Nitric oxide-induced vasodilation of organic nitrate-tolerant rabbit aorta

It is postulated that the organic nitrate vasodilator agents, including glyceryl trinitrate (GTN) and isosorbide dinitrate (ISDN), are prodrugs, such that biotransformation to the active inorganic metabolite, nitric oxide (NO), occurs prior to the onset of vasodilation. Furthermore, it is proposed that organic nitrate tolerance in vascular tissue involves decreased formation of NO. To test this latter hypothesis, we examined vasodilation induced by NO, GTN, and ISDN in non-tolerant, GTN-tolerant, and ISDN-tolerant rabbit aortic rings (RARs). Isolated RARs were contracted submaximally with phenylephrine; the time of onset of relaxation and percent relaxation of tissue were determined in response to NO (0.3 microM), GTN (0.03 microM), and ISDN (0.12 microM) before and after a 1-h treatment with 500 microM GTN, 500 microM ISDN, or buffer only. The data demonstrated that the response to NO was not changed in GTN-tolerant and ISDN-tolerant tissues, in which there was virtually no GTN-induced or ISDN-induced relaxation. These results are consistent with the postulate that organic nitrate vasodilator drugs must undergo biotransformation to NO before vasodilation can occur and that the mechanism of organic nitrate tolerance involves decreased formation of NO.     

Involvement of the endothelial DDAH/ADMA pathway in nitroglycerin tolerance: the role of ALDH-2

Previous studies have shown that nitroglycerin (GTN) tolerance is closely related to an oxidative stress-induced decrease in activity of mitochondrial isoforms of aldehyde dehydrogenase (ALDH-2), and prolonged GTN treatment causes endothelial dysfunction. Asymmetric dimethylarginine (ADMA), a major endogenous NO synthase (NOS) inhibitor, could inhibit NO production and induce oxidative stress in endothelial cells. ADMA and its major hydrolase dimethylarginine dimethylaminohydrolase (DDAH) have recently been thought of as a novel regulatory system of endothelium function. The aim of the present study was to determine whether the DDAH/ADMA pathway is involved in the development of GTN tolerance in endothelial cells. Tolerance, reflected by the decrease in cyclic GMP (cGMP) production, was induced by exposure of human umbilical vein endothelial cells (HUVECs) to GTN (10 microM) for 16 h. While the treatment increased reactive oxygen species (ROS) production/malondialdehyde (MDA) concentration and decreased ALDH-2 activity as well as cGMP production, it markedly increased the level of ADMA in culture medium and decreased DDAH activity in endothelial cells. Exogenous ADMA significantly enhanced ROS production/MDA concentration and inhibited ALDH-2 activity, and overexpression of DDAH2 could significantly suppress GTN-induced oxidative stress and inhibition of ALDH-2 activity, which is also attenuated by L-arginine. Therefore, our results suggest for the first time that the endothelial DDAH/ADMA pathway plays an important role in the development/maintenance of GTN tolerance.     

Possible involvement of nitroglycerin converting step in nitroglycerin tolerance.

Nitroglycerin (GTN) produces a dilation of vascular smooth muscle by releasing NO through a putative GTN-converting step. However, the response to GTN is markedly attenuated after prolonged or repeated exposure, resulting in tolerance. We investigated the mechanisms of GTN tolerance, employing exogenous and endogenous NO in rat aorta. In endothelium-denuded rat aortic strips, the GTN-induced relaxation response was attenuated by preceding exposure to either GTN or sodium nitroprusside (SNP). In contrast, the SNP-induced relaxation response was not affected by this protocol of GTN or SNP preexposure. Preincubation of aortic strips with lipopolysaccharide (LPS) +/- L-arginine for 12 h also caused attenuation of GTN-induced responses such as relaxation, cGMP production and nitrite/nitrate formation. The attenuating effect of LPS abolished in aortic strips co-incubated with LPS and cycloheximide or N(G)-nitro-L-arginine. These results suggest that GTN tolerance is predominantly associated with the reduction of NO release from GTN, which is caused through inhibition of a GTN-converting step due to preceding exposure to NO itself.     

