Basal Catecholamine Secretion Research Articles

Bidirectional regulation of bakuchiol, an estrogenic-like compound, on catecholamine secretion

Excess or deficiency of catecholamine (CA) secretion was related with several diseases. Recently, estrogen and phytoestrogens were reported to regulate the activity of CA system. Bakuchiol is a phytoestrogen isolated from the seeds of Psoralea corylifolia L. (Leguminosae) which has been used in Traditional Chinese medicine as a tonic or aphrodisiac. In the present study, bovine adrenal medullary cells were employed to investigate the effects and mechanisms of bakuchiol on the regulation of CA secretion. Further, its anti-depressant like and anti-stress effects were evaluated by using behavioral despair and chronic immobilization stress models. Our results indicated that bakuchiol showed bidirectional regulation on CA secretion. It stimulated basal CA secretion in a concentration dependent manner (p<0.01), while it reduced 300μM acetylcholine (ACh) (p<0.01), 100μM veratridine (Ver) (p<0.01) and 56mM K+ (p<0.05) induced CA secretion, respectively. We also found that the stimulation of basal CA secretion by bakuchiol may act through estrogen-like effect and the JNK pathway in an extra-cellular calcium independent manner. Further, bakuchiol elevated tyrosine hydroxylase Ser40 and Ser31 phosphorylation (p<0.01) through the PKA and ERK1/2 pathways, respectively. Bakuchiol inhibited ACh, Ver and 56mM K+ induced CA secretion was related with reduction of intracellular calcium rise. In vivo experiments, we found that bakuchiol significantly reduced immobilization time in behavioral despair mouse (p<0.05 or 0.01), and plasma epinephrine (E) and norepinephrine (NE) levels in chronic immobilization stress (p<0.05). Overall, these results present a bidirectional regulation of bakuchiol on CA secretion which indicated that bakuchiol may exert anti-stress and the potential anti-depressant-like effects.

Long‐lasting increase in basal catecholamine secretion from neonatal adrenal medullary chromaffin cells by chronic intermittent hypoxia

We previously reported that intermittent hypoxia (IH) facilitates hypoxia‐evoked catecholamine (CA) secretion from neonatal adrenal medullary chromaffin cells (AMC) in rat pups. Here, we examined the effects of repetitive hypoxia on CA secretion from AMC from IH exposed rat pups. Adrenal medullary slices were harvested from rat pups exposed to either IH or normoxia from ages P0‐P5, 8–9 hours/day. CA secretion was monitored by carbon fiber amperometry. In IH exposed pups, repetitive hypoxia (30s hypoxia followed by 90s normoxia × 5 episodes) facilitated CA secretion from AMC during each episode. Remarkably, basal CA secretion remained elevated for 30 min after terminating repetitive hypoxia. The long‐lasting increase in basal CA secretion was not seen in response to repetitive application of 40 mM K+. In striking contrast, in control pups, CA secretion during repetitive hypoxia was modest and after terminating the stimulus, promptly returned to basal levels. The long‐lasting increase in basal CA secretion in IH pups could be elicited in Ca2+ free medium and was prevented in presence of 10 μM ryanadine, which blocks ryanadine receptors (RyRs). These data suggest that neonatal IH results in long‐lasting activation of basal CA secretion by repetitive hypoxia, and this effect is mediated in part by RyRs and mobilization of intracellular Ca2+ (supported by HL‐90554, HL‐76537, HL‐86493 and HL‐089616).

