Ca2+ displacement by Polymyxin B from sarcolemma isolated by 'gas dissection' from cultured neonatal rat myocardial cells.
Amphiphilic, cationic Polymyxin B is shown to displace Ca2+ from 'gas dissected' cardiac sarcolemma in a dose-dependent, saturable fashion. The Ca2+ displacement is only partially reversible, 57% and 63%, in the presence of 1 mM or 10 mM Ca2+, respectively. Total Ca2+ displaced by a non-specific cationic probe, lanthanum (La3+), at maximal displacing concentration (1 mM) was 0.172 +/- 0.02 nmol/microgram membrane protein. At 0.1 mM, Polymyxin B displaced 42% of the total La3+-displaceable Ca2+ or 0.072 +/- 0.01 nmol/microgram protein. 5 mM Polymyxin displaced Ca2+ in amounts equal to those displaced by 1 mM La3+. Pretreatment of the membranes with neuraminidase (removal of sialic acid) and protease leads to a decrease in La3+-displaceable Ca2+ but to an increase in the fraction displaced by 0.1 mM Polymyxin from 42% to 54%. Phospholipase D (cabbage) treatment significantly increased the La3+-displaceable Ca2+ to 0.227 +/- 0.02 nmol/microgram protein (P less than 0.05), a gain of 0.055 nmol. All of this phospholipid specific increment in bound Ca2+ was displaced by 0.1 mM Polymyxin B. The results suggest that Polymyxin B will be useful as a probe for phospholipid Ca2+-binding sites in natural membranes.
D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D007700:Kinetics; D009206:Myocardium / Q000378:metabolism*; D011112:Polymyxin B / Q000494:pharmacology*; D011113:Polymyxins / Q000494:pharmacology*; D051381:Rats; D012508:Sarcolemma / Q000187:drug effects / Q000378:metabolism*
D011113:Polymyxins; D011112:Polymyxin B; D002118:Calcium