Exercise training normalizes impaired NOS-dependent responses of cerebral arterioles in type 1 diabetic rats

Our goal was to examine whether exercise training (ExT) could normalize impaired nitric oxide synthase (NOS)-dependent dilation of cerebral (pial) arterioles during type 1 diabetes (T1D). We measured the in vivo diameter of pial arterioles in sedentary and exercised nondiabetic and diabetic rats in response to an endothelial NOS (eNOS)-dependent (ADP), an neuronal NOS (nNOS)-dependent [N-methyl-D-aspartate (NMDA)], and a NOS-independent (nitroglycerin) agonist. In addition, we measured superoxide anion levels in brain tissue under basal conditions in sedentary and exercised nondiabetic and diabetic rats. Furthermore, we used Western blot analysis to determine eNOS and nNOS protein levels in cerebral vessels/brain tissue in sedentary and exercised nondiabetic and diabetic rats. We found that ADP and NMDA produced a dilation of pial arterioles that was similar in sedentary and exercised nondiabetic rats. In contrast, ADP and NMDA produced only minimal vasodilation in sedentary diabetic rats. ExT restored impaired ADP- and NMDA-induced vasodilation observed in diabetic rats to that observed in nondiabetics. Nitroglycerin produced a dilation of pial arterioles that was similar in sedentary and exercised nondiabetic and diabetic rats. Superoxide levels in cortex tissue were similar in sedentary and exercised nondiabetic rats, were increased in sedentary diabetic rats, and were normalized by ExT in diabetic rats. Finally, we found that eNOS protein was increased in diabetic rats and further increased by ExT and that nNOS protein was not influenced by T1D but was increased by ExT. We conclude that ExT can alleviate impaired eNOS- and nNOS-dependent responses of pial arterioles during T1D.     

Hindlimb unweighting decreases endothelium-dependent dilation and eNOS expression in soleus not gastrocnemius.

We tested the hypothesis that hindlimb unweighting (HLU) decreases endothelium-dependent vasodilation and expression of endothelial nitric oxide synthase (eNOS) and superoxide dismutase-1 (SOD-1) in arteries of skeletal muscle with reduced blood flow during HLU. Sprague-Dawley rats (300-350 g) were exposed to HLU (n = 15) or control (n = 15) conditions for 14 days. ACh-induced dilation was assessed in muscle with reduced [soleus (Sol)] or unchanged [gastrocnemius (Gast)] blood flow during HLU. eNOS and SOD-1 expression were measured in feed arteries (FA) and in first-order (1A), second-order (2A), and third-order (3A) arterioles. Dilation to infusion of ACh in vivo was blunted in Sol but not Gast. In arteries of Sol muscle, HLU decreased eNOS mRNA and protein content. eNOS mRNA content was significantly less in Sol FA (35%), 1A arterioles (25%) and 2A arterioles (18%). eNOS protein content was less in Sol FA (64%) and 1A arterioles (65%) from HLU rats. In arteries of Gast, HLU did not decrease eNOS mRNA or protein. SOD-1 mRNA expression was less in Sol 2A arterioles (31%) and 3A arterioles (29%) of HLU rats. SOD-1 protein content was less in Sol FA (67%) but not arterioles. SOD-1 mRNA and protein content were not decreased in arteries from Gast. These data indicate that HLU decreases endothelium-dependent vasodilation, eNOS expression, and SOD-1 expression primarily in arteries of Sol muscle where blood flow is reduced during HLU.     

Acute infusion of nicotine impairs nNOS-dependent reactivity of cerebral arterioles via an increase in oxidative stress.

Our goals were to determine whether acute exposure to nicotine alters neuronal nitric oxide synthase (nNOS)-dependent reactivity of cerebral arterioles and to identify a potential role for oxidative stress in nicotine-induced impairment in nNOS-dependent responses of cerebral arterioles. We measured in vivo diameter of cerebral arterioles to nNOS-dependent (N-methyl-d-aspartate and kainate) and -independent (nitroglycerin) agonists before and during acute treatment with nicotine. We found that nNOS-dependent, but not -independent, vasodilatation was impaired during treatment with nicotine. In addition, treatment of the cerebral microcirculation with tempol (1 h before infusion of nicotine) prevented nicotine-induced impairment in nNOS-dependent vasodilatation. Furthermore, the production of superoxide anion (lucigenin chemiluminescence) was increased in parietal cortex tissue of rats by treatment with nicotine, and this increase in superoxide anion production could be inhibited by tempol. Our findings suggest that acute exposure to nicotine impairs nNOS-dependent dilatation of cerebral arterioles by a mechanism that appears to be related to the formation of superoxide anion.     

