Impacto funcional de la inhibición farmacológica de Tmem176b en diferentes subpoblaciones de células dendríticas / Functional impact of pharmacological inhibition of Tmem176b on different subpopulations of dendritic cells

Nuestro grupo ha reportado que la proteína Tmem176b es un canal/transportador iónico intracelular fuertemente expresado en células dendríticas (DCs), que bloquea la respuesta inmune anti-tumoral. Identificamos al compuesto BayK8644 como un inhibidor de Tmem176b. BayK8644 promueve el control tumoral en protocolos profilácticos, pero no en tumores establecidos a través de la activación del inflamasoma y células T CD8+. Entendimos que era necesario profundizar en los mecanismos inmunológicos implicados, con el fin de obtener información que nos permitiera mejorar la respuesta terapéutica. Dada la relevancia de las DCs en la respuesta anti-tumoral y la fuerte expresión de Tmem176b en esos leucocitos, nos planteamos como objetivo general estudiar el impacto de la inhibición de Tmem176b en diferentes sub-poblaciones de DCs (cDC1 y cDC2). Aquí hemos demostrado que la inhibición de Tmem176b con BayK8644 resulta en el bloqueo de una de las funciones más específicas de las cDC1, la presentación cruzada de antígenos. Mostramos que la encapsulación de BayK8644 en NP (NP-BayK8644) previene la inhibición de la presentación cruzada de antígenos mientras mantiene la capacidad de activación del inflamasoma. El tratamiento con NP-BayK8644 de animales que poseen tumores establecidos controló el crecimiento tumoral de una manera dependiente de Tmem176b. El control tumoral se asoció con una mayor infiltración tumoral por células T CD8+ totales y específicas de tumor. Por tanto, la formulación de BayK8644 en NP mejoró la eficacia antitumoral del compuesto libre al evitar la inhibición de la presentación cruzada de antígenos al tiempo que desencadena la activación del inflamasoma. Por otra parte, caracterizamos mecanismos inmunológicos desencadenados por cDC2 luego de la deleción génica y el bloqueo farmacológico de Tmem176b. Mostramos en un modelo in vitro y ex vivo que las DC2 de animales portadores de tumores Tmem176b-/- o WT tratados con BayK8644 promovió la diferenciación de células Th17 de una manera dependiente del inflamasoma. Experimentos de transferencia celular adoptiva mostraron que las células Th17 mejoran los efectos anti-tumorales del bloqueo de PD-1, sugiriendo que el eje cDC2/Th17 podría jugar un papel relevante en la terapia anti-PD1. En suma, caracterizamos el impacto de la inhibición de Tmem176b en diferentes subpoblaciones de DCs. Ese conocimiento aportó elementos para desarrollar formulaciones que mejoren el efecto anti-tumoral del BayK8644. Además describimos mecanismos desencadenados al inhibir a Tmem176b que potenciarían respuestas inmunes durante la terapia oncológica basada en el bloqueo de PD-1

Comparing the effects of small molecules BIX-01294, Bay K8644, RG-108 and valproic acid, and their different combinations on induction of pluripotency marker-genes by Oct4 in the mouse brain

Every cell type is characterized by a specific transcriptional profile together with a unique epigenetic landscape. Reprogramming factors such as Oct4, Klf4, Sox2 and c-Myc enable somatic cells to change their transcriptional profile and convert them to pluripotent cells. Small molecules such as BIX-01294, Bay K8644, RG-108 and valproic acid [VPA] are reported as effective molecules for enhancing induction of pluripotency in vitro, however, their effects during in vivo reprogramming are addressed in this experimental study. In this experimental study, Oct4 expressing lentiviral particles and small molecules BIX-01294, Bay K8644 and RG-108 were injected into the right ventricle of mice brain and VPA was systematically administered as oral gavages. Animals treated with different combinations of small molecules for 7 or 14 days in concomitant with Oct4 exogenous expression were compared for expression of pluripotency markers. Total RNA was isolated from the rims of the injected ventricle and quantitative polymerase chain reaction [PCR] was performed to evaluate the expression of endogenous Oct4, Nanog, c-Myc, klf4 and Sox2 as pluripotency markers, and Pax6 and Sox1 as neural stem cell [NSC] markers. Results showed that Oct4 exogenous expression for 7 days induced pluripotency slightly as it was detected by significant enhancement in expression of Nanog [p<0.05]. Combinatorial administration of Oct4 expressing vector and BIX-01294, Bay K8644 and RG-108 did not affect the expression of pluripotency and NSC markers, but VPA treatment along with Oct4 exogenous expression induced Nanog, Klf4 and c-Myc [p<0.001]. VPA treatment before the induction of exogenous Oct4 was more effective and significantly increased the expression of endogenous Oct4, Nanog, Klf4, c-Myc [p<0.01], Pax6 and Sox1 [p<0.001]. These results suggest VPA as the best enhancer of pluripotency among the chemicals tested, especially when applied prior to pluripotency induction by Oct4

