Evoked Catecholamine Release Research Articles

Gaseous transmitter regulation of hypoxia-evoked catecholamine secretion from murine adrenal chromaffin cells.

Emerging evidence suggests that gaseous molecules, carbon monoxide (CO), and hydrogen sulfide (H2S) generated by heme oxygenase (HO)-2 and cystathionine γ-lyase (CSE), respectively, function as transmitters in the nervous system. Present study examined the roles of CO and H2S in hypoxia-induced catecholamine (CA) release from adrenal medullary chromaffin cells (AMCs). Studies were performed on AMCs from adult (≥6 wk of age) wild-type (WT), HO-2 null, CSE null, and HO-2/CSE double null mice of either gender. CA secretion was determined by carbon fiber amperometry and [Ca2+]i by microflurometry using Fura-2. HO-2- and CSE immunoreactivities were seen in WT AMC, which were absent in HO-2 and CSE null mice. Hypoxia (medium Po2 30-38 mmHg) evoked CA release and elevated [Ca2+]i. The magnitude of hypoxic response was greater in HO-2 null mice and in HO inhibitor-treated WT AMC compared with controls. H2S levels were elevated in HO-2 null AMC. Either pharmacological inhibition or genetic deletion of CSE prevented the augmented hypoxic responses of HO-2 null AMC and H2S donor rescued AMC responses to hypoxia in HO-2/CSE double null mice. CORM3, a CO donor, prevented the augmented hypoxic responses in WT and HO-2 null AMC. CO donor reduced H2S levels in WT AMC. The effects of CO donor were blocked by either ODQ or 8pCT, inhibitors of soluble guanylyl cyclase (SGC) or protein kinase G, respectively. These results suggest that HO-2-derived CO inhibits hypoxia-evoked CA secretion from adult murine AMC involving soluble guanylyl cyclase (SGC)-protein kinase G (PKG)-dependent regulation of CSE-derived H2S.NEW & NOTEWORTHY Catecholamine secretion from adrenal chromaffin cells is an important physiological mechanism for maintaining homeostasis during hypoxia. Here, we delineate carbon monoxide (CO)-sensitive hydrogen sulfide (H2S) signaling as an important mediator of hypoxia-induced catecholamine secretion from murine adrenal chromaffin cells. Heme oxygenase-2 derived CO is a physiological inhibitor of catcholamince secretion by hypoxia and the effects of CO involve inhibition of cystathionine γ-lyase-derived H2S production through soluble guanylyl cyclase-protein kinase G signaling cascade.

Recurrent hypoglycemia inhibits the counterregulatory response by suppressing adrenal activity.

Hypoglycemia activates the counterregulatory response (CRR), a neural-endocrine reflex that restores euglycemia. Although effective if occasionally activated, repeated induction of the CRR leads to a decline in responsiveness and prolonged exposure to hypoglycemia. The mechanism underlying this impairment is not known. We found that the reduction in epinephrine release that characterizes a suppressed CRR involves a long-lasting form of sympatho-adrenal synaptic plasticity. Using optogenetically evoked catecholamine release, we show that recurrent hypoglycemia reduced the secretory capacity of mouse adrenal chromaffin cells. Single activation of the CRR increased the adrenal levels of tyrosine hydroxylase (TH), the rate-limiting enzyme for catecholamine synthesis, but this was prevented by repeated activation. In contrast, the level of neuropeptide Y (NPY), an adrenal cotransmitter, remained elevated after recurrent hypoglycemia. Inhibition of NPY or Y1 signaling, either transgenically or pharmacologically, prevented the attenuation of both TH expression and epinephrine release. These results indicate that impairment of the CRR involves suppressed activity at the adrenal level. Interfering with the peripheral NPY-dependent negative feedback loop may provide a way to avoid the pathophysiological consequences of recurrent hypoglycemia which are common in the diabetic state.

GABAA receptor: a unique modulator of excitability, Ca2+ signaling, and catecholamine release of rat chromaffin cells.