Impaired nitric oxide synthase-dependent dilatation of cerebral arterioles in type II diabetic rats

The goals of this study were to determine the effects of type II diabetes mellitus on nitric oxide synthase-dependent responses of cerebral arterioles and on endothelial nitric oxide synthase (eNOS) protein in cerebral arterioles. We examined dilatation of cerebral (pial) arterioles in 13-15 week old male lean and diabetic obese Zucker rats in response to nitric oxide synthase-dependent agonists (acetylcholine and adenosine diphosphate (ADP)) and a nitric oxide synthase-independent agonist (nitroglycerin). We found that acetylcholine (10 microM) increased cerebral arteriolar diameter by 10 +/- 3% (mean +/- SE) in lean Zucker rats, but by only 2 +/- 2% in diabetic obese Zucker rats (p<0.05). In addition, ADP (100 microM) increased cerebral arteriolar diameter by 20 +/- 2% in lean Zucker rats, but by only 8 +/- 2% in diabetic obese Zucker rats (p<0.05). In contrast, nitroglycerin produced similar vasodilatation in lean and diabetic obese Zucker rats. Thus, impaired dilatation of cerebral arterioles in diabetic obese Zucker rats is not related to non-specific impairment of vasodilatation. Following these functional studies, we harvested cerebral microvessels for Western blot analysis of eNOS protein. We found that eNOS protein was significantly higher in diabetic obese Zucker rats than in lean Zucker rats (p<0.05). Thus, type II diabetes mellitus impairs nitric oxide synthase-dependent responses of cerebral arterioles. In addition, eNOS protein from cerebral blood vessels is increased in diabetic obese Zucker rats.     

Tetrahydrobiopterin, a cofactor for NOS, improves endothelial dysfunction during chronic alcohol consumption

We sought to investigate mechanisms that may account for impaired nitric oxide synthase (NOS)-dependent dilatation of cerebral arterioles during alcohol consumption. Our goals were to examine 1) the effect of exogenous application of a cofactor for NOS, i.e., tetrahydrobiopterin (BH4) on the reactivity of pial arterioles during alcohol consumption; and 2) endothelial NOS (eNOS) protein in nonalcohol-fed and alcohol-fed rats. Sprague-Dawley rats were fed liquid diets with or without alcohol for 2-3 mo. We measured in vivo diameter of pial arterioles in response to NOS-dependent agonists (ACh and ADP) and a NOS-independent agonist (nitroglycerin) before and during application of BH4. Blood vessels were then harvested for Western blot analysis of eNOS protein. In nonalcohol-fed rats, ACh and ADP produced vasodilatation, which was impaired in alcohol-fed rats. Vasodilatation to nitroglycerin was similar in both groups of rats. Application of BH4 did not alter vasodilatation in nonalcohol-fed rats but improved impaired vasodilatation in alcohol-fed rats. Also, eNOS protein in cerebral cortex microvessels, the basilar artery, and aorta was not different between nonalcohol-fed and alcohol-fed rats. Thus impaired NOS-dependent vasodilatation during alcohol consumption does not appear to be related to an alteration in eNOS protein but may be related to a deficiency and/or alteration in the utilization of BH4.     

