Cotinine inhibits catecholamine release evoked by cholinergic stimulation from the rat adrenal medulla

The aim of the present study was to clarify whether cotinine affects the release of catecholamines (CA) from the isolated perfused rat adrenal gland, and to establish the mechanism of its action, in comparison with the response of nicotine. Cotinine (0.3-3 mM), when perfused into an adrenal vein for 60 min, inhibited CA secretory responses evoked by ACh (5.32 mM), DMPP (a selective neuronal nicotinic agonist, 100 microM for 2 min) and McN-A-343 (a selective muscarinic M1-agonist, 100 microM for 2 min) in dose- and time-dependent manners. However, cotinine did not affect CA secretion by high K+ (56 mM). Cotinine itself also failed to affect basal CA output. Furthermore, in the presence of cotinine (1 mM), CA secretory responses evoked by Bay-K-8644 (an activator of L-type Ca2+ channels, 10 microM) and cyclopiazonic acid (an inhibitor of cytoplasmic Ca2+-ATPase, 10 microM) were relative time-dependently attenuated. However, nicotine (30 microM), given into the adrenal gland for 60 min, initially rather enhanced CA secretory responses evoked by ACh and high K+, followed by the inhibition later, while it time-dependently depressed the CA release evoked by McN-A-343 and DMPP. Taken together, these results suggest that cotinine inhibits greatly CA secretion evoked by stimulation of cholinergic (both nicotinic and muscarinic) receptors, but does fail to affect that by the direct membrane-depolarization. It seems that this inhibitory effect of cotinine may be exerted by the cholinergic blockade, which is associated with blocking both the calcium influx into the rat adrenal medullary chromaffin cells and Ca2+ release from the cytoplasmic calcium store. It also seems that there is a big difference in the mode of action between cotinine and nicotine in the rat adrenomedullary CA secretion.     

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 µg/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 µM) and McN-A-343 (a selective muscarinic M₁ receptor agonist, 100 µM). Provinol itself did not affect basal CA secretion. Also, in the presence of provinol (1 µg/ml), the secretory responses of CA evoked by Bay-K-8644 (a voltage-dependent L-type dihydropyridine Ca²⁺ channel activator, 10 µM), cyclopiazonic acid (a cytoplasmic Ca²⁺-ATPase inhibitor, 10 µM) and veratridine (an activator of voltage-dependent Na⁺ channels, 10 µM) were significantly reduced. Interestingly, in the simultaneous presence of provinol (1 µg/ml) plus L-NAME (a selective inhibitor of NO synthase, 30 µM), 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 µg/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.     

Arecoline inhibits catecholamine release from perfused rat adrenal gland

AIM: To study the effect of arecoline, an alkaloid isolated from Areca catechu, on the secretion of catecholamines (CA) evoked by cholinergic agonists and the membrane depolarizer from isolated perfused rat adrenal gland. METHODS: Adrenal glands were isolated from male Sprague-Dawley rats. The adrenal glands were perfused with Krebs bicarbonate solution by means of a peristaltic pump. The CA content of the perfusate was measured directly using the fluorometric method. RESULTS: Arecoline (0.1-1.0 mmol/L) perfused into an adrenal vein for 60 min produced dose- and time-dependent inhibition in CA secretory responses evoked by acetylcholine (ACh) (5.32 mmol/L), 1.1-dimethyl-4-phenyl piperazinium iodide (DMPP) (100 micromol/L for 2 min) and 3-(m-choloro-phenyl-carbamoyl-oxy)-2-butynyl trimethyl ammonium chloride (McN-A-343) (100 micromol/L for 2 min). However, lower doses of arecoline did not affect CA secretion of high K(+) (56 mmol/L); higher doses greatly reduced CA secretion of high K(+). Arecoline also failed to affect basal catecholamine output. Furthermore, in adrenal glands loaded with arecoline (0.3 mmol/L), CA secretory response evoked by Bay-K-8644 (10 micromol/L), an activator of L-type Ca(2+) channels, was markedly inhibited, whereas CA secretion by cyclopiazonic acid (10 micromol/L), an inhibitor of cytoplasmic Ca(2+)-ATPase, was not affected. Nicotine (30 micromol/L), which was perfused into the adrenal gland for 60 min, however, initially enhanced ACh-evoked CA secretory responses. As time elapsed, these responses became more inhibited, whereas the initially enhanced high K(+)-evoked CA release diminished. CA secretion evoked by DMPP and McN-A-343 was significantly depressed in the presence of nicotine. CONCLUSION: Arecoline dose-dependently inhibits CA secretion from isolated perfused rat adrenal gland evoked by activation of cholinergic receptors. At lower doses arecoline does not inhibit CA secretion through membrane depolarization, but at larger doses it does. This inhibitory effect of arecoline may be mediated by blocking the calcium influx into the rat adrenal medullary chromaffin cells without the inhibition of Ca(2+) release from the cytoplasmic calcium store. There seems to be a difference in the mode of action of nicotine and arecoline in rat adrenomedullary CA secretion.     

