Contributions of nitric oxide synthase isozymes to exhaled nitric oxide and hypoxic pulmonary vasoconstriction in rabbit lungs

We investigated the source(s) for exhaled nitric oxide (NO) in isolated, perfused rabbits lungs by using isozyme-specific nitric oxide synthase (NOS) inhibitors and antibodies. Each inhibitor was studied under normoxia and hypoxia. Only nitro-L-arginine methyl ester (L-NAME, a nonselective NOS inhibitor) reduced exhaled NO and increased hypoxic pulmonary vasoconstriction (HPV), in contrast to 1400W, an inhibitor of inducible NOS (iNOS), and 7-nitroindazole, an inhibitor of neuronal NOS (nNOS). Acetylcholine-mediated stimulation of vascular endothelial NOS (eNOS) increased exhaled NO and could only be inhibited by L-NAME. Selective inhibition of airway and alveolar epithelial NO production by nebulized L-NAME decreased exhaled NO and increased hypoxic pulmonary artery pressure. Immunohistochemistry demonstrated extensive staining for eNOS in the epithelia, vasculature, and lymphatic tissue. There was no staining for iNOS but moderate staining for nNOS in the ciliated cells of the epithelia, lymphoid tissue, and cartilage cells. Our findings show virtually all exhaled NO in the rabbit lung is produced by eNOS, which is present throughout the airways, alveoli, and vessels. Both vascular and epithelial-derived NO modulate HPV.     

Inducible nitric oxide synthase aggresome formation is mediated by nitric oxide

Nitric oxide (NO) generated by inducible NO synthase (iNOS) contributes critically to inflammatory injury and host defense. While previously thought as a soluble protein, iNOS was recently reported to form aggresomes inside cells. But what causes iNOS aggresome formation is unknown. Here we provide evidence demonstrating that iNOS aggresome formation is mediated by its own product NO. Exposure to inflammatory stimuli (lipopolysaccharide and interferon-γ) induced robust iNOS expression in mouse macrophages. While initially existing as a soluble protein, iNOS progressively formed protein aggregates as a function of time. Aggregated iNOS was inactive. Treating the cells with the NOS inhibitor N-nitro-l-arginine methyl ester (L-NAME) blocked NO production from iNOS without affecting iNOS expression. However, iNOS aggregation in cells was prevented by L-NAME. The preventing effect of NO blockade on iNOS aggresome formation was directly observed in GFP-iNOS-transfected cells by fluorescence imaging. Moreover, iNOS aggresome formation could be recaptured by adding exogenous NO to L-NAME-treated cells. These studies demonstrate that iNOS aggresome formation is caused by NO. The finding that NO induces iNOS aggregation and inactivation suggests aggresome formation as a feedback inhibition mechanism in iNOS regulation.     

Insulin feedback actions: complex effects involving isoforms of islet nitric oxide synthase

The present study examined the effects of exogenous insulin on C-peptide release in relation to islet activities of neural constitutive nitric oxide synthase (ncNOS) and inducible NOS (iNOS). The dose-response curves for glucose-stimulated insulin and C-peptide release from isolated islets were practically identical: 0.05-0.1 nmol/l insulin stimulated, 1-100 nmol/l had no effect, whereas concentrations >/=250 nmol/l ("high insulin"), inhibited C-peptide release. Both the stimulatory and inhibitory effects were abolished by the phosphatidylinositol 3'-kinase inhibitor wortmannin. Addition of a NOS inhibitor partially reversed the inhibitory action of high insulin, but had no effect on the stimulatory action of low insulin (0.1 nmol/l). Moreover, high insulin markedly increased islet ncNOS activity and induced a strong iNOS activity. As shown biochemically and with confocal microscopy, the stimulatory action of high insulin on NOS activities and the associated inhibition of C-peptide release were reversed by raising cyclic AMP through addition of either glucagon-like peptide 1 (GLP-1) or dibutyryl cyclic AMP (Bt(2)cAMP) to the incubated islets. We conclude that the positive feedback mechanisms of action of insulin are independent of islet NOS activities and remain unclear. The negative feedback action of insulin, however, can be explained by its ability to stimulate both islet ncNOS activity and the expression and activity of iNOS. The effects on iNOS are most likely transduced through phosphatidylinositol 3'-kinase and are counteracted by raising islet cyclic AMP levels.     

