Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have proved that iNOS is regulated by induction of transcription. The induction of iNOS mRNA requires synthesis of an intermediary protein(s) and probably involves action of a protein kinase or kinases. We have cloned and sequenced a 1.7-kilobase pair fragment from the 5'-flanking region of the iNOS gene and proved that this region contains a promoter which confers inducibility of iNOS by LPS and IFN-gamma.
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PMID:Promoter of the mouse gene encoding calcium-independent nitric oxide synthase confers inducibility by interferon-gamma and bacterial lipopolysaccharide. 751 21

To elucidate the role of natriuretic peptides in vascular remodeling, the effects of atrial natriuretic peptide, brain natriuretic peptide, and C-type natriuretic peptide (CNP) on the induction of inducible nitric oxide (NO) synthase (iNOS) in rat aortic smooth muscle cells were examined. Although none of the peptides when applied alone induced the production of nitrite, a stable end product of NO, each peptide dramatically enhanced nitrite production induced by a cytokine combination of interleukin-1 alpha and tumor necrosis factor-alpha. Each natriuretic peptide stimulated intracellular cGMP accumulation in a dose-dependent manner. Time-dependent nitrite production by the cytokines was increased by CNP cotreatment and inhibited by NG-methyl-L-arginine, indicating involvement of the L-arginine-NO pathway. Northern blot analysis showed that the augmented nitrite production was accompanied by an increase in iNOS messenger RNA. A cGMP analog, 8-bromo-cGMP, completely mimicked all of the effects of CNP described above. A cGMP-dependent protein kinase inhibitor, KT5823, paradoxically increased nitrite production and iNOS messenger RNA levels induced by the combination of 8-bromo-cGMP and both cytokines or by the two cytokines only. These data demonstrate the stimulatory effect of cGMP on cytokine-induced iNOS and imply that natriuretic peptides may play a regulatory role in vascular remodeling via the production of large amounts of NO.
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PMID:Natriuretic peptide-augmented induction of nitric oxide synthase through cyclic guanosine 3',5'-monophosphate elevation in vascular smooth muscle cells. 753 63

The present study characterizes mechanisms involved with the induction of nitric oxide (NO) production, nitric oxide synthase (NOS) enzymatic activity and mRNA expression in human articular chondrocytes. Activation of chondrocytes with lipopolysaccharide (LPS) or IL-1 resulted in time- and dose-dependent increases in iNOS mRNA followed by increased NOS enzymatic activity and NO release. The protein tyrosine kinase (PTK) inhibitors herbimycin A or genistein reduced IL-1 or LPS-induced NO release and NOS enzymatic activity. This was associated with inhibition of iNOS mRNA expression as determined by reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. In contrast, inhibitors of protein kinase C (PKC) or protein kinase A (PKA) did not affect these responses. These results were confirmed in experiments with second messenger agonists where neither activation of PKC, nor increases in cyclic adenosine monophosphate (cAMP) or increased intracellular calcium levels were associated with the induction of iNOS mRNA or NO release. These results suggest that PKC, PKA and calcium-dependent signals are not required or sufficient for the stimulation of NO production. However, NO production is dependent on tyrosine kinases due to their role in the expression of iNOS mRNA.
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PMID:Tyrosine kinases are involved with the expression of inducible nitric oxide synthase in human articular chondrocytes. 753 12

