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Detection of the ATP-dependent nonmitochondrial calcium store in a cell surface-derived vesicle fraction from isolated rat hepatocytes.

Abstract：

: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.

journal_name

Exp Cell Res

journal_title

Experimental cell research

authors

Lange J,Schlieps K,Lange K,Brandt U,Knoll-Köhler E

doi

10.1006/excr.1996.0316

subject

Has Abstract

pub_date

1996-11-01 00:00:00

pages

189-96

issue

eissn

0014-4827

issn

1090-2422

pii

S0014-4827(96)90316-X

journal_volume

228

pub_type

杂志文章

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Detection of the ATP-dependent nonmitochondrial calcium store in a cell surface-derived vesicle fraction from isolated rat hepatocytes.