MAHMA NONOate Research Articles

Synthesis and Properties of a pH‐Insensitive Fluorescent Nitric Oxide Cheletropic Trap (FNOCT)

AbstractThe synthesis and properties of the new fluorescent nitric oxide cheletropic trap (FNOCT) 14, designed for the trapping and quantification of nitric oxide (NO) production in chemical and biological systems, is described (Scheme 3). The dicarboxylic acid 14 and the corresponding bis[(acetyloxy)methyl] ester derivative 15 of the FNOCT contain a 2‐methoxy‐substituted phenanthrene group as fluorophoric unit. The fluorescence of the reduced NO adduct of this FNOCT (λexc 320 nm, λem 380 nm) is pH‐independent. Trapping experiments were carried out in aqueous buffer solution at pH 7.4 with nitric oxide being added as a bolus as well as being released from the NO donor compound MAHMA NONOate (= (1Z)‐1‐{methyl[6‐(methylammonio)hexyl]amino}diazen‐1‐ium‐1,2‐diolate), indicating a trapping efficiency of ca. 50%. In a biological application, nitric oxide was scavenged from a culture of lipopolysaccharide‐stimulated rat alveolar macrophages. Under the applied conditions, a production of 11.1 ± 1.5 nmol of NO per hour and per 105 cells was estimated.

Differential effects of ouabain on the vasodilator actions of nitric oxide and S-nitrosothiols in vivo: Relevance to the identity of EDRF/EDHF

Objectives This study examined the role of Na +/K +-ATPase in the vasodilator actions of nitric oxide (NO), S-nitrosothiols and the endothelium-dependent agonist, acetylcholine. Methods The vasodilator responses elicited by intravenous injections of (i) the NO-donors, sodium nitroprusside and MAHMA NONOate, (ii) the S-nitrosothiols, l- S-nitrosocysteine and S-nitrosocoenzyme A, and (iii) acetylcholine, in urethane-anesthetized rats. Results The NO-donors, S-nitrosothiols and acetylcholine elicited dose-dependent depressor responses and reductions in hindquarter (HQR) and mesenteric (MR) vascular resistances. The depressor responses and associated reductions in HQR elicited by NO-donors were markedly attenuated after injection of ouabain. In contrast, the depressor responses and reductions in HQR elicited by the S-nitrosothiols and acetylcholine were not affected. The reductions in MR elicited by all vasodilator agents were exaggerated after injection of ouabain. Finally, the decomposition of sodium nitroprusside, MAHMA NONOate, l- S-nitrosocysteine and S-nitrosocoenzyme A to NO upon addition to rat blood or vascular preparations was not affected by ouabain. Conclusion This study demonstrates that ouabain has opposing effects on NO-mediated vasodilation in resistance arteries in the hindquarter and mesenteric beds of the rat. The similarity of effects of ouabain on the vasodilator actions of acetylcholine, l- S-nitrosocysteine and S-nitrosocoenzyme A as opposed to the NO-donors supports the possibility that endothelium-derived relaxing factor released by acetylcholine in resistance arteries is an S-nitrosothiol.

ACE inhibition restores the vasodilator potency of the endothelium-derived relaxing factor, l- S-nitrosocysteine, in conscious Spontaneously Hypertensive rats

Objective The major aim of this study was to determine whether the angiotensin converting enzyme (ACE) inhibitors, captopril or enalapril, restore the diminished vasodilator potency of the endothelium-dependent agonist, acetylcholine (ACh), and the endothelium-derived relaxing factor (EDRF), l- S-nitrosocysteine (L-SNC), in conscious Spontaneously Hypertensive (SH) rats. Methods The hemodynamic responses elicited by i.v. injections of ACh, L-SNC, and nitric oxide donors such as MAHMA NONOate, were determined in SH rats treated for 7 days with captopril, enalapril, or the direct vasodilator hydralazine. The effects of captopril, enalapril or hydralazine on oxidant stress levels in blood serum and aorta of WKY and SH rats were also determined. Results Captopril, enalapril and hydralazine elicited equivalent falls in mean arterial pressure and systemic vascular resistances in SH rats. ACh- and L-SNC-induced vasodilation were increased in captopril- or enalapril-treated SH rats such that the responses were equal to those in normotensive Wistar Kyoto rats. The attenuated responses of ACh and L-SNC in SH rats were not improved by hydralazine. The vasodilator effects of MAHMA NONOate, which were substantially augmented in SH rats, were not affected by captopril, enalapril or hydralazine. The levels of oxidant stress were markedly reduced in captopril- or enalapril-treated but not hydralazine-treated SH rats. Conclusions The finding that the ACE inhibitors improved the vasodilator potencies of L-SNC and the EDRF released by ACh in SH rats, suggests that the diminished vasodilator potency of these compounds was due to augmented ACE activity, which increased oxidant stress levels. This study provides the first evidence to support the concept that ACE inhibition lowers arterial pressure in SH rats, at least in part, by restoring the vasodilator potency of endothelium-derived L-SNC.

