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)

We investigated whether Rab5, a small guanosine triphosphatase that regulates early endocytic transport in different cell types is involved in the insulin-regulated endocytic pathways in adipocytes. Rab5 was detected in freshly isolated adipocytes and 3T3-L1 adipocytes, but its expression level was not markedly increased with adipocyte differentiation. After subcellular fractionation of adipocytes incubated in the absence of insulin, Rab5 was found to be abundant in plasma membrane and cytosol, but was also present in high and low density microsomes. This subcellular distribution was compatible with a role in early endocytosis. When cells were incubated with insulin, the concentration of Rab5 decreased by about 50% in the internal compartments. In contrast to Rab4, which also leaves the low density microsomes in response to insulin, Rab5 was not found in Glut4-containing vesicles purified by immunoadsorption on antibodies to Glut4. When adipocytes were treated with wortmannin, an inhibitor of phosphatidylinositol 3-kinase, the effect of insulin on Rab5 movement was not affected, whereas the insulin-induced movements of Rab4 and Glut4 were abolished. In parallel, wortmannin inhibited the increase in horseradish peroxidase uptake induced by insulin, an index of fluid phase endocytosis, but did not prevent the endocytosis of the glucose transporters. As a whole, our results suggest that Rab5 is not involved in insulin-stimulated Glut4 exocytosis. These results are compatible with the postulated role of Rab5 in the endocytotic pathway, at a step that does not require phosphatidyl-inositol 3-kinase activation.
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PMID:Insulin induces a change in Rab5 subcellular localization in adipocytes independently of phosphatidylinositol 3-kinase activation. 875 68

Rab7 has been shown to localize to late endosomes and to mediate transport from early to late endosome/lysosome in mammalian cells and in yeast. We developed a novel assay to quantify transport from early to late endosomes using the Xenopus oocyte. Oocytes were pulsed with avidin after which the oocytes were incubated to allow avidin transport to a late compartment. The oocytes were then allowed to internalize biotin-horseradish peroxidase (HRP). The oocytes were then injected with test proteins and incubated further to allow transport of biotin-HRP from early endosomes to late endosomal/lysosomal compartments. Transport was quantified by assessing the formation of HRP-biotin-avidin complexes. Injection of Rab7:wild-type (WT) and Rab7:Q67L, a GTPase defective mutant, stimulated transport. Rab5:WT had no effect. Rab7:WT-stimulated transport was inhibited by nocodazole, suggesting a role for intact microtubules. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, blocked Rab7:WT-stimulated transport, but Rab7:Q67L-stimulated transport was unaffected by the drug. Rab7:Q67L is constitutively activated and may not require phosphatidylinositol 3-kinase activity for activation. Rab7-stimulated transport requires N-ethylmaleimide-sensitive factor (NSF) activity as transport was blocked by N-ethylmaleimide and ATPase defective NSF mutants. Our results indicate that sequentially acting endocytic Rab GTPases utilize similar factors although their modes of action may be different.
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PMID:Rab7 regulates transport from early to late endocytic compartments in Xenopus oocytes. 914 16

Osteoclasts are the primary cells responsible for bone resorption. They are exposed to high ambient concentrations of inorganic phosphate (Pi) during the process of bone resorption and they possess specific Pi-transport system(s) capable of taking up Pi released by bone resorption. By immunochemical studies and PCR, we confirmed previous studies suggesting the presence of an Na-dependent Pi transporter related to the renal tubular "NaPi" proteins in the osteoclast. Using polyclonal antibodies to NaPi-2 (the rat variant), an approximately 95-kD protein was detected, localized in discrete vesicles in unpolarized osteoclasts cultured on glass coverslips. However, in polarized osteoclasts cultured on bone, immunofluorescence studies demonstrated the protein to be localized exclusively on the basolateral membrane, where it colocalizes with an Na-H exchanger but opposite to localization of the vacuolar H-ATPase. An inhibitor of phosphatidylinositol 3-kinase, wortmannin, and an inhibitor of actin cytoskeletal organization, cytochalasin D, blocked the bone-stimulated increase in Pi uptake. Phosphonoformic acid (PFA), an inhibitor of the renal NaPi-cotransporter, reduced NaPi uptake in the osteoclast. PFA also elicited a dose-dependent inhibition of bone resorption. PFA limited ATP production in osteoclasts attached to bone particles. Our results suggest that Pi transport in the osteoclast is a process critical to the resorption of bone through provision of necessary energy substrates.
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PMID:Regulation of sodium-dependent phosphate transport in osteoclasts. 923