The role of gamma-aminobutyric acid (GABA) in adrenal medulla chromaffin cell (CC) function is just beginning to unfold. GABA is stored in catecholamine (CA)-containing dense core granules and is presumably released together with CA, ATP, and opioids in response to physiological stimuli, playing an autocrine-paracrine role on CCs. The reported paradoxical "dual action" of GABAA-R activation (enhancement of CA secretion and inhibition of synaptically evoked CA release) is only one aspect of GABA's multifaceted actions. In this review, we discuss recent physiological experiments on rat CCs in situ which suggest that GABA regulation of CC function may depend on the physiological context: During non-stressful conditions, GABAA-R activation by endogenous GABA tonically inhibits acetylcholine release from splanchnic nerve terminals and decreases spontaneous Ca2+ fluctuations in CCs, preventing unwanted CA secretion. During intense stress, splanchnic nerve terminals release acetylcholine, which depolarizes CCs and allows the Ca2+ influx that triggers the release of CA and GABA. With time, CA secretion declines, due to voltage-independent inhibition of Ca2+channels and desensitization of cholinergic nicotinic receptors. Nonetheless, acute activation of GABAA-R is depolarizing in about 50% of CCs, and thus GABA, acting as an autocrine/paracrine mediator, could help to maintain CA exocytosis under stress. GABAA-R activation is not excitatory in about half of CCs' population because it hyperpolarizes them or elicits no response. This percentage possibly varies, depending on functional demands, since GABAA-R-mediated actions are determined by the intracellular chloride concentration ([Cl-] i ) and therefore on the activity of cation-chloride co transporters, which is functionally regulated. These findings underscore a potential importance of a novel and complex GABA-mediated regulation of CC function and of CA secretion.

Spontaneous and electrically-evoked catecholamine secretion from long-term cultures of bovine adrenal chromaffin cells

Catecholamine release was measured from bovine adrenal medullary chromaffin cell (CC) cultures maintained over a period of three months. Cells were plated over simple biocompatible cell platforms with electrical stimulation capability and at specified times transferred to an acrylic superfusion chamber designed to allow controlled flow of superfusate over the culture. Catecholamine release was measured from the superfusates using fast cyclic voltammetry before, during and after electrical stimulation of the cells. Immunocytochemical staining of CC cultures revealed that they were composed of epinephrine (EP) and/or norepinephrine (NE) type cells. Both spontaneous and evoked-release of catecholamines from CCs were observed throughout the testing period. EP predominated during spontaneous release, whereas NE was more prevalent during electrically-evoked release. Electrical stimulation for 20s, increased total catecholamine release by 60–130% (measured over a period of 500s) compared to that observed for an equivalent 20s period of spontaneous release. Stimulus intensity was correlated with the amount of evoked release, up to a plateau which was observed near the highest intensities. Shorter intervals between stimulation trials did not significantly affect the initial amount of release, and the amount of evoked release was relatively stable over time and did not decrease significantly with age of the culture. The present study demonstrates long-term survival of CC cultures in vitro and describes a technique useful for rapid assessment of cell functionality and release properties of cultured monoaminergic cell types that later can be transplanted for neurotransmitter replacement following injury or disease.

Role of TrkB expression in rat adrenal gland during acute immobilization stress

Expression of tyrosine receptor kinase B (TrkB), a receptor for brain-derived neurotrophic factor (BDNF), is markedly elevated in the adrenal medulla during immobilization stress. Catecholamine release was confirmed in vitro by stimulating chromaffin cells with recombinant BDNF. We investigated the role of TrkB and the localization of BDNF in the adrenal gland during immobilization stress for 60 min. Blood catecholamine levels increased after stimulation with TrkB expressed in the adrenal medulla during 60-min stress; however, blood catecholamine levels did not increase in adrenalectomized rats. Furthermore, expression of BDNF mRNA and protein was detected in the adrenal medulla during 60-min stress. Similarly, in rats undergoing sympathetic nerve block with propranolol, BDNF mRNA and protein were detected in the adrenal medulla during 60-min stress. These results suggest that signal transduction of TrkB in the adrenal medulla evokes catecholamine release. In addition, catecholamine release was evoked by both the hypothalamic–pituitary–adrenal axis and autocrine signaling by BDNF in the adrenal gland. BDNF–TrkB interaction may play a role in a positive feedback loop in the adrenal medulla during immobilization stress.

