Regulation Of Catecholamine Secretion Research Articles

Differential Modulation of Catecholamine and Adipokine Secretion by the Short Chain Fatty Acid Receptor FFAR3 and α2-Adrenergic Receptors in PC12 Cells.

Sympathetic nervous system (SNS) hyperactivity is mediated by elevated catecholamine (CA) secretion from the adrenal medulla, as well as enhanced norepinephrine (NE) release from peripheral sympathetic nerve terminals. Adrenal CA production from chromaffin cells is tightly regulated by sympatho-inhibitory α2-adrenergic (auto)receptors (ARs), which inhibit both epinephrine (Epi) and NE secretion via coupling to Gi/o proteins. α2-AR function is, in turn, regulated by G protein-coupled receptor (GPCR)-kinases (GRKs), especially GRK2, which phosphorylate and desensitize them, i.e., uncouple them from G proteins. On the other hand, the short-chain free fatty acid (SCFA) receptor (FFAR)-3, also known as GPR41, promotes NE release from sympathetic neurons via the Gi/o-derived free Gβγ-activated phospholipase C (PLC)-β/Ca2+ signaling pathway. However, whether it exerts a similar effect in adrenal chromaffin cells is not known at present. In the present study, we examined the interplay of the sympatho-inhibitory α2A-AR and the sympatho-stimulatory FFAR3 in the regulation of CA secretion from rat adrenal chromaffin (pheochromocytoma) PC12 cells. We show that FFAR3 promotes CA secretion, similarly to what GRK2-dependent α2A-AR desensitization does. In addition, FFAR3 activation enhances the effect of the physiologic stimulus (acetylcholine) on CA secretion. Importantly, GRK2 blockade to restore α2A-AR function or the ketone body beta-hydroxybutyrate (BHB or 3-hydroxybutyrate), via FFAR3 antagonism, partially suppress CA production, when applied individually. When combined, however, CA secretion from PC12 cells is profoundly suppressed. Finally, propionate-activated FFAR3 induces leptin and adiponectin secretion from PC12 cells, two important adipokines known to be involved in tissue inflammation, and this effect of FFAR3 is fully blocked by the ketone BHB. In conclusion, SCFAs can promote CA and adipokine secretion from adrenal chromaffin cells via FFAR3 activation, but the metabolite/ketone body BHB can effectively inhibit this action.

Mechanism of action of catestatin as an autocrine modulator regulating catecholamine secretion from chromaffin cells at the of splanchnicoadrenomedullary synapse

ObjectiveThe chromogranin A (CgA)‐derived peptide catestatin (CST: human CgA352–372) is thought to act as an autocrine modulator of catecholamine secretion from the adrenal medulla in vivo. Here, we examined how CST affects nicotine‐ and PACAP‐induced secretion from, and protein resynthesis within, dense core (DC) vesicle (DCV) quanta containing catecholamines and chromogranin/secretogranin proteins in PC12 cells.Methods and ResultsDepletion of norepinephrine (NE) from PC12 cells treated acutely (0.5 hours) with either nicotine (60 μM)‐ or PACAP (0.1 μM) was reversed by CST (2 μM), as was 3H‐NE release elicited by both secretagogues. DCV and DC area and diameter, evaluated by Transmission Electron Microscopy, were both decreased after treatment with either nicotine or PACAP, and this morphological effect was likewise reversed by CST. Nicotine or CST alone increased expression of CgA protein (but not CgB), and, in combination, elicited an additional increase in CgA protein, implying that nicotine and CST utilize separate signaling pathways to activate expression of CgA. In contrast, PACAP increased expression of CgB protein (but not CgA), indicating differential regulation of CgA and CgB proteins by cholinergic and peptidergic stimulation. Although CST augmented expression of tyrosine hydroxylase (TH) protein, it did not induce synthesis of catecholamines, presumably due to its inability to cause post‐translation activation of TH via serine phosphorylation. Increased area and diameter of DCVs, and their DCs after CST, CST+nicotine and CST+PACAP are consistent with increased quantal size. Decreased quantal size is possibly mediated by increased synthesis of granulogenic proteins: CgA after CST treatment; CgA after CST+nicotine treatment; and CgB after CST+PACAP treatment.ConclusionBased on our results obtained in PC12 cells, we suggest that the CgA‐derived peptide CST acts as a paracrine factor during ACh or PACAP stimulation of secretion from chromaffin cells of the adrenal medulla, modulating both the extent of secretory stimulation by splanchnic nerve firing, and the repletion of secretory granule content after bouts of secretory activity.Support or Funding InformationSupported by grants from the VA (I01BX000323 to S.K.M.; I01BX002709 to N.J.G.W), and the NIH (MH002386 to L.E.E.).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Serotonin Transporter Modulates the Quantal Size of Vesicular Release Events in Adrenal Chromaffin Cells

