Effects of exercise training on vasodilatory protein expression and activity in rats

Increased endothelium-dependent vasodilatation is associated with endurance exercise training. The purpose of this study was to test the hypothesis that increased endothelial nitric oxide synthase (eNOS) protein function, but not increased vascular smooth muscle sensitivity to NO, underlies augmented endothelium-dependent dilatation with training. To test these hypotheses, rats ran on a treadmill at 30 m/min (10% grade) for 60 min/day, 5 days/week, over 8–12 weeks (Trn). Training efficacy was demonstrated by greater (P < 0.05) hindlimb muscle citrate synthase activity and left ventricular mass–body mass ratio in Trn compared with sedentary control rats (Sed). Expression of eNOS protein in the aorta was increased with training (Sed, 1.00 ± 0.18 normalized units; Trn, 1.55 ± 0.23; P < 0.05). Aortic NOS activity was, however, unchanged by training (Sed, 1,505 ± 288 fmol/h/mg protein; Trn, 1,650 ± 247; n.s.). Expression of heat shock protein 90 and protein kinase B/Akt was not different between groups, nor was their association with eNOS. In follow-up series of rats, phosphorylated eNOS content (Serine 1177) was similar for Sed and Trn in both the aorta and gastrocnemius feed artery. Aortic NOS activity with eNOS phosphorylation status preserved was also similar between groups. Finally, cGMP concentration with a NO donor did not differ between groups (Sed, 73.0 ± 20.2 pmol/mg protein; Trn, 62.5 ± 12.9; n.s.). These findings indicate that training-induced increases in eNOS protein expression are not coupled to augmented function, illustrating the complexity of eNOS regulation. Further, they show that vascular sensitivity to NO is not altered by exercise training.     

Effect of age and exercise training on protein:protein interactions among eNOS and its regulatory proteins in rat aortas

Purpose

We tested the hypothesis that impaired endothelium-dependent relaxation in aged aorta is due, in part, to altered protein:protein interactions between endothelial nitric oxide synthase (eNOS) and key regulatory proteins resulting in impaired nitric oxide (NO)-mediated relaxation. We also hypothesized that endurance exercise training improves or restores NO-mediated vasorelaxation in aged aorta by reversing the detrimental effects of aging on protein:protein interaction between eNOS and its key regulatory proteins.

Methods

Young (2 month) and old (22 month) rats were exercise trained (Ex) or remained sedentary (Sed) for 10 weeks yielding four groups of rats: (1) young Sed, (2) young Ex, (3) old Sed, and (4) old Ex. Endothelium-dependent relaxation to acetylcholine (ACh) and protein:protein interactions were assessed in aortas. To determine the role of eNOS, endothelium-dependent relaxation to ACh was assessed in the presence of l-NAME. Protein:protein interactions were assessed using co-immunoprecipitation.

Results

Acetylcholine-induced relaxation was impaired in OldSed relative to YoungSed aortas. Training restored ACh-induced vasorelaxation responses so that OldEx were not different from YoungSed. l-NAME abolished the effects of age and exercise training on ACh-induced relaxation responses. Aging resulted in lower Cav1:eNOS and CaM:eNOS interactions but had no effect on Hsp90:eNOS interaction. Exercise training did not alter protein:protein interactions.

Conclusion

Nitric oxide-mediated, endothelium-dependent relaxation is impaired in old aorta, which is associated with reduced Cav1:eNOS and CaM:eNOS interactions. Exercise training restores endothelium-dependent relaxation in old aortas by enhancing NO-mediated vasorelaxation. The beneficial effect of training is not mediated by reversing the detrimental effects of aging on protein:protein interactions between eNOS and its key regulatory proteins.     

