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.

A SAGE approach to identifying novel trans-acting factors involved in the X inactivation process.

X chromosome inactivation ensures the dosage compensation of X-linked genes in XX females compared to their XY male counterpart. It is characterised by the specific recruitment of an inhibitory ribonucleoprotein complex involving the non-coding Xist RNA to the presumptive inactive X chromosome and associated chromatin modifications, which result in the transcriptional silencing of the X chromosome. As an approach to the identification of some of the potential molecular players in this process we have performed comparative transcriptional profiling of mouse 6.5-dpc (days post-coitum) female and male embryos using a modified SAGE (Serial analysis of gene expression) technique which allows the analysis of small quantities of biological material. At 6.5 dpc, a moment when random X inactivation of embryonic tissues has just been achieved, some two hundred transcripts that were significantly enriched in the female gastrula compared to its male counterpart could be identified. The validation of an association with the X inactivation process of a subset of these transcripts has been studied, ex vivo, in differentiating female and male ES cells and in female ES cells in which the establishment of X inactivation is interrupted through the post-transcriptional inhibition of Xist synthesis.

Functional properties of Ca2+-inhibitable type 5 and type 6 adenylyl cyclases and role of Ca2+ increase in the inhibition of intracellular cAMP content.

Among the different adenylyl cyclase (AC) isoforms, type 5 and type 6 constitute a subfamily which has the remarkable property of being inhibited by submicromolar Ca2+ concentrations in addition to Galphai-mediated processes. These independent and cumulative negative regulations are associated to a low basal enzymatic activity which can be strongly activated by Galphas-mediated interactions or forskolin. These properties ensure possible wide changes of cAMP synthesis. Regulation of cAMP synthesis by Ca2+ was studied in cultured or native cells which express naturally type 5 and/or type 6 AC, including well-defined renal epithelial cells. The results underline two characteristics of the inhibition due to agonist-elicited increase of intracellular Ca2+: i) Ca2+ rises achieved through capacitive Ca2+ entry or intracellular Ca2+ release can inhibit AC to a similar extent; and ii) in a same cell type, different agonists inducing similar overall Ca2+ rises elicit a variable inhibition of AC activity. The results suggest that a high efficiency of AC regulation by Ca2+ is linked to a requisite close localization of AC enzyme and Ca2+ rises.

Angiotensin II potentiates vasopressin-dependent cAMP accumulation in CHO transfected cells. Mechanisms of cross-talk between AT1A and V2 receptors.

The V2 vasopressin and the AT1A angiotensin II receptors are respectively coupled to the adenylyl cyclase and the phosphoinositide pathways. The cross-talk between these two receptors and their transduction pathways were investigated in CHO cells transfected with cDNA of both AT1A and V2 receptors. In these cells, angiotensin II induced an increase in intracellular calcium, and vasopressin a rise in intracellular cAMP accumulation. The simultaneous addition of angiotensin II and vasopressin potentiated the production of cAMP by the V2 receptor. This potentiation was dose-dependent and, at a concentration of 10(-7) M angiotensin II, the accumulation of cAMP was 4-fold greater than that induced by 10(-7) M vasopressin alone. Such cross-talk occurred in the presence and absence of cyclic nucleotide phosphodiesterase inhibitors, indicating that inhibition of phosphodiesterase activity was not the principal cause of potentiation. This was confirmed by the absence of calcium-inhibitable isoforms of phosphodiesterases in CHO cells. The addition of angiotensin II to forskolin, which stimulates the adenylyl cyclase, did not modify the production of cAMP. Phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), partially mimicked, and staurosporine, an inhibitor of PKC, partially inhibited the effect of angiotensin II on vasopressin. Chelation of intracellular calcium with BAPTA-AM markedly reduced the potentiation of V2 receptor by angiotensin II. However, increase in intracellular calcium with thapsigargin did not modify the cAMP accumulation induced by vasopressin. It was concluded that, in CHO cells, activation of the AT1A receptor by angiotensin II potentiates the V2 receptor through activation of protein kinase C in the presence of intracellular calcium at a step located between the receptor and the adenylyl cyclase.