Influence of polyphenolic compounds isolated from<Emphasis Type="Italic">Rubus coreanum</Emphasis> on catecholamine release in the rat adrenal medulla

The aim of the present study was to investigate whether polyphenolic compounds isolated from wine brewed fromRubus coreanum MIQUEL (PCRC) may affect the release of catecholamine (CA) from the isolated perfused rat adrenal medulla, and to establish its mechanism of action. PCRC (20<;180 μg/mL) perfused into an adrenal vein for 90 min dose- and time-dependently inhibited the CA secretory responses evoked by acetylcholine (ACh, 5.32 mM), high K+ (a direct membrane-depolarizer, 56 mM), DMPP (a selective neuronal nicotinic N_n receptor agonist, 100 μM) and McN-A-343 (a selective muscarinic M₁ receptor agonist, 100 μM). Also, in the presence of PCRC (60 μg/mL), the secretory responses of CA evoked by 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 PCRC (60 μg/mL) and L-NAME (an inhibitor of NO synthase, 30 μM), 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 the inhibitory effect of PCRC alone. Taken together, these results obtained from the present study demonstrate that PCRC inhibits the CA secretory responses from the isolated perfused adrenal gland of the normotensive rats evoked by stimulation of cholinergic (both muscarinic and nicotinic) receptors as well as by direct membrane-depolarization. It seems that this inhibitory effect of PCRC is exerted by inhibiting both the calcium influx into the rat adrenal medullary chromaffin cells and the uptake of Ca²⁺ into the cytoplasmic calcium store partly through the increased NO production due to the activation of nitric oxide synthase (NOS), which are at least relevant to the direct interaction with the nicotinic receptor itself. It is also thought that PCRC might be effective in prevention of cardiovascular disease. This paper was presented at the 21^st Scientific Meeting of the International Society of Hypertension (ISH), Fukuoka, Japan, October 15–19, 2006.     

α₂-Adrenoceptor-mediated inhibition of catecholamine release from the adrenal medulla of spontaneously hypertensive rats is preserved in the early stages of hypertension

In this study, we evaluated the effect of α(2) -adrenoceptor activation on catecholamine release from the adrenal medulla of pre-hypertensive (6-week-old) and hypertensive (16-week-old) spontaneously hypertensive rats (SHR) and of age-matched normotensive control Wistar Kyoto (WKY) rats. Catecholamine overflow from isolated adrenal medullae was evoked by the nicotinic receptor agonist 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP) in the absence and presence of the α(2) -adrenoceptor agonist medetomidine (MED). The spontaneous outflow of adrenaline was similar between age-matched SHR and WKY rats. However, the spontaneous outflow of noradrenaline was significantly lower in SHR compared with age-matched WKY rats. DMPP (0.1-3 mM) increased the outflow of noradrenaline and adrenaline in a concentration-dependent manner. The E(max) values for adrenaline overflow were similar between strains, but the E(max) values for noradrenaline overflow were significantly lower in SHR. The EC(50) values for noradrenaline and adrenaline overflow were significantly higher in SHR compared with age-matched WKY rats. MED (0.1-300 nM) reduced the DMPP-evoked overflow (DMPP 500 μM) of noradrenaline and adrenaline in a concentration-dependent manner and was capable of totally inhibiting this effect. The inhibitory action of MED was similar between age-matched SHR and WKY rats. In the adrenals, the α(2A)- and α(2B)-adrenoceptor subtypes had the highest mRNA expression levels; the α(2C)-adrenoceptor subtype had the lowest mRNA expression levels. The mRNA levels for the three subtypes were similar between strains. In conclusion, in SHR during the development of hypertension, adrenal α(2) -adrenoceptor inhibitory function is conserved, accompanied by reduced noradrenaline release and unchanged adrenaline release.     

