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
1998; Elsevier BV; Volume: 273; Issue: 24 Linguagem: Inglês
10.1074/jbc.273.24.15192
ISSN1083-351X
AutoresMarie Céleste de Jesus Ferreira, Cécile Héliès-Toussaint, Martine Imbert–Teboul, Claire Bailly, Jean‐Marc Verbavatz, Anne-Christine Bellanger, Danièlle Chabardès,
Tópico(s)Ion Transport and Channel Regulation
ResumoThe 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+. 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+. A new type of G protein-coupled membrane receptor that is activated by increasing concentrations of extracellular ionized calcium ([Ca2+]e) 1The abbreviations used are: [Ca2+]e, extracellular free concentration of Ca2+; AC, adenylyl cyclase; AVP, arginine vasopressin; [Ca2+]i, intracellular free concentration of Ca2+; CTAL, cortical portion of the thick ascending limb; IBMX, 3-isobutyl-1-methylxanthine; IP, inositol phosphates; PDE, phosphodiesterase; PGE2, prostaglandin E2; PTX, Bordetella pertussis toxin; RT-PCR, reverse transcription-polymerase chain reaction; Ro 20-1724, 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone. and polyvalent cations has been cloned recently from bovine parathyroid cells (BoPCaR1, Ref. 1Brown E.M. Gamba G. Riccardi D. Lombardi M. Butters R. Kifor O. Sun A. Hediger M.A. Lytton J. Hebert S.C. Nature. 1993; 366: 575-580Crossref PubMed Scopus (2380) Google Scholar) and rat kidney (RaKCaR, Ref. 2Riccardi D. Park J. Lee W.-S. Gamba G. Brown E.M. Hebert S.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 131-135Crossref PubMed Scopus (437) Google Scholar). When expressed in Xenopus oocytes, these Ca2+-sensing receptors are coupled to phospholipase C stimulation (1Brown E.M. Gamba G. Riccardi D. Lombardi M. Butters R. Kifor O. Sun A. Hediger M.A. Lytton J. Hebert S.C. Nature. 1993; 366: 575-580Crossref PubMed Scopus (2380) Google Scholar, 2Riccardi D. Park J. Lee W.-S. Gamba G. Brown E.M. Hebert S.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 131-135Crossref PubMed Scopus (437) Google Scholar). Increasing [Ca2+]e also stimulates phospholipase C activity in parathyroid cells (3Hawkins D. Enyedi P. Brown E. Endocrinology. 1989; 124: 838-844Crossref PubMed Scopus (47) Google Scholar, 4Kifor O. Diaz R. Butters R. Brown E.M. J. Bone Miner. Res. 1997; 12: 715-725Crossref PubMed Scopus (194) Google Scholar) and inhibits hormone-dependent cAMP accumulation (5Chen C.J. Barnett J.V. Congo D.A. Brown E.M. Endocrinology. 1989; 124: 233-239Crossref PubMed Scopus (119) Google Scholar), but, so far, no interaction between these two transduction pathways has been established. The presence of BoPCaR1 in parathyroid cells explains the crucial role of [Ca2+]e to elicit a negative feedback on parathyroid hormone secretion (6Brown E.M. Physiol. Rev. 1991; 71: 371-411Crossref PubMed Scopus (640) Google Scholar, 7Brown E.M. Hebert S.C. Bone (NY). 1997; 20: 303-309Crossref PubMed Scopus (116) Google Scholar). In rat kidney, a predominant expression of RaKCaR mRNA has been localized in the cortical portion of the thick ascending limb (8Riccardi D. Lee W.-S. Lee K. Segre G.V. Brown E.M. Hebert S.C. Am. J. Physiol. 1996; 271: F951-F956PubMed Google Scholar, 9Yang T. Hassan S. Huang Y.G. Smart A.M. Briggs J.P. Schnermann J.B. Am. J. Physiol. 1997; 272: F751-F758Crossref PubMed Google Scholar), a segment which ensures cAMP-stimulated paracellular Ca2+ reabsorption, from the lumen of the renal tubule to the extracellular fluid compartments (10Morel F. Doucet A. Physiol. Rev. 1986; 66: 377-468Crossref PubMed Scopus (174) Google Scholar). A functional Ca2+ receptor is expressed in the plasma membrane of the rat and mouse cortical thick ascending limb (CTAL) as evidenced by the properties of the dose-dependent increase in the concentration of intracellular calcium ([Ca2+]i) as a function of peritubular [Ca2+]e (11Champigneulle A. Siga E. Vassent G. Imbert-Teboul M. J. Membr. Biol. 1997; 156: 117-129Crossref PubMed Scopus (25) Google Scholar, 12Paulais M. Baudoin-Legros M. Teulon J. Am. J. Physiol. 