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

Angiotensin II (AngII) is a hormone that alters contractility as well as myocyte growth in heart. Since many hormones that regulate cardiac contractility have also been found to modulate intracellular pH (pHi) the goal of this study was to determine if AngII altered pHi in cultured neonatal rat ventricular myocytes. Changes in pHi were monitored in single cells using the fluorescent pH indicator carboxy-seminaphthorhodafluor-1. Application of 100 nM AngII resulted in a rapid, receptor-mediated alkalinization of 0.08 +/- 0.02 pH unit. The Na+/H+ exchanger was not involved since the response was HCO3(-)-dependent and amiloride-insensitive. Ammonia rebound experiments showed AngII increased the initial rate of recovery from an imposed acid load by 3.15-fold and showed that the hormone led to the selective activation of the Na+/HCO3- symport. In contrast, phorbol ester activation of protein kinase C led to the selective activation of Na+/H+ antiport in these cells. Pharmacological studies showed that the alkalinization was independent of the AngII receptor subtype 1 (AT1) phosphoinositide signaling path. In contrast, AngII activation of the symport was blocked by nanomolar AT2 receptor antagonist PD 123319. Superfusion of the myocytes with exogenous arachidonic acid (5 microM) mimicked the AngII-mediated alkalinization, further suggesting that the AT2 signaling pathway underlies the response. In summary, while most of the known actions of AngII in heart are mediated through AT1 receptors, activation of the Na+/HCO3- symport occurs through a distinct alternative path that is likely related to fatty acid production.
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PMID:Angiotensin II activates the Na+/HCO3- symport through a phosphoinositide-independent mechanism in cardiac cells. 765 18

The regulatory mechanism of basolateral Cl- conductance in rabbit renal proximal tubule S3 segments was investigated with conventional and Cl- sensitive microelectrodes. After the basolateral Cl-/HCO3- exchanger was blocked by 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) we increased the bath K+ concentration from 5 mmol/l to 20 mmol/l, which depolarized the cells and thereby increased intracellular Cl- activity ([Cl-]i). This [Cl-]i response was enhanced by +63% in the presence of forskolin (20 mumol/l), by +40% in the presence of dibutyryl adenosine 3',5'-cyclic monophosphate (db-cAMP) (1 mmol/l) and by +44% in the presence of parathyroid hormone (PTH, 10 nmol/l), whereas it was inhibited by a Cl- channel blocker, indanyl-oxyacetic acid (IAA-94, 0.3 mmol/l). In addition, forskolin, PTH and chlorophenylthio-cAMP enhanced the electrogenic response to removal of bath Cl- after the blockade of K+ conductance, and this activation was also sensitive to IAA-94. On the other hand, 2 mumol/l ionomycin and 0.5 mumol/l phorbol myristate failed to activate the [Cl-]i response to elevation of bath K+ concentration and the electrogenic response to Cl- removal, and ionomycin had no effect even in the absence of DIDS. These results indicate that this basolateral Cl- conductance can be activated by cAMP, while neither the increase in cytosolic Ca2+ nor the activation of protein kinase C has direct effects on this conductance.
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PMID:Activation of the basolateral Cl- conductance by cAMP in rabbit renal proximal tubule S3 segments. 766 81

The roles of protein kinase C (PKC) in regulation of the plasmalemmal vacuolar-type H(+)-ATPase (V-ATPase) and Na(+)-H+ exchanger (NHE) of rabbit alveolar macrophages (m phi) were investigated using phorbol 12-myristate 13-acetate (PMA). At an extracellular pH (pHo) of 7.4 (nominal absence of CO2-HCO3-), PMA caused a dose-dependent increase in the rate of cellular H+ extrusion with little change in intracellular pH (pHi). PMA caused a prolonged cytosolic acidification at pHo < or = 6.8. PMA-induced changes in pHi were sensitive to bafilomycin A1, but were insensitive to amiloride. Studies of pHi recovery following intracellular acid challenge showed that both V-ATPase and the NHE were up-regulated by PMA. An inactive analog, 4 alpha-phorbol, had no detectable effects on pHi homeostasis. These data indicate that (a) PKC is involved in regulation of V-ATPase and the NHE of resident alveolar m phi and (b) V-ATPase is the predominant mechanism for pHi homeostasis in unstimulated and PMA-activated m phi.
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PMID:Effects of myristate phorbol ester on V-ATPase activity and Na(+)-H+ exchange in alveolar macrophages. 772 18