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 (10~100µM) 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 N_n receptor agonist, 100µM) and McN-A-343 (a selective muscarinic M₁ receptor agonist, 100µM) in both a time- and dose-dependent fashion. Also, in the presence of resveratrol (30µM), the secretory responses of CA evoked by veratridine 8644 (an activator of voltage-dependent Na⁺ channels, 100µM), Bay-K-8644 (a L-type dihydropyridine Ca²⁺ channel activator, 10µM), and cyclopiazonic acid (a cytoplasmic Ca²⁺-ATPase inhibitor, 10µM) were significantly reduced. In the simultaneous presence of resveratrol (30µM) and L-NAME (an inhibitor of NO synthase, 30µM), 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.     

Influence of naloxone on catecholamine release evoked by nicotinic receptor stimulation in the isolated rat adrenal gland

The present study was designed to investigate the effect of naloxone, a well known opioid antagonist, on the secretion of catecholamines (CA) evoked by cholinergic stimulation and membrane-depolarization in the isolated perfused rat adrenal glands, and to establish its mechanism of action. Naloxone (10(-6) approximately 10(-5) M), perfused into an adrenal vein for 60 min, produced dose- and time-dependent inhibition of CA secretory responses evoked by ACh (5.32 x 10(-3) M), high K+ (5.6 x 10(-2) M), DMPP (10(-4) M) and McN-A-343 (10(-4) M). Naloxone itself also failed to affect the basal CA output. In adrenal glands loaded with naloxone (3 x 10(-6) M), the CA secretory responses evoked by Bay-K-8644, an activator of L-type Ca2+ channels, and cyclopiazonic acid, an inhibitor of cytoplasmic Ca(2+)-ATPase, were also inhibited. In the presence of met-enkephalin (5 x 10(-6) M), a well known opioid agonist, the CA secretory responses evoked by ACh, high K+, DMPP, McN-A-343, Bay-K-8644 and cyclopiazonic acid were also significantly inhibited. Taken together, these results suggest that naloxone greatly inhibits the CA secretion evoked by stimulation of cholinergic (both nicotinic and muscarinic) receptors as well as that by membrane depolarization. It seems that these inhibitory effects of naloxone does not involve opioid receptors, but might be mediated by blocking both the calcium influx into the rat adrenal medullary chromaffin cells and the uptake of Ca2+ into the cytoplasmic calcium store, which are at least partly relevant to the direct interaction with the nicotinic receptor itself.     

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.     

Comparison of green tea extract and epigallocatechin gallate on secretion of catecholamines from the rabbit adrenal medulla

The present study was designed to examine the effects of green tea extract (CUMC6335) and epigallocatechin gallate (EGCG) on secretion of catecholamines (CA) in the isolated perfused rabbit adrenal gland. In the presence of CUMC6335 (200 microg/mL) into an adrenal vein for 60 min, CA secretory responses evoked by ACh (5.32 mM), high K+ (56 mM), DMPP (100 microM for 2 min), and Bay-K-8644 (10 microM for 4 min) from the isolated perfused rabbit adrenal glands were greatly inhibited in a time-dependent fashion. However, EGCG (10 microg/mL) did not affect CA release evoked by ACh, high K+, and Bay-K-8644. CUMC6335 itself failed to affect basal catecholamine output. Taken together, these results demonstrate that CUMC6335 inhibits CA secretion evoked by stimulation of cholinergic nicotinic receptors, as well as the direct membrane depolarization from the isolated perfused rabbit adrenal gland. It is thought that this inhibitory effect of CUMC6335 may be due at least in part to the blocking action of the L-type dihydropyridine calcium channels in the rabbit adrenomedullary chromaffin cells, which is relevant to the cholinergic nicotinic blockade. It seems that there is a big difference in mode of action between CUMC6335 and EGCG.     

