Function In Rat Aorta Research Articles

PPARβ down-regulation is involved in high glucose-induced endothelial injury via acceleration of nitrative stress.

Endothelial injury plays a vital role in vascular lesions from diabetes mellitus (DM). Therapeutic targets against endothelial damage may provide critical venues for the treatment of diabetic vascular diseases. Peroxisome proliferator-activated receptor β (PPARβ) is a crucial regulator in DM and its complications. However, the molecular signal mediating the roles of PPARβ in DM-induced endothelial dysfunction is not fully understood. The impaired endothelium-dependent relaxation and destruction of the endothelium structures appeared in high glucose incubated rat aortic rings. A high glucose level significantly decreased the expression of PPARβ and endothelial nitric oxide synthase (eNOS) at the mRNA and protein levels, and reduced the concentration of nitric oxide (NO), which occurred in parallel with an increase in the expression of inducible nitric oxide synthase (iNOS) and 3-nitrotyrosine. The effect of high glucose was inhibited by GW0742, a PPARβ agonist. Both GSK0660 (PPARβ antagonist) and NG-nitro-l-arginine-methyl ester (NOS inhibitor) could reverse the protective effects of GW0742. These results suggest that the activation of nitrative stress may, at least in part, mediate the down-regulation of PPARβ in high glucose-impaired endothelial function in rat aorta. PPARβ-nitrative stress may hold potential in treating vascular complications from DM.

Contractile function of smooth muscle retained after overnight storage.

The functional responses of different overnight-stored in vitro tissues are not clearly described in any animal model. The influence of overnight storage in an animal model may vary between tissue types. We employed Sprague-Dawley rat as our animal model and investigated the functional changes of rat aorta, trachea, bronchus and bladder that were used (i) immediately after surgical removal (denoted as fresh) and (ii) after storage in aerated (95% O2, 5% CO2) Krebs-Ringer bicarbonate solution at 4 °C for 24 h (denoted as stored). The aorta ring was pre-contracted with phenylephrine, and the functional response of the tissue was investigated using isoprenaline, forskolin and carbachol. Carbachol was also used to increase the tone in trachea, bronchus rings and bladder strips. A clear reduced function of endothelium, with a minor if any effect in the smooth muscle function in rat aorta was observed after overnight storage. The contractile response of overnight-stored rat airway (trachea and bronchus) and bladder smooth muscles remained unchanged. Among all tested tissues, only bronchus showed a reduced response rate (only 40% responded) after storage. In vitro rat tissues that are stored in Krebs solution at 4 °C for 24 h can still be used to investigate smooth muscle responses, however, not endothelium-mediated responses for aorta. The influence of overnight storage on different tissues from an animal model (Sprague-Dawley rat in our study) also provides an insight in maximising the use of sacrificed animals.

Tocotrienol Rich Palm Oil Extract Is More Effective Than Pure Tocotrienols at Improving Endothelium-Dependent Relaxation in the Presence of Oxidative Stress.

Oxidative endothelial dysfunction is a critical initiator of vascular disease. Vitamin E is an effective antioxidant but attempts to use it to treat vascular disorders have been disappointing. This study investigated whether tocotrienols, the less abundant components of vitamin E compared to tocopherols, might be more effective at preserving endothelial function. Superoxide generated by hypoxanthine/xanthine oxidase or rat aorta was measured using lucigenin-enhanced chemiluminescence. The effect of α-tocopherol, α-, δ-, and γ-tocotrienols and a tocotrienol rich palm oil extract (tocomin) on levels of superoxide was assessed. Endothelial function in rat aorta was assessed in the presence of the auto-oxidant pyrogallol. Whilst all of the compounds displayed antioxidant activity, the tocotrienols were more effective when superoxide was produced by hypoxanthine/xanthine oxidase whereas tocomin and α-tocopherol were more effective in the isolated aorta. Tocomin and α-tocopherol restored endothelial function in the presence of oxidant stress but α-, δ-, and γ-tocotrienols were ineffective. The protective effect of tocomin was replicated when the tocotrienols were present with, but not without, α-tocopherol. Tocotrienol rich tocomin is more effective than α-tocopherol at reducing oxidative stress and restoring endothelium-dependent relaxation in rat aortae and although α-, δ-, and γ-tocotrienols effectively scavenged superoxide, they did not improve endothelial function.

