Visualisation of nitric oxide released by nerve stimulation

We have visualised nitric oxide (NO) released from the electrically stimulated myenteric plexus and hypogastric nerve. NO was visualised by a reaction with luminol and hydrogen peroxide to generate photons which were counted using a microscope coupled to a photon counting camera. Electrical stimulation of the tissues induced an increase in photon counts which was frequency-dependent and prevented by inhibition of the NO synthase or by tetrodotoxin. The light emitted during nerve stimulation was not only observed at the nerve terminals but also at the axon and soma. Our results indicate that NO released from the whole nerve cell may affect target cells surrounding all parts of the nitrergic neuron. Thus, NO functions as a unique mechanism of synaptic and non-synaptic communication in the nervous system.     

Rate of nitric oxide production by lower alveolar airways of human lungs

This report describes methods for measuring nitric oxide production by the lungs' lower alveolar airways (VNO), defined as those alveoli and bronchioles well perfused by the pulmonary circulation. Breath holding or vigorous rebreathing for 15-20 s minimizes removal of NO from the lower airways and results in a constant partial pressure of NO in the lower airways (PL). Then the amount of NO diffusing into the perfusing blood will be the pulmonary diffusing capacity for NO (DNO) multiplied by PL and by mass balance equals VNO, or VNO = DNO(PL). To measure PL, 10 normal subjects breath held for 20 s followed by exhalation at a constant flow rate of 0.83 +/- 0.14 (SD) l/s or rebreathed at 59 +/- 15 l/min for 20 s while NO was continuously measured at the mouth. DNO was estimated to equal five times the single-breath carbon monoxide diffusing capacity. By using breath holding, PL equaled 2.9 +/- 0.8 mmHg x 10(-6) and VNO equaled 0.39 +/- 0.12 microl/min. During rebreathing PL equaled 2.3 +/- 0.6 mmHg x 10(-6) and VNO equaled 0.29 +/- 0.11 microl/min. Measurements of NO at the mouth during rapid, constant exhalation after breath holding for 20 s or during rebreathing provide reproducible methods for measuring VNO in humans.     

Endogenous nitric oxide in the airways of different animal species

BACKGROUND: High amounts of endogenous nitric oxide (NO) have been demonstrated in the human upper airway, but the role of nasal NO is still unclear. The present study aims to describe nasal NO excretion in different animal species with special living conditions or anatomy. METHODS: Domestic animals (horse, cow, pig, sheep, dog, cat) and zoo-animals (Rhesus monkey, chimpanzee, gorilla, elephant, fur seal, alpaca, yak, dolphin, camel, capybara, bear, tiger, wolf, giraffe, alligator, Harris' hawk, kangaroo) were studied awake, resting or anaesthetised. NO concentrations were measured by chemiluminescence using different analysers and techniques, including measurements on mixed exhaled air, during continuous or intermittent gas sampling, and on single breaths. RESULTS: Rhesus monkeys (number of individuals N = 5) and pigs (N = 2) were compared and displayed quite different excretion patterns. Allowing NO to accumulate in the nose during timed occlusions yielded peak concentrations in monkeys of 0.46 +/- 0.07 parts per million (ppm, mean +/- SEM), 0.59 +/- 0.08 ppm, 0.70 +/- 0.08 ppm and 1.02 +/- 0.05 ppm NO after 15, 30, 60 and 120 s of occlusion. In pigs, 0.012-0.021 ppm NO were recorded, independent of occlusion time. The chimpanzee was similar to the Rhesus monkey and the highest NO value, 2.9 ppm, was recorded after 4-5 min of occlusion. In single breaths from 3 elephants 0.031-0.082 ppm, from 1 gorilla 0.029 ppm, and from 1 chimpanzee 0.069 +/- 0.003 ppm NO (8 observations) were recorded. CONCLUSIONS: We found considerable species difference in nasal NO excretion with pronounced amounts only in primates and elephants. The physiological implications of these findings remain to be defined.     