Neonatal overfeeding causes higher adrenal catecholamine content and basal secretion and liver dysfunction in adult rats

Rats that are overfed during lactation exhibit neonatal hyperleptinemia and higher visceral adiposity, hypertension, higher liver oxidative stress and insulin resistance in the liver as adults. Previously, we demonstrated that neonatal hyperleptinemia is associated with adrenal medullary hyperfunction, hypertension and liver steatosis in adulthood. Therefore, we hypothesised that adrenal and liver functions are altered in adult obese rats that were overfed during lactation, which would underlie their hypertension and liver alterations. The litter size was reduced from ten to three male pups on the third day of lactation until weaning (SL) to induce early overfeeding in Wistar rats. The control group had ten rats per litter (NL). Rats had free access to standard diet, and water after weaning until the rats were 180 days old. The SL group exhibited higher adrenal catecholamine content (absolute: +35% and relative: +40%), tyrosine hydroxylase (+31%) and DOPA decarboxylase (+90%) protein contents and basal catecholamine secretion in vitro (+57%). However, the hormones of the hypothalamic-pituitary-adrenal cortex axis were unchanged. β₃-adrenergic receptor content in visceral adipose tissue was unchanged in SL rats, but the β₂-adrenergic receptor content in the liver was lower in this group (-45%). The SL group exhibited higher glycogen and triglycerides contents in the liver (+79 and +49%, respectively), which suggested microesteatosis. Neonatal overfeeding led to higher adrenomedullary function, but the liver β₂-adrenergic receptor content was reduced. These results may contribute to the hepatic dysfunction characteristic of liver obesity complications.

Impaired Sympathoadrenal Axis Function Contributes to Enhanced Insulin Secretion in Prediabetic Obese Rats

The involvement of sympathoadrenal axis activity in obesity onset was investigated using the experimental model of treating neonatal rats with monosodium L-glutamate. To access general sympathetic nervous system activity, we recorded the firing rates of sympathetic superior cervical ganglion nerves in animals. Catecholamine content and secretion from isolated adrenal medulla were measured. Intravenous glucose tolerance test was performed, and isolated pancreatic islets were stimulated with glucose and adrenergic agonists. The nerve firing rate of obese rats was decreased compared to the rate for lean rats. Basal catecholamine secretion decreased whereas catecholamine secretion induced by carbachol, elevated extracellular potassium, and caffeine in the isolated adrenal medulla were all increased in obese rats compared to control. Both glucose intolerance and hyperinsulinaemia were observed in obese rats. Adrenaline strongly inhibited glucose-induced insulin secretion in obese animals. These findings suggest that low sympathoadrenal activity contributes to impaired glycaemic control in prediabetic obese rats.

Dual effects of lipophilic extract of Salvia miltiorrhiza (Danshen) on catecholamine secretion in cultured bovine adrenal medullary cells

Ethnopharmacological relevance Salvia miltiorrhiza (Danshen) is a well known traditional Chinese herb, which has been used widely in China for the treatment of cardiovascular diseases in clinic. Aim of this study The aim of the present study is to clarify the effects of lipophilic extract of Salvia miltiorrhiza (LESM) on catecholamine (CA) secretion, a traditional Chinese medicine used widely for the treatment of cardiovascular diseases in China. Materials and methods LESM was evaluated for its effects on CA secretion using HPLC-ECD method. The effects of LESM on 22Na + influx and intracellular calcium ([Ca 2+] i) were also investigated. Results Our results showed that LEMS directly stimulated basal CA secretion in an extracellular Ca 2+-dependent manner. And the stimulation was not affected by combination of hexamethonium (Hex), an inhibitor of nAChR. LESM also directly elevated [Ca 2+] i. In addition, using selective blockers of voltage-dependent Ca 2+ channels, such as nitrendipine (for L-type), ω-agatoxin-IVA (for P-type) and ω-conotoxin-GVIA (for N-type), it was found that nitrendipine suppressed the elevation of [Ca 2+] i induced by LESM, but not ω-agatoxin-IVA or ω-conotoxin-GVIA. Compared with acetylcholine (ACh) only, however, combination of LESM with ACh inhibited the raise of CA secretion, 22Na + influx and [Ca 2+] i in a concentration-dependent manner. Furthermore, LESM also inhibited CA secretion induced by veratridine (Ver), and 56 mM K + at concentrations similar to those for [Ca 2+] i rise. One of the lipophilic active compounds, cryptotanshione (Cryp), also had the same effects on CA secretion with LESM. Conclusions All these findings suggest that LESM exerts dual effects on CA secretion in cultured bovine adrenal medullary cells. LESM exerts antagonistic effects on nAChR, voltage-dependent Na + and Ca 2+ channels, whereas it is an agonist of L-type Ca 2+ channel when it used alone.

