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)

We have investigated the nongenomic as well as the genomic effects of glucocorticoids on phosphate (Pi) uptake in primary rabbit renal proximal tubule cells (PTCs) and have defined the involved signaling pathways. In the present study, cortisol-BSA (cortisol-BSA) (>10(-9) M, 30 min) was found to inhibit Pi uptake in a time- and concentration-dependent manner. However, progesterone-BSA (P(4)-BSA), 17ss-estradiol-BSA (E(2)-BSA), testosterone-BSA (T(4)-BSA), aldosterone, P(4), E(2), and T(4) (10(-9) M, 1 h) had no effect on Pi uptake. In addition, cortisol-BSA (10(-9) M) did not affect either Na(+) uptake or alpha-methylglucopyranoside (alpha-MG) uptake. The cortisol-BSA-induced inhibition of Pi uptake was associated with a decrease in the V(max) for Pi uptake, rather than the K(m). The inhibitory effect of cortisol-BSA was not blocked either by actinomycin D (an inhibitor of transcription), cycloheximide (an inhibitor of translation), or classical glucocorticoid receptor antagonists (RU 486 or P(4)). The cortisol-BSA-induced inhibition of Pi uptake was blocked by two phospholipase C (PLC) inhibitors (neomycin or U73122), and two protein kinase C (PKC) inhibitors (staurosporine or bisindolylmaleimide I) but not by two adenylate cyclase/protein kinase A inhibitors [SQ 22536 (an adenylate cyclase inhibitor) or myristoylated protein kinase A inhibitor amide 14-22]. Furthermore, cortisol-BSA promoted the translocation of PKC from the cytosolic fraction to the membrane fraction, while having no effect on the activity of adenylate cyclase. Our observations may thus be interpreted as indicating that cortisol does indeed inhibit renal Pi uptake via a nongenomic mechanism, which involves the PLC/PKC pathway.
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PMID:Regulation of phosphate uptake in primary cultured rabbit renal proximal tubule cells by glucocorticoids: evidence for nongenomic as well as genomic mechanisms. 1115 43

Alteration of [Ca2+]i by hyperglycemia is implicated in the pathogenesis of diabetic nephropathy. However, the effect of high glucose on Ca2+ regulation in proximal tubule cells is not known. Thus, we examined the mechanisms by which high glucose regulates Ca2+ uptake in primary cultured rabbit renal proximal tubule cells. Glucose increased the Ca2+ uptake in a time- and dose-dependent manner. A stimulatory effect of high glucose on Ca2+ uptake is predominantly observed using 25 mM glucose (high glucose) after 1 h, while 25 mM glucose did not affect cell viability and lactate dehydrogenase release. However, 25 mM mannitol and L-glucose did not affect Ca2+ uptake as compared with controls. Nifedipine and methoxyverapamil (L-type Ca2+ channel blockers) blocked high-glucose-induced stimulation of Ca2+ uptake. High-glucose-induced stimulation of Ca2+ uptake was blocked by pertussis toxin, SQ-22536 (adenylate cyclase inhibitor), myristoylated amide 14-22 (protein kinase A inhibitor), neomycin and U-73122 (phospholipase C inhibitors), and staurosporine and bisindolylmaleimide I (protein kinase C inhibitors). In addition, KN-62 (a Ca2+/calmodulin-dependent protein kinase II inhibitor) and W-7 (a Ca2+/calmodulin antagonist) blocked high-glucose-induced stimulation of Ca2+ uptake. In conclusion, high glucose stimulates the Ca2+ uptake through L-type Ca2+ channels via G-protein-coupled adenylate cyclase/cAMP and phospholipase C/protein kinase C pathways.
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PMID:High glucose stimulates Ca2+ uptake via cAMP and PLC/PKC pathways in primary cultured renal proximal tubule cells. 1117 1