Altered guanylyl-cyclase activity in vitro of pulmonary arteries from fetal lambs with congenital diaphragmatic hernia

Nitric oxide (NO) plays a major role in the modulation of perinatal pulmonary vascular tone. Congenital diaphragmatic hernia (CDH), a major cause of severe persistent pulmonary hypertension of the newborn (PPHN), is often refractory to inhaled NO. Alterations in NO/cyclic guanosine 3',5' monophosphate (cGMP)-mediated pulmonary vasodilatation may contribute to PPHN in CDH. We assessed NO/cGMP-mediated pulmonary vasorelaxation in vitro in 140-d gestational lamb fetuses with surgically created left CDH (term = 147 d) to age-matched controls. Relaxation of fourth generation intralobar pulmonary artery rings in response to the endothelium-dependent vasodilator, acetylcholine (ACh), and to the specific inhibitor of cGMP-phosphodiesterase (PDE), zaprinast, did not differ between the two groups. By contrast, relaxation in response to the calcium ionophore A23187 was impaired in CDH as compared with control animals. Relaxation in response to the NO donor sodium nitroprusside (SNP) (a direct activator of soluble guanylyl cyclase [sGC]) was also impaired in CDH animals as compared with controls. Repeating the challenge increased vasorelaxation in response to SNP in CDH as compared with control animals. Immunohistochemistry revealed the presence of endothelial NO-synthase in the endothelium of pulmonary arteries from both control and CDH animals. We conclude that endothelium-dependent vasodilatation in response to ACh and A23187 was differently affected in the fetal surgical CDH-lamb model. Furthermore, activity of sGC but not that of PDE was impaired in CDH animals. PPHN and decreased inhaled NO responsiveness in CDH may involve decreased sGC activity.     

Exercise training improves ageing-induced decrease in eNOS expression of the aorta

AIMS: Ageing impairs endothelial function such as the regulation of vascular tone. The release of nitric oxide (NO), which has a potent vasodilator effect and antiatherosclerotic property, is decreased in the aorta of aged rats. Exercise training, however, has been reported to increase the expression of endothelial NO synthase (eNOS) in the aorta of young rats. In aged rats, it is not known whether the expression of eNOS is altered by exercise training. We hypothesized that exercise training would improve the ageing-induced decrease in eNOS expression in vessels, and examined the messenger RNA (mRNA) and protein expression of eNOS in the aorta of sedentary-young rats (sedentary-young group; 4 months old), sedentary-aged rats (sedentary-aged group; 23 months old), and swim-trained aged rats (training-aged group; 23 months old, swimming training for 8 weeks, 5 days week(-1), 90 min day(-1)). RESULTS: Body weight was significantly lower, and citrate synthase activity in the epitrochlearis muscle was significantly higher in the training-aged group compared with the sedentary-aged group. The mRNA expression of eNOS in the aorta was significantly higher in the training-aged group than in the sedentary-aged group, while it was significantly lower in both the sedentary-aged and training-aged groups than in the sedentary-young group. The expression of eNOS protein in the aorta was also significantly higher in the training-aged group than in the sedentary-aged group, while it was also significantly lower in the sedentary-aged group, but not in the training-aged group, than in the sedentary-young group. CONCLUSION: The present results revealed that the production of eNOS in the aorta decreases with ageing, and that the decreased production is increased by exercise training in aged rats, which may produce beneficial effects on the impaired cardiovascular system caused by ageing.     

Editorial

Aims: Ageing impairs endothelial function such as the regulation of vascular tone. The release of nitric oxide (NO), which has a potent vasodilator effect and antiatherosclerotic property, is decreased in the aorta of aged rats. Exercise training, however, has been reported to increase the expression of endothelial NO synthase (eNOS) in the aorta of young rats. In aged rats, it is not known whether the expression of eNOS is altered by exercise training. We hypothesized that exercise training would improve the ageing‐induced decrease in eNOS expression in vessels, and examined the messenger RNA (mRNA) and protein expression of eNOS in the aorta of sedentary‐young rats (sedentary‐young group; 4 months old), sedentary‐aged rats (sedentary‐aged group; 23 months old), and swim‐trained aged rats (training‐aged group; 23 months old, swimming training for 8 weeks, 5 days week^?1, 90 min day^?1). Results: Body weight was significantly lower, and citrate synthase activity in the epitrochlearis muscle was significantly higher in the training‐aged group compared with the sedentary‐aged group. The mRNA expression of eNOS in the aorta was significantly higher in the training‐aged group than in the sedentary‐aged group, while it was significantly lower in both the sedentary‐aged and training‐aged groups than in the sedentary‐young group. The expression of eNOS protein in the aorta was also significantly higher in the training‐aged group than in the sedentary‐aged group, while it was also significantly lower in the sedentary‐aged group, but not in the training‐aged group, than in the sedentary‐young group. Conclusion: The present results revealed that the production of eNOS in the aorta decreases with ageing, and that the decreased production is increased by exercise training in aged rats, which may produce beneficial effects on the impaired cardiovascular system caused by ageing.     

