Inhibition Of Catecholamine Secretion Research Articles

Role of Catestatin in the Cardiovascular System and Metabolic Disorders.

Catestatin is a multifunctional peptide that is involved in the regulation of the cardiovascular and immune systems as well as metabolic homeostatis. It mitigates detrimental, excessive activity of the sympathetic nervous system by inhibiting catecholamine secretion. Based on in vitro and in vivo studies, catestatin was shown to reduce adipose tissue, inhibit inflammatory response, prevent macrophage-driven atherosclerosis, and regulate cytokine production and release. Clinical studies indicate that catestatin may influence the processes leading to hypertension, affect the course of coronary artery diseases and heart failure. This review presents up-to-date research on catestatin with a particular emphasis on cardiovascular diseases based on a literature search.

Catestatin regulates core bioenergetic and metabolic functions of the myocardium

ObjectiveHypertension is a global pandemic, affecting more than one billion people. We have recently reported that chromogranin A (CgA) peptide catestatin (CST: human CgA352‐372) decreases blood pressure in hypertensive CST knockout (CST‐KO) mice by inhibiting catecholamine secretion and decreasing inflammation. Using CST‐KO mice we show here that CST regulates core bioenergetic and metabolic functions of the myocardium.Methods and ResultsWe carried out transcriptomic analyses (gene microarray) of the left ventricles of wild‐type (WT) and CST‐KO mice. Analysis revealed altered expression. Downregulated genes, which were most enriched in pathways that impact muscle conduction and contractility include, among other genes, Glo1 (glyoxylase 1). Upregulated genes include, among others, Gm7120 (predicted gene 7120), Astn2 (astrotactin 2), and Defa20 (defensin a‐20). We validated several of these up‐ and downregulated genes by qPCR and found that CST supplementation reversed these aberrant expressions in all cases. Targeted qPCR analysis of sarcomeric genes revealed reduced expression of Tnni3 (cardiac troponin I), Tnnt2 (cardiac troponin T), Myl4(atrial light chain‐1) and Myl7 (atrial light chain‐2) genes in CST‐KO mice. CST treatment increased expression of Tnni3, Tnnt2, Actc1 (cardiac a‐actin), Myl4 and Myl7 in both WT and CST‐KO mice albeit to a much higher level in CST‐KO mice. An analysis of mitochondrial genes revealed reduced expression of Ndufa3 (complex I), Sdhc (complex II) and Atp5j (complex V) genes in CST‐KO mice. CST treatment increased the expression of all three genes in WT and CST‐KO mice. Taken together, these unbiased approaches and qPCR validation studies provide insight into how an inflamed dysfunctional heart in the absence of CST may impact muscle function (contractility), rhythm, mitochondrial bioenergetics, and conduction.CST‐KO mice showed decreased uptake of 2‐deoxy glucose in the left ventricle compared to WT mice. Furthermore, glucose‐6‐phosphate utilization was significantly lower in CST‐KO compared to WT mice. We also found that compared to WT mice, CST‐KO mice show a significantly higher uptake of fatty acids. Furthermore, 14C‐labeled acid soluble metabolites were significantly higher in CST‐KO compared to WT mice, suggesting that increased dependence on fatty acids for energy needs make CST‐KO hearts resistant to insulin.ConclusionsTaken together with the cardiometabolic shifts (decreased glucose utilization with a concomitant increase in the utilization of fatty acids), these findings indicate that CST maintains cardiometabolic homeostasis; it does so at least in part by acting as an insulin‐sensitizing peptide which regulates myocardial choice of substrates for generating energy (i.e., ATP).

The Serotonin Transporter Modulates the Kinetics and Quantal Size of Vesicular Fusion Events in Sympathoadrenal Chromaffin Cells