Although in LLC-PK cells ATP depletion has been shown to result in alterations of cytoskeleton actin and an inhibition of Na+/H+ exchanger activity, there is little information concerning the regulation of this exchanger in the distal luminal membrane by ATP and actin filaments. The present study examined the direct effect of ATP and cytochalasin B on the Na+/H+ exchanger activity in the proximal and distal tubule luminal membranes. The presence of 100 microM ATP in the luminal membrane vesicles from rabbit proximal tubules did not influence the Ethyl Isopropyl Amiloride sensitive Na+ uptake by these membranes. In contrast, the same treatment of luminal membranes from distal tubules significantly enhanced the exchanger activity from 0.22 +/- 0.04 to 0.39 +/- 0.08 pM/microg/10 sec (P < 0.02). When ATP was replaced by its nonhydrolysable form, ATPgammas, the effect on the distal luminal membrane was strongly diminished suggesting that the action of the nucleotide implicates a phosphorylation step. Confirming this hypothesis, addition of 300-microM-Rp cAMP, a protein kinase A inhibitor, completely abolished the effect of ATP. In view of the fact that a tight relationship has been described between ATP, the cytoskeleton complex and the exchanger activity, we studied the effect of cytochalasin B on this activity. The presence of 20 microM cytochalasin B in the distal luminal membrane vesicles induced, as observed with ATP, a significant increase in the Na+ uptake. However, the actions of ATP and cytochalasin B were not additive. These results suggest that firstly, ATP and short actin filaments of the cytoskeleton regulate the distal luminal isoform through an intramembranous mechanism and secondly, a phosphorylation mechanism is, at least partially, implicated in the action of ATP. In contrast, the proximal tubule exchanger is regulated through different mechanisms.
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PMID:Roles of ATP and cytoskeleton in the regulation of Na+/H+ exchanger along the nephron luminal membrane. 1124 55

1. Changes in proximal tubule function have been reported in cystic fibrosis patients. The aim of this study was to investigate proximal tubule function in the Cftr(tm2cam)deltaF508 cystic fibrosis (CF) mouse model. A range of techniques were used including renal clearance studies, in situ microperfusion, RT-PCR and whole-cell patch clamping. 2. Renal Na(+) clearance was similar in wild-type (1.4 +/- 0.3 microl min(-1), number of animals, N = 12) and CF mice (1.6 +/- 0.4 microl min(-1), N = 7) under control conditions. Acute extracellular volume expansion resulted in significant natriuresis in wild-type (7.0 +/- 0.8 microl min(-1), N = 8) and CF mice (9.3 +/- 1.4 microl min(-1), N = 9); no difference between genotypes was observed. 3. In situ microperfusion revealed that fluid absorptive rate (Jv) was similar under control conditions between wild-type (2.2 +/- 0.4 nl mm(-1) min(-1), n = 10) and CF mice (1.9 +/- 0.3 nl mm(-1) min(-1), n = 11). Addition of a forskolin-dibutyryl cAMP (db-cAMP) cocktail to the perfusate caused no significant change in Jv in either wild-type (2.6 +/- 0.7 nl mm(-1) min(-1), n = 10) or Cftr(tm2cam)deltaF508 mice (2.0 +/- 0.5 nl mm(-1) min(-1), n = 10). 4. CFTR expression was confirmed in samples of outer cortex using RT-PCR. However, no evidence for functional CFTR was obtained when outer cortical cells were stimulated with protein kinase A or forskolin-db-cAMP using whole-cell patch clamping. 5. In conclusion, no functional deficit in proximal tubule function was found in Cftr(tm2cam)deltaF508 mice. This may be a consequence of a lack of whole-cell cAMP-dependent Cl(-) conductance in mouse proximal tubule cells.
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PMID:Renal proximal tubule function is preserved in Cftr(tm2cam) deltaF508 cystic fibrosis mice. 1130 63

Dopamine (DA) is a key hormone in mammalian sodium homeostasis. DA induces natriuresis via acute inhibition of the renal proximal tubule apical membrane Na(+)/H(+) exchanger NHE3. We examined the mechanism by which DA inhibits NHE3 in a renal cell line. DA acutely decreases surface NHE3 antigen in dose- and time-dependent fashion without altering total cellular NHE3. Although DA(1) receptor agonist alone decreases surface NHE3, simultaneous DA(2) agonist synergistically enhances the effect of DA(1). Decreased surface NHE3 antigen, caused by stimulation of NHE3 endocytosis, is dependent on intact functioning of the GTPase dynamin and involves increased binding of NHE3 to the adaptor protein AP2. DA-stimulated NHE3 endocytosis can be blocked by pharmacologic or genetic protein kinase A inhibition or by mutation of two protein kinase A target serines (Ser-560 and Ser-613) on NHE3. We conclude that one mechanism by which DA induces natriuresis is via protein kinase A-mediated phosphorylation of proximal tubule NHE3 leading to endocytosis of NHE3 via clathrin-coated vesicles.
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PMID:Dopamine acutely stimulates Na+/H+ exchanger (NHE3) endocytosis via clathrin-coated vesicles: dependence on protein kinase A-mediated NHE3 phosphorylation. 1132 6

