Effect of trifluoperazine and calmodulin on catecholamine secretion by saponin-skinned cultured chromaffin cells

We have examined the effect of trifluoperazine on catecholamine secretion by chemically skinned, cultured adrenal chromaffin cells. These cells require only ATP and calcium for secretion. Catecholamine secretion was unaffected by the drug in the presence or absence of calcium and ATP over the range 0.1 to 10 microM. At 100 microM trifluoperazine, catecholamine release was calcium and ATP independent and represented 70-80% of the total cellular content. High concentrations of exogenous calmodulin had no effect on secretion in the presence or absence of calcium. We conclude that low concentrations of the drug have no effect on secretion, while high concentrations cause non-physiological catecholamine release.     

Asbestos-elicited catecholamine secretion from isolated bovine adrenal chromaffin cells

Standard (UICC) chrysotile B asbestos fibres caused rapid (within minutes) 5-to-8-fold stimulations of catecholamine secretion from isolated bovine adrenal chromaffin cells without affecting their viability (97%). The stimulation of catecholamine secretion by asbestos was selective to chrysotile type fibres, half-maximal stimulation by standard chrysotile B, chrysotile A, crocidolite, amosite and silica fibres being observed at 7, 73, 160, 250 and ? 500 μg per ml, respectively. The secretory effect of chrysotile B was additive to that of acetylcholine and blocked by either the divalent cations, Co²⁺, Ni²⁺ and Mg²⁺ or the ion chelators, EGTA and EDTA. Conversely, neither verapamil, methoxyverapamil, or removal of extracellular calcium affected the asbestos-evoked catecholamine secretion. These data indicate that the selective stimulatory effect of chrysotile type asbestos on adrenal chromaffin cells can be mediated by membrane or intracellular calcium and raise the question of the possible involvement of catecholamines in the pathogenesis of asbestos related diseases.     

DHEA attenuates catecholamine secretion from bovine adrenal chromaffin cells

Dehydroepiandrosterone (DHEA) is a putative anti-stress agent and stress is associated with the secretion of catecholamine from the adrenal gland, but the effects of DHEA on catecholamine secretion are not fully understood. Using bovine chromaffin cells, we found that DHEA inhibited catecholamine secretion and cytosolic Ca²⁺ ([Ca²⁺]_i) rise coupled with nicotinic acetylcholine receptor (nAChR) without exerting an effect on³H-nicotine binding. In the case of high K⁺ stimulation, DHEA effectively suppressed secretion without affecting [Ca²⁺]₁ rise. Trifluoperazine (TFP), a calmodulin inhibitor, was capable of counteracting the inhibition of DHEA on high K⁺-induced secretions. In permeabilized cells, DHEA suppressed the Ca²⁺-induced secretion. These results suggest that DHEA (a) acts as a channel blocker that suppresses Ca²⁺ influx and subsequent secretions associated with nAChR, or (b) affects the intracellular secretion machinery to suppress high K⁺-induced secretions without affecting the high K⁺-induced [Ca²⁺]_i rise.     

Thiophosphorylation prevents catecholamine secretion by chemically skinned chromaffin cells

Skinned cells treated with the adenosine triphosphate analog, adenosine-5'-0-(3-thiotriphosphate) showed calcium-dependent thiophosphorylation of cellular constituents. Catecholamine secretion was inhibited when the analog was used as the substrate to promote secretion. The attenuation of secretion was proportional to the percentage of the analog in mixtures with adenosine triphosphate. Moreover, cells treated with the analog were subsequently unable to secrete when presented with MgATP, their normal substrate, indicating that the secretory systems was locked in the thiophosphorylated state. We hypothesize that phosphorylation is the calcium-dependent step required to prime the secretory system for secretion while dephosphorylation is the event required for exocytosis.     

