Impairment of both nitric oxide-mediated and EDHF-type relaxation in small mesenteric arteries from rats with streptozotocin-induced diabetes

BACKGROUND AND PURPOSE

To investigate whether diabetes affects either or both nitric oxide (NO)-mediated and endothelium-derived hyperpolarizing factor (EDHF)-type relaxation in endothelium-dependent relaxation of mesenteric arteries from streptozotocin-induced diabetic rats.

EXPERIMENTAL APPROACH

Wire myography was employed to examine endothelial function of mesenteric arteries. Superoxide levels were measured by L-012 and lucigenin-enhanced chemiluminescence. Western blotting was used to quantify protein expression levels.

KEY RESULTS

Superoxide levels were significantly increased in diabetic mesenteric arteries compared with normal arteries. Diabetes significantly reduced the sensitivity to the endothelium-dependent relaxant, acetylcholine (ACh) in mesenteric arteries. When the contribution of NO to relaxation was abolished by N-nitro-L-arginine (L-NNA) + a soluble guanylate cyclase inhibitor (ODQ), the sensitivity to ACh was significantly decreased in the diabetic arteries compared with normal arteries, indicating an impaired EDHF-type relaxation despite increased expression of intermediate- and small-conductance calcium-activated potassium channels. Conversely, when the contribution of EDHF was inhibited with TRAM-34 + apamin + iberiotoxin, maximum relaxations to ACh were significantly decreased in diabetic compared with normal arteries, suggesting that the contribution of NO was also impaired by diabetes. Basal levels of NO release, indicated by contraction to L-NNA, were also significantly decreased in diabetic arteries. Western blot analysis demonstrated that diabetic arteries had an increased expression of Nox2, decreased pSer⁴⁷³Akt and a reduced proportion of endothelial NO synthase (eNOS) expressed as a dimer, indicating uncoupling.

CONCLUSION AND IMPLICATIONS

The contribution of both NO and EDHF-type relaxations was impaired in diabetes and was caused by increased oxidative stress, decreased pSer⁴⁷³Akt and/or eNOS uncoupling.     

Contribution of nitric oxide, cyclic GMP and K+ channels to acetylcholine-induced dilatation of rat conduit and resistance arteries

1. We compared the effects of inhibiting nitric oxide synthase (NOS), soluble guanylate cyclase (sGC) and K+ channel activation on dilator responses to acetylcholine (ACh) in rat resistance (hindquarters) and conduit arteries (thoracic aorta). 2. In rat perfused hindquarters, the NO synthase inhibitor N omega-nitro-L-arginine (L-NNA; 1 mmol/L) partially inhibited the ACh-induced dilatation and the combination of L-NNA + haemoglobin (Hb; 20 mumol/L), a NO scavenger, did not further affect the response. Exposure to high K+ (30 mmol/L) also inhibited the response to ACh and this response was further reduced by L-NNA + high K+. Surprisingly, when applied alone 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), an inhibitor of sGC, did not affect responses to ACh, whereas treatment with ODQ + high K+ markedly impaired dilatation. 3. In aortic rings precontracted with phenylephrine (PE; 0.01-1 mumol/L), the maximum relaxation to ACh was significantly reduced by L-NNA (0.1 mmol/L) and further inhibited by L-NNA + Hb (20 mumol/L). At 10 mumol/L, ODQ alone inhibited the maximum relaxation to ACh, which was further reduced by ODQ + high K+ (30 mmol/L). High K+ caused a smaller but significant inhibition of ACh-induced relaxation. 4. These results suggest that NO and cGMP play a relatively greater role in ACh-induced dilatation of the aorta compared with the hindquarters resistance vasculature and are consistent with the hypothesis that a non-NO endothelium-derived hyperpolarizing factor (endothelium-derived hyperpolarizing factor; EDHF) makes a relatively greater contribution to dilatation of resistance vessels than in conduit arteries. The data suggest that when sGC is inhibited, a compensatory mechanism involving K+ channel opening by NO can largely maintain ACh-induced vasodilator responses of resistance vessels. Furthermore, when NO synthesis is blocked, a non-NO EDHF may play a role in ACh-induced dilatation of the resistance vasculature.     