Age impairs Flk-1 signaling and NO-mediated vasodilation in coronary arterioles

Impairment of flow-induced vasodilation in coronary resistance arterioles may contribute to the decline in coronary vasodilatory reserve that occurs with advancing age. This study investigated the effects of age on flow-induced signaling and activation of nitric oxide (NO)-mediated vasodilation in coronary resistance arterioles. Coronary arterioles were isolated from young (approximately 6 mo) and old (approximately 24 mo) male Fischer-344 rats to assess vasodilation to flow, vascular endothelial growth factor (VEGF), and ACh. Flow- and VEGF-induced vasodilation of coronary arterioles was impaired with age (P相似文献   

Cerebral microvascular dilation during hypotension and decreased oxygen tension: a role for nNOS

Endothelial (eNOS) and neuronal nitric oxide synthase (nNOS) are implicated as important contributors to cerebral vascular regulation through nitric oxide (NO). However, direct in vivo measurements of NO in the brain have not been used to dissect their relative roles, particularly as related to oxygenation of brain tissue. We found that, in vivo, rat cerebral arterioles had increased NO concentration ([NO]) and diameter at reduced periarteriolar oxygen tension (Po(2)) when either bath oxygen tension or arterial pressure was decreased. Using these protocols with highly selective blockade of nNOS, we tested the hypothesis that brain tissue nNOS could donate NO to the arterioles at rest and during periods of reduced perivascular oxygen tension, such as during hypotension or reduced local availability of oxygen. The decline in periarteriolar Po(2) by bath manipulation increased [NO] and vessel diameter comparable with responses at similarly decreased Po(2) during hypotension. To determine whether the nNOS provided much of the vascular wall NO, nNOS was locally suppressed with the highly selective inhibitor N-(4S)-(4-amino-5-[aminoethyl]aminopentyl)-N'-nitroguanidine. After blockade, resting [NO], Po(2), and diameters decreased, and the increase in [NO] during reduced Po(2) or hypotension was completely absent. However, flow-mediated dilation during occlusion of a collateral arteriole did remain intact after nNOS blockade and the vessel wall [NO] increased to approximately 80% of normal. Therefore, nNOS predominantly increased NO during decreased periarteriolar oxygen tension, such as that during hypotension, but eNOS was the dominant source of NO for flow shear mechanisms.     

Role of nitric oxide in the coupling of myocardial oxygen consumption and coronary vascular dynamics during pregnancy in the dog

We examined the ability of cardiac endothelial nitric oxide synthase (eNOS) to couple myocardial oxygen consumption (MVo(2)) and oxygen delivery during pregnancy. Awake dogs were studied using echocardiography before and at 40 days, 50 days, and 60 days (60D) of pregnancy and at approximately 14 days postpartum. Left ventricular eNOS, phosphorylated eNOS, and copper, zinc-superoxide dismutase (CuZnSOD or SOD-1) were determined by immunoblotting. MVO(2) of left ventricular tissue samples was measured in vitro in response to increasing doses of bradykinin, enalapril maleate, and amlodipine. We examined the changes in passive diameter and flow-dependant arteriolar dilation of coronary arterioles. Echocardiography indicated increases in cardiac output ( approximately 60%) during pregnancy. Myocardial eNOS (21 +/- 4%), phosphorylated eNOS (19 +/- 3%), and SOD-1 (61 +/- 2.7%) protein levels were significantly increased at 60D. Bradykinin, enalapril maleate, and amlodipine (10(-4) mol/l) decreased MVO(2) in a nitric oxide-dependant manner (by 24 +/- 1.3% in controls and 34 +/- 2.2% at 60D; by 21 +/- 1.1% in controls and 29 +/- 1.1 at 60D; and by 22 +/- 2.5% in controls and 26 +/- 1.0% at 60D, respectively). Arterioles from pregnant dogs showed increased flow-dependant dilation in response to increased shear stress and larger passive diameter. Nitrite production was stimulated by bradykinin and carbachol in microvessels in vitro; pregnancy enhanced nitrite release. Myocardial eNOS, phosphorylated eNOS, and SOD-1 protein expression are increased during pregnancy, and this increase is associated with enhanced nitric oxide-dependant control of MVO(2). Thus increases in eNOS and SOD-1 promote the coupling of oxygen delivery and efficiency in the heart during pregnancy.     