Nitric oxide donors induce calcium-mobilisation from internal stores but do not stimulate catecholamine secretion by bovine chromaffin cells in resting conditions

The potential role of nitric oxide (NO) donors and peroxynitrites on both basal catecholamine (CA) secretion and modulation of calcium levels has been investigated in primary cultures of bovine chromaffin cells. NO donors did not modulate catecholamine secretion, while peroxynitrites induced a time dose-dependent increase in basal CA secretion. Two facts may explain the lack of these compounds on basal CA secretion. NO donors induce, on the one hand, an increase in intracellular calcium levels by depletion of internal IP 3-stores from endoplasmic reticulum. On the other hand, a small calcium influx through N-type voltage-dependent calcium channels (VDCC), which seem not to be coupled to exocytosis of adrenaline and noradrenaline in chromaffin cells. Both effects, calcium-mobilisation from internal stores and calcium entry through N-type VDCC are mediated by cGMP synthesis. In contrast, peroxynitrites induce an increase in basal CA secretion by both a decrease of intracellular catecholamine content and a toxic effect on cellular membrane. All these results, taken together, could explain contradictory results in the literature on the role of NO on basal catecholamine secretion and on modulation of intracellular calcium in chromaffin cells.

Adrenal medullary function and expression of catecholamine-synthesizing enzymes in mice with hypothalamic obesity

The mechanisms underlying the onset of obesity are complex and not completely understood. An imbalance of autonomic nervous system has been proposed to be a major cause of great fat deposits accumulation in hypothalamic obesity models. In this work we therefore investigated the adrenal chromaffin cells in monosodium glutamate (MSG)-treated obese female mice. Newborn mice were injected daily with MSG (4 mg/g body weight) or saline (controls) during the first five days of life and studied at 90 days of age. The adrenal catecholamine content was 56.0% lower in the obese group when compared to lean controls ( P < 0.0001). Using isolated adrenal medulla we observed no difference in basal catecholamine secretion percentile between obese and lean animals. However, the percentile of catecholamine secretion stimulated by high K + concentration was lower in the obese group. There was a decrease in the tyrosine hydroxylase enzyme expression (57.3%, P < 0.004) in adrenal glands of obese mice. Interestingly, the expression of dopamine β-hydroxylase was also reduced (47.0%, P < 0.005). Phenylethanolamine N-methyltransferase expression was not affected. Our results show that in the MSG model, obesity status is associated with a defective adrenal chromaffin cell function. We conclude that in MSG obesity the low total catecholamine content is directly related to a decrease of key catecholamine-synthesizing enzymes, which by its turn may lead to a defective catecholamine secretion.

Neuronal nitric oxide synthase modulates basal catecholamine secretion in bovine chromaffin cells.