The vasodilator potency of the endothelium-derived relaxing factor, l- S-nitrosocysteine, is impaired in conscious spontaneously hypertensive rats

Objective This study compared the hemodynamic responses elicited by the endothelium-derived relaxing factor (EDRF), l- S-nitrosocysteine ( l-SNC), the non-prostanoid EDRF released by acetylcholine (ACh) and nitric oxide (NO)-donors such as MAHMA NONOate, in conscious spontaneously hypertensive (SH) and normotensive Wistar–Kyoto (WKY) rats. Methods The depressor and/or vasodilator responses elicited by intravenous injections of ACh, l-SNC and MAHMA NONOate were determined in adult WKY and SH rats before and after intravenous injection of the NO synthesis inhibitor, N G-nitro- l-arginine methylester ( l-NAME), or the cyclooxygenase inhibitor, indomethacin. Results The responses elicited by ACh and l-SNC were smaller in SH than in WKY rats whereas the responses elicited by MAHMA NONOate were augmented in SH rats. The ACh-induced responses were not diminished after injection of l-NAME in WKY or SH rats. Indomethacin did not affect the responses to any of the vasodilator agents in WKY or SH rats. Addition of l-SNC to whole blood or thoracic aortae from SH rats yielded similar amounts of NO to those of WKY rats. Conclusions The vasodilator potencies of ACh and l-SNC were diminished whereas that of NO was augmented in SH rats. The loss of potency of l-SNC in SH rats was not obviously due to differences in decomposition to NO or the overactivity of cyclooxygenase factors. This study provides the first evidence that diminished endothelium-dependent vasodilation in SH rats may involve a loss of vasodilator potency of endogenous l-SNC.

Differentiation of L- and D-S-Nitrosothiol Recognition Sites In Vivo

The main aim of this study was to determine the effects of the lipophobic electron acceptor, nitroblue tetrazolium (NBT), on the vasodilator responses elicited by femoral vein injections of L- and D-S-nitrosocysteine (L- and D-SNC), L- and D-S-nitroso-beta,beta-dimethylcysteine (L- and D-SNPEN) and the nitric oxide (NO) donor, MAHMA NONOate, in pentobarbital-anesthetized rats. L- and D-SNC, L- and D-SNPEN, and MAHMA NONOate elicited dose-dependent falls in mean arterial blood pressure (MAP), and hindquarter (HQR), renal (RR), and mesenteric (MR) vascular resistances. The L-SNC- and L-SNPEN-induced depressor and vasodilator responses were markedly attenuated after injection of NBT. The D-SNC- and D-SNPEN-induced falls in mean arterial pressure, hindquarter, and mesenteric vascular resistances were also reduced after injection of nitroblue tetrazolium whereas the falls in renal resistances were not affected. However, nitroblue tetrazolium inhibited the L-SNC and L-SNPEN responses much more profoundly than the D-SNC and D-SNPEN responses in each vascular bed. In contrast, the MAHMA NONOate-induced responses were not attenuated by nitroblue tetrazolium. This study demonstrates that nitroblue tetrazolium attenuates L- and D-SNC-and L- and D-SNPEN- mediated but not NO-mediated vasodilation. The lack of effects of NBT on the NO responses suggests that NBT does not interfere with the intracellular mechanisms by which NO relaxes vascular smooth muscle. The more pronounced effects of NBT on the vasodilator effects of L-SNC and L-SNPEN than D-SNC and D-SNPEN suggests that these stereoisomers differentially interact with stereoselective S-nitrosothiol recognition sites in the vasculature and that these sites (or their signaling elements) contain thiol residues that may be susceptible to occupation and/or oxidation (ie, disulfide-bond formation) by nitroblue tetrazolium.

