Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Sodium-hydrogen exchanger regulatory factor isoform-1 (NHERF-1) and NHERF-2 are two structurally related PDZ-domain-containing protein adapters that effectively transduce cyclic AMP (cAMP) signals that inhibit NHE3, the sodium-hydrogen exchanger isoform present at the apical surface of kidney and gut epithelia. The mouse renal proximal tubule expresses both NHERF isoforms, suggesting their redundant functions as regulators of renal electrolyte metabolism. To define the role of NHERF-1 in the physiological control of NHE3, we analyzed NHE3 activity in isolated brush border membrane (BBM) preparations from renal proximal tubules of wild-type (WT) and NHERF-1 (-/-) mice. Basal Na(+)-H(+) exchange was indistinguishable in BBMs from WT and NHERF-1 (-/-) mice (0.96+/-0.08 and 0.95+/-0.10 nmol/mg protein/10 s, respectively). Activation of membrane bound cAMP-dependent protein kinase (PKA) by cAMP inhibited NHE3 activity in WT BBMs (0.55+/-0.07 nmol/mg protein/10 s or 40+/-9%, P<0.01) but had no discernible effect on Na(+)-H(+) exchange in the NHERF-1 (-/-) BBM (0.97+/-0.07 nmol/mg protein/10 s; P=not significant). This was associated with a significant decrease in cAMP-stimulated phosphorylation of NHE3 immunoprecipitated from solubilized NHERF-1 (-/-) BBMs. As the protein levels for NHE3, NHERF-2, PKA and ezrin were not changed in the NHERF-1 (-/-) BBMs, the data suggest a unique role for NHERF-1 in cAMP-mediated inhibition of NHE3 activity in the renal proximal tubule of the mouse.
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PMID:NHERF-1 uniquely transduces the cAMP signals that inhibit sodium-hydrogen exchange in mouse renal apical membranes. 1258 53

1. Optimal dipeptide and peptidomimetic drug transport across the intestinal mucosal surface is dependent upon the co-operative functional activity of the di/tripeptide transporter hPepT1 and the Na(+)/H(+) exchanger NHE3. The ability of the anti-absorptive enteric neuropeptide VIP (vasoactive intestinal peptide) to modulate dipeptide uptake was determined using human intestinal (Caco-2) epithelial cell monolayers. 2. Uptake of glycylsarcosine (Gly-Sar) across the apical membrane of Caco-2 cell monolayers is inhibited by basolateral exposure to either VIP, pituitary adenylate cyclase-activating polypeptide (PACAP), or the VPAC(1) receptor agonist [(11,22,28)Ala]-VIP. Inhibition of Gly-Sar uptake is observed only in the presence of extracellular Na(+). Reverse-transcription polymerase chain reaction (RT-PCR) demonstrates that VPAC(1) mRNA is expressed in Caco-2 cells whereas VPAC(2) mRNA is not detected. 3. The VIP-induced inhibition of Gly-Sar uptake is abolished in the presence of the protein kinase A (PKA) inhibitor H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide.2HCl). 4. (22)Na(+) uptake across the apical membrane is inhibited by the selective NHE3 inhibitor S1611. Experiments with BCECF [2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein]-loaded Caco-2 cells demonstrate that VIP reduces the NHE3-dependent recovery of intracellular pH (pH(i)) after dipeptide-induced acidification. Western blot of Caco-2 cell protein demonstrates expression of the NHE regulatory factor NHERF1 (expression of which is thought to be required for PKA-mediated inhibition of NHE3). 5. VIP has no effect on Gly-Sar uptake in the presence of S1611 suggesting that VIP and S1611 both modulate dipeptide uptake via the same mechanism. 6. These observations demonstrate that VIP (and PACAP) modulate activity of the H(+)/dipeptide transporter hPepT1 in a Na(+)-dependent manner consistent with the modulation being indirect through inhibition of NHE3.
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PMID:Inhibition of intestinal dipeptide transport by the neuropeptide VIP is an anti-absorptive effect via the VPAC1 receptor in a human enterocyte-like cell line (Caco-2). 1259 10