Inhibitory effects of nitric oxide donors on nitric oxide synthesis in rat gastric myenteric plexus

We investigated whether nitric oxide (NO) exerts an inhibition on its own synthesis in the gastric myenteric plexus in rats. Nonadrenergic, noncholinergic relaxations in response to transmural electrical stimulation (TS) were markedly antagonized by NG-nitro-L-arginine methyl ester, (10(-4) M) and abolished by tetrodotoxin (10(-6) M). Pretreatment with various NO donors (3-morpholino-sydnonymide [SIN-1 (3 x 10(-7) to 3 x 10(-6) M)], S-nitroso-N-acetylpenicillamine (10(-6) to 10(-5) M), sodium nitroprusside (10(-8) to 3 x 10(-8) M) and 8-bromoquanosine 3', 5'-cyclic monophosphate [8-bromo-cGMP (10(-6) to 3 x 10(-6) M)]) significantly inhibited TS-evoked nonadrenergic, noncholinergic relaxations in a dose-dependent manner. In contrast, vasoactive intestinal polypeptide (10(-8) M)-induced relaxations were not affected by SIN-1 or 8-bromo-cGMP. TS evoked a significant increase in 3H-citrulline formation, which was completely abolished by calcium-free medium, NG-nitro-L-arginine methyl ester, (10(-4) M) and tetrodotoxin (10(-6) M). 3H-citrulline formation evoked by TS was significantly inhibited by SIN-1 (10(-7) to 10(-5) M) and 8-bromo-cGMP (10(-7) to 10(-5) M) in a dose-dependent manner. The inhibitory effect of SIN-1 was partially prevented by 1H-[1,2, 4]oxadiazolo[3,4-a]quinoxalin-1-one (10(-5) M), a guanylate cyclase inhibitor. We conclude that NO synthesis in the gastric myenteric plexus is negatively regulated by NO and cGMP. This suggests an autoregulatory feedback mechanism of NO synthesis in the gastric myenteric plexus.     

Impairment of neural nitric oxide-mediated relaxation after antigen exposure in guinea pig airways in vitro

A solid-phase erythrocyte adherence assay has been developed for the serological detection of reagin antibodies in syphilis. Capture-S (Immucor, Inc., Norcross, Ga.) is a nontreponemal, qualitative screening test for the detection of immunoglobulin G (IgG) and IgM antilipid antibodies in serum or plasma samples from blood donors. The Capture-S assay utilizes a modified Venereal Disease Research Laboratory antigen bound to microtitration wells and anti-IgG- plus anti-IgM-coated indicator erythrocytes as the detection system. The Capture-S assay was evaluated at six separate sites on 10,942 specimens. For patient samples of clinically diagnosed syphilis categories (n = 366), the Capture-S assay yielded a sensitivity of 80.7% versus 80.3% for the rapid plasma reagin (RPR) card test (Becton Dickinson Microbiology Systems, Cockeysville, Md.). In comparative experiments on patient and donor samples (n = 10,222), the Capture-S assay demonstrated a sensitivity of 94% compared to 91.2% for the RPR card test. The Capture-S and RPR card tests produced essentially equivalent specificities of 99.2% and 99.3%, respectively, for this sample population. For five test sites, the Capture-S and RPR card test demonstrated a 98.3% agreement (10,085 of 10,264) of test results. These evaluations indicate that the Capture-S compares favorably to the RPR card test in assay sensitivity and specificity, with the added benefits of ease of use, accommodation of high-volume testing, and potential for automation.     