The serotonin (5-HT) transporter (SERT) is an important regulator of 5-HT signaling in the CNS. SERT is also expressed in adrenal chromaffin cells (ACCs), part of the sympathetic nervous system that coordinates the physiological response to stress through exocytosis of catecholamines and other transmitters. Here, we used carbon fiber amperometry to show that SERT modulates catecholamine secretion from ACCs by at least two distinct mechanisms. First, SERT accumulates small amounts of 5-HT into ACCs and modulates the ability of 5-HT1A receptors to inhibit the number of vesicular fusion events in isolated ACCs. Additionally, SERT acts via a mechanism independent of 5-HT1A receptor activation to modulate the amount and kinetics of transmitter release from individual vesicular fusion events. The half-width (duration) and charge (amount of oxidizable transmitter released) of individual amperometric spikes (vesicular fusion events) was significantly smaller (∼35%) in SERT−/− cells compared to wild-type. The same effect was recapitulated in wild-type cells by in vitro pharmacological block of SERT (∼48hrs culture with escitalopram) or by restricting the amount of extracellular 5-HT available for uptake (∼48hrs culture dialyzed serum media). Adrenal 5-HT content is determined by SERT-mediated uptake but only accounts for a small fraction (∼0.13%) of the total adrenal gland monoamine content. Although 5-HT content was reduced in SERT−/− adrenal glands, the catecholamine content was unaltered making it unlikely that the reduced spike charge (35% decrease) is simply due to reduced vesicular monoamine content. Ongoing work will dissect the underlying mechanisms by which adrenal SERT / 5-HT regulate catecholamine secretion and hence the sympathoadrenal stress response.

ERK1/2 inhibition increases dopamine release from differentiated PC12 cells

The release of dopamine (DA) is one of the main steps in the control of neuronal functioning and all CNS. It was demonstrated that many factors such as protein kinases and synaptic proteins are tightly involved in the regulation of DA secretion, but the data are contradictory. Here we analysed an effect of ERK1/2 inhibition on DA secretion from differentiated PC12 cells and evaluated the correlation between the activity of kinases/synaptic proteins and the level of released DA. PC12 cells were differentiated by NGF for 6 days. On the 7th day the cells were incubated for 1, 2 and 4 h with 10μM U0126. Obtained data demonstrated a significant accumulation of DA in the media after 4 h incubation with U0126 that accompanied with upregulation of PKG activity. Analysis of exocytosis proteins demonstrated decreased phosphorylation level of synapsin I and content of SNAP25. Taken together our data proposed an inhibitory role of ERK1/2 in the regulation of catecholamine secretion and demonstrated that balance between PKG and ERK1/2 activity could have a substantial impact on the regulation of DA release from the cells.

Membrane Trafficking Protein CDP138 Regulates Fat Browning and Insulin Sensitivity through Controlling Catecholamine Release.

CDP138 is a calcium- and lipid-binding protein that is involved in membrane trafficking. Here, we report that mice without CDP138 develop obesity under normal chow diet (NCD) or high-fat diet (HFD) conditions. CDP138-/- mice have lower energy expenditure, oxygen consumption, and body temperature than wild-type (WT) mice. CDP138 is exclusively expressed in adrenal medulla and is colocalized with tyrosine hydroxylase (TH), a marker of sympathetic nervous terminals, in the inguinal fat. Compared with WT controls, CDP138-/- mice had altered catecholamine levels in circulation, adrenal gland, and inguinal fat. Adrenergic signaling on cyclic AMP (cAMP) formation and hormone-sensitive lipase (HSL) phosphorylation induced by cold challenge but not by an exogenous β3 adrenoceptor against CL316243 were decreased in adipose tissues of CDP138-/- mice. Cold-induced beige fat browning, fatty acid oxidation, thermogenesis, and related gene expression were reduced in CDP138-/- mice. CDP138-/- mice are also prone to HFD-induced insulin resistance, as assessed by Akt phosphorylation and glucose transport in skeletal muscles. Our data indicate that CDP138 is a regulator of stress response and plays a significant role in adipose tissue browning, energy balance, and insulin sensitivity through regulating catecholamine secretion from the sympathetic nervous terminals and adrenal gland.