Dopamine D(1)-mediated inhibition of Na(+),K(+)-ATPase activity in opossum kidney (OK) cells involves the sequential activation of the adenylyl cyclase-protein kinase A (PKA) and the phospholipase C-protein kinase C (PKC) pathways. The present study evaluated the signalling cascades involved in dopamine-mediated inhibition of Na(+)/H(+) exchanger isoform 3 (NHE3) in OK cells. The transport kinetics displayed a simple Michaelis-Menten relationship for extracellular Na(+) of 25+/-6 mM. Dopamine and the dopamine D(1)-like receptor agonist SKF 38393 ((+/-)-1-phenyl-2,3,4,5-tetrahydro-(1H)-3-benzazepine-7,8-diol) inhibited NHE3 activity in a concentration-dependent manner; the dopamine D(2)-like receptor agonist quinerolane was devoid of effect. The SKF 38393-mediated inhibition of NHE3 was prevented either by the dopamine D(1)-like receptor antagonist SKF 83566 ((+/-)-7-Bromo-8-8-hydroxy-3 methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine; 1 microM), overnight treatment with cholera toxin (500 ng/ml), the PKA antagonist H-89 (N-(2-[p-bromocinnamylamino]ethyl)-5 isoquinolinesulfonamide hydrochloride; 10 microM), the PKC antagonist chelerythrine (1 microM), or the phospholipase C inhibitor U-73,122 (1-(6-[(17beta]-3-methoxyestra-1,3,5[10]-trien-17-yl) amino] hexyl)-1H-pyrrole-2,5-dione; 3 microM). In addition, dibutyril cAMP (dB-cAMP; 500 microM) was found to increase phospholipase C activity, both in membranes and in cytosol from OK cells; in contrast, phorbol-12,13-dibutyrate (PDB) (1 microM) did not have a significant effect on phospholipase C activity. Pre-treatment of OK cells with the anti-G(s)alpha antibody, but not the anti-G(q/11)alpha antibody, blunted the inhibitory effect of SKF 38393 on NHE3 activity. It is concluded that dopamine D(1)-mediated inhibition of NHE3 in renal OK cells involves both adenylyl cyclase-PKA and the phospholipase C-PKC pathways, a mechanism similar to that described for Na(+),K(+)-ATPase.
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PMID:Dopamine acutely decreases type 3 Na(+)/H(+) exchanger activity in renal OK cells through the activation of protein kinases A and C signalling cascades. 1504 35

We recently reported that NHE3 exists in multimeric complexes with dipeptidyl peptidase IV (DPPIV) in renal brush-border membranes. To examine the possible role of DPPIV in modulating NHE3 activity, we evaluated whether specific competitive inhibitors that bind to the active site of DPPIV affect NHE3 activity in the OKP line of opossum kidney proximal tubule cells. The DPPIV inhibitors diprotin A and P32/98 significantly reduced NHE3 activity, whereas the inactive isomer P34/98 had no effect. DPPIV inhibitors did not reduce the activity of another brush-border transport process, Na-phosphate cotransport. Effects of DPPIV inhibitors on NHE3 activity were not associated with detectable changes in amount or apparent molecular weight of NHE3 or in NHE3 surface expression. To investigate the signaling mechanisms involved in modulation of NHE3 activity by DPPIV, we used inhibitors of protein kinase pathways known to regulate NHE3. Whereas the PKA inhibitor H-89 failed to block the effect of DPPIV inhibitors, the tyrosine kinase inhibitor genistein alone caused a decrement in NHE3 activity very similar in magnitude to that caused by P32/98. We also found that the effects of genistein and P32/98 on NHE3 activity were not additive. In contrast, forskolin/IBMX and P32/98 had additive inhibitory effects on NHE3 activity. These findings suggested that the effect of DPPIV inhibitors to reduce NHE3 activity results from inhibition of a tyrosine kinase signaling pathway rather than by activation of PKA. We conclude that DPPIV plays an unexpected role in modulating Na+/H+ exchange mediated by NHE3 in proximal tubule cells.
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PMID:Role of dipeptidyl peptidase IV in regulating activity of Na+/H+ exchanger isoform NHE3 in proximal tubule cells. 1521 57