1996; 271: F1052-F1060PubMed Google Scholar). High [Ca2+]e (5 mm) in the mouse CTAL decreases hormone-dependent cAMP accumulation, an effect which has been ascribed to a direct inhibition of adenylyl cyclase (AC) activity (13Takaichi K. Kurokawa K. Miner. Electrolyte Metab. 1986; 12: 342-346PubMed Google Scholar). Experiments using quantitative reverse transcription-polymerase chain reaction (RT-PCR) have shown that the rat CTAL also expresses the Ca2+-inhibitable type 6 AC mRNA (14Chabardès D. Firsov D. Aarab L. Clabecq A. Bellanger A.C. Siaume-Perez S. Elalouf J.M. J. Biol. Chem. 1996; 271: 19264-19271Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). All the Gαs-coupled receptors studied so far in this segment activate a single pool of AC catalytic units (15Morel F. Chabardès D. Imbert-Teboul M. Le Bouffant F. Hus-Citharel A. Montégut M. Kidney Int. 1982; 21: S55-S62Google Scholar, 16Morel F. Imbert-Teboul M. Chabardès D. Kidney Int. 1987; 31: 512-520Abstract Full Text PDF PubMed Scopus (84) Google Scholar), and in addition, electron microscopy studies describe a single cell type in this epithelium (17Kriz W. Kaissling B. Seldin D.W. Giebisch G. The Kidney: Physiology and Pathophysiology. 2nd Ed. Raven Press, Ltd., New York1992: 707-777Google Scholar). These observations lead to the hypothesis that the functional expression of the type 6 AC mRNA accounts for the hormone-dependent cAMP synthesis in the rat CTAL. The aim of the present study was therefore to investigate the functional expression of the AC present in the rat CTAL and the consequences of the possible co-localization in a same cell of a Ca2+-inhibitable AC and of a Ca2+-sensing receptor on the regulation of cAMP synthesis and/or hydrolysis. In order to study the regulation of cAMP levels elicited by potentially similar or different mechanisms of action, we compared the effect of extracellular Ca2+ to those of two agents also active in this segment. The first agent, angiotensin II, induces [Ca2+]i increases in the rat CTAL (11Champigneulle A. Siga E. Vassent G. Imbert-Teboul M. J. Membr. Biol. 1997; 156: 117-129Crossref PubMed Scopus (25) Google Scholar, 18Bouby N. Hus-Citharel A. Marchetti J. Bankir L. Corvol P. Llorens-Cortes C. J. Am. Soc. Nephrol. 1997; 8: 1658-1667PubMed Google Scholar). The pattern of the responses observed demonstrates that a same intracellular Ca2+ pool is released by angiotensin II and extracellular Ca2+ (11Champigneulle A. Siga E. Vassent G. Imbert-Teboul M. J. Membr. Biol. 1997; 156: 117-129Crossref PubMed Scopus (25) Google Scholar). The second agent, prostaglandin E2 (PGE2), inhibits hormone-dependent cAMP synthesis (19Torikai S. Kurokawa K. Am. J. Physiol. 1983; 245: F58-F66PubMed Google Scholar) likely as a result of the interaction of the PGE2 receptor with a GTP-dependent, pertussis toxin-sensitive Gαiprotein as demonstrated in the medullary portion of the rat thick ascending limb (20Firsov D. Aarab L. Mandon B. Siaume-Perez S. de Rouffignac C. Chabardès D. Pfluegers Arch. 1995; 429: 636-646Crossref PubMed Scopus (37) Google Scholar). The experiments were performed on rat CTAL isolated by microdissection, and the results establish that PGE2 (coupled to pertussis toxin-sensitive Gαi protein), angiotensin II, and extracellular Ca2+ (both coupled to phospholipase C pathway) are effective in a same cell to decrease arginine vasopressin-dependent cAMP accumulation. [Ca2+]e, in the physiological range, decreases hormone-dependent cAMP accumulation by more than 50%, an effect which results from both an inhibition of cAMP synthesis and an increase of cAMP hydrolysis. Angiotensin II also regulates both mechanisms, but its ability to inhibit cAMP synthesis is much smaller. The adenylyl cyclase present in this segment has the functional properties previously described for the Ca2+-inhibitable type 6 AC (21Iyengar R. FASEB J. 1993; 7: 768-775Crossref PubMed Scopus (266) Google Scholar, 22Cooper D.M.F. Mons N. Fagan K. Cell. Signalling. 