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

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]GTPgammaS 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]GTPgammaS 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.     

Antioxidant supplementation normalizes elevated protein kinase C activity in the proximal tubules of old rats

Aging is associated with increase in oxidative stress. Earlier, we have shown that higher basal protein kinase C (PKC) activity in the proximal tubules (PTs) of old rats contributes to the hyperphosphorylation of Na,K-ATPase and subsequent decrease in basal Na,K-ATPase activity, resulting in diminished natriuretic response to dopamine in these animals. We hypothesized that the increase in PKC activity in PTs of old rats is caused by increased oxidative stress and that antioxidants administration should reduce/normalize the elevated PKC activity in the renal PTs of old rats. We studied the effect of two antioxidants, namely, alpha-lipoic acid (LA) and tempol, on oxidants level and PKC activity in the PTs of adult (6-month) and old (24-month) Fischer 344 rats. We found that the accumulation of fluorescent dichlorofluorescein (DCF), an indicator of oxidant production, was higher in the PTs of old compared to adult rats. Dietary supplementation with LA for 2 weeks normalized the increased DCF level in old rats. Carboxymethylysine and malondialdehyde, markers of oxidative damage, were elevated in the PTs of old rats, which were normalized to the level of adult rats when tempol was provided in drinking water for 3 weeks. Both LA and tempol treatment also normalized the higher basal PKC activity in the PTs of old rats to the level seen in adult rats. These results suggest that increase in oxidative stress causes an increase in PKC activity, and that antioxidants, while reducing oxidative stress, also normalize PKC activity in the PTs of old rats.     

Effect of albumin on 14C-alpha-Methyl-D-Glucopyranoside uptake in primary cultured renal proximal tubule cells: involvement of PLC, MAPK, and NF-kappaB

A growing body of evidence implicates albumin has an important regulatory function in renal proximal tubule cells (PTCs). In present study, the effect of bovine serum albumin (BSA) on 14C-alpha-methyl-D-glucopyranoside (alpha-MG) uptake and its related signal molecules were examined in the primary cultured rabbit renal PTCs. BSA significantly increased uptake of alpha-MG, a distinctive proximal tubule marker, as well as expression level of Na+/glucose cotransporters (SGLT1 and SGLT2) proteins. The BSA-induced increase of alpha-MG uptake was completely blocked by actinomycin D and cycloheximide. Neomycin or U 73122 (PLC inhibitors), BAPTA/AM or TMB-8 (intracellular Ca2+ mobilization inhibitors) completely abolished BSA-induced increase of alpha-MG uptake. BSA significantly increased IPs accumulation, but did not affect Ca2+ uptake. Effect of BSA on alpha-MG uptake was blocked by PD 98059, but did not SB 203580. BSA increased phosphorylation of p44/42 mitogen activated protein kinase (MAPK) in a time-dependent manner. NAC or catalase (antioxidants) significantly blocked BSA-induced increase of H2O2 formation and alpha-MG uptake. BSA activated NF-kappaB translocation into nucleus. PDTC, SN50, and TLCK (NF-kappaB inhibitors) also completely blocked BSA-induced increase of alpha-MG uptake, NF-kappaB p65 and phospho IkappaB-alpha activation. In conclusion, BSA stimulates alpha-MG uptake and its action is partially correlated with PLC, MAPK, or NF-kappaB signal molecules in primary cultured renal PTCs.     

The sodium pump and cardiotonic steroids-induced signal transduction protein kinases and calcium-signaling microdomain in regulation of transporter trafficking

The Na/K-ATPase was discovered as an energy transducing ion pump. A major difference between the Na/K-ATPase and other P-type ATPases is its ability to bind a group of chemicals called cardiotonic steroids (CTS). The plant-derived CTS such as digoxin are valuable drugs for the management of cardiac diseases, whereas ouabain and marinobufagenin (MBG) have been identified as a new class of endogenous hormones. Recent studies have demonstrated that the endogenous CTS are important regulators of renal Na⁺ excretion and blood pressure. The Na/K-ATPase is not only an ion pump, but also an important receptor that can transduce the ligand-like effect of CTS on intracellular protein kinases and Ca²⁺ signaling. Significantly, these CTS-provoked signaling events are capable of reducing the surface expression of apical NHE3 (Na/H exchanger isoform 3) and basolateral Na/K-ATPase in renal proximal tubular cells. These findings suggest that endogenous CTS may play an important role in regulation of tubular Na⁺ excretion under physiological conditions; conversely, a defect at either the receptor level (Na/K-ATPase) or receptor–effector coupling would reduce the ability of renal proximal tubular cells to excrete Na⁺, thus culminating/resulting in salt-sensitive hypertension.     

