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Acetylcholine induces Ca(2+) oscillations via m3/m4 muscarinic receptors in the mouse oocyte
Kang D, et al .
Changes in intracellular Ca(2+) concentration are required for the activation of mammalian oocytes. They are caused mainly by Ca(2+) release from the endoplasmic reticulum (ER) via Ins P(3) receptors (Ins P(3)R). 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 Ca(2+) oscillations in native mouse oocytes. ACh (10 microM) elicited regular Ca(2+) oscillations similar to those induced by sperm in their temporal characteristics. The Ca(2+) 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 Ca(2+) concentration. 4-DAMP mustard and Tropicamide, specific antagonists of m3AChR and m4AChR, completely abolished ACh-induced Ca(2+ )oscillations. In the ovulated oocytes, the expression of m3/m4 AChR was clearly detected by RT-PCR analysis. Furthermore, ACh-induced Ca(2+) oscillations were also abolished or decreased by PLC inhibitors (U73122 or D609) and an Ins P(3)-receptor antagonist (xestospongin C), confirming that ACh generates Ca(2+) oscillations via the PLC-Ins P(3) (PI) pathway. These results strongly suggest that m3/m4AChR is coupled to the generation of Ca(2+) oscillations mainly via the PI pathway in mouse oocytes.
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