Neurogenic nitric oxide mediates relaxation of canine corpus cavernosum

PURPOSE: The aim of the present study is to analyze mechanisms underlying neurogenic relaxation of the corpus cavernosum which are believed to participate in penile erection. MATERIALS AND METHODS: Mechanical responses to nerve stimulation by electrical pulses and nicotine were measured in strips of canine corpus cavernosum precontracted with phenylephrine. Cyclic guanosine monophosphate (GMP) contents in the strips were also measured by radioimmunoassay. Immunohistochemistry for nitric oxide synthase (NOS) and vasoactive intestinal polypeptide (VIP) was performed. RESULTS: Transmural electrical stimulation and nicotine produced relaxations in the isolated canine corpus. The neurogenic relaxation was abolished by N omega-nitro-L-arginine, a NOS inhibitor, and the inhibition was reversed by L-arginine. Relaxations induced by nerve stimulation and exogenous nitric oxide (NO) were depressed by oxyhemoglobin and methylene blue. Vasoactive intestinal polypeptide (VIP)-induced relaxations were not influenced by these inhibitors. In the controls strips and those made unresponsive to VIP by its repeated application, the responses to nerve stimulation did not differ. The content of cyclic GMP in the tissue increased in response to nicotine, the effect being abolished by the NO synthase inhibitor. Immunohistochemical study demonstrated neurons containing NOS and VIP. CONCLUSIONS: It appears that the relaxation induced by nerve stimulation is mediated solely by NO liberated from the nerve that activates soluble guanylate cyclase and increases the production of cyclic GMP in smooth muscle, whereas VIP does not play a role in the regulation of muscle tone under the experimental conditions used.     

Role of nitric oxide and nitric oxide-independent relaxing factor in contraction and relaxation of rabbit blood vessels

The objective of the present study was to estimate the maximal velocity (Vmax) and Michaelis affinity constant (Km) for the oxidation of pyrene to 1-hydroxypyrene using rat liver post-mitochondrial fractions. The approach involved the determination of the concentrations of 1-hydroxypyrene formed during 5 min incubations of pyrene (initial concentrations: 0.0025-0.5 microM), and correcting for the rate of 1-hydroxypyrene disappearance (2.16 x 10(-5) per (mg protein/l)/min) during the incubation period. The Vmax and Km for pyrene metabolism in the rat corresponded to 0.0577 +/- 0.0108 micromol/min per g liver and 27.73 +/- 13.54 microM, respectively. The intrinsic clearance (CL(int)) of pyrene in the rat estimated in the present study (0.041-0.111 l/min per kg) was within the range of the previously reported CL(int) in humans (0.037-0.125 l/min per kg). The results of this study suggest that CL(int) of pyrene in humans can be predicted from such data obtained in the rat.     

Inhibitory effect of alpha-tocopherol on methemoglobin formation by nitric oxide in normal and acatalasemic mouse hemolysates

The inhibitory effect of alpha-tocopherol on methemoglobin formation in normal and acatalasemic mice was studied by exposing their hemolysates to nitric oxide. Methemoglobin formation in normal and acatalasemic mouse hemolysates exposed to nitric oxide were significantly inhibited by the addition of alpha-tocopherol at final concentrations ranging from 1.2 to 5.8 mM. Negative correlations were observed between the logarithm of alpha-tocopherol concentration and the methemoglobin formation. The formation of methemoglobin in acatalasemic mouse hemolysates was greater than that in normal mouse hemolysates with or without added alpha-tocopherol. The methemoglobin formation in acatalasemic mice was also significantly inhibited by addition of more than 500 units/ml of catalase, and the methemoglobin formation in normal and acatalasemic mice was also inhibited with sodium diethyldithiocarbamate at a final concentration of 1 M.     

Evidence that different mechanisms underlie smooth muscle relaxation to nitric oxide and nitric oxide donors in the rabbit isolated carotid artery