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.

Reduced availability of voltage-gated sodium channels by depolarization or blockade by tetrodotoxin boosts burst firing and catecholamine release in mouse chromaffin cells.

Mouse chromaffin cells (MCCs) of the adrenal medulla possess fast-inactivating Nav channels whose availability alters spontaneous action potential firing patterns and the Ca(2+)-dependent secretion of catecholamines. Here, we report MCCs expressing large densities of neuronal fast-inactivating Nav1.3 and Nav1.7 channels that carry little or no subthreshold pacemaker currents and can be slowly inactivated by 50% upon slight membrane depolarization. Reducing Nav1.3/Nav1.7 availability by tetrodotoxin or by sustained depolarization near rest leads to a switch from tonic to burst-firing patterns that give rise to elevated Ca(2+)-influx and increased catecholamine release. Spontaneous burst firing is also evident in a small percentage of control MCCs. Our results establish that burst firing comprises an intrinsic firing mode of MCCs that boosts their output. This occurs particularly when Nav channel availability is reduced by sustained splanchnic nerve stimulation or prolonged cell depolarizations induced by acidosis, hyperkalaemia and increased muscarine levels. Action potential (AP) firing in mouse chromaffin cells (MCCs) is mainly sustained by Cav1.3 L-type channels that drive BK and SK currents and regulate the pacemaking cycle. As secretory units, CCs optimally recruit Ca(2+) channels when stimulated, a process potentially dependent on the modulation of the AP waveform. Our previous work has shown that a critical determinant of AP shape is voltage-gated sodium channel (Nav) channel availability. Here, we studied the contribution of Nav channels to firing patterns and AP shapes at rest (-50 mV) and upon stimulation (-40 mV). Using quantitative RT-PCR and immunoblotting, we show that MCCs mainly express tetrodotoxin (TTX)-sensitive, fast-inactivating Nav1.3 and Nav1.7 channels that carry little or no Na(+) current during slow ramp depolarizations. Time constants and the percentage of recovery from fast inactivation and slow entry into closed-state inactivation are similar to that of brain Nav1.3 and Nav1.7 channels. The fraction of available Nav channels is reduced by half after 10 mV depolarization from -50 to -40 mV. This leads to low amplitude spikes and a reduction in repolarizing K(+) currents inverting the net current from outward to inward during the after-hyperpolarization. When Nav channel availability is reduced by up to 20% of total, either by TTX block or steady depolarization, a switch from tonic to burst firing is observed. The spontaneous occurrence of high frequency bursts is rare under control conditions (14% of cells) but leads to major Ca(2+)-entry and increased catecholamine release. Thus, Nav1.3/Nav1.7 channel availability sets the AP shape, burst-firing initiation and regulates catecholamine secretion in MCCs. Nav channel inactivation becomes important during periods of high activity, mimicking stress responses.

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

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

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.

Impaired desensitization of a human polymorphic α2B-adrenergic receptor variant enhances its sympatho-inhibitory activity in chromaffin cells

Backgroundα2-adrenergic receptors (ARs) mediate many cellular actions of epinephrine and norepinephrine and inhibit their secretion from adrenal chromaffin cells. Like many other G-protein coupled receptors (GPCRs), they undergo agonist-dependent phopshorylation and desensitization by GPCR Kinases (GRKs), a phenomenon recently shown to play a major role in the sympathetic overdrive that accompanies and aggravates chronic heart failure. A deletion polymorphism in the human α2B-AR gene (Glu301-303) causes impaired agonist-promoted receptor phosphorylation and desensitization in heterologous cell lines. Given the importance of α2-ARs in regulation of catecholamine secretion from chromaffin cells, we sought to investigate, in the present study, the desensitization properties and the sympatho-inhibitory activity of this variant in a chromaffin cell line. For this purpose, we expressed this variant and its wild type counterpart in the well-established chromaffin cell line PC12, and performed receptor phosphorylation and desensitization studies, as well as in vitro catecholamine secretion assays.ResultsBoth the agonist-induced phosphorylation and agonist-dependent desensitization of the human Glu301-303 deletion polymorphic α2B-AR are significantly impaired in PC12 cells, resulting in enhanced signaling to inhibition of cholinergic-induced catecholamine secretion in vitro.ConclusionThis α2B-AR gene polymorphism (Glu301-303 deletion) might confer better protection against conditions characterized and aggravated by sympathetic/catecholaminergic overstimulation in vivo.

