3-morpholinosydnonimine Research Articles

Role of endothelial colony forming cells (ECFCs) Tetrahydrobiopterin (BH4) in determining ECFCs functionality in coronary artery disease (CAD) patients

Nitric oxide (NO.) is critical for functionality of endothelial colony forming cells (ECFCs). Dimerization of endothelial nitric oxide synthase (eNOS) is must to produce NO. and tetrahydrobiopterin (BH4) plays a crucial role in stabilizing this state. We investigated BH4 level in ECFCs and its effect on ECFCs functionality in CAD patients. Intracellular biopterin levels and ECFCs functionality in terms of cell viability, adhesion, proliferation, in vitro wound healing and angiogenesis were assessed. Guanosine Triphosphate Cyclohydrolase-1 (GTPCH-1) expression was studied in ECFCs. Serum total reactive oxygen/nitrogen species was measured and effect of nitrosative stress on ECFC’s biopterins level and functionality were evaluated by treating with 3-morpholino sydnonimine (SIN-1). BH4 level was significantly lower in ECFCs from CAD patients. Cell proliferation, wound closure reflecting cellular migration as well as in vitro angiogenesis were impaired in ECFCs from CAD patients. Wound healing capacity and angiogenesis were positively correlated with ECFC’s BH4. A negative effect of nitrosative stress on biopterins level and cell functionality was observed in SIN-1 treated ECFCs. ECFCs from CAD exhibited impaired functionality and lower BH4 level. Association of BH4 with wound healing capacity and angiogenesis suggest its role in maintaining ECFC’s functionality. Oxidative stress may be a determinant of intracellular biopterin levels.

Development of an Analytical Assay for Electrochemical Detection and Quantification of Protein-Bound 3-Nitrotyrosine in Biological Samples and Comparison with Classical, Antibody-Based Methods.

Reactive oxygen and nitrogen species (RONS) cause oxidative damage, which is associated with endothelial dysfunction and cardiovascular disease, but may also contribute to redox signaling. Therefore, their precise detection is important for the evaluation of disease mechanisms. Here, we compared three different methods for the detection of 3-nitrotyrosine (3-NT), a marker of nitro-oxidative stress, in biological samples. Nitrated proteins were generated by incubation with peroxynitrite or 3-morpholino sydnonimine (Sin-1) and subjected to total hydrolysis using pronase, a mixture of different proteases. The 3-NT was then separated by high performance liquid chromatography (HPLC) and quantified by electrochemical detection (ECD, CoulArray) and compared to classical methods, namely enzyme-linked immunosorbent assay (ELISA) and dot blot analysis using specific 3-NT antibodies. Calibration curves for authentic 3-NT (detection limit 10 nM) and a concentration-response pattern for 3-NT obtained from digested nitrated bovine serum albumin (BSA) were highly linear over a wide 3-NT concentration range. Also, ex vivo nitration of protein from heart, isolated mitochondria, and serum/plasma could be quantified using the HPLC/ECD method and was confirmed by LC-MS/MS. Of note, nitro-oxidative damage of mitochondria results in increased superoxide (O2•–) formation rates (measured by dihydroethidium-based HPLC assay), pointing to a self-amplification mechanism of oxidative stress. Based on our ex vivo data, the CoulArray quantification method for 3-NT seems to have some advantages regarding sensitivity and selectivity. Establishing a reliable automated HPLC assay for the routine quantification of 3-NT in biological samples of cell culture, of animal and human origin seems to be more sophisticated than expected.

Formation of 2-nitrophenol from salicylaldehyde as a suitable test for low peroxynitrite fluxes

