Endothelium-independent Dilator Sodium Nitroprusside Research Articles

The cold receptor TRPM8 activation leads to attenuation of endothelium-dependent cerebral vascular functions during head cooling

Using the cranial window technique, we investigated acute effects of head cooling on cerebral vascular functions in newborn pigs. Head cooling lowered the rectal and extradural brain temperatures to 34.3 ± 0.6°C and 26.1 ± 0.6°C, respectively. During the 3-h hypothermia period, responses of pial arterioles to endothelium-dependent dilators bradykinin and glutamate were reduced, whereas the responses to hypercapnia and an endothelium-independent dilator sodium nitroprusside (SNP) remained intact. All vasodilator responses were restored after rewarming, suggesting that head cooling did not produce endothelial injury. We tested the hypothesis that the cold-sensitive TRPM8 channel is involved in attenuation of cerebrovascular functions. TRPM8 is immunodetected in cerebral vessels and in the brain parenchyma. During normothermia, the TRPM8 agonist icilin produced constriction of pial arterioles that was antagonized by the channel blocker AMTB. Icilin reduced dilation of pial arterioles to bradykinin and glutamate but not to hypercapnia and SNP, thus mimicking the effects of head cooling on vascular functions. AMTB counteracted the impairment of endothelium-dependent vasodilation caused by hypothermia or icilin. Overall, mild hypothermia produced by head cooling leads to acute reversible reduction of selected endothelium-dependent cerebral vasodilator functions via TRPM8 activation, whereas cerebral arteriolar smooth muscle functions are largely preserved.

Modulation of Small Artery Function by Insulin in Young Women: Role of Adiposity.

Vascular dysfunction is common in obesity. Insulin can directly modulate arterial function, but its role is unclear in obesity. We examined the influence of adiposity on direct effects of insulin on human artery responses. 22 healthy women were stratified by median BMI into lower (LA) (n=11) and higher adiposity (HA) (n=11). Small arteries from gluteal biopsies were tested for contractile responses to Noradrenaline (NA), the endothelium-dependent dilator Carbachol and the endothelium-independent dilator sodium nitroprusside were examined before and after incubation with 100 mU/ml human insulin. Contractile responses were similar in the two groups. Insulin reduced NA-induced contraction in HA [3.5 (2.4-4.6) vs. 2.4 (1.4-3.4) mN/mm: p=0.004] but not those from LA [4.1 (2.8-5.3) vs. 3.7 (2.5-5.0) mN/mm: p=0.33]. Endothelium-dependent dilation (EDD) was significantly reduced in arteries from women in the HA (34.7 (18.8-50.6%)) compared to those from women in the LA (62.3 (46.2- 78.4); p=0.013). Insulin improved EDD (change in maximal dilation before/after insulin (%)) in arteries from the HA (37.7 (18.0 to 57.3) but not the LA (6.3 (-6.5 to 19.1), p=0.007. Reduced EDD evident in arteries from HA subjects improve by incubating in insulin. Hyperinsulinaemia may be necessary in maintaining endothelial function in obesity.

Impairment of Protease-Activated Receptor 2-Induced Relaxation of Aortas of Aged Spontaneously Hypertensive Rat.

Hypertension is one of the most prevalent diseases worldwide and can cause harmful complications within the vascular system. Further research on vascular responsiveness to different ligands and diverse receptors in various arteries is required to understand the mechanisms underlying the development of these vascular complications. Here, we investigated the vasorelaxant effect of the protease-activated receptor 2 (PAR2) agonist 2-furoyl-LIGRLO-amide (2-Fly) and two commonest agents, namely endothelium-dependent dilator acetylcholine (ACh) and endothelium-independent dilator sodium nitroprusside (SNP), on the thoracic aorta isolated from aged spontaneously hypertensive rats (SHR) (age, 52±1 weeks). The effects of these agents were compared between aortas isolated from SHR and age-matched normotensive Wistar Kyoto (WKY) rats. Compared with the WKY group, in the SHR group, 2-Fly-induced relaxation was impaired, ACh-induced relaxation was slightly decreased at low concentrations, and SNP-induced relaxation was similar. In addition, 2-Fly-induced aortic relaxation was largely decreased by a PAR2 antagonist (FSLLRY), endothelial denudation, and a nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine (L-NNA) but not by an Akt inhibitor. These results suggested that PAR2-induced relaxations of aortas of aged SHR was impaired, and this impaired aortic relaxation may be attributed to decreased NO bioavailability rather than altered NO sensitivity unrelated to the Akt activity.

