Independent Nitric Oxide Synthase Research Articles

The potential mediation of nitric oxide in the activation of mitochondrion-dependent apoptosis and yak meat tenderness during postmortem aging

Nitric oxide (NO) may play a critical role in the regulation of various physiological and pathophysiological processes due to its effect on the apoptosis pathway. However, few reports have studied the effects of NO on apoptosis and yak meat tenderization. The present study was conducted to explore the molecular mechanisms of Ca2+-independent inducible NO synthase isozyme (iNOS)-derived NO in mitochondrial apoptosis and meat tenderization during postmortem aging. Results indicated that the aminoguanidi hydrochloride (AH) significantly reduced iNOS activities and NO production. Moreover, the inhibitory property of iNOS-derived NO dramatically decreased mitochondrial oxidative stress degree, which further reduced the production of mitochondrial Ca2+ and ATP. The inhibitory property of iNOS-derived NO also influenced the Bcl-2 family members, mitochondrial apoptotic cascade reaction, and meat tenderization by protecting against the degradation of myofibrils and the damage of mitochondrial microstructure. These results demonstrated that the iNOS-derived NO possessed a significant role in the mitochondrial apoptosis pathway and yak meat tenderization during aging.

Са2+- and NF-κB-dependent generation of NO in the photosensitized neurons and satellite glial cells

Photodynamic therapy (PDT) is used for killing of malignant cells in tumors including brain cancer. It can also damage normal neurons and glial cells. Nitric oxide (NO) is known to control PDT-induced cell death. To study the mechanisms that regulate NO generation in photosensitized neurons and glial cells, we used a simple model object - isolated crayfish mechanoreceptor that consists of a single sensory neuron surrounded by glial cells. PDT induced NO generation in glial cells, neuronal dendrites, and, less, in soma and axon. Using modulators of the cytosolic Ca2+ level and nuclear factor-kappa B (NF-κB) activity, we showed that Ca2+ and NF-κB regulate NO generation in the photosensitized neurons and glia. Actually, NO production was stimulated by 4-fold cadmium chloride (CdCl2) concentration in the saline, Ca2+ ionophore ionomycine, or inhibition of Ca2+-ATPase in the endoplasmic reticulum by 2,5-ditert-butylbenzene-1,4-diol (tBuBHQ). Oppositely, CdCl2 or nifedipine, blockers of Ca2+ channels in the plasma membrane, decreased NO generation. NO production was also inhibited by S-methylthiouronium sulfate (SMT), inhibitor of Ca2+-independent inducible NO synthase. SMT also prevented the stimulation of PDT-induced NO generation by NF-κB activator prostratin. This suggests the involvement of both Ca2+-dependent neuronal NO synthase and Ca2+-independent inducible NO synthase, which is regulated by NF-κB, in NO production in the crayfish neurons and glia.

Low-Dose Endotoxin Induces Late Preconditioning, Increases Peroxynitrite Formation, and Activates STAT3 in the Rat Heart

Administration of low-dose endotoxin (lipopolysaccharide, LPS) 24 h before a lethal ischemia induces pharmacological late preconditioning. The exact mechanism of this phenomenon is not clear. Here we aimed to investigate whether low-dose LPS exerts late effects on peroxynitrite formation and activation of Akt, Erk, and STAT3 in the heart. Male Wistar rats were injected with LPS (S. typhimurium; 0.5 mg/kg i.p.) or saline. Twenty-four hours later, hearts were isolated, perfused for 10 min, and then used for biochemical analyses. LPS pretreatment enhanced cardiac formation of the peroxynitrite marker 3-nitrotyrosine. LPS pretreatment also increased cardiac levels of the peroxynitrite precursor nitric oxide (NO) and superoxide. The activities of Ca2+-independent NO synthase and xanthine oxidoreductase increased in LPS-pretreated hearts. LPS pretreatment resulted in significantly enhanced phosphorylation of STAT3 and non-significantly increased phosphorylation of Akt without affecting the activation of Erk. In separate experiments, isolated working hearts were subjected to 30 min global ischemia and 20 min reperfusion. LPS pretreatment significantly improved ischemia-reperfusion-induced deterioration of cardiac function. We conclude that LPS pretreatment enhances cardiac peroxynitrite formation and activates STAT3 24 h later, which may contribute to LPS-induced late preconditioning.