Involvement of G protein-coupled receptor kinases and arrestins in desensitization to follicle-stimulating hormone action.

FSH rapidly desensitizes the FSH-receptor (FSH-R) upon binding. Very little information is available concerning the regulatory proteins involved in this process. In the present study, we investigated whether G protein-coupled receptor kinases (GRKs) and arrestins have a role in FSH-R desensitization, using a mouse Ltk 7/12 cell line stably overexpressing the rat FSH-R as a model. We found that these cells, which express GRK2, GRK3, GRK5, and GRK6 as well as beta-arrestins 1 and 2 as detected by RT-PCR and by Western blotting, were rapidly desensitized in the presence of FSH. Overexpression of GRKs and/or beta-arrestins in Ltk 7/12 cells allowed us to demonstrate 1) that GRK2, -3, -5, -6a, and -6b inhibit the FSH-R-mediated signaling (from 71% to 96% of maximal inhibition depending on the kinase, P < 0.001); 2) that beta-arrestins 1 or 2 also decrease the FSH action when overexpressed (80% of maximal inhibition, P < 0.01) whereas dominant negative beta-arrestin 2 [319-418] potentiates it 8-fold (P < 0.001); 3) that beta-arrestins and GRKs (except GRK6a) exert additive inhibition on FSH-induced response; and 4) that FSH-R desensitization depends upon the endogenous expression of GRKs, since there is potentiation of the FSH response (2- to 3-fold, P < 0.05) with antisenses cDNAs for GRK2, -5, and -6, but not GRK3. Our results show that the desensitization of the FSH-induced response involves the GRK/arrestin system.

Rat G protein-coupled receptor kinase GRK4: identification, functional expression, and differential tissue distribution of two splice variants.

G protein-coupled receptor kinases (GRKs) specifically phosphorylate the agonist-occupied form of G protein-coupled receptors, leading to the homologous mode of desensitization. We report here on the cloning of complementary DNAs that encode two rat GRK4 variants. Rat GRK4A (575 amino acids) displays 76% identity with the long human GRK4 splice variant. Rat GRK4B (545 amino acids) delineates a new variant that is identical to GRK4A except for a 31-amino acid deletion in the N-terminal domain, corresponding to exon VI in the human GRK4 gene. GRKs4A and B are likely produced by alternative splicing from a single gene, the partial characterization of which revealed a structural organization similar to that of the human GRK4 gene. GRK4A messenger RNA (mRNA) is abundant only in testis. A combination of in situ hybridization and quantitative RT-PCR studies demonstrated that GRK4A mRNA level increases during testicular development and predominates in leptotene to late pachytene primary spermatocytes and round spermatids. GRK4B mRNA is poorly expressed in testis and most rat tissues but is heterogeneously distributed in the kidney, with 20-fold enrichment in the outer medulla. GRKs4A and B are both functional protein kinases, as demonstrated in a rhodopsin phosphorylation assay. The differential tissue distribution of GRKA4 and GRK4B suggests that individual GRK4 variants may serve distinct physiological functions.

Beta-adrenergic and muscarinic receptor mRNA accumulation in the sinoatrial node area of adult and senescent rat hearts.

The sinoatrial (SA) node is the cardiac pacemaker and changes in its adrenergic-muscarinic phenotype have been postulated as a determinant of age-associated modifications in heart rate variability. To address this question, right atria were microdissected, the SA node area was identified by acetylcholinesterase staining, and, using a RT-PCR method, the accumulation of mRNA molecules encoding beta1- and beta2-adrenergic (beta1- and beta2-AR) and muscarinic (M2-R) receptor was quantified to define the proportion between beta-AR and M2-R mRNAs within the sinoatrial area of adult (3 months) and senescent (24 months) individual rat hearts. In adult hearts, the highest M2-R/beta-AR mRNA ratio was observed within the sinoatrial area compared with adjacent atrial myocardium, while in the senescent hearts, no difference was observed between sinoatrial and adjacent areas. This change was specific of the sinoatrial area since adult and senescent whole atrial or ventricular myocardium did not differ in their M2-R/beta-AR mRNA ratio, and was associated with a fragmentation of acetylcholinesterase staining of the senescent SA node. Quantitative changes in the expression of genes encoding proteins involved in heart rate regulation specifically affect the sinoatrial area of the senescent heart.