Oligopeptide inhibitors of metalloendoprotease activity inhibit catecholamine secretion from bovine adrenal chromaffin cells by modulating intracellular calcium homeostasis

Synthetic oligopeptide inhibitors of metalloendoprotease activity were found to inhibit catecholamine release from intact bovine adrenal chromaffin cells. The efficiency of these compounds in blocking secretion was dependent on the type and dose of the secretagogues employed. By contrast, catecholamine release from digitonin-permeabilized cells stimulated with micromolar calcium was virtually not affected. Using a different model system mimicking protein-mediated membrane fusion during exocytosis (Bental, M., Lelkes, P.I., Scholma, J., Hoekstra, D., and Wilschut, J. (1984) Biochim. Biophys. Acta 774, 296-300) we found that exposure of chromaffin granules to a genuine metalloendoprotease, thermolysin, impaired their fusion competence with liposomes. The same oligopeptide inhibitors of metalloendoprotease activity that interfered with secretion from the intact cells were also found to cause an increase in 45Ca2+ efflux concomitant with a slight elevation of the free intracellular calcium concentration [( Ca2+]i) to levels not sufficient to elicit secretion. Subsequent stimulation of the cells in the presence of the potent inhibitors resulted in a reduced increase in the cytosolic calcium concentration, as compared to nontreated control cells. The reduction in the secretagogue-evoked rise in [Ca2+]i was also dependent on the time of pretreatment of the cells with the metalloendoprotease inhibitors. Consistently, none of these effects were seen with structurally similar oligopeptides that are not metalloendoprotease substrates/inhibitors. We conclude that potent inhibitors of metalloendoprotease activity and hence, presumably, the enzymes per se modulate stimulus-secretion coupling by interfering with calcium homeostasis rather than directly with membrane fusion.     

Rapid rise in cyclic GMP accompanies catecholamine secretion in suspensions of isolated adrenal chromaffin cells.

Acetylcholine stimulates a five-fold increase in cyclic GMP within seconds of addition to suspensions of isolated adrenal chromaffin cells. The rapid kinetics and dose-response curve for cyclic GMP accumulation are compared with corresponding results for catecholamine secretion and a possible role of cyclic GMP in stimulus-secretion coupling is considered.     

Multiple effects of honokiol on catecholamine secretion from adrenal chromaffin cells

The molecular mechanism of honokiol, extracted from the bark of Magnolia obovata, was studied using bovine adrenal chromaffin cells as a model system. Honokiol inhibits catecholamine secretion induced by carbachol and DMPP and that induced by exposure to high K+ and Ba2+ but to a lesser extent. The inhibitory effects of trifluoperazine and honokiol on carbachol-, high K(+)- and Ba2(+)- induced secretion were not additive. The results suggest that honokiol interferes with the interaction between the acetylcholine receptor and its agonists and that honokiol may also affect the steps in exocytosis after intracellular calcium has been raised, possibly at the site(s) where calmodulin acts.     

The effects of ketamine, phencyclidine and lidocaine on catecholamine secretion from cultured bovine adrenal chromaffin cells

The ability of ketamine, phencyclidine and analogues to alter catecholamine secretion from cultured bovine adrenal chromaffin cells was investigated. Both ketamine and phencyclidine specifically inhibited nicotinic agonist-induced secretion at concentrations which did not alter secretion induced by elevated K+ depolarization. The inhibition of nicotinic agonist-induced secretion was not overcome by increasing concentrations of nicotinic agonist. The effects of stereoisomer pairs of phencyclidine-like drugs - dexoxadrol, levoxadrol and (+)PCMP, (-)PCMP - did not reveal stereospecificity for the inhibition, in contrast to the stereospecific behavioral effects of the drugs. The local anesthetic lidocaine (0.3 mM) also noncompetitively inhibited nicotinic agonist-induced secretion without inhibiting elevated K+-induced secretion. The data indicate that ketamine and phencyclidine at clinically relevant concentrations specifically inhibit the adrenal chromaffin cell nicotinic receptor at a site similar to or identical with the site of action of local anesthetic. Although the nicotinic receptor inhibition is probably not related to the anesthetic and behavioral effects of ketamine and phencyclidine, it is likely that the centrally mediated increase in sympathetic nervous system activity which is characteristic of these drugs is moderated by the peripheral blocking effects on catecholamine secretion from the adrenal medulla.     