Nitric oxide (NO) levels are increased after exposure of cultured proximal tubule cells (PTC) to non-haem iron, potentially contributing to PTC injury in disease states associated with increased iron exposure, including proteinuric renal disease. The mechanisms underlying this observed increase were investigated. After 3 h exposure to 400 microM nitrilotriacetate (NTA)-Fe, inducible nitric oxide synthase (iNOS) mRNA expression was significantly increased, with a corresponding increase in iNOS protein after 12 h. The nuclear binding activity of NFkappaB with 400 microM NTA-Fe was increased, and pyrrolidine dithiocarbamate (PDTC), an antioxidant inhibitor of NFkappaB, prevented both activation of NFkappaB and NO production in response to NTA-Fe. Inhibition of protein tyrosine kinase reduced iNOS mRNA, iNOS protein levels and NO production in response to NTA-Fe. The effect of tyrosine kinase inhibition on NFkappaB activation was variable, with herbimycin but not genistein having an inhibitory effect. Activation of either protein kinase A or C increased iNOS mRNA and protein levels, and NO production in response to NTA-Fe, whereas only the protein kinase C activator phorbol dibutyrate (PDBu) had a stimulatory effect on NFkappaB activation. The protein kinase A activator forskolin did not alter iron-induced activation of NFkappaB. These data suggest that the observed increase in NO production by PTC in response to iron is due to increased transcription of iNOS. The transcriptional regulation of this response is complex and involves NFkappaB, protein tyrosine kinase and the protein kinases A and C.
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PMID:Molecular mechanisms by which iron induces nitric oxide synthesis in cultured proximal tubule cells. 1134 Mar 4

2,3,5-Tris(glutathion-S-yl)hydroquinone [TGHQ] is a potent nephrotoxicant and nephrocarcinogen, and induces a spectrum of mutations in human and bacterial cells consistent with those attributed to reactive oxygen species (ROS). Studies were conducted to determine whether the oxidative stress induced by TGHQ in renal proximal tubule epithelial cells (LLC-PK(1)) modulates transcriptional activities widely implicated in transformation responses, namely 12-O-tetradecanoyl phorbol 13-acetate (TPA) responsive element (TRE)- and nuclear factor kappa B (NF-kappaB)-binding activity. TGHQ increased TRE- and NF-kappaB-binding activity in a concentration- and time-dependent manner. Catalase fully inhibited peak TGHQ-mediated TRE- and NF-kappaB-binding activity. In contrast, although deferoxamine fully inhibited TGHQ-mediated TRE-binding activity, it had only a marginal effect on NF-kappaB-binding activity. Collectively, these data indicate that TGHQ modulates TRE- and NF-kappaB-binding activity in an ROS-dependent fashion. Cycloheximide and actinomycin D fully inhibited TGHQ-mediated TRE-binding activity, but in the absence of TGHQ increased NF-kappaB-binding activity. Although protein kinase C (PKC) is widely implicated in stress response signaling, pretreatment of cells with PKC inhibitors (H-89, calphostin C) did not modulate TGHQ-mediated DNA-binding activities. In contrast, pretreatment of cells with (PD098059), a mitogen activated protein kinase kinase (MEK) inhibitor, markedly reduced TGHQ-mediated TRE-binding activity, but enhanced TGHQ-mediated NF-kappaB-binding activity. We conclude that TGHQ-mediated TRE- and NF-kappaB-binding activities are ROS-dependent. Although there is a common requirement for hydrogen peroxide (H(2)O(2)) in the regulation of these DNA-binding activities, there appears to be divergent regulation after H(2)O(2) generation in renal epithelial cells.
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PMID:Differential regulation of redox responsive transcription factors by the nephrocarcinogen 2,3,5-Tris(glutathion-S-yl)hydroquinone. 1145 27

