Hydrogen peroxide causes uncoupling of dopamine D1-like receptors from G proteins via a mechanism involving protein kinase C and G-protein-coupled receptor kinase 2

Free Radical Biology & Medicine 40 (2006) 13 – 20 www.elsevier.com/locate/freeradbiomed

Original Contribution

Hydrogen peroxide causes uncoupling of dopamine D1-like receptors from G proteins via a mechanism involving protein kinase C and G-protein-coupled receptor kinase 2 Mohammad Asghar 1, Anees Ahmad Banday 1, Riham Z. Fardoun, Mustafa F. Lokhandwala * Heart and Kidney Institute, College of Pharmacy, University of Houston, Houston, TX 77204, USA Received 12 November 2004; revised 8 August 2005; accepted 8 August 2005 Available online 8 September 2005

Abstract Dopamine, via activation of D1-like receptors, inhibits Na,K-ATPase and Na,H-exchanger in renal proximal tubules and promotes sodium excretion. This effect of dopamine is not seen in conditions associated with oxidative stress such as hypertension, diabetes, and aging due to uncoupling of D1-like receptors from G proteins. To identify the role of oxidative stress in uncoupling of the D1-like receptors, we utilized primary cultures from rat renal proximal tubules. Hydrogen peroxide (H2O2), an oxidant, treatment to the cell cultures increased the level of malondialdehyde, a marker of oxidative damage. Further, H2O2 decreased membranous D1-like receptor numbers and proteins, D1-like agonist (SKF 38393)-mediated [35S]GTPgS binding and SKF 38393-mediated inhibition of Na,K-ATPase. Moreover, H2O2 treatment to the cultures caused membranous translocation of G-protein-coupled receptor kinase 2 (GRK 2) and increased serine phosphorylation of D1A receptors accompanied by an increase in protein kinase C (PKC) activity. Interestingly, PKC inhibitors blocked the H2O2-mediated stimulation of GRK 2 and serine phosphorylation of D1A receptors. Further, GRK 2 antisense but not scrambled oligonucleotides attenuated the effect of H2O2 on membranous expression of GRK 2. Moreover, direct activation of PKC with phorbol ester (PMA) resulted in reduction of SKF 38393-mediated [35S]GTPgS binding. We conclude that H2O2 stimulates PKC leading to the activation of GRK 2, which causes serine phopshorylation of D1A receptors and receptor G-protein uncoupling in these cells, resulting in impairment in D1-like receptor function. D 2005 Elsevier Inc. All rights reserved. Keywords: Na,K-ATPase; Renal proximal tubules; Free radical

Introduction Renal dopamine plays an important role in the maintenance of sodium homeostasis, particularly during an increase in sodium intake [1,2]. There are two subtypes of dopamine

Abbreviations: GRKs, G-protein-coupled receptor kinases; GRK-2, Gprotein-coupled receptor kinase 2; GTPgS, guanosine 5-(g-thio)triphosphate; H2O2, hydrogen peroxide; [3H]SCH-23390, R-(+)-2,3,4,5-tetrahydro-3-methyl5-phenyl-1H-3-benzazepin-7-ol hydrochloride; LDH, lactate dehydrogenase; MDA, malondialdehyde; NKA, Na, K-ATPase; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; PMSF, phenylmethylsulfonyl fluoride; PTECs, primary proximal tubular cell cultures; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SKF-38393, (T)-1-phenyl-2,3,4,5tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride; SHR, spontaneously hypertensive rats. * Corresponding author. Fax: (713) 743 1232. E-mail address: [email protected] (M.F. Lokhandwala). 1 Contributed equally to this work. 0891-5849/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2005.08.018