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 (48?mg/kg, i.v.), and after 6?weeks, 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?±?163?AU/mg dry tissue weight). Acetylcholine (ACh)-induced relaxation was similar in aortae from control (pEC₅₀ 7.36?±?0.09, R _max 95?±?3?%) and diabetic rats (pEC₅₀ 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, 100?μM) and an inhibitor of soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 10?μM) in control rats, but under the same conditions, the diabetic aortic rings showed significant relaxation to ACh (pEC₅₀ 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 6?weeks 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.     

Chronic treatment of male rats with daidzein and 17 beta-oestradiol induces the contribution of EDHF to endothelium-dependent relaxation

1. We investigated the effect of chronic (7 days) treatment of male rats with the isoflavone daidzein (0.2 mg kg(-1) sc per day) or 17beta-oestradiol (0.1 mg kg(-1) sc per day) on the contribution of nitric oxide (NO), prostaglandins and endothelium-derived hyperpolarising factor (EDHF) to endothelium-dependent relaxation of isolated aortic rings. 2. The sensitivity and maximum relaxation to acetylcholine (ACh) were significantly greater in aortic rings from rats treated with daidzein or 17beta-oestradiol, in comparison to vehicle-treated rats. Inhibition of nitric oxide synthase with N-nitro-l-arginine (l-NOARG) abolished ACh-induced relaxation in the aortae from vehicle-treated rats, but only attenuated relaxation in aortae from daidzein or 17beta-oestradiol-treated rats. The presence of haemoglobin in addition to l-NOARG did not cause any further inhibition of relaxation. 3. The cyclooxygenase inhibitor indomethacin had no effect on endothelium-dependent relaxation in aortae from any treatment group. Charybdotoxin (ChTX), which blocks large-conductance calcium-activated potassium channels (BK(Ca)) and intermediate-conductance calcium-activated potassium channels (IK(Ca)), plus apamin, which blocks small-conductance calcium-activated potassium channels (SK(Ca)), but not iberiotoxin, which only blocks BK(Ca), attenuated endothelium-dependent relaxation of aortae from daidzein or 17beta-oestradiol-treated rats. Blockade of K(Ca) channels had no effect on the responses to ACh in aortae from vehicle-treated rats. In aortae from daidzein- or 17beta-oestradiol-treated rats, endothelium-dependent relaxation was also attenuated by inhibition of cytochrome P450 (CYP450) epoxygenase with 6-(2-propargylloxyphenyl)hexanoic acid (PPOH) or inhibition of K(IR) channels and Na(+)/K(+)-ATPase with barium and oubain, respectively. 4. This study demonstrates that endothelium-dependent relaxation of male rat aorta is normally entirely mediated by NO, whereas treatment with daidzein or 17beta-oestradiol stimulates a contribution from a non-NO, nonprostaglandin factor acting through the opening of SK(Ca) and IK(Ca) channels, and involving activation of Na/K-ATPase, K(IR) and CYP450 epoxygenase. This pattern of sensitivity to the tested inhibitors is consistent with the contribution of EDHF to relaxation. Thus, EDHF contributes to the enhanced endothelium-dependent relaxation that is observed after chronic treatment with the phytoestrogen daidzein or with 17beta-oestradiol.     

Uterine artery function in a mouse model of pregnancy complicated by diabetes

It has been demonstrated previously that endothelium-dependent vasodilatation is impaired in myometrial arteries from women with gestational diabetes, which may play a role in mediating complications observed in diabetic pregnancies. It is not known which aspects of endothelium-dependent vasodilatation are impaired, thus a mouse model of pregnancy complicated by streptozotocin-induced diabetes was established to investigate underlying mechanisms. Uterine arteries from term-pregnant, diabetic and control C57Bl6/J mice were assessed using acetylcholine (ACh; 10(-10)-10(-5)M) in the presence or absence of a nitric oxide (NO) synthase inhibitor (L-NNA; 10(-5)M), a cyclooxygenase (COX) inhibitor (indomethacin; 10(-5)M) or the two in combination. Sensitivity to ACh was comparable between diabetic and control mice. However, the contribution of endothelium-dependent vasodilators was significantly altered. L-NNA significantly inhibited the relaxation of arteries from diabetic compared to control mice (65+/-11% vs 18+/-6%; p<.05). L-NNA and indomethacin significantly inhibited the relaxation of arteries from diabetic mice compared to control (87+/-5% vs 33+/-14%; p<0.05). These data indicate that endothelium-dependent relaxation of the uterine artery of control, pregnant mice was largely mediated by the non-NO/non-COX component. Surprisingly, arteries from diabetic mice were primarily dependent on NO, which may affect compensatory capacity as the disease progresses.     

