Losartan improves aortic endothelium-dependent relaxation via proline-rich tyrosine kinase 2/Src/Akt pathway in type 2 diabetic Goto-Kakizaki rats

In diabetic states, endothelial dysfunction is related to vascular complications. We hypothesized that insulin-induced relaxation and the associated proline-rich tyrosine kinase 2 (Pyk2)/Src/Akt pathway would be abnormal in aortas from the Goto-Kakizaki (GK) type 2 diabetic rat, which exhibits hyperglycemia/insulin resistance, and that losartan treatment of such rats (25 mg·kg(-1)·day(-1) for 2 wk) would correct these abnormalities. Endothelium-dependent relaxation was by measuring isometric force in helical strips of aortas from four groups, each of 30 rats: normal Wistar (control), GK (diabetic), losartan-treated normal, and losartan-treated GK. Pyk2, Src, and Akt/endothelial nitric oxide synthase (eNOS) signaling-pathway protein levels and activities were assayed mainly by Western blotting and partly by immunohistochemistry. In GK (vs. age-matched control) aortas, various insulin-stimulated levels [nitric oxide production and the phosphorylations of eNOS at Ser(1177), of Akt at Thr(308), of phosphoinositide-dependent kinase-1 (PDK1) at Ser(241), of Src at Tyr(416), and of Pyk2 at Tyr(579)] were all significantly decreased and unaffected by either Src inhibitor (PP2) or Pyk2 inhibitor (AG17), while the insulin-stimulated levels of insulin receptor substrate (IRS)-1 phosphorylation at Ser(307), total-eNOS, and total-Akt were significantly increased. Losartan treatment normalized these altered levels. The insulin-stimulated phosphorylation levels of Src/PDK1/Akt/eNOS, but not of Pyk2, were decreased by PP2 in control and losartan-treated GK, but not in GK, aortas. These results suggest that in the GK diabetic aorta increased phospho-IRS-1 (at Ser(307)) and decreased Pyk2/Src activity inhibit insulin-induced stimulation of the PDK/Akt/eNOS pathway. The observed increase in phospho-IRS-1 (at Ser(307)) may result from increased angiotensin II activity.     

Mechanisms underlying enhanced vasorelaxant response to protease-activated receptor 2-activating peptide in type 2 diabetic Goto–Kakizaki rat mesenteric artery

Protease-activated receptor 2 (PAR2) is a G-protein-coupled receptor that is proteolytically activated by certain endogenous proteases, such as trypsin, tryptase, and factor Xa. PAR2 can also be activated by synthetic peptides if their sequence mimics the tethered ligand exposed after receptor cleavage. Although it is known that PAR2 modulates vascular reactivity, it is unclear whether at the chronic stage of type 2 diabetes there are alterations in PAR2-mediated vascular responses. We investigated this issue by exposing mesenteric artery rings to PAR2-activating peptide (PAR2-AP; SLIGRL-NH₂), the arteries used being obtained from later-stage (32–40-week-old) type 2 diabetic Goto–Kakizaki (GK) rats. The PAR2-AP-induced relaxation was enhanced in GK rats (vs. age-matched Wistar rats), whereas the ACh-induced relaxation was weaker in GK than in Wistar rats. In both groups, the PAR2-AP-induced relaxation was largely blocked by endothelial denudation or by N^G-nitro-l-arginine [nitric oxide (NO) synthase inhibitor] treatment, but it was unaffected by indomethacin (cyclooxygenase inhibitor) treatment. Both the NO production induced by PAR2-AP and the PAR2 protein expression were significantly increased in mesenteric arteries from GK rats (vs. Wistar rats). These data are the first to indicate that the PAR2-AP-induced endothelium-dependent relaxation is enhanced in mesenteric arteries isolated from type 2 diabetic GK rats at the chronic stage, and they further suggest that the enhancement may be due to an increased expression of PAR2 receptors in this artery.     