receptors, which are characterized by their ability to stimulate (via D1-like) or inhibit (via D2-like) adenylyl cyclase [1 –3]. Of the two dopamine receptor subtypes present on both apical and basolateral sides of the proximal tubules, activation of D1-like subtype is involved in natriuretic response to dopamine [4,5]. At the cellular level, the activation of D1-like receptors and subsequent stimulation of second messenger pathways lead to the inhibition of sodium transporters Na,KATPase and Na/Hexchanger and subsequent increase in sodium excretion [1 –3,5,6]. G-protein-coupled receptor kinases (GRKs) play an important role in regulating G-protein-coupled receptor (GPCR) signaling by their ability to phosphorylate and desensitize agonist occupied receptors (reviewed in [7]). GRKs belong to the family of serine/threonine kinases and seem to play a role in the pathophysiology of diseases such as heart failure [8], hypertension [9], and arthritis [10]. There are seven isoforms of GRKs with different receptor specificities [11]. Of these

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isoforms, GRKs 2, 3, and 5 have been shown to phosphorylate D1A receptors, causing a reduction in the agonist affinity in embryonic kidney cells transfected with cDNAs of GRKs and D1A receptors [12]. Numerous studies have shown an increase in oxidative stress in diseases such as hypertension and diabetes as well as during aging. The diminished natriuretic response to dopamine in these conditions is reportedly due to ligand-independent phosphorylation and subsequent uncoupling of D1-like receptors from G proteins [13 –15]. Moreover, available reports also suggest that oxidative stress increases protein kinase C (PKC) activity in many cell types [16,17] including renal proximal tubular cells [18]. Therefore, we wanted to test the hypothesis that oxidative stress causes ligand-independent phosphorylation and uncoupling of D1-like receptors from G proteins via a mechanism involving PKC and GRK 2. To accomplish this, oxidative stress was induced in renal proximal tubular cells with H2O2 and its effect was studied on PKC, GRK 2, and D1like receptor function. Materials and methods R-(+)-SKF-38393 hydrochloride a D1-like receptor agonist—an active enantiomer of (T)-SKF-38393: (T)-1-phenyl2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol hydrochloride—was purchased from Sigma (RBI). [3H]SCH-23390 hydrochloride a D1 receptor antagonist (R-(+)-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin-7-ol hydrochloride) and [35S]GTPgS (guanosine 5V-(g-thio)triphosphate[35S]) were purchased from NEM Life Sciences. Antibodies for GRK 2, D1A receptor and phospho-serine antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), Chemicon (Temecula, CA), and Calbiochem NovaChem Corp. (San Diego, CA), respectively. The antisense and scrambled oligonucleotides were synthesized by Sigma-Genosys (Woodlands, TX). The protein kinase C assay kit was purchased from Promega Corporation (Madison, WI). Inhibitors for PKC (chelerythrine chloride and staurosporine) and protein kinase A (H-89) were purchased from Sigma Chemical Co. and Upstate (Lake Placid, NY), respectively. All other chemicals of highest purity available were purchased from Sigma-Aldrich Co. Cell cultures and induction of oxidative stress The renal proximal tubule (PT) fragments from SpragueDawley rats were purified as previously described [17]. The isolated proximal tubules were resuspended in complete media containing DMEM-F12 (1:1) supplemented with insulin 0.573 ng/ml, transferrin 5 Ag/ml, hydrocortisone 40 ng/ml, selenium 5 ng/ml, 3,3,5-triiodo-l-thyronine (T3) 4 pg/ml, epidermal growth factor 10 ng/ml, sodium bicarbonate 1.2 mg/ml, lglutamine 0.29 mg/L, penicillin 25 IU/ml, streptomycin 25 Ag/ ml, and 10% fetal calf serum. At about 85 –90% confluency (passage 1) the proximal tubular epithelial cell cultures (PTEC) were serum-starved overnight, washed with KrebsHenseleit buffer (KHB), and treated in the absence (vehicle)

