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Previous studies have shown that in contrast to other neuronal cells, Na(+)/Ca(2+) exchange contributes little to Ca(i)(2+) homeostasis in rat cerebellar Purkinje neurons under intracellular perfused conditions and at room temperature [Fierro et al.: J Physiol (Lond) 510: 499-512, 1998]. The purpose of this study was to clarify the role of this transporter in cerebellar Purkinje neurons by using intact cells at nearly physiological body temperature. Using Fluo-3 microfluorometry, we have examined the role of the Na(+)/Ca(2+) exchange in the buffering of calcium loads in cultured rat Purkinje neurons at two temperatures: 20 and 34 degrees C. At 20 degrees C, the recovery of the K(+)-induced [Ca(2+)](i) signal was little affected by the presence of external Na(+) (tau(e) = 35.5 +/- 1.2 s [n = 49]), or by its absence (tau(e) = 36.6 +/- 2.2 s [n = 29]), i.e. in a Li(+)-containing medium. In contrast, at 34 degrees C, the recovery of the [Ca(2+)](i) signal was highly dependent on external Na, i.e. tau(e) = 19.9 +/- 1.2 s (n = 119) and tau(e) = 41.7 +/- 2.6 s (n = 39), in Li(+)-containing media, respectively. A comparison of the rate of clearance of [Ca(2+)](i) in Na(+) or Li(+) media, shows that at a room temperature of 20 degrees C, the Na(+)/Ca(2+) exchange contributes at most to 15-20% of the total [Ca(2+)](i) clearance, compared to 55-65% at 34-36 degrees C. We also demonstrate that under normal physiological conditions forward and reverse Na(+)/Ca(2+) exchanges operate in the same neuron. We conclude that the Na(+)/Ca(2+) exchange is strongly suppressed at room temperature and therefore its role should be reevaluated among different neuronal preparations.

The differential role of Na+/Ca2+ exchange in the regulation of intracellular ionized calcium ([Ca2+]i) in immunological and pharmacologically identified type 1 astrocytes and Purkinje cells was studied in rat cerebellar culture, using Ca2+ (Fluo-3, Fura-2) and Na+ (SBFI) fluorescence measurements. The mean resting [Ca2+]i was significantly higher (191 +/- 8 nM, n=25) in type 1 astrocytes than in Purkinje cells (92 +/- 2.5 nM, n=35). In contrast to Purkinje cells, in unstimulated cerebellar type 1 astrocytes, forward and reverse Na+/Ca2+ modes operate under resting physiological conditions, being responsible for most of the total Ca2+ transplasma membrane fluxes. Four observations support this hypothesis: (1) under resting conditions of temperature and ionic composition, Na+o removal causes a remarkable increase in [Ca2+]i, being inhibited by 2',4' dichlorobenzamil (DCB), and 2-[2-[4-(nitrobenzilloxiphenyl ethyl] isothiourea metanesulfonate (KB-R7943); (2) Ca2+o removal in the presence of Na+o causes an important drop in [Ca2+]i, which is absent in Li+o or NMG+o (N-methyl-D-glucamine) containing medium; (3) the reverse mode exchange inhibitor KB-R7943 mimics the removal of Ca2+o only in the presence of Na+o; and (4) under loaded [Na+]i conditions (ouabain or the activation of taurine-Na+-cotransport), reverse mode exchange increases in both astrocytes and Purkinje cells. In type 1 astrocytes stimulated with endothelin-3 (ET-3), the recovery of the Ca2+i signal occurs largely through the Na+/Ca2+ exchanger. Genistein, a tyrosine kinase inhibitor, completely and reversibly blocks all exchange activity, but not its inactive analogue daidzein, thus suggesting that the Na+/Ca2+ exchanger of cerebellar type 1 astrocytes may be modulated by phosphorylation. Our main conclusion is that in rat cerebellar type 1 astrocytes under resting physiological conditions, most of the total transplasma membrane Ca2+ fluxes take place through the Na+/Ca2+ exchanger, thus accounting for the resting [Ca(2+)]i.

Previous studies have shown that in contrast to other neuronal cells, Na(+)/Ca(2+) exchange contributes little to Ca(i)(2+) homeostasis in rat cerebellar Purkinje neurons under intracellular perfused conditions and at room temperature [Fierro et al.: J Physiol (Lond) 510: 499-512, 1998]. The purpose of this study was to clarify the role of this transporter in cerebellar Purkinje neurons by using intact cells at nearly physiological body temperature. Using Fluo-3 microfluorometry, we have examined the role of the Na(+)/Ca(2+) exchange in the buffering of calcium loads in cultured rat Purkinje neurons at two temperatures: 20 and 34 degrees C. At 20 degrees C, the recovery of the K(+)-induced [Ca(2+)](i) signal was little affected by the presence of external Na(+) (tau(e) = 35.5 +/- 1.2 s [n = 49]), or by its absence (tau(e) = 36.6 +/- 2.2 s [n = 29]), i.e. in a Li(+)-containing medium. In contrast, at 34 degrees C, the recovery of the [Ca(2+)](i) signal was highly dependent on external Na, i.e. tau(e) = 19.9 +/- 1.2 s (n = 119) and tau(e) = 41.7 +/- 2.6 s (n = 39), in Li(+)-containing media, respectively. A comparison of the rate of clearance of [Ca(2+)](i) in Na(+) or Li(+) media, shows that at a room temperature of 20 degrees C, the Na(+)/Ca(2+) exchange contributes at most to 15-20% of the total [Ca(2+)](i) clearance, compared to 55-65% at 34-36 degrees C. We also demonstrate that under normal physiological conditions forward and reverse Na(+)/Ca(2+) exchanges operate in the same neuron. We conclude that the Na(+)/Ca(2+) exchange is strongly suppressed at room temperature and therefore its role should be reevaluated among different neuronal preparations.