Phenylephrine-precontracted Rat Aortic Rings Research Articles

Metabolite profiling, arginase inhibition and vasorelaxant activity of Cornus mas, Sorbus aucuparia and Viburnum opulus fruit extracts

The present study investigated the effects of Cornus mas, Sorbus aucuparia and Viburnum opulus fruit extracts on arginase activity and arterial vasodilation. V. opulus fruit extract exerted the highest vasorelaxant activity in phenylephrine precontracted rat aortic rings (EC50 = 6.31 ± 1.61 μg/mL) and a significant inhibition of arginase (IC50 = 71.02 ± 3.06 μg/mL). By contrast, S. aucuparia and C. mas fruit extracts showed no important anti-arginase activity and a significantly weaker activity in the rat aortic rings relaxation assay (EC50 = 100.9 ± 11.63 and 78.52 ± 8.59 μg/mL, respectively). For all extracts, the main mechanism of vasodilation was proven to be endothelium-dependent. HPLC-ESI-Q-TOF-MS/MS studies revealed a very complex metabolite profiling in all three extracts with chlorogenic acid accounting for 30.89, 0.72 and 2.03 mg/g in V. opulus, C. mas and S. aucuparia fruit extracts, respectively. All extracts were declared non-toxic in the brine shrimp acute toxicity test. Our study highlights potential benefits of V. opulus fruit extract in diseases associated with endothelial dysfunction and impaired vasodilation.

Possible mechanisms involved in the vasorelaxant effect produced by clobenzorex in aortic segments of rats.

Clobenzorex is a metabolic precursor of amphetamine indicated for the treatment of obesity. Amphetamines have been involved with cardiovascular side effects such as hypertension and pulmonary arterial hypertension. The aim of the present study was to investigate whether the direct application of 10–9–10–5 M clobenzorex on isolated phenylephrine-precontracted rat aortic rings produces vascular effects, and if so, what mechanisms may be involved. Clobenzorex produced an immediate concentration-dependent vasorelaxant effect at the higher concentrations (10–7.5–10–5 M). The present outcome was not modified by 10–6 M atropine (an antagonist of muscarinic acetylcholine receptors), 3.1×10–7 M glibenclamide (an ATP-sensitive K+ channel blocker), 10–3 M 4-aminopyridine (4-AP; a voltage-activated K+ channel blocker), 10–5 M indomethacin (a prostaglandin synthesis inhibitor), 10–5 M clotrimazole (a cytochrome P450 inhibitor) or 10–5 M cycloheximide (a general protein synthesis inhibitor). Contrarily, the clobenzorex-induced vasorelaxation was significantly attenuated (P<0.05) by 10–5 M L-NAME (a direct inhibitor of nitric oxide synthase), 10–7 M ODQ (an inhibitor of nitric oxide-sensitive guanylyl cyclase), 10–6 M KT 5823 (an inhibitor of protein kinase G), 10–2 M TEA (a Ca2+-activated K+ channel blocker and non-specific voltage-activated K+ channel blocker) and 10–7 M apamin plus 10–7 M charybdotoxin (blockers of small- and large-conductance Ca2+-activated K+ channels, respectively), and was blocked by 8×10–2 M potassium (a high concentration) and removal of the vascular endothelium. These results suggest that the direct vasorelaxant effect by clobenzorex on phenylephrine-precontracted rat aortic rings involved stimulation of the NO/cGMP/PKG/Ca2+-activated K+ channel pathway.

Pharmacological study of the mechanisms involved in the vasodilator effect produced by the acute application of triiodothyronine to rat aortic rings.

