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

Pulmonary arterial microvascular smooth muscle function governs many aspects of lung physiology and pathophysiology. Acutely, microvascular smooth muscle cells (SMC) modulate pulmonary vascular resistance; chronically, they contribute to vascular remodeling. Recent work has also suggested a possible immune function for pulmonary smooth muscle through cytokine-stimulated nitric oxide production. To facilitate study of the mechanisms underlying these functions, we have developed methods for isolating pulmonary arterial microvessels from the rat and culturing SMC from these vessels. The pulmonary arterial circulation was filled with a suspension of iron oxide in agar, and a subpleural tissue sample was obtained. The vessels were cleared of surrounding lung parenchyma by partial collagenase digestion, and the iron-containing arteries were separated magnetically. The diameter of the harvested arteries confirmed an intraacinar origin, and the cultured cells expressed smooth muscle isoforms of alpha-actin and myosin but did not take up acetylated low density lipoprotein. To assess a possible immune effector role for these cells, confluent monolayers were stimulated with cytokines and endotoxin. At 24 h, immunofluorescent staining for inducible nitric oxide synthase was prominent within these cells. Nitric oxide production, as measured by nitrite levels in the cell-conditioned medium, was also markedly elevated but reduced by adding NG-monomethyl-L-arginine. We conclude that rat pulmonary arterial microvascular SMC can be obtained by the iron oxide infusion method and that these cells express an inducible nitric oxide synthase after cytokine stimulation.
Am J Respir Cell Mol Biol 1994 Jun
PMID:Culture of pulmonary microvascular smooth muscle cells from intraacinar arteries of the rat: characterization and inducible production of nitric oxide. 751 71

Expression of nitric oxide synthase (NOS) was investigated in neurons of lumbar spinal cord of adult rats following subcutaneous injection of formalin (FOR) in one hindpaw. NOS was visualized immunocytochemically using a specific antibody and by the NADPH-diaphorase reaction (NDP). In the untreated rat, NOS immunoreactivity (IR) and NDP were present in neurons of the superficial dorsal horn (sDH) predominantly in layers II-III, and in the deep dorsal horn (dDH) predominantly in layer X. Twenty-four hours following FOR, the numbers of neurons labelled for NOS and NDP and the density of NDP containing nerve fiber varicosities significantly increased in sDH of the ipsilateral L3-L4 segments. NOS-IR and NDP gave a rather congruent distribution of labelled neurons in the dorsal horn. In contrast, distinct NOS-IR but not NDP was visible in large diameter motoneurons and in the lateral spinal nucleus. Double labelling demonstrated that in sDH most of the NDP-reactive neurons show a close spatial relationship to fibers and varicosities immunoreactive for substance P and CGRP. These neuropeptides are considered mediators of synaptic input from nociceptive primary afferents. Colocalization of NDP with c-Jun, JunB, JunD, c-Fos, FosB and Krox-24 transcription factors was investigated in neurons of lumbar spinal cord. c-Jun, JunB, c-Fos and Krox-24 reached their maximal levels of expression 2 h after FOR and returned to basal levels after 10 h. FosB and JunD reached their maximal expression after 5 h, persisted up to 10 h and were still visible in 60%-70% of the maximal number of labelled nuclei after 24 h. This persistent expression of transcription factors might contribute to the up-regulation of NOS expression between 10 h and 24 h. In a low number of NDP neurons, suprabasal immunoreactivity of JunB, c-Fos and Krox-24 proteins was visible up to 10 h, and of JunD and FosB up to 24 h in sDH neurons; c-Jun was not expressed in NDP labelled neurons of sDH, but, similar as JunD, showed basal colocalization in preganglionic sympathetic and parasympathetic neurons. In dDH, colocalization of Jun, Fos and Krox-24 proteins in few neurons was only observed following a second FOR stimulus given 24 h after the first one. Double-staining also demonstrated that many Jun, Fos and Krox labelled neurons are in close proximity to NDP labelled nerve fibers suggesting a functional relationship between expression of immediate-early gene encoded transcription factors and presence of nitric oxide in the rat spinal cord.
Brain Res Mol Brain Res 1994 Mar
PMID:Expression of nitric oxide synthase and colocalisation with Jun, Fos and Krox transcription factors in spinal cord neurons following noxious stimulation of the rat hindpaw. 751 94