Endothelium dysfunction in LDL receptor knockout mice: a role for H2O2

1. In this study, the role of endogenous H(2)O(2) as an endothelium-dependent relaxant factor was characterised in aortas from C57BL/6J and LDL receptor-deficient mice (LDLR(-/-)). 2. Aortic rings from LDLR(-/-) mice showed impaired endothelium-dependent relaxation to acetylcholine (ACh; 0.001-100 micro M) and to the Ca(2+) ionophore A23187 (0.001-3 micro M) compared with aortic rings from control mice. Endothelium-independent relaxation produced by the NO donor, 3-morpholino-sydnonimine (SIN-1) was not different between strains. 3. Pretreatment of vessels with L-NNA (100 micro M) or L-NNA (100 micro M) plus L-NAME (300 micro M) plus haemoglobin (10 micro M) markedly decreased, but did not abolish the relaxation to ACh in control mice. In the aortas from LDLR(-/-) mice treated with L-NNA (100 micro M), ACh induced a contractile effect. Catalase (800 and 2400 U ml(-1)) shifted to the right the endothelium-dependent relaxation to ACh in aortas from control but not from LDLR(-/-) mice. Aminotriazole (50 mM), which inhibits catalase, abolished its effect on control mice. Treatment of vessels with L-NNA and catalase abolished vasorelaxation induced by ACh. Indomethacin (10 micro M) did not modify the concentration-response curve to ACh. Superoxide dismutase (300 U ml(-1)) did not change ACh-induced relaxation in both strains. 4. Exogenous H(2)O(2) produced a concentration-dependent relaxation in endothelium-denuded aortic rings, which was not different between strains. 5. It is concluded that H(2)O(2) greatly contributes to relaxation to ACh in aorta from control mice. Endothelial-dependent relaxation to ACh is impaired in LDLR(-/-) mice. Reduced biosynthesis or increased inactivation of H(2)O(2) is the possible mechanism responsible for endothelial dysfunction in aortas of atherosclerosis-susceptible LDLR(-/-) mice.     

Decreased aortic glutathione levels may contribute to impaired nitric oxide-induced relaxation in hypercholesterolaemia

The aim of this study was to determine if the decrease in aortic total glutathione (GSH) levels in hypercholesterolaemia is related to the impairment of relaxation to acetylcholine (ACh) and exogenous nitric oxide (NO). Isometric tension and vascular GSH levels were measured in thoracic aortic rings from rabbits fed for 12 weeks with 0.5% cholesterol diet. Hypercholesterolaemia decreased aortic GSH levels and impaired relaxation to ACh and NO. To determine if GSH depletion impaired the response to NO, normal rabbit thoracic aorta was incubated with 1,3-bis [2-chloroethyl]-1-nitrosourea (BCNU; 0.2 mmol L(-1)), a GSH reductase inhibitor, or diazine-dicarboxylic acid bis [N, N dimethylamide] (diamide; 1 mmol L(-1)), a thiol oxidizing agent. BCNU or diamide decreased aortic GSH levels and impaired ACh and NO-induced relaxation. The effects of diamide on GSH levels and relaxation were partially prevented by co-incubation with GSH ester (GSE; 2 mmol L(-1)). Increasing GSH with GSE significantly enhanced NO-induced relaxation in aorta from both hypercholesterolaemic and normal rabbits, however relaxation of hypercholesterolaemic rabbit aorta was not restored to normal. These data suggest that other factors, perhaps related to the long-term decrease in GSH levels, are responsible for reduced NO bioactivity in hypercholesterolaemia.     

