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Type 2 diabetes (T2D) is a leading risk factor for a variety of cardiovascular diseases including coronary heart disease and atherosclerosis. Exercise training (ET) has a beneficial effect on these disorders, but the basis for this effect is not fully understood. This study was designed to investigate whether the ET abates endothelial dysfunction in the aorta in T2D. Heterozygous controls (m Lepr(db)) and type 2 diabetic mice (db/db; Lepr(db)) were either exercise entrained by forced treadmill exercise or remained sedentary for 10 wk. Ex vivo functional assessment of aortic rings showed that ET restored acetylcholine-induced endothelial-dependent vasodilation of diabetic mice. Although the protein expression of endothelial nitric oxide synthase did not increase, ET reduced both IFN-γ and superoxide production by inhibiting gp91(phox) protein levels. In addition, ET increased the expression of adiponectin (APN) and the antioxidant enzyme, SOD-1. To investigate whether these beneficial effects of ET are APN dependent, we used adiponectin knockout (APNKO) mice. Indeed, impaired endothelial-dependent vasodilation occurred in APNKO mice, suggesting that APN plays a central role in prevention of endothelial dysfunction. APNKO mice also showed increased protein expression of IFN-γ, gp91(phox), and nitrotyrosine but protein expression of SOD-1 and -3 were comparable between wild-type and APNKO. These findings in the aorta imply that APN suppresses inflammation and oxidative stress in the aorta, but not SOD-1 and -3. Thus ET improves endothelial function in the aorta in T2D via both APN-dependent and independent pathways. This improvement is due to the effects of ET in inhibiting inflammation and oxidative stress (APN-dependent) as well as in improving antioxidant enzyme (APN-independent) performance in T2D.

Endothelium-derived hyperpolarizing factor (EDHF) plays a crucial role in modulating vasomotor tone, especially in microvessels when nitric oxide-dependent control is compromised such as in diabetes. Epoxyeicosatrienoic acids (EETs), potassium ions (K+), and hydrogen peroxide (H2O2) are proposed as EDHFs. However, the identity (or identities) of EDHF-dependent endothelial dilators has not been clearly elucidated in diabetes. We assessed the mechanisms of EDHF-induced vasodilation in wild-type (WT, normal), db/db (advanced type 2 diabetic) mice, and db/db mice null for TNF (dbTNF-/dbTNF-). In db/db mice, EDHF-induced vasodilation [ACh-induced vasodilation in the presence of N(G)-nitro-L-arginine methyl ester (L-NAME, 10 micromol/l) and prostaglandin synthase inhibitor indomethacin (Indo, 10 mumol/l)] was diminished after the administration of catalase (an enzyme that selectively dismutates H2O2 to water and oxygen, 1,000 U/ml); administration of the combination of charybdotoxin (a nonselective blocker of intermediate-conductance Ca2+-activated K+ channels, 10 micromol/l) and apamin (a selective blocker of small-conductance Ca2+-activated K+ channels, 50 micromol/l) also attenuated EDHF-induced vasodilation, but the inhibition of EETs synthesis [14,15-epoxyeicosa-5(Z)-enoic acid; 10 mumol/l] did not alter EDHF-induced vasodilation. In WT controls, EDHF-dependent vasodilation was significantly diminished after an inhibition of K+ channel, EETs synthesis, or H2O2 production. Our molecular results indicate that mRNA and protein expression of interleukin-6 (IL-6) were greater in db/db versus WT and dbTNF-/dbTNF- mice, but neutralizing antibody to IL-6 (anti-IL-6; 0.28 mg.ml(-1).kg(-1) ip for 3 days) attenuated IL-6 expression in db/db mice. The incubation of the microvessels with IL-6 (5 ng/ml) induced endothelial dysfunction in the presence of l-NAME and Indo in WT mice, but anti-IL-6 restored ACh-induced vasodilation in the presence of L-NAME and Indo in db/db mice. In db(TNF-)/db(TNF-) mice, EDHF-induced vasodilation was greater and comparable with controls, but IL-6 decreased EDHF-mediated vasodilation. Our results indicate that EDHF compensates for diminished NO-dependent dilation in IL-6-induced endothelial dysfunction by the activation of H2O2 or a K+ channel in type 2 diabetes.

