Endothelium-intact Aortic Rings Research Articles

Evidence against α2‐adrenoceptors mediating relaxation in rat thoracic aortae: α2‐agonists relaxation depends on interaction with α1‐adrenoceptors

In rat aorta, the presence of functional alpha(2)-adrenoceptors (alpha(2)-AR) was investigated in ring preparations preconstricted with alpha(1)-adrenergic and non- alpha(1)-adrenergic agonists. Particularly, the hypothetical interference of alpha(2)-AR agonists with alpha(1)-AR-mediated vasoconstriction was evaluated. Relaxant and contractile responses to alpha(2)-AR agonists were obtained. In endothelium-intact and endothelium-denuded aortic rings preconstricted with phenylephrine (1 x 10(-6) m), the imidazoline derivatives, clonidine and UK14304, induced relaxations with similar order of potencies (-log EC(50)) and maxima relaxant effects respectively. Pretreatment with the NO synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME) had no effect on the relaxant responses to clonidine and UK14304. In phenylephrine-constricted rings with endothelium, relaxations to clonidine and UK 14304 were not antagonized by the selective alpha(2)-AR antagonist, rauwolscine (< or =1 x 10(-6) m). Clonidine and UK 14304 induced only contractions on endothelium-intact and endothelium-denuded aortic rings contracted with prostaglandin F(2alpha) (3 x 10(-7) m). Moreover, clonidine and UK 14304-induced relaxation of endothelium-denuded arteries precontracted with methoxamine but not with serotonin. Finally, the concentration-contraction curves to clonidine and UK 14304 in endothelium-denuded aortic rings were significantly shifted to the right by the alpha(1D)-AR selective antagonist, BMY 7378, and rauwolscine. The pA(2) and pK(B) values for BMY 7378 and rauwolscine, respectively, against endothelium-independent actions of clonidine and UK 14304 were characteristic of an effect on the alpha(1D)-AR. The other selective alpha(2)-AR agonist tested BHT 933 (an azepine derivative), lacks considerable relaxant and contractile effects in rat aorta. The results provide no evidence for the presence of functional alpha(2)-AR in rat aorta. Respectively, the relaxant and contractile effects of the imidazoline derivatives, clonidine and UK 14304, may be due to an adjustable (in relation to the agonist-dependent active state of the alpha(1)-AR), inhibitory and excitatory, interaction with alpha(1)-ARs.

Vasorelaxant effects of harmine and harmaline extracted from Peganum harmala L. seed's in isolated rat aorta

The present work describes the mechanisms involved in the vasorelaxant effect of harmine and harmaline. These alkaloids induce in a dose-dependent manner the relaxation in the aorta precontracted with noradrenaline or KCl. However, the removal of endothelium or pre-treatment of intact aortic ring with L-NAME (inhibitor of NOSe synthetase) or with indomethacin (non-specific inhibitor of cyclo-oxygenase), reduces significantly the vasorelaxant response of harmaline but not harmine. According to their IC 50 values, prazosin (inhibitor of α-adrenorecepteors) reduces the vasorelaxant effect only of harmaline, whereas, pre-treatment with IBMX (non-specific inhibitor of phosphodiesterase) affects both the harmaline and harmine-responses. Inhibitions of L-type voltage-dependent Ca 2+ channels (VOCs) in endothelium-intact aortic rings with diltiazem depress the relaxation evoked by harmaline as well as by harmine. Pre-treatment with harmaline or harmine (3, 10 or 30 μM) shifted the phenylephrine-induced dose response curves to the right and the maximum response was attenuated indicating that the antagonist effect of both alkaloids on α 1-adrenorecepteors was non-competitive. These two alkaloids also exert an antioxidant activity by scavenging the free radical generated by DPPH. Therefore, the present results suggest that the vasorelaxant effect of harmaline but not harmine is related to its action on the prostacyclin pathway and on the endothelial cells to release NO. However, both alkaloids can act as blockers VOCs, as inhibitors of phosphodiesterase resulting in an increase of the second messenger (cAMP and cGMP) levels and finally reduce the levels of free radicals in tissues.