A novel role for protein phosphatase 2A in the dopaminergic regulation of Na,K-ATPase

Stimulation of dopaminergic type 1 (D(1)) receptors increases lung edema clearance by regulating Na,K-ATPase function in the alveolar epithelium. We studied the role of serine/threonine protein phosphatases in the Na,K-ATPase regulation by D(1) agonists in A549 cells. We found that low doses of the type 1/2A protein phosphatase inhibitor okadaic acid as well as SV40 small t antigen transiently transfected into A549 cells prevented the D(1) agonist-induced increase in Na,K-ATPase activity and translocation from intracellular pools to the plasma membrane. This was associated with a rapid and transient increase in protein phosphatase 2A activity. We conclude that D(1) stimulation regulates Na,K-ATPase activity by promoting recruitment of Na,K-ATPases from intracellular pools into the basolateral membranes of A549 cells via a type 2A protein phosphatase.     

Impairment of Na/K-ATPase signaling in renal proximal tubule contributes to Dahl salt-sensitive hypertension

We have observed that, in renal proximal tubular cells, cardiotonic steroids such as ouabain in vitro signal through Na/K-ATPase, which results in inhibition of transepithelial (22)Na(+) transport by redistributing Na/K-ATPase and NHE3. In the present study, we investigate the role of Na/K-ATPase signaling in renal sodium excretion and blood pressure regulation in vivo. In Sprague-Dawley rats, high salt diet activated c-Src and induced redistribution of Na/K-ATPase and NHE3 in renal proximal tubules. In Dahl salt sensitive (S) and resistant (R) rats given high dietary salt, we found different effects on blood pressure but, more interestingly, different effects on renal salt handling. These differences could be explained by different signaling through the proximal tubular Na/K-ATPase. Specifically, in Dahl R rats, high salt diet significantly stimulated phosphorylation of c-Src and ERK1/2, reduced Na/K-ATPase activity and NHE3 activity, and caused redistribution of Na/K-ATPase and NHE3. In contrast, these adaptations were either much less effective or not seen in the Dahl S rats. We also studied the primary culture of renal proximal tubule isolated from Dahl S and R rats fed a low salt diet. In this system, ouabain induced Na/K-ATPase/c-Src signaling and redistribution of Na/K-ATPase and NHE3 in the Dahl R rats, but not in the Dahl S rats. Our data suggested that impairment of Na/K-ATPase signaling and consequent regulation of Na/K-ATPase and NHE3 in renal proximal tubule may contribute to salt-induced hypertension in the Dahl S rat.     

Dynamic interaction of human vasopressin/oxytocin receptor subtypes with G protein-coupled receptor kinases and protein kinase C after agonist stimulation

Examination of the structure of [Arg(8)]-vasopressin receptors (AVPRs) and oxytocin receptors (OTRs) suggests that G protein-coupled receptor kinases (GRKs) and protein kinase C (PKC) are involved in their signal transduction. To explore the physical association of AVPRs and OTRs with GRKs and PKC, wild types and mutated forms of these receptor subtypes were stably expressed as green fluorescent protein fusion proteins and analyzed by fluorescence, immunoprecipitation, and immunoblotting. Addition of a C-terminal GFP tag did not interfere with ligand binding, internalization, and signal transduction. After agonist stimulation, PKC dissociated from the V(1)R, did not associate with the V(2)R, but associated with the V(3)R and the OTR. After AVP stimulation, only GRK5 briefly associated with AVPRs following a time course that varied with the receptor subtype. No GRK associated with the OTR. Exchanging the V(1)R and V(2)R C termini altered the time course of PKC and GRK5 association. Deletion of the V(1)R C terminus resulted in no PKC association and a ligand-independent sustained association of GRK5 with the receptor. Deletion of the GRK motif prevented association and reduced receptor phosphorylation. Thus, agonist stimulation of AVP/OT receptors leads to receptor subtype-specific interactions with GRK and PKC through specific motifs present in the C termini of the receptors.     