Peroxynitrite Elicits Dysfunction of Stereoselective S-Nitrosocysteine Recognition Sites

The aim of this study was to determine whether induction of tachyphylaxis to peroxynitrite (induced by giving 10 intravenous injections of a 10-micromol/kg dose) differentially affects the vasodilator responses elicited by systemic injections of the L- and D-isomers of S-nitrosocysteine (L-SNC and D-SNC), in pentobarbital-anesthetized rats. L- and D-SNC (12.5-200 nmol/kg, iv) elicited dose-dependent reductions in hindquarter, mesenteric, and renal vascular resistances. The L-SNC-induced vasodilator responses in the hindquarter and renal vascular beds were virtually abolished whereas the vasodilator responses in mesenteric bed were markedly diminished after administration of peroxynitrite. The D-SNC-induced vasodilator responses in the hindquarter and renal beds were slightly attenuated whereas the vasodilator responses in the mesenteric bed were not diminished after administration of peroxynitrite. The vasodilator responses elicited by the nitric oxide donor, MAHMA NONOate (5-50 nmol/kg, iv), were not attenuated by peroxynitrite. The finding that induction of tachyphylaxis to peroxynitrite diminishes the effects of L- and D-SNC but not MAHMA NONOate suggests that the stereoisomers exert their vasodilator effects by mechanisms other than their decomposition to nitric oxide. Moreover, the finding that induction of tachyphylaxis to peroxynitrite causes a more pronounced attenuation of the vasodilator effects of L- than D-SNC supports evidence that the stereoisomers differentially interact with stereoselective S-nitrosothiol recognition sites in the vasculature. Taken together, these novel results support the possibility that peroxynitrite diminishes the vasodilator potencies of L- and D-SNC by oxidation and/or nitration of amino acids in these recognition sites.

Peroxynitrite but not nitric oxide donors destroys epinephrine: HPLC measurement and rat aorta contractility

Nitric oxide (NO) and peroxynitrite (ONOO) have been reported to destroy catecholamines. We compared the ability of NO donors and peroxynitrite to decompose epinephrine in both chemical and pharmacological experiments. Epinephrine (1 μM) was incubated with NO donors (SNAP and MAHMA NONOate) and ONOO at a concentration of 0.1 mM in phosphate buffer (pH 7.4; 0.1 M) or Krebs solution for 10 minutes at 37 °C. HPLC revealed that the concentration of epinephrine in the presence of NO donors was unaltered. In contrast, peroxynitrite decreased epinephrine concentration more than 20 fold. Similar relationships were obtained in the study of rat thoracic aorta ring contraction. The contractile activity (EC 50) of epinephrine in control solutions and after incubation of NE with NO donors did not change. EC 50 was measured at 8–10 nM in control solutions and after preincubation with NO donors. However when epinephrine was preincubated with peroxynitrite, no contractile effect was evoked. Therefore, under these experimental conditions peroxynitrite, but not NO donors, was capable of destroying epinephrine.

Platelet inhibitory effects of the nitric oxide donor drug MAHMA NONOate in vivo in rats

The platelet inhibitory effects of the nitric oxide (NO) donor drug MAHMA NONOate (( Z-1-{ N-methyl- N-[6-( N-methylammoniohexyl)amino]}diazen-1-ium-1,2-diolate) were examined in anaesthetised rats and compared with those of S-nitrosoglutathione (GSNO; an S-nitrosothiol). Bolus administration of the aggregating agent ADP dose-dependently reduced the number of circulating free platelets. Intravenous infusions of MAHMA NONOate (3–30 nmol/kg/min) dose-dependently inhibited the effect of 0.3 μmol/kg ADP. MAHMA NONOate was approximately 10-fold more potent than GSNO. MAHMA NONOate (0.3–10 nmol/kg/min) also reduced systemic artery pressure and was again 10-fold more potent than GSNO. Thus MAHMA NONOate has both platelet inhibitory and vasodepressor effects in vivo. The dose ranges for these two effects overlapped, although blood pressure was affected at slightly lower doses. The platelet inhibitory effects compared favourably with those of GSNO, even though NONO-ates generate free radical NO which, in theory, could have been scavenged by haemoglobin. Therefore platelet inhibition may be a useful therapeutic property of NONOates.

Interactions between neural and hormonal mediators of renal vascular tone in anaesthetized rabbits.