Vasoactive intestinal peptide‐induced relaxation is mediated by nitric oxide activation in the endothelial caveolae of rat aorta

Vasoactive intestinal peptide (VIP) a non-adrenergic non-cholinergic (NANC) transmitter widely distributed in both the central and peripheral nervous system is an endothelium-dependent vasodilator. The contribution of endothelium derived relaxing factors to VIP-induced relaxation remains unclear. We hypothesized that nitric oxide (NO) produced by endothelial NO synthase (eNOS) at the caveolae plays a role in this relaxation. We assessed the effect of VIP on isolated rat aorta rings in vitro which was incubated with either N-nitro-L-Arginine, L-NNA, (a NOS inhibitor), indomethacin (a cycloxygenase inhibitor), or methyl-beta-cyclodextrin (a membrane sequester that disassembles caveolae). In intact and denuded aortic rings precontracted with phenylephrine (PE, 0.1 μmol/L), the addition of VIP (1nmol/L to 1 μmol/L) produced relaxation in a dose-dependent manner. The maximal relaxation induced by VIP in endothelium-intact aortic rings (53 ± 9%, n=8) was impaired after removal of the endothelium (11 ± 2%, n=3, P<0.05), or in the presence of L-NNA (20 ± 11%, n=5, P <0.05) and methyl-β-cyclodextrin (20 ± 8%, n=6, P<0.05). In contrast, VIP-induced relaxation was enhanced in the presence of indomethacin (83 ± 7%, n=5, P<0.05). These data suggest that NO produced by eNOS at the caveolae plays a stimulatory role in VIP-induced relaxation in rat aorta, whereas prostaglandins play an inhibitory role.

Effect of tempol on altered angiotensin II and acetylcholine-mediated vascular responses in thoracic aorta isolated from rats with insulin resistance

The altered vascular responses to various vasopressors and relaxants have been well reported in various animal models of hypertension, insulin resistance and diabetes. Though the role of oxidative stress (increased superoxide levels) associated with these altered vascular responses in hyperglycemic/diabetic state is well documented, the role of the same remains to be largely unknown in vascular dysfunction coupled with prediabetic insulin resistant state. The objective of the present study was therefore to elucidate the role of free radicals particularly superoxides if any associated with vascular dysfunction in diet-induced insulin resistance of rats. In this regard, the effect of tempol (a membrane permeable superoxide dismutase mimetic/free radical scavenger) on the enhanced Ang II-induced contraction and impaired-ACh mediated relaxation in thoracic aorta of rats with insulin resistance was studied. Ang II-induced contraction and ACh-mediated relaxation responses were recorded isometrically in endothelium intact and denuded thoracic aortic ring preparations isolated from male Sprague–Dawley rats which were fed with either normal pellet diet (NPD) (control group) or high fat diet (HFD) (insulin resistant group) for 4 weeks. The HFD-fed rats exhibited characteristic features of insulin resistance syndrome viz., obesity, hyperinsulinaemia, mild hyperglycemia, hypertriglyceridemia, hypercholesterolemia, glucose intolerance and hypertension. Maximal contractile response ( E max) to Ang II was increased in endothelium intact aortic ring preparations obtained from HFD-fed rats as compared to NPD-fed control rats. Denudation of endothelium significantly increased Ang II-mediated E max responses in thoracic aortic rings of NPD-fed rats, whereas it produced only minimal alteration to the E max in the HFD-fed rats. In addition, ACh-mediated relaxation response was impaired in endothelium intact aortic rings isolated from HFD-fed rats. Tempol (30–300 μM) significantly and dose dependently inhibited enhanced vascular responses ( E max) of Ang II in endothelium intact, but not in endothelium denuded aortic ring preparations. Tempol (30 μM) reversed the impaired acetylcholine (ACh)-mediated relaxations in endothelium intact aortic ring preparations of HFD-fed rats. Endothelium independent vasorelaxations (EIV) to sodium nitroprusside (SNP) were similar for both NPD and HFD. In conclusion, our results indicate that superoxide radicals play crucial role in enhanced contractile and impaired vasodilatory responses to Ang II and ACh, respectively, in thoracic aortic rings isolated from diet-induced insulin resistant rats.