Leptin directly stimulates catecholamine secretion and synthesis in cultured porcine adrenal medullary chromaffin cells.

Leptin, a protein encoded by the ob gene, is an adipose tissue-derived signaling factor involved in body weight homeostasis. The hypothalamus is a major site of central action for leptin. However, mounting evidence indicates expression of leptin receptor mRNA in various peripheral organs including the adrenal medulla. Therefore, we investigated the effects of leptin on catecholamine secretion and synthesis in cultured porcine adrenal medullary chromaffin cells. We initially confirmed the expression of leptin receptor (Ob-Rb) mRNA in cultured porcine adrenal medullary cells. Murine recombinant leptin (>==50 nM) strongly induced the release of both epinephrine (E) and norepinephrine (NE) from chromaffin cells. Removal of external Ca(2+) significantly suppressed these effects. Also, leptin (>==1 nM) enhanced nicotine-induced increases in E- and NE. Leptin (1, 10, 100 nM) significantly increased tyrosine hydroxylase (TH) (a rate-limiting enzyme in the biosynthesis of catecholamine) mRNA levels in a concentration-dependent manner. Furthermore, leptin (1, 10, 100 nM) significantly induced increases in cAMP levels, suggesting that the stimulatory effects on TH mRNA are mediated, at least in part, by the cAMP/protein kinase A pathway. These results indicate that leptin directly stimulates catecholamine release and synthesis, which in turn may potentiate the anti-obesity effects of leptin.     

Guanine nucleotide effects on catecholamine secretion from digitonin-permeabilized adrenal chromaffin cells

The nonhydrolyzable GTP analogue guanosine 5'-(beta, gamma-imido)triphosphate (GMP-PNP) produced an ATP-dependent but Ca2+-independent stimulation of [3H]norepinephrine release from permeabilized chromaffin cells. This stimulation of secretion was 25-35% of the secretion induced by 10 microM Ca2+. A similar Ca2+-independent stimulation was produced by other non-hydrolyzable GTP analogues. No effect was seen with a variety of other nucleotides, including GTP. The GMP-PNP effect was specifically inhibited by low concentrations of guanine nucleotides. Addition of cAMP did not mimic the Ca2+-independent GMP-PNP effect, but did slightly enhance Ca2+-dependent secretion. Pretreatment with pertussis toxin had no effect on Ca2+-dependent secretion or on the GMP-PNP effect. There was no detectable diglyceride or inositol phosphate produced during GMP-PNP treatment, and addition of diglyceride and inositol trisphosphate did not induce secretion. Guanosine 5'-(beta-thio)diphosphate (GDP-beta-S), in addition to its ability to inhibit the GMP-PNP effect, partially inhibited Ca2+-dependent secretion. At 10 microM free Ca2+, the effects of GMP-PNP and Ca2+ were nonadditive. In fact, secretion in the presence of both GMP-PNP and 10 microM Ca2+ was slightly less than secretion due to Ca2+ alone. These data suggest that a guanine nucleotide-dependent process interacts in some way with one or more components of the normal Ca2+-dependent secretory pathway. However, it may not be an intrinsic part of the mechanism underlying Ca2+-dependent secretion.     

