Biomedical subjects

C J Lowenstein

Publications and source records attributed to C J Lowenstein.

At least 37 records · Page 2Linked to original sources

PubMed1998

The role of inducible nitric oxide synthase in the host response to Coxsackievirus myocarditis.

The host response to Coxsackievirus infection is complex, including T lymphocytes, B lymphocytes, natural killer cells, and macrophages. Although Coxsackievirus infection induces expression of inducible nitric oxide synthase (NOS2; EC 1.14.13.39) in macrophages, the precise role of NOS2 in the host response to Coxsackievirus myocarditis has been unclear. We show, by using mice homozygous for a disrupted NOS2 allele, that Coxsackievirus replicates to higher titers in NOS2(-/-) mice, that the host lacking NOS2 clears virus more slowly than the wild-type host, and that myocarditis is much more severe in infected NOS2(-/-) mice. These data show that NOS2 is crucial for the host response to Coxsackievirus in the mouse.

Animals↗

PubMed1998

Midkine induces tumor cell proliferation and binds to a high affinity signaling receptor associated with JAK tyrosine kinases.

The G401 cell line derived from a rhabdoid tumor of the kidney secretes the heparin-binding growth factors midkine and pleiotrophin. Both proteins act as mitogens for diverse cells, but only midkine serves as an autocrine mitogen for G401 tumor cells. We show that midkine specifically binds a protein or complex of molecular mass greater than 200 kDa with high affinity (Kd = 0.07 +/- 0.01 nM). Midkine, but not pleiotrophin, stimulates tyrosine phosphorylation of several cellular proteins with molecular mass of 100, 130, and 200+ kDa. Upon midkine binding, the midkine-receptor complex associates with the Janus tyrosine kinases, JAK1 and JAK2. MK stimulates tyrosine phosphorylation of JAK1, JAK2, and STAT1alpha. Our initial characterization of the midkine receptor suggests that midkine autocrine stimulation of tumor cell proliferation is mediated by a cell-surface receptor which in turn might activate the JAK/STAT pathway.

Animals↗

PubMed1998

Involvement of transcriptional mechanisms in the inhibition of NOS2 expression by dexamethasone in rat mesangial cells.

In previous studies we reported that stimulation of rat mesangial cells (RMC) with lipopolysaccharide (LPS) + tumor necrosis factor alpha (TNF-alpha) (L/T) elicits inducible nitric oxide synthase (NOS2) mRNA expression, which is inhibited by dexamethasone (DX). We have now analyzed the mechanisms responsible for this inhibitory effect. Dexamethasone had no destabilizing effect on NOS2 mRNA. Transfection of RMC with several luciferase reporter constructs from the 5' flanking regulatory region of the rat NOS2 gene established the importance of the NF-kappa B site in the transcriptional activation of the NOS2 gene. DNA mobility shift assays showed activation by L/T of the NF-kappa B complex in a time-dependent manner. Dexamethasone specifically inhibited this activation in a process dependent on the glucocorticoid receptor and with a markedly greater effect when it was added prior to L/T. Dexamethasone increased the expression of the I kappa B-alpha transcript and protein in the cytoplasm. While treatment of RMC with L/T induced the transient decrement of cytoplasmic p65 levels and its appearance in the nucleus, preincubation with DX prevented this effect. Co-immunoprecipitation and immunocytochemical studies demonstrated that I kappa B-alpha is associated with p65 in the cytoplasm of RMC after treatment with DX and L/T. These results prove that inhibition of NF-kappa B-mediated transcription is a crucial mechanism by which DX inhibits NOS2 expression, and that this occurs by increasing cytoplasmic I kappa B-alpha levels and sequestering the activating subunits of NF-kappa B in the cytoplasm. The need for previous induction of I kappa B-alpha could provide a molecular explanation for the limited efficacy of these agents in the therapy of septic shock.

Animals↗

PubMed1998

Monosialoganglioside GM1 inhibits neurotoxicity after hypothermic circulatory arrest.

