Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Nitric oxide (NO) has been shown to be a ubiquitous intercellular autacoid in the heart and, in cultured rat ventricular myocytes, to decrease the contractile responsiveness to isoproterenol (ISO). The aim of the present study was to investigate whether exogenous (sodium nitroprusside, SNP) or endogenous nitric oxide generated from L-arginine modulated the response to ISO in cultured chick embryo ventricular myocytes. SNP 1 microM or L-arginine 1 mM had no effect on baseline contractile function. Superfusion with ISO 100 nM significantly increased myocyte amplitude of shortening to 1.31 +/- 0.06 (ratio to baseline amplitude). Initial superfusion with SNP 1 microM or L-arginine 1 mM attenuated the response to ISO to 0.89 +/- 0.05 and 1.09 +/- 0.07 respectively (P < 0.05). Potassium ferrocyanide which is not a NO donor and D-arginine the inactive substrate of NO synthase did not attenuate the response to ISO. Myocyte cGMP content was significantly increased by incubation with SNP 1 microM (31.65 +/- 3 fmol/well) but not by L-arginine 1 mM (11.1 +/- 0.3 fmol/well) as compared to myocytes incubated in control medium (11 +/- 0.9 fmol/well). Preincubation with SNP 1 microM or L-arginine 1 mM significantly attenuated the ISO mediated-increase in cAMP content from 4.33 +/- 0.2 pmol/well (ISO 100 nM alone) to 1.48 +/- 0.36 fmol/well and 1.72 +/- 0.21 pmol/well respectively. Potassium ferrocyanide and D-arginine had no effect on myocyte cGMP or cAMP content. Chick embryo myocytes have measurable and LNMMA-inhibited NO synthase activity as measured by the conversion of [3H] L-arginine to [3H] L-citrulline. In conclusion, these results demonstrate that in cultured chick embryo ventricular myocytes both exogenous and endogenous NO elevate cGMP. This may account for the inhibition of beta-adrenergic agonist-stimulated increases in cAMP and amplitude of shortening via an unidentified intracellular negative coupling.
J Mol Cell Cardiol 1997 Feb
PMID:Effects of exogenous and endogenous nitric oxide on the contractile function of cultured chick embryo ventricular myocytes. 914 Aug 25

There is evidence that nitric oxide (NO) may mediate some of the functional myocardial changes caused by bacterial LPS and inflammatory cytokines. The expression of the inflammatory or inducible NO synthase (iNOS) in human cardiac myocytes, however, has not been well characterized. Therefore, we treated cultured, dedifferentiated human ventricular cardiac myocytes with the combination of TNF-alpha (500 U/ml), IL-1beta (30U/ml), IFNgamma (100 U/ml), and LPS (E.coli 0111:B4, 10 microg/ml). Northern blot analysis revealed a approximately 4.5 kb transcript for inducible NOS (iNOS) in the stimulated human heart cells but not in untreated cells. RT-PCR confirmed that iNOS mRNA was only present in stimulated cells. However, treatment of the myocytes for up to 96 h with cytokines and LPS did not result in NO synthesis as measured by nitrite + nitrate accumulation in the culture medium, and no iNOS enzymatic activity could be detected in the cell lysates. Western blot analysis failed to detect iNOS protein. Thus, despite high and persistent levels of iNOS mRNA in cytokine-treated cells, iNOS protein was absent in this experimental model. GTP-cyclohydrolase I was induced both at the mRNA and protein levels and resulted in increased biopterin levels, indicating sufficient amounts of the cofactor tetrahydrobiopterin (BH4) were present, and that the failure to express an inducible protein was specific to iNOS. To determine if the absence of iNOS protein was due to a novel cardiac iNOS gene or modified iNOS transcript in human myocytes, we cloned an iNOS cDNA from cytokine-treated myocytes. Sequencing and expression of the clone revealed a functional iNOS cDNA with >99% identity to other human iNOS cDNA clones. When human cardiac cells were transduced with a retroviral vector carrying only the coding region of the human hepatocyte iNOS cDNA, both iNOS mRNA and protein could be detected. In conclusion, these cells derived from cultured human cardiac myocytes lacked the capacity to express an endogenous iNOS protein, the basis of which appears to be a cell-specific suppression or failure of iNOS translation.
J Mol Cell Cardiol 1997 Apr
PMID:Dedifferentiated human ventricular cardiac myocytes express inducible nitric oxide synthase mRNA but not protein in response to IL-1, TNF, IFNgamma, and LPS. 916 Aug 67

