Impaired skin microvascular function in children, adolescents, and young adults with type 1 diabetes

OBJECTIVE: Vascular disease in type 1 diabetes is a complex and multifactorial process, which probably begins in childhood in association with the onset of diabetes. To determine the possible factors involved, we measured microvascular responses to endothelium-dependent (acetylcholine) and endothelium-independent (sodium nitroprusside) vasodilators in 56 patients with type 1 diabetes (aged 9-22 years) and 22 control subjects. RESEARCH DESIGN AND METHODS: Skin perfusion was measured at the dorsum of the foot using laser Doppler flowmetry during low-current iontophoresis of acetylcholine and sodium nitroprusside. Maximum vasodilator function was measured during local 44 degrees C skin heating. RESULTS: Vascular responses were significantly reduced in patients with type 1 diabetes compared with responses in control subjects: acetylcholine (P<0.01, analysis of variance [ANOVA]), sodium nitroprusside (P<0.01, ANOVA), and local heating (P<0.02. Mann-Whitney U test). Endothelium-dependent responses were related to duration of diabetes (r = -0.38, P<0.01) and to glycemic control (r = 0.37, P<0.01). Significant correlations were found in the patient group between responses to acetylcholine and sodium nitroprusside (r = 0.28, P<0.05) but not to heating, suggesting that a common factor (e.g., nitric oxide activity) may be responsible for the abnormal vascular responses to these chemicals. CONCLUSIONS: Early changes in microvascular function are present in young patients with type 1 diabetes, long before the initial clinical presentation. These abnormalities may be related to complex interactions between structural abnormalities and functional changes in the endothelium, smooth muscle, and nitric oxide activity.     

Pertussis toxin reduces endothelium-dependent and independent responses to alpha-2- adrenergic stimulation in systemic canine arteries and veins.

A pertussis toxin-sensitive guanine nucleotide regulatory protein (G-protein) is involved in the signal transduction of certain endothelium-dependent responses in mammalian arteries. To determine whether a similar mechanism mediates endothelium-dependent responses in mammalian veins, rings of canine femoral arteries and veins with and without endothelium were suspended for the measurement of isometric force in organ chambers. In femoral arteries, incubation of the rings with pertussis toxin (from Bordetella pertussis, 100 ng/ml for 2 hr) in the presence of indomethacin and propranolol did not reduce significantly endothelium-dependent relaxations to acetylcholine and adenosine diphosphate, thrombin or the calcium ionophore A23187. However, endothelium-dependent relaxations evoked by the alpha-2 adrenergic agonist UK 14,304 were blocked by the pertussis toxin. In venous rings, endothelium-dependent relaxations to acetylcholine were reduced by the toxin, whereas the endothelium-dependent relaxations evoked by adenosine diphosphate, thrombin and A23187 were not affected. UK 14,304 contracted the veins; these contractions were augmented by removal of the endothelium. Pertussis toxin inhibited contractions to UK 14,304 in venous rings without but not with endothelium. Relaxations of arterial and venous smooth muscle to nitric oxide were unaffected by the toxin. Contractions to phenylephrine were not altered by either removal of the endothelium or the toxin in the arteries or veins. These results suggest that the release of endothelium-derived relaxing factor in response to stimulation of purine and thrombin receptors probably does not involve a pertussis toxin-sensitive G-protein in canine femoral arteries or veins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differences in tail vascular bed reactivity in rats with and without heart failure following myocardial infarction

Myocardial infarction (MI) was induced in rats by coronary ligation to compare changes in vascular reactivity from animals that developed heart failure (InfHF) with those that did not (Inf). Infarct size was similar in both groups. In vitro preparations of tail vascular bed were used to investigate the vascular responses to acetylcholine, sodium nitroprusside, and phenylephrine. Acetylcholine-induced relaxation was impaired in the Inf group (53 +/- 2%, n = 6) when compared with Sham (80 +/- 2%, n = 6, P < 0.05). The maximal response (E(max)) to phenylephrine increased in the Inf group (423 +/- 10 mm Hg, n = 9, P < 0.01) and decreased in InfHF (279 +/- 10 mm Hg, n = 7, P < 0.05) when compared with Sham (319 +/- 11 mm Hg, n = 8). Regardless of endothelial integrity, E(max) to phenylephrine increased in the Inf, nitro-l-arginine methyl ester, and indomethacin groups. An increased release of a prostanoid vasodilator was detected in the Inf group. Differently, the InfHF group presented a reduction of the E(max) to phenylephrine and an increment of nitric oxide release. This study demonstrates that MI without heart failure impairs endothelium-dependent relaxation and increases the reactivity to phenylephrine. This increase seems to involve a muscular component. The endothelium participates with an increased release of a vasodilator prostanoid, possibly to compensate the increased smooth muscle response. When heart failure follows MI, the reactivity to phenylephrine decreases, possibly due to an increased nitric oxide release.     