There has been some dispute regarding reaction products formed at physiological peroxynitrite fluxes in the nanomolar range with phenolic molecules, when used to predict the behavior of protein-bound aromatic amino acids like tyrosine. Previous data showed that at nanomolar fluxes of peroxynitrite, nitration of these phenolic compounds was outcompeted by dimerization (e.g. biphenols or dityrosine). Using 3-morpholino sydnonimine (Sin-1), we created low fluxes of peroxynitrite in our reaction set-up to demonstrate that salicylaldehyde displays unique features in the detection of physiological fluxes of peroxynitrite, yielding detectable nitration but only minor dimerization products.By means of HPLC analysis and detection at 380nm we could identify the expected nitration products 3- and 5-nitrosalicylaldehyde, but also novel nitrated products. Using mass spectrometry, we also identified 2-nitrophenol and a not fully characterized nitrated dimerization product. The formation of 2-nitrophenol could proceed either by primary generation of a phenoxy radical, followed by addition of the NO2-radical to the various resonance structures, or by addition of the peroxynitrite anion to the polarized carbonyl group with subsequent fragmentation of the adduct (as seen with carbon dioxide). Interestingly, we observed almost no 3- and 5-nitrosalicylic acid products and only minor dimerization reaction.Our results disagree with the previous general assumption that nitration of low molecular weight phenolic compounds is always outcompeted by dimerization at nanomolar peroxynitrite fluxes and highlight unique features of salicylaldehyde as a probe for physiological concentrations of peroxynitrite.

Protein Tyrosine Nitration and Thiol Oxidation by Peroxynitrite—Strategies to Prevent These Oxidative Modifications

The reaction product of nitric oxide and superoxide, peroxynitrite, is a potent biological oxidant. The most important oxidative protein modifications described for peroxynitrite are cysteine-thiol oxidation and tyrosine nitration. We have previously demonstrated that intrinsic heme-thiolate (P450)-dependent enzymatic catalysis increases the nitration of tyrosine 430 in prostacyclin synthase and results in loss of activity which contributes to endothelial dysfunction. We here report the sensitive peroxynitrite-dependent nitration of an over-expressed and partially purified human prostacyclin synthase (3.3 μM) with an EC50 value of 5 μM. Microsomal thiols in these preparations effectively compete for peroxynitrite and block the nitration of other proteins up to 50 μM peroxynitrite. Purified, recombinant PGIS showed a half-maximal nitration by 10 μM 3-morpholino sydnonimine (Sin-1) which increased in the presence of bicarbonate, and was only marginally induced by freely diffusing NO2-radicals generated by a peroxidase/nitrite/hydrogen peroxide system. Based on these observations, we would like to emphasize that prostacyclin synthase is among the most efficiently and sensitively nitrated proteins investigated by us so far. In the second part of the study, we identified two classes of peroxynitrite scavengers, blocking either peroxynitrite anion-mediated thiol oxidations or phenol/tyrosine nitrations by free radical mechanisms. Dithiopurines and dithiopyrimidines were highly effective in inhibiting both reaction types which could make this class of compounds interesting therapeutic tools. In the present work, we highlighted the impact of experimental conditions on the outcome of peroxynitrite-mediated nitrations. The limitations identified in this work need to be considered in the assessment of experimental data involving peroxynitrite.

The inhibitory effect of S‐nitrosoglutathione on blood–brain barrier disruption and peroxynitrite formation in a rat model of experimental stroke

The hallmark of stroke injury is endothelial dysfunction leading to blood-brain barrier (BBB) leakage and edema. Among the causative factors of BBB disruption are accelerating peroxynitrite formation and the resultant decreased bioavailability of nitric oxide (NO). S-nitrosoglutathione (GSNO), an S-nitrosylating agent, was found not only to reduce the levels of peroxynitrite but also to protect the integrity of BBB in a rat model of cerebral ischemia and reperfusion (IR). A treatment with GSNO (3 μmol/kg) after IR reduced 3-nitrotyrosine levels in and around vessels and maintained NO levels in brain. This mechanism protected endothelial function by reducing BBB leakage, increasing the expression of Zonula occludens-1 (ZO-1), decreasing edema, and reducing the expression of matrix metalloproteinase-9 and E-selectin in the neurovascular unit. An administration of the peroxynitrite-forming agent 3-morpholino sydnonimine (3 μmol/kg) at reperfusion increased BBB leakage and decreased the expression of ZO-1, supporting the involvement of peroxynitrite in BBB disruption and edema. Mechanistically, the endothelium-protecting action of GSNO was invoked by reducing the activity of nuclear factor kappa B and increasing the expression of S-nitrosylated proteins. Taken together, the results support the ability of GSNO to improve endothelial function by reducing nitroxidative stress in stroke.

