Identification and function of ryanodine receptor subtype 3 in non‐pregnant mouse myometrial cells

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Abstract
Subtype 3 of the ryanodine receptor (RYR3) is a ubiquitous Ca2+ release channel which is predominantly expressed in smooth muscle tissues and certain regions of the brain. We show by reverse transcription‐polymerase chain reaction (RT‐PCR) that non‐pregnant mouse myometrial cells expressed only RYR3 and therefore could be a good model for studying the role of endogenous RYR3. Expression of RYR3 was confirmed by Western blotting and immunostaining. Confocal Ca2+ measurements revealed that in 1.7 mm extracellular Ca2+, neither caffeine nor photolysis of caged Ca2+ were able to trigger any Ca2+ responses, whereas in the same cells oxytocin activated propagated Ca2+ waves. However, under conditions of increased sarcoplasmic reticulum (SR) Ca2+ loading, brought about by superfusing myometrial cells in 10 mm extracellular Ca2+, all the myometrial cells responded to caffeine and photolysis of caged Ca2+, indicating that it was possible to activate RYR3. The caffeine‐induced Ca2+ responses were inhibited by intracellular application of an anti‐RYR3‐specific antibody. Immunodetection of RYR3 with the same antibody revealed a rather homogeneous distribution of fluorescence in confocal cell sections. In agreement with these observations, spontaneous or triggered Ca2+ sparks were not detected. In conclusion, our results suggest that under conditions of increased SR Ca2+ loading, endogenous RYR3 may contribute to the Ca2+ responses of myometrial cells.