H2S induces catecholamine secretion in rat adrenal chromaffin cells

Hydrogen sulfide (H2S) is recognized as an important gaseous signaling molecule in mammalian tissues and exerts its modulating functions of different systems via targeting different ion channels and receptors. H2S can be synthesized from l-cysteine by cystathionine β-synthetase (CBS) or cystathionine γ-lyase (CSE). It has been reported recently that H2S can be synthesized and released in rat adrenal medulla chromaffin cells (AMCs) which play a critical role in the regulation of stress response by releasing catecholamine (CA). In the present study, we combined amperometry and whole-cell patch-clamp recording to explore the direct effect of exogenous H2S on CA release in AMCs and the underlying ionic mechanism. Amperometry showed that local application of NaHS, the H2S donor, evoked CA release from AMCs. Furthermore, the CA secretory response to NaHS was totally blocked by removing extracellular Ca2+. Whole-cell patch-clamp experiments showed that H2S-induced CA release is produced by membrane depolarization generated by an inhibition of Ca2+-activated K+ current [IK(Ca) current]. We conclude that H2S is capable of directly inducing CA release by inhibiting the IK(Ca) current. This conclusion indicates that H2S may involve in the response of adrenal medulla to stress by modulating IK(Ca) current and CA release in mammalian animals.

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 β1-, β2- and β3-adrenoceptors on catecholamine release from human adrenal chromaffin cells in culture.We observed that the β-adrenoceptor agonist (isoproterenol) and β2-adrenoceptor agonist (salbutamol) stimulated catecholamine (NE and EP) release from human adrenal chromaffin cells. Furthermore, the β2-adrenoceptor antagonist (ICI 118,551; 100nM) and β3-adrenoceptor antagonist (SR 59230A; 100nM) inhibited the catecholamine release stimulated by isoproterenol and nicotine in chromaffin cells. The β1-adrenoceptor antagonist (atenolol; 100nM) 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 β2- and β3-adrenoceptors modulate the basal and evoked catecholamine release, NE and EP, via an autocrine positive feedback loop in human adrenal chromaffin cells.

Pituitary Adenylate Cyclase-activating Peptide (PACAP) Recruits Low Voltage-activated T-type Calcium Influx under Acute Sympathetic Stimulation in Mouse Adrenal Chromaffin Cells

Low voltage-activated T-type Ca(v)3.2 calcium channels are expressed in neurosecretory chromaffin cells of the adrenal medulla. Previous studies have shown that naïve adrenal chromaffin cells express a nominal Ca(v)3.2-dependent conductance. However, Ca(v)3.2 conductance is up-regulated following chronic hypoxia or long term exposure to cAMP analogs. Thus, although a link between chronic stressors and up-regulation of Ca(v)3.2 exists, there are no reports testing the specific role of Ca(v)3.2 channels in the acute sympathoadrenal stress response. In this study, we examined the effects of acute sympathetic stress on T-type Ca(v)3.2 calcium influx in mouse chromaffin cells in situ. Pituitary adenylate cyclase-activating peptide (PACAP) is an excitatory neuroactive peptide transmitter released by the splanchnic nerve under elevated sympathetic activity to stimulate the adrenal medulla. PACAP stimulation did not evoke action potential firing in chromaffin cells but did cause a persistent subthreshold membrane depolarization that resulted in an immediate and robust Ca(2+)-dependent catecholamine secretion. Moreover, PACAP-evoked secretion was sensitive to block by nickel chloride and was acutely inhibited by protein kinase C blockers. We utilized perforated patch electrophysiological recordings conducted in adrenal tissue slices to investigate the mechanism of PACAP-evoked calcium entry. We provide evidence that stimulation with exogenous PACAP and native neuronal stress stimulation both lead to a protein kinase C-mediated phosphodependent recruitment of a T-type Ca(v)3.2 Ca(2+) influx. This in turn evokes catecholamine release during the acute sympathetic stress response.