Prostaglandin Induces Ca2+ Influx and Cyclic GMP Formation in Mouse Neuroblastoma × Rat Glioma Hybrid NG108–15 Cells in Culture

Various prostaglandins (PGs) (10 nM-30 microM) were added to NG108-15 cells in culture, and changes in the levels of intracellular cyclic GMP and Ca2+ were investigated. Exposure of the cells to PGF2 alpha, PGD2, and PGE2 (10 microM) transiently increased the cyclic GMP content 7.5-, 3.9-, and 3.1-fold, respectively. Furthermore, the increased levels of cyclic GMP correlated well with the rise in cytosolic free Ca2+ concentrations induced by the PGs. Other PGs (10 microM), including metabolites and synthetic analogs, which had no effect on intracellular Ca2+, failed to increase the cyclic GMP content in the cells. When extracellular Ca2+ was depleted from the culture medium, the PG-induced increase in cyclic GMP level was almost completely abolished. In addition, treatment of the cells with quin 2 tetraacetoxymethyl ester dose-dependently inhibited the PG-induced cyclic GMP formation. The increase in cyclic GMP content caused by treatment of the cells with a high K+ level (50 mM) was completely blocked by voltage-dependent Ca2+ entry blockers, such as verapamil (10 microM), nifedipine (1 microM), and diltiazem (100 microM); however, the PG (10 microM)-induced increase in cyclic GMP content was not affected by such Ca2+ entry blockers. These findings indicate that PG-induced cyclic GMP formation may require the rise in intracellular Ca2+ level and that the voltage-dependent Ca2+ channels may not be involved in the PG-induced rise in Ca2+ content.     

Stimulatory effect of dibutyryl cyclic GMP on acid secretion in mouse isolated stomach and on histamine release in gastric mucosal cells.

We previously reported that endogenous nitric oxide (NO) is involved in the peripheral control of gastric acid secretion induced by some secretagogues, and that endogenous NO is involved in the acid secretion process via histamine release from histamine-containing cells. However, the stimulus-secretion coupling in the cells remains to be clarified. In the present study, we investigated the effect of dibutyryl cyclic GMP on gastric acid secretion in mouse isolated stomach and on histamine release in gastric mucosal cells, in comparison with those of dibutyryl cyclic AMP. Dibutyryl cyclic GMP (300 microM) produced a slight but significant increase of gastric acid secretion, which was completely inhibited by the histamine-H2 receptor antagonist famotidine. In contrast, dibutyryl cyclic GMP (1 mM) markedly inhibited histamine-induced acid secretion. Dibutyryl cyclic AMP (100 microM) produced a sustained increase of gastric acid secretion. The pretreatment with famotidine partially inhibited dibutyryl cyclic AMP-induced gastric acid secretion. Dibutyryl cyclic GMP and dibutyryl cyclic AMP significantly increased the histamine release from gastric mucosal cells. These results suggest that both intracellular cyclic GMP and cyclic AMP act as second messengers for histamine release in the histamine-containing cells, probably ECL cells. On the other hand, in gastric parietal cells, cyclic AMP has a stimulatory effect on gastric acid secretion, whereas cyclic GMP has an inhibitory effect.     

Stimulation of the Cyclic GMP Pathway by NO Induces Expression of the Immediate Early Genes c-fos and junB in PC12 Cells

Abstract: Stimulation of several second messenger pathways induces the expression of immediate early genes such as c- fos , c- jun , junB , and junD , but little is known about their induction via the stimulation of the cyclic GMP pathway. Here we looked at the expression of early genes in pheochromocytoma PC12 cells after activation of cytosolic guanylate cyclase by sodium nitroprusside. This compound spontaneously releases NO, a molecule known to be involved in cell communication. We found that expression of c- fos and junB but not of c- jun or junD is increased upon activation of cyclic GMP pathway. c- fos mRNA expression was the most activated (fourfold at 30 min), whereas junB response was more modest (2.2-fold activation at 60 min). Nuclear extracts of stimulated cells show increased binding capacity to the AP1 binding site consistent with the dose-response curve. The activating effect of nitroprusside could be reproduced by dipyridamole, a selective cyclic GMP phosphodiesterase inhibitor and by 8- p -chlorophenylthio-cyclic GMP, a permeant selective cyclic GMP-dependent protein kinase activator, and abolished by KT5823, an inhibitor of that kinase. The results show that NO promotes early gene activation and AP1 binding enhancement through the stimulation of the cyclic GMP pathway.     

