Impaired effectiveness of nitric oxide-donors in resistance arteries of patients with arterial hypertension.

Infusion of nitric oxide (NO) donors is known to induce delayed attacks of migraine and cluster headache or aggravate tension-type headaches in patients suffering from these primary headaches. Previously we have reported that infusion of NO donors in the rat causes delayed neuronal activity in the spinal trigeminal nucleus, which parallels the above clinical observations. Suggesting that endogenous NO production is involved in the generation of primary headaches, we used this animal model of meningeal nociception to determine whether a prolonged increase in NO levels causes an increase in neuronal activity. In anaesthetized rats spinal trigeminal neurons with afferent input from the exposed dura were recorded. Continuous intravenous infusion of the NO donors sodium nitroprusside (25 microg/kg/h) or glycerol trinitrate (250 microg/kg/h) for 2 h induced a persisting increase in neuronal activity but no change in systemic blood pressure. In this activated trigeminal system the calcitonin gene-related peptide (CGRP) receptor antagonist BIBN4096BS (900 microg/kg) was infused. Spinal trigeminal activity was significantly reduced within minutes and to a similar extent as previously reported in animals not treated with NO. Slow continuous NO infusion may be a model of the active headache phase, and inhibition of CGRP receptors can reverse the induced neuronal activity.

arterial chemoreceptors located in the carotid body are an important defense mechanism against systemic hypoxemia. Activation of arterial chemoreceptors increases alveolar ventilation and sympathetic outflow to most vascular beds. During acute exposures to hypoxia, the sympathoexcitatory responses

The assessment of endothelial function in hypertensive patients receiving acetylcholine has revealed conflicting results. Whether an impaired flow response to acetylcholine is explained solely by a diminished endothelial synthesis of nitric oxide (NO) remains unclear as yet. In the present study, we tested the hypothesis that mechanisms other than reduced NO synthesis contribute to the hypertension-associated impairment of endothelium-dependent vasodilation. Therefore, the dilatory response to endogenous and exogenous NO was measured in resistance arteries and cutaneous microvessels in the forearm circulation of 12 normotensive individuals and 17 hypertensive patients. In addition, the overall dilatory capacity was assessed by peak flow during reactive hyperemia after 3 minutes of ischemia. Forearm blood flow was quantified by venous occlusion plethysmography at rest, during application of the NO donor sodium nitroprusside, and during stimulation of endogenous NO synthesis by acetylcholine and bradykinin. Blood flow velocity in the cutaneous microvasculature was measured with laser-Doppler flowmetry in parallel. Resting forearm flow was comparable in both groups (3.1 +/- 0.2 and 3.4 +/- 0.2 mL.min-1.100mL-1 tissue), whereas blood pressure and thus peripheral vascular resistance was significantly elevated in hypertensive compared with normotensive subjects. Hyperemic peak flow was significantly blunted in hypertensive patients. Sodium nitroprusside, acetylcholine, and bradykinin increased flow in a dose-dependent manner to a comparable extent in the control group (13.3 +/- 0.8, 13.6 +/- 1.3, and 14.6 +/- 0.7 mL.min-1.100mL-1 tissue, respectively). In contrast, in hypertensive patients maximum increase in resting flow was significantly reduced (sodium nitroprusside, -36%; acetylcholine, -44%; and bradykinin, -56%). The flow response after stimulation of endogenous NO synthesis by bradykinin was significantly more blunted compared with that of exogenous NO after application of sodium nitroprusside. In the cutaneous microvasculature, bradykinin-induced increases in blood flow velocity were selectively impaired in hypertensive patients, whereas flow response to acetylcholine was preserved. Thus, we conclude that in arterial hypertension endothelium-dependent, NO-mediated dilation of resistance arteries and cutaneous microvessels of the forearm vasculature is heterogeneously impaired, depending on the type of endothelial receptor stimulated. Furthermore, the present data suggest that in hypertensive patients the impairment of NO-dependent dilation of resistance arteries is caused by at least three different mechanisms: (1) a reduced endothelial synthesis of NO due to either a disturbed signal-transduction pathway and/or a reduced activity of NO synthase, (2) an accelerated NO degradation within the vessel wall, and (3) alterations in the vessel architecture resulting in an overall reduced dilatory capacity of resistance arteries.