The antidepressant‐sensitive serotonin transporter (SERT) is a key regulator of serotonin (5‐HT) signaling and availability in the CNS. The robust expression of SERT in adrenal chromaffin cells (ACCs), which comprise the neuroendocrine arm of the sympathetic nervous system, is less well understood. ACCs synthesize and secrete catecholamines (epinephrine, norepinephrine) which mediate the physiological response to stress. They do not synthesize 5‐HT, but do accumulate small amounts through SERT‐mediated uptake (5‐HT content is <0.15% of the epinephrine content). We hypothesized that the chromaffin cells utilize this 5‐HT for autocrine / paracrine control of the sympathoadrenal stress response. Consistent with this hypothesis, we previously reported that 5‐HT1A receptors inhibit catecholamine secretion by reducing the number of secretory vesicles that fuse with the plasma. The objective of the current study was to investigate an additional, receptor‐independent mechanism by which SERT controls adrenal catecholamine secretion.We used carbon fiber amperometry to analyze catecholamine secretion from ACCs that were isolated from either wild type mice, global SERT‐knockout mice (SERT‐/‐), or sympathoadrenal system‐specific SERT knockout mice (SERTΔTH). Transmitter release from individual vesicle fusion events can be resolved as amperometric spikes. There was no difference in the number or time‐course of amperometric spikes evoked by 30mM KCl. The charge of the spikes is directly proportional to the amount of transmitter released by a single vesicle (i.e. quantal size). Spike charge was significantly smaller (~35%) and spike duration (half‐width) was significantly shorter in SERT‐knockout cells compared to matched controls. This was surprising given that HPLC analysis revealed no change in the catecholamine content of adrenal glands isolated from knockout mice compared to wild‐type controls; the 5‐HT content was significantly reduced but this accounted for <0.15% of the total monoamine content. Changes in calcium entry can modulate the quantal size of catecholamine release in ACCs, but patch‐clamp recording revealed there was no significant difference in voltage‐gated calcium channel currents in SERT knockout cells and ratiometric calcium imaging found no difference in KCl‐evoked calcium entry. The decrease in quantal size was mimicked in wild‐type cells by treating for >24 hours with escitalopram (an SSRI antidepressant which blocks SERT), or by depleting extracellular 5‐HT from the culture medium for >24 hours (use of dialyzed serum in the culture media). In contrast, acute (minutes) or short‐term (<6‐8 hrs) treatment with escitalopram or 5‐HT depletion had no effect. Adrenal chromaffin cells lack the rate limiting enzyme for 5‐HT synthesis (tryptophan hydroxylase), but if the product of this enzyme (5‐hydroxytryptophan; 5‐HTP) is provided it can be converted into 5‐HT. Treating ACCs with 5‐HTP for 1‐3 hrs had no effect but 24 hr treatment rescued the reduced spike charge seen in SERT knockout cells. Together, our data suggest that, following SERT‐mediated uptake, intracellular 5‐HT modulates the kinetics and thus fraction of secretory vesicle content that is released during a fusion event.

The Inhibition Effects of Shenmai Injection on Acetylcholine-Induced Catecholamine Synthesis and Secretion by Modulating Nicotinic Acetylcholine Receptor Ion Channels in Cultured Bovine Adrenal Medullary Cells.

Shenmai injection (SMI) has been widely used for the treatment of cardiovascular diseases in China. Cardiovascular disorders are often related to excessive catecholamine (CA) secretion. Here, we report the effects of SMI on CA secretion and synthesis in cultured bovine adrenal medullary cells. We found that SMI significantly reduced CA secretion induced by 300 μM acetylcholine (ACh). Cotreatment with SMI (10 μL/mL) and either of the ACh receptor α-subunit inhibitors, HEX (α3) or DhβE (α4β2), did not produce any further inhibition, indicating that SMI may play a role through α3 and α4β2 channels. Furthermore, SMI reduced tyrosine hydroxylase (TH) activity induced by ACh by inhibiting the phosphorylation of TH at Ser19 and Ser40. TH is phosphorylated at Ser19 by Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) and at Ser40 by protein kinase A (PKA). KN-93 and H89, the antagonists of CaM kinase II and PKA, respectively, inhibited the ACh-induced phosphorylation at Ser19 and Ser40, and the addition of SMI did not augment the inhibitory effect. Taken together, our results show that SMI likely inhibits CA secretion by blocking TH activity at its Ser19 and Ser40 sites.

Catestatin regulates vesicular quanta through modulation of cholinergic and peptidergic (PACAPergic) stimulation in PC12 cells.

