Proof of the mysterious efficacy of ginseng: basic and clinical trials: suppression of adrenal medullary function in vitro by ginseng

The root of Panax ginseng C.A. MEYER has been reported to have an anti-stress action. Therefore, the effects of ginseng components on functions of adrenal medulla, which is one of the most important organs responsive to stress, were investigated in vitro. First, the components of ginseng were mainly divided into two fractions, that is, the saponin-rich and non-saponin fractions. The saponin-rich fraction greatly reduced the secretion of catecholamines from bovine adrenal chromaffin cells stimulated by acetylcholine (ACh), whereas the non-saponin fraction did not affect it at all. The protopanaxatriol-type saponins inhibited the ACh-evoked secretion much more strongly than the protopanaxadiol-type. On the other hand, the oleanane-type saponin, ginsenoside-Ro, had no such effect. Recent reports have demonstrated that the saponins in ginseng are metabolized and absorbed in digestive tracts following oral administration of ginseng. All of the saponin metabolites greatly reduced the ACh-evoked secretion. M4 was the most effective inhibitor among the metabolites. M4 blocked ACh-induced Na(+) influx and ion inward current into the chromaffin cells and into the Xenopus oocytes expressing human alpha3beta4 nicotinic ACh receptors, respectively, suggesting that the saponin metabolites modulate nicotinic ACh receptors followed by the reduction of catecholamine secretion. It is highly possible that these effects of ginsenosides and their metabolites are associated with the anti-stress action of ginseng.     

Characterization of ginseng saponin ginsenoside-Rg(3) inhibition of catecholamine secretion in bovine adrenal chromaffin cells.

Since ginsenoside-Rg(3), one of the panaxadiol saponins isolated from the ginseng root, significantly inhibited the secretion of catecholamines from bovine adrenal chromaffin cells stimulated by acetylcholine (ACh), the properties of ginsenoside-Rg(3) inhibition were investigated. Although ginsenoside-Rg(3) inhibited the secretion evoked by ACh in a concentration-dependent manner, it affected the secretion stimulated by high K(+) or veratridine, an activator of the voltage-sensitive Ca(2+) or Na(+) channels, only slightly. The ACh-induced Na(+) and Ca(2+) influxes into the cells were also reduced by ginsenoside-Rg(3). The inhibitory effect of this saponin on the secretion of catecholamines was not altered by increasing the external concentration of ACh or Ca(2+). The ACh-evoked secretion of catecholamines was completely restored in cells that were preincubated with 10 microM ginsenoside-Rg(3) and then incubated without the saponin, whereas secretion was not completely restored in cells that were preincubated with 30 microM of this compound. Above 30 microM ginsenoside-Rg(3) increased the fluorescence anisotropy of diphenylhexatriene in the cells. Furthermore, the inhibitory effect of ginsenoside-Rg(3) at 30 microM on the ACh-evoked secretion of catecholamines was dependent upon the preincubation time, but this was not the case at 10 microM. These results strongly suggest that ginsenoside-Rg(3) blocks the nicotinic ACh receptor-operated cation channels, inhibits Na(+) influx through the channels, and consequently reduces both Ca(2+) influx and catecholamine secretion in bovine adrenal chromaffin cells. In addition to this action, the ginsenoside at higher concentrations modulates the fluidity of the plasma membrane, which probably contributes to the observed reduction in the secretion of catecholamines.     

Lithium inhibits function of voltage-dependent sodium channels and catecholamine secretion independent of glycogen synthase kinase-3 in adrenal chromaffin cells

