Catecholamine Secretion In Response Research Articles

CHLOROGENIC ACID CAN INHIBIT CATECHOLAMINE SECRETION FROM THE ADRENAL MEDULLA

Objective: Chlorogenic acid (CGA) is one of the most available phenolic acid compounds in foods, such as coffee and tea, which is known to be the most effective agent in lowering blood pressure and helpful in treating hypertension-related disorders. 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 method: he adrenal gland was isolated and perfused with Krebs-bicarbonate. CA was measured directly by using the fluorospectrophotometer. Results: CGA, 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, DMPP, 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+, veratridine, cyclopiazonic acid, Bay-K-8644 were suppressed. However, in the simultaneous presence of CGA and L-NAME, 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. Conclusions: Taken together, we present the first evidence that CGA suppresses the CA secretion induced by activation of cholinoceptors 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 adrenomedullary chromaffin cells as well as by suppressing the Ca2+ release from the cytoplasmic calcium store, through the elevated NO release by the activation of NO synthase, which is associated to the blockade of neuronal cholinonoceptors 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 circulation.

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.

PS-B03-5: HIGH-DOSES OF LOSARTAN RATHER ENHANCE CATECHOLAMINE SECRETION FROM THE PERFUSED ADRENAL GLAND

Objective: Previously, we have found that Losartan (5∼50 μM), an AT1 receptor blocker, dose-dependently inhibits secretion of catecholamines (CA) evoked by cholinergic and angiotensin II (Ang II) receptor stimulation from the perfused rat adrenal medulla. The aim of the present study was to examine the characteristic effects of high-dose losartan on the CA secretion from the isolated perfused rat adrenal gland, and also to establish 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: The perfusion of high-dose losartan (150 ∼ 600 μM) into an adrenal vein for 90 min resulted in great increases in CA secretions evoked by acetylcholine in a dose-dependent fashion. Also, losartan (300 μM) enhanced the CA release evoked by Ang II, high K+ and DMPP (a selective nicotinic receptor agonist), McN-A-343 (a selective muscarinic M1 receptor agonist). Also, in the presence of losartan (300 μM), the CA secretory responses to veratridine (a selective Na+ channel activator), Bay-K-8644 (a Ca2+ channel activator), and cyclopiazonic acid (a cytoplasmic Ca2+-ATPase inhibitor) were significantly potentiated, respectively. The facilitatory CA-releasing effect of Ach evoked by high-dose losartan (300 μM) was time-dependently depressed by pretreatment with nifedipine (Ca2+ channel blocker), while not by pretreatment with sodium nitroprusside (an NO donor). High-dose olmesartan (150 ∼ 600 μM), one of ARBs, also potentiated Ach-evoked CA release in time-dependent fashion. However, in the simultaneous presence of losartan (300 μM) and CGP42112 (an AT2 receptor agonist), the facilitatory responses of losartan on the CA secretion evoked by ACh were rather significantly reduced in comparison with that of losartan-treatment alone. Conclusion: Conclusively, these results demonstrate that high-dose losartan can potentiate the adrenomedullary CA secretion in a calcium-dependent fashion. It seems that this facilitatory effect of high-dose losartan may be mediated by activation of the Ca2+ influx through L-type Ca2+ channels into adrenomedullary cells without inhibition of NO synthesis, which seems to be relevant to AT2 receptor blockade. It also suggests that high-dose ARBs may be a risk factor in causing hypertensive response.

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.

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.