In the rat medullary thick ascending limb (MTAL), hyperosmolality inhibits transepithelial HCO3- absorption (JHCO3-) by inhibiting apical membrane Na+/H+ exchange. To examine signaling mechanisms involved in this regulatory response, MTALs were isolated and perfused in vitro with 25 mM HCO3- solutions (290 mosmol/kg H2O). Osmolality was increased in lumen and bath solutions by addition of 300 mM mannitol or 75 mM NaCl. Addition of mannitol reduced JHCO3- by 60% and addition of NaCl reduced JHCO3- by 50%. With the protein tyrosine kinase (PTK) inhibitor genistein (7 microM) or herbimycin A (1 microM) in the bath, addition of mannitol reduced JHCO3- only by 11% and addition of NaCl reduced JHCO3- only by 15%. Staurosporine (10(-7) M) or forskolin (10(-6) M) in the bath had no effect on inhibition of JHCO3- by hypertonic NaCl. Genistein had no effect on inhibition of JHCO3- by vasopressin (a cyclic AMP-dependent process) or stimulation of JHCO3- by prostaglandin E2 (a protein kinase C-dependent process). Under isosmotic conditions, addition of genistein or herbimycin A to the bath increased JHCO3- by 30% through stimulation of apical membrane Na+/H+ exchange. Addition of the tyrosine phosphatase inhibitor molybdate (50 microM) to the bath reproduced the inhibition of JHCO3- observed with hyperosmolality. These data indicate that 1) the effect of hyperosmolality to inhibit MTAL HCO3- absorption through inhibition of apical membrane Na+/H+ exchange is mediated via a PTK-dependent pathway that functions independent of regulation by cyclic AMP and protein kinase C, and 2) a constitutive PTK activity inhibits apical membrane Na+/H+ exchange and HCO3- absorption under isosmotic conditions. Our results suggest that tyrosine phosphorylation is a critical step in inhibition of the apical Na+/H+ exchanger isoform NHE-3 by hyperosmolality.
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PMID:Hyperosmolality inhibits bicarbonate absorption in rat medullary thick ascending limb via a protein-tyrosine kinase-dependent pathway. 773 Mar 71

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

1. We investigated the regulation of intracellular pH (pHi) in rat brain isolated nerve terminals (synaptosomes), using fluorescence pH indicators and time-resolved fluorescence spectroscopy. 2. The resting pHi was not significantly affected by the presence or absence of HCO3-. Removal of external Na+, in the absence or presence of HCO3- caused a rapid acidification of pHi. The recovery from acid loads was primarily due to the activity of the Na+/H+ exchanger, confirming the relevance of this transport system in synaptosomes. 3. Our data revealed that in synaptosomes the activity of the Na+/H+ exchanger was not regulated by either protein kinase C or kinase A. In contrast, Ca2+ played an important role in the regulation of Na+/H+ exchanger. This was supported by the observation that 4Br-A23187 induced a Na(+)-dependent alkalinization of the resting pHi and greatly enhanced the initial rate and the degree of the recovery from acid loads. 4. In most eukaryotic cells, HCO3(-)-based transport mechanisms play an important role in pHi regulation. In synaptosomes, however, HCO3- transport is not significantly involved in pHi regulation, because the presence or absence of HCO3- does not affect resting pHi nor the rate of pHi recovery to acid loads. Further studies to address the role of Cl- and HCO3- in pHi regulation in synaptosomes are discussed in the companion paper. 5. Increasing the concentration of Ko+ also resulted in a rise of steady-state pHi by a processes that is Ca2+ and HCO3- independent. This alkalinization could be due to either K+/H+ exchanger activity, K(+)-induced depolarization, reduction of delta microH+, or a direct reduction of delta microK+. Calculated H+ driving forces suggest that the reduction in the inwardly directed H+ leak is sufficient to explain this K(+)-induced alkalinization because it changes the delta microH+ by virtue of setting the membrane potential difference (Em) to the K+ equilibrium potential (EK+).
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PMID:Regulation of pH in rat brain synaptosomes. I. Role of sodium, bicarbonate, and potassium. 793 13

