Nitric Oxide Synthase Activity Assay Research Articles

Molecular characterization of the nitric oxide synthase gene and its immunomodulation of nitric oxide production in the triangle shell mussel (Hyriopsis cumingii)

Nitric oxide synthase (NOS) is a critical enzyme that catalyzes nitric oxide biosynthesis and orchestrates various immunological responses mediated by nitric oxide (NO) in host animals. In this study, the NOS gene was identified in the triangle shell mussel (Hyriopsis cumingii) (HcNOS). HcNOS was highly conserved in the characteristic gene structures of NOS. Phylogenetic analysis suggested that HcNOS was a typical invertebrate NOS. Further gene expression analysis, NOS activity assays and nitric oxide content measurements demonstrated the inducibility of HcNOS in responses to lipopolysaccharide (LPS) challenge and during tissue transplantation. Of note, mantle grafting induced a prolonged HcNOS/NO response, suggesting that through the HcNOS/NO system, multiple immunomodulators may play decisive roles in tissue grafting in triangle shell mussels. Thus, HcNOS appears to be a crucial player in responding to both bacterial infection and tissue transplantation.

Isolation of Antibacterial, Nitrosylmyoglobin Forming Lactic Acid Bacteria and Their Potential Use in Meat Processing.

The use of nitrite as a colorant and preservative in meat processing is associated with health risks. This study aimed to isolate nitrite-substituting lactic acid bacteria for use as natural biological colorants and preservatives. Among the 106 strains isolated from fermented foods, two strains with excellent ability to convert myoglobin and metmyoglobin (Met-Mb) to red nitrosylmyoglobin (Mb-NO) were selected. The superior ability to form Mb-NO was confirmed through UV-visible spectrophotometry, Fourier transform infrared spectrometry, electron spin resonance analysis, nitric oxide synthase activity assay, and Met-Mb reductase activity assay. The potent antibacterial activity was confirmed through biofilm and cytomembrane breakage of the indicator bacteria. Though performing 16S rDNA sequencing, they were identified as two different strains of Lactobacillus plantarum. Based on their favorable characteristics, their applications in the meat industry were further evaluated. This study identified a novel dual-function natural biological colorant and preservative to substitute nitrite in meat products. The application of the two strains would decrease the hazardous of nitrite to health.

Arginase activity in mitochondria – An interfering factor in nitric oxide synthase activity assays

Previously, in tightly controlled studies, using three independent, yet complementary techniques, we refuted the claim that a mitochondrial nitric oxide synthase (mtNOS) isoform exists within pure, rat liver mitochondria (MT). Of those techniques, the NOS-catalyzed [ 14C]- l-arginine to [ 14C]- l-citrulline conversion assay (NOS assay) with MT samples indicated a weak, radioactive signal that was NOS-independent [1]. Aliquots of samples from the NOS assays were then extracted with acetone, separated by high performance thin-layer chromatography (HPTLC) and exposed to autoradiography. Results obtained from these samples showed no radioactive band for l-citrulline. However, a fast-migrating, diffuse, radioactive band was observed in the TLC lanes loaded with MT samples. In this manuscript, we identify and confirm that this radioactive signal in MT samples is due to the arginase-catalyzed conversion of [ 14C]- l-arginine to [ 14C]-urea. The current results, in addition to reconfirming the absence of NOS activity in rat liver MT, also show the need to include arginase inhibitors in studies using MT samples in order to avoid confounding results when using NOS activity assays.

Neuroprotection via inhibition of nitric oxide synthase by bis(7)-tacrine

Here we report that bis(7)-tacrine, a novel acetylcholinesterase inhibitor, exerts neuroprotective effects by inhibition of nitric oxide synthase. In cortical neurons at 12 days in vitro, bis(7)-tacrine concentration-dependently reduced cell death induced by glutamate, beta-amyloid and L-arginine, but not by nitric sodium nitroprusside. N-monomethyl-L-arginine, a nitric oxide synthase inhibitor, also prevented the former three types but not the last type of the cytotoxicity; however, nitric oxide scavengers blocked all of these insults, indicating that nitric oxide mediated these neuronal injuries. Furthermore, with nitric oxide synthase activity assays, it was found that bis(7)-tacrine not only suppressed the activation of nitric oxide synthase caused by glutamate in cortical neurons, but also directly inhibited the activity of nitric oxide synthase in vitro.

