Modulation Of Catecholamine Release Research Articles

Analysis and validation of traits associated with a single nucleotide polymorphism Gly364Ser in catestatin using humanized chromogranin A mouse models.

The human prohormone chromogranin A (CHGA), an index member of the granin family is processed to generate catestatin, a peptide that is hypotensive in action and modulates catecholamine release within the sympathoadrenal system. Hypertensive patients with excess sympathetic activity have diminished catestatin. Often the study of physiological consequences of human genetic variation is confounded by elements such as other variations in obligatory linkage disequilibrium with the variant being studied. Also the phenotype of the variant may be influenced by genetic background that varies amongst individuals. This study addresses the effects of a human catestatin polymorphism (rs9658667) using humanized CHGA mouse models. We created pertinent humanized mouse models wherein the mouse Chga gene locus was replaced by the human ortholog wild-type and the variant versions. This allowed for probing of the effects of catestatin variation in vivo with controls for other variations and global genetic background. Both the wild-type and variant human catestatin expressing mouse models were normotensive. The variant catestatin mouse model recapitulated physiological influence of the polymorphism on autonomic traits. These mice had diminished catecholamine, attenuated stress response and increased baroreceptor slopes that would suggest reduced risk of developing hypertension. Elevated plasma glucose, a trait observed in humans was not observed in mice expressing the variant catestatin. This functional genomics approach of creating humanized mouse models to study rs9658667 polymorphism recapitulated and validated many of the human trait associations. This approach can also be applied in the study of other human gene polymorphisms.

The role of pendrin in blood pressure regulation.

Pendrin is a Na(+)-independent Cl(-)/HCO3(-) exchanger found in the apical regions of type B and non-A, non-B intercalated cells within the aldosterone-sensitive region of the nephron, i.e., the distal convoluted tubule (DCT), the connecting tubule (CNT), and the cortical collecting duct (CCD). Type B intercalated cells mediate Cl(-) absorption and HCO3(-) secretion primarily through pendrin-mediated Cl(-)/HCO3(-) exchange. This exchanger is upregulated with angiotensin II administration and in models of metabolic alkalosis, such as following administration of aldosterone or NaHCO3. In the absence of pendrin-mediated HCO3(-) secretion, an enhanced alkalosis is observed following aldosterone or NaHCO3 administration. However, probably of more significance is the role of pendrin in the pressor response to aldosterone. Pendrin mediates Cl(-) absorption and modulates aldosterone-induced Na(+) absorption mediated by the epithelial Na channel (ENaC). Pendrin changes ENaC activity by changing both channel open probability (Po) and surface density (N), at least partly by altering luminal HCO3(-) and ATP concentration. Thus aldosterone and angiotensin II stimulate pendrin expression and function, which stimulates ENaC activity, thereby contributing to the pressor response of these hormones. However, pendrin may modulate blood pressure partly through its extrarenal effects. For example, pendrin is expressed in the adrenal medulla, where it modulates catecholamine release. The increase in catecholamine release observed with pendrin gene ablation likely contributes to the increment in vascular contractile force observed in the pendrin null mouse. This review summarizes the signaling mechanisms that regulate pendrin abundance and function as well as the contribution of pendrin to distal nephron function.

Pendrin localizes to the adrenal medulla and modulates catecholamine release.

Pendrin (Slc26a4) is a Cl(-)/HCO3 (-) exchanger expressed in renal intercalated cells and mediates renal Cl(-) absorption. With pendrin gene ablation, blood pressure and vascular volume fall, which increases plasma renin concentration. However, serum aldosterone does not significantly increase in pendrin-null mice, suggesting that pendrin regulates adrenal zona glomerulosa aldosterone production. Therefore, we examined pendrin expression in the adrenal gland using PCR, immunoblots, and immunohistochemistry. Pendrin protein was detected in adrenal lysates from wild-type but not pendrin-null mice. However, immunohistochemistry and qPCR of microdissected adrenal zones showed that pendrin was expressed in the adrenal medulla, rather than in cortex. Within the adrenal medulla, pendrin localizes to both epinephrine- and norepinephrine-producing chromaffin cells. Therefore, we examined plasma catecholamine concentration and blood pressure in wild-type and pendrin-null mice under basal conditions and then after 5 and 20 min of immobilization stress. Under basal conditions, blood pressure was lower in the mutant than in the wild-type mice, although epinephrine and norepinephrine concentrations were similar. Catecholamine concentration and blood pressure increased markedly in both groups with stress. With 20 min of immobilization stress, epinephrine and norepinephrine concentrations increased more in pendrin-null than in wild-type mice, although stress produced a similar increase in blood pressure in both groups. We conclude that pendrin is expressed in the adrenal medulla, where it blunts stress-induced catecholamine release.

