before from that noted during the generation of inward current by AlF complex revealed that the inward current diminished progressively with hyperpolarizations, as is the case with a nonselective cation current (INS) induced by a muscarinic agonist. Thus, AlF complex seems to be potent with the generation of INS, but not with IIR inhibition. The addition of 3 microM calyculin A significantly retarded the IIR inhibition by GTP gamma S, whereas that of 1 microM okadaic acid, another inhibitor of PPI and PP2A, markedly prevented the decline of IIR by AIF complex. Our observations suggest that the low potency of AlF complex in inhibiting IIR may be due to interference with phosphatase activity and that the activation of G protein suppresses IIR, probably by enhancing the apparent activity of phosphatase, which may explain run down of the current.

Mechanism of activation of nonselective cation channels by putative M4 muscarinic receptor in guinea-pig chromaffin cells.

1. Mechanisms involved in the generation of nonselective cation currents (INS) by muscarinic agonists in the chromaffin cell were investigated by the perforated patch method. 2. Bath application of muscarine (0.1-30 microM) produced an inward INS with or without a transient outward current at -40 mV, whereas oxotremorine (0.06-60 microM) induced INS alone. Rectangular hyperbolas with EC50s of 2.01 and 0.21 microM were fitted to muscarine- and oxotremorine-induced INSS, respectively, and the maximal amplitude of the former was about 3.4 times larger than that of the latter. 3. In 36% of the cells exposed to Ca(2+)-free solution, muscarine INS was suppressed, being 53% of control 20 min after the perfusion, and in four cells that were incubated with Ca(2+)-free solution for 2 h or more, the INS averaged 44% of that induced subsequently in normal solution. In contrast, muscarine INS was enhanced by about 30% when A-23187 was added to normal solution. 4. W-7 and W-5, calmodulin-related agents, were almost equally potent in inhibiting muscarine INS, whereas compound 5, a potent inhibitor of calmodulin-dependent kinase II (CaM kinase II), produced no evident inhibition. 5. HA1004, a weak kinase C inhibitor, induced a reversible suppression of muscarine INS with an IC50 of 163 microM, whereas H-8, another kinase inhibitor, produced an even small degree of inhibition. Administration of phorbol 12, 13-dibutyrate did not mimic muscarinic stimulation of NS channels; rather, it led to a progressive inhibition of INS and this inhibition was almost complete within 20 min. An inactive phorbol ester had no such effect. 6. The muscarinic antagonists, pirenzepine and AF-DX 116, shifted the dose-response curve for the muscarine INs to the right in a parallel manner. The KDS for pirenzepine and AF-DX 116 were estimated to be 13 nM (95% confidence interval, 11-16 nM) and 365 nM (283-470 nM), respectively.7. These results suggest that muscarine efficiently produces INS, probably through binding to the M4 subtype, that intracellular Ca2+ has a facilitating, but not an essential role in the generation of INs, and that neither CaM kinase II nor protein kinase C is involved.

Synaptic transmission from splanchnic nerves to the adrenal medulla of guinea‐pigs.

1. Membrane potentials were recorded with conventional intracellular microelectrodes from chromaffin cells in isolated, bisected adrenal glands from guinea-pigs. 2. All cells were electrically excitable and responded to depolarizing current with all-or-nothing action potentials that were blocked by tetrodotoxin. 3. Input resistance was 180 +/- 14 M omega and this was lower than that reported for isolated chromaffin cells using patch electrodes. 4. All cells responded to transmural stimulation with action potentials that arose from excitatory synaptic potentials in response to the excitation of one or more preganglionic fibres, many having strong synaptic action. Other fibres had weaker synaptic action but in all cases, maximal transmural stimulation caused depolarization well above threshold for action potential initiation. 5. Spontaneous excitatory synaptic potentials were observed whose frequency was greatly increased by repetitive stimulation at 10 or 30 Hz. 6. No evidence was found for the desensitization of nicotinic receptors in response to acetylcholine released from presynaptic nerve terminals. 7. These experiments show that there are many similarities between the responses to splanchnic nerve stimulation of guinea-pig chromaffin cells in situ and sympathetic ganglion cells from the same species.

G protein-mediated inhibition of inwardly rectifying K+ channels in guinea pig chromaffin cells

Properties of inwardly directed rectification and its G protein-mediated inhibition in guinea pig chromaffin cells were studied using the whole cell version of the patch-clamp technique. The current-voltage (I-V) relationship for plateau currents in response to a 50-ms pulse showed an inwardly directed rectification between -80 and -140 mV and a negative slope at more negative potentials in normal solution. Replacement of Na+ with N-methyl-D-glucamine (NMDG) in the perfusate did not alter the plateau I-V relationship between -110 and -130 mV but did abolish the negative slope below -140 mV. The zero current or resting membrane potential in the NMDG solution was in fair agreement with the equilibrium potential for K+. The chord conductance-voltage relationship showed a good fit with the Boltzmann equation and shifted along the voltage axis by an approximate change in driving force on K+ when K+ concentration was increased. External Cs+ and Ba2+ produced a voltage-dependent inhibition of the inwardly directed rectification. These results indicate that inwardly rectifying (IR) K+ channels are mediating an inwardly directed rectification. Intracellular dialysis with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) produced a complete suppression of this IR K+ channel, irrespective of treatment with pertussis toxin. Adding GTP or guanosine 5'-O-(2-thiodiphosphate) to the patch solution resulted in a decrease in GTP gamma S inhibition of the K+ current. Internal application of vanadate was without effect. Time course of the inhibition of the IR K+ current coincided in part with that of inactivation of a nonselective cation current. In conclusion, IR K+ channels in the chromaffin cell are subject to G protein-mediated inhibition.