This study evaluated the transduction pathway associated with type 3 Na(+)/H(+) exchanger (NHE3) activity-induced inhibition during dopamine D(3) receptor activation in immortalized renal proximal tubular epithelial cells from the spontaneously hypertensive rat. The dopamine D(3) receptor agonist 7-OH-DPAT decreased NHE3 activity, which was prevented by the D(2)-like receptor antagonist S-sulpiride, pertussis toxin (PTX; overnight treatment), and the PKC inhibitor chelerythrine, but not by cholera toxin (overnight treatment), the MAPK inhibitor PD-098059, or the p38 inhibitor SB-203580. The PKA inhibitor H-89 abolished the inhibitory effects of forskolin on NHE3 activity, but not that of 7-OH-DPAT. The phospholipase C (PLC) inhibitor U-73122 prevented the inhibitory effects of 7-OH-DPAT, whereas PDBu and 7-OH-DPAT increased PLC activity and reduced NHE3 activity; downregulation of PKC abolished the inhibitory effects of both PDBu and 7-OH-DPAT on NHE activity. The inhibition of NHE3 activity by GTPgammaS and the prevention of the effect of 7-OH-DPAT by PTX suggest an involvement of a G(i/o) protein coupled to the dopamine D(3) receptor. Indeed, the 7-OH-DPAT-induced decrease in NHE3 activity was abolished in cells treated overnight with the anti-G(i)alpha3 antibody, but not in cells treated with antibodies against G(q/11), G(s)alpha, G(beta), and G(i)alpha1,2 proteins. The calcium ionophore A-23187 and the Ca(2+)-ATPase inhibitor thapsigargin increased intracellular Ca(2+) but did not affect NHE3 activity. However, the inhibitory effects of PDBu and 7-OH-DPAT on NHE3 activity were completely abolished by A-23287 and thapsigargin. It is concluded that inhibition of NHE3 activity by dopamine D(3) receptors coupled to G(i)alpha3 proteins is a PLC-PKC-mediated event, modulated by intracellular Ca(2+).
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PMID:Gialpha3 protein-coupled dopamine D3 receptor-mediated inhibition of renal NHE3 activity in SHR proximal tubular cells is a PLC-PKC-mediated event. 1526 66

Inorganic phosphate (P(i)) is reabsorbed in the renal proximal tubule mainly via the type-IIa sodium-phosphate cotransporter (NaPi-IIa). This protein is regulated tightly by different factors, among them dietary P(i) intake and parathyroid hormone (PTH). A number of PDZ-domain-containing proteins have been shown to interact with NaPi-IIa in vitro, such as Na(+)/H(+) exchanger-3 regulatory factor-1 (NHERF1) and PDZK1. PDZK1 is highly abundant in kidney and co-localizes with NaPi-IIa in the brush border membrane of proximal tubules. Recently, a knock-out mouse model for PDZK1 (Pdzk1(-/-)) has been generated, allowing the role of PDZK1 in the expression and regulation of the NaPi-IIa cotransporter to be examined in in vivo and in ex vivo preparations. The localization of NaPi-IIa and other proteins interacting with PDZK1 in vitro [Na(+)/H(+) exchanger (NHE3), chloride-formate exchanger (CFEX)/putative anion transporter-1 (PAT1), NHERF1] was not altered in Pdzk1(-/-) mice. The abundance of NaPi-IIa adapted to acute and chronic changes in dietary P(i) intake, but steady-state levels of NaPi-IIa were reduced in Pdzk1(-/-) under a P(i) rich diet. This was paralleled by a higher urinary fractional P(i) excretion. The abundance of the anion exchanger CFEX/PAT1 (SLC26A6) was also reduced. In contrast, NHERF1 abundance increased in the brush border membrane of Pdzk1(-/-) mice fed a high-P(i) diet. Acute regulation of NaPi-IIa by PTH in vivo and by PTH and activators of protein kinases A, C and G (PKA, PKC and PKG) in vitro (kidney slice preparation) was not altered in Pdzk1(-/-) mice. In conclusion, loss of PDZK1 did not result in major changes in proximal tubule function or NaPi-IIa regulation. However, under a P(i)-rich diet, loss of PDZK1 reduced NaPi-IIa abundance indicating that PDZK1 may play a role in the trafficking or stability of NaPi-IIa under these conditions.
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PMID:Expression and regulation of the renal Na/phosphate cotransporter NaPi-IIa in a mouse model deficient for the PDZ protein PDZK1. 1551 43