A relationship between thyroid hormones and the cardiovascular system has been well established in the literature. The present in vitro study aimed to investigate the mechanisms involved in the vasodilator effect produced by the acute application of 10-8–10-4 M triiodothyronine (T3) to isolated rat aortic rings. Thoracic aortic rings from 80 adult male Wistar rats were isolated and mounted in tissue chambers filled with Krebs-Henseleit bicarbonate buffer in order to analyze the influence of endothelial tissue, inhibitors and blockers on the vascular effect produced by T3. T3 induced a vasorelaxant response in phenylephrine-precontracted rat aortic rings at higher concentrations (10-4.5–10-4.0 M). This outcome was unaffected by 3.1×10-7 M glibenclamide, 10-3 M 4-aminopyridine (4-AP), 10-5 M indomethacin, or 10-5 M cycloheximide. Contrarily, vasorelaxant responses to T3 were significantly (P<0.05) attenuated by endothelium removal or the application of 10-6 M atropine, 10-5 M L-NG-nitroarginine methyl ester (L-NAME), 10-7 M 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), 10-6 M (9S,10R,12R)-2,3,9,10,11,12-Hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i](1,6)benzodiazocine-10-carboxylic acid, methyl ester KT 5823, 10-2 M tetraethylammonium (TEA), or 10-7 M apamin plus 10-7 M charybdotoxin. The results suggest the involvement of endothelial mechanisms in the vasodilator effect produced by the acute in vitro application of T3 to rat aortic rings. Possible mechanisms include the stimulation of muscarinic receptors, activation of the NO-cGMP-PKG pathway, and opening of Ca2+-activated K+ channels.

Rivaroxaban-Mediated Vascular Relaxation As a Potential Cause of Headaches and Dizziness

The availability of the direct Xa inhibitors apixaban, edoxaban and rivaroxaban in clinical practice is leading a paradigm shift in anticoagulation for the management of venous thromboembolism (VTE) and stroke prophylaxis in the context of non-valvular atrial fibrillation (AF). A common side effect experienced by patients receiving rivaroxaban in clinical trials was dizziness and headache, with a reported incidence of between 1 in 10 and 1 in 100 patients. Clinical experience has seen patients reporting rivaroxaban associated headaches and dizziness, leading to discontinuation of therapy. Given that widespread use of these agents is likely to be within an older adult population, it is important to understand the mechanism behind clinical observations which may then allow us to determine why some patients are affected more than others. We hypothesise that rivaroxaban directly causes vascular arteriodilation, resulting in headaches and dizziness requiring discontinuation of treatment.To test this hypothesis the effects of rivaroxaban on phenylephrine pre-contracted rat aortic rings was investigated. Thoracic aorta from male Sprague-Dawley rats (180-220g) were dissected and cut into rings of 2-3mm prior to being mounted under a preload tension of 1.5g in Krebs filled organ baths. Isometric tension of the rings was measured with isometric transducers (Danish Myo Technology, Aarhus, Denmark) digitised using a Power lab system. Following a phenylephrine dose-response curve the aortic rings were pre-contracted with 1 µM phenylephrine before measuring the relaxant effect of rivaroxaban (0.001-0.3 µM) or a similar volume of the vehicle dimethyl sulfoxide (DMSO). Statistical analysis was carried out using two-way ANOVA with Bonferroni's correction, where p<0.05 was considered significant.Exposure of aortic rings to both rivaroxaban and the vehicle DMSO caused relaxation. However, the relaxation observed with rivaroxaban was significantly greater than that observed with the vehicle (Fig 1).Our preliminary data suggests that rivaroxaban may have a direct arteriodilatory effect. This arteriodilatory effect of rivaroxaban may provide a possible explanation for dizziness and headaches experienced by some rivaroxaban treated patients. Further work is required to determine the underlying mechanism of rivaroxaban-mediated vaso-relaxant effect as well as determining if the other Xa inhibitors apixaban and edoxaban also share this physiological effect, and if so, to what extent. [Display omitted] DisclosuresPatel:Bayer plc: Research Funding. Arya:Bayer plc: Research Funding.