Metformin normalizes endothelial function by suppressing vasoconstrictor prostanoids in mesenteric arteries from OLETF rats, a model of type 2 diabetes

We previously reported that in mesenteric arteries from aged Otsuka Long-Evans Tokushima fatty (OLETF) rats (a type 2 diabetes model) endothelium-derived hyperpolarizing factor (EDHF)-type relaxation is impaired while endothelium-derived contracting factor (EDCF)-mediated contraction is enhanced (Matsumoto T, Kakami M, Noguchi E, Kobayashi T, Kamata K. Am J Physiol Heart Circ Physiol 293: H1480-H1490, 2007). Here we investigated whether acute and/or chronic treatment with metformin might improve this imbalance between the effects of the above endothelium-derived factors in mesenteric arteries isolated from OLETF rats. In acute studies on OLETF mesenteric arteries, ACh-induced relaxation was impaired and the relaxation became weaker at high ACh concentrations. Both metformin and 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside [AICAR, an AMP-activated protein kinase (AMPK) activator that is also activated by metformin] 1) diminished the tendency for the relaxation to reverse at high ACh concentrations and 2) suppressed both ACh-induced EDCF-mediated contraction and ACh-stimulated production of prostanoids (thromboxane A2 and PGE2). In studies on OLETF arteries from chronically treated animals, metformin treatment (300 mg.kg(-1).day(-1) for 4 wk) 1) improved ACh-induced nitric oxide- or EDHF-mediated relaxation and cyclooxygenase (COX)-mediated contraction, 2) reduced EDCF-mediated contraction, 3) suppressed production of prostanoids, and 4) reduced superoxide generation. Metformin did not alter the protein expressions of endothelial nitric oxide synthase (eNOS), phospho-eNOS (Ser1177), or COX-1, but it increased COX-2 protein. These results suggest that metformin improves endothelial functions in OLETF mesenteric arteries by suppressing vasoconstrictor prostanoids and by reducing oxidative stress. Our data suggest that within the timescale studied here, metformin improves endothelial function through this direct mechanism, rather than by improving metabolic abnormalities.     

Heat shock protein 90 modulates endothelial nitric oxide synthase activity and vascular reactivity in the newborn piglet pulmonary circulation

Heat shock protein 90 (Hsp90) binding to endothelial nitric oxide synthase (eNOS) is an important step in eNOS activation. The conformational state of bound Hsp90 determines whether eNOS produces nitric oxide (NO) or superoxide (O(2)(*-)). We determined the effects of the Hsp90 antagonists geldanamycin (GA) and radicicol (RA) on basal and ACh-stimulated changes in vessel diameter, cGMP production, and Hsp90:eNOS coimmunoprecipitation in piglet resistance level pulmonary arteries (PRA). In perfused piglet lungs, we evaluated the effects of GA and RA on ACh-stimulated changes in pulmonary arterial pressure (Ppa) and perfusate accumulation of stable NO metabolites (NOx(-)). The effects of GA and RA on ACh-stimulated O(2)(*-) generation was investigated in cultured pulmonary microvascular endothelial cells (PMVEC) by dihydroethidine (DHE) oxidation and confocal microscopy. Hsp90 inhibition with GA or RA reduced ACh-mediated dilation, abolished the ACh-stimulated increase in cGMP, and reduced eNOS:Hsp90 coprecipitation. GA and RA also inhibited the ACh-mediated changes in Ppa and NOx(-) accumulation rates in perfused lungs. ACh increased the rate of DHE oxidation in PMVEC pretreated with GA and RA but not in untreated cells. The cell-permeable superoxide dismutase mimetic M40401 reversed GA-mediated inhibition of ACh-induced dilation in PRA. We conclude that Hsp90 is a modulator of eNOS activity and vascular reactivity in the newborn piglet pulmonary circulation. Uncoupling of eNOS with GA or RA inhibits ACh-mediated dilation by a mechanism that involves O(2)(*-) generation.     

Estradiol-induced expression of N(+)-K(+)-ATPase catalytic isoforms in rat arteries: gender differences in activity mediated by nitric oxide donors