Expression of nitric oxide synthase isoforms and detection of nitric oxide in rat placenta

Nitric oxide (NO) production in therat placenta was monitored and quantified by electron paramagneticresonance (EPR) spectroscopy with hemoglobin and anFe-N-(dithiocarboxy)sarcosine (DTCS) complex as NO-trappingreagents. Expression of nitric oxide synthase (NOS) isoformswas also examined by quantitative RT-PCR analysis. The EPR spectrum ofthe placenta with hemoglobin trapping showed a three-line hyperfinestructure (g = 2.008 and a = 1.66-mT). The EPR signal was diminished after the placenta was homogenized or the NOSinhibitor L-NAME was administered to pregnant rats.Therefore, the specific signal was definitely identified as beingderived from endogenous NO spin-trapped by hemoglobin, and the EPRspectrum showed that the NO adduct existed as a pentacoordinate -NOheme species. The EPR spectrum of the placenta with Fe-DTCS trapping showed a triplet signal (g = 2.038) derived from anNO-Fe-DTCS complex. The height of the triplet signal did not varysignificantly with gestational stage during the last few days ofgestation. At the gestational stages examined, the level of NOS II mRNAexpression was significantly higher than that of NOS III mRNA. NOS IIexpression in term (day 21.5) placenta was significantlyincreased compared with that in preterm (day 19.5) placenta(P < 0.01, n = 4 or 5). These resultssuggest that NOS II is the predominant producer of NO in the placentaand that NOS II-generated NO plays significant roles in the maintenanceof placental functions immediately before birth.

     

Role of nitric oxide synthase isoforms in glucose-stimulated insulin release

The role of islet constitutive nitric oxide synthase (cNOS) in insulin-releasing mechanisms is controversial. By measuring enzyme activities and protein expression of NOS isoforms [i.e., cNOS and inducible NOS (iNOS)] in islets of Langerhans cells in relation to insulin secretion, we show that glucose dose-dependently stimulates islet activities of both cNOS and iNOS, that cNOS-derived nitric oxide (NO) strongly inhibits glucose-stimulated insulin release, and that short-term hyperglycemia in mice induces islet iNOS activity. Moreover, addition of NO gas or an NO donor inhibited glucose-stimulated insulin release, and different NOS inhibitors effected a potentiation. These effects were evident also in K+-depolarized islets in the presence of the ATP-sensitive K+ channel opener diazoxide. Furthermore, our results emphasize the necessity of measuring islet NOS activity when using NOS inhibitors, because certain concentrations of certain NOS inhibitors might unexpectedly stimulate islet NO production. This is shown by the observation that 0.5 mmol/l of the NOS inhibitor N(G)-monomethyl-L-arginine (L-NMMA) stimulated cNOS activity in parallel with an inhibition of the first phase of glucose-stimulated insulin release in perifused rats islets, whereas 5.0 mmol/l of L-NMMA markedly suppressed cNOS activity concomitant with a great potentiation of the insulin secretory response. The data strongly suggest, but do not definitely prove, that glucose indeed has the ability to stimulate both cNOS and iNOS in the islets and that NO might serve as a negative feedback inhibitor of glucose-stimulated insulin release. The results also suggest that hyperglycemia-evoked islet NOS activity might be one of multiple factors involved in the impairment of glucose-stimulated insulin release in type II diabetes mellitus.     

Nitric oxide and nitric oxide synthase isoforms in the normal, hypertrophic, and failing heart

Increased oxidative stress is one of the basic contributors to the development of the cardiovascular complications in diabetes. Both endothelial and vascular smooth muscle cell dysfunctions are the main sign involved in the pathogenesis of diabetic cardiovascular dysfunction. Matrix metalloproteinases (MMPs) are expressed in the vasculature, and participate in tissue remodeling under pathological conditions such as increased oxidative stress, whereas little is known about effect of hyperglycemia on regulation of MMPs in vascular system. Therefore, we aimed to evaluate the effect of an antioxidant, sodium selenate treatment (0.3 mg/kg for 4 weeks) on function of streptozotocin-diabetic rat aorta. Sodium selenate treatment improved significantly impaired isoproterenol-induced relaxation responses and contraction responses of the aortic strips, and exhibited marked protection against diabetes-induced degenerative changes in the smooth muscle cell morphology. Biochemical data showed that sodium selenate treatment induced a significant regulation of MMP-2 activity and protein loss as well as normalization of increased levels of tissue nitrite and protein thiol oxidation. In addition, this treatment restored diabetes-induced increased levels of endothelin-1, PKC, and cAMP production in the aortic tissue. Taken together, our data demonstrate that these beneficial effects of sodium selenate treatment in diabetics are related to be not only inhibition of increased oxidative stress but also prevention of both receptor- and smooth muscle-mediated dysfunction of vasculature, in part, via regulation of MMP-2. Such an observation provides evidence for potential therapeutic usage of selenium compounds for the amelioration of vascular disorders in diabetes.     