BACKGROUND: Prolonged hypothermic circulatory arrest (HCA) causes clinical neurologic injury. This injury involves neuronal apoptosis, or programmed cell death. We have previously demonstrated that HCA causes glutamate excitotoxicity, increased nitric oxide (NO) production, and NO-mediated apoptosis. We hypothesized that monosialoganglioside GM1 inhibits NO synthase. The purpose of this study was to determine whether GM1 inhibits NO production and neuronal apoptosis after HCA. METHODS: Fourteen dogs underwent intracerebral microdialysis to measure excitatory amino acids, glutamate, aspartate, and citrulline, an equal coproduct of NO. They underwent 2 hours of HCA at 18 degrees C and were sacrificed 8 hours after HCA. Group 1 (n = 6) was pretreated with GM1, 30 mg/kg intravenously every day for 3 days, as well as before and after HCA. Group 2 control dogs (n = 8) received vehicle only. Apoptosis was scored from 0 (normal) to 100 (severe injury). RESULTS: Excitatory amino acids, aspartate and glutamate, coagonist glycine, and citrulline levels increased significantly over baseline during HCA and after HCA. GM1 pretreatment did not appreciably alter levels of glutamate, aspartate, and glycine; however, it substantially decreased citrulline and therefore NO production throughout the experiment. GM1 significantly inhibited apoptosis (group 1 vs group 2: 15.56 +/- 13.60 vs 62.92 +/- 6.17; P < .001). CONCLUSIONS: Our results provide the first direct evidence that GM1 inhibits NO synthase to reduce NO production and HCA-induced neuronal apoptosis. GM1 did not affect excitatory glutamate or aspartate levels. GM1 has been used in clinical trials of spinal cord injury and may be efficacious in reducing neurologic injury after HCA.

Animals↗

PubMed1997

Nitric oxide inhibition of coxsackievirus replication in vitro.

Nitric oxide is a radical molecule with antibacterial, -parasitic, and -viral properties. We investigated the mechanism of NO inhibition of Coxsackievirus B3 (CVB3) replication in vitro by determining the effect of NO upon a single replicative cycle of CVB3 grown in HeLa cells. Transfection of inducible NO synthase cDNA into HeLa cells reduces the number of viral particles produced during a single cycle of growth. Similarly, a noncytotoxic concentration of the NO donor S-nitroso-amino-penicillamine reduces the number of viral particles in a dose-dependent manner. To explore the mechanisms by which NO exerts its antiviral effect, we assayed the attachment, replication, and translation steps of the CVB3 life cycle. NO does not affect the attachment of CVB3 to HeLa cells. However, NO inhibits CVB3 RNA synthesis, as shown by a [3H]uridine incorporation assay, reverse transcription-PCR, and Northern analysis. In addition, NO inhibits CVB3 protein synthesis, as shown by [35S]methionine protein labeling and Western blot analysis of infected cells. Thus, NO inhibits CVB3 replication in part by inhibiting viral RNA synthesis by an unknown mechanism.

Cell Survival↗

PubMed1997

Post-transcriptional regulation of inducible nitric oxide synthase mRNA in murine macrophages by doxycycline and chemically modified tetracyclines.

Chemically modified tetracyclines [CMT-3 (IC50 approximately 6-13 microM = approximately 2.5-5 microg/ml) and CMT-8 (IC50 approximately 26 microM = 10 microg/ml), but not CMT-1, -2 or -5], which lack anti-microbial activity, inhibited nitrite production in LPS-stimulated macrophages. Unlike competitive inhibitors of L-arginine which inhibited the specific activity of inducible nitric oxide synthase (iNOS) in cell-free extracts, CMTs exerted no such direct effect on the enzyme. CMTs could, however, be shown to inhibit both iNOS mRNA accumulation and protein expression in LPS-stimulated cells. Tetracyclines (doxycycline and CMT-3) unlike hydrocortisone had no significant effect on murine macrophages transfected with iNOS promoter (tagged to a luciferase reporter gene) in the presence of LPS. However, doxycycline and CMT-3 augmented iNOS mRNA degradation, in LPS-stimulated murine macrophages. These studies show a novel mechanism of action of tetracyclines which harbours properties to increase iNOS mRNA degradation and decrease iNOS protein expression and nitric oxide production in macrophages. This property of tetracyclines may have beneficial effects in the treatment of various diseases where excess nitric oxide has been implicated in the pathophysiology of these diseases.

Animals↗

PubMed1997

Inducible nitric oxide synthase and nitric oxide production by oligodendrocytes.

