The role of cGMP in the relaxation to nitric oxide donors in airway smooth muscle

With real-time confocal microscopy, the effect of three nitric oxide (NO) donors, S-nitroso-N-acetylpenicillamine, S-nitrosoglutathione, and diethylamine NO adduct, on the dynamic intracellular Ca2+ concentration ([Ca2+]i) response of porcine tracheal smooth muscle (TSM) cells to acetylcholine (ACh) was examined. ACh initiated propagating [Ca2+]i oscillations in TSM cells, which were inhibited by NO donors. 8-Bromoguanosine 3',5'-cyclic monophosphate slowed the frequency of [Ca2+]i oscillations but did not completely inhibit oscillations, suggesting that the effects of NO donors are only partially mediated via guanosine 3',5'-cyclic monophosphate-dependent mechanisms. After preexposure to NO donors, ACh induced a small biphasic [Ca2+]i response that was blocked by nifedipine, suggesting a lack of effect on Ca2+ influx through voltage-gated channels. In addition, NO donors did not inhibit Ca2+ influx induced by BAY K 8644. The [Ca2+]i response to caffeine was inhibited by NO donors, indicating inhibition of sarcoplasmic reticulum (SR) Ca2+ release. When Ca2+ influx and SR Ca2+ reuptake were blocked, basal [Ca2+]i increased, and this was inhibited by NO donors, suggesting enhanced Ca2+ efflux. These results indicate that NO donors inhibit [Ca2+]i oscillations by blocking SR Ca2+ release and enhancing Ca2+ extrusion.

should decrease pulmonary artery pressure (PAP) and pulmonary vascular resistance (PVR), without affecting systemic arterial pressure (SAP), and potentially improve oxygenation by redistributing pulmonary blood flow to ventilated areas of lung. INO (INO Therapeutics Inc., Clinton NJ) possesses these properties and has gained approval from the Food and Drug Administration (FDA) for the care of neonates with acute lung injury and pulmonary hypertension (PH), and widespread clinical acceptance (but not FDA approval) for adults with PH with or without lung injury. Delivered as a gas, INO is preferentially distributed to the ventilated areas of the lung, where it produces relaxation of pulmonary vascular smooth muscle via activation of guanylate cyclase and the conversion of guanosine-5-triphosphate to cyclic guanosine monophosphate. 1 Absorbed INO is rapidly inactivated by hemoglobin, thereby preventing systemic effects and confining its vasodilator properties to the pulmonary circulation. 1 The search for inhaled selective pulmonary vasodilators was an active area of research, particularly in Europe and Australia, before the widespread publicity and testing of INO in the early 1990s. However, research on this subject appears to have declined inversely with the growing acceptance and use of INO. Until FDA approval had been granted, INO had been supplied free of charge in the United States on an investigational-drug basis. However, after FDA approval, the cost of treatment with INO became very expensive. This prompted a search at our institution for alternative agents to INO. The purpose of this article is to review the published experience concerning alternative inhaled vasodilators and, when possible, compare their reported efficacy with that of INO.

Nitric oxide (NO) acts as a neurotransmitter and neuromodulator in the nervous system of many vertebrates and invertebrates. The effects of extracellularly applied sodium nitroprusside (SNP) and diethylamine NO (C(2)H(5))(2)N[N(O)NO]-Na(+) (DEA/NO), NO donors, on a glutamate (Glu)-induced K(+) current in identified Onchidium neurons were investigated using voltage clamp and pressure ejection techniques. Bath-applied SNP (10 microM) and DEA/NO (5-10 microM) reduced the Glu-induced K(+) current without affecting the resting membrane conductance and holding current. The Glu-induced K(+) current also was inhibited by the focal application of SNP to the neuron somata. The suppressing effects of NO donors were concentration-dependent and completely reversible. Pretreatment with hemoglobin (50 microM), a nitric oxide scavenger, and 1H-[1,2, 4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 1 microM), a specific inhibitor of NO-stimulated guanylate cyclase, decreased the SNP-induced inhibition of the Glu-induced current. Bath-applied 50 microM 3-isobutyl-1-methylxanthine (IBMX), a nonspecific phosphodiesterase inhibitor, or intracellular injection of 1 mM guanosine 3',5'-cyclic monophosphate (cGMP) inhibited the Glu-induced current, mimicking the effect of NO donors. These results demonstrate that SNP and DEA/NO inhibit the Glu-induced K(+) current and that the mechanism of NO inhibition of the Glu-induced current involves cGMP-dependent protein kinase.

