Chlorotetracycline fluorescence is a quantitative measure of the free internal Ca2+ concentration achieved by active transport. In situ calibration and application to bovine cardiac sarcolemmal vesicles.
Chlorotetracycline (CTC) fluorescence is shown to be a competent and quantitative measure of the free internal calcium concentration, [Ca2+]i, obtained by ATP supported active uptake by bovine cardiac sarcolemmal (SL) vesicles. The fluorescence response of CTC to [Ca2+]i is calibrated by pre-equilibrating the vesicles with known Ca2+ concentrations and then diluting into a Ca2+-free medium containing CTC. The experiments show that CTC comes into equilibrium with the internal Ca2+ more rapidly than the latter can passively leak from the vesicles. The amplitude of the fluorescence increase is proportional to the Ca2+ concentration with which the vesicles are pre-equilibrated. This constitutes a calibration procedure for the use of CTC fluorescence as a quantitative measure of the free internal Ca2+ concentrations achieved in active transport. This method is applied to the determination of the average free Ca2+ concentrations achieved in ATP-energized uptake with sarcolemmal vesicles. Under optimal conditions an initial rate of 13 mM/min (37 nmol/mg/min) is observed. Uptake reaches a maximum corresponding to 70 mM (179 nmol/mg). Half-maximal values are obtained after 5 min of reaction. The mechanism of the CTC response to free internal Ca2+ concentration is discussed and is compared with measurements of vesicle-associated 45Ca2+.
D000818:Animals; D001693:Biological Transport, Active; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000032:analysis*; D002417:Cattle; D002751:Chlortetracycline / Q000032:analysis*; D005453:Fluorescence; D007700:Kinetics; D008722:Methods; D009206:Myocardium / Q000032:analysis*; D012508:Sarcolemma / Q000032:analysis*
D000001:Calcimycin; D002118:Calcium; D002751:Chlortetracycline