Background: Increased levels of cell-free hemoglobin (Hb) and ferric heme in plasma is vasculopathic and contributes to morbidity and mortality in hemolytic and non-hemolytic diseases, including sepsis. Nitric oxide (NO) signals through soluble guanylate cyclase (sGC) to induce vasorelaxation and is rapidly scavenged by plasma Hb/heme. Dysregulated NO signaling and increased oxidative stress underlie sickle cell disease (SCD) pathology and treatments that limit heme toxicity and promote NO signaling are needed. We have discovered a fast catalyzed reductive nitrosylation reaction that converts heme into NO-ferroheme, protecting NO from Hb scavenging and when in complex with albumin, elicits sGC-dependent vasorelaxation. Hypothesis: We hypothesize that reductive nitrosylation can be coopted as a therapeutic to convert pro-oxidative, pro-inflammatory plasma free heme to anti-inflammatory, vasodilatory NO-ferroheme. Approach: NO-ferroheme was measured by ozone-based chemiluminescence in mouse plasma following intravenous infusion of heme, NO donor and reductants to determine if NO-ferroheme is formed in vivo and the effects on mean arterial pressure (MAP) were assessed. To determine therapeutic potential, mice were treated with NO-ferroheme albumin following induction of LPS-mediated lung injury. Total cell extravasation, neutrophil response, and the expression of inflammatory cytokines were assessed. Results: Infusion with heme, NO donor (papaNONOate) and glutathione (GSH) or ascorbate yielded detectable levels of NO-ferroheme (ascorbate - 83.7 nM ± 3 7.7 nM; GSH - 57.2 nM ± 29.8 nM) with low levels of S-nitrosothiols in mouse plasma. Infusion of heme triggered acute hypertension, while infusion of NO donor, ascorbate and heme had a vasodilatory effect (MAP -5 mmHg from baseline). Treatment with 1 umol/kg NO-ferroheme albumin following LPS-mediated lung injury resulted in decreases in total cell extravasation, neutrophil to monocyte ratio and expression of IL-6 , Cxcl9, and Cxcl10 . Conclusions: Free heme is converted to NO-ferroheme in vivo in the presence of excess NO and reductant causing vasorelaxation, reversing the hypertensive effects of heme. Treatment with NO-ferroheme albumin is anti-inflammatory in an LPS model of acute lung injury. Conversion of pro-oxidative and pro-inflammatory heme into anti-inflammatory NO-ferroheme shows promise as a novel strategy to curtail heme-mediated injury in hemolytic conditions.

Nitric oxide (NO) donors, which cause delayed headaches in migraineurs, have been shown to activate central trigeminal neurons with meningeal afferent input in animal experiments. Previous reports indicate that this response may be due to up-regulation of NO-producing cells in the trigeminal brainstem. To investigate this phenomenon further, we determined nitric oxide synthase (NOS)-containing neurons in the rat spinal trigeminal nucleus (STN), the projection site of nociceptive trigeminal afferents, following infusion of the NO donor sodium nitroprusside (SNP). Barbiturate anaesthetized rats were infused intravenously with SNP (50 microg/kg) or vehicle for 20 min or 2 h, and after periods of 3-8 h fixed by perfusion. Cryostat sections of the medulla oblongata containing the caudal STN were histochemically processed for detection of nicotineamide adenine dinucleotide phosphate (NADPH)-diaphorase or immunohistochemically stained for NOS isoforms and examined by light and fluorescence microscopy. The number of neurons positive for these markers was determined. Various forms of neurons positive for NADPH-diaphorase or immunoreactive to neuronal NOS (nNOS) were found in superficial and deep laminae of the STN caudalis and around the central canal. Neurons were not immunopositive for endothelial (eNOS) or inducible (iNOS) NOS isoforms. The number of NADPH-diaphorase-positive neurons increased time dependently after SNP infusion by a factor of more than two. Likewise, the number of nNOS-immunopositive neurons was increased after SNP compared with vehicle infusion. Around the central canal the number of NADPH-diaphorase-positive neurons was slightly increased and the number of nNOS+ neurons not changed after SNP treatment. NO donors increase the number of neurons that produce NO in the STN, possibly by induction of nNOS expression. Increased NO production may facilitate neurotransmitter release and promote nociceptive transmission in the STN. This mechanism may explain the delayed increase in neuronal activity and headache after infusion of NO donors.