Th2 lymphocytes differ from other CD4+ T lymphocytes not only by their effector tasks but also by their T cell receptor (TCR)-dependent signaling pathways. We previously showed that dihydropyridine receptors (DHPR) involved in TCR-induced calcium inflow were selectively expressed in Th2 cells. In this report, we studied whether cGMP-dependent protein kinase G (PKG) activation was implicated in the regulation of DHPR-dependent calcium response and cytokine production in Th2 lymphocytes. The contribution of cGMP in Th2 signaling was supported by the following results: 1) TCR activation elicited cGMP production, which triggered calcium increase responsible for nuclear factor of activated T cell translocation and Il4 gene expression; 2) guanylate cyclase activation by nitric oxide donors increased intracellular cGMP concentration and induced calcium inflow and IL-4 production; 3) reciprocally, guanylate cyclase inhibition reduced calcium response and Th2 cytokine production associated with TCR activation. In addition, DHPR blockade abolished cGMP-induced [Ca2+]i increase, indicating that TCR-induced DHP-sensitive calcium inflow is dependent on cGMP in Th2 cells. Th2 lymphocytes from PKG1-deficient mice displayed impaired calcium signaling and IL-4 production, as did wild-type Th2 cells treated with PKG inhibitors. Altogether, our data indicate that, in Th2 cells, cGMP is produced upon TCR engagement and activates PKG, which controls DHP-sensitive calcium inflow and Th2 cytokine production.

Comparison of the mechanisms underlying the relaxation induced by two nitric oxide donors: Sodium nitroprusside and a new ruthenium complex

Nitric Oxide

Nitric oxide: physiology and pharmacology.

it has long been known that release of intracellular stored Ca [presumably in the sarcoplasmic reticulum (SR)] plays a major role in initiating contraction in airway smooth muscle (ASM) from a variety of species ([6][1]) including human ([17][2]). Therefore, the issue of how extracellular calcium

To compare the effects of nitric oxide (NO) donors, diethylamine/nitric oxide (DEA/NO) and nitroglycerin (NTG), on isolated uterine and aortic tissues from non-pregnant, mid and late pregnant rats. The uterus and thoracic aorta were obtained from non-pregnant (estrous cycle) and pregnant Sprague-Dawley rats on day 14 and day 21. The uterine and aortic rings were incubated in organ chambers filled with Krebs-Henseleit solution bubbled with 5% CO2 in air for isometric tension recordings. Cumulative concentration-response relationships to DEA/NO and NTG were obtained in the aortic rings contracted with phenylephrine and in spontaneously contracting uterine rings. The sensitivity and the maximal inhibitory effects of DEA/NO did not differ in aortic tissues of any group. DEA/NO-induced Maximal inhibition of spontaneous contractions of uterine tissues from mid-pregnant rats was greater (although not significantly) than in the tissues from non-pregnant animals (with similar sensitivity), but it was significantly depressed in tissues from late pregnant rats. The sensitivity to and maximal inhibitory effects of NTG were less in aortic tissues from late pregnant versus mid-pregnant and non-pregnant rats. In uterine tissues from late pregnant rats the effect of NTG was negligible. The inhibitory action of both NO donors was much more pronounced in aortic versus uterine tissues. Uterine smooth muscle is less sensitive than vascular smooth muscle to NO. Uterine smooth muscle from late pregnant animals demonstrates refractoriness to both DEA/NO and NTG, while vascular smooth muscle from late pregnant animals demonstrates refractoriness to NTG, but not to DEA/NO.