Biologically active vallesamine, strychnan, and rhazinilam alkaloids from Alstonia: Pneumatophorine, a nor-secovallesamine with unusual incorporation of a 3-ethylpyridine moiety

Four alkaloids comprising two vallesamine, one strychnan, and one pyranopyridine alkaloid, in addition to 32 other known alkaloids were isolated from two Malayan Alstonia species, Alstonia pneumatophora and Alstonia rostrata. The structures of these alkaloids were determined using NMR and MS analyses, and in one instance, confirmed by X-ray diffraction analysis. The nor-6,7-secovallesamine alkaloid, pneumatophorine, is notable for an unusual incorporation of a 3-ethylpyridine moiety in a monoterpenoid indole. The rhazinilam-type alkaloids (rhazinicine, nor-rhazinicine, rhazinal, and rhazinilam) showed strong cytotoxicity toward human KB, HCT-116, MDA-MB-231, and MRC-5 cells, while pneumatophorine, the uleine alkaloid undulifoline, and the strychnan alkaloids, N4-demethylalstogustine and echitamidine, induced concentration dependent relaxation in phenylephrine-precontracted rat aortic rings.

Magnesium and diltiazem relaxes phenylephrine-precontracted rat aortic rings

Perioperative vasospasm during cardiovascular surgery is a challenging problem. Several vasodilator agents are frequently utilized for its prevention in surgical practice. Magnesium and diltiazem both have known potential vasorelaxant effects. We planned to compare the efficacy of diltiazem and magnesium in relieving phenylephrine-precontracted rat aortic rings. Ten young adult female Wistar albino rats weighing 230-260 g were used in this study. The aortic rings in the organ bath equilibrated and reached their baseline tension. Precontraction was induced by 0.001 mmol/l phenylephrine and cumulative concentration-relaxation curves were obtained by consecutively increasing the addition of either diltiazem (10(-6)-0.1 mmol/l) or magnesium (0.1-10 mmol/l). The mean maximal relaxation responses observed by diltiazem and magnesium on separate aortic rings were 90 ± 3 and 53 ± 2%, respectively. The calculated EC50 of diltiazem was 0.01035 mmol/l, whereas the EC50 of magnesium was 4.064 mmol/l (P < 0.05). Both magnesium and diltiazem produced vasorelaxation on phenylephrine-precontracted rat aortic rings in this study, but the potency of diltiazem regarding the EC50 value was significantly higher than that of magnesium. Magnesium could be a candidate together with diltiazem to inhibit vasospasm on arterial grafts during coronary bypass surgery.

A potent vasoactive cytolysin isolated from Scorpaena plumieri scorpionfish venom

A new vasoactive cytolytic toxin, referred to as Sp-CTx, has been purified from the venom of the scorpionfish Scorpaena plumieri by a combination of gel filtration and anion exchange chromatographies. An estimation of Sp-CTx native molecular mass, performed by size exclusion chromatography, demonstrated that it is a 121 kDa protein. Further physicochemical studies revealed its glycoproteic nature and dimeric constitution, comprising subunits of approximately 65 kDa (MALDI-TOF-MS). Such protein has proved to possess a potent hemolytic activity on washed rabbit erythrocytes (EC 50 0.46 nM), whose effect was strongly reduced after treatment with antivenom raised against stonefish venom – Synanceja trachynis (SFAV). This cross-reactivity has been confirmed by western blotting. Like S. plumieri whole venom (100 μg/mL), Sp-CTx (1–50 nM) caused a biphasic response on phenylephrine pre-contracted rat aortic rings, characterized by an endothelium- and dose-dependent relaxation phase followed by a contractile phase. The vasorelaxant activity has been abolished by l-NAME, demonstrating the involvement of nitric oxide on the response. We report here the first isolation of a cytolytic/vasoactive protein from scorpionfish venom and the data provided suggest structural and functional similarities between Sp-CTx and previously published stonefish hemolytic toxins.