We have previously shown that the chromogranin A (CgA)-derived peptide catestatin (CST: hCgA352-372) inhibits nicotine-induced secretion of catecholamines from the adrenal medulla and chromaffin cells. In the present study, we seek to determine whether CST regulates dense core (DC) vesicle (DCV) quanta (catecholamine and chromogranin/secretogranin proteins) during acute (0.5-h treatment) or chronic (24-h treatment) cholinergic (nicotine) or peptidergic (PACAP, pituitary adenylyl cyclase activating polypeptide) stimulation of PC12 cells. In acute experiments, we found that both nicotine (60 μM) and PACAP (0.1μM) decreased intracellular norepinephrine (NE) content and increased 3H-NE secretion, with both effects markedly inhibited by co-treatment with CST (2μM). In chronic experiments, we found that nicotine and PACAP both reduced DCV and DC diameters and that this effect was likewise prevented by CST. Nicotine or CST alone increased expression of CgA protein and together elicited an additional increase in CgA protein, implying that nicotine and CST utilize separate signaling pathways to activate CgA expression. In contrast, PACAP increased expression of CgB and SgII proteins, with a further potentiation by CST. CST augmented the expression of tyrosine hydroxylase (TH) but did not increase intracellular NE levels, presumably due to its inability to cause post-translational activation of TH through serine phosphorylation. Co-treatment of CST with nicotine or PACAP increased quantal size, plausibly due to increased synthesis of CgA, CgB and SgII by CST. We conclude that CST regulates DCV quanta by acutely inhibiting catecholamine secretion and chronicallyincreasing expression of CgA after nicotinic stimulation and CgB and SgII after PACAPergic stimulation.

Catestatin: A Master Regulator of Cardiovascular Functions

Cardiovascular disease (CVD), the most common cause of death globally, accounts for ~30% of all deaths worldwide. Hypertension is a common contributor to morbidity and mortality from CVD. The plasma concentration of chromogranin A (CgA) is elevated in patients with CVD as well as patients with established human essential hypertension and heart failure (HF). In contrast, the plasma level of the CgA-derived peptide catestatin (CST) is diminished in human essential hypertension. Low conversion of CgA-to-CST has been associated with increased mortality in patients hospitalized with acute HF. Consistent with human findings, the lack of CST in CgA knockout (Chga-KO) mice eventuates in the development of hypertension and supplementation of CST to Chga-KO mice restores blood pressure, implicating CST as a key player in regulating hypertension. In the peripheral system, CST decreases blood pressure by stimulating histamine release, inhibiting catecholamine secretion, or causing vasodilation. Centrally, CST improves baroreflex sensitivity (BRS) and heart rate variability (HRV) by exciting GABAergic neurons in the caudal ventrolateral medulla (CVLM) and pyramidal neurons of the central amygdala; CST also decreases BRS by exciting glutamatergic rostral ventrolateral medulla (RVLM) neurons. In addition, CST provides cardioprotection by inhibiting inotropy and lusitropy; activating mitochondrial KATP channels, and stimulating reperfusion injury salvage kinase (RISK) and survivor activating factor enhancement (SAFE) pathways and consequent inhibition of mitochondrial permeability transition pore (mPTP). CST modulates cardiomyocyte Ca2+ levels by direct inhibition of Ca2+/calmodulin-dependent protein kinase IIδ (CaMKIIδ) activity and consequent reduction in phosphorylation of phospholamban and ryanodine receptor 2, thereby providing support for a direct functional role of CST in the failing myocardium. These multitude of effects establish CST as a master regulator of cardiovascular functions.

Effects of pine nodule extract and its component, SJ-2, on acetylcholine-induced catecholamine secretion and synthesis in bovine adrenal medullary cells