1. The endothelium-dependent relaxants acetylcholine (ACh; 0.03-10 microM) and A23187 (0.03-10 microM), and nitric oxide (NO), applied either as authentic NO (0.01-10 microM) or as the NO donors 3-morpholino-sydnonimine (SIN-1; 0.1-10 microM) and S-nitroso-N-acetylpenicillamine (SNAP; 0.1-10 microM), each evoked concentration-dependent relaxation in phenylephrine stimulated (1-3 microM; mean contraction and depolarization, 45.8+/-5.3 mV and 31.5+/-3.3 mN; n=10) segments of rabbit isolated carotid artery. In each case, relaxation closely correlated with repolarization of the smooth muscle membrane potential and stimulated a maximal reversal of around 95% and 98% of the phenylephrine-induced depolarization and contraction, respectively. 2. In tissues stimulated with 30 mM KCl rather than phenylephrine, smooth muscle hyperpolarization and relaxation to ACh, A23187, authentic NO and the NO donors were dissociated. Whereas the hyperpolarization was reduced by 75-80% to around a total of 10 mV, relaxation was only inhibited by 35% (n=4-7 in each case; P<0.01). The responses which persisted to ACh and A23187 in the presence of 30 mM KCl were abolished by either the NO synthase inhibitor L-NG-nitroarginine methyl ester (L-NAME; 100 microM) or the inhibitor of soluble guanylyl cyclase 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 microM; 10 min; n=4 in each case; P<0.01). 3. Exposure to ODQ significantly attenuated both repolarization and relaxation to ACh, A23187 and authentic NO, reducing the maximum changes in both membrane potential and tension to each relaxant to around 60% of control values (n=4 in each case; P<0.01). In contrast, ODQ almost completely inhibited repolarization and relaxation to SIN-1 and SNAP, reducing the maximum responses to around 8% in each case (n=3-5; P<0.01). 4. The potassium channel blockers glibenclamide (10 microM), iberiotoxin (100 nM) and apamin (50 nM), alone or in combination, had no significant effect on relaxation to ACh, A23187, authentic NO, or the NO donors SIN-1 and SNAP (n=4 in each case; P>0.05). Charybdotoxin (ChTX; 50 nM) almost abolished repolarization to ACh (n=4; P<0.01) and inhibited the maximum relaxation to ACh, A23187 and authentic NO each by 30% (n=4-8; P<0.01). Application of ODQ (10 microM; 10 min) abolished the ChTX-insensitive responses to ACh, A23187 and authentic NO (n=4 in each case; P<0.01 5. When the concentration of phenylephrine was reduced (to 0.3-0.5 microM) to ensure the level of smooth muscle contraction was the same as in the absence of potassium channel blocker, ChTX had no effect on the subsequent relaxation to SIN-1 (n=4; P>0.05). However, in the presence of tone induced by 1-3 microM phenylephrine (51.2+/-3.3 mN; n=4), ChTX significantly reduced relaxation to SIN-1 by nearly 50% (maximum relaxation 53.2+/-6.3%, n=4; P<0.01). 6. These data indicate that NO-evoked relaxation of the rabbit isolated carotid artery can be mediated by three distinct mechanisms: (a) a cyclic GMP-dependent, voltage-independent pathway, (b) cyclic GMP-mediated smooth muscle repolarization and (c) cyclic GMP-independent, ChTX-sensitive smooth muscle repolarization. Relaxation and repolarization to both authentic and endothelium-derived NO in this large conduit artery appear to be mediated by parallel cyclic GMP-dependent and -independent pathways. In contrast, relaxation to the NO-donors SIN-1 and SNAP appears to be mediated entirely via cyclic GMP-dependent mechanisms.     

Femtomolar bradykinin-induced relaxation of isolated bovine coronary arteries, mediated by endothelium-derived nitric oxide

