Endothelium-dependent Contractile Responses Research Articles

Empagliflozin improved systolic blood pressure, endothelial dysfunction and heart remodeling in the metabolic syndrome ZSF1 rat

BackgroundEmpagliflozin (empa), a selective sodium–glucose cotransporter (SGLT)2 inhibitor, reduced cardiovascular mortality and hospitalization for heart failure in patients with type 2 diabetes at high cardiovascular risk independent of glycemic control. The cardiovascular protective effect of empa was evaluated in an experimental model of metabolic syndrome, the obese ZSF1 rat, and its’ lean control.MethodsLean and obese ZSF1 rats were either non-treated or treated with empa (30 mg/kg/day) for 6 weeks. Vascular reactivity was assessed using mesenteric artery rings, systolic blood pressure by tail-cuff sphygmomanometry, heart function and structural changes by echocardiography, and protein expression levels by Western blot analysis.ResultsEmpa treatment reduced blood glucose levels from 275 to 196 mg/dl in obese ZSF1 rats whereas normoglycemia (134 mg/dl) was present in control lean ZSF1 rats and was unaffected by empa. Obese ZSF1 rats showed increased systolic blood pressure, and blunted endothelium-dependent relaxations associated with the appearance of endothelium-dependent contractile responses (EDCFs) compared to control lean rats. These effects were prevented by the empa treatment. Obese ZSF1 rats showed increased weight of the heart and of the left ventricle volume without the presence of diastolic or systolic dysfunction, which were improved by the empa treatment. An increased expression level of senescence markers (p53, p21, p16), tissue factor, VCAM-1, SGLT1 and SGLT2 and a down-regulation of eNOS were observed in the aortic inner curvature compared to the outer one in the control lean rats, which were prevented by the empa treatment. In the obese ZSF1 rats, no such effects were observed. The empa treatment reduced the increased body weight and weight of lungs, spleen, liver and perirenal fat, hyperglycemia and the increased levels of total cholesterol and triglycerides in obese ZSF1 rats, and increased blood ketone levels and urinary glucose excretion in control lean and obese ZSF1 rats.ConclusionEmpa reduced glucose levels by 28% and improved both endothelial function and cardiac remodeling in the obese ZSF1 rat. Empa also reduced the increased expression level of senescence, and atherothrombotic markers at arterial sites at risk in the control lean, but not obese, ZSF1 rat.

Intake of omega-3 formulation EPA:DHA 6:1 by old rats for 2 weeks improved endothelium-dependent relaxations and normalized the expression level of ACE/AT1R/NADPH oxidase and the formation of ROS in the mesenteric artery

Omega-3 polyunsaturated fatty acids (PUFAs) including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to protect the cardiovascular system, in part, by stimulating the endothelial formation of nitric oxide (NO). EPA:DHA 6:1 has been identified as a potent omega 3 PUFA formulation to induce endothelium-dependent vasorelaxation and activation of endothelial NO synthase (eNOS). This study examined whether intake of EPA:DHA 6:1 (500 mg/kg/day) for 2 weeks improves an established endothelial dysfunction in old rats (20 months old), and, if so, the underlying mechanism was subsequently determined.In the main mesenteric artery rings, an endothelial dysfunction characterized by a blunted NO component, an abolished endothelium-dependent hyperpolarization component, and increased endothelium-dependent contractile responses (EDCFs) are observed in old rats compared to young rats. Age-related endothelial dysfunction was associated with increased vascular formation of reactive oxygen species (ROS) and expression of eNOS, components of the local angiotensin system, senescence markers, and cyclooxygenase-2 (COX-2), and the downregulation of COX-1. The EPA:DHA 6:1 treatment improved the NO-mediated relaxation, reduced the EDCF-dependent contractile response and the vascular formation of ROS, and normalized the expression level of all target proteins in the old arterial wall.Thus, the present findings indicate that a 2-week intake of EPA:DHA 6:1 by old rats restored endothelium-dependent NO-mediated relaxations, most likely, by preventing the upregulation of the local angiotensin system and the subsequent formation of ROS.