The Transgenic Overexpression of α-Synuclein and Not Its Related Pathology Associates with Complex I Inhibition

Alpha-synuclein (alphaS) is a protein involved in the cytopathology and genetics of Parkinson disease and is thought to affect mitochondrial complex I activity. Previous studies have shown that mitochondrial toxins and specifically inhibitors of complex I activity enhance alphaS pathogenesis. Here we show that alphaS overexpression specifically inhibits complex I activity in dopaminergic cells and in A53T alphaS transgenic mouse brains. Importantly, our results indicate that the inhibitory effect on complex I activity is not associated with alphaS-related pathology. Specifically, complex I activity measured in purified mitochondria from A53T alphaS transgenic mouse brains was not affected by mouse age; Parkinson disease-like symptoms; levels of alphaS soluble oligomers; levels of insoluble, lipid-associated alphaS; or alphaS intraneuronal depositions in vivo. Likewise, no correlation was found between complex I activity and polyunsaturated fatty acid-induced alphaS depositions in Lewy body-like inclusions in cultured dopaminergic cells. We further show that the effect of alphaS on complex I activity is not due to altered mitochondrial protein levels or affected complex I assembly. Based on the results herein, we suggest that alphaS expression negatively regulates complex I activity as part of its normal, physiological role.

Evoked Catecholamine Release from Sympathetic Nerve Terminals in Cardiac Slices

Neurotransmitter release from presynaptic cells can be recorded by postsynaptic potentials/currents in central nerve system. However, little is known about how to record nerve release in cardiovascular system in fresh tissue or in vivo. We have developed two novel methods for recording catecholamine release induced by nerve action potentials in cardiac slices and in vivo, respectively.

Interleukin‐6 increases intracellular Ca2+ concentration and induces catecholamine secretion in rat carotid body glomus cells

Although abundant evidence indicates mutual regulation between the immune and the central nervous systems, how the immune signals are transmitted to the brain is still an unresolved question. In a previous study we found strong expression of proinflammatory cytokine receptors, including interleukin (IL)-1 receptor I and IL-6 receptor alpha in the rat carotid body (CB), a well-known arterial chemoreceptor that senses a variety of chemostimuli in the arterial blood. We demonstrated that IL-1 stimulation increases intracellular calcium ([Ca(2+)](i)) in CB glomus cells, releases ATP, and increases the discharge rate in carotid sinus nerve. To explore the effect of IL-6 on CB, here we examine the effect of IL-6 on [Ca(2+)](i) and catecholamine (CA) secretion in rat CB glomus cells. Calcium imaging showed that extracellular application of IL-6 induced a rise in [Ca(2+)](i) in cultured glomus cells. Amperometry showed that local application of IL-6 evoked CA release from glomus cells. Furthermore, the CA secretory response to IL-6 was blocked by 200 microM Cd(2+), a well-known Ca(2+) channel blocker. Our experiments provide further evidence for the responsiveness of the CB to proinflammatory cytokines and indicate that the CB might play a role in inflammation sensing and transmission of such information to the brain.

Adeno-Associated Virus Transfer of a Gene Encoding SNAP-25 Resistant to Botulinum Toxin A Attenuates Neuromuscular Paralysis Associated with Botulism

Advances in viral gene therapy have opened new possibilities for treating a range of motor neuron diseases, but these have not yet been translated into clinically applicable therapies because of difficulties in delivery to susceptible/damaged neurons, ambiguities in the identity of gene(s) implicated, and a paucity of means to quantify any physiological improvement. Most of these hurdles can be overcome by using the neuromuscular paralysis induced by botulinum neurotoxin type A (BoNT/A) as a prototype disease. Furthermore, because human botulism, occasionally fatal, causes prolonged muscle disablement as a result of the intraneuronal persistence of the toxin's SNAP-25 (S25)-cleaving protease, development of a genetic approach could lead to a potential treatment for this debilitating disease. Adeno-associated viral delivery of a cleavage-resistant S25 gene (S25-R198T) to chromaffin cells in vitro yielded exocytotically active S25-R198T that diminished subsequent blockade by BoNT/A of evoked catecholamine release. Evaluation in vivo, by administering this virus into rat spinal cord before injecting BoNT/A, showed a decreased inhibition of acetylcholine release as reflected in elevated retention of neuromuscular transmission. A similar, although smaller, protection of synaptic transmission from the toxin was seen after peripherally injecting the therapeutic virus. Such therapy also curtailed nerve sprouting normally induced by BoNT/A. This first demonstration of the utility of a DNA-based therapy for botulism paves the way for further advances in its treatment and for application to genetic disorders of motor neurons.