The ideal pleural sclerosing agent should be easily administered, without significant side effects, inexpensive, and widely available. None of the agents presently used meets all of these criteria. Ethanolamine oleate (ETH) is a sclerosing agent used in the sclerotherapy treatment of varicose veins of the legs and esophagus. The objective of the present study was to assess the efficacy of ETH as a pleural sclerosant in rabbits. An additional objective was to assess if better results were obtained when dextrose 50% (D50) as opposed to saline was used as the diluent. Each group of 10 rabbits received a total volume of 2 ml intrapleurally. The eight treatments were as follows: (1) 2 ml saline; (2) 2 ml D50; (3) 25 mg ETH plus 1.5 ml saline; (4) 25 mg ETH plus 1.5 ml D50; (5) 50 mg ETH plus 1.0 ml saline; (6) 50 mg ETH plus 1 ml D50; (7) 75 mg ETH plus 0.5 ml D50, and (8) 100 mg ETH. The rabbits were sacrificed 28 days after the injection. The intrapleural instillation of ETH resulted in evident pleurodesis, which was dose-dependent; 100 mg ETH induced significantly (p<0.05) more adhesions than did any other treatment. The selection of the diluent had no effect on the pleurodesis. The microscopic examination of the right visceral pleura showed that the mean degree of fibrosis after 100 mg ETH was significantly (p<0.05) greater than that after the other solutions. The mean degree of pleural inflammation, lung inflammation and lung fibrosis was minimal in all the groups. From this study we conclude that undiluted ETH produces pleurodesis in our experimental model. At the doses used, the pleurodesis was less than that produced after talc, tetracycline derivatives or silver nitrate in the same model.     

Exhaled nitric oxide is not elevated in the inflammatory airways diseases of cystic fibrosis and bronchiectasis

Airways inflammation has been associated with increased nitric oxide (NO) in the exhaled breath. It was, therefore, questioned whether exhaled NO could act as an indicator of the severity of airways inflammation in the chronic suppurative lung diseases cystic fibrosis (CF) and bronchiectasis. NO levels in a single exhalation were measured using a chemiluminescence analyser. Thirty-six patients with CF and 16 with bronchiectasis were studied and compared with 22 normal subjects and 35 asthmatic patients. All subjects were nonsmokers and all measurements were made when patients were clinically stable. In addition, exhaled NO was measured in 10 CF patients at the time of onset of an acute infective exacerbation and followed for 7 days during the treatment of the exacerbation in eight of the 10 patients. No significant differences were found in NO levels in patients with CF or bronchiectasis compared with normals (median 4.0, 5.5 and 4.4 parts per billion (ppb), respectively), but all were lower than in asthma patients (10.4 ppb). The NO levels in the CF patients at time of exacerbation were not increased and did not change during treatment. These data show that nitric oxide levels in the exhaled breath of patients with chronic suppurative lung diseases, in contrast to asthma, are not elevated, despite the presence of substantial airways inflammation. Possible explanations include poor diffusion of nitric oxide across increased and viscous airway secretions, removal of nitric oxide by reaction with reactive oxygen species in the inflamed environment and failure of upregulation of epithelial inducible nitric oxide synthase in chronic suppurative conditions.     

Constitutive nitric oxide synthase is expressed and nitric oxide-mediated relaxation is preserved in retrieved human aortocoronary vein grafts

Although little is known about the endothelial cell function of human saphenous vein coronary artery bypass grafts, there is evidence to suggest that receptor-activated, endothelial-dependent relaxation mediated by nitric oxide is impaired. This study examines the expression and function of endothelial cell constitutive nitric oxide synthase (cNOS) of aortocoronary vein bypass grafts and human saphenous veins obtained from 10 patients undergoing repeat coronary artery bypass grafting for recurrent ischemic symptoms. Following precontraction with norepinephrine (10(-5) M), responses to acetylcholine (receptor-mediated, endothelium-dependent), calcium ionophore (A23187; receptor-independent, endothelium-dependent), and sodium nitroprusside (endothelium-independent) were assessed. Following total RNA extraction using phenol/guanidinium isothiocyanate from specimens of human saphenous vein and vein graft, a quantitative RNase Protection Assay (RPA) was performed using a cRNA riboprobe corresponding to a fragment of the human endothelial cell cNOS gene. Histologically, the vein grafts showed both intimal hyperplasia development and focal atherosclerosis formation compared to the saphenous veins. Scanning electron microscopy of the saphenous veins and the vein grafts showed an intact endothelium. Precontracted vein grafts did not relax in response to acetylcholine; in contrast, the saphenous vein relaxed in a dose-dependent manner to reach a maximal relaxation of 19 +/- 4% precontracted tension. Saphenous veins and vein grafts relaxed in response to A23187 with maximal relaxation of 92 +/- 5 and 73 +/- 13%, respectively. Both vessels relaxed in a dose dependent manner to sodium nitroprusside. RPA normalized to beta-actin showed similar levels of expression of endothelial cell cNOS equivalent to 1 pg of sense RNA in both the saphenous vein and vein graft.(ABSTRACT TRUNCATED AT 250 WORDS)     