Multiple mechanisms involving protein phosphorylation are linked to desensitization of muscarinic receptors

Agonists induce phosphorylation of m2 muscarinic receptors (mAChR) in several cell types. This phosphorylation correlates with desensitization. The mechanisms underlying mAChR phosphorylation have been investigated using several in vitro approaches. Protein kinase C phosphorylated the purified and reconstituted m2 mAChR to a stoichiometry of approximately 5 mols P/mol receptor; this phosphorylation resulted in the decreased ability of receptors to activate G-proteins. Although the phosphorylation by PKC was not modulated by agonist binding to the mAChR, heterotrimeric G-proteins were able to completely block the PKC-mediated effects. If significant receptor/G-protein coupling occurs in vivo, agonists would be required to promote dissociation of the G-proteins from the receptors and reveal the phosphorylation sites for PKC. Members of the G-protein coupled receptor kinase (GRK) family also phosphorylated the purified and reconstituted m2 mAChR. In contrast to PKC, the GRKs phosphorylated the m2 mAChR strictly in an agonist-dependent manner. GRK mediated phosphorylation perturbed receptor/G-protein coupling. In addition, phosphorylation allowed for arrestin binding to the m2 mAChR which should further contribute to desensitization. Using a new strategy that does not require purification and reconstitution of receptors for GRK studies, the m3 mAChR were revealed as substrates for the GRKs. For both the m2 and m3 receptor subtypes, the most effective kinases were GRK 2 and 3. Phosphorylation of the receptors by these enzymes was stimulated by low concentrations of G-proteins and by membrane phospholipids. Thus, multiple mechanisms involving protein phosphorylation appear to contribute to the overall process of mAChR desensitization.     

Phosphorylation-independent regulation of metabotropic glutamate receptor signaling by G protein-coupled receptor kinase 2

The accepted paradigm for G protein-coupled receptor kinase (GRK)-mediated desensitization of G protein-coupled receptors involves GRK-mediated receptor phosphorylation followed by the binding of arrestin proteins. Although GRKs contribute to metabotropic glutamate receptor 1 (mGluR1) inactivation, beta-arrestins do not appear to be required for mGluR1 G protein uncoupling. Therefore, we investigated whether the phosphorylation of serine and threonine residues localized within the C terminus of mGluR1a is sufficient to allow GRK2-mediated attenuation of mGluR1a signaling. We find that the truncation of the mGluR1a C-terminal tail prevents mGluR1a phosphorylation and that GRK2 does not contribute to the phosphorylation of an mGluR1 splice variant (mGluR1b). However, mGluR1a-866Delta- and mGluR1b-stimulated inositol phosphate formation is attenuated following GRK2 expression. The expression of the GRK2 C-terminal domain to block membrane translocation of endogenous GRK2 increases mGluR1a-866Delta- and mGluR1b-stimulated inositol phosphate formation, presumably by blocking membrane translocation of GRK2. In contrast, expression of the kinase-deficient GRK2-K220R mutant inhibits inositol phosphate formation by these unphosphorylated receptors. Expression of the GRK2 N-terminal domain (residues 45-185) also attenuates both constitutive and agonist-stimulated mGluR1a, mGluR1a-866Delta, and mGluR1b signaling, and the GRK2 N terminus co-precipitates with mGluR1a. Taken together, our observations indicate that attenuation of mGluR1 signaling by GRK2 is phosphorylation-independent and that the interaction of the N-terminal domain of GRK2 with mGluR1 contributes to the regulation of mGluR1 G protein coupling.     

Effect of adenosine triphosphate on phosphate uptake in renal proximal tubule cells: involvement of PKC and p38 MAPK

ATP has been known to act as an extracellular signal and to be involved in various functions of kidney. Renal proximal tubular reabsorption of phosphate (Pi) contributes to the maintenance of phosphate homeostasis, which is regulated by Na+/Pi cotransporter. However, the effects of ATP on Na+/Pi cotransporters were not elucidated in proximal tubule cells (PTCs). Thus, the effects of ATP on Na+/Pi cotransporter and its related signal pathways are examined in the primary cultured renal PTCs. In the present study, ATP inhibited Pi uptake in a time (> 1 h) and dose (>10(-6)M) dependent manner. ATP-induced inhibition of Pi uptake was correlated with the decrease of type II Na+/Pi cotransporter mRNA. ATP-induced inhibition of Pi uptake may be mediated by P2Y receptor activation, since suramin (non-specific P2 receptor antagonist) and RB-2 (P2Y receptor antagonist) blocked it. ATP-induced inhibition of Pi uptake was blocked by neomycin, U73122 (phospholipase C (PLC) inhibitors), bisindolylmaleimide I, H-7, and staurosporine (protein kinase C (PKC) inhibitors), suggesting the role of PLC/PKC pathway. ATP also increased inositol phosphates (IPs) formation and induced PKC translocation from cytosolic fraction to membrane fraction. In addition, ATP-induced inhibition of Pi uptake was blocked by SB 203580 [a p38 mitogen activated protein kinase (MAPK) inhibitor], but not by PD 98059 (a p44/42 MAPK inhibitor). Indeed, ATP induced phosphorylation of p38 MAPK, which was not blocked by PKC inhibitor. In conclusion, ATP inhibited Pi uptake via PLC/PKC as well as p38 MAPK in renal PTCs.     

