Difference in the Effectiveness of Ca2+ to Evoke Catecholamine Secretion Between Adrenaline-and Noradrenaline-Containing Cells of Bovine Adrenal Medulla

Abstract: Differential adrenaline (Ad) and noradrenaline (NA) secretions evoked by secretagogues were investigated using digitonin-permeabilized adrenal chromaffin cells, cultured adrenal chromaffin cells, and perfused adrenal glands of the ox. In digitonin-permeabilized cells, Ca²⁺ (0.8-160 μM) caused a concentration-dependent increase in catecholamine secretion, which was characterized by a predominance of NA over Ad secretion. Acetylcholine (10-1,000 μM), high K⁺ (14-56 μM), and bradykinin (0.1-1,000 μM) all were confirmed to induce the release of more NA than Ad at all concentrations used. There was no apparent difference in the ratios of NA/Ad between Ca²⁺-induced catecholamine secretion from digitonin-permeabilized cells and those induced by secretagogues from cultured cells. Qualitatively the same result was obtained in the secretory responses to acetylcholine and high K⁺ in perfused adrenal glands. These results indicate that the effectiveness of Ca²⁺ for catecholamine secretion is higher in the secretory apparatus of NA cells than in that of Ad cells of the bovine adrenal medulla. This may be one of the reasons why the secretagogues cause a predominance of NA secretion over Ad secretion in the bovine adrenal medulla.     

Cholinergic receptors and catecholamine secretion from adrenal chromaffin cells of the toad.

1. The effects of cholinergic drugs on catecholamine (CA) secretion from adrenal chromaffin tissue of the toad were studied. 2. CA secretion was induced by ACh or nicotine, but not by muscarine. 3. Hexamethonium inhibited the CA release evoked by ACh or nicotine, while d-tubocurarine only affected the nicotinic response. Atropine did not prevent the secretory response. 4. Muscarine abolished the secretion induced by the agonists, this effect being prevented by atropine or gallamine, but not by pirenzepine. 5. In conclusion, CA secretion in the toad is stimulated by activation of nicotinic receptors. Inhibitory muscarinic receptors are present, most likely of type M2, which may play a regulatory function.     

Benzodiazepine inhibition of the stimulation-evoked catecholamine release from bovine adrenomedullary cells in culture

Effect of benzodiazepines on evoked catecholamine (CA) release from a primary culture of bovine adrenal medullary cells was investigated. Midazolam at high doses (> 10 μ M) inhibited CA release evoked by acetylcholine (ACh), excess K⁺ and veratridine but not by A23187 or caffeine in Ca²⁺ -free media. Other benzodiazepines, diazepam, clonazepam, nitrazepam and R05-4864, as well as 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide (PK11195) and ethyl-β-carboline-3-carboxylate (βCCE) also inhibited ACh-evoked CA release but only at high concentrations. The inhibitory effect of midazolam on ACh-evoked CA release was not affected by R015-1788, a central-type benzodiazepine receptor antagonist which itself had no effect on basal and ACh-evoked CA release. Facilitatory action of Bay K 8644 on CA release evoked by 20 mM K⁺ was reduced by midazolam, PK11195 and R05-4864. Further, ACh-evoked ⁴⁵Ca uptake was markedly reduced by midazolam and R05-4864 in association with the inhibition of CA release. These results suggest that benzodiazepines at high doses, inhibit the evoked CA release from adrenal chromaffin cells possibly through the blockade of Ca²⁺ influx. Possible involvement of receptor subtypes of benzodiazepines in regulating CA secretion is discussed.     