Role of nitric oxide and nitrosative protein damage in cigarette smoke-induced emphysematous lung damage: Attenuation by vitamin C

Emphysema, is a common inflammatory lung disease caused by cigarette smoking involving extensive structural and functional damage of the lung, often causing death. Here, we demonstrate the role of the enzyme, nitric oxide synthase (NOS) and its biologically active product, nitric oxide (NO) in cigarette smoke-induced inflammatory lung damage using a guinea pig model of emphysema as well as human lung cells and deploying established immunoblotting, immunohistochemical and spectroscopic techniques. Our results indicate that cigarette smoke induces severe inflammatory changes in the lung, leading to over-expression of the inducible (iNOS) and endothelial (eNOS) isoforms of NOS as well as its product, NO, which reacts with superoxide ( O 2 ) to form peroxynitrite ( ONOO - - ) , triggering extensive nitration of lung proteins. Nitration renders lung proteins susceptible to proteolytic damage by endogenous proteases causing structural damage to the lung. Such proteolysis of nitrated proteins is also associated with increased apoptosis/necrosis of lung cells as well as lung alveolar airspace characteristic of emphysema. L-NIL, a specific iNOS inhibitor, arrested the observed protein nitration and lung damage indicating the predominant role of iNOS in such nitrosative lung damage. We also found that vitamin C, a potent dietary antioxidant can attenuate the observed cigarette smoke-induced nitrosative lung damage by scavenging the responsible NO, O 2 and ONOO− species. Overall, our work indicates the predominant role of iNOS and NO in the inflammatory lung damage implicated with cigarette smoke-induced emphysema and the potential of vitamin C to attenuate such damage. We will use this new mouse model to investigate the role of BH4-dependent and independent NOS functions in models of vascular function, disease and inflammation.

The effect of N-nitro-L-arginine and aminoguanidine treatment on changes in constitutive and inducible nitric oxide synthases in the spinal cord after sciatic nerve transection

Ca2+-dependent and Ca2+-independent nitric oxide synthase (NOS) activity, and neuronal and inducible NOS immunoreactivity (nNOS-IR and iNOS-IR), were investigated in the rabbit lower lumbar spinal cord after i) sciatic nerve transection and survival of experimental animals for 2 weeks, ii) treatment of animals with N-nitro-L-arginine (NNLA), an inhibitor of nNOS dosed at 20 mg/b.w. for 12 days, and iii) after treatment of animals with the inducible NOS (iNOS) inhibitor, aminoguanidine, dosed at 100 mg/b.w. for 4 and 12 days. Our attention was focused on the dorsal part of L4-L6 segments receiving sensory inputs from the sciatic nerve, and on the ventral part consisting of sciatic nerve motor neurons. Sciatic nerve transection increased Ca2+-dependent NOS activity and the density of nNOS in the dorsal part of the spinal cord on the ipsilateral side. NNLA treatment effectively reduced nNOS-IR in both the dorsal horn and the dorsal column, and decreased Ca2+-dependent NOS activity in the lower lumbar segments. Immunocytochemical analysis disclosed the up-regulation of iNOS immunoreactive staining after peripheral axotomy in alpha-motoneurons. The changes in iNOS expression and Ca2+-independent NOS activity were not significantly corrected by aminoguanidine treatment for 4 days. Long-lasting iNOS inhibition decreased Ca2+-independent NOS activity, but caused motor neuron degeneration and mediated small necrotic foci in the ventrolateral portion of the ventral horn. The results of the present study provide evidence that constitutive NOS inhibition by NNLA is more effective than specific long-lasting inhibition of iNOS by aminoguanidine treatment.

