Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. The hyperosmotic activation of the Na(+)-H+ exchanger was studied in an osteoblast-like rat cell line (RCJ 1.20). The activation was monitored by recording the intracellular pH (pHi) changes employing double excitation of the pH-sensitive fluorescent dye 2'7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM). 2. Exposure of the cells to a hyperosmotic HCO(3-)-free medium at 37 degrees C produced an initial cytosolic acidification of 0.05 pH units followed by a lag period and an alkalinization overshoot of about 0.2 pH units, without a concomitant change of the free cytosolic calcium [Ca2+]i by the use of Fura-2 calcium-sensitive probes. This response was completely inhibited by amiloride (0.33 mM) or by Na+ depletion from the external medium and insensitive to the extracellular Cl- replacement, indicating the involvement of a Na(+)-H+ exchanger in the hyperosmotic response. 3. Hyperosmotic stimuli (200 moSM sucrose) applied in the temperature range of 17-37 degrees C demonstrated a shortening of the lag period preceding alkalinization and an increased rate of proton extrusion upon temperature elevation. The biochemical reaction underlying the lag period and the proton extrusion resulted in apparent activation energies of 19 and 29 kcal mol-1, respectively, as calculated from the appropriate Arrhenius plots. 4. Stimulation of the exchanger under isosmotic conditions by 25 nM 4 beta-phorbol 12-myristate 13-acetate (PMA) and 0.1 mM vanadate resulted in an amiloride-sensitive pHi increase of about 0.08 pH units. The hyperosmotic stress was additive to the stimulatory effects of these agents, suggesting an independent hyperosmotic activation pathway. 5. The hyperosmotic activation of the Na(+)-H+ exchanger was independent of cAMP, cGMP, cytosolic Ca2+ and protein kinase C. Thus, none of the classical transduction mechanisms seem to be involved directly in the hyperosmotic activation of the antiporter. 6. The pHi response induced by the hyperosmotic stress was abolished by two calmodulin inhibitors, W-7 and chlorpromazine (50% inhibition, Ki at 28 and 20 microM, respectively), 20 microM cytochalasin B, but not by 10 microM colchicine. The results suggest the involvement of actin and calmodulin-like structural elements of the cytoskeleton in the transduction process leading to the activation of the Na(+)-H+ exchanger.
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PMID:Hyperosmotic activation of the Na(+)-H+ exchanger in a rat bone cell line: temperature dependence and activation pathways. 133 3

Changes in cytosolic calcium concentration ([Ca2+]i) have been implicated in the regulation of intracellular pH (pHi) in several cell types. In the present study we investigated the regulatory mechanism of Na+/H+ exchange induced by angiotensin II (AII) in cultured rat vascular smooth muscle cells (VSMCs). Serially passaged VSMCs from Sprague-Dawley rat thoracic aorta were grown on coverslips and loaded with the pH-sensitive fluorescent indicator 2',7'-bis-(carboxyethyl)-5,6-carboxyfluorescein (BCECF). In HCO(3-)-free Ringer solution, pH 7.40, the resting pHi was 7.21 +/- 0.02 (n = 21). A biphasic response was seen after exposure of these cells to AII: an initial transient a acidification, followed by sustained alkalization. The magnitude of alkalization was dose-dependent. AII-mediated acidification was completely inhibited by [Sar1-IIe5-Gly8]AII, but amiloride had no effect. In contrast, the alkalization induced by AII was abolished by both amiloride and Na(+)-free medium. In Ca(2+)-free medium, the AII-induced alkalization was partially blocked and verapamil also caused partial inhibition. Since AII activates phospholipase C in VSMCs, we examined whether AII would increase Na+/H+ exchange by activation of protein kinase C. An inhibitor of protein kinase C, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), partially inhibited the alkalization induced by AII. These results indicate that AII stimulates cytoplasmic alkalization via an amiloride-sensitive Na+/H+ exchange system in cultured rat VSMCs, and that this AII-stimulated Na+/H+ exchange is mediated by Ca(2+)-dependent and protein kinase C-dependent mechanisms.
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PMID:Stimulation of Na+/H+ exchange induced by angiotensin II in cultured rat vascular smooth muscle cells: role of Ca2+ and C-kinase. 154 35