Lithium has been proven to be effective in the therapy of bipolar disorder, but its mechanism of pharmacological action is not clearly defined. We examined the effects of lithium on voltage-dependent Na(+) channels, nicotinic acetylcholine receptors, and voltage-dependent Ca(2+) channels, as well as catecholamine secretion in cultured bovine adrenal chromaffin cells. Lithium chloride (LiCl) reduced veratridine-induced (22)Na(+) influx in a concentration-dependent manner, even in the presence of ouabain, an inhibitor of Na(+), K(+)-ATPase. Glycogen synthase kinase-3 (GSK-3) inhibitors (SB216763, SB415286 or the GSK-3 inhibitor IX) did not affect veratridine-induced (22)Na(+) influx, as well as inhibitory effect of LiCl on veratridine-induced (22)Na(+) influx. Enhancement of veratridine (site 2 toxin)-induced (22)Na(+) influx caused by alpha-scorpion venom (site 3 toxin), beta-scorpion venom (site 4 toxin), or Ptychodiscus brevis toxin-3 (site 5 toxin), still occurred in the presence of LiCl in the same manner as in the control cells. LiCl also reduced veratridine-induced (45)Ca(2+) influx and catecholamine secretion. In contrast, LiCl (< or = 30 mM) had no effect on nicotine-induced (22)Na(+) influx, (45)Ca(2+) influx and catecholamine secretion, as well as on high K(+)-induced (45)Ca(2+) influx and catecholamine secretion. Chronic treatment with LiCl at 100mM (but not at < or = 30 mM) significantly reduced cell viability in a time-dependent manner. These results suggest that lithium selectively inhibits Na(+) influx thorough Na(+) channels and subsequent Ca(2+) influx and catecholamine secretion, independent of GSK-3 inhibition.     

Inhibition by pregnenolone sulfate of nicotinic acetylcholine response in adrenal chromaffin cells

To evaluate whether pregnenolone sulfate, an abundant neurosteroid in the brain, modulates nicotinic receptor-mediated responses, the effect of pregnenolone sulfate on acetylcholine-induced catecholamine secretion was investigated in cultured bovine adrenal chromaffin cells. Pregnenolone sulfate inhibited acetylcholine-induced catecholamine secretion (IC(50): 27 microM). In addition, pregnenolone sulfate inhibited acetylcholine-induced Na(+) (IC(50): 12 microM) and Ca(2+) (IC(50): 20 microM) influxes. However, pregnenolone sulfate did not inhibit either catecholamine secretion or Ca(2+) influx stimulated by high K(+). Binding of [3H]nicotine to nicotinic receptors was not altered by pregnenolone sulfate. The inhibitory effect on the acetylcholine-induced secretion was insurmountable by increasing acetylcholine concentrations, but was enhanced by decreasing external Na(+) concentrations. These results suggest strongly that pregnenolone sulfate noncompetitively inhibits nicotinic receptor-operated ion channels, thereby suppressing Na(+) influx through the channels and, consequently, attenuates both Ca(2+) influx and catecholamine secretion. Our results further indicate that pregnenolone sulfate may modulate nicotinic receptor-mediated responses in the brain.     

Differential effect of bovine serum albumin on ginsenoside metabolite-induced inhibition of α3β4 nicotinic acetylcholine receptor expressed in<Emphasis Type="Italic">Xenopus</Emphasis> oocytes

Ginsenosides, major active ingredients of Panax ginseng, that exhibit various pharmacological and physiological actions are transformed into compound K (CK) or M4 by intestinal microorganisms. CK is a metabolite derived from protopanaxadiol (PD) ginsenosides, whereas M4 is a metabolite derived from protopanaxatriol (PT) ginsenosides. Recent reports shows that ginsenosides might play a role as pro-drugs for these metabolites. In present study, we investigated the effect of bovine serum albumin (BSA), which is one of major binding proteins on various neurotransmitters, hormones, and other pharmacological agents, on ginsenoside Rg2-, CK-, or M4-induced regulation of alpha3beta4 nicotinic acetylcholine (ACh) receptor channel activity expressed in Xenopus oocytes. In the absence of BSA, treatment of ACh elicited inward peak current (I(ACh)) in oocytes expressing alpha3beta4 nicotinic ACh receptor. Co-treatment of ginsenoside Rg2, CK, or M4 with ACh inhibited I(ACh) in oocytes expressing (alpha3beta4 nicotinic ACh receptor with reversible and dose-dependent manner. In the presence of 1% BSA, treatment of ACh still elicited I(ACh) in oocytes expressing alpha3beta4 nicotinic ACh receptor and co-treatment of ginsenoside Rg2 or M4 but not CK with ACh inhibited I(ACh) in oocytes expressing alpha3beta4 nicotinic ACh receptor with reversible and dose-dependent manner. These results show that BSA interferes the action of CK rather than M4 on the inhibitory effect of I(ACh) in oocytes expressing alpha3beta4 nicotinic ACh receptor and further suggest that BSA exhibits a differential interaction on ginsenoside metabolites.     