The Involvement of Ser1898 of the Human L-Type Calcium Channel in Evoked Secretion

A PKA consensus phosphorylation site S1928 at the α 11.2 subunit of the rabbit cardiac L-type channel, CaV1.2, is involved in the regulation of CaV1.2 kinetics and affects catecholamine secretion. This mutation does not alter basal CaV1.2 current properties or regulation of CaV1.2 current by PKA and the beta-adrenergic receptor, but abolishes CaV1.2 phosphorylation by PKA. Here, we test the contribution of the corresponding PKA phosphorylation site of the human α 11.2 subunit S1898, to the regulation of catecholamine secretion in bovine chromaffin cells. Chromaffin cells were infected with a Semliki-Forest viral vector containing either the human wt or a mutated S1898A α 11.2 subunit. Both subunits harbor a T1036Y mutation conferring nifedipine insensitivity. Secretion evoked by depolarization in the presence of nifedipine was monitored by amperometry. Depolarization-triggered secretion in cells infected with either the wt α 11.2 or α 11.2/S1898A mutated subunit was elevated to a similar extent by forskolin. Forskolin, known to directly activate adenylyl-cyclase, increased the rate of secretion in a manner that is largely independent of the presence of S1898. Our results are consistent with the involvement of additional PKA regulatory site(s) at the C-tail of α 11.2, the pore forming subunit of CaV1.2.

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.

Insulinostatic activity of cerebellin — Evidence from in vivo and in vitro studies in rats

Cerebellin (CER) is a neuromodulatory hexadecapeptide that originates from the precursor protein precerebellin (Cbln1). Four highly homologous isoforms of Cbln are known (Cbln1–Cbln4), which are expressed in the central nervous system (CNS) and in peripheral tissues. CER modulates synaptic structure formation in the CNS, whereas in the peripheral tissues CER regulates catecholamine secretion. Cbln is also expressed in the pancreas; however, its function in the pancreas is unknown. Here, we demonstrate the role of CER in regulating insulin secretion in vivo and in vitro. We identified Cbln1 and Cbln3 transcripts in rat pancreatic islets and detected Cbln-immunoreactivity, predominantly located in the periphery of the rat endocrine pancreas. In vivo, CER reduced plasma insulin levels in rats after 1 and 2 h. CER decreased insulin secretion from isolated rat pancreatic islets at high (11 mM), but not at low (3.33 mM) glucose concentration. CER inhibited stimulated insulin secretion from clonal rat insulinoma (INS-1) cells, reduced forskolin-induced production of cAMP and intracellular calcium concentration. Our study demonstrates for the first time that Cbln1 and Cbln3 are expressed in the rat endocrine pancreas. Furthermore, we identify CER as an insulinostatic factor, which decreases intracellular cAMP production and calcium in INS-1 cells.

The contribution of voltage-gated Ca2+ currents to K+ channel activation during ovine adrenal chromaffin cell development

Prior to the development of adrenal innervation, the adrenal medulla is capable of responding to low blood oxygen directly. However, this response is lost once adrenal innervation is established. Previous work by our group has outlined mechanisms involved in this direct hypoxic response and the means by which innervation causes the loss of the direct hypoxic response in the ovine adrenal. The current study further investigates mechanisms which may underlie the developmental loss of the direct hypoxic response by concentrating on two aspects of cell function which regulate catecholamine secretion: the contribution of different types of Ca2+ channels to the total Ca2+ current and the contribution of each Ca2+ channel type to K+ channel activation. We identified that Ca2+ current size at −40 to −10mV is increased in amplitude in fetal chromaffin cells. This is not due to the increased prevalence or size of T-type Ca2+ currents present at these voltages. The relative contribution of L-, N- or P/Q-type Ca2+ channels to total Ca2+ current and to activation of the K+ current is unchanged during chromaffin cell development, however K+ current density increases with age. Our results indicate that there is a developmental shift in relative expression of T-type, but not L-, N- or P/Q-type, Ca2+ channels in ovine chromaffin cells. The increased K+ current density in adult cells may result in an altered response to an equal stimulus, while larger Ca2+ current at negative voltages in fetal cells may facilitate Ca2+ entry and catecholamine secretion in response to small depolarisations such as those induced by hypoxia.