We investigated how sympathetic nerve activity and vasoactive hormones interact in controlling renal haemodynamics in pentobarbitone-anaesthetized rabbits. Renal blood flow was progressively reduced by electrical stimulation (0.5-3 Hz) of the renal nerves, during renal arterial infusion of saline, vasoconstrictors (angiotensin II and [Phe2,Ile3,Orn8]-vasopressin), or vasodilators (acetylcholine, adrenomedullin and the nitric oxide donor methylamine hexamethylene NONOate (MAHMA NONOate). A frequency-rich stimulus was also applied to test whether the vasoactive agents affect the dynamic control of renal blood flow by sympathetic nerve activity. The vasodilators tended to increase renal blood flow, but only the effect of MAHMA NONOate was statistically significant. [Phe2,Ile3,Orn8]-vasopressin reduced medullary perfusion (by 61 +/- 12 %) but not renal blood flow or cortical perfusion. Angiotensin II reduced renal blood flow (33 +/- 3 %) and cortical perfusion (14 +/- 5 %) but not medullary perfusion. Steady-state responses of renal blood flow and cortical perfusion during renal nerve stimulation were attenuated during infusion of acetylcholine and [Phe2,Ile3,Orn8]-vasopressin, while angiotensin II attenuated responses of medullary perfusion, and MAHMA NONOate and adrenomedullin had no significant effects. The dynamic response to sympathetic nerve activity (renal blood flow responded as a low pass filter with a pure time delay of ~664 ms) was not altered by the vasoactive agents. We conclude that some vasoactive agents can modulate steady-state renal haemodynamic responses to sympathetic nerve activity in a regionally specific manner, independent of their effects on baseline renovascular tone. However, they have little impact on the dynamic response of renal blood flow to sympathetic nerve activity.

Blockade of voltage-sensitive Ca 2+-channels markedly diminishes nitric oxide- but not l- S-nitrosocysteine- or endothelium-dependent vasodilation in vivo

The aim of this study was to determine the hemodynamic responses elicited by systemic injections of (i) the nitric oxide (NO)-donors, sodium nitroprusside (10 nmol/kg, i.v.) and ( Z)-1-| N-methyl- N-[6( N-methylammoniohexyl)amino]|diazen-1-ium-1,2-diolate (MAHMA NONOate, 25 nmol/kg, i.v.), (ii) the endothelium-derived S-nitrosothiol, l- S-nitrosocysteine (100 nmol/kg, i.v.), and (iii) the endothelium-dependent agonist, acetylcholine (1.0 μg/kg, i.v.), in anesthetized rats, before and after injection of the voltage-sensitive Ca 2+-channel (Ca VS 2+-channel) blocker, nifedipine (500 nmol/kg, i.v.). Before injection of nifedipine, the agents produced similar falls in mean arterial blood pressure, and in hindquarter and mesenteric vascular resistances. The depressor and vasodilator responses elicited by sodium nitroprusside and MAHMA NONOate were markedly attenuated by nifedipine. The falls in mean arterial blood pressure and mesenteric resistance elicited by l- S-nitrosocysteine and acetylcholine were not attenuated but the falls in hindquarter resistance were slightly attenuated by nifedipine. The cyclooxygenase inhibitor, indomethacin (10 mg/kg, i.v.), did not affect the actions of sodium nitroprusside, MAHMA NONOate, l- S-nitrosocysteine or acetylcholine or the effects of nifedipine on the hemodynamic actions of these compounds. The decomposition of sodium nitroprusside (0.2 nmol/ml), MAHMA NONOate (0.5 nmol/ml) and l- S-nitrosocysteine (2 nmol/ml) to NO upon addition to rat blood was not affected by nifedipine (10 μM). These findings suggest that (i) exogenously applied NO relaxes resistance arteries in vivo by inhibition of Ca VS 2+-channels whereas l- S-nitrosocysteine and the non-prostanoid endothelium-derived relaxing factor (EDRF) released by acetylcholine acts by additional mechanisms, and (ii) this EDRF may be an S-nitrosothiol which acts independently of its decomposition to NO.