Voluntary running exercise attenuates the progression of endothelial dysfunction and arterial calcification in ovariectomized rats

Aim: Loss of oestrogen synthesis capacity after menopause contributes to increases in arterial stiffness and calcification. Exercise training improves arterial stiffness and calcification. However, the mechanism of exercise training‐induced improvement of arterial stiffness and calcification remains unclear. Method: We examined the mechanism by using aortas of sham‐operated rats (sham control; SC), ovariectomized rats (OVX control; OC), OVX plus treatment with vitamin D₃ plus nicotine (VDN) rats (OV sedentary; OVSe), which is an animal model of endothelial dysfunction and arterial calcification, and voluntary running wheel exercise for 8 weeks plus OVX plus VDN rats (OV exercise; OVEx). Results: The arterial tissue calcium and endothelin‐1 (ET‐1: a vasoconstrictor peptide and a potent regulator of arterial calcification) levels were significantly higher in OVSe rats compared with the SC and OC rats, whereas these levels in the OVEx rats were significantly lower than in the OVSe rats. Additionally, arterial expression of endothelial nitric oxide synthase (eNOS), which is an enzyme that produces nitric oxide (NO: a vasodilator substance), was reduced in OVSe rats. However, exercise training prevented the decrease in eNOS expression. Moreover, there was a significant positive correlation between arterial calcium level and arterial ET‐1 level. Conclusion: These findings suggest that exercise training‐induced improvement of ET‐1 and NO prevents the impairment of endothelial function after menopause in females, and this improvement may result in less arterial calcification.     

Endothelium-dependent relaxation in response to acetylcholine in pregnant guinea-pig uterine artery

Recently, strong evidence has suggested that nitric oxide (NO) synthesis is significantly increased in the uterine artery during pregnancy, which may mediate the increased blood flow to the uterus that is characteristic of pregnancy. We therefore investigated the nature of the mediators of acetylcholine (ACh)-induced relaxation in pregnant guinea-pig uterine arterial rings. ACh (0.1 nM to 60 microM) induced endothelium-dependent relaxation of phenylephrine-precontracted pregnant guinea-pig uterine artery. N(G)-monomethyl-L-arginine (3-30 microM) antagonized the effect of ACh, with suppression of maximal ACh- induced relaxation, in a concentration-dependent manner. The inhibition of relaxation by N(G)-monomethyl-L-arginine (10 microM) was significantly overcome by L-arginine (10 microM), but not by D-arginine (100 microM). On the contrary, the administration of indomethacin (10 microM) and diethylcarbamazine (100 microM) did not modify the relaxation of guinea-pig uterine artery induced by ACh. The ACh-evoked relaxation was unaltered when K+-rich Krebs-Ringer bicarbonate solution was used to induce tone instead of phenylephrine, or when a nonselective blocker of K+ channels, 4-aminopyridine (6 mM), was applied to phenylephrine-precontracted segments. It is concluded that the relaxation induced by ACh in pregnant guinea-pig uterine artery can be explained entirely by the release of NO from vascular endothelial cells, without involvement of other endothelium-derived relaxing factors, similar to that previously reported for non-pregnant guinea- pig uterine artery. Thus, it seems that increased activity of NO synthase during pregnancy is without significant influence on the ACh action on uterine artery.      

高血脂通过氧化/硝基化双重作用介导大鼠血管内皮功能失调

目的:观察高脂饮食对大鼠血管内皮功能的影响并探讨其具体机制。方法:将30只8周龄雄性SD大鼠随机分为正常饮食组及高脂饮食组,喂养14周,麻醉后下腔静脉采血,测定甘油三酯、胆固醇、空腹血糖及胰岛素水平;分离主动脉,观察胸主动脉对内皮依赖性血管舒张剂乙酰胆碱(ACh)及内皮非依赖性血管舒张剂(SNAP)的舒张反应;检测血管一氧化氮(NO)、过氧化物的生成量及gp91phox的表达水平,NO合酶(NOS)的活性。结果:高脂饮食组大鼠血脂、空腹血糖和胰岛素水平显著升高,胸主动脉对ACh反应明显降低,血管组织gp91phox表达显著上调,过氧化物的生成及诱导型NOS的活性水平显著增加。结论:高脂血症大鼠存在内皮功能障碍,其机制可能与高血脂引起的氧化/硝基化作用密切相关。     

