Donor Diethylamine Research Articles

Nitric oxide decreases the biological activity of norepinephrine resulting in altered vascular tone in the rat mesenteric arterial bed.

Nitric oxide (NO) reacts with catecholamines resulting in their deactivation. In this study, we demonstrated that coincubation of NO donors with sympathetic neurotransmitters decreased the amount of norepinephrine detected but not ATP or neuropeptide Y (NPY). Furthermore, we found that the ability of norepinephrine to increase perfusion pressure in the isolated perfused mesenteric arterial bed of the rat was attenuated by the incubation of norepinephrine with the NO donor diethylamine NONOate. Conversely, the vasoconstrictive ability of NPY and ATP was unaffected by incubation with NONOate. Periarterial nerve stimulation in the presence of the NO synthase (NOS) inhibitor Nomega-nitro-l-arginine methyl ester (l-NAME) resulted in an increase in both perfusion pressure response and norepinephrine levels. This was prevented by l-arginine, demonstrating that the effects of l-NAME were indeed specific to the inhibition of NOS. To confirm that NO was not altering the release of norepinephrine from the sympathetic nerve via presynaptic activation of guanylate cyclase, we repeated the experiments in the presence of the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]-quinoxaloine-one (ODQ). Unlike l-NAME, ODQ infusion did not increase norepinephrine overflow, demonstrating that modulation of norepinephrine by NO at the vascular neuroeffector junction of the rat mesenteric vascular bed is not the result of presynaptic guanylate cyclase activation. These results demonstrate that, in addition to being a direct vasodilatator, NO can also alter vascular reactivity at the sympathetic neuroeffector junction in the rat mesenteric bed by deactivating the vasoconstrictor norepinephrine.

Delayed Recovery of Receptor-Mediated Functional Responses to Acetylcholine in Mouse Isolated Carotid Arteries following Endothelial Denudation in vivo

The time-course of endothelial regrowth and functional recovery following polytetrafluoroethylene filament-induced endothelial denudation in vivo was studied in the left common carotid artery of the mouse. This technique does not result in any intimal hyperplasia, enabling the investigation of endothelial function without any confounding effect of intimal thickening. Endothelial coverage was assessed histologically, and functional recovery was assessed as restoration of receptor-mediated, endothelium-dependent relaxation to acetylcholine in vitro. Re-endothelialization of the carotid artery was complete within 8 days of denudation. However, relaxations to acetylcholine, which are mediated by endothelium-derived nitric oxide, were only partially restored 10 days after the procedure. At this time point, arterial responses to either phenylephrine, the receptor-independent endothelium-dependent dilator cyclopiazonic acid, or the nitric oxide donor diethylamine NONOate, were not significantly different to controls. At 25 days after denudation, acetylcholine-evoked responses remained significantly depressed compared to controls but at 90 days full recovery was observed. These data indicate that following mechanical denudation of the mouse carotid artery, although endothelial re-growth is complete within 8 days, recovery of endothelial cell function – assessed as the ability of the regenerated endothelium to mediate acetylcholine-stimulated relaxation – remains impaired for a prolonged period.

Mammary Blood Flow Does Not Limit Milk Yield in Lactating Goats

There is a close relationship between mammary blood flow (MBF) and milk production, but whether MBF is limiting milk yield has not been determined. Five lactating goats received close arterial (external pudic) infusion of PBS or the nitric oxide donor diethylamine NONOate (0.5 mg/h; NONate) for 6h, according to a crossover design. Goats were hand milked (with oxytocin) every 2h starting 2h before and ending 6h after the end of the infusion. In one goat, a transit time flow probe was implanted around the infused and noninfused artery, whilst in another goat a flow probe was implanted around the infused artery only. Infusion of PBS did not affect MBF or milk production. As with previous results (Lacasse et al., 1996), NONate induced a rapid increase (up to 250% of preinfusion level) in MBF in the infused gland only. Mammary blood flow was still above the preinfusion level at the end of the infusion period. Despite this increase in MBF, NONate did not affect milk production. Milk yield ratio (infused/noninfused gland) averaged 1.20, 1.12, and 1.17 for the preinfusion, infusion and post infusion periods, respectively. Similarly, protein, fat and lactose yields were not affected by PBS or NONate infusion. These results provide no support to the contention that increasing MBF can enhance milk production.