1994; 6: 823-840Crossref PubMed Scopus (61) Google Scholar); in particular, AC activity is inhibited by both Gαi and phospholipase C pathways by different mechanisms. Accordingly, in situ hybridization shows a homogeneous distribution of type 6 AC mRNA in CTAL cells. Taken together, the results establish that the co-expression of a Ca2+ sensing receptor and of a Ca2+-inhibitable AC in the rat CTAL cell allows a specific inhibition by physiological [Ca2+]e of hormone-stimulated cAMP intracellular content. Unless otherwise specified, the compounds were from Merck (Damstardt, Germany), Sigma, and Calbiochem. A probe specific of type 6 AC was chosen in the most divergent region of AC cDNA sequences. A 376-bp fragment (Pvu II-Sph I, nucleotides 3766–4143) of the type 6 AC cDNA, located in the 3′-untranslated region, was subcloned in pGEM3Zf(+) in the corresponding sites (Sma I-Sph I). This sequence was included in the region previously used for RT-PCR experiments (14Chabardès D. Firsov D. Aarab L. Clabecq A. Bellanger A.C. Siaume-Perez S. Elalouf J.M. J. Biol. Chem. 1996; 271: 19264-19271Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). A 1080-bp fragment corresponding to nucleotides 1205 (Eco RI) to 2285 (Pvu II) of the type 5 AC cDNA coding region was subcloned in BSSK+. Sense and antisense cRNA probes were transcribed in vitro with T3, T7, or SP6 RNA polymerase (Promega Biotech, Madison, WI) according to the manufacturer's instructions, in the presence of α-35S-UTP (>3,000 Ci/mmol, Amersham Pharmacia Biotech, Les Ulis, France). Adult rat kidneys were fixed by perfusion with 4% paraformaldehyde in phosphate-buffered saline. Kidney slices were post-fixed overnight in the same solution and then dehydrated with graded series of ethanol and butanol. Tissues were paraffin-embedded, and sections of 5–7 μm were mounted on silane-coated slides. In situ hybridization was performed as described by Shanmugam et al. (23Shanmugam S. Llorens-Cortes C. Clauser E. Corvol P. Gasc J.M. Am. J. Physiol. 1995; 268: F922-F930PubMed Google Scholar). Briefly, slides were deparaffinized in toluene and then rehydrated by a graded series of ethanol (100 to 30%). Pretreatment was continued by boiling in a solution of 0.01 m citric acid in a microwave oven and then treated with 0.1% H2O2 in phosphate-buffered saline. These modifications have been shown to increase specific signal. After a second run of fixation in 4% paraformaldehyde-phosphate-buffered saline, and a proteinase K treatment, slides were covered with hybridization buffer (50% formamide, 21% dextran sulfate (50%), 11% salmon sperm DNA, 11% 20× SSC (1× SSC is equal to 0.15 m NaCl and 0.015m sodium citrate), and 7% 1 m dithiothreitol) containing 0.5 to 1.106 cpm of probe per tissue section. Hybridization was performed overnight at 50 °C in a humidified chamber. Slides were rinsed in 5× SSC, 10 mmdithiothreitol followed by an incubation in 50% formamide, 1× SSC, 12.5 mm dithiothreitol. They were further treated by RNase A (20 μg/ml) and then dehydrated in ethanol containing 300 mm ammonium acetate and dried. The slides were exposed for 3 to 4 weeks to Kodak NTB2 liquid emulsion, counterstained with Evans blue, and examined under the microscope. The experimental conditions used to perform microdissection of intact segments have been detailed previously (20Firsov D. Aarab L. Mandon B. Siaume-Perez S. de Rouffignac C. Chabardès D. Pfluegers Arch. 1995; 429: 636-646Crossref PubMed Scopus (37) Google Scholar, 