Empagliflozin, a sodium-glucose cotransporter inhibitor, improved heart remodeling and mesenteric artery endothelial function in the metabolic syndrome with HFpEF ZSF1 rat: Role of cyclooxygenases

Empagliflozin (EMPA), a selective SGLT2 inhibitor, significantly reduced cardiovascular mortality and hospitalization for heart failure in patients with type 2 diabetes at high cardiovascular risk, but the protective mechanisms remain unclear. This study evaluates the effect of EMPA on the endothelial function, and the heart structure and function in an experimental model of metabolic syndrome with HFpEF, the ZSF1 rat, and its lean control (Ctrl). Rats received either the control diet or the diet containing EMPA (30mg/kg/day) for 6 weeks. Vascular reactivity was assessed in the mesenteric artery using organ chambers, and the heart function and structural changes by echocardiography. In the mesenteric artery, acetylcholine (ACh)-induced endothelium-dependent relaxations were slightly but significantly reduced and endothelium-dependent contractile responses (EDCFs) increased in ZSF1 compared to Ctrl rats. The cyclooxygenase inhibitor indomethacin improved relaxations and abolished EDCFs to ACh in ZSF1 rats. The 6-week EMPA treatment improved the relaxation and blunted EDCFs to ACh in the ZSF1. The weight of the heart and of each part, and the left ventricle area were increased in the ZSF1, and significantly reduced by the EMPA treatment, except for left auricle plus septum weight, which did not reach significance. Left ventricle ejection fraction and cardiac output were similar in all groups. The EMPA treatment reduced the increased body weight and that of lungs, spleen, liver and perirenal fat, and hyperglycemia, and increased blood ketone levels and urinary glucose excretion in ZSF1 rats. The EMPA treatment improved body weight, hyperglycemia, and both endothelial function and cardiac remodeling in the metabolic syndrome with HFpEF ZSF1 rat. The protective vascular effect involves improved endothelium-dependent relaxations and blunted EDCFs most likely by targeting the cyclooxygenase pathway.

EPA:DHA 6:1 prevents angiotensin II-induced hypertension and endothelial dysfunction in rats: role of NADPH oxidase- and COX-derived oxidative stress.

Eicosapentaenoic acid:docosahexaenoic acid (EPA:DHA) 6:1, an omega-3 polyunsaturated fatty acid formulation, has been shown to induce a sustained formation of endothelial nitric oxide (NO) synthase-derived NO, a major vasoprotective factor. This study examined whether chronic intake of EPA:DHA 6:1 prevents hypertension and endothelial dysfunction induced by angiotensin II (Ang II) in rats. Male Wister rats received orally corn oil or EPA:DHA 6:1 (500 mg kg-1 per day) before chronic infusion of Ang II (0.4 mg kg-1 per day). Systolic blood pressure was determined by tail cuff sphingomanometry, vascular reactivity using a myograph, oxidative stress using dihydroethidium and protein expression by immunofluorescence and western blot analysis. Ang II-induced hypertension was associated with reduced acetylcholine-induced relaxations of secondary branch mesenteric artery rings affecting the endothelium-dependent hyperpolarization (EDH)- and the NO-mediated relaxations, both of which were improved by the NADPH oxidase inhibitor VAS-2870. The Ang II treatment induced also endothelium-dependent contractile responses (EDCFs), which were abolished by the cyclooxygenase (COX) inhibitor indomethacin. An increased level of vascular oxidative stress and expression of NADPH oxidase subunits (p47phox and p22phox), COX-1 and COX-2, endothelial NO synthase and Ang II type 1 receptors were observed in the Ang II group, whereas SKCa and connexin 37 were downregulated. Intake of EPA:DHA 6:1 prevented the Ang II-induced hypertension and endothelial dysfunction by improving both the NO- and EDH-mediated relaxations, and by reducing EDCFs and the expression of target proteins. The present findings indicate that chronic intake of EPA:DHA 6:1 prevented the Ang II-induced hypertension and endothelial dysfunction in rats, most likely by preventing NADPH oxidase- and COX-derived oxidative stress.

O38 The omega-3 EPA:DHA 6:1 formulation improves ageing-related blunted endothelium-dependent relaxations and increased contractile responses in the mesenteric artery: Role of oxidative stress and cyclooxygenases

Introduction Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to cause endothelium-dependent nitric oxide (NO)-mediated relaxations of isolated blood vessels, with EPA:DHA 6:1 being a superior formulation. The aim of the present study was to determine whether EPA:DHA 6:1 improves ageing-related endothelial dysfunction, and, if so, to determine the underlying mechanism. Methods Male Wistar rats (20 months-old) received daily 500 mg/kg of either EPA:DHA 6:1 (omega 3), corn oil (control), or water for 2 weeks. Young male Wistar rats (12 weeks-old) were used as a control. Results In the main mesenteric artery, ageing was associated with an endothelial dysfunction characterized by a blunted NO-mediated component, an abolished endothelium-dependent hyperpolarization (EDH)-mediated component, and also by the induction of endothelium-dependent contractile responses (EDCF, in the presence of NO and EDH inhibitors) sensitive to indomethacin (a cyclooxygenase inhibitor) in response to acetylcholine. Endothelial dysfunction was associated with an increased level of vascular oxidative stress and expression of COX-2, but not of COX-1, in the mesenteric artery. The EPA:DHA 6:1 treatment improved both the NO- and EDH-mediated relaxations, and reduced EDCF, vascular oxidative stress and expression of COX-2 in the old mesenteric artery. Conclusion The present findings indicate that intake of EPA:DHA 6:1 prevented the development of the ageing-related endothelial dysfunction in rats. The beneficial effect involves an improvement of both the NO- and the EDH-mediated relaxations as well as a reduction of endothelium-dependent contractile response most likely by preventing vascular oxidative stress.

