Lack of effect of nitric oxide on KCl, acetylcholine and substance P induced contractions in ileal longitudinal muscle of the rat

The aim of this study was to determine whether an excess of nitric oxide (NO) (mimicked by addition of NO donors) might produce by itself changes in the contractile responses to acetylcholine (ACh), substance P (SP) and KCl in the longitudinal muscle of the rat ileum. We also studied the calcium handling properties of this tissue in presence of NO donors. The NO donors assayed sodium nitroprusside (SNP) and 3-morpholinosydnonimine hydrochloride (SIN-1), induced different responses. SNP caused an immediate contraction followed by a sustained relaxation, whereas SIN-1 induced an immediate relaxation followed by a contraction. Even after prolonged incubations (up to 90 min), the NO donors SNP and SIN-1 were unable to modify the ACh- and SP-concentration-response curves, as well as the response to 30 mM KCl. The nifedipine-resistant component of the ACh-induced contraction was not modified in presence of SNP. Cyclopiazonic acid (CPA) induced a contraction that was not modified when the tissue was pre-incubated with SNP. Nifedipine caused a sharp relaxation when added during the CPA-induced contraction and, when added previously, it reduced the CPA-induced contractile response. It is concluded that NO excess is not, by itself, responsible for the altered responses to KCl. ACh and SP. The contractility changes observed in the longitudinal muscle of the rat ileum during inflammation could rather be related to the presence of other inflammatory mediators.     

Cyclooxygenase-derived products, rather than nitric oxide, are endothelium-derived relaxing factor(s) in the ventral aorta of carp (Cyprinus carpio)

In some fish blood vessels, the existence of a NO (nitric oxide) system has been reported. We examined the possibility that this NO system acts as an endothelium-derived relaxing factor (EDRF) in carp aorta using the carp aorta alone and in a combined carp-rat aorta donor-detector system. Use of the typical NO stimulating agent in mammal acetylcholine (ACh) only induced constriction of the carp aorta. This response was not modified by denudation or by NO synthesis inhibition with N-nitro-L-arginine methyl ester. Neither the indirect NO stimulating agents bradykinin and histamine nor the direct NO releasers sodium nitroprusside (SNP) and SIN-1 induced vasorelaxation. Both SNP and ACh elevated the cGMP concentration in rat aorta, but not in carp aorta. In the aorta combination set-up, where carp served as a NO donor and rat aorta served as a NO detector, no relaxation of the rat aorta was observed. The calcium ionophore A23187, a known EDRF producer in mammals, induced relaxation of carp aorta through an endothelium- and cyclooxygenase-dependent mechanism. These results indicate that carp aorta does not produce NO as an EDRF nor does it respond to exogenously supplied NO. The major EDRF in carp is apparently a product(s) of cyclooxygenase metabolism.     

Vascular reactivity in intrapulmonary arteries of chicken embryos during transition to ex ovo life

The present study aimed to characterize pulmonary vascular reactivity in the chicken embryo from the last stage of prenatal development and throughout the perinatal period. Isolated intrapulmonary arteries from non-internally pipped embryos at 19 days of incubation and from internally and externally pipped embryos at 21 days of incubation were studied. Arterial diameter and contractile responses to KCl, endothelin-1, and U-46619 increased with incubation but were unaffected by external pipping. In contrast, the contractions induced by norepinephrine, phenylephrine, and electric field stimulation decreased with development. No developmental changes were observed in endothelium-dependent [acetylcholine (ACh) and cyclopiazonic acid] or endothelium-independent [sodium nitroprusside (SNP)] relaxation. These relaxations were abolished by the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. Endothelium-dependent relaxation was unaffected by blockade of cyclooxygenase or heme oxygenase but was significantly reduced by nitric oxide (NO) synthase inhibitors. Reduction of O2 concentration from 95 to 5% produced a marked reduction in ACh and SNP-induced relaxations. Chicken embryo pulmonary arteries show a marked endothelium-dependent relaxation that is unaffected by transition to ex ovo life. Endothelium-derived NO seems to be the main mediator responsible for this relaxation.     