The expression of mRNA for the calmodulin-dependent form of brain nitric oxide synthase (NOS) was examined in cholinergic cells of the rat brain using a method combining in situ hybridization histochemistry with immunocytochemistry for choline acetyltransferase (ChAT) in the same brain sections. We constructed a riboprobe specific for brain NOS by subcloning a 493 bp fragment of the coding region which displayed low homology to other forms of NOS. The general distribution of NOS mRNA was in excellent agreement with previous studies using the full-length probe or NADPH diaphorase histochemistry. NOS mRNA was observed in many brain structures and relative levels were quantitated using grain counting procedures in a number of cholinergic and non-cholinergic neuronal groups throughout the brain. In the forebrain, ChAT-immunoreactive cells or cell groups were observed in medial septum (MS), vertical limbs of diagonal band (DBV) and horizontal limbs of diagonal band (DBH), nucleus basalis magnocellularis (NBM), substantia innominata (SI), and striatum (ST). In the brainstem, the cholinergic groups studied included those located in the pedunculopontine tegmental nucleus (PPTN), the laterodorsal tegmental nucleus (LDTN), the nucleus parabigeminalis and several motor nuclei. For NOS mRNA quantitation, silver grains overlying ChAT-stained neuronal profiles in sections on emulsion-dipped slides were counted digitally. In the LDTN and PPTN, virtually all the ChAT-positive cells expressed NOS mRNA at high levels. In MS, DBV and SI, about 30-50% of the ChAT-positive cells expressed NOS mRNA at low-to-moderate levels. Less than 20% of ChAT-positive neurons in the other cholinergic populations studied expressed NOS mRNA; the NBM was one of these low-expressing populations. Many scattered non-cholinergic cells expressing NOS mRNA were found in the striatum and cerebral cortex. In other non-cholinergic regions, high NOS mRNA expression was observed in the islands of Calleja, thalamic and hypothalamic nuclei, several amygdaloid nuclei, regions related to the optic tract, the interpeduncular nucleus, and the supramammillary nucleus. The heterogeneous distribution of NOS mRNA implies complex roles for nitric oxide neurotransmission in brain function, including for the cholinergic phenotype. Additionally, given the postulated involvement of nitric oxide in neurodegeneration, the widely varying levels of expression of NOS within identified central cholinergic neurons may relate to differential vulnerability of this phenotype in disease or aging.
Brain Res Mol Brain Res 1994 Apr
PMID:Nitric oxide synthase gene expression in cholinergic neurons in the rat brain examined by combined immunocytochemistry and in situ hybridization histochemistry. 751 28

Two major roles have been defined for nitric oxide (NO): cell-cell communication mediated by the stimulation of cyclic guanosine 3',5'-monophosphate (cGMP) synthesis and cytotoxicity by direct or indirect interaction of the free radical NO with cellular targets. Thus, pathologic states might result from an alteration of NO pathways, e.g., by deregulated activity of NO synthase. To investigate this hypothesis, we introduced the murine-inducible NO synthase (iNOS) sequence into immortalized human bronchial epithelial cells (BEAS-2B). iNOS activity, measured by conversion of [14C]arginine to [14C]citrulline in the presence of 1 mM EGTA, was higher than 100 pmol/min/mg protein in early passages of iNOS-transfected cells but decreased with cell subculturing. No iNOS activity could be detected in control vector-transfected cells. NO stimulated cGMP production in iNOS-transfected cells, and this effect was inhibited by the iNOS inhibitor NG-monomethyl-L-arginine. In addition, NO production induced c-fos expression and did not interfere with clonal cell growth. These results suggest that BEAS-2B cells constitute a suitable model to study the consequences of iNOS activity on signal transduction pathways in bronchial epithelium.
Am J Respir Cell Mol Biol 1994 Aug
PMID:Constitutive expression of inducible nitric oxide synthase in human bronchial epithelial cells induces c-fos and stimulates the cGMP pathway. 751 34