The role of endogenously produced nitric oxide (NO) in the regulation of basal catecholamine (CA) secretion was studied in chromaffin cells. Treatment of chromaffin cells with nitric oxide synthase (NOS) inhibitors produced a dose-dependent increase in basal catecholamine secretion, which paralleled their ability to inhibit NOS activity. This inhibitory profile was similar to that found in neurons, suggesting the constitutive expression of neuronal NOS (nNOS) in these cells, which was confirmed by Western blot analysis. A study of the kinetics and pharmacology of nNOS activity expressed in chromaffin cells in culture indicated that NOS activity is calcium-dependent, increases with time, and is highly dependent on both intracellular concentrations of L-arginine (K(m) approximately 4 microM, V(max) = 908 +/- 60 pmol/hr x 10(6) cells) and transport of L-arginine into the cells (exhibiting two affinity constants of k(1) = 3.2 +/- 0.3 microM and k(2) = 126 +/- 5.5 microM). The effects of NOS inhibitors on CA secretion were mediated by the L-arginine-NO-cGMP pathway, insofar as exogenous L-arginine was able to partially block the increase in CA secretion evoked by them, and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ), a specific inhibitor of guanylate cyclase, and zaprinast, an inhibitor of the cGMP phosphodiesterase, were able to increase and inhibit, respectively, basal CA secretion in a dose-dependent manner. These results suggest that chromaffin cells exhibit a tonic production of NO by nNOS that keeps the basal CA secretion at low levels, and this could be necessary for maintaining a normotensive state.

Calcitonin gene-related peptide (CGRP), acting via CGRP type 1 receptors, inhibits potassium-stimulated aldosterone secretion and enhances basal catecholamine secretion from rat adrenal gland.

Calcitonin gene-related peptide (CGRP) is a potent hypotensive peptide, that acts via two main subtypes of receptors, named CGRP1 and CGRP2. CGRP belongs to a regulatory-peptide family, that includes adrenomedullin (ADM) whose aldosterone antisecretagogue and catecholamine secretagogue actions are well demonstrated. Quantitative autoradiography showed the presence of [125I]CGRP binding sites in both rat adrenal zona glomerulosa (ZG) and medulla. Binding was displaced by the CGRP1-receptor antagonist CGRP(8-37), but not by the CGRP2-receptor agonist [cys(Et)2,7]-alphaCGRP (CGRP2-A). CGRP concentration-dependently inhibited 10 mM-stimulated (but not basal) aldosterone secretion from dispersed rat ZG cells, and enhanced basal catecholamine secretion from rat adrenomedullary fragments. The responses to the maximal effective concentration of CGRP (10-8 M) were blocked by 10-7 M CGRP(8-37). CGRP2-A (10-7 M) neither altered aldosterone response to 10 mM K+ nor enhanced basal catecholamine secretion. The conclusion is drawn that CGRP, like ADM, inhibits agonist-stimulated aldosterone secretion and stimulates basal catecholamine release in the rat, exclusively acting via CGRP1 receptors.

Opioids Suppress Basal and Nicotine-Induced Catecholamine Secretion Via a Stabilizing Effect on Actin Filaments*

Catecholamine secretion and actin filament disassembly are closely coupled in chromaffin cells. Opioid suppression of catecholamine secretion is fast and transient, both characteristics of actin filament involvement. The aim of the present work was to test the hypothesis that opioids suppress catecholamine secretion via an inhibitory effect on actin filament disassembly. For this purpose we used the PC12 rat pheochromocytoma cell line. Norepinephrine and dopamine were measured by enzyme-linked immunosorbent assay or RIA. Polymerized actin was measured by rhodamine-phalloidin and visualized by confocal laser scanning microscopy. Opioids suppressed basal catecholamine secretion. The onset of this effect was fast and transient, peaking at 2 min, and was reversible by opioid antagonists. Synchronously, opioids suppressed actin filament disassembly; this was also reversible by opioid antagonists. Cytochalasin B prevented the inhibitory effect of opioids on catecholamine secretion. In addition, opioids suppressed the stimulatory effect of nicotine on catecholamine secretion and actin depolymerization. Changes in actin cytoskeleton in neuron-like PC12 cells make them resistant to both effects of opioids, i.e. on catecholamine secretion and actin disassembly. In conclusion, our data suggest that the suppressive effect of opioids on basal and nicotine-induced catecholamine secretion may result from an opioid-provoked stabilization of cortical actin. It also appears that basal catecholamine secretion is associated with opioid-sensitive machinery regulating the continuous formation of short-lived areas of cortical actin filament disassembly.

