1. Archive
2. Vol.54, No.3

A Genistein-Sensitive Na/Ca² Exchange Is Responsible for the Resting [Ca²]_i and Most of the Ca² Plasma Membrane Fluxes in Stimulated Rat Cerebellar Type 1 Astrocytes

Abstract: 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, Nao 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) Ca²o removal in the presence of Nao causes an important drop in [Ca²]_i, which is absent in Lio or NMGo (N-methyl-D-glucamine) containing medium; (3) the reverse mode exchange inhibitor KB-R7943 mimics the removal of Ca²o only in the presence of Nao; 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 [Ca²]í.

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