Vasoconstrictive effect of hydrogen sulfide involves downregulation of cAMP in vascular smooth muscle cells

Hydrogen sulfide (H(2)S), a new endogenous mediator, produces both vasorelaxation and vasoconstriction. This study was designed to examine whether cAMP mediates the vasoconstrictive effect of H(2)S. We found that NaHS at a concentration range of 10-100 microM (yields approximately 3-30 microM H(2)S) concentration-dependently reversed the vasodilation caused by isoprenaline and salbutamol, two beta-adrenoceptor agonists, and forskolin, a selective adenylyl cyclase activator, in phenylephrine-precontracted rat aortic rings. Pretreatment with NaHS (10-100 microM) for 5 min also significantly attenuated the vasorelaxant effect of salbutamol and forskolin. More importantly, NaHS (5-100 microM) significantly reversed forskolin-induced cAMP accumulation in vascular smooth muscle cells. However, NaHS produced significant, but weaker, vasoconstriction in the presence of N(G)-nitro-l-arginine methyl ester (100 microM), a nitric oxide synthase inhibitor, or in endothelium-denuded aortic rings. Blockade of ATP-sensitive potassium channels with glibenclamide (10 microM) failed to attenuate the vasoconstriction induced by H(2)S. Taken together, we demonstrated for the first time that the vasoconstrictive effect of H(2)S involves the adenyly cyclase/cAMP pathway.

Sesamin Metabolites Induce an Endothelial Nitric Oxide-Dependent Vasorelaxation through Their Antioxidative Property-Independent Mechanisms: Possible Involvement of the Metabolites in the Antihypertensive Effect of Sesamin

Sesamin, a major lignan in sesame seeds and oil, has been known to lower blood pressure in several types of experimental hypertensive animals. A recent study demonstrated that sesamin metabolites had in vitro radical-scavenging activities. Thus, we determined whether the antioxidative effect of sesamin metabolites modulate the vascular tone and contribute to the in vivo antihypertensive effect of sesamin. We used four demethylated sesamin metabolites: SC-1m (piperitol), SC-1 (demethylpiperitol), SC-2m [(1R,2S,5R,6S)-6-(4-hydroxy-3-methoxyphenyl)-2-(3,4-dihydroxyphenyl)-3,7-dioxabicyclo[3,3,0]octane], and SC-2 [(1R,2S,5R, 6S)-2,6-bis(3,4-dihydroxyphenyl)-3,7-dioxabicyclo-[3,3,0]octane]. SC-1, SC-2m, and SC-2, but not SC-1m, exhibited potent radical-scavenging activities against the xanthine/xanthine oxidase-induced superoxide production. On the other hand, SC-1m, SC-1, and SC-2m produced endothelium-dependent vasorelaxation in phenylephrine-precontracted rat aortic rings, whereas SC-2 had no effect. The SC-1m- and SC-1-induced vasorelaxations were markedly attenuated by pretreatment with a nitric oxide synthase (NOS) inhibitor, NG-nitro-L-arginine (NOARG), or a soluble guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one. Neither SC-1m nor SC-1 changed the expression level of endothelial NOS protein in aortic tissues. The antihypertensive effects of sesamin feeding were not observed in chronically NOARG-treated rats or in deoxycorticosterone acetate-salt-treated endothelial NOS-deficient mice. These findings suggest that the enhancement of endothelium-dependent vasorelaxation induced by sesamin metabolites is one of the important mechanisms of the in vivo antihypertensive effect of sesamin.

A novel capsaicin derivative VOA induced relaxation in rat mesenteric and aortic arteries: involvement of CGRP, NO, cGMP, and endothelium-dependent activities.