Co-localization of vasoactive intestinal peptide (VIP) and enkephalins in chromaffin cells of the adrenal gland of amphibia. Stimulation of corticosteroid production by VIP

Recent studies have shown that biologically active peptides and monoaminergic neurotransmitters coexist in certain neuronal cell populations. Using the immunofluorescence technique, we have examined the localization of enkephalins, vasoactive intestinal peptide (VIP) and tyrosine hydroxylase in the adrenal gland of the frog Rana ridibunda. Most chromaffin cells which stained for tyrosine hydroxylase contained VIP-like immunoreactivity, whereas methionine- (Met-) and leucine- (Leu-) enkephalin-like immunoreactivity was detected in about 40% of the cells revealed by the anti-tyrosine hydroxylase serum. No VIP- or enkephalin-like immunoreactive nerve fibres were observed. Since in the frog, the chromaffin cells are in close contact with the adrenocortical (interrenal) tissue, a possible action of VIP and opiates on corticosteroidogenesis has been investigated. At doses 10(-6) and 10(-5) M, 20-min infusions of synthetic porcine or chicken VIP elicited a significant increase in corticosterone and aldosterone production by perifused frog adrenals, in a dose-dependent manner. As compared to ACTH, VIP was several orders of magnitude less effective in stimulating corticosteroid production. Morphine, Met- and Leu-enkephalins (10(-5) M) had no effect on spontaneous secretion of corticosteroids. In addition, Met- and Leu-enkephalins (10(-5) M) did not alter the production of corticosterone induced by ACTH. THese results suggest that VIP contained in the chromaffin cells of the frog adrenal gland may exert a local action in stimulating corticosteroid production by the interrenal tissue.     

Ryanodine Inhibits Caffeine-Evoked Ca2+ Mobilization and Catecholamine Secretion from Cultured Bovine Adrenal Chromaffin Cells

The effects of ryanodine, a selective inhibitor of the Ca(2+)-induced Ca2+ release mechanism, on caffeine-evoked changes in cytosolic Ca2+ concentration ([Ca2+]i) and catecholamine secretion were investigated using cultured bovine adrenal chromaffin cells. Caffeine (5-40 mM) caused a concentration-dependent transient rise in [Ca2+]i and catecholamine secretion in Ca2+/Mg(2+)-free medium containing 0.2 mM EGTA. Ryanodine (5 x 10(-5) M) alone had no effect on either [Ca2+]i or catecholamine secretion. Although the application of ryanodine plus caffeine caused the same increase in both [Ca2+]i and catecholamine secretion as those induced by caffeine alone, ryanodine (4 x 10(-7) - 5 x 10(-5) M) irreversibly prevented the increase in both [Ca2+]i and catecholamine secretion resulting from subsequent caffeine application over a range of concentrations. The secretory response to caffeine was markedly enhanced by replacement of Na+ with sucrose in Ca2+/Mg(2+)-free medium, and this enhanced response was also blocked by ryanodine. Caffeine was found to decrease the susceptibility of the secretory apparatus to Ca2+ in digitonin-permeabilized cells. These results indicate that caffeine mobilizes Ca2+ from intracellular stores, the function of which is irreversibly blocked by ryanodine, resulting in the increase in catecholamine secretion in the bovine adrenal chromaffin cell.     

Serotonin stimulates corticosteroid secretion by frog adrenocortical tissue in vitro

The mode of action of serotonin (5-HT) in the regulation of frog adrenal steroidogenesis was studied in vitro using the perifusion system technique. Graded doses of 5-HT (from 10(-8) to 10(-6) M) increased both corticosterone and aldosterone production in a dose-dependent manner. Short pulses (20 min) of 10(-6) M 5-HT, administered at 130 min intervals within the same experiment, did not cause any desensitization phenomenon. Indomethacin (IDM; 5 microM), a cyclooxygenase inhibitor which induced a dramatic decrease in the spontaneous secretion of corticosteroids, did not impair the stimulatory effect of 5-HT on corticosterone and aldosterone production. In the absence of calcium, 5-HT (10(-6) M) was still able to stimulate corticosteroid production. Dantrolene (5 x 10(-5) M), a blocker of calcium mobilization from intracellular pools which significantly inhibited the spontaneous production of corticosteroids, did not suppress 5-HT-evoked corticosteroid secretion. These results show that 5-HT, stored in adrenal chromaffin cells, may act as a paracrine factor to stimulate adrenal steroidogenesis in the frog. Our data also indicate that the mechanism of action of 5-HT does not depend on prostaglandin biosynthesis.     