Nitric oxide-dependent vasorelaxation induced by extractive solutions and fractions of Maytenus ilicifolia Mart ex Reissek (Celastraceae) leaves

This study reveals that an ethanolic supernatant obtained from an aqueous extractive solution prepared from residues of methanolic extracts of ground leaves of Maytenus ilicifolia is able to cause a concentration- and endothelium-dependent relaxation in pre-contract rat aorta rings, with EC 50 of 199.7 (190–210) μg/ml. The non-selective nitric oxide synthase inhibitors l-NAME and l-NMMA abolished this effect, while superoxide dismutase and MnTBAP (a non-enzymatic superoxide dismutase mimetic) enhanced it. Further, relaxation induced by this ethanolic supernatant have been strongly inhibited by the guanylate cyclase inhibitors methylene blue and ODQ, as well as by the potassium channel blockers 4-aminopyridine and tetraethylammonium, but was unchanged by the cyclooxigenase inhibitor indomethacin and the membrane receptor antagonists atropine, HOE-140 and pirilamine. Partition of the ethanolic supernatant between H 2O and EtOAc generated a fraction several times more potent, able to fully relax endothelium-intact aorta rings with an EC 50 of 4.3 (3.9–4.8) μg/ml. 13C NMR spectrum of this fraction showed signals typical of catechin. This study reveals that the leaves of M. ilicifolia possess one or more potent substances able to relax endothelium-intact rat aorta rings, an event that appears to involve nitric oxide production, guanylate cyclase activation and potassium channel opening.

Chronic treatment of DA-8159, a new phosphodiesterase type V inhibitor, attenuates endothelial dysfunction in stroke-prone spontaneously hypertensive rat

This study examined the effects of chronic treatment of a new phosphodiesterase type 5 inhibitor, DA-8159, on endothelial dysfunction in stroke-prone spontaneously hypertensive rats (SHR-SP). Six-week-old male SHR-SP were divided into 4 groups; vehicle control, DA-8159 1, 3, and 10 mg/kg/day. During a 32-week experimental period, the animals were administered DA-8159 orally and fed a 4% NaCl-loaded diet. The systolic blood pressure was measured every two weeks throughout the experimental period using the tail-cuff method. At the end of experiments, the vascular function (acetylcholine-induced vasodilation) in the endothelium-intact aortic rings was investigated. In addition, the mortality, the left ventricular hypertrophy index, the plasma parameters and the incidence of a cerebral infarction were assessed. In the DA-8159 treated-rats, the vascular reactivity improved significantly in a dose-dependent manner. Although DA-8159 did not alter the elevation of the systolic blood pressure directly, the 3 and 10 mg/kg/day DA-8159 treatment delayed the early death caused by stroke. DA-8159 significantly reduced the left ventricular heart weight / body weight ratio compared with the vehicle control group. Furthermore, the DA-8159 treatment significantly increased the plasma nitric oxide, cGMP, and the total antioxidative status. The DA-8159 treatment also reduced the occurrence of stroke-associated cerebral damage. These results indicate that DA-8159 can ameliorate an endothelial dysfunction-related vascular injury. Therefore, pharmacological intervention aimed at attenuating an endothelial dysfunction is important and might be useful in both preventing and treating endothelial dysfunction-related complications.

The effect of DA-8159, a novel PDE5 inhibitor, on erectile function in the rat model of hypercholesterolemic erectile dysfunction

This study examined the effects of a new phosphodiesterase type 5 inhibitor, DA-8159, on erectile function associated with hypercholesterolemia. First of all, in order to investigate whether chronic administration of DA-8159 prevents the development of erectile dysfunction associated with hypercholesterolemia, male SD rats were divided into four groups (normal control, hypercholesterolemic control, DA-8159 5 or 20 mg/kg/day). Over a 5-month period, the animals were fed a 2% cholesterol diet and administered DA-8159 orally once a day. After 5 months, the electrostimulation-induced penile erection and the vascular function using acetylcholine-induced vasodilation with endothelium-intact aortic rings were examined. Furthermore, the plasma lipid profiles, endothelin and N(G),N(G)-dimethylarginine (asymmetrical dimethylarginine, ADMA) concentrations were measured. In order to investigate the acute treatment effect of DA-8159 on the erectile function in an established hypercholesterolemic model, additional animals were given a 2% cholesterol diet for 5 months without DA-8159. At the end of 5 months, the rats were divided into three groups (hypercholesterolemic control, DA-8159 0.3 or 1 mg/kg). DA-8159 was administered intravenously 1 min prior to the intracavernous pressure (ICP) measurement. In a chronic treatment study, while the hypercholesterolemic control showed a significantly lower erectile function, vascular reactivity, and increased plasma cholesterol, endothelin and ADMA concentration, the chronic DA-8159 treatment clearly restored the erectile responses by electric stimulation, preserved the potential of thoracic aortic relaxation in a dose-dependent manner, and significantly decreased the plasma endothelin and ADMA concentrations. In an acute treatment study, DA-8159 induced a dose- and frequency-dependent increase in ICP. The ICP/BP ratio and the corresponding AUC values, and the detumescence time were also significantly increased compared to the hypercholesterolemic control. These results suggest that DA-8159 is beneficial for erectile dysfunction in a rat hypercholesterolemic model and provided a rationale for the potential use of DA-8159 for treating erectile dysfunction secondary to hypercholesterolemia.