Redox regulation of S-nitrosocysteine-mediated vasodilation in vivo

This study examined the effects of the lipophobic electron acceptor, nitroblue tetrazolium (2×5 μmol/kg, i.v.) on the vasodilation produced by the putative endothelium-derived S-nitrosothiol, l- S-nitrosocysteine (400 nmol/kg, i.v.), and the nitric oxide (NO) donor, ( Z)-1-| N-methyl- N-[6( N-methylammoniohexyl)amino]|diazen-1-ium-1,2-diolate (MAHMA NONOate, 25 nmol/kg, i.v.), in anesthetized rats. The administration of nitroblue tetrazolium resulted in delayed but long-lasting increases in vascular resistances. The l- S-nitrosocysteine-induced vasodilator responses were markedly diminished whereas the MAHMA NONOate-induced responses were not affected by nitroblue tetrazolium. These results support the possibility that l- S-nitrosocysteine interacts with membrane thiols that are subject to nitroblue tetrazolium-induced oxidation (i.e., disulfide-bridge formation) and that nitroblue tetrazolium-induced vasoconstriction may involve a loss of potency of endothelium-derived S-nitrosothiols.

Apparent association of MK-801-sensitive ion channels with l- S-nitrosocysteine recognition sites in the hindlimb vasculature of the rat

This study demonstrates that the decreases in hindquarter vascular resistance produced by the putative endothelium-derived S-nitrosothiol, l- S-nitrosocysteine, in urethane-anesthetized rats, were attenuated by a lower dose of the N-methyl- d-aspartate (NMDA) receptor ion-channel blocker, dizocilpine (MK-801, 200 μg/kg, i.v.), whereas they were augmented by a higher dose of dizocilpine (500 μg/kg, i.v.). In contrast, l- S-nitrosocysteine-induced decreases in mesenteric vascular resistance were not affected by either dose of dizocilpine. The vasodilator actions of l- S-nitrosocysteine in these beds were not affected by the competitive NMDA receptor antagonist, 2-amino-5-phosphonovaleric acid (2-AP5). The vasodilator actions of the nitric oxide (NO) donor, ( Z)-1-∣ N-methyl- N-[6( N-methylammoniohexyl)amino]∣diazen-1-ium-1,2-diolate (MAHMA NONOate), in these beds were not affected by dizocilpine or by 2-AP5. These findings suggest that l- S-nitrosocysteine recognition sites in hindquarter but not mesenteric beds may be associated with dizocilpine-sensitive ion-channels similar to those in NMDA receptors.

Cyclic GMP-independent relaxation of rat pulmonary artery by spermine NONOate, a diazeniumdiolate nitric oxide donor.

In rat pulmonary artery pre-contracted with phenylephrine, the mechanisms of relaxation to the nitric oxide (NO) donor, spermine NONOate, were investigated. Responses to spermine NONOate were only partially blocked by the soluble guanylate cyclase inhibitor, ODQ (1H:-[1,2,4]Oxadiazolo-[4,3,-a]quinoxalin-1-one) at concentrations up to 30 microM. Ten microM ODQ gave maximal inhibition. Endothelium removal had no effect on the potency of spermine NONOate or its inhibition by ODQ. The protein kinase G inhibitor, Rp-8-Br-cGMPS (100 microM), caused minimal inhibition of spermine NONOate despite causing marked inhibition of glyceryl trinitrate and isosorbide dinitrate. Spermine NONOate (100 microM) caused a 35 fold increase in guanosine 3'5' cyclic monophosphate (cyclic GMP) above basal levels in pulmonary artery rings. ODQ (3 microM) abolished this cyclic GMP production but did not inhibit corresponding relaxant responses. Similar results were seen with another NONOate (MAHMA NONOate; 10 microM). ODQ-resistant relaxation to spermine NONOate (i. e. relaxation seen in the presence of 10 microM ODQ) was inhibited by potassium (80 mM), charybdotoxin (300 nM), iberiotoxin (300 nM), apamin (100 nM), ouabain (1 mM) or thapsigargin (100 nM) but not by 4-aminopyridine (3 mM), glybenclamide (10 microM) or diltiazem (10 microM). Potassium, charybdotoxin, ouabain and thapsigargin also inhibited ODQ-resistant relaxation to FK409 ((+/-)-E:-4-ethyl-2-[E:-hydroxyimino]-5-nitro-3-hexenamide). We conclude that, on rat pulmonary artery, spermine NONOate can produce cyclic GMP-independent relaxation that involves, at least in part, activation of Na(+)/K(+)-ATPase, sarco-endoplasmic reticulum Ca(2+)-ATPase and calcium-activated potassium channels.