24Aarab L. Montégut M. Siaume-Perez S. Imbert-Teboul M. Chabardès D. Pfluegers Arch. 1993; 423: 397-405Crossref PubMed Scopus (23) Google Scholar). Experiments were performed on male Sprague-Dawley rats (120–150 g of body weight, Iffa-Credo, France) that were maintained on a standard diet with free access to water. After anesthesia, the left kidney was perfused with microdissection medium containing 0.16% collagenase (Serva, Boehringer, Mannheim, Germany). After hydrolysis of the kidney (20 min at 30 °C in 0.12% collagenase solution), single CTALs (0.3–1.5 mm length) were microdissected at 4 °C. The standard microdissection medium was composed of the following (in mm): NaCl, 137; KCl, 5; MgSO4, 0.8; Na2HPO4, 0.33; KH2P04, 0.44; MgCl2, 1; NaHCO3, 4; CH3COONa, 10; CaCl2, 0.5; glucose, 5; HEPES, 20, pH 7.4, and 0.1% (w/v) bovine serum albumin (fraction V, protease-free, minimal fatty acid content, Pentex, Miles Inc., Kankakee, IL). This medium was supplemented with 10 μm ibuprofen and 0.5 unit/ml adenosine deaminase (Boehringer Mannheim) to prevent the synthesis of prostaglandins and the release of adenosine, agents known to interact with the regulation of cAMP content in the rat kidney (20Firsov D. Aarab L. Mandon B. Siaume-Perez S. de Rouffignac C. Chabardès D. Pfluegers Arch. 1995; 429: 636-646Crossref PubMed Scopus (37) Google Scholar, 24Aarab L. Montégut M. Siaume-Perez S. Imbert-Teboul M. Chabardès D. Pfluegers Arch. 1993; 423: 397-405Crossref PubMed Scopus (23) Google Scholar). A nominally Ca2+-free medium used in some of our protocols had the same composition except that calcium was omitted; Ca2+-chelating agents were not added to this medium because they have been described to modify the sensitivity to Ca2+ of the Ca2+-inhibitable AC isoforms (25Smith J.A. Griffin M. Mireylees S.E. Long R.G. FEBS Lett. 1993; 327: 137-140Crossref PubMed Scopus (5) Google Scholar). The concentrations of free Ca2+ present in the media were measured by using an electrode sensitive to Ca2+ (Radiometer, Copenhagen, Denmark). The relationship between the theoretical concentrations of Ca2+ added (0.5–5.0 mm) and those measured in the medium was as follows: y = 1.05x −0.07,r = 1.0. The experimental conditions used to measure hormone-dependent cAMP accumulation on an intact single segment (20Firsov D. Aarab L. Mandon B. Siaume-Perez S. de Rouffignac C. Chabardès D. Pfluegers Arch. 1995; 429: 636-646Crossref PubMed Scopus (37) Google Scholar, 24Aarab L. Montégut M. Siaume-Perez S. Imbert-Teboul M. Chabardès D. Pfluegers Arch. 1993; 423: 397-405Crossref PubMed Scopus (23) Google Scholar) will be recalled briefly. Microdissected pieces of CTAL were transferred in 2 μl of incubation medium onto glass slides (1 or 2 pieces per slide) and photographed in order to measure their length. Unless otherwise specified, each sample was preincubated for 10 min at 30 °C in 0.5 mm [Ca2+]eand, after addition of 2 μl of incubation medium containing the agonists to be tested, incubated for a further 4 min at 35 °C. Adenylyl cyclase activity was stimulated by arginine vasopressin (AVP) which binds to V2 receptor in the rat CTAL, the only AVP receptor expressed in this segment (26Firsov D. Mandon B. Morel A. Merot J. Le Maout S. Bellanger A.C. de Rouffignac C. Elalouf J.M. Buhler J.M. Pfluegers Arch. 1994; 429: 79-89Crossref PubMed Scopus (74) Google Scholar). Due to the small number of cells per tubular sample (from about 100 to 600 cells), hormone-dependent cAMP accumulation can be measured only in the presence of a phosphodiesterase inhibitor. Either 50 μm Ro 20-1724, a specific inhibitor of the lowK m cyclic AMP phosphodiesterase (27Beavo J.A. Physiol. Rev. 1995; 75: 725-748Crossref PubMed Scopus (1646) Google Scholar, 28Hoey M. Houslay M.D. Biochem. Pharmacol. 