Interleukin-2 protects against endothelial dysfunction induced by high glucose levels in rats

AIMS: Interleukin-2 (IL-2) can modulate cardiovascular functions, but the effect of IL-2 on vascular endothelial function in diabetes is not known. We hypothesized that IL-2 may attenuate endothelial dysfunction induced by high glucose or diabetes. So the aim of this study was to investigate the effect of IL-2 on endothelium-response of aortas incubated with high glucose or from diabetic rats and its underlying mechanism. METHODS: Acetylcholine (ACh)-induced endothelium-dependent relaxation (EDR), sodium nitroprusside (SNP)-induced endothelium-independent relaxation (EIR), superoxide dismutase (SOD) and nitric oxide synthase (NOS) were measured in aortas isolated from non-diabetic rats and exposed to a high glucose concentration and from streptozotocin-induced diabetic rats. RESULTS: Incubation of aortic rings with high glucose (44 mM) for 4 h resulted in a significant inhibition of EDR, but had no effects on EIR. Co-incubation with IL-2 for 40 min prevented the inhibition of EDR caused by high glucose in a concentration-dependent manner. Similarly, high glucose decreased SOD and NOS activity in aortic tissue. IL-2 (1000 U/ml) significantly attenuated the decrease of SOD and NOS activity caused by high glucose. In addition, EDR declined along with the decrease of serum NO level in aortas from STZ-induced diabetic rats. Injection of IL-2 (5000 and 50,000 U kg(-1) d(-1), s.c.) for 5 weeks prevented the inhibition of EDR and the decrease of serum NO levels caused by diabetes. CONCLUSIONS: IL-2 significantly ameliorated the endothelial dysfunction induced by hyperglycemia, in which the activation of the NO pathway and SOD may be involved.     

Enhanced role for the opening of potassium channels in relaxant responses to acetylcholine after myocardial ischaemia and reperfusion in dog coronary arteries

1. Anaesthetized dogs were subjected to 1 h occlusion of the left circumflex coronary artery followed by 2 h of reperfusion. Relaxant responses were examined in coronary artery rings removed proximal (nonischaemic) or distal (ischaemic) to the site of occlusion. 2. Relaxant responses to acetylcholine (ACh) were similar in nonischaemic and ischaemic artery rings. In addition ACh-induced relaxation of nonischaemic and ischaemic artery rings was equally susceptible to inhibition of nitric oxide (NO) synthase using L-N(G)-nitroarginine (L-NOARG, 10(-4) M), or to inhibition of soluble guanylate cyclase using 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ, 10(-5) M). 3. In nonischaemic arteries, the relaxation to ACh was unaffected by high K+ (67 mM) but in ischaemic arteries, the maximum relaxation to ACh was significantly reduced from 113+/-6 to 60+/-2% (ANOVA, P<0.05). Tetraethylammonium (TEA, 10(-3) M), an inhibitor of large conductance calcium activated potassium (BK(Ca)) channels did not inhibit the response to ACh in nonischaemic arteries but in ischaemic arteries TEA significantly shifted the concentration response curve to ACh to the right (pEC(50); nonischaemic, 7.07+/-0.25; ischaemic, 6.54+/-0.21, P<0.01, ANOVA) without decreasing the maximum relaxation. TEA did not affect the responses to sodium nitroprusside in either nonischaemic or ischaemic arteries. 4. In conclusion, ischaemia/reperfusion did not change the sensitivity of endothelium-dependent relaxation to L-NOARG or ODQ indicating that ischaemia did not affect the contribution of NO or cyclic GMP to ACh-induced relaxation. However, in ischaemic arteries the opening of the BK(Ca) channels contributed to relaxation caused by ACh whereas TEA had no effect in nonischaemic arteries. The factor responsible for the opening of this potassium channel was a factor other than NO and may be endothelium derived hyperpolarizing factor (EDHF).     