Nitric Oxide Synthase in the External Urethral Sphincter of the Sheep: Immunohistochemical and Functional Study

We studied the distribution of neuronal nitric oxide synthase (nNOS) and the effects of nitric oxide (NO) modulating drugs on contractile function of the external urethral sphincter of lambs. Gender differences were evaluated. Longitudinal and transverse sections of the external urethral sphincter from 10 female and 10 male lambs were studied using reduced nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry and nNOS immunocytochemistry. Isometric contractile responses to electrical field stimulation were recorded from external urethral sphincter preparations from 47 female and 45 male lambs and the effects of NO modulating drugs were evaluated. We detected nNOS in the sarcolemma of some but not all striated fibers, where nNOS seems to be concentrated at the neuromuscular junction. In addition, nNOS was present in nerve fibers and intramural ganglia. The density of innervation decreased toward the distal part of the external urethral sphincter and was higher in male preparations. No significant functional effects of the NOS inhibitor NG-nitro-L-arginine (10 mM.) or the NO donors diethylamine and spermine NONOate (Sigma Chemical Co., St. Louis, Missouri) (5 mM. each) on external urethral sphincter isometric contractility were found in either gender. Despite the evidence for nNOS at the sarcolemma and nerve fibers of the external urethral sphincter the physiological relevance of these immunohistochemical findings remains to be determined.

Positive inotropic and lusitropic effects of HNO/NO- in failing hearts: independence from beta-adrenergic signaling.

Nitroxyl anion (HNONO(-)), the one-electron reduced form of nitric oxide (NO), induces positive cardiac inotropy and selective venodilation in the normal in vivo circulation. Here we tested whether HNO/NO(-) augments systolic and diastolic function of failing hearts, and whether contrary to NO/nitrates such modulation enhances rather than blunts beta-adrenergic stimulation and is accompanied by increased plasma calcitonin gene-related peptide (CGRP). HNO/NO(-) generated by Angelis' salt (AS) was infused (10 microg/kg per min, i.v.) to conscious dogs with cardiac failure induced by chronic tachycardia pacing. AS nearly doubled contractility, enhanced relaxation, and lowered cardiac preload and afterload (all P < 0.001) without altering plasma cGMP. This contrasted to modest systolic depression induced by an NO donor diethylamine(DEA)NO or nitroglycerin (NTG). Cardiotropic changes from AS were similar in failing hearts as in controls despite depressed beta-adrenergic and calcium signaling in the former. Inotropic effects of AS were additive to dobutamine, whereas DEA/NO blunted beta-stimulation and NTG was neutral. Administration of propranolol to nonfailing hearts fully blocked isoproterenol stimulation but had minimal effect on AS inotropy and enhanced lusitropy. Arterial plasma CGRP rose 3-fold with AS but was unaltered by DEA/NO or NTG, supporting a proposed role of this peptide to HNO/NO(-) cardiotropic action. Thus, HNO/NO(-) has positive inotropic and lusitropic action, which unlike NO/nitrates is independent and additive to beta-adrenergic stimulation and stimulates CGRP release. This suggests potential of HNO/NO(-) donors for the treatment of heart failure.

Transformation and activation of benzidine by oxidants of the inflammatory response.