Infiltration of Inflammatory Cells Plays an Important Role in Matrix Metalloproteinase Expression and Activation in the Heart during Sepsis

Septicemia is an emerging pathological condition involving, among other effects, refractory hypotension and heart dysfunction. Here we have investigated the contribution of resident nonmyocytic cells to heart alterations after lipopolysaccharide administration. These cells contributed to the rapid infiltration of additional inflammatory cells that enhance the onset of heart disease through the release of inflammatory mediators. Early activation of resident monocytic cells played a relevant role on the infiltration process, mainly of major histocompatibility complex class II- and CD11b-positive cells. This infiltration was significantly impaired in animals lacking the nitric-oxide synthase-2 (NOS-2) gene or after pharmacological in-hibition of NOS-2 or cylooxygenase-2, suggesting a significant contribution of nitric oxide and prostanoids to the infiltration process. Under these conditions, the expression of NOS-2 and cylooxygenase-2 in the whole organ was attenuated because cardiomyocytes failed to express these enzymes. However, cardiomyocytes expressed and activated matrix metalloproteinase-9 through mechanisms regulated, at least in part, by nitric oxide and prostaglandins in an additive way. These results directly link the inflammatory response in the heart and extracellular matrix remodeling by the matrix metalloproteinases released by the cardiomyocytes, suggesting that activation and recruitment of inflammatory cells to the heart is a major early event in cardiac dysfunction promoted by septicemia.

Airway hyperresponsiveness induced by repetitive intraperitoneal injection of lipopolysacharide and the involvement of inflammation and nitric oxide in guinea pigs

Airway hyperresponsiveness (AHR) is involved in bronchial asthma and chronic obstructive pulmonary disease (COPD) and produces respiratory symptoms. Lipopolysaccharide (LPS) has been found to be significantly related to the severity of asthma. However, its clinical mechanism still remains controversial. This study investigated the in vivo effect of repetitive intraperitoneal administration of lipopolysaccharide (LPS) on airway hyperresponsiveness (AHR) in guinea pigs and the possible involvement of inflammation, nitric oxide (NO) and nitric oxide synthase (NOS). There were two exposure groups for intraperitoneal LPS injection: (1) LPS was given at a dose of 1 mg x kg(-1), followed by sterile saline (NS) 1 ml x kg(-1) 8 h later every 24 h; (2) LPS was given at a dose of 0.5 mg x kg(-1) two times with an interval of 8 h every 24 h. Each exposure regime was repeated 4 times. Control animals were given NS and 6 naive guinea pigs were used as baseline control. Determinations were made 24 h after each exposure. Persistent AHR occurred 24 h after the third and fourth exposures to LPS in the first exposure group (at one dose), but occurred earlier after the exposures to LPS in the second exposure group (at divided doses). The numbers of total cells and neutrophils were elevated initially but subsided subsequently in LPS-treated groups. No evidence of morphological changes in the small airways was found 24 h after any of the exposures. The Ca(2+)-dependent and Ca(2+)-independent NOS activities (mainly produced by iNOS) in the BALF, as well as the production of NO, were significantly elevated 24 h after any of third and fourth exposures in LPS-treated groups. Our results demonstrate that repetitive intraperitoneal LPS can induce persistent AHR which occurs earlier when the frequency of injection increase, and an elevation of NO production and iNOS activity may be involved in is systemic-LPS-induced AHR.

Transient expression of NOS-II during development of the murine enteric nervous system

In the enteric nervous system, nitric oxide (NO) is regarded as an important messenger for the non-adrenergic and non-cholinergic neurotransmission. Synthesized mainly by the constitutive nitric oxide synthase (NOS) isoforms NOS I and NOS III, this molecule exerts prejunctional inhibitory effects in the submucosal plexus as well as relaxation of enteric smooth muscles. In order to elucidate the role for NO during enteric development, we looked for the expression of all three NOS-isoforms in the enteric nervous system during mouse development from E8 to E20 using immunohistochemistry. Starting around midgestation, a transient expression of the NOS-II isoform during the very early development of enteric neurones was detected in parallel to that of HNK-1 exclusively in the myenteric plexus. Similar to findings for other neuronal systems, NOS-I and NOS III isoforms could be traced starting significantly later to increase toward the end of embryonic development when NOS II immunoreactivity faded and a strong expression of the vasointestinal peptide could be detected. In contrast to the NOSII expression, the constitutive isoforms can also be detected in the submucosal plexus. Altogether, these findings suggest NOS-II to be exclusively involved during early steps of enteric nervous system development. Absence of downstream signalling elements, such as sGC and cGMP both in neurons and in enteric muscle until the end of the second third of gestation, may indicate different effects executed by NO during development, expressed by Ca(2+) -dependent and Ca(2+) -independent NOS isoforms.

