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.     

Ketamine inhibits 45Ca influx and catecholamine secretion by inhibiting 22Na influx in cultured bovine adrenal medullary cells

The effects of ketamine, an intravenous anesthetic, on 22Na influx, 45Ca influx and catecholamine secretion were investigated in cultured bovine adrenal medullary cells. Ketamine inhibited carbachol-induced 45Ca influx and catecholamine secretion in a concentration-dependent manner with a similar potency (IC50 40 microM). Ketamine also reduced veratridine-induced 45Ca influx and catecholamine secretion (IC50 260 microM) but did not affect high K-induced 45Ca influx and catecholamine secretion. The influx of 22Na caused by carbachol or by veratridine was suppressed by ketamine with a concentration-inhibition curve similar to that of 45Ca influx and catecholamine secretion. Inhibition by ketamine of the carbachol-induced influx of 22Na, 45Ca and secretion of catecholamines was not reversed by the increased concentrations of carbachol. These observations indicate that ketamine, at clinical concentrations, can inhibit nicotinic receptor-associated ionic channels and that the inhibition of Na influx via the receptor-associated ionic channels is responsible for the inhibition of carbachol-induced Ca influx and catecholamine secretion. At higher concentrations, the anesthetic also inhibits voltage-dependent Na channels but has no effect on voltage-dependent Ca channels.     

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.     

Short- and long-term differential effects of neuroprotective drug NS-7 on voltage-dependent sodium channels in adrenal chromaffin cells

In cultured bovine adrenal chromaffin cells, NS-7 [4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy) pyrimidine hydrochloride], a newly-synthesized neuroprotective drug, inhibited veratridine-induced (22)Na(+) influx via voltage-dependent Na(+) channels (IC(50)=11.4 microM). The inhibition by NS-7 occurred in the presence of ouabain, an inhibitor of Na(+),K(+) ATPase, but disappeared at higher concentration of veratridine, and upon the washout of NS-7. NS-7 attenuated veratridine-induced (45)Ca(2+) influx via voltage-dependent Ca(2+) channels (IC(50)=20.0 microM) and catecholamine secretion (IC(50)=25.8 microM). Chronic (>/=12 h) treatment of cells with NS-7 increased cell surface [(3)H]-STX binding by 86% (EC(50)=10.5 microM; t(1/2)=27 h), but did not alter the K(D) value; it was prevented by cycloheximide, an inhibitor of protein synthesis, or brefeldin A, an inhibitor of vesicular transport from the trans-Golgi network, but was not associated with increased levels of Na(+) channel alpha- and beta(1)-subunit mRNAs. In cells subjected to chronic NS-7 treatment, (22)Na(+) influx caused by veratridine (site 2 toxin), alpha-scorpion venom (site 3 toxin) or beta-scorpion venom (site 4 toxin) was suppressed even after the extensive washout of NS-7, and veratridine-induced (22)Na(+) influx remained depressed even at higher concentration of veratridine; however, either alpha- or beta-scorpion venom, or Ptychodiscus brevis toxin-3 (site 5 toxin) enhanced veratridine-induced (22)Na(+) influx as in nontreated cells. These results suggest that in the acute treatment, NS-7 binds to the site 2 and reversibly inhibits Na(+) channels, thereby reducing Ca(2+) channel gating and catecholamine secretion. Chronic treatment with NS-7 up-regulates cell surface Na(+) channels via translational and externalization events, but persistently inhibits Na(+) channel gating without impairing the cooperative interaction between the functional domains of Na(+) channels.     

Inhibition by SEA0400, a selective inhibitor of Na+/Ca2+ exchanger, of Na+ -dependent Ca2+ uptake and catecholamine release in bovine adrenal chromaffin cells

The effects of SEA0400, a selective inhibitor of the Na(+)/Ca(2+) exchanger (NCX), on Na(+)-dependent Ca(2+) uptake and catecholamine (CA) release were examined in bovine adrenal chromaffin cells that were loaded with Na(+) by treatment with ouabain and veratridine. SEA0400 inhibited Na(+)-dependent (45)Ca(2+) uptake and CA release, with the IC(50) values of 40 and 100 nM, respectively. The IC(50) values of another NCX inhibitor KB-R7943 were 1.8 and 3.7 microM, respectively. These results indicate that SEA0400 is about 40 times more potent than KB-R7943 in inhibiting NCX working in the reverse mode. In intact cells, SEA0400 and KB-R7943 inhibited CA release induced by acetylcholine and DMPP. The IC(50) values of SEA0400 were 5.1 and 4.5 microM and the values of KB-R7943 were 2.6 and 2.1 microM against the release induced by acetylcholine and DMPP, respectively, indicating that the potency of SEA0400 is about a half of that of KB-R7943 in inhibiting the nicotinic receptor-mediated CA release. The binding of [(3)H]nicotine with nicotinic receptors was inhibited by SEA0400 (IC(50) = 90 microM) and KB-R7943 (IC(50) = 12 microM). From these results, it is concluded that unlike KB-R7943, SEA0400 has a potent and selective action on NCX in bovine adrenal chromaffin cells.     