It has been previously demonstrated that microglia and astrocytes produce micromolar amounts of nitric oxide in vitro. In this study, we demonstrate that primary rat oligodendrocytes can be stimulated to produce iNOS mRNA as detected by Northern blot and in situ hybridization analysis and a 131-kDa iNOS protein by Western blot analysis; protein was also detected in cells by single- and double-label immunohistochemistry for iNOS and the oligodendrocyte-specific marker CNPase. NO/NOS are produced as a consequence of activation of the gene encoding the inducible nitric oxide synthase as determined by inhibition with actinomycin D and cyclohexamide. The iNOS is functional, leading to calcium/calmodulin-independent NO production in these in vitro cultures.

Animals↗

PubMed1997

Hyaluronan fragments induce nitric-oxide synthase in murine macrophages through a nuclear factor kappaB-dependent mechanism.

Activated macrophages play a critical role in controlling chronic tissue inflammation through the release of a variety of mediators including cytokines, chemokines, growth factors, active lipids, reactive oxygen, and nitrogen species. The mechanisms that regulate macrophage activation in chronic inflammation are poorly understood. A hallmark of chronic inflammation is the turnover of extracellular matrix components, and recent work has suggested that interactions with the extracellular matrix can exert important influences on macrophage effector functions. We have examined the effect of low molecular weight fragments of the extracellular matrix glycosaminoglycan hyaluronan (HA) on the induction of nitric-oxide synthase (iNOS) in macrophages. We found that HA fragments induce iNOS mRNA, protein and activity alone, and markedly synergize with interferon-gamma to induce iNOS gene expression in murine macrophages. In addition, we found that resident tissue alveolar macrophages respond minimally, but inflammatory alveolar macrophages exhibit a marked induction in iNOS expression in response to HA fragments. Finally, we demonstrate that the mechanism of HA fragment-induced expression of iNOS requires activation of the transcriptional regulator nuclear factor kappaB. These data support the hypothesis that HA may be an important regulator of macrophage activation at sites of chronic tissue inflammation.

Animals↗

PubMed1997

Neuronal nitric oxide synthase inhibition reduces neuronal apoptosis after hypothermic circulatory arrest.

BACKGROUND: Neurologic injury, including choreoathetosis and learning and memory deficits, occurs after prolonged hypothermic circulatory arrest (HCA). Apoptosis, or programmed cell death, is a possible cause of the neurologic injury seen after HCA. However, the mechanism of apoptosis is unknown. Hypothermic circulatory arrest causes glutamate excitotoxicity, resulting in increased nitric oxide production. We therefore hypothesized that nitric oxide mediates apoptosis. The purpose of this study was to determine if neuronal nitric oxide synthase inhibition reduces neuronal apoptosis in an established canine model of HCA. METHODS: Fourteen male hound dogs (weight, 20 to 27 kg) were placed on closed-chest cardiopulmonary bypass, subjected to 2 hours of HCA at 18 degrees C, rewarmed to normothermia, and sacrificed 8 hours after HCA. Group 1 (n = 7) dogs were treated with the neuronal nitric oxide inhibitor 7-nitroindazole, 25 mg/kg intraperitoneally, before arrest and every 2 hours until sacrifice. Group 2 (n = 7) dogs received vehicle only. The brains were analyzed histopathologically. Apoptosis, identified by hematoxylin-eosin staining, was confirmed by DNA terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling assay and electron microscopy. Apoptosis was scored by a blinded neuropathologist from 0 (normal) to 100 (severe injury). RESULTS: Apoptosis occurred early after HCA in select neuronal populations, including the hippocampus, stria terminalis, neocortex, and entorhinal cortex. Apoptotic neurons showed a characteristic shrunken cytoplasm and nuclear chromatin condensation. 7-Nitroindazole significantly inhibited apoptosis (group 1 versus 2: 19.17 +/- 14.39 versus 61.11 +/- 5.41; p < .001). CONCLUSIONS: Our results provide evidence that apoptosis is associated with the neurologic injury that occurs after HCA and that nitric oxide mediates the apoptosis that occurs after HCA. Strategies for cerebral protection during HCA may include the inhibition of neuronal nitric oxide synthase.

Animals↗

PubMed1997

Inducible nitric oxide synthase expression in coronary arteries of transplanted human hearts with accelerated graft arteriosclerosis.