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 microM), 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 microM) also time-dependently inhibited the CA secretion evoked by DMPP (100 microM, 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 microg/mL), the secretory responses of CA evoked by veratridine (a selective Na+ channel activator (50 microM), Bay-K-8644 (an L-type dihydropyridine Ca2+ channel activator, 10 microM), and cyclopiazonic acid (a cytoplasmic Ca2+-ATPase inhibitor, 10 microM) were significantly reduced, respectively. Interestingly, in the simultaneous presence of Rb2 (10 microM) and L-NAME (an inhibitor of NO synthase, 30 microM), 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 microM) 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.

Comparison of Inhibitory Effects between Enalapril and Losartan on Adrenal Catecholamine Secretion

BACKGROUND: The present study was attempted to compare enalapril, an angiotensin-converting enzyme inhibitor with losartan an angiotensin II (Ang II) receptor blocker in the inhibitory effects on the secretion of catecholamines (CA) from the perfused model of the rat adrenal gland. METHODS: The adrenal gland was isolated and perfused with Krebs-bicarbonate. CA was measured directly by using the fluorospectrophotometer. RESULTS: Both enalapril and losartan during perfusion into an adrenal vein for 90 minutes inhibited the CA release evoked by acetylcholine (ACh), 1.1-dimethyl-4-phenyl piperazinium (DMPP, a selective Nn agonist), high K+ (a direct membrane-depolarizer), 3-(m-chloro-phenyl-carbamoyl-oxy-2-butynyl-trimethyl ammonium (McN-A-343, a selective M1 agonist), and Ang II in a time-dependent manner. Also, in the presence of enalapril or losartan, the CA release evoked by veratridine (an activator of voltage-dependent Na+ channels), 6-dimethyl-3-nitro-4-(2-trifluoromethyl-phenyl)-pyridine-5-carboxylate (BAY-K-8644, an L-type Ca2+ channel activator), and cyclopiazonic acid (a cytoplasmic Ca2+-ATPase inhibitor) were significantly reduced. Based on the same concentration of enalapril and losartan, for the CA release evoked by ACh, high K+, DMPP, McN-A-343, Ang II, veratridine, BAY-K-8644, and cyclopiazonic acid, the following rank order of inhibitory potency was obtained: losartan > enalapril. In the simultaneous presence of enalapril and losartan, ACh-evoked CA secretion was more strongly inhibited compared with that of enalapril- or losartan-treated alone. CONCLUSIONS: Collectively, these results demonstrate that both enalapril and losartan inhibit the CA secretion evoked by activation of both cholinergic and Ang II type-1 receptors stimulation in the perfused 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, the inhibitory effect of losartan on the CA secretion seems to be more potent than that of enalapril.

Antihypertensive and vasorelaxant effects of ethanol extract of stem barks from Zanthoxylum rhoifolium Lam in rats.