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.     

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.     

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.     

Ca2+ entry via P/Q-type Ca2+ channels and the Na+/Ca2+ exchanger in rat and human neocortical synaptosomes

Rat or human neocortical synaptosomes were used to study the role of voltage-gated Ca(2+) channels and the Na(+)/Ca(2+) exchanger in (45)Ca(2+) influx into nerve terminals. K(+) depolarization-induced (45)Ca(2+) influx through voltage-gated Ca(2+) channels into rat or human synaptosomes was completely blocked by mibefradil 30 microM or Cd(2+) 100 microM but was not affected by tetrodotoxin 1 microM. It was reduced by omega-agatoxin IVA 0.2 microM by 68% in synaptosomes of either species, whereas omega-conotoxin GVIA 0.1 microM and nifedipine 1 microM had no effect. Veratridine-induced (45)Ca(2+) entry into rat neocortical synaptosomes was completely blocked by mibefradil 30 microM, reduced by 80% by Cd(2+) 100 microM, by 90% by tetrodotoxin 1 microM and by 53% by omega-agatoxin IVA 0.2 microM but not by omega-conotoxin GVIA 0.1 microM or nifedipine 1 microM. Na(+)/Ca(2+) exchanger-mediated (45)Ca(2+) uptake into rat neocortical synaptosomes evoked by replacement of Na(+) by choline(+) in the incubation buffer was reduced by KB-R7943 (3-50 microM), an inhibitor of the Na(+)/Ca(2+) exchanger, in a concentration-dependent manner (maximal inhibition by 46% at 50 microM; IC(23%)=7.1 microM). Mibefradil also inhibited the Na(+)/Ca(2+) exchanger-mediated Ca(2+) uptake, although at 3.7 times lower potency (IC(23%)=26 microM). It is concluded that in rat and human neocortical nerve terminals Ca(2+) entry is mediated under physiological conditions by P/Q-type, but not by N- or L-type Ca(2+) channels or the Na(+)/Ca(2+) exchanger. If the cytosolic Na(+) concentration is increased, Ca(2+) is also taken up via the Na(+)/Ca(2+) exchanger. In addition to the ability of mibefradil to block all voltage-operated Ca(2+) channels, this drug is a low potency inhibitor of the Na(+)/Ca(2+) exchanger.     

Chlorpromazine-induced inhibition of catecholamine secretion by a differential blockade of nicotinic receptors and L-type Ca2+ channels in rat pheochromocytoma cells.

We investigated the effect of chlorpromazine (CPZ), a phenothiazine neuroleptic, on catecholamine secretion in rat pheochromocytoma (PC12) cells. CPZ inhibited [3H]norepinephrine ([3H]NE) secretion induced by 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), an agonist of nicotinic acetylcholine receptors (nAChRs) with an IC50 value of 1.0 +/- 0.2 microM. The DMPP-induced rise in cytosolic free Ca2+ concentration [Ca2+]i was inhibited by CPZ with an IC50 of 1.9 +/- 0.1 microM. The DMPP-induced increase in cytosolic free Na+ concentration [Na+]i was also inhibited by CPZ with a similar potency. Furthermore, the binding of [3H]nicotine to PC12 cells was inhibited by CPZ with an IC50 value of 2.7 +/- 0.6 microM, suggesting that the nAChRs themselves are inhibited by CPZ. In addition, both 70 mM K+-induced [3H]NE secretion and [Ca2+]i increase were inhibited by CPZ with IC50 of 7.9 +/- 1.1 and 6.2 +/- 0.3 microM, respectively. Experiments with Ca2+ channel antagonists suggest that L-type Ca2+ channels are mainly responsible for the inhibition. We conclude that CPZ inhibits catecholamine secretion by blocking nAChRs and L-type Ca2+ channels, with the former being more sensitive to CPZ.     

