Stimulatory effect of vasoactive intestinal polypeptide on catecholamine secretion from isolated guinea pig adrenal chromaffin cells

The stimulatory effect of vasoactive intestinal polypeptide (VIP) on catecholamine (CA) secretion from isolated guinea pig adrenal chromaffin cell was studied. VIP (1-10 microM) induced dose-dependent CA secretion, which was slow and continued for at least 30 min. This VIP-induced CA secretion was dependent on the presence of Ca2+ in the medium, but no significant increase in Ca2+ uptake by the cells was observed during their stimulation with VIP. Studies on the intracellular free Ca2+ level ([Ca2+]i) using fura-2 showed that acetylcholine and muscarine induced a marked increase in the [Ca2+]i, but that VIP induced only a slight increase. Thus VIP may induce CA secretion by increasing the sensitivity of the secretion of CA to Ca2+.     

Muscarinic stimulation of guinea pig adrenal chromaffin cells stimulates catecholamine secretion without significant increase in Ca2+ uptake

In isolated guinea pig adrenal chromaffin cells, not only nicotine, but also muscarine stimulated catecholamine (CA) secretion, the stimulation by muscarine being the greater. The secretions of CA by muscarine and nicotine were both dependent on the presence of Ca2+ in the medium, but only the latter was associated with a rapid increase in 45Ca2+ uptake. Experiments with the fluorescent Ca2+ indicator quin 2, showed that muscarine caused an increase in cytoplasmic free Ca2+ concentration [( Ca2+]i). Moreover, the intracellular Ca2+ antagonist 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8) inhibited both CA secretion and increase in [Ca2+]i induced by muscarine. These results indicate that in isolated guinea pig adrenal chromaffin cells, nicotine stimulated CA secretion by increasing Ca2+ uptake by the cells, whereas muscarine stimulated CA secretion by mobilizing Ca2+ from the intracellular pool.     

Stimulation by vasoactive intestinal polypeptide of muscarinic receptor-mediated catecholamine secretion from isolated guinea pig adrenal medullary cells

The effects of vasoactive intestinal polypeptide (VIP) on catecholamine (CA) secretion by isolated guinea pig adrenal medullary cells were studied. VIP (1 microM) alone induced only a slight secretion of CA, but it stimulated ACh-induced CA secretion. At concentrations of 0.01-1 microM, it stimulated muscarine-induced CA secretion, but not nicotine-induced CA secretion. It did not affect high K+ or Ca2+ ionophore-induced CA secretion. The stimulatory effect of VIP on muscarine-induced CA secretion was observed at muscarine concentrations of 2 200 microM and was detectable after 2 min incubation.     

Swelling-induced catecholamine secretion recorded from single chromaffin cells

We have studied osmotically induced catecholamine secretion from bovine adrenal chromaffin cells by combining patch-clamp measurements, electrochemical detection of secretion, and Fura-2 measurements of intracellular free calcium concentration ([Ca²⁺]_i). We find that osmotically induced catecholamine release is exocytotic and calcium dependent. Furthermore, we demonstrate that cell swelling is coupled to such secretion via a volume-activated current, carrying predominantly chloride, which causes a plateau depolarization of the cell membrane potential and thus promotes voltage-activated calcium influx. Therefore, cell volume changes may modulate the secretory activity.     

Somatostatin and substance P inhibit catecholamine secretion from isolated cells of guinea-pig adrenal medulla

Chromaffin cells isolated from guinea-pig adrenal glands secrete catecholamines in response to acetylcholine, nicotine, pilocarpine, veratridine, and high [K⁺]. Both substance P and somatostatin inhibit acetylcholine-induced catecholamine secretion. The maximal inhibition of acetylcholine-induced catecholamine secretion produced by substance P and by somatostatin is approximately 60%: the concentrations of the peptides required for half-maximal inhibition of secretion are approximately 0.8 and 2 μM. respectively. The maximal inhibition of catecholamine secretion produced by somatostatin and that caused by substance P are not additive. The effects of the peptides on secretion are readily reversible. Somatostatin and substance P also inhibit nicotine-induced catecholamine secretion, but they do not inhibit catecholamine secretion stimulated by pilocarpine, veratridine, or high [K⁺]. Thus, these peptides specifically inhibit catecholamine secretion linked to stimulation of nicotinic receptors. The inhibition of acetylcholine-induced catecholamine secretion by somatostatin is noncompetitive with respect to acetylcholine, Na⁺ or Ca²⁺.Immunoreactive somatostatin and substance P are present in guinea-pig adrenal glands. It is suggested that these peptides may play a role in the regulation of catecholamine secretion from the adrenal medulla.     

