D1 dopamine receptor hyperphosphorylation in renal proximal tubules in hypertension
2006; Elsevier BV; Volume: 70; Issue: 6 Linguagem: Inglês
10.1038/sj.ki.5001708
ISSN1523-1755
AutoresPingfeng Yu, Laureano D. Asico, Yingjin Luo, Peter M. Andrews, Gilbert M. Eisner, Ulrich Hopfer, Robin A. Felder, Pedro A. José,
Tópico(s)Renin-Angiotensin System Studies
ResumoA defect in the coupling of the D1 receptor (D1R) to its G protein/effector complex in renal proximal tubules plays a role in the pathogenesis of spontaneous hypertension. As there is no mutation of the D1R gene in the spontaneously hypertensive rat (SHR), we tested the hypothesis that the coupling defect is associated with constitutive desensitization/phosphorylation of the D1R. The following experiments were performed: (1) Cell culture and membrane preparations from rat kidneys and immortalized rat renal proximal tubule cells (RPTCs); (2) immunoprecipitation and immunoblotting; (3) cyclic adenosine 3′,5′ monophosphate and adenylyl cyclase assays; (4) immunofluorescence and confocal microscopy; (5) biotinylation of cell surface proteins; and (6) in vitro enzyme dephosphorylation. Basal serine-phosphorylated D1Rs in renal proximal tubules, brush border membranes, and membranes from immortalized RPTCs were greater in SHRs (21.0±1.5 density units, DU) than in normotensive rats (7.4±2.9 DU). The increased basal serine phosphorylation of D1Rs in SHRs was accompanied by decreased expression of D1R at the cell surface, and decreased ability of a D1-like receptor agonist (fenoldopam) to stimulate cyclic adenosine 3′,5′ monophosphate (cAMP) production. Increasing protein phosphatase 2A activity with protamine enhanced the ability of fenoldopam to stimulate cAMP accumulation (17±4%) and alter D1R cell surface expression in intact cells from SHRs. Alkaline phosphatase treatment of RPTC membranes decreased D1R phosphorylation and enhanced fenoldopam stimulation of adenylyl cyclase activity (26±6%) in SHRs. Uncoupling of the D1R from its G protein/effector complex in renal proximal tubules in SHRs is caused, in part, by increased D1R serine phosphorylation. A defect in the coupling of the D1 receptor (D1R) to its G protein/effector complex in renal proximal tubules plays a role in the pathogenesis of spontaneous hypertension. As there is no mutation of the D1R gene in the spontaneously hypertensive rat (SHR), we tested the hypothesis that the coupling defect is associated with constitutive desensitization/phosphorylation of the D1R. The following experiments were performed: (1) Cell culture and membrane preparations from rat kidneys and immortalized rat renal proximal tubule cells (RPTCs); (2) immunoprecipitation and immunoblotting; (3) cyclic adenosine 3′,5′ monophosphate and adenylyl cyclase assays; (4) immunofluorescence and confocal microscopy; (5) biotinylation of cell surface proteins; and (6) in vitro enzyme dephosphorylation. Basal serine-phosphorylated D1Rs in renal proximal tubules, brush border membranes, and membranes from immortalized RPTCs were greater in SHRs (21.0±1.5 density units, DU) than in normotensive rats (7.4±2.9 DU). The increased basal serine phosphorylation of D1Rs in SHRs was accompanied by decreased expression of D1R at the cell surface, and decreased ability of a D1-like receptor agonist (fenoldopam) to stimulate cyclic adenosine 3′,5′ monophosphate (cAMP) production. Increasing protein phosphatase 2A activity with protamine enhanced the ability of fenoldopam to stimulate cAMP accumulation (17±4%) and alter D1R cell surface expression in intact cells from SHRs. Alkaline phosphatase treatment of RPTC membranes decreased D1R phosphorylation and enhanced fenoldopam stimulation of adenylyl cyclase activity (26±6%) in SHRs. Uncoupling of the D1R from its G protein/effector complex in renal proximal tubules in SHRs is caused, in part, by increased D1R serine phosphorylation. Dopamine receptors in the central nervous system and peripheral tissues have been implicated in the regulation of blood pressure.1.Zeng C. Sanada H. Watanabe H. et al.Functional genomics of the dopaminergic system in hypertension.Physiol Genomics. 