and presence of 50 AM H2O2 for 20 min to induce oxidative stress. In some experiments, cells were pretreated with inhibitors of PKC (chelerythrine chloride and staurosporine, each 1 AM/5 min), PKA (H-89, 5 AM/5 min), and GRK 2 antisense oligonucleotides (1 AM/24 h) before the induction of oxidative stress. PMA treatment of the cells was carried out for 5 min. Subsequently, cells were scraped, homogenized, and processed for membrane preparation. Malondialdehyde (MDA) was determined by measuring MDA levels in butanol extracts of cell homogenate by the method of Urchiyama and Mihara [19]. Protein was determined by commercially available assay kit (Pierce) with bovine serum albumin as standard. [ 3H]SCH 23390 binding The cells were scraped, homogenized in sucrose buffer (in mM, Tris-HCl 10, sucrose 50, pH 7.6) and processed for membrane preparation as reported earlier [20]. Fifty micrograms of membrane protein was incubated with 50 nM [3H]SCH 23390, a D1-like receptor antagonist in 250 Al (final volume) of binding buffer for 120 min at 25-C. Nonspecific binding was determined in the presence of 1 AM unlabeled SCH 23390. [ 35S]GTPcS binding GTPgS binding assay was performed as described in our previous publication [20]. The reaction mixture of 90 Al (final volume) contained 25 mM Hepes, 15 mM MgCl2, 1 mM dithiothrietol, 100 mM NaCl (pH 8.0), 5 Ag protein and ¨100,000 cpm of [35S]GTPgS with or without D1 receptor agonist SKF-38393. Nonspecific binding of [35S]GTPgS, determined in the presence of 100 AM unlabeled GTPgS, was always less than 2% of total binding. Na, K-ATPase assay Vehicle- and H2O2-treated cells were incubated without and with SKF-38393, a D1 receptor agonist, for 10 min at 37-C. A cell suspension (0.05 mg/ml) was used to assay colorimetrically the ouabain (1 mM)-sensitive Na,K-ATPase activity by end-point ATP hydrolysis to inorganic phosphate [15]. Protein kinase C activity PKC activity was determined by a commercially available PKC assay kit as detailed in our previous study [18]. G-protein-coupled receptor kinase 2 downregulation and Western blotting The cells (¨40 – 50% confluency) were incubated with varying concentrations (0.025 –1.0 AM) of GRK 2 antisense oligonucleotides (5V-CTTTTGGAAGATGTCG-3V) using the reagents and protocol from Invitrogen (Carlsbad, CA). The

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antisense oligonucleotides chosen here are against nucleotides 480 –495 of the rat GRK 2 cDNA [21]. Scrambled oligonucleotides of the above nucleotide compositions were used as control. Postincubation (24 h), cell homogenates were resolved on SDS-PAGE, transblotted, and immunoblotted for GRK 2 using specific rabbit GRK 2 antibodies by standard Western blotting. The specificity of GRK 2 antibody was determined by preblocking the antibody with the peptide, which resulted in the disappearance of GRK 2 protein band (data not shown). Immunoprecipitation of D1A receptors D1A receptors were immunoprecipitated using the method of Sanada et al. [22], which has been used in our previous studies [14]. Briefly, cell membranes (1.5 mg protein/ml) were incubated with 10 Ag rabbit dopamine D1A receptor antibody in IP buffer (140 mM NaCl, 3 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, 1 mM orthovanadate, 1% NP-40, 0.5% sodium cholate, 0.1% SDS, 1 mM PMSF, protease inhibitor cocktail, pH 7.4) overnight followed by incubation with protein A/Gagarose beads for 2 h. The ternary complex of D1A receptor-

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antibody-protein A/G agarose was washed with IP buffer and then with 50 mM Tris-HCl, pH 8.0. The complex was dissociated in 2 Laemmli buffer and used for electrophoresis (SDS-PAGE). Detection of serine phosphorylation on D1A receptors The immunoprecipitates (5 Al) were resolved by SDS-PAGE electrophoresis and the proteins were electro-transferred on a PVDF membrane. The membrane was blocked with 4% bovine serum albumin in phosphate-buffered saline with 0.1% Tween 20 (PBST). A specific phosphoserine antibody [23] was used to detect serine phosphorylation on D1A receptors. HRP-conjugated secondary antibody was used to probe phosphoserine antibody and the bands were visualized with the enhanced chemiluminescence reagent kit (Alpha Diagnostics, San Antonio, TX). Statistical analysis Differences between means were evaluated using the unpaired t test or analysis of variance with Newman-Keuls’

