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)

The ATP-sensitive, inwardly rectifying K+ channel, ROMK, has been suggested to be the low-conductance ATP-sensitive K+ channel identified in apical membranes of mammalian renal thick ascending limb (TAL) and cortical collecting duct (CCD). Mutations in the human ROMK gene (KIR 1.2) have been identified in kindreds with neonatal Bartter's syndrome. In the present study, we generated polyclonal antibodies raised against both a COOH-terminal (amino acids 252-391) ROMK-maltose binding protein (MBP) fusion protein and an NH2-terminal (amino acids 34-49) ROMK peptide. Affinity-purified anti-ROMK COOH-terminal antibody detected the 45-kDa ROMK protein in kidney tissues and HEK-293 cells transfected with ROMK1 cDNA. The antibody also recognized 85- to 90-kDa proteins in kidney tissue; these higher molecular weight proteins were abolished by immunoabsorption with ROMK-MBP fusion protein and were also detected on Western blots using anti-ROMK NH2-terminal antibody. Immunofluoresence studies using anti-ROMK COOH-terminal antibody showed intense apical staining along the loop of Henle and distal nephron; staining with preimmune and immunoabsorbed serum was negative. When colocalized with distal nephron markers [the thiazide-sensitive cotransporter (rTSC1), the bumetanide-sensitive cotransporter (rBSC1), the vacuolar type H(+)-ATPase, and neuronal nitric oxide synthase (NOS I)], the ROMK protein was found primarily at the apical border of cells in the TAL, macula densa, distal convoluted tubule, and connecting tubule. Within the CCD, the ROMK protein was expressed in principal cells and was absent from intercalated cells. The tubule localization and polarity of ROMK staining are consistent with the distribution of ROMK mRNA and provide more support for ROMK being the low-conductance K+ secretory channel in the rat distal nephron.
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PMID:Localization of the ROMK protein on apical membranes of rat kidney nephron segments. 937 37

The present study examined the relationship between an important energy-generating enzyme (cytochrome oxidase; CO), a key energy-consuming enzyme (Na+ K+ ATPase) and neurochemicals associated with excitatory glutamatergic synapses (NMDAR1 and neuronal nitric oxide synthase, nNOS) in the adult macaque retina. Polyclonal antibodies against neuronal nitric oxide synthase and N-methyl-D-aspartate receptor subunit I were generated for immunohistochemical examination and labeled sites not previously reported were found. We have also isolated cDNAs for cytochrome oxidase subunits III (mitochondrial-encoded) and IV (nuclear-encoded), as well as for a fragment of neuronal nitric oxide synthase, from a human cDNA library. The distributions of mRNAs of these genes were analyzed by in situ hybridization. We found that three or more of the markers examined coexisted in a number of sites: (a) In the inner segments of photoreceptors, high energy demand for maintaining the dark current was placed by Na+ K+ ATPase. This was partially met by ATP-generating enzymes such as CO. Neuronal NOS was also present there for the synthesis of NO and the cascading event leading to the generation of cGMP and the gating of channels for visual transduction. (b) Both the outer and inner plexiform layers had detectable amounts of all four markers, although the levels varied among them. This was most likely due to the presence of depolarizing glutamatergic synapses arising from photoreceptors and bipolar cells and such synaptic events were energy-demanding. The involvement of NMDA receptors and nNOS in these synaptic layers is strongly implicated in the present study. (c) All four markers were present in the majority of retinal ganglion cells, with some inherent heterogeneity related to intensity and size. Retinal ganglion cells are known to receive excitatory synapses from glutamatergic bipolar cells and are themselves highly active. The presence of both NMDAR1 and nNOS in these cells were verified in the present study and the energy demands related to these synaptic activities were necessarily high. Thus, active ion transporting functions related to synaptic or non-synaptically induced repolarization from the basis for an interrelationship between the neurochemicals/enzymes studied. Finally, (d) all four markers and the gene expression of CO and nNOS in the macaque retina were regulated by neuronal activity.
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PMID:Neurochemical organization of the macaque retina: effect of TTX on levels and gene expression of cytochrome oxidase and nitric oxide synthase and on the immunoreactivity of Na+ K+ ATPase and NMDA receptor subunit I. 966 11