Defining uremic arterial functional abnormalities in patients recently started on haemodialysis: combined in vivo and ex vivo assessment.

Endothelial dysfunction is a key initiating event in vascular disease in chronic kidney disease (CKD) patients and haemodialysis (HD) patients exhibit significant vascular abnormalities. To understand this further, we examined how ex vivo intrinsic function in isolated arteries correlates with in vivo assessments of cardiovascular status in HD patients. Abdominal fat biopsies were obtained from 11 HD patients and 26 non-uremic controls. Subcutaneous arteries were dissected and mounted on a wire myograph, and cumulative concentration-response curves to noradrenalin, endothelin-1, a thromboxane A2 agonist (U46619), angiotensin II, vasopressin, bradykinin (BK), acetylcholine (ACh) and sodium nitroprusside (SNP) were constructed. Pulse wave velocity and blood pressure were measured in HD patients. Enhanced (P<0.05−0.0001) maximal contractile responses (Rmax) to all spasmogens (particularly vasopressin) were observed in arteries from HD patients compared to controls, and this effect was more pronounced in arteries with an internal diameter>600 µm. The potency (pEC50) of U46619 (P<0.01) and vasopressin (P<0.001) was also increased in arteries>600 µm of HD patients. The maximal relaxant response to the endothelium-dependent dilators ACh and BK were lower in HD patients (P<0.01-P<0.0001) (worse for ACh than BK); however the endothelium-independent dilator SNP was similar in both groups. PWV was significantly correlated with the vasoconstrictor response to vasopressin (P = 0.042) in HD patients. HD patients are primed for hypertension and end organ demand ischaemia by a highly sensitised pressor response. The failure of arterial relaxation is mediated by endothelial dysfunction. Intrinsic vascular abnormalities may be important in sensitising HD patients to recurrent cumulative ischaemic end organ injury.

Extracellular but not cytosolic superoxide dismutase protects against oxidant-mediated endothelial dysfunction

Superoxide (O2•−) contributes to the development of cardiovascular disease. Generation of O2•− occurs in both the intracellular and extracellular compartments. We hypothesized that the gene transfer of cytosolic superoxide dismutase (SOD1) or extracellular SOD (SOD3) to blood vessels would differentially protect against O2•−-mediated endothelial-dependent dysfunction. Aortic ring segments from New Zealand rabbits were incubated with adenovirus (Ad) containing the gene for Escherichia coli β-galactosidase, SOD1, or SOD3. Activity assays confirmed functional overexpression of both SOD3 and SOD1 isoforms in aorta 24h following gene transfer. Histochemical staining for β-galactosidase showed gene transfer occurred in the endothelium and adventitia. Next, vessels were prepared for measurement of isometric tension in Kreb's buffer containing xanthine. After precontraction with phenylephrine, xanthine oxidase impaired relaxation to the endothelium-dependent dilator acetylcholine (ACh, max relaxation 33±4% with XO vs. 64±3% without XO, p<0.05), whereas relaxation to the endothelium-independent dilator sodium nitroprusside was unaffected. In the presence of XO, maximal relaxation to ACh was improved in vessels incubated with AdSOD3 (55±2%, p<0.05 vs. control) but not AdSOD1 (34±4%). We conclude that adenoviral-mediated gene transfer of SOD3, but not SOD1, protects the aorta from xanthine/XO-mediated endothelial dysfunction. These data provide important insight into the location and enzymatic source of O2•− production in vascular disease.

Abstract 3684: Short-term Caloric Restriction Reverses Age-associated Vascular Endothelial Dysfunction in Older Mice