Dopamine (DA) inhibition of Na+,K+-ATPase in proximal tubule cells is associated with increased endocytosis of its alpha and beta subunits into early and late endosomes via a clathrin vesicle-dependent pathway. In this report we evaluated intracellular signals that could trigger this mechanism, specifically the role of phosphatidylinositol 3-kinase (PI 3-K), the activation of which initiates vesicular trafficking and targeting of proteins to specific cell compartments. DA stimulated PI 3-K activity in a time- and dose-dependent manner, and this effect was markedly blunted by wortmannin and LY 294002. Endocytosis of the Na+,K+-ATPase alpha subunit in response to DA was also inhibited in dose-dependent manner by wortmannin and LY 294002. Activation of PI 3-K generally occurs by association with tyrosine kinase receptors. However, in this study immunoprecipitation with a phosphotyrosine antibody did not reveal PI 3-K activity. DA-stimulated endocytosis of Na+, K+-ATPase alpha subunits required protein kinase C, and the ability of DA to stimulate PI 3-K was blocked by specific protein kinase C inhibitors. Activation of PI 3-K is mediated via the D1 receptor subtype and the sequential activation of phospholipase A2, arachidonic acid, and protein kinase C. The results indicate a key role for activation of PI 3-K in the endocytic sequence that leads to internalization of Na+,K+-ATPase alpha subunits in response to DA, and suggest a mechanism for the participation of protein kinase C in this process.
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PMID:Phosphatidylinositol 3-kinase-mediated endocytosis of renal Na+, K+-ATPase alpha subunit in response to dopamine. 957 Dec 50

Despite the important physiological role of insulin in the regulation of ionic homeostasis, primarily mediated by the Na+/K(+)-ATPase and Na+/K+/2Cl- cotransporter, the intracellular signalling molecules mediating this effect of insulin have not been elucidated. Treatment of 3T3-L1 fibroblasts with insulin increased total 86Rb+ (K+) uptake from 0.8 +/- 0.04 to 1.02 +/- 0.05 nmol.mg-1.protein-1.min-1 (p < 0.005). These changes in K+ flux, though small, can alter the membrane potential. Uptake occurred through both the Na+/K(+)-ATPase and Na+/K+/2Cl- cotransporter and both were stimulated by insulin. Interestingly, when bumetanide was used to inhibit the Na+/K+/2Cl- cotransporter prior to insulin action, no increase in 86Rb+ uptake via the Na+/K(+)-ATPase was observed. The structurally distinct phosphatidylinositol 3-kinase inhibitors wortmannin (50-200 nmol/l) and LY294002 (50 mumol/l) attenuated both total insulin-stimulated 86Rb+ uptake as well as uptake via the Na+/K(+)-ATPase and Na+/K+/2Cl- cotransporter. Neither the inhibitor of p70.S6 kinase activation, rapamycin (30 ng/ml) nor the mitogen activated protein kinase kinase inhibitor, PD098059 (50 mumol/l), had any effect on insulin's stimulation of K+ influx. A 10 mumol/l concentration of the protein kinase C (PKC) inhibitor bisindolylmaleimide attenuated insulin action but at 1 mumol/l it was ineffective, suggesting involvement of the atypical PKC-zeta isoform. We conclude that insulin-stimulated K+ uptake in 3T3-L1 fibroblasts appears to involve concerted regulation of both the Na+/K(+)-ATPase and Na+/K+/2Cl- cotransporter and we show for the first time that this process is signalled via a pathway involving phosphatidylinositol 3-kinase and PKC-zeta.
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PMID:Insulin stimulation of K+ uptake in 3T3-L1 fibroblasts involves phosphatidylinositol 3-kinase and protein kinase C-zeta. 979 8