Quantitative reverse transcription and polymerase chain reaction analysis of oxytocin and vasopressin receptor mRNAs in the rat uterus near parturition.

Oxytocin receptors (OT-R) are known to be involved in the course of labor since a massive increase in OT-binding sites is observed in the uterus just before parturition. Vasopressin (AVP)-binding sites have also been observed and have been shown to mediate uterotonic responses. To determine exactly which subtypes of OT/AVP receptors are expressed in the rat uterus near parturition, we carried out absolute quantitations of the neurohypophysial hormone receptor (OT-R and the vasopressin receptors V1a-R, V1b-R and V2-R) mRNAs with an assay based on reverse transcription-polymerase chain reaction (RT-PCR) using in vitro transcribed mutated cRNAs as internal standards. The number of mRNA molecules/ng of total RNA was 35 +/- 6, 220 +/- 33 and 39 +/- 9 for OT-R (P < 0.01) and 16 +/- 1, 25 +/- 8 and 31 +/- 5 for V1a-R (P > 0.05) on day (D) 21, 22 and 23 of gestation (post-parturient), respectively. We did not detect V1b-R and V2-R mRNAs in the pregnant uterus. Therefore, the heterogeneity of OT and AVP receptors in the rat uterus can only be assigned to the presence of OT-R and V1a-R neurohypophysial hormone receptor subtypes, whereas V1b-R and V2-R can not be invoked. Only OT-R mRNA levels change in the uterus near parturition.

Capucin: a novel striatal marker down-regulated in rodent models of Huntington disease.

In an initial study, we compared quantitative transcriptome data across mouse brain territories using the serial analysis of gene expression method. Among the novel regional markers that we discovered, we focused on a striatum-enriched transcript with no available experimental cDNA sequence. Here, we report its cloning, gene structure, and detailed distribution in mouse brain. Quantitative RT-PCR and in situ hybridization demonstrated predominant expression in dorsolateral striatum. We therefore named it capucin for caudate-and putamen-enriched sequence. Mouse capucin is a 237-amino-acid protein, without any registered ortholog in mammalian species. It contains no recognizable motif other than two predicted carboxy-terminal transmembrane domains. When expressed in fusion with a fluorescent protein, it localized to the Golgi apparatus in two mammalian cell lines. Interestingly, we observed a significant down-regulation of capucin mRNA levels in two rodent models of Huntington disease, indicating a possible contribution to the pathogenesis of this disorder.

Molecular cloning of two rat GRK6 splice variants.

Desensitization of G protein-coupled receptors is frequently triggered by G protein-coupled receptor kinases (GRKs) that preferentially phosphorylate agonist-occupied receptors. In this study, two GRK6 splice variants were cloned from the rat kidney. One isoform (GRK6a) encodes a 576-amino acid protein that is virtually identical (98% identity) to human GRK6. The second isoform is similar except for a 2-base pair insert that constitutes part of an intron interrupting the 3'-end coding region. This new isoform (GRK6b, 589 amino acids) has therefore a specific COOH-terminal region. A reverse transcription-polymerase chain reaction assay designed to discriminate GRK6 splice variants demonstrated that GRK6b mRNA is widely distributed and expressed at much higher levels than GRK6a mRNA in most peripheral tissues. In contrast, GRK6a predominates in brain. Functional studies, performed with cytosol extracts from transfected Chinese hamster ovary cells, indicated that GRK6a and GRK6b both phosphorylate light-activated rhodopsin as well as a synthetic peptide. The identification of GRK6b extends the family of GRKs. Further studies will be required to establish the tissue and subcellular distribution of this protein and to delineate its physiological role.

Effects of calcitonin on the renal concentrating mechanism.

