High-fat diet-induced obesity leads to increased NO sensitivity of rat coronary arterioles: role of soluble guanylate cyclase activation

The impact of obesity on nitric oxide (NO)-mediated coronary microvascular responses is poorly understood. Thus NO-mediated vasomotor responses were investigated in pressurized coronary arterioles ( approximately 100 microm) isolated from lean (on normal diet) and obese (fed with 60% of saturated fat) rats. We found that dilations to acetylcholine (ACh) were not significantly different in obese and lean rats (lean, 83 +/- 4%; and obese, 85 +/- 3% at 1 microM), yet the inhibition of NO synthesis with N(omega)-nitro-l-arginine methyl ester reduced ACh-induced dilations only in vessels of lean controls. The presence of the soluble guanylate cyclase (sGC) inhibitor oxadiazolo-quinoxaline (ODQ) elicited a similar reduction in ACh-induced dilations in the two groups of vessels (lean, 60 +/- 11%; and obese, 57 +/- 3%). Dilations to NO donors, sodium nitroprusside (SNP), and diethylenetriamine (DETA)-NONOate were enhanced in coronary arterioles of obese compared with lean control rats (lean, 63 +/- 6% and 51 +/- 5%; and obese, 78 +/- 5% and 70 +/- 5%, respectively, at 1 microM), whereas dilations to 8-bromo-cGMP were not different in the two groups. In the presence of ODQ, both SNP and DETA-NONOate-induced dilations were reduced to a similar level in lean and obese rats. Moreover, SNP-stimulated cGMP immunoreactivity in coronary arterioles and also cGMP levels in carotid arteries were enhanced in obese rats, whereas the protein expression of endothelial NOS and the sGC beta1-subunit were not different in the two groups. Collectively, these findings suggest that in coronary arterioles of obese rats, the increased activity of sGC leads to an enhanced sensitivity to NO, which may contribute to the maintenance of NO-mediated dilations and coronary perfusion in obesity.     

Increased caveolae density and caveolin-1 expression accompany impaired NO-mediated vasorelaxation in diet-induced obesity

Diet-induced obesity induces changes in mechanisms that are essential for the regulation of normal artery function, and in particular the function of the vascular endothelium. Using a rodent model that reflects the characteristics of human dietary obesity, in the rat saphenous artery we have previously demonstrated that endothelium-dependent vasodilation shifts from an entirely nitric oxide (NO)-mediated mechanism to one involving upregulation of myoendothelial gap junctions and intermediate conductance calcium-activated potassium channel activity and expression. This study investigates the changes in NO-mediated mechanisms that accompany this shift. In saphenous arteries from controls fed a normal chow diet, acetylcholine-mediated endothelium-dependent vasodilation was blocked by NO synthase and soluble guanylyl cyclase inhibitors, but in equivalent arteries from obese animals sensitivity to these agents was reduced. The expression of endothelial NO synthase (eNOS) and caveolin-3 in rat saphenous arteries was unaffected by obesity, whilst that of caveolin-1 monomer and large oligomeric complexes of caveolins-1 and -2 were increased in membrane-enriched samples. The density of caveolae was increased at the membrane and cytoplasm of endothelial and smooth muscle cells of saphenous arteries from obese rats. Dissociation of eNOS from caveolin-1, as a prerequisite for activation of the enzyme, may be compromised and thereby impair NO-mediated vasodilation in the saphenous artery from diet-induced obese rats. Such altered signaling mechanisms in obesity-related vascular disease represent significant potential targets for therapeutic intervention.     

Hindlimb unweighting alters endothelium-dependent vasodilation and ecNOS expression in soleus arterioles.