Abstract 298: Chronic Environmental Enrichment Improves Aging-related Endothelial Dysfunction in Rats: Role of Oxidative Stress

Aging is associated with an endothelial dysfunction, which promotes cardiovascular diseases. Physical exercise has beneficial effects on the endothelial function. However, the effect of an enriched environment (EE) promoting physical exercise, on the endothelial function has not been explored during aging. We investigated the endothelial dysfunction in Long-Evans female rats housed in standard conditions (SD, 2 rats per cage) until the age of 18 months. Then, half the rats were transferred to EE (12 rats in one large cage containing various objects, which were changed 5 times per week) for 6 months. A group of 2-month old rats served as young controls. Systolic (SBP), diastolic (DBP) blood pressure and heart rate (HR) were determined by plethysmography. Vascular reactivity was assessed in mesenteric arteries using organ chambers, vascular oxidative stress by dihydroethidine staining, and the proteins expression by immunofluorescence. Aging under SD was associated with increased SBP (by 40 mmHg, P <0.001), DBP (by 12 mmHg, P <0.001) and HR (by 110 bpm, P <0.001) compared to young rats. It was also associated with blunted acetylcholine (ACh)-induced endothelium-dependent nitric oxide (NO)-mediated relaxations (EC50 1.08 10 -8 M vs. 3.08 10 -8 M, P <0.05) and endothelium-derived hyperpolarization (EDH)-mediated relaxation (E max 89.5±11.8% vs. 0.28±0.18%, P <0.001), and the induction of endothelium-dependent contractile responses to ACh (E max 0.004±0.008 g vs. 0.71±0.07 g, P <0.001) in mesenteric artery rings. EE reduced SBP and DBP by 18 and 10 mmHg, respectively ( P <0.05), HR by 30 bpm ( P >0.05), and improved NO-mediated responses to a similar level as in young rats ( P <0.001) without affecting the blunted EDH-mediated relaxation and the increased endothelium-dependent contractile responses in old rats. An excessive oxidative stress and the upregulation of endothelial NO synthase (eNOS), nitrotyrosine and arginase I were observed in the aorta of SD old rats compared to young rats ( P <0.001). EE improved all these effects ( P <0.05). In conclusion, EE retarded the aging-related endothelial dysfunction mostly by improving the NO-mediated relaxation. This effect involves normalization of vascular oxidative stress and possibly also eNOS uncoupling.

Suppression of endoplasmic reticulum stress improves endothelium-dependent contractile responses in aorta of the spontaneously hypertensive rat

A contributing factor to increased peripheral resistance seen during hypertension is an increased production of endothelium-derived contractile factors (EDCFs). The main EDCFs are vasoconstrictor prostanoids, metabolites of arachidonic acid (AA) produced by Ca(2+)-dependent cytosolic phospholipase A2 (cPLA2) following phosphorylation (at Ser(505)) mediated by extracellular signal-regulated kinase (ERK1/2) and cyclooxygenase (COX) activations. Although endoplasmic reticulum (ER) stress has been shown to contribute to pathophysiological alterations in cardiovascular diseases, the relationship between ER stress and EDCF-mediated responses remains unclear. We tested the hypothesis that ER stress plays a role in EDCF-mediated responses via activation of the cPLA2/COX pathway in the aorta of the spontaneously hypertensive rat (SHR). Male SHR and Wistar-Kyoto rats (WKY) were treated with ER stress inhibitor, tauroursodeoxycholic acid or 4-phenlybutyric acid (TUDCA or PBA, respectively, 100 mg·kg(-1)·day(-1) ip) or PBS (control, 300 μl/day ip) for 1 wk. There was a decrease in systolic blood pressure in SHR treated with TUDCA or PBA compared with control SHR (176 ± 3 or 181 ± 5, respectively vs. 200 ± 2 mmHg). In the SHR, treatment with TUDCA or PBA normalized aortic (vs. control SHR) 1) contractions to acetylcholine (ACh), AA, and tert-butyl hydroperoxide, 2) ACh-stimulated releases of prostanoids (thromboxane A2, PGF2α, and prostacyclin), 3) expression of COX-1, 4) phosphorylation of cPLA2 and ERK1/2, and 5) production of H2O2. Our findings demonstrate a novel interplay between ER stress and EDCF-mediated responses in the aorta of the SHR. Moreover, ER stress inhibition normalizes such responses by suppressing the cPLA2/COX pathway.