Regulation of Neuronal Nitric Oxide and Cyclic GMP Formation by Ca2+

Nitric oxide (NO) acts as a messenger molecule in the CNS by activating soluble guanylyl cyclase. Rat brain synaptosomal NO synthase was stimulated by Ca2+ in a concentration-dependent manner with half-maximal effects observed at 0.3 microM and 0.2 microM when its activity was assayed as formation of NO and L-citrulline, respectively. Cyclic GMP formation was apparently inhibited, however, at Ca2+ concentrations required for the activation of NO synthase, indicating a down-regulation of the signal in NO-producing cells. Purified synaptosomal guanylyl cyclase was not inhibited directly by Ca2+, and the effect was not mediated by a protein binding to guanylyl cyclase at low or high Ca2+ concentrations. In cytosolic fractions, the breakdown of cyclic GMP, but not that of cyclic AMP, was highly stimulated by Ca2+, and 3-isobutyl-1-methylxanthine did not block this reaction effectively. The effects of Ca2+ on cyclic GMP hydrolysis and on apparent guanylyl cyclase activities were abolished almost completely in the presence of the calmodulin antagonist calmidazolium, whose effect was attenuated by added calmodulin. Thus, a Ca2+/calmodulin-dependent cyclic GMP phosphodiesterase is highly active in synaptic areas of the brain and may prevent elevations of intracellular cyclic GMP levels in activated, NO-producing neurons.     

Comparison of glyceryl trinitrate-induced with pentaerythrityl tetranitrate-induced in vivo formation of superoxide radicals: effect of vitamin C.

Glyceryl trinitrate (GTN) and pentaerythrityl tetranitrate (PETN) are among the most known organic nitrates that are used in cardiovascular therapy as vasodilators. However, anti-ischemic therapy with organic nitrates is complicated by the induction of nitrate tolerance. When nitrates are metabolized to release nitric oxide (NO), there is considerable coproduction of superoxide radicals in vessels leading to inactivation of NO. However, nitrate-induced increase of superoxide radical formation in vivo has not been reported. In this work, the authors studied the in vivo formation of superoxide radicals induced by treatment with PETN or GTN and determined the antioxidant effect of vitamin C. The formation of superoxide radicals was determined by the oxidation of 1-hydroxy-3-carboxy-pyrrolidine (CP-H) to paramagnetic 3-carboxy-proxyl (CP) using electron spin resonance spectroscopy. CP-H (9 mg/kg intravenous bolus and 0.225 mg/kg per minute continuous intravenous GTN or PETN 130 microg/kg) were infused into anesthetized rabbits. Every 5 min, blood samples were obtained from Arteria carotis to measure the CP formation. Both PETN and GTN showed similar vasodilator effects. Formation of CP in blood after infusions of GTN and PETN were 2.0+/-0.4 microM and 0.98+/-0.23 microM, respectively. Pretreatment with 30 mg/kg vitamin C led to a significant decrease in CP formation: 0.27+/-0.14 microM (vitamin C plus GTN) and 0.34+/-0.15 microM (vitamin C plus PETN). Pretreatment of animals with superoxide dismutase (15,000 units/kg) significantly inhibited nitrate-induced nitroxide formation. Therefore, in vivo infusion of GTN or PETN in rabbits increased the formation of superoxide radicals in the vasculature. PETN provoked a minimal stimulation of superoxide radical formation without simultaneous development of nitrate tolerance. The data suggest that the formation of superoxide radicals induced by organic nitrate correlates with the development of nitrate tolerance. The effect of vitamin C on CP formation leads to the conclusion that vitamin C can be used as an effective antioxidant for protection against nitrate-induced superoxide radical formation in vivo.     

Interactions between cell surface protein disulphide isomerase and S-nitrosoglutathione during nitric oxide delivery.