Effects of resveratrol, a grape polyphenol, on catecholamine secretion and synthesis in cultured bovine adrenal medullary cells

We report the effects of resveratrol, a polyphenol found in the skins of red grapes, on catecholamine secretion and synthesis in cultured bovine adrenal medullary cells. Resveratrol suppressed catecholamine secretion and (22)Na(+) and (45)Ca(2+) influx induced by acetylcholine, an agonist of nicotinic acetylcholine receptors, in a concentration-dependent manner (IC(50)=20.4, 11.0, and 62.8 microM, respectively). Resveratrol also inhibited catecholamine secretion induced by veratridine, an activator of voltage-dependent Na(+) channels, and 56 mM K(+), an activator of voltage-dependent Ca(2+) channels, at concentrations similar to those for (45)Ca(2+) influx. Resveratrol directly inhibited the current evoked by acetylcholine in Xenopus oocytes expressing alpha3beta4 neuronal nicotinic acetylcholine receptors (IC(50)=25.9 microM). Furthermore, resveratrol (IC(50)=5.32 microM) attenuated (14)C-catecholamine synthesis induced by acetylcholine. The present findings suggest that resveratrol inhibits acetylcholine-induced catecholamine secretion and synthesis through suppressing ion influx in cultured bovine adrenal medullary cells.     

Effects of extract and ingredients isolated from Magnolia obovata thunberg on catecholamine secretion from bovine adrenal chromaffin cells

The crude extract of magnolia bark, an herbal drug, inhibited the secretion of catecholamines from bovine adrenal chromaffin cells stimulated by acetylcholine (ACh) in a concentration-dependent manner (200-900 microg/mL). The extract also diminished the secretion induced by high K(+), which is a stimulus directly depolarizing the plasma membranes, but its inhibition was weaker than that of ACh-evoked secretion. beta-Eudesmol, honokiol, magnolol, and bornyl acetate, but not alpha- and beta-pinenes, all of which are ingredients of magnolia bark, greatly reduced ACh-evoked secretion. beta-Eudesmol and magnolol also inhibited high K(+)-induced secretion to an extent similar to that of ACh-evoked secretion. However, honokiol and bornyl acetate inhibited the secretion induced by high K(+) much less than the secretion evoked by ACh. ACh-induced Na(+) influx and ACh- or high K(+)-induced Ca(2+) influx into the cells were diminished by beta-eudesmol or honokiol. These results indicate that magnolia bark contains some effective components inhibiting the secretion of catecholamines from bovine adrenal chromaffin cells stimulated by ACh due to the antagonism of Na(+) and Ca(2+) influxes into the cells. However, inhibition by the extract of magnolia bark seems to be attributable to honokiol and bornyl acetate. Furthermore, the results indicate that the inhibitory effect of magnolia bark may be associated with its pharmacological effect on activities of the nervous system.     

Selective inhibition by riluzole of voltage-dependent sodium channels and catecholamine secretion in adrenal chromaffin cells

We examined the effects of riluzole, a neuroprotective drug, on voltage–dependent Na channels, nicotinic receptors, and voltage-dependent Ca channels, as well as catecholamine secretion, in comparison with those of verapamil and nicardipine, in primary cultures of bovine adrenal chromaffin cells. Riluzole inhibited veratridine-induced ²²Na influx via voltage-dependent Na channels even in the presence of ouabain, an inhibitor of Na,K-ATPase. Blockade of Na channels by riluzole was concentration-dependent with an IC₅₀ of 5.3 μM. It was associated with a similar concentration-related reduction of veratridine-induced ⁴⁵Ca influx via voltage-dependent Ca channels, and of catecholamine secretion. Riluzole had no effect on ⁴⁵Ca influx caused by high K, which directly activates voltage-dependent Ca channels, and on nicotine-induced ²²Na influx, which passes through the nicotinic receptors. Verapamil and nicardipine attenuated ²²Na influx caused by veratridine or nicotine at the same concentrations as they suppressed high K-induced ⁴⁵Ca influx. The inhibitory effect of riluzole on veratridine-induced ²²Na influx disappeared at high concentrations of veratridine. A potentiation of veratridine (site 2 toxin)-induced ²²Na influx caused by α-scorpion venom (site 3 toxin), β-scorpion venom (site 4 toxin), or brevetoxin PbTx-3 (site 5 toxin), occurred in the presence of riluzole in the same manner as in control cells. These results suggest that riluzole binds to the veratridine site in voltage–dependent Na channels. It does not impair the cooperative interaction between the functional peptide segments of Na channels, but selectively inhibits gating of Na channels, thereby reducing Ca influx via Ca channels and catecholamine secretion. In contrast, verapamil and nicardipine suppress Na influx both Na channels and nicotinic receptors. Received: 4 November 1997 / Accepted: 11 February 1998     