Intracellular signaling mechanisms mediating catecholamine release upon activation of NPY Y1 receptors in mouse chromaffin cells

The adrenal chromaffin cells synthesize and release catecholamine (mostly epinephrine and norepinephrine) and different peptides, such as the neuropeptide Y (NPY). NPY stimulates catecholamine release through NPY Y1 receptor in mouse chromaffin cells. The aim of our study was to determine the intracellular signaling events coupled to NPY Y1 receptor activation that lead to stimulation of catecholamine release from mouse chromaffin cells. The stimulatory effect of NPY mediated by NPY Y1 receptor activation was lost in the absence of extracellular Ca2+. On the other hand, inhibition of nitric oxide synthase and guanylyl cyclase also decreased the stimulatory effect of NPY. Moreover, catecholamine release stimulated by NPY or by the nitric oxide donor (NOC-18) was inhibited by mitogen-activated protein kinase (MAPK) and protein kinase C inhibitors. In summary, in mouse chromaffin cells, NPY evokes catecholamine release by the activation the NPY Y1 receptor, in a Ca2+-dependent manner, by activating mitogen-activated protein kinase and promoting nitric oxide production, which in turn regulates protein kinase C and guanylyl cyclase activation.

Selective stimulation of catecholamine release from bovine adrenal chromaffin cells by an ionotropic purinergic receptor sensitive to 2-methylthio ATP

Background2-Methylthioadenosine 5'-triphosphate (2-MeSATP), formerly regarded as a specific P2Y (metabotropic) purinergic receptor agonist, stimulates Ca2+ influx and evokes catecholamine release from adrenal chromaffin cells. These cells express P2Y and P2X (ionotropic) purinoceptors, with the latter providing an important Ca2+ influx pathway. Using single cell calcium imaging techniques, we have determined whether 2-MeSATP might be a specific P2X receptor agonist in bovine chromaffin cells and assessed the relative role of P2X and P2Y receptors on catecholamine secretion from these cells.ResultsATP raised the [Ca2+]i in ~50% of the cells. Removing extracellular Ca2+ suppressed the [Ca2+]i-raising ability of 2-MeSATP, observed in ~40% of the ATP-sensitive cells. This indicates that 2-MeSATP behaves as a specific ionotropic purinoceptor agonist in bovine chromaffin cells. The 2-MeSATP-induced [Ca2+]i-rises were suppressed by PPADS. UTP raised the [Ca2+]i in ~40% of the ATP-sensitive cells, indicating that these expressed Ca2+-mobilizing P2Y receptors. UTP-sensitive receptors may not be the only P2Y receptors present, as suggested by the observation that ~20% of the ATP-sensitive pool did not respond to either 2-MeSATP or UTP. The average sizes of the ATP- and 2-MeSATP-evoked [Ca2+]i responses were identical in UTP-insensitive cells. 2-MeSATP stimulated Ca2+ influx and evoked catecholamine release, whereas UTP elicited Ca2+ release from intracellular stores but did not evoke secretion. 2-MeSATP-induced secretion was strongly inhibited by Cd2+ and suppressed by extracellular Ca2+ or Na+ removal. TTX inhibited 2-MeSATP-evoked secretion by ~20%.Conclusion2-MeSATP is a specific P2X purinoceptor agonist and a potent secretagogue in bovine chromaffin cells. Activation of 2-MeSATP-sensitive receptors stimulates Ca2+ influx mainly via voltage-sensitive Ca2+ channels. For the most part, these are activated by the depolarization brought about by Na+ influx across P2X receptor pores.

