Sperm nitric oxide and motility: the effects of nitric oxide synthase stimulation and inhibition

Nitric oxide (NO) is synthesized from L-arginine by a family of enzymes known as the nitric oxide synthases (NOS). We have recently shown a NOS similar to constitutive brain NOS (bNOS) and endothelial NOS (ecNOS) to be present in spermatozoa. The aim of this study is to investigate NO production by human spermatozoa and the effects of stimulation and inhibition of NOS. This was carried out using the Iso-NO, an isolated NO meter and sensor, which provides rapid, accurate and direct measurements of NO. Semen samples with normozoospermic and asthenozoospermic profiles were prepared using a direct swim-up technique. Basal concentrations of NO and stimulated NO production were measured after exposure to the calcium ionophore (A23187; 0.01-10 microM) a potent activator of constitutive NOS. NO production in human spermatozoa was significantly increased by the addition of A23187 30 seconds after stimulation. Furthermore, this response was greatly diminished by pre-incubating the samples with competitive inhibitors of L-arginine, the substrate for NOS, before treatment with calcium ionophore. In the presence of N(G)-nitro-L-arginine methyl ester (L-NAME), N(G)-nitro-L-arginine (L-NA) or N(G)-methyl-L-arginine (L-NMMA; all at 10 microM), NO production was inhibited with a rank order of potency L-NAME > L-NMMA > L-NA which is in accordance with the inhibition of an endothelial type of constitutive NOS.     

Role of soluble guanylate cyclase in the molecular mechanism underlying the physiological effects of nitric oxide

In this review the molecular mechanisms underlying the antihypertensive and antiaggregatory actions of nitric oxide (NO) are discussed. It has been shown that these effects are directly connected with the activation of soluble guanylate cyclase and the accumulation of cyclic 3;,5;-guanosine monophosphate (cGMP). The mechanism of guanylate cyclase activation by NO is analyzed, especially the role and biological significance of the nitrosyl--heme complex formed as a result of interaction of guanylate cyclase heme with NO and the role of sulfhydryl groups of the enzyme in this process. Using new approaches for studying the antihypertensive and antiaggregatory actions of nitric oxide in combination with the newly obtained data on the regulatory role of guanylate cyclase in the platelet aggregation process, the most important results were obtained regarding the molecular bases providing for a directed search for and creation of new effective antihypertensive and antiaggregatory preparations. In studying the molecular mechanism for directed activation of soluble guanylate cyclase by new NO donors, a series of hitherto unknown enzyme activators generating NO and involved in the regulation of hemostasis and vascular tone were revealed.     

Cell-specific effects of pentoxifylline on nitric oxide production and inducible nitric oxide synthase mRNA expression

Cytokine-stimulated astrocytes and macrophages are potent producers of nitric oxide (NO), a free radical proposed to play an important role in organ-specific autoimmunity, including demyelinating diseases of the central nervous system. The aim of this study was to investigate effects of pentoxifylline (PTX), a phosphodiesterase inhibitor with immunomodulatory properties, on NO production and inducible NO synthase (iNOS) mRNA expression in rat astrocytes and macrophages. We have shown that PTX affects cytokine (interferon-gamma, IFN-gamma; interleukin-1, IL-1; tumour-necrosis factor-alpha, TNF-alpha)-induced NO production in both cell types, but in the opposite manner--enhancing in astrocytes and suppressive in macrophages. While PTX did not have any effect on enzymatic activity of iNOS in activated cells, expression of iNOS mRNA was elevated in astrocytes and decreased in macrophages treated with cytokines and PTX. Treatment with PTX alone affected neither NO production nor iNOS mRNA levels in astrocytes or macrophages. This study indicates involvement of different signalling pathways associated with iNOS induction in astrocytes and macrophages, thus emphasizing complexity of regulation of NO synthesis in different cell types.     

Sleep and nitric oxide: effects of 7-nitro indazole, inhibitor of brain nitric oxide synthase

The in vitro amplification method for heterologous gene expression in mammalian cells is based on the stable transfection of cells with long, linear DNA molecules having several copies of complete expression units, coding for the gene of interest, linked to one terminal unit, coding for the selectable marker. DNA concatenamers containing additional expression units can also be prepared: we exploited this feature by co-polymerizing expression units coding for granulocyte colony-stimulating factor (G-CSF) with cassettes for dihydrofolate reductase (DHFR) and for neomycin (Nm) resistance, as selectable markers. We were thus able to obtain high level production of G-CSF in chinese hamster ovary (CHO) dhfr- cells by combining in vitro amplification to just one step of in vivo amplification. This approach required a considerably shorter time than the classical, stepwise amplification by methotrexate.     