1990; 40: 193-202Crossref PubMed Scopus (21) Google Scholar), or 1 mm IBMX, inhibitor of all phosphodiesterases in the rat kidney (28Hoey M. Houslay M.D. Biochem. Pharmacol. 1990; 40: 193-202Crossref PubMed Scopus (21) Google Scholar), was added to the incubation medium. The concentrations of the different agents given in the results are those present during the incubation step. For longer preincubation periods, in experiments performed with Bordetella pertussis toxin and bisindolylmaleimide I, all media were supplemented with essential and nonessential amino acids as well as vitamins (minimum Eagle's medium, Eurobio, Les Ulis, France). The amounts of cAMP were measured on acetylated samples by radioimmunoassay (Sanofi Diagnostics Pasteur, Marnes-La-Coquette, France). The micromethod used allows the determination of 2–80 fmol of cAMP per reaction tube. In our conditions, the basal level of cyclic AMP present in a single piece of tubule is close to, or below, the sensitivity threshold of the assay (20Firsov D. Aarab L. Mandon B. Siaume-Perez S. de Rouffignac C. Chabardès D. Pfluegers Arch. 1995; 429: 636-646Crossref PubMed Scopus (37) Google Scholar, 24Aarab L. Montégut M. Siaume-Perez S. Imbert-Teboul M. Chabardès D. Pfluegers Arch. 1993; 423: 397-405Crossref PubMed Scopus (23) Google Scholar). The results were calculated in femtomoles of cyclic AMP accumulated per mm of tubular length per 4 min incubation time (fmol·mm−1·4 min−1). The first experiments carried out to study the effect of [Ca2+]e showed that AVP-stimulated cAMP levels were similar either in the Ca2+-free medium or in 0.5 mm [Ca2+]e but decreased in 1 mm [Ca2+]e incubation medium. The control incubation medium therefore contained 0.5 mm[Ca2+]e, a concentration previously used in experiments performed on parathyroid cells (3Hawkins D. Enyedi P. Brown E. Endocrinology. 1989; 124: 838-844Crossref PubMed Scopus (47) Google Scholar, 4Kifor O. Diaz R. Butters R. Brown E.M. J. Bone Miner. Res. 1997; 12: 715-725Crossref PubMed Scopus (194) Google Scholar, 5Chen C.J. Barnett J.V. Congo D.A. Brown E.M. Endocrinology. 1989; 124: 233-239Crossref PubMed Scopus (119) Google Scholar). In each experiment, different experimental conditions were tested on replicate CTAL samples microdissected from a same rat kidney (6–9 samples per condition). The mean of the cAMP values obtained in each condition was taken as one single data point, and the results were expressed in absolute value or in percentage of the response to AVP or in percentage of inhibition calculated from the corresponding AVP value. Results are given as the mean values ± S.E. calculated from n different experiments. Unless otherwise indicated, the statistical evaluation of the data was assessed by unpaired Student's t test. Assays were performed by using the microtechnique developed for proximal tubule fragments (29Meneton P. Imbert-Teboul M. Bloch-Faure M. Rajerison R.M. Am. J. Physiol. 1996; 271: F382-F390PubMed Google Scholar) with slight modifications. Briefly, in each experiment, CTAL (150–200 mm of total length) were microdissected from a collagenase-treated kidney. The medium used was supplemented with vitamins and amino acids as described above but contained 2 mm [Ca2+]e. CTAL pieces were radiolabeled in 50 μl of this medium containingmyo -[3H]inositol (1 mCi/ml, Amersham Pharmacia Biotech) for 2 h at 30 °C. After this labeling period, tubules were extensively rinsed in 0.5 mm[Ca2+]e medium, and tubule samples (4–7 mm length each) were incubated at 37 °C during 15 min in 0.5 mm [Ca2+]e medium supplemented with lithium. The reaction was stopped, and the radioactivity associated with phosphoinositides, free inositol, inositol phosphates (IP), and glycerophosphoinositol was separated and counted as described previously (29Meneton P. Imbert-Teboul M. Bloch-Faure M. Rajerison R.M. Am. J. Physiol. 