Calcium-dependent Dephosphorylation Mediates the Hyperosmotic and Lysophosphatidic Acid-dependent Inhibition of Natriuretic Peptide Receptor-B/Guanylyl Cyclase-B
2004; Elsevier BV; Volume: 279; Issue: 47 Linguagem: Inglês
10.1074/jbc.m408247200
ISSN1083-351X
AutoresRegine Potthast, Sarah E. Abbey‐Hosch, Laura K. Antos, Jonathan S. Marchant, Michaela Kühn, Lincoln R. Potter,
Tópico(s)Phosphodiesterase function and regulation
ResumoC-type natriuretic peptide binding to natriuretic peptide receptor-B (NPR-B) stimulates cGMP synthesis, which regulates vasorelaxation, cell proliferation, and bone growth. Here, we investigated the mechanistic basis for hyperosmotic and lysophosphatidic acid-dependent inhibition of NPR-B. Whole cell cGMP measurements and guanylyl cyclase assays indicated that acute hyperosmolarity decreased NPR-B activity in a reversible, concentration- and time-dependent manner, whereas chronic exposure had no effect. Acute hyperosmolarityelevatedintracellularcalciuminaconcentration-dependent fashion that paralleled NPR-B desensitization. A calcium chelator, but not a protein kinase C inhibitor, blocked both calcium elevations and desensitization. Hyperosmotic medium stimulated NPR-B dephosphorylation, and the receptor was rapidly rephosphorylated and resensitized when the hypertonic media was removed. Lysophosphatidic acid also inhibited NPR-B in a calcium- and phosphorylation-dependent process, consistent with calcium being a universal regulator of NPR-B. The absolute requirement of dephosphorylation in this process was demonstrated by showing that a receptor with glutamates substituted at all known NPR-B phosphorylation sites is unresponsive to hyperosmotic stimuli. This is the first study to measure the phosphorylation state of an endogenous guanylyl cyclase and to link intracellular calcium elevations with its dephosphorylation. C-type natriuretic peptide binding to natriuretic peptide receptor-B (NPR-B) stimulates cGMP synthesis, which regulates vasorelaxation, cell proliferation, and bone growth. Here, we investigated the mechanistic basis for hyperosmotic and lysophosphatidic acid-dependent inhibition of NPR-B. Whole cell cGMP measurements and guanylyl cyclase assays indicated that acute hyperosmolarity decreased NPR-B activity in a reversible, concentration- and time-dependent manner, whereas chronic exposure had no effect. Acute hyperosmolarityelevatedintracellularcalciuminaconcentration-dependent fashion that paralleled NPR-B desensitization. A calcium chelator, but not a protein kinase C inhibitor, blocked both calcium elevations and desensitization. Hyperosmotic medium stimulated NPR-B dephosphorylation, and the receptor was rapidly rephosphorylated and resensitized when the hypertonic media was removed. Lysophosphatidic acid also inhibited NPR-B in a calcium- and phosphorylation-dependent process, consistent with calcium being a universal regulator of NPR-B. The absolute requirement of dephosphorylation in this process was demonstrated by showing that a receptor with glutamates substituted at all known NPR-B phosphorylation sites is unresponsive to hyperosmotic stimuli. This is the first study to measure the phosphorylation state of an endogenous guanylyl cyclase and to link intracellular calcium elevations with its dephosphorylation. The mammalian natriuretic peptide family consists of three members: atrial natriuretic peptide (ANP), 1The abbreviations used are: ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; NPR, natriuretic peptide receptor; CNP, C-type natriuretic peptide; GFP, green fluorescent protein; LPA, lysophosphatidic acid; PMA, phorbol 12-myristate 13-acetate; AM, acetoxylmethyl ester; BAPTA, 1,2-bis(2-Aminophenoxy)ethane N,N,N′,N′-tetraacetic acid.1The abbreviations used are: ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; NPR, natriuretic peptide receptor; CNP, C-type natriuretic peptide; GFP, green fluorescent protein; LPA, lysophosphatidic acid; PMA, phorbol 12-myristate 13-acetate; AM, acetoxylmethyl ester; BAPTA, 1,2-bis(2-Aminophenoxy)ethane N,N,N′,N′-tetraacetic acid. brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) (1Levin E.R. Gardner D.G. Samson W.K. N. Engl. J. Med. 1998; 339: 321-328Crossref PubMed Scopus (2027) Google Scholar). ANP and BNP are produced in atrial and ventricular myocytes, respectively. ANP reduces blood pressure by stimulating sodium and water excretion, by stimulating vasorelaxation, and by inhibiting renin and aldosterone secretion. CNP is most highly expressed in the brain (2Sudoh T. Minamino N. Kangawa K. Matsuo H. Biochem. Biophys. Res. Commun. 