The activity of the main base-extruding mechanism in Vero cells, the Na(+)-independent Cl-/HCO-3 antiport, increases 5- to 10-fold when the cytosolic pH (pHi) is increased over a narrow range close to neutrality. We have studied the effect on this regulation of stimulation and inhibition of protein kinase C by short-term and long-term treatment with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). After short-term treatment with TPA to stimulate the kinase, the threshold value for activation of the antiport is shifted to a more acidic pH. After prolonged treatment with TPA to downregulate protein kinase C the sensitivity of the antiport to variation in proton concentration was lowered, possibly by reducing the number of essential proton-binding sites. Concomitantly, the steady state pHi of the cells was increased. The data indicate that protein kinase C is involved in the regulation of the Na(+)-independent Cl-/HCO-3 antiport.
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PMID:Evidence for involvement of protein kinase C in regulation of intracellular pH by Cl-/HCO-3 antiport. 204 87

In many systems, osmotically induced cell shrinkage activates the Na+/H+ exchanger. To assess the role of H(+)-extruding transporters in the response to osmotic shrinkage in vascular smooth muscle (VSM) and Chinese hamster ovary (CHO) cells, intracellular pH (pHi) was measured with 2',7'-bis(carboxy-ethyl)-5(6)- carboxyfluorescein-acetoxymethyl ester (BCECF-AM) after exposing cells to hypertonic medium. In nominally HCO(3-)-free medium, addition of 200 mM sucrose caused pHi to increase 0.33 pH unit on average in VSM cells but only 0.13 pH unit in CHO cells. Permeant solutes failed to increase pHi significantly. Cytochalasin B (1-20 microM), colchicine (1-10 microM), Ca2+ removal, and downregulation of protein kinase C activity did not affect osmotic activation of H+ extrusion in either cell type. Additional work was carried out to determine why osmotic activation of H+ extrusion was less in CHO than in VSM cells. In CHO cells, the osmotically induced delta pHi was only weakly sensitive to amiloride, suggesting that osmotic forces may activate an H+ transport system other than Na+/H+ exchange. In the presence of 10 mM HCO3-, osmotically induced delta pHi decreased by 60% in VSM cells but increased by 50% in CHO cells compared with the delta pHi in HCO(3-)-free medium. Lastly, removal of extracellular Cl- did not affect osmotically induced delta pHi in VSM cells but completely abolished the response in CHO cells. We conclude that in VSM cells osmotically induced changes in pHi are mediated by Na+/H+ exchange, whereas in CHO cells they are most likely mediated by a Na(+)-dependent Cl-/HCO3- exchanger.
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PMID:Osmotic activation of a Na(+)-dependent Cl-/HCO3- exchanger. 784 Jan 43

The role of protein kinase C in the regulation of vacuolar-type H(+)-ATPase (V-ATPase) activity was studied in thioglycolate-elicited mouse peritoneal macrophages. Acid-loaded macrophages suspended in a Na(+)- and HCO(3-)-free K(+)-medium containing Zn2+, a H(+)-conductance blocker, exhibited an initial intracellular pH recovery rate of 0.33 +/- 0.04 pH/min (n = 9). Pretreatment with 12-O-tetradecanoyl phorbol 13-acetate (TPA) or mezerein for as little as 3 min induced a marked (82%) increase in the initial pH recovery rate. Stimulation was prevented by the V-ATPase inhibitor, bafilomycin A1 (200 nM) indicating that the effect of the protein kinase C agonist was via augmentation of proton pump activity. The protein kinase C inhibitor, staurosporine (100 nM) completely blocked the stimulatory effects of TPA and mezerein, suggesting involvement of protein kinase C. In keeping with this notion, the inactive analogue of TPA, 4-phorbol didecanoate did not stimulate recovery from an acid load. Extracellular pH determinations revealed that the observed increase in cytosolic pH recovery rate by the protein kinase C agonists was due to increased extrusion of protons from the cells, likely through V-ATPases located in the plasma membrane. Considered together, these data demonstrate regulation of plasmalemmal V-ATPase-mediated proton extrusion by protein kinase C.
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PMID:Protein kinase C activation accelerates proton extrusion by vacuolar-type H(+)-ATPases in murine peritoneal macrophages. 806 29