We have previously reported that interleukin-1 beta (IL-1) alone induced the transcription of inducible nitric oxide synthase (iNOS) mRNA and nitric oxide (NO) production by isolated neonatal rat cardiac myocytes (CM). The present studies were undertaken to explore the signal transduction pathways involved in IL-1-induced NO production by CM. The addition of IL-1 to CM resulted in a peak rise in both adenosine 3',5'-cyclic monophosphate (cAMP) and protein kinase A (PKA) activities by 10 min followed by rapid declines and return to basal levels within 60 min. The PKA inhibitor KT-5720 completely blocked NO-2 production by IL-1-stimulated CM (P < 0.01; n = 12). The protein kinase C (PKC) inhibitor, calphostin C, had no effect on NO2- production by IL-1 stimulated CM [P = not significant (NS); n = 12]. The addition of PKA+cAMP to cytosols derived from IL-1-treated CM did not directly enhance iNOS enzyme activity (P = NS; n = 3). CM treated with IL-1 alone stained positively for iNOS protein by immunohistochemistry. iNOS staining was absent in CM treated with IL-1+KT-5720. KT-5720 resulted in an earlier disappearance of iNOS mRNA from IL-1-treated CM, as detected by semiquantitative reverse transcriptase-polymerase chain reaction. We report for the first time that PKA (but not PKC) activation is required for IL-1-induced NO production by CM.
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PMID:Protein kinase A activation is required for IL-1-induced nitric oxide production by cardiac myocytes. 876 74

1. A possible interaction between cyclic AMP and nitric oxide (NO) in mediating the relaxant effect of vasoactive intestinal polypeptide (VIP) on intestinal smooth muscle cells has been investigated. The effects of the inhibitor of NO synthesis, NG-nitro-L-arginine methyl ester (L-NAME), have been studied on VIP-, forskolin-, and 8 bromo-cyclic AMP- induced relaxation of cells, dispersed by enzymatic digestion of muscle strips from the circular layer of guinea-pig ileum. 2. VIP alone did not modify the length of isolated muscle cells. By contrast, when the cells were contracted by cholecystokinin octapeptide, CCK8 (10 nM), VIP inhibited this contraction, inducing a concentration-dependent relaxation of the cells. Maximal relaxation was induced by 1 microM VIP (EC50 = 408.2 +/- 16.7 pM). 3. N-ethylmaleimide, inhibitors of adenylate cyclase or somatostatin, abolished the relaxing effect of VIP. (R)-p-cAMPs, an antagonist of cyclic AMP on protein kinase A also inhibited the VIP-induced relaxation by 92.1 +/- 6.3%. Inhibitors of nitric oxide synthase (NOS), L-NAME and L-NMMA, partially inhibited VIP-induced relaxation. The effect of L-NAME was reversed by L-arginine but not by D-arginine. 4. (R)-p-cAMPS and L-NAME also inhibited the cell relaxation induced either by forskolin which directly stimulates adenylate cyclase activity or 8-bromo-cyclic AMP, an analogue of cyclic AMP. 5. When cells were incubated for 30 min with dexamethasone 10 microM, a glucocorticoid known to decrease the synthesis of iNOS, the relaxing effect of a maximal concentration of VIP was decreased by 52 +/- 4% and L-NMMA had no further effect on this residual VIP-induced relaxation. Milrinone, a phosphodiesterase type III inhibitor, potentiated the relaxant effect of VIP. 6. These data demonstrate that the intracellular pathway mediating the relaxant effect of VIP in intestinal smooth muscle cells includes the sequential activation of adenylate cyclase, protein kinase A, activation of NOS and finally production of NO and cyclic GMP. NO could in turn regulate the cyclic AMP-dependent pathway of cell relaxation.
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PMID:VIP-induced relaxation of guinea-pig intestinal smooth muscle cells: sequential involvement of cyclic AMP and nitric oxide. 876 68