In this study, we investigated the role of protein disulphide isomerase (PDI) in rapid metabolism of S-nitrosoglutathione (GSNO) and S-nitrosoalbumin (albSNO) and in NO delivery from these compounds into cells. Incubation of GSNO or albSNO (1 microM) with the megakaryocyte cell line MEG-01 resulted in a cell-mediated removal of each compound which was inhibited by blocking cell surface thiols with 5,5'-dithiobis 2-nitrobenzoic acid (DTNB) (100 microM) or inhibiting PDI with bacitracin (5mM). GSNO, but not albSNO, rapidly inhibited platelet aggregation and stimulated cyclic GMP (cGMP) accumulation (used as a measure of intracellular NO entry). cGMP accumulation in response to GSNO (1 microM) was inhibited by MEG-01 treatment with bacitracin or DTNB, suggesting a role for PDI and surface thiols in NO delivery. PDI activity was present in MEG-01 conditioned medium, and was inhibited by high concentrations of GSNO (500 microM). A number of cell surface thiol-containing proteins were labelled using the impermeable thiol specific probe 3-(N-maleimido-propionyl) biocytin (MPB). Pretreatment of cells with GSNO resulted in a loss of thiol reactivity on some but not all proteins, suggesting selective cell surface thiol modification. Immunoprecipitation experiments showed that GSNO caused a concentration-dependent loss of thiol reactivity of PDI. Our data indicate that PDI is involved in both rapid metabolism of GSNO and intracellular NO delivery and that during this process PDI is itself altered by thiol modification. In contrast, the relevance of PDI-mediated albSNO metabolism to NO signalling is uncertain.     

Multiple potassium channels mediate nitric oxide-induced inhibition of rat vascular smooth muscle cell proliferation.

Several nitric oxide (NO) effects in the cardiovascular system are mediated by soluble guanylate cyclase (sGC) activation but potassium channels (KC) are also emerging as important effectors of NO actions. We investigated the relationship among vascular smooth muscle cell proliferation, NO, cyclic GMP, and KC using the A7r5 smooth muscle cell line derived from rat aorta. NO donors (two nitrosothiols, S-nitroso-acetyl-d,l-penicillamine, SNAP, and S-nitroso-glutathione, GSNO, and an organic nitrate, glyceryl trinitrate, GTN; 1-1000 microM) dose-dependently inhibited cell proliferation. ODQ (a selective inhibitor of sGC; 0.1 and 1 microM) and KT5823 (a selective inhibitor of cGMP-dependent protein kinase, 1 microM) prevented NO effects, confirming that sGC is a key target. In this report, we show that tetraethylammonium (TEA, a non-selective blocker of KC, 300 microM), and 4-aminopyridine (a selective blocker of voltage-dependent KC, 100 microM) prevented SNAP inhibitory effects on cell proliferation, whereas glibenclamide (a selective blocker of ATP-dependent KC, 1 microM) was ineffective. Iberiotoxin (a selective blocker of high conductance calcium-activated KC, 100 nM), as well charybdotoxin (a blocker of high and intermediate conductance calcium-activated KC, 100 nM) and apamine (a selective blocker of small conductance calcium-activated KC, 100 nM), blocked the antiproliferative effect induced by SNAP. NS1619 (an opener of high conductance calcium-activated KC, 1-100 microM), inhibited cell proliferation. In addition, sub-effective concentrations of ODQ (100 nM) and TEA (10 microM) synergized in blocking SNAP antiproliferative effects. Thus, voltage-dependent and calcium-activated but not ATP-dependent KC appear to have a prominent role, besides sGC activation, in NO-induced inhibition of vascular smooth muscle cell proliferation.     