The envelope protein (gp52) of the mouse mammary tumor virus (MMTV) can stimulate RNA synthesis via binding to its cellular receptor on mammary epithelium. This effect was mimicked by either nitric oxide (NO) or 8-bromo-cGMP and was blocked by an NO inhibitor. Furthermore, the effects of gp52 and 8-bromo-cGMP were not additive at maximal concentrations, suggesting that they were using the same signaling route. Finally, gp52 elevated cGMP levels in mammary epithelium. These data suggest that gp52 activates the following transduction pathway in this tissue: gp52-->NO synthase-->NO-->soluble guanylate cyclase cGMP RNA synthesis. In contrast to the mammary gland, gp52 inhibited RNA synthesis in the diaphragm. However, the effect was again mimicked by NO, blocked by an NO inhibitor, and the effects of gp52 and NO were not additive. Therefore, it appears that gp52 is using the NO-cGMP pathway in both tissues, but that muscle tissue may be more susceptible to the toxic effects of NO.
Mol Cell Endocrinol 1997 Apr 25
PMID:Second messengers induced by the envelope protein of a retrovirus. 917 26

In previous work it was shown that the immune cytokine interferon-gamma (IFN-gamma) inhibits hormone secretion in anterior pituitary (AP) cell cultures, an action most likely mediated by folliculostellate (FS) cells. In the present study, we wanted to investigate whether nitric oxide (NO) is involved in this inhibitory action of IFN-gamma. NO synthase (NOS) inhibitors with affinity for the inducible (iNOS) and the constitutive (cNOS) isoform such as N(G)-monomethyl-L-arginine (L-NMMA) and S-methyl-L-thiocitrulline (SMLT) dose-dependently blocked the inhibitory action of IFN-gamma on GHRH-stimulated GH secretion, and partially reversed the inhibitory effect on basal prolactin (PRL) release. In the absence of IFN-gamma these inhibitors significantly augmented basal PRL release and slightly enhanced GHRH-stimulated GH release. L-N6-(1-iminoethyl)lysine (L-NIL), a NOS inhibitor with preferential affinity for iNOS, abrogated the IFN-gamma effect on GHRH-stimulated GH secretion and partially reversed IFN-gamma inhibition of PRL release. However, L-NIL did not exert a stimulatory effect on basal PRL and GHRH-stimulated GH release by its own. 2,4-diamino-6-hydroxypyrimidine (DAHP), a NOS inhibitor by interfering with tetrahydrobiopterin (BH4) cofactor availability, showed the same activity profile as L-NIL. NOS inhibitors blocked or reduced the production of NO as detected by measuring nitrite (NO2-) levels in AP cell cultures and cGMP levels in the NO-reporter cell line RFL-6. The NOS inhibiting action of L-NMMA was confirmed by competition experiments with the natural NOS substrate L-arginine. Thus, in culture medium with lower amounts of L-arginine, L-NMMA blocked the IFN-gamma-induced inhibition of GHRH-stimulated GH release at a lower dose. The inhibition of PRL and GH release by IFN-gamma was markedly reduced in L-arginine-depleted medium. The NO donor sodium nitroprusside (SNP) mimicked the inhibitory action of IFN-gamma on GHRH-stimulated GH and basal PRL release. Similarly to IFN-gamma, SNP did not affect basal GH release. As previously reported, inhibition by IFN-gamma occurred only in AP cell populations containing a minimal proportion of FS cells. As studied in different cell populations obtained by unit gravity sedimentation in a serum albumin gradient, L-NMMA reversed the IFN-gamma effect in the same populations enriched in FS cells. Interestingly, in the absence of IFN-gamma L-NMMA strongly stimulated basal PRL release in the population most enriched in FS cells. It is concluded that IFN-gamma through activation of the iNOS pathway probably in FS cells enhances the production of NO and that this effect is responsible for the inhibitory action of IFN-gamma on GHRH-stimulated GH release and partially for the IFN-gamma-induced decrease in basal PRL release. On the other hand, NO, likely produced by cNOS, appears to exert a tonic inhibitory effect on GHRH-stimulated GH and basal PRL release. It seems therefore that low amounts of NO produced constitutively may take charge of subtle physiological adaptations, and higher levels of NO produced by iNOS under the influence of IFN-gamma may attenuate PRL and GH release during emergency conditions of immune and inflammatory reactions.
Mol Cell Endocrinol 1997 May 16
PMID:Involvement of nitric oxide in the interferon-gamma-induced inhibition of growth hormone and prolactin secretion in anterior pituitary cell cultures. 920 99