高压氧对急性脑创伤后皮层一氧化氮合酶mRNA表达的影响

目的:拟观察高压氧(HBO)治疗对急性创伤性颅脑损伤后皮层NOSmRNA表达的影响,探讨HBO治疗急性脑损伤的机理。方法:采用自由落体法打击模型制备SD大鼠急性脑创伤,伤后1 h、12 h采用0.25 MPaHBO治疗,伤后6 h、24 h取样皮层,应用半定量逆转录聚合酶链反应(RT-PCR)观察神经元型一氧化氮合酶(nNOS)、内皮型一氧化氮合酶(eNOS)和诱导型一氧化氮合酶(iNOS)mRNA表达量变化。结果:0.25MPaHBO治疗各时间组nNOS、eNOS和iNOSmRNA较急性颅脑损伤各时间组显著下降(P<0.01),且HBO治疗24 h组较6 h组下降更明显(P<0.05,P<0.01),0.25 MPa常氧高氮各时间组与急性颅脑损伤各时间组NOSmRNA表达量无统计学意义。结论:HBO治疗可以下调nNOSmRNA、iNOSmRNA和eNOSmRNA的表达量,可能为HBO治疗脑创伤的机理之一。     

Role of neuronal and endothelial nitric oxide synthase in nitric oxide generation in the brain following cerebral ischemia.

Nitric oxide (NO) plays an important role in the pathogenesis of neuronal injury during cerebral ischemia. The endothelial and neuronal isoforms of nitric oxide synthase (eNOS, nNOS) generate NO, but NO generation from these two isoforms can have opposing roles in the process of ischemic injury. While increased NO production from nNOS in neurons can cause neuronal injury, endothelial NO production from eNOS can decrease ischemic injury by inducing vasodilation. However, the relative magnitude and time course of NO generation from each isoform during cerebral ischemia has not been previously determined. Therefore, electron paramagnetic resonance spectroscopy was applied to directly detect NO in the brain of mice in the basal state and following global cerebral ischemia induced by cardiac arrest. The relative amount of NO derived from eNOS and nNOS was accessed using transgenic eNOS(-/-) or nNOS(-/-) mice and matched wild-type control mice. NO was trapped using Fe(II)-diethyldithiocarbamate. In wild-type mice, only small NO signals were seen prior to ischemia, but after 10 to 20 min of ischemia the signals increased more than 4-fold. This NO generation was inhibited more than 70% by NOS inhibition. In either nNOS(-/-) or eNOS(-/-) mice before ischemia, NO generation was decreased about 50% compared to that in wild-type mice. Following the onset of ischemia a rapid increase in NO occurred in nNOS(-/-) mice peaking after only 10 min. The production of NO in the eNOS(-/-) mice paralleled that in the wild type with a progressive increase over 20 min, suggesting progressive accumulation of NO from nNOS following the onset of ischemia. NOS activity measurements demonstrated that eNOS(-/-) and nNOS(-/-) brains had 90% and < 10%, respectively, of the activity measured in wild type. Thus, while eNOS contributes only a fraction of total brain NOS activity, during the early minutes of cerebral ischemia prominent NO generation from this isoform occurs, confirming its importance in modulating the process of ischemic injury.     

Buthionine sulfoximine causes endothelium dependent hyper-relaxation and hypoadiponectinemia

A close relationship between oxidative stress, endothelial dysfunction, and hypoadiponectinemia has been observed. The present study was performed to investigate how glutathione depletion via buthionine sulfoximine (BSO) administration affects endothelial function and adiponectin levels in rats. Acetylcholine (Ach)-induced vasodilation was significantly enhanced in BSO-treated rats, compared with control rats. This was completely abolished by L-NAME, and Ach-induced vasodilation was not observed in the aorta without endothelium. These results suggest that Ach-induced hyper-relaxation of the aorta in BSO-treated rats is completely dependent on the presence of endothelium and mediated by changes in eNOS activity. Catalase significantly inhibited this relaxation to Ach and no effect of catalase on sodium nitroprusside-induced relaxation of the aorta without endothelium was observed in BSO-treated rats. Thus, hyper-relaxation of the aorta in BSO-treated rats is likely caused by H₂O₂ in addition to NO produced by the endothelium via an eNOS-dependent mechanism. Hypoadiponectinemia and decreased levels of adiponectin mRNA in adipose tissue were observed in BSO-treated rats. Protein expression of eNOS and SODs (SOD-1 and SOD-2) in the aorta was increased and plasma NOx levels were decreased in BSO-treated rats. Our results suggest that oxidative stress induced by BSO causes eNOS uncoupling and hyper-relaxation by producing H₂O₂, and that BSO-induced oxidative stress causes hypoadiponectinemia, probably by increasing H₂O₂ production in adipose tissue.     