Role of PAC(1) receptor in adrenal catecholamine secretion induced by PACAP and VIP in vivo.

The present study was conducted to investigate the functional implication of the pituitary adenylate cyclase-activating polypeptide (PACAP) type I (PAC(1)) receptor in the adrenal catecholamine (CA) secretion induced by either PACAP-27 or vasoactive intestinal polypeptide (VIP) in anesthetized dogs. PACAP-27, VIP, and their respective antagonists were locally infused to the left adrenal gland via the left adrenolumbar artery. Plasma CA concentrations in adrenal venous and aortic blood were determined by means of a high-performance liquid chromatograph coupled with an electrochemical detector. Adrenal venous blood flow was measured by gravimetry. The administration of PACAP-27 (50 ng) resulted in a significant increase in adrenal CA output. VIP (5 microg) also increased the basal CA secretion to an extent comparable to that observed with PACAP-27. In the presence of PACAP partial sequence 6--27 [PACAP-(6--27); a PAC(1) receptor antagonist] at the doses of 7.5 and 15 microg, the CA response to PACAP-27 was attenuated by approximately 50 and approximately 95%, respectively. Although the CA secretagogue effect of VIP was blocked by approximately 85% in the presence of PACAP-(6--27) (15 microg), it remained unaffected by VIP partial sequence 10--28 [VIP-(10--28); a VIP receptor antagonist] at the dose of 15 microg. Furthermore, the CA response to PACAP-27 did not change in the presence of the same dose of VIP--(10--28). The results indicate that PACAP-(6--27) diminished, in a dose-dependent manner, the increase in adrenal CA secretion induced by PACAP-27. The results also indicate that the CA response to either PACAP-27 or VIP was selectively inhibited by PACAP-(6--27) but not by VIP-(10--28). It is concluded that PAC(1) receptor is primarily involved in the CA secretion induced by both PACAP-27 and VIP in the canine adrenal medulla in vivo.

Enhanced reactivity of the adrenal medulla in response to pituitary adenylate cyclase activating polypeptide1-27 (PACAP) during insulin-induced hypoglycemia in anesthetized dogs

The present study was to test the hypothesis that the reactivity of the adrenal medulla to pituitary adenylate cyclase activating polypeptide1-27 (PACAP27) is enhanced during insulin-induced hypoglycemia (IIH) in anesthetized dogs. Plasma catecholamine (CA) concentrations in adrenal venous and aortic blood were determined by an HPLC method coupled with electrochemical detection, and the plasma glucose concentration in aortic blood was measured using a glucometer. PACAP27 (25 ng) was administered locally via the adrenolumbar artery to the left adrenal gland. The resulting CA responses were compared before and during IIH following an intravenous bolus injection of insulin (0.15 IU/kg, i.v.). In the first group with normal adrenal innervation, the basal adrenal CA secretion gradually increased, reaching a maximum level 45 min after the insulin injection. The net increase in PACAP27-induced CA secretion was significantly greater 30, 45, and 60 min after the induction of hypoglycemia, compared with the initial net response to PACAP27 observed before insulin injection. In the second group receiving local adrenal denervation, neither the basal CA secretion nor the net CA response to PACAP27 significantly increased despite the presence of IIH, which developed to an extent similar to that found in the first group. In the third group, which was the normoglycemic control group, both the basal CA secretion and the net CA response to PACAP27 remained unchanged during the experimental period. The results indicate that the adrenomedullary reactivity to PACAP27 was significantly enhanced during IIH only when the sympathoadrenal system was activated. The present study suggests that PACAP27 may play a beneficial role in glucose counterregulatory mechanisms in the adrenal medulla during hypoglycemia.

