Abstract
Membrane guanylate cyclase C (GC-C) is the receptor for guanylin, uroguanylin, and heat-stable enterotoxin (STa) in the intestine. GC-C-deficient mice show resistance to STa in intestine but saluretic and diuretic effects of uroguanylin and STa are not disturbed. Here we describe the cellular effects of these peptides using immortalized human kidney epithelial (IHKE-1) cells with properties of the proximal tubule, analyzed with the slow-whole-cell patch clamp technique. Uroguanylin (10 or 100 nm) either hyperpolarized or depolarized membrane voltages (V(m)). Guanylin and STa (both 10 or 100 nm), as well as 8-Br-cGMP (100 microm), depolarized V(m). All peptide effects were absent in the presence of 1 mm Ba(2+). Uroguanylin and guanylin changed V(m) pH dependently. Pertussis toxin (1 microg/ml, 24 h) inhibited hyperpolarizations caused by uroguanylin. Depolarizations caused by guanylin and uroguanylin were blocked by the tyrosine kinase inhibitor, genistein (10 microm). All three peptides increased cellular cGMP. mRNA for GC-C was detected in IHKE-1 cells and in isolated human proximal tubules. In IHKE-1 cells GC-C was also detected by immunostaining. These findings suggest that GC-C is probably the receptor for guanylin and STa. For uroguanylin two distinct signaling pathways exist in IHKE-1 cells, one involves GC-C and cGMP as second messenger, the other is cGMP-independent and connected to a pertussis toxin-sensitive G protein.
Highlights
Membrane guanylate cyclase C (GC-C) is the receptor for guanylin, uroguanylin, and heat-stable enterotoxin (STa) in the intestine
Our study describes for the first time cellular actions of GN, UGN, and STa in human kidney, IHKE-1 cells [22], analyzed with the slow-whole-cell patch clamp technique. mRNA of GC-C was detected in IHKE-1 cells, human kidney, and in isolated human proximal tubules
Since UGN- and STa-induced natriuresis and kaliuresis in vivo are not inhibited in GC-C-deficient mice [21] another so far unknown signaling pathway for UGN, STa, and GN, distinct from GC-C, has to be assumed in the kidney
Summary
Cell Culture—IHKE-1 cells derived from the proximal tubule were cultured in Dulbecco’s modified Eagle’s medium and Ham’s F-12 medium (1:1) containing in addition: 15 mM Hepes (pH 7.3), 1.6 nM epidermal growth factor, 100 nM hydrocortisone, 65 nM transferrin, 0.84 M insulin, 29 nM Na2SeO3, 15 mM NaHCO3, 4 mM L-Gln, 5 ml/liter Ciprobay 100 (Bayer, Leverkusen, Germany), and 1% fetal calf serum as described before [23, 24]. For patch clamp experiments IHKE-1 cells were grown on glass coverslips. Cells were cultured in Dulbecco’s modified Eagle’s medium containing in addition: 1% fetal calf serum, 100 000 IU/liter penicillin, 0.1 g/liter streptomycin, and 1 mM L-Gln in an atmosphere of 5% CO2, 95% air at 37 °C. Monolayers of IHKE-1 cells were incubated with medium containing 1 g/ml PT for 4 or 24 h before experiments were performed. Cells were incubated for another 10 min with culture medium containing IBMX or IBMX and genistein, and UGN, GN, STa, or ANP (as positive control). Cells were washed six times for 5 min each with PBS and incubated with fluorescein isothiocyanate-tagged anti-mouse IgG (Invitrogen, New Delhi, India) diluted 1:100 in blocking buffer for 1 h at room temperature. A p value Ͻ 0.05 was considered significant and is indicated by an asterisk
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