Aromatic amines, such as benzidine (BZ), initiate bladder cancer in humans. Inflammation/infection play an important role in this cancer. This study was designed to assess the influence of inflammatory oxidants, including reactive nitrogen oxygen species (RNOS), on BZ transformation and activation. RNOS were generated under various conditions and reacted with BZ, and the products were examined by HPLC. Conditions that generate nitrogen dioxide radical, NO(2)(-) + myeloperoxidase + H(2)O(2) and ONOO(-), produced primarily a single new product, which was identified by MS as azobenzidine (AZO-BZ). The myeloperoxidase-catalyzed reaction was inhibited by 1 mM cyanide and did not require NO(2)(-). Chloride (100 mM) reduced the myeloperoxidase reaction by 30% with taurine having little effect. In contrast, conditions that generate N(2)O(3), i.e., NO donor diethylamine (DEA) NONOate, produced two products, which were identified by MS as 4'-OH-4-aminobiphenyl (4'-OH-ABP) and 4-aminobiphenyl (ABP). 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, an oxidant of NO thought to produce NO(2)(*), had a biphasic effect on product formation. At a concentration equal to DEA NONOate, a 5-fold increase in BZ nitrosation was observed, while at higher concentrations nitrosation was greatly diminished and formation of AZO-BZ occurred. Glutathione prevented RNOS transformation of BZ. With MPO and ONOO(-), a new product was formed that cochromatographed with 3-(glutathione-S-yl)BZ. Glutathione also prevented nitrosation of BZ but did not form additional BZ products. HOCl-mediated activation of BZ, 4'-OH-ABP, and ABP to bind DNA was assessed. A higher level of binding was observed at pH 5.5 than pH 7.4. BZ elicited the most binding. More binding was observed at both pH values with 4'-OH-ABP than ABP. Thus, components of the inflammatory response are capable of BZ transformation and activation.

Regional distribution of protein and activity of the nitric oxide receptor, soluble guanylyl cyclase, in rat brain suggests multiple mechanisms of regulation

Nitric oxide (NO) is an unconventional neuromodulator that signals by intercellular diffusion. Its effects are often mediated by activation of its cytosolic receptor, the hemoprotein soluble guanylyl cyclase (sGC). Regional distribution of heterodimeric (α/β) sGC at both the activity and protein level and its regulation are still unclear. Here, sGC was analyzed in rat brain by Western blot and NO donor-stimulated cyclic GMP accumulation. sGCα 1 and sGCβ 1 immunoreactive protein signals strongly correlated with each other. However, V max values depended on the type of NO donor used. Sodium nitroprusside, the most widely used compound and formally an NO + donor, was up to 20-fold less effective in stimulating sGC activity than the NO donor diethylamine NONOate. In contrast to the rather even distribution of sGC proteins and SNP-stimulated cGMP accumulation in various regions of rat brain, diethylamine NONOate-stimulated sGC activity varied up to 8-fold between the different brain regions tested. In conclusion, we show that expression of both sGCα 1 and sGCβ 1 subunits is tightly coregulated in rat brain, while yet unknown additional mechanisms affect the V max of sGC.

Nitric Oxide Supplementation Ameliorates Dextran Sulfate Sodium-Induced Colitis in Mice

Nitric oxide (NO) synthesis is up-regulated in inflammatory bowel disease. However, its role in the pathophysiology of this condition is controversial. The aims of this study were to assess whether nitric oxide administration ameliorates experimental colitis and to determine the possible mechanisms underlying its effects on intestinal inflammation. For this purpose, the NO donor diethylamine NONOate (DETA/NO; 0.01, 0.1, 1, 5, or 10 mg/kg/day), or the DETA moiety, was administered daily to mice with dextran sulfate sodium-induced colitis. Daily body weight and colonic pathologic alterations at Day 10 were determined. Leukocyte endothelial cell interactions in colonic venules were assessed with intravital microscopy, and expression of endothelial cell adhesion molecules was determined using radiolabeled antibodies. IL-12 and IFN-γ production were measured in intestinal tissue. Colitis induced a significant loss of body weight, reduction of colon length, and increase in colon weight and myeloperoxidase activity. Administration of 1 mg/kg/day DETA/NO significantly attenuated these pathologic changes. The marked increase in leukocyte rolling and adhesion in colonic venules of colitic mice were significantly reduced by administration of 1 mg/kg/day DETA/NO. Development of colitis was associated with a marked increase in endothelial expression of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and P-selectin. Supplementation with NO significantly attenuated the up-regulation of endothelial intercellular adhesion molecule-1 and P-selectin, but not vascular cell adhesion molecule-1, in colonic tissue. NO abrogated the increase in IL-12 and IFN-γ mRNA expression in the colon of colitic mice. The DETA moiety alone did not have any effect on any of the parameters studied. In conclusion, exogenous NO supplementation significantly ameliorates dextran sulfate sodium-induced colitis. This effect is related to a reduction in leukocyte recruitment and proinflammatory cytokine production.