Enhanced reactivity of the adrenal medulla in response to pituitary adenylate cyclase activating polypeptide&lt;sub&gt;1-27&lt;/sub&gt; (PACAP) during insulin-induced hypoglycemia in anesthetized dogs

The present study was to test the hypothesis that the reactivity of the adrenal medulla to pituitary adenylate cyclase activating polypeptide1-27 (PACAP27) is enhanced during insulin-induced hypoglycemia (IIH) in anesthetized dogs. Plasma catecholamine (CA) concentrations in adrenal venous and aortic blood were determined by an HPLC method coupled with electrochemical detection, and the plasma glucose concentration in aortic blood was measured using a glucometer. PACAP27 (25 ng) was administered locally via the adrenolumbar artery to the left adrenal gland. The resulting CA responses were compared before and during IIH following an intravenous bolus injection of insulin (0.15 IU/kg, i.v.). In the first group with normal adrenal innervation, the basal adrenal CA secretion gradually increased, reaching a maximum level 45 min after the insulin injection. The net increase in PACAP27-induced CA secretion was significantly greater 30, 45, and 60 min after the induction of hypoglycemia, compared with the initial net response to PACAP27 observed before insulin injection. In the second group receiving local adrenal denervation, neither the basal CA secretion nor the net CA response to PACAP27 significantly increased despite the presence of IIH, which developed to an extent similar to that found in the first group. In the third group, which was the normoglycemic control group, both the basal CA secretion and the net CA response to PACAP27 remained unchanged during the experimental period. The results indicate that the adrenomedullary reactivity to PACAP27 was significantly enhanced during IIH only when the sympathoadrenal system was activated. The present study suggests that PACAP27 may play a beneficial role in glucose counterregulatory mechanisms in the adrenal medulla during hypoglycemia.

The effects of chronic hypoxia on the acute adrenergic stress response in the rainbow trout (Oncorhynchus mykiss).

We have investigated the effects of chronic hypoxia on the acute adrenergic stress response of adult rainbow trout (Oncorhynchus mykiss). The goal of this study was to determine whether a prior 5-d exposure of fish to lowered environmental oxygen levels (60 or 80 Torr) would influence the nature of catecholamine secretion from chromaffin tissue in situ. Using a saline-perfused posterior cardinal vein preparation, it was demonstrated that the basal (unstimulated) secretion of noradrenaline and adrenaline was increased at 60-Torr hypoxia. In response to cholinergic (carbachol-elicited) stimulation, noradrenaline and adrenaline secretion were significantly affected by prior exposure to hypoxia. The construction of dose response curves revealed that noradrenaline secretion was enhanced at the lowest doses of carbachol (1 - 5 x 10(-7) mol kg(-1)) and that this was reflected by an approximate 10-fold reduction in the ED50 (the dose of carbachol eliciting half-maximal noradrenaline secretion). The effect of chronic hypoxia on in situ carbachol-evoked adrenaline secretion was similar but less pronounced. The results of this study suggest that during chronic moderate hypoxia, increased basal catecholamine secretion and enhanced responsiveness of chromaffin cells to cholinergic stimulation, as well aiding the ongoing stress, may assist the physiological adaptations to subsequent bouts of more severe acute stress.

Effect of Glutamate Receptor Agonists on Catecholamine Secretion in Bovine Chromaffin Cells