Blockade of voltage-sensitive Na(+) channels by the 5-HT(1A) receptor agonist 8-OH-DPAT: possible significance for neuroprotection

The present study was undertaken to determine whether 5-hydroxytryptamine(1A) (5-HT(1A)) receptor agonists interact with voltage-sensitive Na(+) or N- and P/Q-type Ca(2+) channels to reduce the influx of Na(+) and/or Ca(2+). The 5-HT(1A) receptor agonist 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) inhibited both [3H]batrachotoxinin binding to neurotoxin site 2 of the Na(+) channel in rat cortical membranes (IC(50)=5.1 microM) and veratridine-stimulated Na(+) influx into rat synaptosomes (EC(50)=20. 8 microM). The 5-HT(1A) receptor agonist flesinoxan and the 5-HT(1A) receptor antagonist N-(2-(4-(2-methoxyphenyl)-1-piperazinyl)ethyl)-N-(2-pyridinyl) cyclohexanecarboxamide (WAY-100635) also displaced [3H]batrachotoxinin binding with similar affinities to 8-OH-DPAT, but were much less effective in reducing veratridine-stimulated Na(+) influx. All three serotonergic agents also increased [3H]saxitoxin binding to neurotoxin site 1 of the Na(+) channel. In contrast, none of these agents interacted with radioligand binding to N- or P/Q-type Ca(2+) channels. These data show that 8-OH-DPAT directly interacts with voltage-sensitive Na(+) channels to reduce Na(+) influx so providing an additional mechanism to explain how it functions as a neuroprotectant.     

Mesaconitine-induced relaxation in rat aorta: role of Na+/Ca2+ exchangers in endothelial cells

Previously, we reported that mesaconitine, an aconite alkaloid, increased intracellular Ca(2+) concentration ([Ca(2+)](i)) level in endothelium and caused relaxation in rat aorta via nitric oxide production. In the present study, we investigated the mechanisms of increase in the [Ca(2+)](i) level induced by mesaconitine in rat aorta and in human umbilical vein endothelial cells (HUVECs). Treatment with the low Na(+) buffer delayed the 30 microM mesaconitine-, but not 10 microM acetylcholine-, induced relaxation in rat aorta. Treatments with an inhibitor of Na(+)/Ca(2+) exchangers (20 microM 3',4'-dichlorobenzamil) and a reversed mode (Ca(2+) influx) inhibitor of the exchangers (30 microM 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate, KBR7943) showed similar effects. In HUVECs, 30 microM mesaconitine increased the [Ca(2+)](i) level in the presence of extracellular CaCl(2) and NaCl, and the response was inhibited by KBR7943. Mesaconitine increased intracellular Na(+) concentration level in HUVECs. The [Ca(2+)](i) response by mesaconitine was inhibited by 100 microM D-tubocurarine (an inhibitor of nicotinic acetylcholine receptors), but was not inhibited in the glucose-free buffer and by inhibitors of Na(+)/H(+) exchangers. These findings suggest that mesaconitine stimulated Ca(2+) influx via the Na(+)/Ca(2+) exchangers in endothelial cells and caused relaxation in the aorta. The possibility of D-tubocurarine-sensitive Na(+) channels as target(s) of mesaconitine is discussed.     

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     

Memantine inhibits efferent cholinergic transmission in the cochlea by blocking nicotinic acetylcholine receptors of outer hair cells.

Memantine is a blocker of Ca(2+)-permeable glutamate and nicotinic acetylcholine receptors (nAChR). We investigated the action of memantine on cholinergic synaptic transmission at cochlear outer hair cells (OHCs). At this inhibitory synapse, hyperpolarization of the postsynaptic cell results from opening of SK-type Ca(2+)-activated K(+) channels via a highly Ca(2+)-permeable nAChR containing the alpha 9 subunit. We show that inhibitory postsynaptic currents recorded from OHCs were reversibly blocked by memantine with an IC(50) value of 16 microM. RT-PCR revealed that a newly cloned nAChR subunit, alpha 10, is expressed in OHCs. In contrast to homomeric expression, coexpression of alpha 9 and alpha 10 subunits in Xenopus laevis oocytes resulted in robust acetylcholine-induced currents, indicating that the OHC nAChR may be an alpha 9/alpha 10 heteromer. Accordingly, nAChR currents evoked by application of the ligand to OHCs and currents through alpha 9/alpha 10 were blocked by memantine with a similar IC(50) value of about 1 microM. Memantine block of alpha 9/alpha 10 was moderately voltage dependent. The lower efficacy of memantine for inhibition of inhibitory postsynaptic currents (IPSCs) most probably results from a blocking rate that is slow with respect to the short open time of the receptor channels during an IPSC. Thus, synaptic transmission in OHCs is inhibited by memantine block of Ca(2+) influx through nAChRs. Importantly, prolonged receptor activation and consequently massive Ca(2+) influx, as might occur under pathological conditions, is blocked at low micromolar concentrations, whereas the fast IPSCs initiated by short receptor activation are only blocked at concentrations above 10 microM.     