Evidence against tetrahydrobiopterin depletion of vascular tissue exposed to nitric oxide/superoxide or nitroglycerin

Several cardiovascular disorders, including atherosclerosis and tolerance to the antianginal drug nitroglycerin (GTN), may be associated with the generation of superoxide anions, which react with nitric oxide (NO) to yield peroxynitrite. According to a widely held view, oxidation of tetrahydrobiopterin (BH 4) by peroxynitrite causes uncoupling of endothelial NO synthase (eNOS), resulting in reduced NO bioavailability and endothelial dysfunction under conditions of oxidative stress. In this study we determined the levels of reduced biopterins and endothelial function in cultured cells exposed to peroxynitrite and GTN as well as in blood vessels isolated from GTN-tolerant guinea pigs and rats. BH 4 was rapidly oxidized by peroxynitrite and 3-morpholino sydnonimine (SIN-1) in buffer, but this was prevented by glutathione and not observed in endothelial cells exposed to SIN-1 or GTN. Prolonged treatment of the cells with 0.1 mM GTN caused slow N G-nitro- l-arginine-sensitive formation of reactive oxygen species without affecting eNOS activity. Endothelial function and BH 4/BH 2 levels were identical in blood vessels of control and GTN-tolerant animals. Our results suggest that peroxynitrite-triggered BH 4 oxidation does not occur in endothelial cells or GTN-exposed blood vessels. GTN seems to trigger minor eNOS uncoupling that is unrelated to BH 4 depletion and without observable consequence on eNOS function.

Arginase contributes to endothelial cell oxidative stress in response to plasma from women with preeclampsia

Preeclampsia is a hypertensive disorder characterized by vascular oxidative stress. Decreased availability of the vasodilator nitric oxide (NO) has been postulated to be involved in the pathophysiology of this disorder. Arginase, an enzyme that competes with nitric oxide synthase (NOS) for l-arginine, not only reduces NO formation but also increases superoxide production by NOS. In placenta of preeclamptic women, arginase upregulation has been shown to be increased and contributes to superoxide formation via uncoupling of NOS. However, the role of arginase in the maternal vasculature is not clear. We hypothesized that arginase would be upregulated in the maternal vasculature of women with preeclampsia and contribute to oxidative stress within the endothelium. We observed increased arginase expression in the maternal vasculature of women with preeclampsia compared with normotensive pregnant women. Furthermore, human umbilical vein endothelial cells treated with 2% plasma from preeclamptic women show increased arginase II expression and activity that was reduced by a peroxynitrite scavenger. Also, both 3-morpholino sydnonimine and exogenous peroxynitrite increased arginase expression and activity. Preeclamptic plasma treatment increased superoxide and peroxynitrite levels. Superoxide levels were significantly reduced after arginase and NOS inhibition with [(S)-(2-boronoethyl)-l-cysteine] and N(omega)-nitro-l-arginine methyl ester, respectively, but peroxynitrite levels were in fact increased after arginase inhibition. Moreover, in the presence of preeclamptic plasma, l-arginine supplementation increased peroxynitrite formation during arginase inhibition. Increased arginase expression in preeclampsia can induce uncoupling of NOS as a source of superoxide in the maternal vasculature in preeclampsia. However, l-arginine supplementation in the face of oxidative stress could lead to a further increase in peroxynitrite.

Increased Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1 Expression in the Maternal Vasculature of Women With Preeclampsia