Fig. 1. (A) H2O2 exposure reduced [3H]SCH 23390 binding in the membranes of renal proximal tubular epithelial cell cultures. *Significantly different from vehicle-treated cells. (B) SKF 38393-induced increase in [35S]GTPgS binding in the membranes from vehicle- and H2O2-treated renal proximal tubular epithelial cell cultures. *Significantly different from control. #Significantly different from H2O2-treated cells at respective concentrations. Lines represent mean T SE of 5 – 6 different cell cultures (from separate rats) performed in triplicate. (C) H2O2 reduced membranous D1A receptors [left panel: representative blot of D1A receptor (lanes 1, vehicle; 2 H2O2), right panel: densitometric analysis of the blots from 3 separate experiments]. The numbers in the scale bar in the blot represent MW (kDa). Data were analyzed by Students t test (A and C) and ANOVA (B) followed by Newman-Keuls multiple test, p < 0.05 was considered statistically significant.

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shown in Fig. 2, SKF 38393 (1 AM) significantly inhibited the activity of NKA in vehicle-treated but not in H2O2treated cells. H2O2 did not affect the basal Na,K-ATPase activity (vehicle vs H2O2: 413 T 12.1 vs 406 T 20.3 nmol Pi/mg protein/h). H2O2 caused GRK 2 membrane translocation in PTECs

Fig. 2. SKF 38393-induced inhibition of Na,K-ATPase activity in vehicle- and H2O2-treated renal proximal tubular epithelial cell cultures. *Significantly different from basal. Bars represent mean T SE of 5 – 6 different cell cultures (from separate rats) performed in triplicate. Data were analyzed by ANOVA followed by Newman-Keuls multiple test, p < 0.05 was considered statistically significant.

multiple test, as appropriate, p < 0.05 was considered statistically significant. Results Hydrogen peroxide caused lipid peroxidation as evidenced by increased malondialdehyde content in cells exposed to H2O2 compared to vehicle-treated cells (H2O2 vs vehicle, 8.84 T 0.3 vs 3.85 T 0.2 nmol/mg protein). As assessed by lactate dehydrogenase (LDH) release and the trypan blue exclusion test, 50 AM H2O2 did not produce any cytotoxic effect. In H2O2-treated cells LDH release was 0.8%, suggesting 99.2% cell viability.

As shown in Fig. 3A lane 4, maximum depletion of GRK 2 proteins was observed in cells incubated with 1 AM antisense oligonecleotides compared to nonincubated cells (Fig. 3A, lane 1). The level of GRK 2 proteins was not affected in cells incubated with scrambled oligonucleotides (Fig. 3A, lanes 5– 7). The specificity of GRK 2 antibody was confirmed by preblocking the antibody with the peptide, which resulted in the disappearance of the GRK 2 protein band (data not shown). Further, in cells not incubated with GRK 2 antisense oligonucleotides, H2O2 caused an increase in the membranous GRK 2 protein abundance compared to vehicle treatment (Fig. 3B, lane 2). Interestingly, H2O2 treatment failed to increase the GRK 2 protein level in the membranes of cells incubated with antisense oligonucleotides (Fig. 3B, lane 3), but increased it in cells incubated with scrambled oligonucleotides (Fig. 3B, lane 4).

