Direct Membrane Depolarization Research Articles

Improving focality and consistency in micromagnetic stimulation.

The novel micromagnetic stimulation (μMS) technology aims to provide high resolution on neuronal targets. However, consistency of neural activation could be compromised by a lack of surgical accuracy, biological variation, and human errors in operation. We have recently modeled the activation of an unmyelinated axon by a circular micro-coil. Although the coil could activate the axon, its performance sometimes lacked focality and consistency. The site of axonal activation could shift by several experimental factors, including the reversal of the coil current, displacement of the coil, and changes in the intensity of the stimulation. Current clinical practice with transcranial magnetic stimulation (TMS) has suggested that figure-eight coils could provide better performance in magnetic stimulation than circular coils. Here, we estimate the performance of μMS by a figure-eight micro-coil, by exploring the impact of the same experimental factors on its focality and consistency in axonal activation. We derived the analytical expression of the electric field and activating function generated by the figure-eight micro-coil, and estimated the location of axonal activation. Using NEURON modeling of an unmyelinated axon, we found two different types (A and B) of axon activation by the figure-eight micro-coil, mediated by coil currents of reversed direction. Type A activation is triggered by membrane hyperpolarization followed by depolarization; Type B activation is triggered by direct membrane depolarization. Consequently, the two types of stimulation are governed by distinct ion channel mechanisms. In comparison to the circular micro-coil, the figure-eight micro-coil requires significantly less current for axonal activation. Under figure-eight micro-coil stimulation, the site of axonal activation does not change with the reversal of the coil current, displacement of the coil, or changes in the intensity of the stimulation. Ultimately, the figure-eight micro-coil provides a more efficient and consistent site of activation than the circular micro-coil in μMS.

S-13-4: INHIBITORY IMPACTS OF CHLOROGENIC ACID ON ADRENAL CATECHOLAMINE SECRETION

(Objective) Chlorogenic acid (CGA) is one of the most available phenolic acid compounds in foods, such as coffee and tea. Thus, the present study was aimed to determine the characteristics of CGA on secretion of catecholamines (CA) in the perfused model of the rat adrenal medulla, and also to validify its mechanism of action. (Design and Methods) The adrenal gland was isolated and perfused with Krebs-bicarbonate. CA was measured directly by using the fluorospectrophotometer. (Results) CGA (30∼300 μM), administered into an adrenal vein for 90 min, reduced ACh-induced CA secretion in a dose- and time-dependent manner. CGA also time-dependently depressed the CA secretion induced by McN-A-343 (a selective muscarinic M1 receptor agonist), DMPP (a selective neuronal nicotinic receptor agonist), and angiotensin II. CGA itself failed to influence spontaneous CA secretion. Also, During the perfusion of CGA for 90 min, the CA secretory responses induced by high K+ (56 mM, a direct membrane depolarizer), veratridine (a voltage-dependent Na+ channel activator, cyclopiazonic acid (a cytoplasmic Ca2+-ATPase inhibitor, Bay-K-8644 (an L-type dihydropyridine Ca2+ channel activator were suppressed. However, in the simultaneous presence of CGA and L-NAME (an inhibitor of NO synthase), the CA secretory responses induced by ACh, Ang II, Bay-K-8644 and veratridine was restored nearly to the control level compared to those of the inhibitory effects of CGA-treatment alone. Virtually, the adrenal NO release was significantly enhanced by CGA-treatment. Also, in the coexistence of CGA and olmesartan, ACh-induced CA secretion was markedly reduced compared to that of olmesartan-treatment alone. (Conclusion) Taken together, we present the first evidence that CGA suppresses the CA secretion induced by activation of cholinergic receptors as well as AT1-receptors. This CGA-induced inhibitory effect seems to be exerted by reducing both the influx of Na+ and Ca2+ through their ionic channels into the adrenomedullary chromaffin cells as well as by suppressing the Ca2+ release from the cytoplasmic calcium store, at least through the elevated NO release by the activation of NO synthase, which is associated to the blockade of neuronal cholinergic receptors and AT1-receptors. Based on these results, the ingestion of CGA may be helpful to alleviate or prevent the cardiovascular diseases, via reduction of adrenal CA secretion and consequent decreased CA level in the circulation. Co-administration of CGA and Olmesartan (ARB) may be clinically beneficial for treatment of cardiovascular diseases, including hypertension and angina pectoris.

