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:2.7.1.1 (hexokinase)
5,274 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Physiologically, a postprandial glucose rise induces metabolic signal sequences that use several steps in common in both the pancreas and peripheral tissues but result in different events due to specialized tissue functions. Glucose transport performed by tissue-specific glucose transporters is, in general, not rate limiting. The next step is phosphorylation of glucose by cell-specific hexokinases. In the beta-cell, glucokinase (or hexokinase IV) is activated upon binding to a pore protein in the outer mitochondrial membrane at contact sites between outer and inner membranes. The same mechanism applies for hexokinase II in skeletal muscle and adipose tissue. The activation of hexokinases depends on a contact site-specific structure of the pore, which is voltage-dependent and influenced by the electric potential of the inner mitochondrial membrane. Mitochondria lacking a membrane potential because of defects in the respiratory chain would thus not be able to increase the glucose-phosphorylating enzyme activity over basal state. Binding and activation of hexokinases to mitochondrial contact sites lead to an acceleration of the formation of both ADP and glucose-6-phosphate (G-6-P). ADP directly enters the mitochondrion and stimulates mitochondrial oxidative phosphorylation. G-6-P is an important intermediate of energy metabolism at the switch position between glycolysis, glycogen synthesis, and the pentose-phosphate shunt. Initiated by blood glucose elevation, mitochondrial oxidative phosphorylation is accelerated in a concerted action coupling glycolysis to mitochondrial metabolism at three different points: first, through NADH transfer to the respiratory chain complex I via the malate/aspartate shuttle; second, by providing FADH2 to complex II through the glycerol-phosphate/dihydroxy-acetone-phosphate cycle; and third, by the action of hexo(gluco)kinases providing ADP for complex V, the ATP synthetase. As cytosolic and mitochondrial isozymes of creatine kinase (CK) are observed in insulinoma cells, the phosphocreatine (CrP) shuttle, working in brain and muscle, may also be involved in signaling glucose-induced insulin secretion in beta-cells. An interplay between the plasma membrane-bound CK and the mitochondrial CK could provide a mechanism to increase ATP locally at the KATP channels, coordinated to the activity of mitochondrial CrP production. Closure of the KATP channels by ATP would lead to an increase of cytosolic and, even more, mitochondrial calcium and finally to insulin secretion. Thus in beta-cells, glucose, via bound glucokinase, stimulates mitochondrial CrP synthesis. The same signaling sequence is used in the opposite direction in muscle during exercise when high ATP turnover increases the creatine level that stimulates mitochondrial ATP synthesis and glucose phosphorylation via hexokinase. Furthermore, this cytosolic/mitochondrial cross-talk is also involved in activation of muscle glycogen synthesis by glucose. The activity of mitochondrially bound hexokinase provides G-6-P and stimulates UTP production through mitochondrial nucleoside diphosphate kinase. Pathophysiologically, there are at least two genetically different forms of diabetes linked to energy metabolism: the first example is one form of maturity-onset diabetes of the young (MODY2), an autosomal dominant disorder caused by point mutations of the glucokinase gene; the second example is several forms of mitochondrial diabetes caused by point and length mutations of the mitochondrial DNA (mtDNA) that encodes several subunits of the respiratory chain complexes. Because the mtDNA is vulnerable and accumulates point and length mutations during aging, it is likely to contribute to the manifestation of some forms of NIDDM.(ABSTRACT TRUNCATED)
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PMID:Mitochondria and diabetes. Genetic, biochemical, and clinical implications of the cellular energy circuit. 854 53

