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:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Protein kinase C (PKC) and Na+/K+-ATPase in hepatocytes from the anoxia-tolerant goldfish (Carassius auratus) and the anoxia-intolerant rainbow trout (Oncorhynchus mykiss) were studied to determine their role in the anoxic response of these cells. PKC and Na+/K+-ATPase activities were measured for up to 90 min in the absence (normoxia) and presence (chemical anoxia) of 2 mmol l-1 sodium cyanide. PKC activity of normoxic cells from both species remained constant for the entire experimental period. Addition of cyanide had no effect on PKC activity of trout cells, which was maintained at 25 % of maximal PKC activity. In goldfish hepatocytes, PKC activity remained constant at 56 % of maximal PKC activity for 30 min but fell to 27 % after 90 min of anoxic exposure. ATPase activity was measured in hepatocytes exposed to 100 nmol l-1 phorbol-12,13-dibutyrate (PdBu), a treatment which enhanced PKC activity to its maximum level. In trout cells, there was no significant change in Na+/K+-ATPase activity whereas in goldfish hepatocytes a significant increase to about 150 % of the respective controls was observed. On the basis of the experimental evidence that in hepatocytes of goldfish (1) PKC and Na+/K+-ATPase activities decreased in parallel during chemical anoxia and (2) a stimulation of PKC activity by PdBu increased Na+/K+-ATPase activity, we postulate that PKC activity in goldfish, but not in trout, may be implicated in the Na+/K+-ATPase inhibition observed under anoxia.
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PMID:Effect of chemical anoxia on protein kinase C and Na+, K+-ATPase in hepatocytes of goldfish (Carassius auratus) and rainbow trout (Oncorhynchus mykiss) 931 15

Functional coupling of Na+,K+-ATPase pump activity to a basolateral membrane (BLM) K+ conductance is crucial for sustaining transport in the proximal tubule. Apical sodium entry stimulates pump activity, lowering cytosolic [ATP], which in turn disinhibits ATP-sensitive K+ (KATP) channels. Opening of these KATP channels mediates hyperpolarization of the BLM that facilitates Na+ reabsorption and K+ recycling required for continued Na+,K+-ATPase pump turnover. Despite its physiological importance, little is known about the regulation of this channel. The present study focuses on the regulation of the BLM KATP channel by second messengers and protein kinases using membrane patches from dissociated, polarized Ambystoma proximal tubule cells. The channel is regulated by protein kinases A and C, but in opposing directions. The channel is activated by forskolin in cell-attached (c/a) patches, and by PKA in inside-out (i/o) membrane patches. However, phosphorylation by PKA is not sufficient to prevent channel rundown. In contrast, the channel is inhibited by phorbol ester in c/a patches, and PKC decreases channel activity (nPo) in i/o patches. The channel is pH sensitive, and lowering cytosolic pH reduces nPo. Increasing intracellular [Ca2+] ([Ca2+]i) in c/a patches decreases nPo, and this effect is direct since [Ca2+]i inhibits nPo with a Ki of approximately 170 nM in i/o patches. Membrane stretch and hypotonic swelling do not significantly affect channel behavior, but the channel appears to be regulated by the actin cytoskeleton. Finally, the activity of this BLM KATP channel is coupled to transcellular transport. In c/a patches, maneuvers that inhibit turnover of the Na+,K+-ATPase pump reduce nPo, presumably due to a rise in intracellular [ATP], although the associated cell depolarization cannot be ruled out as the possible cause. Conversely, stimulation of transport (and thus pump turnover) leads to increases in nPo, presumably due to a fall in intracellular [ATP]. These results show that the inwardly rectifying KATP channel in the BLM of the proximal tubule is a key element in the feedback system that links cellular metabolism with transport activity. We conclude that coupling of this KATP channel to the activity of the Na+,K+-ATPase pump is a mechanism by which steady state NaCl reabsorption in the proximal tubule may be maintained.
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PMID:Regulation of an inwardly rectifying ATP-sensitive K+ channel in the basolateral membrane of renal proximal tubule. 941 42

