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Biomedical subjects

L J Ignarro

Publications and source records attributed to L J Ignarro.

At least 37 records · Page 2Linked to original sources
PubMed2000

Adenoviral gene transfer of endothelial nitric oxide synthase (eNOS) to the penis improves age-related erectile dysfunction in the rat.

Nitric oxide (NO) is the principal mediator of penile erection. NO is synthesized by a variety of nitric oxide synthases (NOS). It has been demonstrated that a decrease in NOS activity, as observed in aging, is associated with a diminished erectile response. The objective of this study was to determine if adenoviral-mediated gene transfer of eNOS could reverse age-related erectile dysfunction in the rat. Two groups of animals were transfected with adenoviruses: (1) aged rats (60 weeks) with AdRSVbetagal; and (2) aged rats (60 weeks) with AdRSVeNOS. Five days after transfection, these study animals underwent cavernosal nerve stimulation (CNS) to assess erectile function and their responses were compared with young (20 weeks) control rats. Cross-sections of the rat penises transfected with AdRSVeNOS were examined after trichrome staining. Adenoviral transduction efficiency of beta-galactosidase reporter gene was measured by a galacto-light chemiluminescent reporter gene assay in cavernosal tissues of rats administered AdRSVbetagal. The transgene expression of eNOS was examined by RT-PCR in rats transfected with AdRSVbetagal and AdRSVeNOS. eNOS and iNOS protein levels were measured by Western blot analysis, and cGMP levels were assessed in cavernosal tissue by enzyme immunoassay. Adenoviral expression of the beta-galactosidase reporter gene was observed in cavernosal tissue for up to 30 days, with peak expression registered at 5 days after intracavernosal administration of AdRSVbetagal. Cross-sections of the rat penises transfected with the AdRSVeNOS revealed no pathological (morphological or histological) changes. Five days after administration of AdRSVeNOS, eNOS protein, mRNA and cGMP levels in the corpora cavernosa were significantly increased (P<0. 05), while iNOS protein levels remained unchanged (P>0.05). In conclusion, enhanced expression of eNOS employing an adenoviral vector significantly increased the erectile response to cavernosal nerve stimulation in the aged rat, similar to the response observed in younger rats. These data suggest that in vivo adenoviral gene transfer of eNOS can physiologically improve erectile function in the aged rat.

Adenoviridae↗
PubMed2000

IL-4 and IL-13 upregulate arginase I expression by cAMP and JAK/STAT6 pathways in vascular smooth muscle cells.

The objectives of this study were to determine whether rat aortic smooth muscle cells (RASMC) express arginase and to elucidate the possible mechanisms involved in the regulation of arginase expression. The results show that RASMC contain basal arginase I (AI) activity, which is significantly enhanced by stimulating the cells with either interleukin (IL)-4 or IL-13, but arginase II (AII) expression was not detected under any condition studied here. We further investigated the signal transduction pathways responsible for AI induction. AI mRNA and protein levels were enhanced by addition of forskolin (1 microM) and inhibited by H-89 (30 microM), suggesting positive regulation of AI by a protein kinase A pathway. Genistein (10 microgramg/ml) and sodium orthovanadate (Na(3)VO(4); 10 microM) were used to investigate the role of tyrosine phosphorylation in the control of AI expression. Genistein inhibited, whereas Na(3)VO(4) enhanced the induction of AI by IL-4 or IL-13. Along with immunoprecipitation and immunoblot analyses, these data implicate the JAK/STAT6 pathway in AI regulation. Dexamethasone (Dex) and interferon (IFN)-gamma were investigated for their effects on AI induction. Dex (1 microM) and IFN-gamma (100 U/ml) alone had no effect on basal AI expression in RASMC, but both reduced AI induction by IL-4 and IL-13. In combination, Dex and IFN-gamma abolished AI induction by IL-4 and IL-13. Finally, both IL-4 and IL-13 significantly increased RASMC DNA synthesis as monitored by [(3)H]thymidine incorporation, demonstrating that upregulation of AI is correlated with an increase in cell proliferation. Blockade of AI induction by IFN-gamma, H-89, or genistein also blocked the increase in cell proliferation. These observations are consistent with the possibility that upregulation of AI might play an important role in the pathophysiology of vascular disorders characterized by excessive smooth muscle growth.

Animals↗
PubMed2000

Gastrointestinal motility during pregnancy: role of nitrergic component of NANC nerves.