2004; 19: 233-246Crossref PubMed Scopus (104) Google Scholar,2.Hussain T. Lokhandwala M.F. Renal dopamine receptor function in hypertension.Hypertension. 1998; 32: 187-197Crossref PubMed Scopus (169) Google Scholar Dopamine exerts its action via two families of dopamine receptors that belong to the superfamily of G protein-coupled receptors (GPCR). The D1-like receptor family (D1A and D1B in rodents, D1 and D5 in humans) is linked to stimulation of adenylyl cyclase, via the stimulatory G protein, GαS. The D2-like receptor family (D2, D3, and D4) is linked to inhibition of adenylyl cyclase, via the inhibitory G proteins, Gαi and GO. There are remarkable parallels in the abnormal dopamine signaling, via D1-like receptors, in rodent models of genetic hypertension (e.g., spontaneously hypertensive rat (SHR)) and human essential hypertension. Disruption of either one of the D1-like receptors (D1 or D5) in mice leads to the development of hypertension.3.Albrecht F.E. Drago J. Felder R.A. et al.Role of the D1A dopamine receptor in the pathogenesis of genetic hypertension.J Clin Invest. 1996; 97: 2283-2288Crossref PubMed Scopus (165) Google Scholar,4.Hollon T.R. Bek M.J. Lachowicz J.E. et al.Mice lacking D5 dopamine receptors have increased sympathetic tone and are hypertensive.J Neurosci. 2002; 22: 10801-10810PubMed Google Scholar D1-like receptor inhibition of NHE3, Na+/HCO3−, Cl−/HCO3−, and Na+/K+ATPase activities in the renal proximal tubule and medullary thick ascending limb of Henle is impaired in the SHR and Dahl salt-sensitive rat.1.Zeng C. Sanada H. Watanabe H. et al.Functional genomics of the dopaminergic system in hypertension.Physiol Genomics. 2004; 19: 233-246Crossref PubMed Scopus (104) Google Scholar, 2.Hussain T. Lokhandwala M.F. Renal dopamine receptor function in hypertension.Hypertension. 1998; 32: 187-197Crossref PubMed Scopus (169) Google Scholar, 5.Kunimi M. Seki G. Hara C. et al.Dopamine inhibits renal Na+:HCO3-cotransporter in rabbits and normotensive rats but not in spontaneously hypertensive rats.Kidney Int. 2000; 57: 534-543Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 6.Nishi A. Eklöf A-C. Bertorello A.M. Aperia A. Dopamine regulation of renal Na+, K+-ATPase activity is lacking in Dahl salt-sensitive rats.Hypertension. 1993; 21: 767-771Crossref PubMed Google Scholar, 7.Pedrosa R. Jose P.A. Soares-da-Silva P. Defective D1-like receptor-mediated inhibition of the Cl-/HCO3-exchanger in immortalized SHR proximal tubular epithelial cells.Am J Physiol Renal Physiol. 2004; 286: F1120-F1126Crossref PubMed Scopus (48) Google Scholar The impaired D1-like receptor inhibitory effect on renal sodium transport in hypertension has been shown to be caused, in part, by impaired production of cytoplasmic second messengers.1.Zeng C. Sanada H. Watanabe H. et al.Functional genomics of the dopaminergic system in hypertension.Physiol Genomics. 2004; 19: 233-246Crossref PubMed Scopus (104) Google Scholar, 2.Hussain T. Lokhandwala M.F. Renal dopamine receptor function in hypertension.Hypertension. 