We have previously shown that crosslinkage of a receptor protein on catfish nonspecific cytotoxic cells (NCC) with anti-receptor monoclonal antibody or with a synthetic peptide activates cytotoxicity and initiates signalling responses. Receptor linked signalling was associated with the production of increased levels of expression of 50-60 and 20-30 kDa phosphoproteins determined by immunoprecipitation with anti-phosphoserine and anti-phosphotyrosine mabs. These proteins are components of a macromolecular protein complex (>200 kDa) determined by reducing and nonreducing SDS-PAGE. The calcium ionophore A23187 treatment produced the same pattern of phosphoprotein expression as peptide or mab. Maximum phosphoserine expression occurred at 15'-30' post-mab binding. We now show that synthetic peptide or mab treatment initiated the same serine and tyrosine phosphorylation profiles. The PKC specific inhibitor MDL 29,152 produced 50% inhibition of NCC lysis of IM-9 target cells, and completely inhibited serine phosphorylation of peptide activated cells but had no effect on tyrosine phosphorylation of the phosphointermediates. Genistein pretreatment of NCC inhibited cytotoxicity and tyrosine phosphorylation. Sequential immunprecipitation of the phosphointermediate demonstrated that the phosphorylated serine and tyrosine residues were on the same 50-60 kDa protein. These data indicate that both proximal and distal signalling events required for NCC activation may be associated with ATPase phosphorylation.
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PMID:Receptor associated phosphorylation following monoclonal antibody or synthetic peptide binding to nonspecific cytotoxic cells. 949 68

Cultured vascular smooth muscle cells (VSMC) from rat thoracic aortas were exposed to hyperosmotic media to determine the effects on Na, K-ATPase alpha1- and beta1-mRNA expression. Hyperosmotic media (500 mOsm/kgH2O) supplemented with glucose or mannitol increased alpha1-mRNA levels threefold at 24 hr and beta1-mRNA levels sevenfold at 12 hr. In sharp contrast, hyperosmotic urea medium had no effect at any time. Both the protein synthesis inhibitor cycloheximide and the RNA transcription inhibitor actinomycin D reduced alpha1- and beta1-mRNA upregulation induced by hyperosmotic glucose or mannitol media. Protein kinase C (PKC) inhibitors (staurosporine A or calphostin C) or tyrosine kinase (TK) inhibitors (genistein or herbimycin A) had no effect on the alpha1-mRNA upregulation induced by hyperosmotic glucose or mannitol media. Hyperosmotic glucose or mannitol media (500 mOsm/kgH2O) significantly increased alpha1- and beta1-subunit protein levels and Na, K-ATPase activity, whereas hyperosmotic urea medium had no effect. Transfection experiments with the 5'-flanking sequences of the alpha1- or beta1-subunit genes linked to the luciferase reporter gene revealed that hyperosmolar glucose medium increased luciferase activity 2.9- and 3.7-fold, respectively. Similarly, hyperosmotic mannitol medium increased such activity 2.7- and 3.4-fold, respectively. These results demonstrate that: (i) hyperosmolality induced by the poorly permeating solutes (glucose and mannitol) stimulates alpha1- and beta1-mRNA accumulation, alpha1- and beta1-subunit protein accumulation, and Na, K-ATPase activity, whereas the rapidly permeating solute (urea) has no effect; (ii) the upregulation of alpha1- and beta1-mRNA in response to hyperosmotic glucose or mannitol media requires, at least in part, de novo synthesis of intermediate regulatory proteins; (iii) the hyperosmolality-induced alpha1-mRNA upregulation occurs through PKC- and TK-independent mechanisms, whereas the hyperosmolality-induced beta1-mRNA upregulation occurs through activation of PKC and TK; and (iv) hyperosmolality induced by glucose or mannitol increases promoter activities of the alpha1- and beta1-subunit genes.
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PMID:Effects of hyperosmolality on Na, K-ATPase gene expression in vascular smooth muscle cells. 954 96

In the present study we show that expression of the neural PKC-substrate B-50 (growth-associated protein [GAP-43]) in Rat-1 fibroblasts induced the formation of filopodial extensions during spreading. This morphological change was accompanied by an enhanced formation of peripheral actin filaments and by accumulation of vinculin immunoreactivity in filopodial focal adhesions, colocalizing with B-50. In time lapse experiments, the B-50-induced filopodial extensions were shown to stay in close contact with the substratum and appeared remarkably stable, resulting in a delayed lamellar spreading of the fibroblasts. The morphogenetic effects of the B-50 protein were entirely dependent on the integrity of the two N-terminal cysteines involved in membrane association (C3C4), but were not significantly affected by mutations of the PKC-phosphorylation site (S41) or deletion of the C terminus (177-226). Cotransfection of B-50 with dominant negative Cdc42 or Rac did not prevent B-50-induced formation of filopodial cells, whereas this process could be completely blocked by cotransfection with dominant negative Rho or Clostridium botulinum C3-transferase. Conversely, constitutively active Rho induced a similar filopodial phenotype as B-50. We therefore propose that the induction of surface extensions by B-50 in spreading Rat-1 fibroblasts depends on Rho-guanosine triphosphatase function.
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PMID:B-50/GAP-43-induced formation of filopodia depends on Rho-GTPase. 961 74