Forearm nitric oxide balance, vascular relaxation, and glucose metabolism in NIDDM patients

Endothelium-dependent and -independent vascular responses were assessed in 10 NIDDM patients and 6 normal subjects with no evidence of atherosclerotic disease. Changes in forearm blood flow and arteriovenous (AV) serum nitrite/nitrate (NO2-/NO3-) concentrations were measured in response to intra-arterial infusion of acetylcholine (ACh) (7.5, 15, 30 microg/min, endothelium-dependent response) and sodium nitroprusside (SNP) (0.3, 3, 10 microg/min, endothelium-independent response). Insulin sensitivity (determined by minimal model intravenous glucose tolerance test) was lower in NIDDM patients (0.82 +/- 0.20 vs. 2.97 +/- 0.29 10(4) min x microU(-1) x ml(-1); P < 0.01). Baseline forearm blood flow (4.8 +/- 0.3 vs. 4.4 +/- 0.3 ml x 100 ml(-1) tissue x min(-1); NS), mean blood pressure (100 +/- 4 vs. 92 +/- 4 mmHg; NS), and vascular resistance (21 +/- 1 vs. 21 +/- 1 units; NS), as well as their increments during ACh and SNP, infusion were similar in both groups. No difference existed in baseline NO2-/NO3- concentrations (4.09 +/- 0.33 [NIDDM patients] vs. 5.00 +/- 0.48 micromol/l [control subjects]; NS), their forearm net balance (0.31 +/- 0.08 [NIDDM patients] vs. 0.26 +/- 0.08 micromol/l x 100 ml(-1) tissue x min(-1); NS), and baseline forearm glucose uptake. During ACh infusion, both NO2- and NO3- concentrations and net balance significantly increased in both groups, whereas glucose uptake increased only in control subjects. When data from NIDDM and control groups were pooled together, a correlation was found between the forearm AV NO2- and NO3- differences and blood flow (r = 0.494, P = 0.024). On the contrary, no correlation was evident between NO2- and NO3- concentrations or net balance and insulin sensitivity. In summary, 1) no difference existed in basal and ACh-stimulated NO generation and endothelium-dependent relaxation between uncomplicated NIDDM patients and control subjects; 2) in both NIDDM and control groups, forearm NO2- and NO3- net balance following ACh stimulation was related to changes in the forearm blood flow; and 3) ACh-induced increase in forearm blood flow was associated with an increase in glucose uptake only in control subjects but not in NIDDM patients. In conclusion, our results argue against a role of impaired NO generation and blood flow regulation in determining the insulin resistance of uncomplicated NIDDM patients; rather, it supports an independent insulin regulation of hemodynamic and metabolic effects.     

Expression of three forms of nitric oxide synthase in peripheral nerve regeneration

Nitric oxide (NO) is a short-lived molecule with messenger and cytotoxic functions in nervous, cardiovascular, and immune systems. Nitric oxide synthase (NOS), the enzyme responsible for NO synthesis, exists in three different forms: the neuronal (nNOS), present in discrete neuronal populations; the endothelial (eNOS), present in vascular endotheliun, and the inducible isoform (iNOS), expressed in various cell types when activated, including macrophages and glial cells. In this study, we have investigated the possible involvement of NO in Wallerian degeneration and the subsequent regeneration occurring after sciatic nerve ligature, using histochemistry and immunocytochemistry for the three NOS isoforms, at different postinjury periods. Two days after lesion, the three NOS isoforms are overexpressed, reaching their greatest expression during the second week. nNOS is upregulated in dorsal root ganglion neurons, centrifugally transported and accumulated in growing axons. eNOS is overexpressed in vasa nervorum of the distal stump and around ligature, and iNOS is induced in recruited macrophages. These findings indicate that different cellular sources contribute to maintain high levels of NO at the lesion site. The parallelism between NOS inductions and well-known repair phenomena suggests that NO, acting in different ways, may exert a beneficial effect on nerve regeneration.     