Contribution of 20-HETE to vasodilator actions of nitric oxide in the cerebral microcirculation.

The present study examined the contributions of a rise in cGMP versus a fall in 20-HETE levels to the vasodilator response to nitric oxide (NO) in the cerebral circulation of the rat. Intact rat middle cerebral and basilar arteries were bathed in physiological saline solution containing indomethacin (5 micromol/L) and baicalein (0.5 micromol/L) and pressurized at 90 mm Hg. Relaxations to sodium nitroprusside (SNP) were studied before and after addition of [1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one] (ODQ, a guanylyl cyclase blocker), 8R,9S, 11S-(-)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8, 11-epoxy-1H,8H,11H-2,7b,11a-trizadibenzo-(a,g)-cycloocta-(c, d, e)-trinden-1-one (KT5823, a protein kinase G blocker), and 20-hydroxyeicosatetraenoic acid (20-HETE). Cerebral blood flow was measured by using a laser Doppler flow probe over a thin cranial window in anesthetized rats, and the effects of intracerebroventricular infusion of 1-hexamine, 6-(2-hydroxy-1-methyl-2-nitrosohydrazino)N-methyl (MAHMA nonoate) and dibromododecenyl methylsulfimide (DDMS) were determined. SNP-induced dilation of serotonin-preconstricted (0.2 micromol/L) middle cerebral arteries (10(-7) to 10(-3) mol/L) was attenuated in arteries treated with ODQ (10 micromol/L) or KT5823 (1 micromol/L) by 52% and 27%, respectively. Preventing the NO-induced fall in intracellular 20-HETE, by adding 20-HETE (100 nmol/L) to the bath, reduced the dilation to SNP by 62%. Simultaneous administration of ODQ and 20-HETE markedly attenuated the SNP-induced dilation by 90%. In basilar arteries, ODQ (10 micromol/L) alone completely blocked the response to SNP. Infusion of MAHMA nonoate (10 nmol/min ICV) in anesthetized rats increased cerebral blood flow by 52% before and 8% after blockade of the endogenous production of 20-HETE with DDMS (50 pmol/min). These results suggest that NO dilates cerebral arteries through both cGMP-dependent and cGMP-independent pathways and that inhibition of 20-HETE formation contributes to the cerebral vasodilator response to NO both in vitro and in vivo.

In vivo evidence that l- S-nitrosocysteine may exert its vasodilator effects by interaction with thiol residues in the vasculature

This study examined the effects of the lipophobic thiol chelator, para-hydroxymercurobenzoic acid (25 and 50 μmol/kg, i.v.) on the falls in mean arterial blood pressure and regional vascular resistances produced by l- S-nitrosocysteine (400 nmol/kg, i.v.) and the nitric oxide (NO)-donors, ( Z)-1-| N-methyl- N-[6( N-methylammoniohexyl)amino]|diazen-1-ium-1,2-diolate (MAHMA NONOate, 25 nmol/kg, i.v.) and sodium nitroprusside (10 μg/kg, i.v.), in urethane-anesthetized rats. The l- S-nitrosocysteine-induced responses were markedly diminished whereas the MAHMA NONOate- and sodium nitroprusside-induced responses were minimally affected by para-hydroxymercurobenzoic acid. These results suggest that the vasodilator actions of l- S-nitrosocysteine involves the interaction with membrane thiols in vascular smooth muscle of resistance arteries and that para-hydroxymercurobenzoic acid does not markedly affect NO-mediated vasodilation.

In vitro comparison of two NONOates (novel nitric oxide donors) on rat pulmonary arteries

