Relaxation Of Rat Aortic Rings Research Articles

Uridine adenosine tetraphosphate induces contraction and relaxation in rat aorta

Uridine adenosine tetraphosphate (Up 4A) has been recently reported as an endothelium-derived vasoconstrictor and plasma levels of this dinucleotide are increased in juvenile hypertensive subjects. This study aimed to evaluate the vascular actions of Up 4A, typify the putative purinergic receptors that might mediate these effects and characterize the intracellular signaling pathways that may govern Up 4A responses. Up 4A induced a modest endothelium-dependent relaxation of rat aortic rings contracted with phenylephrine. From baseline, Up 4A induced concentration-dependent contractions that were significantly potentiated by endothelium removal or nitric oxide synthase inhibition. The contractile response induced by Up 4A was not tachyphylactic and was significantly reduced in the presence of P1 or P2X receptor antagonists, L-type Ca 2+ channel blocker and Rho-kinase inhibitor. Up 4A-induced contraction apparently involves superoxide anion formation since it was significantly reduced by treatment with apocynin or tempol. This study presents the unique findings that the endogenous compound Up 4A is able to induce relaxation in addition to contraction of rat aorta. Up 4A-induced contraction is modulated by nitric oxide production, mediated by P1 and P2X receptor activation, and involves L-type Ca 2+ channels, Rho-kinase pathway and superoxide formation.

K ATP channel openers: Tissue selectivity of original 3-alkylaminopyrido- and 3-alkylaminobenzothiadiazine 1,1-dioxides

The present study was designed to further evaluate the biological effects and tissue selectivity of new 3-alkylaminobenzo- and 3-alkylaminopyridothiadiazine 1,1-dioxides bearing identical branched alkylamino chains at the 3-position. These original compounds were compared with their parent molecules; namely the K ATP channel openers diazoxide and pinacidil. All tested 3-alkylamino-substituted derivatives provoked a marked, concentration-dependent inhibition of the glucose-induced insulin secretion from rat pancreatic islets. The 3-alkylaminopyridothiadiazine 1,1-dioxides evoked a weak vasorelaxant activity whilst their 7-halo-substituted benzothiadiazine counterparts elicited pronounced, concentration-dependent, relaxations of rat aortic rings. The myorelaxant effect of the different drugs was tightly correlated with their capacity to increase 86Rb outflow ( 42K substitute) from prelabelled and perifused rat aortic rings. EC 50/IC 50 ratios (vascular/pancreatic) revealed a pronounced selectivity of the 3-alkylaminopyridothiadiazine 1,1-dioxides towards the pancreatic endocrine tissue. Structure–activity relationships suggest that, besides the requirement of an electronegative pole at the 7-position of the heterocycle, a minimal steric hindrance confers an optimal insulin-secreting cell selectivity. Lastly, radioisotopic, electrophysiological and pharmacological investigations indicate that the marked vasorelaxant properties of the 3-alkylaminobenzothiadiazine 1,1-dioxides are related to the activation of smooth muscle K ATP channels.

Potency of authentic nitric oxide in inducing aortic relaxation

The reported EC 50 of vasorelaxation for authentic nitric oxide (NO) is from the nM to μM range. The cause of this large difference is undetermined. In this study, NO electrodes were used to monitor the actual NO concentration in the organ chamber during the recording of the relaxation of rat aortic rings. It was demonstrated that both the O 2 concentration in the solution and the rate of stirring the solution markedly affected the actual NO concentration, while the vasorelaxation response to NO was also changed with the composition of the buffer solution. It was observed that the apparent EC 50 of aortic relaxation for authentic NO is 340 ± 40 nM and 81 ± 4 nM in the conventional organ chamber containing Krebs–Ringer buffer bubbled with a high content (95% O 2 + 5% CO 2) and a low content (20% O 2 + 5% CO 2 + 75% N 2) of O 2 gas mixture, respectively. The apparent EC 50 was further reduced to 45 ± 2 nM after the Krebs–Ringer buffer, a bicarbonate buffer, was replaced by a phosphate buffer solution bubbled with a low content of O 2 gas mixture (20% O 2 + 80% N 2) or with air. By using an organ chamber, which makes low concentrations of NO more stable in the solution, it was determined that the apparent EC 50 was 9.7 ± 0.4 nM and the threshold of NO concentration in dilating the aortas was ∼0.3 nM. This modified organ chamber will be useful for quantitatively measuring EC 50 of vasorelaxation for authentic NO under physiological and pathological conditions.