Voluntary wheel running restores endothelial function in conduit arteries of old mice: direct evidence for reduced oxidative stress, increased superoxide dismutase activity and down-regulation of NADPH oxidase

Habitual aerobic exercise is associated with enhanced endothelium-dependent dilatation (EDD) in older humans, possibly by increasing nitric oxide bioavailability and reducing oxidative stress. However, the mechanisms involved are incompletely understood. EDD was measured in young (6–8 months) and old (29–32 months) cage control and voluntary wheel running (VR) B6D2F1 mice. Age-related reductions in maximal carotid artery EDD to acetylcholine (74 vs. 96%, P < 0.01) and the nitric oxide (NO) component of EDD (maximum dilatation with ACh and l -NAME minus that with ACh alone was −28% vs. −55%, P < 0.01) were restored in old VR (EDD: 96%, NO: −46%). Nitrotyrosine, a marker of oxidative stress, was increased in aorta with age, but was markedly lower in old VR ( P < 0.05). Aortic superoxide dismutase (SOD) activity was greater ( P < 0.01), whereas NADPH oxidase protein expression ( P < 0.01) and activity ( P = 0.05) were lower in old VR vs. old cage control. Increasing SOD (with 4-hydroxy-2,2,6,6-tetramethylpiperidine 1-oxyl) and inhibition of NADPH oxidase (with apocynin) improved EDD and its NO component in old cage control, but not old VR mice. VR increased endothelial NO synthase (eNOS) protein expression ( P < 0.05) and activation (Ser1177 phosphorylation) ( P < 0.05) in old mice. VR did not affect EDD in young mice. Our results show that voluntary aerobic exercise restores the age-associated loss of EDD by suppression of oxidative stress via stimulation of SOD antioxidant activity and inhibition of NADPH oxidase superoxide production. Increased eNOS protein and activation also may contribute to exercise-mediated preservation of NO bioavailability and EDD with ageing.     

Aortic function is compromised in a rat model of polycystic ovary syndrome

BACKGROUND: Arterial mechanical parameters are modified in women with polycystic ovary syndrome (PCOS), before and during pregnancy. This study tested the hypothesis that aortic mechanics and endothelial function are modified in the mifepristone-treated rat model of PCOS. METHODS: Female rats injected daily with mifepristone or vehicle for 7-9 days were assessed by ultrasound to allow estimation of aortic stiffness index and compliance. The influence of acetylcholine (ACh) and sodium nitroprusside (SNP) on dissected phenylephrine-contracted aortic rings was assessed. RESULTS: Aortic compliance was reduced by 67% in mifepristone-treated rats versus controls (P<0.05), while stiffness index was increased 2.3-fold (P<0.02). ACh-induced dilation was less in aortic rings from mifepristone-treated rats (P=0.022) and was less sensitive to the nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) (P<0.001), while SNP-induced dilation was greater (P=0.001). CONCLUSIONS: Aortic mechanics in vivo and endothelial function in vitro were consistently perturbed in mifepristone-treated rats. Aortic ring behaviour suggested that NO release was depressed or degradation elevated, with a compensatory increase in NO sensitivity and/or activation of a non-NO-mediated relaxation mechanism. The mifepristone-treated rat is a valid model for investigation of the vascular deficits seen in PCOS.     