Backgound: Oxidative stress-mediated, nitric oxide (NO)-dependent vascular endothelial dysfunction develops with aging, increasing the risk of cardiovascular diseases. As such, it is important to identify interventions that can prevent/restore the loss of endothelial function with aging. Caloric restriction (CR) is associated with several physiological benefits and increased longevity in rodents. Some of these benefits are linked to increases in SIRT-1, an enzyme activating endothelial NO synthase (eNOS), and reductions in oxidative stress. We tested the hypothesis that short-term CR would restore vascular endothelial function by improving NO bioavailability and reducing oxidative stress in old B6D2F1-mice, and that this would be associated with increased expression of SIRT-1 and eNOS. Methods: Old (30 months) male B6D2F1-mice were either fed ad libitum (AL, n=6) or were restricted to 70% (3.2g) of their normal food intake for 6 weeks after 2 weeks of progressive restriction (n=6). Carotid artery dilation was assessed ex vivo in response to the endothelium-dependent dilator acetylcholine (ACh) [before and after incubation with the eNOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) or the superoxide dismutase mimetic, TEMPOL] and the endothelium-independent dilator sodium nitroprusside (SNP). Results: ACh-induced dilation was markedly impaired in the AL mice, but was completely restored in the CR mice (63±10% vs. 98±1%, P&lt;0.01). L-NAME decreased ACh dilation by 97% in CR, but only 56% in AL mice, indicating increased NO bioavailability in the CR mice. TEMPOL restored ACh-mediated dilation in AL mice (to 97±1%), but had no effect in CR mice. Aortic protein expression of SIRT-1 and eNOS (western blotting) were 80 –90% higher in CR vs. AL mice (eNOS: 1.9±0.1 vs. 1.0±0.4 P&lt;0.05; SIRT-1: 1.8±0.2 vs. 1.0±0.2, P=0.05). Dilation to SNP was similar in the CR and AL mice (97±1% vs. 96±2%, P=0.63). Conclusion: Short-term CR selectively restores endothelium-dependent dilation in older B6D2F1-mice by increasing NO bioavailability and reducing oxidative stress. These effects are associated with increased vascular expression of SIRT-1 and eNOS. CR may be an effective intervention for reversing age-associated vascular endothelial dysfunction.

Comparison of CGRP and NO Responses in the Human Peripheral Microcirculation of Migraine and Control Subjects

Calcitonin gene-related peptide (CGRP) and nitric oxide (NO) are two molecules shown to have a role in migraine pathophysiology. Our objective was to test the hypothesis that migraine subjects are particularly sensitive to these signal molecules. The cutaneous microvascular responses to endothelial and non-endothelial dependent dilators were tested using laser Doppler flowmetry in combination with iontophoresis. The blood flow responses to iontophoretic administration of the endothelium-dependent vasodilator acetylcholine (ACh), or to the endothelium-independent dilators sodium nitroprusside (SNP) and CGRP, and to local warming (44 degrees C) were compared in this controlled trial. The design was that of two arms: patients diagnosed with migraine without aura (n = 9) for >10 years were compared with nine healthy subjects matched for age and gender (seven female and two male, age range 30-60 years). Iontophoretic administration resulted in local vasodilation. ACh induced a relaxation of 1225 +/- 245% (relative to baseline) in controls and 1468 +/- 368% (P > 0.05) in migraine. The responses to SNP were 873 +/- 193% in controls and 1080 +/- 102% (P > 0.05) in migraine subjects. The responses to CGRP were 565 +/- 89% in controls and 746 +/- 675% (P > 0.05) in migraine patients. The responses to local heating which induced maximum dilation did not differ between the groups (1976 +/- 314% for controls and 1432 +/- 226% in migraine; P > 0.05. We conclude that there is no change in the microvascular responsiveness of the subcutaneous microvasculature in migraine.

Anti‐inflammatory treatment restores endothelium‐dependent dilation in older mice by increasing nitric oxide bioavailability

Chronic low‐grade inflammation may contribute to impaired endothelium‐dependent dilation (EDD) with aging, but evidence is limited. To gain further insight, we studied isolated carotid arteries from young (Y, 4–7 mo) and older (O, 29–34 mo) B6D2F1 mice (n=6–8/group). Maximal carotid artery dilation to acetylcholine (ACh), an endothelium‐dependent dilator, was 24% lower in O (76±3 vs. 99±1%, mean±SE, P=0.001). Administration of the nitric oxide (NO) synthase inhibitor NG‐nitro‐L arginine methyl ester (L‐NAME) decreased ACh dilation less (P=0.02) in O (−20% to 56±8%) vs. Y (−51% to 48±8%). Treatment with the anti‐inflammatory compound sodium salicylate (200 mg/kg/d via diet for 5 d, n=5/group) resulted in similar ACh dilation (P=0.98) in O and Y mice before (O: 97±2 vs. Y: 99±1%) and after (O: 45±15 vs. Y: 43±9%) L‐NAME. Inhibition of superoxide with TEMPOL markedly improved EDD in untreated, but not in sodium salicylate treated O mice or in Y mice. Carotid artery dilation to the endothelium‐independent dilator sodium nitroprusside was not different in Y and O and was not affected by sodium salicylate (P=0.93). These preliminary results support the hypothesis that the development of vascular inflammation plays a key role in impaired EDD with aging in B6D2F1 mice through an oxidative stress‐mediated reduction in NO bioavailability. Supported by NIH AG006537, AG013038 and AG022241