The purpose of this study was to test the hypothesis that endothelium-dependent dilation is impaired in soleus resistance arteries from hindlimb-unweighted (HLU) rats. Male Sprague-Dawley rats (300-350 g) were exposed to HLU (n = 14) or weight-bearing control (Con, n = 14) conditions for 14 days. After the 14-day treatment period, soleus first-order (1A) arterioles were isolated and cannulated with micropipettes to assess vasodilator responses to an endothelium-dependent dilator, ACh (10(-9)-10(-4) M), and an endothelium-independent dilator, sodium nitroprusside (SNP, 10(-9)-10(-4) M). Arterioles from HLU rats were smaller than Con arterioles (maximal passive diameter = 140 +/- 4 and 121 +/- 4 microm in Con and HLU, respectively) but developed similar spontaneous myogenic tone (43 +/- 3 and 45 +/- 3% in Con and HLU, respectively). Arteries from Con and HLU rats dilated in response to increasing doses of ACh, but dilation was impaired in arterioles from HLU rats (P = 0.03), as was maximal dilation to ACh (85 +/- 4 and 65 +/- 4% possible dilation in Con and HLU, respectively). Inhibition of nitric oxide (NO) synthase (NOS) with N(omega)-nitro-L-arginine (300 microM) reduced ACh dilation by approximately 40% in arterioles from Con rats and eliminated dilation in arterioles from HLU rats. The cyclooxygenase inhibitor indomethacin (50 microM) did not significantly alter dilation to ACh in either group. Treatment with N(omega)-nitro-L-arginine + indomethacin eliminated all ACh dilation in Con and HLU rats. Dilation to sodium nitroprusside was not different between groups (P = 0.98). To determine whether HLU decreased expression of endothelial cell NOS (ecNOS), mRNA and protein levels were measured in single arterioles with RT-PCR and immunoblot analysis. The ecNOS mRNA and protein expression was significantly lower in arterioles from HLU rats than in Con arterioles (20 and 65%, respectively). Collectively, these data indicate that HLU impairs ACh dilation in soleus 1A arterioles, in part because of alterations in the NO pathway.     

Mechanisms of flow and ACh-induced dilation in rat soleus arterioles are altered by hindlimb unweighting.

The purpose of this study was to test the hypothesis that endothelium-dependent dilation (flow-induced dilation and ACh-induced dilation) in rat soleus muscle arterioles is impaired by hindlimb unweighting (HLU). Male Sprague-Dawley rats (approximately 300 g) were exposed to HLU or weight-bearing control (Con) conditions for 14 days. Soleus first-order (1A) and second-order (2A) arterioles were isolated, cannulated, and exposed to step increases in luminal flow at constant pressure. Flow-induced dilation was not impaired by HLU in 1A or 2A arterioles. The cyclooxygenase inhibitor indomethacin (Indo; 50 microM) did not alter flow-induced dilation in 1As or 2As. Inhibition of nitric oxide synthase (NOS) with N(omega)-nitro-L-arginine (L-NNA; 300 microM) reduced flow-induced dilation by 65-70% in Con and HLU 1As. In contrast, L-NNA abolished flow-induced dilation in 2As from Con rats but had no effect in HLU 2As. Combined treatment with L-NNA + Indo reduced tone in 1As and 2As from Con rats, but flow-induced dilation in the presence of L-NNA + Indo was not different from responses without inhibitors in either Con or HLU 1As or 2As. HLU also did not impair ACh-induced dilation (10(-9)-10(-4) M) in soleus 2As. L-NNA reduced ACh-induced dilation by approximately 40% in Con 2As but abolished dilation in HLU 2As. Indo did not alter ACh-induced dilation in Con or HLU 2As, whereas combined treatment with L-NNA + Indo abolished ACh-induced dilation in 2As from both groups. We conclude that flow-induced dilation (1As and 2As) was preserved after 2 wk HLU, but HLU decreased the contribution of NOS in mediating flow-induced dilation and increased the contribution of a NOS- and cyclooxygenase-independent mechanism (possibly endothelium-derived hyperpolarizing factor). In soleus 2As, ACh-induced dilation was preserved after 2-wk HLU but the contribution of NOS in mediating ACh-induced dilation was increased.     