Sodium-dependent inhibition by PN200-110 enantiomers of nicotinic adrenal catecholamine release.

1. Dimethylphenylpiperazinium (DMPP) or high K concentrations evoke catecholamine release from perfused cat adrenal glands; in both cases the secretory response was significantly enhanced in the absence of Na. Tetrodotoxin did not modify the nicotinic secretory response. 2. The (+)- and (-)-enantiomers of the dihydropyridine Ca channel blocker PN200-110 show a high degree of stereoselectivity in the inhibition of catecholamine secretion evoked by high K or by DMPP in the presence of Na, the (+)-enantiomer being 57 and 80 times more potent, respectively, than the (-)-enantiomer. Both, noradrenaline and adrenaline release were equally depressed by PN200-110. 3. The IC50 values for (+)- and (-)-PN200-110 for blockade of the secretory response induced by K or DMPP in the presence of Na are in the same range. In the absence of Na, (-)-PN200-110 did not affect DMPP-evoked secretion; however, the (+)-enantiomer partially inhibited it. 4. The results suggest that the physiological catecholamine release from chromaffin cells is preceded by Na entry through the nicotinic receptor-associated ionophore; this causes cell depolarization, opening of voltage-dependent, dihydropyridine-sensitive Ca channels and Ca entry into the cell. In the absence of Na, additional Ca influx through an alternative pathway (the nicotinic cholinoceptor ionophore?) might also activate secretion.     

DMPP-evoked increases in postganglionic sympathetic nerve activity and blood pressure occurs by two mechanisms in the rat

1 Intravenous administration of the ganglionic nicotinic receptor agonist DMPP (1,1-dimethyl-4-phenylpiperazinium iodide) into urethane-anaesthetized rats evoked dose-dependent increases in mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA). 2 The ganglionic nicotinic receptor antagonists pentolinium and hexamethonium either alone or combined did not inhibit the increase in RSNA and MAP evoked by 50 to 200 μg kg^?1 doses of DMPP. The increase in renal sympathetic nerve activity evoked by DMPP occurred as a brief burst in firing. 3 The increase in MAP, but not RSNA, evoked by DMPP in the presence of pentolinium was inhibited by the selective α₁-adrenergic receptor antagonist prazosin. 4 The non-selective α-adrenoceptor and NPY receptor antagonist benextramine also inhibited the increase in MAP without inhibiting the increase in RSNA. Surprisingly, the combination of benextramine and pentolinium, or benextramine and hexamethonium, completely blocked the DMPP-evoked increase in RSNA and thus the increase in MAP. 5 The uptake₁ antagonist desipramine combined with pentolinium did not affect the DMPP-evoked increases in MAP or RSNA when compared to the responses evoked in the presence of pentolinium alone. 6 Adding the selective M₁ muscarinic receptor antagonist telenzepine to pentolinium and prazosin did not inhibit the increase in RSNA evoked by a 100 μg kg^?1 dose of DMPP. 7 While the DMPP-evoked increase in MAP in the presence of ganglionic nicotinic receptor antagonists is primarily dependent upon activation of α₁-adrenoceptors, the increase in RSNA occurs via activation of ganglionic nicotinic receptors and activation of a mechanism susceptible to blockade by benextramine.     

Inhibitory mechanism of pinacidil on catecholamine secretion from the rat perfused adrenal gland evoked by cholinergic stimulation and membrane depolarization