Circumferential deformation and shear stress induce differential responses in saphenous vein endothelium exposed to arterial flow.

Adaptation of saphenous vein to the hemodynamic stresses of the arterial circulation is critical to the maturation of vein bypass grafts. We have investigated early adaptive responses of venous endothelium by placing excised human saphenous vein in a bypass circuit with either venous or arterial flow conditions, using external stenting to resolve the effects of longitudinal (shear) from circumferential stress. Endothelial protein concentrations were assessed by immunostaining area (ratio of protein/CD31) and Western blotting of endothelial cell lysates (staining ratio protein/vWf). In both unstented and stented veins nitric oxide synthase increased after 90 min of arterial flow: twofold increase of immunostaining area (P = 0.001), four- to fivefold increase by Western blotting (P = 0.02), and increased A23187mediated maximum endothelium-dependent relaxation of vein rings (P = 0.01). In unstented veins, ICAM-1 concentration was increased after 45 min of arterial flow: twofold increase by immunostaining (P = 0.001) and Western blotting (P = 0.038), with maximum fibrinogen-mediated endothelium-dependent relaxation increasing from 55.9+/-4.9 to 97+/-2.1% (P = 0.01). In contrast, in unstented veins there was a threefold decrease of VCAM-1 and no change in P-selectin after arterial flow for 45 and 90 min, respectively. However, no changes in ICAM-1 and VCAM-1 were observed in stented veins. The flow-induced alterations in nitric oxide synthase, ICAM-1, and VCAM-1 were abolished when 3 mM tetraethylammonium ion (K+ channel blocker) was included in the vein perfusate. The very rapid changes in ICAM-1 and VCAM-1 expression are a response to circumferential stress, whereas the slower upregulation of nitric oxide synthase is a response to longitudinal (shear) stress. Similar changes could influence the adhesiveness of endothelium in newly implanted saphenous vein bypass grafts.     

The C242T p22phox polymorphism and endothelium-dependent vasodilation in subjects with hypercholesterolaemia

Oxidative stress plays a major pathogenetic role in cardiovascular disease. The C242T variant of the CYBA gene encoding the p22phox subunit of the NAD(P)H oxidase, a major source of superoxide production, has been shown to be associated with coronary artery disease and with vascular superoxide production in human veins ex vivo. Since superoxide degrades nitric oxide (NO), we hypothesized that the C242T variant influences endothelium-dependent vasodilation of the human forearm vasculature in vivo. In the present study, 90 subjects with elevated cholesterol levels were stratified for the C242T polymorphism of the CYBA p22phox gene. Endothelium-dependent and -independent vasodilation were assessed by plethysmographic monitoring of forearm blood flow responses to intra-arterial infusion of acetylcholine and sodium nitroprusside respectively. N(G)-Monomethyl-L-arginine (L-NMMA) was infused to analyse NO-mediated basal vascular tone. Baseline parameters (age, gender, blood pressure, body mass index, cholesterol level) were similar across the genotypes. No differences in forearm blood flow responses to the intra-arterial infusion of acetylcholine, sodium nitroprusside or L-NMMA were found across the CYBA p22phox genotypes. Our sample size of n =90 had a power of >80% (beta=0.20) with a P value of <0.05 (alpha=0.05) to detect a difference greater than 156% in the forearm blood flow response to acetylcholine across genotypes (S.D. 336%; average increase in forearm blood flow=514%). In conclusion, at a power of 80%, our study excludes a major effect of the C242T CYBA p22phox polymorphism on acetylcholine-mediated endothelium-dependent vasodilation and basal NO-mediated vascular tone of the human forearm circulation in subjects with hypercholesterolaemia.     