Dynamic Association of Nitric Oxide Downstream Signaling Molecules with Endothelial Caveolin-1 in Rat Aorta

Classically, nitric oxide (NO) formed by endothelial NO synthase (eNOS) freely diffuses from its generation site to smooth muscle cells where it activates soluble guanylyl cyclase (sGC), producing cGMP. Subsequently, cGMP activates both cGMP- and cAMP-dependent protein kinases [cGMP-dependent protein kinase (PKG) and cAMP-dependent protein kinase (PKA), respectively], leading to smooth muscle relaxation. In endothelial cells, eNOS has been localized to caveolae, small invaginations of the plasma membrane rich in cholesterol. Membrane cholesterol depletion impairs acetylcholine (ACh)-induced relaxation due to alteration in caveolar structure. Given the nature of NO to be more soluble in a hydrophobic environment than in water, and assuming that colocalization of components in a signal transduction cascade seems to be a critical determinant of signaling efficiency by eNOS activation, we hypothesize that sGC, PKA, and PKG activation may occur at the plasma membrane caveolae. In endothelium-intact rat aortic rings, the relaxation induced by ACh, by the sGC activator 3-(5'-hydroxymethyl-2'furyl)-1-benzyl indazole (YC-1), and by 8-bromo-cGMP was impaired in the presence of methyl-beta-cyclodextrin, a drug that disassembles caveolae by sequestering cholesterol from the membrane. sGC, PKG, and PKA were colocalized with caveolin-1 in aortic endothelium, and this colocalization was abolished by methyl-beta-cyclodextrin. Methyl-beta-cyclodextrin efficiently disassembled caveolae in endothelium. In summary, our results provide evidence of compartmentalization of sGC, PKG, and PKA in endothelial caveolae contributing to NO signaling cascade, giving new insights by which the endothelium mediates vascular smooth muscle relaxation.

Inhibition of poly(ADP-ribose) polymerase prevents vascular hyporesponsiveness induced by lipopolysaccharide in isolated rat aorta

Recent studies clearly show that there is a relationship between endotoxemia and impaired vascular responsiveness. The aim of this study was to investigate whether treatment with the new potent PARP inhibitor PJ34 could prevent the vascular hyporesponsiveness induced by lipopolysaccharide (LPS). Endotoxemia was induced in rats by LPS injection (20 mg kg −1, i.p.). Administration of LPS caused a decrease in mean blood pressure and an increase in heart rate. In endothelium-denuded rings of thoracic aorta from untreated rats, contractile responses to KCl and phenylephrine decreased after LPS injection. Furthermore, there was a significant loss of endothelium-dependent vasodilatation in response to acetylcholine in LPS-treated rats. The animals pretreated with PJ34 (10 mg kg −1, i.p., 30 min before LPS injection), the effect of LPS on vascular responsiveness was lower than the untreated ones. Pretreating the animals with PJ34 before the LPS challenge prevented the decline in mean blood pressure. However, this did not result in significant changes to the heart rate. The inhibitory effect of LPS treatment on both KCl- and phenylephrine-induced contraction responses was significantly antagonized by PJ34. Additionally, pretreatment of the rats with PJ34 attenuated the LPS-induced endothelial dysfunction in endothelium-intact aorta rings. This study demonstrates that PARP activation in the vascular system is an important contributory factor to the impaired vascular responsiveness associated with endotoxic shock. Hence, the pharmacological inhibition of PARP pathway might be an effective intervention to prevent endotoxin-induced vascular hyporesponsiveness.