Phosphorylation-dependent regulation of nonselective cation channels in guinea pig chromaffin cells.

In guinea pig chromaffin cells, acetylcholine induces a nonselective cation current by activating a muscarinic receptor, probably m4. We investigated activation and deactivation processes of this current. The calmodulin antagonists trifluoperazine and calmidazolium reversibly suppressed the muscarinic activation of the nonselective cation current in a dose-dependent manner; the former was about six times less potent than the latter. H-7, a kinase inhibitor, also reversibly inhibited the muscarinic current with a concentration eliciting 50% of maximal inhibition of 277 microM. Internal application of vanadate, a nonspecific phosphatase inhibitor, enhanced the muscarinic current and markedly slowed the time course of its deactivation, but the phosphatase 1 and 2A inhibitors okadaic acid and calyculin A had no effects. Lowering Mg2+ concentration in the patch solution mimicked the effects of vanadate on the muscarinic current. Internal dialysis with vanadate or low-Mg2+ solution gradually led to development of an inward current, and the related voltage dependence was similar to that seen with the muscarinic cation current. These results suggest that kinase and Mg(2+)-dependent phosphatase are responsible for activation and deactivation of nonselective cation channels. The channels seem to be under the influence of both enzymes, even in the absence of external signals.

P-037 - Effects of tetrahydroaminoacridine on catecholamine secretion and membrane currents induced by secretagogues in adrenal chromaffin cells of guinea pig

P-037 - Effects of tetrahydroaminoacridine on catecholamine secretion and membrane currents induced by secretagogues in adrenal chromaffin cells of guinea pig

Muscarine induces two distinct current responses in adrenal chromaffin cells of the guinea-pig.

Muscarine-induced membrane responses were studied in dissociated chromaffin cells of the guinea-pig adrenal medulla, using the whole-cell version of the patch-clamp technique. Bath application of muscarine (1-10 microM) produced two distinct current responses at a holding potential of -40 mV. One is an inward current associated with an increase in current noise. This current response was sustained during stimulation and had a reversal potential of 4.5 +/- 3.4 mV (n = 6) with a negative slope conductance below about -30 mV in 12.5 mM K(+)-containing perfusate. The other is a transient outward current. This was evoked at membrane potentials more positive than -60 mV and completely suppressed by addition of 2 mM TEA to the bath solution, suggesting a possible involvement of the Ca2(+)-dependent K+ channel. Generation of the outward current response was suppressed for at least 60-90 s following 25 s muscarinic stimulation and was facilitated by activation of the nicotinic receptor. The maximum inward current seemed to be produced by 3 microM, whereas the threshold concentration required for generation of the outward current was somewhere between 3 and 10 microM. The outward current was evoked less often in cells treated with 2% collagenase for 1 h than in those treated with 0.2% for 30 min. The results suggest that guinea-pig chromaffin cells have two muscarinic receptors: one is coupled with a cation nonselective channel and the other may be related to a Ca2(+)-dependent K+ channel.

Both nicotinic and muscarinic receptors mediate catecholamine secretion by isolated guinea-pig chromaffin cells

We have studied the roles of nicotinic and muscarinic receptors in the acetylcholine-evoked secretion of catecholamine from guinea-pig chromaffin cells. Isolated guinea-pig chromaffin cells secrete catecholamine in response to acetylcholine, nicotine, and a variety of muscarinic agonists. Optimal concentrations of acetylcholine (50–200 μM) induce the release of 10–25% of the catecholamine content of the cells in 10 min. Maximal secretion evoked by nicotine or by muscarinic agonists is 5–12% of the catecholamine content of the cells. Secretion evoked by optimal concentrations of nicotine (50 μM) and muscarine (200 μM) are additive, and together these agonists cause catecholamine release equivalent to that produced by optimal concentrations of acetylcholine. Atropine causes a biphasic inhibition of acetylcholine-induced catecholamine secretion; low concentrations of atropine (0.02–0.1 μM) inhibit by 35–45% the catecholamine secretion evoked by 100 μM acetylcholine. Increasing the atropine concentration from 0.1 to 5 μM causes no further decrease in acetylcholine-evoked release, but at concentrations above 5 μM, a second distinct phase of inhibition appears. At 100 μM, atropine reduces acetylcholine-evoked secretion by 85%. At 0.1 μM, atropine significantly inhibits secretion induced by muscarinic, but not nicotinic, agonists. Tubocurarine (50 μM) does not block muscarinic stimulation of release, but inhibits acetylcholine- and nicotine-evoked release by 70 and 80%, respectively. Our experiments indicate that nicotinic and muscarinic stimulation represent distinct mechanisms for the activation of catecholamine release from guinea-pig chromaffin cells. Acetylcholine causes secretion by both of these pathways. Nicotinic stimulation accounts for approximately 60–70% of the release caused by 100 μM acetylcholine; the remaining 30–40% of the acetylcholine-evoked release is mediated by muscarinic receptors.