α-Synuclein Overexpression in PC12 and Chromaffin Cells Impairs Catecholamine Release by Interfering with a Late Step in Exocytosis

Alpha-synuclein (alpha-syn), a protein implicated in Parkinson's disease pathogenesis, is a presynaptic protein suggested to regulate transmitter release. We explored how alpha-syn overexpression in PC12 and chromaffin cells, which exhibit low endogenous alpha-syn levels relative to neurons, affects catecholamine release. Overexpression of wild-type or A30P mutant alpha-syn in PC12 cell lines inhibited evoked catecholamine release without altering calcium threshold or cooperativity of release. Electron micrographs revealed that vesicular pools were not reduced but that, on the contrary, a marked accumulation of morphologically "docked" vesicles was apparent in the alpha-syn-overexpressing lines. We used amperometric recordings from chromaffin cells derived from mice that overexpress A30P or wild-type (WT) alpha-syn, as well as chromaffin cells from control and alpha-syn null mice, to determine whether the filling of vesicles with the transmitter was altered. The quantal size and shape characteristics of amperometric events were identical for all mouse lines, suggesting that overexpression of WT or mutant alpha-syn did not affect vesicular transmitter accumulation or the kinetics of vesicle fusion. The frequency and number of exocytotic events per stimulus, however, was lower for both WT and A30P alpha-syn-overexpressing cells. The alpha-syn-overexpressing cells exhibited reduced depression of evoked release in response to repeated stimuli, consistent with a smaller population of readily releasable vesicles. We conclude that alpha-syn overexpression inhibits a vesicle "priming" step, after secretory vesicle trafficking to "docking" sites but before calcium-dependent vesicle membrane fusion.

Sympathoneural and adrenomedullary functional effects of ??2C-adrenoreceptor gene polymorphism in healthy humans

alpha2-Adrenoreceptors restrain sympathetic nervous outflows and inhibit release of noradrenaline from sympathetic nerves. In-frame deletion of the alpha2C-adrenoreceptor subtype (alpha2CDel322-325) increases the risk of congestive heart failure. Increased delivery of catecholamines to cardiovascular receptors might explain this increased risk. Twenty-nine healthy African-Americans genotyped for alpha2-adrenoreceptor subtype polymorphisms underwent 3H-noradrenaline and 3H-adrenaline intravenous infusion and arterial blood sampling for measurements of rates of entry of endogenous noradrenaline and adrenaline into arterial plasma (total body spillovers) by the tracer dilution technique. Eleven subjects were homozygotes for the alpha2CDel322-325 polymorphism, nine heterozygotes, and nine non-carriers. Subjects were studied during supine rest and during and after i.v. infusion of the alpha2-adrenoreceptor antagonist, yohimbine. At rest, homozygotes for the alpha2CDel322-325 polymorphism had higher total body noradrenaline spillover than did heterozygotes (t=2.90, df=18, P=0.023) or non-carriers (t=3.22, df=18, P=0.010). Adrenaline spillover was higher in homozygotes than non-carriers (t=2.61, df=18, P=0.045). Administration of yohimbine produced larger, more sustained increments in noradrenaline spillover, heart rate, and anxiety in homozygotes than in the other groups. In healthy people, alpha2CDel322-325 polymorphism is associated with increased sympathetic nervous and adrenomedullary hormonal activities, both during supine rest and during pharmacologically evoked catecholamine release. Polymorphisms of the alpha2C-adrenoreceptor may help explain individual differences in predisposition to a variety of disorders of catecholaminergic function, such as cardiovascular disorders, depression or anxiety disorders.

Tetrapeptides on N- and C-terminal regions of mastoparan inhibit catecholamine release from chromaffin cells by blocking nicotinic acetylcholine receptor.