Endothelium-dependent relaxation of rat aorta induced by resveratrol

It has been noted that there are a number of biologically active phenolic compounds present in wine, particularly red wine. Such compounds include, for example, catechin, epicatechin, quercetin, rutin, trans-resveratrol and cis-resveratrol. The resveratrol isomers, in particular, have been found to promote vascular relaxation. The mechanisms by which resveratrol causes vasodilatation are uncertain. The aim of this study was to investigate the mechanism(s) of resveratrolinduced vasorelaxation in rat aorta with endothelium present. Rat aortic rings were precontracted with phenylephrine. Resveratrol induced relaxation of the rat aortic rings. L-NAME, an inhibitor of NO synthase, abolished relaxation of rat aorta, but methylene blue, an inhibitor of guanylate cyclase, did not abolish relaxation induced by resveratrol. Highly selective blocker of ATP-sensitive K+ channels, glibenclamide as well as a nonselective blocker of big Ca-sensitive K+ channels, charybdotoxin did not block resveratrol-induced relaxation of rat aorta. 4-Aminopiridine, which is a non selective blocker of voltage-gated K+ (Kv) channels, and margatoxin which inhibits Kv1 channels abolished relaxation of rat aortic rings induced by resveratrol. In conclusion, we have shown that resveratrol potently relaxed rat aortic rings with endothelium present. It seems that NO and smooth muscle KV channels are included in this relaxation. This finding is of clinical importance in both veterinary and human medicine.

Potassium Channels‐Mediated Vasorelaxation of Rat Aorta Induced by Resveratrol

Resveratrol, a phenolic substance present in grapes and a variety of medical plants, has been reported to induce vasorelaxation, however the mechanisms are uncertain. In this paper we investigate the possible participation of K(+) channels in the endothelium-independent vasodilatation of rat aorta induced by resveratrol. Resveratrol induced concentration-dependent relaxation of rings with endothelium and without endothelium. We used different potassium channel inhibitors to determine whether the K(+) channels mediated endothelium-independent relaxation of rat aorta induced by resveratrol. Highly selective blocker of ATP-sensitive K(+) channels, glibenclamide, as well as non-selective blockers of K(+) channels, tetraethylammonium, did not block resveratrol-induced relaxation of rat aortic rings. Charybdotoxin, a blocker of calcium-sensitive K(+) channels did not affect the resveratrol-induced relaxation. 4-Aminopiridine, non-selective blocker of voltage-gated K(+) (Kv) channels, and margatoxin that inhibits Kv1 channels abolished relaxation of rat aortic rings induced by resveratrol. In conclusion, we have shown that resveratrol potently relaxed rat aortic rings with denuded endothelium. It seems that 4-aminopiridine and margatoxin-sensitive K(+) channels located in the smooth muscle of rat aorta mediated this relaxation.

Endothelium-dependent and -independent relaxation induced by pinocembrin in rat aortic rings

The aim of present study was to evaluate the vasorelaxant effects of the flavonone pinocembrin and its possible mechanisms in isolated rat aortic rings. Pinocembrin (5 ∼ 100 μM) induced relaxation in aortic rings pre-contracted with norepinephrine (NE, 1 μM) or KCl (60 mM), with pEC 50 value 4.37 ± 0.02 and 4.52 ± 0.04. Pretreatment with pinocembrin (30 or 50 μM) also inhibited contractile responses to NE and KCl. The vasorelaxant effect of pinocembrin relied on intact endothelium partially, and incubation with n ω-nitro- l-arginine methyl ester ( l-NAME, 100 μM) or methylene blue (10 μM) significantly inhibited the effect, however indomethacin (5 μM) had no influence on the action. In endothelium-denuded rings, the vasorelaxant effect of pinocembrin was reduced by glibenclamide (10 μM), tetraethylammonium (5 mM) and 4-aminopyridine (100 μM). Pinocembrin also reduced NE-induced transient contraction in Ca 2+-free solution and inhibited contraction induced by increasing external calcium in Ca 2+-free medium plus 60 mM KCl. Our results suggest that pinocembrin induces relaxation in rat aortic rings through an endothelium-dependent pathway, involving NO-cGMP, and also through an endothelium-independent pathway, opening K + channels and blockade of Ca 2+ channels.