The objective of this study was to investigate the effects of insulin and insulin-like growth factor I on transepithelial Na(+) transport across porcine glandular endometrial epithelial cells grown in primary culture. Insulin and insulin-like growth factor I acutely stimulated Na(+) transport two- to threefold by increasing Na(+)-K(+) ATPase transport activity and basolateral membrane K(+) conductance without increasing the apical membrane amiloride-sensitive Na(+) conductance. Long-term exposure to insulin for 4 d resulted in enhanced Na(+) absorption with a further increase in Na(+)-K(+) ATPase transport activity and an increase in apical membrane amiloride-sensitive Na(+) conductance. The effect of insulin on the Na(+)-K(+) ATPase was the result of an increase in V(max) for extracellular K(+) and intracellular Na(+), and an increase in affinity of the pump for Na(+). Immunohistochemical localization along with Western blot analysis of cultured porcine endometrial epithelial cells revealed the presence of alpha-1 and alpha-2 isoforms, but not the alpha-3 isoform of Na(+)-K(+) ATPase, which did not change in the presence of insulin. Insulin-stimulated Na(+) transport was inhibited by hydroxy-2-naphthalenylmethylphosphonic acid tris-acetoxymethyl ester [HNMPA-(AM)(3)], a specific inhibitor of insulin receptor tyrosine kinase activity, suggesting that the regulation of Na(+) transport by insulin involves receptor autophosphorylation. Pretreatment with wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase as well as okadaic acid and calyculin A, inhibitors of protein phosphatase activity, also blocked the insulin-stimulated increase in short circuit and pump currents, suggesting that activation of phosphatidylinositol 3-kinase and subsequent stimulation of a protein phosphatase mediates the action of insulin on Na(+)-K(+) ATPase activation.
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PMID:Insulin stimulates transepithelial sodium transport by activation of a protein phosphatase that increases Na-K ATPase activity in endometrial epithelial cells. 1049 74

In order to understand the fundamental and putative roles of PrP(c) in the central nervous system, neuronal cell lines were established. Cells were immortalized by recombinant retrovirus vector-mediated transduction of SV40 T-antigen gene. Among these, two cell lines were selected based on their RT-PCR expressions of neuron-specific neurofilament (NF-H, NF-M) and cell morphology. These cell lines showed the properties of neuronal progenitor cells in antigenicity, morphology and responses to differentiating agents. Expression of PrP(c) was detected by immunocytochemical analysis. These cell lines responded to differentiating agents such as dibutyl cyclic AMP (dcAMP) and phorbol 12-myristate 13-acetate (PMA) before developing into neuronal-like cells. Neurite extensions were observed 20 min after incubation with the differentiating agents. Treatment with nerve growth factor (NGF) and insulin induced cell differentiation and enhanced expression of PrP gene (Prnp) mRNA and protein. The latter phenomenon was not inhibited by wortmannin, which is a specific inhibitor of phosphatidylinositol 3-kinase. These results suggest that PrP(c) plays an important role in the differentiation-mediated classic signaling pathway of neuronal cell.
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PMID:Enhanced expression of cellular prion protein gene by insulin or nerve growth factor in immortalized mouse neuronal precursor cell lines. 1067 79

