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

Abstract

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 beta 3 sequence. Receptor products were confirmed by sequencing and are identical to reported mouse beta 1- and beta 2-receptor sequence. Receptor binding of[3H]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 22Na+ 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 45Ca2+ 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.