Ryanodine-sensitive, thapsigargin-insensitive calcium uptake in rat ventricle homogenates

Thapsigargin is a natural product that specifically inhibits all known SERCA calcium pumps with high affinity. We investigated the effects of thapsigargin on cardiac sarcoplasmic reticulum (SR) by measuring the oxalate-supported calcium uptake rate in the unfractionated homogenate and in the isolated SR fraction. The uptake rate in both the isolated SR and unfractionated homogenate are stimulated about two-fold by preincubation with high concentrations of ryanodine, which closes the SR efflux channel. Thapsigargin stoichiometrically and completely inhibited the calcium uptake rate in the isolated SR, both in the presence and absence of SR channel blockade. In contrast, thapsigargin nearly completely inhibited the homogenate calcium uptake only in the absence of SR channel blockade; in the presence of blockade, about 20% of the uptake activity was insensitive to thapsigargin. This result unmasks a thapsigargin-insensitive, ryanodine-sensitive component of calcium uptake in the heart. This activity is in an oxalate-permeable pool and is inhibited by cyclopiazonic acid, another inhibitor of the SERCA calcium pumps. There was no TG-insensitive activity in the rat EDL muscle homogenate. The absence of thapsigargin-insensitive uptake activity in the isolated SR can be attributed to its inactivation during the isolation of the SR. The oxalate permeability and ryanodine sensitivity suggest that the TG-insensitive calcium uptake activity is closely related to the classical SR. The different thapsigargin sensitivities suggests the existence of two kinds of intracellular calcium pumps in the heart.