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Background— The present study investigates the vascular reactivity and the involvement of nitric oxide and prostanoids in regulating vasoconstriction of small mesenteric arteries from patients with Crohn’s disease (CD) to understand the vascular component of this pathology. Methods and Results— An increased production of proinflammatory cytokines (tumor necrosis factor-α and interleukins 1β, 6, and 8) has been observed in biopsy specimens of inflammatory intestinal mucosa. However, contractile responses of small mesenteric arteries from CD patients in response to norepinephrine were not changed ex vivo when compared with controls. Exposure to either the nitric oxide synthase inhibitor N G -nitro- l -arginine or the cyclooxygenase (COX) inhibitor indomethacin did not modify contractions induced by norepinephrine in either control or CD patients. However, in the latter, the specific COX-2 inhibitor N-(2-cyclohexyloxy-4-nitrophenyl) methanesulfonamide significantly attenuated norepinephrine-induced vasoconstriction. Furthermore, immunohistochemical analysis showed marked COX-2 expression in the whole arterial wall of vessels from CD patients. Vessels from control patients exhibited weak COX-2 staining in the adventitial and endothelial layers only. Conclusions— The above results provide direct evidence for COX-2 expression in small mesenteric arteries from CD patients. They also shed new light on the involvement of vasoconstrictor metabolites of COX in regulating contraction of these arteries. Of particular interest is the balance between vasoconstrictor products from COX-2 and unidentified vasodilatory products that maintained vascular reactivity in a physiological range despite an increase of circulatory cytokines in patients with CD.

The two portions of rat vas deferens differed in the postjunctional sensitivity to noradrenaline. Alpha1-adrenoceptor-linked phosphoinositide breakdown was analysed in this tissue. The noradrenaline-induced [3H]inositol phosphate accumulation was similar in both ends although the pEC50 was higher in the epididymal (5.97+/-0.07) than in the prostatic (5.47+/-0.15, P0.01) portion. [3H]Prazosin showed similar density of binding sites in both portions. Tissue pretreated with pertussis toxin did not change [3H]inositol phosphate accumulation. Finally, Western blot analysis indicated a smaller concentration of Gq/11 protein in the prostatic half (-29+/-5%, P0.01). These results suggest that the different sensitivity to noradrenaline could be due to the higher availability of this sort of G protein in the epididymal portion.

This study was designed to evaluate the effects of aminoguanidine, a selective inhibitor of the inducible isoform of nitric oxide synthase (iNOS), on the reactivity and intracellular calcium ([Ca2+]i) mobilization induced by noradrenaline in the perfused tail artery from aged WAG/Rij rats. Global mean internal diameter was 350±15 microns and wall thickness 161±3 microns. The influence of the endothelium on these responses was also analysed. The intracellular dye fura-2 for [Ca2+]i measurements was used. Noradrenaline-induced vasoconstriction decreased progressively from 3 to 20 and 30 months. Removal of the endothelium attenuated vasoconstriction in 20 and 30 month-old rats (P

1. We have studied the alpha 1-adrenoceptor-mediated responses in intact tail artery rings from 3-4 and 20-22 months old Sprague-Dawley rats, focusing on possible endothelial alterations. The influence of nitric oxide released by the endothelium, the number of alpha 1-adrenoceptors and the functional receptor reserve were evaluated to determine their contribution to the contractile response mediated by this receptor. The state of the endothelial layer was assessed by confocal microscopy. 2. Noradrenaline (1 nM-100 microM) induced concentration-dependent vasoconstriction. The maximum contractions to noradrenaline (P < 0.05) and to 75 mM KCl (P < 0.01) were higher in young than in old animals. 3. The density (Bmax) of alpha 1-adrenoceptors and the dissociation constant (KD) obtained in [3H]-prazosin binding experiments were unchanged by age. 4. The apparent affinity (pKA) and the percentage of functional receptors (qx 100) remaining after phenoxybenzamine (0.03 microM) were similar in both age groups. 5. After partial alpha 1-adrenoceptor inactivation with phenoxybenzamine, NG-nitro-L-arginine methylester (30 microM) significantly potentiated the E/[A] curve to noradrenaline in young rats. However, only responses to 0.1 to 1 microM noradrenaline were significantly potentiated in old animals. In addition, 94% of the vessels from young, but only 52% from old rats were relaxed by 80-100% of the noradrenaline (0.03 microM) contraction, with 1 microM acetylcholine. 6. No modifications in the area (micron2) or in the number of endothelial nuclei (per mm2) were observed between age groups. An elongation of the nuclei of endothelial cells was observed in the old animals. 7. These data suggest that the noradrenaline-induced contraction is decreased in old rats probably due to differences in either the contractile machinary or postreceptor mechanisms. These alterations may be accompanied by an impairment of the release or production of NO from endothelial cells.

