Contribution of ryanodine receptor subtype 3 to Ca2+ responses in Ca2+-overloaded cultured rat portal vein myocytes

Using an antisense strategy, we have previously shown that in vascular myocytes, subtypes 1 and 2 of ryanodine receptors (RYRs) are required for Ca2+ release during Ca2+ sparks and global Ca2+ responses, evoked by activation of voltage-gated Ca2+ channels, whereas RYR subtype 3 (RYR3) has no contribution. Here, we investigated the effects of increased Ca2+ loading of the sarcoplasmic reticulum (SR) on the RYR-mediated Ca2+ responses and the role of the RYR3 by injecting antisense oligonucleotides targeting the RYR subtypes, RYR3 expression was demonstrated by immunodetection in both freshly dissociated and cultured rat portal vein myocytes, Confocal Ca2+ measurements revealed that the number of cells showing spontaneous Ca2+ sparks was strongly increased by superfusing the vascular myocytes in 10 mM Ca2+-containing solution. These Ca2+ sparks were blocked after inhibition of RYR1 or RYR2 by treatment with antisense oligolucleotides but not after inhibition of RYR3, In contrast, inhibition of RYR3 reduced the global Ca2+ responses induced by caffeine and phenylephrine, indicating that RYR3 participated together with RYR1 and RYR2 to these Ca2+ responses in Ca2+-overloaded myocytes. Ca2+ transients evoked by photolysis of caged Ca2+ with increasing flash intensities were also reduced after inhibition of RYR3 and revealed that the [Ca2+](i) sensitivity of RYR3 would be similar to that of RYR1 and RYR2. Our results show that, under conditions of increased SR Ca2+ loading, the RYR3 becomes activable by caffeine and local increases in [Ca2+](i).