Inhibitory effect of 2,3-butanedione monoxime (BDM) on Na(+)/Ca(2+) exchange current in guinea-pig cardiac ventricular myocytes.

1. The effect of 2,3-butanedione monoxime (BDM), a 'chemical phosphatase', on Na(+)/Ca(2+) exchange current (I(NCX)) was investigated using the whole-cell voltage-clamp technique in single guinea-pig cardiac ventricular myocytes and in CCL39 fibroblast cells expressing canine NCX1. 2. I(NCX) was identified as a current sensitive to KB-R7943, a relatively selective NCX inhibitor, at 140 mM Na(+) and 2 mM Ca(2+) in the external solution and 20 mM Na(+) and 433 nM free Ca(2+) in the pipette solution. 3. In guinea-pig ventricular cells, BDM inhibited I(NCX) in a concentration-dependent manner. The IC(50) value was 2.4 mM with a Hill coefficients of 1. The average time for 50% inhibition by 10 mM BDM was 124+/-31 s (n=5). 4. The effect of BDM was not affected by 1 microM okadaic acid in the pipette solution, indicating that the inhibition was not via activation of okadaic acid-sensitive protein phosphatases. 5. Intracellular trypsin treatment via the pipette solution significantly suppressed the inhibitory effect of BDM, implicating an intracellular site of action of BDM. 6. PAM (pralidoxime), another oxime compound, also inhibited I(NCX) in a manner similar to BDM. 7. Isoprenaline at 50 microM and phorbol 12-myristate 13-acetate (PMA) at 8 microM did not reverse the inhibition of I(NCX) by BDM. 8. BDM inhibited I(NCX) in CCL39 cells expressing NCX1 and in its mutant in which its three major phosphorylatable serine residues were replaced with alanines. 9. We conclude that BDM inhibits I(NCX) but the mechanism of inhibition is not by dephosphorylation of the Na(+)/Ca(2+) exchanger as a 'chemical phosphatase'.

Direction-independent block of bi-directional Na+/Ca2+ exchange current by KB-R7943 in guinea-pig cardiac myocytes.

1. We investigated the inhibitory effect of KB-R7943 on 'bi-directional' Na+/Ca2+ exchange current (iNCX) with the reversal potential of iNCX (ENCX) in the middle of the ramp voltage pulse employed. 2. Bi-directional iNCX was recorded with 'full' ramp pulses given every 10 s from the holding potential of -60 mV over the voltage range between 30 and -150 mV under the ionic conditions of 140 mM [Na]o, 20 mM [Na]i, 1 mM [Ca]o and 433 nM [Ca]i with calculated ENCX at -50 mV. 3. KB-R7943 (0.1 - 100 mirconM) concentration-dependently inhibited the current, which reversed near the calculated ENCX, indicating that the blocked current was iNCX. 4. The inhibition levels were not significantly different between outward and inward iNCX measured at 0 and -120 mV, respectively. IC50 of KB-R7943 was approximately 1 micronM for both directions of iNCX. 5. Under the bi-directional ionic conditions, only an outward or inward iNCX was induced by positive or negative 'half' ramp pulses, respectively, from the holding potential of -60 mV. KB-R7943 inhibited both direction of iNCX and the concentration-inhibition relations were superimposable to the ones obtained by 'full' ramp pulses. 6. These results indicate that KB-R7943 inhibits iNCX direction-independently under bi-directional conditions. This conclusion is different from that of our previous results obtained from iNCX under uni-directional ionic conditions, where KB-R7943 inhibited iNCX direction-dependently. The difference could be attributed to slow dissociation of the drug from the exchanger.

Effect of Na+/Ca2+ exchange inhibitor, KB-R7943 on ouabain-induced arrhythmias in guinea-pigs.