Paradoxical roles of different nitric oxide synthase isoforms in colonic injury

Nitric oxide (NO) is a free radical that is largely produced by three isoforms of NO synthase (NOS): neuronal (nNOS), endothelial (eNOS), and inducible (iNOS). NO regulates numerous processes in the gastrointestinal tract; however, the overall role that NO plays in intestinal inflammation is unclear. NO is upregulated in both ulcerative colitis and Crohn's disease as well as in animal models of colitis. There have been conflicting reports on whether NO protects or exacerbates injury in colitis or is simply a marker of inflammation. To determine whether the site, timing, and level of NO production modulate the effect on the inflammatory responses, the dextran sodium sulfate model of colitis was assessed in murine lines rendered deficient in iNOS, nNOS, eNOS, or e/nNOS by targeted gene disruption. The loss of nNOS resulted in more severe disease and increased mortality, whereas the loss of eNOS or iNOS was protective. Furthermore, concomitant loss of eNOS reversed the susceptibility found in nNOS-/- mice. Deficiencies in specific NOS isoforms led to distinctive alterations of inflammatory responses, including changes in leukocyte recruitment and alterations in colonic lymphocyte populations. The present studies indicate that NO produced by individual NOS isoforms plays different roles in modulating an inflammatory process.     

Structural characterization of nitric oxide synthase isoforms reveals striking active-site conservation

Crystal structures of human endothelial nitric oxide synthase (eNOS) and human inducible NOS (iNOS) catalytic domains were solved in complex with the arginine substrate and an inhibitor S-ethylisothiourea (SEITU), respectively. The small molecules bind in a narrow cleft within the larger active-site cavity containing heme and tetrahydrobiopterin. Both are hydrogen-bonded to a conserved glutamate (eNOS E361, iNOS E377). The active-site residues of iNOS and eNOS are nearly identical. Nevertheless, structural comparisons provide a basis for design of isozyme-selective inhibitors. The high-resolution, refined structures of eNOS (2.4 A resolution) and iNOS (2.25 A resolution) reveal an unexpected structural zinc situated at the intermolecular interface and coordinated by four cysteines, two from each monomer.     

Presence and activity of nitric oxide synthase isoforms in ischemia-reperfusion-injured flaps

Nitric oxide is produced from the amino acid L-arginine by nitric oxide synthase, which has three known isoforms: (1) endothelial nitric oxide synthase and (2) brain nitric oxide synthase, both of which are constitutive nitric oxide synthase; and (3) inducible nitric oxide synthase. The authors' hypothesis is that after reperfusion injury, endothelial cell dysfunction leads to disruption of nitric oxide synthase-mediated nitric oxide production and that this may in part explain the deleterious effects of ischemia-reperfusion injury on tissue survival and blood reflow in flaps. An experiment was designed to study the effects of ischemia-reperfusion injury on the bioactivity of all three isoforms of nitric oxide synthase. Buttock skin flaps and latissimus dorsi myocutaneous flaps were elevated in eight pigs. Flaps on one side of the animal were randomized to receive 6 hours of arterial ischemia, whereas flaps on the other side served as controls. At 6 hours of ischemia and at 1, 4, and 18 hours after reflow, tissue biopsy specimens were obtained and were processed for both constitutive nitric oxide synthase and inducible nitric oxide synthase enzyme activity on the basis of the L-citrulline assay. In addition, specimens were processed for Western blot analysis of the three isoforms. The authors' results revealed three key findings: first, there was a statistically significant (p < 0.001) decrease in constitutive nitric oxide synthase activity of ischemia-reperfusion-injured flaps as compared with controls in both skin and muscle for all time intervals measured. Second, Western blot analyses of endothelial nitric oxide synthase and brain nitric oxide synthase showed a significant decrease in the signal intensity in ischemic and reperfused tissue as compared with controls. Third, the inducible nitric oxide synthase isoform's activity and protein remained undetectable in both tissue types for all time points measured. The authors' data demonstrated that following ischemia-reperfusion injury in the pig flap model there was a disruption of constitutive nitric oxide synthase expression and activity, which may lead to decreased nitric oxide production. The significant decrease in nitric oxide synthase activity found in the current study may partly explain the mechanism of tissue damage in flaps subjected to ischemia-reperfusion injury. Knowledge of the kinetics of nitric oxide synthase activity under conditions of ischemia-reperfusion injury has important implications for the choice and timing of delivery of therapeutic agents whose goal is to increase the bioavailability of nitric oxide in reperfused tissue.     