Crataegus special extract WS®1442 prevents aging-related endothelial dysfunction

Aging is associated with a markedly increased incidence of cardiovascular diseases due, in part, to the development of vascular endothelial dysfunction. The present study has evaluated whether the Crataegus special extract WS®1442 prevents the development of aging-related endothelial dysfunction in rats, and, if so, to determine the underlying mechanisms. Wistar rats received either a control diet or the same diet containing 100 or 300mg/kg/day of WS®1442 from week 25 until week 65. Vascular reactivity was assessed in mesenteric artery rings using organ chambers, oxidative stress by dihydroethidine staining and cyclooxygenase-1 (COX-1) and -2 (COX-2) expression by immunohistochemistry. Acetylcholine-induced endothelium-dependent relaxations in mesenteric artery rings were blunted in 65-week-old rats compared to 16-week-old rats. This effect was associated with a marked reduction of the endothelium-derived hyperpolarizing factor (EDHF) component whereas the nitric oxide (NO) component was not affected. Aging was also associated with the induction of endothelium-dependent contractile responses to acetylcholine. Both aging-related impairment of endothelium-dependent relaxations and the induction of endothelium-dependent contractile responses were improved by the Crataegus treatment and by COX inhibitors. An excessive vascular oxidative stress and an upregulation of COX-1 and COX-2 were observed in the mesenteric artery of old rats compared to young rats, and these effects were improved by the Crataegus treatment. In conclusion, chronic intake of Crataegus prevented aging-related endothelial dysfunction by reducing the prostanoid-mediated contractile responses, most likely by improving the increased oxidative stress and the overexpression of COX-1 and COX-2.

Impaired hemodynamics and endothelial vasomotor function via endoperoxide-mediated vasoconstriction in the carotid artery of spontaneously hypertensive rats

The fact that endothelium removal increases diameter and compliance in the common carotid artery (CCA) of spontaneously hypertensive rats (SHR) and that improving CCA endothelium-dependent vasorelaxation has been shown to normalize a reduced systolic blood flow through the SHR CCA compared with normotensive Wistar-Kyoto rats (WKY) suggests that endothelial vasomotor dysfunction may be linked to altered large artery hemodynamics in hypertension. The experiments herein were designed to further investigate WKY and SHR CCA hemodynamics and endothelium-dependent vasomotor functions. It was hypothesized that CCA blood flow and conductance would be reduced throughout the cardiac cycle in SHR and that endothelium-dependent contractile activity would impair SHR CCA vasorelaxation. We report that mean, maximal systolic, and diastolic blood flow was reduced in SHR vs. WKY CCA, as was vascular conductance. Pressure was augmented in SHR CCA and accompanied by late systolic flow augmentation so that total flow during systole was indeed no different between strains, possibly explained by earlier lower body wave reflection. While ACh stimulation in isolated precontracted WKY CCA caused a robust nitric oxide (NO)-mediated vasorelaxation, endothelium-dependent, cyclooxygenase (COX)-mediated contractile activity stimulated by high ACh concentration impaired NO- and non-NO/non-COX-mediated vasorelaxation in precontracted SHR CCA. In quiescent CCA, this endothelium-dependent contractile response was COX-1 and thromboxane-prostanoid receptor mediated and modulated by the availability of NO. These data collectively suggest that endothelium-dependent, COX-mediated endoperoxide signaling in the CCA of SHR may elicit vasoconstriction, which could shift the mechanical properties of this conduit artery and contribute to reduced CCA blood flow in vivo.

Effects of the flavonoid quercetin and its methylated metabolite isorhamnetin in isolated arteries from spontaneously hypertensive rats.

Chronic oral quercetin exerts antihypertensive effects in spontaneously hypertensive rats (SHR). In the present study, the vasodilator effects of the flavonoid quercetin and its main metabolite isorhamnetin were analysed in isolated thoracic aorta, iliac artery and on the isolated perfused mesenteric resistance vascular bed from SHR and normotensive Wistar Kyoto rats (WKY). In noradrenaline-precontracted vessels from SHR there was an inverse correlation between the relaxant potency (pIC50) of quercetin (4.76 +/- 0.02, 5.08 +/- 0.12, 5.30 +/- 0.18, in aorta, iliac arteries and mesentery, respectively) and isorhamnetin (4.90 +/- 0.11, 5.38 +/- 0.15 and 5.80 +/- 0.10, respectively) and the diameter of the vessel studied. Both flavonoids were more potent in endothelium-denuded aortae and iliac arteries from SHR than from normotensive WKY rats. In addition, in aortae from SHR both flavonoids restored the endothelial-dependent vasodilation. Isorhamnetin, but not quercetin, also reduced the endothelium-dependent contractile responses induced by acetylcholine. These direct vasodilator effects, together with the improvement of endothelial function, are good candidates to explain the blood pressure reduction and vascular protective effects of quercetin in animal models of hypertension and possibly in human cardiovascular diseases.