Reactive oxygen species (ROS) have been proposed to mediate vasodilation in the microcirculation. We investigated the role of ROS in arachidonic acid (AA)-induced coronary microvascular dilation. Porcine epicardial coronary arterioles (110 +/- 4 microm diameter) were mounted onto pipettes in oxygenated Krebs buffer. Vessels were incubated with vehicle or 1 mM Tiron (a nonselective ROS scavenger), 250 U/ml polyethylene-glycolated (PEG)-superoxide dismutase (SOD; an O2- scavenger), 250 U/ml PEG-catalase (a H2O2 scavenger), or the cyclooxygenase (COX) inhibitors indomethacin (10 microM) or diclofenac (10 microM) for 30 min. After endothelin constriction (30-60% of resting diameter), cumulative concentrations of AA (10(-10)-10(-5)M) were added and internal diameters measured by video microscopy. AA (10-7 M) produced 37 +/- 6% dilation, which was eliminated by the administration of indomethacin (4 +/- 7%, P < 0.05) or diclofenac (-8 +/- 8%, P < 0.05), as well as by Tiron (-4 +/- 5%, P < 0.05), PEG-SOD (-10 +/- 6%, P < 0.05), or PEG-catalase (1 +/- 4%, P < 0.05). Incubation of small coronary arteries with [3H]AA resulted in the formation of prostaglandins, which was blocked by indomethacin. In separate studies in microvessels, AA induced concentration-dependent increases in fluorescence of the oxidant-sensitive probe dichlorodihydrofluorescein diacetate, which was inhibited by pretreatment with indomethacin or by SOD + catalase. We conclude that in porcine coronary microvessels, COX-derived ROS contribute to AA-induced vasodilation.

Noncyclooxygenase metabolites of arachidonic acid (AA) have been proposed to mediate endothelium-dependent vasodilation in the coronary microcirculation. Therefore, we examined the formation and bioactivity of AA metabolites in porcine coronary (PC) microvascular endothelial cells and microvessels, respectively. The major noncyclooxygenase metabolite produced by microvascular endothelial cells was 12(S)-hydroxyeicosatetraenoic acid (HETE), a lipoxygenase product. 12(S)-HETE release was markedly increased by pretreatment with 13(S)-hydroperoxyoctadecadienoic acid but not by the reduced congener 13(S)-hydroxyoctadecadienoic acid, suggesting oxidative upregulation of 12(S)-HETE output. 12(S)-HETE produced potent relaxation and hyperpolarization of PC microvessels (EC(50), expressed as -log[M] = 13.5 +/- 0.5). Moreover, 12(S)-HETE potently activated large-conductance Ca(2+)-activated K(+) currents in PC microvascular smooth muscle cells. In contrast, 12(S)-HETE was not a major product of conduit PC endothelial AA metabolism and did not exhibit potent bioactivity in conduit PC arteries. We suggest that, in the coronary microcirculation, 12(S)-HETE can function as a potent hyperpolarizing vasodilator that may contribute to endothelium-dependent relaxation, particularly in the setting of oxidative stress.

Previous studies showed that nitric oxide (NO) plays an important role in coronary arteriolar dilation to increases in myocardial oxygen consumption (MVO(2)). We sought to evaluate coronary microvascular responses to endothelium-dependent and to endothelium-independent vasodilators in an in vivo model. Microvascular diameters were measured using intravital microscopy in 10 normal (N) and 9 hyperglycemic (HG; 1 wk alloxan, 60 mg/kg iv) dogs during suffusion of acetylcholine (1, 10, and 100 microM) or nitroprusside (1, 10, and 100 microM) to test the effects on endothelium-dependent and -independent dilation. During administration of acetylcholine, coronary arteriolar dilation was impaired in HG, but was normal during administration of nitroprusside. To examine a physiologically important vasomotor response, 10 N and 7 HG control, 5 HG and 5 N during superoxide dismutase (SOD), and 5 HG and 4 N after SQ29,548 (SQ; thromboxane A(2)/prostaglandin H(2) receptor antagonist) dogs were studied at three levels of MVO(2): at rest, during dobutamine (DOB; 10 microg. kg(-1). min(-1) iv), and during DOB with rapid atrial pacing (RAP; 280 +/- 10 beats/min). During dobutamine, coronary arterioles dilated similarly in all groups, and the increase in MVO(2) was similar among the groups. However, during the greater metabolic stimulus (DOB+RAP), coronary arterioles in N dilated (36 +/- 4% change from diameter at rest) significantly more than HG (16 +/- 3%, P < 0.05). In HG+SQ and in HG+SOD, coronary arterioles dilated similarly to N, and greater than HG (P < 0.05). MVO(2) during DOB+RAP was similar among groups. Normal dogs treated with SOD and SQ29,548 were not different from untreated N dogs. Thus, in HG dogs, dilation of coronary arterioles is selectively impaired in response to administration of the endothelium-dependent vasodilator acetylcholine and during increases in MVO(2).