In this study, the effects of glutamate and glutamate receptor agonists in cultured chromaffin cells from bovine adrenal medulla were investigated. It was found that glutamate increases basal catecholamine (CA) secretion in a dose-dependent manner. This effect is mimicked by specific agonists of the four known glutamate receptors N-methyl-D-aspartate (NMDA), quisqualate/(RS)-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate (KA), and trans-(+)-1-amino-1,3-cyclopentane dicarboxylic acid (t-ACPD), which increased both basal and nicotine-evoked CA secretion. The NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid, 6-cyano-7-nitroquinoxaline-2,3-dione, an antagonist of KA and AMPA receptors, and L-(+)-2-amino-3-phosphonopropionic acid, an antagonist of the t-ACPD receptor, inhibited the stimulatory effect of related glutamate agonists. Hexamethonium, an antagonist of the nicotinic receptor, failed to influence glutamate agonists except for a 15% inhibition of KA. The increase in CA secretion produced by a 100 µM concentration of glutamate agonists was about 20–60% of that obtained with 10 µM of nicotine, an agonist of the physiological stimulatory cholinergic receptor. The increase in CA secretion produced by glutamate was accompanied by both an increase in bisoxonol fluorescence, suggesting membrane depolarization, and by an increase in intracellular Ca²⁺ concentrations. Results obtained with image analysis on single cells indicated that the percentage of cells which respond to the stimulation of 50 µM of glutamate is 42%. From these results, we conclude that glutamate, through its four known glutamate receptors, can increase both basal and nicotine-evoked CA secretion in chromaffin cells by a process which involves membrane depolarization and an increase in intracellular calcium levels.

1233 Tracer analysys of m6, a mouse growth cone rich protein, utilising flag epitope

For determination of whether myosin light-chain kinase (MLCK) is involved in the secretory mechanism of adrenal chromaffin cells, the effect of a preferential inhibitor of the enzyme, 1-(5-chlor-naphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9), on catecholamine secretion from cultured bovine adrenal chromaffin cells was studied. ML-9 did not affect basal catecholamine secretion, but inhibited catecholamine secretion stimulated by acetylcholine, high K+, veratridine or palytoxin. At similar concentrations to those inhibiting the secretion of catecholamine, ML-9 also inhibited increased [45Ca]2+ uptake by the cells induced by these stimulants. However, it did not inhibit catecholamine secretion induced by the Ca2+ ionophore A23187. Moreover, it did not affect catecholamine secretion from digitonin-permeabilized cells induced by a micromolar Ca2+ concentration in the presence of Mg ATP. These results indicate that ML-9 inhibits catecholamine secretion from adrenal chromaffin cells by inhibiting the transmembrane Ca2+ uptake mechanism, but not by inhibiting the intracellular Ca2+- dependent mechanism. The possible role of MLCK in stimulus-secretion coupling in adrenal chromaffin cells is discussed.

A reassessment of the modulatory role of cyclic AMP in catecholamine secretion by chromaffin cells

1. The role of adenosine 3':5'-cyclic monophosphate (cyclic AMP) in the regulation of catecholamine (CA) secretion in chromaffin cells remains equivocal from previous studies. 2. In the present study the effect of this cyclic nucleotide on basal CA secretion, as well as on intracellular calcium and membrane potential has been examined. 3. Forskolin and the permeable cyclic AMP analogue, 8-(4-chlorphenylthio)-adenosine-3'-5' monophosphate cyclic (pClpcAMP), increased basal CA secretion in a dose-dependent manner. The EC50s were 0.43 +/- 0.10 microM for forskolin and 39 +/- 9 microM for pClpcAMP. Other agonists with adenylate cyclase activity such as stimulants of adenosine receptors, beta-adrenoceptors, GABAB receptors and intestinal vasoactive peptide (VIP), also increased basal CA secretion in a highly significant manner. However, when they were added together with forskolin, CA secretion was not affected although an additive increase in cyclic AMP levels was produced. 4. Statistical analysis of the correlation between cyclic AMP levels and CA secretion evoked by these cyclic AMP increasing compounds showed that a significant direct correlation between both parameters existed only when low levels of cyclic AMP were produced by secretagogue stimulation. When the increase in intracellular cyclic AMP concentrations exceeded approximately 8 times the basal cyclic AMP levels the correlation was not significant. These results indicate a dual dose-dependent effect of cyclic AMP on basal CA secretion. 5. The stimulatory effect of low cyclic AMP on basal CA secretion was accompanied by an increase in membrane potential and in intracellular calcium concentrations ([Ca2+]j), the latter mainly being due to an increase in intracellular Ca2+ entry through L-type voltage-dependent Ca2" channels.6. The possible mechanisms involved in these cyclic AMP effects are discussed.