Segmental heterogeneity in relaxation response to nitric oxide (NO) was examined using NO donor sodium nitroprusside (SNP) in second- (medium) and fourth-generation (small) ovine isolated intralobar pulmonary arteries. In vessels precontracted with serotonin, NO donors SNP and S-nitroso-N-acetylpenicillamine (SNAP) were more potent in relaxing medium, in comparison to the small, arteries. Soluble guanylyl cyclase (sGC) inhibitor [1,2,4]oxadiazolo-[4,3-a]quinoxaline-1-one (ODQ 3 microM) caused a profound inhibition of SNP relaxation in small as compared with medium-sized arteries. However, both basal and SNP (10 microM)-stimulated intracellular cyclic guanosine monophosphate (cGMP) content was identical in these 2 arterial segments. The Na,K-ATPase inhibitor ouabain (1 microM) had a marked inhibitory effect on SNP-mediated relaxation in both segments. There was no segmental difference in SNP (10 microM)-stimulated plasma membrane Na,K-ATPase activity and ouabain-sensitive Rb-uptake. 4-AP (1 mM), a relatively selective inhibitor of Kv channels, decreased the potency of SNP relaxation by about 10-fold in the medium-sized vessels. On the other hand, 4-AP was without effect on the vasodilator potency of SNP in small vessels. Interestingly, in the presence of 4-AP, SNP was equipotent in dilating both medium (pD2 = 5.80 +/- 0.07; Emax = 84 +/- 1.6%, n = 7) and small (pD2 = 5.74 +/- 0.15; Emax = 83 +/- 2.5%, n = 7) pulmonary arteries. In conclusion, the results of the present study suggest that Kv channels determine the segmental heterogeneity of NO-mediated relaxation in ovine pulmonary artery.

1. The membrane conductance changes underlying the membrane hyperpolarizations induced by nitric oxide (NO), S-nitroso-L-cysteine (NC) and sodium nitroprusside (SNP) were investigated in the circular smooth muscle cells of the guinea-pig proximal colon, by use of standard intracellular microelectrode recording techniques. 2. NO (1%), NC (2.5-25 microM) and SNP (1-1000 microM) induced membrane hyperpolarization in a concentration-dependent manner, the hyperpolarizations to NO and NC developing more rapidly than those to SNP. The slower-developing responses to SNP were mimicked by the membrane permeable analogue of guanosine 3':5' cyclic-monophosphate (cyclic GMP), 8-bromo-cyclic GMP (500 microM), and by isoprenaline (10 microM). 3. The hyperpolarizations to NC and SNP were reduced in a low Ca2+ (0.25 mM) saline and upon the addition of haemoglobin (20 microM), but were not effected by NG-nitro-L-arginine (L-NOARG) (100 microM) or omega-conotoxin GVIA (100 nM). the hyperpolarizations to SNP were also significantly reduced by methylene blue (50 microM). 4. Apamin (250 nM) depolarized the membrane potential approximately 10 mV and reduced the initial transient component of the hyperpolarization to NO (1%) and NC (25 microM), but had no effects on the hyperpolarizations to SNP and cyclic GMP. Tetraethylammonium (TEA) (5-15 mM), had little effect on the membrane responses to NO(1%), NC(2.5-25 microM), SNP(100(-1000) microM) or cyclic GMP(500 microM). However, TEA (5-15 mM) reduced the membrane hyperpolarizations to SNP (10 microM) and isoprenaline (10 microM) in a concentration-dependent manner. The hyperpolarization to isoprenaline (10 microM) remaining in the presence of 15 mM TEA was blocked by ouabain (10 microM). 5. The amplitude of electronic potentials (1 s duration) elicited during NO donor hyperpolarizations were little changed or only slightly reduced (5-25%). However, the amplitude of the electrotonic potentials elicited during maintained electrically-induced hyperpolarizations of similar amplitude were significantly increased (30-150%), suggesting that the non-linear membrane properties of the proximal colon partially mask an increase in membrane conductance elicited during the NO donor hyperpolarizations. 6. Membrane hyperpolarization in the presence of an NO donor, 8-bromo-cyclic GMP, isoprenaline, or upon application of a maintained hyperpolarizing electrical current, often evoked oscillations of the membrane potential. These oscillations were prevented by Cs+ (1 mM). 7. These results indicate that NO and NC hyperpolarize the circular muscle of the proximal colon by activating at least two TEA-resistant membrane K+ conductances, one of which is sensitive to apamin blockade. The K+ conductance increases activated by SNP or 8-bromo-cyclic GMP were little effected by apamin, perhaps suggesting a common mechanism. In contrast, the hyperpolarization to isoprenaline appears to involve the activation of TEA-sensitive Ca2(+)-activated K+ ('BK') channels, as well as a Na:K ATPase. Finally, the 'background' membrane conductance of the circular muscle cells of the proximal colon decreased upon membrane hyperpolarization to reveal oscillations of the membrane potential which may well represent 'pacemaker' or 'slow wave' activity.