We tested the hypothesis that previously demonstrated gender differences in ACh-induced vascular relaxation could involve diverse Na(+)-K(+)-ATPase functions. We determined Na(+)-K(+)-ATPase by measuring arterial ouabain-sensitive 86Rb uptake in response to ACh. We found a significant increase of Na+ pump activity only in aortic rings from female rats (control 206 +/- 11 vs. 367 +/- 29 nmol 86Rb/K.min(-1).g wt tissue(-1); P < 0.01). Ovariectomy eliminated sex differences in Na(+)-K(+)-ATPase function, and chronic in vivo hormone replacement with 17beta-estradiol restored the ACh effect on Na(+)-K(+)-ATPase. Because ACh acts by enhancing production of NO, we examined whether the NO donor sodium nitroprusside (SNP) mimics the action of ACh on Na(+)-K(+)-ATPase activity. SNP increased ouabain-sensitive 86Rb uptake in denuded female arteries (control 123 +/- 7 vs. 197 +/- 12 nmol 86Rb/K.min(-1).g wt tissue(-1); P < 0.05). Methylene blue (an inhibitor of guanylate cyclase) and KT-5823 (a cGMP-dependent kinase inhibitor) blocked the stimulatory action of SNP. Exposure of female thoracic aorta to the Na+/K+ pump inhibitor ouabain significantly decreased SNP-induced and ACh-mediated relaxation of aortic rings. At the molecular level, Western blot analysis of arterial tissue revealed significant gender differences in the relative abundance of catalytic isoforms of Na(+)-K(+)-ATPase. Female-derived aortas exhibited a greater proportion of alpha2-isoform (44%) compared with male-derived aortas. Furthermore, estradiol upregulated the expression of alpha2 mRNA in male arterial explants. Our results demonstrate that enhancement of ACh-induced relaxation observed in female rats may be in part explained by 1) NO-dependent increased Na(+)-K(+)-ATPase activity in female vascular tissue and 2) greater abundance of Na(+)-K(+)-ATPase alpha2-isoform in females.     

Nitric-oxide-dependent pial arteriolar dilation in the female rat: effects of chronic estrogen depletion and repletion

In this study, we compared endothelial nitric oxide synthase (eNOS)-mediated cerebral vasodilating responses in intact female rats, chronically ovariectomized (OVX) rats, and OVX rats treated for 2 weeks with 17beta-estradiol (E(2)). Under anesthesia, using intravital microscopy and a closed cranial window system, pial arteriolar diameter changes were monitored during sequential cortical suffusions of an eNOS-dependent dilator [acetylcholine (ACh)] and a direct NO donor [S-nitrosoacetylpenicillamine (SNAP)]. In separate rats from the same groups, we compared eNOS and caveolin-1 (CAV-1) protein abundance in pial arterioles (via immunofluorescence analyses). In untreated and low-dose E(2)-treated (1.0 microg x kg(-1) x day(-1)) OVX rats, ACh-induced vasodilations were virtually absent. High-dose E(2) treatment (100 microg x kg(-1) x day(-1)) restored ACh-induced pial arteriolar dilations to levels seen in intact females. The vasodilations elicited by SNAP and ADO were unaffected by chronic estrogen changes, indicating no direct estrogen influence on vascular smooth muscle (VSM) reactivity. Pial arteriolar eNOS protein abundance was diminished by ovariectomy and restored by high-dose E(2) treatment. Pial arteriolar CAV-1 expression was higher in OVX versus intact and E(2)-treated OVX females. These results suggest that long-term changes in estrogen directly influence brain eNOS functional activity. The estrogen-related changes in eNOS-dependent vasodilating function appear to be related, in part, to a capacity for E(2) to increase eNOS protein expression and, in part, to an E(2)-associated diminution in endothelial CAV-1 expression.     

Mechanisms underlying the chronic pioglitazone treatment-induced improvement in the impaired endothelium-dependent relaxation seen in aortas from diabetic rats

The objectives of this study were to determine the effects of chronic treatment with pioglitazone, a peroxisome proliferator-activated receptor gamma agonist, on the impaired endothelium-dependent relaxation seen in aortas from established streptozotocin (STZ)-induced diabetic rats, and to identify some of the molecular mechanisms involved. Starting at 8 weeks of diabetes, pioglitazone (10 mg/kg) was administered to STZ-induced diabetic rats for 4 weeks. In untreated STZ rats (vs age-matched control rats): (1) ACh-induced relaxation, cGMP accumulation, phosphorylation of the cGMP-dependent protein kinase substrate vasodilator-stimulated phosphoprotein at Ser-239 [an established biochemical end-point of nitric oxide (NO)/cGMP signaling], and Cu/Zn-superoxide dismutase (SOD) expression and SOD activity were all reduced; (2) aortic superoxide generation, nitrotyrosine expression, and NAD(P)H oxidase activity were increased; (3) plasma endothelin-1 (ET-1) and aortic c-Jun (AP-1 component) protein expressions were increased. Pioglitazone treatment markedly corrected the above abnormalities. Collectively, these results suggest that pioglitazone treatment improves endothelium-dependent relaxation by reducing oxidative stress via increased SOD activity, decreased NAD(P)H oxidase activity, and a decreased ET-1 level, and that this decreased ET-1 level may be attributable to an inhibition of the AP-1 signaling pathway.     