Nitric oxide implication in the control of neurosecretion by chromaffin cells.

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 microM) 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 microM; EC50 = 16 +/- 3 microM), it produces a dose-dependent inhibition of the CA secretion induced by high doses of nicotine (> or = 30 microM; IC50 = 52 +/- 6 microM). 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]citrulline 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.

GABAB receptors modulate catecholamine secretion in chromaffin cells by a mechanism involving cyclic AMP formation

1. The function of gamma-aminobutyric acidB (GABAB) receptors in modulation of catecholamine secretion by chromaffin cells and the possible mechanism involved in this action have been examined. 2. The GABAB agonists (-)-baclofen and 3-aminopropylphosphinic acid (3-APPA) were found to induce a dose-dependent increase of basal catecholamine secretion. The EC50s were 151 +/- 35 microM and 225 +/- 58 microM for baclofen and 3-APPA, respectively. This stimulatory effect was specific since it could be blocked by 0.5 mM of the specific GABAB antagonist CGP-35348. 3. In contrast, preincubation of chromaffin cells with the GABAB agonists was found to inhibit, in a dose-dependent manner, the catecholamine secretion evoked by 10 microM nicotine and 200 microM muscimol. 4. The effects of GABAB agonists on both basal and evoked catecholamine secretion were found to be accompanied by parallel changes in intracellular calcium concentration ([Ca2+]i). GABAB agonists produced a dose-dependent increase in [Ca2+]i which was partially blocked by CGP 35348, but they produced a strong inhibition of the [Ca2+]i increase induced by nicotine and muscimol. 5. The GABAB agonists also produced a dose-dependent increase in intracellular cyclic AMP levels, there being a direct correlation between both increase in catecholamine secretion and in intracellular cyclic AMP levels. 6. The pretreatment of chromaffin cells with pertussis toxin doubled the catecholamine secretion and increased by four times the intracellular cyclic AMP levels evoked by GABAB agonists. 7. The possible involvement of adenylate cyclase in the mechanism of GABAA receptor modulation of catecholamine secretion is discussed.

Substance P has biphasic effects on catecholamine secretion evoked by electrical stimulation of perfused rat adrenal glands in vitro

The adrenal medulla is innervated by the splanchnic nerve which contains substance P-immunoreactive fibres originating in the dorsal root ganglia but whose function in the adrenal medulla is not known. In the present study, we have examined the effect of exogenous substance P on catecholamine secretion and [ 3H]-choline overflow evoked by field stimulation of the perfused rat adrenal gland. Substance P had biphasic effects on catecholamine secretion evoked by field stimulation: at the lower concentrations (10 −7−3 × 10 −6 M), substance P facilitated catecholamine secretion whereas at higher concentrations (3 × 10 −5 M), substance P inhibited catecholamine secretion. The effects of substance P were long-lasting: substance P at all concentrations present during the second of three 6-min stimulations increased catecholamine secretion during the third stimulation. The enhancement of catecholamine secretion by substance P during field stimulation was not blocked by atropine but was substantially blocked by hexamethonium plus atropine. Substance P at all concentrations inhibited the evoked [ 3H]-choline overflow with a maximal inhibition of 60% (at 10 −6 M). In the absence of stimulation, substance P (10 −7−3 × 10 −5 M) had no effect on the basal catecholamine secretion or basal [ 3H]-choline overflow. These results indicate that substance P has modulatory effects on adrenal catecholamine secretion and [ 3H]-choline overflow evoked by electrical stimulation. Presynaptically, substance P inhibits transmitter overflow from cholinergic nerve terminals and post-synaptically substance P has biphasic effects on catecholamine secretion dependent on the substance P concentration. These results suggest a functional role for substance P-containing fibres innervating the adrenal medulla.