Pendrin localizes to the adrenal medulla where it modulates catecholamine release (701.1)

Pendrin is an aldosterone‐sensitive, Na+‐independent Cl‐/HCO3‐ exchanger expressed in intercalated cells within the renal cortex where it modulates blood pressure. Since pendrin gene ablation stimulates the release of renin, but not aldosterone, we hypothesized that pendrin is expressed in the adrenal gland and modulates adrenal function. Thus, we examined rat and mouse adrenal pendrin mRNA and protein abundance by PCR, immunoblot and immunohistochemistry and examined adrenal responses in wild type and pendrin null mice. Pendrin protein was detected in adrenal lysates from wild type, but not pendrin null mice. In both mice and rats, we observed pendrin mRNA and protein in the adrenal medulla but not in the adrenal cortex. Since the adrenal medulla produces catecholamines, further experiments examined plasma epinephrine responses to 5 and 20 min of immobilization stress in wild type and pendrin null mice. Basal epinephrine concentration was similar in wild type and pendrin null mice. Immobilization stress increased epinephrine in both groups, but responses were ~50% higher in pendrin null vs wild type after either 5 or 20 min of immobilization stress (20 min: KO, 1562 ± 263, n=4, vs WT, 1041 ± 129 pg/ml, n=6, P < 0.05). We conclude that pendrin is expressed in the adrenal medulla where it may blunt stress‐induced epinephrine release.Grant Funding Source: Supported by NIH

Commentary: Chromaffin Cell Plasticity in Stress, Inflammation, Development, and Disease

Chromaffin cells secrete catecholamine in response to acetylcholine stimulation released from splanchnic neurons. Various hormone and peptides co-released with catecholamine and acetylcholine have been shown to cause paracrine or autocrine regulation of stimulus–secretion coupling in chromaffin cells in normal conditions and during stress. In this meeting, several talks focused on this subject in three different sessions chaired by Youssef Anouar and Corey Smith and Kathrin Engisch. Several papers presented focused on the responses induced by pituitary adenylate cyclase activating polypeptide (PACAP), a neuropeptide coreleased with acetylcholine from presynaptic terminals. Lee Eiden (USA) gave two talks: first, he reported a thorough microarray analysis of the transcriptomes induced by TNF and PACAP, respectively. Secondly, he reported the genes that were upregulated during restraint stress and hypoglycemia in adrenal medulla. Among them was galanin, a peptide implicated in the modulation of catecholamine release. The galanin transcript, as well as transcripts encoding catecholamine synthesizing enzymes (TH and PNMT) were upregulated by PACAP via a PKA-independent, ERK1/2dependent pathway, during stress. It appears that PACAP regulates catecholamine secretion at different levels. S.-A. Chan (USA), from Corey Smith’s group, reported that PACAP stimulates a phosphorylation-dependent recruitment of T-type Ca channels leading to an increase of catecholamine secretion. Eiden together with Smith have presented their work in a review on PACAP as a regulator of stimulus– secretion–synthesis coupling at the sympathoadrenal synapse during stress (below). Stephen Bunn (New Zealand) highlighted the importance of bidirectional signaling between the immune and neuroendocrine systems. He found that treatment of chromaffin cells with the cytokine IL-6 activated the ERK1/2 signal transduction pathway and induced significant changes in gene transcription. An extensive review of this work implicating the physiological significance of cytokine signaling on chromaffin cells during stress is presented by Bunn and Eiden (below). In addition to the classical model used for the study of the molecular mechanisms involved in stimulus-secretion coupling, chromaffin cells have also been used as a model for the study of cell survival and differentiation. Youssef Anouar (France) provided new evidence to support that PACAPregulated selenoprotien Texpression is involved in chromaffin cell survival during development. Vladimir Vukicevic (Germany) reported the isolation and characterization of the progenitors from adrenal medulla. He demonstrated that these cells can self-renew and differentiate into cells with characteristics of dopaminergic neurons. A review of the regulation of the progenitor state of chromaffin cells is presented below. Indeed, the presence of such progenitor/stem cells in the adrenal medulla is very exciting and raises the possibility of a potential source of dopaminergic neurons for treating neurodegenerative diseases such as Parkinson’s disease.