Increased nitric oxide activity compensates for increased oxidative stress to maintain endothelial function in rat aorta in early type 1 diabetes

Hyperglycaemia and oxidative stress are known to acutely cause endothelial dysfunction in vitro, but in the initial stages of diabetes, endothelium-dependent relaxation is preserved. The aim of this study was to investigate how endothelium-dependent relaxation is maintained in the early stages of type 1 diabetes. Diabetes was induced in Sprague-Dawley rats with a single injection of streptozotocin (48mg/kg, i.v.), and after 6weeks, endothelium-dependent and endothelium-independent relaxations were examined in the thoracic aorta in vitro. Lucigenin-enhanced chemiluminescence was used to measure superoxide generation from the aorta. Diabetes increased superoxide generation by the aorta (2,180 ± 363 vs 986 ± 163AU/mg dry tissue weight). Acetylcholine (ACh)-induced relaxation was similar in aortae from control (pEC(50) 7.36 ± 0.09, R (max) 95 ± 3%) and diabetic rats (pEC(50) 7.33 ± 0.10, R (max) 88 ± 5%). The ACh-induced relaxation was abolished by the combined presence of the nitric oxide synthase inhibitor N-nitro-L-arginine (L-NNA, 100μM) and an inhibitor of soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10μM) in control rats, but under the same conditions, the diabetic aortic rings showed significant relaxation to ACh (pEC(50) 6.75 ± 0.15, R (max) 25 ± 4%, p < 0.05). In diabetic aortae, the addition of haemoglobin, which inactivates nitric oxide, to L-NNA + ODQ abolished the response to ACh. The addition of the potassium channel blockers, apamin and TRAM-34, to L-NNA + ODQ also abolished the relaxation response to ACh. Diabetes significantly elevated plasma total nitrite/nitrate and increased expression of endothelial nitric oxide synthase (eNOS) and calmodulin in aortae. These data indicate that after 6weeks of diabetes, despite increased oxidant stress, endothelium-dependent relaxation is maintained due to the increased eNOS expression resulting in increased NO synthesis. In diabetic arteries, NO acts both through and independently of cGMP pathways to cause relaxation.

Identification and functional implication of a Rho kinase-dependent moesin-EBP50 interaction in noradrenaline-stimulated artery

Ezrin, radixin, and moesin (ERM) proteins are known to be substrates of Rho kinase (ROCK), a key player in vascular smooth muscle regulation. Their function in arteries remains to be elucidated. The objective of the present study was to investigate ERM phosphorylation and function in rat aorta and mesenteric artery and the influence of ERM-binding phosphoprotein 50 (EBP50), a scaffold partner of ERM proteins in several cell types. In isolated arteries, ERM proteins are phosphorylated by PKC and ROCK with different kinetics after either agonist stimulation or KCl-induced depolarization. Immunoprecipitation of EBP50 in noradrenaline-stimulated arteries allowed identification of its interaction with moesin and several other proteins involved in cytoskeleton regulation. This interaction was inhibited by Y27632, a ROCK inhibitor. Moesin or EBP50 depletion after small interfering RNA transfection by reverse permeabilization in intact mesenteric arteries both potentiated the contractility in response to agonist stimulation without any effect on contractile response induced by high KCl. This effect was preserved in ionomycin-permeabilized arteries. These results indicate that, in agonist-stimulated arteries, the activation of ROCK leads to the binding of moesin to EBP50, which interacts with several components of the cytoskeleton, resulting in a decrease in the contractile response.