1. In the present study we examined whether interleukin-1 beta (IL-1 beta) increases the activity of adenylyl cyclase in vascular smooth muscle cells and determined its role in the cytokine-induced expression of the inducible nitric oxide synthase (iNOS) and activation of nuclear transcription factor-kappa B (NF-kappa B). In addition the interaction between cyclic AMP- and cyclic GMP-elevating agonists on the IL-1 beta-stimulated expression of iNOS was examined. 2. Exposure of vascular smooth muscle cells to IL-1 beta stimulated the formation of cyclic AMP but not of cyclic GMP. The intracellular level of cyclic AMP reached a maximum within 1 h and then gradually declined over the next 5 h. This IL-1 beta (60 u ml-1)-stimulated formation of cyclic AMP was modest (about 3 fold at 60 u ml-1 for 1 h) compared to that evoked by isoprenaline (about 9 fold at 3 x 10(-6) M for 2 min). 3. The IL-1 beta (60 u ml-1 for 24 h)-stimulated accumulation of nitrite, which was taken as an index of NO production, was concentration-dependently increased by preferential inhibitors of cyclic AMP-dependent phosphodiesterases (rolipram and trequinsin). This effect was reproduced by a specific activator of the cyclic AMP-dependent protein kinase(s) A, Sp-8-CPT-cAMPS (10(-4) M) but was prevented by a specific inhibitor of cyclic AMP-dependent protein kinase(s) A, Rp-8-CPT-cAMPS (10(-4) M). These compounds alone [rolipram (10(-6) M), trequinsin (3 x 10(-6) M) and Sp-8-CPT-cAMPS (10(-4) M)] slightly but significantly increased the release of nitric oxide while Rp-8-CPT-cAMPS elicited no such effect. 4. Inducible NOS protein was expressed in IL-1 beta (30 u ml-1, 24 h)-stimulated smooth muscle cells as assessed by Western blot analysis. The level of iNOS protein was markedly increased in smooth muscle cells which had been exposed to IL-1 beta in combination with either rolipram (3 x 10(-6) M) or Sp-8-CPT-cAMPS (10(-4) M) but was reduced in those exposed to IL-1 beta and Rp-8-CPT-cAMPS (10(-4) M). A weak expression of iNOS protein was found in smooth muscle cells which had been exposed to either Sp-8-CPT-cAMPS or rolipram alone for 24 h while Rp-8-CPT-cAMPS elicited no such effect. 5. Exposure of smooth muscle cells to IL-1 beta (30 u ml-1) for 30 min increased the level of NF-kappa B-DNA complexes in nuclear extracts as detected by electrophoretic mobility shift assay. Similar levels of NF-kappa B-DNA complexes were found in cells which had been exposed to IL-1 beta in combination with either Sp-8-CPT-cAMPS (10(-4) M), trequinsin (10(-6) M) or rolipram (10(-6) M). None of the modulators alone affected the basal level of NF-kappa B binding activity. 6. NO-donors [sodium nitroprusside (SNP) 10(-4) M; dinitrosyl-iron-di-L-cysteine-complex (DNIC), 10(-4) M; 3-morpholino-sydnonimine (SIN-1), 10(-4) M] and atrial natriuretic factor (10(-6) M) significantly increased the IL-1 beta (30 or 60 u ml-1, 24 h)-stimulated expression of iNOS protein and activity as assessed indirectly by the conversion of oxyhaemoglobin to methaemoglobin. In the absence of IL-1 beta, SNP (10(-4) M, 24 h) but not the other cyclic GMP-dependent vasodilators caused a modest expression of iNOS protein. No such effect was found in smooth muscle cells exposed to SNP in combination with Rp-8-CPT-cAMPS (10(-4) M) while an increased level of iNOS protein was found in those exposed to SNP in combination with either Sp-8-CPT-cAMPS (10(-4) M) or rolipram (3 x 10(-6) M). 7. Exposure of vascular smooth muscle cells to either S-nitroso-L-cysteine (Cys-SNO, 10(-4) M), SNP (10(-4) M) or SIN-1 (10(-4) M) for 35 min affected minimally the basal activation of NF-kappa B but abolished that evoked by IL-1 beta (30 u ml-1 added during the last 30 min). However, addition of Cys-SNO following the stimulation with IL-1 beta (during the last 5 min of the 30 min exposure period) reduced the level of NF-kappa B-DNA complexes only slightly. 8. These data indicate that the cyclic AMP-dependent pathway plays a decisi
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PMID:Effect of cyclic GMP-dependent vasodilators on the expression of inducible nitric oxide synthase in vascular smooth muscle cells: role of cyclic AMP. 890 45