Effect of redox agents on the response of rat aorta to nitric oxide and sodium nitroprusside

<b>Objective:</b> To study the redox regulation of vascular responses to endogenous nitric oxide (NO) and NO derived from nitrovasodilator sodium nitroprusside (SNP) in isolated rat aorta. <br><b> Materials and Methods:</b> To determine the influence of reducing [ascorbic acid (1 mM) and reduced glutathione (GSH) (1 mM)] and oxidizing agents [oxidized glutathione (GSSG) (1 mM) and CuSO4 (1 and 5 µM)] on the vasodilation caused by acetylcholine (ACh; 10-11-10-5 M) and SNP (10-9-10-4 M). Isometric tensions were measured in isolated aorta by a force transducer and recorded in a computer, using Chart V4.1.2 software. <br><b> Results:</b> ACh and SNP produced relaxation of rat aortic rings that was dependent on concentration. The rings were preconstricted with L-phenylephrine (1 µM). It was observed that oxidizing and reducing agents caused opposite effects on vasodilation induced by NO in rat aorta. Ascorbic acid and GSH potentiated the responses to NO, causing a leftward shift in the concentration-response curve of ACh with significant increase in the pD2 and the Emax. GSSG and CuSO4 inhibited relaxation caused by ACh and shifted the concentration-response curve to the right. In concentration-responses induced by SNP, ascorbic acid significantly increased the pD2 and Emax values from 5.85 ± 0.08 to 6.24 ± 0.05 and 80.83 ± 1.37% to 89.26 ± 1.49%, respectively. However, CuSO4 significantly decreased these values from 5.85 ± 0.02 to 4.56 ± 0.10 and 77.18 ± 0.82% to 53.52 ± 1.60%, respectively. Potentiation of NO response by reducing agents may be related to either increased availability of nitroxyl anion (NO-) or reduction in superoxide anion radical (O2·-). The opposite could be true for the oxidizing agents. <br><b> Conclusion:</b> The findings of this study suggest that reducing agents like ascorbic acid can improve the vascular responses to NO under oxidative stress.

Hydralazine decreases sodium nitroprusside-induced rat aortic ring relaxation and increased cGMP production by rat aortic myocytes

Association of hydralazine with nitrovasodilators has long been known to be beneficial in the vasodilator treatment of heart failure. We previously found that hydralazine appeared to reduce the increase in cGMP induced by sodium nitroprusside in cultured rat aortic myocytes. In order to further explore this seemingly paradoxical interaction, we extended our initial observations in rat aortic myocytes and also determined the influence of hydralazine on sodium nitroprusside-induced relaxation of rat aortic rings. Hydralazine produced a concentration-dependent inhibition of sodium nitroprusside stimulation of cGMP production and caused a rightward shift of concentration–relaxation curves in aortic rings. A possible mechanism of the hydralazine–nitroprusside interaction could be the interference with bioactivation of the nitrovasodilator to release nitric oxide. Recent evidence indicates that vascular NADH oxidase, an enzyme known to be inhibited by hydralazine, could be involved in this process. Accordingly, hydralazine was found to inhibit NADH oxidase activity in rat aortic myocytes at concentrations similar to those reducing sodium nitroprusside responses. It was concluded that antagonism of sodium nitroprusside action by hydralazine could be a consequence of interference with bioactivation of the former, apparently through inhibition of vascular NADH oxidase.