The Na(+)/phosphate cotransporter NaPi-IIa (SLC34A1) is the major transporter mediating the reabsorption of P(i) in the proximal tubule. Expression and activity of NaPi-IIa is regulated by several factors, including parathyroid hormone, dopamine, metabolic acidosis, and dietary P(i) intake. Dopamine induces natriuresis and phosphaturia in vivo, and its actions on several Na(+)-transporting systems such as NHE3 and Na(+)-K(+)-ATPase have been investigated in detail. Using freshly isolated mouse kidney slices, perfused proximal tubules, and cultured renal epithelial cells, we examined the acute effects of dopamine on NaPi-IIa expression and localization. Incubation of isolated kidney slices with the selective D(1)-like receptor agonists fenoldopam (10 microM) and SKF-38393 (10 microM) for 1 h induced NaPi-IIa internalization and reduced expression of NaPi-IIa in the brush border membrane (BBM). The D(2)-like selective agonist quinpirole (1 microM) had no effect. The D(1) and D(2) agonists did not affect the renal Na(+)/sulfate cotransporter NaSi in the BBM of the proximal tubule. Studies with isolated perfused proximal tubules demonstrated that activation of luminal, but not basolateral, D(1)-like receptors caused NaPi-IIa internalization. In kidney slices, inhibition of PKC (1 microM chelerythrine) or ERK1/2 (20 microM PD-098089) pathways did not prevent the fenoldopam-induced internalization. Inhibition with the PKA blocker H-89 (10 microM) abolished the effect of fenoldopam. Immunoblot demonstrated a reduction of NaPi-IIa protein in BBMs from kidney slices treated with fenoldopam. Incubation of opossum kidney cells transfected with NaPi-IIa-green fluorescent protein chimera shifted fluorescence from the apical membrane to an intracellular pool. In summary, dopamine induces internalization of NaPi-IIa by activation of luminal D(1)-like receptors, an effect that is mediated by PKA.
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PMID:Activation of dopamine D1-like receptors induces acute internalization of the renal Na+/phosphate cotransporter NaPi-IIa in mouse kidney and OK cells. 1554 13

Vasopressin and ANG II, which are known to play a major role in renal water and sodium reabsorption, are mainly coupled to the cAMP/PKA and phosphoinositide pathways, respectively. There is evidence for cross talk between these intracellular signaling pathways. We therefore hypothesized that vasopressin-induced water reabsorption could be attenuated by ANG II AT(1) receptor blockade in rats. To address this, three protocols were used: 1) DDAVP treatment (20 ng/h sc for 7 days, n = 8); 2) DDAVP (20 ng/h sc for 7 days) and candesartan (1 mg.kg(-1).day(-1) sc for 7 days) cotreatment (n = 8); and 3) vehicle infusion as the control (n = 8). All rats were maintained on a NaCl-deficient diet (0.1 meq Na(+).200 g body wt(-1).day(-1)) during the experiment. DDAVP treatment alone resulted in a significant decrease in urine output (3.1 +/- 0.2 ml/day) compared with controls (11.5 +/- 2.2 ml/day, P < 0.05), whereas the urine output was significantly increased in response to DDAVP and candesartan cotreatment (9.8 +/- 1.0 ml/day, P < 0.05). Consistent with this, rats cotreated with DDAVP and candesartan demonstrated decreased urine osmolality (1,319 +/- 172 mosmol/kgH(2)O) compared with rats treated with DDAVP alone (3,476 +/- 182 mosmol/kgH(2)O, P < 0.05). Semiquantitative immunoblotting revealed significantly decreased expression of medullary aquaporin-2 (AQP2) and AQP2 phosphorylated in the PKA phosphorylation consensus site Ser-256 (p-AQP2) in response to DDAVP and candesartan cotreatment compared with DDAVP treatment alone. In addition, cortical and medullary AQP1 was also downregulated. Fractional sodium excretion (FE(Na)) and plasma potassium levels were markedly increased, and the expressions of the cortical type 3 Na(+)/H(+) exchanger (NHE3), thiazide-sensitive Na-Cl cotransporter (NCC), and Na-K-ATPase were significantly decreased in response to DDAVP and candesartan cotreatment. Moreover, medullary type 1 bumetanide-sensitive Na-K-2Cl cotransporter expression showed a marked gel mobility shift from 160 to approximately 180 kDa corresponding to enhanced glycosylation, whereas expression was unchanged. In conclusion, ANG II AT(1) receptor blockade in DDAVP-treated rats was associated with decreased urine concentration and decreased AQP2 and AQP1 expression. Moreover, FE(Na) was increased in parallel with decreased expression of NHE3, NCC, and Na-K-ATPase. These results suggest that ANG II AT(1) receptor activation plays a significant role in regulating aquaporin and sodium transporter expression and modulating urine concentration in vivo.
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PMID:Angiotensin II AT1 receptor blockade decreases vasopressin-induced water reabsorption and AQP2 levels in NaCl-restricted rats. 1558 68