The nitric oxide (NO) signalling pathway is thought to play a direct role in regulating the contractile properties of cardiac muscle both in vitro and in vivo. The inducible isoform of NO synthase (iNOS) mediates a sustained increase in NO production in response to cytokines in the cardiac myocytes; however, the regulation of NO synthesis in these cells remains poorly understood. Tetrahydrobiopterin (BH4) is an essential cofactor for NO formation. Cytokines induce the de novo synthesis of BH4 in cardiac myocytes, an event that is essential for the induction of NO synthesis. Activation of NO formation by cytokines in cardiac myocytes requires transcriptional induction of the genes that encode iNOS and guanosine triphosphate cyclohydrolase I (GTPCH), the first and rate-limiting enzyme in de novo BH4 synthesis. Given that nuclear factor kappa B (NF-kappa B) mediates the induction of iNOS gene expression in various cell types, the role of NF-kappa B in the induction of iNOS in cytokine-stimulated rat neonatal cardiac myocytes was assessed by examining the effects of pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappa B activation, on the abundance of iNOS mRNA and NO synthesis. The effects of PDTC on GTPCH mRNA abundance and biopterin synthesis were also investigated. PDTC inhibited in a dose-dependent manner both NO and BH4 synthesis induced by a combination of interleukin-1 alpha (IL-1 alpha) and interferon-gamma (IFN gamma), with a half-maximal inhibitory concentration of 22 muM. PDTC also prevented the accumulation of iNOS and GTPCH mRNAs induced by IL-1 alpha and IFN gamma. Cytokine-induced NO and BH4 synthesis was also inhibited by tosyl-lysine-chloromethyl ketone. another inhibitor of NF-kappa B activation. Results suggest that PDTC inhibits cytokine-induced NO and BH4 synthesis by inhibiting the expression of iNOS and GTPCH genes. Thus, the induction of both genes necessary for NO synthesis in cardiac myocytes appears to be regulated, at least in part, by a common mechanism: NF-kappa B activation.
J Mol Cell Cardiol 1997 Jun
PMID:Role of nuclear factor kappa B in cytokine-induced nitric oxide and tetrahydrobiopterin synthesis in rat neonatal cardiac myocytes. 922 Mar 44

Biosynthesis of nitric oxide (NO) and tetrahydrobiopterin (BH4) was investigated during cytokine-mediated activation of chicken macrophages. Monocyte derived macrophages and HD11 cells, a chicken macrophage cell line, constitutively synthesize BH4. Treatment of these cells with chicken macrophage activation factor (ChMAF) causes up to 10-fold increases of intracellular BH4 and of nitrite concentrations in the cell culture supernatant. Elevated BH4 levels correlate with an increase in GTP-cyclohydrolase I (GTP-CH) activity. Kinetic studies show a joint upregulation of GTP-CH activity and NO synthase activity first detectable 4 hr after stimulation. A corresponding increase in the mRNA for GTP-CH was detected by Northern blot analysis with a chicken GTP-CH specific cDNA probe. These results demonstrate that cytokine-induced BH4 synthesis by chicken macrophages is at least partially regulated through increased GTP-CH gene expression. The functional relevance of BH4 formation for NO production is shown by experiments using 2,4-diamino-6-hydroxypyrimidine (DAHP) as a specific inhibitor of GTP-CH. Monocyte derived macrophages stimulated in the presence of DAHP show a significant decrease in NO synthesis. The effect of DAHP was reversed by adding sepiapterin, which allows synthesis of BH4 through a salvage pathway.
Comp Biochem Physiol B Biochem Mol Biol 1997 Jun
PMID:Coordinate induction of tetrahydrobiopterin synthesis and nitric oxide synthase activity in chicken macrophages: upregulation of GTP-cyclohydrolase I activity. 922 80