Bone medullary arterioles from ovariectomized rats have smaller baseline diameters but normal eNOS expression and NO-mediated dilation

This study was designed to test the hypothesis that endogenous estrogens decrease the expression of endothelial nitric oxide synthase (eNOS) in resistance-size bone arterioles, thereby reducing endothelium-dependent vasodilator function. Sexually mature female rats were ovariectomized to reduce endogenous estrogens. Age-matched female rats served as controls. Seven to ten days after ovariectomy, bone marrow tissue was collected from the femoral canal. Immuno-histochemistry was performed to detect expression of estrogen receptors, alpha and beta and eNOS. eNOS protein content in medullary bone arterioles was compared using Western blot analysis. Endothelial cell function was assessed by quantitating the dilation of isolated, pressurized bone arterioles in response to acetylcholine. The results indicate that the endothelium of bone arterioles from ovariectomized and control rats express ER-alpha, ER-beta and eNOS. eNOS protein content in the two groups of arterioles did not differ. However, the baseline diameter of arterioles from ovariectomized rats (63+/-4 microm) was significantly smaller than the diameter of arterioles from control rats (75+/-3 microm, p<0.05). The two groups of arterioles dilated equally in response to acetylcholine. L-NAME, an inhibitor of eNOS, almost completely abolished the dilator responses to acetylcholine, but not to sodium nitroprusside. L-Arginine restored acetylcholine-induced dilation after L-NAME treatment. Thus, arteriole dilation to acetylcholine appears to be mediated almost exclusively by NO. The smaller diameter of arterioles from ovariectomized rats suggests that endogenous estrogens exert a significant dilator influence on bone arterioles. However, the dilator influence does not appear to be mediated by an increase in eNOS expression or enhanced NO-dependent vasodilation. These results indicate that estrogens do not decrease eNOS expression or diminish NO-mediated dilation of bone medullary arterioles.     

Nitric Oxide Synthase Dysfunction Contributes to Impaired Cerebroarteriolar Reactivity in Experimental Cerebral Malaria

Cerebrovascular dysfunction plays a key role in the pathogenesis of cerebral malaria. In experimental cerebral malaria (ECM) induced by Plasmodium berghei ANKA, cerebrovascular dysfunction characterized by vascular constriction, occlusion and damage results in impaired perfusion and reduced cerebral blood flow and oxygenation, and has been linked to low nitric oxide (NO) bioavailability. Here, we directly assessed cerebrovascular function in ECM using a novel cranial window method for intravital microscopy of the pial microcirculation and probed the role of NOS isoforms and phosphorylation patterns in the impaired vascular responses. We show that pial arteriolar responses to endothelial NOS (eNOS) and neuronal NOS (nNOS) agonists (Acetylcholine (ACh) and N-Methyl-D-Aspartate (NMDA)) were blunted in mice with ECM, and could be partially recovered by exogenous supplementation of tetrahydrobiopterin (BH4). Pial arterioles in non-ECM mice infected by Plasmodium berghei NK65 remained relatively responsive to the agonists and were not significantly affected by BH4 treatment. These findings, together with the observed blunting of NO production upon stimulation by the agonists, decrease in total NOS activity, augmentation of lipid peroxidation levels, upregulation of eNOS protein expression, and increase in eNOS and nNOS monomerization in the brain during ECM development strongly indicate a state of eNOS/nNOS uncoupling likely mediated by oxidative stress. Furthermore, the downregulation of Serine 1176 (S1176) phosphorylation of eNOS, which correlated with a decrease in cerebrovascular wall shear stress, implicates hemorheological disturbances in eNOS dysfunction in ECM. Finally, pial arterioles responded to superfusion with the NO donor, S-Nitroso-L-glutathione (GSNO), but with decreased intensity, indicating that not only NO production but also signaling is perturbed during ECM. Therefore, the pathological impairment of eNOS and nNOS functions contribute importantly to cerebrovascular dysfunction in ECM and the recovery of intrinsic functionality of NOS to increase NO bioavailability and restore vascular health represents a target for ECM treatment.     