Nitric oxide relaxes rat tail artery smooth muscle by cyclic GMP-independent decrease in calcium sensitivity of myofilaments

The effects of authentic nitric oxide (NO, 10(-6) M) and NO-donors such as sodium nitroprusside (SNP, 10(-5) M) and glyceryl trinitrate (GTN, 10(-4) M) on contractile force and free intracellular calcium level ([Ca2+]i) were studied on precontracted with high potassium chloride (KCl, 70 mM) isolated rings of rat tail artery. The sensitivity of contractile myofilaments to Ca2+ was measured using chemically permeabilized (alpha-toxin, beta-escin, Triton X-100) vascular rings. [Ca2+]i and contractile activity were measured simultaneously. The relationship of [Ca2+]i and tension developed was studied in endothelium-denuded rings and controlled calcium response was evaluated in both endothelium-denuded and permeabilized vascular rings. Both authentic NO and NO-donors decreased [Ca2+]i and high potassium-induced tension with a different time course. Inhibitor of soluble guanylyl cyclase (sGC) LY83583 (10(-5) M) did not affect SNP-induced relaxation whereas the other sGC inhibitor ODQ (10(-6) M) attenuated SNP-induced relaxation. Both inhibitors had no effect on NO- and SNP-induced reduction in [Ca2+]i. On the contrary, GTN induced neither relaxation nor decrease in [Ca2+]i on application of both LY83583 and ODQ. Tail artery rings permeabilized with alpha-toxin, beta-escin, but not with Triton X-100 were relaxed by authentic NO and NO-donors, but to a less extent than non-permeabilized rings. Dithioerythritol (DTE, 5 x 10(-3) M) that maintains sulfhydryl (SH) groups in reduced state preventing their nitrosylation attenuated NO-induced relaxation in both non-permeabilized and permeabilized tail artery rings. The cyclic heptapeptide mycrocystin-LR (MC-LR) (10(-5) M), an inhibitor of type 1 and 2A phosphatases, induced sustained increase in tension of beta-escin permeabilized rings in low Ca2+ (10(-8) M) solution. The tension was not affected by authentic NO and SNP. We conclude that authentic NO and SNP relax rat tail artery smooth muscle (SM) in the presence of inhibitors of sGC via cyclic guanosine monophosphate (cGMP)-independent pathway, whereas relaxation induced by GTN is inhibited. The data demonstrate that cGMP-dependent pathway in vascular smooth muscle is ubiquitous, but not the only way of relaxation induced by NO. NO can modulate vascular tone directly by reducing sensitivity of contractile myofilaments to [Ca2+]i and may involve activation of protein phosphatase(s).     

The complex trans-[RuCl([15]aneN4NO]2+ induces rat aorta relaxation by ultraviolet light irradiation.

The aim of the present study was to investigate the possible endogenous storage of photosensitive nitric oxide, and also to examine the relaxant effect of NO released from the compound by UV light irradiation. Aorta was isolated from rats and the endothelium was mechanically removed. Denuded aortic rings pre-contracted with prostaglandin F(2alpha) responded with relaxation to UV light irradiation. The first stimulation produced the greatest response that decreased until complete disappearance. After this, the addition of the compound in the absence of light did not produce any response. However, in the presence of UV light irradiation, the complex trans-[RuCl([15]aneN4)NO]2+ induced 100% relaxation. After incubation with the nitric oxide scavenger, oxyhaemoglobin, this relaxation was completely abolished. In PGF2(2alpha)-pre-contracted aortas, the time to reach maximum relaxation was only 50s. Taken together, these results suggest that preformed endogenous nitric oxide stores exist in the denuded rat aorta, and that they are sensitive to UV light. The photo-induction of the complex trans-[RuCl([15]aneN4NO]2+ induces complete aorta relaxation, which is due to release of nitric oxide in the extracellular medium.     