Angiotensin II stimulates production of nitric oxide in guinea pig airways via AT1 receptor activation

Angiotensin II (Ang II) and nitric oxide (NO) regulate a variety of physiological functions. In this study, we suggest that inhaled Ang II produces an initial bronchodilation apparently by stimulating NO production via AT1 receptors. Also, we demonstrate that following the initial bronchodilation, Ang II causes bronchoconstriction in the guinea pig, also via AT1 receptors. Both of the findings are important for our understanding of airway functions induced by Ang II.     

Purification, characterisation and distribution of ovine neuronal nitric oxide synthase

Nitric oxide (NO) is an ubiquitous intercellular messenger molecule synthesised from the amino acid L-arginine by the enzyme nitric oxide synthase (NOS). A number of NOS iso-enzymes have been identified, varying in molecular size, tissue distribution and possible biological role. To further understand the role of NO in the regulation of neuroendocrine function in the sheep, we have purified and characterised ovine neuronal NOS (nNOS) using anion exchange, affinity and size-exclusion chromatography. SDS-PAGE reveals that ovine nNOS has an apparent denatured molecular weight of 150 kDa which correlates well with the other purified nNOS forms such as rat, bovine and porcine. The native molecular weight predicted by size-exclusion chromatography was 200 kD which is in close agreement with that found for porcine and rat nNOS. Internal amino acid sequences generated from tryptic digests of the purified ovine nNOS are highly homologous to rat nNOS. There was no significant difference in the cofactor dependence and kinetic characteristics of ovine nNOS when compared to rat and bovine nNOS, (K(m) for L-arginine 2.8, 2.0 and 2.3 microM respectively). A polyclonal anti-peptide antibody directed toward the C-terminal end of the rat nNOS sequence showed full cross-reactivity with the purified ovine nNOS. Immunohistochemical and Western analysis using this antiserum demonstrate the expression of nNOS in the cortex, cerebellum, hypothalamus and pituitary of the sheep. The lack of staining in the neural and anterior lobes of the pituitary seems to suggest that NOS plays a varied role in the control of endocrine systems between species.     

Ultrastructural localization of nerve terminals containing nitric oxide synthase in rat adrenal gland

The effect of hyperosmolality (300, 320 mosmol/kg H2O) and angiotensin II (A II, 10(-8) and 10(-6) M) was tested on a total of 64 neurons within the periventricular part of the anteroventral third ventricle (AV3V) region in brain slice preparations obtained from ovariectomized (OVX) rats with or without chronic treatment of estradiol-17 beta (E2). Hypertonic perfusion with a 320 mosmol/kg H2O but not a 300 mosmol/kg H2O medium caused a significant increase in the firing discharge rate in OVX animals. Perfusion with either hypertonic medium had no effect in E2-treated rats. The neuronal firing discharge rate of neurons in OVX rats was increased following perfusion with 10(-6) M A II, but not affected following perfusion with 10(-8) M A II. In E2-treated rats, perfusion with neither 10(-8) nor 10(-6) M A II had any effect. These data suggest a dynamic relationship between the ovarian endocrine function and the central mechanisms regulating dipsogenic behavior and/or release of vasopressin in the female rat.     