Effect of in vivo nitrate tolerance on hypersensitivity to NO donors after NO-synthase blockade

Animals treated with nitric oxide synthase (NOS) inhibitors exhibit marked hypersensitivity to the blood pressure lowering effects of exogenous nitric oxide (NO) donors. We used this model as a sensitive index to evaluate the relative importance of reduced biotransformation of glyceryl trinitrate (GTN) to NO in the development of nitrate tolerance. NOS-blockade hypertension using N(G)-nitro-L-arginine methyl ester (L-NAME) caused a marked enhancement of the mean arterial pressure (MAP) decrease mediated by GTN in nontolerant rats. However, even large doses of GTN were unable to change the MAP in GTN-tolerant, NOS-blockade hypertensive animals. In contrast, the MAP responses to the spontaneous NO donor sodium nitroprusside (SNP) were completely unaltered in either tolerant rats or tolerant NOS-blockade hypertensive animals, indicating that NO-dependent vasodilatory mechanisms remain intact despite the development of GTN tolerance. The MAP-lowering effects of GTN in NOS-blockade hypertensive animals were restored 48 h after cessation of chronic GTN exposure. These alterations in the pharmacodynamic response to GTN during tolerance development and reversal were associated with parallel changes in the pattern of GTN metabolite formation, suggesting that the activity of one or more enzymes involved in nitrate metabolism was altered as a consequence of chronic GTN exposure. These findings suggest that the vasodilation resulting from the vascular biotransformation of GTN to NO (or a closely related species) is severely compromised in nitrate-tolerant animals, and that although other mechanisms may contribute to the vascular changes observed following the development of GTN tolerance, decreased GTN bioactivation is likely the most important.     

Substance P increases cyclic GMP levels on coronary postcapillary venular endothelial cells

The vasodilating effect of substance P (SP) at the microvascular level is endothelium-dependent. In the present study we evaluated whether SP activates nitric oxide (NO) production by venular endothelial cell. We evaluated NO activation by measuring cyclic GMP levels in cultured endothelial cells isolated from coronary postcapillary venules of bovine origin (CVEC). Our results indicate that 5 min exposure of CVEC to 10 nM SP doubled basal cyclic GMP levels. Cell treatment with the NO synthase inhibitor L-NMMA reduced the basal levels of cyclic GMP and abolished the effect of SP but did not modify the increase in cyclic GMP in response to exogenous NO. These data indicate that a) microvascular endothelium responds in an autocrine fashion to NO with increased cyclic GMP levels, b) SP activates cyclic GMP pathway through NO production.     

Role of Intercellular and Intracellular Communication by Nitric Oxide in Coupling of Muscarinic Receptors to Activation of Guanylate Cyclase in Neuronal Cells

Abstract: Muscarinic receptor-mediated cyclic GMP formation and release of nitric oxide (NO) (or a precursor thereof) were compared in mouse neuroblastoma N1E-115 cells. [³H]Cyclic GMP was assayed in cells prelabeled with [³H]guanine. Release of NO upon the addition of muscarinic agonists to unlabeled neuroblastoma cells (NO donor cells) was quantitated indirectly by its ability to increase the [³H]cyclic GMP level in labeled cells whose muscarinic receptors were inactivated by irreversible alkylation (NO detector cells). Carbachol increased NO release in a concentration-dependent manner, with half-maximal stimulation at 173 μ M (compared to 96 μ M for direct activation of cyclic GMP formation). The maximal effect of carbachol in stimulating release of NO when measured indirectly was lower than that in elevating [³H]cyclic GMP directly in donor cells. Hemoglobin was more effective in blocking the actions of released NO than in attenuating direct stimulation of [³H]cyclic GMP synthesis. There was a good correlation between the ability of a series of muscarinic agonists to release NO or to activate [³H]cyclic GMP formation directly, and the potency of pirenzepine in inhibiting the two responses. Furthermore, there was a similar magnitude of desensitization of both responses by prolonged receptor activation or stimulation of protein kinase C. NO release was also regulated in relation to the cellular growth phase. A model is proposed in which a fraction of NO generated upon receptor activation does not diffuse extracellularly and stimulates cyclic GMP synthesis within the same cell where it is formed (locally acting NO). The remainder of NO that is extruded extracellularly might travel to neighboring cells (neurotransmitter NO) or might be taken back into the cells of origin (homing NO).     

Evaluation of the pentose phosphate pathway from 14CO2 data. Fallibility of a classic equation when applied to non-homogeneous tissues.