Administration of ethanol extract of stem bark from Z. rhoifolium (EEtOH-ZR) induced hypotension associated with a dual effect in heart rate in normotensive rats. This response was highlighted in spontaneously hypertensive rats (SHR). In rat superior mesenteric artery rings, the cumulative addition of EEtOH-ZR (0.1–750 µg/mL) on a phenylephrine-induced pre-contraction (10-5 M) promoted a vasorelaxant effect by a concentration-dependent manner and independent of vascular endothelium. A similar effect was obtained on KCl-induced pre-contractions (80 mM). EEtOH-ZR attenuated contractions induced by cumulative addition of CaCl2 (10-6–3 × 10-2 M) in depolarizing medium without Ca2+ only at 500 or 750 µg/mL. Likewise, on S-(–)-Bay K 8644-induced pre-contractions (10-7 M), the EEtOH-ZR-induced vasorelaxant effect was attenuated. EEtOH-ZR (27, 81, 243 or 500 µg/mL) inhibited contractions induced by cumulative addition of phenylephrine (10-9 - 10-5 M) in endothelium-denuded preparations or by a single concentration (10-5 M) in a Ca2+-free medium. The involvement of K+ channels was evaluated by tetraethylammonium (3 mM); the EEtOH-ZR-induced vasorelaxation was not attenuated. Thus, calcium influx blockade through voltage-operated calcium channels (CaVL) and inhibition of calcium release from intracellular stores are probably underlying EEtOH-ZR-induced cardiovascular effects.

Influence of PD 123319 (AT2-Receptor Antagonist) on Catecholamine Secretion in the Perfused Rat Adrenal Medulla

BACKGROUND: The aim of this study was to examine whether PD 123319 (an angiotensin II type 2 [AT2] receptor antagonist) can influence the release of catecholamines (CA) from the perfused model of the rat adrenal medulla. METHODS: The adrenal gland was isolated by the modification of Wakade method, and perfused with normal Krebs-bicarbonate solution. The content of CA was measured using the fluorospectrophotometer. RESULTS: During perfusion of PD 123319 (range, 5 to 50 nM) into an adrenal vein for 90 minutes the CA secretory responses evoked by acetylcholine (ACh), high K+, 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), and McN-A-343 was dose- and time-dependently inhibited. Furthermore, loading with PD 123319 for 90 minutes also markedly inhibited the CA secretory responses evoked by 4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoro-methyl-phenyl)-pyridine-5-carboxylate (Bay-K-8644), cyclopiazonic acid, veratridine, and angiotensin II (Ang II). PD 123319 did not affect basal CA output. Simultaneous perfusion of PD 123319 and CGP 42112 perfused into an adrenal vein for 90 minutes rather more potently inhibited the CA seretory responses evoked by Ach, high K+, DMPP, Bay-K-8644, veratridine, and Ang II compared to the inhibitory effect by PD123319-treated alone. CONCLUSIONS: Taken together, these results show that PD 123319 inhibits the CA secretion evoked by both cholinergic and Ang II receptor stimulation from the perfused rat adrenal medulla. This inhibitory effect of PD 123319 seems to be exerted by blocking the influx of both Na+ and Ca2+ through their voltage-dependent channels into the rat adrenomedullary chromaffin cells as well as by reducing the Ca2+ release from its cytoplasmic calcium store, which may be relevant to AT2 receptor blockade. Based on these present data, it is thought that PD 123319 has different activity from previously known AT2 antagonist activity in the perfused adrenal medulla, and that AT2 receptors may be involved in the rat adrenomedullary CA secretion.

Influence of Fimasartan (a Novel AT1 Receptor 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 microM) 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 microM) and McN-A-343 (100 microM). Fimasartan failed to affect basal CA output. Furthermore, in adrenal glands loaded with fimasartan (15 microM), the CA secretory responses evoked by Bay-K-8644 (10 microM, an activator of L-type Ca2+ channels), cyclopiazonic acid (10 microM, an inhibitor of cytoplasmic Ca(2+)-ATPase), and veratridine (100 microM, an activator of Na+ channels) as well as by angiotensin II (Ang II, 100 nM), were markedly inhibited. In simultaneous presence of fimasartan (15 microM) and L-NAME (30 microM, 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 microM) alone. Also there was no difference in NO release between before and after treatment with fimasartan (15 microM). 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.