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.     

Acute effects of inhaled nitric oxide in children

At present, there are only two laser Doppler perfusion imaging systems (LDIs) manufactured for medical applications: a 'stepwise' and a 'continuous' scanning LDI. The stepwise scanning LDI has previously been investigated and compared with coloured microsphere determined standardised flow. The continuous scanning LDI is investigated and compared with the stepwise scanning LDI for its ability to measure in vivo, hypoaemic, ligament tissue blood flow changes. The continuous scanning system was supplied with two lasers, red and near infrared (NIR), allowing for additional assessment of the effect of wavelength on imaging ligament perfusion. Perfusion images were obtained from surgically exposed rabbit medial collateral ligaments (MCL). Continuous and stepwise LDI scans were compared using correlation and linear regression analysis of image. averages and standard deviations. Using the same method of analysis, LDI measurements using red and NIR lasers indicated a high degree of correlation, at least over the ranges of perfusion assessed, indicating that red and NIR lasers measure similar regions of flow in the rabbit MCL. These experiments confirm that both LDI techniques provide a valid in vivo measure of dynamic changes in connective tissue perfusion and could have significant impact on the understanding and treatment of joint injury and arthritis.     

A common mechanism for the interaction of nitric oxide with the oxidized binuclear centre and oxygen intermediates of cytochrome c oxidase

The reactions of nitric oxide (NO) with fully oxidized cytochrome c oxidase (O) and the intermediates P and F have been investigated by optical spectroscopy, using both static and kinetic methods. The reaction of NO with O leads to a rapid (approximately 100 s-1) electron ejection from the binuclear center to cytochrome a and CuA. The reaction with the intermediates P and F leads to the depletion of these species in slower reactions, yielding the fully oxidized enzyme. The fastest optical change, however, takes place within the dead time of the stopped-flow apparatus (approximately 1 ms), and corresponds to the formation of the F intermediate (580 nm) upon reaction of NO with a species that we postulate is at the peroxide oxidation level. This species can be formulated as either Fe5+ = O CuB2+ or Fe4+ = O CuB3+, and it is spectrally distinct from the P intermediate (607 nm). All of these reactions have been rationalized through a mechanism in which NO reacts with CuB2+, generating the nitrosonium species CuB1+ NO+, which upon hydration yields nitrous acid and CuB1+. This is followed by redox equilibration of CuB with Fea/CuA or Fea3 (in which Fea and Fea3 are the iron centers of cytochromes a and a3, respectively). In agreement with this hypothesis, our results indicate that nitrite is rapidly formed within the binuclear center following the addition of NO to the three species tested (O, P, and F). This work suggests that nitrosylation at CuB2+ instead of at Fea32+ is a key event in the fast inhibition of cytochrome c oxidase by NO.     

Electrochemical nitric oxide and nitrogen dioxide analyzer for use with inhaled nitric oxide

The use of inhaled nitric oxide (NO) in research and clinical applications requires the monitoring of NO and its autooxidation product nitrogen dioxide (NO2) in inspired gas and in the ambient environment. We describe an inexpensive electrochemical NO and NO2 analyzer that uses a critical orifice constant-flow controller and a microprocessor crossover correction for the measurement of NO and NO2 in the concentration range relevant to the use of inhaled NO. The analyzer proved to have good accuracy and precision for NO and NO2 in the range of concentrations relevant to studies of inhaled NO. In this range, the performance was similar to that of a chemiluminescence analyzer, and the response characteristics were not affected by varying the O2 concentration of the mixtures analyzed.     

Involvement of endogenous nitric oxide in the mechanism of bradykinin-induced peripheral hyperalgesia