Effect of L-arginine on the relaxation caused by sodium nitroprusside on isolated rat renal artery

In the present study we investigated the mechanism of nitric oxide induced relaxation of renal arteries, with or without endothelium, taken from normotensive and spontaneously hypertensive (SH) rats. With this purpose in mind, the effects of the nitric oxide donor, sodium nitroprusside (SNP), with and without L-arg in the medium, on isolated rat renal artery relaxation were studied. Relaxing effect of SNP was higher in normotensive (10(-5) M of SNP caused 220% of relaxation in the cases with endothelium and 240% without endothelium), in comparison with SH rats (100% of relaxation with endothelium and 150% without). L-arg antagonized the relaxing effect of SNP in the examined renal arteries, more in normotensive (100-160% with endothelium and 110-195% without) than in hypertensive ones (0-10% with endothelium and 35-75% without) at SNP concentrations 10(-7) - 10(-5) M, respectively (*P < 0.05; **P < 0.001). L-arg did not significantly change relaxing effect of SNP in the isolated renal arteries with endothelium taken from SH rats, which show that L-arg, by modifying the chemical versatility of NO into redox active forms -nitrosonium (NO+) and -nitroxyl (NO-), produces different relaxing effects in normotensive and hypertensive isolated arteries of rats, with or without endothelium, potentiating the role of nitroxyl induced relaxation in SH rats.     

NO-induced modulation of calcium-oscillations in pulmonary vascular smooth muscle

The effect of the nitric oxide (NO) donor sodium nitroprusside (SNP) on both [Ca(2+)](i)and mechanical activity was studied in the rat isolated pulmonary artery (RPA). In freshly isolated myocytes loaded with 1 microM indo-lacetoxymethyl ester for 30 min, short (40-60 s) application of ATP (100 microM) or ET-1 (0.1 microM) induced 3-6 cyclic rises in [Ca(2+)](i)(Ca-oscillations) of decreasing amplitude. Preincubation of cells with SNP (10-250 microM) for 10 min had no effect on the resting [Ca(2+)](i)value, but progressively abolished the oscillations. A similar effect was obtained with 8-bromo-cGMP (100-500 microM). SNP (0.001-100 microM) concentration-dependently relaxed ATP (10 mM, n = 4) and ET-1 (0.1 microM, n = 4)-precontracted RPA. 1H-[1,2,4]oxadiazolol [4,3,-a]quinoxalin-1-one (ODQ, 10 microM), a potent inhibitor of the cytosolic guanylyl cyclase, fully reversed the effect of SNP on ATP-induced [Ca(2+)](i)oscillations as well as on ATP-precontracted RPA. In contrast, N-[2-(methylamino)ethyl]-5-isoquinolinesulfonamide (H8, 10 microM), a potent inhibitor of cGMP-dependent protein kinase (PKG), did not alter the effect of SNP. Caffeine (5 mM) induced only one transient [Ca(2+)](i)-increase (n = 24), the amplitude of which was altered neither by SNP nor by 8-bromo-cGMP. Our results show that the relaxing effect of NO in RPA is related, at least in part, to its action on the Ca-signalling pathway. NO interacts with inositol trisphosphate pathway without interacting with the ryanodine-sensitive receptor. Finally, the effect of NO involves an increase in cGMP but appears independent of activation of PKG.     