Neuronal nitric oxide synthase-derived hydrogen peroxide is a major endothelium-dependent relaxing factor

Endothelium-dependent vasorelaxation in large vessels is mainly attributed to Nomega-nitro-L-arginine methyl ester (L-NAME)-sensitive endothelial nitric oxide (NO) synthase (eNOS)-derived NO production. Endothelium-derived hyperpolarizing factor (EDHF) is the component of endothelium-dependent relaxations that resists full blockade of NO synthases (NOS) and cyclooxygenases. H2O2 has been proposed as an EDHF in resistance vessels. In this work we propose that in mice aorta neuronal (n)NOS-derived H2O2 accounts for a large proportion of endothelium-dependent ACh-induced relaxation. In mice aorta rings, ACh-induced relaxation was inhibited by L-NAME and Nomega-nitro-L-arginine (L-NNA), two nonselective inhibitors of NOS, and attenuated by selective inhibition of nNOS with L-ArgNO2-L-Dbu-NH2 2TFA (L-ArgNO2-L-Dbu) and 1-(2-trifluoromethylphehyl)imidazole (TRIM). The relaxation induced by ACh was associated with enhanced H2O2 production in endothelial cells that was prevented by the addition of L-NAME, L-NNA, L-ArgNO2-L-Dbu, TRIM, and removal of the endothelium. The addition of catalase, an enzyme that degrades H2O2, reduced ACh-dependent relaxation and abolished ACh-induced H2O2 production. RT-PCR experiments showed the presence of mRNA for eNOS and nNOS but not inducible NOS in mice aorta. The constitutive expression of nNOS was confirmed by Western blot analysis in endothelium-containing vessels but not in endothelium-denuded vessels. Immunohistochemistry data confirmed the localization of nNOS in the vascular endothelium. Antisense knockdown of nNOS decreased both ACh-dependent relaxation and ACh-induced H2O2 production. Antisense knockdown of eNOS decreased ACh-induced relaxation but not H2O2 production. Residual relaxation in eNOS knockdown mouse aorta was further inhibited by the selective inhibition of nNOS with L-ArgNO2-L-Dbu. In conclusion, these results show that nNOS is constitutively expressed in the endothelium of mouse aorta and that nNOS-derived H2O2 is a major endothelium-dependent relaxing factor. Hence, in the mouse aorta, the effects of nonselective NOS inhibitors cannot be solely ascribed to NO release and action without considering the coparticipation of H2O2 in mediating vasodilatation.     

Exercise training activates large-conductance calcium-activated K+ channels and enhances nitric oxide production in rat mesenteric artery and thoracic aorta

Exercise training has reversible beneficial effects on cardiovascular diseases, e.g. hypertension, which may result from a decrease in systemic vascular resistance. The purpose of this study was to investigate possible mechanisms associated with the changes in vascular reactivity in large and small arteries with vasoconstrictors and vasodilators in rats after exercise. Wistar-Kyoto rats were trained for 8 weeks (Ex group) on a treadmill and compared with sedentary counterparts (Sed group). After the measurement of blood pressure and heart rate at 8 weeks, rat mesenteric arteries and thoracic aortas were excised and prepared as rings for this study. In addition, special care was taken not to damage the endothelium of the preparations. Our results showed that exercise training for 8 weeks (1) not only prevented an increase in blood pressure but also caused a fall in heart rate, (2) attenuated the contractions induced by both prostaglandin F(2alpha) (PGF(2alpha)) and high K(+) in the mesenteric artery, but reduced the PGF(2alpha)-induced contraction in the aorta only, (3) enhanced the relaxation elicited by acetylcholine (ACh) in both mesenteric arteries and aortas, and (4) increased nitrate [an indicator of nitric oxide (NO) formation] in plasma. The enhancement of ACh-induced relaxation in the mesenteric arteries in the Ex group was suppressed by pretreatment with N(omega) -nitro-L-arginine methyl ester (L-NAME), tetraethylammonium (TEA; a nonselective inhibitor of K(+) channels) or charybdotoxin [CTX; a selective inhibitor of large-conductance calcium-activated K(+) (BK(Ca)) channels], whereas in the aorta that response was attenuated by TEA or CTX and almost completely abolished by L-NAME. However, with a combination of L-NAME plus CTX in the mesenteric artery, ACh-induced relaxation was completely abolished in the Sed group, but not in the Ex group. These results suggest that in addition to NO, activation of BK(Ca) channels in the vascular beds, at least in part, also contributes to vasodilatation in animals with exercise training.     