Effect of sildenafil on the isolated rat aortic rings

Sildenafil, a highly selective inhibitor of PDE 5, is effective in the treatment of erectile dysfunction. Penile erection involves relaxation of smooth muscle of corpus cavernosum and its associated arterioles. The objective of this study was to investigate the effect of sildenafil on nitric oxide/cyclic guanosine monophosphate (NO/cGMP)-dependent relaxation of rat aortic rings. The contribution of sildenafil to the vasorelaxation of rat aortic rings was also investigated. Sildenafil produced significant potentiation of acetylcholine (ACh, 2 x 10(-6) m)-induced relaxation at concentration > or =1 x 10(-8) m. Addition of sildenafil (1 x 10(-7) m) to aortic rings failed to alter the effect of N(G)-nitro-L-arginine (l-NNA, 3 x 10(-5) m) or methylene blue (MB, 3 x 10(-5) m) on ACh response. Similarly, sildenafil (1 x 10(-7) m) augmented significantly the vasorelaxation induced by sodium nitroprusside over the range of 1 x 10(-9)-1 x 10(-8) m. When added to phenylephrine (3 x 10(-6) m)-precontracted rat aortic rings, sildenafil (1 x 10(-9)-1 x 10(-4) m) induced concentration-dependent relaxation reaching a maximum of 96.48 +/- 1.44%. These relaxations were not significantly attenuated by previous incubation with L-NNA (3 x 10(-5) m) or MB (3 x 10(-5) m). Denudation did not significantly affect the vasorelaxant effect of sildenafil. Sildenafil may act in the rat aortic rings through the amplification of NO/cGMP pathway. It may augment both basal endothelial NO function and exogenous NO-dependent vasodilatation. However, sildenafil may act by a mechanism independent of NO/cGMP pathway and this mechanism contributes to its smooth muscle relaxant effect.

Autoregulatory Role of Endothelium-derived Nitric Oxide (NO) on Lipopolysaccharide-induced Vascular Inducible NO Synthase Expression and Function

Nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) is responsible for sepsis-induced hypotension and plays a major contributory role in the ensuing multiorgan failure. The present study aimed to elucidate the role of endothelial NO in lipopolysaccharide (LPS)-induced iNOS expression, in isolated rat aortic rings. Exposure to LPS (1 mug/ml, 5 h) resulted in a reversal of phenylephrine precontracted tone in aortic rings (70.7 +/- 3.2%). This relaxation was associated with iNOS expression and NF-kappaB activation. Positive immunoreactivity for iNOS protein was localized in medial and adventitial layers of LPS-treated aortic rings. Removal of the endothelium rendered aortic rings resistant to LPS-induced relaxation (8.9 +/- 4.5%). Western blotting of these rings demonstrated an absence of iNOS expression. However, treatment of endothelium-denuded rings with the NO donor, diethylamine-NONOate (0.1 mum), restored LPS-induced relaxation (61.6 +/- 6.6%) and iNOS expression to levels comparable with arteries with intact endothelium. Blockade of endothelial NOS (eNOS) activation using geldanamycin and radicicol, inhibitors of heat shock protein 90, in endothelium-intact arteries suppressed both LPS-induced relaxation and LPS-induced iNOS expression (9.0 +/- 8.0% and 2.0 +/- 6.2%, respectively). Moreover, LPS treatment (12.5 mg/kg, intravenous, 15 h) of wild-type mice resulted in profound elevation of plasma [NO(x)] measurements that were reduced by approximately 50% in eNOS knock-out animals. Furthermore, LPS-induced changes in vascular reactivity and iNOS expression evident in wild-type tissues were profoundly suppressed in tissues taken from eNOS knockout animals. Together, these data suggest that eNOS-derived NO, in part via activation of NF-kappaB, regulates iNOS-induction by LPS. This study provides the first demonstration of a proinflammatory role of vascular eNOS in sepsis.

Endothelium-independent Vasorelaxant Effect of the Phyto-oestrogen Biochanin A on Rat Thoracic Aorta.