Effect of exercise training volume on arterial contractility and BKCa channel activity in rat thoracic aorta smooth muscle cells

Large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels play a critical role in regulating cellular excitability and vascular tone. Exercise training showed reversible beneficial effects on cardiovascular systems with an improvement of vascular functions. This study investigated the effects of exercise training volume on vascular function and BK_Ca channel activity in thoracic aorta smooth muscle cells (SMCs) in 20 sedentary (SED) and 40 training rats, submitted to a treadmill training protocol (20 m/min, 60 min/day, 12 weeks). Training rats were divided into two groups, exercising 3 days/week (EX1) and 5 days/week (EX2). Since intensity and duration of exercise were identical between training groups, the training volume was higher in EX2 than in EX1. Exercise training not only decreased heart rate, but also attenuated pressor responses induced by angiotensin II or norepinephrine (NE). The maximal vascular contraction induced by 10^?5 M NE was significantly decreased after training. In precontracted thoracic aorta with NE (10^?5 M), activation of the BK_Ca channels by NS1619 significantly decreased the tension. The sensitivity of tissue to NS619 (pD2) was significantly correlated with volume of training (SED < EX1 < EX2). Inside-out patch clamp recording on aortic SMCs showed that exercise training significantly increased the open probability, decreased the mean closed time and increased the mean open time of BK_Ca channels. This effect was more significant in the EX2 group than in the EX1 group. These data suggest that there is a dose effect for exercise training volume for the activation of BK_Ca channels in vascular SMCs, which contributes to improvement of the arterial function in thoracic aortas.     

Hyperglycaemia inhibits thymidine incorporation and cell growth via protein kinase C,mitogen-activated protein kinases and nitric oxide in human umbilical vein endothelium

An elevated extracellular concentration of D-glucose (i.e. hyperglycaemia) inhibits cell proliferation and incorporation of the endogenous nucleoside thymidine into DNA in human umbilical vein endothelial cells (HUVECs). Cells in their log-phase of growth (3.7 +/- 0.3 days, n = 27) incubated for 30 min with 25 mM D-glucose, but not with equimolar concentrations of L-glucose or D-mannitol, exhibited reduced [3H]thymidine incorporation and cell growth rate, with no change in cell viability (> 98 %), total DNA, protein content or cell volume. Incubation with D-glucose activated protein kinase C (PKC), endothelial NO synthase (eNOS), p42 and p44 mitogen-activated protein kinases (p42/44(mapk)), but inhibited superoxide dismutase (SOD). Incubation with D-glucose also increased cGMP and cAMP levels. The effect of D-glucose was blocked by the PKC inhibitor calphostin C, the MAP kinase kinase 1/2 (MEK1/2) inhibitor PD-98059, the eNOS inhibitor L-NAME, the protein kinase G (PKG) inhibitor KT-5823 and the protein kinase A (PKA) inhibitor KT-5720. In the presence of 5 mM D-glucose, [3H]thymidine incorporation and cell growth were reduced by the PKC activator phorbol 12-myristate 13-acetate (PMA), the NO donor S-nitroso-N-acetyl-L,D-penicillamine (SNAP), dibutyryl cGMP, dibutyryl cAMP and the Ca2+ ionophore A-23187. The effect of A-23187 was blocked by calphostin C and PD-98059. D-Glucose-dependent inhibition of thymidine incorporation and cell proliferation is associated with increased PKC, eNOS, and MEK1/2, but decreased SOD activity, and higher intracellular levels of cGMP, cAMP and Ca2+ in HUVECs. These are cellular mechanisms which may reduce endothelial cell growth in pathological conditions such as in diabetes mellitus or hyperglycaemia.     

Relative significance of the nitric oxide (NO)/cGMP pathway and K+ channel activation in endothelium-dependent vasodilation in the femoral artery of developing piglets

Mechanisms mediating endothelium-dependent vasodilation were investigated in femoral artery rings from <2-day-old (newborn) and 2-week-old piglets. Based on previous results we hypothesized an age difference in the relative contribution of nitric oxide(NO)-cyclic 3',5'-guanosine monophosphate (cGMP) and K+ channel-activation to acetylcholine (ACh)-induced vasodilation. Changes in vascular tone were studied in organ baths in the absence or presence of NO synthase(NOS) inhibition or K+ channel blockade and the intra-arterial accumulation of cGMP in response to ACh was measured with radioimmunoassay (RIA). In control experiments, relaxant responses to ACh were equal in the two age groups. In the presence of the NOS-inhibitors N G-monomethyl-L-arginine acetate (L-NMMA; 100 microM) or NG-nitro-L-arginine (L-NOARG; 1-100 microM), however, relaxation was significantly more reduced in femoral artery rings from 2-week-old than from newborn, with lower pD2 values in the older age group. Inhibition of large (BKCa) conductance calcium-sensitive K+ channels with tetraethylammonium chloride (TEA; 1 mM), gave a significant rightward shift in the concentration-response curves to ACh which was of the same magnitude in both age groups. The ACh-induced vasodilation was abolished in both age groups by high K+ (20 mM) in combination with L-NOARG (100 microM). The relative increase in cGMP levels after addition of ACh (10 nM) was significantly larger in rings from newborn compared with 2-week-old piglets (12- vs. four-fold). In summary, sensitivity to NOS inhibition increased with age while the effect of K+ channel blockade with TEA was the same in femoral artery rings from newborn to 2-week-old piglets. Lower sensitivity to NOS inhibition and a larger increase in cGMP in response to ACh could indicate a higher efficacy of the NO/cGMP pathway in this vessel in the newborn piglet.     