Inhibitory effects of tramadol on nicotinic acetylcholine receptors in adrenal chromaffin cells and in Xenopus oocytes expressing alpha 7 receptors

1. Tramadol has been used clinically as an analgesic; however, the mechanism of its analgesic effects is still unknown. 2. We used bovine adrenal chromaffin cells to investigate effects of tramadol on catecholamine secretion, nicotine-induced cytosolic Ca(2+) concentration ([Ca(2+)](i)) increases and membrane current changes. We also investigated effects of tramadol on alpha7 nicotinic acetylcholine receptors (AChRs) expressed in Xenopus oocytes. 3. Tramadol concentration-dependently suppressed carbachol-induced catecholamine secretion to 60% and 27% of the control at the concentration of 10 and 100 microM, respectively, whereas it had little effect on veratridine- or high K(+)-induced catecholamine secretion. 4. Tramadol also suppressed nicotine-induced ([Ca(2+)](i)) increases in a concentration-dependent manner. Tramadol inhibited nicotine-induced inward currents, and the inhibition was unaffected by the opioid receptor antagonist naloxone. 5. Tramadol inhibited nicotinic currents carried by alpha7 receptors expressed in Xenopus oocytes. 6. Tramadol inhibited both alpha-bungarotoxin-sensitive and -insensitive nicotinic currents in bovine adrenal chromaffin cells. 7. In conclusion, tramadol inhibits catecholamine secretion partly by inhibiting nicotinic AChR functions in a naloxone-insensitive manner and alpha7 receptors are one of those inhibited by tramadol.     

Amphetamine enhances Ca2+ entry and catecholamine release via nicotinic receptor activation in bovine adrenal chromaffin cells

Amphetamine, a psychostimulant, has been shown to act as a channel blocker of muscle nicotinic receptors and to induce a Ca(2+)-dependent secretion from adrenal chromaffin cells. In this study, the relationship between amphetamine and nicotinic receptors was studied using bovine adrenal chromaffin cells as a model system. Our results show that D-amphetamine sulfate alone induced an increase in the cytosolic Ca(2+) concentration ([Ca(2+)](c)) and [3H]norepinephrine release in a dose-dependent and extracellular Ca(2+)-dependent manner. Two common nicotinic receptor antagonists, hexamethonium and mecamylamine, suppressed the D-amphetamine sulfate-induced [Ca(2+)](c) rise and [3H]norepinephrine release. In addition, D-amphetamine sulfate inhibited the 1,1-dimethyl-4-phenyl-piperazinium iodide (DMPP)-induced [Ca(2+)](c) rise and [3H]norepinephrine release, but not the high K(+)- or veratridine-induced [Ca(2+)](c) increase and [3H]norepinephrine release. Antagonists, including alpha-bungarotoxin and choline, that are more specific for alpha7 nicotinic receptors were capable of inhibiting the D-amphetamine sulfate-induced [Ca(2+)](c) rise, while D-amphetamine sulfate was found to be capable of inhibiting the [Ca(2+)](c) rise induced by the alpha7-nicotinic receptor agonists, epibatidine and choline. Moreover, D-amphetamine sulfate dose-dependently suppressed [3H]nicotine binding to chromaffin cells. We, therefore, conclude that D-amphetamine sulfate acts as a nicotinic receptor agonist to induce [Ca(2+)](c) increase and [3H]norepinephrine release in bovine adrenal chromaffin cells.     

The action of pentobarbitone on stimulus-secretion coupling in adrenal chromaffin cells.