1996; 271: F382-F390PubMed Google Scholar). In several experiments, it was checked that there was a good correlation between inositol phosphate formation expressed either per unit of tubule length or as a percentage of the total radioactivity counted (r = 0.93 ± 0.03, n = 4), an observation in agreement with that previously observed with proximal tubule fragments (29Meneton P. Imbert-Teboul M. Bloch-Faure M. Rajerison R.M. Am. J. Physiol. 1996; 271: F382-F390PubMed Google Scholar). Consequently, IP production was expressed as percentage of the total radioactivity counted. In each experiment, different experimental conditions (5 replicate samples each) were tested, and the mean of the IP values measured in each condition was taken as one single data point. The results are given as the mean values ± S.E. calculated from n different experiments. The statistical evaluation of the results was assessed by paired Student's t test. Intracellular Ca2+ concentration was measured in single CTAL samples microdissected from collagenase-treated kidneys by using the calcium-sensitive fluorescence probe fura-2 as described previously (11Champigneulle A. Siga E. Vassent G. Imbert-Teboul M. J. Membr. Biol. 1997; 156: 117-129Crossref PubMed Scopus (25) Google Scholar). Briefly, CTAL were loaded for 60 min with 10 μm fura-2 AM. Each CTAL was then transferred to a superfusion chamber fixed on the stage of an inverted fluorescence microscope (Zeiss IM 35, Oberkochen, Germany). The tubule was superfused at 37 °C at a rate of 10–12 ml/min, corresponding to about 10 exchanges per min. The composition of the superfusion medium was similar to that used in cAMP experiments, except that serum albumin, ibuprofen, and adenosine deaminase were not added since the superfusion medium was flushed continuously. After a 5- to 10-min equilibration period, agonists were added to either the 0.5 mm [Ca2+]e medium or to the Ca2+-free medium and superfused over tubule. Due to the dead space of the superfusion set up, the time necessary to achieve a full equilibration of agonist concentration in the chamber was of 15–20 s. The tubular portion selected for fluorescence measurement included about 30 cells. Double wavelength measurements of fura-2 fluorescence were recorded every 2 s. Calculations of [Ca2+]i were performed as described previously (11Champigneulle A. Siga E. Vassent G. Imbert-Teboul M. J. Membr. Biol. 1997; 156: 117-129Crossref PubMed Scopus (25) Google Scholar). The results obtained from different tubules microdissected from several rats were expressed as mean values ± S.E. The localization of type 6 AC mRNA in the rat kidney was examined by in situ hybridization at the light microscope. The most intense labeling in the kidney cortex was found in thick ascending limb, including CTAL (Fig. 1), collecting duct, and glomeruli (not shown). Fig. 1 shows strong labeling of CTALs (a, c, and arrowheads ), whereas proximal tubules (a , c , and *) are weakly labeled. In CTAL, labeling was equally intense in all cells. No labeling was observed when the sense cRNA probe was used (Fig. 1,b and d ). In contrast to type 6 AC, no significant labeling for type 5 AC was found in CTAL (not shown). This is consistent with previous quantitative RT-PCR results showing that the type 6 AC mRNA was more abundant in CTAL than in proximal tubule and that type 5 AC mRNA was not detected in CTAL (14Chabardès D. Firsov D. Aarab L. Clabecq A. Bellanger A.C. Siaume-Perez S. Elalouf J.M. J. Biol. Chem. 1996; 271: 19264-19271Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). AVP was used at 10 nm, a concentration that induces a maximal stimulation of cAMP accumulation in intact cells (Ref. 19Torikai S. Kurokawa K. Am. J. Physiol. 