Effects of chronic treatment with a low dose of nicorandil on the function of the rat aorta during ageing

1. It is known that ATP-sensitive potassium (K(ATP)) channels regulate the membrane potential of smooth muscle cells and vascular tone. Because their activity is altered during ageing, many pharmacological treatments aimed at improving K(ATP) channel and cardiovascular functions have been evaluated. Nicorandil, a K(ATP) channel opener, nitric oxide (NO) donor and anti-oxidant, induces vasodilation, decreases blood pressure and exhibits cardioprotection in ageing, as well as after ischaemia-reperfusion. 2. In the present study, using tension myography and biochemical and histological techniques, we investigated the effects of chronic (2 months) low-dose nicorandil (0.1 mg/kg per day) treatment on the function of rat aorta during ageing (in 4-, 12- and 24-month old rats). 3. The results showed that chronic nicorandil treatment significantly improves mechanical relaxation and noradrenaline-induced vasoconstriction in aged rats. At all ages, the nicorandil-induced vasodilation was primarily mediated by its NO donor group. Nicorandil treatment resulted in an additional 0.5-1 elastic lamella in the aorta and decreased total protein, collagen and elastin content in the aortic wall at all ages. However, in 4-month-old rats, nicorandil significantly increased the elastin : total protein ratio by 19%. 4. In contrast with results of previous studies that used high doses of nicorandil (i.e. 60 mg/kg per day), low-dose nicorandil treatment in the present study did not lead to a progressive desensitization to nicorandil and may be beneficial in improving arterial function in ageing or cardiovascular diseases.

Effects of hyperglycemia on the modulation of vascular function by perivascular adipose tissue

To study the acute and chronic effect of hyperglycemia on perivascular adipose tissue (PVAT) function in rat aorta. Alterations in PVAT function in rat aorta incubated with 22 mmol/l D-glucose for 30 min and in aorta from streptozotocin (STZ)-induced diabetic rats were studied. Incubation with D-glucose caused an attenuation of contraction in response to phenylephrine, both in the presence and absence of endothelium, whereas removal of PVAT eliminated this attenuation effect. The presence of PVAT did not affect concentration-related relaxation response of the aorta to carbamylcholine in STZ rats. There was also no difference in the relaxation response of the aorta to carbamylcholine between STZ and control rats. The presence of PVAT, however, caused a higher attenuation of the concentration-dependent contraction to phenylephrine in aorta from STZ rats with intact endothelium as compared with that from control rats. Incubation of the aorta from control rats with Nomega-nitro-L-arginine or carboxy-2-phenyl-4,4,5,5-tetra-methyl-imidazoline-1-oxyl-3-oxide potentiated the contraction of the vessels to phenylephrine, and this potentiation effect was higher in the vessels from STZ rats than control rats when N-nitro-L-arginine was used. Removal of PVAT reduced this potentiation effect and eliminated the difference between the vessels from control and STZ rats. Under both acute and chronic conditions, hyperglycemia enhanced the relaxation response of the vessels mediated by PVAT. These new findings provide important information on the mechanism underlying the postprandial effect of hyperglycemia on blood pressure control and the presence of hypotension under chronic hyperglycemia in a type-1 model of diabetes.

Activation of protein kinase B/Akt and endothelial nitric oxide synthase mediates agmatine-induced endothelium-dependent relaxation