The action of pentobarbitone on stimulus-secretion coupling was studied in bovine isolated adrenal medullary cells. Pentobarbitone inhibited catecholamine release evoked by 500 microM carbachol with half maximal inhibition (IC50) around 50 microM. It also inhibited catecholamine release induced by depolarization with 77 mM potassium (IC50 100 microM). These effects of pentobarbitone were observed with concentrations that lie within the range encountered during general anaesthesia. Evoked secretion required the presence of calcium in the extracellular medium and was associated with an influx of Ca2+ through voltage-sensitive channels. Pentobarbitone inhibited 45Ca influx in response to both carbachol (IC50 50 microM) and K+-depolarization (IC50 150 microM). The action of pentobarbitone on the relationship between intracellular free Ca and exocytosis was examined using electropermeabilised cells which were suspended in solutions containing a range of concentrations of ionised calcium between 10(-8) and 10(-4)M. Catecholamine secretion was measured in the presence of 0, 50, 200 or 500 microM pentobarbitone. The anaesthetic had no effect on the activation of exocytosis by intracellular free calcium. When catecholamine secretion in response to potassium or carbachol was modulated by varying extracellular calcium or by adding pentobarbitone to the incubation medium, the amount of catecholamine secretion for a given Ca2+ entry was the same. Pentobarbitone inhibited the secretion and 45Ca uptake induced by carbachol in a non-competitive manner. The secretion evoked by nicotinic agonists was associated with an increase in 22Na influx. Pentobarbitone inhibited this influx with an IC50 of 100 microM. We concluded that: (a) Pentobarbitone inhibits the catecholamine secretion from bovine adrenal chromaffin cells induced by nicotinic agonists by non-competitive inhibition of the nicotinic receptor. (b) The decrease in Ca influx caused by pentobarbitone accounts fully for the decrease in secretion in response to depolarization with potassium.     

Possible role of a neuropeptide PACAP (pituitary adenylate cyclase-activating polypeptide) on stimulus-secretion coupling in catecholamine neuron

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide first isolated from ovine hypothalamic tissue. This peptide stimulates adenylate cyclase activation. However, few details were known of the function of this peptide on stimulus-secretion coupling in neuronal cells. The authors have investigated the role of PACAP on catecholamine biosynthesis and secretion using cultured bovine adrenal chromaffin cells as a model for catecholamine-containing neurons. PACAP38, the 38-amino acid form of PACAP, increased cAMP formation in bovine adrenal chromaffin cells. In addition, PACAP38 increased [Ca2+]i associated with PI turnover and Ca2+ influx into the cells. The synthesis of catecholamine and the phosphorylation of tyrosine hydroxylase, a rate-limiting enzyme of catecholamine biosynthesis, stimulated by the maximal effective concentration of dibutyryl cAMP or a high concentration (56 mM) of K+ were further enhanced by PACAP38. Thus PACAP38 stimulated the pathway of catecholamine biosynthesis mainly by both activation of cAMP- and Ca2(+)-dependent protein kinases. On catecholamine secretion from the cells, the effect of PACAP38 was markedly potentiated by addition of ouabain, an inhibitor of Na+/K+ ATPase. This markedly potentiated secretion was greatly reduced with Na+ omitted-sucrose medium. PACAP38 increased 22Na+ influx into the cells treated with ouabain. Thus PACAP38 with ouabain stimulated catecholamine secretion by accumulation of intracellular Na+, resulting in an increase in Ca2+ influx. These results indicate that the neuropeptide PACAP has an important role in stimulus-secretion coupling in adrenal chromaffin cells.     

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.     

The role of Na+ in muscarinic receptor-mediated catecholamine secretion in the absence of extracellular Ca2+ in cat perfused adrenal glands.

1. The role of Na+ in muscarinic receptor-mediated catecholamine secretion, which is independent of extracellular Ca2+, was investigated by observing the effect of veratridine and ouabain in perfused adrenal glands of the cat. 2. Veratridine (10(-4) M) markedly enhanced catecholamine secretion evoked by acetylcholine (ACh, 10(-4) M) during perfusion with Ca2(+)-free Locke solution containing hexamethonium (10(-3) M). The enhancement tended to be larger for noradrenaline secretion than for adrenaline secretion. Qualitatively the same result was obtained in the response to pilocarpine (5 x 10(-4) M). 3. Ouabain (10(-4) M) also enhanced ACh- and pilocarpine-induced catecholamine secretions, especially noradrenaline secretion in the absence of extracellular Ca2+. 4. Tetrodotoxin (10(-6) M) blocked the enhancing effect of veratridine on ACh-induced catecholamine secretion, but not that of ouabain in the absence of extracellular Ca2+. 5. When NaCl was replaced with sucrose, there was no secretory response to ACh regardless of the presence or absence of veratridine or ouabain. However, when ouabain, but not veratridine, was infused with Na+ before the replacement of NaCl, the response to ACh was substantially augmented. 6. These results indicate that Na+ is essential in the initiation of muscarinic receptor-mediated catecholamine secretion and its enhancement by veratridine and ouabain in the absence of extracellular Ca2+. Both drugs seem to increase the intracellular concentration of Na+ through different mechanisms and result in increases in the efficiency of Ca2+ mobilization from intracellular Ca2+ pools linked to muscarinic receptors.     