Nitric oxide is a highly reactive molecule that has been implicated in host defense and tissue injury. In the present studies, we determined whether rat type II alveolar epithelial cells have the capacity to produce this mediator. We found that type II cells synthesize significant quantities of nitric oxide after treatment with the inflammatory cytokines, interferon-gamma (IFN-gamma) and/or interleukin-1 beta (IL-1 beta), or with the combination of IFN-gamma and tumor necrosis factor-alpha. In contrast to rat alveolar macrophages, type II cells were unresponsive to lipopolysaccharide. Production of nitric oxide by type II cells in response to IFN-gamma was dose dependent, reaching a maximum at 100 U/ml, and blocked by NG-monomethyl-L-arginine (L-NMA), a nitric oxide synthase inhibitor. Northern blot analysis demonstrated that nitric oxide production by type II cells was due to expression of mRNA for an inducible form of nitric oxide synthase (iNOS). Following brief exposure of rats to irritant-inducing doses of ozone (2 ppm, 3 h), type II cells were found to produce significantly more nitric oxide than were cells from control animals. This was due to increased expression of iNOS mRNA. Cells from ozone-treated rats were also sensitized to produce more nitric oxide in response to IFN-gamma and IL-1 beta. This was associated with a marked increase in expression of iNOS mRNA and enzyme protein in the cells. We also found that ozone inhalation caused enhanced production of hydrogen peroxide, as well as spontaneous and IFN-gamma-induced cytostasis of type II cells toward P815 mouse mastocytoma cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Am J Respir Cell Mol Biol 1994 Aug
PMID:Production of nitric oxide by rat type II pneumocytes: increased expression of inducible nitric oxide synthase following inhalation of a pulmonary irritant. 751 35

Rat neurotensin (NT) receptor (NTR) cDNA was subcloned into the pRC-CMV expression vector and transfected into 293 cells, and cellular clones that stably expressed the NTR were isolated and characterized. [3H]NT binding to membranes prepared from the NTR cDNA-transfected cells displayed specificity and saturability, with an apparent Kd of 1.25 nM and a Bmax of 43.4 pmol/mg of protein (approximately 3.5 x 10(6) binding sites/cell). NT stimulated an increase in [3H]inositol phosphate levels in the NTR-expressing cells up to 2500% of basal levels. The response was time and dose dependent, with an EC50 of 10.4 nM. NT also stimulated cAMP formation in these cells, with an EC50 of 27.0 nM. In addition, NT evoked an increase in the level of intracellular calcium. Approximately 60% of the calcium rise was attributable to the release of intracellular stores and 40% was attributable to calcium influx. Although NTR occupancy has been shown to stimulate cGMP formation in several brain preparations and cell lines, NT was unable to mediate cGMP synthesis in the NTR-expressing 293 cells. We found that 293 cells have guanylate cyclase activity but have undetectable levels of nitric oxide synthase (NOS) activity. Because it was possible that the production of nitric oxide is required as the mediator of NT-induced cGMP synthesis, we subcloned NOS cDNA into the pCEP4 expression vector and transiently expressed it in the NTR cells. We report that NT increased cGMP levels up to 375% of basal levels when NOS cDNA was coexpressed and that the increase was completely inhibited by the NOS inhibitor N omega-nitro-L-arginine. NT-induced cGMP accumulation was time and dose dependent, with an EC50 of 1.7 nM. To our knowledge, this is the first report of NT mediating cGMP formation with a cloned receptor and the first evidence that NT-induced cGMP accumulation requires the production of nitric oxide.
Mol Pharmacol 1994 Jul
PMID:The cloned neurotensin receptor mediates cyclic GMP formation when coexpressed with nitric oxide synthase cDNA. 752 Jan 23

Rat aortic smooth muscle cells produced large quantities of nitric oxide (NO) after exposure to interleukin-1 beta, and this was depressed in the presence of the protein kinase C inhibitor bisindolylmaleimide. Intracellular cAMP levels were elevated mildly in cytokine-treated smooth muscle cells, and the presence of forskolin enhanced both the cAMP levels and NO production. Inhibition of GTP:cyclohydrolase I by 2,4-diamino-6-hydroxypyrimidine attenuated NO production by interleukin-1 beta-treated cells. GTP:cyclohydrolase is the regulatory enzyme for de novo tetrahydrobiopterin synthesis, and the latter is a required cofactor for NO synthase activity. Treatment of smooth muscle cells with forskolin induced GTP:cyclohydrolase mRNA expression, and simultaneous treatment of cells with forskolin and phorbol esters elicited NO production. Angiotensin II and arginine-vasopressin, acknowledged agonists for protein kinase C, elicited production of NO by forskolin-treated smooth muscle cells. These observations confirm the importance of GTP:cyclohydrolase activity for NO production by cultured smooth muscle cells and implicate both adenylyl cyclase and protein kinase C in this process.
Mol Pharmacol 1994 Aug
PMID:Simultaneous activation of adenylyl cyclase and protein kinase C induces production of nitric oxide by vascular smooth muscle cells. 752 13