In the medullary thick ascending limb (MTAL) of the rat kidney, prostaglandin E2 (PGE2) reverses inhibition of HCO3 absorption by arginine vasopressin (AVP). This effect of PGE2 is blocked by chelerythrine or staurosporine and mimicked by phorbol ester, suggesting a critical role for protein kinase C (PKC). The present study was designed to examine directly regulation of PKC isoforms by PGE2 in the inner stripe of the outer medulla and in microdissected MTALs. Immunoblots with isoform-specific anti-PKC antibodies detected alpha-, beta II-, delta-, epsilon-, and zeta-isoforms in both inner stripe and MTAL. The beta I- and gamma-isoforms were not detected. Translocation and activation of PKC were assessed by immunoblot analysis and by direct measurement of enzyme activity using an immune complex kinase assay. In inner stripe tissue incubated with 10(10) M AVP, PGE2 10(6) M for 20 min) induced translocation of PKC-delta from the cytosolic fraction to the membrane fraction. This translocation was associated with an 85% increase in PKC-delta activity in the membrane fraction and a 70% decrease in PKC-delta activity in the cytosolic fraction. PGE2 had no effect on the subcellular distribution or the activities of the other isoforms. Activation of PKC-delta was confirmed directly in microdissected MTALs, in which PGF2 caused a near complete loss of PKC-delta from the cytosolic fraction. PGE2 did not induce translocation of PKC-delta in the absence of AVP. These results demonstrate that 1) the MTAL expresses Ca(2+)-dependent (alpha, beta II) and Ca(2+)-independent (delta, epsilon, zeta) PKC isoforms; 2) PGE2 causes selective activation of PKC-delta, which likely mediates the action of PGE2 to reverse AVP inhibition of HCO-3 absorption; and 3) PGE2 activation of PKC-delta requires the presence of AVP, which may explain the fact that PGE2 influences HCO-3 transport only when AVP is present.
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PMID:PKC isoforms in rat medullary thick ascending limb: selective activation of the delta-isoform by PGE2. 917 73

The addition of phorbol 12-myristate 13-acetate (PMA) to renal LLC-PK1-F+ cells caused a rapid decrease in the level of phosphoenolpyruvate carboxykinase (PCK) mRNA and reversed the stimulatory effects of exposure to acidic medium (pH 6.9, 10 mM HCO-3) or cAMP. In contrast, prolonged treatment with PMA increased the levels of PCK mRNA. The two effects correlated with the membrane translocation and downregulation of the alpha-isozyme of protein kinase C and were blocked by pretreatment with specific inhibitors of protein kinase C. The rapid decrease in PCK mRNA caused by PMA occurred with a half-life (t1/2 = 1 h) that is significantly faster than that measured during recovery from acid medium or following inhibition of transcription (t1/2 = 4 h). The effect of PMA was reversed by staurosporine, which apparently acts by inhibiting a signaling pathway other than protein kinase C. Staurosporine had no effect on the half-life of the PCK mRNA, but it stimulated the activity of a chloramphenicol acetyltransferase gene that was driven by the initial 490 base pairs of the PCK promoter and transiently transfected into LLC-PK1-F+ cells. This effect was additive to that of cAMP, and neither stimulation was reversed by PMA. The stimulatory effect of staurosporine was mapped to the cAMP response element (CRE-1) and P3(II) element of the PCK promoter. The data indicate that, in LLC-PK1-F+ cells, activation of protein kinase C decreases the stability of the PCK mRNA, whereas transcription of the PCK gene may be suppressed by a kinase that is inhibited by staurosporine.
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PMID:PMA and staurosporine affect expression of the PCK gene in LLC-PK1-F+ cells. 972 8

The effect of hypotonicity on H+-ATPase activity was examined in cultured inner medullary collecting duct (mIMCD-3) cells. mIMCD-3 cells were grown to confluence, loaded with 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), and assayed for H+-ATPase activity measured as the Na+- and K+-independent intracellular pH (pHi) recovery following an acid load. Exposure of mIMCD-3 cells to a hypotonic solution (150 mosmol/kgH2O) increased pHi recovery by approximately 350% (P < 0.0001). This effect was inhibited by diethylstilbestrol (an inhibitor of H+-ATPase) and was not dependent on external K+, indicating lack of involvement of H+-K+-ATPase. H+-ATPase activation was acute, independent of cell calcium, and was not secondary to Cl- channel activation. The magnitude of H+-ATPase upregulation was dependent on the osmolarity of the media, with maximum stimulation at 150 mosmol/kgH2O. H+-ATPase upregulation in hypotonicity was significantly blocked in the presence of staurosporine or calphostin C or in cells pretreated with phorbol 12-myristate 13-acetate (PMA), indicating involvement of protein kinase C. Hypotonicity inhibited the Na+/H+ exchanger activity in mIMCD-3 cells, indicating that its stimulatory effect is specific to H+-ATPase. In conclusion, a novel regulatory mechanism of H+-ATPase by hypotonicity is described. The increased H+-ATPase activity in hypotonicity may be responsible for increased HCO-3 reabsorption and maintained acid-base homeostasis in hyposmolar states.
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PMID:Activation of H+-ATPase by hypotonicity: a novel regulatory mechanism for H+ secretion in IMCD cells. 975 20