1. When NG-nitro-L-arginine methyl ester (L-NAME, 0.1-10 nmol) or NG-monomethyl-L-arginine (L-NMMA, 10 nmol-1 mumol) was intradermally administered with bradykinin (BK, 3 nmol) into the instep of rat hind-paws, a dose-related suppression of BK-induced hyperalgesia, assessed by the paw-pressure test, was produced. 2. L-Arginine (1 mumol) but not D-arginine (1 mumol) reversed the suppressive effects of L-NAME (10 nmol) and L-NMMA (1 mumol) on BK-induced hyperalgesia. 3. Concomitant intradermal administration of BK (3 nmol) with haemoglobin (1 nmol) significantly suppressed BK-induced hyperalgesia in the paw-pressure test. The BK-induced hyperalgesia was abolished by concomitant intradermal administration of either a guanylate cyclase inhibitor, methylene blue (10 nmol), or LY83583 (1 nmol). In addition, KT5823 (1 nmol) or Rp-8-bromoguanosine-3':5'-cyclic monophosphothioate (Rp-8-Br-cGMPS; 1 nmol), an inhibitor of cyclic GMP-dependent protein kinase, also significantly suppressed BK-induced hyperalgesia. 4. The carrageenin-induced hyperalgesia was significantly attenuated by L-NAME in a dose-dependent manner. 5. L-Arginine (1 mumol), sodium nitroprusside (1 mumol), dibutyryl cyclic GMP (1 mumol) or 8-bromo cyclic GMP (1 mumol) all failed to produce any significant relieving effect on the nociceptive threshold of rodent hind-paws. Concomitant administrations of each agent with a sub-threshold dose (0.1 nmol) of BK induced significant hyperalgesia. 6. Rp-adenosine 3':5'-cyclic monophosphothioate (Rp-cAMPS; 1 nmol), an inhibitor of cyclic AMP-dependent protein kinase, significantly suppressed BK-induced mechanical hyperalgesia. Concomitant administration of forskolin (1 nmol) with 8-bromo cyclic GMP (100 nmol) induced significant hyperalgesia. 7. In the superfusion experiment of a blister base on the instep of rodent hind-paws, intradermally administered BK (3 nmol) significantly increased the outflow of both cyclic GMP and cyclic AMP from the blister base. Concomitant administrations of L-NAME (10 nmol) with BK significantly reduced the BK-induced outflow of cyclic GMP without affecting the cyclic AMP content. 8. These results suggest that the NO-cyclic GMP pathway is involved in the mechanism of BK-induced hyperalgesia, and an activation of both cyclic GMP-and cyclic AMP-second messenger system plays an important role in the production of peripherally induced mechanical hyperalgesia.     

Involvement and dual effects of nitric oxide in septic shock

Bacterial endotoxin (LPS) releases many mediators such as interleukins, tumour necrosis factor, oxygen free radicals, toxic eicosanoids, platelet activating factor, and nitric oxide (NO). LPS is a potent inducer of inducible nitric oxide synthase (iNOS). Large amounts of NO (made by iNOS) and peroxynitrite, among other factors, are responsible for the late phase of hypotension, vasoplegia, cellular suffocation, apoptosis, lactic acidosis and multiorgan failure in endotoxic shock. Indeed, experimental and clinical use of NOS inhibitors, which do not differentiate clearly between constitutive endothelial NOS (ceNOS) and iNOS, prevents LPS-induced hypotension. However, many detrimental effects of such NOS inhibitors are also reported, including increases in pulmonary resistance, decreases in cardiac output and organ perfusion, and even an increase in mortality of experimental animals. We believe that, in lungs, NO made by ceNOS plays a protective role against the pneumotoxic effects of LPS-released lipids such as thromboxane, leukotrienes and PAF. This is why selective iNOS inhibitors like aminoguanidine or thiourea derivatives might be preferred over nonselective NOS inhibitors for the treatment of septic shock. However, since iNOS-derived NO seems to have more than just a destructive action, the selective iNOS inhibition may be not as beneficial as expected. Accordingly, inhalation of NO gas or NO-donors in septic shock might be a complementary treatment to the use of NOS inhibitors.     

Scavenging effects of dopamine agonists on nitric oxide radicals

It has recently been considered that free radicals are closely involved in the pathogenesis of Parkinson's disease (PD), and the level of nitric oxide radical (.NO), one of the free radicals, is reported to increase in PD brain. In the present study, we established a direct detection system for .NO in an in vitro .NO-generating system using 3-(2-hydroxy-1-methylethyl-2-nitrosohydrazino)-N-methyl-1-propa namine as an .NO donor and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (carboxy-PTIO) by electron spin resonance (ESR) spectrometry and examined the quenching effects of the dopamine agonists pergolide and bromocriptine on the amount of.NO generated. .NO appeared to be scavenged by pergolide and, to a lesser extent, by bromocriptine. In the competition assay, the 50% inhibitory concentration values for pergolide and bromocriptine were estimated to be approximately 23 and 200 microM, respectively. It was previously reported that in vivo treatment of pergolide and bromocriptine completely protected against the decrease in levels of striatal dopamine and its metabolites in the 6-hydroxydopamine-injected mouse. Considering these findings, pergolide and probably bromocriptine may also protect against dysfunction of dopaminergic neurons because of its multiple effects; not only does it stimulate the presynaptic autoreceptors, but it also directly scavenges .NO radicals and hence protects against .NO-related cytotoxicity. This ESR spectrometry method using carboxy-PTIO may be useful for screening other drugs that can quench .NO.     