The pathogenesis of diabetic neuropathy remains unclear, although several factors have been implicated in its pathogenesis. We have examined possible roles of decreased production of nitric oxide, ion channel dysfunction and decreased capacity of nerve regeneration. STZ-induced diabetic rats showed decreases in nociceptive threshold and NADPH-diaphorase positive neurons, nNOS level and cGMP content of DRG at 12 weeks after induction of diabetes. The rats injected by L-NAME, potent nNOS inhibitor, showed decreased nociceptive threshold, although D-NAME, inactive in nNOS inhibition, did not. These results suggest that decreased NO production might be involved in hyperalgesia in diabetic rats. Both hyperglycemia and decreased Na/K-ATPase activity are thought to be characteristic features of diabetic neuropathy. To investigate the presence of ion channel abnormality in diabetic nerves, a Vaseline-gap voltage clamp technique was applied for a single myelinated fibers under 30 mM high glucose plus 0.1 mM ouabain. Since K current was increased, a Ca activated K channel blocker was applied and this increase was shown to be suppressed. Furthermore, Ca channel blockers all suppressed increased K currents, suggesting that the condition induced an increase of Ca influx, thereby increasing Ca activated K currents through K channels. The data are important in that diabetic condition may induce both Ca influx, leading to nerve degeneration, and increased K current, resulting in decreased nerve conduction. Nerve regeneration has been known to be disturbed in diabetic condition. We have shown a decrease in nerve elongation rate in diabetic rats after crush of sciatic nerve, although this decrease was not ameliorated by ARI. Furthermore, Wallerian degeneration was shown to be delayed in diabetic nerves, leading to delayed nerve regeneration. Hyperphosphorylation of both medium and high molecular weight neurofilaments that might be induced by protein kinases including CDK 5 may be involved in the mechanism.
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PMID:[New trend in pathogenesis of diabetic neuropathy]. 1037 17

Our previous studies showed a differential distribution of the glutamatergic terminals in cytochrome oxidase-rich and -poor regions of the visual cortex. The NMDA type of glutamate receptors have been proposed to be involved in the activation of nitric oxide synthase to produce nitric oxide, the neurotransmitter. In the present study, we hypothesized that the expressions of glutamate receptor, NMDA receptors (NMDAR1) and neuronal nitric oxide synthase (nNOS) were colocalized and were also correlated with that of cytochrome oxidase (CO) in a subset of neurons. We used primary cultures of postnatal rat visual cortical neurons as a model system, so that we could examine both the somatic and dendritic expressions of these neurochemicals in individual neurons. We found a difference in the sequence of developmental expressions of NMDAR1, nNOS, CO, and Na(+)/K(+) ATPase. Triple labeling showed that all nNOS-positive neurons were immunoreactive for NMDAR1, and a subpopulation of them had high CO activity. The expression of NMDAR1 was positively correlated with CO activity. This is consistent with our previous finding that CO activity is strongly governed by excitatory glutamatergic synapses. After 40 hours of depolarizing potassium chloride treatment, CO activity was increased, and NMDAR1and nNOS levels were up-regulated in parallel. One week of tetrodotoxin significantly decreased the expression of NMDAR1, nNOS, and CO activity. Our results demonstrate that NMDA receptors and nNOS do co-exist in a subset of neurons that have high CO activity and their expressions are under the control of neuronal activity.
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PMID:Expression and regulation of NMDA receptor subunit R1 and neuronal nitric oxide synthase in cortical neuronal cultures: correlation with cytochrome oxidase. 1080 Feb 3

An immortal nonhormone-producing cell line with a characteristic star-shaped morphology, named Tpit/F1, was derived from an anterior pituitary gland of a temperature-sensitive large T antigen transgenic mouse. To characterize Tpit/F1 cells, we performed cytological studies, which revealed that Tpit/F1 cells express the messenger RNAs of neruonal nitric oxide (NO) synthase, S-100 protein, basic fibroblast growth factor, and pituitary-restricted transcription factor. The Tpit/F1 cells response to pituitary adenylate cyclase-activating peptide comprised the stimulated secretion of interleukin-6. Furthermore, glucocorticoids stimulate glutamine synthase production by Tpit/F1 cells. Considering these cytological characteristics together with their morphology, we deduced that Tpit/F1 cells are derived from pituitary folliculo-stellate (FS) cells. Our cytophysiological analyses of Tpit/F1 cells revealed that intracellular Ca2+ increased dose dependently on ATP administration (0-100 microM), and that this effect did not require the presence of extracellular Ca2+ and was not abolished by treatment with gadolinium, a Ca2+ channel blocker. The ATP-induced increase in intracellular Ca2+ ([Ca2+]i) was completely abolished by treatment with the Ca2+-adenosine triphosphatase (Ca2+-ATPase) inhibitor thapsigargin, which suggests that ATP increases [Ca2+]i by mobilizing internally stored Ca2+ followed by an influx of Ca2+. Moreover, UTP was equipotent with ATP in causing the [Ca2+]i increase in Tpit/F1 cells. Also, the Ca2+ response was prevented by the phospholipase C inhibitor, U-73122, but not by its inactive analog, U-73343. From these results we therefore concluded that ATP acts on Tpit/F1 cells via P2Y2-purinoceptors. Interestingly, both neuronal nitric oxide synthase messenger RNA and NO secretion were increased by ATP administration (10 and 100 microM). These results suggest the biological significance of the topological colocalization of FS cells and endocrine cells. Namely, ATP is cosecreted with hormones from endocrine cells and stimulates NO production by FS cells, and the released NO may regulate neighboring endocrine cell and blood vessels.
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PMID:Cytological characterization of a pituitary folliculo-stellate-like cell line, Tpit/F1, with special reference to adenosine triphosphate-mediated neuronal nitric oxide synthase expression and nitric oxide secretion. 1101 14