Differential effects of diet-induced obesity on BKCa {beta}1-subunit expression and function in rat skeletal muscle arterioles and small cerebral arteries

Mechanisms underlying obesity-related vascular dysfunction are unclear. This study examined the effect of diet-induced obesity on expression and function of large conductance Ca(2+)-activated potassium channel (BK(Ca)) in rat pressurized small resistance vessels with myogenic tone. Male Sprague-Dawley rats fed a cafeteria-style high fat diet (HFD; ～30% energy from fat) for 16-20 wk were ～30% heavier than controls fed standard chow (～13% fat). Obesity did not alter BK(Ca) α-subunit function or α-subunit protein or mRNA expression in vessels isolated from the cremaster muscle or middle-cerebral circulations. In contrast, BK(Ca) β(1)-subunit protein expression and function were significantly reduced in cremaster muscle arterioles but increased in middle-cerebral arteries from obese animals. Immunohistochemistry showed α- and β(1)-subunits were present exclusively in the smooth muscle of both vessels. Cremaster muscle arterioles from obese animals showed significantly increased medial thickness, and media-to-lumen ratio and pressurized arterioles showed increased myogenic tone at 30 mmHg, but not at 50-120 mmHg. Myogenic tone was not affected by obesity in middle-cerebral arteries. The BK(Ca) antagonist iberiotoxin constricted both cremaster muscle and middle-cerebral arterioles from control rats; this effect of iberiotoxin was abolished in cremaster muscle arteries only from obese rats. Diet-induced obesity has contrasting effects on BK(Ca) function in different vascular beds, through differential effects on β(1)-subunit expression. However, these alterations in BK(Ca) function had little effect on overall myogenic tone, suggesting that the mechanisms controlling myogenic tone can be altered and compensate for altered BK(Ca) expression and function.     

High-fat diet-induced reduction in nitric oxide-dependent arteriolar dilation in rats: role of xanthine oxidase-derived superoxide anion

Obesity frequently leads to the development of hypertension. We hypothesized that high-fat diet (HFD)-induced obesity impairs the endothelium-dependent dilation of arterioles. Male Wistar rats were fed with normal (control) or HFD (60% of saturated fat, for 10 wk). In rats with HFD, body weight, mean arterial blood pressure, and serum insulin, cholesterol, and glucose were elevated. In isolated gracilis muscle arterioles (diameter: approximately 160 microm) of HFD, rat dilations to ACh (at 1 microM, maximum: 83 +/- 3%) and histamine (at 10 microM, maximum: 16 +/- 4%) were significantly (P < 0.05) decreased compared with those of control responses (maximum: 90 +/- 2 and 46 +/- 4%, respectively). Dilations to the NO donor sodium nitroprusside were similar in the two groups. Inhibition of NO synthesis by N(omega)-nitro-l-arginine methyl ester reduced ACh- and histamine-induced dilations in control arterioles but had no effect on microvessels of HFD rats. The superoxide dismutase mimetic Tiron or xanthine oxidase inhibitor allopurinol enhanced ACh (maximum: 90 +/- 2 and 93 +/- 2%, respectively)- and histamine (maximum: 30 +/- 7 and 37 +/- 8%, respectively)-induced dilations in HFD arterioles, whereas the NAD(P)H oxidase inhibitor apocynin had no significant effect. Correspondingly, in carotid arteries of HFD rats, an enhanced superoxide production was shown by lucigenin-enhanced chemiluminescence, in association with an increased xanthine oxidase, but not NAD(P)H oxidase activity. In addition, a marked xanthine oxidase immunostaining was detected in the endothelial layer of the gracilis arterioles of HFD, but not in control rats. These findings suggest that, in obese rats, NO mediation of endothelium-dependent dilation of skeletal muscle arterioles is reduced because of an enhanced xanthine oxidase-derived superoxide production. These alterations demonstrate substantial dysregulation of arteriolar tone by the endothelium in HFD-induced obesity, which may contribute to disturbed tissue blood flow and development of increased peripheral resistance.     

