br Discussion According to the

Discussion According to the generally accepted notion which originated from the study of ATP-depleted 2-APB the changes in Ca2+ homeostasis induced by ATP depletion and leading to the activation of the Gárdos effect are due to the inhibition of Ca2+ pumping, and the subsequent entry of Ca2+ through the membrane `resting' permeability mechanism(s). Also, the effect of NaF was explained by its inhibition of glycolysis, the major catabolic pathway in human RBC [2]. This way of thinking was adopted when the study of the effect of NaVO3 on the RBC Ca2+ homeostasis has been initiated 8, 13where similar numerical values of the 45Ca2+ influx were observed. Apparently, this is not the case when NaF is used as an inducer (Fig. 1). The Ca2+ influx induced by NaF is not only much more extensive but also much faster than that induced by NaVO3. It should be mentioned that its time course resembles the NaF-induced and Ca2+-dependent Na+ influx which we described previously [18]and both processes clearly precede the onset of the Gárdos effect which is delayed by about 5 min after addition of Ca2+ to both NaF- and NaVO3-treated RBC (1, 8, unpublished observation). In addition, NaF- and NaVO3-induced Ca2+ influxes exhibit striking differences (Fig. 2) in the sensitivity to inhibitors. The inhibitory action of Cu2+ and DTNB is largely reduced in NaF-treated RBC as compared with NaVO3 treatment, suggesting only a minor role of HS groups in facilitating the NaF-induced Ca2+ influx. On the other hand, the effect of CSA suggests a selective involvement of calcineurin or cyclophilin in mediating the NaF-induced Ca2+ influx. It should be mentioned at this point that Co2+ inhibits both processes with approximately equal efficiency (not shown). The experiment with the most explanatory power is the effect of TTX which is an inhibitor of the NaF-induced Ca2+ influx only. This (together with the time course) strongly suggests that Ca2+ shares the influx pathway with Na+[18]in the same manner as suggested by Baker for the nerve and/or chromaffin cell Na+ channel almost three decades ago 20, 21. The delayed appearance of the Ca2+-dependent K+ efflux which follows Na+ and Ca2+ influxes is similar to the time course of these ions during the action potential in nerve cells. Phenomenologically, the effect of NaF and Ca2+ could be regarded as an extremely slow chemically induced action potential with negligible inactivation (if any). The stimulatory effect of Na+ substitution on the NaF-induced 45Ca2+ influx (Fig. 1) could hence be explained by the elimination of the competition of Na+ for the common transport pathway. It is conspicuous that the inhibitory efficiencies of several inhibitors tested (nifedipine, TTX, CSA) were less than those observed in other (e.g. excitable) cells. We could not provide any experiments which could explain these results. The recent theoretical model by Bray et al. [29]based on the study of bacterial chemotaxis suggests that the sensitivity of the signalling pathway is a variable of receptor clustering. This concept, which is amenable to experimental analysis, may be useful for explaining the differences in inhibitor sensitivities also in animal cells. The results shown in Fig. 1, Fig. 2 indicate that the properties of the Ca2+ influx induced by NaVO3 and NaF are different. The interspecies difference experiment shown in Fig. 3 compellingly suggests that both effects are mediated by independent mechanisms. In experiments published in our recent papers 15, 16we have shown that NaVO3-induced Ca2+ influx may be preceded by changes in inositol phospholipid metabolism rather than by changes in G-protein activation whereas the effect of NaF implies the involvement of G-protein. The extensive shifts in ion concentrations and the effects of inhibitors presented above enable us to answer the question whether the Gárdos effect induced by NaF is a inique event or has some homology in other cells and/or organs. Such extensive shifts in concentrations of Ca2+, K+, and Na+ occur in brain during ischemia and spreading depression [22], or severe hypoglycemia [23]and were recently found to be antagonized (among other inhibitors) by TTX 24, 25, 26. Although the details of the mechanisms in RBC and brain have still to be found in the future, perturbations of Ca2+ homeostasis seem to have central roles in triggering these events.