Endogenous nitric oxide (NO) reduces sympathetic vasoconstriction by attenuating neuronal excitability in the brain stem and inhibition of postganglionic neurotransmission. We studied whether this modulation of sympathetic circulatory control by NO may be altered during chronic administration of NO donor drugs in pigs. Nitrate tolerance was induced by oral administration of isosorbide dinitrate (ISDN, 4 mg/kg per day for 4 weeks) in eight pigs. Four of them were chronically instrumented for the measurement of mean arterial blood pressure and cardiac output in the conscious state. ISDN treatment caused hemodynamic tolerance to NO donors and significantly increased the hypotensive responses to pharmacologic ganglionic blockade in conscious pigs. In general anesthesia, ISDN-treated animals and age-matched controls (n=5) had similar baseline renal sympathetic nerve activity and in both groups neither inhibition of NO synthases (NOS) nor administration of NO donors to the brain stem by intracerebroventricular (i.c.v.) infusions caused significant changes in baseline renal sympathetic nerve activity. However, whereas sympathoexcitatory responses to glutamate (0.5 mL, 0.1 mol/L, i.c.v.) or electrical stimulation of somatic nerve afferents were significantly potentiated by central NOS inhibition and attenuated by NO donors in controls, these treatments no longer had significant effects in ISDN-treated pigs. Furthermore, reflex sympathetic activation in response to intravenous NO donor treatment was more pronounced in nitrate tolerant animals, which suggests loss of central sympathoinhibitory effects of NO. Subsequent histology on brain stem slices with NADPH-diaphorase as NOS marker revealed significant reduction of NOS density in ISDN-treated pigs. Long-term administration of organic nitrates reduces the number of NO-producing neurons in the brain stem and causes loss of inhibitory effects of NO on sympathetic excitability. This component of tolerance to organic nitrates may be important in patients confronted frequently with sympathetic activation caused by mental and/or physical stressors.

Activation of M1 macrophages in nonalcoholic steatohepatitis (NASH) is produced by several external or endogenous factors: inflammatory stimuli, oxidative stress, and cytokines are known. However, any direct role of oxidative stress in causing M1 polarization in NASH has been unclear. We hypothesized that CYP2E1-mediated oxidative stress causes M1 polarization in experimental NASH, and that nitric oxide (NO) donor administration inhibits CYP2E1-mediated inflammation with concomitant attenuation of M1 polarization. Because CYP2E1 takes center stage in these studies, we used a toxin model of NASH that uses a ligand and a substrate of CYP2E1 for inducing NASH. Subsequently, we used a methionine and choline-deficient diet-induced rodent NASH model where the role of CYP2E1 in disease progression has been shown. Our results show that CYP2E1 causes M1 polarization bias, which includes a significant increase in interleukin-1β (IL-1β) and IL-12 in both models of NASH, whereas CYP2E1-null mice or diallyl sulfide administration prevented it. Administration of gadolinium chloride (GdCl3), a macrophage toxin, attenuated both the initial M1 response and the subsequent M2 response, showing that the observed increase in cytokine levels is primarily from macrophages. Based on the evidence of an adaptive NO increase, the NO donor administration in vivo that mechanistically inhibited CYP2E1 catalyzed the oxidative stress during the entire study in NASH-abrogated M1 polarization and NASH progression. The results obtained show the association of CYP2E1 in M1 polarization, and that inhibition of CYP2E1 catalyzed oxidative stress by an NO donor (DETA NONOate [(Z)-1-[N-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate]) can be a promising therapeutic strategy in NASH.

Nitrovasodilators, such as glyceroltrinitrate (GTN), which produce nitric oxide (NO) in the organism, are known to cause delayed headaches in migraineurs, accompanied by increased plasma levels of calcitonin gene-related peptide (CGRP) in the cranial venous outflow. Increases in plasma CGRP and NO metabolites have also been found in spontaneous migraine attacks. In a rat model of meningeal nociception, infusion of NO donors induced activity of neurons in the spinal trigeminal nucleus. Isoflurane-anaesthetised rats were intravenously infused with GTN (250 µg/kg) or saline for two hours and fixed by perfusion after a further four hours. Cryosections of dissected trigeminal ganglia were immunostained for detection of CGRP and neuronal NO synthase (nNOS). The ganglion neurons showing immunofluorescence for either of these proteins were counted. The proportions of CGRP- and nNOS- as well as double-immunopositive neurons were increased after GTN infusion compared to saline treatment in all parts of the trigeminal ganglion (CGRP) or restricted to the ophthalmic region (nNOS). The size of immunopositive neurons was not significantly different compared to controls. High levels of NO may induce the expression or availability of CGRP and nNOS. Similar changes may be involved in nitrovasodilator-induced and spontaneous headache attacks in migraineurs.

