Acetylcholine induces Ca2+ oscillations via m3/m4 muscarinic receptors in the mouse oocyte.
Changes in intracellular Ca2+ concentration are required for the activation of mammalian oocytes. They are caused mainly by Ca2+ release from the endoplasmic reticulum (ER) via Ins P3 receptors (Ins P3R). Several studies have reported that acetylcholine (ACh) is capable of triggering early activation events in mouse oocytes over-expressed with the m1 muscarinic ACh receptor (m1AChR). Here we examined which subtypes of the mAChR (m1 to m4) are involved in the generation of Ca2+ oscillations in native mouse oocytes. ACh (10 microM) elicited regular Ca2+ oscillations similar to those induced by sperm in their temporal characteristics. The Ca2+ oscillations were abolished by application with atropine, the mAChR inhibitor. Within 1 min after treatment of ACh, intracellular Fluo-3 fluorescence intensity increased from 794+/-119 to 2023+/-755 (increase to 250% of original value), indicating a strong rise of cytosolic Ca2+ concentration. 4-DAMP mustard and Tropicamide, specific antagonists of m3AChR and m4AChR, completely abolished ACh-induced Ca2+ oscillations. In the ovulated oocytes, the expression of m3/m4 AChR was clearly detected by RT-PCR analysis. Furthermore, ACh-induced Ca2+ oscillations were also abolished or decreased by PLC inhibitors (U73122 or D609) and an Ins P3-receptor antagonist (xestospongin C), confirming that ACh generates Ca2+ oscillations via the PLC-Ins P3 (PI) pathway. These results strongly suggest that m3/m4AChR is coupled to the generation of Ca2+ oscillations mainly via the PI pathway in mouse oocytes.
Kang
Dawon
D
Department of Physiology, College of Medicine and Institute of Health Sciences, Gyeongsang National University, 90 Chilam-Dong, 660-751 Jinju, Korea.
Park
Jae-Yong
JY
Han
Jaehee
J
Bae
In-Ha
IH
Yoon
Sook-Young
SY
Kang
Sang Soo
SS
Choi
Wan Sung
WS
Hong
Seong-Geun
SG
D000109:Acetylcholine / Q000502:physiology*; D000818:Animals; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D005260:Female; D051379:Mice; D008813:Mice, Inbred ICR; D009865:Oocytes / Q000187:drug effects / Q000502:physiology*; D043587:Receptor, Muscarinic M3 / Q000819:agonists / Q000502:physiology*; D043588:Receptor, Muscarinic M4 / Q000819:agonists / Q000502:physiology*
D043587:Receptor, Muscarinic M3; D043588:Receptor, Muscarinic M4; D000109:Acetylcholine