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 microm 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 La(3+)-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 La(3+)-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.
Institut für Pharmakologie, Freie Universität Berlin, Germany.
J
J
Lange
K
K
Schlieps
K
K
Lange
U
U
Brandt
E
E
Knoll-Köhler
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000199:Actins / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002458:Cell Fractionation; D002478:Cells, Cultured; D006224:Cricetinae; D005260:Female; D051273:Glucose Transporter Type 2; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008854:Microscopy, Electron; D008855:Microscopy, Electron, Scanning; D008871:Microvilli / Q000378:metabolism* / Q000648:ultrastructure; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D015388:Organelles / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D010539:Permeability; D051381:Rats; D017208:Rats, Wistar
D000199:Actins; D051273:Glucose Transporter Type 2; D009004:Monosaccharide Transport Proteins; D000255:Adenosine Triphosphate; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D002118:Calcium