Regulator of G Protein Signaling-4 Controls Fatty Acid and Glucose Homeostasis

Circulating free fatty acids are a reflection of the balance between lipogenesis and lipolysis that takes place mainly in adipose tissue. We found that mice deficient for regulator of G protein signaling (RGS)-4 have increased circulating catecholamines, and increased free fatty acids. Consequently, RGS4-/- mice have increased concentration of circulating free fatty acids; abnormally accumulate fatty acids in liver, resulting in liver steatosis; and show a higher degree of glucose intolerance and decreased insulin secretion in pancreas. We show in this study that RGS4 controls adipose tissue lipolysis through regulation of the secretion of catecholamines by adrenal glands. RGS4 controls the balance between adipose tissue lipolysis and lipogenesis, secondary to its role in the regulation of catecholamine secretion by adrenal glands. RGS4 therefore could be a good target for the treatment of metabolic diseases.

Corticotropin-Releasing Factor (CRF) and the Urocortins Differentially Regulate Catecholamine Secretion in Human and Rat Adrenals, in a CRF Receptor Type-Specific Manner

Corticotropin-releasing factor (CRF) affects catecholamine production both centrally and peripherally. The aim of the present work was to examine the presence of CRF, its related peptides, and their receptors in the medulla of human and rat adrenals and their direct effect on catecholamine synthesis and secretion. CRF, urocortin I (UCN1), urocortin II (UCN2), and CRF receptor type 1 (CRF1) and 2 (CRF2) were present in human and rat adrenal medulla as well as the PC12 pheochromocytoma cells by immunocytochemistry, immunofluorescence, and RT-PCR. Exposure of dispersed human and rat adrenal chromaffin cells to CRF1 receptor agonists induced catecholamine secretion in a dose-dependent manner, an effect peaking at 30 min, whereas CRF2 receptor agonists suppressed catecholamine secretion. The respective effects were blocked by CRF1 and CRF2 antagonists. CRF peptides affected catecholamine secretion via changes of subplasmaliminal actin filament polymerization. CRF peptides also affected catecholamine synthesis. In rat chromaffin and PC12 cells, CRF1 and CRF2 agonists induced catecholamine synthesis via tyrosine hydroxylase. However, in human chromaffin cells, activation of CRF1 receptors induced tyrosine hydroxylase, whereas activation of CRF2 suppressed it. In conclusion, it appears that a complex intraadrenal CRF-UCN/CRF-receptor system exists in both human and rat adrenals controlling catecholamine secretion and synthesis.

Analysis of the late steps of exocytosis: biochemical and total internal reflection fluorescence microscopy (TIRFM) studies.

1. Time with Julie in his laboratory at the NIH in the early 1970s is remembered. The experience led to a life-long interest in the regulation of catecholamine secretion. Here are summarized aspects of this work. 2. The relationship between ATP-dependent priming of exocytosis and the polyphosphoinositides is reviewed. In addition, studies are summarized in which total internal reflection fluorescent microscopy (TIRFM) was used to visualize secretory granule behavior before exocytosis and individual exocytotic events. 3. Quantitative optical analysis indicates that chromaffin granule motion is highly restricted but regulated. Granules can undergo significant motion in the 100 ms prior to fusion and interactions with the plasma membrane leading to fusion can occur within this time. The small motions may permit granules adjacent to the plasma membrane to repetitively sample microdomains of the plasma membrane, thereby increasing the probability of fruitful interactions that lead to fusion.

Identification of MrgX2 as a human G-protein-coupled receptor for proadrenomedullin N-terminal peptides

Proadrenomedullin N-terminal 20 peptide (PAMP[1–20]/PAMP-20) and its truncated analog, PAMP[9–20]/PAMP-12, are endogenous peptides that elicit hypotension through inhibiting catecholamine secretion from sympathetic nerve endings and adrenal chromaffin cells. Although the binding sites for PAMP are widely distributed, the nature of its receptor has been elusive. In an effort to identify potential PAMP receptor(s), we found that a human G-protein-coupled receptor, MrgX2, was specifically activated by PAMP. Although a previous study revealed that MrgX2 was a receptor for cortistatin, a neuropeptide involved in sleep regulation and locomotor activity, our present data indicated that the rank order of the agonistic effect against MrgX2 was “PAMP-12 ≥ cortistatin > PAMP-20”. These activities were confirmed by the inhibition of the forskolin-elevated cAMP accumulation, Ca 2+ mobilization, and [ 35S]guanosine 5′-(γ-thio)triphosphate binding assays. These findings suggest that MrgX2 couples with not only G αq but also G αi, consistent with previous reports on the pharmacological profile of PAMP signaling. Furthermore, by immunostaining, we found that MrgX2 was expressed in the adrenal chromaffin cells as well as the dorsal root ganglia. From these results, we concluded that MrgX2 is a potential human PAMP-12 receptor that regulates catecholamine secretion from adrenal glands. The present discovery will eventually lead to a better understanding of the pathophysiological role of proadrenomedullin peptides.