PS-B03-6: COMPARISON OF SUPPRESSIVE EFFECTS ON CATECHOLAMINE SECRETION BETWEEN OLMESARTAN AND ENALAPRIL

Objective: The present study was designed to compare olmesartan, an angiotensin II receptor blocker (ARB) with enalapril, an angiotensin converting enzyme inhibitor (ACEI) in the suppressive effects on the secretion of catecholamines (CA) from the perfused model of the rat adrenal gland. Design and Methods: The adrenal gland was isolated and perfused with Krebs-bicarbonate. CA was measured directly by using the fluorospectrophotometer. Results: Both olmesartan (50 μM) and enalapril (50 μM) during perfusion into an adrenal vein for 90 min inhibited the CA secretory responses evoked by ACh (5.32 μM), DMPP (a selective neuronal nicotinic Nn receptor agonist, 100 μM), high K+ (a direct membrane-depolarizer, 56 μM), and McN-A-343 (a selective muscarinic M1 receptor agonist, 100 μM) in a time-dependent manner. Also, in the presence of enalapril or olmesartan, the secretory responses of CA evoked by veratridine (an activator of voltage-dependent Na+ channels, 100 μM), Bay-K-8644 (a L-type dihydropyridine Ca2+ channel activator, 10 μM), and cyclopiazonic acid (a cytoplasmic Ca2+-ATPase inhibitor, 10 μM) were significantly reduced. Based on the same concentration (50 μM) of enalapril and olmesartan, for the CA release evoked by Ach, high K+, DMPP, McN-A-343, angiotensin II, veratridine, Bay-K-8644 and cyclopiazonic acid, the following rank order of inhibitory potency was obtained: olmesartan > enalapril. In the simultaneous presence of enalapril and olmesartan, Ach-evoked CA secretory responses were more strongly inhibited in comparison with that of enalapril- or olmesartan-treated alone. Conclusion: Collectively, these results demonstrate that both enalapril and olmesartan inhibit the CA secretory responses evoked by activation of both cholinergic and angiotensin II receptors stimulation as well as by direct membrane depolarization in the perfused model of the isolated rat adrenal medulla. When these two drugs were used in combination, their effects were enhanced, which may also be of clinical benefit. Based on concentration used in this study, it seems that the inhibitory effect of olmesartan on the CA secretion is more potent than that of enalapril.

COMPARISON OF INHIBITORY EFFECTS EVOKED BY OLMESARTAN AND ENALAPRIL ON CATECHOLAMINE SECRETION

Objective: The present study was designed to compare enalapril, an angiotensin converting enzyme inhibitor (ACEI) with olmesartan an angiotensin II receptor blocker (ARB) in the inhibitory effects on the secretion of catecholamines (CA) from the perfused model of the rat adrenal gland. Design and method: The adrenal gland was isolated and perfused with Krebs-bicarbonate. CA was measured directly by using the fluorospectrophotometer. Results: Both enalapril (50 microM) and olmesartan (50 microM) during perfusion into an adrenal vein for 90 min inhibited the CA secretory responses evoked by ACh (5.32 microM), DMPP (a selective neuronal nicotinic Nn receptor agonist, 100 microM), high K+ (a direct membrane-depolarizer, 56 mM), and McN-A-343 (a selective muscarinic M1 receptor agonist, 100 microM) in a time-dependent manner. Also, in the presence of enalapril or olmesartan, the secretory responses of CA evoked by veratridine (an activator of voltage-dependent Na+ channels, 100 microM), Bay-K-8644 (a L-type dihydropyridine Ca2+ channel activator, 10 microM), and cyclopiazonic acid (a cytoplasmic Ca2+-ATPase inhibitor, 10 microM) were significantly reduced. Based on the same concentration (50 microM) of enalapril and olmesartan, for the CA release evoked by Ach, high K+, DMPP, McN-A-343, angiotensin II, veratridine, Bay-K-8644 and cyclopiazonic acid, the following rank order of inhibitory potency was obtained: olmesartan> enalapril. In the simultaneous presence of enalapril and olmesartan, Ach-evoked CA secretory responses were more strongly inhibited in comparison with that of enalapril- or olmesartan-treated alone. Conclusions: Collectively, these results demonstrate that both enalapril and olmesartan inhibit the CA secretory responses evoked by activation of both cholinergic and angiotensin II receptors stimulation as well as by direct membrane depolarization in the perfused model of the isolated rat adrenal medulla. When these two drugs were used in combination, their effects were enhanced, which may also be of clinical benefit. Based on concentration used in this study, it seems that the inhibitory effect of olmesartan on the CA secretion is more potent than that of enalapril.

Histamine modulates spinal motoneurons and locomotor circuits.