Characterization of catecholamine release from deer adrenal medullary chromaffin cells

Isolated adrenal medullary chromaffin cells maintained in culture have been widely used to study neurosecretory events. Many of these studies have been conducted using cells obtained from the bovine adrenal. In this study we have cultured chromaffin cells from an alternative large animal model, the deer, and have conducted the first characterization of secretion from this preparation. Cervine chromaffin cells, preloaded with [3H]noradrenalin, displayed a strong secretory response to the cholinergic agonist carbachol, with a maximal secretion of approximately 28% cell content over 15 min. This response was reproduced by nicotinic but not muscarinic agonists and was similarly inhibited by nicotinic but not muscarinic antagonists. Nicotine-evoked secretion measured over a 15 min time period was inhibited approximately 50% by the L-type Ca2+-channel antagonist nifedipine and approximately 20% by N-type (omega-conotoxin GVIA) or N, P/Q-type (omega-conotoxin MVIIC) antagonists. In contrast the response was unaffected by omega-agatoxin IVA, a P/Q-type antagonist. In addition to nicotinic receptor stimulation, activation of PACAP or histamine H1 receptors resulted in a concentration-dependent increase in secretion. PACAP was approximately two-fold more effective than histamine although both were weaker secretagogues than nicotine. In contrast, cervine chromaffin cells did not respond to angiotensin II or bradykinin, two agents known to stimulate secretion from bovine chromaffin cells. These data provide an initial characterization of the secretory response from cervine adrenal medullary chromaffin cells indicating that there are marked similarities but also potentially significant differences between them and their far more extensively described bovine counterparts.     

Reinvestigation of the effect of orexin A on catecholamine release from adrenal chromaffin cells

Orexins have been shown to be implicated in the regulation of adrenal medulla functions. However, there are still inconsistent investigations on the effects of orexins on catecholamine release from chromaffin cells in varying species. In the present study, using the carbon-fiber amperometry, we investigated whether orexin A would stimulate catecholamine release from rat and mouse adrenal chromffin cells. Puff application of orexin A dose-dependently induced amperometric currents in the cultured rat chromaffin cells, which was completely blocked by the selective OX1R antagonist SB-334867 or by the removal of extracellular calcium. Likewise, in the mouse adrenal medulla slices, orexin A also induced catecholamine release mainly through the activation of OX1R. These results gain insight into our understanding of the pharmacological relevance of orexin system in modulating neuroendocrine functions.     

Catecholamine secretion from rat foetal adrenal chromaffin cells and hypoxia sensitivity

The adrenal medulla chromaffin cells (AMCs) secrete catecholamines in response to various types of stress. We examined the hypoxia-sensitivity of catecholamine secretion by rat foetal chromaffin cells in which the innervation by the splanchnic nerve is not established. The experiments were performed in primary cultured cells from two different ages of foetuses (F15 and F19). Membrane potential of AMCs was monitored with the patch clamp technique, and the catecholamine secretion was detected by amperometry. We found that: (1) AMCs from F19 foetuses showed hypoxia-induced catecholamine release. (2) This hypoxia-induced secretion is produced by membrane depolarization generated by an inhibition of Ca²⁺-activated K⁺ current [I _K(Ca)] current. (3) Chromaffin precursor cells from F15 foetuses secrete catecholamine. The quantal release is calcium-dependent, but the size of the quantum is reduced. (4) In the precursor cells, a hypoxia-induced membrane hyperpolarization is originated by an ATP-sensitive K⁺ current [I _K(ATP)] activation. (5) During the prenatal period, at F15, the percentage of the total outward current for I _K(ATP) and I _K(Ca) was 50 and 29.5%, respectively, whereas at F19, I _K(ATP) is reduced to 14%, and I _K(Ca) became 64% of the total current. We conclude that before birth, the age-dependent hypoxia response of chromaffin cells is modulated by the functional activity of K_ATP and K_Ca channels.     

Mechanism through which GABAA receptor modulates catecholamine secretion from bovine chromaffin cells.