Chronic nitric oxide synthase blockade desensitizes the heart to the negative metabolic effects of nitric oxide

The consequences of chronic nitric oxide synthase (NOS) blockade on the myocardial metabolic and guanylyl cyclase stimulatory effects of exogenous nitric oxide (NO) were determined. Thirty-three anesthetized open-chest rabbits were randomized into four groups: control, NO donor S-nitroso-N-acetyl-penicillamine (SNAP, 10(-4 )M), NOS blocking agent N(G)-nitro-L-arginine methyl ester (L-NAME, 20 mg/kg/day) for 10 days followed by a 24 hour washout and L-NAME for 10 days followed by a 24 hour washout plus SNAP. Myocardial O(2) consumption was determined from coronary flow (microspheres) and O(2) extraction (microspectrophotometry). Cyclic GMP and guanylyl cyclase activity were determined by radioimmunoassay. There were no baseline metabolic, functional or hemodynamic differences between control and L-NAME treated rabbits. SNAP in controls caused a reduction in O(2) consumption (SNAP 5.9+/-0.6 vs. control 8.4+/-0.8 ml O(2)/min/100 g) and a rise in cyclic GMP (SNAP 18.3+/-3.8 vs. control 10.4+/-0.9 pmol/g). After chronic L-NAME treatment, SNAP caused no significant changes in O(2) consumption (SNAP 7.1+/-0.8 vs. control 6.4+/-0.7) or cyclic GMP (SNAP 14.2+/-1.8 vs. control 12.1+/-1.3). In controls, guanylyl cyclase activity was significantly stimulated by SNAP (216.7+/-20.0 SNAP vs. 34.4+/-2.5 pmol/mg/min base), while this increase was blunted after L-NAME (115.9+/-24.5 SNAP vs. 24.9+/-4.7 base). These results demonstrated that chronic NOS blockade followed by washout blunts the response to exogenous NO, with little effect on cyclic GMP or myocardial O(2) consumption. This was related to reduced guanylyl cyclase activity after chronic L-NAME. These results suggest that, unlike many receptor systems, the NO-cyclic GMP signal transduction system becomes downregulated upon chronic inhibition.     

Expression of nitric oxide synthase isoforms in normal ventilatory and limb muscles

Hussain, Sabah N. A., Qasim El-Dwairi, Mohammed N. Abdul-Hussain, and Dalia Sakkal. Expression of nitric oxidesynthase isoforms in normal ventilatory and limb muscles.J. Appl. Physiol. 83(2): 348-353, 1997.Nitric oxide (NO), an important messenger molecule withwidespread actions, is synthesized by NO synthases (NOS). In thisstudy, we investigated the correlation between fiber type and NOSactivity among ventilatory and limb muscles of various species. We alsoassessed the presence of the three NOS isoforms in normal skeletalmuscles and how various NOS inhibitors influence muscle NOS activity.NOS activity was detected in various muscles; however, NOS activity inrabbits and rats varied significantly among different muscles.Immunoblotting of muscle samples indicated the presence of both theneuronal NOS and the endothelial NOS isoforms but not thecytokine-inducible NOS isoform. However, these isoforms were expressedto different degrees in various muscles. Although the neuronal NOSisoform was detectable in the canine diaphragm, very weak expressionwas detected in rabbit, rat, and mouse diaphragms. The endothelial NOSisoform was detected in the rat and mouse diaphragms but not in thecanine and rabbit diaphragms. We also found thatN^G-nitro-L-arginine methyl ester,7-nitroindazole, andS-methylisothiourea werestronger inhibitors of muscle NOS activity than was aminoguanidine. These results indicate the presence of different degrees ofconstitutive NOS expression in normal ventilatory and limb muscles ofvarious species. Our data also indicate that muscle NOS activity is not determined by fiber type distribution but by other not yet identified factors. The functional significance of this expression remains to beassessed.