Spinal motoneurons and locomotor networks are regulated by monoamines, among which, the contribution of histamine has yet to be fully addressed. The present study investigates histaminergic regulation of spinal activity, combining intra- and extracellular electrophysiological recordings from neonatal rat spinal cord in vitro preparations. Histamine dose-dependently and reversibly generated motoneuron depolarization and action potential firing. Histamine (20 µM) halved the area of dorsal root reflexes and always depolarized motoneurons. The majority of cells showed a transitory repolarization, while 37% showed a sustained depolarization maintained with intense firing. Extracellularly, histamine depolarized ventral roots (VRs), regardless of blockage of ionotropic glutamate receptors. Initial, transient glutamate-mediated bursting was synchronous among VRs, with some bouts of locomotor activity in a subgroup of preparations. After washout, the amplitude of spontaneous tonic discharges increased. No desensitization or tachyphylaxis appeared after long perfusion or serial applications of histamine. On the other hand, histamine induced single motoneuron and VR depolarization, even in the presence of tetrodotoxin (TTX). During chemically induced fictive locomotion (FL), histamine depolarized VRs. Histamine dose-dependently increased rhythm periodicity and reduced cycle amplitude until near suppression. This study demonstrates that histamine induces direct motoneuron membrane depolarization and modulation of locomotor output, indicating new potential targets for locomotor neurorehabilitation.

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.

How does artemisinin act inside a cell?

Artemisinins are a set of miraculous antimalarial drugs. In addition to their potent antimalarial activities, artemisinins are found to possess some other biological properties such as cancer inhibition. The biological mechanism of artemisinin has long been controversial. However, it is generally believed that the endoperoxidic bond within artemisinin is key to its functions. The action of artemisinin is a two-step process: artemisinin first has to be activated by a reductive process to unleash the killing power of the endoperoxidic bond, and then exerts its actions. Nevertheless, the details of these processes are unclear and hotly debated. What molecules provide the necessary reduction force to activate artemisinin? Is it iron, heme or an uncertain component such as electron transport chain in the mitochondrial membrane? Evidences exist for all these possibilities, but none is conclusive. After artemisinin is activated, does it target a specific protein/biological process, or relatively non-specifically many proteins/biological processes? Does it affect vacuole or mitochondria or some other organelles? Heme/iron-mediated artemisinins activation has been widely reported and well supported by many pieces of research evidences, and almost certainly exist in many types of cells. Direct mitochondrial membrane depolarization has also been detected in Baker’s yeast and malarial parasites, but not in mammalian cells. This review is intended to summarize our current understanding of the action mechanism of artemisinin. It appears that artemisinin is associated with various biological properties, and depending on the cell types, one action might be more predominant than the others. This is further supported by the observation that the potencies of artemisinin derivatives are often not well correlated in different types of action assays. The critical issue in the future is to reveal more of the biological properties of artemisinins and pinpoint which property is more functionally relevant under a particular setting of biological action.

GLP-1R Signaling Directly Activates Arcuate Nucleus Kisspeptin Action in Brain Slices but Does not Rescue Luteinizing Hormone Inhibition in Ovariectomized Mice During Negative Energy Balance

Kisspeptin (Kiss1) neurons in the hypothalamic arcuate nucleus (ARC) are key components of the hypothalamic-pituitary-gonadal axis, as they regulate the basal pulsatile release of gonadotropin releasing hormone (GnRH). ARC Kiss1 action is dependent on energy status, and unmasking metabolic factors responsible for modulating ARC Kiss1 neurons is of great importance. One possible factor is glucagon-like peptide 1 (GLP-1), an anorexigenic neuropeptide produced by brainstem preproglucagon neurons. Because GLP fiber projections and the GLP-1 receptor (GLP-1R) are abundant in the ARC, we hypothesized that GLP-1R signaling could modulate ARC Kiss1 action. Using ovariectomized mice, we found that GLP-producing fibers come in close apposition with ARC Kiss1 neurons; these neurons also contain Glp1r mRNA. Electrophysiological recordings revealed that liraglutide (a long-acting GLP-1R agonist) increased action potential firing and caused a direct membrane depolarization of ARC Kiss1 cells in brain slices. We determined that brainstem preproglucagon mRNA is decreased after a 48-h fast in mice, a negative energy state in which ARC Kiss1 expression and downstream GnRH/luteinizing hormone (LH) release are potently suppressed. However, activation of GLP-1R signaling in fasted mice with liraglutide was not sufficient to prevent LH inhibition. Furthermore, chronic central infusions of the GLP-1R antagonist, exendin(9–39), in ad libitum–fed mice did not alter ARC Kiss1 mRNA or plasma LH. As a whole, these data identify a novel interaction of the GLP-1 system with ARC Kiss1 neurons but indicate that CNS GLP-1R signaling alone is not critical for the maintenance of LH during fasting or normal feeding.