We evaluated the anti-inflammatory and neuroprotective effects of the selective neuronal nitric oxide synthase inhibitor 7-nitroindazole and aminoguanidine, which predominantly inhibits inducible nitric oxide synthase, during the early phase of experimental bacterial meningitis in the newborn piglet. Meningitis was induced by intracisternal injection of 10(8) colony-forming units of Escherichia coli in 100 microl of saline. 7-Nitroindazole significantly attenuated the meningitis-induced acute inflammatory responses such as increased intracranial pressure, decreased cerebrospinal fluid (CSF) glucose concentration, and CSF leukocytosis at 2 h. However, meningitis-induced CSF leukocytosis at 4 h and increased CSF lactate and tumor necrosis factor alpha levels were not significantly attenuated. Reduced cerebral cortical cell membrane Na(+),K(+)-ATPase activity and increased lipid peroxidation products, indicative of meningitis-induced brain cell membrane dysfunction, were also significantly improved with 7-nitroindazole treatment. In contrast, although aminoguanidine significantly attenuated the increase in the CSF tumor necrosis factor alpha level, it failed to attenuate the acute inflammation and the ensuing brain injury in bacterial meningitis. In summary, 7-nitroindazole, but not aminoguanidine, significantly attenuated the acute inflammatory responses and brain injury during the early phase of neonatal bacterial meningitis.
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PMID:7-Nitroindazole, but not aminoguanidine, attenuates the acute inflammatory responses and brain injury during the early phase of Escherichia coli meningitis in the newborn piglet. 1147 50

Nitric oxide (NO*) is produced endogenously from NOS isoforms bound to sarcolemmal (SL) and sarcoplasmic reticulum (SR) membranes. To investigate whether locally generated NO* directly affects the activity of enzymes mediating ion active transport, we studied whether knockout of selected NOS isoforms would affect the functions of cardiac SL (Na+ + K+)-ATPase and SR Ca2+-ATPase. Cardiac SL and SR vesicles containing either SL (Na+ + K+)-ATPase or SR Ca2+-ATPase were isolated from mice lacking either nNOS or eNOS, or both, and tested for enzyme activities. Western blot analysis revealed that absence of single or double NOS isoforms did not interrupt the protein expression of SL (Na+ + K+)-ATPase and SR Ca2+-ATPase in cardiac muscle cells. However, lack of NOS isoforms in cardiac muscle significantly altered both (Na+ + K+)-ATPase activity and SR Ca2+-ATPase function. Our experimental results suggest that disrupted endogenous NO* production may change local redox conditions and lead to an unbalanced free radical homeostasis in cardiac muscle cells which, in turn, may affect key enzyme activities and membrane ion active transport systems in the heart.
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PMID:Lack of nitric oxide synthase depresses ion transporting enzyme function in cardiac muscle. 1207 80

Nitric oxide (NO.) generated from nitric oxide synthase (NOS) isoforms bound to cellular membranes may serve to modulate oxidative stresses in cardiac muscle and thereby regulate the function of key membrane-associated enzymes. Ischemia is known to inhibit the function of sarcolemmal enzymes, including the (Na+ + K+)-ATPase, but it is unknown whether concomitant injury to sarcolemma (SL)-associated NOS isoforms may contribute to this process by reducing the availability of locally generated NO. Here we report that nNOS, as well as eNOS (SL NOSs), are tightly associated with cardiac SL membranes in several different species. In isolated perfused rat hearts, global ischemia caused a time-dependent irreversible injury to cardiac SL NOSs and a disruption of SL NO. generation. Pretreatment with low concentrations of the NO. donor 1-hydroxy-2-oxo-3-(N-3-methyl-aminopropyl)-3-methyl-1-triazene (NOC-7) markedly protected both SL NOS and (Na+ + K+)-ATPase functions against ischemia-induced inactivation. Moreover, ischemia impaired SL Na+/K+ binding, and NOC-7 significantly prevented ischemic injury to the ion binding sites on (Na+ + K+)-ATPase. These novel findings indicate that NO. can protect cardiac SL NOSs and (Na+ + K+)-ATPase against ischemia-induced inactivation and suggest that locally generated NO. may serve to regulate SL Na+/K+ ion active transport in the heart.
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PMID:Nitric oxide protects cardiac sarcolemmal membrane enzyme function and ion active transport against ischemia-induced inactivation. 1290 95