Cytokine-stimulated expression of inducible type of nitric oxide synthase (iNOS) seems to be regulated by various signal pathways in a cell-specific manner. In this study, we examined how it was regulated in L6 rat skeletal muscle cells. In L6 cells, the combination of interleukin-1 beta and interferon-gamma induced a marked accumulation of nitrite, a stable metabolite of nitric oxide. In parallel with this reaction, iNOS mRNA expression was achieved at a maximum between 3 and 6 h, and iNOS protein was detectable at 6 h and peaked at 24 h after stimulation. Tyrosine kinase inhibitors, herbimycin A, and genistein suppressed cytokine-induced iNOS expression and nitrite production. Forskolin, an adenosine 3',5'-cyclic monophosphate-dependent protein kinase (PKA) activator, and phorbol 12-myristate 13-acetate, a protein kinase C (PKC)-activating phorbol ester, enhanced these cytokine-induced reactions. These results indicate that iNOS expression by cytokines is mediated via a protein tyrosine kinase-dependent pathway and is positively modulated by both PKA- and PKC-dependent pathways in this cell type.
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PMID:Regulation of inducible nitric oxide synthase expression in L6 rat skeletal muscle cells. 903 8

Recent work has demonstrated that endotoxin or cytokines induce nitric oxide synthase in heart or cardiac myocytes. We investigated the functional significance of inducible nitric oxide synthase (iNOS) in indo 1-loaded beating myocytes with regard to intracellular Ca2+ concentration ([Ca2+]i) and cell contraction. Lipopolysaccharide (LPS; 10 micrograms/ml) time dependently induced iNOS mRNA and protein in cultured neonatal rat cardiac myocytes. Nitrite concentration in the medium and intracellular guanosine 3',5'-cyclic monophosphate (cGMP) contents after 24-h exposure to LPS increased in proportion to the levels of iNOS induction in these cells. Myocytes treated with both NG-monomethyl-L-arginine and LPS for 24 h expressed iNOS protein, but nitrite production was significantly inhibited. Subsequent perfusion with 100-fold molar excess L-arginine of these myocytes elicited decreases in peak systolic [Ca2+]i (790 +/- 42 to 551 +/- 27 nM, P < 0.05), relative amplitude of cell contraction (100 to 72.4 +/- 5.5%, P < 0.05), and spontaneous beating rate (146 +/- 13 to 85 +/- 22 beats/min, P < 0.05). Pretreatment with methylene blue or KT-5823 inhibited these negative myocardial effects. These results suggest that LPS induces iNOS in cardiac myocytes and that the increased nitric oxide produced by iNOS has cardiac depressant effects through the activation of cGMP-dependent protein kinase.
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PMID:Cardiac inducible nitric oxide synthase negatively modulates myocardial function in cultured rat myocytes. 903 20