Nicotine impairs endothelium-dependent dilatation in human veins in vivo

BACKGROUND: Cigarette smoking is associated with impaired endothelium-dependent dilatation in human veins and arteries. An in vivo study in animals suggests that nicotine may contribute to this abnormality. We tested the hypothesis that local administration of nicotine at a dose reproducing the plasma concentration observed during smoking would impair endothelium-dependent vasodilatation in human veins in vivo. METHODS: We studied 11 healthy nonsmokers with the dorsal hand vein compliance technique. After 70% to 80% preconstriction with phenylephrine, endothelium-dependent venous relaxation was assessed by infusion of bradykinin (1 to 278 ng/min), a potent vasodilator acting primarily in this model through endothelial release of nitric oxide and prostanoids. Sodium nitroprusside (0.0001 to 3166 ng/min) was used to test endothelium-independent relaxation. Dose-response curves were constructed before and during nicotine coinfusion at a rate of 40 ng/min, reproducing a plasma concentration of 15 ng/mL. RESULTS: After a 10-minute preinfusion, nicotine administration was associated with a loss in sensitivity to bradykinin (P < .001). After 30 and 60 minutes of preinfusion with nicotine, the venorelaxant effect of bradykinin was further reduced (P < .001). A similar inhibition of the response to bradykinin by nicotine persisted in the presence of indomethacin (INN, indomethacin). Coinfusion of nicotine did not attenuate sodium nitroprusside-induced venodiiation. CONCLUSION: The results show that acute local exposure to nicotine in vivo is associated with an impaired response to endothelium-derived nitric oxide in human veins. This finding may provide further insight into the pathophysiology of smoking-induced endothelial dysfunction.     

Biphasic response to acetylcholine in human veins in vivo: the role of the endothelium

1. The dose-response to acetylcholine has been examined in dorsal hand veins of healthy volunteers before and after removal of the endothelium. 2. Measurements were made in single dorsal hand veins during local infusions of acetylcholine. The vein was irrigated with distilled water to remove the endothelium. Dilator studies were performed in vessels preconstricted by a continuous infusion of noradrenaline. 3. In the endothelium-intact vessel the dose-response to acetylcholine was biphasic; low doses produced venodilatation with higher doses causing venoconstriction. 4. Dilatation to low doses of acetylcholine was abolished by prior irrigation with distilled water, consistent with denudation of the endothelium by this process. Irrigation augmented the constriction seen in response to higher doses of acetylcholine. 5. This is the first demonstration of an endothelium-dependent biphasic dose-response to acetylcholine in man. The results raise questions as to the possible physiological actions of endogenous acetylcholine and as to the use of the acetylcholine dose-response curve as a marker of endothelial function.     