Extract of pine nodules (matsufushi) formed by bark proliferation on the surface of trees of Pinus tabulaeformis or Pinus massoniana has been used as an analgesic for joint pain, rheumatism, neuralgia, dysmenorrhea and other complaints in Chinese traditional medicine. Previous studies have reported that pine oil extract has analgesic and antitumor effects and a dissolving effect on gallstones (Anticancer research, 7:1153-1159, 1987). Extract of the oil from Siberian pine (Pinus sibirica) has been found to have an anti-inflammatory effect (J Nat Med., 62:436-440, 2008). The pharmacological effects of matsufushi extract, however, have not been studied in the sympathetic nervous system. Adrenal medullary cells are derived from embryonic neural crests and share many properties with sympathetic postganglionic neurons. In the present study, we report the effects of matsufushi extract and its components on catecholamine secretion and synthesis in cultured bovine adrenal medullary cells. We found that matsufushi extract (0.0003-0.005 %) and its component, SJ-2 (5-hydroxy-3-methoxy-trans-stilbene) (0.3-100 µM) concentration-dependently inhibited catecholamine secretion induced by acetylcholine, a physiological secretagogue. Matsufushi extract (0.0003-0.005 %) and SJ-2 (0.3-100 µM) also inhibited 45Ca2+ influx induced by acetylcholine in a concentration-dependent manner, similar to its effect on catecholamine secretion. They also suppressed 14C-catecholamine synthesis and tyrosine hydroxylase activity induced by acetylcholine. In Xenopus oocytes expressing α3β4 nicotinic acetylcholine receptors, matsufushi extract (0.00003-0.001 %) and SJ-2 (1-100 µM) directly inhibited the current evoked by acetylcholine. The present findings suggest that SJ-2, as well as matsufushi extract, inhibits acetylcholine-induced catecholamine secretion and synthesis by suppression of nicotinic acetylcholine receptor-ion channels in bovine adrenal medullary cells.

Effects of Sho-ju-sen, a Japanese herbal medicine, on catechoamine secretion induced by acetylcholine in cultured bovine adrenal medullary cells

Sho-ju-sen is a Japanese herbal medicine which has been used as a nourishing tonic. It is composed of a water extract of Kumazasa leaves (Sasa kurinensis Makino et Sibata) and ethanol extracts of Japanese red pine needles (Pinus densiflora Sieb. Et Zucc) and ginseng roots (Panax ginseng C. A. Meyer). Previous studies showed that Sho-ju-sen has antidepressant and anxiolytic effects in mice (Phytother Res., 15: 142-147, 2001 and 18: 173-176, 2004). Here we report that the effects of Sho-ju-sen on catecholamine secretion in cultured bovine adrenal medullary cells. Adrenal medullary cells have provided a good material for the detailed analysis of a drug's actions on catecholamine secretion and have been used as a model of the sympathetic neurons. We found that Sho-ju-sen (0.5 - 1 %) and extract of Kumazasa leaves (0.5-1 %), but not ethanol extract of Japanese red pine needles or ginseng roots, concentration-dependently inhibited the secretion of catecholamines induced by acetylcholine, a physiological secretagogue and agonist of nicotinic acetylcholine receptors. In Xenopus oocytes expressing α3β4 nicotinic acetylcholine receptors, Sho-ju-sen (0.5 - 1 %) directly inhibited Na+ current evoked by acetylcholine. Sho-ju-sen (0.3 - 1 %) also inhibited catecholamine secretion induced by veratridine, an activator of voltage-dependent Na+ channels, but not by 56 mM K+ which depolarizes cell membranes. The present findings demonstrate that Sho-ju-sen inhibits acetylcholine- and veratridine-induced catecholamine secretion by suppression of nicotinic acetylcholine receptor-ion channels and voltage-dependent Na+ channels in adrenal medulla cells and probably sympathetic neurons, and also suggest that Sho-ju-sen has an anti-stress effect.

Sigma-1 receptor ligands inhibit catecholamine secretion from adrenal chromaffin cells due to block of nicotinic acetylcholine receptors.