Acute increases in intraluminal pressure improve vasodilator responses in aged soleus muscle feed arteries

Purpose

We tested the hypothesis that exposure to an acute increase in intraluminal pressure, to mimic pressure associated with a bout of exercise, improves nitric oxide (NO)-mediated endothelium-dependent dilation in aged soleus muscle feed arteries (SFA) and that improved endothelial function would persist after a 2 h recovery period.

Methods

SFA from young (4-month) and old (24-month) Fischer 344 rats were cannulated and pressurized at 90 (P90) or 130 (P130) cmH₂O for 60 min. At the end of the treatment period, pressure in the P130 SFA was lowered to 90 cmH₂O for examination of endothelium-dependent [flow or acetylcholine (ACh)] and endothelium-independent [sodium nitroprusside (SNP)] vasodilation. To determine the role of NO, vasodilator responses were assessed in the presence of N ^ω-nitro-l-arginine (L-NNA). To determine whether the effects of pressure persisted following a recovery period at normal pressure, SFA were pressurized to 130 cmH₂O for 60 min and subsequently lowered to 90 cmH₂O for 2 h before assessing function.

Results

ACh- and flow-induced dilations were impaired in old SFA. Treatment with increased pressure for 60 min improved ACh- and flow-induced dilations in old SFA. SNP-induced dilation was improved in old and young SFA. The beneficial effect of pressure treatment on ACh- and flow-induced dilation in old SFA was blocked by L-NNA and was not present following a 2 h recovery period.

Conclusion

These results indicate that an acute increase in intraluminal pressure improves NO-mediated endothelium-dependent dilation in aged SFA; however, the beneficial effect does not persist after 2 h.     

Calcineurin is not involved in some mitochondrial enzyme adaptations to endurance exercise training in rat skeletal muscle

The purpose of this study was to test the hypothesis that calcineurin, a calcium-dependent protein phosphatase recently implicated in the signaling of skeletal muscle hypertrophy and fiber type conversion, is required to induce some mitochondrial enzyme adaptations to endurance exercise training in skeletal muscle. Three- to four-week-old male Sprague-Dawley rats with an initial body weight ranging from 45 to 55 g were used in this study. The rats were randomly assigned to groups injected with either a specific calcineurin inhibitor, cyclosporin A (CsA), (group CI) or vehicle (group VI). CsA was subcutaneously injected into the rats at a rate of 50 mg·kg^–1 body weight per day for 10 days. The CI and VI groups were further assigned to sedentary (SED) or exercise training (EX) groups. In the EX group, the rats were trained for 10 days (90 min·day^–1, 14–20 m·min^–1, 10% grade). The citrate synthase (CS) activities in the soleus and plantaris muscles of the EX group rats were significantly higher than those of the SED group rats (p<0.001). Furthermore, 3--hydroxyacyl-CoA dehydrogenase (3-HAD) activities in the soleus and plantaris muscles were significantly higher in the EX group rats than in the SED group rats (p<0.001). However, there were no significant differences in CS and 3-HAD activities between the VI and CI groups. The interactions between CsA injection and exercise training were not statistically significant in any of the parameters. These results may suggest that calcineurin is not involved in some mitochondrial enzyme adaptations to endurance exercise training.S. Terada and H. Nakagawa contributed equally to this work.     