GABAA receptor-mediated increase of cytosolic Ca2+ in isolated bovine adrenal chromaffin cells

We have studied the effects of GABA on cytosolic free Ca2+ concentration ([Ca2+]i) as a means of investigating the role of GABA in adrenal catecholamine (CA) secretion. It was demonstrated that GABA caused an elevation of [Ca2+]i via the GABAA receptor in a concentration-dependent manner, which was well correlated with an increase of 45Ca uptake, an increase of CA release and a depolarization of chromaffin cells assessed with bis-oxonol fluorescence. Since the GABA-induced rise of [Ca2+]i was absolutely dependent on the presence of extracellular Ca2+ and partly sensitive to nifedipine, at least one entry route for Ca2+ facilitated by GABA via a voltage-sensitive Ca2+ channel was suggested. When extracellular Cl- was lowered, GABA-induced CA release, depolarization, and rise of [Ca2+]i were all markedly enhanced. It is possible that GABA plays a modulatory role in the regulation of adrenal CA secretion as a facilitatory modulator.     

Alamethicin-evoked catecholamine release from cat adrenal glands

Alamethicin enhances the rate of catecholamine output from perfused cat adrenal glands in a concentration-dependent manner. At 37 degrees C, catecholamine released went from 4.29 +/- 0.25 to 20.51 +/- 0.63 micrograms/stimulus at ionophore concentrations ranging from 20 to 100 micrograms/ml. Secretion was abolished at 22 degrees C or in the absence of extracellular Ca. The time-course of secretion (quick activation followed by a decline) evoked by alamethicin considerably differs from the catecholamine release pattern seen with A23187, X537A or ionomycin, which evoke a slowly developing, non-inactivating secretory response. In fact, its transient secretion pattern resembles that of nicotinic or high-K stimulation of cat adrenal glands, thus suggesting that alamethicin might form Ca permeable artificial channels in chromaffin cell plasma membranes.     

Regulation of chromaffin cell secretion and protein kinase C activity by chronic phorbol ester treatment

Bovine adrenal chromaffin cells were exposed to phorbol esters to determine the effects of reduced levels of protein kinase C on secretion of hormones. Treatment with active phorbol esters such as 4 beta-phorbol 12, 13-didecanoate (PDD) reduced levels of protein kinase C activity with a maximal 80-90% reduction in activity after 16-24 h treatment (greater than or equal to 500 nM PDD). Treatment with PDD also inhibited catecholamine secretion from chromaffin cells evoked by nicotine, barium, and scorpion venom (50-70%, t1/2 approximately 6 h) and by veratridine (80%, t1/2 less than 15 min). Secretion induced by these agents in phorbol ester-treated cells returned to that of untreated cells by 3-4 days despite no recovery of protein kinase C activity. Potassium-evoked secretion was not inhibited by phorbol ester treatment. Catecholamine secretion from digitonin-permeabilized cells was more sensitive to calcium between 1 and 24 h, but not greater than or equal to 48 h, after addition of phorbol ester. The results suggest that phorbol esters inhibit secretion by activation of protein kinase C resulting in inhibition of ion channels or receptors but not of the secretory machinery itself; hence, protein kinase C may usually machinery itself; hence, protein kinase C may usually attenuate secretory responses in the adrenal chromaffin cell.     