The presence of the P2Y2 (P(2U)-purinergic) receptor on the apical surface of airway tissue raises the possibility that aerosolized UTP might be used therapeutically to induce Cl- secretion in individuals with cystic fibrosis. However, the duration of the effects of UTP may be limited by enzymatic degradation. We therefore have analyzed the metabolism of UTP and its metabolite UDP on polarized human nasal epithelium (HNE), and have compared the pharmacological activities of these two uridine nucleotides. HPLC analysis of medium bathing the mucosal surface of HNE cells revealed the presence of an ecto-nucleotidase(s) that hydrolyzed [3H]UTP and [3H]UDP with t1/2 values (at 1 microM nucleotide) of 14 and 27 min, respectively. An ecto-nucleoside diphosphokinase activity also was observed, which promoted conversion of [3H]UDP into [3H]UTP in the presence of ATP. The effects of UDP on [3H]inositol phosphate accumulation, intracellular calcium levels ([Ca2+]i), and Cl- secretory rates (I(Cl-)) were quantitated in HNE cells in the presence of hexokinase and glucose to ensure that no UTP (or ATP) contaminated UDP solutions during the assays. Although UDP does not activate the human P2Y2 receptor, mucosal addition of UDP promoted [3H]inositol phosphate accumulation with an EC50 of 190 nM. Mucosal addition of UTP stimulated [3H]inositol phosphate accumulation with an EC50 of 280 nM. The maximal effects of mucosal UDP on [3H]inositol phosphate, [Ca2+]i, and I(Cl-) responses were approximately one-half of those observed with mucosal UTP. Serosal application of UTP promoted a 50% greater [3H]inositol phosphate and calcium response than did mucosal application of UTP. In contrast, UDP had no effect when added to the serosal medium. Repetitive mucosal applications of UDP to HNE cells resulted in a progressive loss, i.e., desensitization, of the [Ca2+]i and I(Cl-) response to UDP, whereas the corresponding responses to UTP remained unchanged. Our results provide evidence for the existence of a UDP receptor on HNE cells that is pharmacologically distinct from the P2Y2 receptor. The relative stability of UDP on the airway surface and the apparent predominant mucosal expression of this putative UDP receptor make it a potential target for cystic fibrosis treatment.
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PMID:UDP activates a mucosal-restricted receptor on human nasal epithelial cells that is distinct from the P2Y2 receptor. 912 41

ATP is released from most cell types and functions as an extracellular signaling molecule through activation of members of the two large families of P2X and P2Y receptors. Although three mammalian P2Y receptors have been cloned that are selectively activated by uridine nucleotides, direct demonstration of the release of cellular UTP has not been reported. Pharmacological studies of the P2Y4 receptor expressed in 1321N1 human astrocytoma cells indicated that this receptor is activated by UTP but not by ATP. Mechanical stimulation of 1321N1 cells also resulted in release of a molecule that markedly activated the expressed P2Y4 receptor. This nucleotide was shown to be UTP by two means. First, high performance liquid chromatography analysis of the medium from [33P]H3PO4-loaded 1321N1 cells illustrated that mechanical stimulation resulted in a large increase in a radioactive species that co-eluted with authentic UTP. This species was degraded by incubation with the nonspecific pyrophosphohydrolase apyrase or with hexokinase and was specifically lost by incubation with the UTP-specific enzyme UDP-glucose pyrophosphorylase. Second, a sensitive assay that quantitates UTP mass at low nanomolar concentrations was devised based on the nucleotide specificity of UDP-glucose pyrophosphorylase. Using this assay, mechanical stimulation of 1321N1 cells was shown to result in an increase of medium UTP levels from 2.6 to 36.4 pmol/10(6) cells within 2 min. This increase was paralleled by a similar augmentation of extracellular ATP levels. A calcein-based fluorescence quenching method was utilized to confirm that none of the increases in medium nucleotide levels could be accounted for by cell lysis. Taken together, these results directly demonstrate the mechanically induced release of UTP and illustrate the efficient coupling of this release to activation of P2Y4 receptors.
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PMID:Direct demonstration of mechanically induced release of cellular UTP and its implication for uridine nucleotide receptor activation. 930 92