Increased nitric oxide synthase activity and Hsp90 association in skeletal muscle following chronic exercise

Exercise training results in dynamic changes in skeletal muscle blood flow and metabolism. Nitric oxide (NO) influences blood flow, oxidative stress, and glucose metabolism. Hsp90 interacts directly with nitric oxide synthases (NOS), increasing NOS activity and altering the balance of superoxide versus NO production. In addition, Hsp90 expression increases in various tissues following exercise. Therefore, we tested the hypothesis that exercise training increases Hsp90 expression as well as Hsp90/NOS association and NOS activity in skeletal muscle. Male, Sprague–Dawley rats were assigned to either a sedentary or exercise trained group (n = 10/group). Exercise training consisted of running on a motorized treadmill for 10 weeks at 30 m/min, 5% grade for 1 h. Western blotting revealed that exercise training resulted in a 1.9 ± 0.1-fold increase in Hsp90 expression in the soleus muscle but no increase in neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase, or endothelial nitric oxide synthase (eNOS). Exercise training also resulted in a 3.4 ± 1.0-fold increase in Hsp90 association with nNOS, a 2.3 ± 0.4-fold increase association with eNOS measured by immunoprecipitation as well as a 1.5 ± 0.3-fold increase in eNOS phosphorylation at Ser-1179. Total NOS activity measured by the rate of conversion of L-[¹⁴C]arginine to L-[¹⁴C]citrulline was increased by 1.42 ± 0.9 fold in soleus muscle following exercise training compared to controls. In summary, a 10-week treadmill training program in rats results in a significant increase in total NOS activity in the soleus which may be due, in part, to increased NOS interaction with Hsp90 and phosphorylation. This interaction may play a role in altering muscle blood flow and skeletal muscle redox status.     

Constitutive nitric oxide production in bovine aortic and brain microvascular endothelial cells: a comparative study

Vascular endothelium constitutively generates nitric oxide (NO) in large vessels and induces a relaxation of smooth muscle cells. However, little is known about the production of NO in microvessels, where smooth muscle layers are thin or absent. In this study, we have compared the constitutive production of NO in bovine brain microvascular endothelial cells (BBECs) with that in bovine aortic endothelial cells (BAECs). ATP, acetylcholine (ACh) and A23187 induced Ca²⁺ transients both in BBECs and BAECs. In contrast, although ATP and A23187 evoked a similar degree of [Ca²⁺]_i increase in both types of cell, they failed to induce NO production in BBECs, as measured with an NO-sensitive fluorescent dye DAF-2, whereas in BAECs there was an increase in DAF-2 fluorescence. Hypotonic stress induced ATP release and subsequent NO production in BAECs, but not in BBECs. We have developed an in vitro model vessel system that consists of aortic smooth muscle cells embedded in a collagen gel lattice and overlaid with endothelial cells. Precontracted gels showed relaxation in response to ACh, when BAECs were overlaid. However, ACh-induced relaxation was not observed in BBEC-overlaid gels. Expression of eNOS protein as well as cellular uptake of l- [³H]arginine were significantly lower in BBECs than in BAECs. These results indicate that Ca²⁺-dependent NO production is at an undetectable level in BBEC, for which at least two factors, i.e. low levels of eNOS expression and l- arginine uptake, are responsible.     

Cellular signaling and NO production

The endothelium can evoke relaxations (dilatations) of the underlying vascular smooth muscle, by releasing vasodilator substances. The best characterized endothelium-derived relaxing factor is nitric oxide (NO), which is synthesized by the endothelial isoform of nitric oxide synthase (eNOS). Endothelium-dependent relaxations involve both pertussis-toxin-sensitive G_i (e.g., responses to serotonin, sphingosine 1-phosphate, alpha₂-adrenergic agonists, and thrombin) and pertussis-toxin-insensitive G_q (e.g., adenosine diphosphate and bradykinin) coupling proteins. eNOS undergoes a complex pattern of intracellular regulation, including post-translational modifications involving enzyme acylation and phosphorylation. eNOS is reversibly targeted to signal-transducing plasmalemmal caveolae where the enzyme interacts with a number of regulatory proteins, many of which are modified in cardiovascular disease states. The release of nitric oxide by the endothelial cell can be up- (e.g., by estrogens, exercise, and dietary factors) and down-regulated (e.g. oxidative stress, smoking, and oxidized low-density lipoproteins). It is reduced in the course of vascular disease (e.g., diabetes and hypertension). Arteries covered with regenerated endothelium (e.g. following angioplasty) selectively lose the pertussis-toxin-sensitive pathway for NO release which favors vasospasm, thrombosis, penetration of macrophages, cellular growth, and the inflammatory reaction leading to atherosclerosis. The unraveling of the complex interaction of the pathways regulating the presence and the activity of eNOS will enhance the understanding of the perturbations in endothelium-dependent signaling that are seen in cardiovascular disease states, and may lead to the identification of novel targets for therapeutic intervention.     