Preeclampsia is a hypertensive disorder unique to pregnancy, in which the placenta may release factors into the maternal circulation resulting in systemic effects. Small dense low-density lipoprotein (LDL; which is susceptible for oxidation) is increased in preeclampsia. Lectin-like oxidized LDL receptor-1 (LOX-1) is a receptor for oxidized LDL. However, the expression levels and the regulation of LOX-1 in the maternal vasculature of women with preeclampsia are unknown. We hypothesized that there is an increased LOX-1 expression in arteries from women with preeclampsia. We further hypothesized that circulating factors in the plasma of women with preeclampsia would upregulate the LOX-1 expression in vascular endothelial cells and contribute to vascular endothelial oxidative stress. We observed abundant LOX-1 expression and the presence of oxidized LDL in arteries from women with preeclampsia, which was negligible in arteries from normotensive pregnant women. Human umbilical vein endothelial cells treated for 24 hours with 2% plasma from preeclamptic women increased LOX-1 expression and oxidized LDL uptake, as well as induced oxidative stress, as evidenced by increased NADPH oxidase activity and superoxide and peroxynitrite levels. These effects were significantly reduced by pretreatment with blocking antibody or small interfering RNA to LOX-1, as well as 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron (III), chloride (FeTPPS), a peroxynitrite scavenger. Exogenous peroxynitrite and 3-morpholino sydnonimine (SIN-1) increased LOX-1 protein and mRNA expression. In conclusion, increased LOX-1 expression in the systemic vasculature of preeclampsia women provides a fundamental insight into the pathology of preeclampsia and likely contributes to the induction and maintenance of vascular oxidative stress.

Detection of Superoxide and Peroxynitrite in Model Systems and Mitochondria by the Luminol Analogue L-012

In the present study we investigated the specificity and sensitivity of the chemiluminescence (CL) dye and luminol analogue 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4-(2H,3H) dione (L-012) to detect reactive oxygen species (ROS) such as superoxide, peroxynitrite and hydrogen peroxide in cell free systems as well as in isolated mitochondria. The results obtained by L-012 were compared with other CL substances such as luminol, lucigenin, coelenterazine and the fluorescence dye dihydroethidine. The results indicate that the L-012-derived chemiluminescence induced by superoxide from hypoxanthine/xanthine oxidase (HX/XO) or by 3-morpholino sydnonimine (SIN-1)-derived peroxynitrite largely depends on the incubation time. Irrespective of the experimental conditions, L-012-derived CL in response to HX/XO and SIN-1 was 10–100 fold higher than with other CL dyes tested. In a cell-free system, authentic peroxynitrite yielded a higher L-012-enhanced CL signal than authentic superoxide and the superoxide-induced signal in cell-free as well as isolated mitochondria increased in the presence of equimolar concentrations of nitrogen monoxide (NO). The superoxide signal/background ratio detected by L-012-enhanced CL in isolated mitochondria with blocked respiration was 7 fold higher than that obtained by the superoxide sensitive fluorescence dye dihydroethidine. We conclude that L-012-derived CL may provide a sensitive and reliable tool to detect superoxide and peroxynitrite formation in mitochondrial suspensions.

NMDA-induced acetylcholine release in mouse striatum: role of NO synthase isoforms.

Striatal cholinergic interneurons are stimulated by glutamatergic inputs from thalamus and cortex via NMDA receptors. The present microdialysis study was designed to characterize the role of nitric oxide (NO) in this process and to identify the NO synthase (NOS) isoform responsible for this effect. For this purpose, we studied the effects of NMDA and 3-morpholino sydnonimine (SIN-1) perfusions on the release of acetylcholine (ACh) in mouse striatum. In wild-type C57/Bl6 mice, perfusion of NMDA (100 micro m) induced a two-fold stimulation of ACh release. This effect was attenuated in mice lacking endothelial NOS but was completely absent in mice lacking neuronal NOS. Local perfusion of SIN-1 (300 micro m), an NO donor, increased ACh release by more than two-fold in all three mouse lines. We conclude that NO synthesized by neuronal NOS provides a nitrergic link in the glutamatergic stimulation of striatal cholinergic interneurons.