The effect of protein kinase C activating phorbol ester, phorbol-12-myristate-13-acetate (PMA), on purinergic agonists- and thapsigargin-induced Ca2+ signals in Fura-2 loaded rat peritoneal macrophages was investigated. PMA (100 ng/ml) was shown to inhibit 200 muM ATP- or 200 microM UTP-evoked Ca2+ entry in macrophages. Protein kinase C activation by PMA also inhibits the store-dependent or "capacitative" Ca2+ influx stimulated by emptying the intracellular Ca2+ stores with endoplasmic Ca(2+)-ATPase inhibitor thapsigargin (0.5 microM). Inhibition of entry by PMA was fully prevented by protein kinase C inhibitor 50 microM H-7. These data are compatible with the important role played by protein kinase C in the control of Ca2+ entry in rat peritoneal macrophages.
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PMID:[Role of protein kinase C in the regulation of Ca-responses induced by purinergic agonists and thapsigargin in rat peritoneal macrophages]. 969 42

Protein kinase C (PKC) modulates the activity and phosphorylation of the catalytic alpha-subunit of sodium-potassium-adenosine triphosphatase (Na+/K+ ATPase) in normal arteries. Because PKC is altered in cirrhotic aortae, Na+/K+ ATPase may also be altered in these arteries. The aim of the present study was to investigate alpha-subunit activity and phosphorylation in aortae from normal and cirrhotic rats, under baseline conditions and during exposure to PKC modulators. Alpha-subunit activity was assessed by measuring the amount of 32P released by hydrolysis of [gamma-32P]ATP in freshly isolated cell membranes (in the absence of PKC modulators only) and membrane depolarization caused by ouabain-induced alpha-subunit inhibition in isolated aortae (in the absence and presence of PKC modulators). Alpha-subunit phosphorylation was assessed by incorporation of 32P into alpha-subunits. Staurosporine, a PKC inhibitor, and phorbol 12,13-dibutyrate (PDBU), a PKC activator, were used. In addition, alpha-subunit expression was studied by Western blot analysis. In the absence of PKC modulators, the amount of 32P released by hydrolysis of [gamma-32P]ATP and ouabain-induced membrane depolarization were significantly lower in cirrhotic than in normal aortae. Staurosporine suppressed ouabain-induced membrane depolarization in cirrhotic and normal arteries. Ouabain-induced membrane depolarization was similar in cirrhotic aortae exposed to PDBU and in normal arteries studied under baseline conditions. Alpha-subunit phosphorylation was significantly lower in cirrhotic than in normal aortae, in aortae under baseline conditions, and in arteries exposed to staurosporine. Phosphorylation of the alpha-subunit was similar in cirrhotic aortae exposed to PDBU and in normal arteries under baseline conditions. Western blot analysis showed that the amount of alpha-subunit did not significantly differ between cirrhotic and normal aortae. In conclusion, a decrease in baseline Na+/K+ ATPase alpha-subunit activity occurs in aortae from cirrhotic rats as a result of reduced basal PKC activity. This PKC-dependent decreased alpha-subunit activity may be caused by a reduction in PKC-induced alpha-subunit phosphorylation.
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PMID:Effects of protein kinase C modulators on Na+/K+ adenosine triphosphatase activity and phosphorylation in aortae from rats with cirrhosis. 973 56