We investigated the relationship between nitric oxide (NO) and Na(+),K(+)-ATPase (NKA) in the gill of anadromous Atlantic salmon. Cells containing NO-producing enzymes were revealed by means of nitric oxide synthase (NOS) immunocytochemistry and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) histochemistry, which can be used as an indicator of NOS activity, i.e. NO production. Antibodies against the two constitutive NOS isoforms, neuronal and endothelial NOS, both produced immunoreactivity restricted to large cells at the base and along the secondary lamellae. NADPHd-positive cells showed a corresponding distribution. Antibodies against the inducible NOS isoform only labeled small cells located deep in the filament. Using in situ hybridization and NKA immunoreactivity, cells expressing Na(+),K(+)-ATPase alpha-subunit mRNA were found to have a similar distribution to the NOS cells. Double labeling for NOS immunoreactivity and NKA alpha-subunit mRNA revealed cellular colocalization of NKA alpha-subunit mRNA and nNOS protein in putative chloride cells at the base of the lamellae and interlamellar space. Along the lamellae, some NOS- or NKA-immunoreactive cells possessed a relatively lower expression of NKA alpha-subunit mRNA in smolts. A clear increase in NADPHd staining in the gill was demonstrated from parr to smolt. The regulatory role of NO on gill NKA activity was studied in vitro using sodium nitroprusside (SNP; 1 mmol l(-1)) and PAPA-NONOate (NOC-15; 0.5 mmol l(-1)) as NO donors. Both SNP and NOC-15 inhibited gill NKA activity by 30% when compared to controls. The study shows that NO systems are abundant in the gill of Atlantic salmon, that NO may be produced preferentially by a constitutive NOS isoform, and suggests that NO influence on gill functions is mediated via intracellular, possibly both auto/paracrine, inhibition of Na(+),K(+)-ATPase activity in chloride cells. Furthermore, the increase in NADPHd in the gill during smoltification suggests a regulatory role of NO in the attenuation of the smoltification-related increase in Na(+),K(+)-ATPase activity prior to entering seawater.
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PMID:Nitric oxide synthase in the gill of Atlantic salmon: colocalization with and inhibition of Na+,K+-ATPase. 1576 2

The role of the neuronal NO synthase (nNOS or NOS1) enzyme in the control of cardiac function still remains unclear. Results from nNOS(-/-) mice or from pharmacological inhibition of nNOS are contradictory and do not pay tribute to the fact that probably spatial confinement of the nNOS enzyme is of major importance. We hypothesize that the close proximity of nNOS and certain effector molecules like L-type Ca(2+)-channels has an impact on myocardial contractility. To test this, we generated a new transgenic mouse model allowing conditional, myocardial specific nNOS overexpression. Western blot analysis of transgenic nNOS overexpression showed a 6-fold increase in nNOS protein expression compared with noninduced littermates (n=12; P<0.01). Measuring of total NOS activity by conversion of [(3)H]-l-arginine to [(3)H]-l-citrulline showed a 30% increase in nNOS overexpressing mice (n=18; P<0.05). After a 2 week induction, nNOS overexpression mice showed reduced myocardial contractility. In vivo examinations of the nNOS overexpressing mice revealed a 17+/-3% decrease of +dp/dt(max) compared with noninduced mice (P<0.05). Likewise, ejection fraction was reduced significantly (42% versus 65%; n=15; P<0.05). Interestingly, coimmunoprecipitation experiments indicated interaction of nNOS with SR Ca(2+)ATPase and additionally with L-type Ca(2+)- channels in nNOS overexpressing animals. Accordingly, in adult isolated cardiac myocytes, I(Ca,L) density was significantly decreased in the nNOS overexpressing cells. Intracellular Ca(2+)-transients and fractional shortening in cardiomyocytes were also clearly impaired in nNOS overexpressing mice versus noninduced littermates. In conclusion, conditional myocardial specific overexpression of nNOS in a transgenic animal model reduced myocardial contractility. We suggest that nNOS might suppress the function of L-type Ca(2+)-channels and in turn reduces Ca(2+)-transients which accounts for the negative inotropic effect.
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PMID:Conditional neuronal nitric oxide synthase overexpression impairs myocardial contractility. 1727 13


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