Endothelium-Dependent Stretch-Induced Contractile Responses of Vascular Smooth Muscles in Experimental Diabetes Mellitus, and the Nitric Oxide System

Endothelium-Dependent Stretch-Induced Contractile Responses of Vascular Smooth Muscles in Experimental Diabetes Mellitus, and the Nitric Oxide System

Involvement of protein kinase C and Na+/K+-ATPase in the contractile response induced by myricetin in rat isolated aorta.

The role of PKC and Na+/K+-ATPase in the vascular smooth muscle responses induced by the bioflavonoid myricetin was investigated. KCl induced a concentration-dependent relaxation in arteries exposed to K+-free solution that was mainly mediated by an activation of Na+/K+-ATPase. Myricetin (50 microM) partially inhibited this vasorelaxant effect induced by KCl in intact rings, being unaffected in the endothelium-denuded rings. This inhibitory effect induced by myricetin was suppressed by the PGH2-TXA2 receptor antagonist, SQ 29,548, and the PKC inhibitor, staurosporine. Myricetin also induced an endothelium-dependent contractile response which was increased in the presence of PMA and reduced by staurosporine. In conclusion, myricetin both modulates Na+/K+-ATPase-induced vasodilatation acting as a functional inhibitor of Na+/K+-ATPase activity and activates protein kinases, including PKC, to induce contraction. These effects appear to be related to the activation of PGH2-TXA2 receptors on vascular smooth muscle by the TXA2 released from endothelium.NA:noradrenalineNA+/K+-ATPase pump:sodium-potassium-activated ATPasePKC:protein kinase CPMA:phorbol 12-myristate 13-acetateTXA2:thromboxane A2The role of PKC and Na+/K+-ATPase in the vascular smooth muscle responses induced by the bioflavonoid myricetin was investigated. KCl induced a concentration-dependent relaxation in arteries exposed to K+-free solution that was mainly mediated by an activation of Na+/K+-ATPase. Myricetin (50 microM) partially inhibited this vasorelaxant effect induced by KCl in intact rings, being unaffected in the endothelium-denuded rings. This inhibitory effect induced by myricetin was suppressed by the PGH2-TXA2 receptor antagonist, SQ 29,548, and the PKC inhibitor, staurosporine. Myricetin also induced an endothelium-dependent contractile response which was increased in the presence of PMA and reduced by staurosporine. In conclusion, myricetin both modulates Na+/K+-ATPase-induced vasodilatation acting as a functional inhibitor of Na+/K+-ATPase activity and activates protein kinases, including PKC, to induce contraction. These effects appear to be related to the activation of PGH2-TXA2 receptors on vascular smooth muscle by the TXA2 released from endothelium.

Involvement of thromboxane A2 in the endothelium-dependent contractions induced by myricetin in rat isolated aorta.

1. The present study was undertaken to analyse the mechanism of the contractile response induced by the bioflavonoid myricetin in isolated rat aortic rings. 2. Myricetin induced endothelium-dependent contractile responses (maximal value=21+/-2% of the response induced by 80 mM KCl and pD2=5.12+/-0.03). This effect developed slowly, reached a peak within 6 min and then declined progressively. 3. Myricetin-induced contractions were almost abolished by the phospholipase A2 (PLA2) inhibitor, quinacrine (10 microM), the cyclo-oxygenase inhibitor, indomethacin (10 microM), the thromboxane synthase inhibitor, dazoxiben (100 microM), the putative thromboxane A2 (TXA2)/prostaglandin endoperoxide receptor antagonist, ifetroban (3 microM). These contractions were abolished in Ca2+-free medium but were not affected by the Ca2+ channel blocker verapamil (10 microM). 4. In cultured bovine endothelial cells (BAEC), myricetin (50 microM) produced an increase in cytosolic free calcium ([Ca2+]i) which peaked within 1 min and remained sustained for 6 min, as determined by the fluorescent probe fura 2. This rise in [Ca2+]i was abolished after removal of extracellular Ca2+ in the medium. 5. Myricetin (50 microM) significantly increased TXB2 production both in aortic rings with and without endothelium and in BAEC. These increases were abolished both by Ca2+-free media and by indomethacin. 6. Taken together, these results suggests that myricetin stimulates Ca2+ influx and subsequently triggers the activation of the PLA2 and cyclo-oxygenase pathways releasing TXA2 from the endothelium to contract rat aortic rings. The latter response occurs via the activation of Tp receptors on vascular smooth muscle cells.