H2O2 reduced D1-like receptor numbers, receptor proteins, and G-protein coupling in PTEC membranes The specific binding of [3H]SCH 23390, a D1-like receptor ligand, and D1A receptor proteins was reduced by 58 and 38%, respectively, in the membranes from H2O2treated cells compared to vehicle-treated cells (Figs. 1A and C), indicating a marked decrease in D1-like receptor numbers. As shown in Fig. 1B, SKF 38393 (0.1 –10 AM) elicited a concentration-dependent stimulation of [35S]GTPgS binding in the membranes from vehicle-treated cells. However, the stimulatory effect of SKF 38393 was absent in membranes from H2O2-treated cells. There was no significant difference in basal [35S]GTPgS binding (fmol [35S]GTPgS bound/mg protein) in the membranes from vehicle- (109.4 T 4.3) and H2O2- (102.2 T 5.3) treated cells. H2O2 abolished SKF 38393-induced inhibition of Na, K-ATPase activity in PTECs In addition to D1-like receptor G-protein coupling, the functional responsiveness of D1-like receptor was assessed by SKF 38393-mediated inhibition of NKA activity. As

Fig. 3. (A) antisense oligonucleotides reduced GRK 2 protein expression in renal proximal tubular epithelial cell cultures. Lane 1, cells not incubated with oligonucleotides; lanes 2 to 4, cells incubated with varying concentrations of GRK 2 antisense oligonucleotides, 0.025 AM (lane 2), 0.1 AM (lane 3), 1 AM (lane 4); lanes 5 and 6, cells incubated with varying concentrations of scrambled oligonucleotides, 0.025 AM (lane 5), 0.1 AM (lane 6), 1 AM (lane 7). (B) H2O2 increases GRK 2 proteins in the membranes of renal proximal tubular epithelial cell cultures. Lane 1, cells not incubated with oligonucleotides treated with vehicle; lane 2, cells not incubated with oligonucleotides treated with H2O2; lane 3, GRK 2 antisense oligonucleotide incubated cells treated with H2O2; lane 4, scrambled oligonucleotide incubated cells treated with H2O2.

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Fig. 4. (A) Bar diagram representation of protein kinase C activity (vehicle, 2.625 T 0.44; H2O2, 4.21 T 0.23; H2O2 + chelerythrine chloride, 2.28 T 0.60; H2O2 + H89, 4.186 T 0.35; PMA, 7.51 T 0.65; PMA + chelerythrine chloride, 3.63 T 0.23 ng peptide phosphorylated/mg protein/min). (B and C) Western blots of GRK 2. Upper panels: representative blot of GRK2 [lanes 1, vehicle in B and C; 2, H2O2 in B and C; 3, H2O2 + chelerythrine chloride in B and PMA in C; 4, H2O2 + staurosporine in B and PMA + chelerythrine chloride in C; 5, H2O2 + H-89 in C. Lower panels: densitometric analysis of the protein bands. (D) Bar diagram presentation of SKF 38393 (10 AM)-mediated GTPgS binding in the membranes of vehicle-, H2O2-, and PMA-treated cell cultures. Bars in A, B, C, and D represent results mean T SE from 3 separate experiments (n = 3 animals). *Significantly different from vehicle-treated cells. #Significantly different from H2O2 in A and B, PMA in C, and vehicle in the presence of 10 AM SKF 38393 in D. Data were analyzed by ANOVA followed by Newman-Keuls multiple test, p < 0.05 was considered statistically significant.

H2O2-induced GRK 2 translocation requires protein kinase C activation PKC activity increased in the cells treated with H2O2 compared to vehicle-treated cells (Fig. 4A). H2O2-induced increase in PKC activity decreased in the presence of PKC inhibitor (chelerythrine chloride) but not protein kinase A

inhibitor (H-89) (Fig. 4A). Further, H2O2 caused an increase in membranous abundance of GRK 2, which was attenuated with PKC inhibitors (both chelerythrine chloride and staurosporine) (Fig. 4B) but not with PKA inhibitor (H-89) (Fig. 4C). Also, direct activation of PKC with PMA (Fig. 4A) increased membranous abundance of GRK2, which decreased in the presence of PKC inhibitor (chelerythrine