Salutary properties of YC-1 in the cardiovascular and hematological systems.

The soluble guanylate cyclase (sGC)/cyclic guanosine monophosphate (cGMP) second messenger system provides a complex and highly regulated mechanism for signal transduction events and ensuing functional responses through a cascade of serine/threonine protein kinase-dependent pathways. Nitric oxide (NO) and carbon monoxide (CO), two unique diatomic gases endogenously produced by the respective enzymes nitric oxide synthase (NOS) and heme oxygenase (HO), stimulate cellular sGC and synthesize cGMP within the vasculature. Emerging evidence suggests that the independent NOS and HO systems provide reciprocal and complimentary approaches that act to regulate cardiovascular and hematological homeostasis as well as provide protection to the vasculature in response to inimical stimuli or following the onset of vasoproliferative disease. Recent results from our laboratory and others suggest that the newly identified, chemically synthesized benzyl indazole derivative YC-1 is capable of exerting multifunctional and broad-ranging effects in the cardiovascular and hematological systems. YC-1 has been demonstrated to possess redundant biochemical mechanisms that confer significant stimulation upon NO- and CO-regulated, cyclase-dependent events. Ultimately, these acute molecular processes eventuate in YC-1-dependent modulation of platelet and vascular smooth muscle cell (SMC) and endothelial cell (EC) function under both eutrophic and deleterious conditions. Based on accumulating evidence, YC-1 has been suggested to serve as a potential therapeutic adjuvant to be used in interventional medicine, and these results may indicate the existence of an endogenous " YC-1-like" compound that would be the focus of much anticipated investigation. The purpose of this review, therefore, is to provide update information on the mechanisms and physiologic and pathophysiologic roles of the pivotal new multifunctional agent YC-1 in the cardiovascular and hematological systems, and to provide evidence for its potential use as a clinically relevant salutary agent.

Cortical calcium increase following traumatic brain injury represents a pitfall in the evaluation of Ca 2+-independent NOS activity

In this report, our findings highlighted the presence of a high level of calcium in the cortex following traumatic brain injury (TBI) in a rat model of fluid percussion-induced brain injury. This calcium increase represents a pitfall in the assessment of Ca 2+-independent nitric oxide synthase (NOS) activity supposed to play a role in the secondary brain lesion following TBI. The so-called Ca 2+-independent NOS activity measured in the injured cortex 72 h after TBI had the pharmacological profile of a Ca 2+-dependent NOS and was therefore inhibited with a supplement of calcium chelator. The remaining activity was very low and iNOS protein was hardly immunodetected on the same sample used for NOS activity assay. The concentration of calcium chelator used in the assay should be revised and adjusted consequently to make sure that the calcium-free condition is achieved for the assay. Otherwise, the findings tend towards an overestimation of Ca 2+-independent and underestimation of Ca 2+-dependent NOS activities. The revised Ca 2+-independent NOS activity assay was then tested, in relation with the amount of iNOS protein, in a model of LPS-induced neuroinflammation. Taken together, precautions should be taken when assessing the Ca 2+-independent enzymatic activity in cerebral tissue after a brain insult.