Utility of a nitric oxide electrode for monitoring the administration of nitric oxide in biologic systems

Von Hippel-Lindau (VHL) disease is an inherited autosomal dominant neoplastic disorder causing central nervous system haemangioblastomas. The VHL gene (3p25-3p26) is known to be a tumour suppressor gene, with its inactivation being responsible for a predisposition to tumour development. As far as we know, the present report of VHL disease manifestation in identical twins is unique. Genetic inquiry into the family background did not reveal this disease among their progenitors. For presymptomatic diagnosis of 17 presently unaffected family members, constitutional DNA of the twins was screened for VHL germline mutations, using loss of heterozygosity studies and exon-specific DNA sequencing. To determine the influence of somatic mutations of the VHL gene in tumourigenesis, DNA of five surgically removed intracerebral haemangioblastomas of the identical twins was analyzed in comparison with their constitutional DNA by DNA sequencing of the complete VHL coding region. However, no allelic losses were found for the VHL gene or for various other tumour suppressor genes (p53, BRCA1, BRCA2, DCC, and MCC). Furthermore, no mutations were found in the constitutional DNA of either twin sister or in the DNA of all five tumour lesions. Based on our observations, we conclude that in certain VHL families, presymptomatic molecular diagnosis of the disease is not feasible and requires close clinical surveillance of all individuals at risk.     

Localization of nitric oxide synthases and nitric oxide production in the rat mammary gland

We investigated nitric oxide (NO) production and the presence of nitric oxide synthase (NOS) in the mammary gland by use of an organ culture system of rat mammary glands. Mammary glands were excised from the inguinal parts of female Wistar-MS rats primed by implantation with pellets of 17beta-estradiol and progesterone and were diced into approximately 3-mm cubes. Three of these cubes were cultured with 2 ml of 10% FCS/DMEM plus carboxy-PTIO (an NO scavenger, 100 microM) in the presence or absence of LPS (0.5 microgram/ml) for 2 days. The amount of NO produced spontaneously by the cultured mammary glands was relatively minute at the end of the 2-day culture period, and the NO production was significantly enhanced by the presence of LPS. This enhancement of NO production was completely eliminated by addition of hydrocortisone (3 microM), an inhibitor of inducible NOS (iNOS), to the incubation medium. Immunoblot analyses with specific antisera against NOS isoforms such as iNOS, endothelial NOS (eNOS), and brain NOS (bNOS) showed immunoreactive bands of iNOS (122 +/- 2 kD) and eNOS (152 +/- 3 kD) in extracts prepared from the mammary glands in the culture without LPS. The immunoreactive band of iNOS was highly intense after the treatment of mammary glands with LPS, whereas the corresponding eNOS immunoreactive band was faded. The immunohistochemical study of anti-iNOS antiserum on frozen sections of the cultured mammary glands showed that an immunoreactive substance with the antiserum was localized to the basal layer (composed of myoepithelial cells of alveoli and lactiferous ducts) of the mammary epithelia and to the endothelium of blood vessels that penetrated into the interstitium of the mammary glands. Histochemical staining for NADPH-diaphorase activity, which is identical to NOS, showed localization similar to that of iNOS in the mammary glands. Similar observations were noted in the immunohistochemistry of eNOS. In contrast, the immunoreactive signal with the bNOS antiserum was barely detected in the epithelial parts of alveoli and lactiferous ducts of the mammary glands. These observations demonstrate that three isoforms of NOS are present not only in the endothelium of blood vessels but also in the parenchymal cells (the glandular epithelium) of the rat mammary gland, such as epithelial cells and myoepithelial cells, and suggest that NO may have functional roles in the physiology of the mammary glands.     

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.     