1998; 32: 187-197Crossref PubMed Scopus (169) Google Scholar, 5.Kunimi M. Seki G. Hara C. et al.Dopamine inhibits renal Na+:HCO3-cotransporter in rabbits and normotensive rats but not in spontaneously hypertensive rats.Kidney Int. 2000; 57: 534-543Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 6.Nishi A. Eklöf A-C. Bertorello A.M. Aperia A. Dopamine regulation of renal Na+, K+-ATPase activity is lacking in Dahl salt-sensitive rats.Hypertension. 1993; 21: 767-771Crossref PubMed Google Scholar, 7.Pedrosa R. Jose P.A. Soares-da-Silva P. Defective D1-like receptor-mediated inhibition of the Cl-/HCO3-exchanger in immortalized SHR proximal tubular epithelial cells.Am J Physiol Renal Physiol. 2004; 286: F1120-F1126Crossref PubMed Scopus (48) Google Scholar Despite the impairment of D1-like receptors to exert their agonist effects in hypertension, there are no mutations in the coding region of the D1R or D5R in patients with essential hypertension or in genetically hypertensive rats.8.Sanada H. Jose P.A. Hazen-Martin D. et al.Dopamine-1 receptor defect in renal proximal tubular cells in essential hypertension.Hypertension. 1999; 33: 1036-1042Crossref PubMed Scopus (132) Google Scholar Instead, in hypertension, the D1R in the renal proximal tubule and medullary thick ascending limb of Henle is uncoupled from its G protein/effector complex,5.Kunimi M. Seki G. Hara C. et al.Dopamine inhibits renal Na+:HCO3-cotransporter in rabbits and normotensive rats but not in spontaneously hypertensive rats.Kidney Int. 2000; 57: 534-543Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 6.Nishi A. Eklöf A-C. Bertorello A.M. Aperia A. Dopamine regulation of renal Na+, K+-ATPase activity is lacking in Dahl salt-sensitive rats.Hypertension. 1993; 21: 767-771Crossref PubMed Google Scholar, 7.Pedrosa R. Jose P.A. Soares-da-Silva P. Defective D1-like receptor-mediated inhibition of the Cl-/HCO3-exchanger in immortalized SHR proximal tubular epithelial cells.Am J Physiol Renal Physiol. 2004; 286: F1120-F1126Crossref PubMed Scopus (48) Google Scholar, 8.Sanada H. Jose P.A. Hazen-Martin D. et al.Dopamine-1 receptor defect in renal proximal tubular cells in essential hypertension.Hypertension. 1999; 33: 1036-1042Crossref PubMed Scopus (132) Google Scholar, 9.Hussain T. Lokhandwala M.F. Renal dopamine DA1 receptor coupling with GS and Gq/11 proteins in spontaneously hypertensive rats.Am J Physiol. 1997; 272: F339-F346PubMed Google Scholar, 10.Xu J. Li X.X. Albrect F.E. et al.D1 receptor, Gsα, and Na+/H+ exchanger interactions in the kidney in hypertension.Hypertension. 2000; 36: 395-399Crossref PubMed Scopus (85) Google Scholar a state that is associated with increased basal phosphorylation of the D1R.8.Sanada H. Jose P.A. Hazen-Martin D. et al.Dopamine-1 receptor defect in renal proximal tubular cells in essential hypertension.Hypertension. 1999; 33: 1036-1042Crossref PubMed Scopus (132) Google Scholar,11.Felder R.A. Sanada H. Xu J. et al.G protein-coupled receptor kinase 4 gene variants in human essential hypertension.Proc Natl Acad Sci USA. 2002; 99: 3872-3877Crossref PubMed Scopus (230) Google Scholar This finding is of interest because the functional status of dopamine receptors is determined, in part, by their state of phosphorylation, a process common to members of the GPCR superfamily.12.Pitcher J.A. Payne E.S. Csortos C. et al.The G-protein-coupled receptor phosphatase: a protein phosphatase type 2A with a distinct subcellular distribution and substrate specificity.Proc Natl Acad Sci USA. 1995; 92: 8343-8347Crossref PubMed Scopus (154) Google Scholar, 13.Carman C.V. Benovic J.L. 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Regulation of D1 dopamine receptors with mutations of protein kinase phosphorylation sites: attenuation of the rate of agonist-induced desensitization.Mol Pharmacol. 