1. We investigated protective effects of KB-R7943, a Na+/Ca2+ exchange (NCX) inhibitor, on ouabain-induced tonotropy and arrhythmias in isolated whole atria and ouabain-induced changes in electrocardiogram (ECG) in the guinea-pig. 2. KB-R7943 (10 and 30 microM) suppressed the tonotropic effect of ouabain, and prolonged the onset time of extra-systole induced by ouabain in isolated atria. 3. The intravenous injection of KB-R7943 (1 and 3 mg kg-1) significantly increased the doses of ouabain required to induce ventricular premature beats (VPB), ventricular tachycardia (VT), ventricular fibrillation (VF) and cardiac arrest (CA) in anaesthetized guinea-pigs. 4. Lidocaine (Na+channel inhibitor) and R56865 (Na+ and Ca2+ overload inhibitor) also suppressed the ouabain-induced tonotropic effect and extra-systole in isolated atria, but Hoe-694 (Na+/H+ exchange inhibitor) or diltiazem (Ca2+ channel inhibitor) did not affect them. 5. Lidocaine also increased the doses of ouabain required to induce VPB, VT, VF and CA in anaesthetized guinea-pigs. 6. From these results, we conclude that KB-R7943 suppresses ouabain-induced arrhythmias through inhibition of the reverse-mode NCX.

Calcium-dependent inhibition of the sodium-calcium exchange current by KB-R7943.

The inhibitory effect of KB-R7943 (previously called No 7943) on the outward sodium-calcium exchange current was re-examined by applying the drug after inducing the outward exchange current rather than before the current onset as has been done previously. The outward exchange current was induced by raising the extracellular calcium ion concentration, [Ca2+]o, from 0.15 mM to 1.8 mM instead of from 0 mM to 1 mM, as has been done previously. Aftertreatment KB-R7943 inhibited the outward exchange current, and the concentration of KB-R7943 required to inhibit 50% of the exchange current [IC50] was approximately 3 microM. This value is 10 times higher than that obtained by pretreatment of the drug at 1 mM [Ca2+]o. The new IC50 of 3 microM is close to the values for the calcium current (8 microM) and the inward rectifier potassium current (7 microM) but is still significantly lower than that for the inward sodium-calcium exchange current (17 microM).

Inhibitory effect of lomerizine, a diphenylpiperazine Ca2+-channel blocker, on Ba2+ current through voltage-gated Ca2+ channels in PC12 cells.

We investigated the effect of lomerizine, an anti-migraine drug, on the Ba2+ current through voltage-gated Ca2+ channels in rat pheochromocytoma (PC12) cells using a whole-cell voltage-clamp technique. Lomerizine inhibited the Ba2+ current with an IC50 value of 1.9 microM. Lomerizine and nicardipine were >4 times more potent than flunarizine, diltiazem, verapamil and dimetotiazine. The time course of inactivation induced by lomerizine was similar to that induced by nicardipine and flunarizine. These data indicate that lomerizine may inhibit the Ca2+ channel in a similar manner to nicardipine and flunarizine, and its potency is almost equal to that of nicardipine.

A novel antagonist, No. 7943, of the Na+/Ca2+ exchange current in guinea-pig cardiac ventricular cells.

1. The effects of No. 7943 on the Na+/Ca2+ exchange current and on other membrane currents were investigated in single cardiac ventricular cells of guinea-pig with the whole-cell voltage-clamp technique. 2. No. 7943 at 0.1-10 microM suppressed the outward Na+/Ca2+ exchange current in a concentration-dependent manner. The suppression was reversible and the IC50 value was approximately 0.32 microM. 3. No. 7943 at 5-50 microM suppressed also the inward Na+/Ca2+ exchange current in a concentration-dependent manner but with a higher IC50 value of approximately 17 microM. 4. In a concentration-response curve, No. 7943 raised the K(m)Ca2+ value, but did not affect the Imax value, indicating that No. 7943 is a competitive antagonist with external Ca2+ for the outward Na+/ Ca2+ exchange current. 5. The voltage-gated Na+ current, Ca2+ current and the inward rectifier K+ current were also inhibited by No. 7943 with IC50S of approximately 14, 8 and 7 microM, respectively. 6. In contrast to No. 7943, 3', 4'-dichlorobenzamil (DCB) at 3-30 microM suppressed the inward Na+/Ca2+ exchange current with IC50 of 17 microM, but did not affect the outward exchange current at these concentrations. 7. We conclude that No. 7943 inhibits the outward Na+/Ca2+ exchange current more potently than any other currents as a competitive inhibitor with external Ca2+. This effect is in contrast to DCB which preferentially inhibits the inward rather than the outward Na+/Ca2+ exchange current.

