COMPARISON OF TYPE-2 INOSITOL 1,4,5-TRISPHOSPHATE RECEPTOR DISTRIBUTION AND SUBCELLULAR CA2+ RELEASE SITES THAT SUPPORT CA2+ WAVES IN CULTURED ASTROCYTES

We have examined the mechanisms that underlie Ca2+ wave propagation in cultured cortical astrocytes. Norepinephrine evoked Ca2+ waves in ast rocytes that began at discrete initiation loci and propagated througho ut the cell by regenerative amplification at a number of cellular site s, as shown by very high Ca2+ release rates at these regions. We have hypothesized previously that domains displaying elevated Ca2+ release kinetics in astrocytes may correspond to sites of high inositol 1,4,5- trisphosphate receptor (InsP(3)R) density. To examine this possibility , we compared the distribution pattern of endoplasmic reticulum (ER) a nd InsP(3)Rs with Ca2+ release kinetics in subcellular regions during propagation of norepinephrine-evoked waves. 3,3'-Dihexyloxacarbocyanin e iodide staining revealed that the ER in astrocytes exists as a meshw ork of membranes extending throughout the cells, including fine proces ses. A specific antibody directed against type 2 InsP(3)Rs (InsP(3)R2) detected a 260-kDa band in western blotting of astrocyte membranes. I mmunocytochemistry using this antibody stained the entire ER system in a punctate, variegated manner. When Ca2+ responses and InsP(3)R2 immu nofluorescence were compared in the same cell, domains of elevated Ca2 + response kinetics (high amplitude and rapid rate of rise) showed sig nificant positive correlation with high local intensity of InsP(3)R2 s taining. It appears, therefore, that specializations in the ER respons ible for discrete local Ca2+ release sites that support regenerative w ave propagation include increased levels of InsP(3)R2 expression.