This study evaluated whether increased release of nitric oxide (NO) from the nitrergic component of the nonadrenergic, noncholinergic (NANC) nerves may be partly responsible for the decrease in gastrointestinal motility observed during pregnancy. Segments of fundal strip, ileum, and colon were obtained from nonpregnant rats, rats in midpregnancy (days 9-11), and rats in late pregnancy (days 18-20). NANC activity was studied by assessing changes in tone after application of electric field stimulation (EFS). The role of NO was determined by observing the effects of EFS in the presence and absence of the NO synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) and the reversibility of the effects of L-NAME by L-arginine. The magnitude of change in cGMP levels in the tissues after application of EFS was also assessed. Our studies indicate that there was increased magnitude of relaxation of isolated strips of rat gastric fundus and rat colon, after application of EFS to tissues obtained only from animals in late pregnancy. These results paralleled the changes in cGMP levels in tissues. NOS activity in the gastric fundus was significantly increased in animals in late pregnancy compared with nonpregnant controls. Our studies suggest that the delay in gastric emptying and increase in colonic transit time observed in rats during pregnancy may be caused in part by increased activity of the nitrergic component of the NANC nerves innervating these organs.

Acetylcholine↗
PubMed1999

Gene transfer of endothelial nitric oxide synthase to the penis augments erectile responses in the aged rat.

Nitric oxide (NO), a mediator involved in penile erection, is synthesized by the nitric oxide synthase (NOS) family of enzymes. It has been shown that NOS activity decreases with age. To determine whether adenoviral-mediated overexpression of endothelial NOS (eNOS) could enhance erectile responses, we administered a recombinant adenovirus containing the eNOS gene (AdCMVeNOS) into the corpora cavernosum of the aged rat. Adenoviral expression of the beta-galactosidase reporter gene was observed in cavernosal tissue 1 day after intracavernosal administration of AdCMVbetagal; 1 day after administration of AdCMVeNOS, transgene expression was confirmed by immunoblot staining of eNOS protein, and cGMP levels were increased. The increase in cavernosal pressure in response to cavernosal nerve stimulation was enhanced in animals transfected with eNOS, and erectile responses to acetylcholine and zaprinast were enhanced at a time when the erectile response to the NO donor sodium 1-(N,N-diethylamino)diazen-1-ium-1,2-diolate was not altered. These results suggest that in vivo gene transfer of eNOS, alone or in combination with a type V phosphodiesterase inhibitor, may constitute a new therapeutic intervention for the treatment of erectile dysfunction.

Acetylcholine↗
PubMed1999

Nitric oxide inhibits ornithine decarboxylase by S-nitrosylation.

Ornithine decarboxylase (ODC) is the initial enzyme in the polyamine synthetic pathway, and polyamines are required for cell proliferation. We have shown previously that nitric oxide (NO) inhibits ODC activity in Caco-2 cells and in crude cell lysate preparations. In this study we examined the mechanism by which NO inhibits the activity of purified ODC. NO, in the form of S-nitrosocysteine (CysNO), S-nitrosoglutathione (GSNO), or 1, 1-diethyl-2-hydroxy-2-nitroso-hydrazine (DEA/NO), inhibited enzyme activity in a concentration-dependent manner. CysNO (1 microM) inhibited ODC activity by approximately 90% and 3 microM GSNO by more than 70%. DEA/NO was less potent, inhibiting enzyme activity by 70% at a concentration of 30 microM. Inhibition of enzyme activity by CysNO, GSNO, or DEA/NO was reversible by addition of dithiothreitol or glutathione. Cuprous ion (Cu (I)) also reversed the inhibitory effect of these NO donor agents. The data presented here support the hypothesis that NO inhibits ODC activity via S-nitrosylation of a critical cysteine residue(s) on ODC.

Catalytic Domain↗
PubMed1999

Increased nitric oxide concentrations in posterior hypothalamus and central sympathetic function on nitrate tolerance following subcutaneous nitroglycerin.