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Fig. 5. PKC inhibitor attenuates H2O2-induced increase in serine phosphorylation of D1A receptors. Upper panel: representative blot of D1A receptors immunoblotted for serine phosphorylation using phospho-serine antibody (lanes 1, vehicle; 2, H2O2; 3, staurosporine + H2O2). Lower panel: densitometric analysis of serine phosphorylation on D1A receptors. Bars represent mean T SE of 3 different cell cultures (from separate rats). *Significantly different from vehicle. #Significantly different from H2O2. p < 0.05 value was considered significant (t test).

chloride) (Fig. 4C). PMA treatment to the cells also caused reduction in SKF 38393 (D1-like agonist)-mediated GTPgS binding (Fig. 4D). H2O2-induced increase in serine phosphorylation of D1A receptors via PKC As shown in Fig. 5, serine phosphorylation of D1A receptors increased in the cells treated with H2O2 (lane 2) compared to that of vehicle-treated cells (lane 1). The effect of H2O2 was abolished when the cells were pretreated with PKC inhibitor, staurosporine (lane 3). Discussion The results presented here show that exposure of PTECs to hydrogen peroxide (H2O2) induced lipid peroxidation, reduced D1-like receptor numbers and D1A receptor proteins, caused D1-like receptor G-protein uncoupling, elevated basal protein kinase C activity, and increased GRK 2 membrane expression. Functionally, D1-like receptor agonist SKF 38389 was unable to elicit G-protein stimulation resulting in its failure to inhibit Na,K-ATPase activity. Reactive oxygen species, such as hydroxyl radicals and superoxide anions, are generated in both acute and chronic pathologies of the renal and cardiovascular system, such as ischemia, stroke, and/or hypertension [24 – 27]. Oxidative radicals react with phospholipids in the cell membrane, thereby causing peroxidation of fatty acids [28]. H2O2 used in the present studies is probably more harmful than superoxide anion S O2 , because of its diffusive nature across the plasma membrane and enters the inner compartments of a cell [29]. Also, in addition to causing lipid peroxidation it can generate most potent oxidant, hydroxy radicals, via the Fenton reaction

[30,31]. Lipid peroxidation generates substantial amounts of biologically active aldehydes, in particular, 4-hydroxynonenal (4-NHE) and malondialdehyde, which can disturb cell function by acting as stable, diffusible mediators of oxidative damage [32]. Herein, we also observed significant lipid peroxidation as evidenced by increased active aldehydes, MDA, in H2O2S exposed cells. However, since we did not determine the O2 S and OH levels in H2O2-exposed cells it is not clear whether lipid peroxidation was induced by H2O2 alone or in combination with other free radicals. There is evidence suggesting that oxygen species such as superoxide, hydrogen peroxide, and the hydroxyl radicals may play a role in the development of organ damage associated with cardiovascular diseases in general and hypertension in particular [26 – 28]. A number of recent studies in various models of hypertension (human essential hypertension, spontaneously hypertensive rats, Dahl salt-sensitive hypertensive rats, obese Zucker rats) have also demonstrated a significantly enhanced level of oxidative stress in kidney and endothelial cells as well as in venular segments of the circulation [24,25,27,33– 35]. Of the many redox-sensitive pathways that are compromised in hypertension, renal D1-like receptor signaling seems to be of great interest. There is increasing evidence suggesting that dopamine plays an important role in the regulation of renal function and blood pressure [1 –3,36]. Since abnormal renal sodium handling has been known to be one of the major factors involved in the initiation of high blood pressure in several genetic and nongenetic hypertensive models, it is suggested that impaired renal sodium handling in hypertension may be partly due to defective renal D1-like receptor signaling [1,6,36]. Although a great deal of work has been done to identify a unifying link for D1-like receptor dysfunction in various human and animal hypertensive models, the molecular mechanism responsible for such defect is still elusive. One of the factors, which coexist with D1-like receptor defect in both hypertension and in aging, is oxidative stress [1,6,24,27,37]. Also, it has been shown that in brain, oxidants abolish D1-like receptor high-affinity sites and thiol-reducing agents preserve and protect such sites from oxidation [38,39]. Conversely, Yasunari et al. reported that dopamine and SKF 38393 via D1-like receptors inhibit platelet-derived growth factor-induced increases in oxidative stress of vascular smooth muscle cells [40], thus suggesting a more active involvement of D1-like receptors in redox-sensitive signaling cascades in various tissues. In order to investigate the mechanism of functional vulnerability of renal D1-like receptor to oxidative stress and to establish a role of oxidative stress in causing renal D1-like receptor dysfunction, the PTECs were directly exposed to H2O2. We observed that H2O2 decreased D1-like receptor numbers and subsequently blunted SKF 38393-induced inhibition of Na, K-ATPase. Although H2O2 caused a 58% decrease in D1-like receptor ligand binding, it seems unlikely that a decrease in receptor alone could account for complete loss of SKF 38393-induced Na, K-ATPase inhibition. Interestingly, in cells treated with H2O2, SKF-38393 also failed to stimulate GTPgS binding, suggesting the failure of the remaining D1-like receptors to couple with G proteins and