Mastoparan (MP), a tetradecapeptide in wasp venom, has been reported to evoke catecholamine release, but also reported to inhibit secretory response upon nicotinic stimulation in adrenal chromaffin cells. To elucidate the inhibitory mechanism of MP, we examined the effect of two MP fragments (INLK-NH2 and KKIL-NH2) on catecholamine release in bovine adrenal chromaffin cells. These MP fragments inhibited catecholamine release induced by nicotinic stimulation in a noncompetitive manner. These fragments did not affect catecholamine release evoked by high [K+] or by other secretagogues, neither caused catecholamine release by themselves. Replacement by hydrophobic and basic amino acids of the MP fragments enhanced the inhibitory effects on ACh-evoked catecholamine release. Among 23 analogs of the MP fragments, (Nle)3-R-NH2 showed the most potent inhibition with IC50 = 541 microM. These results suggested that the MP fragments selectively inhibit the secretory response to nicotinic stimulation by attacking nAChR on the site(s) made up of hydrophobic and acidic amino acids but other than ACh-binding sites. This mechanism may explain the inhibitory action of MP on nicotine-evoked catecholamine release.

Cardiorespiratory adjustments during hypercarbia in rainbow trout Oncorhynchus mykiss are initiated by external CO(2) receptors on the first gill arch.

Experiments were performed to test the hypothesis that the marked ventilatory and cardiovascular responses to hypercarbia in rainbow trout Oncorhynchus mykiss arise from specific stimulation of chemoreceptors localised to the first gill arch. This was accomplished by measuring cardiorespiratory variables during acute hypercarbia (20 min at P(CO(2))=8 mmHg; 1 mmHg=0.133 kPa) in fish subjected to selective bilateral extirpation of the first gill arch. The cardiovascular responses to hypercarbia in the intact fish included a significant bradycardia (from 75.0+/-1.6 to 69.0+/-2.0 beats min(-1); means +/- S.E.M.; N=16), an increase in dorsal aortic blood pressure (from 30.8+/-1.5 to 41.9+/-2.5 mmHg; N=16) and a rise in systemic vascular resistance (from 1.1+/-0.1 to 1.4+/-0.1 mmHg ml(-1) kg(-1) min(-1); N=16). Removal of the first gill arch or pre-treatment with the muscarinic receptor antagonist atropine prevented the hypercarbic bradycardia without affecting the pressure or resistance responses. Correlation analysis, however, revealed shallow but significant inverse relationships between water P(CO(2)) and cardiac frequency in both atropinised (r(2)=0.75) and gill-extirpated (r(2)=0.90) fish, suggesting a direct mild effect of CO(2) on cardiac function. The ventilatory response to hypercarbia in the intact fish consisted of an increase in ventilation amplitude (from 0.62+/-0.06 to 1.0+/-0.13 cm; N=16) with no change in breathing frequency. Removal of the first gill arch lowered resting breathing frequency and prevented the statistically significant elevation of breathing amplitude. Gill extirpation, however, did not totally abolish the positive correlation between water P(CO(2)) and ventilation amplitude (r(2)=0.84), suggesting the presence of additional (although less important) chemoreceptive sites that are not confined to the first gill arch. Plasma catecholamine levels were elevated during hypercarbia, and this response was unaffected by prior gill extirpation. To assess whether the CO(2) chemoreceptors of the first gill arch were sensing water and/or blood P(CO(2)), bolus injections of CO(2)-enriched water or saline were made into the buccal cavity or caudal vein, respectively. Injections of CO(2)-enriched water to preferentially stimulate external receptors evoked catecholamine release and cardiorespiratory responses that closely resembled the responses to hypercarbia. As in hypercarbia, extirpation of the first gill arch prevented the bradycardia and the increase in ventilation amplitude associated with externally injected CO(2)-enriched water. Except for a slight decrease in cardiac frequency (from 73.0+/-2.8 to 70.3+/-3.5 beats min(-1); N=11), injection of CO(2)-enriched saline to preferentially stimulate internal chemoreceptors did not affect any measured variable. Taken together, these data indicate that, in rainbow trout, the bradycardia and hyperventilation associated with hypercarbia are triggered largely by external CO(2) chemoreceptors confined to the first gill arch.

Nitric oxide modulates evoked catecholamine release from canine adrenal medulla.