1. Vascular contraction induced by phenylephrine was studied in tail artery rings from spontaneously hypertensive (SHR) and Wistar Kyoto rats (WKY) with particular focus on the role of endothelium. The influence of receptor reserve and the density of alpha 1-adrenoceptors on the possible differences observed were also analysed. 2. Phenylephrine (0.01-100 microM) induced concentration-dependent vasoconstrictions. The maximum response (alpha, P < 0.001) was greater but the pEC50 (P < 0.05) smaller in rings from SHR than from WKY rats irrespective of the presence or absence of endothelium. 3. Removal of endothelial cells resulted in a decrease of the maximum contraction with no modification in the pEC50 in arteries from both WKY and SHR. 4. The density of alpha 1-adrenoceptors (Bmax) and the dissociation constant (KD) were found to be the same for preparations from SHR and WKY rats in [3H]-prazosin binding experiments. 5. The apparent affinity (pKA) determined by the nested hyperbolic method and the operational model was similar in tail arteries from the two rat strains, irrespective of the presence or absence of endothelium. However, in endothelium-denuded rings, the pKA value was enhanced when compared with intact rings, in both SHR and WKY rats. 6. In rings from hypertensive rats, the operational parameter maximum possible effect (Em) was greater and the agonist efficacy (tau) was smaller than in rings from normotensive rats. When the endothelium was removed log tau and Em diminished in preparations from both rat strains. 7. In summary, the increased maximum responsiveness to phenylephrine in rings from SHR could be due to enhancement in Em. The log tau values indicate a deterioration in the transduction of the stimulus provided by the agonist in tail arteries from hypertensive animals. This study also suggests that the absence of endothelium modifies the alpha 1-adrenoceptor-mediated vasoconstriction probably by altering the transduction signalling mechanisms. The importance of analysing the degree of endothelium functionality when comparing results from different groups of rats is stated.

1. The effects of neuropeptide Y (NPY) were examined on adrenergic and non-adrenergic, non-cholinergic (NANC) neurotransmission in the rat anococcygeus muscle. 2. NPY (0.1-0.3 microM) greatly potentiated the contractile responses induced by field stimulation. Prazosin (0.1 microM) completely abolished the stimulation-induced responses either in the absence or presence of NPY. 3. NPY (0.1-0.3 microM) enhanced only the contractile responses to low doses of noradrenaline (NA, 0.003-0.01 microM). Responses to tyramine were unaffected by the same concentrations of NPY. 4. In superfused anococcygeus, previously loaded with [3H]-NA, NPY (0.1-0.3 microM) failed to modify the basal, as well as the stimulation-evoked, release of tritium at 2 and 4 Hz. 5. NANC relaxations induced by electrical stimulation were significantly reduced, in a concentration-related manner, by 0.1-0.3 microM NPY. 6. L-NG-nitro-arginine (L-NOARG, 30 microM) enhanced the stimulation (0.25-1 Hz)-induced motor responses. In the presence of L-NOARG (30 microM), NPY (0.1 microM) did not modify the motor responses induced by field stimulation (0.25-0.5 Hz). L-Arginine did not reverse the NPY-induced potentiation of stimulation-induced motor responses. 7. The relaxations of anococcygeus muscle induced by sodium nitroprusside (SNP, 0.01-0.3 microM) were diminished by NPY (0.1-0.3 microM). 8. Our study suggests that NPY, at concentrations devoid of contractile effect, potentiates the motor responses of rat anococcygeus muscle as a consequence, at least in part, of the inhibition of NANC relaxing responses by a different mechanism from L-NOARG.