The effects of salmon calcitonin on the renal concentrating mechanism were investigated in homozygous DI Brattleboro rats. The levels of peptide hormones believed to produce the same physiological responses as antidiuretic hormone on the thick ascending limb (glucagon, parathyroid hormone, and calcitonin) and the cortical collecting ducts (calcitonin) were reduced by acute thyroparathyroidectomy and somatostatin administration. In these hormone-deprived animals, the corticomedullary concentration gradient was almost abolished; the (F/P)osmol at the tip of the juxtamedullary nephrons was 1.19 +/- 0.05. Calcitonin administration restored the gradient [(F/P)osmol = 1.85 +/- 0.14] and simultaneously absolute and fractional water excretion fell significantly despite the concomitant rise in the glomerular filtration rate. It is concluded that 1) in the hormone-deprived animal, calcitonin administration consistently enhances the corticomedullary concentration gradient, and 2) the effects of hormone deprivation and calcitonin administration on the urinary concentrating mechanism are compatible with direct stimulation by calcitonin of electrolyte reabsorption along the thick ascending limb and/or of the water permeability of the cortical collecting ducts.

Glucagon inhibits water and NaCl transports in the proximal convoluted tubule of the rat kidney.

The effects of glucagon on water and electrolyte transport in the kidney were investigated on hormone-deprived rats, i.e. thyroparathyroidectomized diabetes insipidus Brattleboro rats infused with somatostatin. Glucagon consistently inhibited the reabsorption of water and Na+, Cl-, K+ and Ca2+ along the proximal tubule accessible to micropuncture, leaving the reabsorption of inorganic phosphate (Pi) untouched. In the loop, besides its previously described stimulatory effects on Na+, Cl-, K+, Ca2+ and Mg2+ reabsorption, glucagon strongly inhibited Pi reabsorption, very probably in the proximal straight tubule. These effects resulted in a significant phosphaturia and considerable reductions of Mg2+ and Ca2+ excretions. The effects of glucagon at both the whole kidney and the nephron levels are very similar to those previously described for calcitonin. In the absence of an adenylate cyclase system sensitive to glucagon and calcitonin in the rat proximal tubule, and from the analogy of their physiological effects with those elicited by parathyroid hormone, it is suggested that glucagon and calcitonin exert their inhibitory effects on Na and Pi reabsorption in the proximal tubule through another pathway, which could be the phosphoinositide regulatory cascade.

Effects of dDAVP on rat juxtamedullary nephrons: stimulation of medullary K recycling.

The effects of 1-desamino-8-D-arginine vasopressin (dDAVP) on the handling of water and electrolytes by the juxtamedullary nephrons were studied on rats with reduced circulating levels of antidiuretic hormone (ADH), parathyroid hormone, calcitonin, and glucagon, all of which stimulate the adenylate cyclase system of the thick ascending limb and the distal tubule. In such hormone-deprived rats and in hormone-deprived + dDAVP rats, the concentration of Na, Cl, and total solutes was lower in the ascending than in the descending limbs, whereas the inulin concentration was similar at both sites. dDAVP did not alter the fraction of NaCl remaining in the thin limbs, but tended to reduce that of Mg and Ca. On the other hand, dDAVP significantly increased the fraction of filtered K remaining from 65.8 +/- 5.2 to 107.3 +/- 15.8%. A direct correlation was observed between the fraction of filtered K remaining at the tip of the juxtamedullary loops and the fractional excretion rate of K in urine. Since dDAVP enhances distal K net secretion, as previously shown in our laboratory, these results indicate that the medullary recycling of K from nephron terminal segments to Henle's loop of juxtamedullary nephrons is stimulated by this peptide.

Additive effects of glucagon and vasopressin on renal Mg reabsorption and urine concentrating ability in the rat.

