CONTROL OF CULTURED EPITHELIAL (MDCK) CELL TRANSPORT FUNCTION: IDENTIFICATION OF a Β‐ADRENOCEPTOR COUPLED TO ADENYLATE CYCLASE

Abstract

SUMMARYA catecholamine-sensitive adenylate cyclase activity was observed in cell homogenates of cultured renal epithelial (MDCK) cells. 10 4UM isoprenaline gave a 2-85-fold increase in adenylate cyclase activity above basal levels. A series of adrenoceptor agonists gave a relative ptoency series of isoprenaline > adrenaline > noradrenaline (K values of 19 x 10-7, 16 x 10-6 and 19 x l0-, M respectively), consistent with activation of a fl-adrenoceptor. Intracellular accumulation of cyclic AMP was also stimulated by 1O /M isoprenaline, peak values being observed after 2 min, followed by a decline to lower maintained levels. The phosphodiesterase inhibitor isobutylmethylzanthine (1 mM

Chloride secretion stimulated by prostaglandin E1 and by forskolin in a canine renal epithelial cell line.

“…Hassid (1983) in particular has demonstrated that exogenous PGE2 stimulates intracellular cyclic AMP accumulation in the presence of IBMX; in the present experiments, using MDCK cells of a defined phenotype (Rugg & Simmons, 1984b), this finding has been confirmed. In addition it appears that the response is relatively specific for the prostaglandin E series.…”

“…Data is presented that suggest that PGE1, most probably via generation of intracellular cyclic AMP, stimulates an inwards short-circuit current in voltage clamped MDCK epithelial layers mounted in Ussing chambers, consistent with basal to apical Cl-secretion and not with an inhibition of Na+ absorption. Preliminary communications of some of the present findings have been published (Simmons, 1981a;Rugg & Simmons. 1984a).…”

“…For experimeintal purposes MDCK cells were grown as epithelial layers upon millipore filters using high-density seedinlg. exactly as previously described (Barker & Simmons, 1981)' or as subconfluent layers grow-n upoIn plastic Petri dishes (Rugg & Simmons, 1984b (pH 7.4 at 37°C) with 1 % v/v donor horse serum, as previously described (Barker & SimimIlloins. 1981).…”

A cultured human renal epithelioid cell line responsive to vasoactive intestinal peptide

“…VIP activates renal adenylate cyclase and the pattern of VIPstimulated adenylate cyclase along the rabbit nephron is unique amongst renal-acting hormones (Griffiths et al 1988a). (Rugg & Simmons, 1984;Griffiths & Simmons, 1987). Renal cortical homogenates from cat, dog and human renal cortex from nephrectomy samples (performed, in humans, for therapeutic reasons) were prepared for comparative purposes as described previously (Griffiths & Simmons, 1987 ,1 ofI M-HCI.…”

ATTRIBUTION OF [3H]BUMETANIDE BINDING TO THE Na+K+Cl ‘CO‐TRANSPORTER’ IN RABBIT RENAL CORTICAL PLASMA MEMBRANES: A CAVEAT

“…1 1) was determined using L-leucine-p-nitroanilide as substrate in a 0 1 M phosphate buffer (pH 7-2). Adenylate cyclase activity was measured exactly as described by Rugg & Simmons (1984), except that the final incubation pH was 7-5. Maximal activity of the catalytic unit was taken as that activity (minus basal levels) stimulated by 100/uM-forskolin.…”