Structural and functional properties of adult rat heart myocytes lysed with digitonin.
Low concentrations of digitonin disrupt the sarcolemma of adult rat heart myocytes selectively and completely. When the digitonin lysis is carried out in the presence of 10 mM Mg-ATP, the permeabilized cells retain the rod-cell morphology typical of heart cells in situ and show spontaneous phasic contractions. The rate of contraction is a function of the free Ca2+ concentration from a pCa of 7.2 to 5.2. Higher levels of free Ca2+ result in hypercontracture of the myocytes into round cells with characteristically distorted morphology. The sarcoplasmic reticulum of digitonin-lysed myocytes takes up Ca2+ in an ATP-dependent reaction that is inhibited and reversed by caffeine and strongly enhanced by procaine or ruthenium red. The Ca2+ accumulation has a Km of 0.6 microM Ca2+, depends on Pi (Km of 13 mM), and is strongly inhibited by bicarbonate ion. The hypercontracture of digitonin-lysed myocytes is a function of both the pCa and the Mg-ATP concentration of the suspending medium. Hypercontracture requires ATP. Hypercontracture due to Ca2+ overload occurs at lower Ca2+ concentrations when Mg-ATP is decreased from 10 to 1 mM. However, at low concentrations of Mg-ATP (in the range from 1 to 10 microM), hypercontracture also occurs and is essentially Ca2+-independent. Since hypercontracture of heart myocytes appears analogous to the formation of contraction bands in situ, these observations may be relevant to the phenomena of oxygen paradox and of Ca2+ paradox in intact myocardial tissue.
R A
RA
Altschuld
W C
WC
Wenger
K G
KG
Lamka
O R
OR
Kindig
C C
CC
Capen
V
V
Mizuhira
R S
RS
Vander Heide
G P
GP
Brierley
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D004072:Digitonin / Q000494:pharmacology*; D006321:Heart / Q000187:drug effects; D008854:Microscopy, Electron; D008855:Microscopy, Electron, Scanning; D009200:Myocardial Contraction / Q000187:drug effects*; D009206:Myocardium / Q000648:ultrastructure*; D011343:Procaine / Q000494:pharmacology; D051381:Rats; D012430:Ruthenium Red / Q000494:pharmacology; D012508:Sarcolemma / Q000648:ultrastructure; D012519:Sarcoplasmic Reticulum / Q000378:metabolism
D012430:Ruthenium Red; D002110:Caffeine; D011343:Procaine; D000255:Adenosine Triphosphate; D004072:Digitonin; D002118:Calcium