Co-expression of a Ca2+-inhibitable adenylyl cyclase and of a Ca2+-sensing receptor in the cortical thick ascending limb cell of the rat kidney. Inhibition of hormone-dependent cAMP accumulation by extracellular Ca2+.

The Ca2+-sensing receptor protein and the Ca2+-inhibitable type 6 adenylyl cyclase mRNA are present in a defined segment of the rat renal tubule leading to the hypothesis of their possible functional co-expression in a same cell and thus to a possible inhibition of cAMP content by extracellular Ca2+. By using microdissected segments, we compared the properties of regulation of extracellular Ca2+-mediated activation of Ca2+ receptor to those elicited by prostaglandin E2 and angiotensin II. The three agents inhibited a common pool of hormone-stimulated cAMP content by different mechanisms as follows. (i) Extracellular Ca2+, coupled to phospholipase C activation via a pertussis toxin-insensitive G protein, induced a dose-dependent inhibition of cAMP content (1.25 mM Ca2+ eliciting 50% inhibition) resulting from both stimulation of cAMP hydrolysis and inhibition of cAMP synthesis; this latter effect was mediated by capacitive Ca2+ influx as well as release of intracellular Ca2+. (ii) Angiotensin II, coupled to the same transduction pathway, also decreased cAMP content; however, its inhibitory effect on cAMP was mainly accounted for by an increase of cAMP hydrolysis, although angiotensin II and extracellular Ca2+ can induce comparable release of intracellular Ca2+. (iii) Prostaglandin E2, coupled to pertussis toxin-sensitive G protein, inhibited the same pool of adenylyl cyclase units as extracellular Ca2+ but by a different mechanism. The functional properties of the adenylyl cyclase were similar to those described for type 6. The results establish that the co-expression of a Ca2+-inhibitable adenylyl cyclase and of a Ca2+-sensing receptor in a same cell allows an inhibition of cAMP accumulation by physiological concentrations of extracellular Ca2+.

Localization of mRNAs encoding Ca2+-inhibitable adenylyl cyclases along the renal tubule. Functional consequences for regulation of the cAMP content.

Expression of Ca2+-inhibitable types V and VI adenylyl cyclases was studied by reverse transcription-polymerase chain reaction in rat renal glomeruli and nephron segments isolated by microdissection. Quantitation of each mRNA was achieved using a mutant cRNA which differed from the wild type by substituting two bases to create a new restriction site in the corresponding cDNA. Type VI mRNA was present all along the nephron but was more abundant in distal than in proximal segments. The expression of type V mRNA was restricted to the glomerulus and to the initial portions of the collecting duct. Expression of the Ca2+-insensitive type IV mRNA studied on the same samples was evidenced only in the glomerulus. The functional relevance of the expression of Ca2+-inhibitable isoforms was studied by measuring cAMP content in the microdissected outer medullary collecting duct which expressed both type V mRNA (2367 +/- 178 molecules/mm tubular length; n = 8) and type VI mRNA (5658 +/- 543 molecules/mm, n = 8). Agents known to increase intracellular Ca2+ in this segment induced a Ca2+-dependent inhibition on either arginine vasopressin- or glucagon-stimulated cAMP level. The characteristics of these inhibitions suggest a functional and differential expression of types V and VI adenylyl cyclases in two different cell types of the rat outer medullary collecting duct.

Quantitative RT-PCR analysis of calcitonin receptor mRNAs in the rat nephron.

A quantitative assay based on the method of reverse transcription and polymerase chain reaction (RT-PCR) was developed to study the expression of calcitonin (CT) receptors in microdissected rat nephron segments. Steady-state mRNA levels of two CT-receptor spliced variants (CT1a and CT1b) were measured using a mutant cRNA as internal standard. CT1a, but not the CT1b isoform, was detected in the kidney cortex, outer medulla, and papilla. Among the tested segments, predominant expression of CT1a mRNA was found in the cortical thick ascending limb of Henle's loop (754 +/- 87 mRNA molecules/mm tubular length; n = 8). Lower expression levels were measured in the medullary thick ascending limb (460 +/- 62 molecules/mm tubule length; n = 7) and in the cortical collecting duct (327 +/- 61 molecules/mm tubule length; n = 6). A weak expression was also detected in the outer medullary collecting duct and the glomerulus. No expression was found in the proximal convoluted tubule, pars recta, and thin descending and thin ascending limb of Henle's loop. We conclude that only the CT1a-receptor mRNA is present in the rat kidney, with a significant level of expression in the cortical and medullary thick ascending limb and in the cortical collecting duct.