The novel Na(+)/Ca(2+) exchange inhibitor KB-R7943 also blocks native and expressed neuronal nicotinic receptors

We studied the effects of the novel Na(+)/Ca(2+) exchange inhibitor KB-R7943, 2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulphonate, on the native nicotinic receptors present at the bovine adrenal chromaffin cells, as well as on rat brain alpha(3)beta(4) and alpha(7) nicotinic acetylcholine receptors (AChRs) expressed in XENOPUS: oocytes. As expected, KB-R7943 blocked the Na(+)-gradient dependent (45)Ca(2+) uptake into chromaffin cells (IC(50) of 5.5 microM); but in addition, the compound also inhibited the (45)Ca(2+) entry and the increase of cytosolic Ca(2+) concentration, [Ca(2+)](c), stimulated by 5 s pulses of ACh (IC(50) of 6.5 and 1.7 microM, respectively). In oocytes expressing alpha(3)beta(4) and alpha(7) nicotinic AChRs, voltage-clamped at -60 mV, inward currents elicited by 1 s pulses of 100 microM ACh (I(ACh)) were blocked by KB-R7943 with an IC(50) of 0.4 microM and a Hill coefficient of 0.9. Blockade of alpha(3)beta(4) currents by KB-R7943 was noncompetitive; moreover, the blocker (0.3 microM) became more active as the ACh concentration increased (34 versus 66% blockade at 30 microM and 1 mM ACh, respectively). Inhibition of alpha(3)beta(4) currents by 0.3 microM KB-R7943 was more pronounced at hyperpolarized potentials. If given within the ACh pulse (10 microM), the inhibition amounted to 33, 64 and 80% in oocytes voltage-clamped at -40, -60 and -100 mV, respectively. The onset of blockade was faster and the recovery slower at -100 mV; the reverse was true at -40 mV. In conclusion, KB-R7943 is a potent blocker of nicotinic AChRs; moreover, it displays many features of an open-channel blocker at the rat brain alpha(3)beta(4) AChR. These results should be considered when KB-R7943 is to be used to study Ca(2+) homeostasis in cells expressing nicotinic AChRs and the Na(+)/Ca(2+) exchanger.     

Trichosporin-B-III, an alpha-aminoisobutyric acid-containing peptide, causes Ca(2+)-dependent catecholamine secretion from adrenal medullary chromaffin cells.

We examined the effect of trichosporin-B-III, an alpha-aminoisobutyric acid-containing antibiotic peptide consisting of 19 amino acid residues and a phenylalaninol, on catecholamine secretion from cultured bovine adrenal chromaffin cells. Incubation of the cells with trichosporin-B-III (3-20 microM) caused an increase in the secretion of catecholamines. The secretion induced by trichosporin-B-III at low concentrations (3 and 5 microM) was completely dependent on external Ca2+, whereas that induced by higher concentrations (10 and 20 microM) was partly independent of Ca2+. Trichosporin-B-III at low concentration (5 microM) did not increase the release of lactate dehydrogenase, a marker enzyme of cytoplasm, from the cells. In contrast, the peptide at higher concentration (10 microM) increased the release of the enzyme. Trichosporin-B-III also caused both 45Ca2+ influx into the cells and an increase in the intracellular free Ca2+ concentration. The increases in catecholamine secretion and 45Ca2+ influx behaved similarly in relation to trichosporin-B-III concentration (3-10 microM). The time courses of the increases in secretion, 45Ca2+ influx, and intracellular free Ca2+ concentration induced by trichosporin-B-III were also quite similar. Trichosporin-B-III-induced (at 5 microM) secretion was not affected by the elimination of Na+ from the incubation medium or by the addition of tetrodotoxin, a blocker of highly selective voltage-dependent Na+ channels, or hexamethonium, a blocker of nicotinic acetylcholine receptors. On the other hand, both diltiazem (2-200 microM) and nicardipine (1-200 microM), blockers of voltage-dependent Ca2+ channels, inhibited the secretion induced by trichosporin-B-III (5 microM) in a concentration-dependent manner. Trichosporin-B-III-induced (at 5 microM) secretion also was suppressed by the addition of Mn2+ (5 mM) to the medium. The diltiazem (20 microM) inhibition of trichosporin-B-III-induced (at 5 microM) secretion was reversed by increasing the external Ca2+ concentration. These results indicate that trichosporin-B-III causes the secretion of catecholamines from bovine adrenal chromaffin cells by two mechanisms, Ca2+ dependent and Ca2+ independent (only at high concentrations of trichosporin-B-III). Furthermore, these results strongly suggest that trichosporin-B-III, in Ca(2+)-dependent secretion, activates endogenous voltage-dependent Ca2+ channels, or itself forms the channels in the membranes, and induces Ca2+ influx into the cells.     