Transfection studies using mutant constructs have implicated one or both protein kinase A (PKA) consensus phosphorylation sites [serines 552 and 605 in rat Na(+)/H(+) exchanger type 3 (NHE3)] as critical for mediating inhibition of NHE3 in response to several stimuli including dopamine. However, whether one or both of these sites is actually phosphorylated in endogenous NHE3 in proximal tubule cells is unknown. The purpose of this study was to generate phosphospecific antibodies so that the state of phosphorylation of these serine residues in endogenous NHE3 could be assessed in vitro and in vivo. To this end, polyclonal and monoclonal phosphospecific peptide antibodies were generated against each PKA consensus site. Phosphospecificity was established by ELISA and Western blot assays. We then used these antibodies in vitro to evaluate the effect of dopamine on phosphorylation of the corresponding PKA sites (serines 560 and 613) in NHE3 endogenously expressed in opossum kidney cells. Baseline phosphorylation of both sites was detected that was significantly increased by dopamine. Next, we determined the baseline phosphorylation state of each serine in rat kidney NHE3 in vivo. We found that serine 552 of NHE3 is phosphorylated to a much greater extent than serine 605 at baseline in vivo. Moreover, we detected a distinct subcellular localization for NHE3 phosphorylated at serine 552 compared with total NHE3. Specifically, NHE3 phosphorylated at serine 552 localized to the coated pit region of the brush-border membrane, where NHE3 is inactive, while total NHE3 was found throughout the brush-border membrane. These findings strongly suggest that phosphorylation of NHE3 plays a role in its subcellular trafficking in vivo. In conclusion, we successfully generated phosphospecific antibodies that should be useful to assess the phosphorylation of endogenous NHE3 at its two PKA consensus sites under a variety of physiological conditions in vitro and in vivo.
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PMID:Use of phospho-specific antibodies to determine the phosphorylation of endogenous Na+/H+ exchanger NHE3 at PKA consensus sites. 1600 90

The sodium-hydrogen exchanger 3 (NHE3) isoform is the major regulated sodium transporter in the proximal convoluted tubule of the kidney. Study of the regulation of NHE3 by hormonal stimuli has identified a number of PDZ adaptor proteins that form an apical/subapical membrane scaffold that binds NHE3 and facilitates down-regulation of its activity in response to cAMP and activation of protein kinase A. The precise relation of proximal tubule adaptor proteins such as sodium-hydrogen exchanger regulatory factor-1 (NHERF-1), NHERF-2, and PDZ domain-containing-protein-1 (PDZK1) with each other and with protein targets such as NHE3 has been evolving with the development of specific reagents and genetically altered animals. In this review, we trace the discovery of NHERF-1 and NHERF-2, and update our current understanding of the relation between these proteins and the regulation and trafficking of NHE3.
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PMID:NHERF and regulation of the renal sodium-hydrogen exchanger NHE3. 1574 80


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