Actions of NO donors and endogenous nitrergic transmitter on the longitudinal muscle of rat ileum in vitro: mechanisms involved

The aim of this work has been to characterize and to compare the responses of the rat ileal longitudinal muscle to the nitric oxide (NO) donors, sodium nitroprusside (SNP) and morpholinosydnonimine hydrochloride (SIN-1). SNP (10(-5)-10(-3) M) caused a contraction followed by a relaxation, both components being concentration-dependent. In contrast, SIN-1 (10(-5)-10(-4) M) caused a relaxation followed by a contraction. Neither the neural blocker tetrodotoxin (TTX) nor atropine were able to change the response to SNP, whereas nifedipine abolished its contractile component. In contrast, TTX and nifedipine diminished both the relaxation and the contraction in response to SIN-1, whereas atropine decreased only the contractile component. The specific guanylate cyclase inhibitor oxadiazolo-quinoxalin-1-one (ODQ) decreased the relaxation induced by SNP but did not modify that caused by SIN-1. The K+ channel blockers charybdotoxin, apamin and tetraethylamonium were unable to modify the response to SNP. In contrast, both TEA and apamin significantly decreased the relaxation induced by SIN- 1. The relaxation resulting from electrical field stimulation (EFS) of enteric nerves in non-adrenergic non-cholinergic conditions is mainly but not exclusively nitrergic, as incubation with the NO synthase inhibitor L-NNA markedly decreases such relaxation. EFS-induced relaxation is also sensitive to ODQ. We conclude that SNP acts mainly on smooth muscle cells activating L-type Ca2+ channels, which result in contraction, and activates the soluble guanylate cyclase, which results in relaxation. In contrast SIN-1 has mixed--neuronal and muscular--effects, the contraction being caused both by acetylcholine release from neurons and by direct activation of L-type Ca2+ channels on smooth muscle cells. SIN-1-induced relaxation is cGMP-independent and it is likely to occur as a consequence of both, neuronal release of inhibitory transmitter(s) and by activation of apamin sensitive K+ channels. The effect of the nitrergic transmitter released from enteric nerves is different from those caused by SIN-1 but shows similarities with those caused by SNP.     

Hypotensive and vasorelaxing effects of the new NO-donor [Ru(terpy)(bdq)NO(+)](3+) in spontaneously hypertensive rats

Drugs that release nitric oxide (NO) usually have limitations due to their harmful effects. Sodium nitroprusside (SNP) induces a rapid hypotension that leads to reflex tachycardia, which could be an undesirable effect in patients with heart disease, a common feature of hypertension. The nitrosyl ruthenium complex [Ru(terpy)(bdq)NO(+)](3+) (TERPY) is a NO donor that is less potent than SNP in denuded aortic rings. This study evaluated the hypotension and vasorelaxation induced by this NO donor in Wistar (W) and spontaneously hypertensive rats (SHR) and compared to the results obtained with SNP. Differently from the hypotension induced by SNP, the action of TERPY was slow, long lasting and it did not lead to reflex tachycardia in both groups. The hypotension induced by the NO-donors was more potent in SHR than in W. TERPY induced relaxation with similar efficacy to SNP, although its potency is lower in both strains. The relaxation induced by TERPY is similar in W and SHR, but SNP is more potent and efficient in SHR. The relaxation induced by TERPY is partially dependent on guanylate cyclase in SHR aorta. The NO released from the NO donors measured with DAF-2 DA by confocal microscopy shows that TERPY releases similar amounts of NO in W and SHR, while SNP releases more NO in SHR aortic rings.     

Role of phospholamban in NO/EDRF-induced relaxation in rat aorta.

The role of endothelium-derived nitric oxide (NO) to cause smooth muscle phospholamban (PLB) phosphorylation was studied in the isolated perfused rat aorta precontracted with norepinephrine using a back-phosphorylation technique. NO-induced relaxation was associated with increased PLB-phosphorylation while norepinephrine as such was ineffective. Removal of endothelium significantly reduced PLB-phosphorylation in indomethacin treated vessels. Stimulation of NO-formation by ATP augmented PLB-phosphorylation in intact vessels but was ineffective in denuded aortas. The results indicate that PLB-phosphorylation of vascular smooth muscle plays an important role in mediating NO-dependent relaxation by enhancing Ca(++)-uptake into sarcoplasmic reticulum.     