Inducible nitric oxide synthase (iNOS) is a high-output isoform of NOS that produces nitric oxide (NO), a nonspecific immune effector molecule. In some animal models of autoimmunity, the induction of iNOS has been shown to lead to inflammation and tissue damage, and it has been suggested that iNOS is an immune mediator in humans as well. Using in situ hybridization and immunohistochemical techniques, we demonstrate that iNOS mRNA and protein are present in the coronary arteries of transplanted human hearts with accelerated graft arteriosclerosis (AGA). iNOS is expressed in cells morphologically consistent with macrophages in the neointima of 7 of 10 of the transplanted vessels with AGA that were examined. In serial sections, these same cells express the macrophage marker CD68. In contrast, iNOS is absent from five native coronary arteries with atherosclerosis and absent from two normal coronary arteries. Although iNOS is expressed in macrophages in AGA, its role in the pathogenesis of AGA is unknown.

Adolescent↗

PubMed1996

Regulation of endothelial nitric-oxide synthase during hypoxia.

The mechanism by which nitric-oxide (NO) production increases during hypoxia is unknown. To explore the effect of hypoxia upon endothelial nitric-oxide synthase (ecNOS) activity and expression, we exposed bovine aortic endothelial cells (BAEC) to hypoxia (1% O2) for 0-24 h and measured levels of ecNOS mRNA, protein, and activity. The amount of ecNOS mRNA increases to more than twice the basal level after 6 h of hypoxia. Incubation of BAEC with actinomycin D during hypoxia prevents this increase, demonstrating that higher levels of mRNA observed during hypoxia are due to increased synthesis, not to increased stability of ecNOS mRNA. Levels of ecNOS protein increase throughout 24 h of hypoxia to more than twice normoxic levels. Although ecNOS expression increases within 2 h of hypoxia, total activity remains unchanged. To explore the transcriptional regulation of ecNOS, we constructed a reporter plasmid containing the ecNOS promoter region upstream of the luc gene and transfected this reporter plasmid into BAEC. In this system, hypoxia induces a linear increase over time in the expression of luciferase driven by the ecNOS promoter. It is concluded that hypoxia induces an increase in transcription of ecNOS in endothelial cells, activating the regulatory region of ecNOS by undefined transcription factors.

Aerobiosis↗

PubMed1996

Nitric oxide inhibits viral replication in murine myocarditis.

Nitric oxide (NO) is a radical molecule that not only serves as a vasodilator and neurotransmitter but also acts as a cytotoxic effector molecule of the immune system. The inducible enzyme making NO, inducible NO synthase (iNOS), is transcriptionally activated by IFN-gamma and TNF-alpha, cytokines which are produced during viral infection. We show that iNOS is induced in mice infected with the Coxsackie B3 virus. Macrophages expressing iNOS are identified in the hearts and spleens of infected animals with an antibody raised against iNOS. Infected mice have increased titers of virus and a higher mortality when fed NOS inhibitors. Thus, viral infection induces iNOS in vivo, and NO inhibits viral replication. NO is a novel, nonspecific immune defense against viruses in vivo.

Amino Acid Sequence↗

PubMed1996

Arrest of endotoxin-induced hypotension by transforming growth factor beta1.

Septic shock is a cytokine-mediated process typically caused by a severe underlying infection. Toxins generated by the infecting organism trigger a cascade of events leading to hypotension, to multiple organ system failure, and frequently to death. Beyond supportive care, no effective therapy is available for the treatment of septic shock. Nitric oxide (NO) is a potent vasodilator generated late in the sepsis pathway leading to hypotension; therefore, NO represents a potential target for therapy. We have previously demonstrated that transforming growth factor (TGF) beta1 inhibits inducible NO synthase (iNOS) mRNA and NO production in vascular smooth muscle cells after its induction by cytokines critical in the sepsis cascade. Thus, we hypothesized that TGF-beta1 may inhibit iNOS gene expression in vivo and be beneficial in the treatment of septic shock. In a conscious rat model of septic shock produced by Salmonella typhosa lipopolysaccharide (LPS), TGF-beta1 markedly reduced iNOS mRNA and protein levels in several organs. In contrast, TGF-beta1 did not decrease endothelium-derived constitutive NOS mRNA in organs of rats receiving LPS. We also performed studies in anesthetized rats to evaluate the effect of TGF-beta1 on the hemodynamic compromise of septic shock; after an initial 25% decrease in mean arterial pressure, TGF-beta1 arrested LPS-induced hypotension and decreased mortality. A decrease in iNOS mRNA and protein levels in vascular smooth muscle cells was demonstrated by in situ hybridization and NADPH diaphorase staining in rats treated with TGF-beta1. Thus these studies suggest that TGF-beta1 inhibits iNOS in vivo and that TGF-beta1 may be of future benefit in the therapy of septic shock.