UDP-induced relaxation is enhanced in aorta from female obese Otsuka Long–Evans Tokushima Fatty rats

Uridine 5′-diphosphate (UDP) plays an important role in controlling vascular tone; however, UDP-mediated response in metabolic syndromes, including obesity and type 2 diabetes in females, remains unclear. In this study, we investigated UDP-mediated response in the aorta of female obese Otsuka Long–Evans Tokushima Fatty (OLETF) rats and control Long–Evans Tokushima Otsuka (LETO) rats. In OLETF rat aortas precontracted by phenylephrine (PE) (vs. LETO), (1) UDP-induced relaxation was increased, whereas acetylcholine (ACh)-induced relaxation was decreased; (2) no UDP- or ACh-induced relaxations were observed in endothelial denudation, whereas UDP-induced small contraction was observed; and (3) N^G-nitro-L-arginine [L-NNA, a nitric oxide (NO) synthase inhibitor] eliminated UDP-induced relaxation and small contraction, whereas caused contrasting responses by ACh, including slight relaxations (LETO) and contractions (OLETF). Indomethacin, a cyclooxygenase inhibitor, eliminated the difference in UDP- and ACh-induced relaxations between the groups by increased UDP-induced relaxation in the LETO group and increased ACh-induced relaxation in the OLETF group. MRS2578, a P2Y₆ receptor antagonist, eliminated the difference in UDP-induced relaxations between the groups by decreasing UDP-induced relaxation in the OLETF group. MRS2578 had no effect on UDP-induced contraction in endothelium-denuded aortas. Therefore, these findings demonstrate opposite trends of relaxations by UDP and ACh in OLETF and LETO rat aortas. These differences may be attributed to the imbalance between NO and vasoconstrictor prostanoids upon stimulations. Increased UDP-induced relaxation in OLETF rat aorta may be caused by the activation of endothelial MRS2578-sensitive P2Y₆ receptor.     

Alterations in vascular endothelial function in the aorta and mesenteric artery in type II diabetic rats

We used the partial protection exerted by suitable dosages of nicotinamide against the beta-cytotoxic effect of streptozotocin (STZ) to create an experimental diabetic syndrome in adult rats that appears closer to type II diabetes mellitus than other available animal models. The dosage of 230 mg/kg of nicotinamide given intraperitoneally 15 min before STZ administration (65 mg/kg i.v.) yielded animals with hyperglycemia (187.8 +/- 17.8 vs. 103.8 +/- 2.8 mg/dL in controls; P < 0.001) and preservation of plasma insulin levels. This study assessed the relationship between endothelial dysfunction and agonist-induced contractile responses in such rats. In the thoracic aorta, the acetylcholine (ACh) induced relaxation was significantly reduced and the noradrenaline (NA) induced contractile response was significantly increased in diabetic rats compared with age-matched control rats. In the superior mesenteric artery, the ACh-induced relaxation was similar in magnitude between diabetic and age-matched control rats; however, the ACh-induced endothelium-derived hyperpolarizing factor (EDHF) type relaxation was significantly weaker in diabetic rats than in the controls. The phenylephrine (PE) induced contractile response was not different between the two groups. The plasma concentration of NOx (NO2- + NO3-) was significantly lower in diabetic rats than in control rats. We conclude that vasomotor activities in conduit arteries are impaired in this type II diabetes model.     

Role of superoxide and angiotensin II suppression in salt-induced changes in endothelial Ca2+ signaling and NO production in rat aorta

Male Sprague-Dawley rats were maintained on a low-salt (LS) diet (0.4% NaCl) or changed to a high-salt (HS) diet (4% NaCl) for 3 days. Increases in intracellular Ca2+ ([Ca2+]i) in response to methacholine (10 microM) and histamine (10 microM) were significantly attenuated in aortic endothelial cells from rats fed a HS diet, whereas thapsigargin (10 microM)-induced increases in [Ca2+]i were unaffected. Methacholine-induced nitric oxide (NO) production was eliminated in endothelial cells of aortas from rats fed a HS diet. Low-dose ANG II infusion (5 ng x kg(-1) x min(-1) iv) for 3 days prevented impaired [Ca2+]i signaling response to methacholine and histamine and restored methacholine-induced NO production in aortas from rats on a HS diet. Adding Tempol (500 microM) to the tissue bath to scavenge superoxide anions increased NO release and caused N(omega)-nitro-L-arginine methyl ester-sensitive vascular relaxation in aortas from rats fed a HS diet but had no effect on methacholine-induced Ca2+ responses. Chronic treatment with Tempol (1 mM) in the drinking water restored NO release, augmented vessel relaxation, and increased methacholine-induced Ca2+ responses significantly in aortas from rats on a HS diet but not in aortas from rats on a LS diet. These findings suggest that 1) agonist-induced Ca2+ responses and NO levels are reduced in aortas of rats on a HS diet; 2) increased vascular superoxide levels contribute to NO destruction, and, eventually, to impaired Ca2+ signaling in the vascular endothelial cells; and 3) reduced circulating ANG II levels during elevated dietary salt lead to elevated superoxide levels, impaired endothelial Ca2+ signaling, and reduced NO production in the endothelium.     