Nitric Oxide Implication in the Control of Neurosecretion by Chromaffin Cells

Abstract: In this work, we have studied the effects of pure nitric oxide (NO) on the regulation of catecholamine (CA) secretion by chromaffin cells, as well as the possible presence of its synthesizing enzyme l -arginine:NO synthase (NOS) in these cells. Our results show that NO produces a large stimulation of basal CA secretion. This effect was calcium- and concentration-dependent (EC₅₀ = 64 ± 8 µ M ) and was not due to nonspecific damage of the tissue by NO. NO also modulates the CA secretion evoked by nicotine in a dose-dependent manner. Although it has a stimulatory effect on the CA secretion evoked by low doses of nicotine (<3 µ M ; EC₅₀ = 16 ± 3 µ M ), it produces a dose-dependent inhibition of the CA secretion induced by high doses of nicotine (≥30 µ M ; IC₅₀ = 52 ± 6 µ M ). The mechanism by which NO modulates CA secretion seems to be through the increase in the cyclic GMP levels, because there was a close correlation between the CA secretion and the cyclic GMP levels. The presence of a specific activity of NOS in chromaffin cells has been demonstrated by two independent methods: release of [¹⁴C]citruiline from [¹⁴C]arginine and formation of an NO-hemoglobin complex. NOS activity was about 0.5 pmol/min/mg of protein. It was calcium- and mainly calmodulin-dependent and could be specifically blocked by the NOS inhibitor N -methyl- l -arginine. These results suggest that NO could be an important intracellular messenger in the regulation of neurosecretion in chromaffin cells.     

Studies on the Inhibitory Action of Opiate Compounds in Isolated Bovine Adrenal Chromaffin Cells: Noninvolvement of Stereospecific Opiate Binding Sites

In isolated bovine adrenal chromaffin cells, beta-endorphin, dynorphin, and levorphanol caused a dose-dependent inhibition of catecholamine (CA) secretion elicited by acetylcholine (ACh), with an ID50 of 50, 1.3, and 4.3 microM, respectively. The inhibition by the opiate compounds was specific for the release evoked by ACh and nicotinic drugs and was noncompetitive with ACh. Stereospecific binding sites for the opiate agonist [3H]etorphine were found in homogenates of bovine adrenal medulla (KD = 0.59 nM). beta-Endorphin, dynorphin, levorphanol, and naloxone were potent inhibitors of the binding of [3H]etorphine with an ID50 of 12, 0.4, 5.2, and 6.2 nM, respectively. However, [3,5-I2Tyr1]-beta-endorphin, [3,5-I2Tyr1]-dynorphin, and dextrorphan, three opiate compounds with no or little activity in the guinea pig ileum assay, were relatively ineffective in inhibiting the binding of [3H]etorphine (ID50 700, 600, and 10,000 nM, respectively). On the other hand, these three compounds were equipotent with beta-endorphin, dynorphin, and levorphanol, respectively, in inhibiting the ACh-evoked release of CA from the adrenal chromaffin cells (ID50 of 10, 1.5, and 6 microM, respectively). Inhibition of CA release was also obtained with naloxone (ID50 = 14) microM) and naltrexone (ID50 greater than 10(-4) M), two classical antagonists of opiate receptors, and this effect was additive to that of beta-endorphin. These data indicate that the opiate modulation of CA release from adrenal chromaffin cells is not related to the stimulation of the high affinity stereospecific opiate binding sites of the adrenal medulla. The physiological function of these sites remains to be determined.     

Direct and Continuous Detection of ATP Secretion from Primary Monolayer Cultures of Bovine Adrenal Chromaffin Cells