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transduce downstream signal. Thus it is likely that D1-like receptor G-protein uncoupling led to the loss of D1-like receptor function. The possibility that H2O2 directly or indirectly via lipid peroxidation can modulate Na,K-ATPase activity or G protein function is ruled out because we did not observe any differences in basal Na, K-ATPase activity or GTPgS binding in H2O2- and vehicle-treated cells. These results are in agreement with studies performed in proximal tubules from SHR. In these studies D1-like receptor G-protein uncoupling was attributed to lipid peroxidation and subsequent loss of high-affinity binding sites [24]. Evidence to date indicates that oxidative stress can activate PKC by increases in DAG via hydrolysis of phosphatidylcholine (by activating phospholipase D) or tyrosine phosphorylation [16,41]. It has also been postulated that oxidative modification, which occurs probably in the regulatory domain of PKC, may generate active enzyme [42]. At present there are no reports regarding the oxidative modification of PKC by H2O2, while on the other hand, Konishi et al. have shown that almost all PKC isoforms, namely a, h, g of cPKC, y and ( of nPKC, and s of aPKC, are tyrosine-phosphorylated and catalytically activated by H2O2 [41]. In agreement, our results also showed a significant increase in PKC activity in cells exposed to H2O2 compared to vehicle-treated cells. The molecular mechanism responsible for this H2O2-induced increase in PKC activity was not investigated as it is out of the scope for the present studies. The important finding of this study is that in PTECs, H2O2 increased membranous abundance of GRK 2. The effect is specific because in cells incubated with GRK 2 antisense oligonucleotides, H2O2 failed to increase the level of membranous GRK 2. It should be noted that in the present study the cells were exposed to H2O2 for 20 min, which rules out the possibility of de novo synthesis of GRK 2, which may have caused its increased expression on the membranes. The observation that PKC inhibitors but not PKA inhibitor abolished the H2O2-induced increase in membranous GRK 2 suggests that PKC activation is a prerequisite for GRK 2 stimulation. In support of our results several recent studies have shown that receptor phosphorylation by GRKs is modulated by the activity of other kinases that directly phosphorylate the GRKs and alter a variety of their properties [7]. It has been demonstrated that PKC can also affect the desensitization of GPCRs by phosphorylating GRK 2 and altering its activity [7]. Similarly, GRK 2 phosphorylation can also be induced when cells are treated with either calcium ionophore or with phorbol esters, both of which potently activate PKC [7]. We have also found that direct activation of PKC with PMA (a phorbol ester) increases membranous GRK 2 abundance, which seems to play a role in D1-like receptor G-protein uncoupling (Fig. 4). Our studies in animal models of type 2 diabetes (obese Zucker rats) and in aging (old Fischer 344 rats), which are associated with oxidative stress, suggest that D1A receptors in the proximal tubules are already phosphorylated in the basal state and are therefore essentially desensitized [14,43]. In agreement with these studies we also observed a marked

