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.
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.
Rojas
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Laboratorio de Permeabilidad Iónica, Centro de Biofísica y Bioquímica, IVIC Apartado 21827, Caracas 1020 A, Venezuela.
Ramos
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Dipolo
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D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D002531:Cerebellum / Q000187:drug effects / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D019833:Genistein / Q000494:pharmacology*; D011689:Purkinje Cells / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D019831:Sodium-Calcium Exchanger / Q000037:antagonists & inhibitors / Q000378:metabolism*
D019831:Sodium-Calcium Exchanger; D019833:Genistein; D002118:Calcium