     

Phenylarsine oxide inhibits nitric oxide synthase in pulmonary artery endothelial cells

The role of protein tyrosine phosphorylation during regulation of NO synthase (eNOS) activity in endothelial cells is poorly understood. Studies to define this role have used inhibitors of tyrosine kinase or tyrosine phosphatase (TP). Phenylarsine oxide (PAO), an inhibitor of TP, has been reported to bind thiol groups, and recent work from our laboratory demonstrates that eNOS activity depends on thiol groups at its catalytic site. Therefore, we hypothesized that PAO may have a direct effect on eNOS activity. To test this, we measured (i) TP and eNOS activities both in total membrane fractions and in purified eNOS prepared from porcine pulmonary artery endothelial cells and (ii) sulfhydryl content and eNOS activity in purified bovine aortic eNOS expressed in Escherichia coli. High TP activity was detected in total membrane fractions, but no TP activity was detected in purified eNOS fractions. PAO caused a dose-dependent decrease in eNOS activity in total membrane and in purified eNOS fractions from porcine pulmonary artery endothelial cells, even though the latter had no detectable TP activity. PAO also caused a decrease in sulfhydryl content and eNOS activity in purified bovine eNOS. The reduction in eNOS sulfhydryl content and the inhibitory effect of PAO on eNOS activity were prevented by dithiothreitol, a disulfide-reducing agent. These results indicate that (i) PAO directly inhibits eNOS activity in endothelial cells by binding to thiol groups in the eNOS protein and (ii) results of studies using PAO to assess the role of protein tyrosine phosphorylation in regulating eNOS activity must be interpreted with great caution.     

The three isoforms of nitric oxide synthase distinctively affect mouse nocifensive behavior

Nitric oxide synthases (NOSs) have been shown to modulate thermal hyperalgesia and mechanical hypersensitivity in inflammatory and neuropathic pain. However, little is known about the effect of NOSs on baseline function of sensory nerve fibers. Using genetic deficiency and pharmacologic inhibition of NOSs, we examined the impact of the three isoforms NOS1, NOS2, and NOS3 on baseline nocifensive behavior by measuring current vocalization threshold in response to electrical stimulation at 5, 250, 2000 Hz that preferentially stimulate C, Aδ, and Aβ fibers. In response to 5, 250 and 2000 Hz, NOS1-deficient animals had significantly higher current vocalization thresholds compared with wild-type. Genetic deficiency of NOS2 was associated with higher current vocalization thresholds in response to 5 Hz (C-fiber) stimulation. In contrast, NOS3-deficient animals had an overall weak trend toward lower current vocalization thresholds at 5 Hz and significantly lower current vocalization threshold compared with wild-type animals at 250 and 2000 Hz. Therefore, NOSs distinctively affect baseline mouse current vocalization threshold and appear to play a role on nocifensive response to electrical stimulation of sensory nerve fibers.     

Effects of capsaicin on nitric oxide synthase isoforms in prepubertal rat ovary

Nitric oxide (NO) has emerged as an important intra-ovarian regulatory factor. We investigated effects of low dose capsaicin (CAP) treatment on the different NOS isoforms in prepubertal rat ovaries. Fifteen 21-day-old female Sprague-Dawley rats were divided randomly into three groups. The first group received no treatment, the second group received 0.5 mg/kg/day CAP dissolved in the vehicle, and the third group was treated with the vehicle only. The animals were euthanized by ether inhalation after 15 days and their ovaries were excised. Ovaries were fixed in 10% neutral buffered formalin and embedded in paraffin. Sections were processed for standard immunohistochemistry using the labeled streptavidin-biotin technique for expression of nNOS, eNOS and iNOS. We demonstrated that CAP induced expression of NOS isotypes including eNOS, iNOS and nNOS in prepubertal rat ovaries. CAP may lead to release of NO either directly from nerves or indirectly by evoking release from other cells via the action of neuropeptides that are released from afferent terminals and are involved in regulating female reproductive function.     