Biochemical and cellular experiments in fibroblasts have established the requirement for a member of the PDZ motif Na(+)/H(+) exchanger regulatory factor family of proteins (NHERF and NHERF2) in cAMP-mediated phosphorylation and inhibition of NHE3 activity. NHERF interacts with the actin cytoskeleton through the scaffolding protein ezrin to target a multiprotein signal complex to the plasma membrane. Recent experiments have focused on elements of this model. First, using specific antibodies, NHERF was identified in the renal proximal tubule, where it colocalized with ezrin and NHE3. NHERF2 was seen in glomeruli, the renal vasculature, and collecting duct cells, where it colocalized with ROMK. This distinct nephron localization suggests different physiologic roles for NHERF and NHERF2. Second, the signal-complex model of protein kinase A regulation of NHE3 developed in fibroblasts has been extended to epithelial cells by the development of a dominant-negative opossum kidney cell line expressing an ezrin binding domain-deficient truncation of NHERF. Preliminary studies indicate that these cells have normal basal Na+/H+ exchanger activity but a blunted inhibitory response to cAMP. Third, biochemical, biophysical, and cell experiments have indicated that NHERF binds to itself in a head-to-head configuration, raising the possibility that dimerization may alter the availability of active NHERF. The potential role of the NHERF proteins in the kidney has been expanded by recent studies indicating their involvement in the membrane targeting, trafficking, sorting, and regulation of a range of other transporters, receptors, and signaling proteins. NHERF and related PDZ-containing proteins may serve as adapters for regulation of renal transporters.
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PMID:Acute regulation of NHE3 by protein kinase A requires a multiprotein signal complex. 1147 25

Phosphoenolpyruvate carboxykinase (PEPCK) is a key regulatory enzyme in renal gluconeogenesis. Activation of various PEPCK(-2300)Luc reporter constructs in LLC-PK(1)-F+ cells, a gluconeogenic line of porcine renal proximal tubule-like cells, by protein kinase A (PKA) is mediated, in part, through the cAMP-response element (CRE)-1 of the PEPCK promoter. Incubation of a CRE-1 containing oligonucleotide with nuclear extracts from LLC-PK(1)-F+ cells produced multiple bands, all of which were blocked by antibodies that are specific for C/EBPbeta but not for C/EBPalpha or C/EBPdelta. Treatment of cells with cAMP did not affect the expression of C/EBPbeta, but the observed binding activity was increased nearly threefold. Mutation of CRE-1 to a Gal-4 binding site reduced the PKA-dependent activation of PEPCK(-2300)Luc to 40% of that observed with the wild-type construct. Coexpression of a chimeric protein containing a Gal-4 binding domain and the transactivation domain of C/EBPbeta, but not of C/EBPalpha or CRE binding protein (CREB), restored full activation by PKA. A deletion construct that lacks the activation domain of C/EBPbeta functions as a dominant negative inhibitor. Thus the binding of C/EBPbeta to the CRE-1 may contribute to the cAMP-dependent activation of the PEPCK promoter in kidney cells.
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PMID:C/EBPbeta contributes to cAMP-activated transcription of phosphoenolpyruvate carboxykinase in LLC-PK(1)-F+ cells. 1155 11

Although renal K(+) channels along the nephron have been explored in various animal species, little is known about the K(+) channels in human proximal tubule cells. Using the patch-clamp technique, we investigated the properties of an inwardly rectifying K(+) channel present in the surface membrane of cultured human proximal tubule cells of normal kidney origin. This channel was the most frequently observed K(+) channel in cell-attached patches, and cytoplasmic ATP was required to maintain channel activity in inside-out patches. Its single channel conductance was about 42 pS for inward currents and 7 pS for outward currents under the symmetrical K(+) condition. The ATP effect on channel activity was dose-dependently stimulatory within a range of 0.1 to 10 mM, and a nonhydrolyzable ATP analog, AMP-PNP (3 mM), had no effect on channel activity in either the presence or absence of ATP (1 mM). The channel activity observed in cell-attached patches was reduced to 30 to 50% of controls by a membrane-permeable nonspecific protein kinase inhibitor, K252a (1 microM), or a potent protein kinase A inhibitor, KT5720 (500 nM). In contrast, a membrane-permeable cAMP analog, 8Br-cAMP (100 microM), induced a twofold increase in channel activity. The addition of a catalytic subunit of protein kinase A (PKA-CS, 100 U/ml) to the bath in inside-out patches stimulated channel activity in the presence of 1 mM ATP. Furthermore, the channel activity maintained with 1 mM ATP in inside-out patches was suppressed by internal acidification and enhanced by alkalization. These results suggest that the activity of the inwardly rectifying K(+) channel in cultured human proximal tubule cells was ATP-dependent and regulated at least in part by cAMP/PKA-mediated phosphorylation processes and intracellular pH.
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PMID:An ATP-regulated and pH-sensitive inwardly rectifying K(+) channel in cultured human proximal tubule cells. 1156 89


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