Functional reconstitution of vascular smooth muscle cells with cGMP-dependent protein kinase I isoforms.

The cGMP-dependent protein kinase type I (cGKI) is a major mediator of NO/cGMP-induced vasorelaxation. Smooth muscle expresses two isoforms of cGKI, cGKIalpha and cGKIbeta, but the specific role of each isoform in vascular smooth muscle cells (VSMCs) is poorly understood. We have used a genetic deletion/rescue strategy to analyze the functional significance of cGKI isoforms in the regulation of the cytosolic Ca(2+) concentration by NO/cGMP in VSMCs. Cultured mouse aortic VSMCs endogenously expressed both cGKIalpha and cGKIbeta. The NO donor diethylamine NONOate (DEA-NO) and the membrane-permeable cGMP analogue 8-bromo-cGMP inhibited noradrenaline-induced Ca(2+) transients in wild-type VSMCs but not in VSMCs genetically deficient for both cGKIalpha and cGKIbeta. The defective Ca(2+) regulation in cGKI-knockout cells could be rescued by transfection of a fusion construct consisting of cGKIalpha and enhanced green fluorescent protein (EGFP) but not by a cGKIbeta-EGFP construct. Fluorescence imaging indicated that the cGKIalpha-EGFP fusion protein was concentrated in the perinuclear/endoplasmic reticulum region of live VSMCs, whereas the cGKIbeta-EGFP protein was more homogeneously distributed in the cytoplasm. These results suggest that one component of NO/cGMP-induced smooth muscle relaxation is the activation of the cGKIalpha isoform, which decreases the noradrenaline-stimulated cytosolic Ca(2+) level.

Role of cyclic GMP‐dependent protein kinase in the contractile response to exogenous nitric oxide in rat cardiac myocytes

Nitric oxide (NO) can directly modulate cardiac contractility by accelerating relaxation and reducing diastolic tone. The intracellular mechanisms underlying these contractile effects are poorly understood. Here we investigate the role of cyclic GMP-dependent protein kinase (PKG) in the contractile response to exogenous NO in rat ventricular myocytes. Isolated ventricular myocytes were stimulated electrically and contractility was assessed by measuring cell shortening. Some cells were loaded with the fluorescent Ca(2+) probe indo-1 AM for simultaneous assessment of the intracellular Ca(2+) transient. The NO donor diethylamine NONOate (DEA/NO, 10 microM) significantly increased resting cell length, reduced twitch amplitude and accelerated time to 50 % relaxation (to 100.8 +/- 0.2, 83.7 +/- 3.0 and 88.9 +/- 3.7 % of control values, respectively). The contractile effects of DEA/NO occurred without significant changes in the amplitude or kinetics of the intracellular Ca(2+) transient, suggesting that the myofilament response to Ca(2+) was reduced. These effects were abolished by inhibition of either guanylyl cyclase (with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; ODQ, 10 microM) or PKG (with Rp-8-Br-cGMPs, 10 microM) suggesting that, at the concentration investigated, the effects of DEA/NO were mediated exclusively by PKG, following activation of guanylyl cyclase and elevation of cGMP. Direct activation of PKG with 8-pCPT-cGMP (10 microM) mimicked the effects of DEA/NO (resting cell length and time to 50 % relaxation were 100.6 +/- 0.1 and 90.5 +/- 1.5 % of control values, respectively).The reduced myofilament Ca(2+) responsiveness was not attributable to an intracellular acidosis since the small reduction in pH(i) induced by DEA/NO was found to be uncoupled from its contractile effects. However, hearts treated with DEA/NO (10 microM) showed a significant increase (1.4-fold; P < 0.01) in troponin I phosphorylation compared to control, untreated hearts. These results suggest that the reduction in myofilament Ca(2+) responsiveness produced by DEA/NO results from phosphorylation of troponin I by PKG.