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.

Diphasic Effects of Astragalus membranaceus BUNGE (Leguminosae) on Vascular Tone in Rat Thoracic Aorta

This study was designed to investigate the effects of the aqueous ethanol extract of Astragalus membranaceus BUNGE (Leguminosae) on rat thoracic aorta. Isometric tension was recorded in response to drugs in organ bath. In endothelium-intact aortic rings, A. membranaceus extract induced a significant dose-dependent relaxation of the rings precontracted by phenylephrine, which could be inhibited by preincubation with L-N(omega)-nitro-arginine methyl ester or methylthioninium chloride. In endothelium-denuded ones, the extract could dose-dependently relax the rings contracted by phenylephrine, not by KCl; and it could also attenuate contractile response to phenylephrine, not to caffeine or phorbol-12,13-diacetate in Ca(2+)-free medium; but it failed to affect the CaCl(2)-induced enhancement of contractile response to phenylephrine in Ca(2+)-free medium. These results indicate that nitric oxide signaling and Ca(2+)-handling pathway are involved in the A. membranaceus extract-induced vasodilatation.

Vasodilatation Produced by Orientin and Its Mechanism Study

In this paper we investigated the vascular activity and possible mechanism of Orientin, from bamboo leaves (Phyllostachys nigra), in isolated thoracic aortic rings from New Zealand rabbit. Among the four compounds, studied, only Orientin relaxed phenylephrine-induced contractions with an IC50 value of 2.28 microM in the endothelium intact and with an IC50 value around 7.27 microM in the endothelium removed aortic rings. The vasorelaxant effect of Orientin on endothelium-intact thoracic aortic rings was attenuated by the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine methyl ester, but not by indomethacin (a cyclooxygenase inhibitor), tetraethylammonium chloride (K+ channels inhibitor) or propranolol (beta-receptor inhibitor). Furthermore, Orientin inhibited norepinephrine (NE), CaCl2 and KCl-induced vasoconstriction concentration dependently in a non-competitive manner, and also reduced both the initial fast release and the sustained phases of phenylephrine-induced contractions. Orientin can stimulate NO production from endothelial cells. Orientin also increased cyclic guanosine 3,5-cyclic monophosphate (cGMP) levels without changes in adenosine-3',5'-cyclic phosphoric acid (cAMP) in rabbit aorta. The results showed that Orientin relaxed thoracic aortic rings by the nitric oxide-cGMP pathway, and in the vascular smooth muscle inhibited the contraction induced by the activation of receptor-operating and voltage-dependent Ca2+ channels. Cyclooxygenase pathway, potassium channels, beta-receptors and cAMP pathway, on the other hand, had no apparent roles. The inhibition of both intracellular Ca2+ release and extracellular Ca2+ influx may be one of the main vasorelaxant mechanisms of Orientin.

Novel natriuretic peptides from the venom of the inland taipan ( Oxyuranus microlepidotus): isolation, chemical and biological characterisation

Three natriuretic-like peptides (TNP-a, TNP-b, and TNP-c) were isolated from the venom of Oxyuranus microlepidotus (inland taipan) and were also present in the venoms of Oxyuranus scutellatus canni (New Guinea taipan) and Oxyuranus scutellatus scutellatus (coastal taipan). They were isolated by HPLC, characterised by mass spectrometry and Edman analysis, and consist of 35–39 amino acid residues. These molecules differ from ANP/BNP through replacement of invariant residues within the 17-membered ring structure and by inclusion of proline residues in the C-terminal tail. TNP-c was equipotent to ANP in specific GC-A assays or aortic ring assays whereas TNP-a and TNP-b were either inactive (GC-A over-expressing cells and endothelium-denuded aortic rings) or weakly active (endothelium-intact aortic rings). TNP-a and TNP-b were also unable to competitively inhibit the binding of TNP-c in endothelium-denuded aortae (GC-A) or endothelium-intact aortae (NPR-C). Thus, these naturally occurring isoforms provide a new platform for further investigation of structure–function relationships of natriuretic peptides.

Interaction between endothelial heme oxygenase-2 and endothelin-1 in altered aortic reactivity after hypoxia in rats.

