title: Control of cultured epithelial (MDCK) cell transport function: identification of a beta-adrenoceptor coupled to adenylate cyclase.
abstract: A catecholamine-sensitive adenylate cyclase activity was observed in cell homogenates of cultured renal epithelial (MDCK) cells. 10 microM isoprenaline gave a 2.85-fold increase in adenylate cyclase activity above basal levels. A series of adrenoceptor agonists gave a relative potency series of isoprenaline greater than adrenaline greater than noradrenaline (K1(9) values of 1.9 X 10(-7), 1.6 X 10(-6) and 1.9 X 10(-5) M respectively), consistent with activation of a beta-adrenoceptor. Intracellular accumulation of cyclic AMP was also stimulated by 10 microM isoprenaline, peak values being observed after 2 min, followed by a decline to lower maintained levels. The phosphodiesterase inhibitor isobutylmethylzanthine (1 mM) augmented isoprenaline-stimulated cyclic AMP accumulation. In epithelial preparations of MDCK cells grown upon Millipore filters and mounted in Ussing chambers isoprenaline was only effective in elevating intracellular cyclic AMP contents when applied to the basal cell surfaces. Direct measurement of beta-adrenoceptor density and subtype was determined by (+/-)-3-[125I]iodocyanopindolol binding to MDCK cell homogenates. Binding consisted of a saturable component (Vmax = 14.9 fmol/mg cell protein) of high molar affinity (Kd = 10.8 pM) and a non-saturable component which showed a linear dependence on iodocyanopindolol concentration. In addition to the high-affinity binding site, dissociation kinetics revealed a low-affinity component (Kd = 450 pM) comprising 24% of saturable binding. Competition of (+/-)-3-[125I]iodocyanopindolol binding with beta-adrenoceptor agonists and antagonists was entirely consistent with the existence of a beta 2-adrenoceptor. Examination of various MDCK cultures and clones revealed the existence of MDCK cultures whose adenylate cyclase activity was unresponsive to catecholamine stimulation; this correlated with a reduced or undetectable level of (+/-)-3-[125I]iodocyanopindolol binding. The control of transepithelial chloride transport in MDCK epithelia by catecholamines is discussed.
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism*; D001692:Biological Transport; D002395:Catecholamines / Q000494:pharmacology; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D020120:Iodocyanopindolol; D007668:Kidney / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D010641:Phenotype; D010869:Pindolol / Q000031:analogs & derivatives / Q000378:metabolism; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*
chemical_list: D002395:Catecholamines; D011943:Receptors, Adrenergic, beta; D020120:Iodocyanopindolol; D010869:Pindolol; D000242:Cyclic AMP; D000262:Adenylyl Cyclases