A Novel Pathway Underlying the Inhibitory Effects of Melatonin on Isolated Rat Urinary Bladder Contraction

The aim of the present study was to elucidate the direct effects of melatonin on bladder activity and to determine the mechanisms responsible for the detrusor activity of melatonin in the isolated rat bladder. We evaluated the effects of melatonin on the contractions induced by phenylephrine (PE), acetylcholine (ACh), bethanechol (BCh), KCl, and electrical field stimulation (EFS) in 20 detrusor smooth muscle samples from Sprague-Dawley rats. To determine the mechanisms underlying the inhibitory responses to melatonin, melatonin-pretreated muscle strips were exposed to a calcium channel antagonist (verapamil), three potassium channel blockers [tetraethyl ammonium (TEA), 4-aminopyridine (4-AP), and glibenclamide], a direct voltage-dependent calcium channel opener (Bay K 8644), and a specific calcium/calmodulin-dependent kinase II (CaMKII) inhibitor (KN-93). Melatonin pretreatment (10(-8)~10(-6) M) decreased the contractile responses induced by PE (10(-9)~10(-4) M) and Ach (10(-9)~10(-4) M) in a dose-dependent manner. Melatonin (10(-7) M) also blocked contraction induced by high KCl ([KCl]ECF; 35 mM, 70 mM, 105 mM, and 140 mM) and EFS. Melatonin (10(-7) M) potentiated the relaxation response of the strips by verapamil, but other potassium channel blockers did not change melatonin activity. Melatonin pretreatment significantly decreased contractile responses induced by Bay K 8644 (10(-11)~10(-7) M). KN-93 enhanced melatonin-induced relaxation. The present results suggest that melatonin can inhibit bladder smooth muscle contraction through a voltage-dependent, calcium-antagonistic mechanism and through the inhibition of the calmodulin/CaMKII system.

Electrophysiological effects of hydrogen sulfide on human atrial fibers / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>It has been reported that endogenous or exogenous hydrogen sulfide (H(2)S) exerts physiological effects in the vertebrate cardiovascular system. We have also demonstrated that H(2)S acts as an important regulator of electrophysiological properties in guinea pig papillary muscles and on pacemaker cells in sinoatrial nodes of rabbits. This study was to observe the electrophysiological effects of H(2)S on human atrial fibers.</p><p><b>METHODS</b>Human atrial samples were collected during cardiac surgery. Parameters of action potential in human atrial specialized fibers were recorded using a standard intracellular microelectrode technique.</p><p><b>RESULTS</b>NaHS (H(2)S donor) (50, 100 and 200 µmol/L) decreased the amplitude of action potential (APA), maximal rate of depolarization (V(max)), velocity of diastolic (phase 4) depolarization (VDD) and rate of pacemaker firing (RPF), and shortened the duration of 90% repolarization (APD(90)) in a concentration-dependent manner. ATP-sensitive K(+) (K(ATP)) channel blocker glibenclamide (Gli, 20 µmol/L) partially blocked the effects of NaHS (100 µmol/L) on human atrial fiber cells. The L-type Ca(2+) channel agonist Bay K8644 (0.5 µmol/L) also partially blocked the effects of NaHS (100 µmol/L). An inhibitor of cystathionine γ-lyase (CSE), DL-propargylglycine (PPG, 200 µmol/L), increased APA, V(max), VDD and RPF, and prolonged APD(90).</p><p><b>CONCLUSIONS</b>H(2)S exerts a negative chronotropic action and accelerates the repolarization of human atrial specialized fibers, possibly as a result of increases in potassium efflux through the opening of K(ATP) channels and a concomitant decrease in calcium influx. Endogenous H(2)S may be generated by CSE and act as an important regulator of electrophysiological properties in human atrial fibers.</p>

Protective effect of calcium channel agonist BayK8644 on lungs against ischemia-reperfusion injury in rabbits / 中南大学学报(医学版)

OBJECTIVE@#To explore the protective effect of calcium channel agonist BayK8644 preconditioning on the lungs against ischemia-reperfusion injury (I/R) and its mechanism in rabbits.@*METHODS@#Forty rabbits were randomly divided into 4 groups (10 in each group): a sham-operated group (Sham), an I/R group (I/R), an ischemic preconditioning (IP) group (IP), and a BayK8644 preconditioning group(BayK8644).The wet to dry weight (W/D) ratio, superoxide dismutase (SOD) activity, myleoperoxidase (MPO), and malondialdehyde (MDA) contents of the lung tissues were measured after the operation. Morphological and ultrastructural changes of the lung tissue were observed by light and electron microscope. The expression of pulmonary surfactant-associated protein-A (SP-A) was examined with immunohistochemistry.@*RESULTS@#The W/D ratio, MPO and MDA contents of the lung tissue in the BayK8644 group and IP group were significantly lower than those in the I/R group (P0.05).@*CONCLUSION@#Appropriate BayK8644 preconditioning can induce transient Ca²+ influx, and elicit strong protection against the lung ischemia-reperfusion injury, which can simulate the endogenous protective effect of ischemic preconditioning.