Effects of capsaicin on nitric oxide synthase isoforms in prepubertal rat ovary

Abstract

Nitric oxide (NO) has emerged as an important intra-ovarian regulatory factor. We investigated effects of low dose capsaicin (CAP) treatment on the different NOS isoforms in prepubertal rat ovaries. Fifteen 21-day-old female Sprague-Dawley rats were divided randomly into three groups. The first group received no treatment, the second group received 0.5 mg/kg/day CAP dissolved in the vehicle, and the third group was treated with the vehicle only. The animals were euthanized by ether inhalation after 15 days and their ovaries were excised. Ovaries were fixed in 10% neutral buffered formalin and embedded in paraffin. Sections were processed for standard immunohistochemistry using the labeled streptavidin-biotin technique for expression of nNOS, eNOS and iNOS. We demonstrated that CAP induced expression of NOS isotypes including eNOS, iNOS and nNOS in prepubertal rat ovaries. CAP may lead to release of NO either directly from nerves or indirectly by evoking release from other cells via the action of neuropeptides that are released from afferent terminals and are involved in regulating female reproductive function.     

Similar but not the same: normobaric and hyperbaric pulmonary oxygen toxicity, the role of nitric oxide

Pulmonary manifestations of oxygen toxicity were studied and quantified in rats breathing >98% O(2) at 1, 1.5, 2, 2.5, and 3 ATA to test our hypothesis that different patterns of pulmonary injury would emerge, reflecting a role for central nervous system (CNS) excitation by hyperbaric oxygen. At 1.5 atmosphere absolute (ATA) and below, the well-recognized pattern of diffuse pulmonary damage developed slowly with an extensive inflammatory response and destruction of the alveolar-capillary barrier leading to edema, impaired gas exchange, respiratory failure, and death; the severity of these effects increased with time over the 56-h period of observation. At higher inspired O(2) pressures, 2-3 ATA, pulmonary injury was greatly accelerated but less inflammatory in character, and events in the brain were a prelude to a distinct lung pathology. The CNS-mediated component of this lung injury could be attenuated by selective inhibition of neuronal nitric oxide synthase (nNOS) or by unilateral transection of the vagus nerve. We propose that extrapulmonary, neurogenic events predominate in the pathogenesis of acute pulmonary oxygen toxicity in hyperbaric oxygenation, as nNOS activity drives lung injury by modulating the output of central autonomic pathways.     

Rapid kinetic studies of electron transfer in the three isoforms of nitric oxide synthase.

The nitric oxide synthases (NOSs) consist of a flavin-containing reductase domain, linked to a heme-containing oxygenase domain, by a calmodulin (CaM) binding sequence. The flavin-containing reductase domains of the NOS isoforms possess close sequence homology to NADPH-cytochrome P450 reductase (CPR). Additionally, the oxygenase domains catalyze monooxygenation of L-arginine through a cytochrome P450-like cysteine thiolate-liganded heme bound in the active site. With these considerations in mind, we conducted studies in an attempt to gain insight into the intermediates involved in flavoprotein-to-heme electron transfer in the NOSs. Static, steady-state, and stopped-flow kinetic studies indicated that nNOS must be reduced to a more than one-electron-reduced intermediate before efficient electron transfer can occur. Therefore, the possibility exists that the oxygenase domains of the NOS isoforms may receive their electrons from the reductase domains by a mechanism resembling the CPR-P450 interaction. Furthermore, the rate-limiting step in electron transfer appears to be the transfer of electrons from the flavoprotein to the oxygenase domain facilitated by the binding of CaM at increased intracellular Ca(2+) concentrations. Thus, modulation of electron transfer rates appears to be regulated at the level of the flavoprotein domains of the NOS isoforms.     

Inhibitory effects of NO on carotid body: contribution of neural and endothelial nitric oxide synthase isoforms

We tested the hypothesis that nitric oxide (NO) produced within the carotid body is a tonic inhibitor of chemoreception and determined the contribution of neuronal and endothelial nitric oxide synthase (eNOS) isoforms to the inhibitory NO effect. Accordingly, we studied the effect of NO generated from S-nitroso-N-acetylpenicillamide (SNAP) and compared the effects of the nonselective inhibitor N(omega)-nitro-l-arginine methyl ester (l-NAME) and the selective nNOS inhibitor 1-(2-trifluoromethylphenyl)-imidazole (TRIM) on chemosensory dose-response curves induced by nicotine and NaCN and responses to hypoxia (Po(2) approximately 30 Torr). CBs excised from pentobarbitone-anesthetized cats were perfused in vitro with Tyrode at 38 degrees C and pH 7.40, and chemosensory discharges were recorded from the carotid sinus nerve. SNAP (100 microM) reduced the responses to nicotine and NaCN. l-NAME (1 mM) enhanced the responses to nicotine and NaCN by increasing their duration, but TRIM (100 microM) only enhanced the responses to high doses of NaCN. The amplitude of the response to hypoxia was enhanced by l-NAME but not by TRIM. Our results suggest that both isoforms contribute to the NO action, but eNOS being the main source for NO in the cat CB and exerting a tonic effect upon chemoreceptor activity.     