The peroxisome proliferator-activated receptor-alpha (PPARalpha) plays a major role in the control of cardiac energy metabolism. The role of PPARalpha on cardiac functions was evaluated by using PPARalpha knockout (PPARalpha -/-) mice. Hemodynamic parameters by sphygmomanometric measurements show that deletion of PPARalpha did not affect systolic blood pressure and heart rate. Echocardiographic measurements demonstrated reduced systolic performance as shown by the decrease of left ventricular fractional shortening in PPARalpha -/- mice. Telemetric electrocardiography revealed neither atrio- nor intraventricular conduction defects in PPARalpha -/- mice. Also, heart rate, P-wave duration and amplitude, and QT interval were not affected. However, the amplitude of T wave from PPARalpha -/- mice was lower compared with wild-type (PPARalpha +/+) mice. When the myocardial function was measured by ex vivo Langendorff's heart preparation, basal and beta-adrenergic agonist-induced developed forces were significantly reduced in PPARalpha-null mice. In addition, Western blot analysis shows that the protein expression of beta1-adrenergic receptor is reduced in hearts from PPARalpha -/- mice. Histological analysis showed that hearts from PPARalpha -/- but not PPARalpha +/+ mice displayed myocardial fibrosis. These results suggest that PPARalpha-null mice have an alteration of cardiac contractile performance under basal and under stimulation of beta1-adrenergic receptors. These effects are associated with myocardial fibrosis. The data shed light on the role of PPARalpha in maintaining cardiac functions.

To investigate the expression of inducible cyclooxygenase (COX-2) and inducible nitric oxide synthase (iNOS) and the role of vasodilatory prostanoids and endogenous nitric oxide (NO) in small omental arteries harvested from patients with severe liver diseases.Ex vivo study of resistance arteries. SETTING. Intensive care unit.Twenty patients undergoing liver transplantation for fulminant hepatic failure (FHF, n=6), cirrhogenous viral hepatitis (CH, n=6) and limited hepatocarcinoma (controls, n=8).Western blot and immunohistochemical labeling for assessment of COX-2 and iNOS expression and localization and ex vivo vascular reactivity studies.Significant upregulation of COX-2 and iNOS expressions were detected in arteries from FHF and CH patients with a greater increase in the former than in the latter. Ex vivo contractile responses to norepinephrine and the thromboxane A(2) analog, U46619, were not significantly different between patients with severe liver dysfunction and controls. Exposure to either the NO-synthase inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME), the cyclooxygenase inhibitor, indomethacin, or their combination did not significantly modify contractions of agonists in controls and CH patients. In FHF, the specific COX-2 inhibitor, N-(2-cyclohexyloxy-4-nitrophenyl) methanesulfonamide (1 micro m/l), but not L-NAME, significantly enhanced the maximal effect ( p0.01) and the sensitivity ( p0.01) to norepinephrine.COX-2 and iNOS are upregulated in omental arteries from patients with cirrhogenous hepatitis and fulminant hepatic failure. Whereas neither NO nor vasodilatory prostaglandins seem to play a major role in counteracting arterial contractility of arteries from control patients, COX-2 derivatives are involved in lowering the arterial contractility of vessels harvested from FHF patients.

1 The present study focuses on the role of endothelium on α1-adrenoceptor-mediated vasoconstriction in aorta from Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). To define and quantify changes in the parameters governing agonism at α1-adrenoceptor by hypertension and/or endothelium, the operational model of analysis was used. 2 In either endothelium intact or denuded aorta, the sensitivity (P