Aging induces muscle-specific impairment of endothelium-dependent dilation in skeletal muscle feed arteries.

We tested the hypothesis that aging decreases endothelium-dependent vasodilation in feed arteries perfusing rat skeletal muscle. In addition, we tested the hypothesis that attenuated vasodilator responses are associated with decreased endothelial nitric oxide synthase (eNOS) and superoxide dismutase-1 (SOD-1) expression. Soleus feed arteries (SFA) and gastrocnemius feed arteries (GFA) were isolated from young (4 mo) and old (24 mo) male Fischer 344 rats. Feed arteries from the right hindlimb were cannulated with two glass micropipettes for examination of endothelium-dependent [acetylcholine (ACh)] and endothelium-independent [adenosine (Ado) or sodium nitroprusside (SNP)] vasodilator function. Feed arteries from the left hindlimb were frozen and used to assess eNOS and SOD-1 protein and mRNA expression. In SFA, endothelium-dependent dilation to ACh was reduced in old rats (0.9 +/- 0.04 vs. 0.8 +/- 0.03), whereas dilator responses to Ado and SNP were similar in SFA of young and old rats. In GFA, vasodilator responses to ACh, Ado, and SNP were not altered by age. eNOS and SOD-1 protein expression declined with age in SFA (-71 and -54%, respectively) but not in GFA. eNOS and SOD-1 mRNA expression were not altered by age in SFA or GFA. Collectively, these data indicate aging induces muscle-specific impairment of endothelium-dependent vascular function in SFA.     

Regulation of nitric oxide-dependent vasodilation in coronary arteries of estrogen receptor-alpha-deficient mice

Estrogen has been shown to increase endothelium-dependent vasodilation and expression of endothelial nitric oxide (NO) synthase (eNOS); however, the role of estrogen receptors in mediating estrogen effects on endothelial function remains to be elucidated. The purpose of this study was to test the hypothesis that estrogen modulates NO-dependent vasodilation of coronary arteries through its action on estrogen receptor-alpha (ER-alpha) to increase protein levels of eNOS and Cu/Zn superoxide dismutase (SOD-1). Vasodilation to acetylcholine (ACh) and sodium nitroprusside was assessed in isolated coronary arteries from intact and ovariectomized female wild-type (WT) and ER-alpha knockout (ERalphaKO) mice. Protein levels for eNOS and SOD-1 were also evaluated. Vasodilation to ACh was not significantly altered in ERalphaKO mice compared with WT mice. Ovariectomy reduced responsiveness to ACh in ERalphaKO mice but not WT mice. Responses to sodium nitroprusside were not altered by disruption of ER-alpha or by ovariectomy. Supplementation with estrogen restored ACh-induced vasodilation in ovariectomized ERalphaKO mice. eNOS protein was reduced in ERalphaKO mice compared with WT mice. Ovariectomy caused a further reduction in eNOS protein in ERalphaKO mice, but this reduction was reversed by estrogen treatment. SOD-1 protein levels were increased by disruption of ER-alpha. Ovariectomy reduced SOD-1 protein in ERalphaKO mice, but this reduction was partially reversed by estrogen replacement. These results suggest that estrogen modulation of eNOS protein content is mediated in part through ER-alpha. NO-dependent responses are preserved in ERalphaKO mice, possibly through increased SOD-1 expression and enhanced bioavailability of NO.     

Influence of exercise on dilatation of the basilar artery during diabetes mellitus.