Adenosine responses in experimental vein bypass grafts

Purpose: Veins develop unique endothelial and smooth muscle cell physiologic phenotypes after implantation as vein grafts in the arterial circulation. Receptor-mediated relaxation is reduced or absent in these grafts. This study examines the responses of vein grafts to adenosine, a known potent endogenous vasodilator, and compares these responses with the native veins and arteries. Methods: The presence of adenosine receptors (A 1 and A 2) by radioligand binding and the in vitro responses to the adenosine analogue N-ethyl-carboxyamido-adenosine were assessed in precontracted common carotid jugular vein bypass grafts placed in New Zealand white rabbits for 28 days. Results were compared with those obtained in precontracted jugular veins and carotid arteries. Both endothelialized and de-endothelialized vessels were studied. The contribution of nitric oxide (NO) and prostanoid production to relaxation was also determined by preincubation with the specific inhibitors <!--**Check here**-->l-monomethylarginine and indomethacin, respectively. Finally, the in vitro relaxation in response to the respective A 1 and A 2 adenosine receptor agonists R-phenyl-isopropyl-adenosine and CGS-21680 was also examined. Results: The results show that the adenosine-induced responses of the vein grafts differ from those of the jugular vein and carotid artery. First, in contrast to the carotid artery, vein graft adenosine-mediated relaxation is NO and prostanoid dependent, similar to the response of the jugular vein. Second, A 1 receptor activation in the vein graft produces an endothelium-dependent contractile response. Third, the A 2 receptor-mediated responses in the vein grafts appear to be independent of the endothelium. Fourth, radioligand studies show the presence of both receptor subtypes (A 1 and A 2) on the vein grafts with a ratio (A 1/A 2 = 1.4) closer to that of the jugular vein (A 1/A 2 = 1.8) than to that seen in the carotid artery (A 1/A 2 = 0.5). Conclusions: Vein graft adenosine responses appear to be unique in that they neither maintain a venous phenotype nor acquire an arterial phenotype. In particular, endothelial A 1 receptor-mediated responses change from relaxation to contraction, and receptor activated NO-mediated relaxation is preserved within the vein grafts probably via A 2 receptor signalling. (J Vasc Surg 1998;28:929-38.)

Functional cross-talk between endothelial muscarinic and alpha2-adrenergic receptors in rabbit cerebral arteries.

Interactions between two classes of receptors have been observed in several cell lines and preparations. The aim of this work was to assess the impact of simultaneous stimulation of endothelial muscarinic and alpha2-adrenergic receptors (alpha2-AR) on vascular reactivity. Rabbit middle cerebral arteries were isolated and changes in isometric tension were recorded in the presence of indomethacin. Inhibition of nitric oxide (NO) synthase with Nomega-nitro-L-arginine (L-NOARG, 100 micromol l(-1)) revealed alpha-AR-dependent contractions. Pre-addition of acetylcholine (ACH, 1 micromol l(-1)) augmented oxymetazoline (OXY, 10 micromol l(-1), alpha2-AR agonist)-, but decreased phenylephrine (PE, 10 micromol(-1), alpha1-AR agonist)-induced contraction (P<0.05). The effects of ACH were endothelium-dependent. Vessels were precontracted with 40 mmol l(-1) KCl-physiological salt solution (PSS) in the absence of L-NOARG, or PE or OXY in the presence of L-NOARG. In the presence of high external K+ or PE, ACH induced a potent relaxation (P<0.05). In the presence of OXY, however, ACH mediated contraction (P<0.05). After pertussis toxin (PTX, inactivator of Galpha(i/o) proteins) pre-treatment, alpha2-AR-dependent contractions were abolished. Forty mmol l(-1) KCl-PSS induced contraction was not altered by PTX whereas ACH-induced relaxation was augmented (P<0.05). To investigate if endothelin-1 (ET-1) intervened in the endothelium-dependent contractile response to ACH in the presence of OXY-dependent tone, vessels were incubated in the presence of BQ123 (1 micromol l(-1)), an ETA receptor antagonist. OXY-mediated tone was not affected by BQ123; however, ACH-induced contraction was reversed to a relaxation (P<0.05). These data indicate that activation of endothelial alpha2-AR triggers an endothelium-dependent, ET-1 mediated, contraction to ACH. This suggests that activation of alpha2-AR affects muscarinic receptor/G protein coupling leading to an opposite biological effect.