Mechanisms of beta-adrenergic receptor-mediated venodilation in humans

OBJECTIVES: Recent studies suggest that stimulation of beta-adrenergic receptors results in both endothelium-dependent and endothelium-independent venodilation, but results of former studies are inconsistent. This study was designed to elucidate the underlying mechanisms of isoproterenol (INN, isoprenaline)-induced venodilation by investigation of dorsal hand vein responses. METHODS: In phenylephrine-constricted veins, isoproterenol (2-514 ng/min) was infused with and without oral pretreatment with 1 g acetylsalicylic acid (n = 7) or 5 mg of the selective beta(1)-adrenergic receptor antagonist bisoprolol (n = 7). In addition, isoproterenol was coinfused with the nitric oxide inhibitor N(G)-monomethyl-l-arginine (l-NMMA) (6.3 micromol/min [n = 6]), with selective blockers of calcium (Ca(++))-dependent potassium (K(+)) channels (tetraethylammonium, 300 microg/min [n = 6]) and adenosine triphosphate (ATP)-sensitive K(+) channels (glyburide [INN, glibenclamide], 20 microg/min [n = 6]) or with the cyclic guanosine monophosphate inhibitor methylene blue (13 microg/min [n = 6]). Finally, L-NMMA was coinfused with potassium chloride (20 mmol/L) to inhibit hyperpolarization (n = 6). RESULTS: Isoproterenol induced dose-dependent venodilation to 67.4% +/- 6.8%. Oral pretreatment with bisoprolol (P =.340) or acetylsalicylic acid (P =.760) did not affect isoproterenol-induced venodilation. Coinfusion of isoproterenol and L-NMMA relaxed the veins to the same extent as in the presence of isoproterenol alone. Neither inhibition of ATP-sensitive K(+) channels (P =.196) nor blockade of Ca(++)-dependent K(+) channels (P =.640) modulated isoproterenol-induced venodilation. In contrast, methylene blue reduced the maximum response to isoproterenol by about one third (68.5% +/- 4.3% versus 41.7% +/- 5.5%, P =.001). Infusion of L-NMMA alone raised vein size to 38.8% +/- 6.5%, yielding an L-NMMA-sensitive increase of 20% (P =.001), which was antagonized by coinfusion of potassium chloride to 17.1% +/- 6.7% (P =.02). CONCLUSIONS: Isoproterenol dilates human hand veins exclusively via beta(2)-adrenergic receptors without involvement of endothelium-derived epoprostenol. Although a contribution of endothelium-derived nitric oxide appears unlikely, the venodilating effect of L-NMMA could have obscured the nitric oxide component of isoproterenol. beta(2)-Adrenergic receptor-mediated dilation is mediated in part by cyclic guanosine monophosphate-dependent mechanisms, whereas ATP- and Ca(++)-dependent K(+) channels are not involved, excluding a significant contribution of smooth muscle cell hyperpolarization. In addition, high concentrations of the nitric oxide synthase blocker L-NMMA dilate human hand veins via activation of endothelium-derived hyperpolarizing factors.     

Enhancement on reactive oxygen species and COX‐1 mRNA levels modulate the vascular relaxation induced by sodium nitroprusside in denuded mice aorta

This study aimed to investigate the modulation of nitric oxide/reactive oxygen species in sodium nitroprusside relaxation in mice aorta. Sodium nitroprusside induced relaxation in endothelium‐intact (e+) and endothelium‐denuded (e?) aortas with greater potency in e+ than in e?. The nitric oxide synthase inhibitor did not alter the sodium nitroprusside relaxation in both e+ and e? aortas. However, the superoxide anion scavenger abolished the difference in sodium nitroprusside potency between e+ and e?. Sodium nitroprusside reduced dihydroethidium‐derived fluorescent products in both groups; however, the difference between intact and denuded mice aorta remains. The glutathione levels and basal antioxidant activity of superoxide dismutase were reduced in e? aorta when compared with e+, and these values were not altered by sodium nitroprusside. Confirming these results, the levels of lipid peroxidation in e+ were significantly lower when compared to e?, and these values were not altered by sodium nitroprusside. The sodium nitroprusside potency in the presence of a nonselective COX inhibitor or the EP/DP prostaglandin receptor antagonist in endothelium denuded was similar to that in intact mice aorta. Based on these results, we performed the COX‐1 and COX‐2 mRNA level studies, and in denuded mice aorta, there was an upregulation in COX‐1 mRNA levels. Taken together, our findings show that in the absence of endothelium, there is an enhancement of superoxide levels, leading to GSH consumption and higher levels of lipid peroxidation, showing an intense redox status. Furthermore, in denuded mice aorta, there was an upregulation of COX‐1 mRNA expression, leading to vasoconstrictor prostanoids synthesis. The interaction of vasoconstrictor prostanoids with its receptors EP/DP negatively modulates the vascular relaxation induced by SNP in denuded mice aorta.     

Enhanced prostanoid-mediated vasorelaxation in pulmonary arteries isolated during experimental endotoxemia.