Adrenal chromaffin cells (ACCs) are the neuroendocrine arm of the sympathetic nervous system and key mediators of the physiological stress response. Acetylcholine (ACh) released from preganglionic splanchnic nerves activates nicotinic acetylcholine receptors (nAChRs) on chromaffin cells causing membrane depolarization, opening voltage-gated Ca2+ channels (VGCC), and exocytosis of catecholamines and neuropeptides. The serotonin transporter is expressed in ACCs and interacts with 5-HT1A receptors to control secretion. In addition to blocking the serotonin transporter, some selective serotonin reuptake inhibitors (SSRIs) are also agonists at sigma-1 receptors which function as intracellular chaperone proteins and can translocate to the plasma membrane to modulate ion channels. Therefore, we investigated whether SSRIs and other sigma-1 receptor ligands can modulate stimulus-secretion coupling in ACCs. Escitalopram and fluvoxamine (100nM to 1μM) reversibly inhibited nAChR currents. The sigma-1 receptor antagonists NE-100 and BD-1047 also blocked nAChR currents (≈50% block at 100nM) as did PRE-084, a sigma-1 receptor agonist. Block of nAChR currents by fluvoxamine and NE-100 was not additive suggesting a common site of action. VGCC currents were unaffected by the drugs. Neither the increase in cytosolic [Ca2+ ] nor the resulting catecholamine secretion evoked by direct membrane depolarization to bypass nAChRs was altered by fluvoxamine or NE-100. However, both Ca2+ entry and catecholamine secretion evoked by the cholinergic agonist carbachol were significantly reduced by fluvoxamine or NE-100. Together, our data suggest that sigma-1 receptors do not acutely regulate catecholamine secretion. Rather, SSRIs and other sigma-1 receptor ligands inhibit secretion evoked by cholinergic stimulation because of direct block of Ca2+ entry via nAChRs.

Serotonin and Serotonin Transporters in the Adrenal Medulla: A Potential Hub for Modulation of the Sympathetic Stress Response.

Serotonin (5-HT) is an important neurotransmitter in the central nervous system where it modulates circuits involved in mood, cognition, movement, arousal, and autonomic function. The 5-HT transporter (SERT; SLC6A4) is a key regulator of 5-HT signaling, and genetic variations in SERT are associated with various disorders including depression, anxiety, and autism. This review focuses on the role of SERT in the sympathetic nervous system. Autonomic/sympathetic dysfunction is evident in patients with depression, anxiety, and other diseases linked to serotonergic signaling. Experimentally, loss of SERT function (SERT knockout mice or chronic pharmacological block) has been reported to augment the sympathetic stress response. Alterations to serotonergic signaling in the CNS and thus central drive to the peripheral sympathetic nervous system are presumed to underlie this augmentation. Although less widely recognized, SERT is robustly expressed in chromaffin cells of the adrenal medulla, the neuroendocrine arm of the sympathetic nervous system. Adrenal chromaffin cells do not synthesize 5-HT but accumulate small amounts by SERT-mediated uptake. Recent evidence demonstrated that 5-HT1A receptors inhibit catecholamine secretion from adrenal chromaffin cells via an atypical mechanism that does not involve modulation of cellular excitability or voltage-gated Ca2+ channels. This raises the possibility that the adrenal medulla is a previously unrecognized peripheral hub for serotonergic control of the sympathetic stress response. As a framework for future investigation, a model is proposed in which stress-evoked adrenal catecholamine secretion is fine-tuned by SERT-modulated autocrine 5-HT signaling.

Inhibitory effects of pine nodule extract and its component, SJ-2, on acetylcholine-induced catecholamine secretion and synthesis in bovine adrenal medullary cells

Extract of pine nodules (matsufushi) formed by bark proliferation on the surface of trees of Pinus tabulaeformis or Pinus massoniana has been used as an analgesic for joint pain, rheumatism, neuralgia, dysmenorrhea and other complaints in Chinese traditional medicine. Here we report the effects of matsufushi extract and its components on catecholamine secretion and synthesis in cultured bovine adrenal medullary cells. We found that matsufushi extract (0.0003–0.005%) and its component, SJ-2 (5-hydroxy-3-methoxy-trans-stilbene) (0.3–100 μM), but not the other three, concentration-dependently inhibited catecholamine secretion induced by acetylcholine, a physiological secretagogue. Matsufushi extract (0.0003–0.005%) and SJ-2 (0.3–100 μM) also inhibited 45Ca2+ influx induced by acetylcholine in a concentration-dependent manner, similar to its effect on catecholamine secretion. They also suppressed 14C-catecholamine synthesis and tyrosine hydroxylase activity induced by acetylcholine. In Xenopus oocytes expressing α3β4 nicotinic acetylcholine receptors, matsufushi extract (0.00003–0.001%) and SJ-2 (1–100 μM) directly inhibited the current evoked by acetylcholine. The present findings suggest that SJ-2, as well as matsufushi extract, inhibits acetylcholine-induced catecholamine secretion and synthesis by suppression of nicotinic acetylcholine receptor-ion channels in bovine adrenal medullary cells.