The vasorelaxant effects of N-[4-O-[2-methoxy, phenoxyethylaminobutyl]-3-methoxy benzyl]-nonamide (VOA), a novel capsaicin derivative, and associated releasing activities of nitric oxide (NO) and calcitonin gene-related peptide (CGRP) were investigated in this study. Systemic administration of VOA decreased blood pressure and heart rate in a dose-dependent manner in both normotensive as well as spontaneously hypertensive rats. Nw-nitro-L-arginine methyl ester (L-NAME), glibenclamide, and capsazepine inhibited VOA-induced hypotension. In phenylephrine-precontracted rat aortic rings and mesenteric arteries with intact endothelium, VOA caused a concentration-dependent relaxation. This relaxation was reduced after endothelium was removed or pretreated with L-NAME, methylene blue, 1 H-[1,2,4]oxidazolol [4,3-a] quinoxalin-1-one, tetraethylammonium, glibenclamide, CGRP (8-37), or capsazepine, respectively. In endothelially denuded vessel rings, tetraethylammonium, glibenclamide, CGRP (8-37), and capsazepine also reduced VOA-induced relaxation. In high potassium (80 mmol/L)-precontracted rat aortic rings with intact endothelium, VOA failed to induce relaxation. VOA induced a concentration-dependent increase of CGRP-like enzyme immunoreactivity, which was also significantly inhibited by capsazepine. In human umbilical vein endothelial cells, VOA increased NO release and guanosine-3', 5'-cyclic monophosphate level, which were significantly inhibited by L-NAME. The Western blot analysis on human umbilical vein endothelial cells indicated that VOA increased the expression of endothelium nitric oxide synthase. In conclusion, VOA might exert its relaxation effects in rat vascular smooth muscle through the CGRP/KATP channel and the NO/ cGMP pathway.

Restricted ability of human mast cell tryptase to activate proteinase-activated receptor-2 in rat aorta.

We investigated the potential of human mast cell tryptase to induce relaxation of rat aorta. Trypsin and the selective PAR2-activating peptide (PAR2-AP) SLIGRL-NH2 stimulated robust relaxation of phenylephrine-precontracted rat aortic rings. However, human lung tryptase (1-100 nM) either in the presence or absence of heparin failed to induce any significant relaxation. Notwithstanding, incubation of the aorta with tryptase (100 nM), following the addition of a peptide corresponding to the cleavage/activation sequence of rat PAR2 (rPAR2), resulted in relaxation of precontracted tissue due to the proteolytic release of the PAR2-AP SLIGRL/ from the parent peptide. Thus, tryptase was enzymatically active in the bioassay system. Preincubation of aorta with neuraminidase to remove cell-surface sialic acid unmasked the ability of tryptase to induce relaxation of the aorta, but had no effect on relaxation induced by trypsin, SLIGRL-NH2, or acetylcholine (Ach). Like trypsin and SLIGRL-NH2, the tryptase-induced relaxation was inhibited by either removal of the endothelium or pretreatment of the tissue with NG-nitro-L-arginine methyl ester (L-NAME), suggesting an endothelium-derived nitric oxide mechanism. Interestingly, tryptase in the presence of heparin failed to induce relaxation of precontracted neuraminidase-treated rat aorta. We conclude that tryptase-induced relaxation of rat aorta, most likely via PAR2, is tightly regulated by heparin and cell-surface sialic acid.

Relaxant effect of 2-methyl-thio-adenosine diphosphate on rat thoracic aorta: effect of clopidogrel