Inhibitory modulation of optogenetically identified neuron subtypes in the rostral solitary nucleus.

Inhibition is presumed to play an important role in gustatory processing in the rostral nucleus of the solitary tract (rNST). One source of inhibition, GABA, is abundant within the nucleus and comes both from local, intrasolitary sources and from outside the nucleus. In addition to the receptor-mediated effects of GABA on rNST neurons, the hyperpolarization-sensitive currents, Ih and IA, have the potential to further modulate afferent signals. To elucidate the effects of GABAergic modulation on solitary tract (ST)-evoked responses in phenotypically defined rNST neurons and to define the presence of IA and Ih in the same cells, we combined in vitro recording and optogenetics in a transgenic mouse model. This mouse expresses channelrhodopsin 2 (ChR2) in GAD65-expressing GABAergic neurons throughout the rNST. GABA positive (GABA+) neurons differed from GABA negative (GABA-) neurons in their response to membrane depolarization and ST stimulation. GABA+ neurons had lower thresholds to direct membrane depolarization compared with GABA- neurons, but GABA- neurons responded more faithfully to ST stimulation. Both IA and Ih were present in subsets of GABA+ and GABA- neurons. Interestingly, GABA+ neurons with Ih were more responsive to afferent stimulation than inhibitory neurons devoid of these currents, whereas GABA- neurons with IA were more subject to inhibitory modulation. These results suggest that the voltage-gated channels underlying IA and Ih play an important role in modulating rNST output through a circuit of feedforward inhibition.

Inhibitory effects of ginsenoside-rb2 on nicotinic stimulation-evoked catecholamine secretion.

The aim of the present study was to investigate whether ginsenoside-Rb2 (Rb2) can affect the secretion of catecholamines (CA) in the perfused model of the rat adrenal medulla. Rb2 (3~30 µM), perfused into an adrenal vein for 90 min, inhibited ACh (5.32 mM)-evoked CA secretory response in a dose- and time-dependent fashion. Rb2 (10 µM) also time-dependently inhibited the CA secretion evoked by DMPP (100 µM, a selective neuronal nicotinic receptor agonist) and high K+ (56 mM, a direct membrane depolarizer). Rb2 itself did not affect basal CA secretion (data not shown). Also, in the presence of Rb2 (50 µg/mL), the secretory responses of CA evoked by veratridine (a selective Na+ channel activator (50 µM), Bay-K-8644 (an L-type dihydropyridine Ca2+ channel activator, 10 µM), and cyclopiazonic acid (a cytoplasmic Ca2+-ATPase inhibitor, 10 µM) were significantly reduced, respectively. Interestingly, in the simultaneous presence of Rb2 (10 µM) and L-NAME (an inhibitor of NO synthase, 30 µM), the inhibitory responses of Rb2 on ACh-evoked CA secretory response was considerably recovered to the extent of the corresponding control secretion compared with the inhibitory effect of Rb2-treatment alone. Practically, the level of NO released from adrenal medulla after the treatment of Rb2 (10 µM) was greatly elevated compared to the corresponding basal released level. Collectively, these results demonstrate that Rb2 inhibits the CA secretory responses evoked by nicotinic stimulation as well as by direct membrane-depolarization from the isolated perfused rat adrenal medulla. It seems that this inhibitory effect of Rb2 is mediated by inhibiting both the influx of Ca2+ and Na+ into the adrenomedullary chromaffin cells and also by suppressing the release of Ca2+ from the cytoplasmic calcium store, at least partly through the increased NO production due to the activation of nitric oxide synthase, which is relevant to neuronal nicotinic receptor blockade.