Cortical calcium increase following traumatic brain injury represents a pitfall in the evaluation of Ca2+-independent NOS activity

In this report, our findings highlighted the presence of a high level of calcium in the cortex following traumatic brain injury (TBI) in a rat model of fluid percussion-induced brain injury. This calcium increase represents a pitfall in the assessment of Ca2+-independent nitric oxide synthase (NOS) activity supposed to play a role in the secondary brain lesion following TBI. The so-called Ca2+-independent NOS activity measured in the injured cortex 72 h after TBI had the pharmacological profile of a Ca2+-dependent NOS and was therefore inhibited with a supplement of calcium chelator. The remaining activity was very low and iNOS protein was hardly immunodetected on the same sample used for NOS activity assay. The concentration of calcium chelator used in the assay should be revised and adjusted consequently to make sure that the calcium-free condition is achieved for the assay. Otherwise, the findings tend towards an overestimation of Ca2+-independent and underestimation of Ca2+-dependent NOS activities. The revised Ca2+-independent NOS activity assay was then tested, in relation with the amount of iNOS protein, in a model of LPS-induced neuroinflammation. Taken together, precautions should be taken when assessing the Ca2+-independent enzymatic activity in cerebral tissue after a brain insult.

Nitric oxide voltammetric measurements in the rat brain after gamma irradiation.

The effects of a lethal gamma irradiation were investigated on cerebral NO-ergic system by using a voltammetric method in freely moving rats. It is reported that the cortical NO concentration increases right from the end of the radiation exposure (15 Gy) and reaches a maximal magnitude (+120%) 24 h later. A dose-effect relationship from 2 to 15 Gy for gamma-ray exposure has also been observed. The effects, obtained with either an NO synthase inhibitor nonselective for the different NO synthase isoforms or an NO synthase inhibitor selective for the constitutive isoform, suggest that the radiation-induced increase in NO is likely to be dependent on the inducible NO synthase isoform. Moreover, experiments performed under ex vivo conditions showed that the cortical mRNA level for Ca(++)-independent NO synthase, the brain NOS activity, and urinary nitrites/nitrates increased significantly 24 h after gamma-ray exposure. These results demonstrate that a supralethal whole-body irradiation alters the NO-ergic pathways. The increase in NO obtained under such conditions might constitute a good index of central nervous system radiosensitivity during the acute phase of the radiation syndrome.

Hyperlipidemia induced by a cholesterol-rich diet leads to enhanced peroxynitrite formation in rat hearts.

We investigated the influence of experimental hyperlipidemia on the formation of cardiac NO, superoxide, and peroxynitrite (ONOO(-)) in rat hearts. Wistar rats were fed 2% cholesterol-enriched diet or normal diet for 8 weeks. Separate groups of normal and hyperlipidemic rats were injected twice intraperitoneally with 2 x 20 micromol/kg FeTPPS (5,10,15,20-tetrakis-[4-sulfonatophenyl]-porphyrinato-iron[III]), a ONOO(-) decomposition catalyst, 24 h and 1 h before isolation of the hearts. A cholesterol diet significantly decreased myocardial NO content, however, myocardial Ca(2+)-dependent and Ca(2+)-independent NO synthase activity and NO synthase protein level did not change. Myocardial superoxide formation and xanthine oxidase activity were significantly increased; however, cardiac superoxide dismutase activity did not change in the cholesterol-fed group. Dityrosine in the perfusate, a marker of cardiac ONOO(-) formation, and plasma nitrotyrosine, a marker for systemic ONOO(-) formation, were both elevated in hyperlipidemic rats. In cholesterol-fed rats, left ventricular end-diastolic pressure (LVEDP) was significantly elevated as compared to controls. Administration of FeTPPS normalized LVEDP in the cholesterol-fed group. We conclude that cholesterol-enriched diet-induced hyperlipidemia leads to an increase in cardiac ONOO(-) formation and a decrease in the bioavailability of NO which contributes to the deterioration of cardiac performance and may lead to further cardiac pathologies.