The aim of these experiments was to study acute regulation of proximal tubule H+/HCO3- transport systems in acid-base disorders. Proximal tubular suspensions were prepared from rabbit kidney cortex and incubated in acidic (pH 6.9), control (pH 7.4), or alkalotic (pH 8.0) media for 45 minutes and gassed with 5% CO2. Brush border membrane (BBM) and basolateral membrane (BLM) vesicles were isolated from the tubular suspensions and studied for the activity of the pH-regulating transport systems. Influx of amiloride-sensitive 22Na at 10 seconds (pHo 7.5, pHi 6.0) into BBM vesicles was 36% higher in the acidotic and 51% lower in alkalotic groups compared to control. HCO3-dependent 22Na uptake was increased by 55% in BLM vesicles from acidotic and remained unchanged in ones from alkalotic tubules. The presence of 250 nmol/L staurosporine during incubation in acidic medium reduced the activities of Na+/H+ exchange and Na+/HCO3- cotransport by 19% and 17%, respectively. 36Chloride influx into BBM vesicles (mediated via Cl-/Cl- exchange) remained unchanged in vesicles harvested from tubules in acidic and alkalotic media. However, 36chloride influx into BLM vesicles (mediated via Cl-/Cl- exchange) decreased by 23% in the acidic and increased by 37% in the alkalotic group. Staurosporine had no effect on Cl/base exchange in BLM vesicles isolated from control, acidotic, or alkalotic tubules. We conclude that in vitro acid-base disorders are associated with complex and preferential adaptive changes in certain pH-regulating processes in kidney proximal tubules. Some of these effects are partially mediated via protein kinase C.
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PMID:Acute regulation of Na+/H+ exchange, Na+:HCO3- cotransport, and C1-/base exchange in acid base disorders. 803 6

Transport defects by retinal pigment epithelial (RPE) and other cells are observed in experimental models of diabetes mellitus. Recent studies have established that glucose concentration, per se, is the critical risk factor in the pathogenesis of diabetic complications. This study was designed to test whether transport alterations could be produced in the simplest model of diabetes, sustained exposure of cultured cells to a high-glucose environment. The regulatory transport responses to acute changes in cell volume were measured in order to assess the effects of glucose on a range of transport processes. Continuous lines of nontransformed human retinal pigment epithelial (hRPE) cells were grown for two weeks with either 5.6 low glucose (LG) or 26.0 high glucose (HG) mM in paired experiments. The cell volumes of suspended cells were studied in hypo-, iso- and hypertonic solutions containing the same ionic composition. Hypotonic swelling triggered a regulatory volume decrease (RVD), inhibited by reducing the chemical driving force for K+ efflux, or blocking K+ channels (with Ba2+) or Cl- channels (with NPPB). Thus, the RVD of the hRPE cells likely reflects efflux of K+ and Cl- through parallel channels. Shrinkage caused a regulatory volume increase (RVI), which was inhibited by blocking Na+/H+ (with dimethylamiloride) or Cl-/HCO3- exchange (with DIDS). Bumetanide inhibited the RVI significantly only when the K+ concentration was increased above the baseline level. Therefore, the RVI under our baseline conditions likely reflects primarily Na+/H+ and Cl-/HCO3- antiport exchange. Growth in high-glucose medium had no substantial effect on the RVD, but reduced the rate constant of the RVI by approximately 50%. The RVI was unaffected by growth in high-mannitol medium. Stimulation of protein kinase C (PKC) with DiC8 increased the RVI of HG-cells, but not of LG-cells. The DiC8-induced stimulation was bumetanide insensitive and abolished by 1 mM amiloride. Other transport effects of PKC (on the RVD) were unaltered in the HG-cells. We conclude that sustained elevation of extracellular glucose, per se, can downregulate the Na+/H+ antiport of target cells, an effect noted in streptozotocin-treated rats, and that this downregulation does not reflect interruption of the PKC-signaling pathway.
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PMID:Prolonged incubation with elevated glucose inhibits the regulatory response to shrinkage of cultured human retinal pigment epithelial cells. 807 83