HIGH-DOSES OF LOSARTAN RATHER ACCELERATE THE SECRETION OF ADRENAL CATECHOLAMINES

Objective: Previously, we have found that Losartan (5∼50 microM), an AT1 receptor blocker, dose-dependently inhibits secretion of catecholamines (CA) evoked by cholinergic and angiotensin II (Ang II) receptor stimulation from the perfused rat adrenal medulla. The aim of the present study was to examine the characteristic effects of high-dose losartan on the CA secretion from the isolated perfused rat adrenal gland, and also to establish its mechanism of action. Design and method: The adrenal gland was isolated and perfused with Krebs-bicarbonate. CA was measured directly by using the fluorospectrophotometer. Results: The perfusion of high-dose losartan (150 ∼ 600 microM) into an adrenal vein for 90 min resulted in great increases in CA secretions evoked by acetylcholine in a dose-dependent fashion. Also, losartan (300 microM) enhanced the CA release evoked by Ang II, high K+ and DMPP (a selective nicotinic receptor agonist), McN-A-343 (a selective muscarinic M1 receptor agonist). Also, in the presence of losartan (300 microM), the CA secretory responses to veratridine (a selective Na+ channel activator), Bay-K-8644 (a Ca2+ channel activator), and cyclopiazonic acid (a cytoplasmic Ca2+-ATPase inhibitor) were significantly potentiated, respectively. The facilitatory CA-releasing effect of Ach evoked by high-dose losartan (300 microM) was time-dependently depressed by pretreatment with nifedipine (Ca2+ channel blocker), while not by pretreatment with sodium nitroprusside (an NO donor). High-dose olmesartan (150 ∼ 600 microM), one of ARBs, also potentiated Ach-evoked CA release in time-dependent fashion. However, in the simultaneous presence of losartan (300 microM) and CGP42112 (an AT2 receptor agonist), the facilitatory responses of losartan on the CA secretion evoked by ACh were rather significantly reduced in comparison with that of losartan-treatment alone. Conclusions: Conclusively, these results demonstrate that high-dose losartan can potentiate the adrenomedullary CA secretion in a calcium-dependent fashion. It seems that this facilitatory effect of high-dose losartan may be mediated by activation of the Ca2+ influx through L-type Ca2+ channels into adrenomedullary cells without inhibition of NO synthesis, which seems to be relevant to AT2 receptor blockade. It also suggests that high-dose ARBs may be a risk factor in causing hypertensive response.

A sodium background conductance controls the spiking pattern of mouse adrenal chromaffin cells in situ.

Key points Mouse chromaffin cells in acute adrenal slices exhibit two distinct spiking patterns, a repetitive mode and a bursting mode.A sodium background conductance operates at rest as demonstrated by the membrane hyperpolarization evoked by a low Na+‐containing extracellular saline.This sodium background current is insensitive to TTX, is not blocked by Cs+ ions and displays a linear I‐V relationship at potentials close to chromaffin cell resting potential. Its properties are reminiscent of those of the sodium leak channel NALCN.In the adrenal gland, Nalcn mRNA is selectively expressed in chromaffin cells.The study fosters our understanding of how the spiking pattern of chromaffin cells is regulated and adds a sodium background conductance to the list of players involved in the stimulus‐secretion coupling of the adrenomedullary tissue. Chromaffin cells (CCs) are the master neuroendocrine units for the secretory function of the adrenal medulla and a finely‐tuned regulation of their electrical activity is required for appropriate catecholamine secretion in response to the organismal demand. Here, we aim at deciphering how the spiking pattern of mouse CCs is regulated by the ion conductances operating near the resting membrane potential (RMP). At RMP, mouse CCs display a composite firing pattern, alternating between active periods composed of action potentials spiking with a regular or a bursting mode, and silent periods. RMP is sensitive to changes in extracellular sodium concentration, and a low Na+‐containing saline hyperpolarizes the membrane, regardless of the discharge pattern. This RMP drive reflects the contribution of a depolarizing conductance, which is (i) not blocked by tetrodotoxin or caesium, (ii) displays a linear I‐V relationship between −110 and −40 mV, and (iii) is carried by cations with a conductance sequence g Na > g K > g Cs. These biophysical attributes, together with the expression of the sodium‐leak channel Nalcn transcript in CCs, state credible the contribution of NALCN. This inaugural report opens new research routes in the field of CC stimulus‐secretion coupling, and extends the inventory of tissues in which NALCN is expressed to neuroendocrine glands.

Lack of p11 expression facilitates acidity-sensing function of TASK1 channels in mouse adrenal medullary cells.