Cardioprotection by kappa-opioid receptor agonist U50488H is mediated by opioidergic regulation but not by calcium current modulation / 대한마취과학회지

BACKGROUND: Because the kappa-opioid receptor (OR) agonist U50488H stimulates opioidergic regulation and inhibits L-type Ca2+ channels, this study was aimed at assessing the roles of OR and L-type Ca2+ channels on U50488H-induced cardioprotection. METHODS: Langendorff-perfused rat hearts were subjected to 30 min of regional ischemia and 2 h of reperfusion. Isolated hearts were treated with U50488H with or without the kappa-OR antagonist nor-binaltorphimine (nor-BNI) or the Ca2+ channels activator BAY K 8644. Infarct size was measured with 2,3,5-triphenyltetrazolium chloride staining. RESULTS: U50488H treatment at reperfusion: (1) significantly reduced infarct size (11.3 +/- 1.3%) compared to control hearts (27.7 +/- 1.1%, P 0.05 vs. U50488H); (2) significantly increased left ventricular developed pressure (65.3 +/- 4.8%) after 2 h of reperfusion compared to control hearts (44.8 +/- 3.6%, P 0.05 vs. control) but not by BAY K 8644 (64.3 +/- 5.6%, P < 0.01 vs. control); and (3) significantly decreased heart rate (P < 0.01 vs. control), an effect that was completely abrogated by both nor-BNI and BAY K 8644. CONCLUSIONS: U50488H significantly limits myocardial infarction and stunning in isolated rat hearts after ischemia-reperfusion induction. The infarct size limitation and contractility improvement observed with U50488H treatment during reperfusion are entirely mediated by OR stimulation and not by Ca2+ channel modulation.

Cardioprotection by kappa-opioid receptor agonist U50488H is mediated by opioidergic regulation but not by calcium current modulation / 대한마취과학회지

BACKGROUND: Because the kappa-opioid receptor (OR) agonist U50488H stimulates opioidergic regulation and inhibits L-type Ca2+ channels, this study was aimed at assessing the roles of OR and L-type Ca2+ channels on U50488H-induced cardioprotection. METHODS: Langendorff-perfused rat hearts were subjected to 30 min of regional ischemia and 2 h of reperfusion. Isolated hearts were treated with U50488H with or without the kappa-OR antagonist nor-binaltorphimine (nor-BNI) or the Ca2+ channels activator BAY K 8644. Infarct size was measured with 2,3,5-triphenyltetrazolium chloride staining. RESULTS: U50488H treatment at reperfusion: (1) significantly reduced infarct size (11.3 +/- 1.3%) compared to control hearts (27.7 +/- 1.1%, P 0.05 vs. U50488H); (2) significantly increased left ventricular developed pressure (65.3 +/- 4.8%) after 2 h of reperfusion compared to control hearts (44.8 +/- 3.6%, P 0.05 vs. control) but not by BAY K 8644 (64.3 +/- 5.6%, P < 0.01 vs. control); and (3) significantly decreased heart rate (P < 0.01 vs. control), an effect that was completely abrogated by both nor-BNI and BAY K 8644. CONCLUSIONS: U50488H significantly limits myocardial infarction and stunning in isolated rat hearts after ischemia-reperfusion induction. The infarct size limitation and contractility improvement observed with U50488H treatment during reperfusion are entirely mediated by OR stimulation and not by Ca2+ channel modulation.