Inverse PCR-mediated cloning of the promoter for the rat vasopressin V2 receptor gene.

The adenylyl cyclase-coupled vasopressin V2 receptor has been cloned recently and shown, in rats, to be produced from the predominant form of two alternate spliced variants. To begin to unravel the transcriptional regulation of this receptor, we have isolated the 5' flanking region of the rat vasopressin V2 receptor gene and characterized its promoter sequence. The method of inverse polymerase chain reaction (PCR), which allows the amplification of DNA fragments adjacent to a segment of known sequence, was used as an alternative approach to genomic DNA library screening. Using a probe encompassing part of the coding region, first we identified by Southern blot analysis, a single BstX I hybridizing fragment of 2.3 kilobases (kb). This size predicted a BstX I restriction site 1.5 kb upstream to the gene coding region. Cloning of this fragment was accomplished through circularization of BstX I restriction digests and inverse PCR-mediated amplification. Sequence analysis of the gene 5' flanking domain enabled the design of oligonucleotide primers with the usual forward/reverse orientation, and additional clones were generated from native genomic DNA using a high fidelity thermoresistant DNA polymerase. Reverse transcription-PCR (RT-PCR) and primer extension analysis mapped the major transcription start site 422 nucleotides upstream to the translation initiation codon. The promoter region lacks a TATA box but contains a CAAT box and a consensus binding site for transcription factor Sp1. Multiple potential binding sites for the transcription factor PEA3 are clustered in two DNA portions located 0.6 kb and 1 kb upstream to the coding region. In addition, sequences homologous to glucocorticoid response elements are present and might be responsible for the regulation by adrenal steroids of vasopressin-dependent adenylyl cyclase activity in the kidney.

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

Expression and regulation of vasopressin V2 and V1a 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 (V2L) form encodes the adenylyl cyclase-coupled receptor. The short (V2S) 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 V2L, V2S nor V1A 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 V2L 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 V2S over V2L mRNA is constant (15%) in all nephron segments; hence high V2S levels are only observed in the collecting tubule. The V1A 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% V2L and V2S mRNAs, but did not alter the V1A mRNA level. We conclude that this study provides the quantitation, on a molar basis, of vasopressin receptor mRNAs in kidney tubules and demonstrates the occurrence of two V2 mRNA spliced variants which are similarly down-regulated.

Predominant expression of beta 1-adrenergic receptor in the thick ascending limb of rat kidney. Absolute mRNA quantitation by reverse transcription and polymerase chain reaction.

Beta 1- and beta 2-adrenergic receptor (beta-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 beta 1- and beta 2-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 beta 1-AR and 159 +/- 26 for beta 2-AR (P < 0.01). Lower levels were obtained in the medullary thick ascending, beta 1-AR mRNA still being predominant. The pharmacological properties of beta-ARS was also studied in the CTAL. Cyclic AMP accumulation was stimulated by beta-agonist with a rank order of potency of isoproterenol > norepinephrine > epinephrine. This observation, and the higher efficiency of a beta 1 than of a beta 2 antagonist to inhibit isoproterenol-induced cAMP accumulation, establish the typical beta 1-AR sensitivity of the CTAL. No detectable contribution of atypical or beta 3-ARs to adenylyl cyclase stimulation could be found. In conclusion, this study, which shows markedly different levels of beta 1- and beta 2-AR mRNAS in the CTAL, provides a molecular basis for the predominant expression of the beta 1 receptor subtype in this nephron segment.