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.

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.

Transcriptome of a mouse kidney cortical collecting duct cell line: effects of aldosterone and vasopressin.

Aldosterone and vasopressin are responsible for the final adjustment of sodium and water reabsorption in the kidney. In principal cells of the kidney cortical collecting duct (CCD), the integral response to aldosterone and the long-term functional effects of vasopressin depend on transcription. In this study, we analyzed the transcriptome of a highly differentiated mouse clonal CCD principal cell line (mpkCCD(cl4)) and the changes in the transcriptome induced by aldosterone and vasopressin. Serial analysis of gene expression (SAGE) was performed on untreated cells and on cells treated with either aldosterone or vasopressin for 4 h. The transcriptomes in these three experimental conditions were determined by sequencing 169,721 transcript tags from the corresponding SAGE libraries. Limiting the analysis to tags that occurred twice or more in the data set, 14,654 different transcripts were identified, 3,642 of which do not match known mouse sequences. Statistical comparison (at P < 0.05 level) of the three SAGE libraries revealed 34 AITs (aldosterone-induced transcripts), 29 ARTs (aldosterone-repressed transcripts), 48 VITs (vasopressin-induced transcripts) and 11 VRTs (vasopressin-repressed transcripts). A selection of the differentially-expressed, hormone-specific transcripts (5 VITs, 2 AITs and 1 ART) has been validated in the mpkCCD(cl4) cell line either by Northern blot hybridization or reverse transcription-PCR. The hepatocyte nuclear transcription factor HNF-3-alpha (VIT39), the receptor activity modifying protein RAMP3 (VIT48), and the glucocorticoid-induced leucine zipper protein (GILZ) (AIT28) are candidate proteins playing a role in physiological responses of this cell line to vasopressin and aldosterone.

Gene expression profiles in normal and Otx2-/- early gastrulating mouse embryos.

The mouse Otx2 gene is a homeobox transcription factor required as early as gastrulation for the proper development of the head. We compared gene expression profiles in wild-type and Otx2(-/-) 6.5 days postcoitum embryos by using a serial analysis of gene expression assay adapted to microdissected structures. Among a broader list, the study of six genes found to be differentially expressed allows defining a role for Otx2 in the orchestration of cell movements leading to the adequate organization of the embryo before gastrulation.

Serial microanalysis of renal transcriptomes.

Large-scale gene expression studies can now be routinely performed on macroamounts of cells, but it is unclear to which extent current methods are valuable for analyzing complex tissues. In the present study, we used the method of serial analysis of gene expression (SAGE) for quantitative mRNA profiling in the mouse kidney. We first performed SAGE at the whole-kidney level by sequencing 12,000 mRNA tags. Most abundant tags corresponded to transcripts widely distributed or enriched in the predominant kidney epithelial cells (proximal tubular cells), whereas transcripts specific for minor cell types were barely evidenced. To better explore such cells, we set up a SAGE adaptation for downsized extracts, enabling a 1, 000-fold reduction of the amount of starting material. The potential of this approach was evaluated by studying gene expression in microdissected kidney tubules (50,000 cells). Specific gene expression profiles were obtained, and known markers (e.g., uromodulin in the thick ascending limb of Henle's loop and aquaporin-2 in the collecting duct) were found appropriately enriched. In addition, several enriched tags had no databank match, suggesting that they correspond to unknown or poorly characterized transcripts with specific tissue distribution. It is concluded that SAGE adaptation for downsized extracts makes possible large-scale quantitative gene expression measurements in small biological samples and will help to study the tissue expression and function of genes not evidenced with other high-throughput methods.

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.

[Regulation of intracellular cAMP content in kidney tubules: role of adenylyl cyclases inhibitable by calcium]. / Régulation du contenu intracellulaire d'AMPc dans le tubule rénal: rôle des adénylyl cyclases inhibables par le calcium.

Adenylyl cyclase (AC) isoforms 5 and 6 can be inhibited by submicromolar concentrations of Ca2+. Quantitative RT-PCR allowed to study the corresponding messengers (mRNA) along the rat nephron. The results demonstrate a significant expression of AC 6 mRNA all along the nephron and of AC 5 mRNA in the glomerulus and the collecting tubule located in the cortex and the outer medulla. Regulation of cAMP synthesis and of intracellular cAMP content in defined renal cell types established the functional expression of AC 5 and AC 6. In particular, adenylyl cyclase activity is strongly stimulated by hormones and can be inhibited by several factors which either increase intracellular Ca2+ concentration or are coupled to G alpha 1. In each renal cell studied, the expression of 5 and 6 isoforms allow to integrate specific, multiple and independent inhibitory pathways which contribute to decrease intracellular cAMP content.

