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

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.