Our goal was to examine whether exercise training alleviates impaired nitric oxide synthase (NOS)-dependent dilatation of the basilar artery in Type 1 diabetic rats. To test this hypothesis, we measured in vivo diameter of the basilar artery in sedentary and exercised nondiabetic and diabetic rats in response to NOS-dependent (acetylcholine) and -independent (nitroglycerin) agonists. To determine the potential role for nitric oxide in vasodilatation in sedentary and exercised nondiabetic and diabetic rats, we examined responses after NG-monomethyl-l-arginine (l-NMMA). We found that acetylcholine produced dilatation of the basilar artery that was similar in sedentary and exercised nondiabetic rats. Acetylcholine produced only minimal vasodilatation in sedentary diabetic rats. However, exercise alleviated impaired acetylcholine-induced vasodilatation in diabetic rats. Nitroglycerin produced dilatation of the basilar artery that was similar in sedentary and exercised nondiabetic and diabetic rats. l-NMMA produced similar inhibition of acetylcholine-induced dilatation of the basilar artery in sedentary and exercised nondiabetic and diabetic rats. Finally, we found that endothelial NOS (eNOS) protein in the basilar artery was higher in diabetic compared with nondiabetic rats and that exercise increased eNOS protein in the basilar artery of nondiabetic and diabetic rats. We conclude that 1) exercise can alleviate impaired NOS-dependent dilatation of the basilar artery during diabetes mellitus, 2) the synthesis and release of nitric oxide accounts for dilatation of the basilar artery to acetylcholine in sedentary and exercised nondiabetic and diabetic rats, and 3) exercise may exert its affect on cerebrovascular reactivity during diabetes by altering levels of eNOS protein in the basilar artery.     

Urinary matrix metalloproteinase activities: biomarkers for plaque angiogenesis and nephropathy in diabetes

Diabetic complications of nephropathy and accelerated atherosclerosis are associated with vascular remodeling and dysregulated angiogenesis. Matrix metalloproteinases (MMP) modify extracellular matrix during vascular remodeling and are excreted in urine of patients with vascular malformation or tumor angiogenesis. We hypothesized that urinary MMP activities would be sensitive biomarkers for vascular remodeling in diabetic complications. Activities of MMP-2, MMP-9, and its complex with neutrophil gelatinase-associated lipocalin (NGAL/MMP-9) were measured by substrate gel zymography in urine from nondiabetic (ND) and type 1 diabetic (T1D) rodents that were susceptible to both T1D-induced plaque angiogenesis and nephropathy, or nephropathy alone. Additionally, these urine activities were measured in ND and T1D adolescents. Urinary MMP-9, MMP-2, and NGAL/MMP-9 activities were increased and more prevalent in T1D compared with ND controls. Urinary MMP-2 activity was detected in mice with T1D-induced plaque neovascularization. In nephropathy models, urinary NGAL/MMP-9 and MMP-9 activities appeared before onset of albuminuria, whereas MMP-2 was absent or delayed. Finally, urinary MMP activities were increased in adolescents with early stages of T1D. Urinary MMP activities may be sensitive, noninvasive, and clinically useful biomarkers for predicting vascular remodeling in diabetic renal and vascular complications.     

Short-term training enhances endothelium-dependent dilation of coronary arteries, not arterioles.

Our objective was to test the hypothesis that short-term exercise training (STR) of pigs increases endothelium-dependent dilation (EDD) of coronary arteries but not coronary arterioles. Female Yucatan miniature swine ran on a treadmill for 1 h, at 3.5 mph, twice daily for 7 days (STR; n = 28). Skeletal muscle citrate synthase activity was increased in STR compared with sedentary controls (Sed; n = 26). Vasoreactivity was evaluated in isolated segments of conduit arteries (1-2 mm ID, 3-4 mm length) mounted on myographs and in arterioles (50-100 microm ID) isolated and cannulated with micropipettes with intraluminal pressure set at 60 cmH(2)O. EDD was assessed by examining responses to increasing concentrations of bradykinin (BK) (conduit arteries 10(-12)-10(-6) M and arterioles 10(-13)-10(-6) M). There were no differences in maximal EDD or BK sensitivity of coronary arterioles from Sed and STR hearts. In contrast, sensitivity of conduit arteries (precontracted with PGF(2alpha)) to BK was increased significantly (P < 0.05) in STR (EC(50), 2.33 +/- 0.62 nM, n = 12) compared with Sed animals (EC(50), 3.88 +/- 0.62 nM, n = 13). Immunoblot analysis revealed that coronary arteries from STR and Sed animals had similar levels of endothelial nitric oxide synthase (eNOS). In contrast, eNOS protein was increased in STR aortic endothelial cells. Neither protein nor mRNA levels of eNOS were different in coronary arterioles from STR compared with Sed animals. STR did not alter expression of superoxide dismutase (SOD-1) protein in any artery examined. We conclude that pigs exhibit increases in EDD of conduit arteries, but not in coronary arterioles, at the onset of exercise training. These adaptations in pigs do not appear to be mediated by alterations in eNOS or SOD-1 expression.     