Hypoxic pulmonary endothelial cells release a diffusible contractile factor distinct from endothelin.

Hypoxia (0% O2) evokes a late-phase, endothelium-dependent contractile response in porcine isolated pulmonary arteries that may be caused by a cyclooxygenase-independent, endothelium-derived contractile factor. The aim of this study was to further analyze the mechanism underlying this hypoxic response. Proximal porcine pulmonary arterial rings were suspended for isometric tension recording in organ chambers. Hypoxia (0% O2) caused a late-phase, endothelium-dependent contractile response that was not inhibited by the endothelin (ET)A-receptor antagonist BQ-123 (10(-6) M), by the ETB-receptor antagonist BQ-788 (10(-7) M), or by their combination. In contrast, ET-1 caused a concentration-dependent contraction of arterial rings that was inhibited by BQ-123 (10(-6) M) and a relaxation that was abolished by BQ-788 (10(-7) M) or by endothelial cell removal. Therefore, the endothelium-dependent contraction to hypoxia is not mediated by ET. Hypoxia caused only relaxation in endothelium-denuded rings. However, when a pulmonary valve leaflet, a rich source of pulmonary endothelial cells, was placed into the lumen of endothelium-denuded rings, hypoxia caused a late-phase contractile response that was similar to that observed in arterial rings with native endothelium. This hypoxic contraction persisted in the presence of indomethacin (10(-5) M) and N-nitro-L-arginine methyl ester (3 x 10(-5) M) to block cyclooxygenase and nitric oxide synthase, respectively. These results suggest that hypoxic contraction of pulmonary arteries is mediated by a diffusible, contractile factor released from hypoxic endothelial cells. This contractile mediator is distinct from ET.

Dual endothelium-dependent vascular activities of proteinase-activated receptor-2-activating peptides: evidence for receptor heterogeneity.

1. The vascular actions of the proteinase-activated receptor-2-activating peptides (PAR2APs), SLIGRL-NH2 (SL-NH2) and SLIGKV-NH2 (KV-NH2) as well as the reverse-sequence peptide, LSIGRL-NH2 (LS-NH2) and an N-acylated PAR2AP derivative, trans-cinnamoyl-LIGRLO-NH2 (tcLI-NH2), were studied in rat intact and endothelium-denuded artery ring preparations, primarily from the pulmonary artery (RPA). 2. In RPA rings with but not without a functional endothelium, SL-NH2 (but not LS-NH2) caused either an endothelium-dependent relaxation (at concentrations: < 10 microM) or (at higher concentrations: > 10 microM), an endothelium-dependent contraction. No contractile response was observed in endothelium-denuded preparations, that otherwise contracted in response to the PAR1AP, TFLLR-NH2. 3. The endothelium-dependent contractile response to SL-NH2 was not blocked by the alpha-adrenoceptor antagonist prazosin, the endothelin antagonist BQ123, the angiotensin II antagonist DuP753, by tetrodotoxin; nor by the enzyme inhibitors, N(omega)-nitro-L-arginine-methylester (NO-synthase), indomethacin (cyclo-oxygenase), SKF-525A (epoxygenase) and MK886 (leukotriene synthesis inhibitor). 4. In the relaxation assay, KV-NH2 was 5 fold less potent than SL-NH2, whereas in the contractile assay KV-NH2 was about equipotent with SL-NH2. However, the maximal contractile response to KV-NH2 was lower than that of SL-NH2. 5. The PAR2AP analogue, tcLI-NH2, was as active as SL-NH2 in the relaxation assay but was inactive as a contractile agonist in the endothelium-intact RPA. 6. The relaxant responses caused by SL-NH2 and trypsin, as well as the contractile response caused by SL-NH2, did not desensitize in the course of repeated exposures of the tissue to agonist; whereas the contractile response to trypsin, only observed at concentrations greater than 30 u ml(-1), was desensitized by previous exposure of the tissue to either thrombin or trypsin. 7. In a contractile assay, where the tissue was desensitized to a concentration of trypsin that would otherwise cause a relaxant response, the preparation still contracted in response to SL-NH2. However, the trypsin-desensitized preparations were no longer contracted by thrombin. 8. From the cross-desensitization by thrombin of the contractile response to trypsin (and vice versa), we concluded that the contractile effect of trypsin was due to activation of the thrombin receptor and not PAR2. 9. We concluded that the endothelium-dependent contraction caused by high concentrations of SL-NH2 is due to an as yet unidentified contracting factor; whereas the endothelium-dependent relaxation response observed at low concentrations of SL-NH2 (< or = 10 microM) is mediated by nitric oxide. 10. The distinct structure activity profiles for the contractile response (potency of KV-NH2 < or = SL-NH2) compared with the relaxant response (potency of KV-NH2 << SL-NH2); the contractile responsiveness to SL-NH2 of an endothelium-intact RPA preparation, that did not contract in response to trypsin; and the lack of contractile activity of the PAR2AP analogue tcLI-NH2, that was as active as SL-NH2 in the relaxation assay all argue in favour of receptor heterogeneity in the vasculature for the PAR2APs. It remains to be determined if the distinct endothelial receptor responsible for the contractile action of SL-NH2 can be proteolytically activated, like PAR1 and PAR2.

