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. The effects of acetylcholine (ACh) on the free intracellular calcium concentration ([Ca 2+ ] i ) of microdissected glomeruli were investigated using fura-2 fluorescence digital imaging and two-photon confocal microscopy. ACh caused a concentration-dependent [Ca 2+ ] i  increases with an initial peak followed by a sustained plateau, which was suppressed by reduced extracellular Ca 2+  concentrations. The [Ca 2+ ] i  plateau was not affected by the L-type Ca 2+  channel blocker nicardipine, whereas gadolinium and lanthanum (both at 1 μM) blocked the plateau. Diphenylacetoxy- N -methylpiperidine methiodide (100 nM), an M 3 /M 5  receptor antagonist, and pirenzepine (1 μM), an M 1  receptor antagonist, completely inhibited the effect of ACh. [Ca 2+ ] i  measurements using two-photon excitation of fluo-3 and staining of the cells with calcein/acetoxymethyl ester, for observation of the capillary network together with the glomerular cells, showed that [Ca 2+ ] i  was increased in single podocytes. Immunohistochemical studies did not demonstrate M 3  receptor expression in glomerular cells. M 1  receptors could be detected only in the parietal sheet of Bowman's capsule, whereas M 5  receptors were found only in podocytes. The data show that ACh increases [Ca 2+ ] i  in podocytes of intact glomeruli, most likely via muscarinic M 5  receptors.

Acetylcholine Increases the Free Intracellular Calcium Concentration in Podocytes in Intact Rat Glomeruli via Muscarinic M5 Receptors