1990; 168: 863-870Crossref PubMed Scopus (958) Google Scholar), endothelial cells (3Suga S. Nakao K. Itoh H. Komatsu Y. Ogawa Y. Hama N. Imura H. J. Clin. Investig. 1992; 90: 1145-1149Crossref PubMed Scopus (489) Google Scholar), and chondrocytes (4Chusho H. Tamura N. Ogawa Y. Yasoda A. Suda M. Miyazawa T. Nakamura K. Nakao K. Kurihara T. Komatsu Y. Itoh H. Tanaka K. Saito Y. Katsuki M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4016-4021Crossref PubMed Scopus (365) Google Scholar, 5Yasoda A. Ogawa Y. Suda M. Tamura N. Mori K. Sakuma Y. Chusho H. Shiota K. Tanaka K. Nakao K. J. Biol. Chem. 1998; 273: 11695-11700Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). It relaxes vascular smooth muscle cells (6Clavell A.L. Stingo A.J. Wei C.M. Heublein D.M. Burnett Jr., J.C. Am. J. Physiol. 1993; 264: R290-R295PubMed Google Scholar) and inhibits their proliferation (7Furuya M. Yoshida M. Hayashi Y. Ohnuma N. Minamino N. Kangawa K. Matsuo H. Biochem. Biophys. Res. Commun. 1991; 177: 927-931Crossref PubMed Scopus (174) Google Scholar), a process that has been exploited to inhibit vascular restenosis and to stimulate vascular regeneration (8Yamahara K. Itoh H. Chun T.H. Ogawa Y. Yamashita J. Sawada N. Fukunaga Y. Sone M. Yurugi-Kobayashi T. Miyashita K. Tsujimoto H. Kook H. Feil R. Garbers D.L. Hofmann F. Nakao K. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3404-3409Crossref PubMed Scopus (139) Google Scholar). In addition, CNP stimulates the proliferation of chondrocytes, which promotes the growth of long bones (4Chusho H. Tamura N. Ogawa Y. Yasoda A. Suda M. Miyazawa T. Nakamura K. Nakao K. Kurihara T. Komatsu Y. Itoh H. Tanaka K. Saito Y. Katsuki M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 4016-4021Crossref PubMed Scopus (365) Google Scholar, 5Yasoda A. Ogawa Y. Suda M. Tamura N. Mori K. Sakuma Y. Chusho H. Shiota K. Tanaka K. Nakao K. J. Biol. Chem. 1998; 273: 11695-11700Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar).Natriuretic peptide receptors are structurally related cell surface guanylyl cyclases called natriuretic peptide receptor-A (NPR-A) and natriuretic peptide receptor-B (NPR-B) or guanylyl cyclase-A and guanylyl cyclase-B (9Schulz S. Waldman S.A. Vitam. Horm. 1999; 57: 123-151Crossref PubMed Scopus (29) Google Scholar, 10Potter L.R. Hunter T. J. Biol. Chem. 2001; 276: 6057-6060Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 11Kuhn M. Circ. Res. 2003; 93: 700-709Crossref PubMed Scopus (218) Google Scholar). They consist of an extracellular ligand binding domain, a single membrane-spanning region and intracellular kinase homology, dimerization, and carboxyl-terminal guanylyl cyclase domains. ANP and BNP activate NPR-A, whereas CNP activates NPR-B. Binding of these receptors by their respective ligands stimulates the synthesis of the intracellular second messenger, cGMP.The mechanisms regulating NPR-A and NPR-B activity are incompletely understood. It is known, however, that receptor phosphorylation is required for natriuretic peptide activation and that prolonged exposure to natriuretic peptides or acute exposure to activators of protein kinase C causes receptor dephosphorylation and desensitization (12Potter L.R. Garbers D.L. J. Biol. Chem. 1992; 267: 14531-14534Abstract Full Text PDF PubMed Google Scholar, 13Potter L.R. Hunter T. J. Biol. Chem. 2000; 275: 31099-31106Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). NPR-A and NPR-B also are inhibited by hormones (arginine, vasopressin, angiotensin II, and lysophosphatidic acid) or growth factors (platelet-derived and fibroblast growth factors) that antagonize the actions of natriuretic peptides (14Nambi P. Whitman M. Gessner G. Aiyar N. Crooke S.T. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 8492-8495Crossref PubMed Scopus (58) Google Scholar, 15Haneda M. Kikkawa R. Maeda S. Togawa M. Koya D. Horide N. Kajiwara N. Shigeta Y. Kidney Int. 1991; 40: 188-194Abstract Full Text PDF PubMed Scopus (99) Google Scholar, 16Chrisman T.D. Garbers D.L. J. Biol. Chem. 1999; 274: 4293-4299Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 17Abbey S.E. Potter L.R. J. Biol. Chem. 2002; 277: 42423-42430Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 18Abbey S.E. Potter L.R. Endocrinology. 2003; 144: 240-246Crossref PubMed Scopus (41) Google Scholar). Protein kinase C was initially implicated in this so-called heterologous desensitization, but recent studies indicate that it is dispensable for this response (17Abbey S.E. Potter L.R. J. Biol. Chem. 2002; 277: 42423-42430Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 18Abbey S.E. Potter L.R. Endocrinology. 2003; 144: 240-246Crossref PubMed Scopus (41) Google Scholar).While characterizing how hyperosmotic medium inhibits NPR-B, we found that agents or conditions that elevate intracellular calcium stimulate the dephosphorylation of NPR-B and that both calcium elevations and dephosphorylation are required for the inhibition. We suggest that this is a universal mechanism by which environmental conditions and antagonistic hormones inhibit natriuretic peptide signaling. This mechanism may underlie the desensitization of NPR-A and NPR-B that accompanies pathological conditions such as diabetes and congestive heart failure (19Sechi L.A. Valentin J.P. Griffin C.A. Lee E. Bartoli E. Humphreys M.H. Schambelan M. J. Clin. Investig. 1995; 95: 2451-2457Crossref PubMed Scopus (41) Google Scholar, 20Smits P. Hersbach F.M. Jansen T.L. Thien T. Lutterman J.A. Diabetes. 1993; 42: 1454-1461Crossref PubMed Scopus (19) Google Scholar, 21Tsutamoto T. Kanamori T. Morigami N. Sugimoto Y. Yamaoka O. Kinoshita M. Circulation. 1993; 87: 70-75Crossref PubMed Scopus (140) Google Scholar).EXPERIMENTAL PROCEDURESMaterials—Rat CNP, GF-109203X, and BAPTA-AM were purchased from Sigma-Aldrich. Lysophosphatidic acid (LPA) was acquired from Avanti Polar Lipids (Alabaster, AL). Fluo-4-AM was purchased from Molecular Probes (Eugene, OR). The NIH3T3 cells (CRL-1658) and the A10 rat aortic smooth muscle cells (CRL-1476) were acquired from American Type Culture Collection (Manassas, VA).Cell Culture and Preparation of Crude Membranes—NIH3T3 fibroblast (18Abbey S.E. Potter L.R. Endocrinology. 2003; 144: 240-246Crossref PubMed Scopus (41) Google Scholar) and A10 aortic smooth muscle cells (17Abbey S.E. Potter L.R. J. Biol. Chem. 2002; 277: 42423-42430Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) were cultured as described previously. HEK 293T cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum. Crude membranes were prepared as reported previously (18Abbey S.E. Potter L.R. Endocrinology. 2003; 144: 240-246Crossref PubMed Scopus (41) Google Scholar).Confocal Calcium Imaging and Transfections—NIH3T3 cells were plated on poly-d-lysine-coated 35-mm glass-bottom culture dishes (Mattek Corp., Ashland, MA). For the GFP transfections, cells were grown to 50% confluence and transfected with 1 μg of a plasmid expressing green fluorescent protein under the control of a cytomegalovirus promoter using the FuGENE 6 transfection reagent (Roche Diagnostics). For calcium imaging experiments, untransfected NIH3T3 cells at ∼90% confluence were first washed with modified HEPES buffer, pH 7.3 (109.5 mm NaCl, 5.9 mm KCl, 1.2 mm MgCl2, 1.5 mm CaCl2, 11.6 mm HEPES, 11.5 mm glucose) and then incubated with HEPES buffer supplemented with 2 μm fluo-4-AM, a high affinity cell-permeable calcium indicator, at room temperature for 1 h. Cells were bathed in modified HEPES buffer containing no dye for 30 min to allow dye de-esterification. The untransfected and GFP-transfected cells were superfused at ∼3 ml/min at room temperature with modified HEPES buffer supplemented with varying concentrations of NaCl. Video-rate calcium imaging was performed using a self-constructed confocal microscope built around an Olympus IX70 inverted microscope. Fluorophores (fluo-4-AM and GFP) were excited using a 488-nm argon laser, and changes in fluorescence emission were monitored with a 530 ± 15-nm band pass filter. Images were recorded every 33 ms, and the resulting image stacks were analyzed by the Metamorph software package (Universal Imaging).Whole Cell Cyclic GMP Stimulations and Guanylyl Cyclase Assays— NIH3T3 