Commentary: Ion Channels, Fusion Pores and Exocytosis

The key function of adrenal chromaffin cells is to secrete catecholamines in response to stress. Under stress, chromaffin cells are stimulated by cholinergic synaptic inputs resulting in catecholamine release. This stimulus-secretion coupling is mediated by activation of Ca signaling through ion channels followed by fusion pore formation and exocytosis, and subsequent endocytosis. Several sessions at this symposium highlighted advances in the understanding of the role of ion channels in exocytosis and endocytosis, the mechanics of fusion pore formation and the role of Ca syntillas and gap junctional communication in modulating catecholamine release. As well, new technologies used to investigate stimulus–secretion coupling in catecholamine release were presented. The functions of different types of calcium channels in exocytosis and endocytosis, as well as other ion channels in chromaffin cell function were discussed in two sessions: “Voltage-gated calcium channels and cell function” organized by Emilio Carbone (Italy) and “Structure and function of channels” organized by Jiu-Ping Ding (China). Among the speakers discussing the role of calcium channels in exocytosis/endocytosis were Luis Gandia (Spain) who gave an update on the role L-type Ca channels (LTCCs; Cav1) play in the control of Ca-dependent endocytosis. Gandia’s group brought new evidence that endocytosis in bovine chromaffin cells is likely associated with prolonged Ca entry during cell depolarization and is likely associated with slowly inactivating Ca channels rather than due to a colocalization of LTCCs with the clathrin-mediated endocytotic machinery. Indeed, immunostaining of L-, P/Q, and Ntype channels showed no colocalization with clathrin, and application of roscovitine (a blocker of N and P/Q type channel inactivation) restored endocytosis when LTTCs were blocked by nifedipine. Thus, “compensatory” and “excess” endocytosis is driven by the Ca entry through slowly inactivating Ca channels. More extensive discussions on the role of different calcium channel subtypes on endocytosis in chromaffin cells is presented in a review by Luis Gandia below. Antonio Miguel G. de Diego (Spain) reviewed the possibility that LTCCs could exert control on exocytosis induced by acute hypoxia in rat embryo chromaffin cells. According to this, LTCCs (besides T-type channels) are also likely to be involved in the control of catecholamine release at the embryonic stage, when chromaffin cells are particularly sensitive to O2 homeostasis and control cardiovascular responses. Emilio Carbone gave an overview of the ion channels controlling the spontaneous pacemaking of E. Carbone Department of Neuroscience, NIS Center, National Institute of Neuroscience, University of Torino, 10125 Torino, Italy

The Novel Plasminogen Receptor, Plasminogen ReceptorKT (Plg-RKT), Regulates Catecholamine Release