A common characteristic of tumor cells is the constant overexpression of glycolytic and glutaminolytic enzymes. In tumor cells the hyperactive hexokinase and the partly inactive pyruvate kinase lead to an expansion of all phosphometabolites from glucose 6-phosphate to phosphoenolpyruvate. In addition to the glycolytic phosphometabolites, synthesis of their metabolic derivatives such as P-ribose-PP, NADH, NADPH, UTP, CTP, and UDP-N-acetyl glucosamine is also enhanced during cell proliferation. Another phosphometabolite derived from P-ribose-PP, AMP, inhibits cell proliferation. The accumulation of AMP inhibits both P-ribose-PP-synthetase and the increase in concentration of phosphometabolites derived from P-ribose-PP. In cells with low glycerol 3-phosphate and malate-aspartate shuttle capacities the inhibition of the lactate dehydrogenase by low NADH levels leads to an inhibition of glycolytic ATP production. Several tumor-therapeutic drugs reduce NAD and NADH levels, thereby inhibiting glycolytic energy production. The role of AMP, NADH, and NADPH levels in the success of chemotherapeutic treatment is discussed.
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PMID:The role of phosphometabolites in cell proliferation, energy metabolism, and tumor therapy. 938 92

1. Previous studies have shown that ATP and UTP are able to stimulate phospholipase C (PLC) and proliferation in cultured aortic smooth muscle cells. Here we set out to characterize the receptor responsible, and investigate a possible role for p42 and p44 mitogen activated protein kinase (MAPK) in the proliferative response. 2. The phospholipase C response of spontaneously hypertensive rat (SHR) derived aortic smooth muscle cells in culture showed that the response to ATP was partial compared to the response to UTP. 3. Further studies characterized the responses of the SHR derived cells. UTP was the only full agonist with the SHR cells; UDP gave a partial response while ADP, 2-methythio-ATP and alpha,beta-methylene ATP were essentially ineffective. The response to UDP was almost lost in the presence of hexokinase, consistent with this being due to extracellular conversion to UTP. These observations are inconsistent with the response being mediated by either P2Y1 or P2Y6 receptors. 4. When increasing concentrations of ATP were present with a maximally effective concentration of UTP, the size of the response diminished, consistent with UTP and ATP acting at a single population of receptors for which ATP was a partial agonist. This is inconsistent with a response mainly at P2Y2 receptors. 5. 1321N1 cells transfected with human P2Y4 receptors gave a similar agonist response profile, with ATP being partial compared to UTP, loss of response to UDP with hexokinase treatment, and with the response to UTP diminishing in the presence of increasing concentrations of ATP. 6. Use of the reverse transcriptase-polymerase chain reaction confirmed the presence of mRNA encoding P2Y4 receptors in SHR derived vascular smooth muscle cells. Transcripts for P2Y2, P2Y4 and P2Y6 receptors, but not P2Y1 receptors, were detected. 7. Stimulation of SHR derived cells with UTP enhanced the tyrosine phosphorylation of both p42 and p44 MAPK, and the incorporation of [3H]-thymidine into DNA. Both these responses were diminished in the presence of an inhibitor of activation of MAPK. 8 These results lead to the conclusion that in SHR derived cultured aortic smooth muscle cells, PLC responses to extracellular UTP and ATP are predominantly at P2Y4 receptors, and suggest that these receptors are coupled to mitogenesis via p42/p44 MAPK.
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PMID:Evidence that P2Y4 nucleotide receptors are involved in the regulation of rat aortic smooth muscle cells by UTP and ATP. 969 Aug 62