Noncholinergic penile erection in mice lacking the gene for endothelial nitric oxide synthase.

With the current understanding that nitric oxide (NO) mediates penile erection, the endothelial isoform of NO synthase (eNOS) has been implicated in this function. We undertook this study applying transgenic mice with targeted deletion of the eNOS gene (eNOS-/- mice) as an experimental approach to evaluate the importance of eNOS in cholinergically stimulated erectile function in vivo. Combined pharmacostimulation with intracavernosal carbachol (3 ng) administration and submaximal cavernous nerve (CN) electrical stimulation (16 Hz, 5 millisecond, 1 V) simultaneous with intracavernosal pressure (ICP) monitoring, and both biochemical assay of NO synthase activity and Western blot analysis of eNOS protein content in penile tissue, were performed on eNOS-/- mice and wild-type controls. Combined intracavernosal carbachol administration and submaximal CN electrical stimulation raised the recorded ICP, elicited by CN electrical stimulation alone in wild-type mice (from 35.7 +/- 2.7 to 48.1 +/- 5.5 mm Hg, P < .05) but not in eNOS-/ - mice (from 54.9 +/- 6.3 to 51.0 +/- 9.5 mm Hg, not significant [NS]). Pretreatment with the nonselective nitric oxide synthase inhibitor nitro-L-arginine methyl ester (L-NAME; 100 mg intracavernosally) blocked electrically stimulated ICP responses in eNOS-/- mice to baseline levels (37.8 +/- 4.4 vs 12.7 +/- 4.0 mm Hg, P < .05). In penes of eNOS-/- mice, approximately 60% NO synthase activity of wild-type penis levels was retained (NS), and eNOS protein was absent. We concluded that eNOS-/- mice preserve erectile function on the basis of a noncholinergic but NO-dependent mechanism and that eNOS physiologically mediates penile erection under cholinergic stimulation.

Citrulline is not the major product using the standard "NOS activity" assay on renal cortical homogenates.

A standard approach to assessing nitric oxide synthase (NOS) activity in tissue homogenates is 1) removal of small-molecular-weight substances by size-exclusion chromatography, 2) adding back of substrates/cofactors in precise concentrations with a radioactive isotope of arginine (Arg), and 3) quantification of labeled citrulline (Cit) after separation of Arg and Cit by cation-exchange column chromatography. Using this approach and L-[2,3-3H]Arg, we found that the major product(s) was not Cit in cortical homogenates prepared from rat, mouse, and human kidneys. The product(s) mimicked Cit, insofar as it passed freely through cation-exchange columns and comigrated with Cit on both one-dimensional and two-dimensional straight-phase thin-layer chromatography systems. However, it was clearly resolved from Cit by precolumn derivatization and reverse-phase HPLC. The maximum velocity and Michaelis-Menten constant were approximately 100 pmol x mg protein(-1) x min(-1) and 100 microM, respectively, in renal cortical homogenates from rats. The enzyme activity was the same in the presence or absence of cofactors including Ca2+, calmodulin, tetrahydrobiopterin, and NADPH. It was only modestly inhibited by L-Arg analogs and was mainly in the supernatant after a 100,000 g centrifugation. These enzyme characteristics contrasted markedly with those simultaneously obtained for NOS activity in placental homogenates. Thus results from the conventional NOS activity assay should be viewed cautiously.