Effect of Aging, MnSOD Deficiency, and Genetic Background on Endothelial Function

The goal of this study was to compare vascular function, superoxide levels, and MnSOD protein expression in young (4 to 7 months) and old (22 to 24 months) MnSOD+/+ and MnSOD-deficient (MnSOD+/-) mice. Relaxation of aorta in vitro to the endothelium-dependent dilator acetylcholine (ACh) was similar in young MnSOD+/+ (n=9) and young MnSOD+/- (n=6) mice. This response was impaired in old MnSOD+/+ (n=8) mice and old MnSOD+/- mice (n=14), with dysfunction being greater in old MnSOD-deficient mice (eg, 100 micromol/L ACh produced 77+/-3% [mean+/-SE], 77+/-3%, 70+/-4%, and 57+/-4% relaxation in young MnSOD+/+, young MnSOD+/-, old MnSOD+/+, and old MnSOD+/- mice, respectively). The endothelial dysfunction was similar in mice on both C57BL/6 and CD-1 genetic backgrounds. In contrast to ACh, responses to the endothelium-independent dilator sodium nitroprusside were enhanced in old MnSOD+/+ and MnSOD+/- mice compared with both groups of young mice (P<0.05). Superoxide levels, as measured using lucigenin-enhanced chemiluminescence, were increased more than 2-fold in old MnSOD+/- mice compared with old MnSOD+/+ and young mice (P<0.05). These data provide the first direct evidence that MnSOD haploinsufficiency results in increased vascular oxidative stress and endothelial dysfunction with aging.

Maternal undernutrition reduces aortic wall thickness and elastin content in offspring rats without altering endothelial function

Epidemiological studies suggest a link between fetal/early infant nutrition and adult coronary artery disease. In the present study, we examined the effects of altering nutrition during gestation, lactation and juvenile life on aortic structure and function in rats. Wistar rat dams were fed either a control or low-protein diet throughout pregnancy, or a low-protein diet for the final 7 days of gestation only. At 21 days post-partum, male pups were weaned on to a control, low-protein or high-fat diet. At 12 weeks, the offspring rats were killed. In 46 rats, aortic sections were mounted and stained to assess media thickness and elastin content. In a further 38 rats, aortic rings were suspended in an organ bath and vascular reactivity was tested with dose-response curves to the endothelium-dependent dilator acetylcholine and the endothelium-independent dilator sodium nitroprusside. Rats exposed to maternal protein restriction while in utero had a significantly decreased aortic wall thickness compared with control rats (P=0.005). Total elastin content of the aorta was also decreased by both maternal low-protein (P=0.02) and early postnatal low-protein (P=0.01) diets. Neither maternal nor postnatal low-protein or high-fat diets, however, resulted in any significant changes in arterial dilator function. In conclusion, fetal undernutrition in rats, induced via a maternal low-protein diet, causes a decrease in aortic wall thickness and elastin content without altering aortic dilator function. These changes in vascular structure may amplify aging-related changes to the vasculature and contribute to the pathophysiology of the putative link between impaired fetal growth and adult cardiovascular disease.