Two cyclic nucleotide phosphodiesterase (PDE) activities were identified in pig aortic endothelial cells, a cyclic GMP-stimulated PDE and a cyclic AMP PDE. Cyclic GMP-stimulated PDE had Km values of 367 microM for cyclic AMP and 24 microM for cyclic GMP, and low concentrations (1 microM) of cyclic GMP increased the affinity of the enzyme for cyclic AMP (Km = 13 microM) without changing the Vmax. This isoenzyme was inhibited by trequinsin [IC50 (concn. giving 50% inhibition of substrate hydrolysis) = 0.6 microM for cyclic AMP hydrolysis in the presence of cyclic GMP; IC50 = 0.6 microM for cyclic GMP hydrolysis] and dipyridamole (IC50 = 5 microM for cyclic AMP hydrolysis in the presence of cyclic GMP; IC50 = 3 microM for cyclic GMP hydrolysis). Cyclic AMP PDE exhibited a Km of 2 microM for cyclic AMP and did not hydrolyse cyclic GMP. This activity was inhibited by trequinsin (IC50 = 0.2 microM), dipyridamole (IC50 = 6 microM) and, selectively, by rolipram (IC50 = 3 microM). Inhibitors of cyclic GMP PDE (M&B 22948) and of low Km (Type III) cyclic AMP PDE (SK&F 94120) only weakly inhibited the two endothelial PDEs. Incubation of intact cells with trequinsin and dipyridamole induced large increases in cyclic GMP, which were completely blocked by LY-83583. Rolipram, SK&F 94120 and M&B 22948 did not significantly influence cyclic GMP accumulation. Dipyridamole enhanced the increase in cyclic GMP induced by sodium nitroprusside. Cyclic AMP accumulation was stimulated by dipyridamole and trequinsin with and without forskolin. Rolipram, although without effect alone, increased cyclic AMP in the presence of forskolin, whereas M&B 22948 and SK&F 94120 had no effects on resting or forskolin-stimulated levels. These results suggest that cyclic GMP-stimulated PDE regulates cyclic GMP levels and that both endothelial PDE isoenzymes contribute to the control of cyclic AMP.     

Nitric oxide can differentially modulate striatal neurotransmitter concentrations via soluble guanylate cyclase and peroxynitrite formation

In vivo microdialysis was used to investigate whether nitric oxide (NO) modulates striatal neurotransmitter release in the rat through inducing cyclic GMP formation via soluble guanylate cyclase or formation of peroxynitrite (ONOO(-)). When NO donors, S-nitroso-N-acetyl-DL-penicillamine (SNAP; 1 mM) or (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1, 2-diolate (NOC-18; 1 mM), were retrodialysed for 15 min, acetylcholine (ACh), serotonin (5-HT), glutamate (Glu), gamma-aminobutyric acid (GABA), and taurine levels were significantly increased, whereas those of dopamine (DA), dihydroxyphenylacetic acid (DOPAC), and 5-hydroxyindoleacetic acid (5-HIAA) were decreased. Only effects on ACh, 5-HT, and GABA showed calcium dependency. Inhibition of soluble guanylate cyclase by 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one (ODQ; 100 and 200 microM) dose-dependently reduced NO donor-evoked increases in ACh, 5-HT, Glu, and GABA levels. Coperfusion of SNAP or NOC-18 with an ONOO(-) scavenger, L-cysteine (10 mM) resulted in enhanced concentrations of Glu and GABA. On the other hand, DA concentrations increased rather than decreased, and no reductions in DOPAC and 5-HIAA occurred. This increase in DA and the potentiation of Glu and GABA were calcium-dependent and prevented by ODQ. Similar to NO, infusions of ONOO(-) (10 or 100 microM) decreased DA, DOPAC, and 5-HIAA. Overall, these results demonstrate that NO increases ACh, 5-HT, Glu, and GABA levels primarily through a cyclic GMP-dependent mechanism. For DA, DOPAC, and 5-HIAA, effects are determined by levels of ONOO(-) stimulated by NO donors. When these are high, they effectively reduce extracellular concentrations through oxidation. When they are low, DA concentrations are increased in a cyclic GMP-dependent manner and may act to facilitate Glu and GABA release further. Thus, changes in brain levels of antioxidants, and the altered ability of NO to stimulate cyclic GMP formation during ageing, or neurodegenerative pathologies, may particularly impact on the functional consequences of NO on striatal dopaminergic and glutamatergic function.     

