Predominant expression of β1-adrenergic receptor in the thick ascending limb of rat kidney: Absolute mRNA quantitation by reverse transcription and polymerase chain reaction

Abstract

β1- and β2-adrenergic receptor (β-ARs) expression in the thick ascending limb of rat kidney was studied at the level of mRNA and receptor coupling to adenylyl cyclase. Absolute quantitation of β1- and β2-AR mRNAs in microdissected nephron segments was performed with an assay based on reverse transcription and polymerase chain reaction, using in vitro transcribed mutant RNAs as internal standards. In the cortical thick ascending limb (CTAL), the number of mRNA molecules/mm of tubular length was 2,806±328 (n = 12) for β1-AR and 159±26 for β2-AR (P < 0.01). Lower levels were obtained in the medullary thick ascending, β1-AR mRNA still being predominant. The pharmacological properties of β1-ARS was also studied in the CTAL. Cyclic AMP accumulation was stimulated by β-agonist with a rank order of potency of isoproterenol >norepinephrine >epinephrine. This observation, and the higher efficiency of a β1 than of a β2 antagonist to inhibit isoproterenol-induced cAMP accumulation, establish the typical β1-AR sensitivity of the CTAL. No detectable contribution of atypical or β3-ARs to adenylyl cyclase stimulation could be found. In conclusion, this study, which shows markedly different levels of β1- and β2-AR mRNAS in the CTAL, provides a molecular basis for the predominant expression of the β1 receptor subtype in this nephron segment.