The present study was to examine the distributions of nitric oxide (NO) in the brain regions and peripheral vessels following subcutaneously administered nitroglycerin (NTG) and determine the noradrenergic activity and the role of central sympathetic function in acute nitrate tolerance. Tolerance to NTG was produced by subcutaneous (sc) administration of 4.0 mg NTG as four separate hourly pulse injections of 1.0 mg each in male (5-8 months) Sprague-Dawley rats. Rats in sham-treated group received sc injections of saline. Rats were killed by sodium pentobarbital (150 mg/kg, ip) at 10 min after last sc injection. The brain, gracilis muscle, aorta, superior mesenteric artery, coronary artery, and pulmonary vessels were quickly removed. Concentrations of nitrite (NO2-), nitrate (NO3-), and total NO2- plus NO3- (NOx-) were quantified in the micropunches of the anterior hypothalamus, the posterior hypothalamus (PH), the nucleus tractus solitarius, the lateral reticular nucleus, and the vessels in a blinded fashion. The central actions of acute tolerance to NTG were also determined using blockades of sympathetic functions in conscious rats. Four separate hourly pulse sc injections of 1.0 mg NTG produced a marked shift of the dose-response curve for arterial pressure depression induced by intravenous injection of the challenge doses of NTG. The same doses of sc NTG caused increases in NOx- [92+/-16% (mean +/- SE)] and NO3- productions (77+/-15%) in the PH, but did not significantly change in other brain regions (n = 6). NOx- and NO3- productions were significantly enhanced in the superior mesenteric artery, aorta, coronary artery, and pulmonary vessels following sc NTG, but were not altered in gracilis muscle by the treatment. The tolerance responses to arterial pressure depression were attenuated by intravenous administration of either prazosin (300 microg/kg), an alpha1-adrenoceptor antagonist, or chlorisondamine (10 mg/kg), a sympathetic ganglion blockading agent (n = 5-6). The results suggest that acute NTG tolerance predominately increases NO production in the PH. NO production was also markedly enhanced in the large and middle vessels but not in small vessels during acute NTG tolerance. The arterial pressure tolerance to NTG was reversed by blockade of central sympathetic function. We conclude that NO formation is increased in the PH following systemically administered NTG and NO in the PH may facilitate central sympathetic functions which contribute to nitrate tolerance.

Animals↗
PubMed1999

Effects of estrogen replacement therapy on plasma levels of nitric oxide in postmenopausal women.

OBJECTIVE: Our purpose was to assess the effects of estrogen replacement therapy on plasma levels of nitric oxide in postmenopausal women. STUDY DESIGN: The study, designed as a randomized, double-blind placebo-controlled crossover trial, involved 28 healthy postmenopausal women who had previously undergone hysterectomy. Women received either transdermal estradiol (50 g/day) (estradiol group) or placebo (placebo group) for 6 months continuously. At the end of month 6 the treatment allocations were opened, and then the treatments were exchanged for 1 month. The serum concentration of estradiol was measured at baseline before treatment and at the end of months 6 and 7. The plasma concentration of the stable oxidation products of nitric oxide was assessed before treatment and monthly until month 7. RESULTS: The mean baseline concentrations of nitric oxide metabolites in the estradiol and placebo groups were similar (mean and SD: 19+/-4.3 vs 21+/-5.6 micromol/L, respectively). At subsequent measurements from months 1 to 6, the mean concentration of nitric oxide metabolites increased significantly in the estradiol group alone, in which the concentration ranged between 33 6.4 and 36 8.5 micromol/L. At the end of month 7 the mean level of nitric oxide metabolites in women previously treated with estradiol fell to baseline value (19 2.6 micromol/L), whereas in the placebo group the level increased significantly (34 4.4 micromol/L). CONCLUSION: Estrogen replacement therapy induces a sustained increase in plasma levels of nitric oxide in postmenopausal women; the suspension of estrogen replacement therapy is followed by a significant reduction in nitric oxide levels. The results of this study suggest that a nitric oxide-related mechanism may help to explain the cardioprotective effect of estrogen replacement therapy in the postmenopausal period.

Administration, Cutaneous↗
PubMed1999

Different plasma levels of nitric oxide in arterial and venous blood.

Experimental investigations suggest that a basal release of nitric oxide (NO) occurs in arterial but not in venous endothelium. We therefore decided to compare plasma levels of NO in the arterial and venous circulation. Parallel blood samples were drawn from the radial artery and brachial vein in 15 healthy drug-free women. Nitric oxide levels were assessed by measuring plasma levels of nitrite and nitrate, the two stable oxidation products of NO metabolism. Plasma levels of NO metabolites in arterial blood were significantly higher than in the paired venous blood samples (45.1 +/- 17.7 versus 22.5 +/- 8.5 mumol l-1, respectively, mean +/- SD). The results of this preliminary study strongly suggest that the endothelial release of NO is probably different in arteries and veins in vivo; this is also consistent with previous literature indicating that basal release of NO occurs mainly from the endothelium of arteries but not from that of veins.

Arteries↗
PubMed1999

Nitric oxide as a signaling molecule in the vascular system: an overview.