Adenosine acting on A1 receptors protects NO-triggered rebound potentiation and LTP in rat hippocampal slices.

Exposure of hippocampal slices to nitric oxide (NO) results in a depression of CA1 synaptic transmission. Under 0.2-Hz stimulation, washout of NO leads to a persistent potentiation that depends on N-methyl-D-aspartate (NMDA) receptors and endogenous NO formation and that occludes tetanus-induced long-term potentiation (LTP). The experiments were initially aimed at determining the relationship between the NO-induced synaptic depression and rebound potentiation. The adenosine A1 antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) partially inhibited the depression produced by the NO donor diethylamine NONOate (300 microM). It also led to a complete block of both the rebound potentiation and the subsequent tetanus-induced LTP. LTP was preserved in the presence of DPCPX if the stimulation frequency was reduced to 0.033 Hz or if the NO application was omitted. The NO-triggered rebound potentiation was restored if the experiment (DPCPX followed by exogenous NO) was conducted in the presence of an NMDA antagonist. The restored potentiation was completely blocked by the NO synthase inhibitor, L-nitroarginine. It is concluded that the NO-induced depression is partially mediated by increased release of endogenous adenosine acting on A1 receptors. Moreover, tonic A1 receptor activation by adenosine protects LTP and the rebound potentiation from being disabled by untimely NMDA receptor activity. Hence, the NO-induced depression and rebound potentiation are linked in the sense that the depression helps to preserve the capacity of the synapses to undergo potentiation. Finally, the results give the first example of exogenous NO eliciting an enduring potentiation of hippocampal synaptic transmission that is dependent on endogenous NO formation, but not on NMDA receptors.

Structural and kinetic modifications of aldose reductase by S-nitrosothiols.

Modification of aldose reductase (AR) by the nitrosothiols S-nitroso-N-acetyl penicillamine (SNAP) and N-(beta-glucopyranosyl)-N(2)-acetyl-S-nitrosopenicillamide (glyco-SNAP) resulted in a 3-7-fold increase in its k(cat) and a 25-40-fold increase in its K(m) for glyceraldehyde. In comparison with the native protein, the modified enzyme was less sensitive to inhibition by sorbinil and was not activated by SO(2-)(4) anions. The active-site residue, Cys-298, was identified as the main site of modification, because the site-directed mutant in which Cys-298 was replaced by serine was insensitive to glyco-SNAP. The extent of modification was not affected by P(i) or O(2), indicating that it was not due to spontaneous release of nitric oxide (NO) by the nitrosothiols. Electrospray ionization MS revealed that the modification reaction proceeds via the formation of an N-hydroxysulphenamide-like adduct between glyco-SNAP and AR. In time, the adduct dissociates into either nitrosated AR (AR-NO) or a mixed disulphide between AR and glyco-N-acetylpenicillamine (AR-S-S-X). Removal of the mixed-disulphide form of the protein by lectin-column chromatography enriched the preparation in the high-K(m)-high-k(cat) form of the enzyme, suggesting that the kinetic changes are due to the formation of AR-NO, and that the AR-S-S-X form of the enzyme is catalytically inactive. Modification of AR by the non-thiol NO donor diethylamine NONOate (DEANO) increased enzyme activity and resulted in the formation of AR-NO. However, no adducts between AR and DEANO were formed. These results show that nitrosothiols cause multiple structural and functional changes in AR. Our observations also suggest the general possibility that transnitrosation reactions can generate both nitrosated and thiolated products, leading to non-unique changes in protein structure and function.