Chronic nitric oxide synthase inhibition blunts endothelium-dependent function of conduit coronary arteries, not arterioles

Current literature suggests that chronic nitric oxide synthase (NOS) inhibition has differential effects on endothelium-dependent dilation (EDD) of conduit arteries vs. arterioles. Therefore, we hypothesized that chronic inhibition of NOS would impair EDD of porcine left anterior descending (LAD) coronary arteries but not coronary arterioles. Thirty-nine female Yucatan miniature swine were included in the study. Animals drank either tap water or water with N(G)-nitro-L-arginine methyl ester (L-NAME; 100 mg/l), resulting in control and chronic NOS inhibition (CNI) groups, respectively. Treatment was continued for 1-3 mo (8.3 +/- 0.6 mg x kg(-1) x day(-1)). In vitro EDD of coronary LADs and arterioles was assessed via responses to ADP (LADs only) and bradykinin (BK), and endothelium-independent function was assessed via responses to sodium nitroprusside (SNP). Chronic NOS inhibition diminished coronary artery EDD to ADP and BK. Incubating LAD rings with L-NAME decreased relaxation responses of LADs from control pigs but not from CNI pigs such that between-group differences were abolished. Neither indomethacin (Indo) nor sulfaphenazole incubation significantly affected relaxation responses of LAD rings to ADP or BK. Coronary arteries from CNI pigs showed enhanced relaxation responses to SNP. In contrast to coronary arteries, coronary arterioles from CNI pigs demonstrated preserved EDD to BK and no increase in dilation responses to SNP. L-NAME, Indo, and L-NAME + Indo incubation did not result in significant between-group differences in arteriole dilation responses to BK. These results suggest that although chronic NOS inhibition diminishes EDD of LAD rings, most likely via a NOS-dependent mechanism, it does not affect EDD of coronary arterioles.     

Interval sprint training enhances endothelial function and eNOS content in some arteries that perfuse white gastrocnemius muscle.

The purpose of this study was to test the hypothesis that interval sprint training (IST) selectively increases endothelium-dependent dilation (EDD) and endothelial nitric oxide synthase and/or superoxide dismutase-1 protein content in arteries and/or arterioles that perfuse the white portion of rat gastrocnemius muscle (WG). Male Sprague-Dawley rats completed 10 wk of IST (n = 62) or remained sedentary (Sed) (n = 63). IST rats performed six 2.5-min exercise bouts, with 4.5 min of rest between bouts (60 m/min, 15% incline), 5 days/wk. EDD was assessed from acetylcholine (ACh)-induced increases in muscle blood flow measured in situ and by ACh-induced dilation of arteries and arterioles [first to third order (1A-3A)] that perfuse red gastrocnemius muscle (RG) and WG. Artery protein content was determined with immunoblot analysis. ACh-induced increases in blood flow were enhanced in WG of IST rats. eNOS content was increased in conduit arteries, gastrocnemius feed artery, and fourth-order arterioles from WG and fifth-order arterioles of RG but not in 2As from RG. EDD was examined in 2As and 3As from a subset of IST and Sed rats. Arterioles were canulated with micropipettes, and intraluminal pressure was set at 60 cmH2O. Results indicate that passive diameter (measured in 0 calcium PSS) of WG 2As was similar in IST and Sed, whereas diameter of WG 3As was greater in IST (96 +/- 8 microm) than Sed (73 +/- 4 microm). WG 2As and 3As of IST rats exhibited greater spontaneous tone, but sensitivity to stretch, phenylephrine, and sodium nitroprusside was similar to Sed arterioles. ACh-induced dilation was enhanced by IST in WG 2As but not in RG 2As or WG 3As. We conclude that IST induces vascular adaptations nonuniformly among arteries that perfuse WG muscle.     