External acidity induces catecholamine secretion by inhibiting TASK1-like channels in rat adrenal medullary (AM) cells. TASK channels can function as a heteromer or homomer in the TASK subfamily. In this study, we elucidate the molecular identity of TASK1-like channels in mouse AM cells using gene knockout. Genetic deletion of TASK1, but not TASK3, abolished the depolarizing inward current and catecholamine secretion in response to acidity, whereas it did not affect the resting current level. Immunocytochemistry revealed that AM cells exhibited predominantly TASK1-like and little TASK3-like immunoreactivity. A proximity ligation assay showed that TASK1/3 heteromeric channels were not formed in AM cells or PC12 cells. However, the exogenous expression of p11 in PC12 cells resulted in the heteromeric formation of TASK isoforms, which were mainly located in the cytoplasm, and p11 was not expressed in rat adrenal medullae or PC12 cells. In AM cells, genetic deletion of TASK1 resulted in enhancement of the immunoreactivity of the TALK2 channel, but not TASK3. The results indicate that TASK1 homomeric channels function as acidity sensors in AM cells, and that function is facilitated by the lack of p11 expression.-Inoue, M., Matsuoka, H., Lesage, F., Harada, K. Lack of p11 expression facilitates acidity-sensing function of TASK1 channels in mouse adrenal medullary cells.

Guinea Pig as a Model to Study the Carotid Body Mediated Chronic Intermittent Hypoxia Effects.

Clinical and experimental evidence indicates a positive correlation between chronic intermittent hypoxia (CIH), increased carotid body (CB) chemosensitivity, enhanced sympatho-respiratory coupling and arterial hypertension and cardiovascular disease. Several groups have reported that both the afferent and efferent arms of the CB chemo-reflex are enhanced in CIH animal models through the oscillatory CB activation by recurrent hypoxia/reoxygenation episodes. Accordingly, CB ablation or denervation results in the reduction of these effects. To date, no studies have determined the effects of CIH treatment in chemo-reflex sensitization in guinea pig, a rodent with a hypofunctional CB and lacking ventilatory responses to hypoxia. We hypothesized that the lack of CB hypoxia response in guinea pig would suppress chemo-reflex sensitization and thereby would attenuate or eliminate respiratory, sympathetic and cardiovascular effects of CIH treatment. The main purpose of this study was to assess if guinea pig CB undergoes overactivation by CIH and to correlate CIH effects on CB chemoreceptors with cardiovascular and respiratory responses to hypoxia. We measured CB secretory activity, ventilatory parameters, systemic arterial pressure and sympathetic activity, basal and in response to acute hypoxia in two groups of animals: control and 30 days CIH exposed male guinea pigs. Our results indicated that CIH guinea pig CB lacks activity elicited by acute hypoxia measured as catecholamine (CA) secretory response or intracellular calcium transients. Plethysmography data showed that only severe hypoxia (7% O2) and hypercapnia (5% CO2) induced a significant increased ventilatory response in CIH animals, together with higher oxygen consumption. Therefore, CIH exposure blunted hyperventilation to hypoxia and hypercapnia normalized to oxygen consumption. Increase in plasma CA and superior cervical ganglion CA content was found, implying a CIH induced sympathetic hyperactivity. CIH promoted cardiovascular adjustments by increasing heart rate and mean arterial blood pressure without cardiac ventricle hypertrophy. In conclusion, CIH does not sensitize CB chemoreceptor response to hypoxia but promotes cardiovascular adjustments probably not mediated by the CB. Guinea pigs could represent an interesting model to elucidate the mechanisms that underlie the long-term effects of CIH exposure to provide evidence for the role of the CB mediating pathological effects in sleep apnea diseases.

Hydrogen sulphide facilitates exocytosis by regulating the handling of intracellular calcium by chromaffin cells.