Discoordinate regulation of renal nitric oxide synthase isoforms in ovariectomized mRen2. Lewis rats

Estrogen depletion markedly exacerbates hypertension in female congenic mRen2. Lewis rats, a model of tissue renin overexpression. Because estrogen influences nitric oxide synthase (NOS) and NO may exert differential effects on blood pressure, the present study investigated the functional expression of NOS isoforms in the kidney of ovariectomized (OVX) mRen2. Lewis rats. OVX-mRen2. Lewis exhibited an increase in systolic blood pressure (SBP) of 171 +/- 5 vs. 141 +/- 7 mmHg (P < 0.01) for intact littermates. Renal cortical mRNA and protein levels for endothelial NOS (eNOS) were reduced 50-60% (P < 0.05) and negatively correlated with blood pressure. In contrast, cortical neuronal NOS (nNOS) mRNA and protein levels increased 100 to 300% (P < 0.05). In the OVX kidney, nNOS immunostaining was more evident in the macula densa, cortical tubules, and the medullary collecting ducts compared with the intact group. To determine whether the increase in renal nNOS expression constitutes a compensatory response to the reduction in renal eNOS, we treated both intact and OVX mRen2. Lewis rats with the selective nNOS inhibitor L-VNIO from 11 to 15 wk of age. The nNOS inhibitor reduced blood pressure in the OVX group (185 +/- 3 vs. 151 +/- 8 mmHg, P < 0.05), but pressure was not altered in the intact group (146 +/- 4 vs. 151 +/- 4 mmHg). In summary, exacerbation of blood pressure in the OVX mRen2. Lewis rats was associated with the discoordinate regulation of renal NOS isoforms. Estrogen sensitivity in this congenic strain may involve the influence of NO through the regulation of both eNOS and nNOS.     

Levodopa modulating effects of inducible nitric oxide synthase and reactive oxygen species in glioma cells

Neurological injury and Parkinson disease (PD) are often associated with the increase of nitric oxide (NO) and free radicals from resident glial cells in the brain. In vitro, exposure to L-3-4-dihydroxyphenylalanine (L-DOPA), one of the main therapeutic agents for the treatment of PD, can lead to neurotoxicity. In this study, lipopolysaccharide (LPS) and interferon-gamma (IFN-g) were used to stimulate C6 glioma cells in the presence of varying concentrations of L-DOPA (1 microM-1 mM). The results indicated a slight augmentation of NO(2)(-) production at low concentrations of L-DOPA (<100 microM) and complete inhibition of NO(2)(-) at higher concentrations (500 microM, 1 mM), (p < 0.001). Western blot analysis corroborated that L-DOPA effects on iNOS was at the level of its protein expression. Total reactive oxygen species (ROS) were detected using 2', 7'-dichlorofluorescein diacetate fluorescence dye (2', 7'-DCFC) and there was an increase of intensity with the increasing concentrations of L-DOPA. Furthermore, large amounts of superoxide (O(2)(-)) and hydrogen peroxide (H(2)O(2)) were generated from the autoxidation of L-DOPA. C6 cells contain high levels of catalase, with inadequate levels of superoxide dismutase (SOD); therefore, there was an accumulation of O(2)(-), tantamount to elevation in 2'7'-DCFC intensity. Simultaneous accumulation of O(2)(-) and NO(2)(-) would propel formation of peroxynitrite (ONOO-). SOD completely attenuated the autoxidation of L-DOPA and significantly reversed the inhibitory effects on iNOS at high concentrations. The data obtained confirmed that the observed effects on iNOS were not due to the activation of the D(1) or beta1 adrenergic receptors by L-DOPA. It was concluded from this study that L-DOPA contributed to the modulation of iNOS and to the increase of O(2)(-) production in the stimulated glioma cells in vitro.