Subtype differences in pre-coupling of muscarinic acetylcholine receptors

Based on the kinetics of interaction between a receptor and G-protein, a myriad of possibilities may result. Two extreme cases are represented by: 1/Collision coupling, where an agonist binds to the free receptor and then the agonist-receptor complex "collides" with the free G-protein. 2/Pre-coupling, where stable receptor/G-protein complexes exist in the absence of agonist. Pre-coupling plays an important role in the kinetics of signal transduction. Odd-numbered muscarinic acetylcholine receptors preferentially couple to G(q/11), while even-numbered receptors prefer coupling to G(i/o). We analyzed the coupling status of the various subtypes of muscarinic receptors with preferential and non-preferential G-proteins. The magnitude of receptor-G-protein coupling was determined by the proportion of receptors existing in the agonist high-affinity binding conformation. Antibodies directed against the C-terminus of the α-subunits of the individual G-proteins were used to interfere with receptor-G-protein coupling. Effects of mutations and expression level on receptor-G-protein coupling were also investigated. Tested agonists displayed biphasic competition curves with the antagonist [(3)H]-N-methylscopolamine. Antibodies directed against the C-terminus of the α-subunits of the preferential G-protein decreased the proportion of high-affinity sites, and mutations at the receptor-G-protein interface abolished agonist high-affinity binding. In contrast, mutations that prevent receptor activation had no effect. Expression level of preferential G-proteins had no effect on pre-coupling to non-preferential G-proteins. Our data show that all subtypes of muscarinic receptors pre-couple with their preferential classes of G-proteins, but only M(1) and M(3) receptors also pre-couple with non-preferential G(i/o) G-proteins. Pre-coupling is not dependent on agonist efficacy nor on receptor activation. The ultimate mode of coupling is therefore dictated by a combination of the receptor subtype and the class of G-protein.     

B2 receptor-mediated dual effect of bradykinin on proximal tubule Na+ -ATPase: sequential activation of the phosphoinositide-specific phospholipase Cbeta/protein kinase C and Ca2+ -independent phospholipase A2 pathways

In a previous paper we showed that bradykinin (BK), interacting with its B2 receptor, inhibits proximal tubule Na+ -ATPase activity but does not change (Na+ +K+)ATPase activity. The aim of this paper was to investigate the molecular mechanisms involved in B2-mediated modulation of proximal tubule Na+ -ATPase by BK. To abolish B1 receptor-mediated effects, all experiments were carried out in the presence of (Arg-Pro-Pro-Gly-Phe-Ser-Pro-Leu), des-Arg9-[Leu8]-BK (DALBK), a specific antagonist of B1 receptor. A dual effect on the Na+ -ATPase activity through the B2 receptor was found: short incubation times (1-10 min) stimulate the enzyme activity; long incubation times (10-60 min) inhibit it. The stimulatory effect of BK is mediated by activation of phosphoinositide-specific phospholipase C beta (PI-PLCbeta)/protein kinase C (PKC); its inhibitory action is mediated by Ca2+ -independent phospholipase A2 (iPLA2). Prior activation of the PI-PLCbeta/PKC pathway is required to activate the iPLA2-mediated inhibitory phase. These results reveal a new mechanism by which BK can modulate renal sodium excretion: coupling between B2 receptor and activation of membrane-associated iPLA2.     