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increase in D1A receptor serine phosphorylation in cells exposed to H2O2 compared to vehicle, thus suggesting that hyperphosphorylation of D1A receptors could be responsible for its uncoupling from G proteins. It is of interest that as observed in this study GRK activity and GRK 2 expression are also increased in lymphocytes of patients with essential hypertension [44]. The observation that PKC inhibitor not only prevented H2O2-dependent GRK 2 activation but completely abolished H2O2-induced D1A receptor phosphorylation provides strong evidence that oxidative stress can modulate the D1A receptor phosphorylation state by activating GRK 2 via the PKC signaling pathway. In conclusion, this is the first study to report that in PTECs, H2O2 increases GRK 2 activity via PKC causing an increase in serine phosphorylation of D1A receptors leading to receptor Gprotein uncoupling. Furthermore, H2O2-mediated reduction in D1-like receptor numbers (loss of functional receptors) and uncoupling from G proteins were responsible for the failure of SKF-38393 to inhibit Na,K-ATPase activity. It is likely that oxidative stress which is present in hypertension and aging may contribute to D1-like receptor G-protein uncoupling and diminished natriuretic response to dopamine seen in these conditions. Acknowledgments This study was supported in part by National Institutes of Health Grants AG-15031 and AG-25056 from National Institute on Aging. References [1] Jose, P. A.; Eisner, G. M.; Felder, R. A. Renal dopamine receptors in health and hypertension. Pharmacol. Ther. 80:149 – 182; 1998. [2] Aperia, A. C. Intrarenal dopamine: a key signal in the interactive regulation of sodium metabolism. Annu. Rev. Physiol. 62:621 – 647; 2000. [3] Missale, C.; Nash, S. R.; Robinson, S. W.; Jaber, M.; Caron, M. G. Dopamine receptors: from structure to function. Physiol. Rev. 78: 189 – 225; 1998. [4] Felder, C. C.; McKelvey, A. M.; Gitler, M. S.; Eisner, G. M.; Jose, P. A. Dopamine receptor subtypes in renal brush border and basolateral membranes. Kidney Int. 36:183 – 193; 1989. [5] Hegde, S. S.; Jadhav, A. L.; Lokhandwala, M. F. Role of kidney dopamine in the natriuretic response to volume expansion in rats. Hypertension 13:828 – 834; 1989. [6] Hussain, T.; Lokhandwala, M. F. Renal dopamine receptor function in hypertension. Hypertension 32:187 – 197; 1998. [7] Kohout, T. A.; Lefkowitz, R. J. Regulation of G protein-coupled receptor kinases and arrestins during receptor desensitization. Mol. Pharmacol. 63:9 – 18; 2003. [8] Petrofski, J. A.; Koch, W. J. The beta-adrenergic receptor kinase in heart failure. J. Mol. Cell. Cardiol. 35:1167 – 1174; 2003. [9] Felder, R. A.; Sanada, H.; Xu, J.; Yu, P. Y.; Wang, Z.; Watanabe, H.; Asico, L. D.; Wang, W.; Zheng, S.; Yamaguchi, I.; Williams, S. M.; Gainer, J.; Brown, N. J.; Hazen-Martin, D.; Wong, L. J.; Robillard, J. E.; Carey, R. M.; Eisner, G. M.; Jose, P. A. G protein-coupled receptor kinase 4 gene variants in human essential hypertension. Proc. Natl. Acad. Sci. USA 99:3872 – 3877; 2002. [10] Lombardi, M. S.; Kavelaars, A.; Schedlowski, M.; Bijlsma, J. W.; Okihara, K. L.; Van de Pol, M.; Ochsmann, S.; Pawlak, C.; Schmidt, R. E.; Heijnen, C. J. Decreased expression of and activity of G-

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