A novel isothiourea derivative selectively inhibits the reverse mode of Na+/Ca2+ exchange in cells expressing NCX1.

No.7943 (2-[2-[4-(4-nitrobenzyloxy)phenyl]ethyl]isothiourea methanesulfonate), a selective inhibitor of the Na+/Ca2+ exchanger (NCX1), has been newly synthesized. It dose-dependently inhibited Na+i-dependent 45Ca2+ uptake and Na+i-dependent [Ca2+]i increase in cardiomyocytes, smooth muscle cells, and NCX1-transfected fibroblasts (IC50 = 1.2-2.4 microM). Inhibition was observed without prior incubation with the agent and was completely reversed by washing cells with buffer for 1 min. Interestingly, No.7943 was much less potent in inhibiting Na+o-dependent 45Ca2+ efflux and Na+o-induced [Ca2+]i decline (IC50 = >30 microM), indicating that it selectively blocks the reverse mode of Na+/Ca2+ exchange in intact cells. In cardiac sarcolemmal preparations consisting mostly of inside-out vesicles, the agent inhibited Na+i-dependent 45Ca2+ uptake and Na+o-dependent 45Ca2+ efflux with similar, but slightly lower, potencies (IC50 = 5.4-13 microM). Inhibition was noncompetitive with respect to Ca2+ and Na+ in both cells and sarcolemmal vesicles. These results suggest that No.7943 primarily acts on external exchanger site(s) other than the transport sites in intact cells, although it is able to inhibit the exchanger from both sides of the plasma membrane. No.7943 at up to 10 microM does not affect many other ion transporters nor several cardiac action potential parameters. This agent at these concentrations also did not influence either diastolic [Ca2+]i or spontaneous beating in cardiomyocytes. Furthermore, No.7943 markedly inhibited Ca2+ overloading into cardiomyocytes under the Ca2+ paradox conditions. Thus, No.7943 is not only useful as a tool with which to study the transport mechanism and physiological role of the Na+/Ca2+ exchanger but also has therapeutic potential as a selective blocker of excessive Ca2+ influx mediated via the Na+/Ca2+ exchanger under pathological conditions.

Effects of Ca2+ channel blockers on cortical hypoperfusion and expression of c-Fos-like immunoreactivity after cortical spreading depression in rats.

1. We examined the effects of two Ca2+ channel blockers, lomerizine (KB-2796) and flunarizine, on the cortical hypoperfusion (measured by hydrogen clearance and laser Doppler flowmetry methods) and cortical c-Fos-like immunoreactivity that follow KCl-induced cortical spreading depression in anaesthetized rats. Cortical spreading depression was induced by application of 1 M KCl for 30 s to the cortical surface, 3.0 mm posterior to the area of cerebral blood flow measurement. 2. In control rats, KB-2796 (0.3 and 1 mg kg-1, i.v.) dose-dependently increased cerebral blood flow significantly at 30 min and 15 min, respectively, after its administration. Flunarizine (1 mg kg-1, i.v.) significantly increased cerebral blood flow 15 min after its administration. In contrast, dimetotiazine (3 mg kg-1, i.v.), a 5-HT2 and histamine H1 antagonist, failed to affect cerebral blood flow significantly. 3. After KCl application to the cortex, cerebral blood flow monitored by the laser Doppler flowmetry method increased transiently, for a few minutes, then fell and remained approximately 20 to 30% below control for at least 60 min. Cerebral blood flow monitored by the hydrogen clearance method was also approximately 20 to 30% below baseline for at least 60 min after KCl application. KB-2796 (0.3 and 1 mg kg-1, i.v.) and flunarizine (1 and 3 mg kg-1, i.v.) administered 5 min before KCl application inhibited the cortical hypoperfusion that followed KCl application, but dimetotiazine (1 and 3 mg kg-1, i.v.) did not. 4. An indicator of neuronal activation, c-Fos-like immunoreactivity, was detected in the ipsilateral, but not in the contralateral frontoparietal cortex 2 h after KCl application. No c-Fos-like immunoreactivity was seen on either side of the brain in the hippocampus, thalamus, striatum or cerebellum. 5. KB-2796 (1 mg kg-1, i.v.) and flunarizine (3 mg kg-1, i.v.), but not dimetotiazine (3 mg kg-1, i.v.), significantly attenuated the expression of c-Fos-like immunoreactivity in the ipsilateral frontoparietal cortex. 6. These findings suggest that the inhibitory effects of KB-2796 and flunarizine on the cortical hypoperfusion and expression of c-Fos-like immunoreactivity induced by spreading depression are mediated via the effects of Ca(2+)-entry blockade, which may include an increase in cerebral blood flow and the prevention of excessive Ca2+ influx into brain cells. KB-2796 and flunarizine may prove useful as inhibitors of cortical spreading depression in migraine.