1999; 56: 675-683PubMed Google Scholar Upon removal of the agonist, the attenuated responsiveness of the receptor is reversed by a dephosphorylation process subserved by protein phosphatases, probably of the 2A or 2B type, in the case of D1-like receptors.16.Yu P. Asico L.D. Eisner G.M. et al.Renal protein phosphatase 2A activity and spontaneous hypertension in rats.Hypertension. 2000; 36: 1053-1058Crossref PubMed Scopus (41) Google Scholar, 17.Efendiev R. Yudowski G.A. Zwiller J. et al.Relevance of dopamine signals anchoring dynamin-2 to the plasma membrane during Na+,K+-ATPase endocytosis.J Biol Chem. 2002; 277: 44108-44114Crossref PubMed Scopus (36) Google Scholar, 18.Adlersberg M. Hsiung S.C. Glickstein S.B. et al.Regulation of dopamine D1-receptor activation in vivo by protein phosphatase 2B (calcineurin).J Neurochem. 2004; 90: 865-873Crossref PubMed Scopus (14) Google Scholar The dephosphorylation leads to cell surface membrane recycling and resensitization of GPCRs. We have reported that the uncoupling of the D1R in renal proximal tubules in the SHR may be a consequence of a defective resensitization process, caused by decreased activity of protein phosphatase2A (PP2A).16.Yu P. Asico L.D. Eisner G.M. et al.Renal protein phosphatase 2A activity and spontaneous hypertension in rats.Hypertension. 2000; 36: 1053-1058Crossref PubMed Scopus (41) Google Scholar However, the phosphorylation state and the cell surface membrane localization of the D1R in kidneys of SHRs have not been reported. Therefore, the current studies were designed to determine the phosphorylation state and subcellular distribution of D1 receptors (D1Rs) in renal proximal tubules of WKY and SHRs. As the D1R is phosphorylated at serine residues,8.Sanada H. Jose P.A. Hazen-Martin D. et al.Dopamine-1 receptor defect in renal proximal tubular cells in essential hypertension.Hypertension. 1999; 33: 1036-1042Crossref PubMed Scopus (132) Google Scholar, 11.Felder R.A. Sanada H. Xu J. et al.G protein-coupled receptor kinase 4 gene variants in human essential hypertension.Proc Natl Acad Sci USA. 2002; 99: 3872-3877Crossref PubMed Scopus (230) Google Scholar, 14.Tiberi M. Nash S.R. Bertrand L. et al.Differential regulation of dopamine D1A receptor responsiveness by various G protein-coupled receptor kinases.J Biol Chem. 1996; 271: 3771-3778Crossref PubMed Scopus (148) Google Scholar, 15.Jiang D. Sibley D.R. Regulation of D1 dopamine receptors with mutations of protein kinase phosphorylation sites: attenuation of the rate of agonist-induced desensitization.Mol Pharmacol. 1999; 56: 675-683PubMed Google Scholar, 19.Kim O.J. Gardner B.R. 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Picard L. et al.Human cytomegalovirus carries serine/threonine protein phosphates PP1 and a host-cell derived PP2A.J Virol. 1996; 70: 1415-1423PubMed Google Scholar can enhance D1R function in SHRs. To determine the extent of phosphorylation of the D1R, ex vivo and in vitro studies were performed using freshly obtained rat kidney cortices, freshly prepared BBMs, and immortalized RPTCs because renal D1Rs are predominantly located in proximal tubules.1.Zeng C. Sanada H. Watanabe H. et al.Functional genomics of the dopaminergic system in hypertension.Physiol Genomics. 2004; 19: 233-246Crossref PubMed Scopus (104) Google Scholar, 7.Pedrosa R. Jose P.A. Soares-da-Silva P. Defective D1-like receptor-mediated inhibition of the Cl-/HCO3-exchanger in immortalized SHR proximal tubular epithelial cells.Am J Physiol Renal Physiol. 2004; 286: F1120-F1126Crossref PubMed Scopus (48) Google Scholar, 8.Sanada H. Jose P.A. Hazen-Martin D. et al.Dopamine-1 receptor defect in renal proximal tubular cells in essential hypertension.Hypertension. 