Involvement of G protein-coupled receptor kinase 4 and 6 in rapid desensitization of dopamine D1 receptor in rat IEC-6 intestinal epithelial cells

Dopamine-induced inhibition of Na(+)-K(+)-ATPase has been suggested to play a role in the regulation of Na(+) absorption at the intestinal level, and these effects were mediated by dopamine D(1)-like receptors. The aim of this work was to evaluate the effect of the activation of the D(1)-like receptors on the activity of the Na(+)/H(+) exchanger (NHE) in the rat intestinal epithelial cell line IEC-6. The presence of D(1) receptors was confirmed by immunoblotting. The dopamine D(1)-like receptor agonist SKF-38393 produced a concentration-dependent inhibition of NHE activity and stimulation of adenylyl cyclase (AC), this being antagonized by the D(1) selective antagonist SKF-83566. Effects of SKF-38393 on NHE and AC activities were maximal at 5 min of exposure to the agonist and rapidly diminished with no effect at 25 min. Exposure of cells for 25 min to dibutyryl-cAMP (0.5 mM) or to the AC activator forskolin (3 microM) effectively inhibited NHE activity. Pretreatment of cells with heparin (1 microM), a nonselective G protein-coupled receptor kinase (GRK) inhibitor, prevented the loss of effects on NHE activity after 25 min exposure to SKF-38393. The presence of GRK4, GRK6A, and GRK6B was confirmed by immunoblotting. Overnight treatment with the anti-GRK4-6 antibody complexed with Lipofectin was also effective in preventing loss of the effects of SKF-38393 on NHE and AC activities. It is concluded that dopamine D(1) receptors in IEC-6 rapidly desensitize to D(1)-like agonist stimulation and GRK4 and 6 appear to be involved in agonist-mediated responsiveness and desensitization.     

Cell signaling microdomain with Na,K-ATPase and inositol 1,4,5-trisphosphate receptor generates calcium oscillations

Recent studies indicate novel roles for the ubiquitous ion pump, Na,K-ATPase, in addition to its function as a key regulator of intracellular sodium and potassium concentration. We have previously demonstrated that ouabain, the endogenous ligand of Na,K-ATPase, can trigger intracellular Ca2+ oscillations, a versatile intracellular signal controlling a diverse range of cellular processes. Here we report that Na,K-ATPase and inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) form a cell signaling microdomain that, in the presence of ouabain, generates slow Ca2+ oscillations in renal cells. Using fluorescent resonance energy transfer (FRET) measurements, we detected a close spatial proximity between Na,K-ATPase and InsP3R. Ouabain significantly enhanced FRET between Na,K-ATPase and InsP3R. The FRET effect and ouabain-induced Ca2+ oscillations were not observed following disruption of the actin cytoskeleton. Partial truncation of the NH2 terminus of Na,K-ATPase catalytic alpha1-subunit abolished Ca2+ oscillations and downstream activation of NF-kappaB. Ouabain-induced Ca2+ oscillations occurred in cells expressing an InsP3 sponge and were hence independent of InsP3 generation. Thus, we present a novel principle for a cell signaling microdomain where an ion pump serves as a receptor.     

Cross-regulation of VPAC(2) receptor desensitization by M(3) receptors via PKC-mediated phosphorylation of RKIP and inhibition of GRK2

In gastrointestinal smooth muscle cells, VPAC(2) receptor desensitization is exclusively mediated by G protein-coupled receptor kinase 2 (GRK2). The present study examined the mechanisms by which acetylcholine (ACh) acting via M(3) receptors regulates GRK2-mediated VPAC(2) receptor desensitization in gastric smooth muscle cells. Vasoactive intestinal peptide induced VPAC(2) receptor phosphorylation, internalization, and desensitization in both freshly dispersed and cultured smooth muscle cells. Costimulation with ACh in the presence of M(2) receptor antagonist (i.e., activation of M(3) receptors) inhibited VPAC(2) receptor phosphorylation, internalization, and desensitization. Inhibition was blocked by the selective protein kinase C (PKC) inhibitor bisindolylmaleimide, suggesting that the inhibition was mediated by PKC, derived from M(3) receptor activation. Similar results were obtained by direct activation of PKC with phorbol myristate acetate. In the presence of the M(2) receptor antagonist, ACh induced phosphorylation of Raf kinase inhibitory protein (RKIP), increased RKIP-GRK2 association, decreased RKIP-Raf-1 association, and stimulated ERK1/2 activity, suggesting that, upon phosphorylation by PKC, RKIP dissociates from its known target Raf to associate with, and block the activity of, GRK2. In muscle cells expressing RKIP(S153A), which lacks the PKC phosphorylation site, RKIP phosphorylation was blocked and the inhibitory effect of ACh on VPAC(2) receptor phosphorylation, internalization, and desensitization and the stimulatory effect on ERK1/2 activation were abolished. This study identified a novel mechanism of cross-regulation of G(s)-coupled receptor phosphorylation and internalization by G(q)-coupled receptors. The mechanism involved phosphorylation of RKIP by PKC, switching RKIP from association with Raf-1 to association with, and inhibition of, GRK2.     