Inhibition of aggregation of rabbit and human platelets induced by adrenaline and 5-hydroxytryptamine by KB-R7943, a Na(+)/Ca(2+) exchange inhibitor

1. We investigated the effect of KB-R7943, a Na(+)/Ca(2+) exchange inhibitor, on the aggregation response induced by adrenaline and 5-hydroxytryptamine (5-HT), alone or in combination in human and rabbit platelets in the presence or absence of ouabain. 2. KB-R7943 inhibited aggregation induced by the combination of adrenaline and 5-HT in a concentration-dependent manner. The IC(50) values of KB-R7943 were 4.2+/-2.0 or 3.0+/-0.7 microM with washed rabbit platelets with or without ouabain pretreatment, respectively. 3. In platelet-rich human plasma, the aggregation was biphasic. The IC(50) value of KB-R7943 was 17.2+/-4.4 microM for the first phase aggregation. 4. KB-R7943 did not inhibit the first phase of aggregation induced by adrenaline alone, or the monophasic aggregation induced by 5-HT alone. 5. The aggregation of rabbit platelets depended on the presence of K(+) in the medium, and K(+)-dependent and K(+)-independent Ca(2+) influx were observed in resting platelets. Ouabain treatment increased only the K(+)-dependent but not the K(+)-independent Ca(2+) influx. 6. KB-R7943 inhibited K(+)-dependent Ca(2+) influx with or without ouabain pretreatment, but not K(+)-independent Ca(2+) influx. 7. From these results, we conclude that KB-R7943 inhibits the adrenaline plus 5-HT induced aggregation of rabbit and human platelets by inhibiting K(+)-dependent Na(+)/Ca(2+) exchange (NCKX). Our results suggest that NCKX plays an important role in platelet aggregation.     

Nanomolar concentrations of lead selectively block neuronal nicotinic acetylcholine responses in mouse neuroblastoma cells.

The effects of inorganic lead (Pb2+) on the ion currents mediated by (1) neuronal nicotinic acetylcholine (ACh) receptors, (2) serotonin 5-HT3 receptors, as well as (3) voltage-dependent Ca2+ and Na+ channels have been investigated in voltage clamped mouse neuroblastoma cells. The nicotinic ACh receptor-ion channel complex appeared more sensitive to Pb2+ than the other ion channels investigated. Low concentrations of Pb2+ (1 nM - 3 microM) reduced the peak amplitude of the ACh-induced inward current to 74%-10% of the control value in a concentration-dependent manner. However, between 10 microM and 100 microM Pb2+ the blocking effect was reversed, while the decay of the ACh-induced inward current was delayed. These effects of Pb2+ on the nicotinic receptor-mediated inward current can be described by the sum of two sigmoidal concentration-effect curves with an IC50 value of 19 nM and an EC50 of 21 microM and with slope factors of -0.5 and 0.8, respectively. The current mediated by 5-HT3 receptors was less potently blocked by Pb2+ (IC50 = 49 microM; slope factor = -0.3). In addition, Pb2+ blocked the ion current through voltage-dependent Ca2+ channels. The IC50 value of the concentration-effect curve of block of transient type Ca2+ channels by Pb2+ is 4.8 microM and the slope factor is -0.9. Voltage-dependent Na+ channels were not affected by Pb2+ up to 100 microM. At concentrations greater than 1 microM, Pb2+ also induced a noninactivating inward current. The present results show that modification of neuronal nicotinic receptor function may contribute to neurotoxic effects of Pb2+ poisoning.     

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.