Changes of vasoactive peptides and effects of inhaled nitric oxide after pneumonectomy

To clarify the role of vasoactive peptides in the physiologic response to pneumonectomy, we investigated the changes of atrial (A-type) natriuretic peptide (ANP). C-type natriuretic peptide (CNP), and endothelin-1 (ET-1) levels in the lung and blood after pneumonectomy and the effects of inhaled nitric oxide (NO; 5 ppm) after pneumonectomy in beagle dogs. The concentrations of these peptides in the lung and blood were measured by radioimmunoassay. The dogs in group A (n = 10) were observed without NO inhaling after right pneumonectomy, and the dogs in group B (n = 5) were observed with NO inhaling from 120 to 180 min after right pneumonectomy. After the thoracotomy, right lung tissue was resected for the pre-operative histological control. Tissue from the left lung was obtained at 120 min (5 dogs in group A), at 180 min (5 dogs in group A), and after 60 min of NO inhalation (group B) for the post-operative histological material. Peripheral blood was collected from the femoral artery. The pulmonary arterial pressure (PAP) was significantly increased after pneumonectomy, but rapidly decreased to the same level as the pre-operative stage after NO inhalation. Increases of plasma ANP, lung ANP and lung CNP levels occurred after pneumonectomy, while the ET-1 level was unchanged. Inhaled NO rapidly reduced the plasma ANP, lung ANP and lung CNP. These results indicate that both ANP and CNP act to maintain normotensive homeostatic balance in the pulmonary circulation.     

Cardiovascular effects of nitric oxide and adenosine in the nucleus tractus solitarii of rats

It has been shown that nitric oxide (NO) is synthesized in the central nervous system as well as in vascular endothelial cells. We recently reported that NO was involved in central cardiovascular regulation, modulated the baroreflex, and was involved in a reciprocal release with excitatory amino acids in the nucleus tractus solitarii (NTS) of rats. We also reported previously that adenosine increased the release of glutamate in the NTS. The purpose of the present study was to investigate the possible interaction of NO and adenosine in the NTS. Male Sprague-Dawley rats were anesthetized with urethane, and blood pressure was monitored intra-arterially. Unilateral microinjection of L-arginine (3.3 nmol/60 nL) into the NTS produced decreases in blood pressure and heart rate. Microinjection of adenosine (2.3 nmol/60 nL) also produced depressive and bradycardic effects. These cardiovascular effects were attenuated by prior administration of the specific adenosine receptor antagonist DPSPX (0.92 nmol). Similarly, prior administration of NO synthase inhibitor NG-monomethyl-L-arginine or NG-nitro-L-arginine methyl ester significantly attenuated the depressive and bradycardic effects of adenosine. These results demonstrate a reciprocal attenuation of adenosine receptor antagonist and NO synthase inhibitor on L-arginine and adenosine responses, respectively, in the NTS and implicate an interaction between NO and adenosine in central cardiovascular regulation.     

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.     

Protective effects of intravascular pressure and nitric oxide in ischemic lung injury

Cessation of blood flow during ischemia will decrease both distending and shear forces exerted on endothelium and may worsen ischemic lung injury by decreasing production of nitric oxide (NO), which influences vascular barrier function. We hypothesized that increased intravascular pressure (Piv) during ventilated ischemia might maintain NO production by increasing endothelial stretch or shear forces, thereby attenuating ischemic lung injury. Injury was assessed by measuring the filtration coefficient (Kf) and the osmotic reflection coefficient for albumin (sigmaalb) after 3 h of ventilated (95% O2-5% CO2; expiratory pressure 3 mmHg) ischemia. Lungs were flushed with physiological salt solution, and then Piv was adjusted to achieve High Piv (mean 6.7 +/- 0.4 mmHg, n = 15) or Low Piv (mean 0.83 +/- 0.4 mmHg, n = 10). NG-nitro-L-arginine methyl ester (L-NAME; 10(-5) M, n = 10), NG-nitro-D-arginine methyl ester (D-NAME; 10(-5) M, n = 11), or L-NAME (10(-5) M)+L-arginine (5 x 10(-4) M, n = 6) was added at the start of ischemia in three additional groups of lungs with High Piv. High Piv attenuated ischemic injury compared with Low Piv (sigmaalb 0.67 +/- 0.04 vs. 0. 35 +/- 0.04, P < 0.05). The protective effect of High Piv was abolished by L-NAME (sigmaalb 0.37 +/- 0.04, P < 0.05) but not by D-NAME (sigmaalb 0.63 +/- 0.07). The effects of L-NAME were overcome by an excess of L-arginine (sigmaalb 0.56 +/- 0.05, P < 0.05). Kf did not differ significantly among groups. These results suggest that Piv modulates ischemia-induced barrier dysfunction in the lung, and these effects may be mediated by NO.