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 (AT1) receptor blocker, can influence the CA release from the isolated perfused model of the rat adrenal medulla. Olmesartan (5~50 micrometer) 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 micrometer) and McN-A-343 (100 micrometer). Olmesartan did not affect basal CA secretion. Also, in adrenal glands loaded with olmesartan (15 micrometer), the CA secretory responses evoked by Bay-K-8644 (10 micrometer, an activator of voltage-dependent L-type Ca2+ channels), cyclopiazonic acid (10 micrometer, an inhibitor of cytoplasmic Ca2+ -ATPase), veratridine (100 micrometer, an activator of voltage-dependent Na+ channels), and Ang II (100 nM) were markedly inhibited. However, at high concentrations (150~300 micrometer), 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 Ca2+ into the rat adrenomedullary chromaffin cells as well as by inhibiting the Ca2+ release from the cytoplasmic calcium store, which is thought to be relevant to the AT1 receptor blockade, in addition to its enhancement on the CA secreton.

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 microgram/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 NN receptor agonist, 100 micrometer) and McN-A-343 (a selective muscarinic M1 receptor agonist, 100 micrometer). Provinol itself did not affect basal CA secretion. Also, in the presence of provinol (1 microgram/ml), the secretory responses of CA evoked by Bay-K-8644 (a voltage-dependent L-type dihydropyridine Ca2+ channel activator, 10 microgram), cyclopiazonic acid (a cytoplasmic Ca2+-ATPase inhibitor, 10 microgram) and veratridine (an activator of voltage-dependent Na+ channels, 10 microgram) were significantly reduced. Interestingly, in the simultaneous presence of provinol (1 microgram/ml) plus L-NAME (a selective inhibitor of NO synthase, 30 micrometer), 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 microgram/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 Ca2+ release from the cytoplasmic calcium store at least partly through the increased NO production due to the activation of nitric oxide synthase.

Effects of Losartan on Catecholamine Release in the Isolated Rat Adrenal Gland

The aim of this study was to determine whether losartan, an angiotensin II (Ang II) type 1 (AT1) receptor could influence the CA release from the isolated perfused model of the rat adrenal medulla. Losartan (5~50 micrometer) 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 micrometer) and McN-A-343 (100 micrometer). Losartan failed to affect basal CA output. Furthermore, in adrenal glands loaded with losartan (15 micrometer) for 90 min, the CA secretory responses evoked by Bay-K-8644 (10 micrometer, an activator of L-type Ca2+ channels), cyclopiazonic acid (10 micrometer, an inhibitor of cytoplasmic Ca2+-ATPase), veratridine (100 micrometer, an activator of Na+ channels), and Ang II (100 nM) were markedly inhibited. However, at high concentrations (150~300 micrometer), losartan rather enhanced the CA secretion evoked by ACh. Collectively, these experimental results suggest that losartan at low concentrations inhibits the CA secretion evoked by cholinergic stimulation (both nicotininc and muscarinic receptors) as well as by membrane depolarization from the rat adrenal medulla, but at high concentration it rather inhibits ACh-evoked CA secretion. It seems that losartan has a dual action, acting as both agonist and antagonist to nicotinic receptors of the rat adrenal medulla, which might be dependent on the concentration. It is also thought that this inhibitory effect of losartan may be mediated by blocking the influx of both Na+ and Ca2+ into the rat adrenomedullary chromaffin cells as well as by inhibiting the Ca2+ release from the cytoplasmic calcium store, which is thought to be relevant to the AT1 receptor blockade, in addition to its enhancement of the CA release.