1. The present study attempted to investigate the effect of potassium channel openers on secretion of catecholamines (CA) evoked by cholinergic stimulation and membrane depolarization from rat isolated perfused adrenal gland. 2. The perfusion of pinacidil (30-300 microM) into an adrenal vein for 20 min produced dose-dependent inhibition of CA secretion evoked by acetylcholine (ACh; 5.32 mM), high K+ (56 mM), 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP; 100 microM for 2 min), 3-(m-chloro-phenyl-carbamoyl-oxy)-2-butynyl trimethyl ammonium chloride (McN-A-343; 100 microM for 2 min), cyclopiazonic acid (CPA; 10 microM for 4 min) and methyl-1,4-dihydro-2,6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyri dine-5-carboxylate (Bay-K-8644; 10 microM for 4 min). 3. In the presence of minoxidil (100 microM), which is also known to be a potassium channel activator, CA secretory responses evoked by ACh, high potassium, DMPP, McN-A-343, Bay-K-8644 and CPA were also significantly depressed. 4. In adrenal glands preloaded with pinacidil (100 microM) in the presence of glibenclamide (GB; 1 microM), a specific blocker of ATP-regulated potassium channels, CA secretory responses evoked by ACh, high potassium, DMPP, McN-A-343, Bay-K-8644 and CPA were restored to a considerable extent of the control release as compared with that of pinacidil only. 5. These results suggest that pinacidil causes marked inhibition of CA secretion evoked by stimulation of cholinergic (both nicotinic and muscarinic) receptors, as well as by membrane depolarization, indicating that this effect may be mediated by inhibiting influx of extracellular calcium and release of intracellular calcium in the rat adrenomedullary chromaffin cells. Furthermore, these findings suggest that these potassium channel opener-sensitive membrane potassium channels also play a modulatory role in regulating CA secretion.     

Inhibitory mechanism of bromocriptine on catecholamine release evoked by cholinergic stimulation and membrane depolarization from the rat adrenal medulla

The purpose of this study was to determine whether bromocriptine affects the catecholamines (CA) secretion evoked in isolated perfused rat adrenal glands, by cholinergic stimulation, membrane depolarization and calcium mobilization, and to establish the mechanism of its action. The perfusion of bromocriptine (1-10 microM) into an adrenal vein, for 60 min, produced relatively dose-dependent inhibition in the secretion of catecholamines (CA) evoked by acetylcholine (ACh, 5.32 mM), DMPP (100 microM for 2 min), McN-A-343 (100 microM for 2 min), cyclopiazonic acid (CPA, 10 microM for 4 min) and Bay-K-8644 (10 microM for 4 min). High K+ (56 mM)-evoked CA release was also inhibited, although not in a dose-dependent fashion. Also, in the presence of apomorphine (100 microM), which is also known to be a selective D2-agonist, the CA secretory responses evoked by ACh, high potassium, DMPP, McN-A-343, Bay-K-8644 and cyclopiazonic acid were also significantly depressed. However, in adrenal glands preloaded with bromocriptine (3 microM) in the presence of metoclopramide (15 microM), a selective D2-antagonist, the CA secretory responses evoked by ACh, high potassium, DMPP, McN-A-343, Bay-K-8644 and cyclopiazonic acid considerably recovered as compared to that of bromocriptine only. Taken together, these results suggest that bromocriptine can inhibit the CA secretion evoked by stimulation of cholinergic receptors, as well as by membrane depolarization, in the perfused rat adrenal medulla. It is thought this inhibitory effect of bromocriptine may be mediated by inhibiting the influx of extracellular calcium and the release from intracellular calcium stores, through the activation of dopaminergic D2-receptors located in the rat adrenomedullary chromaffin cells. Furthermore, these findings also suggest that the dopaminergic D2-receptors may play an important role in regulating adrenomedullary CA secretion.     

Inhibition and facilitation by pimobendan,a calcium sensitizer,of catecholamine secretion from bovine adrenal chromaffin cells

The effects of pimobendan, a Ca(2+) sensitizer with inhibitory action against cyclic-GMP-inhibited phosphodiesterase (PDE-III), on catecholamine (CA) secretion were studied in bovine adrenal chromaffin cells. In intact cells, pimobendan (10 - 100 microM) inhibited CA secretion stimulated by acetylcholine (10 and 30 microM) and 1,1-dimethyl-4-phenyl-piperazinium (DMPP) (3 and 10 microM), but facilitated CA secretion stimulated by high K(+) (30 mM), histamine (3 microM), and angiotensin-II (3 microM). Histamine and angiotensin-II had no effect on CA secretion in Ca(2+)-free medium. The inhibition or facilitation by pimobendan of the stimulation-evoked CA secretion was not affected by H-89 (1 microM) and H-8 (30 microM), inhibitors of cyclic-AMP-dependent protein kinase. Milrinone (10 and 30 microM) and amrinone (100 and 300 microM), inhibitors of PDE-III, did not affect the stimulation-evoked CA secretion. In beta-escin-permeabilized cells, pimobendan (10 - 100 microM) did not affect CA secretion stimulated by Ca(2+) (0.1 - 10 microM) in the presence and absence of MgATP (2 mM). These results indicate that pimobendan has dual effects, inhibition and facilitation, on CA secretion. The inhibition may be due to an inhibitory action on nicotinic receptors and the facilitation may be due to a facilitatory action on stimulation-induced Ca(2+) influx. Neither Ca(2+) sensitizing nor PDE-III inhibiting actions seem to be related to these effects.     