In retrospect, basic research in the fields of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) during the past two decades appears to have followed a logical course, beginning with the findings that NO and cGMP are vascular smooth muscle relaxants, that nitroglycerin relaxes smooth muscle by metabolism to NO, progressing to the discovery that mammalian cells synthesize NO, and finally the revelation that NO is a neurotransmitter mediating vasodilation in specialized vascular beds. A great deal of basic and clinical research on the physiologic and pathophysiologic roles of NO in cardiovascular function has been conducted since the discovery that endothelium-derived relaxing factor (EDRF) is NO. The new knowledge on NO should enable investigators in this field to develop novel and more effective therapeutic strategies for the prevention, diagnosis, and treatment of numerous cardiovascular disorders. The goal of this review was to highlight the early research that led to our current understanding of the pathophysiologic role of NO in cardiovascular medicine. Furthermore, we discussed the possible mechanism of some drugs interfering with NO signaling cascade.

Animals↗
PubMed1998

Antisense knockdown of inducible nitric oxide synthase inhibits induction of experimental autoimmune encephalomyelitis in SJL/J mice.

We used an antisense oligodeoxynucleotide (ODN) complementary to inducible nitric oxide synthase (iNOS) to inhibit experimental autoimmune encephalomyelitis (EAE) in female SJL/J mice, an animal model for multiple sclerosis. The antisense ODN was administered intraventricularly to mice daily for 10 days beginning at the time of adoptive transfer of myelin basic protein-specific T lymphocytes. The antisense ODN treatment significantly reduced the clinical score of EAE and blocked iNOS mRNA and protein synthesis, as well as iNOS enzyme activity within the central nervous system. The levels of nitric oxide and cyclic guanosine monophosphate were also significantly reduced by the antisense ODN treatment. Neither sense nor random ODN affected clinical EAE or iNOS expression. Moreover, the protein and enzyme activity level of constitutive neuronal nitric oxide synthase was not affected by the antisense ODN. Thus, we have shown that the iNOS antisense ODN specifically blocked iNOS expression and ameliorated the induction of EAE.

Animals↗
PubMed1998

Certain S-substituted isothioureas not only inhibit NO synthase catalytic activity but also decrease translation and stability of inducible NO synthase protein.

In an attempt to identify potent inhibitors of inducible (type II) NO synthase (iNOS) for use in cell culture systems, we found that two S-substituted isothioureas were very potent in cell culture but one such compound also interfered with the induction of NO synthase. S-Ethylisothiourea (EITU) and S-aminoethylisothiourea (AEITU) were found to be much more potent than NG-methylarginine, NG-nitroarginine methy lester, or aminoguanidine as inhibitors of NO production by cultured RAW 264.7 cell macrophages activated by lipopolysaccharide (LPS). The approximate EC50 values as inhibitors of NO production, assessed by 24-h accumulation in cell culture media, were 10 microM (EITU), 30 microM (AEITU), 300 microM (NG-methylarginine), and 1000 microM (aminoguanidine). EITU was found to inhibit NO production by activated macrophages without interfering with the induction of iNOS. More specifically, EITU failed to influence transcription of iNOS mRNA (Northern blot analysis), translation of iNOS protein (pulse experiments), or degradation of translated iNOS protein (pulse-chase experiments). In contrast, however, AEITU interfered markedly with the induction of iNOS by mechanisms attributed to inhibition of translation of iNOS mRNA into functional protein as well as acceleration of degradation of already translated iNOS protein. These observations indicate that AEITU should not be used in cell culture experiments where the intent is solely to assess the consequences of inhibition of iNOS catalytic activity.

Animals↗
PubMed1998

Effects of short-term transdermal estradiol administration on plasma levels of nitric oxide in postmenopausal women.

OBJECTIVE: To assess the effects of short-term transdermal E2 administration on nitric oxide (NO) plasma levels in postmenopausal women. DESIGN: Randomized, placebo-controlled trial. SETTING: Healthy volunteers in an academic research environment. PATIENT(S): Twenty-eight healthy postmenopausal women. INTERVENTION(S): Transdermal administration of E2 (100 microg/d) or placebo on days 1 and 4 of a 1-week treatment regimen. MAIN OUTCOME MEASURE(S): Serum concentrations of E2 and plasma concentrations of NO stable oxidation products were assessed on day 1, before placement of the patch, and subsequently on days 2, 3, and 6. RESULT(S): The mean concentration of NO metabolites on days 2, 3, and 6 was significantly greater in the E2 group (40.08+/-15.42 micromol/L, 38.05+/-18.82 micromol/L, and 42.03+/-16.81 micromol/L on days 2, 3, and 6, respectively) compared with both baseline levels (23.07+/-5.79 micromol/L) and the placebo group (23.51+/-4.06 micromol/L, 21.64+/-4.72 micromol/L, and 21.81+/-4.46 micromol/L on days 2, 3, and 6, respectively). CONCLUSION(S): During a 1-week treatment regimen with transdermal E2, plasma levels of NO in postmenopausal women were significantly higher than baseline levels on days 2, 3, and 6. This suggests that the effect of estrogens on NO synthesis is rapid and that it is maintained with repeated administration.