Neurotransmitter release by catecholaminergic cells is negatively regulated by prohormone cleavage products formed from plasmin-mediated proteolysis. Here, we investigated the expression and subcellular localization of Plg-R(KT), a novel plasminogen receptor, and its role in catecholaminergic cell plasminogen activation and regulation of catecholamine release. Prominent staining with anti-Plg-R(KT) mAb was observed in adrenal medullary chromaffin cells in murine and human tissue. In Western blotting, Plg-R(KT) was highly expressed in bovine adrenomedullary chromaffin cells, human pheochromocytoma tissue, PC12 pheochromocytoma cells, and murine hippocampus. Expression of Plg-R(KT) fused in-frame to GFP resulted in targeting of the GFP signal to the cell membrane. Phase partitioning, co-immunoprecipitation with urokinase-type plasminogen activator receptor (uPAR), and FACS analysis with antibody directed against the C terminus of Plg-R(KT) were consistent with Plg-R(KT) being an integral plasma membrane protein on the surface of catecholaminergic cells. Cells stably overexpressing Plg-R(KT) exhibited substantial enhancement of plasminogen activation, and antibody blockade of non-transfected PC12 cells suppressed plasminogen activation. In functional secretion assays, nicotine-evoked [(3)H]norepinephrine release from cells overexpressing Plg-R(KT) was markedly decreased (by 51 ± 2%, p < 0.001) when compared with control transfected cells, and antibody blockade increased [(3)H]norepinephrine release from non-transfected PC12 cells. In summary, Plg-R(KT) is present on the surface of catecholaminergic cells and functions to stimulate plasminogen activation and modulate catecholamine release. Plg-R(KT) thus represents a new mechanism and novel control point for regulating the interface between plasminogen activation and neurosecretory cell function.

Expression and Role of the BDNF Receptor-TrkB in Rat Adrenal Gland under Acute Immobilization Stress

We reported that plasma brain-derived neurotrophic factor (BDNF) was maximally elevated following a 60-min period of acute immobilization stress and that salivary glands were the main source of plasma BDNF under this stress condition. However, the expression pattern of the BDNF receptor, Tyrosine receptor kinase B (TrkB), under this condition has yet to be determined. We therefore investigated the effect of this stress on the expression level of TrkB in various rat organs using real-time PCR. No significant differences were found between controls and 60 min-stressed rats with respect to TrkB level in various organs. Only adrenal glands showed significantly increased TrkB mRNA levels after 60 min of stress. TrkB mRNA and protein were observed to localize in chromaffin cells. In addition, we investigated whether BDNF-TrkB interaction influences the release of stress hormones from PC12 cells, derived from chromaffin cells. Truncated receptor, TrkB-T1, was identified in PC12 cells using RT-PCR. Exposure of PC12 cells to BDNF induced the release of catecholamine. This BDNF-evoked release was totally blocked by administration of the K252a in which an inhibitor of Trk receptors. Thus, BDNF-TrkB interactions may modulate catecholamine release from adrenal chromaffin cells under acute stress conditions.

Calcium Signaling and Exocytosis in Adrenal Chromaffin Cells

At a given cytosolic domain of a chromaffin cell, the rate and amplitude of the Ca2+ concentration ([Ca2+]c) depends on at least four efficient regulatory systems: 1) plasmalemmal calcium channels, 2) endoplasmic reticulum, 3) mitochondria, and 4) chromaffin vesicles. Different mammalian species express different levels of the L, N, P/Q, and R subtypes of high-voltage-activated calcium channels; in bovine and humans, P/Q channels predominate, whereas in felines and murine species, L-type channels predominate. The calcium channels in chromaffin cells are regulated by G proteins coupled to purinergic and opiate receptors, as well as by voltage and the local changes of [Ca2+]c. Chromaffin cells have been particularly useful in studying calcium channel current autoregulation by materials coreleased with catecholamines, such as ATP and opiates. Depending on the preparation (cultured cells, adrenal slices) and the stimulation pattern (action potentials, depolarizing pulses, high K+, acetylcholine), the role of each calcium channel in controlling catecholamine release can change drastically. Targeted aequorin and confocal microscopy shows that Ca2+ entry through calcium channels can refill the endoplasmic reticulum (ER) to nearly millimolar concentrations, and causes the release of Ca2+ (CICR). Depending on its degree of filling, the ER may act as a sink or source of Ca2+ that modulates catecholamine release. Targeted aequorins with different Ca2+ affinities show that mitochondria undergo surprisingly rapid millimolar Ca2+ transients, upon stimulation of chromaffin cells with ACh, high K+, or caffeine. Physiological stimuli generate [Ca2+]c microdomains in which the local subplasmalemmal [Ca2+]c rises abruptly from 0.1 to approximately 50 microM, triggering CICR, mitochondrial Ca2+ uptake, and exocytosis at nearby secretory active sites. The fact that protonophores abolish mitochondrial Ca2+ uptake, and increase catecholamine release three- to fivefold, support the earlier observation. This increase is probably due to acceleration of vesicle transport from a reserve pool to a ready-release vesicle pool; this transport might be controlled by Ca2+ redistribution to the cytoskeleton, through CICR, and/or mitochondrial Ca2+ release. We propose that chromaffin cells have developed functional triads that are formed by calcium channels, the ER, and the mitochondria and locally control the [Ca2+]c that regulate the early and late steps of exocytosis.