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 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 Ca²⁺ channels), cyclopiazonic acid (10 µM, an inhibitor of cytoplasmic Ca²⁺ -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 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.     

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 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). Losartan failed to affect basal CA output. Furthermore, in adrenal glands loaded with losartan (15 µM) for 90 min, 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), veratridine (100 µM, an activator of Na⁺ channels), and Ang II (100 nM) were markedly inhibited. However, at high concentrations (150~300 µM), 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.     

Comparison of catecholamine release in the isolated adrenal glands of SHR and WKY rats

1 The present study was designed to investigate the secretion of catecholamines (CA) evoked by stimulation of cholinergic receptors and membrane depolarization from the isolated perfused adrenal gland of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKYR) at adult age. 2 The wet weight of adrenal gland in SHR was greater than that in WKYR. The CA releasing responses evoked by acetylcholine (5.32 x 10-3 m), and high potassium (5.6 x 10-2 m), a membrane depolarizer, were significantly lower in WKYR than in SHR. 3 The secretory responses of CA evoked by DMPP (10-4 m for 2 min), a selective agonist of neuronal nicotinic receptors, and McN-A-343 (10-4 m for 2 min), a selective agonist of neuronal muscarinic receptors, were also significantly lower in WKYR than in SHR. 4 The CA release evoked by Bay-K-8644 (10-5 m), a dihydropyridine-sensitive Ca2+ channel activator, and cyclopiazonic acid (10-5 m), a selective inhibitor of Ca2+-ATPase in the endoplasmic reticulum, were also significantly greater in SHR than WKYR. 5 Taken together, these experimental results demonstrate that the CA secretion evoked by stimulation of cholinergic (nicotinic and muscarinic) receptors as well as membrane depolarization is enhanced more greatly in the perfused adrenal glands of SHR than in those of WKYR. It is suggested that the augmented CA release in SHR compared with WKYR was involved in essential hypertensive pathogenesis.     

Stimulation of the hypothalamo-pituitary-adrenal axis in the rat by the type 4 phosphodiesterase (PDE-4) inhibitor, denbufylline.

1. Otilonium, a clinically useful spasmolytic, behaves as a potent blocker of neuronal nicotinic acetylcholine receptors (AChR) as well as a mild wide-spectrum Ca2+ channel blocker in bovine adrenal chromaffin cells. 2. 45Ca2+ uptake into chromaffin cells stimulated with high K+ (70 mM, 1 min) was blocked by otilonium with an IC50 of 7.6 microM. The drug inhibited the 45Ca2+ uptake stimulated by the nicotinic AChR agonist, dimethylphenylpiperazinium (DMPP) with a 79 fold higher potency (IC50 = 0.096 microM). 3. Whole-cell Ba2+ currents (IBa) through Ca2+ channels of voltage-clamped chromaffin cells were blocked by otilonium with an IC50 of 6.4 microM, very close to that of K(+)-evoked 45Ca2+ uptake. Blockade developed in 10-20 s, almost as a single step and was rapidly and almost fully reversible. 4. Whole-cell nicotinic AChR-mediated currents (250 ms pulses of 100 microM DMPP) applied at 30 s intervals were blocked by otilonium in a concentration-dependent manner, showing an IC50 of 0.36 microM. Blockade was induced in a step-wise manner. Wash out of otilonium allowed a slow recovery of the current, also in discrete steps. 5. In experiments with recordings in the same cells of whole-cell IDMPP, Na+ currents (INa) and Ca2+ currents (ICa), 1 microM otilonium blocked 87% IDMPP, 7% INa and 13% ICa. 6. Otilonium inhibited the K(+)-evoked catecholamine secretory response of superfused bovine chromaffin cells with an IC50 of 10 microM, very close to the IC50 for blockade of K(+)-induced 45Ca2+ uptake and IBa. 7. Otilonium inhibited the secretory responses induced by 10 s pulses of 50 microM DMPP with an IC50 of 7.4 nM. Hexamethonium blocked the DMPP-evoked responses with an IC50 of 29.8 microM, 4,000 fold higher than that of otilonium. 8. In conclusion, otilonium is a potent blocker of nicotinic AChR-mediated responses. The drugs also blocked various subtypes of neuronal voltage-dependent Ca2+ channels at a considerably lower potency. Na+ channels were unaffected by otilonium. This extraordinary potency of otilonium in blocking nicotinic AChR, unrecognised until now, might account in part for its well known spasmolytic effects.     