The effect of a selective inhibition of potassium channels on the relaxation induced by nitric oxide in the human pregnant myometrium

The aim of this study was to investigate whether apamin-sensitive K(+)channels play a role in the NO induced relaxation of the human pregnant myometrium. Concentration-response curves for sodium nitroprusside (SNP) (10(-9)-10(-4)M) were constructed in the absence and presence of 10(-8)M apamin and 10(-7)M charybdotoxin (CTX). Preincubation with apamin resulted in a significant attenuation of the relaxation caused by SNP, while pre-treatment with CTX insignificantly decreased the SNP induced relaxation. Our findings suggest that apamin-sensitive K(+)channels exist in the human pregnant myometrium and play a role in modulation of the myometrium response to NO donors     

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.     

Acute responses to phytoestrogens in small arteries from men with coronary heart disease

The aim of this study was to investigate acute vasodilator responses to phytoestrogens and selective estrogen receptor-alpha (ERalpha) agonist in isolated small arteries from men with established coronary heart disease (CHD) and with a history of myocardial infarction versus healthy male control subjects. As to methodology, small arteries obtained from subcutaneous fat biopsies and mounted on a wire myograph were preconstricted with norepinephrine, and dilator responses to increasing nanomolar-micromolar concentrations of the phytoestrogens resveratrol and genistein (predominantly ERbeta agonists) and to propyl-[1H]-pyrazole-1,3,5-triyl-trisplenol (PPT, a selective ERalpha agonist) were determined. These were compared with responses to reference compound 17beta-estradiol (17beta-E2). Concentration-response curves were constructed before and after nitric oxide (NO) synthase inhibition with Nomega-nitro-L-arginine methyl ester. As a result, relaxation induced by the investigated compounds was similar in men with CHD and control men, but in both groups PPT and genistein-induced relaxation was greater than that of resveratrol and 17beta-E2. NO contributed to both phytoestrogens and PPT-induced relaxation but not to 17beta-E2 responses in arteries from control men. This NO-mediated component of relaxation was absent in arteries from men with established CHD. In conclusion, phytoestrogens, at concentrations achievable by ingestion of phytoestrogen-rich food products, evoke dilatation ex vivo of small peripheral arteries from normal men and those with established CHD. The contribution of NO to dilatory responses by these compounds is pertinent to arteries from control males, whereas other NO-independent dilatory mechanism(s) are involved in arteries from CHD.     

Inhibition of nitrovasodilator- and acetylcholine-induced relaxation and cyclic GMP accumulation by the cytochrome P-450 substrate, 7-ethoxyresorufin.

We examined the effect of the cytochrome P-450 substrate, 7-ethoxyresorufin (7-ER), and its corresponding product, resorufin, on nitrovasodilator- and endothelium-dependent relaxation of isolated rat aorta. The EC50 value for glyceryl trinitrate (GTN) induced relaxation was increased over 100-fold by 7-ER and less than 3-fold by resorufin. The EC50 value for sodium nitroprusside (SNP) induced relaxation was increased approximately 12-fold by 7-ER, acetylcholine (ACh) induced relaxation was abolished, and relaxation induced by isopropylnorepinephrine was not significantly affected. GTN-, SNP-, and ACh-induced increases in cyclic GMP accumulation were inhibited by 7-ER, as were basal cyclic GMP levels in endothelium-intact, but not endothelium-denuded tissues. 7-ER decreased GTN biotransformation in intact aorta and decreased the regioselective formation of glyceryl-1,2-dinitrate. The activation by GTN and SNP of aortic guanylyl cyclase in broken cell preparations was not affected by 7-ER, indicating that the inhibitory effect of 7-ER is probably not due to a direct interaction with guanylyl cyclase. The inhibitory effect of 7-ER on GTN-induced relaxation was not altered by the addition of superoxide dismutase, suggesting that 7-ER does not act by increasing superoxide anion concentration (which would serve to increase the degradation of nitric oxide (NO) formed during vascular GTN biotransformation). Our data provide further evidence for the role of the cytochrome P-450--cytochrome P-450 reductase system in the biotransformation of GTN to an activator (presumably nitric oxide) of guanylyl cyclase. The data are consistent with a mode of action of 7-ER involving either competitive inhibition of vascular cytochrome P-450 or uncoupling of vascular cytochrome P-450 reductase from cytochrome P-450. The data also suggest that the cytochrome P-450 system facilitates NO release from SNP and that 7-ER has an inhibitory effect on endothelial nitric oxide synthase.     