Epidemiological data suggest that the incidence of cardiovascular disease is reduced in people who have a high intake of phytoestrogens. The plant-derived estrogen biochanin A is known to cause vasodilation, but its mechanisms of action remain unclear. This study was undertaken to investigate the effects and mechanisms of biochanin A on rat thoracic aorta. Isolated aortic rings were suspended in individual organ baths and isometric tension was measured. Biochanin A induced significant relaxation in rings with or without endothelium. Contractile responses induced by phenylephrine (PE), KCl and CaCl<inf>2</inf>were antagonized by 10-7~10-4mol/L biochanin A. The transient contraction elicited by PE was significantly attenuated by 10-5mol/L biochanin A in Ca2+-free medium. The relaxant effect of biochanin A was significantly inhibited by pretreatment with the K+channel antagonists tetraethylammonium and glibenclamide in endothelium-denuded aorta. We conclude that biochanin A induces an endothelium-independent relaxation in rat aortic rings. The underlying mechanism may involve the blockage of Ca2+entry through both voltage-dependent and receptor-operated Ca2+channels, the inhibition of intracellular Ca2+release, and the activation of large-conductance Ca2+-activated K+channels and ATP-sensitive K+channels.

C-reactive protein does not relax vascular smooth muscle: effects mediated by sodium azide in commercially available preparations

C-reactive protein (CRP), an acute-phase protein and newly recognized indicator of cardiovascular risk, may have direct actions on the vascular wall. Previous studies suggest that CRP is a vasodilator that activates smooth muscle K(+) channels. We examined the reported vasoactive properties of CRP and further explored its mechanisms of action. CRP decreased blood pressure in rats and increased coronary flow in open-chest dogs at a constant coronary perfusion pressure. CRP relaxed rat aortic rings and mesenteric small arteries that were contracted with phenylephrine. Relaxation was not affected by endothelial denudation or inhibition of nitric oxide (NO) synthase but was blocked by inhibition of soluble guanylate cyclase or K(+) channels. CRP solutions remained effective, i.e., elicited vasodilation, even after boiling or enzymatic digestion, which suggests the presence of a nonprotein contaminant. Sodium azide (NaN(3), 0.1%) is the preservative used for commercially available CRP and a potential source of NO. NaN(3) elicited the same cardiovascular effects as CRP preparations at equal concentrations, and its actions were blocked by inhibition of guanylate cyclase and K(+) channels. NaN(3)-free CRP, prepared by gel-filtration centrifugation and confirmed by electrophoresis, had no effect on vascular tone. Inhibition of vascular smooth muscle catalase with 3-amino-1,2,4-triazole completely prevented the effects of NaN(3) and NaN(3)-containing CRP solutions. We demonstrate that the acute vasoactive properties of commercially available CRP preparations are attributable to NaN(3) (and subsequent production of NO by catalase); therefore, this study suggests a reappraisal of the acute role of CRP in regulating vascular tone.

VASCULAR AND ANTI‐OXIDANT ACTIONS OF FLAVONOLS AND FLAVONES

1. Flavonols and flavones are plant-derived polyphenolic compounds that are commonly consumed in the diet. Epidemiological studies indicating that high dietary intake of flavonols reduces the risk of mortality due to coronary heart disease have provoked interest in the mechanism of this cardioprotective effect. 2. We have investigated the structure-activity relationships of a range of flavonols and flavones with regard to their vascular relaxant and anti-oxidant activity. In rat isolated thoracic aorta, the synthetic flavonol 3',4'-dihydroxyflavonol (DiOHF) was found to be a significantly more potent vasorelaxant than the naturally occurring compounds chrysin, apigenin, luteolin, quercetin and fisetin. Similarly, DiOHF was significantly more potent than those compounds in the inhibition of calcium-induced contraction of the rat aorta. 3. 3',4'-Dihydroxyflavonol was also found to significantly inhibit superoxide radical generation in a cell-free system in the presence of xanthine/xanthine oxidase or by rat isolated aorta in the presence of NADPH. In the presence of oxidant stress generated by pyrogallol or xanthine/xanthine oxidase, endothelium-dependent relaxation of rat aortic rings was impaired. 3',4'-Dihydroxyflavonol was able to significantly improve endothelium-dependent relaxation in the presence of those oxygen radical generators. 4. In addition, DiOHF was found to significantly improve dilatation in the rat hindquarters vasculature after exposure to ischaemia and reperfusion. 3',4'-Dihydroxyflavonol was found to be equally effective whether applied before ischaemia or during ischaemia just before reperfusion. 5. In conclusion, DiOHF is an effective vasodilator and anti-oxidant that is able to prevent vascular reperfusion injury. We suggest that DiOHF may be useful as an adjunct to thrombolytic therapy in the management of reperfusion injury.