Enhanced NMDA receptor-mediated intracellular calcium signaling in magnocellular neurosecretory neurons in heart failure rats

An enhanced glutamate excitatory function within the hypothalamic supraoptic and paraventricluar nuclei is known to contribute to increased neurosecretory and presympathetic neuronal activity, and hence, neurohumoral activation, during heart failure (HF). Still, the precise mechanisms underlying enhanced glutamate-driven neuronal activity in HF remain to be elucidated. Here, we performed simultaneous electrophysiology and fast confocal Ca²⁺ imaging to determine whether altered N-methyl-d-aspartate (NMDA) receptor-mediated changes in intracellular Ca²⁺ levels (NMDA-ΔCa²⁺) occurred in hypothalamic magnocellular neurosecretory cells (MNCs) in HF rats. We found that activation of NMDA receptors resulted in a larger ΔCa²⁺ in MNCs from HF when compared with sham rats. The enhanced NMDA-ΔCa²⁺ was neither dependent on the magnitude of the NMDA-mediated current (voltage clamp) nor on the degree of membrane depolarization or firing activity evoked by NMDA (current clamp). Differently from NMDA receptor activation, firing activity evoked by direct membrane depolarization resulted in similar changes in intracellular Ca²⁺ in sham and HF rats. Taken together, our results support a relatively selective alteration of intracellular Ca²⁺ homeostasis and signaling following activation of NMDA receptors in MNCs during HF. The downstream functional consequences of such altered ΔCa²⁺ signaling during HF are discussed.

Influence of Fimasartan (a Novel AT1Receptor Blocker) on Catecholamine Release in the Adrenal Medulla of Spontaneously Hypertensive Rats

The aim of this study was to determine whether fimasartan, a newly developed AT1 receptor blocker, can affect the CA release in the isolated perfused model of the adrenal medulla of spontaneously hypertensive rats (SHRs). Fimasartan (5~50 µM) perfused into an adrenal vein for 90 min produced dose- and time-dependently inhibited the CA secretory responses evoked by ACh (5.32 mM), high K+ (56 mM, a direct membrane depolarizer), DMPP (100 µM) and McN-A-343 (100 µM). Fimasartan failed to affect basal CA output. Furthermore, in adrenal glands loaded with fimasartan (15 µM), the CA secretory responses evoked by Bay-K-8644 (10 µM, an activator of L-type Ca2+ channels), cyclopiazonic acid (10 µM, an inhibitor of cytoplasmic Ca2+-ATPase), and veratridine (100 µM, an activator of Na+ channels) as well as by angiotensin II (Ang II, 100 nM), were markedly inhibited. In simultaneous presence of fimasartan (15 µM) and L-NAME (30 µM, an inhibitor of NO synthase), the CA secretory responses evoked by ACh, high K+, DMPP, Ang II, Bay-K-8644, and veratridine was not affected in comparison of data obtained from treatment with fimasartan (15 µM) alone. Also there was no difference in NO release between before and after treatment with fimasartan (15 µM). Collectively, these experimental results suggest that fimasartan inhibits the CA secretion evoked by Ang II, and cholinergic stimulation (both nicotininc and muscarinic receptors) as well as by membrane depolarization from the rat adrenal medulla. It seems that this inhibitory effect of fimasartan may be mediated by blocking the influx of both Na+ and Ca2+ through their ion channels into the rat adrenomedullary chromaffin cells as well as by inhibiting the Ca2+ release from the cytoplasmic calcium store, which is relevant to AT1 receptor blockade without NO release.

276 COMPARISON OF INHIBITORY EFFECTS BETWEEN LOSARTAN, OLMESARTAN AND FIMASARTAN ON CATECHOLAMINE RELEASE IN THE PERFUSED ADRENAL MEDULLA

Recently, we have found that both losartan (Noh et al. 2009) and olmesartan (Lim et al., 2010) inhibit the secretion of catecholamines (CA) from the perfused rat adrenal medulla. Therefore, the aim of the present study was to compare the inhibitory effects of three major ARBs (losartan, olmesartan and fimasartan) each other on the CA release from the isolated perfused model of the rat adrenal medulla. Losartan (LST, 15 μM), olmesartan (OST, 15 μM) and fimasartan (FST, 15 μM), perfused into an adrenal vein for 90 min, inhibited the CA secretory responses evoked by ACh (5.32 mM) and high K+ (56 mM, a direct membrane depolarizer), DMPP (100 μM, a selective neuronal nicotinic receptor agonist) and McN-A-343 (100 μM, a selective muscarinic M1 receptor agonist) in a time-dependent fashion, respectively. Also, in adrenal glands loaded with LST (15 μM), OST (15 μM) and FST (15 μM), the CA secretory responses evoked by Bay-K-8644 (10 μM, an activator of L-type Ca2+ channels), cyclopiazonic acid (10 μM, an inhibitor of cytoplasmic Ca2+-ATPase), and veratridine (100 μM, an activator of voltage-dependent Na+ channels) as well as by angiotensin ll (100 nM) were time-dependently reduced, respectively. Based on the same concentration (15 μM) of three ARBs, for the CA release evoked by Ach, high K+, DMPP, and McN-A-343, the following rank order of inhibitory potency was obtained: LST<OST<FST. Also, for the CA release evoked by Bay-K-8644, cyclopiazonic acid, and angiotensin ll, the following rank order of inhibitory potency was obtained: LST<OST<FST. However, for the CA release evoked by veratridine, the following rank order of inhibitory potency was obtained: LST<OST=FST. All three ARBs themselves did not affect basal CA output. Collectively, these experimental results suggest that all three ARBs inhibit the CA secretion evoked by cholinergic stimulation (both nicotininc and muscarinic receptors) as well as direct membrane depolarization from the perfused rat adrenal medulla. It seems that this inhibitory effect of all three ARBs may be mediated by blocking the influx of both Na+ and Ca2+ through their voltage-dependent ion channels into the adrenomedullary chromaffin cells as well as by inhibiting the Ca2+ release from its cytoplasmic calcium store. Based on the same concentration used in this study, it is also suggested that there is difference in potency of the inhibitory effects on the CA release between three ARBs, indicating that fimasartan seems to be more potent among three ARBs.