Oxidation and Nitrosation in the Nitrogen Monoxide/Superoxide System

Based on the previous report of McCord and co-workers (Crow, J. P., Beckman, J. S., and McCord, J. M. (1995) Biochemistry 34, 3544-3552), the zinc dithiolate active site of alcohol dehydrogenase (ADH) has been studied as a target for cellular oxidants. In the nitrogen monoxide ((*NO)/superoxide (O(2)) system, an equimolar generation of both radicals under peroxynitrite (PN) formation led to rapid inactivation of ADH activity, whereas hydrogen peroxide and ( small middle dot)NO alone reacted too slowly to be of physiological significance. 3-Morpholino sydnonimine inactivated the enzyme with an IC(50) value of 250 nm; the corresponding values for PN, hydrogen peroxide, and (*NO) were 500 nm, 50 microm, and 200 microm. When superoxide was generated at low fluxes by xanthine oxidase, it was quite effective in ADH inactivation (IC(50) (XO) approximately 1 milliunit/ml). All inactivations were accompanied by zinc release and disulfide formation, although no strict correlation was observed. From the two zinc thiolate centers, only the zinc Cys(2)His center released the metal by oxidants. The zinc Cys(4) center was also oxidized, but no second zinc atom could be found with 4-(2-pyridylazo)resorcinol (PAR) as a chelating agent except under denaturing conditions. Surprisingly, the oxidative actions of PN were abolished by a 2-3-fold excess of (*)NO under generation of a nitrosating species, probably dinitrogen trioxide. We conclude that in cellular systems, low fluxes of (*)NO and O(2) generate peroxynitrite at levels effective for zinc thiolate oxidations, facilitated by the nucleophilic nature of the complexed thiolate group. With an excess of (*)NO, the PN actions are blocked, which may explain the antioxidant properties of (*)NO and the mechanism of cellular S-nitrosations.

The Effect of the Nitric Oxide Synthesis Inhibitorl-NAME on Amitriptyline-Induced Hypotension in Rats

Hypotension induced by tricyclic antidepressants is multifactorial. Previous animal experiments suggest a contribution from nitric oxide production. Our study aimed to evaluate the role of nitric oxide in amitriptyline-induced hypotension using N-nitro-L-arginine methyl ester, a nitric oxide synthesis inhibitor, and 3-morpholino sydnonimine, a nitric oxide donor, in anesthetized rats. Amitriptyline intoxication was induced by the continuous infusion of amitriptyline 0.625 mg/kg/min throughout the experiment in anesthetized rats. Fifteen and 25 minutes after amitriptyline infusion began, two bolus doses of 10 mg/kg of N-nitro-L-arginine methyl ester (n = 8) or an equivalent volume of 5% dextrose solution (n = 8) was administered to each rat (Protocol 1). To investigate whether the effect of N-nitro-L-arginine methyl ester on blood pressure is counteracted by 3-morpholino sydnonimine, after the same protocol of amitriptyline infusion and 5 minutes after an N-nitro-L-arginine methyl ester bolus, a bolus of 3000 nmol/kg of 3-morpholino sydnonimine was administered (n = 8) to each rat (Protocol 2). To investigate the effect of N-nitro-L-arginine methyl ester on 3-morpholino sydnonimine induced hypotension, a group of rats received a continuous infusion of 0.54 mg/kg/h of 3-morpholino sydnonimine until 50% reduction was observed in mean arterial blood pressure followed by a bolus dose of 10 mg/kg of N-nitro-L-arginine methyl ester (n = 6) or 5% dextrose solution (n = 6) (Protocol 3). Outcome measures included mean arterial blood pressure, heart rate, and QRS duration in electrocardiogram. Student's t test and survival analysis were used for selected comparisons. For all parameters, the treatment groups were similar at baseline and at postamitriptyline periods before therapy was rendered. Amitriptyline infusion significantly reduced mean arterial blood pressure by 50.8 +/- 2.2% and prolonged QRS by 23.9 +/- 7.2% after 15 minutes. In Protocol 1, N-nitro-L-arginine methyl ester significantly increased mean arterial blood pressure compared to dextrose-treated control animals within 30 minutes (77.9 +/- 8.5% vs. 49.7 +/- 5.0% mmHg, p < 0.01, 95% CI 57.1-98.7%). QRS duration progressively increased during the amitriptyline infusion; however, there was no significant difference in QRS width between N-nitro-L-arginine methyl ester and control groups at any time point. N-nitro-L-arginine methyl ester increased survival time compared to controls (33.4 +/- 4.1 vs. 19.9 +/- 2.7 minutes, p < 0.01, 95% CI 25.4-41.3) but did not affect mortality. In Protocol 2 of continuous infusion of amitriptyline, 3-morpholino sydnonimine counteracted the N-nitro-L-arginine methyl ester-induced increase in mean arterial blood pressure. In both protocols, heart rate decreased significantly during amitriptyline infusion but there was no difference between treatment and control groups. In Protocol 3, N-nitro-L-arginine methyl ester bolus reversed 3-morpholino sydnonimine-induced hypotension compared to dextrose bolus. (83.8 +/- 5.7% vs. 54.6 +/- 4.8%, p < 0.01, 95% CI 69.2-98.4). N-nitro-L-arginine methyl ester is found to be effective in temporarily improving hypotension and prolonging survival time but does not affect overall mortality. Because this effect was antagonized by 3-morpholino sydnonimine, nitric oxide production appears to contribute to the pathophysiology of amitriptyline-induced hypotension.