Cerebral microvascular nNOS responds to lowered oxygen tension through a bumetanide-sensitive cotransporter and sodium-calcium exchanger

Na(+) cotransporters have a substantial role in neuronal damage during brain hypoxia. We proposed these cotransporters have beneficial roles in oxygen-sensing mechanisms that increase periarteriolar nitric oxide (NO) concentration ([NO]) during mild to moderate oxygen deprivation. Our prior studies have shown that cerebral neuronal NO synthase (nNOS) is essential for [NO] responses to decreased oxygen tension and that endothelial NO synthase (eNOS) is of little consequence. In this study, we explored the mechanisms of three specific cotransporters known to play a role in the hypoxic state: KB-R7943 for blockade of the Na(+)/Ca(2+) exchanger, bumetanide for the Na(+)-K(+)-2Cl(-) cotransporter, and amiloride for Na(+)/H(+) cotransporters. In vivo measurements of arteriolar diameter and [NO] at normal and locally reduced oxygen tension in the rat parietal cortex provided the functional analysis. As previously found for intestinal arterioles, bumetanide-sensitive cotransporters are primarily responsible for sensing reduced oxygen because the increased [NO] and dilation were suppressed. The Na(+)/Ca(2+) exchanger facilitated increased NO formation because blockade also suppressed [NO] and dilatory responses to decreased oxygen. Amiloride-sensitive Na(+)/H(+) cotransporters did not significantly contribute to the microvascular regulation. To confirm that nNOS rather than eNOS was primarily responsible for NO generation, eNOS was suppressed with the fusion protein cavtratin for the caveolae domain of eNOS. Although the resting [NO] decreased and arterioles constricted as eNOS was suppressed, most of the increased NO and dilatory response to oxygen were preserved because nNOS was functional. Therefore, nNOS activation secondary to Na(+)-K(+)-2Cl(-) cotransporter and Na(+)/Ca(2+) exchanger functions are key to cerebral vascular oxygen responses.     

SOD-1 expression in pig coronary arterioles is increased by exercise training

Coronary arterioles of exercise-trained (EX) pigs have enhanced nitric oxide (NO.)-dependent dilation. Evidence suggests that the biological half-life of NO. depends in part on the management of the superoxide anion. The purpose of this study was to test the hypothesis that expression of cytosolic copper/zinc-dependent superoxide dismutase (SOD)-1 is increased in coronary arterioles as a result of exercise training. Male Yucatan pigs either remained sedentary (SED, n = 4) or were EX (n = 4) on a motorized treadmill for 16-20 wk. Individual coronary arterioles ( approximately 100-microm unpressurized internal diameter) were dissected and frozen. Coronary arteriole SOD-1 protein (via immunoblots) increased as a result of exercise training (2.16 +/- 0.35 times SED levels) as did SOD-1 enzyme activity (measured via inhibition of pyrogallol autooxidation; approximately 75% increase vs. SED). In addition, SOD-1 mRNA levels (measured via RT-PCR) were higher in EX arterioles (1.68 +/- 0.16 times the SED levels). There were no effects of exercise training on the levels of SOD-2 (mitochondrial), catalase, or p67(phox) proteins. Thus chronic aerobic exercise training selectively increases the levels of SOD-1 mRNA, protein, and enzymatic activity in porcine coronary arterioles. Increased SOD-1 could contribute to the enhanced NO.-dependent dilation previously observed in EX porcine coronary arterioles by improving management of superoxide in the vascular cell environment, thus prolonging the biological half-life of NO.