Phosphodiesterase type 5 inhibitor sildenafil citrate does not potentiate the vasodilative properties of nebivolol in rat aorta

BACKGROUND: Sildenafil citrate (SIL) is contraindicated in patients with coronary heart disease who are treated with nitric oxide (NO) donators such as organic nitrates, as it potentiates NO-mediated vasodilation. The present study investigated whether SIL also affects the vasodilatory effects of nebivolol (NEB), a selective beta1-adrenoceptor blocker with an additional, endothelium-dependent NO-liberating property, in comparison to the combination SIL/glycerol trinitrate (GTN). METHODS AND RESULTS: Experiments were performed in isolated vessel rings of rat aorta (Wistar rats, 8-12 weeks), which had been pre-contracted with phenylephrine (10(-5) M). Isometric tension was measured by a force transducer, and cumulative concentration-response curves were obtained for each drug. The rank order of vasodilatory potency as measured by the concentration needed to achieve 50% relaxation (EC50) was GTN (0.08 microM) > SIL (1.25 microM) > or = NEB (3.5 microM). In the presence of both therapeutic (1 nM) and high (1 microM) concentrations of SIL, vasodilation of GTN was potentiated as indicated by a significant increase in vasodilatory potency (EC50 GTN + low SIL: 0.019 microM, EC50 GTN + high SIL: 0.002 microM; both P < 0.01 vs. GTN). In contrast, SIL did not potentiate the vasodilatory effect of NEB (EC50 NEB + low SIL: 5.01 microM, EC50 NEB + high SIL: 3.2 microM; n.s. vs. NEB). CONCLUSIONS: These data demonstrate that SIL does not potentiate NEB-induced vasodilation in vitro. These findings indicate that the interaction between SIL and NO-donators/organic nitrates does not apply to the NO-liberating properties of NEB. Our findings suggest that SIL may safely be used in hypertensive patients treated with NEB.     

Stimulation of Cyclic GMP Production via a Nitrosyl Factor in Sensory Neuronal Cultures by Algesic or Inflammatory Agents

Abstract: Capsaicin stimulates cyclic GMP production via nitric oxide (NO) (or another nitrosyl factor) in dorsal root ganglion (DRG) neurons maintained in culture. The purpose of the present study was to characterize further capsaicin stimulation of cyclic GMP production in DRG cells maintained in culture, investigate other algesic and/or inflammatory agents for effects on cyclic GMP production, and examine cells responsible for NO production and cyclic GMP production. Capsaicin stimulation of cyclic GMP production in DRG cells was dose dependent, receptor mediated, and attenuated by hemoglobin. Prostaglandin E₂, substance P, and calcitonin gene-related peptide did not affect basal, capsaicin-stimulated, or bradykinin-stimulated cyclic GMP production. Other inflammatory or algesic agents, including serotonin, histamine, ATP, glutamate, aspartate, and NMDA, did not affect cyclic GMP production. Pretreatment of DRG cells with lipopolysaccharide increased basal cyclic GMP production in neuronal but not in nonneuronal cultures and facilitated stimulation of cyclic GMP production by l -arginine. Capsaicin pretreatment of neuronal DRG cultures, which destroys capsaicin-sensitive (small diameter) afferent neurons, attenuated capsaicin- and bradykinin-stimulated cyclic GMP production but did not affect basal or sodium nitroprusside-stimulated cyclic GMP production. These results indicate that capsaicin elicits production of a nitrosyl factor via capsaicin-sensitive (small diameter) neurons. Capsaicin evoked cyclic GMP production in nonneuronal DRG cultures in the presence but not in the absence of apposed neuronal DRG cultures. Overall, these findings suggest that specific exogenous (or endogenous) substances may stimulate production of a nitrosyl factor(s) by a subset of DRG neurons, and nitrosyl factors produced by these neurons may affect cyclic GMP production in neighboring neuronal or non-neuronal cells.     