Increased matrix metalloproteinase activity after canine cardiopulmonary bypass is suppressed by a nitric oxide scavenger

Objectives: We tested whether nitric oxide scavenging with a ruthenium-based compound (AMD6221) would improve hemodynamics and alter nitric oxide synthase and matrix metalloproteinase activities in a canine model of cardiopulmonary bypass. Methods: Dogs were randomized to either cardiopulmonary bypass (n = 12) or control (n = 12) groups. They were further randomized to receive a continuous infusion of AMD6221 or placebo. Cardiopulmonary bypass was maintained for 90 minutes, and then, 4 hours later, dogs were killed. Cardiac, lung, and brain sections were snap frozen in liquid nitrogen for determination of nitric oxide synthase, matrix metalloproteinase 2, and matrix metalloproteinase 9 activities. Results: After cardiopulmonary bypass, 3 of 6 placebo-treated (cardiopulmonary bypass-placebo) and 0 of 6 AMD6221-treated (cardiopulmonary bypass-6221) animals required phenylephrine infusion to maintain a predetermined blood pressure (P <.05). Total fluid administration was lower in the cardiopulmonary bypass-6221 group compared with that in the cardiopulmonary bypass-placebo group (983 ± 134 vs 1617 ± 254 mL, respectively; P <.005). After cardiopulmonary bypass, matrix metalloproteinase 2 and matrix metalloproteinase 9 activities in the lung, left ventricle, and left atrium were decreased in the cardiopulmonary bypass-6221 group compared with that in the cardiopulmonary bypass-placebo group (P <.05). Ca2+-independent nitric oxide synthase activity and matrix metalloproteinase 2 activity in the brain were also lower (P <.05) in the cardiopulmonary bypass-SCV group. Finally, neutrophil expression of CD18, an adhesion complex, was lower at 4 hours after cardiopulmonary bypass in the cardiopulmonary bypass-6221 group compared with that in the cardiopulmonary bypass-placebo group (38 ± 27 vs 81 ± 11; P <.05). Conclusions: We found that (1) infusion of an nitric oxide scavenger, AMD6221, was associated with improved predefined hemodynamics; (2) cardiopulmonary bypass increased activities of Ca2+-independent nitric oxide synthase and matrix metalloproteinases in multiple organs; and (3) AMD6221 could ameliorate the increased generation of nitric oxide and increased matrix metalloproteinase activities.J Thorac Cardiovasc Surg 2003;125:661-8

Enhanced NO and superoxide generation in dysfunctional hearts from endotoxemic rats.

Free radicals have been implicated in the etiology of cardiac dysfunction during sepsis, but the actual species responsible remains unclear. We studied the alterations in myocardial nitric oxide (NO), superoxide, and peroxynitrite generation along with cardiac mechanical function and efficiency in hearts from lipopolysaccharide (LPS)-treated rats. Six hours after LPS (4 mg/kg ip) or saline (control) treatment, hearts were isolated and perfused for 1 h with recirculating Krebs-Henseleit buffer and paced at 300 beats/min. Cardiac work, O(2) consumption, and cardiac efficiency were markedly depressed in LPS hearts compared with controls. Plasma nitrate/nitrite level was elevated in LPS rats, and ventricular NO production was enhanced as measured by electron spin resonance spectroscopy, Ca(2+)-independent NO synthase (NOS) activity, and inducible NOS immunohistochemistry. Ventricular superoxide production was also enhanced in LPS-treated hearts as seen by lucigenin chemiluminescence and xanthine oxidase activity. Increased nitrotyrosine staining (immunohistochemistry) and higher lipid hydroperoxides levels were also detected in LPS-treated hearts, indicating oxygen radical-induced stress. Enhanced generation of both NO and superoxide, and thus peroxynitrite, occur in dysfunctional hearts from endotoxemic rats.

Ca2+-Independent Activity of Nitric Oxide Synthase

Ca2+-independent forms of nitric-oxide synthase have significant activity when the endogenous calmodulin subunit is Ca2+ free. Further activation is seen when Ca2+ is added. We have examined the activation of a Ca2+-independent nitric-oxide synthase variant and its two point mutants that are more dependent on Ca2+ for activation using mutant calmodulins containing non-functional Ca2+-binding sites. These studies provide evidence that the Ca2+-independent activity of these enzymes can be exerted through specific adapted interactions between the enzyme and the Ca2+-binding site 2 of calmodulin. Further, the results suggest that EGTA-sensitive metals other than Ca2+ complexed to calmodulin may be involved in maximal activation of these nitric-oxide synthase variants.