1999; 33: 1036-1042Crossref PubMed Scopus (132) Google Scholar, 9.Hussain T. Lokhandwala M.F. Renal dopamine DA1 receptor coupling with GS and Gq/11 proteins in spontaneously hypertensive rats.Am J Physiol. 1997; 272: F339-F346PubMed Google Scholar, 10.Xu J. Li X.X. Albrect F.E. et al.D1 receptor, Gsα, and Na+/H+ exchanger interactions in the kidney in hypertension.Hypertension. 2000; 36: 395-399Crossref PubMed Scopus (85) Google Scholar, 11.Felder R.A. Sanada H. Xu J. et al.G protein-coupled receptor kinase 4 gene variants in human essential hypertension.Proc Natl Acad Sci USA. 2002; 99: 3872-3877Crossref PubMed Scopus (230) Google Scholar, 16.Yu P. Asico L.D. Eisner G.M. et al.Renal protein phosphatase 2A activity and spontaneous hypertension in rats.Hypertension. 2000; 36: 1053-1058Crossref PubMed Scopus (41) Google Scholar, 25.Yu P. Yang Z. 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Felder R.A. et al.Role of the D1A dopamine receptor in the pathogenesis of genetic hypertension.J Clin Invest. 1996; 97: 2283-2288Crossref PubMed Scopus (165) Google Scholar, 5.Kunimi M. Seki G. Hara C. et al.Dopamine inhibits renal Na+:HCO3-cotransporter in rabbits and normotensive rats but not in spontaneously hypertensive rats.Kidney Int. 2000; 57: 534-543Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 7.Pedrosa R. Jose P.A. Soares-da-Silva P. Defective D1-like receptor-mediated inhibition of the Cl-/HCO3-exchanger in immortalized SHR proximal tubular epithelial cells.Am J Physiol Renal Physiol. 2004; 286: F1120-F1126Crossref PubMed Scopus (48) Google Scholar, 8.Sanada H. Jose P.A. Hazen-Martin D. et al.Dopamine-1 receptor defect in renal proximal tubular cells in essential hypertension.Hypertension. 1999; 33: 1036-1042Crossref PubMed Scopus (132) Google Scholar, 9.Hussain T. Lokhandwala M.F. Renal dopamine DA1 receptor coupling with GS and Gq/11 proteins in spontaneously hypertensive rats.Am J Physiol. 1997; 272: F339-F346PubMed Google Scholar, 10.Xu J. Li X.X. Albrect F.E. et al.D1 receptor, Gsα, and Na+/H+ exchanger interactions in the kidney in hypertension.Hypertension. 2000; 36: 395-399Crossref PubMed Scopus (85) Google Scholar, 11.Felder R.A. Sanada H. Xu J. et al.G protein-coupled receptor kinase 4 gene variants in human essential hypertension.Proc Natl Acad Sci USA. 2002; 99: 3872-3877Crossref PubMed Scopus (230) Google Scholar, 16.Yu P. Asico L.D. Eisner G.M. et al.Renal protein phosphatase 2A activity and spontaneous hypertension in rats.Hypertension. 2000; 36: 1053-1058Crossref PubMed Scopus (41) Google Scholar, 25.Yu P. Yang Z. Jones J.E. et al.D1 dopamine receptor signaling involves caveolin-2 in HEK-293 cells.Kidney Int. 2004; 66: 2167-2180Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 26.Albrecht F.E. Xu J. Moe O.W. et al.Regulation of NHE3 activity by G protein subunits in renal brush-border membranes.Am J Physiol Regul Integr Comp Physiol. 2000; 278: R1064-R1073PubMed Google Scholar, 27.Zeng C. Yang Z. Wang Z. et al.Interaction of AT1 and D5 dopamine receptors in renal proximal tubule cells.Hypertension. 2005; 45: 804-810Crossref PubMed Scopus (75) Google Scholar, 32.Zaas D.W. Duncan M.J. Li G. et al.Pseudomonas invasion of type I pneumocytes is dependent on the expression and phosphorylation of caveolin-2.J Biol Chem. 2005; 280: 4864-4872Crossref PubMed Scopus (64) Google Scholar Basal levels of serine-phosphorylated D1R were greater in renal cortical membranes, BBMs, and immortalized RPTCs from SHRs than from WKY rats (Figure 1, Table 1). The D1-like agonist, fenoldopam, increased the quantity of serine-phosphorylated D1R in WKY rats but not in SHRs (Figure 1, Table 1). Pretreatment with carbidopa (renal cortical membranes and BBMs) had no effect on basal serine phosphorylation of D1Rs (not shown). Therefore, the results from the non-treated and treated groups were pooled together.Table 1Serine-phosphorylated D1Rs in cortical and BBMs from kidneys and immortalized RPTCs from WKY and SHRs were quantified by co-immunoprecipitation/immunoblotting as described in 'Materials and Methods'Kidney cortexBBMRPTCWKYSHRWKYSHRWKYSHRControl/vehicle7±3*P<0.05, WKY control vs WKY fenoldopam-treated and SHR control, n=4–5.21±1.58±4*P<0.05, WKY control vs WKY fenoldopam-treated and SHR control, n=4–5.36±414±1.9*P<0.05, WKY control vs WKY fenoldopam-treated and SHR control, n=4–5.31±2.2Fenoldopam (5 μM)55±317±625±431±538±2.124±3.5ANOVA Student—Newman–KeulsThe results in density units from Figure 1 are shown.Abbreviations: BBMs, brush border membranes; D1Rs, D1 receptors; RPTC, renal proximal tubule cell.* P<0.05, WKY control vs WKY fenoldopam-treated and SHR control, n=4–5. Open table in a new tab ANOVA Student—Newman–Keuls The results in density units from Figure 1 are shown. Abbreviations: BBMs, brush border membranes; D1Rs, D1 receptors; RPTC, renal proximal tubule cell. In order to determine the relative subcellular distribution of D1Rs, we studied immortalized RPTCs in culture. Using live cells labeled with a cell-impermeant and non-cleavable biotin (sulfo-N-hydrosuccinamide (NHS)-biotin) reagent,25.Yu P. Yang Z. Jones J.E. et al.D1 dopamine receptor signaling involves caveolin-2 in HEK-293 cells.Kidney Int. 2004; 66: 2167-2180Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar,32.Zaas D.W. Duncan M.J. Li G. et al.Pseudomonas invasion of type I pneumocytes is dependent on the expression and phosphorylation of caveolin-2.J Biol Chem. 2005; 280: 4864-4872Crossref PubMed Scopus (64) Google Scholar we found that the cell surface membrane fluorescence intensity was greater in WKY than in SHRs (Figure 2a). The cell surface membrane proteins (red) and D1Rs (green) showed extensive colocalization (yellow) in cells from WKY rats, but were markedly reduced in cells from SHRs. Indeed, most of D1Rs were internal to the cell surface membranes in SHRs. We also compared the fluorescence intensity in permeabilized and non-permeabilized cells. The D1R can be visualized in non-permeabilized cells because the polyclonal D1R antibody recognizes the third extracellular loop of the receptor. In non-permeabilized cells, fluorescence intensity was again greater in WKY than in SHRs (Figure 2b). However, after cell permeabilization, the total cellular expression of D1Rs was similar in WKY and SHRs (Figure 2b), consistent with our previous report that the expression of D1Rs, quantified by immunoblotting, in renal cortex or in RPTCs was similar in WKY and SHRs.10.Xu J. Li X.X. Albrect F.E. et al.D1 receptor, Gsα, and Na+/H+ exchanger interactions in the kidney in hypertension.Hypertension. 2000; 36: 395-399Crossref PubMed Scopus (85) Google Scholar To verify the visual impression of decreased cell surface membrane D1Rs in RPTCs from SHRs, cells were cultured in Transwells to maintain apical and basolateral polarity. Cell surface membrane proteins were biotinylated with cell impermeant and non-cleavable sulfo-NHS-biotin, immunoprecipitated with anti-D1R antibodies, and immunoblotted with avidin-conjugated peroxidase (Figure 2c). D1R abundance at the apical membranes was greater in cells from WKY rats (67.8±4.4 density units, DU) than those from SHRs (32.1±4.7 DU) (Figure 2c), corroborating the immunofluorescence studies in Figure 2a and b. In contrast, D1Rs were mainly located at basolateral membranes in cells from SHRs (67.1±4.5 DU) relative to cells from WKY rats (33.8±.4.6 DU). Comparison of protein abundance in BBMs between WKY and SHRs is not precise because proteins can be redistributed within renal brush border microvilli33.Yang L.E. Maunsbach A.B. Leong P.K. McDonough A.A. Differential traffic of proximal tubule Na+ transporters during hypertension or PTH: NHE3 to base of microvilli vs. NaPi2 to endosomes.Am J Physiol Renal Physiol. 