In cystic fibrosis, the mutation of the CFTR protein causes reduced transepithelial Cl(-) secretion. As recently proposed, beside its role of Cl(-) channel, CFTR may regulate the activity of other channels such as a Ca(2+)-activated Cl(-) channel. Using a calcium imaging system, we show, in adenovirus-CFTR infected Chinese Hamster Ovary (CHO) cell monolayers, that CFTR can act as a regulator of intracellular [Ca(2+)](i) ([Ca(2+)](i)), involving purino-receptors. Apical exposure to ATP or UTP produced an increase in ([Ca(2+)](i) in noninfected CHO cell monolayers (CHO-WT), in CHO monolayers infected with an adenovirus-CFTR (CHO-CFTR) or infected with an adenovirus-LacZ (CHO-LacZ). The transient [Ca(2+)](i) increase produced by ATP or UTP could be mimicked by activation of CFTR with forskolin (20 microm) in CHO-CFTR confluent monolayers. However, forskolin had no significant effect on [Ca(2+)](i) in noninfected CHO-WT or in CHO-LacZ cells. Pretreatment with purino-receptor antagonists such as suramin (100 microm) or reactive blue-2. (100 microm), and with hexokinase (0.28 U/mg) inhibited the [Ca(2+)](i) response to forskolin in CHO-CFTR infected cells. Taken together, our experiments provide evidence for purino-receptor activation by ATP released from the cell and regulation of [Ca(2+)](i) by CFTR in CHO epithelial cell membranes.
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PMID:Regulation of intracellular Ca(2+) by CFTR in Chinese hamster ovary cells. 1050 33

Dilatory responses of extracellular nucleotides were examined in the precontracted isolated rat mesenteric artery. Dilatation mediated by endothelium-derived hyperpolarising factor (EDHF) was studied in the presence of Nomega-nitro-L-arginine (L-NOARG) and indomethacin, and was most potently induced by the selective P2Y(1) receptor agonist adenosine 5'-O-thiodiphosphate (ADPbetaS), while 2-methylthioadenosine triphosphate (2-MeSATP) and adenosine triphosphate (ATP) were almost inactive. However, after P2X receptor desensitisation (with alphabeta-methylene-adenosine triphosphate, alphabeta-MeATP), 2-MeSATP and ATP potently stimulated EDHF-mediated dilatation. This can be explained by simultaneous activation of endothelial P2Y receptors that release EDHF, and depolarising P2X receptors on smooth muscle cells. Uridine triphosphate (UTP) also induced potent dilatation, suggesting EDHF release via P2Y(2)/P2Y(4) receptors. Uridine diphosphate (UDP) had only minor dilatory effects, and when pretreated with hexokinase it was almost inactive, suggesting a minor role for P2Y(6) receptors. The nitric oxide (NO) mediated dilatation was studied in the presence of charybdotoxin, apamin and indomethacin. ADPbetaS, 2-MeSATP, ATP and UTP were all potent relaxant agonists suggesting NO release via P2Y(1) and P2Y(2)/P2Y(4) receptors, while UDP was much less potent and efficacious. P2X receptor desensitisation had only minor effect on the NO-mediated dilatations. In conclusion, both EDHF and NO-mediated dilatation can be induced by activation of P2Y(1) and P2Y(2)/P2Y(4) receptors. P2X receptor stimulation of smooth muscle cells selectively counteracts the dilatory effect of EDHF.
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PMID:P2X receptors counteract the vasodilatory effects of endothelium derived hyperpolarising factor. 1070 21

