Identification and function of ryanodine receptor subtype 3 in non-pregnant mouse myometrial cells.
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.
Mironneau
J
J
Laboratoire de Signalisation et Interactions Cellulaires, CNRS UMR 5017, Université Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux, France. jean.mironneau@umr5017.u-bordeaux2.fr
Macrez
N
N
Morel
J L
JL
Sorrentino
V
V
Mironneau
C
C
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D002110:Caffeine / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects; D002478:Cells, Cultured; D004492:Edetic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005260:Female; D051379:Mice; D008810:Mice, Inbred C57BL; D009215:Myometrium / Q000166:cytology / Q000502:physiology; D010121:Oxytocin / Q000494:pharmacology; D010782:Photolysis; D019837:Ryanodine Receptor Calcium Release Channel / Q000276:immunology / Q000502:physiology*
C452519:1-(1,4,5-dimethoxy-2-nitrophenyl) ethylenediaminetetraacetic Acid; D000906:Antibodies; D019837:Ryanodine Receptor Calcium Release Channel; D002110:Caffeine; D010121:Oxytocin; D004492:Edetic Acid