Calcitonin gene-related peptide-dependent vascular relaxation of rat aorta: An additional mechanism for nitroglycerin

Cytotoxicity of nitric oxide donors in smooth muscle cells is dependent on phenotype, and mainly due to apoptosis

We have examined the mechanisms of action of a broad spectrum of nitric oxide (NO) donors, including several S-nitrosothiols, sodium nitroprusside (SNP) and nitroglycerine (GTN), in relation to their relaxant activity of urethral smooth muscle. For all the compounds examined, NO release (in solution and in the presence of urethral tissue), relaxation responses, elevations in cGMP levels and the effect of thiol modulators were evaluated and compared with the effect of NO itself. Whilst all NO donors, except GTN, released NO in solution due to photolysis or chemical catalysis, this release was not correlated with their relaxant activity in sheep urethral preparations, which were furthermore not affected by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (cPTIO; 0.3 mM). A substantial NO-generating activity was found for S-nitroso-L-cysteine (CysNO) and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) in the presence of urethral cytosolic fractions, suggesting metabolic activation to NO in the cytosol of the target tissue. In contrast, NO generation from S-nitroso-N-acetyl-L-cysteine (N-ac-CysNO), S-nitrosoglutathione (GSNO) and SNP were reduced by the presence of urethral homogenate and/or subcellular fractions, suggesting direct NO transfer to tissue constituents. NO donors and NO gas induced dissimilar degrees of cGMP accumulation in urethral tissue, while they were essentially equipotent as urethral relaxants. Furthermore, 1H-[1,2,4] -oxadiazole-[4,3-a]-quinoxalin-1-one (ODQ; 10 microM) inhibited both relaxation and cGMP accumulations, but with different potency for the different compounds. Oxidation of sarcolemmal thiol groups with 5-5'-dithio-bis[2-nitrobenzoic acid] (DTNB; 0.5 mM) enhanced relaxations to GSNO, an effect that was reversed by dithiotreitol (DTT; 1 mM), suggesting a direct effect through nitrosylation/oxidation reactions at the cell membrane, while relaxations to NO and to all the other compounds were not affected by these treatments. Finally, photodegradation of SNP induced the formation of a stable intermediate that still evoked NO-cGMP-mediated relaxations. This indicates that the assumption that SNP is fully depleted of NO by exposure to light should be revised. It can be concluded that important differences exist in the mechanisms by which distinct NO donors relax urethral smooth muscle and they cannot be regarded simply as NO-releasing prodrugs.

The vascular relaxation induced by nitric oxide (NO) donors is impaired in aortas from renal hypertensive rats (2K-1C). Our hypothesis was that the lower NO effect in aortas from 2K-1C rats could be related with the higher degradation of NO and/or caveolae changes in vascular smooth muscle cells (VSMCs), considering that NO can be rapidly degraded and caveolae seems to play important role in the reduction of cytosolic Ca 2+ concentration [Ca 2+ ]c. The present study aimed to investigate the alterations on aorta relaxation induced by NO in 2K-1C rat aorta. At first, we studied the influence of oxidative stress on the effect of NO released from the NO donors [Ru(NH.NHq)(terpy)NO + ] 3+ (TERPY) and sodium nitroprusside (SNP) in aortas from normotensive (2K) and 2K-1C rats. The relaxation induced by both NO donors was impaired in aortas from 2K-1C rats and the reduction on [Ca 2+ ]c induced by TERPY was also impaired in 2K-1C VSMCs. However, in aortas treated with antioxidants the relaxation to both NO donors and the reduction on [Ca 2+ ]c to TERPY were normalized. The basal concentration of superoxide (O 2 -) was greater in 2K-1C than in 2K, which was reduced by the antioxidants. The basal cytosolic NO concentration ([NO]c) and the NO released from TERPY were lower in aortas from 2K-1C rats. We studied the influence of caveolae on the effects of NO released from the NO donors, in aortas from 2K and 2K-1C rats. We verified that caveolae disassemble with ciclodextrin impaired the relaxation to NO donors and the reduction on [Ca 2+ ]c to TERPY only in aortas from 2K rats. The number of caveolae is reduced in aortic VSMCs and in the endothelial cells from 2K-1C rats. We studied the effect of TERPY on arterial pressure from 2K and 2K-1C rats. TERPY reduced the arterial pressure only in 2K-1C rats, which effect was longer than that produced by SNP. The hypotensive effect of SNP was greater in 2K-1C than in 2K rats. Taken together, our results indicate that the higher concentration of O 2 -and the reduced number of caveolae on aortas from 2K-1C rats could contribute to impaired aorta relaxation of 2K-1C rats.