Gene transfer of extracellular superoxide dismutase protects against vascular dysfunction with aging

Aging is an independent risk factor for cardiovascular disease, but mechanisms leading to vascular dysfunction have not been fully elucidated. Recent studies suggest that oxidative stress may increase in blood vessels during aging. Levels of superoxide are influenced by the activity of SODs. The goal of this study was to examine the effect of extracellular superoxide dismutase (ECSOD) on superoxide levels and vascular function in an animal model of aging. Aortas from young (4-8 mo old) and old (29-31 mo old) Fischer 344 rats were examined in vitro. Relaxation of aorta to ACh was impaired in old rats compared with young rats; e.g., 3 muM ACh produced 57 +/- 4% (mean +/- SE) and 84 +/- 2% relaxation in old and young rats, respectively (P < 0.0001). Three days after gene transfer of adenovirus expressing human ECSOD (AdECSOD), the response to ACh was not affected in young rats but was improved in old rats. There was no difference in relaxation to the endothelium-independent dilator sodium nitroprusside between young, aged, and AdECSOD-treated old rats. Superoxide levels (lucigenin-enhanced chemiluminescence) were significantly increased in aged rats compared with young rats. After gene transfer of ECSOD to aged rats, superoxide levels in aorta were similar in old and young rats. Gene transfer of an ECSOD with the heparin-binding domain deleted had no effect on vascular function or superoxide levels in old rats. These results suggest that 1) vascular dysfunction associated with aging is mediated in part by increased levels of superoxide, 2) gene transfer of ECSOD reduces vascular superoxide and dysfunction in old rats, and 3) beneficial effects of ECSOD in old rats require the heparin-binding domain of ECSOD.

Nitric oxide mediates protective effect of endothelin receptor antagonism during myocardial ischemia and reperfusion.

Endothelin (ET) receptor antagonism protects from ischemia-reperfusion injury. We hypothesized that the cardioprotective effect is related to nitric oxide (NO) bioavailability. Buffer-perfused rat and mouse hearts were subjected to ischemia and reperfusion. At the onset of ischemia, the rat hearts received vehicle, the dual endothelin type A/type B (ETA/ETB) receptor antagonist bosentan (10 microM), the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA; 100 microM), the combination of bosentan and L-NMMA or the combination of bosentan, L-NMMA, and the NO substrate L-arginine (1 mM). Hearts from wild-type and endothelial NO synthase (eNOS)-deficient mice received either vehicle or bosentan. Myocardial performance, endothelial function, NO outflow, and eNOS expression were monitored. Bosentan significantly improved myocardial function during reperfusion in rats and in wild-type mice, but not in eNOS-deficient mice. The functional protection afforded by bosentan was inhibited by L-NMMA, whereas it was restored by L-arginine. Myocardial expression of eNOS (immunoblotting) increased significantly in bosentan-treated rat hearts compared with vehicle hearts. Recovery of NO outflow during reperfusion was enhanced in the bosentan-treated rat heart. The endothelium-dependent vasodilator adenosine diphosphate increased coronary flow by 18 +/- 9% at the end of reperfusion in the bosentan group, whereas it reduced coronary flow by 7 +/- 5% in the vehicle group (P < 0.001). The response to the endothelium-independent dilator sodium nitroprusside was not different between the two groups. In conclusion, the dual ETA/ETB receptor antagonist bosentan preserved endothelial and cardiac contractile function during ischemia and reperfusion via a mechanism dependent on endothelial NO production.

Influence of sialic acid content of low-density lipoprotein particles on endothelium-dependent vasorelaxation in rabbit aorta

Low-density lipoprotein (LDL) concentration in plasma is an important predictor for atherosclerosis, and desialylated LDL has been proposed to be particularly atherogenic. Atherosclerosis is also associated with vascular endothelial dysfunction. We therefore wished to test the hypothesis that removal of sialic acid residues from LDL increases its ability to inhibit endothelium-dependent vasorelaxation. We studied vasorelaxant responses to acetylcholine (ACh) in isolated rabbit aortic rings as a model of endothelium-dependent relaxation, in the presence or absence of LDL treated either with saline or with neuraminidase, to cleave sialic acid residues. Vasorelaxant responses to ACh were inhibited by 300 μg protein per ml saline-treated LDL ( E max 77.5±4.5 vs. 89.7±2.2% in the absence of LDL, P<0.05). This inhibitory effect was not altered by neuraminidase treatment of LDL. In contrast, 300 μg protein per ml LDL, either saline- or neuraminidase-treated, did not affect vasorelaxant responses to the endothelium-independent dilator sodium nitroprusside. We found no correlation between sialic acid content of saline-treated LDL and its ability to inhibit endothelium-dependent vasorelaxation, in rabbit aortic rings, at a concentration of 300 μg protein per ml. Our results therefore suggest that sialic acid content is not an important determinant of the effect of LDL on vascular endothelium-dependent relaxation.