Chronic administration of genistein improves aortic reactivity of streptozotocin-diabetic rats: mode of action

Diabetes mellitus is associated with major cardiovascular risk factors which are responsible for excess morbidity and mortality. Soy isoflavones like genistein are beneficial for correcting the hyperglycemia and preventing some diabetic complications. Thus, the effect of chronic administration of genistein was studied on aortic reactivity of streptozotocin (STZ)-diabetic rats. Male diabetic rats received genistein 1 mg/kg/day (i.p.) for 4 weeks 3 days after diabetes induction. Contractile responses to KCl and phenylephrine (PE) and relaxation responses to acetylcholine (ACh) and isosorbide dinitrate (ISD) were obtained from aortic rings. Maximum contractile response of endothelium-intact rings to KCL and PE was significantly lower in genistein-treated diabetic rats relative to untreated diabetic ones. Endothelium removal abolished the significant difference between genistein-treated and untreated diabetic groups regarding contractile response to KCl and PE. Meanwhile, endothelium-dependent relaxation to ACh was significantly higher in genistein-treated diabetic rats as compared to diabetic ones. Pretreatment of rings with N(omega)-L-arginine methyl ester (L-NAME) and indomethacin (INDO) significantly attenuated the observed responses. Meanwhile, one-month diabetes resulted in an elevation of malondialdehyde (MDA) and decreased superoxide dismutase (SOD) activity in aortic tissue and genistein treatment attenuated the increased MDA content and reduced activity of SOD. Therefore, chronic treatment of diabetic rats with genistein could prevent the abnormal functional changes in vascular reactivity in diabetic rats through nitric oxide- and prostaglandin-dependent pathways and via attenuating oxidative stress in the wall of aortic tissue.     

Biological evaluation of the nitric oxide-trapping agent, N-methyl-D-glucamine dithiocarbamate-Fe2+, as a probe of nitric oxide activity released from control and diabetic rat endothelium.

We utilized the nitric oxide (NO) scavenger N-methyl-D-glucamine dithiocarbamate-Fe2+ (MGD-Fe) to characterize the role of NO in basal and acetylcholine (ACh)-stimulated relaxation arising from the endothelium of control vs diabetic rat aortic rings. In phenylephrine-contracted rings, MGD-Fe produced an additional increment in tension that was indomethacin-insensitive (i.e., excluding a role of prostanoids in this action). This MGD-Fe-sensitive component was more pronounced in control rings than diabetic rings and of the same magnitude achieved in rings without MGD-Fe treatment after removal of endothelium or treatment with the NO synthase inhibitor L-nitroarginine (L-NA). This suggests complete scavenging of basal NO by MGD-Fe and supports reduced basal NO in diabetic rings. ACh fully relaxed both control and diabetic rings. This relaxation was abolished by removal of the endothelium and was inhibited by L-NA (by 100% and 90% in control and diabetic rings, respectively). In contrast, MGD-Fe only partially inhibited ACh-induced relaxation in control (65+/-5% inhibition) and diabetic (41+/-11% inhibition) rings. The MGD-Fe-resistant component was not further modified by indomethacin. Addition of L-arginine (L-ARG) (but not D-arginine (D-ARG) enhanced the ACh-induced relaxation of MGD-Fe-treated diabetic (but not control) rings. These data provide evidence about endothelium-dependent relaxation in control and diabetic rings which cannot be discerned by use of L-NA alone. This study suggests that ACh produces a NO synthase-dependent vasodilation, a portion of which is due to free NO radical (*NO) or due to NO in a form or location that is unavailable for scavenging by MGD-Fe.     

Captopril restores endothelium-dependent relaxation of rat aortic rings after exposure to homocysteine

This study was designed to investigate the effects of captopril, an angiotensin-converting enzyme inhibitor, on inhibition of endothelium-dependent relaxation induced by homocysteine in isolated rat aorta. Isometric tension recordings were used to assess inhibitory effects of homocysteine and protective effects of captopril on endothelium-dependent relaxation of aortic rings. Exposure of aortic rings to homocysteine (0.3 approximately 3 mmol/L) for 30 min induced a significant concentration-dependent inhibition of endothelium-dependent relaxation response to acetylcholine (ACh), but did not affect endothelium-independent relaxation response to sodium nitroprusside. Pre-incubation of aortic rings with captopril (3 approximately 30 micromol/L) for 15 min and co-incubation of aortic rings with homocysteine (1 mmol/L) for another 30 min attenuated the inhibition of homocysteine in a dose-dependent manner. Moreover, superoxide dismutase (SOD, 200 U/mL), a scavenger of superoxide anions, reduced homocysteine-induced inhibition. L-Arginine (3 mmol/L), a precursor of nitric oxide (NO), also attenuated the impairment of vasorelaxation induced by homocysteine. However, in the combined presence of SOD and L-arginine, the inhibitory effect of homocysteine was reversed, which was very similar to the effect of 30 micromol/L captopril. These results suggest that captopril can prevent the inhibition of endothelium-dependent relaxation induced by homocysteine in isolated rat aorta, which may be related to scavenging oxygen free radicals and enhancing NO production.     

