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

. 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.