Peripheral resistance vessel dysfunction in Marfan syndrome

Background Patients with Marfan syndrome show a hereditary abnormality of elastin metabolism that may cause aortic enlargement and dissection. We have hypothesized that abnormal elastin may alter peripheral vascular structure and function. Methods Forearm blood flow (FBF) (in milliliters per minute per 100 mL) response to the endothelium-dependent dilator acetylcholine (0.75 to 4.5 μg/min per 100 mL), the endothelium-independent dilator sodium nitroprusside (0.05 to 0.3 μg/min per 100 mL), and structure-related maximum dilator response (10-minute occlusion-induced reactive hyperemia) were measured by plethysmograph in 10 patients with Marfan syndrome (mean age 44 years) and 10 healthy age- and sex-matched controls. Patients with the complications of hypercholesterolemia, diabetes mellitus, or heart failure were excluded from the study. Results Basal FBF (mean ± SE) did not differ between the 2 groups (2.7 ± 0.3 vs 2.3 ± 0.4). Maximum FBF response to acetylcholine in patients with Marfan syndrome was significantly lower than that of healthy controls (8.5 ± 2.1 vs 15.4 ± 1.7 mL/min per 100 mL; P < .05). Reactive hyperemia was also lower in patients with Marfan syndrome (at peak 23.0 ± 2.5 vs 29.5 ± 2.3 mL/min per 100 mL; P < .05). Reactive hyperemia was also lower in patients with Marfan syndrome (at peak 23.0 ± 2 groups (10.3 ± 1.1 vs 10.2 ± 1.5 mL/min per 100 mL; P = not significant). Conclusion These observations suggest that endothelium-dependent dilation and maximum dilator reserve capacity are both abnormal in peripheral resistance vessels of patients with Marfan syndrome. These peripheral vasomotion abnormalities may have a detrimental impact on the cardiovascular system in this disorder.

Vasomotor responses of soleus feed arteries from sedentary and exercise-trained rats.

Our goals were to determine the nature of endothelium-dependent and -independent vascular responses in isolated soleus feed arteries (SFA) and to test the hypothesis that these responses would be altered by exercise training. Exercise-trained rats ran 30 m/min, up a 15% grade, 1 h/day, 5 days/wk for 10-12 wk, while sedentary control rats were confined to normal cage activity. SFA were isolated, cannulated, and pressurized at 90 cmH2O. After a 1-h equilibration period, the dose-response relationships to constrictors, endothelium-dependent dilators, and endothelium-independent dilators were examined. SFA developed spontaneous tone, demonstrated myogenic reactivity by maintaining vessel diameter in the face of large changes in intraluminal pressure, and constricted in a dose-dependent manner to norepinephrine and potassium chloride. SFA dilated in a dose-dependent manner to the endothelium-dependent dilators acetylcholine and increased flow and to the endothelium-independent dilator sodium nitroprusside. SFA did not dilate to the putative endothelium-dependent dilators bradykinin, substance P, and clonidine or to adenosine. Dilation to acetylcholine was attenuated markedly by arginine analogs and less by 20 mM KCl, but it was unaltered by indomethacin. These results indicate that SFA respond to a number of vasoactive substances, consistent with the hypothesis that SFA participate in the control of vascular resistance. However, exercise training does not appear to elicit a stimulus adequate to alter vasomotor responses in SFA.

Effect of isoflurane on the β-adrenergic and endothelium-dependent relaxation of pig cerebral microvessels after cardiopulmonary bypass