Nitric oxide (NO), a potent nonadrenergic, noncholinergic mediator of gastrointestinal smooth muscle, causes relaxation of the category I pump like sphincter of Oddi (SO) (eg, opossum, rabbit) and category II resistor like SO (eg, pig, human). Topical administration of a NO donor induces SO relaxation in humans, and parenteral administration of sodium nitroprusside (SNP) decreases sphincter contractility in pig SO. The aim of this study is to evaluate the effect of intrasphincteric SNP injection on pig SO. Under general anesthesia, two pigs received intrasphincteric saline injection (1 ml) and six pigs received intrasphincteric SNP (0.5 microg/ml) injection into the SO. All injections were administered into the major papilla using a 5-mm sclerotherapy needle through the duodenoscope. Endoscopic biliary manometry was performed using the standard station pull-through technique and SO pressures were recorded before and after injection. Intrasphincteric saline injection did not significantly change the mean SO motility index (MI) (197 vs 198). However, intrasphincteric SNP reduced both the mean SO basal pressure (P = 0.002) and the mean SO MI (226 vs 109; P = 0.002). The effect of intrasphincteric SNP lasted up to 45 min and did not cause significant lowering of systemic blood pressure. This is the first study to demonstrate that intrasphincteric SNP results in significant reduction in both SO basal pressure and SO MI in the porcine model. The endoscopic intrasphincteric administration of NO donor drugs is technically feasible and without observed systemic side effects.

Nicotine intensifies experimental gastric ulceration by reducing gastric mucosal blood flow (GMBF) and mucus. As both these parameters can be improved by nitric oxide (NO), we evaluated the impact of a NO donor in ethanol-induced gastric mucosal injury in rats administered nicotine. A nicotine solution or water was administered for 20 days to Sprague-Dawley rats. NO donor (isosorbide dinitrate) was given 60 and 10 min before preparation of ex vivo gastric chambers and exposure to ethanol. Chronic nicotine intake significantly reduced GMBF and gastric mucus content. Nicotine intensifies ethanol-induced gastric injury and short-term administration of NO donor failed to antagonize the ulcerogenic action from either nicotine or alcohol. In another study, rats drank nicotine solution for 20 days, after which the nicotine was withdrawn and replaced by water for 10 additional days. NO donor was provided during these last 10 days. The gastric effects of nicotine persisted for at least 10 days after nicotine was withdrawn but then these effects could be abolished by prolonged NO treatment. Nicotine reduces plasma nitrite level, but gastric mucosal MPO activity remained unchanged. Our data suggest that nicotine cessation plus a longer period of NO donor administration can completely abolish the gastric effects of nicotine.

Discovered and patented as an explosive by Alfred Nobel in the 1860s, nitroglycerin has been formulated for use in the treatment of symptomatic CAD for over 140 years. In fact, later in life, Nobel himself was prescribed the medication for angina, but refused to take it because of the associated side effect of headache. See page 1729 With glycerol trinitrate (GTN) as the prototype, nitrates represent one of the safest and most rapidly effective pharmacological means to reduce acute symptoms of myocardial ischemia attributable to obstructive coronary disease. This has led, over the years, to the development of long-acting oral and topical preparations. However, efficacy with chronic administration is more difficult to achieve because of the development of therapeutic resistance, generally occurring a few days after initiating treatment. This phenomenon known as nitrate tolerance has been the stimulus for intense investigation of the metabolic fate of nitroglycerin with the idea that modulation of its biotransformation could improve efficacy of chronic treatment. The mechanism of GTN-induced dilation is complex and was not identified until more than 100 years after its discovery. GTN is not a direct vasodilator, rather it must be converted to dinitrate products for vasoactivity. Biotransformation to the active metabolite nitric oxide (NO) occurs in parallel with the formation of glycerol-1,2-dinitrate and involves a dithiol-dependent process.1 It was not until recently that the principal enzyme responsible for biotransformation of GTN was identified. Chen et al1 showed that mitochondrial aldehyde dehydrogenase (ALDH-2) metabolizes GTN to glycerol-1,2-dinitrate and nitrite. This was confirmed by Sydow et al2 using mitochondrial-deficient cultured endothelial cells, although a cytosolic source of ALDH-2 has also been suggested.3 The mitochondrial enzyme converts nanomolar concentrations of GTN to active nitrodilator metabolites in vivo and in vitro, as shown by direct measurements coupled with the use …