Exercise training enhances flow-induced vasodilation in skeletal muscle resistance arteries of aged rats: role of PGI2 and nitric oxide

Flow-induced vasodilation is attenuated with old age in rat skeletal muscle arterioles. The purpose of this study was to determine whether diminished cyclooxygenase (COX) signaling contributes to the age-induced attenuation of flow-induced vasodilation in gastrocnemius muscle arterioles and to determine whether, and through which mechanism(s), exercise training restores this deficit in old rats. Fischer 344 rats (3 and 22 mo old) were assigned to a sedentary or exercise-trained group. First-order arterioles were isolated from the gastrocnemius muscles, cannulated, and pressurized to 70 cm H(2)O. Diameter changes were determined in response to graded increases in intraluminal flow in the presence and absence of nitric oxide synthase (NOS) inhibition [10(-5) M N(G)-nitro-L-arginine methyl ester (L-NAME)], COX inhibition (10(-5) M indomethacin), or combination NOS (10(-5) M L-NAME) plus COX (10(-5) M indomethacin) inhibition. Aging reduced flow-induced vasodilation in gastrocnemius muscle arterioles. Exercise training restored responsiveness to flow in arterioles of aged rats and enhanced flow-induced vasodilation in arterioles from young rats. L-NAME inhibition of flow-induced vasodilation was greater in arterioles from old rats compared with those from young rats and was increased after exercise training in arterioles from both young and old rats. Although the indomethacin-sensitive portion of flow-induced dilation was not altered by age or training, both COX-1 mRNA expression and PGI(2) production increased with training in arterioles from old rats. These data demonstrate that exercise training restores flow-induced vasodilation in gastrocnemius muscle arterioles from old rats and enhances flow-induced vasodilation in gastrocnemius muscle arterioles from young rats. In arterioles from both old and young rats, the exercise training-induced enhancement of flow-induced dilation occurs primarily through a NOS mechanism.     

High salt intake reduces endothelium-dependent dilation of mouse arterioles via superoxide anion generated from nitric oxide synthase

In skeletal muscle arterioles of normotensive rats fed a high salt diet, the bioavailability of endothelium-derived nitric oxide (NO) is reduced by superoxide anion. Because the impact of dietary salt on resistance vessels in other species is largely unknown, we investigated endothelium-dependent dilation and oxidant activity in spinotrapezius muscle arterioles of C57BL/6J mice fed normal (0.45%, NS) or high salt (7%, HS) diets for 4 wk. Mean arterial pressure in HS mice was not different from that in NS mice, but the magnitude of arteriolar dilation in response to different levels of ACh was 42-57% smaller in HS mice than in NS mice. Inhibition of nitric oxide synthase (NOS) with N(G) monomethyl L-arginine (L-NMMA) significantly reduced resting diameters and reduced responses to ACh (by 45-63%) in NS mice but not in HS mice. Arteriolar wall oxidant activity, as assessed by tetranitroblue tetrazolium reduction or hydroethidine oxidation, was greater in HS mice than in NS mice. Exposure to the superoxide scavenger 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO) + catalase reduced this oxidant activity to normal and restored normal arteriolar responsiveness to ACh in HS mice but had no effect in NS mice. L-NMMA also restored arteriolar oxidant activity to normal in HS mice. ACh further increased arteriolar oxidant activity in HS mice but not in NS mice, and this effect was prevented with L-NMMA. These data suggest that a high salt diet promotes increased generation of superoxide anion from NOS in the murine skeletal muscle microcirculation, thus impairing endothelium-dependent dilation through reduced NO bioavailability.     

Dilation of rat diaphragmatic arterioles by flow and hypoxia: roles of nitric oxide and prostaglandins.