Improvement of EDHF by chronic ACE inhibition declines rapidly after withdrawal in rats with myocardial infarction

Heart failure after myocardial infarction (MI) is associated with endothelial dysfunction. There is conflicting evidence on the exact nature of this endothelial dysfunction and how endothelium-dependent vasodilation is affected by angiotensin-converting enzyme inhibitor (ACE-I) therapy. Furthermore, consequences of acute ACE-I withdrawal are largely unknown. Therefore, we studied the contribution of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) to the effects of ACE-I therapy and its withdrawal on endothelial function in MI rats. Rats were subjected to coronary ligation to induce MI and were assigned to quinapril or vehicle from 2 weeks to 8 months post-MI. In parallel, MI rats treated for 14 months with quinapril were subjected to treatment withdrawal for 0, 4, and 6 weeks. Acetylcholine (ACh)-induced relaxation and underlying endothelium-derived mediators were studied in isolated aortic rings. Long-term quinapril (8 months) resulted in markedly improved endothelium-dependent vasodilation in rats with myocardial infarction, which could be attributed to marked improvement in non-NO/prostanoid-mediated relaxation (ie, EDHF). After 14 months of follow-up, maximum vasodilation was still preserved by quinapril. Withdrawal after 14 months of treatment caused significantly impaired ACh-induced EDHF-mediated relaxation within 4 weeks. A marked reduction in EDHF-mediated relaxation caused this impairment. NO-mediated relaxation was unaffected. These findings highlight the importance of EDHF impairment in development of endothelial dysfunction after myocardial infarction and the possibility of improving EDHF-mediated vasodilation with chronic ACE inhibitor therapy. In addition, withdrawal of chronic ACE inhibition after MI should be considered carefully, as profound endothelial dysfunction may develop rapidly.     

Temocapril, an angiotensin converting enzyme inhibitor, protects against diabetes-induced endothelial dysfunction

The effect of chronic treatment with the angiotensin converting enzyme inhibitor, temocapril, on prevention of endothelial dysfunction was evaluated in an experimental model of diabetes mellitus. Endothelium-dependent relaxation to acetylcholine was impaired while endothelium-independent relaxation to nitroglycerin was unaltered in diabetic aortic ring segments. Treatment of diabetic animals with temocapril prevented the impaired endothelium-dependent relaxation without altering responses to nitroglycerin. Acetylcholine-induced relaxation was largely due to nitric oxide (NO)-mediated relaxation; however, a small but significant portion of relaxation in aortic rings from temocapril-treated diabetic rats was resistant to inhibition by the nitric oxide synthase (NOS) inhibitor, L-nitroarginine.     

Chronic epigallocatechin-gallate improves aortic reactivity of diabetic rats: Underlying mechanisms

Diabetes mellitus is associated with major cardiovascular risk factors which are responsible for excess morbidity and mortality. Green tea catechins including epigallocatechin-3-gallate (EGCG) could exert beneficial health effects to ameliorate cardiovascular and metabolic diseases. Thus, the effect of chronic administration of EGCG was studied on aortic reactivity of streptozotocin (STZ)-diabetic rats. Male diabetic rats received EGCG 25 mg/kg/day for 8 weeks 1 week after diabetes induction. Contractile responses to KCl and phenylephrine (PE) and relaxation responses to acetylcholine (ACh) and sodium nitroprusside (SNP) were obtained from aortic rings. Maximum contractile response of endothelium-intact rings to KCL and PE was significantly lower in EGCG-treated diabetic rats relative to untreated diabetic ones. Endothelium removal abolished the significant difference between EGCG-treated and untreated diabetic groups regarding contractile response to KCl and PE. Meanwhile, endothelium-dependent relaxation to ACh was significantly higher in EGCG-treated diabetic rats as compared to diabetic ones. Pretreatment of rings with N(omega)-L-arginine methyl ester (L-NAME) and indomethacin (INDO) significantly attenuated the observed responses. Meanwhile, two-month diabetes resulted in an elevation of malondialdehyde (MDA) and decreased superoxide dismutase (SOD) activity in aortic tissue and EGCG treatment attenuated the increased MDA content and reduced activity of SOD. Therefore, chronic treatment of diabetic rats with EGCG could prevent the abnormal functional changes in vascular reactivity in diabetic rats through nitric oxide- and prostaglandin-dependent pathways and via attenuation of aortic lipid peroxidation.     