Lack of endothelium-dependent relaxation in coronary resistance arteries of cholesterol-fed rabbits.

The endothelium-dependent contractile responses of subepicardial coronary resistance arteries (286 +/- 18 microns ID, n = 22) from rabbits fed either a 0.5 or 2.0% cholesterol-enriched diet or a control diet for 10-12 wk were determined under isometric conditions at the optimum length for active force production (Lo). After the development of tone with 29 mM K+-Krebs, arteries from control rabbits treated with acetylcholine (0.1-10 microM) showed a concentration-dependent relaxation, with a maximum decrease in tone of 63%. In contrast, coronary arteries from animals fed 0.5 and 2.0% cholesterol contracted to acetylcholine (approximately 210% increase in tone). A similar phenomenon was seen with arteries precontracted with 10 nM 9,11-methanoepoxy-prostaglandin H2 (U 46,619), a thromboxane A2 mimetic. The contractile responses to acetylcholine occurred in arteries in which the endothelium was structurally intact and which were devoid of plaque. Arteries from cholesterol-fed animals were poorly responsive to ADP (0.01-10.0 microM), whereas arteries from normal animals relaxed. All arteries relaxed to an equal degree when exposed to acidified nitrite, which produces nitric oxide (NO). The data suggest that as a result of hypercholesterolemia, there may be a dysfunction in the synthesis or release of endothelium-derived relaxing factor (EDRF) by the endothelial cells of coronary resistance arteries, rather than an abnormality of the smooth muscle cells per se.

Muscarinic receptor subtype (M1) identification on rabbit pulmonary vascular endothelium in vivo.

We have investigated the presence of M1-subtype muscarinic receptors on pulmonary vascular endothelium in vivo. Utilizing multiple indicator-dilution techniques, we studied [3H]-pirenzepine ([3H]-PNZ; a selective M1 receptor antagonist) binding during a single transpulmonary pass in anesthetized, artificially ventilated rabbits, before and after administration of 4.3 mumol/kg of the nonselective muscarinic receptor antagonist atropine. [14C]-Dextran (MW = 70,000-90,000) served as the intravascular indicator. Before atropine administration, approximately 20% of [3H]-PNZ was lost (i.e., presumed bound to PNZ-specific and nonspecific sites) during a single pass through the pulmonary microvasculature. A significant decrease in [3H]-PNZ binding occurred 90 min after atropine (to approximately 60% of total initial binding), but not after saline, reflecting the loss of specific binding to M1 receptors. These data are in support of previous findings from our laboratory indicating a pressor response to M1 receptor stimulation in the rabbit pulmonary circulation as well as an endothelium-dependent contractile response to M1 receptor stimulation in vitro.

Altered vasodilator and endothelium‐dependent responses in hypertension

AbstractA compromised ability to relax has been described as a characteristic of vascular smooth muscle from animal models of hypertension. Since the discovery that many vasodilators act via the release of an endothelium‐derived relaxant factor (EDRF), studies have been designed to assess whether endothelium‐dependent vs. ‐independent relaxations are impaired in the hypertensive state. A decreased maximal relaxation for such endothelium‐dependent vasodilators as acetylcholine, A23187, and histamine exists in both large conduit and resistance vessels from hypertensive animals compared to their normotensive counterparts. Endothelium‐independent vasodilators (i.e., sodium nitroprusside) typically achieve similar maximal percent relaxations in hypertensive vs. normotensive vascular preparations. This defect for endothelium‐dependent vasodilators has been described in vessels from genetic, renal, mineralocorticoid, and coarctation models of hypertension. An altered endothelium‐dependent relaxation could be a corollary of the documented structural changes which occur in hypertension although it remains to be determined if there exists an abnormal coupling between EDRF and its putative receptor in vascular smooth muscle. Several complexities hamper interpretation of altered endothelium‐dependent responses in hypertersion such as regional vascular differences, suitability of reference endothelium‐independent vasodilators, and endothelium‐dependent contractile responses elicited by acetylcholine.