Previous studies demonstrated that in rat kidney, arginine-vasopressin (AVP), glucagon (GLU), calcitonin and parathyroid hormone exert similar effects on the thick ascending limb of Henle's loop (TALH). To ascertain the physiological significance of such multiple hormonal control of TALH function, it is necessary to establish whether one hormone can exert its effect on TALH, even when other hormones are present. We therefore compared renal responses to submaximal (1 ng/min) and maximal (10 ng/min) doses of glucagon in rats deprived of endogenous AVP, GLU, calcitonin and parathyroid hormone with the responses of similar rats given a maximal dose of AVP (40 pg/min). Administration of glucagon or AVP alone reduced Mg fractional excretion, but the reduction was more marked when both hormones were given together. Consequently, their effects were additive, at submaximal and maximal doses. In the presence of AVP + glucagon, urinary osmolality was also higher than in the presence of AVP alone (Umax: 1242 +/- 49 vs. 936 +/- 50 mosmol/kg; p less than 0.001). This latter effect may indicate that AVP and glucagon also exert additive effects on Na reabsorption in the TALH.

Sensitivities of rat kidney thick ascending limbs and collecting ducts to vasopressin in vivo.

Clearance experiments were performed to characterize the sensitivity to vasopressin of the thick ascending limbs and collecting duct system of the rat kidney. The response of the thick ascending limbs was evaluated by measuring the Mg2+ excretion rate in the urine, since the [arginine-8] vasopressin-mediated effects on Mg2+ excretion are the direct result of a stimulation of Mg2+ reabsorption in this nephron segment, and the response of the collecting ducts was evaluated by changes in urine flow. To avoid the effects of parathyroid hormone, glucagon, and calcitonin, which stimulate Mg2+ reabsorption in the thick ascending limb and distal tubule, and of calcitonin, which increases the permeability of the cortical collecting ducts to water, experiments were performed on Brattleboro D. I. rats (with hereditary diabetes insipidus, due to a lack of [Arg8]vasopressin) acutely deprived of endogenous parathyroid hormone, calcitonin, and glucagon. Vasopressin infused at rates up to 5 pg/min did not reduce the Mg2+ fractional excretion rate, whereas at 5 pg/min water excretion was decreased by 50%. The half-maximal reduction of Mg2+ excretion occurred at vasopressin infusion rates 4-6 times higher than those necessary to diminish the water excretion rate to the same extent. We conclude that in vivo, two segments involved in the production of concentrated urine have different sensitivities to vasopressin and that this difference in sensitivity is very similar for the biological response in vivo and the adenylate cyclase activation in vitro. We suggest that both the magnitude and the nature of the effects of [Arg8]vasopressin on the kidney may vary according to the required antidiuretic response.

Effects of glucagon and PTH on the loop of Henle of rat juxtamedullary nephrons.

We investigated by micropuncture the effects of glucagon and parathyroid hormone (PTH) on thin limbs of juxtamedullary nephrons of rats with reduced plasma concentration of endogenous glucagon, PTH, antidiuretic hormone (ADH) and calcitonin, all four hormones enhancing the adenylate-cyclase activity in the thick ascending limbs and the distal nephron. Such a hormonal depletion suppresses the corticomedullary concentration gradient, making favourable conditions for studying the influence of these hormones on the renal concentrating mechanism. Administration of glucagon (4.4 ng/min-1) or PTH (5 mU/min-1) to these hormone-deprived rats elicited the expected decrease in urinary Mg and Ca fractional excretion without modifying either fractional or absolute excretion of water. At the tip of the loop, glucagon enhanced the loop fluid osmolality by 20%, but left the delivery of water unchanged. The Na and Cl concentrations increased significantly with the osmolality, resulting in a positive correlation between the fractional delivery of either ion and the loop fluid osmolality. PTH increased the fraction of filtered phosphate delivered to the thin limbs, as expected, but, in contrast to glucagon, did not alter either the Na, Cl, or total solute fractional deliveries. The Mg, Ca and K deliveries were unaffected by glucagon and PTH. In conclusion, glucagon, which activates the cyclase system of both the medullary and cortical portion of the thick ascending limb, enhances the delivery of salt to the tip of the loop by net sodium chloride addition to the descending limb. PTH which activates the adenyl-cyclase system only in the cortical thick ascending limb cannot enhance such NaCl delivery. NaCl, when added, might therefore originate from the medullary thick ascending limb.

Effects of human calcitonin on water and electrolyte movements in rat juxtamedullary nephrons: inhibition of medullary K recycling.