Animals↗

PubMed1996

Induction of neuronal nitric oxide after hypothermic circulatory arrest.

BACKGROUND: Although hypothermic circulatory arrest (HCA) has become routine practice in cardiac surgery, it is associated with substantial neurotoxicity. We tested the hypothesis that increased nitric oxide production during HCA participates in neuronal death. We previously described a canine survival model of HCA that produces a consistent neurologic deficit and histopathologic pattern of selective neuronal death. METHODS: Adult male hound dogs (n = 17) were subjected to 2 hours of HCA at a brain temperature of 18 degrees C and reperfused to normothermia; they were sacrificed at various intervals up to 74 hours. Using in vivo cerebral microdialysis, dogs (n = 5) were given a simultaneous infusion of artificial cerebrospinal fluid containing L-[14C]arginine or L-[14C]arginine and L-nitroarginine methyl ester (a nitric oxide synthase inhibitor) in contralateral hemispheres while undergoing 2 hours of HCA and reperfusion to normothermia. RESULTS: L-[14C]citrulline recovery, a coproduct of nitric oxide, significantly increased during HCA in the hemisphere without the inhibitor (at 300 minutes: control, 236 +/- 94 fmol/min versus L-nitroarginine methyl ester, 6 +/- 6 fmol/min; p < 0.05). Citrulline production in vitro from canine cortical homogenates in the presence of calcium (n = 12) was significantly greater 8 and 20 hours after reperfusion (5.11 +/- 0.54 x 10(-7) mmol.mg-1.min-1 and 7.52 +/- 0.59 x 10(-7) mmol.mg-1.min-1, respectively) than before HCA (1.51 +/- 0.09 x 10(-7) mmol.mg-1.min-1; p < 0.05). Nitric oxide metabolites in the serum were also increased significantly early after reperfusion (baseline, 6.72 +/- 0.95 mmol/L; at 4 hours, 17.58 +/- 1.46 mmol/L; p < 0.05). Immunocytochemical staining of the cortex with neuronal nitric oxide synthase-specific monoclonal antibodies (Transduction Labs) revealed increased neuronal nitric oxide synthase expression 6 to 18 hours after HCA. Darkfield analysis demonstrated neuronal nitric oxide synthase localization to neuronal processes with widespread formation of dense plexi of nitric oxide synthase fibers. CONCLUSIONS: We conclude that neurotoxicity after HCA involves a significant, early induction in neuronal nitric oxide synthase expression in neuronal processes leading to widespread augmented nitric oxide production in the brain.

Animals↗

PubMed1996

Transcriptional regulation of iNOS by IL-1 beta in cultured rat pulmonary artery smooth muscle cells.

Transcriptional regulation of iNOS by IL-1 beta in cultured rat pulmonary artery smooth muscle cells. Am. J. Physiol. 271 (Lung Cell. Mol. Physiol. 15): L166-L171, 1996.-Interleukin-1 beta (IL-1 beta) is the critical cytokine affecting peripheral vascular expression of inducible nitric oxide synthase (iNOS). Accordingly, we sought to determine a role for IL-1 beta in stimulating iNOS transcription in cultured rat pulmonary artery smooth muscle cells (RPASMC). Treatment of RPASMC with IL-1 beta caused a concentration-dependent increase in iNOS gene expression by Northern and Western blotting. To demonstrate IL-1 beta-mediated transcriptional activation, we used transient liposome-mediated transfection of RPASMC with promoter-luciferase constructs containing deletional mutations of the murine macrophage iNOS 5' flanking promoter region. IL-1 beta increased promoter activity approximately two- to threefold over baseline in fragments ranging from -1592 (full-length) to -242 bp. Activity was lost, however, when the promoter fragment was shorter than -242 bp. IL-1 beta-mediated increases in steady-state iNOS mRNA were sensitive to pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappa B activation. Nuclear proteins from IL-1 beta-stimulated cells demonstrated PDTC-sensitive binding to an oligonucleotide containing the sequence for the NF-kappa B binding element present in the region between -242 and -42 bp. These data document that IL-1 beta, by itself, increases iNOS expression in RPASMC by transcriptional activation, mediated in part by NF-kappa B.

Animals↗

PubMed1995

Tumor necrosis factor-alpha is required in the protective immune response against Mycobacterium tuberculosis in mice.