Inhibiting long chain fatty Acyl CoA synthetase increases basal and agonist-stimulated NO synthesis in endothelium.

Endothelial nitric oxide synthase (eNOS) activation/deactivation is associated with cyclic depalmitoylation/repalmitoylation of specific Cys residues. The mechanism of depalmitoylation has been identified recently, but repalmitoylation remains undefined. We hypothesized that long chain fatty acyl CoA synthetase (LCFACoAS) modulates endothelial nitric oxide synthase repalmitoylation by limiting palmitoyl CoA availability. Human coronary endothelial cells were treated with triacsin-C, an inhibitor of long chain fatty acyl CoA synthetase, for 24 h. Media nitrite accumulation, eNOS activity, and eNOS palmitoylation were measured. Methacholine-induced NO synthesis or vascular relaxation were measured in endothelium-intact rat aortae in the presence and absence of triacsin-C. Triacsin-C significantly reduced incorporation of [3H] palmitate into immunoreactive endothelial nitric oxide synthase and over a concentration range of 0.1 to 10 microM, increased media nitrite accumulations 2- to 2.5-fold over baseline. Total in vitro catalytic activity of nitric oxide synthase in triacsin-C treated cells did not differ significantly from control. Triacsin-C significantly increased methacholine-induced NO synthesis in the isolated rat aorta, and significantly enhanced methacholine-induced relaxation of rat aortic rings. These data are consistent with the interpretation that inhibition of palmitoylation increases endothelial nitric oxide synthase activity without changing endothelial nitric oxide synthase expression, suggesting that inhibiting palmitoylation increases the catalytically active fraction of endothelial nitric oxide synthase.

Human free apolipoprotein A-I and artificial pre-beta-high-density lipoprotein inhibit eNOS activity and NO release

Little is known about the effects of human free apolipoprotein A-I (Free-Apo A-I) and pre-beta-high density lipoprotein (pre-beta-HDL) on the endothelium function. In this study, we have investigated the effects of Free-Apo A-I and artificial pre-beta-HDL on endothelial NO synthase (eNOS) activity and on NO production by endothelial cells. Free-Apo A-I drastically inhibited NO production in human umbilical cord vein endothelial cells (HUVECs) and eNOS activity in bovine aortic endothelial cells (BAECs). Pre-beta-HDL and serum from human apolipoprotein A-I transgenic rabbits inhibited eNOS activity in BAECs but HDL3 did not. Free-Apo A-I displaced eNOS from BAEC plasma membrane towards intracellular pools without affecting eNOS activity and eNOS mass in BAEC crude homogenates. Free-Apo A-I and HDL3 did not decrease either caveolin bound to BAEC plasma membrane or caveola cholesterol content. As previously described, we showed that HDL3 directly induced endothelium-dependent relaxation of rings from rat aorta. We observed that pre-beta-HDL significantly decreased endothelium-dependent relaxation of rat aortic rings ex vivo.

Relaxant effect of oxime derivatives in isolated rat aorta: role of nitric oxide (NO) formation in smooth muscle