Role of intracellular pH in secretion from adrenal medulla chromaffin cells

The role of intracellular pH in stimulus-secretion coupling was investigated in cultured bovine adrenal medullary chromaffin cells. NH4Cl (1-25 mM) did not affect basal catecholamine or ATP release but markedly inhibited nicotine- or high K+-induced release by up to 60%. The inhibition had a rapid onset (less than 1 min) and was maximal at about 5 mM NH4Cl. The effect of NH4Cl was largely sustained over 20 min and was reversed upon NH4Cl removal. Sodium propionate did not affect secretion but partially reversed the inhibition by NH4Cl in a concentration-dependent manner. Methylamine (10 mM) produced a similar, but slower, inhibition than NH4Cl. Monensin (1-10 microM) inhibited catecholamine secretion by 30-60%, and its effect was reduced in the presence of NH4Cl. Using the fluorescent Ca2+ probe Fura-2, we found that the increase of [Ca2+]i following stimulation was not altered by concentrations of NH4Cl which inhibited secretion maximally. Measurement of cytosolic pH (pHi) with the fluorescent probe 2',7'-bis-carboxyethyl-5(6)-carboxyfluorescein (BCECF) revealed an alkalinization by NH4Cl (2.5-25 mM) of 0.1-0.23 pH units and acidification by sodium propionate (10-20 mM) of 0.2-0.25 pH units, with intermediate combined effects. Monensin (1 microM) caused a cytosolic acidification of 0.26 pH units. All pHi changes were partly recovered in 15 min. Fluorescence quenching measurements using the weakly basic fluorescent probe acridine orange indicated the accumulation of the probe into acidic compartments, presumably the chromaffin granules, which was strongly reduced by both NH4Cl and monensin. From these findings we conclude that the pH of the chromaffin granule modulates secretion by affecting some step in the secretory process unrelated to the rise in [Ca2+]i.     

Osmotic strength differentiates between two types of calcium transport pathways regulating catecholamine secretion from cultured bovine chromaffin cells

Calcium transport and catecholamine secretion was measured in cultured bovine chromaffin cells. Calcium ions which entered the cells following stimulation with either nicotine or 50 mM KCl (high potassium) triggered catecholamine release, but then inactivated the secretory process. The nicotine and the high potassium-induced calcium transport mechanisms were mechanistically distinct, but functionally dependent on each other. The specific evidence is that whereas the high potassium-induced Ca2+ influx was found to be inhibited by hyperosmotic medium, the nicotine-stimulated calcium influx was unaffected under these conditions. High potassium and nicotine-stimulated catecholamine release were also differently affected by hyperosmotic medium. While potassium-stimulated catecholamine release was profoundly inhibited by hyperosmolarity, nicotine-stimulated release was only moderately inhibited. Sequential treatments of cells with nicotine and high potassium, under isotonic physiological conditions, indicate that there is a functional, biochemical communication between the otherwise mechanistically distinct calcium channels. Calcium ions which were found to inactivate these channels may be the basis for such communication.     

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.     

Peripheral O2 chemoreceptors mediate humoral catecholamine secretion from fish chromaffin cells

This study addressed the hypothesis that the secretion of catecholamines from trout (Oncorhynchus mykiss) chromaffin cells, during hypoxia, is triggered by stimulation of O(2) chemoreceptors located within the gills. Sodium cyanide was administered into the inspired water (external cyanide) or injected into the gill circulation (internal cyanide) to pharmacologically stimulate external (water sensing) or internal (blood sensing) O(2) chemoreceptors, respectively. Both of these treatments caused an elevation of circulating catecholamine levels. The response to external, but not internal, cyanide was abolished by removal of the first gill arch. Hypoxia produced an increase in circulating catecholamine levels that was unaffected by removal of the first gill arch or by denervation of the pseudobranch. Cyanide and hypoxia both caused the well-documented cardiorespiratory reflexes normally observed in this species. This study demonstrates, for the first time, that gill O(2) chemoreceptors can initiate the reflex that leads to catecholamine release from the chromaffin cells and that stimulation of internally oriented O(2) receptors on all gill arches appears to be the physiologically important mechanism for initiating release.     

Pertussis toxin facilitates secretagogue-induced catecholamine release from cultured bovine adrenal chromaffin cells

Pretreatment of cultured bovine adrenal chromaffin cells with pertussis toxin facilitated nicotine-induced catecholamine release. This facilitation was correlated with the ability of the toxin to catalyze the ADP-ribosylation of an approximately 40-kDa membrane protein. The actions of the toxin were reversed by isonicotinamide, an inhibitor of ADP-ribosylation. Catecholamine release due to high K+ and muscarine was also enhanced by pertussis toxin. In all cases, 45Ca2+ uptake was unaltered in cells treated with the toxin. These results suggest that ADP-ribosylation of a 40-kDa membrane protein facilitates catecholamine release from bovine chromaffin cells without affecting 45Ca2+ uptake.     