Structural and kinetic modifications of aldose reductase by S-nitrosothiols

Modification of aldose reductase (AR) by the nitrosothiolsS-nitroso-N-acetyl penicillamine (SNAP) and N-(β-glucopyranosyl)-N2-acetyl-S-nitrosopenicillamide (glyco-SNAP) resulted in a 3–7-fold increase in its kcat and a 25–40-fold increase in its Km for glyceraldehyde. In comparison with the native protein, the modified enzyme was less sensitive to inhibition by sorbinil and was not activated by SO2−4 anions. The active-site residue, Cys-298, was identified as the main site of modification, because the site-directed mutant in which Cys-298 was replaced by serine was insensitive to glyco-SNAP. The extent of modification was not affected by Pi or O2, indicating that it was not due to spontaneous release of nitric oxide (NO) by the nitrosothiols. Electrospray ionization MS revealed that the modification reaction proceeds via the formation of an N-hydroxysulphenamide-like adduct between glyco-SNAP and AR. In time, the adduct dissociates into either nitrosated AR (AR-NO) or a mixed disulphide between AR and glyco-N-acetylpenicillamine (AR-S-S-X). Removal of the mixed-disulphide form of the protein by lectin-column chromatography enriched the preparation in the high-Km–high-kcat form of the enzyme, suggesting that the kinetic changes are due to the formation of AR-NO, and that the AR-S-S-X form of the enzyme is catalytically inactive. Modification of AR by the non-thiol NO donor diethylamine NONOate (DEANO) increased enzyme activity and resulted in the formation of AR-NO. However, no adducts between AR and DEANO were formed. These results show that nitrosothiols cause multiple structural and functional changes in AR. Our observations also suggest the general possibility that transnitrosation reactions can generate both nitrosated and thiolated products, leading to non-unique changes in protein structure and function.

Contractility of late pregnant rat myometrium is refractory to activation of soluble but not particulate guanylate cyclase

Objective: Our purpose was to compare the effects of agents activating particulate or soluble guanylate cyclases on the spontaneous contractile activity of the isolated pregnant rat uterus. Study Design: Uterine rings from midpregnant (14-day) and late pregnant (21-day) rats were suspended in organ chambers to record spontaneous contractile activity. Concentration-response curves were obtained for the following natriuretic peptides: atrial, brain, and C-type; concentration-response curves were also obtained for diethylamine nitric oxide, 3-morpholino-sydnominine, and authentic nitric oxide. Results: All 3 natriuretic peptides inhibited spontaneous uterine contractions equally at midgestation and late gestation. The inhibitory effects of the nitric oxide donors diethylamine nitric oxide, 3-morpholino-sydnominine, and authentic nitric oxide were attenuated in uterine tissues from animals in late stages of pregnancy. Conclusion: Agents activating either soluble or particulate guanylate cyclase inhibit contractions of uterine rings from midgestation rats, whereas the effects of soluble guanylate cyclase are attenuated at late pregnancy. Thus spontaneous uterine contractions are under the control of both soluble and particulate guanylate cyclases; the former is dependent on gestational age but the latter is not. (Am J Obstet Gynecol 2001;185:158-62.)

Effects of potassium channel inhibitors on the relaxation induced by the nitric oxide donor diethylamine nitric oxide in isolated human cerebral arteries.

The goal of this study was to investigate whether K+ channels are involved in nitric oxide (NO)-induced relaxation of isolated human cerebral arteries. Successive concentration-response curves relating to the use of the NO donor diethylamine NO (DEA/NO) were established in the absence and presence of different K+ channel inhibitors after mounting human cerebral arteries onto a wire myograph. The arteries were obtained from macroscopically intact tissue that had been removed during brain tumor operations. A high K+ concentration partially inhibited the relaxant effects of DEA/NO. Different K+ channel inhibitors (tetraethylammonium [TEA], 10(-3) M; charybdotoxin, 10(-7) M; glibenclamide, 10(-6) M; 4-aminopyridine [4-AP], 10(-3) M; BaCl2, 5 x 10(-5) M; and apamin, 10(-6) M) alone failed to affect the responses to DEA/NO. However, a combination of TEA, glibenclamide, 4-AP, and BaCl2 partially blocked the relaxant effects of DEA/NO. In addition, the effects of DEA/NO were inhibited by the thromboxane A2 analog U46619 (3 x 10(-7) M). Inhibitors of the large-conductance or small-conductance Ca++-activated K+ channels, the adenosine triphosphate-sensitive K+ channels, and the delayed-rectifier or inward-rectifier K+ channels failed to alter the effects of DEA/NO when only one K+ channel blocker was used. However, a regimen of a combination of K+ channel blockers that possess selectivity for different channels demonstrated that different K+ channel types are involved; these channels may function in a redundant manner and compensate for each other. Selective thromboxane A2 agonists are capable of inhibiting the relaxant response to the NO donor.

