Molecular and functional identification of beta-adrenergic receptors in distal convoluted tubule cells

Renal nerve stimulation or circulating catecholamines activate the beta-adrenergic receptors that mediate direct effects on tubular transport. Three subtypes of beta-adrenergic receptors have been characterized: beta(1), beta(2), and beta(3) beta-Adrenergic-receptor effects on Na+ and Ca2+ transport in distal convoluted tubules (DCT) have not been established. The focus of this study was to 1) identify the subtypes of beta-adrenergic receptors in DCT cells and 2) examine functional responses to beta-receptor activation on adenosine 3',5'-cyclic monophosphate (cAMP) formation and Na+ and Ca2+ entry. To determine the subtypes of beta-receptors present, RNA isolated from immortalized mouse DCT cells was reverse transcribed, and the cDNA was amplified using primers designed to reported sequences for beta(1)-, beta(2)- and beta(3)-receptor subtypes. Products of the appropriate sizes were obtained with beta(1)- and beta(2)-primers. No product was observed with primers to the ps sequence. Receptor products were confirmed by sequencing and are identical to reported mouse beta(1)- and beta(2)-receptor sequence. Receptor binding of [H-3]dihydroalprenolol was 123 +/- 13 fmol/mg protein, and a 3:1 ratio of beta(1)- to beta(2)-receptors was observed with DCT cell membranes. Isoproterenol, a beta-receptor agonist, increased cAMP formation 8.5-fold. Pretreatment with the antagonist propranolol abolished agonist-induced cAMP accumulation. Isoproterenol significantly increased Na-22(+) uptake to 345 +/- 23 compared with a basal rate of 256 +/- 12 nmol min(-1) mg protein(-1) and was blocked with propranolol and beta(1)- and beta(2)-selective antagonists. Isoproterenol had no effect on Ca-45(2+) entry into DCT cells. In summary, DCT cells express three times more beta(1)-than beta(2)-receptors and express no detectable beta(3)-adrenergic receptors. beta-Receptors couple to adenylyl cyclase, and activation of beta-adrenergic receptors increases Na+ but not Ca2+ entry in DCT cells.