Modulation of catecholamine release from rat striatal slices by the fixed combination of aspirin, paracetamol and caffeine

The fixed combination of aspirin, paracetamol (acetaminophen) and caffeine has been used successfully to treat different kinds of pain including migraine attacks. Even when this formulation has been marketed for a long time, the exact molecular mechanisms underlying its therapeutic effectiveness have not been completely elucidated. In the present investigation, we have studied the effects of the fixed combination of aspirin, paracetamol and caffeine (APC) on the release of dopamine and noradrenaline from rat striatal slices in an attempt to find potential new mechanisms of action of this widely used analgesic combination. We found that APC produced a significant reduction in extracellular dopamine and a dramatic increase in norepinephrine release from the slices incubated with different concentrations of APC (dose relationship 1:1:0.2, corresponding to the dose-relationship of Thomapyrin ®). These findings suggest that the modulation of catecholaminergic neurotransmission is a new pharmacological effect of APC which could explain the mechanism of action of this formulation, considering that the independent effect of either compound alone does not explain the potent antinociceptive properties when observed in combination.

Insulin-like growth factors decrease catecholamine content in PC12 rat pheochromocytoma cells.

Insulin-like growth factors (IGFs) stimulate proliferation and differentiation of PC12 rat pheochromocytoma cells and modulate catecholamine release in bovine adrenal medullary cells. Dexamethasone increases catecholamine synthesis in PC12 cells. We therefore studied the effects of IGFs and dexamethasone on catecholamine content in PC12 cells. Dopamine (DA) and norepinephrine (NE) content of PC12 cells were measured after incubation for 72 h with IGFs (100 ng/ml) and/or dexamethasone (500 nM). IGF-I (100 ng/ml) and IGF-II (100 ng/ml) decreased DA and NE content to approximately 35% and approximately 25% of control, respectively. [Leu27]IGF-II, which binds to the IGF-I receptor with markedly decreased affinity, did not reduce catecholamine levels, indicating that the effect is likely to be mediated by the IGF-I receptor. Dexamethasone (500 nM) increased levels of DA and NE to 173 +/- 20% and 331 +/- 48% of controls, respectively. Coincubation with IGFs did not significantly affect the stimulation of DA by dexamethasone, but abolished the rise in NE. Levels of tyrosine hydroxylase mRNA, protein and activity were increased following incubation with dexamethasone, but were unchanged by IGFs. These results indicate that IGFs decrease catecholamine content in PC12 cells via the IGF-I receptor. Complex regulation involving multiple synthetic and/or degradative steps is implicated in this process.