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.

beta2-adrenergic but not vasopressin V2 receptor mRNAs are expressed in the stria vascularis of the rat inner ear.

Activation of beta2-adrenergic receptors (beta2-AR) and of V2-vasopressin receptors (V2-VR) has been shown to stimulate cAMP production in the stria vascularis. Expression of these receptors in this epithelial structure has never been investigated at the mRNA level. A quantitative assay based on the reverse transcriptase-polymerase chain reaction (RT-PCR) method was developed to study the expression of beta2-AR, vasopressin (V1a,V1b, V2) and oxytocin receptors in rat microdissected stria vascularis fragments. Steady-state mRNA levels were measured using mutant cRNAs as internal standards. We consistently found evidence of the expression of beta2-AR transcripts in the stria vascularis; however, no such evidence of V2-VR mRNA expression was found in studies of the same samples. As our method is sensitive enough to detect 200 mRNA molecules, V2-VR mRNA is thought to be present, if at all, at a concentration that is 40 times lower than that of the beta2-AR transcripts. V1a-VR mRNA was found to be present in trace amounts and is the only subtype of the vasopressin-oxytocin receptor family expressed in this epithelium. These data demonstrate, at the mRNA level, the expression of beta2-AR in the stria vascularis, and indicate that V2-VR transcripts are not present in this structure.

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.

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.

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.

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.

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.

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 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.

Polarized targeting of a shaker-like (A-type) K(+)-channel in the polarized epithelial cell line MDCK.

Functional Kv 1-4 channels were stably expressed in filter-grown MDCK cells which form a polarized epithelium with two distinct plasma membrane domains: a basolateral and an apical cell surface. The Shaker-related Kv 1-4 channels mediated in MDCK cells fast transient (A-type) voltage-activated outward currents having similar properties to the ones reported for Kv 1-4 in the Xenopus oocytes expression system. Immunoblot analysis with specific anti-Kv 1-4 antibodies showed that two Kv 1-4 protein forms are expressed in MDCK cells which most likely represent the glycosylated and non-glycosylated Kv 1-4 protein, respectively. Using immunocytochemistry and confocal microscopy we showed that the Kv 1-4 channels are specifically localized in the basolateral membranes of MDCK cells. Thus, the MDCK cells may provide an important model system to analyse the polarized transport of ion channels such as Kv 1-4, which are distinctly expressed in the mammalian central nervous system.

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.

Molecular analysis of beta-adrenergic receptor subtypes in rat collecting duct: effects on cell cAMP and Ca2+ levels.

Expression and functional properties of beta-adrenergic receptors (beta-ARs) were studied in rat collecting tubules isolated by microdissection. Reverse transcription-polymerase chain reaction experiments demonstrated that the beta 1- and beta 2-AR mRNAs, but not the beta 3-subtype, are expressed in the cortical collecting duct (CCD). Quantitation of mRNAs, carried out using mutant RNAs as internal standards, further showed that beta 1- and beta 2-ARs transcripts are present at comparable amounts in CCD (3,000-4,000 copies/mm of tubular length), but reach 6-8 times lower levels in the outer medullary collecting duct (OMCD: beta 1, 480 +/- 180; beta 2, 590 +/- 110 copies/mm of tubular length). Functional studies, carried out in CCD, corroborated the expression of these two receptor subtypes. The rank order of potency of beta-agonists for stimulating adenosine 3',5'-cyclic monophosphate (cAMP) accumulation was isoproterenol > norepinephrine = epinephrine, and similar efficiencies were found for a beta 1- and a beta 2-antagonist to inhibit isoproterenol-dependent cAMP formation. Fura 2 fluorescence measurements revealed that isoproterenol (10 microM) induces a biphasic rise of intracellular free Ca2+ concentration ([Ca2+]i), consisting of an initial fast increase (delta [Ca2+]i = 122 nM) followed by a plateau phase (delta [Ca2+]i = 58 nM). In the absence of basolateral Ca2+, the initial peak was still observed, suggesting intracellular Ca2+ release. Norepinephrine and epinephrine, as well as selective beta 1- and beta 2-agonists, also increased [Ca2+]i in CCD. Only slight [Ca2+]i variations were produced by isoproterenol in the OMCD (delta [Ca2+]i = 21 nM) and the cortical thick ascending limb (delta [Ca2+]i = 25 nM). These results show that both beta 1- and beta 2-ARs are expressed in the collecting tubule and that they predominate in the CCD. The two receptor subtypes contribute to cAMP accumulation induced by beta-agonists. They also trigger [Ca2+]i variations, indicating their possible coupling to several transduction pathways in the rat CCD.