Effects of propofol on endothelial cells subjected to a peroxynitrite donor (SIN‐1)

We investigated the effect of propofol on endothelial cells subjected to the peroxynitrite (ONOO-) donor 3-morpholino sydnonimine (SIN-1). Cells were incubated overnight with 0.5, 1.0 or 2.0 mM SIN-1, with or without 10-3 M propofol (Diprivan). Cytotoxicity, assessed by measuring the release of pre-incorporated 51Cr, increased when the concentration of SIN-1 increased, and was significantly decreased by 10-3 M propofol (90%, 78% and 28% of protection against 0.5, 1.0 and 2.0 mM SIN-1, respectively). Cell protection against 1 mM SIN-1 was tested with 0.03-1.0 mM propofol and this was compared to tyrosine, a target molecule for peroxynitrite. Propofol protected cells in a dose-dependent manner (r = 0.98; p < 0.001) and was as effective as tyrosine. Finally, using high-performance liquid chromatography, we demonstrated that propofol reacted with ONOO- more rapidly than did tyrosine, inhibiting nitrotyrosine formation. In the absence of propofol, 3.5 mM ONOO- with 1 mM tyrosine yielded 39.6% nitrotyrosine, but nitrotyrosine was not produced when 5 mM propofol was added. We conclude that propofol protects endothelial cells against the toxicity of ONOO-. The anti-oxidant properties of propofol can be partially attributed to its scavenging effect on peroxynitrite, a property that might be relevant in pathological situations involving a significant contribution of peroxynitrite to tissue damage.

Epinephrine prevents nitric oxide/peroxynitrite induced apoptosis of neuronal cells through ?-adrenergic receptor activation

In the present study, we demonstrate that the NO-donors sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP) and 3-morpholino sydnonimine (SIN-1) dose-dependently caused rat pheochromocytoma (PC-12) and human neuroblastoma (NB-1) cells death, determined by tryphan blue dye exclusion. That death was apoptosis, as determined by terminal-deoxynucleotide transferase-mediated dUTP-digoxigenin nick end-labeling (TUNEL) method and propidium iodide (PI) staining using fluorescence-activated cells sorting (FACS). Epinephrine exhibited pronounced inhibitory effect on this NO donors-induced cell death. Similar protecting effect was also found when cells were pretreated with β-adrenergic receptor agonist isoproterenol, adenylate cyclase activator forskolin, nerve growth factor (NGF) or antioxidant N-acetyl-L-cysteine (NAC). However, cycloheximide the inhibitor of protein synthesis, the transcription inhibitor actinomycin D (act D) or anti-NGF abolished the rescuing effect of epinephrine. Furthermore, SNP and SIN-1, which form peroxynitrite in the culture media were more effective to retract neurites of NB-1 cells compared to SNAP. These results suggest that NGF plays pivotal role in epinephrine-supported cytoprotection, and probably peroxynitrite is involved in the retraction of neurites of the neuronal cells.

A Novel Action of 5-Hydroxytryptamine as a Peroxynitrite Scavenger

Peroxynitrite is a strong oxidant formed by the reaction of a superoxide anion radical and nitric oxide. It plays an important role in inducing apoptosis, damaging the mitochondrial respiratory chain and causing the occurrence of nitrotyrosine in cortical neurons. We have demonstrated that 5-hydroxytryptamine (5-HT) does not inhibit peroxynitrite formation by 3-morpholinosydnonimine with the release of a superoxide anion radical and nitric oxide, however, it does markedly scavenge peroxynitrite itself. 5-HT is directly involved in the scavenging of peroxynitrite, and might play a role in retarding the development of brain disease.