Modulation of catecholamine release and cortisol secretion by social interactions in the rainbow trout, Oncorhynchus mykiss

The objective of the present study was to investigate the effect of social status on the ability of rainbow trout to secrete the stress hormones, cortisol, and catecholamines. Rainbow trout were confined in pairs for six days to permit the formation of dominance hierarchies. An in situ saline-perfused posterior cardinal vein (PCV) preparation was then used to assess cortisol secretion or release of the catecholamine hormones, adrenaline and noradrenaline, in response to the inclusion of appropriate secretagogues in the perfusate. Fish identified as subordinate on the basis of their behaviour showed a characteristic elevation of circulating plasma cortisol concentrations when compared with dominant fish. When the interrenal cells were stimulated in situ with adrenocorticotropic hormone, subordinate fish displayed a significantly lower rate of cortisol secretion than dominant fish. However, social status had no significant effect on either adrenaline or noradrenaline secretion rates upon stimulation of the chromaffin cells in situ with acetylcholine. These results suggest that the chronic elevation of plasma cortisol associated with subordinate social status in rainbow trout reduces the sensitivity of the cortisol-secreting interrenal cells, presumably through negative feedback mechanisms.

Modulation of catecholamine release by endogenous adenosine in the rat adrenal medulla.

Adenosine was shown to inhibit norepinephrine (NE) release from sympathetic nerve endings. The purpose of this study was to examine whether endogenous adenosine restrains NE and epinephrine release from the adrenal medulla. The effects of an adenosine receptor antagonist, 1,3-dipropyl-8-(p-sulfophenyl) xanthine (DPSPX), on epinephrine and NE release induced by intravenous administration of insulin in conscious rats were examined. Plasma catecholamines were measured by HPLC with an electrochemical detector. DPSPX significantly increased plasma catecholamine in both control rats and rats treated with insulin. The effect of DPSPX on plasma catecholamine was significantly greater in rats treated with insulin. Additional experiments were performed in adrenalectomized rats to investigate the contribution of the adrenal medulla to the effect of DPSPX on plasma catecholamine. The effect of DPSPX and insulin on epinephrine in adrenalectomized rats was significantly reduced compared with that of the controls. Finally, we tested whether endogenous adenosine restrains catecholamine secretion partially through inhibiting the renin-angiotensin system. The effect of DPSPX on plasma catecholamine in rats pretreated with captopril (an angiotensin-converting enzyme inhibitor) was reduced. These results demonstrate that under basal physiological conditions, endogenous adenosine tonically inhibits catecholamine secretion from the adrenal medulla, and this effect is augmented when the sympathetic system is stimulated. The effect of endogenous adenosine on catecholamine secretion from the adrenal medulla is achieved partially through the inhibitory effect of adenosine on the renin-angiotensin system.

Modulation of Catecholamine Release by Endogenous Adenosine in the Rat Adrenal Medulla

Adenosine was shown to inhibit norepinephrine (NE) release from sympathetic nerve endings. The purpose of this study was to examine whether endogenous adenosine restrains NE and epinephrine release from the adrenal medulla. The effects of an adenosine receptor antagonist, 1,3-dipropyl-8-(p-sulfophenyl) xanthine (DPSPX), on epinephrine and NE release induced by intravenous administration of insulin in conscious rats were examined. Plasma catecholamines were measured by HPLC with an electrochemical detector. DPSPX significantly increased plasma catecholamine in both control rats and rats treated with insulin. The effect of DPSPX on plasma catecholamine was significantly greater in rats treated with insulin. Additional experiments were performed in adrenalectomized rats to investigate the contribution of the adrenal medulla to the effect of DPSPX on plasma catecholamine. The effect of DPSPX and insulin on epinephrine in adrenalectomized rats was significantly reduced compared with that of the controls. Finally, we tested whether endogenous adenosine restrains catecholamine secretion partially through inhibiting the renin-angiotensin system. The effect of DPSPX on plasma catecholamine in rats pretreated with captopril (an angiotensin-converting enzyme inhibitor) was reduced. These results demonstrate that under basal physiological conditions, endogenous adenosine tonically inhibits catecholamine secretion from the adrenal medulla, and this effect is augmented when the sympathetic system is stimulated. The effect of endogenous adenosine on catecholamine secretion from the adrenal medulla is achieved partially through the inhibitory effect of adenosine on the renin-angiotensin system.

Angiotensin II modulates catecholamine release into interstitial fluid of canine myocardium in vivo.