We examined the direct vasomotor effect of isoflurane as well as its effect on endothelium-dependent and beta-adrenergic vasodilation of cerebral microcirculation following either normothermic cardiopulmonary bypass (CPB) or profoundly hypothermic CPB with circulatory arrest. Pigs were placed on CPB; the systemic temperature was either maintained at 37 degrees C or lowered to 15 degrees C with 60 minutes of circulatory arrest. After 2 hours of CPB, the animals were separated from CPB; 15 minutes later the brain was quickly harvested in cold Krebs solution. Control animals were not instrumented and their brains were similarly harvested. Arteries of approximately 100 microm were dissected and changes in diameter monitored by in vitro videomicroscopy. Following preconstriction with the thromboxane analogue U46619 1 micromol/L, percent relaxation to the endothelium-dependent dilator adenosine diphosphate (ADP) 10(-9) to 10(-4) mol/L, the endothelium-independent dilator sodium nitroprusside (SNP) 10(-9) to 10(-4) mol/L, or the beta-adrenergic agonist isoproterenol 10(-12) to 10(-4) mol/L was measured either in the presence or absence of isoflurane 2%. Additionally, with or without preconstriction with U46619 1 micromol/L, vessel diameter changes were monitored with increasing concentrations of isoflurane 0-3%. Dose-response curves were compared by two-way analysis of variance. Vasodilation to ADP or isoproterenol, but not SNP, was attenuated after normothermic CPB (N-CPB) or profoundly hypothermic CPB (PH-CPB). Although isoflurane attenuated vasodilation of control vessels to ADP or isoproterenol, isoflurane did not further attenuate vasodilation to ADP or isoproterenol after N-CPB or PH-CPB. The direct vasomotor effect of isoflurane depended on the preexisting tone of the vessels, constricting vessels without preconstriction and dilating them after preconstriction. These findings may have implications on the incidence of neuropsychological dysfunction after CPB and use of isoflurane.

Coronary vascular and endothelial reactivity changes in transgenic mice overexpressing atrial natriuretic factor.

Recent advances in genetic methods permit the introduction of random and defined mutations into the mouse germ line, producing novel mouse strains, some of which affect the heart and vasculature. A TTR-ANF transgenic strain of mice, which constitutively expresses a fusion gene consisting of the transthyretin promoter and the ANF structural gene, has been shown to result in a lifelong elevation of plasma atrial natriuretic factor (ANF) and a chronically lowered arterial blood pressure. However, no established method for efficient functional analysis of possible alterations in coronary vascular function in mice has been reported. In the present study, we describe an isolated mouse coronary artery preparation that permits an effective and reproducible evaluation of coronary endothelial and vascular function. Both left main and right coronary arteries (resting luminal diam 70-90 microns) were isolated and pressurized, and changes in luminal diameter were determined by videomicroscopy. The coronary pressure-luminal diameter relationship was not significantly different between TTR-ANF transgenic and nontransgenic littermates. Relaxation of coronaries to 0.1-100 nM ANF was significantly reduced in transgenic [maximum effect (Emax) = 43 +/- 10% (mean +/- SE) of 11 vessels] compared with nontransgenic (Emax = 73 +/- 7%, n = 15) mice. Similarly, the relaxant response to an endothelium-dependent dilator, acetylcholine, but not to endothelium-independent dilators sodium nitroprusside and isoproterenol, was significantly decreased in transgenic (Emax = 46 +/- 10%, n = 12) compared with nontransgenic (Emax = 85 +/- 5%, n = 14) mice. In contrast, the dose-dependent vasoconstriction to endothelin-1, KCl and the thromboxane mimetic U-46619 was not significantly different between the two groups. These results indicate that lifelong ANF elevation in mice is associated with a decreased responsiveness of coronary vasorelaxation to ANF, possibly resulting from receptor downregulation and/or desensitization. Endothelium-dependent relaxation was also significantly depressed in transgenic mouse coronary arteries, but smooth muscle-specific dilation and constriction were not affected. The present findings are consistent with previous studies of TTR-ANF transgenic mice showing that ANF regulates arterial blood pressure and vascular function.

Restoration of endothelium-dependent vasodilation after reperfusion injury by tetrahydrobiopterin.