Changes in functional responsiveness to alpha1-adrenoceptor activation with noradrenaline and in [3H]prazosin binding in the epididymal portion of vas deferens from normotensive Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR) were investigated. The operational model fitting and the nested hyperbolic method were used to analyze the effects of irreversible receptor alkylation by phenoxybenzamine (0.1 microM) on the alpha1-adrenoceptor mediated contractile responses to noradrenaline of vasa deferentia from SHR and WKY rats. Saturation isotherms for [3H]prazosin revealed a significant increase (P < 0.05) in the Bmax in SHR vas deferens (145 +/- 19 fmol/mg protein) compared with vas deferens from normotensive controls (75 +/- 12 fmol/mg protein) without changes in the K(D). No differences in the proportion of high and low affinity binding sites for WB-4101 and 5-methylurapidil were observed. The maximum contractile response, alpha, (P < 0.001) and the pEC50 (P < 0.05) values for noradrenaline were greater for SHR than for WKY rat tissues. The apparent affinity (pK(A)) determined by the nested hyperbolic method and by the operational model of agonism was found to be similar in the two strains. In agreement with relative pEC50, the efficacy (tau) value for SHR was greater than for WKY rats. However, the difference in the tau estimates did not reach statistical significance. In summary, in the epididymal portion of SHR vas deferens, the increased maximum contractile response to noradrenaline is due to an increase of Em. Taken together, the tau values and the results from binding experiments lead to the assumption that the transducer constant K(E) must be greater in SHR than in WKY rats, suggesting a deterioration in the transduction of the stimulus provided by the agonist in hypertensive animals.

1. The relative importance of intracellular and extracellular Ca2+ on alpha 1-adrenoceptor-mediated contraction by noradrenaline and St-587 has been studied and correlated with the binding characteristics in intact tail artery from Sprague-Dawley rats. 2. Noradrenaline and St-587 behaved as full agonists inducing a concentration-dependent vasoconstriction. 3. Nifedipine (1 microM and 10 microM) blocked by 50% (P < 0.001) and 75% (P < 0.001) respectively, the maximum contraction (Emax) induced by St-587. Nevertheless, to reach 40% inhibition of Emax on noradrenaline responses (P < 0.01), 10 microM nifedipine was necessary. 4. Both agonists induced a concentration-dependent accumulation of inositol phosphates. Noradrenaline behaved as a full agonist and St-587 as a partial agonist for this response. 5. [3H]-prazosin binding to intact tail artery rings was saturable and of high affinity (KD = 4.44 +/- 0.46 nM; Bmax = 36.35 +/- 4.22 fmol mg-1 tissue). 6. Competition curves for inhibition of specific [3H]-prazosin binding by WB-4101 suggest that the rat tail artery contains two alpha 1-adrenoceptor subtypes in an approximate ratio of 60:40. 7. After irreversible alkylation of alpha 1B-adrenoceptors with 100 microM chloroethylclonidine (CEC), nifedipine (1 microM) influenced to a greater extent the St-587- than the noradrenaline-induced contraction. 8. Our results indicate that the degree of participation of intracellular and extracellular Ca2+ sources, on the alpha 1-adrenoceptor-mediated contraction, depends on the agonist used. The two alpha 1-adrenoceptor subtypes observed in binding experiments seem to be unrelated to the Ca2+ sources used for contraction.

