Detection of the ATP-Dependent Nonmitochondrial Calcium Store in a Cell Surface-Derived Vesicle Fraction from Isolated Rat Hepatocytes

In preceding studies, the IP₃-sensitive Ca²⁺store of the hamster insulinoma cell line, HIT, was detected in cell surface protrusions such as microvilli and related membrane structures [Lange, K., and Brandt, U. (1993)FEBS Lett.320, 183–188; and (1993)FEBS Lett.325, 205–209]. In this study, these experiments were extended on rat hepatocytes. We used the previously described shearing technique for isolating cell surface-derived vesicle fractions from freshly isolated and 48-h-cultured rat hepatocytes. As shown by Western blot analysis, these vesicles contained the hepatocyte-specific glucose transporter, GluT2, and actin, which are both typical microvillar components. Scanning electron microscopy revealed that a spherical vesicle population of uniform size (about 1 μm in diameter) originates from the hepatocyte microvilli. This vesicle fraction exhibited ATP-dependent and thapsigargin-sensitive Ca²⁺storage activity with properties identical to those of the known microsomal systems and of HIT cell surface-derived vesicles, except that the ATP-dependent Ca²⁺pool was insensitive to IP₃. Like HIT surface vesicles, hepatocyte surface vesicles rapidly took up ATP via a 4,4′-diisocyanostilbene-2,2′-disulfonic acid (DIDS)-sensitive anion pathway. Inhibition of ATP influx into the vesicles by DIDS also completely inhibited ATP-dependent Ca²⁺storage. Moreover, determination of efflux kinetics of Ca²⁺from passively (in the absence of ATP) loaded vesicles revealed a La³⁺-sensitive but IP₃-independent Ca²⁺pathway which rapidly equilibrated intravesicular free Ca²⁺with the external medium. Permeabilization of the vesicles with saponin (0.005%) opened an additional efflux pathway for Ca²⁺which is not La³⁺-sensitive. However, saponin treatment of vesicles preloaded with Ca²⁺in the presence of ATP did not affect the thapsigargin-sensitive vesicular Ca²⁺store but only released a small portion (about 20%) of the vesicular Ca²⁺that is not part of the thapsigargin-sensitive Ca²⁺pool. Also, the size of the saponin-releasable Ca²⁺pool was not affected by depletion of the thapsigargin-sensitive Ca²⁺store. These findings indicate that hepatocyte surface vesicles are readily permeable for Ca²⁺and ATP via cation and anion pathways. Consequently, Ca²⁺storage into these vesicles does not occur by concentrative Ca²⁺pumping but rather appears to be due to an internal, ATP-dependent mechanism of Ca²⁺sequestration. The presented data are in accord with the previously reported colocalization of the ATP-dependent Ca²⁺store and its functionally coupled, store-regulated Ca²⁺influx pathway in special cell surface organelles, the microvilli.