Responses of hepatic glucose output to electro-acupuncture stimulation of the hindlimb in anaesthetized rats

Responses of hepatic glucose output (HGO) to electro-acupuncture (EA) stimulation of the hindlimb were investigated in anaesthetized rats, focusing on involvement of the somatic afferent and autonomic efferent nerves. HGO was measured with a microdialysis probe implanted into the left lateral lobe of the liver. Stainless steel needles with a diameter of 0.25 mm were inserted into the right tibialis anterior muscle and connected to an electrical stimulator. The EA stimulation was delivered for 10 min at 10 mA, 20 Hz. Atropine was injected in order to block the action of the parasympathetic nerves, whereas phentolamine and propranolol were injected in order to block the action of the sympathetic nerves. Furthermore, adrenal sympathetic nerves were crushed bilaterally to block the reflex secretion of adrenal medullary hormones. The EA stimulation significantly increased HGO for 20 min after the onset of stimulation. The increases of HGO were abolished by severing the femoral and sciatic nerves, demonstrating that the responses are elicited via activation of somatic afferent nerves. Furthermore, the increases were diminished after severance of the adrenal sympathetic nerves, which regulate catecholamine secretion from the adrenal medulla. The increases were totally abolished after pretreatment with phentolamine, an α-adrenergic blocker, and propranolol, a β-adrenergic blocker. On the other hand, the increases of HGO in response to the EA stimulation were augmented after pretreatment with atropine, a muscarinic cholinergic blocker. The present results demonstrate that EA stimulation to a hindlimb can reflexly increase HGO via activation of somatic afferents and, thereby, sympathetic efferents, including sympathetic efferents to the adrenal medulla. The present results further show that the increases of HGO in responses to EA stimulation are simultaneously reflexly inhibited via the parasympathetic nerves.

Catecholamine Response is Attenuated During Moderate Intensity Exercise in Response to the “Lactate Clamp”.

0149 Plasma catecholamines are known to respond to feedforward and feedback mechanisms. Norepinephrine (NE) and epinephrine (E) rise in response to stresses, such as exercise, that compromise blood glucose homeostasis. Both NE and E have been correlated with blood pH, blood lactate, and ATP demand as assessed by relative exercise intensity. PURPOSE: To assess the hypothesis that the lactate anion (La-) is involved in feedback control of catecholamine release plasma concentrations of norepinephrine and epinephrine were determined at rest and during exercise at 55% of VO2peak with (LC) and without (CON) exogenous lactate infusion. METHODS: Six apparently healthy recreationally active young men were recruited to participate in this study (26 ± 2 yr, 82 ± 2 kg, 50.7 ± 2.1 ml·kg−1·min−1 VO2peak). Participants were studied on 5 occasions. Exercise intensities were 65% of VO2peak for the first trial, and 55% of VO2peak for all remaining trials. The blood lactate profile of the first (65%) trial was matched during the subsequent LC trials, target [lactate] > 4 mM. Participants were studied after an overnight fast; exogenous lactate was infused in a forearm vein; blood was sampled from an arterialized hand vein during 90 min of rest and 90 min of exercise, and catecholamines were determined using HPLC with electrochemical detection. RESULTS: Plasma concentrations of NE and E increased from rest (NE: 241 ± 98 pg/ml, E: 63 ± 25 pg/ml) to exercise (NE: 1498 ± 611 pg/ml, E: 207 ± 187 pg/ml; P <0.05). There was also a condition effect during exercise with NE and E lower during the LC trial (NE: LC = 1211 ± 494 pg/ml, CON = 1785 ± 728 pg/ml, E: LC = 145 ± 59 pg/ml CON = 269 ± 109, P <0.05). As reported previously, LC did not affect endogenous lactate Ra, but raised total [La-], Ra and Rd to levels seen in the 65% trial. Neither glucose, insulin (I), or glucagon (G) concentrations or the I/G were affected, but RER was decreased, and blood pH raised by LC. CONCLUSIONS: NE and E responses were attenuated during moderate intensity (55% of VO2peak) exercise with the exogenous lactate infusion. Results indicate an effect of lactate or an associate parameter of acid-base balance on regulation of catecholamine secretion during physical activity. Supported by NIH grant: AR-42906