Zinc potentiation of neurotransmission and inhibition of background cationic conductance in rat cultured hippocampal neurones.

1. The facilitation by zinc (Zn2+) of neurotransmission and the mechanisms underlying it were electrophysiologically investigated in rat cultured hippocampal neurones using whole-cell voltage- and current-clamp techniques. 2. Under whole-cell voltage clamp with an intracellular solution containing CsCl as a major salt, inward postsynaptic currents were observed at -40 mV in a cell culture where a neuronal network had been formed. The postsynaptic currents appeared to be mediated by gamma-aminobutyric acid (GABA) because the inward currents were abolished when intracellular CsCl was replaced with caesium phosphate and they were blocked by bicuculline (10 microM), an antagonist to GABA-gated channels. The currents were, however, also blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 30 microM), an antagonist to non-NMDA glutamate-gated channels, suggesting a contribution of a glutamatergic mechanism to the generation of the currents. Zn2+ (10 and 100 microM) potentiated the postsynaptic currents. 3. In addition to the potentiation of the postsynaptic currents, Zn2+ shifted net membrane current at -60 mV in an outward direction. The current-voltage relationship obtained under various ionic conditions indicated that Zn2+ inhibits a current component which is mainly carried by extracellular Na+. 4. Under whole-cell current clamp, Zn2+ (10 microM) induced a small hyperpolarization (up to 20 mV), which was accompanied by potentiation of the postsynaptic potentials and spike potentials. Tests were carried out to examine whether changes in resting potential by different protocols mimic responses observed with Zn2+. Hyperpolarization induced by current injection through patch pipettes increased the amplitude of postsynaptic currents, but did not enhance the appearance of spike potentials. In contrast, when extracellular K+ concentration was decreased from 5 to 2.5 mM, cells were hyperpolarized and spike potentials of large amplitude appeared. 5. The results suggest that Zn2+ potentiates neurotransmission and inhibits a background cationic current mainly carried by extracellular Na+ under physiological conditions. The inhibition of the Na+ permeation may increase membrane excitability and thereby contribute to the potentiation of neurotransmission.

[Recent advances in apolipoprotein measurement].

Owing to the development of protein measurement methods, apolipoprotein can be measured more rapidly and precisely. It has been only in recent years that apolipoprotein measurement widely prevails in the clinical field. Various apolipoprotein measurement systems are available for the clinical laboratory in Japan. Formerly, apolipoprotein was manually measured mainly by a single radial immunodiffusion system, but recently more sophisticated automated systems for apolipoprotein measurement have been developed and applied. The more the need to detect apolipoprotein in the clinical laboratory and the larger the scale for apolipoprotein measurement, the more progress to be made in rapidity and cost performance. That is, the advances in apolipoprotein measurement depend on the requirement of its test by the clinicians. In the global trend, standardization for apolipoprotein measurement is being discussed and is in progress. The effort of standardization is fulfilled in some results of external standardization surveys on apolipoprotein measurement. In this report, we introduced the advances in apolipoprotein measurement which supported the standardization process in Japan.

Dual modulation by adenosine of ATP-activated channels through GTP-binding proteins in rat pheochromocytoma PC12 cells.

Effects of adenosine on inward current activated by extracellular ATP were examined in rat pheochromocytoma PC12 cells. Adenosine induced two types of modulation on the current activated by 30 microM ATP; a low concentration of adenosine (1 microM) inhibited the current whereas a high concentration (> 10 microM) enhanced the current. Neither the inhibition nor the enhancement was observed in cells pretreated with pertussis toxin (PTX), or in cells dialyzed with guanosine 5'-O-(2-thiotriphosphate) trilithium salt (GDP beta S). In contrast, dialysis with K-252a, a protein kinase inhibitor, abolished the inhibition, but not the enhancement. Adenosine induced similar inhibition and enhancement on ATP-evoked increase in intracellular free Ca2+ concentration. The results suggest that adenosine produces dual modulation on the ATP-activated channels through different mechanisms involving PTX-sensitive GTP-binding proteins.