The pulmonary vasorelaxant properties of two NONOates (diazeniumdiolates) were examined because this novel group of nitric oxide (NO) donors may be useful in pulmonary hypertension. MAHMA NONOate (( Z)-1-{ N-Methyl- N-[6-( N-methylammoniohexyl)amino]} diazen-1-ium-1,2-diolate) and spermine NONOate (( Z)-1-{ N-[3-aminopropyl]- N-[4-(3-aminopropylammonio)butyl]-amino}diazen-1-ium-1,2-diolate) decomposed at different rates (half-lives 1.3 min and 73 min, respectively; 37°C, pH 7.3) but generated the same total amount of NO. They fully relaxed submaximally contracted ring preparations of main and intralobar pulmonary arteries from rats. Responses were inhibited by the guanylate cyclase inhibitor, ODQ (1 H-[1,2,4]Oxadiazolo[4,3- a]quinoxalin-1-one). Potency was not affected by choice of contractile spasmogen (phenylephrine, endothelin-1, thromboxane-mimetic) or endothelium removal, and tolerance did not develop; thus the drugs had properties important for use in pulmonary hypertension. MAHMA NONOate was 10–40-fold more potent than spermine NONOate but responses to spermine NONOate were more sustained (spermine NONOate>60 min; MAHMA NONOate<7 min). It is concluded that the differences in potency and time-course reflect the different rates of NO generation by these NONOates.

Attenuation of vascular relaxation after development of tachyphylaxis to peroxynitrite in vivo.

Peroxynitrite, formed endogenously by the near diffusion-limited reaction of nitric oxide with superoxide anion, induces vascular relaxation. This effect is subject to rapid tachyphylaxis, suggesting that peroxynitrite may alter subsequent vasorelaxant responses. The present study examined the effects of peroxynitrite on mean arterial pressure and hindquarter, renal, and mesenteric vascular resistances in pentobarbital-anesthetized rats. Peroxynitrite induced dose-dependent decreases in mean arterial pressure and hindquarter and mesenteric vascular resistances. The repetitive administration of peroxynitrite resulted in the rapid development of tachyphylaxis, with subsequent doses producing progressively smaller effects. After the development of tachyphylaxis to peroxynitrite, the hemodynamic effects produced by the systemic administration of acetylcholine and prostacyclin were significantly attenuated, whereas the hemodynamic responses to bradykinin and the nitric oxide donor (Z)-1-¿N-methyl-N-[6(N-methylammoniohexyl)amino]¿diazen-1-++ +ium-1, 2-diolate (MAHMA NONOate) remained unchanged. These results demonstrate that 1) peroxynitrite is a potent vasorelaxant in vivo, 2) peroxynitrite-mediated vasodilatation is subject to the development of rapid tachyphylaxis, and 3) peroxynitrite alters the vascular smooth muscle response to prostacyclin, perhaps via inactivation of vascular smooth muscle ATP-sensitive potassium channel function.

Stimulation of mucin exocytosis from human epithelial cells by nitric oxide: evidence for a cGMP-dependent and a cGMP-independent pathway.

The aim of this work was to investigate the role of nitric oxide (NO) on the macromolecular exocytotic function of human epithelial cells. We tested the effects of two NO-generating drugs, i.e. 1-hexanamine 6-(2-hydroxy-1-methyl-2-nitrosohydrazine)-N-methyl (MAHMA NONOate) and sodium nitroprusside (SNP), on mucin exocytosis from the human colonic epithelial HT29-Cl.16E cell line. Our results show that MAHMA NONOate and SNP elicited a rapid mucin exocytotic response through a cGMP-dependent and a cGMP-independent pathway respectively. Indeed, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ), a newly available specific inhibitor of soluble guanylate cyclase, inhibited both cGMP accumulation and subsequent mucin exocytosis evoked by MAHMA NONOate. By contrast, SNP did not alter intracellular cGMP levels, and SNP-mediated mucin exocytosis was not inhibited by ODQ. As expected from two NO donors acting through distinct pathways, the combined action of MAHMA NONOate and SNP led to an additive effect on mucin exocytosis. SNP was likely to act through S-nitrosylation of a cellular target, because cysteine, a reductive thiol that provides decoy targets for SNP through the formation of nitrosocysteine, abolished the early stimulatory effect of SNP on mucin exocytosis. Finally, the fact that in the presence of cysteine SNP was able to trigger a late, ODQ-inhibitable, mucin exocytotic response demonstrates the ability of NO to shift its intracellular signalling pathway depending on the changes of the redox state of the milieu.

Neuronal NOS-derived NO plays permissive role in cerebral blood flow response to hypercapnia.