Methylene blue inhibits stimulatory effect of sodium nitroprusside but not of 3-morpholino sydnonimine on prolactin secretion in freely moving male rats

Previously we reported that intracerebroventricular (i.c.v.) administration of nitric oxide (NO) donors, sodium nitroprusside (SNP) (1–10 μg), and 3-morpholino-sydnonimine (SIN-1) (10–100 μg), induced dose-dependent increases in plasma prolactin levels of freely moving male rats, suggesting a role of NO in the control of prolactin secretion. The present results show that i.c.v. pretreatment with methylene blue (MB) (30 μg), a guanylyl cyclase inhibitor, significantly reduced the effects of microinjections of SNP (3 and 5 μg), however, this did not modify the stimulatory action of SIN-1 (30 μg) on plasma prolactin levels of conscious male rats. Alone, MB did not modify basal prolactin levels. These results suggest different mechanisms of action of SNP and SIN-1 to stimulate prolactin secretion in vivo. Activation of soluble guanylyl cyclase seems to mediate the neuroendocrine action of NO released from SNP but not of SIN-1. Different cellular distribution of NO generating activity from these donors as well as the possible generation of other radicals simultaneously with NO from SIN-1 could explain these differences.

Effect of K+ channel blocking drugs and nitric oxide synthase inhibition on the response to hypoxia in rat pulmonary artery rings.

1. The aims of this study were to investigate the effects of potassium (K+) channel blockers and the nitric oxide (NO) synthase inhibitor, L-nitroarginine (L-NOARG), on the response produced by acute hypoxia in rat intrapulmonary artery rings in vitro. 2. In rat phenylephrine-precontracted pulmonary artery rings, hypoxia (pO2 = 7 mmHg) induced a response which consisted of a rapidly developing initial contraction (phase 1), a transient relaxation (phase 2) and a slowly developing sustained contraction (phase 3) over 30 min. The NOS inhibitor, L-NOARG (300 microM), attenuated phase 1 and 3, and amplified phase 2 of the response to hypoxia. The voltage-gated K+ channel blocker 4-aminopyridine (4-AP) (10 mM) also abolished phase 3 and magnified phase 2 of the response to hypoxia. 3. The hypoxic response was not modified by the calcium-activated K+ channel (KCa) blockers, tetraethylammonium (TEA) (20 mM) or charybdotoxin (50 or 200 nM), nor by the ATP-dependent K+ channel (KATP), blocker, glibenclamide (10 microM). 4. L-NOARG (300 microM) and 4-AP (10 mM) also abolished carbachol-induced endothelium-dependent NO-mediated relaxation. Relaxation produced by the NO releasing agent 3-morpholino sydnonimine (SIN-1) was reduced by 4-AP (10 mM) and TEA (20 mM). 5. The data suggest that NO production is reduced during severe hypoxia in rat intrapulmonary artery rings and that this underlies the sustained phase of the hypoxic contraction. The data also suggests that 4-AP-sensitive K+ channels play an important role in the release and or action of NO, and therefore, in the response to hypoxia.

Role of nitric oxide in the regulation of osteoblast metabolism.

Nitric oxide has recently been shown to inhibit the proliferation of several types of cells, including osteoclasts. Both osteoclasts and osteoblasts have inducible nitric oxide synthase and produce nitric oxide. Although the direct effect of nitric oxide on osteoblasts in general and osteoblast proliferation in particular has not been delineated, the authors performed studies to clarify the role of nitric oxide on osteoblast proliferation and metabolism. Cultures of human osteoblasts were exposed to 0.1, 1.0, and 10 microM of the nitric oxide releasing agent 3-morpholino sydnonimine (SIN-1) for 7 days. Cells were evaluated for proliferation and production of alkaline phosphatase and osteocalcin. Osteoblasts exhibited decreased proliferation relative to control cultures at 1.0 and 10 microM concentrations of SIN-1 (p < 0.05). Concentrations of 1.0 and 10 microM of SIN-1 effected a decreased production of osteocalcin and alkaline phosphatase (p < 0.05). The results of these studies indicate that nitric oxide may play a critical role by which osteoblasts exhibit self-regulation of mineral metabolism.

