CaCl was from the Radiochemical
45CaCl2 was from the Radiochemical Centre (Amersham, UK), NaVO3 was from Reachim (Moscow, Russia). Tetrodotoxin was purchased from Calbiochem, DTNB from Merck, Darmstadt, Germany. Nifedipine was synthesized in the Institute of Drug Research, Modra, Slovakia and was kindly provided by Dr. Zdeno Mahrla. Cyclosporin A was kindly provided by Prof. V. Betina (Slovak University of Technology, Bratislava). Menthol and camphor were purchased from Medika (Bratislava, Slovakia). Other chemicals (all of analytical grade) were purchased from Lachema (Brno, Czech Republic).
Results The dependence of the 45Ca2+ influx induced by NaVO3 and NaF on the extracellular Ca2+ showed saturability (Fig. 1) with KM(Ca)=0.5 mmol/l for both NaVO3 and NaF. The time courses of the 45Ca2+ influx induced by either agent (Fig. 1) showed marked differences. That induced by NaVO3 reached the half-maximal value in several tens of minutes and gradually increased until the end of measurement, in accordance with previously published data 8, 16. On the other hand, NaF-induced Ca2+ influx reached the maximal values within about 30 s and then (in most experiments) slightly decreased. The NaF-induced 45Ca2+ influx was different from that induced by NaVO3 also in the extent (expressed as μmol Ca2+/lcells) at the end of experiments (1 and 60 min, respectively). The former exceeded the latter by an order of magnitude and in some experiments values were obtained (50–3000 μmol/lcells) that suggested equilibration of Ca2+ across the RBC membrane (and possible binding and/or precipitation in the cytoplasm) upon the action of NaF in some experiments. In NaVO3-treated PF-CBP1 hydrochloride sale values between 5 and 80 μmol/lcells were obtained (values from several tens of experiments with either inducer). The prominent feature of the Ca2+ influx induced in NaVO3(and ATP-depleted RBC ) is its inhibition by isotonic substitution of Na+ for K+ but not for choline. In NaF-treated cells, the Ca2+ influx was influenced by Na+ substitution in the opposite direction (Fig. 2). Moreover, the effect was not specific for K+ because the substitution of Na+ by choline+ had a similar, even more pronounced, effect (not shown). It was found previously that the NaVO3-induced Ca2+ influx displays sensitivity to several inhibitors, e.g. to HS reagents and divalent cations creating mercaptide bonds which exert a biphasic action on the Ca2+ influx . NaF-induced Ca2+ influx was less sensitive to these compounds, represented by Cu2+ and DTNB (Fig. 2). At submillimolar concentrations, the maximal inhibition by Cu2+ was less than 20% and by DTNB less than 30% (75 and 65%, for the NaVO3-induced Ca2+ influx, with IC50=9 μmol/l and 12 μmol/l, respectively). In accordance with the data of Stimpel et al. , the NaVO3-induced Ca2+ influx was sensitive to dihydropyridine Ca2+ antagonists. It was found to be fully suppressed by nifedipine as an inhibitor (up to 140 μmol/l) (Fig. 2) (IC50=50 μmol/l). On the other hand, the Ca2+ influx induced by NaF was less sensitive to nifedipine (Fig. 2) and, in the same concentration range, the degree of inhibition did not reach 20%. NaF was found to increase the membrane permeability for Na+ ions which was sensitive to the nerve poison tetrodotoxin (TTX) . The Ca2+ influx elicited by NaF was also sensitive to TTX in the same concentration range (IC50=21 μmol/l) (Fig. 2). However, the NaVO3-induced Ca2+ influx was not sensitive to TTX (Fig. 2). Also other agents which are known to affect the function of CNS, menthol and camphor, in millimolar concentrations, in part inhibited the NaF-induced Ca2+ influx but not that induced by NaVO3 (not shown). The calcineurin inhibitor and RBC cyclophilin ligand 27, 28cyclosporin A (CSA) was another compound which inhibited the effect of NaF (IC50=28 μmol/l) but it exerted a rather stimulatory effect on the NaVO3-induced Ca2+ influx (Fig. 2). It is known that NaVO3 is unable to elicit the Ca2+ influx and the Gárdos effect in pig RBC (Fig. 3). However, NaF under conditions similar to those used for human RBC was still able to induce the 45Ca2+ influx, although to a lesser extent, in pig RBC (Fig. 3).