European Journal of Pharmacology. 176 (1990) 241-243 Elsevier

241

EJP 20549

Short communication

Endothelin receptors in cultured renal epithelial cells D i e t e r N e u s e r , Siegfried Zaiss a n d J o h a n n e s - P e t e r Stasch Institute of Pharmacology, Bayer A G, P, O.B. 101709, 5600 Wuppertal 1, F.R.G. Received 16 November 1989, accepted 5 December 1989

We d e m o n s t r a t e d the presence of specific binding sites for endothelin in the renal epithelial cell lines, M D C K and L L C - P K 1. Endothelin binding induced mobilisation of intracellular calcium, as shown by an increase in 45Ca2+ efflux. This suggests a direct effect of endothelin on renal reabsorption in addition to the effects on the renal vasculature.

Endothelin; MDCK cells: LLC-PK 1 cells; Ca 2+ (intracellular): Renal reabsorption

1. Introduction

Endothelin-1, a 21-amino-acid peptide isolated from the supernatant of cultured endothelial cells, shows potent hypertensive effects in vivo and vasoconstrictor activity in vitro (Yanagisawa et al., 1988). In isolated vascular smooth muscle cells endothelin induces a biphasic increase in cytosolic free (Ca2+)~, an increase which consists of an initial (rapid and transient) and a second (sustained plateau) phase, The transient increase in (Ca2+)~ is caused by the release of calcium from intracellular stores (Miasivo et al., 1988), while the sustained increase is probably mediated by activation of L-type Ca 2+ channels (Van Renterghem et al., 1988). Endothelin also has vascular effects in the renal artery, which suggests a role in the control of renal blood flow. On the other hand, the localisation of binding sites in renal non-vascular tissue, renal tubules and papilla, suggests a possible role for endothelin in the regulation renal function (Neuser et al., 1989; Jones et al., 1989). In the present study we investigated the interaction of endothelin with the non-vascular renal

Correspondence to: D. Neuser, Institute of Pharmacology, Bayer AG, P.O.B. 10 17 09, 5600 Wuppertal 1, F.R.G.

epithelial cell lines, the LLC-PK~ and the M D C K cell line. LLC-PK~ cells have been used as model for the proximal tubular epithelium, while M D C K cells are assumed to be of distal tubular or cortical collecting duct origin (Gstraunthaler et al., 1985). In order to demonstrate whether renal epithelial cells respond to endothelin binding with mobilisation of intracellular calcium, we examined the effects of endothelin o n 45Ca2+ efflux.

2. Materials and methods

2.1. Cell culture M D C K and LLC-PK~ cells (American Type Culture Collection) were grown in incubation medium (Dulbecco's MEM, 200 mM glutamine, 500 /~g/ml penicillin, 5000 p,g/ml streptomycin. 10 mM HEPES. 10% fetal calf serum, pH 7.3). For binding experiments, cells were seeded at a density of 2 × 105 cells/ well and incubated overnight.

2.2. Binding experiments Confluent cell layers were washed twice with assay medium (Dulbecco's MEM, 0.5% BSA, pH 7.4, 37°C). The cells were incubated in a final

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Endothelin receptors in cultured renal epithelial cells.

We demonstrated the presence of specific binding sites for endothelin in the renal epithelial cell lines. MDCK and LLC-PK1. Endothelin binding induced...
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