Pyrilamine inhibits nicotine-induced catecholamine secretion

Function of nicotine, which induces activation of all parts of the body including our brain, has been receiving much attention for a long period of time and also been actively studied by researchers for its pharmacological actions in the central nervous system. The modulation of nicotine concentration and the inhibition of nicotine binding on target receptors in the brain are the key factors for smoking addiction therapy. In previous studies showed that influx of nicotine at the blood–brain barrier was through the pyrilamine-sensitive organic cation transporters. But the direct interacting mechanism of pyrilamine on the nicotine binding target receptors has not yet been clarified. The aim of the present study is to investigate the direct binding mechanisms of a pyrilamine on the nicotinic acetylcholine receptors (nAChRs). We found that pyrilamine shares the same ligand binding pocket of nicotine (NCT) on nAChRs but interacts with more amino acid residues than NCT does. The extended part of pyrilamine interacts with additional residues in the ligand binding pocket of nAChRs which are located nearby the entrance of the binding pocket. The catecholamine (CA) secretion induced by nAChR agonist (NCT′) was significantly inhibited by the pyrilamine pretreatment. Real time carbon-fiber amperometry confirmed the inhibition of the NCT′-induced exocytosis by pyrilamine in a single cell level. We also found that pyrilamine inhibited the NCT′-induced [Ca2+]i. In contrast, pyrilamine did not affect the increase in calcium induced by high K+. Overall, these data suggest that pyrilamine directly docks into the ligand binding site of nAChRs and specifically inhibits the nAChR-mediated effects thereby causing inhibition of CA secretion. Therefore, pyrilamine may play an important role to explore new treatments to aid smoking cessation.

Chromogranin B: intra‐ and extra‐cellular mechanisms to regulate catecholamine storage and release, in catecholaminergic cells and organisms

Chromogranin B (CHGB) is the major matrix protein in human catecholamine storage vesicles. CHGB genetic variation alters catecholamine secretion and blood pressure. Here, effective Chgb protein under-expression was achieved by siRNA in PC12 cells, resulting in ~ 48% fewer secretory granules on electron microscopy, diminished capacity for catecholamine uptake (by ~ 79%), and a ~ 73% decline in stores available for nicotinic cholinergic-stimulated secretion. In vivo, loss of Chgb in knockout mice resulted in a ~ 35% decline in chromaffin granule abundance and ~ 44% decline in granule diameter, accompanied by unregulated catecholamine release into plasma. Over-expression of CHGB was achieved by transduction of a CHGB-expressing lentivirus, resulting in ~ 127% elevation in CHGB protein, with ~ 122% greater abundance of secretory granules, but only ~ 14% increased uptake of catecholamines, and no effect on nicotinic-triggered secretion. Human CHGB protein and its proteolytic fragments inhibited nicotinic-stimulated catecholamine release by ~ 72%. One conserved-region CHGB peptide inhibited nicotinic-triggered secretion by up to ~ 41%, with partial blockade of cationic signal transduction. We conclude that bi-directional quantitative derangements in CHGB abundance result in profound changes in vesicular storage and release of catecholamines. When processed and released extra-cellularly, CHGB proteolytic fragments exert a feedback effect to inhibit catecholamine secretion, especially during nicotinic cholinergic stimulation.

Inhibition of catecholamine secretion by iron-rich and iron-deprived multiwalled carbon nanotubes in chromaffin cells