The main aim of this study was to determine the functional effect of 2-methyl-thio-adenosine diphosphate (2MeS-ADP) on vascular purinoceptors, in comparison with that of a characterised agonist of the P2Y1 receptor, 2-methyl-thio-adenosine triphosphate (2MeS-ATP), and of the P2Y2 receptor, uridine triphosphate (UTP). On phenylephrine-precontracted rat aortic rings, mounted isometrically in organ baths, we found that 2MeS-ADP (10 −9 to 10 −6 M) induced concentration-dependent relaxation of rings with a functional endothelium. Mechanical removal of the endothelium abolished the relaxant effect of 2MeS-ADP. The 2MeS-ADP-induced relaxation of phenylephrine-precontracted rings was inhibited by Nω-nitro- l-arginine methyl ester ( l-NAME) (100 μM) but not by indomethacin (100 μM) or aspirin (1 mM), indicating that the 2MeS-ADP-induced relaxation was nitric oxide (NO) synthase-mediated but not cyclooxygenase-dependent. Repeated stimulation with 2MeS-ADP resulted in desensitisation of the receptor. Under these conditions, the relaxant effect of 2MeS-ATP was abolished. On the contrary, UTP-induced relaxation was not affected, showing that 2MeS-ADP and 2MeS-ATP but not UTP shared the same receptor. Suramin (100 μM), a non-specific P2 inhibitor, abolished the effect of 2MeS-ADP, 2MeS-ATP and UTP. In contrast, pyridoxal-phosphate-6-azophenyl-2′-4′-disulphonic acid (PPADS) and adenosine-3′-phosphate-5′-phosphosulphate (A3P5PS) abolished only the vasodilator responses to 2MeS-ADP and 2MeS-ATP and did not affect the relaxant effect of UTP, showing that 2MeS-ADP acted through the P2Y1 receptor. Clopidogrel, a potent platelet ADP receptor antagonist, at a dose that strongly inhibited ADP-induced platelet aggregation ex vivo, did not modify the relaxant responses to 2MeS-ADP or 2MeS-ATP. In conclusion, these results showed that 2MeS-ADP induces endothelium-dependent, NO-mediated relaxation of rat aortic rings. This effect, resistant to clopidogrel treatment, occurred through activation of the P2Y1 receptor.

Cardiovascular effects of a novel, potent and selective phosphodiesterase 5 inhibitor, DMPPO: in vitro and in vivo characterization.

1. The aim of this study was to investigate the cardiovascular effects of a novel, potent and specific phosphodiesterase 5 (PDE 5) inhibitor, 1,3 dimethyl-6-(2-propoxy-5-methane sulphonylamidophenyl)-pyrazolo[3,4-d]pyrimidin-4-(5H)-one (DMPPO) in phenylephrine-precontracted rat aortic rings and different in vivo rat preparations. 2. DMPPO elicited a concentration-dependent relaxation of rat aortic rings with functional endothelium. DMPPO-induced relaxation was abolished by endothelium removal or pretreatment with the soluble guanylate cyclase inhibitor, methylene blue (10 microM). 3. In aortic rings without endothelium, the potency (pD2= -log10 EC50) of atrial natriuretic peptide (ANP) to induce relaxation increased from 8.13 +/- 0.05 in the absence of DMPPO, to 8.32 +/- 0.05 and 8.52 +/- 0.08 in the presence of 30 nM and 100 nM DMPPO, respectively. Similarly, the potency of sodium nitroprusside (SNP) in inducing relaxation increased from 7.38 +/- 0.07 in the absence of the PDE 5 inhibitor to 8.07 +/- 0.11 and 8.15 +/- 0.08 in the presence of 30 nM and 100 nM DMPPO, respectively. In contrast, relaxation to the adenylate cyclase activator, forskolin, was unchanged by DMPPO (100 nM). 4. In rings without endothelium, DMPPO (100 nM) increased by 2.5 fold intracellular levels of guanosine 3':5'-cyclic monophosphate (cyclic GMP). Moreover, DMPPO (100 nM) potentiated the increases in cyclic GMP levels induced by ANP (30 nM) by 3 fold and SNP (30 nM) by 2.7 fold. Adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels were not modified by DMPPO. 5. In anaesthetized normotensive or spontaneously hypertensive rats (SHR), DMPPO (2 and 5 mg kg-1, i.v.) lowered blood pressure without affecting heart rate. Similarly, in conscious SHR, orally administered DMPPO (5 mg kg-1) induced a 25 mmHg decrease in blood pressure for at least 7 h without modifying heart rate. Meanwhile, urinary cyclic GMP was increased by 50% whereas cyclic AMP remained unchanged. 6. In normotensive anaesthetized rats, sodium nitroprusside (SNP) (i.v. bolus) induced a decrease in blood pressure which rapidly returned to baseline. In DMPPO (1 mg kg-1, i.v.)-treated rats, the hypotensive effects of SNP (10 to 100 micrograms kg-1) were prolonged over time whereas the peak effect was unchanged. 7. In pithed rats, phenylephrine (i.v. bolus) induced dose-dependent increases in blood pressure. Pretreatment with DMPPO (5 mg kg-1, i.v.) partially inhibited the pressor response to phenylephrine (0.3 to 100 micrograms kg-1). 8. In conclusion, the potent and selective PDE 5 inhibitor, DMPPO, produces relaxation in isolated vessels in the presence of a cyclic GMP drive and reduces blood pressure of intact animals. Its high oral bioavailability and long duration of action should make it a useful tool to study the role of cyclic GMP in various biological systems.