Metabolic acidosis in vivo, as well as in vitro (1 h at pH 6.8 followed by 2 h at pH 7.4) stimulates H+-ATPase-dependent H+ secretion in outer medullary collecting ducts from the inner stripe (OMCDi) (S. Tsuruoka and G. J. Schwartz. J. Clin. Invest. 99: 1420-1431, 1997). Another group has shown that the adaptation to metabolic acidosis in vivo is mediated by an apical polarization of H+ pumps without an increase in total H+ pump mRNA or protein (B. Bastani, H. Purcell, P. Hemken, D. Trigg, and S. Gluck. J. Clin. Invest. 88: 126-136, 1991). To further address the mechanism of adaptation, we measured net HCO-3 absorption before and after applying protein/RNA synthesis and signal transduction inhibitors during the 1 h of low pH and a cytoskeletal inhibitor during the entire 3-h incubation. Net HCO-3 transport, measured by microcalorimetry, increased approximately 33% after in vitro acidosis. This increase was prevented by application during the first hour of anisomycin (10 microM) or actinomycin D (4 microM), but not by anisomycin applied during the 2-h incubation at pH 7.4. Similar results were obtained with the cell calcium chelator, 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM, 20 microM), the calmodulin antagonist, calmidazolium (30 nM), the endoplasmic reticulum Ca-ATPase inhibitor, thapsigargin (100 nM), and the protein kinase C (PKC) inhibitor, staurosporine (100 nM), applied during the 1 h at pH 6.8, but not with BAPTA-AM or thapsigargin used during the 2-h incubation at pH 7. 4. Colchicine (10 microM) applied during the entire 3-h incubation also prevented this adaptive increase in H+ secretion, whereas lumicolchicine (10 microM, the inactive congener) did not. Colchicine also reversibly prevented any adaptive increases in transepithelial positive voltage. Thus the adaptation to acidosis in vitro required RNA and protein synthesis, changes in intracellular calcium and PKC activity, and intact microtubules. Time was required for the adaptation to occur, as the increase in HCO-3 transport was small after <3-h incubation. Protein synthesis and changes in cell calcium were critical during the initial period of low pH but not once the acid stimulus had been removed. Exocytosis of H+ pumps appears to occur continually during the entire 3-h incubation. These data would suggest that the synthesis and regulation of proteins involved in shuttling H+ pumps in cytoplasmic vesicles to the apical membrane via exocytosis are important for the OMCDi to adapt to low pH in vitro and probably to metabolic acidosis in vivo.
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PMID:Adaptation of the outer medullary collecting duct to metabolic acidosis in vitro. 984 16

The role of ANG II in the regulation of ion reabsorption by the renal thick ascending limb is poorly understood. Here, we demonstrate that ANG II (10(-8) M in the bath) inhibits HCO-3 absorption by 40% in the isolated, perfused medullary thick ascending limb (MTAL) of the rat. The inhibition by ANG II was abolished by pretreatment with eicosatetraynoic acid (10 microM), a general inhibitor of arachidonic acid metabolism, or 17-octadecynoic acid (10 microM), a highly selective inhibitor of cytochrome P-450 pathways. Bath addition of 20-hydroxyeicosatetraenoic acid (20-HETE; 10(-8) M), the major P-450 metabolite in the MTAL, inhibited HCO-3 absorption, whereas pretreatment with 20-HETE prevented the inhibition by ANG II. The addition of 15-HETE (10(-8) M) to the bath had no effect on HCO-3 absorption. The inhibition of HCO-3 absorption by ANG II was reduced by >50% in the presence of the tyrosine kinase inhibitors genistein (7 microM) or herbimycin A (1 microM). We found no role for cAMP, protein kinase C, or NO in the inhibition by ANG II. However, addition of the exogenous NO donor S-nitroso-N-acetylpenicillamine (SNAP; 10 microM) or the NO synthase (NOS) substrate L-arginine (1 mM) to the bath stimulated HCO-3 absorption by 35%, suggesting that NO directly regulates MTAL HCO-3 absorption. Addition of 10(-11) to 10(-10) M ANG II to the bath did not affect HCO-3 absorption. We conclude that ANG II inhibits HCO-3 absorption in the MTAL via a cytochrome P-450-dependent signaling pathway, most likely involving the production of 20-HETE. Tyrosine kinase pathways also appear to play a role in the ANG II-induced transport inhibition. The inhibition of HCO-3 absorption by ANG II in the MTAL may play a key role in the ability of the kidney to regulate sodium balance and extracellular fluid volume independently of acid-base balance.
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PMID:Angiotensin II inhibits HCO-3 absorption via a cytochrome P-450-dependent pathway in MTAL. 1033 55


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