The assay of the toxic effects of carbon nanotubes (CNTs) on human health is a stringent need in view of their expected increasing exploitation in industrial and biomedical applications. Most studies so far have been focused on lung toxicity, as the respiratory tract is the main entry of airborne particulate, but there is also recent evidence on the existence of toxic effects of multiwalled carbon nanotubes (MWCNTs) on neuronal and neuroendocrine cells (Belyanskaya et al., 2009; Xu et al., 2009; Gavello et al., 2012). Commercial MWCNTs often contain large amounts of metals deriving from the catalyst used during their synthesis. Since metals, particularly iron, may contribute to the toxicity of MWCNTs, we compared here the effects of two short MWCNTs samples (<5μm length), differing only in their iron content (0.5 versus 0.05% w/w) on the secretory responses of neurotransmitters in mouse chromaffin cells.We found that both iron-rich (MWCNT+Fe) and iron-deprived (MWCNT−Fe) samples enter chromaffin cells after 24h exposure, even though incorporation was attenuated in the latter case (40% versus 78% of cells). As a consequence of MWCNT+Fe or MWCNT−Fe exposure (50–263μg/ml, 24h), catecholamine secretion of chromaffin cells is drastically impaired because of the decreased Ca2+-dependence of exocytosis, reduced size of ready-releasable pool and lowered rate of vesicle release. On the contrary, both MWCNTs were ineffective in changing the kinetics of neurotransmitter release of single chromaffin granules and their quantal content. Overall, our data indicate that both MWCNT samples dramatically impair secretion in chromaffin cells, thus uncovering a true depressive action of CNTs mainly associated to their structure and degree of aggregation. This cellular “loss-of-function” is only partially attenuated in iron-deprived samples, suggesting a minor role of iron impurities on MWCNTs toxicity in chromaffin cells exocytosis.

Dual effects of nobiletin, a citrus polymethoxy flavone, on catecholamine secretion in cultured bovine adrenal medullary cells

Nobiletin, a compound of polymethoxy flavones found in citrus fruits, possesses a wide range of pharmacological activities. Here we report the effects of nobiletin on catecholamine secretion in cultured bovine adrenal medullary cells. Nobiletin (1.0-100 microM) concentration-dependently stimulated catecholamine secretion and (45)Ca(2+) influx. Its stimulatory effect of nobiletin on catecholamine secretion was abolished by deprivation of extracellular Ca(2+) and partially inhibited by specific inhibitors of voltage-dependent Ca(2+) channels and Na(+)/Ca(2+) exchangers. On the other hand, nobiletin suppressed catecholamine secretion and (22)Na(+) and (45)Ca(2+) influx induced by acetylcholine, an agonist of nicotinic acetylcholine receptors, in a concentration-dependent manner. It also inhibited catecholamine secretion, (22)Na(+) influx and/or (45)Ca(2+) influx induced by veratridine, an activator of voltage-dependent Na(+) channels, and 56 mM K(+), an activator of voltage-dependent Ca(2+) channels. In Xenopus oocytes expressing alpha3beta4 neuronal acetylcholine receptors, nobiletin directly inhibited the current evoked by acetylcholine in a concentration-dependent manner similar to that observed in catecholamine secretion. The present findings suggest that nobiletin, by itself, stimulates catecholamine secretion via activation of voltage-dependent Ca(2+) channels or Na(+)/Ca(2+) exchangers, whereas it inhibits catecholamine secretion induced by acetylcholine through the suppression of Na(+) influx and Ca(2+) influx in cultured bovine adrenal medullary cells.

P419 FENOFIBRATE ATTENUATES NICOTINE-INDUCED VASCULAR ENDOTHELIAL DYSFUNCTION IN THE RAT

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

Antagonistic effects of two herbs in Zuojin Wan, a traditional Chinese medicine formula, on catecholamine secretion in bovine adrenal medullary cells

In order to research the target of superior efficacy and lesser side effects, combination of herbal materials has been applied to phytotherapy for thousands of years in China and some other countries. Zuojin Wan (ZJW), a famous traditional Chinese medicine formula, is used in treating gastric diseases in China. It is composed of two herbs, Rhizoma Coptidis (RC) and Fructus Evodiae (FE) in the ratio of 6: 1(w/w). In the present study, we examined the effects of ZJW, RC, FE and active components isolated from these herbs on catecholamine (CA) secretion and intracellular calcium ([Ca(2+)](i)) in cultured bovine adrenal medullary cells. Extracts of ZJW and RC and berberine, palmatine and jatrorrhizine, components of RC, all inhibited CA secretion and rise in [Ca(2+)](i) induced by acetylcholine (ACh), veratridine (Ver) and/or 56 mM K(+). On the other hand, extract of FE, evodiamine and rutaecarpine, components of FE, stimulated CA secretion and rise in [Ca(2+)](i) induced by ACh. Furthermore, different proportions of RC and FE caused different responses in CA secretion. The present findings suggest that two herbs in ZJW have opposite effects, i.e., inhibitory effect of RC and stimulatory effect of FE, on CA secretion induced by acetylcholine in cultured bovine adrenal medullary cells.