Possible role of nitric oxide on adipocyte lipolysis in exercise-trained rats

A possible role of nitric oxide (NO) on adipocyte lipolysis was studied in exercise-trained (9 weeks of running) rats. Lipolysis in adipose tissue tended to be greater in trained rats than in control rats. A treatment of adipose tissue with 5 mM N(G)-nitro-L-arginine methyl ester (L-NAME) showed that basal and isoproterenol-stimulated lipolysis were both significantly greater in trained rats than in control rats. In contrast, in isolated adipocytes L-NAME had no effect on lipolysis in either group of rats, though the lipolysis of isolated adipocytes was significantly greater in trained rats than in control rats. Training significantly reduced nitrite/nitrate production in adipocytes, but not in tissue. On the other hand, training increased the protein expression of endothelial nitric oxide synthase (eNOS), but not that of inducible NOS (iNOS) in the extracts of tissue homogenates. In tissue homogenates, eNOS activity but not iNOS activity was significantly greater in trained rats than in control rats. In cellular extracts, training significantly reduced the activities of both NOS's, but the mRNA expressions of both NOS's were not different between groups. The NO donors, S-nitroso-N-acetyl-penicillamine (SNAP) and 1-propamine, 3-(2-hydroxy-2-nitroso-1-propyl-hydrazine) (PAPA-NONOate), significantly inhibited adipocyte lipolysis in response to isoproterenol in both groups. This inhibitory effect of SNAP, but not that of PAPA-NONOate, was greater in the adipocytes of trained rats than in those of the control rats. Thus it is possible that NO is involved in the regulation of lipolysis and that exercise training enhances the responsiveness of adipocytes to extracellular NO with the reduced production of nitrite/nitrate in adipocytes because of decreased activities of NOS's. On the other hand, it is also possible that exercise increases either the activity or the protein expression of eNOS in adipose tissue.     

Hypoxia increases Hsp90 binding to eNOS via PI3K-Akt in porcine coronary artery endothelium

This study examines the molecular mechanisms by which hypoxia regulates phosphorylated endothelial nitric oxide synthase (eNOS) activity and NO production in porcine coronary artery endothelial cells (PCAEC). Exposure to hypoxia (pO(2)=10 mmHg) for periods up to 3 h resulted in a time-dependent increase in eNOS protein expression and an early (15 min) and sustained increase in eNOS phosphorylation at Ser-1177. Exposure to hypoxia for 30 min led to a doubling in eNOS activity (control=6.2+/-4.4 vs hypoxia=14.1+/-5.0 fmol cGMP/microg protein, P<0.05) and NO release (control=5.9+/-0.8 vs hypoxia=11.8+/-1.2 nM/microg protein, P<0.05). Hypoxia also led to a significant increase in Akt phosphorylation and upregulation of Hsp90 binding to eNOS. Pretreatment of cells with either 1 microg/ml geldanamycin (a specific inhibitor of Hsp90) or 500 nM wortmannin (a specific PI3 kinase inhibitor) suppressed hypoxia-stimulated Akt and eNOS phosphorylation and significantly attenuated hypoxia-stimulated Hsp90 binding to eNOS. Both eNOS activity and NO production were inhibited by geldanamycin and wortmannin. Although hypoxia led to early activation of p42/44 mitogen-activated protein kinases (MAPK), inhibition of their pathway by PD98059 did not suppress hypoxia-stimulated eNOS phosphorylation and eNOS activity. These data demonstrate that hypoxia leads to increased eNOS phosphorylation via stimulated Hsp90 binding to eNOS and activation of the PI3-Akt pathway. We conclude that a coordinated interaction between Hsp90 and PI3-Akt may be an important mechanism by which eNOS activity and NO production is upregulated in hypoxic heart.