The ability of agmatine, formed from L-arginine by the enzyme arginine decarboxylase (ADC), to modulate vasomotor function in rat aorta was investigated in the present study. Agmatine-mediated modulation of vasomotor tone was studied in organ chambers, protein expression quantified by Western blot analysis and cyclic guanosine 5'-monophosphate (cGMP) levels measured by radioimmunoassay. Agmatine (10(-10) to 10(-3) M) produced concentration-dependent relaxations (82+/-5%) in phenylephrine-contracted endothelium intact rat aorta. Relaxations to agmatine were diminished on denudation of endothelium and nitric oxide synthase (NOS) inhibition by L-Nomega-nitro arginine or soluble guanylate cyclase inhibition by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (P<0.001) abolished agmatine-mediated relaxations, while relaxations were insensitive to inducible NOS inhibition by 1400W. Agmatine-treated aorta demonstrated increased protein expression of phosphorylated S473-Akt and phosphorylated S1177-endothelial nitric oxide synthase (eNOS), and elevated the levels of cyclic GMP (P<0.01). Agmatine-mediated potentiation of relaxations and elevation of cGMP levels was sensitive to phosphatidylinositol 3'-kinase inhibitor, wortmannin. Relaxations to agmatine were also affected by pre-treatment with tetraethylammonium (P<0.01) or apamin (P<0.05), and were not affected by charybdotoxin. Relaxations to agmatine were partially affected by pre-treatment of aortic rings with barium chloride (P<0.05), and glybenclamide (P<0.05). Results obtained suggest that agmatine activates protein kinase B/Akt to phosphorylate eNOS and elevate cyclic GMP levels to produce vasodilatation of aorta. Agmatine-mediated relaxations in rat aorta seems to be mediated mainly by endothelial NO-mediated activation of small conductance Ca2+-activated K+ channels, and partly by ATP-sensitive and inward rectifying K+ channels.

Raloxifene Elicits Combined Rapid Vasorelaxation and Long-Term Anti-Inflammatory Actions in Rat Aorta

Previous studies reported the ability of raloxifene to acutely relax arterial and venous vessels, but the underlying mechanisms are controversial. Anti-inflammatory effects of the drug have been reported in nonvascular tissues. Therefore, the aim of this study was to investigate the nature of short- and long-term effects of raloxifene on selected aspects of vascular function in rat aorta. Isometric tension changes in response to raloxifene were recorded in aortic rings from ovariectomized female rats that underwent estrogen replacement, whereas long-term experiments were performed in isolated aortic smooth muscle cells (SMCs). Raloxifene (0.1 pM-0.1 microM) induced acute vasorelaxation through endothelium- and nitric oxide (NO)-dependent, prostanoid-independent mechanisms. The relaxant response to raloxifene was significantly weaker than that to 17beta-estradiol and was sensitive to neither the nonselective estrogen receptor antagonist ICI 182,780 [7,17-[9[(4,4,5,5,5-pentafluoropentyl)sulfinyl]nonyl]estra-1,3,5(10)-triene-3,17-diol] nor a selective estrogen receptor (ER) alpha antagonist. This rapid vasorelaxant effect was retained in aortic rings from rats treated with 0.1 mg/kg, but not 1 mg/kg, lipopolysaccharide, 4 h before sacrifice. In cultured aortic SMCs, raloxifene treatment (1 nM-1 microM) for 24 h reduced inducible NO synthase activation in response to cytokines. This effect was prevented by the selective ERalpha antagonist and was associated with up-regulation of ERalpha protein levels, which dropped markedly upon cytokine stimulation. These findings illustrate the relevance of classic ER-dependent pathways to the vascular anti-inflammatory effects rather than to the nongenomic vasorelaxation induced by raloxifene and may assist in the design of novel ER isoform-selective estrogen-receptor modulators targeted to the vascular system.

Anandamide vehicles: a comparative study

Among the studies that investigate the vasorelaxation induced by anandamide, one of the most frequent differences is the use of distinct solvents that could modify vascular function and explain the controversial results described. The aims of this study were: to evaluate the influence of different cannabinoid vehicles in vascular function of rat aorta, and to compare the vasorelaxation induced by anandamide dissolved in different vehicles. Vehicles were: ethanol (70%), Tween 80/ethanol (2:1 and 1:1), 1:1:18 (Tween 80/ethanol/saline) and dimethylsulphoxide (DMSO) 0.5%. All the vehicles tested, except DMSO 0.5%, modified the vascular and/or the endothelial function in rat aorta rings. Anandamide caused a time- and concentration-dependent vasorelaxation in all the experimental groups except in ethanol group, but the mechanisms involved in its vasorelaxation appear to be different depending on the vehicle used. The results obtained with vehicles containing Tween 80 suggest a non-endothelial component in the vasorelaxation caused by anandamide, while those obtained with DMSO at 0.5% suggest an endothelial component in this vasorelaxation.