Endothelium-derived hyperpolarizing factor does not contribute to the decrease in endothelium-dependent relaxation in the aorta of streptozotocin-induced diabetic rats

• 1.1. We examined the contribution of endothelium-derived hyperpolarizing factor (EDHF) to the impairment of endothelium-dependent relaxation caused by acetylcholine (ACh) in the aorta of streptozotocin-induced diabetic rats, by using N^ω-nitro-l-arginine methylester (L-NAME) and tetraethylammonium chloride (TEA) to inhibit nitric oxide (NO) and EDHF, respectively.
• 2.2. ACh-induced relaxation of the aorta decreased in diabetic rats. In contrast, sodium nitroprusside-induced relaxation was the same in diabetic rats and control rats.
• 3.3. Treatment with 5 × 10⁻⁷ M L-NAME resulted in a right shift of the dose-response curves of ACh-induced relaxation in the aorta. The shift was greater in the control aorta.
• 4.4. Treatment with 5 × 10⁻⁴ M TEA resulted in a similar right shift in both the control and diabetic aorta.
• 5.5. Therefore, while endothelium-derived NO appears to contribute to the impairment of ACh-induced endothelium-dependent relaxation in the aorta of diabetic rats, EDHF does not

.     

Divergent effects of vitamin C on relaxations of rabbit aortic rings to acetylcholine and NO-donors

1. Vitamin C may influence NO-dependent relaxation independently of effects on oxidant stress. 2. We investigated effects of vitamin C (0.1 -- 10 mmol l(-1)) on relaxation of pre-constricted rabbit aortic rings to acetylcholine (ACh), authentic NO and the NO-donors glyceryl trinitrate (GTN), nitroprusside (NP) and S-nitroso-N-acetyl-penicillamine (SNAP). DETCA (2 -- 6 mmol l(-1)), a cell permeable inhibitor of endogenous Cu-Zn superoxide dismutase (SOD) was used to increase intracellular superoxide anion (O(2)(-)). 3. Vitamin C reduced the response to ACh (71 +/- 7% inhibition of maximum relaxation at 10 mmol l(-1)) and inhibited relaxation to authentic NO. Vitamin C inhibited relaxation to GTN but potentiated relaxations to NP and SNAP, causing a parallel shift to a lower concentration range of the log dose-response curve by approximately one log unit at the highest dose. 4. Vitamin C increased the concentration of NO in bath solution (plus EDTA, 1.0 mmol l(-1)) following the addition of SNAP from 53 +/- 14 to 771 +/- 101 nmol l(-1) over the range 0.1-3.0 mmol l(-1). 5. DETCA inhibited relaxation to ACh (71 +/- 9% inhibition of maximum relaxation). This inhibition was abolished by a cell permeable SOD mimetic, but not by vitamin C. DETCA inhibited relaxation to SNAP but not that to NP nor to GTN. 6. Vitamin C inhibits endothelium-dependent relaxations of rabbit aortic rings to ACh and authentic NO and does not reverse impaired relaxation resulting from increased intracellular oxidant stress. Vitamin C potentiates relaxation to the NO-donors NP and SNAP by a mechanism that could involve release of NO from nitrosothiols.     