cells plated in 12-well dishes were grown to 90% confluence and incubated for at least 4 h in serum-free medium. The medium was aspirated and replaced with 0.5 ml of Dulbecco's modified Eagle's medium containing 20 mm HEPES, 0.5 mm 1-methyl-3-isobutylxanthine, and various concentrations of NaCl. After a 30-min incubation, the medium was replaced with the same medium containing CNP. Cells were stimulated for 3 min and stopped by aspirating the medium and adding 1 ml of ice-cold 80% ethanol. All incubations were at 37 °C. Cyclic GMP concentrations in the ethanol extract were estimated as reported (17Abbey S.E. Potter L.R. J. Biol. Chem. 2002; 277: 42423-42430Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Guanylyl cyclase assays were performed for 3 min as described previously (17Abbey S.E. Potter L.R. J. Biol. Chem. 2002; 277: 42423-42430Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar).Immunoprecipitations—Serum-starved cells from 10-cm plates were exposed to various treatments and solubilized in 1 ml of immunoprecipitation buffer (50 mm HEPES, pH 7.4, 150 mm NaCl, 10 mm NaH2PO4, 50 mm NaF, 1 mm EDTA, 0.1 μm microcystin, 1% Triton X-100, 10% glycerol, 1× protease inhibitor mixture from Roche Diagnostics, 0.1% SDS, 0.5% sodium deoxycholate). After rotation for 15 min with 30 μl of a 50% protein A slurry at 4 °C the extract was cleared by centrifugation at 20,000 × g for 15 min at 2 °C. 0.8 ml of the cleared extract from each treatment was incubated with 3 μl of polyclonal antiserum from rabbit 6328 for 1 h at 4 °C. Rabbit 6328 was immunized with the synthetic peptide CGERKGPPGLL that corresponds to the last 10 carboxyl-terminal amino acids of NPR-B. Thirty μl of a 50% protein A slurry were added to the extract and incubated for an additional hour. The protein A immunocomplex was pelleted by low speed centrifugation and washed three times with 1 ml of immunoprecipitation buffer. NPR-B was released from the immunocomplex by boiling for 3 min in the presence of 30 μl of 2× reducing SDS sample buffer and fractionated by SDS-PAGE on a 4–12% gradient resolving gel.Gel Staining—Polyacrylamide gels were immersed in a fixing solution containing 50% methanol and 10% acetic acid and then incubated overnight at room temperature with rocking. The gel was then rinsed twice for 10 min in 100 ml of water. Staining was performed in the dark by adding 20 ml of a phosphoprotein-specific dye to the gel (Pro-Q diamond phosphoprotein gel stain, Molecular Probes) for 2 h with gentle agitation. The gel was destained by incubation with 100 ml of a solution of 20% acetonitrile and 50 mm sodium acetate (pH 4.0) for 3 h in the dark. The destaining solution was changed twice within the 3-h incubation period. The stained phosphoproteins were visualized at 532 nm using a Fluoroimage analyzer (FLA-5000, Fujifilm). To detect proteins, the same gel was washed three times with distilled water for 10 min and incubated overnight with 20 ml of SYPRO ruby protein gel stain (Molecular Probes). The next day the gel was rinsed twice with a solution of 10% methanol and 7% acetic acid for 30 min each. The stained proteins were detected at 473 nm as described above. Phosphoprotein staining was quantitated after correction of protein amounts using the Fuji Image Gauge software.RESULTSHyperosmolarity Acutely Inhibits NPR-B in NIH3T3 Cells—To examine the effect of hyperosmolarity on the activity of NPR-B, we incubated NIH3T3 cells with medium alone or medium containing 10 or 100 mm final concentrations of Tris-HCl or HEPES buffers to increase the extracellular osmotic pressure. After 30 min, we prepared crude membranes from these cells and assayed them for CNP-dependent and Triton X-100-dependent guanylyl cyclase activities. The former treatment measures the physiologic activation of NPR-B, whereas the latter artificial conditions indicate the total amount of guanylyl cyclase present in any given membrane preparation. CNP-dependent but not Triton X-100-dependent cyclase activity was markedly reduced in membranes from cells exposed to 100 mm concentrations of either buffer, whereas activities measured in membranes from cells exposed to 10 mm concentrations were only slightly decreased (Fig. 1A).To determine whether other general osmolytes inhibit