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.

Provinol inhibits catecholamine secretion from the rat adrenal medulla.

The aim of the present study was to examine the effect of provinol, which is a mixture of polyphenolic compounds from red wine, on the secretion of catecholamines (CA) from isolated perfused rat adrenal medulla, and to elucidate its mechanism of action. Provinol (0.3~3 microg/ml) perfused into an adrenal vein for 90 min dose- and time-dependently inhibited the CA secretory responses evoked by ACh (5.32 mM), high K(+) (a direct membrane-depolarizer, 56 mM), DMPP (a selective neuronal nicotinic N(N) receptor agonist, 100 microM) and McN-A-343 (a selective muscarinic M(1) receptor agonist, 100 microM). Provinol itself did not affect basal CA secretion. Also, in the presence of provinol (1 microg/ml), the secretory responses of CA evoked by Bay-K-8644 (a voltage-dependent L-type dihydropyridine Ca(2+) channel activator, 10 microM), cyclopiazonic acid (a cytoplasmic Ca(2+)-ATPase inhibitor, 10 microM) and veratridine (an activator of voltage-dependent Na(+) channels, 10 microM) were significantly reduced. Interestingly, in the simultaneous presence of provinol (1 microg/ml) plus L-NAME (a selective inhibitor of NO synthase, 30 microM), the CA secretory responses evoked by ACh, high K(+), DMPP, McN-A-343, Bay-K-8644 and cyclpiazonic acid recovered to the considerable extent of the corresponding control secretion in comparison with the inhibition of provinol-treatment alone. Under the same condition, the level of NO released from adrenal medulla after the treatment of provinol (3 microg/ml) was greatly elevated in comparison to its basal release. Taken together, these data demonstrate that provinol inhibits the CA secretory responses evoked by stimulation of cholinergic (both muscarinic and nicotinic) receptors as well as by direct membrane-depolarization from the perfused rat adrenal medulla. This inhibitory effect of provinol seems to be exerted by inhibiting the influx of both calcium and sodium into the rat adrenal medullary cells along with the blockade of Ca(2+) release from the cytoplasmic calcium store at least partly through the increased NO production due to the activation of nitric oxide synthase.

Polyphenols of Rubus coreanum Inhibit Catecholamine Secretion from the Perfused Adrenal Medulla of SHRs

The present study was attempted to investigate whether polyphenolic compounds isolated from wine, which is brewed from Rubus coreanum Miquel (PCRC), may affect the release of catecholamines (CA) from the isolated perfused adrenal medulla of the spontaneously hypertensive rats (SHRs), and to establish its mechanism of action. PCRC (20~180 microg/ml) perfused into an adrenal vein for 90 min relatively dose-dependently inhibited the CA secretory responses to ACh (5.32 mM), high K(+) (56 mM), DMPP (100 microM) and McN-A-343 (100 microM). PCRC itself did not affect basal CA secretion (data not shown). Also, in the presence of PCRC (60 microg/ml), the CA secretory responses to veratridine (a selective Na(+) channel activator (10 microM), Bay-K-8644 (a L-type dihydropyridine Ca(2+) channel activator, 10 microM), and cyclopiazonic acid (a cytoplasmic Ca(2+) -ATPase inhibitor, 10 microM) were significantly reduced, respectively. In the simultaneous presence of PCRC (60 microg/ml) and L-NAME (an inhibitor of NO synthase, 30 microM), the inhibitory responses of PCRC on the CA secretion evoked by ACh, high K(+), DMPP, and Bay-K-8644 were considerably recovered to the extent of the corresponding control secretion compared with that of PCRC-treatment alone. The level of NO released from adrenal medulla after the treatment of PCRC (60 microg/ml) was greatly elevated compared with the corresponding basal level. Taken together, these results demonstrate that PCRC inhibits the CA secretion from the isolated perfused adrenal medulla of the SHRs evoked by stimulation of cholinergic receptors as well as by direct membrane-depolarization. It seems that this inhibitory effect of PCRC is mediated by blocking the influx of calcium and sodium into the adrenal medullary chromaffin cells of the SHRs as well as by inhibition of Ca(2+) release from the cytoplasmic calcium store at least partly through the increased NO production due to the activation of NO synthase.