The in vitro responses to ACh, flow, and hypoxia were studied in arterioles isolated from the diaphragms of rats. The endothelium was removed in some vessels by low-pressure air perfusion. In endothelium-intact arterioles, pressurized to 70 mmHg in the absence of luminal flow, ACh (10(-5) M) elicited dilation (from 103 +/- 10 to 156 +/- 13 microm). The response to ACh was eliminated by endothelial ablation and by the nitric oxide synthase antagonists NG-nitro-L-arginine (L-NNA; 10(-5) M) and NG-nitro-L-arginine methyl ester (L-NAME, 10(-5) M) but not by indomethacin (10(-5) M). Increases in luminal flow (5-35 microl/min in 5 microl/min steps) at constant distending pressure (70 mmHg) elicited dilation (from 98 +/- 8 to 159 +/- 12 microm) in endothelium-intact arterioles. The response to flow was partially inhibited by L-NNA, L-NAME, and indomethacin and eliminated by endothelial ablation and by concurrent treatment with L-NAME and indomethacin. The response to hypoxia was determined by reducing the periarteriolar PO2 from 100 to 25-30 Torr by changing the composition of the gas used to bubble the superfusing solution. Hypoxia elicited dilation (from 110 +/- 9 to 165 +/- 12 microm) in endothelium-intact arterioles but not in arterioles from which the endothelium had been removed. Hypoxic vasodilation was eliminated by treatment with indomethacin and was not affected by L-NAME or L-NNA. In rat diaphragmatic arterioles, the response to ACh is dependent on endothelial nitric oxide release, whereas the response to hypoxia is mediated by endothelium-derived prostaglandins. Flow-dilation requires that both nitric oxide and cyclooxygenase pathways be intact.     

NAD(P)H oxidase-derived reactive oxygen species contribute to age-related impairments of endothelium-dependent dilation in rat soleus feed arteries

We tested the hypothesis that age-related endothelial dysfunction in rat soleus muscle feed arteries (SFA) is mediated in part by NAD(P)H oxidase-derived reactive oxygen species (ROS). SFA from young (4 mo) and old (24 mo) Fischer 344 rats were isolated and cannulated for examination of vasodilator responses to flow and acetylcholine (ACh) in the absence or presence of a superoxide anion (O(2)(-)) scavenger (Tempol; 100 μM) or an NAD(P)H oxidase inhibitor (apocynin; 100 μM). In the absence of inhibitors, flow- and ACh-induced dilations were attenuated in SFA from old rats compared with young rats. Tempol and apocynin improved flow- and ACh-induced dilation in SFA from old rats. In SFA from young rats, Tempol and apocynin had no effect on flow-induced dilation, and apocynin attenuated ACh-induced dilation. To determine the role of hydrogen peroxide (H(2)O(2)), dilator responses were assessed in the absence and presence of catalase (100 U/ml) or PEG-catalase (200 U/ml). Neither H(2)O(2) scavenger altered flow-induced dilation, whereas both H(2)O(2) scavengers blunted ACh-induced dilation in SFA from young rats. In old SFA, catalase improved flow-induced dilation whereas PEG-catalase improved ACh-induced dilation. Compared with young SFA, in response to exogenous H(2)O(2) and NADPH, old rats exhibited blunted dilation and constriction, respectively. Immunoblot analysis revealed that the NAD(P)H oxidase subunit gp91phox protein content was greater in old SFA compared with young. These results suggest that NAD(P)H oxidase-derived reactive oxygen species contribute to impaired endothelium-dependent dilation in old SFA.     

Nebivolol-induced vasodilation of renal afferent arterioles involves β3-adrenergic receptor and nitric oxide synthase activation

Nebivolol is a β(1)-adrenergic blocker that also elicits renal vasodilation and increases the glomerular filtration rate (GFR). However, its direct actions on the renal microvasculature and vasodilator mechanism have not been established. We used the in vitro blood-perfused juxtamedullary nephron technique to determine the vasodilator effects of nebivolol and to test the hypothesis that nebivolol induces vasodilation of renal afferent arterioles via an nitric oxide synthase (NOS)/nitric oxide (NO)/soluble guanylate cyclase (sGC)/cGMP pathway and the afferent arteriolar vasodilation effect may be mediated through the release of NO by activation of NOS via a β(3)-adrenoceptor-dependent mechanism. Juxtamedullary nephrons were superfused with nebivolol either alone or combined with the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or the NOS inhibitor N(ω)-nitro-l-arginine (l-NNA) or the β-blockers metoprolol (β(1)), butoxamine (β(2)), and SR59230A (β(3)). Nebivolol (100 μmol/l) markedly increased afferent and efferent arteriolar diameters by 18.9 ± 3.0 and 15.8 ± 1.8%. Pretreatment with l-NNA (1,000 μmol/l) or ODQ (10 μmol/l) decreased afferent vasodilator diameters and prevented the vasodilator effects of nebivolol (2.0 ± 0.2 and 2.4 ± 0.6%). Metoprolol did not elicit significant changes in afferent vasodilator diameters and did not prevent the effects of nebivolol to vasodilate afferent arterioles. However, treatment with SR59230A, but not butoxamine, markedly attenuated the vasodilation responses to nebivolol. Using a monoclonal antibody to β(3)-receptors revealed predominant immunostaining on vascular and glomerular endothelial cells. These data indicate that nebivolol vasodilates both afferent and efferent arterioles and that the afferent vasodilator effect is via a mechanism that is independent of β(1)-receptors but is predominantly mediated via a NOS/NO/sGC/cGMP-dependent mechanisms initiated by activation of endothelial β(3)-receptors.     