Potentiation by adenosine of ATP-evoked dopamine release via a pertussis toxin-sensitive mechanism in rat phaeochromocytoma PC12 cells.

1. The effects of adenosine on adenosine 5'-triphosphate (ATP)-evoked dopamine release from rat phaeochromocytoma PC12 cells was investigated to determine whether adenosine exerts a regulatory effect on the ATP-evoked response. Adenosine potentiated ATP (30 microM)-evoked dopamine release in a concentration-dependent manner over a concentration-range of 1 to 100 microM. Adenosine (100 microM) shifted the concentration-dependence of the ATP-evoked response to the left without affecting the maximal response. 2. Aminophylline, a non-selective adenosine receptor antagonist, and CP66713, a selective antagonist at the A2 subclass of adenosine receptors, abolished the adenosine-induced potentiation. Furthermore, 8-cyclopentyltheophylline, a selective antagonist at the adenosine A1 receptor partially inhibited the adenosine-evoked potentiation. CGS22492, a selective A2 receptor agonist, potentiated ATP-evoked dopamine release whereas N6-cyclohexyladenosine (CHA), a selective A1 receptor agonist, had no effect. 3. Pertussis toxin (PTX), a bacterial exotoxin which catalyzes the ADP-ribosylation of guanosine 5'-triphosphate (GTP)-binding proteins (G-proteins), inhibited the adenosine-induced potentiation of dopamine release. Dibutyryl cyclic AMP (db cyclic AMP), an analogue of cyclic AMP, had no effect on the release on the ATP-evoked response. 4. Adenosine potentiated the ATP-evoked rise in intracellular Ca2+ concentration ([Ca]i) in PC12 cells. This potentiation was also observed with CGS 22492 but not with CHA. PTX completely inhibited the adenosine-induced potentiation of the rise in [Ca]i. 5. On the basis of these findings, we suggest that the adenosine-induced potentiation of ATP-evoked dopamine release was due to an increase in [Ca]i in the cells. Although the potentiation is most likely mediated by a subclass of A2 receptors, the subclass may be different from those previously reported since the potentiation was sensitive to PTX and was not reproduced by db cyclic AMP.

Non-competitive antagonism by hirsuteine of nicotinic receptor-mediated dopamine release from rat pheochromocytoma cells.

Effects of hirsuteine, an indole alkaloid extracted from Uncaria genus, on nicotine- and high K-induced responses were investigated in rat pheochromocytoma PC12 cells. Hirsuteine (300 nM-10 microM) inhibited dopamine release evoked by 100 microM nicotine in a concentration-dependent manner. Hirsuteine did not produce a parallel shift of the concentration-response relationship curve for nicotine, but reduced maximal dopamine release. Dopamine release evoked by 60 and 155 mM KCl was also inhibited by hirsuteine, but the concentration necessary for significant inhibition was higher (more than 10 microM). Under whole cell voltage-clamp, hirsuteine reversibly inhibited inward currents activated by 100 microM nicotine. The current inhibition was slightly accelerated by hyperpolarization. The results suggest that hirsuteine non-competitively antagonizes nicotine-evoked dopamine release by blocking ion permeation through nicotinic receptor channel complexes. The blockade of Ca channels, which are activated during nicotine-evoked depolarization, may not play a major role in the antagonism.

Mechanisms underlying facilitation by dopamine of ATP-activated currents in rat pheochromocytoma cells.