This study tested the hypothesis that exogenous infusion of angiotensin II (ANG II) leads to the release of catecholamines [norepinephrine (NE) and epinephrine (EPI)] into the cardiac interstitial fluid (ISF) space of dogs with adrenals intact (AI) (n = 7) and with adrenals clamped (AC) (n = 5). LV ISF samples were collected at 3-min intervals during administration of ANG II (100 microM ANG II at 1 ml/min for 10 min) to right atrial neurons via their local arterial blood supply and during electrical stimulation of the stellate ganglia of open-chest anesthetized dogs. In AI dogs, ANG II caused ISF NE to increase fivefold (P < 0.05) without a significant increase in coronary sinus (CS) NE. Electrical stimulation (5 ms, 4 Hz, 8-14 V, and 10 min) of the stellate ganglia caused a similar increase in ISF NE (P < 0.05), accompanied by a sevenfold increase in CS NE (P < 0.05). ISF EPI increased greater than sixfold during ANG II infusion (P < 0.05) and during stellate stimulation. However, during ANG II infusions, aorta plasma EPI levels increased fourfold in AI dogs, whereas in AC dogs, CS NE and EPI levels were unaffected during ANG II infusions. Nevertheless, baseline ISF NE and EPI did not differ and increased to a similar extent during ANG II infusions in AI versus AC dogs. Thus exogenously administered ANG II increases the amount of NE liberated into the ISF independent of the adrenal contribution, the amount matching that induced by electrical stimulation of all cardiac sympathetic efferent neurons. In contrast, NE spillover into the CS occurred only during electrical stimulation of stellate ganglia. NE release and uptake mechanisms within the myocardium are differently affected, depending on how the final common pathway of the sympathetic efferent nervous system is modified.

Chromatin-derived acidic peptides modulate catecholamine release in the hypothalamus☆

We have studied the neuromodulatory effects of three synthetic peptides, structurally related to chromatin-derived acidic peptides (ACPs): ACP-1 (Asp-Asp-Ser-Asp-Glu-Glu-Asn), corresponding to the C-terminal fragment of the largest subunit of eukaryotic RNA polymerase II; a more lipophilic derivative, ACP-2 (Ala-Ile-Ser-Pro-Asp-Asp-Ser-Asp-Glu-Glu-Asn); and its phosphorylated form ACP-3 (Ala-Ile-Ser-Pro-Asp-Asp-Ser(P)-Asp-Glu-Glu-Asn). Rat hypothalamic synaptosomes, loaded with [ 3H]norepinephrine or [ 3H]dopamine, were perfused with the above peptides, both basally and during a depolarizing stimulus. We have found: ACP-1 inhibited both dopamine and norepinephrine release; ACP-2 inhibited dopamine release, without affecting norepinephrine release; ACP-3 was almost ineffective, except for a weak dopamine inhibiting effect only at a higher concentration.

Mechanisms of carvedilol action in human congestive heart failure.

The precise mechanism by which beta-adrenoceptor blockers exert their beneficial actions in patients with heart failure remains unclear. Several possibilities have been proposed, including heart rate reduction, beta2-adrenoceptor-mediated modulation of catecholamine release, antagonism of the receptor-mediated toxic actions of norepinephrine on the myocardium, and favorable effects on myocardial energetics. In the present study we evaluated the effect of 3 months of carvedilol therapy on hemodynamics, total systemic and cardiac norepinephrine spillover (isotope dilution method), and myocardial metabolism (myocardial oxygen consumption and carbon dioxide release) in 10 patients with severe congestive heart failure. Although carvedilol treatment was associated with a significant improvement in left ventricular ejection fraction (17+/-1% to 28+/-3%; P<0.01) and left ventricular stroke work (87+/-13 to 119+/-21 g. m per beat; P<0.05), this effect was unrelated to changes in total systemic or cardiac norepinephrine spillover. The rise in left ventricular stroke work was accompanied by a modest rise in myocardial oxygen consumption per beat (0.33+/-0.04 to 0.42+/-0.04; P=0.05), although contractile efficiency was unchanged. The favorable effects of carvedilol on ventricular function in the failing heart are not explained by alterations in norepinephrine release or by changes in myocardial contractile efficiency.