Endothelium-dependent and -independent vasodilation of isolated rat aorta induced by caffeine.

Caffeine (10(-4)-10(-3) M) induced concentration-dependent relaxations of phenylephrine-precontracted rat aortic rings with endothelium. Endothelial denudation significantly, but only partially, attenuated caffeine-induced relaxation. Pretreatment with NG-nitro-L-arginine, oxyhemoglobin, and methylene blue attenuated the relaxations to an extent similar to endothelial denudation. Guanosine 3',5'-cyclic monophosphate (cGMP) and adenosine 3',5'-cyclic monophosphate (cAMP) contents of aortic strips with endothelium increased significantly after exposure to caffeine (10(-3) M). Endothelial denudation attenuated caffeine-induced cGMP increase. Pretreatment with ryanodine (2 x 10(-5) M), which has been shown to combine with receptors on endoplasmic reticulum (ER) of endothelium, attenuated caffeine-induced relaxation and cGMP content increase of rings with endothelium. Pretreatment with caffeine potentiated sodium nitroprusside-induced relaxations and cGMP increase of rings without endothelium. These results demonstrated that caffeine-induced relaxation comprises two components. In the endothelium-dependent mechanism, caffeine promotes nitric oxide synthesis in endothelium by release of Ca2+ from ER through a ryanodine-sensitive Ca2+ channel, and the suppression of cGMP degradation also contributes to the relaxation. In the endothelium-independent mechanism, caffeine acts as a 3',5'-cyclic-nucleotide phosphodiesterase inhibitor.

Vasorelaxant effect of isoliquiritigenin, a novel soluble guanylate cyclase activator, in rat aorta.