Shear stress induces eNOS mRNA expression and improves endothelium-dependent dilation in senescent soleus muscle feed arteries.

We tested the hypothesis that increased intraluminal shear stress induces endothelial nitric oxide (NO) synthase (eNOS) mRNA expression and improves endothelium-dependent dilation in senescent soleus muscle feed arteries (SFA) by increasing NO production. SFA were isolated from young (4 mo) and old (24 mo) male Fischer 344 rats and cannulated with two resistance-matched glass micropipettes. SFA were exposed to no flow (NF), low flow (LF), intermediate flow (IF), or high flow (HF) for 4 h. Mean intraluminal shear stress ranged from 0 to 82 dyn/cm(2). At the end of the 4-h treatment period, eNOS mRNA expression was assessed in each SFA. eNOS mRNA expression was significantly lower in old NF SFA than in young NF SFA. In old SFA, eNOS mRNA expression was induced by IF (+154%) and HF (+136%), resulting in a level of expression that was not different from that of young SFA. In a separate series of experiments, SFA were pretreated with NF or HF for 4 h, and endothelial function was assessed by examining vasodilator responses to ACh. ACh-induced dilation was less in old NF SFA than young NF SFA. Pretreatment with HF improved ACh-induced dilation in old SFA such that the response was similar to that of young SFA. In the presence of N(omega)-nitro-L-arginine to inhibit NOS, ACh-induced dilation was inhibited in old HF SFA such that the response was no longer greater than that of old NF SFA. These results indicate that increased intraluminal shear stress induces eNOS mRNA expression and improves endothelium-dependent dilation in senescent SFA by increasing NO production.     

Short-term insulin treatment and aortic expressions of IGF-1 receptor and VEGF mRNA in diabetic rats

We investigated the relationship between the changes in vascular responsiveness and growth factor mRNA expressions induced by 1-wk treatment with high-dose insulin in control and established streptozotocin (STZ)-induced diabetes. Aortas from diabetic rats, but not those from insulin-treated diabetic rats, showed impaired endothelium-dependent relaxation in response to ACh (vs. untreated controls). The ACh-induced nitrite plus nitrate (NOx) level showed no significant difference between controls and diabetics. Insulin treatment increased NOx only in diabetics. In diabetics, insulin treatment significantly increased the aortic expressions of endothelial nitric oxide synthase (eNOS) mRNA and VEGF mRNA. The expression of IGF-1 mRNA was unaffected by diabetes or by insulin treatment. In contrast, the mRNA for the aortic IGF-1 receptor was increased in diabetics and further increased in insulin-treated diabetics. In aortic strips from age-matched control rats, IGF-1 caused a concentration-dependent relaxation. This relaxation was significantly stronger in strips from STZ-induced diabetic rats. These results suggest that in STZ-diabetic rats, short-term insulin treatment can ameliorate endothelial dysfunction by inducing overexpression of eNOS and/or VEGF mRNAs possibly via IGF-1 receptors. These receptors were increased in diabetes, perhaps as result of insulin deficiency.     