Oxygen concentration regulates NO-dependent relaxation of aortic smooth muscles

Nitric oxide (NO) functions as an endothelium-derived relaxation factor and regulates vascular resistance. Recent studies in this laboratory (Arch. Biochem. Biophys. 323, 27-32, 1995) revealed that the lifetime of NO significantly increased at physiologically low levels of oxygen concentrations and, hence, this gaseous radical strongly inhibited mitochondrial electron transport for a fairly long duration at low oxygen concentrations. The present work describes the effect of oxygen concentration on NO-induced relaxation and guanylate cyclase (GC) activity of endothelium-denuded aorta of the rat. Both NO and 2,2'-hydroxynitrosohydrazono)bis-ethanamine (NOC18), an NO donor, induced the relaxa-tion of endothelium-denuded helical segments of rat aorta which were contracted by norepinephrine. NO-dependent relaxation of arterial specimens was enhanced by lowering oxygen concentration in the medium with concomitant increase in their cGMP levels. Anoxia induced the relaxation of the aorta by some NO-enhanceable and methylene blue-insensitive mechanism. These results suggested that local concentrations of oxygen might play important roles in the regulation of NO-dependent GC activity and vascular tonus of resistance arteries.     

A novel mechanism of vascular relaxation induced by sodium nitroprusside in the isolated rat aorta

Sodium nitroprusside (SNP) is an endothelium-independent relaxant agent and its effect is attributed to its direct action on the vascular smooth muscle (VSM). Endothelium modulates the vascular tone through the release of vasoactive agents, such as NO. The aim of this study was to investigate the contribution of the endothelium on SNP vasorelaxation, NO release and Ca²⁺ mobilization. Vascular reactivity experiments showed that endothelium potentiates the SNP-relaxation in rat aortic rings and this effect was abolished by l-NAME. SNP-relaxation in intact endothelium aorta was inhibited by NOS inhibitors for the constitutive isoforms (cNOS). Furthermore, endogenous NO is involved on the SNP-effect and this endogenous NO is released by cNOS. Moreover, Ca²⁺ mobilization study shows that l-NAME inhibited the reduction of Ca²⁺-concentration in VSM cells and reduced the increase in Ca²⁺-concentration in endothelial cells induced by SNP. This enhancement in Ca²⁺-concentration in the endothelial cells is due to a voltage-dependent Ca²⁺ channels activation. The present findings indicate that the relaxation and [Ca²⁺]_i decrease induced by SNP in VSM cells is potentiated by endothelial production of NO by cNOS-activation in rat aorta.     

Oxygen concentration regulates NO-dependent relaxation of aortic smooth muscles

Nitric oxide (NO) functions as an endothelium-derived relaxation factor and regulates vascular resistance. Recent studies in this laboratory (Arch. Biochem. Biophys. 323, 27–32, 1995) revealed that the lifetime of NO significantly increased at physiologically low levels of oxygen concentrations and, hence, this gaseous radical strongly inhibited mitochondrial electron transport for a fairly long duration at low oxygen concentrations. The present work describes the effect of oxygen concentration on NO-induced relaxation and guanylate cyclase (GC) activity of endothelium-denuded aorta of the rat. Both NO and 2,2′-hydroxynitrosohydrazono)bis-ethanamine (NOC18), an NO donor, induced the relaxa-tion of endothelium-denuded helical segments of rat aorta which were contracted by norepinephrine. NO-dependent relaxation of arterial specimens was enhanced by lowering oxygen concentration in the medium with concomitant increase in their cGMP levels. Anoxia induced the relaxation of the aorta by some NO-enhanceable and methylene blue-insensitive mechanism. These results suggested that local concentrations of oxygen might play important roles in the regulation of NO-dependent GC activity and vascular tonus of resistance arteries.     