Hypoxia-evoked vasodilatation is a fundamental regulatory mechanism that is often attributed to adenosine. The identity of the O(2) sensor is unknown. Nitric oxide (NO) inhibits endothelial mitochondrial respiration and ATP generation by competing with O(2) for its binding site on cytochrome oxidase. We proposed that in vivo this interaction allows endothelial cells to release adenosine when O(2) tension falls or NO concentration increases. Using anaesthetised rats, we confirmed that the increase in femoral vascular conductance (FVC, hindlimb vasodilatation) evoked by systemic hypoxia is attenuated by NO synthesis blockade with L-NAME, but restored when baseline FVC is restored by infusion of NO donor. This "restored" hypoxic response, like the control hypoxic response, is inhibited by the adenosine A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. Similarly, the FVC increase evoked by adenosine infusion was attenuated by L-NAME but restored by infusion of NO donor. However, when baseline FVC was restored after L-NAME with 8-bromo-cGMP, the FVC increase evoked by adenosine infusion was restored, but not in response to systemic hypoxia, suggesting that adenosine was no longer released by hypoxia. Infusion of NO donor at a given rate after treatment with L-NAME evoked a greater FVC increase during systemic hypoxia than during normoxia, both responses being reduced by 8-cyclopentyl-1,3-dipropylxanthine. Finally, both bradykinin and NO donor released adenosine from superfused endothelial cells in vitro; L-NAME attenuated only the former response. We propose that in vivo, shear-released NO increases the apparent K(m) of endothelial cytochrome oxidase for O(2), allowing the endothelium to act as an O(2) sensor, releasing adenosine in response to moderate falls in O(2).

Experiments were carried out to explore the possible role played by the nitric oxide (NO) system in the organum vasculosum laminae terminalis (OVLT) of rat brain in arterial pressure regulation. Intracerebroventricular (ICV) or intra-OVLT administration of NO donors such as hydroxylamine, sodium nitro-prusside or s-nitro-acetylpenicillamine caused an up to 55 mmHg decrease in blood pressure (BP) but an increase in NO release (measured by porphyrin/nafion coated carbon fibre electrodes in combination with voltammetry) in the OVLT. In contrast, ICV or intra-OVLT administration of N(G)-nitro-L-arginine methyl ester (L-NAME; a constitutive NO synthase inhibitor) caused an up to 45 mmHg increase in BP but a fall in NO release in the OVLT. Compared with the BP responses induced by ICV injection of NO donors or NO synthase inhibitors, the OVLT route of injection required a much lower dose of NO donors or NO synthase inhibitors to produce a similar BP effect. The depressor effects induced by ICV or intra-OVLT administration of NO donors were attenuated by pretreatment with intra-OVLT injection of methylene blue (an inhibitor of guanylate cyclase), haemoglobin (a NO scavenger), L-NAME or spinal transection. On the other hand, the L-NAME-induced pressor effects were attenuated by pretreatment with intra-OVLT injection of L-arginine or spinal transection. The data suggest that activation of cyclic GMP-dependent NO synthase in the OVLT of rat brain causes cyclic GMP-dependent decreases in arterial pressure via inhibiting the sympathetic efferent activity.

EFFECT OF INTRAVESICAL NITRIC OXIDE THERAPY ON CYCLOPHOSPHAMIDE-INDUCED CYSTITIS

Nitric oxide (NO) is administered via infusion of donors such as nitroglycerin or in inhaled form for treatment of ischemia and pulmonary hypertension, respectively. In rabbits, the NO donor, DETANONOate, decreases whole blood clotting function as assessed by thromboelastographic variables (R, reaction time; α, angle; and G, a measure of clot strength). I hypothesized that DETANONOate-derived NO would adversely affect coagulation protein and platelet function. Blood obtained from ear arteries of conscious rabbits (n = 8) anticoagulated with sodium citrate. The blood was then incubated with 0 or 10mM DETANONOate for 30 min. After incubation and recalcification, thromboelastography was performed for 60 min under four conditions: 1) 0mM DETANONOate, 2) 0mM DETANONOate with platelet inhibition with cytochalasin D, 3) 10mM DETANONOate, and 4) 10mM DETANONOate with platelet inhibition. DETANONOate significantly (P < 0.05) increased R and decreased α and G in samples with or without platelet inhibition, compared with samples not exposed to DETANONOate. Lastly, the percentage of total G (GT) attributable to platelet function (GP) was significantly more in the absence of DETANONOate (GP = 92.3% ± 1.6%; mean ± sd) than after exposure to DETANONOate (GP = 90.2% ± 2.3%). DETANONOate-derived NO significantly decreased coagulation protein function and platelet function. Coagulation protein function may be similarly affected in clinical situations involving the administration of NO or NO donors. Implications In rabbit whole blood, nitric oxide (NO) decreases hemostatic function by decreasing both coagulation protein function and platelet function. Coagulation protein function may be similarly affected in clinical situations involving the administration of NO or NO donors.