Novel nitric oxide donors reverse endothelin-1-mediated constriction in human blood vessels.

Cardiovascular disease is associated with elevated circulating plasma levels of endothelin-1 (ET-1). Our aim was to compare the ability of the nitric oxide donors (NO-donors) 3-morpholinylsydnonimine (SIN-1) and S-nitroso-N-acetylpenicillamine (SNAP) with the novel nitric oxide donors (NONOates) diethylamine NONOate (DEA/NO), and diethylenetriamine NONOate (DETA/NO) in order to physiologically antagonize ET-1-mediated constriction of human internal mammary arteries (IMA) in vitro. Both SNAP and DETA/NO caused a significant rightward shift in the ET-1 concentration-response curve. All four NO-donors were found to completely reverse an established contraction to a submaximal concentration ET-1 (decreasing order potency: SNAP >DEA/NO > SIN-1 > DETA/NO). These data suggest that the NONOates DEA/NO and DETA/NO can physiologically antagonize the effects of ET-1 in human arteries and may prove to be useful therapeutic agents in the treatment of cardiovascular disease.

Novel Nitric Oxide Donors Reverse Endothelin-1-Mediated Constriction in Human Blood Vessels

Summary: Cardiovascular disease is associated with elevated circulating plasma levels of endothelin-1 (ET-1). Our aim was to compare the ability of the nitric oxide donors (NO-donors) 3-morpholinylsydnonimine (SIN-1) and S-nitroso-N-acetylpenicillamine (SNAP) with the novel nitric oxide donors (NONOates) diethylamine NONOate (DEA/NO), and diethylenetriamine NONOate (DETA/NO) in order to physiologically antagonize ET-1-mediated constriction of human internal mammary arteries (IMA) in vitro. Both SNAP and DETA/NO caused a significant rightward shift in the ET-1 concentration-response curve. All four NO-donors were found to completely reverse an established contraction to a submaximal concentration of ET-1 (decreasing order of potency; SNAP > DEA/NO > SIN-1 > DETA/NO). These data suggest that the NONOates DEA/NO and DETA/NO can physiologically antagonize the effects of ET-1 in human arteries and may prove to be useful therapeutic agents in the treatment of cardiovascular disease.

Roles of potassium channels and nitric oxide in modulation of uterine contractions in rat pregnancy

Objective: We sought to study the involvement of potassium channels in the inhibition by nitric oxide of spontaneous contractions in isolated uterine rings from midterm and term pregnant rats. Study Design: Uterine rings from Sprague-Dawley rats at midterm and term gestation were used for isometric tension recording. The inhibition of spontaneous contractile activity by potassium channel openers and nitric oxide was studied in the absence and presence of potassium channel inhibitors. Results: The adenosine triphosphate–dependent potassium channel opener levcromakalim inhibited spontaneous contractions in rings from both midterm and term pregnant rats in a concentration-dependent manner, and the effects were significantly attenuated by pretreatment with selective inhibitor of the adenosine triphosphate–dependent potassium channel inhibitor glibenclamide. The opener of calcium-dependent potassium channel NS 1619 inhibited spontaneous contractions in rings from midterm but significantly less so in rings from term pregnant rats in a concentration-dependent manner, and the effect was significantly attenuated by pretreatment with potassium channel inhibitors tetraethylammonium and tetrabutylammonium but not with glibenclamide. Rings from midterm and term pregnant rats were more sensitive to the inhibitory effect of levcromakalim compared with NS 1619. Nitric oxide donor diethylamine–nitric oxide inhibited spontaneous contractions in rings from midterm but significantly less in rings from term pregnant rats in a concentration-dependent manner, and the effect was attenuated by tetraethylammonium and tetrabutylammonium but not by glibenclamide. Conclusions: There is gestational age–dependent refractoriness to calcium-dependent potassium but not adenosine triphosphate–dependent potassium channel opener–induced inhibition of spontaneous contractile activity of isolated rat uterine rings. Nitric oxide inhibits uterine contractions by opening of calcium-dependent potassium channels in pregnant rat myometrium. Refractoriness to nitric oxide toward term may result from decreased probability to open or number of calcium-dependent potassium channels. (Am J Obstet Gynecol 1999;181:649-55.)