G Protein-coupled receptor kinase 2 regulator of G protein signaling homology domain binds to both metabotropic glutamate receptor 1a and Galphaq to attenuate signaling

Heterotrimeric guanine nucleotide-binding (G) protein-coupled receptor kinases (GRKs) are cytosolic proteins that contribute to the adaptation of G protein-coupled receptor signaling. The canonical model for GRK-dependent receptor desensitization involves GRK-mediated receptor phosphorylation to promote the binding of arrestin proteins that sterically block receptor coupling to G proteins. However, GRK-mediated desensitization, in the absence of phosphorylation and arrestin binding, has been reported for metabotropic glutamate receptor 1 (mGluR1) and gamma-aminobutyric acid B receptors. Here we show that GRK2 mutants impaired in Galphaq/11 binding (R106A, D110A, and M114A), bind effectively to mGluR1a, but do not mediate mGluR1a adaptation. Galphaq/11 is immunoprecipitated as a complex with mGluR1a in the absence of agonist, and either agonist treatment or GRK2 overexpression promotes the dissociation of the receptor/Galphaq/11 complex. However, these mGluR1a/Galphaq/11 interactions are not antagonized by the overexpression of either GRK2 mutants defective in Galphaq/11 binding or RGS4. We have also identified a GRK2-D527A mutant that binds Galphaq/11 in an AlF4(-)-dependent manner but is unable to either bind mGluR1a or attenuate mGluR1a signaling. We conclude that the mechanism underlying GRK2 phosphorylation-independent attenuation of mGluR1a signaling is RH domain-dependent, requiring the binding of GRK2 to both Galphaq/11 and mGluR1a. This serves to coordinate GRK2 interactions with Galphaq/11 and to disrupt receptor/Galphaq/11 complexes. Our findings indicate that GRK2 regulates receptor/G protein interactions, in addition to its traditional role as a receptor kinase.     

Nuclear factor-kappaB inhibition by pyrrolidinedithiocarbamate attenuates gastric ischemia-reperfusion injury in rats

Pyrrolidinedithiocarbamate (PDTC) is a potent antioxidant and an inhibitor of nuclear factor-kappaB (NF-kappaB). The present study examined the impact of PDTC preconditioning on gastric protection in response to ischemia-reperfusion (I/R) injury to the rat stomach. Male Wistar rats were recruited and divided into 3 groups (n = 7). One group was subjected to gastric ischemia for 30 min and reperfusion for 1 hour. The second group of rats was preconditioned with PDTC (200 mg/kg body mass i.v.) 15 min prior to ischemia and before reperfusion. The third group of rats was sham-operated and served as the control group. Gastric I/R injury increased serum lactate dehydrogenase level, vascular permeability of gastric mucosa (as indicated by Evans blue dye extravasation) and gastric content of inflammatory cytokine; tumor necrosis factor-alpha (TNF-alpha). Moreover, oxidative stress was increased as indicated by elevated lipid peroxides formation (measured as thiobarbituric acid reactive substances) and depleted reduced glutathione in gastric tissues. NF-kappaB translocation was also detected by electrophoretic mobility shift assay. Microscopically, gastric tissues subjected to I/R injury showed ulceration, hemorrhages, and neutrophil infiltration. Immunohistochemical studies of gastric sections revealed increased expression of p53 and Bcl-2 proteins. PDTC pretreatment reduced Evans blue extravasation, serum lactate dehydrogenase levels, gastric TNF-alpha levels, and thiobarbituric acid reactive substances content, and increased gastric glutathione content. Moreover, PDTC pretreatment abolished p53 expression and inhibited NF-kappaB translocation. Finally, histopathological changes were nearly restored by PDTC pretreatment. These results clearly demonstrate that NF-kappaB activation and pro-apoptotic protein p53 induction are involved in gastric I/R injury. PDTC protects against gastric I/R injury by an antioxidant, NF-kappaB inhibition, and by reduction of pro-apoptotic protein p53 expression, which seems to be downstream to NF-kappaB, thus promoting cell survival.