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

The present study was attempted to investigate whether polyphenolic compounds isolated from wine, which is brewed from Rubus coreanum Miquel (PCRC), may affect the release of catecholamines (CA) from the isolated perfused adrenal medulla of the spontaneously hypertensive rats (SHRs), and to establish its mechanism of action. PCRC (20~180 microgram/ml) perfused into an adrenal vein for 90 min relatively dose-dependently inhibited the CA secretory responses to ACh (5.32 mM), high K+ (56 mM), DMPP (100 micrometer) and McN-A-343 (100 micrometer). PCRC itself did not affect basal CA secretion (data not shown). Also, in the presence of PCRC (60 microgram/ml), the CA secretory responses to veratridine (a selective Na+ channel activator (10 micrometer), Bay-K-8644 (a L-type dihydropyridine Ca2+ channel activator, 10 micrometer), and cyclopiazonic acid (a cytoplasmic Ca2+ -ATPase inhibitor, 10 micrometer) were significantly reduced, respectively. In the simultaneous presence of PCRC (60 microgram/ml) and L-NAME (an inhibitor of NO synthase, 30 micrometer), the inhibitory responses of PCRC on the CA secretion evoked by ACh, high K+, DMPP, and Bay-K-8644 were considerably recovered to the extent of the corresponding control secretion compared with that of PCRC-treatment alone. The level of NO released from adrenal medulla after the treatment of PCRC (60 microgram/ml) was greatly elevated compared with the corresponding basal level. Taken together, these results demonstrate that PCRC inhibits the CA secretion from the isolated perfused adrenal medulla of the SHRs evoked by stimulation of cholinergic receptors as well as by direct membrane-depolarization. It seems that this inhibitory effect of PCRC is mediated by blocking the influx of calcium and sodium into the adrenal medullary chromaffin cells of the SHRs as well as by inhibition of Ca2+ release from the cytoplasmic calcium store at least partly through the increased NO production due to the activation of NO synthase.

Ginkgolide B inhibits carotid sinus baroreflex in anesthetized male rats / 生理学报

The effects of ginkgolide B on the carotid sinus baroreflex (CSB) were studied in the perfused isolated carotid sinus of 30 anesthetized Sprague-Dawley male rats. The results were as follows. (1) By perfusing with ginkgolide B (0.1, 1, 10 μmol/L), the functional curve of the baroreflex was shifted to the right and upward. There was a marked decrease in peak slope (PS) and reflex decrease (RD) in mean arterial pressure (P<0.01), while the threshold pressure (TP), equilibrium pressure (EP) and saturation pressure (SP) were significantly increased (P<0.05, P<0.01). Among the functional parameters of CSB, the changes in PS, RD, TP, EP and SP were dose-dependent. (2) Pretreatment with Bay K8644 (500 nmol/L), an agonist of L-type calcium channel, completely eliminated the effects of ginkgolide B (1 μmol/L) on the CSB. (3) Pretreatment with tetraethylammonium (TEA, 1 mmol/L), an inhibitor of potassium channel, completely abolished the above effects of ginkgolide B (1 μmol/L) on the CSB. These results suggest that ginkgolide B inhibits the CSB in anesthetized rats, which is mediated by decreased calcium influx and increased potassium efflux in baroreceptor nerve endings.

Resveratrol inhibits electrical activity of paraventricular nucleus neurons in rat hypothalamic slices / 生理学报

To study the role of resveratrol in the discharges of neurons in paraventricular nucleus (PVN) in hypothalamic slices, extracellular single-unit discharge recording technique was used. The effects of resveratrol were examined with glass microelectrodes in the rat PVN neurons at resting potential level. The results were as follows: (1) In response to the application of resveratrol (0.05, 0.5, 5.0 μmol/L, n=29) to the superfusate for 2 min, the spontaneous discharge rate (SDR) of neurons in 28/29 (96.6%) hypothalamic slices significantly decreased in a dose-dependent manner; (2) Pretreatment with L-glutamate (0.2 mmol/L) led to a marked increase in the SDR in all 8/8 (100%) slices in an epileptiform pattern. The increased discharges were suppressed by the application of resveratrol (5.0 mmol/L) in all 8 slices; (3) In 8 slices, perfusion of the selective L-type calcium channel agonist, Bay K8644 (0.1 μmol/L), induced a significant increase in the discharge rate in 8/8 (100%) slices. Resveratrol (5.0 μmol/L) significantly attenuated the increased SDR in all 8 slices; (4) Pretreatment with the nitric oxide synthase (NOS) inhibitor N(ω)-nitro-L-arginine methyl ester (L-NAME, 50 μmol/L) increased SDR in 7/8 (87.5%) slices, but did not affect the inhibitory effect of resveratrol (5.0 μmol/L). These results suggest that resveratrol inhibits the electrical activity of PVN neurons and exerts neuroprotective actions on central neurons. The inhibitory effect of resveratrol is possibly related to the blockade of L-type calcium channel, but not due to NO release.