1. The vasorelaxant activity of isoliquiritigenin, isolated from Dalbergia odorifera T, was investigated in the phenylephrine-precontracted rat aorta by measuring tension, guanylate and adenylate cyclase activities, guanosine 3':5'-cyclic monophosphate (cyclic GMP) and adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels. 2. Isoliquiritigenin concentration-dependently relaxed rat aorta contracted with phenylephrine, KCl, U-46619, endothelin and 5-hydroxytryptamine, with EC50s of 7.4 +/- 1.6, 10.5 +/- 2.3, 14.3 +/- 3.3, 11.8 +/- 2.0 and 13.6 +/- 3.7 microM, respectively. 3. Isoliquiritigenin caused endothelium-independent relaxation of phenylephrine-precontracted rat aortic rings. Neither NG-monomethyl-L-arginine (L-NMMA) (an inhibitor of the L-arginine-NO pathway) nor oxyhaemoglobin (which binds NO) modified the relaxant effect of isoliquiritigenin. The relaxant action of isoliquiritigenin also persisted in intact aorta in the presence of indomethacin or glibenclamide. However, methylene blue, an inhibitor of soluble guanylate cyclase, abolished relaxation induced by isoliquiritigenin. 4. Incubation of rat aorta with isoliquiritigenin not only increased aortic cyclic GMP content but also caused small increases in aortic cyclic AMP content, and greatly potentiated the increases in cyclic AMP observed in the presence of forskolin. The maximum increase in cyclic GMP by isoliquiritigenin was reached earlier than the increase in cyclic AMP. This result suggests that the increases in cyclic GMP caused by isoliquiritigenin might stimulate the accumulation of cyclic AMP. 5. Concentration-dependent increases in soluble guanylate cyclase activity were observed in isoliquiritigenin (1-100 microM)- or sodium nitroprusside (SNP)-treated rat aortic smooth muscle cells, while adenylate cyclase activity was unchanged in isoliquiritigenin (100 microM)-treated cells. 6. Relaxation and cyclic AMP formation of rat aorta caused by isoliquiritigenin was potentiated in the presence of forskolin (10 nM), which had little effect when given alone. 2',5'-Dideoxyadenosine (DDA,200 microM), an adenylate cyclase inhibitor, diminished the relaxation and cyclic AMP formation of rat aorta by isoliquiritigenin only in the presence of forskolin. DDA did not affect the increases in cyclic GMP formation induced by isoliquiritigenin. These results suggest that elevated levels of cyclic GMP may mediate the majority of the relaxation of the phenylephrine-precontracted aorta induced byisoliquiritigenin, while the synergistic interaction with a low concentration of forskolin depends on an enhanced accumulation of cyclic AMP.7. Relaxation of phenylephrine-precontracted rat aorta and carbachol-precontracted guinea-pig trachea by rolipram (phosphodiesterase, PDE IV inhibitor) was markedly enhanced by isoliquiritigenin, while response to cilostamide (PDE III inhibitor) was not significantly changed by isoliquiritigenin.8. It is concluded that isoliquiritigenin exerts a vasorelaxant effect by activating soluble guanylatecyclase and increasing cyclic GMP. Synergistic effects of isoliquiritigenin and forskolin on muscle relaxation and cyclic AMP accumulation indicate that inhibition of cyclic AMP breakdown by cyclic GMP via the inhibition of PDE III (cyclic GMP-inhibited PDE) is the dominant mechanism.

Alteration of lysophosphatidylcholine content in low density lipoprotein after oxidative modification: relationship to endothelium dependent relaxation.

The aim was to examine the formation of lipid peroxidation products and the alteration in phospholipid content in low density lipoprotein (LDL) after oxidative modification by CuSO4, and subsequently, to determine the ability of the modified LDL to impair endothelium dependent relaxation in rat aortic rings. Blood samples were obtained from normal human volunteers. LDL was prepared by sequential ultracentrifugation and it was oxidatively modified in the presence of 5 microM CuSO4. Lipid peroxidation products (thiobarbituric acid reactive substances, TBARS), and alterations in electrophoretic mobility and phospholipid content were determined in normal (native) and oxidised LDL. Endothelium dependent relaxation was produced by acetylcholine (10(-8)-10(-5) M) in phenylephrine precontracted rat aortic rings. LDL incubated for 24 h with 5 microM CuSO4 at 20 degrees C and 37 degrees C with constant agitation displayed higher amounts of TBARS than the respective native LDL. While the amounts of TBARS in LDL modified at 20 degrees C and 37 degrees C were similar, the former condition resulted in statistically smaller changes of phospholipid contents. LDL with higher lysophosphatidylcholine content showed greater impairment of endothelium dependent relaxation in rat aortic rings than LDL with lower lysophosphatidylcholine content. The raised lysophosphatidylcholine level in oxidatively modified LDL was related to the ability of the LDL to impair endothelium dependent relaxation. However, lipid peroxidation products assessed by TBARS did not relate to the phospholipid changes in LDL and therefore cannot be used to predict the vascular effects of LDL after oxidative modification.