A method was developed for direct and continuous detection of secretion of ATP from primary monolayer cultures of bovine adrenal chromaffin cells. ATP, which is costored with catecholamines within adrenal chromaffin cells, was released into the incubation medium, where it reacted with firefly luciferin-luciferase producing light detected by a photomultiplier located directly below the culture well. Acetylcholine, nicotine, the Ca2+ ionophore A23187, BaCl2, and KCl induced release of ATP. Induction of release of ATP by acetylcholine was dose dependent, with a threshold at 10(-7) M and a maximum at 10(-4) M. The dose-response curve for nicotine was bell shaped, with a threshold at 10(-7) M, a maximum at 10(-5) M, and diminished release at higher concentrations, an observation indicative of desensitization. Investigation of the initial rates of ATP secretion revealed that 10(-4) M nicotine actually induced release of ATP at a faster rate than 10(-5) M nicotine. However, the rate of ATP release evoked by 10(-4) M nicotine began to decline by 6 s, a result indicating the onset of receptor desensitization, whereas release induced by 10(-5) M nicotine continued unabated. Induction of release of ATP by acetylcholine or nicotine was biphasic, with a rapid, initial phase of release followed by a plateau at 0.5-1.5 min and a second phase of release beginning at 1.5-2 min, reaching a maximum by 2-3 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of catecholamine release in human adrenal chromaffin cells by β-adrenoceptors

The adrenal gland plays a fundamental role in the response to a variety of stress situations. After a stress condition, adrenal medullary chromaffin cells release, by exocytosis, high quantities of catecholamine (epinephrine, EP; norepinephrine, NE), especially EP. Once in the blood stream, catecholamines reach different target organs, and induce their biological actions through the activation of different adrenoceptors. Adrenal gland cells may also be activated by catecholamines, through hormonal, paracrine and/or autocrine system. The presence of functional adrenoceptors on human adrenal medulla and their involvement on catecholamines secretion was not previously evaluated. In the present study we investigated the role of β(1)-, β(2)- and β(3)-adrenoceptors on catecholamine release from human adrenal chromaffin cells in culture. We observed that the β-adrenoceptor agonist (isoproterenol) and β(2)-adrenoceptor agonist (salbutamol) stimulated catecholamine (NE and EP) release from human adrenal chromaffin cells. Furthermore, the β(2)-adrenoceptor antagonist (ICI 118,551; 100 nM) and β(3)-adrenoceptor antagonist (SR 59230A; 100 nM) inhibited the catecholamine release stimulated by isoproterenol and nicotine in chromaffin cells. The β(1)-adrenoceptor antagonist (atenolol; 100 nM) did not change the isoproterenol- neither the nicotine-evoked catecholamine release from human adrenal chromaffin cells. Moreover, our results show that the protein kinase A (PKA), protein kinase C (PKC), mitogen-activated protein kinase (MAPK) and phospholipase C (PLC) are intracellular mechanisms involved in the catecholamine release evoked by salbutamol. In conclusion, our data suggest that the activation of β(2)- and β(3)-adrenoceptors modulate the basal and evoked catecholamine release, NE and EP, via an autocrine positive feedback loop in human adrenal chromaffin cells.     

Inhibition of Ca(2+)-dependent catecholamine release by myosin light chain kinase inhibitor, wortmannin, in adrenal chromaffin cells.

To elucidate the possible involvement of myosin light chain kinase (MLCK) in the mechanism of exocytosis, we studied effects of MLCK inhibitor, wortmannin, on the secretory function of bovine adrenal chromaffin cells. Preincubation of chromaffin cells with wortmannin inhibited both acetylcholine- and high K(+)-evoked catecholamine (CA) release. The IC50 for high K(+)-evoked CA release was 1 microM. When the cells were permeabilized with digitonin after wortmannin preincubation, Ca(2+)-dependent exocytosis was inhibited in a dose-dependent manner (IC50, 1 microM). These findings suggest the implication of MLCK in the Ca(2+)-triggered process in the machinery of exocytosis.     

Cholinergic receptors and catecholamine secretion from adrenal chromaffin cells of the toad

1. The effects of cholinergic drugs on catecholamine (CA) secretion from adrenal chromaffin tissue of the toad were studied.2. CA secretion was induced by ACh or nicotine, but not by muscarine.3. Hexamethonium inhibited the CA release evoked by ACh or nicotine, while d-tubocurarine only affected the nicotinic response. Atropine did not prevent the secretory response.4. Muscarine abolished the secretion induced by the agonists, this effect being prevented by atropine or gallamine, but not by pirenzepine.5. In conclusion, CA secretion in the toad is stimulated by activation of nicotinic receptors. Inhibitory muscarinic receptors are present, most likely of type M₂, which may play a regulatory function.     