New Mechanisms for Inhaled NO

New Mechanisms for Inhaled NO

Functional and molecular characterization of nitric oxide synthase in the endometrium and myometrium of pregnant sheep during the last third of gestation

Objective: This study was undertaken to characterize the biochemical and expression profiles of the nitric oxide synthase isoforms present in the sheep uterus during late gestation. Study Design: Myometrium and endometrium were obtained from 28 time-mated pregnant sheep that were under halothane general anesthesia. Tissues were kept frozen at –80°C until they were homogenized for the measurement of (1) nitric oxide synthase activity according to the carbon 14–labeled arginine-citrulline conversion assay, (2) nitric oxide synthase protein mass according to Western blot analysis, and (3) nitric oxide synthase messenger ribonucleic acid according to the ribonuclease protection assay. The nitric oxide synthase activity assay included 8 parallel treatments for biochemical characterization, in particular with the arginase inhibitors ornithine and (+)-S-2-amino-5-iodoacetamidopentanoic acid. Results: The biochemical characterization of nitric oxide synthase indicated that the predominant form of nitric oxide synthase in endometrium and myometrium (80%-90%) was calcium-calmodulin dependent. In endometrium 50% of reduced nicotinamide adenine dinucleotide–dependent arginine metabolism was accounted for by the presence of alternative arginine metabolic pathways. Expressions of type 1 and type 3 nitric oxide synthase were demonstrated in endometrium and myometrium by Western blot and ribonuclease protection assay. Although no significant decrease in nitric oxide synthase activity or protein mass was observed, a significant decrease in myometrial type 1 nitric oxide synthase messenger ribonucleic acid occurred in sheep not in labor at 140 days’ gestation ( P < .05 by analysis of variance; term is 144 ± 5 days). Conclusion: In the gravid sheep uterus the predominant nitric oxide synthase isoforms are type 1 in myometrium and type 3 in endometrium. Despite a decrease in type 1 nitric oxide synthase messenger ribonucleic acid, enzymatic activity and type 1 nitric oxide synthase protein mass do not decrease before parturition. (Am J Obstet Gynecol 1999;181:116-25.)

Endogenous nitric oxide suppresses rat myometrial connexin 43 gap junction protein expression during pregnancy.

Nitric oxide (NO) synthase (NOS) is active in the gravid uterus, and its activity decreases prior to the onset of parturition. We tested the hypothesis that NO helps maintain uterine quiescence by suppressing the expression of genes necessary for parturition. Pregnant rats (18 days gestation) were treated with inducible NOS (iNOS) inhibitor N-iminoethyl-L-lysine (NIL) or endothelial NOS inhibitor nitro-L-arginine methyl ester (L-NAME); 24 h later, uteri were analyzed for myometrial connexin 43 (Cx43) protein by immunoblotting and mRNA by Northern analysis. Myometrial oxytocin receptors (OTR) were measured by radioligand binding, and decidual prostaglandin H synthase (PGHS) protein by immunoblotting. Uterine NOS blockade was verified by NOS activity assay. We found that NIL, but not L-NAME, significantly increased myometrial Cx43 protein to parturitional levels with treatment at 19 but not 17 days gestation. Steady state mRNA concentrations were not changed at 24 h. NOS inhibition did not increase the concentrations of OTR, or PGHS protein, nor did it decrease maternal serum progesterone. We conclude that endogenous uterine NO from iNOS suppresses myometrial Cx43 gap junction protein expression during rat pregnancy. Although the exact mechanism is unknown, an increase of uterine wall stretch due to inhibition of relaxation could account for increased Cx43 gene transcription.

Portal hypertension triggers local activation of inducible nitric oxide synthase gene in colonic mucosa

Recently a new clinical entity “portal hypertensive colopathy” has been reported. It involves vascular abnormalities and bleeding. Because nitric oxide may mediate these changes, we studied whether portal hypertension affects nitric oxide synthase in portal hypertensive colonic mucosa. In portal hypertensive and sham-operated rats the following studies were done: (1) colonic mucosal blood flow, (2) quantitative histologic examination, (3) reverse transcription—polymerase chain reaction for nitric oxide synthase mRNA, (4) nitric oxide synthase activity assay, and (5) immunostaining for nitric oxide synthase. In portal hypertensive rats, colonic mucosal blood flow and the number of submucosal veins were significantly increased in comparison to sham-operated rats. The mRNA expression and enzyme activity for inducible nitric oxide synthase (but not constitutive nitric oxide synthase) were significantly increased in portal hypertensive rats. Fluorescence signal intensity for inducible nitric oxide synthase in endothelia of mucosal and submucosal veins was significantly higher in portal hypertensive rats than in sham-operated rats. Portal hypertension activates inducible nitric oxide synthase gene and protein in colonic mucosal vessels. The excess of nitric oxide generated by overexpressed inducible nitric oxide synthase may play an important role in the development of vascular and hemodynamic abnormalities characterizing portal hypertensive colopathy.