NPR-B, we incubated serum-starved cells with 100 mm NaCl, 200 mm sorbitol, or 200 mm urea so that 200 meq of each osmolyte were added per treatment. As shown in Fig. 1B, membranes prepared from cells exposed to either NaCl or sorbitol had similarly reduced CNP-dependent guanylyl cyclase activities. In contrast, no significant reductions were observed in membranes prepared from cells incubated with the same number of milliosmoles of urea. The lack of effect of urea is because of its high cell membrane permeability (22Garcia-Perez A. Burg M.B. Physiol. Rev. 1991; 71: 1081-1115Crossref PubMed Scopus (484) Google Scholar). Hence, its osmotic gradient is short lived. In further support of a general osmotic inhibitory mechanism, we found that the effects of submaximal concentrations of NaCl (50 mm) and sorbitol (100 mm) were additive, whereas incubations with 50 mm NaCl and 100 mm urea resulted in an inhibitory response that was about half that observed with 100 mm NaCl alone.To further examine the sensitivity of the NPR-B receptor to hyperosmolarity, we conducted guanylyl cyclase assays on membranes derived from NIH3T3 cells that were incubated for 30 min with 0, 10, 25, 50, 100, and 150 mm NaCl in addition to the normal 110 mm NaCl contained in the medium. We found that the additional NaCl inhibited CNP-dependent activity in a concentration-dependent manner (Fig. 1C). Slight reductions were observed with NaCl incubations of 25 mm, and marked desensitization was detectable after exposure of cells to 50 mm or higher additional NaCl concentrations. The maximum concentration tested, 150 mm, reduced hormone-dependent but not detergent-dependent activity to about half of the amount detected in untreated cells.To characterize the time course of the inhibition, cells were serum-starved overnight and then incubated for 0, 5, 10, 20, 30, or 60 min with 100 mm additional NaCl. Membranes were prepared from these cells and assayed for hormone-dependent or detergent-dependent guanylyl cyclase activities (Fig. 1D). The effect of NaCl on hormone-dependent activity was rapid, having a t½ of ∼15 min. Again, the hyperosmotic conditions had no effect on cyclase activity measured in the presence of detergent.Chronic Hyperosmolarity Does Not Increase NPR-B Activity—Because previous studies have shown that the ANP-dependent activity of NPR-A is enhanced after long term exposure to hyperosmotic medium (23Katafuchi T. Mizuno T. Hagiwara H. Itakura M. Ito T. Hirose S. J. Biol. Chem. 1992; 267: 7624-7629Abstract Full Text PDF PubMed Google Scholar), we investigated the effect of a long term (days) NaCl incubation on NPR-B activity in the NIH3T3 fibroblasts. In these experiments, cells were exposed to 25 mm, 50 mm, or 75 mm additional NaCl for 2 days. However, neither CNP-dependent nor detergent-dependent NPR-B guanylyl cyclase activities were affected by these treatments (data not shown), suggesting that different mechanisms underlie the regulation of NPR-A and NPR-B in response to chronic hyperosmotic stress.Acute Hyperosmolarity Inhibits NPR-B in A10 Cells—We next asked whether the osmotic regulation of NPR-B is specific to NIH3T3 cells or is a general characteristic of this receptor by investigating the osmotic regulation of NPR-B in the A10 vascular smooth muscle cell line. We have shown previously that NPR-B is expressed and regulated appropriately in these cells (17Abbey S.E. Potter L.R. J. Biol. Chem. 2002; 277: 42423-42430Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). As with the NIH3T3 fibroblasts, we found that acute but not chronic exposure to hyperosmotic medium inhibited CNP-dependent NPR-B activity in vascular smooth muscle cells (Fig. 1, E and F). Hence, both the acute osmotic effect and the lack of a chronic osmotic effect are general characteristics of this receptor.Hyperosmolarity Inhibits CNP-dependent cGMP Elevations—Because the previous experiments were conducted by directly measuring the guanylyl cyclase activity of NPR-B in broken cell preparations, we examined the hyperosmotic effect in the more physiologic environment of an intact cell. Whole cells were incubated with 50, 100, or 150 mm additional NaCl for 30 min as shown in Fig. 1A, but instead of measuring cyclase activity in crude membrane preparations, we treated