Action of vasoactive intestinal peptide (VIP) on amphibian adrenocortical function,in vitro

Vasoactive intestinal peptide (VIP) is located in chromaffin cells of the frog adrenal gland and is able to stimulate corticosteroid secretion in amphibians. In the present study we have investigated the possible involvement of prostaglandins, microfilaments and calcium in the mechanism of action of VIP on frog adrenocortical tissue. Rana ridibunda interrenal dice were perifused with amphibian culture medium for more than 10 hours. Corticosterone and aldosterone concentrations were measured in the effluent perifusate using sensitive and specific radioimmunoassay methods. In the presence of indomethacin (5 μM), a specific blocker of prostaglandin biosynthesis, the spontaneous secretion of corticosteroids was markedly reduced (80%) but the stimulatory effect of VIP was not altered. The administration of the microfilament disrupting agent cytochalasin B (50 μM) inhibited both spontaneous and VIP-induced corticosteroid secretion. In the absence of calcium, the spontaneous level of corticosteroid was reduced to about 60% but VIP was still able to stimulate corticosteroid secretion. From these data we conclude that the integrity of the cytoskeleton is required for the secretory response of adrenocortical cells to VIP, whereas neither prostaglandins nor calcium are involved in VIP-induced adrenocortical stimulation.     

Light-evoked recovery from wortmannin-induced inhibition of catecholamine secretion and synaptic transmission.

Wortmannin (WT) is known to inhibit catecholamine (CA) secretion in chromaffin cells. This effect was found to be sensitive to UV light in experiments designed to perform simultaneous monitoring of changes in [Ca2+]i and CA secretion in perfused rat adrenal medullas. When the change in [Ca2+]i was measured using calcium green-1 (490 nm excitation), a 35-min treatment with 10 microM WT caused a 69% inhibition of CA secretion evoked by excess (30 mM) extracellular K+ and a moderate inhibition of the [Ca2+]i response. In contrast, the same treatment of fura-2-loaded cells with WT caused only an 11% inhibition of the high-K+-evoked secretion and no significant attenuation of the [Ca2+]i response. However, during interruption of fluorometry with fura-2, the inhibitory effect of WT developed at a rate similar to that exhibited in calcium green-1-loaded cells. The WT-induced inhibition of high-K+- or bradykinin-evoked secretory responses, which was otherwise irreversible, was reversed by exposing WT-treated chromaffin cells to 380-nm light. When WT was reapplied to the cells of which the secretory ability had been restored by light irradiation, the secretory response was inhibited with a time course similar to that shown during the initial treatment with WT. The photosensitive effect of WT was also demonstrated using bullfrog sympathetic ganglia in which WT-induced inhibition of synaptic transmission was reversed by irradiation with 380-nm light. These results suggest that UV light removes the inhibitory effects of WT by disrupting the covalent bond formed between WT and a target molecule which remains to be determined, although myosin light chain kinase has been reported as the target molecule in both cases examined in this study.     

The benzodiazepine agonist clonazepam potentiates the effects of gamma-aminobutyric acid on alpha-MSH release from neurointermediate lobes in vitro

The action of the central-type benzodiazepine-receptor agonist clonazepam on alpha-MSH release has been studied in vitro using perifused frog neurointermediate lobes. High concentrations of clonazepam (3.16 X 10(-5) and 10(-4) M) caused an inhibition of alpha-MSH release and this effect was reversed by the central-type benzodiazepine-receptor antagonist Ro 15-1788. High doses of GABA (10(-5) and 10(-4) M) induced a biphasic effect on pars intermedia cells: a brief stimulation followed by a sustained inhibition of alpha-MSH secretion. Administration of clonazepam (10(-5) M) in the presence of various concentrations of GABA (10(-6) to 10(-4) M) led to a potentiation of both stimulatory and inhibitory phases of alpha-MSH secretion induced by GABA. Ro 15-1788 completely abolished the potentiating effect of clonazepam. Thus our results indicate that endogenous benzodiazepine receptors may modulate the effects of GABA on alpha-MSH secretion.     