Primary spontaneous coronary artery dissections in atherosclerotic patients

Clonidine, clinically used in the treatment of hypertension, is a central α2-adrenergic agonist that reduces blood pressure and slows heart rate by reducing sympathetic stimulation. Considering the structural similarity between clonidine and hydrophobic heterocyclic nitric oxide synthase (NOS) inhibitors, the effect of clonidine on the nitric oxide (NO) pathway was investigated. This was verified by determination of NOS activity in vitro and by analysis of inducible Ca2+-independent NOS (NOS-II) mRNA expression and measurement of nitrite levels in rat C6 glioma cells, taken as a cellular model. Clonidine inactivated neuronal Ca2+-dependent NOS (NOS-I) competitively without affecting NOS-II and endothelial Ca2+-dependent NOS (NOS-III) activity. However, the value of Ki for clonidine binding to NOS-I depended on tetrahydrobiopterin (BH4) concentration, as reported for NOS inhibition by other nitrogen heterocyclic compounds. In particular, the value of Ki for clonidine binding to NOS-I increased (from [7.9 ± 0.4] × 10−5 M to [8.0 ± 0.4] × 10−3 M) as BH4 concentration was increased (between 3.0 × 10−7 M and 1.0 × 10−3 M), at pH 7.5 and 37.0°. In addition, clonidine (1.0 × 10−4 M) enhanced NOS-II mRNA expression in rat C6 glioma cells, as induced by Escherichia coli lipopolysaccharide (LPS) plus interferon-γ (IFN-γ). Finally, clonidine (1.0 × 10−4 M to 1.0 × 10−3 M) dose dependently increased the levels of LPS/IFN-γ-induced nitrites, the breakdown product of NO, in supernatants of rat C6 glioma cells. As reported for other NOS inhibitors, clonidine was also able to regulate NOS-I and NOS-II inversely.

BACILLUS CALMETTE-GUERIN INDUCES LONG-TERM LOCAL FORMATION OF NITRIC OXIDE IN THE BLADDER VIA THE INDUCTION OF NITRIC OXIDE SYNTHASE ACTIVITY IN UROTHELIAL CELLS

Bladder instillation of bacillus Calmette-Guerin (BCG) is effective therapy for recurrent superficial bladder cancer and carcinoma in situ. BCG induces nitric oxide synthase activity in the bladder. Nitric oxide is formed from L-arginine by nitric oxide synthase. We investigated nitric oxide formation and its localization in bladder cancer patients treated with intravesical BCG instillation. The L-citrulline conversion assay was done to assess nitric oxide synthase activity in BCG treated T24 human bladder cancer cells and cultured normal human urothelial cells. Nitrite and nitrate in cell culture medium, urine and plasma were measured by capillary electrophoresis. Nitric oxide formation in the bladder was measured by chemiluminescence. A 24-hour treatment with BCG induced calcium independent nitric oxide synthase activity in T24 cells in a dose dependent manner. Nitrite and nitrate production by T24 cells also increased in a dose dependent manner after 24-hour BCG treatment. BCG treatment of cultured normal human urothelial cells resulted in the induction of calcium dependent and independent nitric oxide synthase activity. Nitrite in the urine of patients receiving BCG for the first time was increased 5-fold 24 hours after instillation. Furthermore, BCG increased luminal nitric oxide in the bladder. The increase was noted after a single treatment and sustained for 6 months. No changes in plasma nitrite or nitrate were observed after BCG treatment. BCG induces the local formation of nitric oxide in the bladder, whereas no evidence for systemic nitric oxide formation was noted. Increased nitric oxide production in the bladder is likely due to the induction of nitric oxide synthase activity in urothelial cells.