Cloning of rat and human inducible penile nitric oxide synthase. Application for gene therapy of erectile dysfunction.

Erectile dysfunction is mainly due to the inability of the cavernosal smooth muscle of the penis to undergo complete relaxation. In the aging rat model, erectile dysfunction is accompanied by a reduction of penile smooth muscle compliance and, in very old animals, by a decrease in penile nitric oxide synthase (NOS), which is responsible for the synthesis of the mediator of penile erection, nitric oxide (NO). We have investigated whether the stimulation of penile NOS expression by local induction or gene therapy can mitigate erectile dysfunction in the aged rat. A mix of iNOS (inducible NOS) inducers was continuously delivered to the penises of 5- ("adult"), 20- ("old"), and 30- ("very old") mo-old rats for 3-6 days, and the erectile response to electrical field stimulation of the cavernosal nerve was measured. The erectile dysfunction observed in old and very old rats as compared to adult animals was ameliorated by treatment with iNOS inducers. Penile iNOS was detectable in the penis of these rats by Western blot, NADPH diaphorase, and NOS activity assays. Inducible NOS was inducible in vitro in both rat and human corpora cavernosal tissue and in rat penile smooth muscle cells (RPSMC), as shown by Western blots. However, NO synthesis in cavernosal tissue upon iNOS protein induction remained low, indicating that the increased NOS levels were under physiological control. The iNOS cDNA was cloned from induced RPSMC mRNA and generated by reverse transcriptase polymerase chain reaction (RT-PCR) from induced human penile smooth muscle cells and corporal tissue. The coding regions from both the rat (RPiNOS) and human (HPiNOS) penile iNOS showed several amino acid differences from their analogous isoform in nonpenile tissues. RPiNOS cDNA injected into the penis mitigated the aging-associated erectile dysfunction. The iNOS construct was detected in cavernosal tissue by PCR, and its expression by RT-PCR and Western blots. These results open the way for the possible use of NOS isoforms in the management of erectile dysfunction.

Vasoactive intestinal peptide (VIP)-induced enzyme secretion in rat pancreatic tissue is not associated with activation of nitric oxide synthase (NOS) and increase in cyclic GMP level

Nitric oxide (NO) is thought to be a second messenger involved in secretion. Upon stimulating pancreatic acinar cells with cholecystokinin- pancreozymin (CCK-PZ), NO formation has been shown to be associated with increased levels of cGMP (Seoet al., 1995). To elucidate the signaling pathway of VIP-induced enzyme secretion, we investigated the NO and cGMP synthesis steps as potential steps where two signal pathways triggered by CCK-PZ and VIP interact. The results obtained in this work provide evidence that increase in pancreatic enzyme secretion by treatment with VIP has no relationship with NOS activity and cGMP level. This conclusion was derived from the following findings that VIP treatment of rat pancreatic tissue increased amylase release as well as protein output in a dose- and time-dependent manner, whereas NOS activity and cGMP synthesis were not affected by VIP treatment as monitored by NOS activity assay and determining cGMP level, which was further confirmed by a NOS-inhibitor study. Consequently, CCK-PZ or VIP increases enzyme secretion in rat pancreatic tissue, but the two hormones are different in their mode of action. Together the results suggest that signaling pathway of VIP-induced enzyme secretion might either bypass the NO and cGMP synthesis steps or lie on a distinct pathway from CCK-PZ-induced pathway.