Insulin and insulin-like growth factor I (IGF-I) influence numerous metabolic and mitogenic processes; these hormones also have vasoactive properties. This study examined mechanisms involved in insulin- and IGF-I-induced dilation in canine conduit and microvascular coronary segments. Tension of coronary artery segments was measured after constriction with PGF(2alpha). Internal diameter of coronary microvessels (resting diameter = 112.6+/-10.1 microm) was measured after endothelin constriction. Vessels were incubated in control (Krebs) solution and were treated with N(omega)-nitro-L-arginine (L-NA), indomethacin, or K(+) channel inhibitors. After constriction, cumulative doses of insulin or IGF-I (0.1-100 ng/ml) were administered. In conduit arteries, insulin produced modest maximal relaxation (32 +/- 5%) compared with IGF-I (66+/-12%). Vasodilation was attenuated by nitric oxide synthase (NOS) and cyclooxygenase inhibition and was blocked with KCl constriction. Coronary microvascular relaxation to insulin and IGF-I was not altered by L-NA, indomethacin, tetraethylammonium chloride, glibenclamide, charybdotoxin, and apamin; however, tetrabutylammonium chloride attenuated the response. In conclusion, insulin and IGF-I cause vasodilation in canine coronary conduit arteries and microvessels. In conduit vessels, NOS/cyclooxygenase pathways are involved in the vasodilation. In microvessels, relaxation to insulin and IGF-I is not mediated by NOS/cyclooxygenase pathways but rather through K(+)-dependent mechanisms.

In the canine coronary microcirculation, acetylcholine (ACh)-induced vasodilation of large (> or = 100 microns) epicardial arterioles (LgA), but not small (< 100 microns) epicardial arterioles (SmA), is blocked by nitric oxide (NO) synthase inhibitors in vivo. We hypothesized that the ACh-induced vasodilation of SmA is mediated by a cytochrome P-450 metabolite of arachidonic acid (AA). Epicardial coronary microvascular diameters in dogs were measured at baseline and after treatment with topically applied ACh (1, 10, and 100 microM), AA (1, 5, and 10 microM), or sodium nitroprusside (SNP; 10-100 microM). Coronary microvascular diameters were compared among control dogs (group OO); dogs pretreated with N omega-nitro-L-arginine (L-NNA; 70 microM topically) (group NO); dogs pretreated with L-NNA plus clotrimazole (Clo; 1.6 microM topically) or 17-octadecynoic acid (ODYA; 2 microM topically), cytochrome P-450 monooxygenase inhibitors (groups NC and NY, respectively); dogs pretreated with Clo alone (group OC); and dogs pretreated with L-NNA plus Clo with AA as the agonist (group AA). ACh-induced vasodilation of LgA was abolished by L-NNA alone, whereas in SmA, L-NNA was without effect. Clo alone did not inhibit ACh-induced dilation in either SmA or LgA. However, the combinations of L-NNA plus either Clo or ODYA abolished ACh- and AA-induced dilation of SmA (100 microM ACh: NC, 3 +/- 5%; NY, 8 +/- 2%; 10 microM AA: 6 +/- 3%) but did not affect responses to SNP. These results suggest that the ACh-induced vasodilation of SmA is mediated in part by cytochrome P-450 metabolites of AA and provide the first evidence that the cytochrome P-450 pathway contributes to the regulation of coronary resistance vessels in vivo.

Unlabelled: Vascular responses to endothelium-dependent vasodilators are greatly impaired in vivo, while isolated blood vessels from animals with diabetes mellitus demonstrate less consistent degrees of impairment. Glycation of proteins, such as hemoglobin, has been implicated in the vascular abnormalities associated with diabetes., Objective: The purpose of this study was to test the hypothesis that glycosylated hemoglobin is capable of reducing endothelium-dependent vasodilator responses, possibly explaining impaired dilation observed in vivo., Methods: To test this hypothesis, the effect of glycosylated hemoglobin (GH) on vascular responses was studied in several vascular beds, including ventricular microvessels and coronary, mesenteric, femoral, and renal arteries. Coronary arterioles were isolated and mounted between two glass pipettes in a pressurized (30 cmH2O) organ chamber. Isolated artery segments were studied using a standard isometric ring technique., Results: In ventricular microvessels, 10 nM nGH (non-GH) and GH both attenuated the relaxation to Ach. A lower concentration, 1 nM nGH or GH, did not alter dilation to Ach. In coronary, femoral, mesenteric and renal artery segments, endothelium-dependent responses were not altered by the presence of 10 or 100 nM nGH or GH., Conclusion: In coronary microvessels, and coronary, femoral, mesenteric and renal arteries, GH is not responsible for the impaired endothelial function associated with diabetes mellitus.