A deficit in the endothelial production of nitric oxide (NO) is associated with the sequelae of reperfusion injury. Because endothelial NO synthesis depends on the cofactor tetra-hydrobiopterin (BH4), we hypothesized that depletion of this cofactor underlies the reduction of endothelium-dependent dilation in reperfusion injury. After occlusion of the left anterior descending coronary artery of a pig for 60 minutes followed by 90 minutes of reperfusion (ischemia/reperfusion), hearts were removed and the arterioles were isolated, cannulated, pressurized, and placed on an inverted microscope stage. Dose responses to the endothelium-independent dilator sodium nitroprusside and the endothelium-dependent dilators serotonin, A23187, and substance P were obtained under control conditions, after incubation with sepiapterin (intracellularly converted to BH4) or synthetic BH4 6-methyltetrahydropterin (MH4), and again after their washout. After ischemia/reperfusion, sodium nitroprusside maximally dilated arterioles (99 +/- 3%), whereas relaxation to serotonin, A23187, and substance P was significantly reduced (19 +/- 9%, 44 +/- 9%, and 54 +/- 8%, respectively). During incubation with sepiapterin (1 mumol/L) or MH4 (10 mumol/L), endothelium-dependent dilation was significantly enhanced (P < .05), whereas the response to sodium nitroprusside was unaltered. After washout, the vasodilatory responses were not significantly different from the initial ischemia/reperfusion responses. Sepiapterin and MH4 did not affect vasodilatory responses in vessels obtained from nonischemic control hearts. As after ischemia/reperfusion, incubation of control vessels with 2,4-diamino-6-hydroxypyrimidine, an inhibitor of GTP cyclohydrolase I, decreased endothelium-dependent vasodilation, which was restored in the presence of sepiapterin or MH4. These data indicate that exogenous administration of sepiapterin or MH4 restores the response to endothelium-dependent vasodilators in pig coronary arterioles after ischemia/ reperfusion. We therefore conclude that ischemia/reperfusion alters the availability or production of BH4, which contributes to blunted endothelial nitroxidergic vasodilation.

Direct vasomotor effects of isoflurane in subepicardial resistance vessels from collateral-dependent and normal coronary circulation of pigs.

The purpose of the current investigation was to determine and compare the direct effect of isoflurane on normal resistance coronary arteries and similarly sized coronary arteries in collateral-dependent (CD) circulation. A differential vasomotor effect of isoflurane might contribute to flow redistribution, either adverse or favorable, between normal and CD regions. The authors used a swine model of chronic coronary occlusion. Six weeks after ameroid occluder placement around the left circumflex artery, the heart was removed. Myocardial blood flow, measured in vivo before the removal, was lower during rapid atrial pacing in the CD region than in the normally perfused region. These in vivo studies established the existence and location of CD circulation. Subepicardial microvessels, measuring approximately 100 microns, were dissected from both the CD region and the normal region. Either with or without preconstriction of the vessels with the thromboxane analogue U46619 1 microM, direct vasomotor effect of isoflurane was examined in vitro by video microscopy. In addition, vasomotor responses to endothelin-1, the endothelium-dependent dilator adenosine 5' diphosphate, and the endothelium-independent dilator sodium nitroprusside were compared between CD and normal resistance vessels. Isoflurane caused greater concentration-dependent constriction of the normal vessels than of the CD vessels (P < 0.05); this constrictive effect was masked by preconstriction of the vessels. Vasodilation of ADP was less in CD vessels than in normal vessels (P < 0.001), but vasodilation to sodium nitroprusside was not significantly different (P = 0.45). Vasoconstriction to endothelin-1 was greater in CD vessels than in normal vessels (P < 0.01). The vasoconstrictive effect of isoflurane normally observed in coronary resistance vessels is impaired in resistance vessels that supply a CD area. The basis of this may be related to endothelial dysfunction.

Vasodilatory responses of isolated arteries of cirrhotic rats.

1. There is currently considerable interest in the role of locally produced vasodilators such as nitric oxide and adenosine in the pathogenesis of the peripheral vasodilatation of cirrhosis. However, the signal transduction pathways involving guanylate cyclase and adenylate cyclase have not been clearly delineated in the isolated blood vessel. 2. We therefore aimed to examine the in vitro vasorelaxant effects of the endothelium-dependent dilator bethanechol, the endothelium-independent dilator sodium nitroprusside and adenosine, as drugs that work via activation of guanylate and adenylate cyclases, in isolated aortic and superior mesenteric arterial rings from cirrhotic and control rats. 3. Cirrhosis was induced by chronic bile duct ligation and section of 24-28 days' duration, while controls underwent sham operation. The vessels were precontracted with the alpha 1-adrenoceptor agonist phenylephrine, then relaxed by incremental doses of the three drugs. 4. Marked attenuation of vasoconstriction induced by phenylephrine in isolated aortic and mesenteric arterial rings from cirrhotic rats compared with the control vessels was observed. 5. There were no significant differences in relaxation between the cirrhotic and control vessels to the three drugs. We conclude that in vitro vasodilatory responses mediated through signal transduction pathways involving guanylate cyclase and adenylate cyclase remain unchanged in a rat model of biliary cirrhosis.