Arginine-Modulated Receptor-Activated Calcium Influx via a NO/Cyclic GMP Pathway in Human SK-N-SH Neuroblastoma Cells

Abstract: The effects of arginine on calcium mobilization in human SK-N-SH neuroblastoma cells were examined. It was found that arginine potentiated an increase in carbachol-induced Ca²⁺ from the external Ca²⁺ influx as opposed to an internal Ca²⁺ release from intracellular pools. The potentiation effect of arginine on carbachol-induced calcium mobilization was mimicked by either 8-bromo cyclic GMP or sodium nitroprusside. In addition, it was found that arginine induced NO production and an increase in cyclic GMP. Moreover, arginine-induced potentiation, NO production, and cyclic GMP increases were all suppressed after the preincubation of cells with N -methyl- l -arginine or N -nitro- l -arginine, nitric oxide synthase inhibitor. It is suggested that the NO production and subsequent cyclic GMP elevation induced by arginine are responsible for the potentiation of carbachol-induced Ca²⁺ increase. Our results show the existence of a NO/cyclic GMP pathway and an interconnection of NO and Ca²⁺ signaling pathways in human SK-N-SH neuroblastoma cells. We also observed that NO, which is produced by endothelial CPAE cells, has a modulating effect on cyclic GMP elevation in human SK-N-SH neuroblastoma cells. The intercellular communication role of NO and its cell-diffusing character may also affect the regulation of nonneuronal cells in their interactions with neuronal cells.     

Adaptive resistance to nitric oxide in motor neurons

Nitric oxide (NO) is a free radical produced actively by mammalian cells, including neurons. Low levels of NO can function in intercellular signaling, but high levels are cytotoxic. This cytotoxic potential suggests that cells at risk for NO damage, such as neurons, might have NO resistance mechanisms to prevent cell death, and adaptive resistance to NO-releasing compounds has been reported for some non-neuronal cell types. Here we show that immortalized mouse motor neurons (NSC34 cells) respond to sub-lethal fluxes of pure NO by activating adaptive resistance mechanisms that counteract cytotoxic NO exposure. This adaptive NO resistance is reversible and is paralleled by the induction of the oxidative stress enzyme heme oxygenase 1 (HO-1). An inhibitor of both HO-1 and heme-dependent guanylate cyclase (tin-protoporphyrin IX) greatly sensitized NO-pretreated NSC34 cells to the NO challenge. However, readdition of cyclic GMP (in the form of the 8-bromo derivative) restored rather little resistance, and a more selective guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxaline-1-one (at 10 microM), did not have the sensitizing effect. Therefore, the inducible HO-1 pathway contributes substantially to adaptive NO resistance, while cyclic GMP seems to play at most a small role. A similar adaptive resistance to NO was observed in primary rat spinal chord motor neurons. The activation of NO resistance in motor neurons may counteract age- or disease-related neurodegeneration.     

YC-1 enhances the responsiveness of tolerant vascular smooth muscle to glyceryl trinitrate

A major limitation of the use of organic nitrates in cardiovascular medicine is the development of tolerance, which has been attributed, in part, to a decrease in their metabolic activation in the vascular smooth muscle cell. Recently, 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) was shown to potentiate vascular smooth muscle responsiveness to glyceryl trinitrate (GTN), sodium nitroprusside, and the nitric oxide donor NOC 18, in organic nitrate-naive vascular smooth muscle. We used GTN-tolerant rabbit aortic rings (RARs) to test the hypothesis that a non-vasorelaxant concentration of YC-1 enhances the ability of the prototypical organic nitrate GTN to relax vascular smooth muscle and elevate intravascular cGMP under conditions of GTN tolerance. Treatment with YC-1 (3 microM) produced a left shift of the GTN concentration-response curve and decreased the EC50 value for GTN-induced relaxation in both GTN-tolerant and non-tolerant RARs (P < 0.05). Intravascular cGMP elevation induced by GTN was enhanced in the presence of YC-1 in GTN-tolerant and non-tolerant RARs (P < 0.05). These observations indicate that YC-1, or similarly acting drugs, may be useful in overcoming the tolerance that develops during sustained GTN therapy, and that its mechanism may involve enhanced cGMP formation.