Nitric oxide counteracts 5-hydroxytryptamine- and cholera toxin-induced fluid secretion and enhances the effect of oral rehydration solution

The effects of pharmacological modulation of the nitric oxide (NO) pathway on intestinal fluid transport were studied in a model of ligated jejunal loops of anaesthetized rats in vivo. Close intraarterial infusion of 5-hydroxytryptamine (5-HT) (0.16 μg/min) induced net fluid secretion. Intravenous infusion of the NO synthase inhibitor N ω-nitro- l-arginine methyl ester ( l-NAME) (0.55 mg/kg per min) reversed net fluid absorption in controls to net secretion and significantly enhanced 5-HT-induced fluid secretion. 5-HT-induced net fluid secretion was inhibited by intravenous infusion of l-arginine (8.88 mg/kg per min), sodium nitroprusside (22.2 μg/kg per min), or 3-morpholino sydnonimine (SIN-1) (22.2 μg/kg per min). Intraluminal instillation of cholera toxin (0.5 μg/ml) induced net secretion, which was significantly enhanced by l-NAME and reduced by l-arginine. Another series of experiments was performed using a model of luminally perfused jejunal loops. Cholera toxin (10 μg/ml) induced profuse net fluid secretion also in this model. l-Arginine and sodium nitroprusside significantly enhanced net fluid absorption compared to controls and abolished the secretory effect of cholera toxin. Luminal perfusion with oral rehydration solution enhanced net absorption of fluid in controls and reversed cholera toxin-induced secretion to absorption. Intravenous infusion, but not intraluminal administration, of l-arginine significantly enhanced the antisecretory effect of oral rehydration solution. These results give further support to the existence of an intestinal NO-mediated proabsorptive tone, which also downregulates fluid secretion elicited by different enterotoxins or mediators of secretion. Intravenous administration of exogenous sources of NO counteracts intestinal fluid accumulation and augments the antisecretory effect of oral rehydration solution, findings which may lead to therapeutic consequences.

Neural mechanisms of blood flow regulation during synaptic activity in cerebellar cortex.

1. We investigated the neural mechanisms of the increases in blood flow produced by synaptic activity using the parallel fiber (PF) system of the cerebellum as a model. The midline cerebellum was exposed in anesthetized rats and the PFs were stimulated with tungsten microelectrodes. Cerebellar blood flow (BFcrb) was recorded using a laser-Doppler probe, whereas field potentials were recorded using glass micropipettes. PF stimulation produced increases in BFcrb that were related to the frequency and intensity of stimulation (+60 +/- 9%, mean +/- SE, at 100 microA and 30 Hz; n = 6). The greatest increases were confined to a band stretching along the major axis of the stimulated folium and corresponding to the beam of activated PFs. The increase in evoked by PF stimulation was associated with a corresponding increase in glucose utilization, assessed by the 2-deoxyglucose method. The increases in BFcrb and the field potentials evoked by PF stimulation were abolished by tetrodotoxin (1 microM; n = 6). Ringer solution containing 12 mM Mg2+ and 0 mM Ca2+ blocked synaptic activity in the PFs and abolished the increases in flow (P > 0.05 from baseline; n = 5). The broad-spectrum glutamate receptor antagonist kynurenate (5 mM) prevented depolarization of Purkinje cells and interneurons and abolished the increase in BFcrb evoked by PF stimulation (P > 0.05; n = 6). Treatment with tetrodotoxin, Mg2+, or kynurenate did not affect the increase in BFcrb elicited by systemic hypercapnia or by topical application of the nitric oxide donor 3-morpholino sydnonimine (P > 0.05 from Ringer solution). We conclude that the increases in flow produced by synaptic activity are linked to glutamate-induced depolarization of Purkinje cells and interneurons. These findings provide evidence that activation of glutamate receptors participates in the mechanisms of functional hyperemia, and they support the validity of the PF system as a model for study of the relationship between synaptic activity and blood flow in the CNS.