Objectives: Thromboxane has been shown to contribute to coronary constriction in conduit coronary arteries during platelet aggregation at the site of a critical stenosis. Previous studies from our laboratory suggest that following a critical coronary stenosis, persistent vasomotor tone occurs. We tested the hypothesis that thromboxane is responsible for that increased tone., Methods: To test this hypothesis, 14 mongrel dogs of either sex were anesthetized and subjected to a critical coronary stenosis where distal coronary perfusion pressure was reduced to 36 +/- 2 mm Hg. During the coronary stenosis, SQ 29,548 (thromboxane/endoperoxide receptor antagonist) was administered intravenously (0.2 mg/kg and 2.0 mg/kg). Coronary microvascular responses were observed by directly visualizing the epicardial microcirculation. Diameters were measured using intravital microscopy coupled to stroboscopic epi-illumination and jet ventilation to compensate for cardiac and respiratory-induced motion., Results: Coronary microvessels were divided into small (< 150 microns) and large (> 150 microns) arterioles. During SQ 29,548 administration, small coronary arterioles demonstrated no additional dilation during a critical coronary stenosis. In contrast, coronary microvessels > 150 microns demonstrated a dose-dependent vasodilation to SQ 29,548 (0.2 mg/kg: 6 +/- 2%; 2.0 mg/kg: 11 +/- 4%; P < 0.05 vs. no change). A time control study in six additional animals demonstrated no significant microvascular diameter changes following a critical stenosis over the time course of SQ 29,548 administration., Conclusion: Endoperoxides contribute to poststenotic microvascular vasoconstriction in vessels 150-300 microns.

Previous studies in which an isolated heart or in situ constant pressure preparation was used suggested a minimal role for adenosine in autoregulatory control of coronary circulation. These results, however, are controversial, and the role of adenosine in autoregulation of flow in heart is uncertain. To test the hypothesis that adenosine mediates microvascular dilation in response to reduction in perfusion pressure (PP), we performed experiments in 41 open-chest chloralose-anesthetized dogs. Internal diameters (ID) of epicardial small arterioles < 100 mumol were measured with an intravital microscope and stroboscopic epiillumination synchronized to cardiac cycle. PP was reduced by graded stenoses of the left anterior descending coronary artery (LAD, mild stenosis PP = 60 mm Hg; critical stenosis PP = 40 mm Hg) and complete occlusion. 8-Phenyltheophylline (8-PT 10 microM) or adenosine deaminase (ADA 10 U/min) was topically superfused onto the heart. Arteriolar dilation induced by topically applied adenosine < or = 10 microM was completely blocked by 8-PT. Without 8-PT (vehicle group), mild critical stenosis and complete occlusion caused arteriolar dilation (percentage of change in diameter 8.6 +/- 2.6, 16.0 +/- 2.7, and 13.6 +/- 4.8%). 8-PT did not inhibit this dilation (8.5 +/- 2.8, 16.1 +/- 4.6, 15.1 +/- 5.7%, NS vs. vehicle group). Topically applied ADA significantly inhibited intravenously (i.v.) administered adenosine-induced arteriolar dilation. Without ADA, arteriolar dilation occurred (16.6 +/- 3.0, 28.2 +/- 4.3, 15.4 +/- 6.2%, at each PP). However, ADA did not inhibit dilation induced by gradual stenoses (10.6 +/- 1.4, 24.2 +/- 4.3, 17.5 +/- 6.9%, at each PP, NS vs. vehicle group).(ABSTRACT TRUNCATED AT 250 WORDS)

The purpose of this study was to elucidate the contribution of endothelium-derived relaxing factor (EDRF) derived from arginine to acetylcholine (ACh)-induced coronary arteriolar vasodilatation in vivo. Experiments were performed in 62 open-chest anesthetized dogs. Internal diameters of small arterioles (less than 120 microns) and large arterioles (greater than 120 microns) were measured using an intravital microscope and stroboscopic epiillumination synchronized to the cardiac cycle. Topically administered NG-monomethyl-L-arginine (L-NMMA, 3 x 10(-4) M) constricted small arterioles (-10.7 +/- 3.1% from control diameter, P less than 0.05), but L-NMMA did not produce vasoconstriction in large arterioles. ACh, in the absence of L-NMMA, caused a dose-dependent vasodilatation in both small and large arterioles. In large arterioles, L-NMMA completely abolished the ACh-induced vasodilatation (10(-5) M topical ACh: from 13.3 +/- 3.0 to -2.0 +/- 1.5%, P less than 0.05; 10(-4) M ACh: from 20.9 +/- 3.9 to -3.0 +/- 1.9%, P less than 0.01). In small arterioles, L-NMMA only partially inhibited the vasodilatation (10(-5) M ACh: from 35.4 +/- 4.0 to 19.0 +/- 2.7%, P less than 0.05; 10(-4) M ACh: from 42.5 +/- 4.8 to 22.6 +/- 3.1%, P less than 0.05). L-Arginine (10(-3) M topically) reversed L-NMMA inhibition of ACh-induced vasodilatation. Persistent dilatation of small arterioles also occurred when NG-nitro-L-arginine rather than L-NMMA was administered.(ABSTRACT TRUNCATED AT 250 WORDS)