The time- and dose-dependent effects of wortmannin on transepithelial electrical resistance (Rte) and forskolin-stimulated chloride secretion in T84 monolayer cultures were studied. In both instances, maximal effects developed over 2 h and were stable thereafter. Inhibition of forskolin-stimulated chloride secretion, as measured by the short-circuit current (Isc) technique, had an IC50 of 200-500 nM, which is 100-fold higher than for inhibition of phosphatidylinositol 3-kinase (PI3K), but similar to the IC50 for inhibition of myosin light chain kinase (MLCK) and mitogen-activated protein kinases (MAPK). Previous work demonstrated that 500 nM wortmannin did not inhibit the cAMP activation of apical membrane chloride channels. We show here that 500 nM wortmannin has no affect on basolateral Na/K/2Cl-cotransporter activity, but inhibits basolateral membrane Na/K-ATPase activity significantly. The MLCK inhibitors ML-7 and KT5926 were without affect on forskolin-stimulated Isc. Similarly, the p38- and MEK-specific MAPK inhibitors SB203580 and PD98059 did not reduce forskolin-stimulated Isc. In contrast, the non-specific MAPK inhibitor apigenin reduced forskolin-stimulated Isc and basolateral membrane Na/K-ATPase activity similar to wortmannin. In isolated membranes from T84 cells, wortmannin did not inhibit Na/K-ATPase enzymatic activity directly. We conclude that one or more MAPK may regulate the functional expression of basolateral membrane Na/K-ATPase by controlling the abundance of enzyme molecules in the plasma membrane.
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PMID:Wortmannin inhibition of forskolin-stimulated chloride secretion by T84 cells. 1093 May 8

Mechanisms responsible for increased jejunal transport rates observed in tissues treated with orally administered insulin-like growth factor-I (IGF-I) were studied in 5-day-old colostrum-deprived piglets. Human recombinant IGF-I (3.5 mg. kg(-1). day(-1)) or control vehicle was given orogastrically for 4 days. Disaccharidase activity, fructose uptake, and Na+-glucose cotransporter SGLT-1 protein abundance were similar between groups. Oral IGF-I produced greater rates of enterocyte Na+-K+-ATPase activity with no significant differences in Na+-K+-ATPase abundance. Cellular mechanisms responsible for transport changes were studied in Ussing chambers. In control tissues, the presence of IGF-I in mucosal solutions increased basal short-circuit current (I(sc)), potential difference, D-glucose-stimulated I(sc), and Na+-K+-ATPase activity; these changes were abolished by preincubation of tissues with wortmannin, a phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor. The results suggest that the effect of IGF-I on jejunal ion and nutrient transport involves activation of PI 3-kinase and stimulation of Na+-K+-ATPase activity in enterocytes.
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PMID:Involvement of PI 3-kinase in IGF-I stimulation of jejunal Na+-K+-ATPase activity and nutrient absorption. 1120 44

We previously demonstrated that expression of both the alpha1- and alpha2-subunits of Na+-K+-ATPase is elevated after a 2- to 4-day cyclic stretch in aortic smooth muscle cells. In this study, we determined the effect of short-term (2-30 min) cyclic stretch on the activity of the Na pump and investigated possible mechanisms that may be involved in the action of stretch. Na pump activity was significantly increased above the baseline activity between 2 and 30 min of stretch. This effect of stretch was reversible within 1 h. Intracellular Na was also elevated at corresponding time points. Blocking the entry of Na with Gd and amiloride did not affect the stretch-induced increase in Na pump activity. Inhibition of protein kinase A (PKA) activity attenuated the effect of stretch on the Na pump. Furthermore, inhibition of polymerization of actin and phosphatidylinositol 3-kinase (PI3K) activity prevented the action of stretch on Na pump activity. We conclude that the stimulation of the Na pump in response to cyclic stretch requires the integrity of the actin cytoskeleton as well as the activity of PI3K, which has a role in intracellular vesicular trafficking. PKA may also be involved in this effect of stretch on Na pump.
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PMID:Effect of short-term cyclic stretch on sodium pump activity in aortic smooth muscle cells. 1166 68


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