Endothelial function and vascular oxidative stress in long-lived GH/IGF-deficient Ames dwarf mice

Hypopituitary Ames dwarf mice have low circulating growth hormone (GH)/IGF-I levels, and they have extended longevity and exhibit many symptoms of delayed aging. To elucidate the vascular consequences of Ames dwarfism we compared endothelial O2(-) and H2O2 production, mitochondrial reactive oxygen species (ROS) generation, expression of antioxidant enzymes, and nitric oxide (NO) production in aortas of Ames dwarf and wild-type control mice. In Ames dwarf aortas endothelial O2(-) and H2O2 production and ROS generation by mitochondria were enhanced compared with those in vessels of wild-type mice. In Ames dwarf aortas there was a less abundant expression of Mn-SOD, Cu,Zn-SOD, glutathione peroxidase (GPx)-1, and endothelial nitric oxide synthase (eNOS). NO production and acetylcholine-induced relaxation were also decreased in aortas of Ames dwarf mice. In cultured wild-type mouse aortas and in human coronary arterial endothelial cells treatment with GH and IGF significantly reduced cellular O2(-) and H2O2 production and ROS generation by mitochondria and upregulated expression of Mn-SOD, Cu,Zn-SOD, GPx-1, and eNOS. Thus GH and IGF-I promote antioxidant phenotypic changes in the endothelial cells, whereas Ames dwarfism leads to vascular oxidative stress.     

Restoration of impaired endothelium-dependent coronary vasodilation in failing heart: role of eNOS phosphorylation and CGMP/cGK-I signaling

In congestive heart failure (CHF), coronary vascular relaxation is associated with endothelial dysfunction and nitric oxide (NO) deficiency. This study explored the reversibility of this process in hearts recovering from CHF and its related mechanisms. Dogs were chronically instrumented to measure cardiac function and coronary blood flow (CBF). Heart failure was induced by right ventricular pacing at 240 beats/min for 3-4 wk, and cardiac recovery (CR) was allowed by the termination of cardiac pacing for 3-4 wk after the development of CHF, in which left ventricular contractile function was restored by 80-90%. The endothelium-dependent CBF response to bradykinin and acetylcholine was depressed in CHF and fully restored in CR. Myocardial NOx (nitrate/nitrite), endothelial NO synthase (eNOS) mRNA expression, total protein, and phosphorylated eNOS decreased significantly in failing hearts. However, myocardial NOx recovered to 78% of control and phosphorylated eNOS was fully restored in CR, despite the fact that eNOS mRNA expression and protein levels remained lower than control. Furthermore, the endothelium-independent CBF response to nitroglycerin did not change in CHF; however, it increased by 75% in CR, in conjunction with a near threefold increase in the phosphorylation of vasodilation-stimulated phosphoprotein (VASP) at Ser(239) in recovering hearts. Thus the complete restoration of endothelium-dependent coronary vascular relaxation during cardiac recovery from CHF was mediated by 1) a restoration of phosphorylated eNOS for partial recovery of the NO production and 2) an increase in cGMP/cGMP-dependent protein kinase-I pathway signaling activity for the enhancement of coronary vascular smooth muscle relaxation in response to NO.     

Role of oxidative stress in age-related reduction of NO-cGMP-mediated vascular relaxation in SHR

The incidence of hypertension increases during the late stages of aging; however, the vascular mechanisms involved are unclear. We investigated whether the late stages of aging are associated with impaired nitric oxide (NO)-mediated vascular relaxation and enhanced vascular contraction and whether oxidative stress plays a role in the age-related vascular changes. Aging (16 mo) male spontaneously hypertensive rats (SHR) nontreated or treated for 8 mo with the antioxidant tempol (1 mM in drinking water) or vitamin E (E; 5,000 IU/kg chow) and vitamin C (C; 100 mg. kg-1. day-1 in drinking water) and adult (12 wk) male SHR were used. After the arterial pressure was measured, aortic strips were isolated from the rats for measurement of isometric contraction. The arterial pressure and phenylephrine (Phe)-induced vascular contraction were enhanced, and the ACh-induced vascular relaxation and nitrite/nitrate production were reduced in aging compared with adult rats. In aging rats, the arterial pressure was nontreated (188 +/- 4), tempol-treated (161 +/- 6), and E + C-treated (187 +/- 1 mmHg). Phe (10-5 M) caused an increase in active stress in nontreated aging rats (14.3 +/- 1.0) that was significantly (P < 0.05) reduced in tempol-treated (9.0 +/- 0.7) and E + C-treated rats (9.8 +/- 0.6 x 104 N/m2). ACh produced a small relaxation of Phe contraction in nontreated aging rats that was enhanced (P < 0.05) in tempol- and E + C-treated rats. l-NAME (10-4 M), inhibitor of NO synthase, or ODQ (10-5 M), inhibitor of cGMP production in smooth muscle, inhibited ACh relaxation and enhanced Phe contraction in tempol- and E + C-treated but not the nontreated aging rats. ACh-induced vascular nitrite/nitrate production was not different in nontreated, tempol- and E + C-treated aging rats. Relaxation of Phe contraction with sodium nitroprusside, an exogenous NO donor, was smaller in aging than adult rats but was not different between nontreated, tempol- and E + C-treated aging rats. Thus, during the late stages of aging in SHR rats, an age-related inhibition of a vascular relaxation pathway involving not only NO production by endothelial cells but also the bioavailability of NO and the smooth muscle response to NO is partially reversed during chronic treatment with the antioxidants tempol and vitamins E and C. The data suggest a role for oxidative stress in the reduction of vascular relaxation and thereby the promotion of vascular contraction and hypertension during the late stages of aging.     