Effects of a High-Cholesterol Diet on Vascular and Endothelial Function in Rat Aorta

We have examined the effects of high cholesterol (1%) or normal diet for 8 weeks on vascular and endothelial responsiveness of rat aorta. The cholesterol diet produced a small but significant elevation of plasma cholesterol levels (vehicle: 0.95±0.13 mmol/l, n = 11; cholesterol fed: 1.40 ± 0.12 mmol/l, n = 14; p < 0.05). There were significant differences between control and high cholesterol groups in the contractile response to noradrenaline in rat aortic rings, both in terms of maximum response and potency. Both the potency of noradrenaline (NA) (pD₂ values; vehicle diet: 7.84 ± 0.08; cholesterol diet: 8.27 ± 0.09; p < 0.01) and the maximum response (vehicle diet: 0.78 ± 0.05 g; cholesterol diet: 0.95 ± 0.06 g; p < 0.05) were significantly greater in the high-cholesterol group. There were no significant differences between control and high cholesterol groups in endothelium-independent relaxations to sodium nitroprusside (SNP). When responses were correlated with plasma cholesterol levels, there was a significant negative correlation with maximum endothelium-dependent relaxation to ACh (r = 0.53, n = 16, p < 0.05), so that the maximum relaxation decreased with increasing plasma levels of cholesterol. There were no significant correlations between cholesterol levels and endothelium-independent relaxation to SNP or between cholesterol levels and vascular contractions to NA. In conclusion, cholesterol-fed normal Wistar rats show functional changes in vascular responsiveness even with a relatively small elevation of plasma cholesterol levels.

Long-term cold storage effect of univesity of wisconsin (uw) solution on vascular endothelial function of rat aorta.

Long-term cold storage effect of univesity of wisconsin (uw) solution on vascular endothelial function of rat aorta.

L-NAME Hypertension Alters Endothelial and Smooth Muscle Function in Rat Aorta

Nitric oxide is an important regulator of vascular function and blood pressure. Chronic administration of nitric oxide inhibitors provides a new model of hypertension with pronounced target organ damage. We investigated the effects of oral treatment with N omega-nitro-L-arginine methyl ester (L-NAME) for 6 weeks on vascular reactivity of the aorta in Wistar-Kyoto rats. Certain rats received verapamil or trandolapril in addition to L-NAME. Systolic blood pressure increased in the L-NAME group (by approximately or equal to 80 mm Hg systolic) but not in controls or rats treated with verapamil or trandolapril. Isometric tension changes of aortic rings were recorded. Endothelium-dependent relaxations to acetylcholine were reduced in the L-NAME group (58 +/- 6% versus 104 +/- 1% in placebo, P < .05) but were normalized by treatment with verapamil or trandolapril. In contrast, endothelium-independent relaxations to sodium nitroprusside were not significantly reduced in L-NAME hypertension but were slightly enhanced by trandolapril therapy (P < .05) in the L-NAME group only. In quiescent rings, acetylcholine caused endothelium-dependent contractions in particular after in vitro incubation with L-NAME. These contractions tended to be enhanced in L-NAME hypertension (23 +/- 4% versus 14 +/- 3% in the placebo group; P = NS) and were significantly reduced after treatment with verapamil or trandolapril (P < .05). Concentrations to norepinephrine and angiotensin I and II were unaffected by L-NAME hypertension, whereas those to endothelin-1 were reduced (P < .05).(ABSTRACT TRUNCATED AT 250 WORDS)