Relaxant effects of sodium nitroprusside and NONOates in goat middle cerebral artery: delayed impairment by global ischemia-reperfusion.

Global cerebral ischemia and subsequent reperfusion induce early impairment of the vasodilator responses to hypercapnia and vasoactive substances. Nitric oxide (NO) is involved in the regulation of cerebral blood flow (CBF) in both health and disease. The present study was designed to assess possible changes in the cerebrovascular reactivity to NO donors induced by cerebral ischemia-reperfusion in goats. Female goats (n = 9) were subjected to 20 min global cerebral ischemia under halothane/N2O anesthesia. Sixteen additional goats were sham-operated as a control group. One week later the effects of ischemia-reperfusion on relaxations to NO donors sodium nitroprusside (SNP), diethylamine/NO (DEA/NO), diethylenetriamine/NO (DETA/NO), and spermine/NO (SPER/NO) were studied in rings of middle cerebral artery (MCA) isolated in an organ bath for isometric tension recording. SNP, DEA/NO, DETA/NO, and SPER/NO induced concentration-dependent relaxations of MCA precontracted with KCl (DEA/NO > SPER/NO > SNP > DETA/NO) or with endothelin-1 (DEA/NO > SNP > SPER/NO > DETA/NO). Relaxations were always higher in endothelin-1-precontracted arteries. One week after cerebral ischemia concentration-response curves to SNP and DEA/NO were displaced to the right, indicating a reduction in relaxant potency of NO donors. The classical nitrovasodilator SNP and NONOates induce relaxation of isolated goat MCA which is partially inhibited by arterial depolarization. Global cerebral ischemia followed by reperfusion induces delayed impairment of the relaxant effects of NO on cerebrovascular smooth muscle, which results in reduced vasodilatory potency of NO donors in large cerebral arteries.     

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.     

Involvement of Ca2+/calmodulin-dependent protein kinase II in endothelial NO production and endothelium-dependent relaxation

Nitric oxide (NO) is synthesized from l-arginine by the Ca(2+)/calmodulin-sensitive endothelial NO synthase (NOS) isoform (eNOS). The present study assesses the role of Ca(2+)/calmodulin-dependent protein kinase II (CaMK II) in endothelium-dependent relaxation and NO synthesis. The effects of three CaMK II inhibitors were investigated in endothelium-intact aortic rings of normotensive rats. NO synthesis was assessed by a NO sensor and chemiluminescence in culture medium of cultured porcine aortic endothelial cells stimulated with the Ca(2+) ionophore A23187 and thapsigargin. Rat aortic endothelial NOS activity was measured by the conversion of l-[(3)H]arginine to l-[(3)H]citrulline. Three CaMK II inhibitors, polypeptide 281-302, KN-93, and lavendustin C, attenuated the endothelium-dependent relaxation of endothelium-intact rat aortic rings in response to acetylcholine, A23187, and thapsigargin. None of the CaMK II inhibitors affected the relaxation induced by NO donors. In a porcine aortic endothelial cell line, KN-93 decreased NO synthesis and caused a rightward shift of the concentration-response curves to A23187 and thapsigargin. In rat aortic endothelial cells, KN-93 significantly decreased bradykinin-induced eNOS activity. These results suggest that CaMK II was involved in NO synthesis as a result of Ca(2+)-dependent activation of eNOS.