Acetylcholine Increases the Free Intracellular Calcium Concentration in Podocytes in Intact Rat Glomeruli via Muscarinic M 5 Receptors ROLAND NITSCHKE,* ANNA HENGER, † SIGRID RICKEN,* VICTORIA MU ¨ LLER,* MICHAEL KO ¨ TTGEN,* MARTIN BEK, † and HERMANN PAVENSTA ¨ DT † *Institute of Physiology and † Department of Medicine, Division of Nephrology, Albert-Ludwigs-Universita ¨t Freiburg, Freiburg, Germany. Abstract. The effects of acetylcholine (ACh) on the free intra- cellular calcium concentration ([Ca 2+ ] i ) of microdissected glo- meruli were investigated using fura-2 fluorescence digital im- aging and two-photon confocal microscopy. ACh caused a concentration-dependent [Ca 2+ ] i increase 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 nicardi- pine, whereas gadolinium and lanthanum (both at 1 M) blocked the plateau. Diphenylacetoxy-N-methylpiperidine me- thiodide (100 nM), an M 3 /M 5 receptor antagonist, and pirenz- epine (1 M), 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 (ACh) modulates renal medullary circulation, induces vasodilation in isolated perfused kidneys, increases renal plasma flow, and produces natriuresis and diuresis (1). The effects of ACh can be modulated by locally produced hormones; for example, inhibition of cyclooxygenases by in- domethacin reversed the effects of ACh on renal plasma flow, natriuresis, and diuresis (2,3). ACh is probably released from cholinergic renal nerves, as has been directly demonstrated in kidney cortex slices (4,5). Knowledge regarding the role of ACh in glomeruli is limited. ACh increases glomerular intra- cellular cGMP accumulation and produces glomerular contrac- tions, probably via a Ca 2+ -dependent mechanism (6,7). Lebrun et al. (8) demonstrated that the parietal sheet of Bowman’s capsule and single intact glomeruli exhibit intracellular cal- cium concentration ([Ca 2+ ] i ) increases in response to ACh. However, it was unclear, because of the limitations of conven- tional fluorescence assays, which of the three resident cell types of the glomeruli, i.e., podocytes, endothelial cells, or mesangial cells, responded to ACh (8). Each of the three glomerular cell types can be propagated in cell culture. It has been demonstrated that cultured glomerular endothelial cells do not respond to ACh with [Ca 2+ ] i increases (9) and, to our knowledge, ACh-mediated [Ca 2+ ] i increases in mesangial cells or podocytes have not been reported. However, the interpreta- tion of possible effects of ACh in cultured cells is difficult, because many cell types that possess muscarinic receptors in vivo lose their muscarinic receptors under in vitro conditions. In addition, changes in the expression of muscarinic receptor subtypes may occur under cell culture conditions (10). To overcome these problems, we recently developed a technique that allows measurement of [Ca 2+ ] i in intact glomeruli (11). The aim of this study was to investigate whether ACh affects [Ca 2+ ] i in intact glomeruli and whether podocytes are involved in the [Ca 2+ ] i response to ACh. Materials and Methods Isolation and Preparation of Glomeruli The method for the preparation of isolated glomeruli was previ- ously described in detail (12). In brief, rat glomeruli were obtained using the sieving technique (the sieve sizes used were, in descending order, 150, 100, and 50 mesh size). A single glomerulus (with intact capsule) was transferred into a bath chamber mounted on the stage of an inverted microscope. The glomerulus was maintained at room temperature and immobilized at the vascular pole with a holding glass pipette. After incubation with 1 g/liter collagenase IV (collagenase A, catalog number C 5138; activity, 369 U/mg solid; Sigma Chemical Co., Deisenhofen, Germany) for 1 to 2 min and subsequent wash-off of the collagenase, the capsule was mechanically stripped off with a Received July 13, 1999. Accepted September 12, 2000. Correspondence to Prof. Dr. Hermann Pavensta ¨dt, Department of Medicine, Division of Nephrology, Albert-Ludwigs-Universita ¨t Freiburg, Hugstetter- Strasse 55, D-79106 Freiburg, Germany. Phone: 0761-270-3270; Fax: 0761- 270-3245; E-mail: nitschro@ruf.uni-freiburg.de 1046-6673/1204-0678 Journal of the American Society of Nephrology Copyright © 2001 by the American Society of Nephrology J Am Soc Nephrol 12: 678 –687, 2001