The aim of this study was to determine whether increased expression of heme oxygenase (HO) contributes to impairment of aortic contractile responses after hypoxia through effects on reactivity to endothelin-1 (ET-1). Thoracic aortas from normoxic rats and rats exposed to hypoxia (10% O2) for 16 or 48 h were mounted in organ bath myographs for contractile studies, fixed in paraformaldehyde, or frozen in liquid nitrogen for protein extraction. In rings from normoxic rats, the HO inhibitor tin protoporphyrin IX (SnPP IX, 10 microM) did not alter the response to phenylephrine or ET-1. In rings from rats exposed to 16-h hypoxia, maximum tension generated in response to these agonists was higher in endothelium-intact but not -denuded rings in the presence of SnPP IX. In rings from rats exposed to 48-h hypoxia SnPP IX increased contraction in endothelium-intact but not -denuded rings. In endothelium-intact aortic rings from rats exposed to 16-h hypoxia incubated with endothelin A receptor-specific antagonist BQ-123 (10(-7) M), SnPP IX did not alter phenylephrine-induced contraction. Aortic ET-1 protein levels, measured by radioimmunoassay, were increased in rats exposed to hypoxia for 16 and 48 h. Western blotting showed that HO-1 and HO-2 protein were increased after 16 h of hypoxia and returned to near-control levels after 48 h. Increase in HO-1 protein was detected in endothelium-intact and -denuded rings. Removal of endothelium abolished the increase in HO-2 immunoreactivity. Immunohistochemistry localized expression of HO-1 protein to vascular smooth muscle, whereas HO-2 was only detected in endothelium. HO-2 is expressed by aortic endothelial cells early during hypoxic exposure and impairs ET-1-mediated potentiation of contraction to alpha-adrenoceptor stimulation.

Vasorelaxation by amentoflavone isolated from Selaginella tamariscina.

In the courses of in vitro screening for the vasorelaxant effect of the various extracts from medicinal plants, an ethyl acetate-soluble extract of Selaginella tamariscina was found to exhibit distinctive vasorelaxant activity. Further purifications of the extract as guided by in vitro vasorelaxant assay afforded an active biflavonoid, amentoflavone. Amentoflavone induced concentration-dependent relaxation of the phenylephrine-precontracted aorta, which disappeared by removal of functional endothelium. Pretreatment of the aortic tissues with N(G)-nitro- L-arginine methyl ester (L-NAME), methylene blue, or 1 H- -oxadiazolo[4,3- a]quinoxalin-1-one (ODQ) inhibited the relaxation induced by amentoflavone. Amentoflavone-induced relaxations were also markedly attenuated by addition of tetraethylammonium (TEA) or verapamil. However, the relaxant effect of amentoflavone was not blocked by pretreatment with indomethacin, glibenclamide, atropine, or propranolol. Incubation of endothelium-intact aortic rings with amentoflavone increased the production of cGMP, but this effect was blocked by endothelium-denudation or pretreatment with L-NAME or ODQ. These results suggest that amentoflavone relaxes vascular smooth muscle via endothelium-dependent nitric oxide-cGMP signaling, with possible involvement of non-specific K (+) and Ca (2+) channels. Abbreviations. EDRF:endothelium-derived relaxing factor EDHF:endothelium-derived hyperpolarizing factor NO:nitric oxide cGMP:guanosine 3',5'-cyclic monophosphate DMSO:dimethyl sulfoxide L-NAME: N(G)-nitro- L-arginine methyl ester ODQ:1 H-[1,2,4]-oxadiazole-[4,3- a]-quinoxalin-1-one IBMX:3-isobutyl-1-methylxanthine K (Ca):Ca (2+)-dependent K (+) channel K (ATP):adenosine triphosphate (ATP)-sensitive K (+) channel TEA:tetraethylammonium

Heat-shock response is associated with enhanced contractility of vascular smooth muscle in isolated rat aorta.