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.     

Vasodilator mechanisms in the coronary circulation of endothelial nitric oxide synthase-deficient mice

Previous studies have demonstrated that responses to endothelium-dependent vasodilators are absent in the aortas from mice deficient in expression of endothelial nitric oxide synthase (eNOS -/- mice), whereas responses in the cerebral microcirculation are preserved. We tested the hypothesis that in the absence of eNOS, other vasodilator pathways compensate to preserve endothelium-dependent relaxation in the coronary circulation. Diameters of isolated, pressurized coronary arteries from eNOS -/-, eNOS heterozygous (+/-), and wild-type mice (eNOS +/+ and C57BL/6J) were measured by video microscopy. ACh (an endothelium-dependent agonist) produced vasodilation in wild-type mice. This response was normal in eNOS +/- mice and was largely preserved in eNOS -/- mice. Responses to nitroprusside were also similar in arteries from eNOS +/+, eNOS +/-, and eNOS -/- mice. Dilation to ACh was inhibited by N(G)-nitro-L-arginine, an inhibitor of NOS in control and eNOS -/- mice. In contrast, trifluoromethylphenylimidazole, an inhibitor of neuronal NOS (nNOS), decreased ACh-induced dilation in arteries from eNOS-deficient mice but had no effect on responses in wild-type mice. Indomethacin, an inhibitor of cyclooxygenase, decreased vasodilation to ACh in eNOS-deficient, but not wild-type, mice. Thus, in the absence of eNOS, dilation of coronary arteries to ACh is preserved by other vasodilator mechanisms.     

Selected Contribution: Aging impairs nitric oxide and prostacyclin mediation of endothelium-dependent dilation in soleus feed arteries.

We tested the hypothesis that endothelium-dependent dilation in soleus muscle feed arteries (SFA) is impaired by aging due to attenuated nitric oxide (NO)-mediated vasodilation. SFA were isolated from young (4 mo) and old (24 mo) male Fischer 344 rats and cannulated with two glass micropipettes for examination of endothelium-dependent [flow or acetylcholine (ACh)] and endothelium-independent [sodium nitroprusside (SNP)] vasodilator function. Flow- and ACh-induced dilation was significantly attenuated by age, whereas dilation to SNP was not compromised. To determine the mechanism(s) by which aging affected dilator responses to flow and ACh, dilation was assessed in the presence of Nomega-nitro-L-arginine (L-NNA; to inhibit NO synthase), indomethacin (Indo; to inhibit cyclooxygenase), and L-NNA + Indo. In the presence of L-NNA, Indo, or L-NNA + Indo, flow-induced dilation was inhibited in young SFA, resulting in a response to flow that was no longer greater than old SFA. In the presence of L-NNA or Indo, ACh-induced dilation was not significantly inhibited in young or old SFA; however, double blockade with L-NNA + Indo inhibited ACh-induced dilation in young SFA such that the response to ACh was no longer greater than old SFA. Collectively, these data indicate that aging impairs vasodilator responses in SFA by attenuating NO- and prostacyclin-mediated, endothelium-dependent, dilation.     