The actions and mechanism of GABAergic modulation of catecholamine secretion from isolated bovine chromaffin cells were investigated. The GABAA receptor agonist muscimol induced a fast rise in cytosolic [Ca2+]. The mean peak increase was 290 +/- 30 nM over basal levels. The increase in cytosolic [Ca2+] was abolished in the absence of extracellular [Ca2+] and was blocked by the GABAA antagonist bicuculline and the dihydropiridine nifedipine. Muscimol also elicited the release of catecholamines and increased the bisoxonol fluorescence indicating a cell depolarization. The [Ca2+] entry was well correlated with muscimol-evoked catecholamine secretion. When cells were treated with muscimol and a second secretagogue, a biphasic behavior was revealed. Muscimol enhanced the catecholamine release evoked by low concentrations of nicotine or K+, whereas release obtained at high concentrations of nicotine or K+ was actually inhibited. When the muscimol effect on membrane potential was studied in the presence of low K+ or nicotine concentrations, an enhancement of the bisoxonol fluorescence was observed. This effect was reversed at high concentrations of both K+ and nicotine. Measurement of 36Cl- fluxes showed an increase in membrane permeability to Cl- during muscimol stimulation. The influx or efflux in Cl- was dependent on membrane potential. In normal conditions, with a K+ concentration of 5.4 mM, a Cl- efflux was observed by both radiometric techniques, with 36Cl- and by the use of the chloride-sensitive fluorescent probe 6-methoxy-N-(3-sulphopropil)quinolinium, as indicator of intracellular Cl-. At high nicotine (20 mM) or K+ concentrations (105 mM) a Cl- influx was observed using 6-methoxy-N-(3-sulphopropil)quinolinium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Palytoxin: a potent stimulator of catecholamine release from cultured bovine adrenal chromaffin cells.

The effect of palytoxin (PTX), a potent marine toxin, on catecholamine release from cultured bovine adrenal chromaffin cells was examined. PTX at concentrations of over 10(-9) M induced catecholamine release dose-dependently. About 40-50% of the total cellular catecholamine was released during 20-min incubation with 3 x 10(-8) M PTX. PTX-induced catecholamine release was dependent on both extracellular Na+ and Ca2+, and was inhibited by organic and inorganic Ca2+ channel blockers, but not by tetrodotoxin. PTX-induced increase in 45Ca2+ influx into the cells, which was associated with catecholamine release, was also inhibited by these Ca2+ channel blockers. These results indicated that PTX-induced catecholamine release was mediated by activation of Na(+)-dependent, tetrodotoxin (TTX) insensitive voltage-dependent Ca2+ channels.     

Pertussis toxin attenuates clonidine inhibition of catecholamine release in adrenal chromaffin cells

Characteristics of the inhibitory action of clonidine on catecholamine release in bovine adrenal chromaffin cells were investigated. Clonidine at 3 x 10(-5) M inhibited acetylcholine (ACh)-evoked release by about 50%, but not catecholamine release evoked by high K+. Another alpha 2-agonist alpha-methyladrenaline was ineffective at inhibiting ACh-evoked release. The inhibition by clonidine of ACh-evoked release was not reversed by alpha 2-antagonists. Treatment of these cells with pertussis toxin reversed the inhibitory effect of clonidine, while it did not affect the inhibitory action of hexamethonium and of nifedipine. Therefore, clonidine inhibition of catecholamine release in these cells seems not to be mediated by the alpha 2-adrenoceptor, but might be mediated by a specific receptor for clonidine.     

Inhibitory effect of polymyxin B on catecholamine secretion from cultured bovine adrenal medullary cells

Incubation of cultured bovine adrenal medullary cells with 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of Ca2+/phospholipid-dependent protein kinase (protein kinase C), was associated with increased secretion of catecholamine (CA) from the cells. Polymyxin B (PMB, 30-300 microM), a preferential inhibitor of protein kinase C, inhibited the TPA-induced secretion of CA. PMB also inhibited CA secretion induced by other secretagogues, the Ca2+ ionophore ionomycin (10 microM), 56 mM K+ or acetylcholine (ACh). Ionomycin, 56 mM K+ or ACh increased the concentration of intracellular free Ca2+ ([Ca2+]i) (measured using the fluorescent calcium indicator quin2), whereas TPA did not increase [Ca2+]i. PMB blocked the increase in [Ca2+]i induced by 56 mM K+ or ACh at concentrations similar to those inhibiting the secretion of CA. In contrast, PMB did not affect ionomycin-induced increase in [Ca2+]i. These results strongly suggest that CA secretion induced by TPA or ionomycin is mediated via activation of protein kinase C. The results further indicate that in 56 mM K+- or ACh-evoked CA secretion, PMB inhibits the secretion by blocking Ca2+ influx into the cells.     

The involvement of specific phospholipase C isozymes in catecholamine release from digitonin permeabilized bovine adrenal medullary chromaffin cells

The role of phospholipase C (PLC) in exocytosis has been investigated using digitonin permeabilized, [(3)H]noradrenaline ([(3)H]NA) loaded, bovine adrenal medullary chromaffin cells. The PLC inhibitor U-73122 caused a concentration-dependent suppression of Ca(2+)-evoked [(3)H]NA release but increased basal release (that occurring in the absence of Ca(2+)). Preincubation with antibodies against PLCgamma1 or PLCbeta3 (but not PLCdelta1, delta2, beta1 and beta2) also inhibited [(3)H]NA release evoked by Ca(2+) and increased basal release, indicating that only specific PLC isozymes are involved in these actions. Interestingly, PLCgamma1 (but not PLCbeta3) antibodies inhibited the ability of Ca(2+) to increase PLC activity in these permeabilized cells. These data therefore suggest that PLCgamma1 activity may have a specific role in regulating the exocytotic response from the adrenal chromaffin cell.     