Stress proteins have been implicated in pathological cardiovascular conditions. We hypothesized that a heat-shock response modulates contractility of vascular smooth muscles. Rat aortic ring preparations were mounted in organ baths, exposed to 42 degrees C for 45 min, and subjected to contractions. Expression of HSP70 and phosphorylation of myosin light chain were examined with immunoblots. Heat shock enhanced contractile response to KCl in parallel with HSP70 expression in rat aortic rings from 8 h but not 1 h after the end of heat shock. Heat shock also augmented vascular contractility to phenylephrine whether endothelium was intact or denuded. Treatment of heat shock-preconditioned aortic rings with Bay K8644, a calcium channel activator, but not treatment with phorbol dibutyrate (1 micromol/l), a protein kinase C activator, enhanced contractions of the rings as compared with those of the control. The levels of phosphorylation of myosin light chains after administration of phenylephrine in heat shock-preconditioned tissues were statistically significantly higher than those in control tissues. Pretreatment with wortmannin (300 nmol/l), an inhibitor of myosin light chain kinase, decreased both contractility and phosphorylation of myosin light chains in parallel. However, heat-shock response did not affect relaxation responses to either acetylcholine in endothelium-intact aortic rings or sodium nitroprusside in endothelium-denuded rings. These results suggest that the heat-shock response is associated with enhanced vascular smooth muscle contractility through a modulation of thick-filament regulation.

Role of oxidative stress and nitric oxide in regulation of spontaneous tone in aorta of DOCA-salt hypertensive rats.

1. The roles of nitric oxide (NO), superoxide anion (O(2)(-)), and hydrogen peroxide (H(2)O(2)) in the modulation of spontaneous tone were investigated in isolated aorta from deoxycorticosterone acetate (DOCA)-salt hypertensive rats. 2. Increases in preload from 1 to 5 g were accompanied by increases in spontaneous tone in aortic rings from DOCA-salt hypertensive rats but not from SHAM-normotensive rats. 3. Tone was higher in endothelium-denuded aortic rings than in endothelium-intact vessels. Inhibition of nitric oxide synthase (NOS) with 300 microM N(G)-nitro-L-arginine methyl ester (l-NAME) increased spontaneous tone. 4. Basal O(2)(-) generation was higher in aortic rings from DOCA-salt hypertensive rats than in those from SHAM-normotensive rats. Stretch increased O(2)(-) levels even further in the DOCA-salt group. In rings isolated from DOCA-salt hypertensive rats, administration of the O(2)(-) scavenger, superoxide dismutase (SOD, 150 U ml(-1)), or the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase inhibitor, apocynin (100 microM), completely abolished the development of spontaneous tone in endothelium-intact aortic rings but not in endothelium-denuded or in L-NAME-treated rings. SOD and apocynin decreased the generation of O(2)(-) in endothelium-intact, endothelium-denuded, and L-NAME-treated aortic rings. 5. Oral treatment of DOCA-salt hypertensive rats with the O(2)(-) scavengers, tempol or tiron, or with apocynin for 3 weeks prevented the development of hypertension and abolished the increases in O(2)(-) generation and spontaneous tone. 6. Administration of catalase (1000 U ml(-1)) to aortic rings increased spontaneous tone in vessels from DOCA-salt hypertensive rats. 7. Administration of the cyclooxygenase (COX) inhibitor, valeroyl salicylate, or the thromboxane/prostaglandin antagonist, SQ 29548, to aortic rings abolished tone. 8. The results suggest that NO plays a major role in preventing the generation of spontaneous tone in isolated aortic rings from DOCA-salt hypertensive rats. NADPH-oxidase-derived O(2)(-) enhanced spontaneous tone by inactivating NO. Endogenous H(2)O(2) appears to mitigate the increase in tone. In addition, a COX component may also contribute to spontaneous tone.

Evidence for the Mechanisms Underlying the Effects of Pimaradienoic Acid Isolated from the Roots of Viguiera arenaria on Rat Aorta