Recent evidence suggested that nitric oxide (NO) acts as an important factor in a variety of physiological and pathological roles, including reproductive functions. The purpose of the present study was to investigate whether NO might significantly induce any change in steroidogenesis in cultured porcine granulosa cells (PGC). An NO donor, NOC18, significantly suppressed the oestradiol release from basal (unstimulated) and gonadotrophin-stimulated PGC in a 2 h culture. NOC18 also significantly inhibited the aromatase activity of basal and gonadotrophin-stimulated PGC as measured by a modified tritiated water method. However, the cGMP analogue, 8-bromo-cGMP, had no significant effect on the accumulation of oestradiol and progesterone in basal and gonadotrophin-stimulated PGC during 24 h culture. An NO synthase (NOS) inhibitor, NG-monomethyl-L-arginine (LNMMA), significantly stimulated the basal oestradiol release and dose-dependently enhanced the oestradiol and progesterone release from follicle stimulating hormone (FSH)-stimulated PGC in a 24 h culture. However, NG-monomethyl-D-arginine, which does not inhibit NOS, did not enhance the release of oestradiol and progesterone under the same experimental conditions. LNMMA also significantly suppressed the nitrite concentrations in the media as measured by chemiluminescence. These results demonstrate for the first time that NO inhibits oestradiol secretion independent of cGMP by inhibiting P450 aromatase activity in moderately mature PGC.
Mol Hum Reprod 1997 Apr
PMID:Nitric oxide inhibits steroidogenesis in cultured porcine granulosa cells. 923 55

Nitric oxide synthase (NOS) is responsible for the biological production of nitric oxide (NO) in several organs. NOS activity has also been localized in the reproductive tract, although direct evidence for its presence in the human or bovine oviduct is still lacking. In the present study, four different techniques were used to identify the presence of NOS activity in human (n = 11) and bovine (n = 9) oviduct: (i) conversion of [3H]-L-arginine to [3H]-L-citrulline; (ii) production of nitrite/nitrate (NO2/NO3; stable NO metabolites); (iii) identification of NADPH-diaphorase activity; and (iv) immunostaining with antiserum to endothelial NOS. Cytosolic extracts from human ampullary segments of the Fallopian tube, obtained from post-partum patients (n = 4), converted [3H]-L-arginine to [3H]-L-citrulline (21.0 +/- 8.8 fmol/mg protein/min). This conversion rate was significantly (P < 0.05) reduced in the presence of either EDTA or N-monomethyl-L-arginine monoacetate (L-NMMA), an inhibitor of NOS activity. When bovine (n = 3) ampullary segments were incubated for 36 h in Hanks' balanced salt solution, the concentration of NO2/NO3 in the medium was increased (P < 0.05) if segments were pretreated with lipopolysaccharide (LPS; an inducer of inducible NOS), but not after treatment with LPS + L-NMMA. Additionally, epithelial cells cultured from ampullary segments showed positive staining both for NADPH-diaphorase activity and with antiserum to endothelial NOS. The results of the present study provide direct evidence for the presence of both the Ca(2+)-dependent constitutive form of NOS, as well as the inducible form of NOS activity in human and bovine oviduct. Since the oviduct plays a key role in the reproductive process, it is possible that the two forms of NOS may be involved in the physiological regulation of oviduct function.
Mol Hum Reprod 1996 Aug
PMID:Identification of nitric oxide synthase in human and bovine oviduct. 923 73