The effect of CdSe-ZnS quantum dots on calcium currents and catecholamine secretion in mouse chromaffin cells

Semiconductor nanocrystal quantum dots (QDs) possess an enormous potential of applications in nanomedicine, drug delivery and bioimaging which derives from their unique photoemission and photostability characteristics. In spite of this, however, their interactions with biological systems and impact on human health are still largely unknown. Here we used neurosecretory mouse chromaffin cells of the adrenal gland for testing the effects of CdSe-ZnS core-shell quantum dots (5-36 nM) on Ca(2+) channels functionality and Ca(2+)-dependent neurosecretion. Prolonged exposure (24 h) to commonly used concentrations of CdSe-ZnS QDs (≥16 nM) showed that the semiconductor nanocrystal is effectively internalized into the cells without affecting cell integrity (no changes of membrane resistance and cell capacitance). QDs reduced the size of Ca(2+) currents by ～28% in a voltage-independent manner without affecting channel gating. Correspondingly, depolarization-evoked exocytosis, measured at +10 mV, where Ca(2+) currents are maximal, was reduced by 29%. CdSe-ZnS QDs reduced the size of the readily releasable pool (RRP) of secretory vesicles by 32%, the frequency of release by 33% and the overall quantity of released catecholamines by 61%, as measured by carbon fibers amperometry. In addition, the Ca(2+)-dependence of exocytosis was reduced, whereas the catecholamine content of single granules, as well as the kinetics of release, remained unaltered. These data suggest that exposure to CdSe-ZnS QDs impairs Ca(2+) influx and severely interferes with the functionality of the exocytotic machinery, compromising the overall catecholamine supply from chromaffin cells.     

Sodium ions and the secretion of catecholamines

1. Perfusion of bovine adrenal glands with a potassium-free Tyrode solution results in an increased spontaneous release of catecholamines and an increased response to stimulation with carbamylcholine.2. Perfusion with a Tyrode solution containing 70 mM-KCl causes a marked secretion of catecholamines and a subsequent inhibition of secretion in response to stimulation with carbamylcholine.3. Perfusion with sodium-free media abolishes or severely reduces the secretory response of the gland to carbamylcholine and to 70 mM-KCl although the basal secretion shows an initial rise.4. Perfusion with sodium-deficient media permits some retention of the secretory response.5. A possible role for intracellular sodium ions in the secretion of catecholamines is discussed.     

Observations on the muscarinic activation of catecholamine secretion in the chicken adrenal

Cells were isolated by collagenase digestion of chicken adrenal glands. Catecholamine secretion could be stimulated by acetylcholine, carbamylcholine, potassium or veratridine. Methacholine, muscarine and oxotremorine were also effective secretagogues whereas nicotine was not. Secretion evoked by acetylcholine was blocked by low concentrations of atropine but was relatively insensitive to hexamethonium. Atropine-sensitive secretion required both external sodium and calcium, was unaffected by tetrodotoxin, blocked by methoxy verapamil and nifedipine, and potentiated by BAY-K-8644. These data suggest that muscarinic activation of these cells facilitates tetrodotoxin insensitive depolarization, thereby opening conventional voltage-sensitive calcium channels. The mechanism by which calcium activates catecholamine secretion was investigated in cells that had been made permeable by exposure to brief intense electric fields. Catecholamine release required Mg-adenosine 5' triphosphate, was half-maximally activated by 1 microM Ca2+ and could be inhibited by high concentrations of Mg2+. At low Ca2+ concentrations, release was potentiated by 12-O-tetradecanoylphorbol 13-acetate, dioctanoylglycerol, guanosine 5'-O-(3-thiotriphosphate) and 5'-guanylylimidodiphosphate, all of which increased the apparent affinity of exocytosis for Ca2+.     

Effects of pro-opiomelanocortin fragments on release of catecholamines from adrenal chromaffin cells

The effects of pro-opiomelanocortin (POMC) peptide fragments on the basal and agonist-induced release of catecholamines (CAs) from monolayer cultures of purified bovine adrenal chromaffin cells were tested. None of the 5 peptides tested, i.e. ß-MSH, ACTH_1–39, γ-MSH_1–13. γ₃-MSH and N-terminal POMC fragment, had any effect on basal CA release. However, ß-MSH (10^?5 M), γ-MSH_1–13 (10^?6–10^?5 M), γ₃-MSH (10^?5 M) and N-terminal POMC fragment (10^?5 M) inhibited the nicotine-induced release of CAs from the chromaffin cells. The possible physiological significance of this inhibitory neuromodulation is discussed.