Effect of placental tissue on inhibition of uterine contraction by nitric oxide donors

Objective: Our purpose was to test the hypothesis that placental tissue modulates the effect of nitric oxide on spontaneous uterine contractility in pregnant rats. Study Design: Rings (approximately 4 mm) of uterus taken from rats on day 14 (midpregnancy, n = 6), day 18 (late pregnancy, n = 4), and day 22 (term, n = 4) of gestation were placed in organ chambers filled with Krebs-bicarbonate buffer bubbled with 5% carbon dioxide in air (37°C, pH approximately 7.4) for isometric tension recording. In some rings a piece of placenta was left attached to the uterine wall. In the other rings the fetuses, placentas, and membranes were removed completely. Change of spontaneous contractions of the rings (percentage change of basal integral activity for 10 minutes) in response to cumulative concentrations of the nitric oxide donors diethylamine–nitric oxide and nitroglycerin (10 –6 mol/L to 10 –4 mol/L) were compared between rings with and without placenta. Results: Diethylamine–nitric oxide and nitroglycerin inhibited spontaneous uterine contractions in rings from midpregnancy, in both the absence and the presence of placenta. In rings from midpregnancy, the maximal inhibition of contractions by diethylamine–nitric oxide but not by nitroglycerin was significantly ( P < .05) higher in the presence (26.7% ± 3.5% of basal activity) than in the absence (39.6% ± 3.3%) of placenta. Inhibition of contraction by nitric oxide donors in rings from late and term pregnancy was less than in midpregnancy, and the presence of placental tissue did not influence the responses. Conclusions: The presence of placental tissue enhances inhibition of uterine contractility by agents that spontaneously release nitric oxide, such as diethylamine–nitric oxide, but not by nitroglycerin, which requires metabolic transformation for nitric oxide to be released. Refractoriness to nitric oxide near or at term does not depend on the presence or absence of placental tissue. (Am J Obstet Gynecol 1999;181:415-8.)

Optical biosensing of nitric oxide using the metalloprotein cytochrome c'.

The metalloprotein cytochrome c' was extracted and purified from the bacterium Paracoccus denitrificans in order to develop a specific biosensing system for nitric oxide (NO). The metalloprotein was encapsulated in a porous silicate sol-gel glass to enable spectroscopic changes in the haem centre as a function of NO ligation to be quantified using absorption measurements. Spectroscopic evidence suggested that, between 2 and 4 d after encapsulation, the cytochrome c' protein changed conformation in the locality of the haem moiety, possibly from a five to a six coordinate haem centre. Such conformational changes were also observed when the cytochrome c' was stored in solution, although over a 2-3 month period. The conformational changes occurring in the protein altered the spectral characteristics of the reduced, oxidised and nitrosyl complex of the cytochrome c' and appear to change the binding affinity of the protein towards NO. However, the encapsulated (reconformed) cytochrome c' was shown to retain its selectivity towards NO with good reproducibility (seven consecutive measurements of NO produced an intensity value with a relative standard deviation of 0.28%). An NO calibration curve, using the in situ release of NO from the donor diethylamine NONOate, was obtained for the encapsulated cytochrome c' with an approximate working range of 10-400 mumol l-1.