Permeabilization by streptolysin-o reveals a role for calcium-dependent protein kinase c isoforms alpha and beta in the response of cultured cardiomyocytes to hyposmotic challenge.
Immunocytochemical techniques indicate that the uninhibited activity of protein kinase C alpha and protein kinase C beta are necessary for a normal regulatory volume decrease (RVD) response of cultured chick embryo cardiomyocytes subjected to a hyposmotic environment. Antibodies against protein kinase C isoforms alpha, beta, gamma and epsilon were introduced into the cultured myocytes using a developed streptolysin-O (SLO) permeabilization technique that allows the targeted cells to accumulate large biomolecules without perturbing their normal physiological state. The loaded cells were then tested for their ability to RVD when submitted to hypo-osmotic stimulus. Results show that exposing the cultured cells to SLO in the presence of antibodies against protein kinase C alpha and beta, prior to volume challenge, significantly slows the RVD rate. Additional experiments that combined anti-alpha and anti-beta antibodies in the same exposure media did not result in a significantly different rate than the anti-alpha or anti-beta rates alone. The evidence gained in this study is in agreement with previous work in the cultured chick embryo cardiomyocyte that report the involvement of a calcium dependent protein kinase C in the signal transduction pathway of the RVD.
Boyle
R T
RT
Division of Physiology, Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710, USA. rtboyle@inbrapenet.com.br
Lieberman
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M
D000818:Animals; D000906:Antibodies; D001426:Bacterial Proteins; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects; D002478:Cells, Cultured; D002642:Chick Embryo; D007038:Hypotonic Solutions / Q000494:pharmacology; D007527:Isoenzymes / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D018613:Microscopy, Confocal; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000201:enzymology; D009206:Myocardium / Q000166:cytology / Q000201:enzymology*; D011493:Protein Kinase C / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; D013301:Streptolysins / Q000494:pharmacology*; D014882:Water-Electrolyte Balance / Q000187:drug effects / Q000502:physiology*
D000906:Antibodies; D001426:Bacterial Proteins; D007038:Hypotonic Solutions; D007527:Isoenzymes; D013301:Streptolysins; C025964:streptolysin O; D007770:L-Lactate Dehydrogenase; C087857:protein kinase C gamma; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; D000252:Calcium-Transporting ATPases; D002118:Calcium