Influence of quinine on catecholamine release evoked by cholinergic stimulation and membrane depolarization from the rat adrenal gland

The present study was attempted to investigate the effect of quinine on secretion of catecholamines (CA) evoked by cholinergic stimulation and membrane depolarization from the isolated perfused rat adrenal gland. The perfusion of quinine (15-150 microM) into an adrenal vein for 60 min produced dose- and time-dependent inhibition in CA secretion evoked by ACh (5.32 x 10(-3) M), high K+ (5.6 x 10(-2) M), DMPP (10(-4) M for 2 min), McN-A-343 (10(-4) M for 2 min), cyclopiazonic acid (10(-5) M for 4 min) and Bay-K-8644 (10(-5) M for 4 min). Also, under the presence of pinacidil (10(-4) M), which is also known to be a selective potassium channel activator, CA secretory responses evoked by ACh, high potassium, DMPPF McN-A-343, Bay-K-8644 and cyclopiazonic acid were also greatly reduced. When preloaded along with quinine (5 x 10(-5) M) and glibenclamide (10(-6) M), a specific blocker of ATP-regulated potassium channels, CA secretory responses evoked by ACh, high potassium, DMPP, McN-A-343, Bay-K-8644 and cyclopiazonic acid were recovered as compared to those of quinine-treatment only. Taken together, these results demonstrate that quinine inhibits CA secretion evoked by stimulation of cholinergic (both nicotinic and muscarinic) receptors as well as by membrane depolarization through inhibiting influx of extracellular calcium and release in intracellular calcium in the rat adrenomedullary chromaffin cells. These findings suggest that activation of potassium channels may be involved at least in inhibitory action of quinine on CA secretion from the rat adrenal gland.     

Studies on secretion of catecholamine evoked by caffeine from the isolated perfused rat adrenal gland

The influence of caffeine on secretion of catecholamines (CA) was examined in the isolated perfused rat adrenal gland. Caffeine (0.3 mM) perfused into an adrenal vein of the gland produced a marked increase in secretion of CA. This secretory effect of CA evoked by perfusion of caffeine for one minute was considerably prolonged, lasting for more than 90 minutes. The tachyphylaxis to releasing effect of CA induced by caffeine was observed by repeated perfusion of this drug. The caffeine-evoked CA secretion was markedly inhibited by pretreatment with ouabain, trifluoperazine, TMB-8 and perfusion with calcium-free Krebs solution containing 5 mM EGTA, but was not affected by perfusion of calcium-free Krebs solution without other addition. CA secretion evoked by caffeine was not reduced significantly by pretreatment with chlorisondamine but after the first collection of perfusate for 3 min was clearly inhibited. Interestingly, the caffeine-evoked CA secretion was considerably potentiated by pretreatment with atropine or pirenzepine, but after the first collection for 3 min it was markedly decreased. These experimental results suggest that caffeine causes a marked increase in secretion of CA from the isolated perfused rat adrenal gland by an extracellular calcium-independent exocytotic mechanism. The secretory effect of caffeine may be mainly due to mobilization of calcium from an intracellular calcium pool in the rat chromaffin cells and partly due to stimulation of both muscarinic and nicotinic receptors.