the cells with 20 nm CNP and measured intracellular cGMP concentrations. CNP-dependent cGMP elevations in the whole cellsweremarkedlyinhibitedbyNaClexposureinaconcentration-dependent manner (IC50 ∼50 mm) (Fig. 2, top panel). Remarkably, the maximal concentration tested, 150 mm, suppressed CNP-dependent cGMP production to less than 15% of control values.Fig. 2Hyperosmolarity decreases CNP-dependent NPR-B activity in NIH3T3 cells. Top panel, cells were incubated with increasing amounts of NaCl for 30 min. The medium was aspirated, and the cells were stimulated with 20 nm CNP for 3 min. Cyclic GMP concentrations were estimated by radioimmunoassay. Values are the mean of six separate determinations. Vertical bars within symbols represent the S.E. Middle panel, cells were incubated with 100 mm additional NaCl for 30 min. The hypertonic medium was replaced with normotonic medium, and the cells were incubated for an additional 30 min and then stimulated with 20 nm CNP for 3 min. Bottom panel, cells were incubated with 100 mm additional NaCl for 30 min. Then the hyperosmotic medium was removed and replaced by a normotonic medium for 5, 10, 15, and 30 min. Crude membranes were prepared at the indicated time points and assayed for CNP-dependent (squares) and Triton X-100-dependent (circles) guanylyl cyclase activities. Values are the mean of five separate experiments that were assayed in duplicate (n = 10). The vertical bars within each column represent the S.E.View Large Image Figure ViewerDownload (PPT)The Hyperosmotic Effect Is Reversible—To confirm that the acute suppression of the CNP-dependent activity was not because of irreversible cellular damage, we incubated whole cells with hypertonic medium for 30 min and then replaced the medium with Dulbecco's modified Eagle's medium lacking exogenous salt and measured the CNP-dependent cGMP response after an additional 30-min incubation. In this "washout" experiment, we found that removal of the hypertonic medium resulted in a marked recovery of the CNP-dependent cGMP response (Fig. 2, middle panel). In a similar experiment, we incubated cells in hypertonic medium for 30 min and then replaced it with medium lacking additional salt for 5, 10, 15, or 30 min. After the indicated times, we prepared membranes from these cells and assayed them for guanylyl cyclase activity (Fig. 2, bottom panel). CNP-dependent guanylyl cyclase activity increased from the inhibited levels as early as 5 min after the removal of the hypertonic medium and was completely recovered by 30 min. Detergent-dependent cyclase activity was unaffected by the presence or absence of exogenous NaCl. These data indicate that the hyperosmotic inhibition of NPR-B is reversible. It also suggests that the reductions in cyclase activity do not result from receptor degradation as was reported for the osmotic inhibition of NPR-A (24Iimura O. Kusano E. Ishida F. Oono S. Ando Y. Asano Y. Pfluegers Arch. Eur. J. Physiol. 1995; 430: 81-87Crossref PubMed Scopus (16) Google Scholar).Calcium Elevations Are Required for Hyperosmotic and LPA-dependent Inhibition of NPR-B—We recently demonstrated that the arginine-, vasopressin-, and sphingosine-1-phosphate-dependent desensitization of NPR-B requires elevated calcium concentrations (17Abbey S.E. Potter L.R. J. Biol. Chem. 2002; 277: 42423-42430Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 25Abbey-Hosch S.E. Cody A.N. Potter L.R. Hypertension. 2004; 43: 1103-1109Crossref PubMed Scopus (29) Google Scholar). Therefore, we asked whether calcium also mediates the hyperosmotic effect. To determine whether hyperosmotic medium elevates intracellular calcium, we employed a confocal imaging approach based on the calcium indicator fluo-4-AM. As shown in the top panel of Fig. 3, superfusion of the NIH3T3 cells with modified HEPES buffer containing 150 mm additional NaCl caused a sustained increase in intracellular calcium as evidenced by increased fluo-4-AM fluorescence at the calcium-bound wavelength. We also performed parallel experiments with GFP-transfected fibroblasts to confirm that changes in fluorescence represented bona fide calcium signals rather than simply a change in fluorophore distribution resulting from altered cell morphology. We examined the concentration dependence