Possible muscarinic regulation of catecholamine secretion mediated by cyclic GMP in isolated bovine adrenal chromaffin cells

Catecholamine secretion and cyclic GMP levels were measured in chromaffin cells isolated from bovine adrenal medulla. Acetylcholine (ACh) and nicotine, but not muscarine, induced 8- to 10-fold increases in catecholamine secretion, with respective ED₅₀ values of 10 and 2 M. Cyclic GMP levels were also increased from 3- to 5-fold in the presence of ACh, and this stimulation was mimicked by muscarine but not by nicotine. Half-maximum stimulations of cyclic GMP levels with ACh and muscarine were observed at 0.1 and 0.3 M respectively. The order of potency of various cholinergic drugs for cyclic GMP stimulation was as follows: ACh > oxotremorine > methacholine > muscarine > carbamylcholine > furthretonium > arecholine > bethanechol. Pilocarpine, McN-A-343, and AHR-602 were inactive at concentrations between 10^?8 and 10^?3 M. Isobutylmethylxanthine (1 mM), a specific phosphodiesterase inhibitor, caused a 7-fold increase in cyclic GMP and potentiated 3-fold the stimulation of cyclic GMP by ACh. The nicotine-induced catecholamine secretion was inhibited 19 and 33 per cent by the co-stimulation of the muscarinic receptor with 0.2 and 0.5 M ACh, respectively. Isobutylmethylxanthine (1 mM) also caused a 44 per cent inhibition of nicotine-induced catecholamine secretion, and its effect was additive to that of ACh. Atropine (0.1 M) selectively abolished the inhibition caused by ACh. Similar inhibitions were also obtained in the presence of exogenous dibutyryl cyclic GMP or 8-bromo cyclic GMP. These data indicate that the nicotinic stimulation of catecholamine secretion from bovine adrenal chromaffin cells may be regulated by cyclic GMP via the stimulation of a muscarinic receptor.     

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.     

Interferon-α reduces catecholamine secretion from bovine adrenal chromaffin cells stimulated by acetylcholine

A long-term pretreatment (72h) of bovine adrenal chromaffin cells with recombinant human interferon (IFN)-α-2b (1500 units/ml) produced a decrease in the secretion of catecholamines from the cells stimulated by acetylcholine (ACh) (25μmol/l) but not that with human fibloblast IFN-β (3000 units/ml) or recombinant human IFN-γ (3000 units/ml). IFN-α-2b inhibited the ACh-induced secretion in a concentration- (30–1500 units/ml) and time-dependent manner (18–72h). The content of catecholamines in the cells treated with IFN-α-2b for 72h did not change. The inhibitory effect of IFN-α-2b on the secretion was abolished when the cells were simultaneously treated with anti-IFN-α antibody, and it was overcome by the increase in the external ACh concentration. IFN-α-2b also inhibited ACh-induced Ca²⁺ influx into the cells in a concentration-dependent manner similar to that of the IFN-α-2b inhibiting ACh-induced secretion. On the other hand, IFN-α-2b failed to reduce the secretion from the cells induced by high K⁺. These results strongly suggest that IFN-α-2b reduces the ACh-induced secretion of catecholamines from bovine adrenal chromaffin cells due to modulating the gene expression of the nicotinic ACh receptor-operated cation channels rather than due to directly affecting the channels. The results further indicate that the IFN-α-2b inhibition may be associated with the psychiatric side effects of IFN-α (depression, neurasthenica and somnolence, etc.), and that immune systems may regulate the function of (autonomic) nervous systems or adrenal medulla via IFN-α in vivo. Received: 6 March 1997 / Accepted: 31 July 1997     

Inhibition of nicotinic receptor-mediated catecholamine secretion by dryobalanops aromatica in bovine adrenal chromaffin cells