The aim of the present study was to determine whether neuronal nitric oxide synthase (nNOS)-derived nitric oxide (NO) plays a permissive role in the regulation of cerebral blood flow (CBF) response to hypercapnia. To this end, we examined whether the administration of NO donors could reestablish the regional CBF (rCBF) response to hypercapnia after nNOS inhibition with 7-nitroindazole (7-NI). Rats were anesthetized with 1% halothane, and rCBF in the cortex was measured by laser-Doppler flowmetry. The administration of 7-NI (40 mg/kg ip) decreased resting rCBF by 17 +/- 5% (n = 6, P < 0.05) and attenuated the rCBF response to hypercapnia by 30 +/- 8% in comparison with the response seen in rats treated with the vehicle (peanut oil) alone. Intracerebroventricular administration of NO donors, sodium nitroprusside (SNP; n = 7) and (Z)-1-[N-methyl-N-[6(N-methylammoniohexyl)aminol]]diazen+ ++-1-ium-1,2-diolate (MAHMA NONOate; n = 6) in a dose of 0.1-1 nmol/min after 7-NI restored both resting rCBF to baseline and the vasodilatory response to hypercapnia. In contrast, intravenous infusion of SNP (0.05-0.5 nmol/min, n = 6) or intracerebroventricular administration of an NO-independent vasodilator, the stable prostaglandin I2 analog iloprost (0.01-0.1 nmol/min, n = 6), after 7-NI failed to restore the vasodilatory response to hypercapnia, despite the fact that it restored the resting rCBF to baseline. nNOS activity, assessed by the conversion of labeled arginine to citrulline, was inhibited by 70 +/- 7% after the administration of 7-NI. These findings confirm that the selective inhibition of nNOS decreases resting rCBF and attenuates the rCBF response of hypercapnia. They further indicate that the repletion of intraparenchymal NO allows the hypercapnic cerebrocortical vasodilation to occur. Therefore, it is suggested that the nNOS-derived NO plays a permissive role in the CBF response to hypercapnia.

Inhibition of 20-HETE production contributes to the vascular responses to nitric oxide.

Nitric oxide (NO) inhibits a variety of heme-containing enzymes, including NO synthase and cytochrome P4501A1 and 2B1. The present study examined whether NO inhibits the production of 20-hydroxyeicosatetraenoic acid (20-HETE) by cytochrome P4504A enzymes and whether blockade of the production of this substance contributes to the vascular effects of NO. Sodium nitroprusside (SNP; 10(-5), 10(-4), and 10(-3) mol/L) reduced the production of 20-HETE by renal microsomes incubated with arachidonic acid to 71 +/- 5%, 29 +/- 4%, and 4 +/- 2% of control, respectively (n = 5). Similar results were obtained with the use of 1-propanamine, 3-(2-hydroxy-2-nitroso-1-propylhydrazino) (n = 3). To determine whether inhibition of 20-HETE contributes to the vasodilatory effects of NO, the effects of dibromo-dodecenyl-methylsulfimide (DDMS), a selective inhibitor of the formation of 20-HETE, on the response to SNP (10(-7) to 10(-3) mol/L) were examined in rat renal arterioles preconstricted with phenylephrine (n = 5). SNP increased vascular diameter in a concentration-dependent manner to 82 +/- 4% of control. After DDMS (25 mumol/L), SNP (10(-3) mol/L) increased vascular diameter by only 17 +/- 3%. The effects of DDMS on the mean arterial pressure (MAP) and renal blood flow (RBF) responses to infusion of an NO donor and a synthase inhibitor were also examined in thiobutabarbital-anesthetized, Sprague-Dawley rats. Infusion of MAHMA NONOate at 1, 3, 5, and 10 nmol/min reduced MAP by 16 +/- 2, 30 +/- 3, 40 +/- 5, and 48 +/- 5 mm Hg and lowered renal vascular resistance (RVR) by 15 +/- 3%, 26 +/- 2%, 30 +/- 3%, and 34 +/- 4% of control. After DDMS (10 mg/kg, n = 7 rats), the MAP and RVR responses to 1-hexamine, 6-(2-hydroxy-1-methyl-2-nitrohydrazino)N-methyl (MAHMA NONOate) averaged only 20% of those seen during control. In other experiments, MAP increased by 32 +/- 4% and RBF fell to 56 +/- 5% of control after administration of N-nitro-L-arginine (L-NArg) (10 mg/kg IV). After DDMS (10 mg/kg, n = 7 rats), MAP increased by only 19 +/- 4% and RBF fell by only 7 +/- 4% after L-NArg. These results indicate that NO inhibits cytochrome P4504A enzymes and that inhibition of the production of 20-HETE contributes to the vasodilatory effects of NO.