Phorbol ester stimulates a protein kinase C-mediated agatoxin-TK-sensitive calcium permeability pathway in human red blood cells.
Calcium entry into mature erythrocytes (red blood cells; RBCs) is associated with multiple changes in cell properties. At low intracellular Ca(2+), efflux of potassium and water predominates, leading to changes in erythrocyte rheology. At higher Ca(2+) content, activation of kinases and phosphatases, rupture of membrane-to-skeleton bridges, stimulation of a phospholipid scramblase and phospholipase C, and induction of transglutaminase-mediated protein cross-linking are also observed. Because the physiologic relevance of these latter responses depends partially on whether Ca(2+) entry involves a regulated channel or nonspecific leak, we explored mechanisms that initiate controlled Ca(2+) influx. Protein kinase C (PKC) was considered a prime candidate for the pathway regulator, and phorbol-12 myristate-13 acetate (PMA), a stimulator of PKC, was examined for its influence on erythrocyte Ca(2+). PMA was found to stimulate a rapid, dose-dependent influx of calcium, as demonstrated by the increased fluorescence of an entrapped Ca(2+)-sensitive dye, Fluo-3/AM. The PMA-induced entry was inhibited by staurosporine and the PKC-selective inhibitor chelerythrine chloride, but was activated by the phosphatase inhibitors okadaic acid and calyculin A. The PMA-promoted calcium influx was also inhibited by omega-agatoxin-TK, a calcium channel blocker specific for Ca(v)2.1 channels. To confirm that a Ca(v)2.1-like calcium channel exists in the mature erythrocyte membrane, RBC membrane preparations were immunoblotted with antiserum against the alpha(1A) subunit of the channel. A polypeptide of the expected molecular weight (190 kDa) was visualized. These studies indicate that an omega-agatoxin-TK-sensitive, Ca(v)2.1-like calcium permeability pathway is present in the RBC membrane and that it may function under the control of kinases and phosphatases.
Andrews
Dina A
DA
Department of Veterinary Pathobiology, Purdue University, 1243 Veterinary Pathology Building, West Lafayette, IN 47907-1243, USA. andrews@purdue.edu
Yang
Lu
L
Low
Philip S
PS
D000328:Adult; D060848:Agatoxins; D000470:Alkaloids; D053119:Benzophenanthridines; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000097:blood*; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D020864:Calcium Channels, N-Type / Q000187:drug effects* / Q000378:metabolism; D020013:Calcium Signaling; D048429:Cell Size / Q000187:drug effects; D004075:Diglycerides / Q000494:pharmacology; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004912:Erythrocytes / Q000187:drug effects*; D006801:Humans; D017136:Ion Transport / Q000187:drug effects; D007476:Ionophores / Q000494:pharmacology; D008387:Marine Toxins; D019319:Okadaic Acid / Q000494:pharmacology; D009996:Osmotic Fragility; D010080:Oxazoles / Q000494:pharmacology; D010617:Phenanthridines / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology*; D013111:Spider Venoms / Q000494:pharmacology*; D019311:Staurosporine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D060848:Agatoxins; D000470:Alkaloids; D053119:Benzophenanthridines; D000001:Calcimycin; D002118:Calcium; D002121:Calcium Channel Blockers; D020864:Calcium Channels, N-Type; D004075:Diglycerides; D004121:Dimethyl Sulfoxide; D004791:Enzyme Inhibitors; D007476:Ionophores; D008387:Marine Toxins; D019319:Okadaic Acid; D010080:Oxazoles; D010617:Phenanthridines; D010749:Phosphoprotein Phosphatases; D011493:Protein Kinase C; D013111:Spider Venoms; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate; C083855:omega-agatoxin-Aa4b; C044297:1,2-didecanoylglycerol; C059041:calyculin A; C016299:chelerythrine