1. The modulation by NG-nitro-L-arginine methylester (L-NAME) of alpha 1-adrenoceptor-mediated contraction was investigated on isolated segments of rat tail artery and aorta. The influence of L-NAME on inositol phosphates accumulation by alpha 1-adrenoceptor agonists was also investigated to elucidate the intracellular mechanism responsible for this modulation. 2. In aorta but not in tail artery L-NAME (30 microM) enhanced the sensitivity (3.3 times) and the maximum contraction (Emax) induced by the full agonist, phenylephrine. 3. St-587, a partial alpha 1-adrenoceptor agonist, behaved as a weak agonist in the aorta (22.2% of phenylephrine Emax). However, when the same agonist was studied in tail artery rings a maximum contraction that was 78.4% of the phenylephrine induced Emax was reached. 4. L-NAME increased (3.3 times) the Emax for St-587 contraction in the aorta but not in the tail artery. Sensitivity to St-587 was slightly but significantly (P < 0.001) enhanced (1.9 times) by L-NAME in tail artery segments. 5. Contractile responses to phenylephrine after partial alkylation with phenoxybenzamine were analyzed by the nested hyperbolic null method. To elicit 50% of Emax for contraction only 1.1% of the receptors in the tail artery and 21% of the receptors in the aorta need to be occupied. These results indicate a higher receptor reserve for the tail artery than the aorta. 6. In the tail artery but not in the aorta, St-587 activates phosphoinositide turnover. The presence of L-NAME was without effect on inositol phosphates accumulation induced by this partial alpha 1-adrenoceptor agonist. 7. The maximum contraction induced by phenylephrine, after partial alpha-adrenoceptor alkylation, was enhanced by L-NAME in tail artery rings. However, the NO synthase inhibitor was unable to modify the phenylephrine-induced accumulation of inositol phosphates in the presence of phenoxybenzamine. 8. These results indicate that the differences in St-587-induced contraction and the modulation by L-NAME of alpha 1-adrenoceptor-mediated contraction observed between the tail artery and aorta are associated with differences in receptor reserve. In addition, our biochemical studies indicate that the potentiating effect of L-NAME is independent of intracellular calcium release via phosphatidylinositol turnover.

BACKGROUND: The peroxisome proliferator-activated receptor alpha (PPARalpha) plays an important role in the metabolism of lipoproteins and fatty acids, and seems to protect against the development of atherosclerosis. To evaluate the possible protective role of PPARalpha on cardiovascular function, the effect of the PPARalpha agonist, fenofibrate was assessed with respect to ischaemia/reperfusion injury and endothelial function in mice. RESULTS: Fenofibrate treatment reduces myocardial infarction size and improves post-ischaemic contractile dysfunction. Hearts from PPARalpha null mice exhibit increased susceptibility to ischaemic damages and were refractory to protection by fenofibrate treatment suggesting that the beneficial effects of fenofibrate were mediated via PPARalpha. Furthermore, fenofibrate improves endothelium- and nitric oxide-mediated vasodilatation in aorta and mesenteric vascular bed. A decreased inhibitory effect of reactive oxygen species in the vessel wall accounts for enhanced endothelial vasodilatation. However, the latter cannot be explained by an increase in nitric oxide synthase expression nor by an increase sensitivity of the arteries to nitric oxide. CONCLUSIONS: Altogether the present data suggest that fenofibrate exerts cardioprotective effect against ischaemia and improves nitric oxide-mediated response probably by enhancing antioxidant capacity of the vessel wall. These data underscore new therapeutic perspectives for PPARalpha agonists in ischaemic myocardial injury and in cardiovascular diseases associated with endothelial dysfunction.

BACKGROUND: The peroxisome proliferator-activated receptor alpha (PPARalpha) plays an important role in the metabolism of lipoproteins and fatty acids, and seems to protect against the development of atherosclerosis. To evaluate the possible protective role of PPARalpha on cardiovascular function, the effect of the PPARalpha agonist, fenofibrate was assessed with respect to ischaemia/reperfusion injury and endothelial function in mice. RESULTS: Fenofibrate treatment reduces myocardial infarction size and improves post-ischaemic contractile dysfunction. Hearts from PPARalpha null mice exhibit increased susceptibility to ischaemic damages and were refractory to protection by fenofibrate treatment suggesting that the beneficial effects of fenofibrate were mediated via PPARalpha. Furthermore, fenofibrate improves endothelium- and nitric oxide-mediated vasodilatation in aorta and mesenteric vascular bed. A decreased inhibitory effect of reactive oxygen species in the vessel wall accounts for enhanced endothelial vasodilatation. However, the latter cannot be explained by an increase in nitric oxide synthase expression nor by an increase sensitivity of the arteries to nitric oxide. CONCLUSIONS: Altogether the present data suggest that fenofibrate exerts cardioprotective effect against ischaemia and improves nitric oxide-mediated response probably by enhancing antioxidant capacity of the vessel wall. These data underscore new therapeutic perspectives for PPARalpha agonists in ischaemic myocardial injury and in cardiovascular diseases associated with endothelial dysfunction.