2004; 287: F896-F906Crossref PubMed Scopus (74) Google Scholar and D1Rs are present in both brush border and subapical membranes. We next studied the immunohistochemistry of rat kidney sections. We found that the cellular distribution of D1Rs in renal cortical tubules was different between WKY and SHRs. In the renal cortex of WKY rats, D1Rs were mainly located at microvillous brush border and apical membranes. In contrast, in SHRs, D1Rs were distributed throughout the cytosol with minimal expression at brush border and apical membranes (Figure 3). Therefore, the kidney section studies confirmed the results obtained in immortalized RPTCs (Figure 2a, b and c). We also stained for rab GTPase 5 (Rab5), a marker protein for early or sorting endosome. Rab5-positive endosomes have been shown to serve as sorting compartments for both receptor recycling and degradation pathways.34.Zerial M. McBride H. Rab proteins as membrane organizers.Nat Rev Mol Cell Biol. 2001; 2: 107-117Crossref PubMed Scopus (2586) Google Scholar, 35.Rosenfeld J.L. Knoll B.J. Moore R.H. Regulation of G-protein-coupled receptor activity by rab GTPases.Receptors Channels. 2002; 8: 87-97Crossref PubMed Scopus (42) Google Scholar, 36.Seachrist J.L. Ferguson S.S. Regulation of G protein-coupled receptor endocytosis and trafficking by Rab GTPases.Life Sci. 2003; 74: 225-235Crossref PubMed Scopus (168) Google Scholar Several studies have shown that internalized GPCRs, such as the β2-adrenergic receptor, and the AT1R, colocalize with Rab5 in endocytic vesicles.35.Rosenfeld J.L. Knoll B.J. Moore R.H. Regulation of G-protein-coupled receptor activity by rab GTPases.Receptors Channels. 2002; 8: 87-97Crossref PubMed Scopus (42) Google Scholar,36.Seachrist J.L. Ferguson S.S. Regulation of G protein-coupled receptor endocytosis and trafficking by Rab GTPases.Life Sci. 2003; 74: 225-235Crossref PubMed Scopus (168) Google Scholar We found that in the kidney cortex, Rab5 was distributed throughout the cytosol in both rat strains. However, some Rab5 expression was also observed in apical membranes in WKY. In contrast, the colocalization of D1R with Rab5 in small vesicles (yellow color) in SHRs was mainly located at the basal membranes (Figure 3). The results indicated that more D1Rs colocalized in Rab5-positive compartment vesicles in SHRs relative to WKY rats under basal conditions. In preliminary studies, we found that the D1R co-immunoprecipitated with PP2A catalytic subunit (PP2Ac) but not with PP1 or PP2B in both WKY and SHRs (data not shown). The data indicated that PP2A may directly interact with D1R and that PP2A may play a more important role than other protein phosphatases in D1R regulation. PP2A has been reported to be important in the dephosphorylation and resensitization of the D1R.16.Yu P. Asico L.D. Eisner G.M. et al.Renal protein phosphatase 2A activity and spontaneous hypertension in rats.Hypertension. 2000; 36: 1053-1058Crossref PubMed Scopus (41) Google Scholar,17.Efendiev R. Yudowski G.A. Zwiller J. et al.Relevance of dopamine signals anchoring dynamin-2 to the plasma membrane during Na+,K+-ATPase endocytosis.J Biol Chem. 2002; 277: 44108-44114Crossref PubMed Scopu
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