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Expression and characterization of protein kinase C in isolated rabbit parietal cells
- Source :
- International Journal of Molecular Medicine.
- Publication Year :
- 1999
- Publisher :
- Spandidos Publications, 1999.
-
Abstract
- We investigated the expression, characterization and distribution of protein kinase C (PKC) isozymes in isolated rabbit parietal cells (PC). Cellular extracts of PC were analyzed by Western blot using isozyme-specific antibodies. The Ca2+-independent PKC-epsilon was detected in cytosolic, membrane and cytoskeletal fractions of basal and histamine-stimulated PC, whereas the Ca2+-dependent PKC-alpha was confined to the cytosolic and membrane fractions. Cytosolic and membrane fractions were partially purified by DEAE cellulose column chromatography with elution of increasing NaCl concentration. Eluates of 0.15 M and 0.3 M NaCl PC fractions were identified as PKC-alpha and -epsilon isoforms, respectively. Phorbol 12-myristate 13-acetate (TPA) treatment of PC for 15, 30 and 60 sec decreased significantly cytosolic PKC-alpha and increased membrane-associated PKC-alpha. In contrast to the distribution of PKC-alpha, TPA did not alter membrane or cytosolic level of PKC-epsilon. Comparison of the dose-response curves between TPA-induced hydrogen (H+) secretion, as measured by aminopyrine (AP) uptake, and the membrane-associated PKC-alpha suggests that translocation of PKC-alpha is not involved in the H+ secretory process in PC. Furthermore, a PKC inhibitor, staurosporine, produced a concentration-dependent enhancement of histamine-stimulated H+ secretion. These findings suggest that PKC-alpha plays a negative modulatory role, rather than an obligatory role, in H+ secretion. The localization and distribution of PKC-epsilon into the cytoskeletal fraction of PC also suggests that this isozyme may be involved in the cellular regulation of reversible morphological transformation during stimulation.
- Subjects :
- Male
Protein Kinase C-alpha
Protein Kinase C-epsilon
In Vitro Techniques
Biology
Isozyme
chemistry.chemical_compound
Parietal Cells, Gastric
Western blot
1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine
Genetics
medicine
Animals
Staurosporine
Secretion
Enzyme Inhibitors
Aminopyrine
Cytoskeleton
Protein Kinase C
Protein kinase C
medicine.diagnostic_test
General Medicine
Molecular biology
Isoenzymes
Cytosol
chemistry
Phorbol
Tetradecanoylphorbol Acetate
Calcium
Rabbits
Protons
Cimetidine
Histamine
medicine.drug
Subjects
Details
- ISSN :
- 1791244X and 11073756
- Database :
- OpenAIRE
- Journal :
- International Journal of Molecular Medicine
- Accession number :
- edsair.doi.dedup.....8b5cedd3de5aecf7e16aae98830b89a0