640 INFLUENCE OF FIMASARTAN ON CATECHOLAMINE RELEASE IN THE PERFUSED ADRENAL MEDULLA OF SHRS

The aim of this study was to determine whether fimasartan, a newly developed AT1 receptor blocker, can affect the CA release from the isolated perfused model of the adrenal medulla of spontaneously hypertensive rats (SHRs). Fimasartan (5∼50 μM) perfused into an adrenal vein for 90 min produced dose- and time-dependently inhibited the CA secretory responses evoked by ACh (5.32 mM), high K+ (56 mM, a direct membrane depolarizer), DMPP (100 μM) and McN-A-343 (100 μM). Fimasartan failed to affect basal CA output. Furthermore, in adrenal glands loaded with fimasartan (15 μM), the CA secretory responses evoked by Bay-K-8644 (10 μM, an activator of L-type Ca2+ channels), cyclopiazonic acid (10 μM, an inhibitor of cytoplasmic Ca2+-ATPase), and veratridine (100 μM, an activator of Na+ channels) as well as by angiotensin II (100 nM), were markedly inhibited. In simultaneous presence of fimasartan (15 μM) and L-NAME (30 μM, an inhibitor of NO synthase), the CA secretion evoked by ACh, high K+, DMPP and McN-A-343 was not affected in comparison of data obtained from treatment with fimasartan (15 μM) alone. Also there was no difference in NO release between before and after treatment with fimasartan (15 μM). Collectively, these experimental results suggest that fimasartan inhibits the CA secretion evoked by cholinergic stimulation (both nicotininc and muscarinic receptors) as well as by membrane depolarization from the rat adrenal medulla. It seems that this inhibitory effect of fimasartan may be mediated by blocking the influx of both Na+ and Ca2+ through their ion channels into the rat adrenomedullary chromaffin cells as well as by inhibiting the Ca2+ release from the cytoplasmic calcium store, which is relevant to AT1 receptor blockade without NO release.