Gasotransmitter hydrogen sulphide (H2S) has emerged as a regulator of multiple physiological and pathophysiological processes throughout. Here, we have investigated the effects of NaHS (fast donor of H2S) and GYY4137 (GYY, slow donor of H2S) on the exocytotic release of catecholamines from fast-perifused bovine adrenal chromaffin cells (BCCs) challenged with sequential intermittent pulses of a K+-depolarizing solution. Both donors caused a concentration-dependent facilitation of secretion. This was not due to an augmentation of Ca2+ entry through voltage-activated Ca2+ channels (VACCs) because, in fact, NaHS and GYY caused a mild inhibition of whole-cell Ca2+ currents. Rather, the facilitation of exocytosis seemed to be associated to an augmented basal [Ca2+]c and the K+-elicited [Ca2+]c transients; such effects of H2S donors are aborted by cyclopiazonic acid (CPA), that causes endoplasmic reticulum (ER) Ca2+ depletion through sarcoendoplasmic reticulum Ca2+ ATPase inhibition and by protonophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP), that impedes the ability of mitochondria to sequester cytosolic Ca2+ during cell depolarization. Inasmuch as CPA and FCCP reversed the facilitation of secretion triggered by K+ in the presence of NaHS and GYY, is seems that such facilitation is tightly coupled to Ca2+ handling by the ER and mitochondria. On the basis of these results, we propose that H2S regulates catecholamine secretory responses triggered by K+ in BCCs by (i) mobilisation of ER Ca2+ and (ii) interference with mitochondrial Ca2+ circulation. In so doing, the clearance of the [Ca2+]c transient will be delayed and the Ca2+-dependent trafficking of secretory vesicles will be enhanced to overfill the secretory machinery with new vesicles to enhance exocytosis.

Hypoxia-regulated catecholamine secretion in chromaffin cells.

Adrenal catecholamine (CAT) secretion is a general physiological response of animals to environmental stressors such as hypoxia. This represents an important adaptive mechanism to maintain homeostasis and protect vital organs such as the brain. In adult mammals, CAT secretory responses are triggered by activation of the sympathetic nervous system that supplies cholinergic innervation of adrenomedullary chromaffin cells (AMC) via the splanchnic nerve. In the neonate, the splanchnic innervation of AMC is immature or absent, yet hypoxia stimulates a non-neurogenic CAT secretion that is critical for adaptation to extra-uterine life. This non-neurogenic, hypoxia-sensing mechanism in AMC is gradually lost or suppressed postnatally along a time course that parallels the development of splanchnic innervation. Moreover, denervation of adult AMC results in a gradual return of the direct hypoxia-sensing mechanism. The signaling pathways by which neonatal AMC sense acute hypoxia leading to non-neurogenic CAT secretion and the mechanisms that underlie the re-acquisition of hypoxia-sensing properties by denervated adult AMC, are beginning to be understood. This review will focus on current views concerning the mechanisms responsible for direct acute hypoxia sensing and CAT secretion in perinatal AMC and how they are regulated by innervation during postnatal development. It will also briefly discuss plasticity mechanisms likely to contribute to CAT secretion during exposures to chronic and intermittent hypoxia.

Tight mitochondrial control of calcium and exocytotic signals in chromaffin cells at embryonic life.

Calcium buffering by mitochondria plays a relevant physiological function in the regulation of Ca(2+) and exocytotic signals in mature chromaffin cells (CCs) from various adult mammals. Whether a similar or different role of mitochondrial Ca(2+) buffering is present in immature CCs at early life has not been explored. Here we present a comparative study in rat embryonic CCs and rat mother CCs, of various physiological parameters that are known to be affected by mitochondrial Ca(2+) buffering during cell activation. We found that the clearance of cytosolic Ca(2+) transients ([Ca(2+)]c) elicited by high K(+) was 7-fold faster in embryo CCs compared to mother CCs. This strongly suggests that at embryonic life, the mitochondria play a more significant role in the clearance of [Ca(2+)]c loads compared to adult life. Consistent with this view are the following results concerning the transient suppression of mitochondrial Ca(2+) buffering by protonophore FCCP, in embryonic CCs compared to mother CCs: (i) faster and greater inactivation of inward calcium currents, (ii) higher K(+)-elicited [Ca(2+)]c transients with 25-fold faster clearance, (iii) higher increase of basal catecholamine release and (iv) higher potentiation of K(+)-evoked secretion. These pronounced differences could be explained by two additional features (embryo versus mother CCs): (a) slower recovery of mitochondrial resting membrane potential after the application of a transient FCCP pulse and (b) greater relative density of the mitochondria in the cytosol. This tighter control by the mitochondria of Ca(2+) and exocytotic signals may be relevant to secure a healthy catecholamine secretory response at early life.

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.

