Molecular analysis of vasopressin receptors in the rat nephron. Evidence for alternative splicing of the V₂ receptor

Abstract

Expression and regulation of vasopressin V₂ and V_1a receptors were studied at the mRNA level in the rat kidney. Two V₂ mRNA variants were identified and shown to arise from a single gene by alternative splicing using one donor and two different acceptor sites. The long (V_2L) form encodes the adenylyl cyclase-coupled receptor. The short (V_2S) form lacks the nucleotide sequence encoding the putative seventh transmembrane domain and undergoes a frame shift in its 3'end coding region; it is inactive on the cyclase pathway in transfected cells. Measurement of mRNAs, carried out by quantitative reverse transcription-polymerase chain reaction (RT-PCR) on microdissected nephrons, demonstrated that neither V_2L, V_2S nor V_1A mRNAs are expressed in glomeruli and proximal tubules (<100 mRNA copies/glomerulus or mm of tubular length), whereas they are present in the ascending limb of Henle's loop and in the collecting tubule. The V_2L mRNA, which is always predominant in these structures, is expressed throughout the collecting tubule at 10 times higher levels (30,000 copies/mm) than in the thin and thick ascending limbs. The ratio of the V_2S over V_2L mRNA is constant (15%) in all nephron segments; hence high V_2S levels are only observed in the collecting tubule. The V_1A mRNA is slightly expressed in the thin ascending limb, absent in the thick ascending limb and reaches its maximum in the cortical collecting duct (4,000 copies/mm), before gradually decreasing to undetectable levels in the terminal collecting duct. Finally, in vivo administration of a vasopressin V₂ agonist decreased by 50% V_2L and V_2S mRNAs, but did not alter the V_1A mRNA level. We conclude that this study provides the quantitation, on a molar basis, of vasopressin receptor mRNAs in kidney tubules and demonstrates the occurence of two V₂ mRNA spliced variants which are similarly down-regulated.