Human eNOS gene delivery attenuates cold-induced elevation of blood pressure in rats

We previously showed that chronic cold exposure inhibits endothelial nitric oxide synthase (eNOS) expression and decreases nitric oxide (NO) production. The aim of the present study was to evaluate the possible role of the NO system in the development of cold-induced hypertension (CIH) by testing the hypothesis that adenoviral delivery of human eNOS gene increases NO production and attenuates CIH in rats. The effect of in vivo delivery of adenovirus carrying human eNOS full-length cDNA (rAdv.heNOS) on CIH was tested using four groups of Sprague-Dawley rats (6 rats/group). Blood pressure (BP) did not differ among the four groups during the control period at room temperature (24 degrees C). Two groups of rats received intravenous injection of rAdv.heNOS (1 x 10(9) plaque-forming units/rat), and the other two groups received the same dose of rAdv.LacZ to serve as controls. After gene delivery, one rAdv.heNOS-treated group and one rAdv.LacZ-treated group were exposed to cold (6 degrees C) while the remaining groups were kept at 24 degrees C. We found that the BP of the rAdv.LacZ group increased significantly within 1 wk of exposure to cold and reached a peak level at week 5 (152.2 +/- 6.4 mmHg). In contrast, BP (118.7 +/- 8.4 mmHg) of the cold-exposed rAdv.heNOS group did not increase until 5 wk after exposure to cold. The rAdv.heNOS increased plasma and urine levels of NO significantly in cold-exposed rats, which indicates that eNOS gene transfer increased NO production. Notably, rAdv.heNOS decreased plasma levels of norepinephrine and plasma renin activity in cold-exposed rats, which suggests that eNOS gene transfer may decrease the activities of the sympathetic nervous system and the renin-angiotensin system. Immunohistochemical analysis showed that the transferred human eNOS was expressed in both endothelium and adventitia of mesenteric arteries. We conclude that 1) eNOS gene transfer attenuates CIH by increasing NO production and inhibiting the sympathetic nervous system and the renin-angiotensin system; and 2) the NO system appears to mediate this nongenetic, nonpharmacological, nonsurgical model of hypertension.     

Short-term increases in intraluminal pressure reverse age-related decrements in endothelium-dependent dilation in soleus muscle feed arteries.

We tested the hypothesis that short-term increases in intraluminal pressure improve endothelium-dependent dilation and increase endothelial nitric oxide (NO) synthase (eNOS) expression in senescent soleus muscle feed arteries (SFA). SFA isolated from young (4 mo) and old (24 mo) Fischer 344 rats were cannulated and pressurized at 90 (p90) or 130 (p130) cmH(2)O for 4 h. At the end of the 4-h protocol, pressure in p130 SFA was lowered to 90 cmH(2)O for examination of endothelium-dependent (flow- or ACh-induced) vasodilation. Flow- and ACh-induced dilations were blunted in old p90 SFA relative to young p90 SFA. Pretreatment with increased pressure (p130) improved flow- and ACh-induced dilations in old SFA, such that vasodilator responses were similar to those in young SFA. In the presence of N(omega)-nitro-l-arginine (l-NNA) or l-NNA + indomethacin (Indo), flow-induced dilation was inhibited in old p130 SFA, such that the response was not greater than the response in old p90 SFA. In old p130 SFA, ACh-induced dilation was inhibited by l-NNA + Indo (not l-NNA alone). In a separate experiment, SFA were pressurized at 70, 90, 110, or 130 cmH(2)O for 4 h, and eNOS mRNA and protein content were assessed. Increased pressure induced eNOS mRNA expression in young (not old) SFA. eNOS protein content was not altered in young or old SFA. These results indicate that short-term increases in intraluminal pressure improve endothelium-dependent dilation in senescent SFA, in part by enhancing NO bioavailability; however, the beneficial effect was not associated with increased eNOS expression.