Understanding the immunological mechanisms of protection and pathogenesis in tuberculosis remains problematic. We have examined the extent to which tumor necrosis factor-alpha (TNF alpha) contributes to this disease using murine models in which the action of TNF alpha is inhibited. TNF alpha was neutralized in vivo by monoclonal antibody; in addition, a mouse strain with a disruption in the gene for the 55 kDa TNF receptor was used. The data from both models established that TNF alpha and the 55 kDa TNF receptor are essential for protection against tuberculosis in mice, and for reactive nitrogen production by macrophages early in infection. Granulomas were formed in equal numbers in control and experimental mice, but necrosis was observed only in mice deficient in TNF alpha or TNF receptor. TNF alpha and the 55 kDa TNF receptor are necessary conditions for protection against murine M. tuberculosis infection, but are not solely responsible for the tissue damage observed.

Amino Acid Oxidoreductases↗

PubMed1995

Induction of NO synthase in rat cardiac microvascular endothelial cells by IL-1 beta and IFN-gamma.

There are important phenotypic differences between endothelial cells of large vessels and the microvasculature and among microvascular endothelial cells isolated from different tissues and organs. In contrast to most macrovascular endothelial cells, we demonstrate that cultured cardiac microvascular endothelial cells (CMEC) have no detectable constitutive NO synthase (NOS) activity but have a robust increase in NOS activity in response to specific inflammatory cytokines. To determine the identity of the inducible NOS (iNOS) isoform(s) induced by cytokines, we used reverse-transcription polymerase chain reaction techniques to clone and sequence a 217-bp cDNA fragment from CMEC cultures pretreated with interleukin-1 beta (IL-1 beta) and interferon-gamma (IFN-gamma) that was identical to the corresponding portion of the murine macrophage iNOS cDNA. By use of this CMEC iNOS cDNA as a probe in Northern analyses, IL-1 beta, but not IFN-gamma, increased iNOS mRNA content in CMEC, although IFN-gamma markedly potentiated iNOS induction in these cells. In IL-1 beta- and IFN-gamma-pretreated CMEC, dexamethasone only minimally suppressed the rise in iNOS mRNA, protein abundance, or maximal iNOS enzyme activity in whole cell lysates but suppressed nitrite production by 60% in intact CMEC. Dual labeling of cytokine-pretreated CMEC in primary culture with an anti-iNOS antiserum and a fluorescein-labeled lectin specific for the microvascular endothelium of rat heart (GS-1) confirmed the presence of iNOS expression in these cells. iNOS was also detected in microvascular endothelium in situ in ventricular muscle from lipopolysaccharide-, but not sham-injected, rat hearts.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗

PubMed1995

Contractile responsiveness of ventricular myocytes to isoproterenol is regulated by induction of nitric oxide synthase activity in cardiac microvascular endothelial cells in heterotypic primary culture.

Unlike large-vessel endothelial cells in cell culture, cardiac microvascular endothelial cells (CMEC) isolated from adult rat ventricular muscle exhibit little detectable constitutive nitric oxide (NO) synthase activity after isolation in vitro but respond to specific combinations of inflammatory mediators with an increase in inducible NO synthase (iNOS; type 2 NO synthase) activity. CMEC iNOS is induced by soluble inflammatory mediators in lipopolysaccharide-activated rat alveolar macrophage-conditioned medium at 24 hours, and this induction can be partially prevented by either interleukin-1 (IL-1) receptor antagonist or a polyclonal anti-rat tumor necrosis factor-alpha (TNF-alpha) antiserum. Interferon-gamma (IFN-gamma), which by itself does not induce iNOS in CMEC, potentiates and accelerates iNOS induction by IL-1 beta. Transforming growth factor-beta (TGF-beta) decreases iNOS activity, protein content, and mRNA abundance in IL-1 beta- and IFN-gamma-pretreated CMEC. To determine whether NO released by CMEC would affect myocyte contractile function in vitro, freshly isolated ARVM were allowed to settle onto confluent, serum-starved CMEC that had been pretreated for 24 hours with IL-1 beta, a cytokine that alone does not affect myocyte contractile function in vitro. Baseline contractile amplitude, at 2 Hz and 37 degrees C, of myocytes in heterotypic culture with IL-1 beta-pretreated CMEC was not different from that of myocytes in control, homotypic myocyte cultures. However, cocultured myocytes exhibited decreased contractile responsiveness to 2 nmol/L isoproterenol compared with control cells, and this could be reversed by the addition of 1 mmol/L NG-monomethyl-L-arginine, an inhibitor of NOS.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