of the response by incubating the cells with 25, 50, 100, or 150 mm additional NaCl. In all cells the -fold change in fluo-4-AM fluorescence was greater than that observed with GFP (Fig. 3, middle panel). The net difference between these curves (% change in fluo-4-AM fluorescence–% change in GFP fluorescence) is displayed in the bottom panel of Fig. 3, along with the concentration response curve for CNP-evoked guanylyl cyclase activity to highlight the correlation between calcium levels and desensitization. Small elevations in media osmolarity (25 mm NaCl) did not cause significant cytoplasmic calcium elevations or NPR-B desensitization. However, over the higher range of 50 to 150 mm supplemental NaCl, the amplitude of the calcium signals and NPR-B desensitization were generally correlated.Fig. 3Hypertonic medium increases cytoplasmic calcium in concert with NPR-B desensitization. Top panel, NIH3T3 cells loaded with fluo-4-AM (black) or transiently transfected with GFP (gray) were superfused with modified HEPES buffer containing 150 mm additional NaCl. The resulting changes in fluorescence emission were monitored from single cells as a function of time. Middle panel, additional extracellular NaCl increases intracellular calcium concentrations in a concentration-dependent manner. Cumulative data are from the fluorescence traces of fluo-4-AM-loaded NIH3T3 and GFP-transfected NIH3T3 cells expressed as a ratio of fluorescence intensity (F) versus initial fluorescence intensity (Fo) at any time. Bottom panel, the lower curve (triangles) represents the net fluorescence change evoked by NaCl obtained by subtracting the mean amplitudes of the GFP signal from the fluo-4-AM signal at each point (triangles). The upper curve (squares) represents the CNP-dependent NPR-B desensitization after treatment of NIH3T3 cells with the indicated additional NaCl concentrations.View Large Image Figure ViewerDownload (PPT)To examine the absolute requirement of intracellular calcium elevations in the osmotic and LPA-dependent desensitization of NPR-B, we tested whether the cell-permeable calcium chelator, BAPTA-AM, could prevent either process. Consistent with a common calcium-dependent mechanism, pretreatment of the cells with 50 μm BAPTA-AM completely blocked the ability of LPA and hyperosmolarity to inhibit NPR-B (Fig. 4, top panel). In contrast, the protein kinase C inhibitor, GF-109203X, was ineffective, although it blocked the PMA-dependent inhibition (Fig. 4, bottom panel). These data indicate that elevated intracellular calcium but not protein kinase C activation is required for these inhibitory processes.Fig. 4NaCl- and LPA-dependent desensitization of NPR-B require intracellular calcium elevations. Top panel, NIH3T3 cells were pretreated with 50 μm BAPTA-AM or with buffer for 30 min. Cells were then incubated with or without 10 μm LPA or 100 mm additional NaCl for 30 min. Crude membranes were prepared and assayed for CNP-dependent and detergent-dependent guanylyl cyclase activities. Bottom panel, serum-starved NIH3T3 cells were pretreated with or without 1 μm GF-109203X (GFX), for 1 h. The cells were then incubated with or without 1 μm PMA or 100 mm NaCl for an additional 30 min. Crude membranes were prepared and assayed as described for the top panel. For each experiment, values represent the average of six separate experiments that were assayed in duplicate (n = 12). The vertical bars within each column represent the S.E.View Large Image Figure ViewerDownload (PPT)Hyperosmolarity and LPA Cause NPR-B Dephosphorylation—Because the hormone responsiveness of NPR-B is directly proportional to its phosphorylation state (26Potter L.R. Biochemistry. 1998; 37: 2422-2429Crossref PubMed Scopus (85) Google Scholar), we tested whether the calcium-elevating treatments stimulate NPR-B dephosphorylation. The phosphate content of NPR-B was determined by immunoprecipitating the receptor, fractionating the immunocomplex by SDS-PAGE, and staining the gel with a phosphoprotein-specific dye. In control experiments, we found that this staining closely paralleled the 32P content of NPR-B isolated from metabolically labeled cells. 2D. Smirnov and L. R. Potter, unpublished data. The intensity of th
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