SARCOPLASMIC-RETICULUM CA-2+ UPTAKE AND THAPSIGARGIN SENSITIVITY IN PERMEABILIZED RABBIT AND RAT VENTRICULAR MYOCYTES

Ca2+ uptake by the sarcoplasmic reticulum (SR) and free [Ca2+] were me asured simultaneously with indo 1 and a Ca2+-selective minielectrode i n suspensions of permeabilized rabbit or rat ventricular myocytes (alm ost-equal-to 10 mg/mL protein). In the presence of 25 mumol/L rutheniu m red and 10 mmol/L oxalate, the K(m) for Ca2+ uptake by the SR was al most-equal-to 250 nmol/L in rabbit and rat ventricular myocytes. The m aximal Ca2+ uptake rate was 2.4 times higher in rat than in rabbit. Ad dition of 5 nmol thapsigargin (TG) per milligram cell protein abolishe d Ca2+ uptake completely in both species. The [TG] necessary for a hal f-maximal reduction of the uptake rate (K1/2) was 55 pmol/mg cell prot ein for rabbit and 390 pmol/mg cell protein for rat. Assuming that the number of pump sites is two times the concentration of TG necessary t o inhibit half of the Ca2+ pump activity (ie, the TG affinity is very high), the density of pump sites is 7.7 mumol/kg wet wt for rabbit and 54.6 mumol/kg wet wt for rat. Despite a fivefold decrease of the Ca2 uptake rate by a submaximal [TG], the permeabilized myocytes were sti ll able to lower the free [Ca2+] to < 150 nmol/L from a peak value > 1 0 mumol/L. The relative inhibition of Ca2+ uptake by TG did not depend on the free [Ca2+]. Addition of more than 5 nmol TG per milligram cel l protein abolished Ca2+ uptake by the SR completely in <15 seconds an d 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.