Administration, Cutaneous↗
PubMed1998

NG-hydroxy-L-arginine and nitric oxide inhibit Caco-2 tumor cell proliferation by distinct mechanisms.

The objective of this study was to elucidate the role and mechanism of nitric oxide (NO) synthase (NOS) in modulating the growth of the Caco-2 human colon carcinoma cell line. The two novel observations reported here are, first, that NG-hydroxy-L-arginine (NOHA) inhibits Caco-2 tumor cell proliferation, likely by inhibiting arginase activity, and, second, that NO causes cytostasis by mechanisms that might involve inhibition of ornithine decarboxylase (ODC) activity. Both arginase and ODC are enzymes involved in the conversion of arginine to polyamines required for cell proliferation. Cell growth was monitored by cell count, cell protein analysis, and DNA synthesis. NOHA (1-30 microM) and NO in the form of DETA/NO (1-30 microM) inhibited cell proliferation by 30-85%. The cytostatic effect of NOHA was prevented by addition of excess ornithine, putrescine, spermidine, or spermine to cell cultures, whereas the cytostatic effect of NO (DETA/NO) and alpha-difluoromethylornithine (ODC inhibitor) was unaffected by ornithine but was prevented by putrescine, spermidine, or spermine. The cytostatic effect of NOHA appeared to be independent of its conversion to NO, and the effect of NO appeared to be independent of cGMP. NOHA inhibited urea production by Caco-2 cells and inhibited arginase catalytic activity (85% at 3 microM), whereas NO (DEA/NO and SNAP) inhibited ODC activity (>/=60% at 30 microM) without affecting arginase activity. Coculture of Caco-2 cells with lipopolysaccharide/cytokine-activated rat aortic endothelial cells markedly slowed Caco-2 cell proliferation, and this was blocked by NOS inhibitors. These observations that NOHA and NO may inhibit sequential steps in the arginine-polyamine pathway suggest a novel biological role for NOS in the inhibition of cell proliferation of certain tumor cells and possibly other cell types.

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

Inducible nitric-oxide synthase and nitric oxide production in human fetal astrocytes and microglia. A kinetic analysis.

The understanding of the induction and regulation of inducible nitric-oxide synthase (iNOS) in human cells may be important in developing therapeutic interventions for inflammatory diseases. In the present study, we not only demonstrated that human fetal mixed glial cultures, as well as enriched microglial cultures, synthesize iNOS and nitric oxide (NO) in response to cytokine stimulation, but also assessed the kinetics of iNOS and NO synthesis in human fetal mixed glial cultures. The iNOS mRNA was expressed within 2 h after stimulation and decreased to base line by 2 days. Significant levels of iNOS protein appeared within 24 h after stimulation and remained elevated during the culture period. A dramatic increase in NO production and NO-mediated events, such as the induction of cyclic guanosine monophosphate (cGMP), NADPH diaphorase activity, and nitrotyrosine occurred 3 days after stimulation, a delay of 48 h from the time of the first expression of iNOS enzyme. This delay of NO production was altered by the addition of tetrahydrobiopterin, but not by the addition of L-arginine, heme, flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), or NADPH. These findings suggest that a post-translational regulatory event might be involved in iNOS-mediated NO production in human glia.

Astrocytes↗
PubMed1997

Evidence for the presence of an unusual nitric oxide- and citrulline-producing enzyme in rat kidney.

We have found an enzymatic activity obtained from rat kidney capable of producing citrulline and NOx. (nitrate and nitrite) which was resistant to inhibition by conventional arginine analogues. This enzyme activity does not require any calcium or calmodulin and was found to be induced during pregnancy. This unique enzyme was found to be tissue and species specific. Another unique feature of this enzyme is that it did not bind to 2'5'-ADP-sepharose under standard conditions. Western blot analysis of the 100,000 g kidney supernatant using monoclonal antibody for macrophage inducible nitric oxide synthase failed to produce a band for inducible nitric oxide synthase. HPLC and capillary ion analysis for nitrate and nitrite (NOx) showed clear peaks for [3H] L-citrulline and NOx, respectively, which were not changed either in the absence of calcium and calmodulin or in the presence of 300 microM S-ethylisothiourea, which has been shown to be a very potent and selective inhibitor of inducible nitric oxide synthase with a Ki of about 14.7 nM. These results suggest the possible existence of another isoform of nitric oxide synthase with very distinct properties from the known isoforms.

Animals↗