Effect of the aqueous extract from a medicinal plant Dryobalanops aromatica(Dipterocarpaceae) on catecholamine secretion was investigated in bovine adrenal chromaffin cells. The aqueous extract inhibited [(3)H]norepinephrine ([(3)H]NE) secretion induced by 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), a nicotinic acetylcholine receptor (nAChR) agonist, with a half-maximal inhibitory concentration (IC(50)) of 8.4 +/- 1.7 microgml(-1). Increases in cytosolic calcium ([Ca(2+)](i)) and sodium ([Na(+)](i)) induced by DMPP were also inhibited by the extract. However, the binding of [(3)H]nicotine to nAChRs was not affected by the addition of the extract in receptor binding competition analysis, suggesting that active components in the extract and nicotine do not share the binding site in the nAChR. On the other hand, [Ca(2+)](i)increases induced by high K(+), ionomycin, bradykinin, angiotensin II, and thapsigargin were not inhibited by the extract. The data suggest that the extract from D. aromatica specifically inhibits catecholamine secretion by blocking nAChR in a noncompetitive manner.     

Effects of hypoxia on stimulus-release coupling mechanisms in cultured bovine adrenal chromaffin cells

Summary To clarify the effects of hypoxia on stimulus-release coupling, we have examined the effects of hypoxia on nicotine-induced catecholamine (noradrenaline and adrenaline) release from, and ²²Na⁺ influx, ⁴⁵Ca²⁺ influx and cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) in, cultured bovine adrenal chromaffin cells. Experiments were carried out in media pre-equilibrated with 21% O₂/79% N₂ (control) or with 0% O₂/100% N₂ (hypoxia). Cells were stimulated with either nicotine (activating nicotinic acetylcholine (ACh) receptors) or a high K⁺ concentration (55 mmol/1 KCI; directly activating voltage-dependent Ca²⁺ channels). Hypoxia reduced both nicotine- and high K⁺-induced catecholamine releases from the cells, but the reduction of the former (to about 30% of the control value) was more pronounced than that of the latter (to about 40% of the control value). Nicotine-induced ²²Na⁺ influx, which is considered to reflect the function of nicotinic ACh receptors, was inhibited by hypoxia. Both nicotine- and high K⁺-induced ⁴⁵Ca²⁺ influx into the cells were reduced by hypoxia, but the reduction of the former was more pronounced than that of the latter. Nicotine- and high K⁺-induced increases in [Ca²⁺]_i were reduced by hypoxia to about 30% and 40% of the control values, respectively. These results suggest that hypoxia reduces cation influxes (Na⁺ and Ca²⁺) through both the ligand-gated cation channels of the nicotinic ACh receptor and the voltage-dependent Ca²⁺ channels. Correspondence to K. Lee at the present address     

Inhibitory effects of caffeine on secretagogue-induced catecholamine secretion from adrenal chromaffin cells of the guinea-pig.

1. The inhibitory action of caffeine on catecholamine secretion induced by secretagogues was investigated in perfused adrenal glands and dispersed chromaffin cells of the guinea-pig. 2. Caffeine (10 mM) caused a reversible inhibition of catecholamine secretion evoked by acetylcholine (ACh, 50 microM), KCl (56 mM, high K+) and veratridine (100 microM) and that induced by muscarinic receptor activation in the absence of extracellular Ca2+ in perfused adrenal glands. 3. In dispersed chromaffin cells, caffeine caused a dose-dependent inhibition of the secretory responses to 100 microM ACh and veratridine. Forskolin (30 microM), dibutyryl cyclic AMP (1 mM) and 8-bromo cyclic AMP (1 mM) did not mimic the action of caffeine. 4. In the voltage-clamp, whole-cell recording mode (at a holding potential of -60 mV or -70 mV), ACh (100 microM) evoked an inward current, and depolarizing pulses elicited inward Na+, Ca2+ and outward K+ currents. All these responses were partially inhibited by caffeine (20 mM). 5. ACh rapidly increased the intracellular concentration of Ca2+ ([Ca2+]i) in fura-2-loaded cells in either the presence or the absence of external Ca2+, though its magnitude was decreased by about 50% in Ca(2+)-free conditions. Caffeine (20 mM) inhibited these ACh-induced increases in [Ca2+]i. 6. In permeabilized chromaffin cells, caffeine (20 mM) caused an inhibition of catecholamine secretion evoked by Ca2+ (10 microM). 7. These results suggest that caffeine inhibits evoked catecholamine secretion through mechanisms such as the blockade of voltage-dependent Na+ and Ca2+ currents and ACh receptor current, and reduction of the release of intracellularly stored Ca2+ and/or Ca(2+)-sensitivity of the secretory apparatus.