Nicotonic and muscarinic components in acetylcholine stimulation of porcine adrenal medullary cells

Adrenal medullary chromaffin cells secrete catecholamines (CA) in response to cholinergic receptor activation by acetylcholine (ACh) released from splacnic nerve terminals. In cultured bovine chromaffin cells nicotinic receptors play a preponderant (> 90%) role in the control of CA release. By contrast, we found and report here that up to 40% of the ACh-evoked CA secretion from cultured porcine chromaffin cells can be associated with muscarinic receptor activation. The following results support our belief that in porcine adrenal medullary cells ACh (100 M) evoked CA secretion is mediated by both nicotinic and muscarinic cholinergic receptors. 1) Hexamethonium (100 M), a nicotinic receptor antagonist, inhibited ACh-induced CA secretion to ca. 40% of the control release and atropine (1 M), a muscarinic receptor antagonist, inhibited to ca. 60% of the control value. 2) We also found that ACh (100 M) evoked intracellular Ca2+ concentration ([Ca2+]i) rise was inhibited by these receptor antagonists to a different extent, and reversibly reduced by lowering the concentration of Ca2+ in the external medium ([Ca2+]o). This last maneuver ([Ca2+]o < 0.1 M) per se caused a marked reduction in the peak phase of the [Ca2+]i rise evoked by ACh (40% of the control response). Switching the external medium back to physiologic [Ca2+]o in the continued presence of ACh caused a partial recovery of the elevated [Ca2+]i. This [Ca2+]o-dependent [Ca2+]i rise was blocked by hexamethonium (100 M) but not by atropine (1 M). Conversely, the ACh-evoked [Ca2+]i rise in low external [Ca2+]o was blocked by atropine but not by hexamethonium. From these data we conclude that in porcine adrenal medullary cells an important fraction (ca. 0.4) of both ACh-induced CA secretion and peak [Ca2+]i rise is due to muscarinic receptor activation.     

Phospholipase C-mediated signalling is not required for histamine-induced catecholamine secretion from bovine chromaffin cells

A possible role for signalling through phospholipase C in histamine-induced catecholamine secretion from bovine adrenal chromaffin cells has been investigated. Secretion evoked by histamine over 10 min was not prevented by inhibiting inositol-1,4,5-trisphosphate receptors with 2-APB, by blocking ryanodine receptors with a combination of ryanodine and caffeine, or by depleting intracellular Ca(2+) stores by pretreatment with thapsigargin. Inhibition of protein kinase C with Ro31-8220 also failed to reduce secretion. Inhibition of phospholipase C with ET-18-OCH(3) reduced both histamine- and K(+) -induced inositol phosphate responses by 70-80% without reducing their secretory responses. Stimulating phospholipase C with Pasteurella multocida toxin did not evoke secretion or enhance the secretory response to histamine. The secretory response to histamine was little affected by tetrodotoxin or by substituting extracellular Na(+) with N -methyl-d-glucamine(+) or choline(+), or by substituting external Cl(-) with nitrate(-). Blocking various K(+) channels with apamin, charybdotoxin, Ba(2+), tetraethylammonium, 4-aminopyridine, tertiapin or glibenclamide failed to reduce the ability of histamine to evoke secretion. These results indicate that histamine evokes secretion by a mechanism that does not require inositol-1,4,5-trisphosphate-mediated mobilization of stored Ca(2+), diacylglycerol-mediated activation of protein kinase C, or activation of phospholipase C. The results are consistent with histamine acting by depolarizing chromaffin cells through a phospholipase C-independent mechanism.