Various oxime derivatives were evaluated as nitric oxide (NO) donors in arteries. Relaxation of rat aortic rings was used for bioassay of NO production, and electron paramagnetic resonance spectroscopy for demonstration of NO elevation. In rings with or without endothelium or adventitia, hydroxyguanidine and hydroxyurea were almost inactive, whereas formamidoxime, acetaldoxime, acetone oxime, acetohydroxamic acid and formaldoxime elicited relaxation. Active compounds increased NO levels in endothelium-denuded rings. Formaldoxime was the most potent agent for both relaxation and NO elevation in aortic rings, and it also increased NO in human aortic smooth muscle cells. In endothelium-denuded rings, relaxation was inhibited by a NO scavenger (2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide) and by inhibitors of soluble guanylyl-cyclase (1 H[1,2,4,]oxadiazolo[4,3- a]quinoxalin-1-one) or cyclic GMP-dependent protein kinases (Rp-8-bromo cyclic GMP monophosphorothioate). Neither N ω-nitro- l-arginine methylester (a NO synthases inhibitor) nor proadifen (a cytochrome P450 inhibitor) decreased the effect of oxime derivatives. However, 7-ethoxyresorufin (7-ER, an inhibitor of P4501A 1 which can also inhibit various NADPH-dependent reductases) abolished the relaxant effect of these compounds, without affecting the one of glyceryl trinitrate (GTN) or 2-( N, N-diethylamino)-diazenolate-2-oxide. 7-ER also abolished formaldoxime-induced NO increase in aortic rings. In rings tolerant to GTN, formaldoxime-induced relaxation and NO elevation were not different from those obtained in control rings. In conclusion, some oxime derivatives release NO by 7-ER-sensitive pathways in aortic smooth muscle, thus eliciting vasorelaxation. Pathways of NO formation are likely distinct from NO synthases and from those responsible for GTN biotransformation. Oxime derivatives could be useful for NO delivery in arteries in which endothelial NO synthase activity is impaired.

Amlodipine activates the endothelial nitric oxide synthase by altering phosphorylation on Ser1177 and Thr495.

The Ca2+ antagonist amlodipine increases the generation of nitric oxide (NO) from native and cultured endothelial cells. The aim of this investigation was to determine whether or not the activation of the endothelial NO synthase (eNOS) by this Ca2+ antagonist is related to alterations in eNOS phosphorylation. In isolated, pre-contracted, endothelium-intact porcine coronary arteries, amlodipine elicited an NO-mediated relaxation and a leftward shift in the concentration-relaxation curve to bradykinin. Moreover, the Ca2+ antagonist increased the generation of NO from native endothelial cells, as detected by electron spin resonance spectroscopy and stimulated an 8-fold increase in cyclic GMP levels in cultured endothelial cells. In unstimulated endothelial cells, eNOS was not phosphorylated on Ser1177 but was phosphorylated on Thr495. Amlodipine elicited the phosphorylation of Ser1177 and attenuated Thr495 phosphorylation, with a time course similar to that of eNOS activation. The amlodipine-induced relaxation of porcine coronary arteries was attenuated by the B2 kinin receptor antagonist, icatibant, but this antagonist did not affect amlodipine-induced changes in eNOS phosphorylation in cultured endothelial cells. Moreover, amlodipine elicited the NO-mediated relaxation of rat aortic rings which do not express the B2 receptor. Amlodipine time-dependently attenuated the phosphorylation of protein kinase C (PKC) in endothelial cells, with a time course similar to the changes in eNOS phosphorylation, and prevented the phorbol-12-myristate-13-acetate-induced activation of PKC. The PKC inhibitor, Ro 31-8220, also elicited the phosphorylation of Ser1177 and the dephosphorylation of Thr495 in cultured cells and induced a leftward shift in the concentration-relaxation curve to bradykinin in rings of porcine coronary artery. The Ca2+ antagonist, amlodipine, enhances endothelial NO generation by inducing changes in the phosphorylation of eNOS. Although the activation of eNOS was related to the activation of the B2 kinin receptor in the porcine coronary artery, a B2 receptor-independent mechanism involving the inhibition of PKC appears to account for the effects observed in the rat aorta as well as in cultured endothelial cells.

Captopril restores endothelium-dependent relaxation of rat aortic rings after exposure to homocysteine.