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.     

乙酰胆碱不依赖NO的释放引起耳蜗螺旋动脉平滑肌细胞的超极化

目的：乙酰胆碱（ACh）不仅是神经递质,也是一种有效的血管舒张物质参与许多血管床的调节活动。本实验观察ACh引起耳蜗螺旋动脉平滑肌细胞超极化的离子机制以及NO在超极化反应中的可能作用。方法：在豚鼠离体耳蜗螺旋动脉标本上,运用细胞内微电极技术记录外源性的ACh引起的反应。结果：在保持灌流液中含有5mmol／L K^＋以及最小纵向张力的情况下,ACh（0．1—10μmol／L）引起低静息膜电位细胞明显的超极化反应,而引起高静息膜电位细胞明显的去极化反应。ACh引起的平滑肌细胞超极化反应是浓度依赖性的（ACh的浓度是1μmol／L和10／μmol／L时,分别引起超极化的幅度是22和30mV,n=7）。ACh引起的超极化反应能被阿托品（atropine,0．1～1μmol／L,n=6）或DAMP（50～100nmol／L,n=6,一种选择性的地受体的拮抗剂）所阻断,同时也可被BAPTA—AM（10μmol／L,n=7,一种可通过细胞膜的Ca^2＋螯合剂）或eharybdotoxin＋apamin（50-100nmol／L,n=4,两种Ca^2＋激活K^＋通道的阻断剂）所阻断,但是Nω-nitro-L-arginine methyl ester（L-NAME,300μmol／L,n=8,一种NO合成酶的完全抑制剂,n≥5）或glipizide（10μmol／L,ATP敏感性的K^＋通道阻断剂,n=4）或indomethacin（10μmol／L,环氧合酶的抑制剂,n=4）不能阻断ACh引起的超极化反应。结论：ACh通过激活内皮细胞的M3受体,开放钙依赖的钾通道．进而引起耳蜗螺旋动脉平滑肌细胞产生超极化反应,并且这一超极化反应与内皮细胞NO的产生和释放无关。     

Heme oxygenase-mediated endothelial dysfunction in DOCA-salt, but not in spontaneously hypertensive, rat arterioles

Vascular heme oxygenase (HO) metabolizes heme to form carbon monoxide. Carbon monoxide inhibits nitric oxide synthase and promotes endothelium-dependent vasoconstriction. We reported HO-1-mediated endothelial dysfunction in Dahl salt-sensitive hypertension. Previous studies suggested that salt-sensitive hypertensive rats, but not spontaneously hypertensive rats (SHR), display endothelial dysfunction. This study examines the hypothesis that HO-1-mediated arteriolar endothelial dysfunction develops in deoxycorticosterone acetate (DOCA)-salt hypertensive (DOCA) rats, but not in SHR. Uninephrectomized (isoflurane anesthesia) male Sprague-Dawley rats received DOCA injections and saline drinking solution for 4 wk. Rats subjected to sham surgery received vehicle injections and tap water. Blood pressure was elevated in DOCA rats and SHR compared with sham and Wistar-Kyoto (WKY) groups. Aortic HO-1 expression and blood carboxyhemoglobin levels were elevated in the DOCA group, but not in SHR. In isolated gracilis muscle arterioles, ACh caused concentration-related vasodilation in all groups, with attenuated maximum responses in DOCA, but not in SHR, arterioles. Acute pretreatment with an inhibitor of HO, chromium mesoporphyrin, restored ACh-induced responses in DOCA arterioles to sham levels. ACh responses remained the same in SHR and WKY arterioles after chromium mesoporphyrin treatment. These data show that HO-1 levels and activity are increased and arteriolar responses to ACh are decreased in DOCA rats, but not in SHR. Furthermore, in DOCA arterioles, an inhibitor of HO restores ACh-induced vasodilation to sham levels. These results suggest that elevated HO-1 levels and activity, not resulting from hypertension per se, contribute to endothelial dysfunction in DOCA rats.