Perinatal hyperoxia exposure impairs hypoxia-induced depolarization in rat carotid body glomus cells

Chronic post-natal hyperoxia reduces the hypoxic ventilatory response by reducing the carotid body sensitivity to acute hypoxia as demonstrated by a reduced afferent nerve response, reduced calcium response of carotid body glomus cells and reduced catecholamine secretion in response to acute hypoxia. The present study examined whether hyperoxia alters the electrophysiological characteristics of glomus cells. Rats were treated with hyperoxia for 1 week starting at P1 or P7 and for 2 weeks starting at P1 followed by harvesting and dissociation of their carotid bodies for whole cell, perforated-patch recording. As compared to glomus cells from normoxia animals, hyperoxia treated cells showed a significant reduction in the magnitude of depolarization in response to hypoxia and anoxia, despite little change in the depolarizing response to 20mMK+. Resting cell membrane potential in glomus cells from rats exposed to hyperoxia from P1 to P15 and studied at P15 was slightly depolarized compared to other treatment groups and normoxia-treated cells, but conductance normalized to cell size was not different among groups. We conclude that postnatal hyperoxia impairs carotid chemoreceptor hypoxia transduction at a step between hypoxia sensing and membrane depolarization. This occurs without a major change in baseline electrophysiological characteristics, suggesting altered signaling or alterations in the relative abundance of different leak channel isoforms.

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.

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.

H2S induces catecholamine secretion in rat adrenal chromaffin cells

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

Hydrogen sulfide mediates catecholamine secretion elicited by hypoxia in the carotid body

We previously demonstrated that H2S is a physiologic transmitter in the carotid body, enhancing responses to hypoxia. Carotid bodies express the H2S producing enzyme cystathionine γ‐lyase (CSE). Genetic ablation of CSE or inhibition by DL‐propargylglycine (PAG) eliminated carotid body sensory and ventilatory responses to hypoxia. In the present study we examine the role of H2S in the glomus cell's response to hypoxia. Catecholamine secretion was monitored by carbon fiber amperometry and [Ca2+]i with fura 2. In rat glomus cells PAG attenuated the secretory response to hypoxia, but did not alter secretion elicited by high‐K+. Experiments with CSE−/− mice produced similar results; glomus cells from CSE−/− mice showed little catecholamine secretion in response to hypoxia, but exhibited robust secretion when exposed to high‐K+. Secretion is tightly regulated by [Ca2+]i. PAG treated rat glomus cells exhibited reduced elevation of [Ca2+]i in response to hypoxia. Exposing rat glomus cells to the H2S donor NaHS produced an elevation in [Ca2+]i that mimicked the elevation of [Ca2+]i elicited by hypoxia. More importantly, glomus cells from CSE−/− mice had markedly attenuated elevation of [Ca2+]i in response to hypoxia. These results suggest that hypoxia evoked catecholamine secretion from glomus cells is mediated by H2S via a Ca2+ dependent mechanism. Supported by NIH HL‐90554, HL‐86493.

Cytosolic organelles shape calcium signals and exo–endocytotic responses of chromaffin cells

The concept of stimulus–secretion coupling was born from experiments performed in chromaffin cells 50 years ago. Stimulation of these cells with acetylcholine enhances calcium (Ca2+) entry and this generates a transient elevation of the cytosolic Ca2+ concentration ([Ca2+]c) that triggers the exocytotic release of catecholamines. The control of the [Ca2+]c signal is complex and depends on various classes of plasmalemmal calcium channels, cytosolic calcium buffers, the uptake and release of Ca2+ from cytoplasmic organelles, such as the endoplasmic reticulum, mitochondria, chromaffin vesicles and the nucleus, and Ca2+ extrusion mechanisms, such as the plasma membrane Ca2+-stimulated ATPase, and the Na+/Ca2+ exchanger. Computation of the rates of Ca2+ fluxes between the different cell compartments support the proposal that the chromaffin cell has developed functional calcium tetrads formed by calcium channels, cytosolic calcium buffers, the endoplasmic reticulum, and mitochondria nearby the exocytotic plasmalemmal sites. These tetrads shape the Ca2+ transients occurring during cell activation to regulate early and late steps of exocytosis, and the ensuing endocytotic responses. The different patterns of catecholamine secretion in response to stress may thus depend on such local [Ca2+]c transients occurring at different cell compartments, and generated by redistribution and release of Ca2+ by cytoplasmic organelles. In this manner, the calcium tetrads serve to couple the variable energy demands due to exo–endocytotic activities with energy production and protein synthesis.

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.