We investigated the effects of an alpha 1-adrenoceptor (phenylephrine) and a purinoceptor agonist (ATP), both of which accelerate the phosphoinositide turnover, on the Na-H antiport activity of rat single cardiac cells using the pH-sensitive fluorescent indicator seminaphthorhodafluor-1 (SNARF-1). Both phenylephrine, in the presence of a beta-adrenoceptor blocker, and ATP enhanced the ability of the cell to regulate its intracellular pH (pHi) after an imposed acid load. This effect was observed in HCO3-free N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) and prevented by Na-H antiport inhibitors ethylisopropylamiloride (EIPA) or amiloride. Similar results were obtained when cells were bathed in an acidic extracellular medium. Hence, the alpha 1-adrenoceptor and purinoceptor agonists activate the Na-H antiport even when it is partially inhibited by extracellular protons. To further evaluate the effects of the two neurohormones, the rate of proton efflux was estimated as a function of the magnitude of the imposed acid load. The results indicate that the agonist-induced modulation of the Na-H antiport is caused by an acceleration of its exchange activity and by a shift of its dependence on pHi toward more alkaline pH values. The agonist-mediated stimulation of the antiport was also observed in partially depolarized cells and was not dependent on intracellular Ca. Phorbol 12-myristate 13-acetate was not able to reproduce the effects of the agonists on the Na-H antiport. Conversely, the inhibitors of protein kinase C did not prevent the activation of the antiport by the neurohormones. Thus our data suggest that neither a Ca-calmodulin-dependent kinase nor protein kinase C is responsible for the alpha 1-adrenoceptor- and purinoceptor-mediated stimulation of the antiport.
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PMID:Alpha 1-adrenoceptor and purinoceptor agonists modulate Na-H antiport in single cardiac cells. 809 73

We examined the effects of calcitonin gene-related peptide (CGRP), forskolin, phorbol 12-myristate 13-acetate (PMA), and ionomycin on the intracellular pH (pHi) dependence of Na-H exchange in UMR-106 cells. In the nominal absence of CO2-HCO3-, each agent increased pHi, measured with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). From the rate of pHi recovery (dpHi/dt) from an acid load, and intracellular buffering power, we computed the pHi dependence of the total acid-extruding flux (JTotal). All four agents increased JTotal. From dpHi/dt data obtained in the presence of ethylisopropyl amiloride (EIPA, a blocker of Na-H exchange), we determined the EIPA-resistant component of JTotal (JEIPA/R). We estimated the Na-H exchange flux (JNa-H) as the difference JTotal-JEIPA/R-CGRP, forskolin, and PMA produced similar increases in the slope of the JNa-H vs. pHi-relationship. The net effect of these agents, as well as ionomycin, was to increase JNa-H over a broad pHi range. Ionomycin alkaline shifted the JEIPA/R vs. pHi relationship; the other agents had no effect. Our results indicate that CGRP increased JTotal by stimulating Na-H exchange, with little effect on EIPA-resistant processes. A signaling pathway involving only adenosine 3',5'-cyclic monophosphate, only protein kinase C, or only Ca2+ cannot account for the effects of CGRP on both pHi and pHi dependence of JNa-H. Thus, CGRP probably affects UMR-106 pHi physiology via more than one pathway.
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PMID:Effects of CGRP, forskolin, PMA, and ionomycin on pHi dependence of Na-H exchange in UMR-106 cells. 817 55


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