1. Using equivalent short circuit current (ISC) measurements we examined the effect of extracellular ATP on transepithelial ion transport in M-1 mouse cortical collecting duct cells. Apical addition of ATP produced a rapid transient peak increase in ISC. This was followed by a fall below basal ISC due to a reduction in the amiloride-sensitive ISC component. 2. The ATP-induced ISC increase was preserved in the presence of apical amiloride while it was reduced in the absence of extracellular Cl- and in the presence of the apical Cl- channel blockers diphenylamine-2-carboxylic acid (DPC, 1 mM), DIDS (300 microM) and niflumic acid (100 microM). 3. The stimulatory effect of apical ATP on ISC was concentration dependent with an EC50 of about 0.6 microM. Basolateral ATP elicited a similar ISC response. Experiments using the ATP scavenger hexokinase demonstrated that the ATP effects were elicited via separate apical and basolateral receptors. 4. ATP and UTP applied to either the apical or the basolateral bath equi-potently stimulated ISC while 'purified' ADP and UDP had no effect consistent with P2Y2 purinoceptors, the expression of which was confirmed using RT-PCR. 5. Intracellular calcium concentration ([Ca2+]i) measurements using fura-2 demonstrated that ATP and UTP elicited a rise in [Ca2+]i with EC50 values of 1.1 and 0.6 microM, respectively. The shape and time course of the calcium response were similar to those of the ISC response. The peak ISC response was preserved in the nominal absence of extracellular calcium but was significantly reduced in cells pre-incubated with the calcium chelator BAPTA AM. 6. We conclude that in M-1 cells extracellular ATP reduces amiloride-sensitive Na+ absorption and stimulates Cl- secretion via calcium-activated Cl- channels through activation of P2Y2 purinoreceptors located in the apical and basolateral membrane.
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PMID:ATP stimulates Cl- secretion and reduces amiloride-sensitive Na+ absorption in M-1 mouse cortical collecting duct cells. 1074 85

A strong and coordinated upregulation of the glycolytic, glutaminolytic and pentose phosphate pathway enzymes occurs during the onset of lactation in the normal mouse mammary gland. Induction of apoptosis by removing the pups led to an inactivation of the same enzymes with different time courses. While the ATP-consuming glycolytic 6-phosphofructo 1-kinase and mitochondrial bound hexokinase still remained high on days one and two of involution, the ATP-regenerating pyruvate kinase was immediately reduced. The enzymes of the pentose phosphate and glutaminolytic pathway were inactivated on the first two days of involution. In accordance with such an inactivation of the enzymes ATP, GTP, UTP, ADP, NAD NADH and lactate concentrations decreased. The synthetic product of UTP, UDP-N-acetylglucosamine, increased. AMP was found in the milk, not in the epithelial cells. The inactivation of the enzymes was caused by partial proteolysis or by a loss of the intact proteins from the cytosol without signs of proteolysis.
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PMID:Energy metabolism in the involuting mammary gland. 1075 39

In cystic fibrosis airway epithelia, mutation of the CFTR protein causes a reduced response of Cl(-) secretion to secretagogues acting via cAMP. Using a Ca(2+) imaging system, the hypothesis that CFTR activation may permit ATP release and regulate [Ca(2+)](i) via a receptor-mediated mechanism, is tested in this study. Application of external nucleotides produced a significant increase in [Ca(2+)](i) in normal (16HBE14o(-) cell line and primary lung culture) and in cystic fibrosis (CFTE29o(-) cell line) human airway epithelia. The potency order of nucleotides on [Ca(2+)](i) variation was UTP >> ATP > UDP > ADP > AMP > adenosine in both cell types. The nucleotide [Ca(2+)](i) response could be mimicked by activation of CFTR with forskolin (20 microm) in a temperature-dependent manner. In 16HBE14o(-) cells, the forskolin-induced [Ca(2+)](i) response increased with increasing temperature. In CFTE29o(-) cells, forskolin had no effect on [Ca(2+)](i) at body temperature-forskolin-induced [Ca(2+)](i) response in CF cells could only be observed at low experimental temperature (14 degrees C) or when cells were cultured at 26 degrees C instead of 37 degrees C. Pretreatment with CFTR channel blockers glibenclamide (100 microm) and DPC (100 microm), with hexokinase (0.5 U/mg), and with the purinoceptor antagonist suramin (100 microm), inhibited the forskolin [Ca(2+)](i) response. Together, these results demonstrate that once activated, CFTR regulates [Ca(2+)](i) by mediating nucleotide release and activating cell surface purinoceptors in normal and CF human airway epithelia.
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PMID:CFTR regulation of intracellular calcium in normal and cystic fibrosis human airway epithelia. 1101 59


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