ROLE OF SARCOPLASMIC ENDOPLASMIC-RETICULUM CA2+-ATPASES IN MEDIATING CA2+ WAVES AND LOCAL CA2+-RELEASE MICRODOMAINS IN CULTURED GLIA/

We have characterized the sarcoplasmic-endoplasmic reticulum Ca2+-ATPa se (SERCA) pumps in cultured rat cortical type-1 astrocytes, type-2 as trocytes and oligodendrocytes. Perfusion with 10 mu M cyclopiazonic ac id (CPA) or 1 mu M thapsigargin evoked a large and persistent elevatio n in cytosolic [Ca2+] in normal Ca2+-containing medium and a small and transient increase in nominally Ca2+-free medium. Subtraction of the response in Ca2+-free medium from that in the control revealed-a slow- onset Ca2+-entry response to SERCA inhibition, which began after most of the store depletion had occurred. Thapsigargin- and CPA-induced res ponses propagated as Ca2+ waves, which began in several distinct cellu lar sites and travelled throughout the cell and through nearby cells, in confluent cultures. Propagation was supported by specialized Ca2+-r elease sites where the amplitude of the response was significantly hig her and the rate of rise steeper. Such higher Ca2+-release kinetics we re observed at these sites during Ins(1,4,5)P-3 mediated Ca2+ waves in the same cells. Fluorescently tagged thapsigargin labelled SERCA pump s throughout glial cell bodies and processes. In oligodendrocyte proce sses, multiple domains with elevated SERCA staining were always associ ated with mitochondria. Our results are consistent with a model in whi ch only a single Ca2+ store, expressing Ins(1,4,5)P-3 receptors and SE RCAs sensitive to both thapsigargin and CPA, is present in rat cortica l glia, and indicate that inhibition of SERCA activates both Ca2+ rele ase as a wavefront and Ca2+ entry via store-operated channels. The spa tial relationship between SERCAs and mitochondria is likely to be impo rtant for regulating microdomains of elevated Ca2+ release kinetics.