The present study examines the effects of the diterpene ent-pimara-8(14),15-dien-19-oic acid (PA) on rat thoracic aorta. PA (10^–5, 3 × 10^–5 and 10^–4 mol/l) caused concentration-dependent inhibition of phenylephrine (Phe)-induced contraction in either endothelium-intact or endothelium-denuded aortic rings. PA attenuated the contraction induced by CaCl₂ in Ca²⁺-free solution containing Phe (10^–7 mol/l) or KCl (30 mmol/l). This diterpene did not interfere with Ca²⁺ release from intracellular stores mediated by either Phe (10^–6 mol/l) or caffeine (30 mmol/l). PA (10^–6 to 3 × 10^–4 mol/l) concentration dependently relaxed Phe-pre-contracted rings with intact (92.64 ± 7.60%) or denuded endothelium (98.82 ± 1.56%). Pre-incubation of denuded aortic rings with N^G-nitro-L-arginine methyl ester (10^–4 mol/l), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (10^–6 mol/l) or indomethacin (10^–5 mol/l) reduced PA-induced relaxation (percentage of relaxation: 77.50 ± 3.95, 78.56 ± 2.81, 77.11 ± 6.22, respectively). However, the relaxant responses induced by PA on Phe-pre-contracted rings were unaffected by tetraethylammonium (1 and 5 mmol/l). PA also relaxed KCl-pre-contracted rings with intact (97.44 ± 3.66%) or denuded endothelium (95.95 ± 3.72%). Collectively, these results support the notion that the effects elicited by PA on vascular smooth muscle are endothelium-independent and involve extracellular Ca²⁺ influx blocked. In addition, PA effects are partly dependent on the release of nitric oxide from the vascular smooth muscle through an activation of guanylyl cyclase-dependent mechanism and are related to the release of metabolites derived from the arachidonic acid pathway. Finally, our results demonstrated that the PA relaxant action is not related with the opening of potassium (K⁺) channels.

NO/cGMP signal pathway involved in the disturbance of calcium homeostasis in vascular smooth muscle during the late phase of sepsis

To evaluate the alterations in calcium metabolism of the vascular smooth muscle of rat thoracic aorta in the late phase of sepsis and to investigate the involvement of nitric oxide (NO)/cyclic-GMP(cGMP) signal transduction pathway in the sepsis-induced vascular hyporeactivity. Male Sprague-Dawley rats were subjected to sepsis by cecal ligation and puncture (CLP). Eighteen hours post CLP, rat aortic rings were removed for measurement of contractile responses to vasoconstrictors by using organ bath technique. In endothelium intact aortic rings from CLP rats, concentration-contraction curves to phenylephrine (PE) and high KCl were significantly decreased when compared with those from control rats. The transient contraction induced by PE in calcium-free Krebs solution and the concentration-dependent contraction to CaCl(2)in KCl-depolarized medium were also markedly reduced. The hyporeactivity to vasoconstrictors was completely reversed by pretreatment either with aminoguanidine (AMG), a selective inducible nitric oxide synthase inhibitor, or with 1H [1,2,4] oxadiazolo[4,3-a] quininoxalin-1-one(ODQ), an inhibitor of NO-sensitive guanylyl cyclase. A generalized impairment in calcium handling in vascular smooth muscle,including the calcium influx through the voltage-operated and receptor-operated channels and calcium release from intracellular calcium stores, is involved in vascular hyporeactivity during the late phase of sepsis. The NO/cGMP signal transduction pathway might be involved in this defect in vascular smooth muscle.

Zidovudine-induced alterations in the heart and vascular smooth muscle of the rat.

We investigated the effects of zidovudine (AZT) on cardiac and vascular smooth muscle function and morphology in rats. Four adult male Wistar-Kyoto rats received AZT in drinking water for 240 days; four rats served as controls. Echocardiographic examination and systolic blood pressure (SBP) measurement were performed. At the end of treatment the rats were sacrificed, their hearts were weighed and vascular smooth muscle contractile and relaxing properties were evaluated in vitro on endothelium-intact aortic rings. Morphological studies were performed on cardiac and aortic myocytes by light and electron microscopy. AZT-treated rats (AZT-Rs) showed higher SBP, greater heart weight and, as revealed by echocardiography, greater interventricular septum thickness. Electron microscopy revealed mitochondrial swelling in myocardiocytes in AZT-Rs. Reduced response to contractile stimuli and enhanced relaxation in response to charbacol were observed in the aortic rings of AZT-Rs. The aortic myocytes of AZT-Rs contained apparently unaffected ultrastructural features, but light microscopy suggested their marked enlargement. AZT treatment for 240 days in rats induces a modest increase in SBP, hypertrophy of the interventricular septum and modified vascular smooth muscle responsiveness. The role of mitochondria in these AZT-induced cardiovascular alterations remains to be established.