Rapid Inactivation of NOS-I by Lipopolysaccharide Plus Interferon-γ-induced Tyrosine Phosphorylation

Abstract

Human astrocytoma T67 cells constitutively express a neuronal NO synthase (NOS-I) and, following administration of lipopolysaccharide (LPS) plus interferon-␥ (IFN␥), an inducible NOS isoform (NOS-II). Previous results indicated that a treatment of T67 cells with the combination of LPS plus IFN␥, by affecting NOS-I activity, also inhibited NO production in a very short time. Here, we report that under basal conditions, a NOS-I protein of about 150 kDa was weakly and partially tyrosine-phosphorylated, as verified by immunoprecipitation and Western blotting. Furthermore, LPS plus IFN␥ increased the tyrosine phosphorylation of NOS-I, with a concomitant inhibition of its enzyme activity. The same effect was observed in the presence of vanadate, an inhibitor of phosphotyrosine-specific phosphatases. On the contrary, genistein, an inhibitor of protein-tyrosine kinases, reduced tyrosine phosphorylation of NOS-I, enhancing its enzyme activity. Finally, using reverse transcriptase-polymerase chain reaction, we have observed that a suboptimal induction of NOS-II mRNA expression in T67 cells was enhanced by vanadate (or L-NAME) and inhibited by genistein. Because exogenous NO has been found to suppress NOS-II expression, the decrease of NO production that we have obtained from the inactivation of NOS-I by LPS/IFN␥-induced tyrosine phosphorylation provides the best conditions for NOS-II expression in human astrocytoma T67 cells.

Nitric oxide mediates anti‐inflammatory action of extracorporeal shock waves

“…As a consequence, iNOS gene expression takes place after LPS/cytokine stimulation, provided that the cNOS-generated NO is reduced below a threshold value in a short time [15,17]. In this respect, we have recently reported that iNOS inducers (e.g., LPS and IFNc) elicit a rapid inactivation of nNOS and a decrease of basal NO levels [24], an event mediated by arachidonic acid-dependent tyrosine phosphorylation of nNOS [15,25].…”

“…In order to control the amount of RNA in each lane, the gel was stained with ethidium bromide before and after blotting. RNA was hybridized with the cDNA specific for rat iNOS [15] previously labeled by means of a DECAprime II DNA Labeling Kit (1.0-2.0· 109 cpm/lg; Ambion, Austin, TX, USA). The intensity of hybridization was visualized by autoradiography and mRNA expression was quantified using the public domain NIH Image 1.61 program (developed at the U.S. National Institutes of Health and available on Internet at http://rsb.info.-nih.gov/nih-image/).…”

“…Total cellular RNA was purified as described above and reverse transcribed into cDNA, using MMLV reverse transcriptase and oligo dT (12)(13)(14)(15)(16)(17)(18) as primers. cDNA was amplified for the iNOS gene (450 bp) or TNF-a gene (200 bp) using specific primers (iNOS forward: 5 0 -TCCTTGCATCCTCATCGGGCC-3 0 , iNOS reverse: 5 0 -TCGTGA-TAGCGCTTCTGGCTCT-3 0 ; TNFa forward: GAGCTGAGAGA-TAACCAGCTGGTG-3 0 , TNFa reverse 5 0 -CAGATAGATGGGC-TCATACCAGGG-3 0 ).…”

Flavopiridol downregulates the expression of both the inducible NO synthase and p27kip1 in malignant cells from B-cell chronic lymphocytic leukemia

“…Alternatively, post-transcriptional modifications cannot be eliminated; as reported for downregulation of survivin, 21 flavopiridol, through its properties of protein kinase inhibitor, could inactivate iNOS, as the activity as well as the expression of NOS enzymes can be regulated by phosphorylation-dephosphorylation. 22,23 Experiments are in progress to test the different possibilities.…”

Inhibition of Nitric-oxide Synthase-I (NOS-I)-dependent Nitric Oxide Production by Lipopolysaccharide plus Interferon-γ Is Mediated by Arachidonic Acid

“…A possible candidate of the downstream kinase leading to NOS-I phosphorylation and inactivation is a tyrosine kinase, because AA is a known activator of this pathway (20) and our previous work showed that LPS/IFN␥-induced inhibition of NOS-I activity is mediated by tyrosine phosphorylation (7). To address this question, experiments were performed using the human A172 astrocytoma cell line, which expresses higher levels of NOS-I than C6 cells.…”