Selective inhibition of nitric oxide synthase type I by clonidine, an anti-hypertensive drug

Clonidine, clinically used in the treatment of hypertension, is a central α 2-adrenergic agonist that reduces blood pressure and slows heart rate by reducing sympathetic stimulation. Considering the structural similarity between clonidine and hydrophobic heterocyclic nitric oxide synthase (NOS) inhibitors, the effect of clonidine on the nitric oxide (NO) pathway was investigated. This was verified by determination of NOS activity in vitro and by analysis of inducible Ca 2+-independent NOS (NOS-II) mRNA expression and measurement of nitrite levels in rat C6 glioma cells, taken as a cellular model. Clonidine inactivated neuronal Ca 2+-dependent NOS (NOS-I) competitively without affecting NOS-II and endothelial Ca 2+-dependent NOS (NOS-III) activity. However, the value of K i for clonidine binding to NOS-I depended on tetrahydrobiopterin (BH 4) concentration, as reported for NOS inhibition by other nitrogen heterocyclic compounds. In particular, the value of K i for clonidine binding to NOS-I increased (from [7.9 ± 0.4] × 10 −5 M to [8.0 ± 0.4] × 10 −3 M) as BH 4 concentration was increased (between 3.0 × 10 −7 M and 1.0 × 10 −3 M), at pH 7.5 and 37.0°. In addition, clonidine (1.0 × 10 −4 M) enhanced NOS-II mRNA expression in rat C6 glioma cells, as induced by Escherichia coli lipopolysaccharide (LPS) plus interferon-γ (IFN-γ). Finally, clonidine (1.0 × 10 −4 M to 1.0 × 10 −3 M) dose dependently increased the levels of LPS/IFN-γ-induced nitrites, the breakdown product of NO, in supernatants of rat C6 glioma cells. As reported for other NOS inhibitors, clonidine was also able to regulate NOS-I and NOS-II inversely.

Activation of Ca(2+)-independent nitric oxide synthase by 17beta-estradiol in post-ischemic rat heart.

Nitric oxide (NO) donors or facilitation of endogenous NO production is cardioprotective. This study sought to determine whether enhanced myocardial NO production might contribute to estrogen-induced cardioprotection. Ca(2+)-dependent and Ca(2+)-independent NOS activities (pmol min(-1) mg(-1) protein), NOS protein expression (quantitative immunoblot), cGMP content (pmol mg(-1) protein) and LV work (Joules) were measured in hearts isolated from ovariectomized rats that were either untreated or treated chronically with 17beta-estradiol (0.25 mg, 21 day release formulation). After 14 days, serum levels of 17beta-estradiol were 6+/-1 and 135+/-16 pg ml(-1) in untreated and 17beta-estradiol-treated animals, respectively. After 60 min aerobic working mode perfusion, Ca(2+)-dependent NOS (untreated, 1.47+/-0.36; 17beta-estradiol 1.13+/-0.25) and Ca(2+)-independent NOS (untreated, 0.45+/-0.24; 17beta-estradiol, 0.41+/-0.21) activities, eNOS and iNOS proteins and cGMP content (untreated, 0.64+/-0.08; 17beta-estradiol, 0.76+/-0.12) were not different in the two groups. After 60 min low-flow (0.5 ml min(-1)) ischemia and 30 min reperfusion, Ca(2+)-dependent NOS activities were again similar (untreated, 1.25+/-0.23; 17beta-estradiol, 0.78+/-0.27). However, after reperfusion, Ca(2+)-independent NOS activity (untreated, 0. 39+/-0.10; 17beta-estradiol, 1.36+/-0.36) was 3.5-fold higher (P=0. 008) and cGMP content (untreated, 0.30+/-0.03; 17beta-estradiol, 0. 49+/-0.07) was 1.6-fold higher (P=0.017) in hearts from 17beta-estradiol-treated animals. Although pre-ischemic function was similar, recovery of post-ischemic LV work was 2-fold greater (P=0.024) in the 17beta-estradiol group. The ability of ischemia and reperfusion in combination with chronic 17beta-estradiol to increase Ca(2+)-independent NOS activity and cGMP content supports a role for enhanced myocardial NO signaling in 17beta-estradiol-induced cardioprotection.