This article reviews related studies from the authors' laboratory, which focus on the regulation of vascular Na+,K+-ATPase in hypertension. Earlier studies, including the authors', suggested that Na-pump activity in cardiovascular tissues is subject to regulation during hypertension; most of these studies report a stimulation of the vascular enzyme during established stages of hypertension. To test hypothesis that in vascular smooth muscle, strain resulting from elevated pressure may be a signal initiating a cascade of events leading to increased expression of Na+,K+-ATPase, the authors used cell culture and the Flexercell Strain Unit to apply cyclical stretch to rat aortic smooth muscle cells (ASMC) for several days. These studies demonstrated that mechanical strain induces the upregulation of both the alpha-1 and alpha-2 subunits of Na+,K+-ATPase. Mechanisms underlying these changes appear to involve a transient increase in intracellular sodium entering the cell through stretch-activated channels. Calcium entering the cell via L-type channels did not affect stretch-induced upregulation of the alpha isoforms. In addition, protein kinase C inhibition resulted in inhibition of the Na-pump during stretch, but not under nonstretch conditions. The authors conclude that the stretch component of vascular pressure upregulates the Na+,K+-ATPase catalytic subunits. Intracellular sodium may be a signal for this regulation. In addition, phosphorylation by PKC may be important in stretch-induced short-term regulation of the vascular Na-pump.
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PMID:Effect of mechanical strain on expression of Na+,K+-ATPase alpha subunits in rat aortic smooth muscle cells. 974 62

Using SK-N-SH cells, we observe that muscarinic acetylcholine receptor activation by methacholine (MCh) rapidly and selectively diminishes l-NE transport capacity (Vmax) with little or no change in norepinephrine (NE) Km and without apparent effects on membrane potential monitored directly under current clamp. Over the same time frame, MCh exposure reduces the density of [3H]nisoxetine binding sites (Bmax) in intact cells but not in total membrane fractions, consistent with a loss of transport capacity mediated by sequestration of transporters rather than changes in intrinsic transport activity or protein degradation. Similar changes in NE transport and [3H]nisoxetine binding capacity are observed after phorbol ester (beta-PMA) treatment. Inhibition of PKC by antagonists and downregulation of PKC by chronic treatment with phorbol esters abolishes beta-PMA-mediated effects but produce only a partial blockade of MCh-induced effects. Neither muscarinic acetylcholine receptor nor PKC activation require extracellular Ca++ to diminish NET activity. In contrast, treatment of cells with the Ca++/ATPase antagonist, thapsigargin in Ca++-free medium, eliminates the staurosporine-insensitive component of MCh regulation. These findings were further corroborated by the ability of [1, 2-bis(o-amino-phenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester application in Ca++-free medium to abolish NET regulation by MCh. Although they may contribute to basal NET expression, we could not implicate CaMKII-, PKA- or nitric oxide-linked pathways in MCh regulation. Together, these findings 1) provide evidence in support of G-protein coupled receptor-mediated regulation of catecholamine transport, 2) reveal intracellular Ca++-sensitive, PKC-dependent and -independent pathways that serve to regulate NET expression and 3) indicate that the diminished capacity for NE transport evident after mAChR and PKC activation involves a redistribution of NET protein.
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PMID:Acute regulation of norepinephrine transport: I. protein kinase C-linked muscarinic receptors influence transport capacity and transporter density in SK-N-SH cells. 980 4

While several studies have investigated the regulation of the Na, K-ATPase consisting of the alpha1 and beta1 subunits, there is little evidence that intracellular messengers influence the other Na pump isozymes. We studied the effect of different protein kinases and arachidonic acid on the rat Na,K-ATPase isoforms expressed in Sf-9 insect cells. Our results indicate that PKA, PKC, and PKG are able to differentially modify the function of the Na,K-ATPase isozymes. While PKC activation leads to inhibition of all isozymes, PKA activation stimulates the activity of the Na,K-ATPase alpha3 beta1 and decreases that of the alpha1 beta1 and alpha2 beta1 isozymes. In contrast, activation of PKG diminishes the activity of the alpha1 beta1 and alpha3 beta1 isozymes, without altering that of alpha2 beta1. Treatment of cells with arachidonic acid reduced the activities of all the isozymes. The changes in the catalytic capabilities of the Na pump isozymes elicited by PKA and PKC are reflected by changes in the molecular activity of the Na,K-ATPases. One of the mechanisms by which PKA and PKC affect Na pump isozyme activity is through direct phosphorylation of the alpha subunit. In the insect cells, we found a PKA- and PKC-dependent phosphorylation of the alpha1, alpha2 and alpha3 polypeptides. In conclusion, several intracellular messengers are able to modulate the function of the Na,K-ATPase isozymes and some of them in a specific fashion. Because the Na,K-ATPase isozymes have kinetic properties that are unique, this isozyme-specific regulation may be important in adapting Na pump function to the requirements of each cell.
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PMID:Differential regulation of Na,K-ATPase isozymes by protein kinases and arachidonic acid. 980 55


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