The IP3-sensitive calcium store of HIT cells is located in a surface-derived vesicle fraction

Electron microscopic and biochemical techniques were used to study the cellular localization of the ATP-dependent, IP3-sensitive, Ca2+ store in the glucose- and phosphatidylinositol(PI) agonist-sensitive hamster insulinoma cell line HIT-T15. Scanning electron microscopy revealed conspicuous shape changes of the microvilli following stimulation of these cells with bombesin or thapsigargin. These changes closely resemble those previously shown to accompany stimulation of hexose transport in adipocytes with insulin [J. Cell. Physiol. 142 (1990) 1-14]. Using a hydrodynamic shearing technique for the isolation of microvilli, two cell surface-derived vesicle fractions were prepared containing 80% of the total cellular Ca(2+)-storing activity. In contrast, subcellular fractionation using normal homogenization with a glass/teflon homogenizer yielded the well-known distribution of the Ca(2+)-storing activity which is then predominantly recovered within the microsomal fraction. The surface-derived vesicle fraction was clearly distinguished from the microsomal fraction by its high content of Na+/K(+)-ATPase and an immunoreactive fragment of the GluT-1 glucose transporter isoform which both are not detectable in the microsomal fraction isolated from homogenates from sheared cells. The Ca2+ uptake properties of the cell surface-derived vesicle fractions including the vanadate, A23187, and thapsigargin sensitivity were found to be identical with those described for the microsomal Ca2+ stores of various cell types. Inositol 1,4,5-trisphosphate (IP3) at 1 microM induced a maximal release of 35-40% of the stored Ca2+ from these vesicles.