This study was designed to investigate the effects of captopril, an angiotensin-converting enzyme inhibitor, on inhibition of endothelium-dependent relaxation induced by homocysteine in isolated rat aorta. Isometric tension recordings were used to assess inhibitory effects of homocysteine and protective effects of captopril on endothelium-dependent relaxation of aortic rings. Exposure of aortic rings to homocysteine (0.3 approximately 3 mmol/L) for 30 min induced a significant concentration-dependent inhibition of endothelium-dependent relaxation response to acetylcholine (ACh), but did not affect endothelium-independent relaxation response to sodium nitroprusside. Pre-incubation of aortic rings with captopril (3 approximately 30 micromol/L) for 15 min and co-incubation of aortic rings with homocysteine (1 mmol/L) for another 30 min attenuated the inhibition of homocysteine in a dose-dependent manner. Moreover, superoxide dismutase (SOD, 200 U/mL), a scavenger of superoxide anions, reduced homocysteine-induced inhibition. L-Arginine (3 mmol/L), a precursor of nitric oxide (NO), also attenuated the impairment of vasorelaxation induced by homocysteine. However, in the combined presence of SOD and L-arginine, the inhibitory effect of homocysteine was reversed, which was very similar to the effect of 30 micromol/L captopril. These results suggest that captopril can prevent the inhibition of endothelium-dependent relaxation induced by homocysteine in isolated rat aorta, which may be related to scavenging oxygen free radicals and enhancing NO production.

Effects of hydrazine derivatives on vascular smooth muscle contractility, blood pressure and cGMP production in rats: comparison with hydralazine

Hydralazine is a hydrazine derivative used clinically as a vasodilator and antihypertensive agent. Despite numerous studies with the drug, its mechanism of action has remained unknown; guanylate cyclase activation and release of endothelial relaxing factors are thought to be involved in its vasodilator effect. Other hydrazine derivatives are known to stimulate guanylate cyclase and could therefore share the vasodilator activity of hydralazine, although such possibility has not been assessed systematically. In the present study, hydralazine, hydrazine, phenylhydrazine, and isoniazid were evaluated for vascular smooth muscle relaxation in rat aortic rings with and without endothelium, as well as after incubation with the guanylate cyclase inhibitor methylene blue. They were also tested for enhancement of cyclic guanosine monophosphate (cGMP) production by cultured rat aortic smooth muscle cells and for hypotension in the anesthetized rat. All hydrazines relaxed aortic rings, an action unaffected by endothelium removal and, in all cases except hydralazine, antagonized by methylene blue. Only phenylhydrazine increased cGMP production and only hydralazine markedly lowered blood pressure. It was concluded that hydralazine vascular relaxation is independent of endothelium and is not related to guanylate cyclase activation. The other hydrazines studied also elicit endothelium-independent relaxation, but the effect is related to guanylate cyclase. The marked hypotensive effect of hydralazine contrasts with its modest relaxant activity and is not shared by the other hydrazines. The fact that hydrazine and isoniazid produce methylene blue-sensitive relaxation, yet do not enhance cGMP production suggests the need for activating factors present in aortic rings but not in isolated cells.

Lithocholylcholine, a bile acid/acetylcholine hybrid, is a muscarinic receptor antagonist.

Previous work from our laboratory indicates that bile acids, specifically lithocholic acid conjugates, interact with muscarinic receptors on gastric chief cells. Structural similarities between acetylcholine and lithocholyltaurine suggest a potential molecular basis for their interaction with the same receptor. We synthesized a hybrid molecule consisting of the steroid nucleus of lithocholyltaurine and the choline moiety of acetylcholine. The new molecule, lithocholylcholine, is hydrolyzed by acetyl-cholinesterase. Lithocholylcholine inhibited binding of a cholinergic radioligand to Chinese hamster ovary cells expressing each of the five muscarinic receptor subtypes. The binding affinities (K(i); micromolar) of lithocholylcholine for these receptors were: M3 (1.0) > M1 (2.7) > M2 (4.1) = M4 (4.9) > M5 (6.2). Lithocholylcholine inhibited intracellular signaling pathways mediated by interaction with M1, M2, and M3 muscarinic receptors. Regarding M3 receptors, lithocholylcholine was 10-fold more potent than lithocholyltaurine in terms of binding affinity and inhibition of acetylcholine-induced increases in inositol phosphate formation and mitogen-activated protein kinase phosphorylation. In a functional assay, lithocholylcholine inhibited acetylcholine-induced relaxation of rat aortic rings. These observations indicate that lithocholylcholine is a muscarinic receptor antagonist and provide further evidence that bile acids may have gastrointestinal signaling functions that extend beyond their effects on sterol metabolism, lipid absorption, and cholesterol elimination. Hybrid molecules created from bile acids and acetylcholine may be used to develop selective muscarinic receptor ligands.