203 INFLUENCE OF SELF-FERMENTED PINE NEEDLE FRACTIONS ON CATECHOLAMINE RELEASE FROM THE RAT ADRENAL MEDULLA

Previously, we have found that self-fermented pine needle (Pinus densiflora) extract inhibits secretion of catecholamines (CA) from the perfused rat adrenal medulla (Shin & Lim, 2010), and also the contractile responses of isolated rat aortic strips induced by phenylephrine and high K+ (Yu et al., 2009). Therefore, the aim of the present study was to investigate the effects of several fractions obtained from methylene chloride (CH2Cl2) extract of self-fermented pine needle on the acetylcholine (ACh)-evoked CA release from the isolated perfused model of the rat adrenal medulla and to establish the mechanism of the most active fraction (Fr.)-induced inhibitory action on the CA release. We obtained 6 fractions from CH2Cl2 extract of self-fermented pine needle. For the ACh (5.32 mM)-evoked CA release, the following rank order of inhibitory potency was obtained: Fr.4>Fr.8-11>>Fr.3>Fr.6=Fr.7>Fr.1.2. Fr.4 (60 μg/mL) perfused into an adrenal vein for 90 min produced relatively time-dependent inhibition of the CA secretory responses to ACh (5.32 mM), high K+ (56 mM), DMPP (100 μM) and McN-A-343 (100 μM). Fr.4 itself did not affect basal CA secretion (data not shown). Also, in the presence of Fr.4 (60 μg/mL), the CA secretory responses to angiotensin II (AngII, 0.1 μM), veratridine (a selective Na+ channel activator, 10 μM), Bay-K-8644 (a L-type dihydropyridine Ca2+ channel activator, 10 μM), and cyclopiazonic acid (a cytoplasmic Ca2+-ATPase inhibitor, 10 μM) were significantly reduced, respectively. In the simultaneous presence of Fr.4 (60 μg/mL) and L-NAME (an inhibitor of NO synthase, 30 μM), the inhibitory responses of Fr.4 on the CA secretion evoked by ACh, high K+, DMPP, McN-A-343, Bay-K-8644, cyclopiazonic acid, AngII and veratridine were considerably recovered to the extent of the corresponding control secretion compared with that of Fr.4-treatment alone. The level of NO released from adrenal medulla after the treatment of Fr.4 (60 μg/mL) was greatly elevated compared with the basal level. Taken together, these results demonstrate that Fr.4 inhibits the CA secretion from the isolated perfused rat adrenal medulla evoked by stimulation of cholinergic receptors as well as by direct membrane-depolarization. It seems that this inhibitory effect of Fr.4 is mediated by blocking the influx of Ca2+ and Na+ into the adrenomedullary chromaffin cells as well as by inhibition of Ca2+ release from the cytoplasmic calcium store, which is evoked at least partly through the increased NO production due to the activation of NO synthase. Based on these results, it is also thought that Fr. 4 isolated from CH2Cl2 extract of pine needle may contain beneficial antihypertensive components to prevent or treat hypertension.

Inhibitory Effects of Total Ginseng Saponin on Catecholamine Secretion from the Perfused Adrenal Medulla of SHRs

There seems to be some controversy about the effect of total ginseng saponin (TGS) on the secretion of catecholamines (CA) from the adrenal gland. Therefore, the present study aimed to determine whether TGS can affect the CA release in the perfused model of the adrenal medulla isolated from spontaneously hypertensive rats (SHRs). TGS (15-150 μg/mL), perfused into an adrenal vein for 90 min, inhibited the CA secretory responses evoked by acetylcholine (ACh, 5.32 mM) and high K+ (56 mM, a direct membrane depolarizer) in a dose- and time-dependent fashion. TGS (50 μg/mL) also time-dependently inhibited the CA secretion evoked by 1.1-dimethyl-4 -phenyl piperazinium iodide (DMPP; 100 μM, a selective neuronal nicotinic receptor agonist) and McN-A-343 (100 μM, a selective muscarinic M1 receptor agonist). TGS itself did not affect basal CA secretion (data not shown). Also, in the presence of TGS (50 μg/mL), the secretory responses of CA evoked by veratridine (a selective Na+ channel activator (50 μM), Bay-K-8644 (an L-type dihydropyridine Ca2+ channel activator, 10 μM), and cyclopiazonic acid (a cytoplasmic Ca2+-ATPase inhibitor, 10 μM) were significantly reduced, respectively. Interestingly, in the simultaneous presence of TGS (50 μg/mL) and Nω-nitro-L-arginine methyl ester hydrochloride [an inhibitor of nitric oxide (NO) synthase, 30 μM], the inhibitory responses of TGS on the CA secretion evoked by ACh, high K+, DMPP, McN-A-343, Bay-K-8644, cyclopiazonic acid, and veratridine were considerably recovered to the extent of the corresponding control secretion compared with the inhibitory effect of TGS-treatment alone. Practically, the level of NO released from adrenal medulla after the treatment of TGS (150 μg/mL) was greatly elevated compared to the corresponding basal released level. Taken together, these results demonstrate that TGS inhibits the CA secretory responses evoked by stimulation of cholinergic (both muscarinic and nicotinic) receptors as well as by direct membrane-depolarization from the isolated perfused adrenal medulla of the SHRs. It seems that this inhibitory effect of TGS is mediated by inhibiting both the influx of Ca2+ and Na+ into the adrenomedullary chromaffin cells and also by suppressing the release of Ca2+ from the cytoplasmic calcium store, at least partly through the increased NO production due to the activation of nitric oxide synthase, which is relevant to neuronal nicotinic receptor blockade, without the enhancement effect on the CA release. Based on these effects, it is also thought that there are some species differences in the adrenomedullary CA secretion between the rabbit and SHR.