Effect of calmidazolium and phorbol ester on catecholamine secretion from adrenal chromaffin cells

Carbamylcholine-stimulated catecholamine release from adrenal chromaffin cells was completely inhibited by pretreatment of the cells for 10 min with 1 μM calmidazolium. Catecholamine release due to 55 mM K⁺ and ionophore A23187 was also inhibited by calmidazolium but less effectively than release due to carbamylcholine. Inhibition of release appeared to be due to an effect of calmidazolium on a step distal to Ca²⁺ entry, since the carbamylcholine-stimulated rise in the concentration of intracellular free calcium, monitored using quin-2, was unaffected by calmidazolium. The possibility was considered that calmidazolium inhibited secretion through an effect on protein kinase C rather than calmodulin. However, the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), had no demonstrable effect on catecholamine release, arguing against a significant role for protein kinase C in secretion from adrenal chromaffin cells. These results give further support to the notion that calmodulin plays a role in the secretory process in chromaffin cells.     

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.     

Stimulation of catecholamine synthesis in cultured bovine adrenal medullary cells by leptin

Recently, we characterized leptin receptors in bovine adrenal medullary cells (Yanagihara et al. 2000). Here we report the stimulatory effect of leptin on catecholamine synthesis in the cells. Incubating cells with leptin (10 nM) for 20 min increased the synthesis of 14C-catecholamines from [14C]tyrosine, but not from L-3,4-dihydroxyphenyl [3-14C]alanine. The stimulation of catecholamine synthesis in the cells by leptin was associated with the phosphorylation and activation of tyrosine hydroxylase, the rate-limiting enzyme of catecholamine biosynthesis. The incubation of cells with leptin resulted in a rapid activation of the mitogen-activated protein kinases (MAPKs). An inhibitor of MAPK kinase, U0126, nullified the stimulatory effect of leptin on the synthesis of 14C-catecholamines. Leptin potentiated the stimulatory effect of acetylcholine on 14C-catecholamine synthesis, whereas leptin failed to enhance the phosphorylation and activation of tyrosine hydroxylase induced by acetylcholine. These findings suggest that leptin stimulates catecholamine synthesis via the activation of tyrosine hydroxylase by two different mechanisms, i.e., one is dependent on tyrosine hydroxylase phosphorylation mediated through the MAPK pathway and the second is independent of enzyme phosphorylation.     

Calmodulin is involved in catecholamine secretion from digitonin-permeabilized bovine adrenal medullary chromaffin cells.

The role of calmodulin in exocytotic secretion was studied using digitonin-permeabilized bovine adrenal medullary chromaffin cells. Addition of calmodulin to the permeabilized cells increased Ca(2+)-dependent norepinephrine release in a dose-dependent manner. Unlike calmodulin, addition of caldesmon, actin or bovine serum albumin did not increase the release. Calmodulin increased the release at Ca2+ concentrations of more than 10(-6) M and its effect increased with increase in Mg2+ concentration. Th release of norepinephrine enhanced by calmodulin was inhibited by tetanus toxin, which specifically inhibits exocytotic secretion. These results indicate directly that calmodulin plays an important role in exocytotic secretion from chromaffin cells.     

Nicotinic receptor-mediated catecholamine secretion from individual chromaffin cells. Chemical evidence for exocytosis

Nicotinic receptor-mediated secretion of catecholamines from individual cultured bovine adrenal medullary chromaffin cells was measured and characterized with a voltametric microelectrode placed adjacent to the cells. Nicotine-induced secretion is associated with a large increase in chemical spikes that is temporally resolved into the apparent secretion of discrete packets of attomole quantities of easily oxidized molecules. These data are consistent with direct chemical measurement of single exocytotic events.