Alveolar macrophages (AM) exposed to cytokines or bacterial lipopolysaccharide (LPS) produce the free radical nitric oxide (NO.) by an inducible nitric oxide synthase (iNOS). They also release reactive oxygen free radicals following exposure to silica dust. The purpose of the present study was to determine whether NO. is produced by rat AM and/or recruited leukocytes following the intratracheal (IT) instillation of silica. Male Sprague-Dawley rats (175 to 225 g) were IT instilled with either silica dust (10 mg/100 g body wt) or LPS (0.25 mg/100 g body wt). After 24 h, bronchoalveolar lavage cells (BALC) and lavaged lung tissue were assayed for iNOS mRNA. Cell counts of BALC and iNOS-dependent (N omega-nitro-L-arginine methyl ester [L-NAME]-inhibitable) chemiluminescence generated by AM were also determined. Northern blot analysis demonstrated that the steady-state levels of BALC iNOS mRNA were significantly increased by 3-fold following IT silica and by 7-fold following IT LPS. Partially enriched fractions of either AM or leukocytes from silica-treated rats both exhibited significantly elevated iNOS mRNA in Northern analysis. iNOS-dependent chemiluminescence was significantly increased in AM by 36-fold following IT silica and by 89-fold following IT LPS. Differential counts of BALC showed that AM numbers did not change in any of the treatments; however, red blood cells increased by 30-fold following IT silica and by 23-fold following IT LPS. Total leukocytes (polymorphonuclear leukocytes plus lymphocytes) increased by 58-fold following IT silica and by 274-fold following IT LPS.(ABSTRACT TRUNCATED AT 250 WORDS)
Am J Respir Cell Mol Biol 1994 Oct
PMID:Intratracheal instillation of silica up-regulates inducible nitric oxide synthase gene expression and increases nitric oxide production in alveolar macrophages and neutrophils. 752 85

Acute hypoxia causes pulmonary hypertension in the fetus and newborn that is contrasted by systemic hypotension or normotension. To better understand the role of nitric oxide (NO) in this specific pulmonary vascular response, we determined the acute effects of decreased oxygenation on NO production in ovine fetal pulmonary and systemic (mesenteric) endothelial cells. NO was assessed by measuring cGMP accumulation in fetal vascular smooth muscle (VSM) cells during co-culture incubations of endothelium and VSM (40 s) in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine. Changes in cGMP were dependent on the endothelium and on NO synthase and guanylate cyclase activity. At high O2 (680 mm Hg), basal NO was detectable and NO increased 6- to 10-fold with bradykinin or A23187. In pulmonary endothelium, basal NO fell 58% at pO2 = 150 mm Hg and 51% at 40 mm Hg versus 680 mm Hg, while NO with bradykinin fell 56% and 63%, respectively. NO with A23187, however, was unchanged at 150 mm Hg, but it fell 56% at 40 mm Hg. In contrast, in systemic endothelium basal and stimulated NO production were not altered at lower O2. Findings were similar using pulmonary or systemic detector VSM cells, and exogenous L-arginine had no effect. Thus, decreased O2 acutely attenuates NO production specifically in fetal pulmonary endothelial cells. This process is not related to changes in O2 or L-arginine availability as substrates for NO synthase; alternatively, it may be partially mediated by specific effects of O2 on pulmonary endothelial cell calcium homeostasis.
Am J Respir Cell Mol Biol 1994 Oct
PMID:Oxygen modulates nitric oxide production selectively in fetal pulmonary endothelial cells. 752 86

Epidermal keratinocytes (EK) are exposed to multiple inflammatory stimuli and paracrine factors secreted by various dermal cells (lymphocytes, mast-cells, macrophages, fibroblasts) during wounding, cutaneous allergy and infections. We have previously demonstrated that following stimulation with interleukin-4 (IL-4) or interferon-gamma, human EK express the low affinity receptor for IgE (Fc epsilon RII/CD23) on their surface. In the present study, we showed that the ligation of CD23 by IgE/anti-IgE immune complexes or specific monoclonal antibody, induces a dose-dependent release of interleukin-6 and tumor necrosis factor-alpha from EK. CD23-ligation activates the nitric oxide-dependent pathway, as demonstrated by the high levels of nitrites released in cell supernatants, and the accumulation of intracellular cyclic nucleotides in EK. These second messengers are required for IgE-dependent stimulation of cytokine production by these cells, as this is completely abolished by cAMP or NO synthase antagonists. Human epithelial keratinocytes may thus participate in IgE-mediated immune responses, through their ability to express functional CD23 antigen.
Cell Mol Biol (Noisy-le-grand) 1994 May
PMID:IgE-dependent activation of Fc epsilon RII/CD23+ normal human keratinocytes: the role of cAMP and nitric oxide. 924 2


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>