1983; 245: F58-F66PubMed Google Scholar and data not shown) and thus allows us to study the maximal amount of functional AC proteins present in the rat CTAL. AVP-dependent cAMP accumulation was about 2-fold higher in the presence of 1 mm IBMX, which reflects AVP-stimulated cAMP synthesis only, than in the presence of 50 μm Ro 20-1724, which allows the measurement of cAMP accumulation that integrates both the synthesis and a partial catabolism of cAMP (Fig.2, left panel ). In the presence of increasing [Ca2+]e, there was a dose-dependent inhibition of AVP-dependent cAMP levels with half-maximal inhibition of about 1.2 and 2.0 mm [Ca2+]e in the presence of Ro 20-1724 and IBMX, respectively (Fig. 2, right panel ). With Ro 20-1724, a steep inverse relationship was observed, and thus a small variation of [Ca2+]e induced a high variation of hormone-dependent cAMP accumulation. The shape of the curve obtained with either IBMX or Ro 20-1724 shows that [Ca2+]e below 1.5 mm had a small effect on cAMP synthesis but increased cAMP hydrolysis; conversely, at higher [Ca2+]e the inhibition was mediated mainly by a decrease of cAMP synthesis. Altogether in this study, the inhibition induced by 1.25 mm[Ca2+]e in the presence of Ro 20-1724 was of 63.3 ± 3.9% (p < 0.001 versus 10 nm AVP, n = 11) and that induced by 1.5 mm [Ca2+]e in the presence of IBMX was of 30.5 ± 3.5% (p < 0.01 versus 10 nm AVP, n = 8). The implication of the Ca2+ receptor RaKCaR to explain the inhibitory effect of [Ca2+]e was supported by the results obtained with an agonist of this receptor, neomycin (1Brown E.M. Gamba G. Riccardi D. Lombardi M. Butters R. Kifor O. Sun A. Hediger M.A. Lytton J. Hebert S.C. Nature. 1993; 366: 575-580Crossref PubMed Scopus (2380) Google Scholar, 2Riccardi D. Park J. Lee W.-S. Gamba G. Brown E.M. Hebert S.C. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 131-135Crossref PubMed Scopus (437) Google Scholar). In 0.5 mm [Ca2+]e, 100 μmneomycin inhibited by 82.2 ± 5.8% (n = 3) and 43.5 ± 5.7% (n = 5) cAMP accumulated by 10 nm AVP in the presence of Ro 20-1724 and IBMX, respectively. In the presence of IBMX, 2.5 mm[Ca2+]e inhibited cAMP synthesis stimulated by either 10 μm forskolin or 10 nm AVP with a comparable efficiency (62.3 ± 7.2% of inhibition and 73.5 ± 2.5% in forskolin and AVP experimental groups, respectively,n = 3). These results suggest that [Ca2+]e inhibited AC activity at post-receptor sites. In our experimental conditions, no detectable [Ca2+]i variations were obtained with 1.25 mm [Ca2+]e (TableI), and the response to 1.5 mm [Ca2+]e was characterized in most tubules by a low but sustained [Ca2+]i increase without a clear-cut peak phase (Fig. 3and Table I). With 2.5 mm [Ca2+]e, the response was a transient peak followed by a lower sustained plateau (Fig. 3) that reflects Ca2+ entry (11Champigneulle A. Siga E. Vassent G. Imbert-Teboul M. J. Membr. Biol. 1997; 156: 117-129Crossref PubMed Scopus (25) Google Scholar). The simultaneous superfusion of 10 nm AVP and [Ca2+]edid not modify [Ca2+]i variations (data not shown). Unlike the peak, the plateau was blocked by nonspecific Ca2+ channel blockers such as La3+ and Ni2+, whereas addition of voltage-sensitive channel blockers, i.e. verapamil and nifedipine, did not modify [Ca2+]i (Ref. 11Champigneulle A. Siga E. Vassent G. Imbert-Teboul M. J. Membr. Biol. 1997; 156: 117-129Crossref PubMed Scopus (25) Google Scholar and data not shown).Table IEffect of agonists on inositol phosphates (IPs) production and on [Ca2+]i in the CTALExperimental conditionIPs production (stimulation factor)Δ[Ca2+]i Increases (nm)PeakPlateau1.25 mm[Ca2+]e1.52 ± 0.051-aMean value statistically different from the corresponding control. [Ca2+]i variations (Δ[Ca2+]i) are expressed as increases over basal value observed during the peak and plateau phases, respectively. Basal value of [Ca2+]i calculated from 28 tubules was of 99.2 ± 7.2 nm. Data are the mean val
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