Influence of Ketamine on Catecholamine Secretion in the Perfused Rat Adrenal Medulla

The aim of the present study was to examine the effects of ketamine, a dissociative anesthetics, on secretion of catecholamines (CA) secretion evoked by cholinergic stimulation from the perfused model of the isolated rat adrenal gland, and to establish its mechanism of action, and to compare ketamine effect with that of thiopental sodium, which is one of intravenous barbiturate anesthetics. Ketamine (30~300 micrometer), perfused into an adrenal vein for 60 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 NN receptor agonist, 100 micrometer) and McN-A-343 (a selective muscarinic M1 receptor agonist, 100 micrometer). Also, in the presence of ketamine (100 micrometer), the CA secretory responses evoked by veratridine (a voltage-dependent Na+ channel activator, 100 micrometer), Bay-K-8644 (an L-type dihydropyridine Ca2+ channel activator, 10 micrometer), and cyclopiazonic acid (a cytoplasmic Ca2+-ATPase inhibitor, 10 micrometer) were significantly reduced, respectively. Interestingly, thiopental sodium (100 micrometer) also caused the inhibitory effects on the CA secretory responses evoked by ACh, high K+, DMPP, McN-A-343, veratridine, Bay-K-8644, and cyclopiazonic acid. Collectively, these experimental results demonstrate that ketamine inhibits the CA secretion evoked by stimulation of cholinergic (both nicotinic and muscarinic) receptors and the membrane depolarization from the isolated perfused rat adrenal gland. It seems likely that the inhibitory effect of ketamine is mediated by blocking the influx of both Ca2+ and Na+ through voltage-dependent Ca2+ and Na+ channels into the rat adrenal medullary chromaffin cells as well as by inhibiting Ca2+ release from the cytoplasmic calcium store, which are relevant to the blockade of cholinergic receptors. It is also thought that, on the basis of concentrations, ketamine causes similar inhibitory effect with thiopental in the CA secretion from the perfused rat adrenal medulla.

Resveratrol Inhibits Nicotinic Stimulation-Evoked Catecholamine Release from the Adrenal Medulla

Resveratrol has been known to possess various potent cardiovascular effects in animal, but there is little information on its functional effect on the secretion of catecholamines (CA) from the perfused model of the adrenal medulla. Therefore, the aim of the present study was to determine the effect of resveratrol on the CA secretion from the isolated perfused model of the normotensive rat adrenal gland, and to elucidate its mechanism of action. Resveratrol (10~100micrometer) during perfusion into an adrenal vein for 90 min inhibited the CA secretory responses evoked by ACh (5.32 mM), high K+ (a direct membrane-depolarizer, 56 mM), DMPP (a selective neuronal nicotinic Nn receptor agonist, 100micrometer) and McN-A-343 (a selective muscarinic M1 receptor agonist, 100micrometer) in both a time- and dose- dependent fashion. Also, in the presence of resveratrol (30micrometer), the secretory responses of CA evoked by veratridine 8644 (an activator of voltage-dependent Na+ channels, 100micrometer), Bay-K-8644 (a L-type dihydropyridine Ca2+ channel activator, 10micrometer), and cyclopiazonic acid (a cytoplasmic Ca2+ -ATPase inhibitor, 10micrometer) were significantly reduced. In the simultaneous presence of resveratrol (30micrometer) and L-NAME (an inhibitor of NO synthase, 30micrometer), the CA secretory evoked by ACh, high K+, DMPP, McN-A-343, Bay-K-8644 and cyclopiazonic acid were recovered to a considerable extent of the corresponding control secretion compared with the inhibitory effect of resveratrol alone. Interestingly, the amount of nitric oxide (NO) released from the adrenal medulla was greatly increased in comparison to its basal release. Taken together, these experimental results demonstrate that resveratrol can inhibit the CA secretory responses evoked by stimulation of cholinergic nicotinic receptors, as well as by direct membrane-depolarization in the isolated perfused model of the rat adrenal gland. It seems that this inhibitory effect of resveratrol is exerted by inhibiting an influx of both ions through Na+ and Ca2+ channels into the adrenomedullary cells as well as by blocking the release of Ca2+ from the cytoplasmic calcium store, which are mediated at least partly by the increased NO production due to the activation of NO synthase.