Ca21 Uptake and Thapsigargin Sensitivity in Permeabilized Rabbit and Rat Ventricular Myocytes

Ca'+ uptake by the sarcoplasmic reticulum (SR) and free [Ca2"] were measured simultaneously with indo 1 and a Ca'+-selective minielectrode in suspensions of permeabilized rabbit or rat ventricular myocytes ('-'10 mg/mL protein). In the presence of 25 ,umol/L ruthenium red and 10 mmol/L oxalate, the K. for Ca2 uptake by the SR was '250 nmol/L in rabbit and rat ventricular myocytes. The maximal Ca ` uptake rate was 2.4 times higher in rat than in rabbit. Addition of 5 nmol thapsigargin (TG) per milligram cell protein abolished Ca2+ uptake completely in both species. The [TG] necessary for a half-maximal reduction of the uptake rate (K1,2) was 55 pmol/mg cell protein for rabbit and 390 pmol/mg cell protein for rat. Assuming that the number of pump sites is two times the concentration ofTG necessary to inhibit half of the Ca21 pump activity (ie, the TG affinity is very high), the density of pump sites is 7.7 ,mol/kg wet wt for rabbit and 54.6 ,umol/kg wet wt for rat. Despite a fivefold decrease of the Ca2+ uptake rate by a submaximal [TG], the permeabilized myocytes were still able to lower the free [Ca2+] to <150 nmol/L from a peak value > 10 ,umol/L. The relative inhibition of Ca ` uptake by TG did not depend on the free [Ca2+]. Addition of more than 5 nmol TG per milligram cell protein abolished Ca2+ uptake by the SR completely in <15 seconds and reduced the uptake rate by 95% in 5 seconds. We conclude that TG can rapidly and completely inhibit the SR Ca2 pump and that there may be more pump sites in rat than in rabbit ventricle. (Circ Res. 1993;73:820-828.)