Lack of endothelium-derived hyperpolarizing factor (EDHF) up-regulation in endothelial dysfunction in aorta in diabetic rats

It is not known whether the impairment of nitric oxide (NO)-dependent vasodilation of the aorta of diabetic rats is associated with any changes in the endothelial production of vasoactive prostanoids and endothelium-derived hyperpolarizing factor (EDHF). Therefore, we analyzed the contribution of NO, vasoactive prostanoids and EDHF to the decreased endothelium-dependent vasorelaxation in Sprague-Dawley rats at 4 and 8 weeks after diabetes mellitus induced by streptozotocin (STZ). The acetylcholine-induced (Ach) endothelium-dependent relaxation was significantly decreased in the thoracic aorta 8 weeks after the STZ-injection (Ach 10(-6) M: 73.1 +/- 7.4% and 56.7 +/- 7.9% for control and diabetic rats, respectively). The sodium nitroprusside-induced (NaNP) endothelium-independent vasodilation was also impaired in the diabetic rats (8 weeks after STZ) (NaNP 10(-8) M: 74.2 +/- 11.4% and 35.9 +/- 9.4% for control and diabetic rats, respectively). In contrast, the basal NO production, as assessed by the N omega-nitro-L-arginine methyl ester (L-NAME)-induced vasoconstriction was not modified in diabetes. Moreover, the amount of 6-keto-PGF(1 alpha) (stable metabolite of prostacyclin / prostaglandin I2 / PGI2 ), 12-L-hydroxy-5,8,10-heptadecatrienoic acid (12-HHT) and thromboxane B2 (TxB2 ) (stable metabolite of thromboxane A2 - TxA2) were significantly increased in the 8 weeks diabetic rat aorta. The EDHF-pathway did not change in the aortic endothelium during the development of STZ-induced diabetes. Our results indicate that STZ-induced diabetes mellitus did not modify the basal NO production, but induced the impairment of acetylcholine- and sodium nitroprusside-induced vasodilation in the thoracic aorta. In parallel with the impairment of NO-dependent vasodilation, the basal PGI2, 12-HHT and TxA2 synthesis were increased. The EDHF-pathway did not contribute to the endothelium-dependent relaxation either in control or diabetic aorta. The above alterations in the endothelial function may play an important role in the development of endothelial dysfunction and vascular complications of diabetes.     

Changes in superoxide dismutase mRNA expression by streptozotocin-induced diabetes.

1. Experiments were designed to investigate the involvement of superoxide anions in the attenuated endothelium-dependent relaxation of the rat aorta from streptozotocin (STZ)-induced diabetic rats. 2. The endothelium-dependent relaxation responses to acetylcholine (ACh, 10(-7) M) in helical strips of the aorta precontracted with noradrenaline (NA, 5 x 10(-3) approximately 3 x 10(-7) M) were significantly decreased in STZ-induced diabetic rats. The recovery phase of the relaxation after single administration of ACh in the STZ-induced diabetic rats was more rapid than those in control vessels. 3. Preincubation of aortic strips with superoxide dismutase (SOD, 60 u ml-1) normalized the recovery phase of the relaxation of diabetic aorta after single administration of ACh, whereas catalase (150 u ml-1) or indomethacin (10(-5) M) had no effects on the relaxation. 4. SOD (180 u ml-1) caused relaxation in NA precontracted aortic strips and the degree of the SOD-induced relaxation was significantly greater in diabetic aorta as compared with age-matched control vessels. 5. When the changes in mRNA expressions of Mn-SOD or Cu-Zn-SOD were observed, Mn-SOD mRNA expression was markedly decreased, and Cu-Zn-SOD was slightly decreased in diabetic aorta. 6. These results suggest that the rapid destruction of NO by superoxide anions may occur in the STZ-induced diabetic rats, and this may be due to a decrease in mRNA expression of Mn-SOD or Cu-Zn-SOD.     

Attenuation of endothelium-dependent relaxation in aorta from diabetic rats

Endothelium-dependent relaxation was examined in aortic ring preparations obtained from rats with streptozotocin-induced diabetes. The endothelium-dependent relaxation which was produced by acetylcholine and histamine in aortic rings precontracted with norepinephrine was significantly attenuated in aortic rings from diabetic rats when compared with the relaxation in rings from age-matched control animals. However, the relaxation induced by sodium nitroprusside (an endothelium-independent relaxant agent) in diabetic preparations was comparable to the control. These results show that diabetes leads to an impairment of the endothelium-dependent relaxation of aorta.