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.

Processing of chromogranin A by plasmin provides a novel mechanism for regulating catecholamine secretion.

Chromogranin A (CgA) is the major soluble protein in the core of catecholamine-storage vesicles and is also distributed widely in secretory vesicles throughout the neuroendocrine system. CgA contains the sequences for peptides that modulate catecholamine release, but the proteases responsible for the release of these bioactive peptides from CgA have not been established. We show here that the major fibrinolytic enzyme, plasmin, can cleave CgA to form a series of large fragments as well as small trichloroacetic acid-soluble peptides. Peptides generated by plasmin-mediated cleavage of CgA significantly inhibited nicotinic cholinergic stimulation of catecholamine release from PC12 cells and primary bovine adrenal chromaffin cells. We also show that the zymogen, plasminogen, as well as tissue plasminogen activator bind saturably and with high capacity to catecholaminergic (PC12) cells. Occupancy of cell surface binding sites promoted the cleavage of CgA by plasmin. Positive and negative modulation of the local cellular fibrinolytic system resulted in substantial alterations in catecholamine release. These results suggest that catecholaminergic cells express binding sites that localize fibrinolytic molecules on their surfaces to promote plasminogen activation and proteolytic processing of CgA in the environment into which CgA is secreted to generate peptides which may regulate neuroendocrine secretion. Interactions between CgA and plasmin(ogen) define a previously unrecognized autocrine/paracrine system that may have a dramatic impact upon catecholamine secretion.

Modulation of catecholamine release by α2-adrenoceptors and I 1-imidazoline receptors in rat brain

The physiological and pharmacological effects of imidazoli(di) ne derivatives, such as Clonidine, have been related not only to the interaction with α 2-adrenoceptors but also to their activity on non-adrenoceptor sites termed imidazoline receptors. The modulation of catecholamine release by imidazoline drugs was studied by monitoring extracellular levels of norepinephrine (NE), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) with microdialysis in cingulate cortex of rats, with or without irreversible α 2-adrenoceptor blockade. NE and DA levels were in the 1 nM range whereas DOPAC and HVA levels were ≅100 nM. NE and DA levels were increased when the uptake blocker desipramine (1 μM) or KCl (100 mm) were added to the perfusion medium. Clonidine induced a dose-dependent (0.3–1.2 mg/kg i.p.) decrease in NE (max 61%) and DA (max 40%) levels that was reversed by the c 2-adrenoceptor antagonist RX821002. After α 2-adrenoceptor irreversible blockade with the alkylating agent 7V-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), [ 3H] clonidine binding to α 2-adrenoceptors was reduced by 94 ± 1%. Under such conditions, clonidine elicited a paradoxical dose-dependent (0.6–2.4 mg/kg i.p.) increase of NE (max 56%) without modifications in DA, DOPAC and HVA levels. The stimulatory effect of clonidine was prevented by the imidazoline receptor antagonist idazoxan (10 mg/kg i.p.) but not by RX821002 (5 mg/kg i.p.). In rats pretreated with EEDQ, cirazoline (I 1/I 2-imidazoline receptor agonist), moxonidine (Iimidazoline receptor agonist), but not guanabenz (I 2-imidazoline receptor agonist) (1.2–2.4 mg/kg i.p.) elicited an increase of NE levels in a similar manner to clonidine (11–82%). Idazoxan also abolished these responses to cirazoline or moxonidine. In contrast to systemic administration, local perfusion of clonidine (10–100 μM) through the microdialysis probe under α 2-adrenoceptor alkylating conditions, did not modify extracellular levels of NE and DA suggesting an indirect mechanism. The results demonstrate that clonidine and related imidazoli(di) ne drugs are able not only to inhibit NE release in rat cerebral cortex involving an α 2-adrenoceptor mechanism, but also to induce a paradoxical NE release through an indirect extracortical mechanism. The findings evidence that the indirect modulation of NE levels by imidazoline drugs is mainly due to a functional activity on I 1-imidazoline receptors.