Mechanisms underlying facilitation by dopamine of extracellular adenosine 5'-triphosphate (ATP)-activated current were investigated in rat pheochromocytoma PC12 cells using the whole-cell voltage-clamp techniques. Dopamine (10 and 100 microM) augmented the peak amplitude of an inward current elicited by ATP (3-100 microM). The activation time course of the ATP-evoked current was accelerated by dopamine; the presence of 10 microM dopamine shifted the dependence of activation rate constants on the concentration of ATP toward a lower concentration range two fold. Dopamine also accelerated the inactivation and the deactivation, which was determined from the current decay upon washout of ATP. Intracellular mediators responsible for the dopamine-induced facilitation was estimated by loading various compounds in patch pipettes. Facilitation was not observed when K-252a (1 microM), a protein kinase inhibitor, was included in the intracellular solution. In addition, facilitation was also attenuated by intracellular adenosine 5'-O-(thiotriphosphate)tetralithium salt (ATP gamma S (1 mM) or alpha-beta-methylene ATP (1 mM). Inclusion of adenosine 3',5'-cyclic monophosphate sodium salt (cAMP, 100 microM), guanosine 3',5'-cyclic monophosphate sodium salt (cGMP, 100 microM), 12-O-tetradecanoylphorbol-13-acetate (TPA, 1 microM) or phorbol-12,13-dibutyrate (1 microM) in the intracellular solution did not affect the facilitation. Guanosine 5'-O-(thiotriphosphate)tetralithium salt (GTP gamma S, 500 microM) or guanosine 5'-O(2-thiodiphosphate)-trilithium salt (GDP beta S, 500 microM) did not modify the facilitation either.(ABSTRACT TRUNCATED AT 250 WORDS)

Dopamine receptor agonists and antagonists enhance ATP-activated currents.

The effects of dopamine and related compounds on ATP-activated channels were investigated in pheochromocytoma PC12 cells. Dopamine (10 microM) enhanced an inward current activated by 100 microM ATP. A similar enhancement of the ATP-activated current was observed with apomorphine (10 microM), a non-selective dopamine receptor agonist, with (+)-SKF-38393 (10 microM), a selective dopamine D1 receptor agonist, and with (-)-quinpirole (10 microM), a selective dopamine D2 receptor agonist. Moreover, (+)-SCH-23390 (30 microM), a dopamine D1 receptor antagonist, and (-)-sulpiride (30 microM), a dopamine D2 receptor antagonist, also enhanced the ATP-activated current. The results suggest that ATP-activated channels are modulated by dopaminergic mechanisms, and that this modulation cannot be attributed to any single class of dopamine receptors.

Extracellular adenosine 5'-triphosphate-evoked glutamate release in cultured hippocampal neurons.

Characteristics of extracellular ATP-evoked electrical responses in rat hippocampal neurons were investigated. Extracellular ATP (100 microM) induced a rapid depolarization followed by repetitive firings of spikes in these cells under whole-cell current-clamp. In whole-cell voltage-clamp experiments, ATP activated 2 types of inward currents that were inhibited by P2-purinoceptor blocker suramin (300 microM). One is a small (about -20 pA) sustained current which is insensitive to tetrodotoxin (TTX), and the other is a large (-100 to -300 pA) transient current which abolished in the presence of 3 microM TTX. The ATP-induced transient current was blocked by 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX; 30 microM), a non-N-methyl-D-aspartate (non-NMDA) receptor antagonist. ATP failed to induce the transient current in the cell which showed the desensitization to quisqualic acid (QA; 10 microM), a non-NMDA receptor agonist. These findings suggest that ATP directly activates small sustained currents, and indirectly induces the transient currents by evoking glutamate release.

Inhibition of ion channels by hirsutine in rat pheochromocytoma cells.

Effects of hirsutine, an alkaloid that produces a potent ganglion blocking effect, were investigated using rat pheochromocytoma PC12 cells. Hirsutine (1 to 10 microM) suppressed dopamine-release evoked by 100 microM nicotine. In voltage-clamped cells, hirsutine (1 to 10 microM) inhibited the inward current activated by 100 microM nicotine. Hirsutine was equipotent to hexamethonium in blocking the nicotine-activated current. The voltage-dependency of the nicotine activated current was not modified by hirsutine. Effects of hirustine on other ion channels were tested to determine its selectivity. Inward currents mediated through ATP-activated channels were scarcely affected by hirsutine (up to 100 microM). However, hirustine (10 microM) inhibited Ba currents passing through Ca channels and K currents activated by depolarizing voltage steps. The results suggest that hirsutine potently blocks nicotinic receptor-channels, but hirsutine also inhibits voltage-gated Ca and K channels. Roles of the inhibition of these channels in the pharmacological effects of hirsutine were discussed.