Recent structural studies indicate that the substrate- and O2-binding distal pocket of the P450 enzymes are not identical. Thus, P450terp (CYP108) from the alpha-terpineol-metabolizing Pseudomonad differs from P450cam (CYP-101) (C. A. Hasemann et al., J. Mol. Biol. 236, 1169, 1994). In contrast, the distal pockets of P450terp and P450BMP (CYP102 heme domain; Bacillus megaterium) are more closely similar, including novel hydrogen-bonding interactions between the distal H2O ligand and the I helix (C. A. Hasemann et al., Structure, 3, 41-62, 1995). To evaluate the significance of these differences, we have compared solution magnetic circular dichroism (MCD) spectra of P450terp with spectra of other P450 enzymes (e.g., P450cam, P450BMP, P450BM-3holo, and P450BM1), as well as with spectra of chloroperoxidase and NO synthase. Spectra of native P450terp are more similar to those of P450BMP and those of mammalian P450LM-2 than to those of P450cam. Upon substrate-binding, the MCD spectra of ferric P450terp and all other thiolate-ligated heme systems examined to date display a strong Soret band that is distinctly unique relative to the typical Soret MCD pattern(s) of catalases or other 5-coordinate ferric heme systems. This intense negative MCD feature thus appears diagnostic for cysteinate-linked ferric hemes. In the case of ferrous P450s, the intensity of the Soret-region MCD trough varies between substrate-bound and substrate-free enzymes (despite the fact that the substrate is NOT in direct contact with the heme moiety). A novel finding of particular interest is the clear spectral shifts of the Soret MCD band between the substrate-bound and substrate-free forms of ferrous-CO-P450terp. No such observation has been made previously. Furthermore, the band positions for BOTH types of P450terp are red-shifted from known bands of ferrous-CO-P50cam. These data thus indicate a surprising sensitivity of MCD spectra to active-site polarity and to H2O occupancy, concurring with reports of distal pocket effects on CO-binding rates and equilibrium constants. Comparative analysis of the spectral properties of P450terp with MCD spectra of other P450 enzymes, as well as with chloroperoxidase and NO synthase, demonstrates both the expected similarities and the significant differences that reflect active-site structural features. The detailed spectral analysis of P450terp relative to other P450 enzymes presented herein includes the first observation of a substrate-induced spectral shift for a ferrous-CO-P450. Furthermore, testable structural predictions for P450-BM-1 and for the novel NO synthase enzyme (neither of which has been crystallized to date) are made herein. This work thus provides insights into structurally defined P450s and may also lead to understanding of other P450 enzymes.
 ...
PMID:Active site analysis of P450 enzymes: comparative magnetic circular dichroism spectroscopy. 928 14

Aspirin and aspirin-like drugs are the most commonly indicated agents for the treatment of inflammation. Mechanisms of action for these drugs, however, are not clearly understood. In this study, we examined the effects of aspirin on production of nitric oxide (NO), a proinflammatory mediator, and show that aspirin inhibits NO production by transformed pancreatic beta cells (RINm5F) and rat islets in a concentration-dependent manner with an IC50 value of approximately 3 mM. Therapeutic concentrations of aspirin (1-5 mM) that block NO production affected neither nuclear factor-kappaB activation nor inducible NO synthase (iNOS) mRNA transcription but potently inhibited iNOS protein expression by both RINm5F cells and rat islets. The effects of aspirin on islet function were examined by measuring glucose-stimulated insulin secretion in the presence of various concentrations of aspirin. Aspirin (1-5 mM) did not affect insulin secretion at basal or glucose-stimulated conditions, whereas higher concentrations of aspirin (10-20 mM) significantly increased basal insulin secretion. Aspirin at high concentrations of 10 and 20 mM inhibited de novo protein synthesis as demonstrated by inhibition of [35S]methionine incorporation into total islet protein and by inhibition of rabbit reticulocyte expression by Brome mosaic virus mRNA, suggesting that inhibition of iNOS expression at these high concentrations of aspirin may be due to the impairment of the translational machinery. These findings indicate that inhibition of iNOS expression and NO production may explain, in part, the beneficial effects of aspirin as an anti-inflammatory agent at therapeutic concentrations, whereas inhibition of de novo protein synthesis may possibly explain clinical and side effects of aspirin in the inflamed tissues and organs such as stomach and kidney that may accumulate high concentrations of aspirin.
Mol Pharmacol 1997 Sep
PMID:Effects of aspirin on nitric oxide formation and de novo protein synthesis by RINm5F cells and rat islets. 928 1


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