Down-regulation of neuronal nitric oxide synthase by nitric oxide after oxygen-glucose deprivation in rat forebrain slices

The precise role that nitric oxide (NO) plays in the mechanisms of ischemic brain damage remains to be established. The expression of the inducible isoform (iNOS) of NO synthase (NOS) has been demonstrated not only in blood and glial cells using in vivo models of brain ischemia-reperfusion but also in neurons in rat forebrain slices exposed to oxygen-glucose deprivation (OGD). We have used this experimental model to study the effect of OGD on the neuronal isoform of NOS (nNOS) and iNOS. In OGD-exposed rat forebrain slices, a decrease in the calcium-dependent NOS activity was found 180 min after the OGD period, which was parallel to the increase during this period in calcium-independent NOS activity. Both dexamethasone and cycloheximide, which completely inhibited the induction of the calcium-independent NOS activity, caused a 40-70% recovery in calcium-dependent NOS activity when compared with slices collected immediately after OGD. The NO scavenger oxyhemoglobin produced complete recovery of calcium-dependent NOS activity, suggesting that NO formed after OGD is responsible for this down-regulation. Consistently, exposure to the NO donor (Z)-1-[(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-iu m-1,2-diolate (DETA-NONOate) for 180 min caused a decrease in the calcium-dependent NOS activity present in control rat forebrain slices. Furthermore, OGD and DETA-NONOate caused a decrease in level of both nNOS mRNA and protein. In summary, our results indicate that iNOS expression down-regulates nNOS activity in rat brain slices exposed to OGD. These studies suggest important and complex interactions between NOS isoforms, the elucidation of which may provide further insights into the physiological and pathophysiological events that occur during and after cerebral ischemia.     

Pneumococci stimulate the production of the inducible nitric oxide synthase and nitric oxide by murine macrophages

The role of nitric oxide (NO) in the pathophysiology of gram-positive sepsis is uncertain. In inflammatory conditions, high-output NO production is catalyzed by the enzyme inducible nitric oxide synthase (iNOS). The ability of 2 strains of pneumococci, pneumococcal cell wall preparations, and purified pneumococcal capsule (Pnu-Imune 23) to trigger the production of iNOS protein and NO in RAW 264.7 murine macrophages was tested. Live pneumococci, oxacillin-killed pneumococci, and pneumococcal cell wall preparations stimulated the production of iNOS and NO by RAW 264.7 cells in the presence, but not the absence, of low concentrations of recombinant murine interferon-gamma. In contrast, purified pneumococcal capsule induced little or no iNOS or NO production by these cells. Thus, pneumococci stimulate high-output NO production by murine macrophages. The potential role of NO in the pathogenesis of pneumococcal sepsis deserves further study.     

Monophosphoryl lipid A stimulated up-regulation of nitric oxide synthase and nitric oxide release by human monocytes in vitro

Monophosphoryl lipid A (MPL) is a derivative of lipopolysaccharide (LPS) with reduced toxicity which has been shown to modulate various immune functions in monocytes. We examined whether human monocytes can be stimulated to produce nitric oxide (NO) and its catalytic enzyme nitric oxide synthase (NOS). Monocytes were stimulated with LPS or MPL and both NOS and NO (as nitrite) production were measured. MPL at high doses (> 100 micrograms/ml) stimulated monocytes to release NO that was significantly greater than both the control and LPS-treated monocytes (p < 0.05). NO release by control cells and the LPS treated cells was not significantly different. Both arginase and N-monomethyl arginine (NMLA) inhibited the MPL stimulated release of NO (p < 0.01). MPL significantly increased inducible NOS (iNOS) expression as measured by both fluorescent microscopy and flow cytometry (p < 0.05). Similarly, both soluble NOS (sNOS) and particulate NOS (pNOS) activity were significantly up-regulated by MPL (p < 0.05). Significant correlations were found between pNOS expression and sNOS release (r = 0.72, p < 0.0001) and between 12 h NO release and sNOS production (r = 0.44, p < 0.005). These experiments confirm that human monocytes can be stimulated with MPL to produce NO in vitro and suggest that up-regulation of pNOS does not preclude NO release.