Nitric oxide has various actions, acting in a neurotransmitter-like role and also as a paracrine messenger between vascular endothelial and smooth muscle cells. This study was done to determine whether endogenous nitric oxide has a role in modulating evoked catecholamine release from the canine adrenal medulla. Isolated adrenal glands were perfused with Krebs-Ringer solution as a control, or with Krebs-Ringer solution containing either N(G)-monomethyl-L-arginine (L-NMMA; 3x10(-4) M) to non-selectively inhibit nitric oxide synthase or 7-nitroindazole (10(-4) M), a relatively selective inhibitor of neuronal nitric oxide synthase. Catecholamine release was evoked using the nicotinic cholinergic agonist 1,1-dimethyl-4-phenylpiperazinium iodine. From the collected perfusate epinephrine, norepinephrine, and dopamine were measured by high performance liquid chromatography. Previous studies have shown that in the presence of L-NMMA, basal releases of epinephrine, norepinephrine and dopamine are increased. 7-Nitroindazole had no effect on basal catecholamine release, suggesting that nitric oxide from an endothelial source was responsible for the inhibition of basal catecholamine release from the adrenal medulla. Epinephrine and norepinephrine releases were augmented when either of the nitric oxide synthase inhibitors was added during submaximal nicotinic stimulation, indicating that endogenous nitric oxide inhibited release of epinephrine and norepinephrine. Both neuronal and endothelial nitric oxide synthases appeared to be responsible for this inhibition. In summary, these studies suggest that nitric oxide, from both neuronal and endothelial sources, modulates evoked catecholamine release from canine adrenal medulla, while nitric oxide from an endothelial source is most likely responsible for modulation of catecholamine release under basal conditions.

Neurotransmitter release from bovine adrenal chromaffin cells is modulated by capacitative Ca2+entry driven by depleted internal Ca2+stores

Two potential mechanisms by which the intracellular Ca2 stores might modulate catecholamine release from bovine adrenal chromaffin cells were investigated: (i) that the cytosolic Ca2+transient caused by Ca2+release from the intracellular stores recruits additional chromaffin granules to a readily releasable pool that results in augmented catecholamine release when this is subsequently evoked, and (ii) that the Ca2+influx that follows depletion of intracellular stores (i.e. store-operated Ca2+entry) triggers release per se thereby augmenting evoked catecholamine release. When histamine or caffeine were applied in Ca2+-free perfusion media, a transient elevation of intracellular free Ca2+occurred owing to mobilization of Ca2+from the stores. When Ca2+was later readmitted to the perfusing fluid there followed a prompt and maintained rise in intracellular Ca2+concentrations of magnitude related to the degree of store mobilization. In parallel experiments, increased catecholamine secretion was measured under the conditions when Ca2+influx following store-mobilization occurred. Furthermore, the size of the catecholamine release increment correlated with the degree of Ca2+influx. Store-operated Ca2+entry evoked by mobilization with histamine and/or caffeine did not augment nicotine-evoked secretion per se; that is, it augmented evoked catecholamine release only to the extent that it increased basal catecholamine release. The nicotine-evoked catecholamine release was sensitive to cytosolic BAPTA, which, at the concentration used (50μM BAPTA-AM), reduced release by approximately 25%. However, the increment in basal catecholamine release which followed Ca2+influx triggered by Ca2+store mobilization was not reduced by intracellular BAPTA. This finding is inconsistent with the hypothesis that the elevated cytosolic Ca2+from store mobilization recruits additional vesicles of catecholamine to the sub-plasmalemmal release sites to augment subsequently evoked secretion. This position is supported by the observation that histamine (10μM) in Ca2+-free medium caused a pronounced elevation of cytosolic free Ca2+, but this caused no greater catecholamine release when Ca2+was re-introduced than did prior exposure to Ca2+-free medium alone, which caused no elevation of cytosolic free Ca2+. It is concluded that intracellular Ca2+stores can modulate secretion of catecholamines from bovine chromaffin cells by permitting Ca2+influx through a store-operated entry pathway. The results do not support the notion that the Ca2+released from intracellular stores plays a significant role in the recruitment of vesicles into the ready-release pool under the experimental conditions reported here.