The effects of synthetic human calcitonin (HCT) on water and electrolyte deliveries to the thin limbs of Henle's loop of juxtamedullary nephrons were investigated by micropuncture in the rat. To avoid undesirable interference with exogenous calcitonin, experiments were performed in hormone-deprived rats with reduced circulating calcitonin, antidiuretic hormone, parathyroid hormone and glucagon, all four of which stimulate the adenylate-cyclase activity in the thick ascending limb and the distal tubule. Administration of HCT (1.0 mU/min X 100 g body wt) to such rats significantly reduced the urinary fractional excretion rate of water, Mg, Ca and K. At the tip of the longlooped nephrons, the fractional delivery of water diminished in the presence of HCT, although the glomerular filtration rate of these nephrons was unaltered. Simultaneously, the loop fluid osmolality rose significantly. HCT, however, did not alter the fraction of total filtered solutes remaining in the thin limbs, nor the NaCl fractional delivery. As previously observed in this laboratory with dDAVP, the reduced fractional delivery of water at the hairpin turn was accompanied by a decrease in Mg and Ca deliveries in rats given HCT, indicating that the handling of these two ions along the descending limb may be linked in part to the water movements in this nephron segment. The fractional deliveries of K at the hairpin turn and in urine were significantly correlated, and both decreased in the presence of HCT. Since, as shown previously, HCT reduces the net addition of K along the superficial distal tubule, it is concluded that calcitonin inhibits the medullary recycling of K between the nephron terminal segments and the loop of Henle of juxtamedullary nephrons.

Re-evaluation of the expression of the gastric H,K-ATPase alpha subunit along the rat nephron.

Under normal conditions, the rat collecting duct displays an H, K-ATPase activity with kinetic and pharmacological properties very close to those of the gastric H,K-ATPase. However, whether the collecting duct H,K-ATPase and the gastric enzyme are identical remains controversial. Therefore, we re-evaluated the expression of the mRNAs encoding the gastric H,K-ATPase alpha subunit in the rat nephron. For this purpose, gastric H,K-ATPase mRNAs were quantitated by RT-PCR at the level of microdissected nephron segments using known amounts of gastric H,K-ATPase cRNA as external standards. Results indicate that gastric H,K-ATPase mRNAs are undetectable (<1 copy per cell) in the glomerulus and along the proximal tubule, thick ascending limb of Henle's loop and collecting duct, although a faint expression ( approximately 400 copies per micro;g total RNA) is measurable in whole-kidney preparations. Gastric H,K-ATPase mRNA is also absent along the nephron of K-depleted rats and of rats with chronic metabolic acidosis and alkalosis. Taken with other data from the literature, these results suggest that the collecting duct of normal rats might express an H,K-ATPase similar, but not identical, to the gastric isoform.

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.

Quantitative RT-PCR analysis of mRNAs encoding a colonic putative H, K-ATPase alpha subunit along the rat nephron: effect of K+ depletion.

The rat nephron displays two ouabain-sensitive K-ATPases: one, which is present in proximal tubules and thick ascending limbs of normal rats, is specifically activated by K+ and is down-regulated by K+ depletion, whereas the other one appears in collecting ducts of K+-depleted rats and is activated by either Na+ or K+. To determine which of these two ATPases is similar to colonic-type H,K-ATPase, we quantitated by reverse transcriptase-polymerase chain reaction (RT-PCR) the mRNAs encoding the colonic H,K-ATPase alpha subunit in microdissected nephron segments. In normal rats, statistically significant amounts of colonic H,K-ATPase mRNAs were detected exclusively in cortical thick ascending limbs and cortical collecting ducts (200-500 copies/mm). Because these levels of expression were low (1-1.2 copies/target cell), they probably have no physiological relevance. In rats fed a K+-depleted diet for 2 weeks, expression of colonic H,K-ATPase was markedly enhanced in cortical and medullary collecting ducts (5000-12,000 copies/mm or 30-40 copies per cell), whereas it remained low in all other nephron segments. Thus, colonic H,K-ATPase alpha subunit is specifically expressed in cortical and outer medullary collecting ducts of K+-depleted rats where it likely accounts for the ouabain-sensitive K-ATPase activity.