Inhibitory Effects of Olmesartan on Catecholamine Secretion from the Perfused Rat Adrenal Medulla

The present sutdy aimed to determine whether olmesartan, an angiotensin II (Ang II) type 1 (AT(1)) receptor blocker, can influence the CA release from the isolated perfused model of the rat adrenal medulla. Olmesartan (5~50 µM) perfused into an adrenal vein for 90 min produced dose- and time-dependent inhibition of the CA secretory responses evoked by ACh (5.32 mM), high K(+) (56 mM, a direct membrane-depolarizer), DMPP (100 µM) and McN-A-343 (100 µM). Olmesartan did not affect basal CA secretion. Also, in adrenal glands loaded with olmesartan (15 µM), the CA secretory responses evoked by Bay-K-8644 (10 µM, an activator of voltage-dependent L-type Ca(2+) channels), cyclopiazonic acid (10 µM, an inhibitor of cytoplasmic Ca(2+) -ATPase), veratridine (100 µM, an activator of voltage-dependent Na(+) channels), and Ang II (100 nM) were markedly inhibited. However, at high concentrations (150~300 µM), olmesartan rather enhanced the ACh-evoked CA secretion. Taken together, these results show that olmesartan at low concentrations inhibits the CA secretion evoked by cholinergic stimulation (both nicotininc and muscarinic receptors) as well as by direct membrane depolarization from the rat adrenal medulla, but at high concentrations it rather potentiates the ACh-evoked CA secretion. It seems that olmesartan has a dual action, acting as both agonist and antagonist at nicotinic receptors of the isolated perfused rat adrenal medulla, which might be dependent on the concentration. It is also thought that this inhibitory effect of olmesartan may be mediated by blocking the influx of both Na(+) and Ca(2+) into the rat adrenomedullary chromaffin cells as well as by inhibiting the Ca(2+) release from the cytoplasmic calcium store, which is thought to be relevant to the AT(1) receptor blockade, in addition to its enhancement on the CA secreton.

Synchronized Network Oscillations in Rat Tuberoinfundibular Dopamine Neurons: Switch to Tonic Discharge by Thyrotropin-Releasing Hormone

The pituitary hormone, prolactin, triggers lactation in nursing mothers. Under nonlactating conditions, prolactin secretion is suppressed by powerful inhibition from hypothalamic tuberoinfundibular dopamine (TIDA) neurons. Although firing pattern has been suggested as integral to neuroendocrine control, the electrical behavior of TIDA cells remains unknown. We demonstrate that rat TIDA neurons discharge rhythmically in a robust 0.05 Hz oscillation. The oscillation is phase locked between neurons, and while it persists during chemical synaptic transmission blockade, it is abolished by gap junction antagonists. Thyrotropin-releasing hormone (TRH) potently stimulates prolactin release, an effect assumed to take place in the pituitary. In TIDA cells, TRH caused a transition from phasic to tonic firing through combined pre- and postsynaptic effects. These findings suggest a model for prolactin regulation where a TIDA network switch from oscillations to sustained discharge converts dopamine from an antagonist at high concentrations to a functional agonist as dopamine output from the network decreases.

Neuroendocrine Proopiomelanocortin Neurons Are Excited by Hypocretin/Orexin

Hypocretin/orexin, produced by a group of neurons in the lateral hypothalamus/perifornical area, enhances cognitive arousal and also may play a crucial role in modulating the neuroendocrine system. How hypocretin modulates the endocrine system remains an open question. Hypocretin cells innervate the mediobasal hypothalamus where they can potentially influence the activity of specific cell populations within the arcuate nucleus. Here, we examine whether hypocretin modulates the median eminence-projecting proopiomelanocortin (POMC) neurons identified by selective green fluorescent protein expression and antidromic stimulation or retrograde Evans blue dye tracing in transgenic mice. We find that POMC neurons, in general, and, in addition, those that project their axons to the median eminence, were robustly activated by hypocretin in a dose-dependent manner. These excitatory actions included a threefold increase in spike frequency and direct membrane depolarization of up to 22 mV (mean, 17.9+/-7.2 mV). Direct postsynaptic depolarization was decreased at more positive membrane potentials, inhibited by the sodium-calcium exchanger antagonist KB-R7943, and reduced by lowering the bath temperature, or by buffering the postsynaptic calcium with BAPTA, suggesting that the primary mechanism for hypocretin-mediated excitation is the activation of the sodium-calcium exchanger. Hypocretin also enhanced excitatory inputs to POMC cells via a presynaptic mechanism and indirectly increased the release of GABA onto these cells in a spike-dependent manner. However, these synaptic actions were not necessary to cause postsynaptic membrane depolarization and spiking. Thus, in contrast to previous suggestions that hypocretin inhibited POMC cells, our results demonstrate robust direct excitation of POMC neurons by hypocretin.

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.