1. The effect of different protein kinase inhibitors on the expression of the inducible isoform of nitric oxide (NO) synthase (iNOS) was investigated in cultured vascular smooth muscle cells (VSMC) isolated from the rat aorta. 2. The non-selective protein kinase C (PKC) inhibitor, staurosporine, but not the more selective PKC inhibitors, calphostin C and Ro 31-8820, or the tyrosine kinase inhibitors, genistein and erbstatin analogue (erbstatin A), elicited a distinct (up to six fold) up-regulation of iNOS gene expression in these cells, as demonstrated by a parallel increase in iNOS mRNA and protein abundance as well as an accumulation of nitrite (NO2-) in the conditioned medium. Actinomycin D and cycloheximide inhibited the effect of staurosporine, suggesting an involvement of both DNA transcription and de nova protein synthesis. 3. Staurosporine also synergistically potentiated the stimulating effect of interleukin-1 beta (IL-1 beta), but not that of the adenylyl cyclase activator, forskolin, on NO2- production and iNOS protein abundance. Staurosporine, on the other hand, had no effect on the IL-1 beta-mediated increase in iNOS mRNA abundance. The effect of staurosporine on both basal and IL-1 beta-stimulated NO2- production was concentration-dependent with an apparent maximum at 3 nM. Among the other protein kinase inhibitors tested, only calphostin C also enhanced the stimulant effect of IL-1 beta approximately two fold, while genistein, erbstatin A and Ro 31-8220 inhibited rather than potentiated it. 4. Staurosporine did not influence basal activity of the transcription factors CREB and nuclear factor kappa B (NF-kappa B), but increased that of C/EBP. Moreover, staurosporine significantly augmented the activation of C/EBP by IL-1 beta and forskolin. 5. These findings suggest that in cultured VSMC a staurosporine-sensitive protein kinase exists, which is unlikely to be related to PKC, that prevents iNOS gene expression presumably by suppressing basal C/EBP activity. They also indicate that NF-kappa B and a member of the C/EBP family of transcription factors, presumably C/EBP beta, act synergistically under basal conditions and possibly also following exposure to IL-1 beta in the up-regulation of iNOS gene expression in these cells. Targeting of the activation of C/EBP beta may thus represent an interesting approach to interfere selectively with the cytokine-induced over-production of NO in acute and chronic inflammatory conditions.
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PMID:Induction by staurosporine of nitric oxide synthase expression in vascular smooth muscle cells: role of NF-kappa B, CREB and C/EBP beta. 913 19

Expression of cytokines by T lymphocytes is a highly balanced process, involving stimulatory and inhibitory intracellular signalling pathways. In the present work, we attempted to clarify the role of cAMP on interferon-gamma (IFN-gamma), interleukin (IL)-10, IL-4 and IL-13 expression as well as on the inducible nitric oxide synthase (iNOS) expression. Treatment of phytohaemagglutinin (PHA)/phorbol 12-myristate 13-acetate (PMA)-activated Jurkat cells with either dibutyryl-cyclic adenosine monophosphate (cAMP) or pentoxifylline induced a strong inhibition of IFN-gamma mRNA expression as measured by reverse transcription (RT)-polymerase chain reaction (PCR), without affecting IL-10 expression. Both cholera toxin and prostaglandin E2 (PGE2) induced a strong inhibition of IFN-gamma mRNA expression, whereas IL-10 mRNA expression was significantly enhanced. This differential regulation of IFN-gamma and IL-10 expression was related to intracellular cAMP concentration. IL-13 and IL-4 mRNA expressions were not inhibited. We developed a new method based on immunofluorescence for intracellular cytokine detection followed by optical and computerized image processing, and our results showed that IFN-gamma protein was strongly inhibited when cells were treated with PGE2 or dibutyryl (db)-cAMP, whereas IL-10 protein was enhanced. This suggests that cAMP exerts its action at both the transcriptional and protein levels. iNOS mRNA expression was markedly elevated in the presence of PGE2. The generation of nitric oxide using sodium nitroprusside (SNP) induced a dramatic decrease of IFN-gamma, while IL-10 was enhanced; and conversely the inhibition of iNOS activity using 1-NG-monomethyl arginine (1-NMMA) induced a clear inhibition of IL-10 and IL-4, while IFN-gamma was enhanced. These results provide evidence that the protein kinase A (PKA) activation pathway plays a prominent role in the balance between the type 1 and type 2 cytokine profile in PHA/PMA-activated Jurkat cells. Data also suggest that iNOS expression is under the control of PKA activation, and that NO seems to be able to assume the polarization of activated T cells to the type 2 profile.
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PMID:Differential regulation of IFN-gamma, IL-10 and inducible nitric oxide synthase in human T cells by cyclic AMP-dependent signal transduction pathway. 930 24


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