Endotoxemia secondary to gram-negative sepsis has been shown to inhibit endothelium-dependent vasomotion in numerous vascular beds, including guinea pig aortae and coronary arteries. We tested the hypothesis that in vivo endotoxin impairs endothelium-dependent nitric oxide-mediated relaxation responses of pulmonary arteries isolated from guinea pigs given intraperitoneal injections of Escherichia coli endotoxin lipopolysaccharide (LPS) or saline (control) 16 h before sacrifice. Pulmonary rings from the main artery and primary branches were isolated and studied in vitro using conventional isometric techniques. Interestingly, endotoxemia resulted in enhanced pulmonary artery relaxation in response to the endothelium-dependent receptor agonists acetylcholine (10(-10) -10(-5) M) and adenosine diphosphate (ADP; 10(-9) -10(-5) M), as compared with control responses (p < .05). Nitric oxide synthase inhibitors N-monomethyl-L-arginine (300 microM) and N-nitro-L-arginine methyl ester (100 microm) reduced acetylcholine- and adenosine diphosphate-mediated relaxation in both groups (p < .05); however, vasodilation responses in arteries from LPS animals remained enhanced relative to those of control arteries. In contrast to nitric oxide synthase inhibitors, the cyclooxygenase inhibitor indomethacin markedly inhibited acetylcholine- and adenosine diphosphate-mediated relaxation responses of pulmonary arteries isolated from LPS-treated animals (p < .05) but not control arteries; indomethacin effectively reversed LPS-induced enhanced vasodilation of pulmonary arteries. Relaxation responses to the receptor-independent calcium ionophore (A23187) and to the direct smooth muscle vasodilator sodium nitroprusside (+ N-nitro-L-arginine methyl ester) were not significantly altered by LPS treatment (p > .05). These data suggest that in pulmonary arteries, unlike aortae and coronary arteries isolated from the same model, in vivo LPS enhances agonist-mediated endothelium-dependent vasodilation responses to acetylcholine and adenosine diphosphate. Underlying mechanisms appear to involve increased dependency upon vasodilator prostanoids and decreased dependency on nitric oxide synthesis/release for LPS-induced alterations in pulmonary relaxation responses.     

Hypercholesterolemia increases endothelial superoxide anion production.

Indirect evidence suggests accelerated degradation of endothelium-derived nitric oxide (ENDO) by superoxide anion (O2-) in hypercholesterolemic vessels (HV). To directly measure O2- production by normal vessels (NV) and HV, we used an assay for O2- based on the chemiluminescence (CL) of lucigenin (L). HV (1 mo cholesterol-fed rabbits) produced threefold more O2- than NV (1.47 +/- 0.20 nM/mg tissue/min, n = 7 vs. 0.52 +/- 0.05 nmol/mg tissue/min, n = 8, P < 0.001). Endothelial removal increased O2- production in NV (0.73 +/- 0.08, n = 6, P < 0.05), while decreasing it in HV (0.76 +/- 0.15, n = 5, P < 0.05). There was no difference between denuded HV and denuded NV. Oxypurinol, a noncompetitive inhibitor of xanthine oxidase, normalized O2- production in HV, but had no effect in NV. In separate isometric tension studies treatment with oxypurinol improved acetylcholine induced relaxations in HV, while having no effect on responses in normal vessels. Oxypurinol did not alter relaxations to nitroprusside. Thus, the endothelium is a source of O2- in hypercholesterolemia probably via xanthine oxidase activation. Increased endothelial O2- production in HV may inactivate endothelium-derived nitric oxide and provide a source for other oxygen radicals, contributing to the early atherosclerotic process.     

Type 2 diabetic individuals have impaired leg blood flow responses to exercise: role of endothelium-dependent vasodilation

OBJECTIVE: Diabetic individuals have impaired endothelium-dependent forearm vasodilatory responses to ischemia, acetylcholine, and other endothelium-dependent agonists. The functional significance of impaired endothelium-dependent dilation in diabetic individuals is uncertain but is most likely to be manifest during leg muscle exercise and may have relevance to peripheral vascular disease and leg ischemia, which is prevalent in diabetic individuals. The current study aimed to determine the relationship between leg blood flow (LBF) responses to endothelium-dependent vasodilation and dynamic large muscle exercise. RESEARCH DESIGN AND METHODS: LBF responses (thermodilution) to intrafemoral arterial infusions of an endothelium-dependent (acetylcholine) and endothelium-independent (sodium nitroprusside) vasodilator and a standardized 25-min cycling bout at 60% VO(2peak) were compared in nine male type 2 diabetic subjects and nine age-, sex-, VO(2peak)-, and weight-matched control subjects. RESULTS: LBF responses to acetylcholine and exercise but not sodium nitroprusside were significantly (P < 0.05) attenuated in patients with diabetes compared with healthy control subjects. The percentage increase in LBF in response to exercise and acetylcholine were significantly correlated (r = 0.54, P = 0.02). Furthermore, resting plasma glucose was significantly related to the LBF response to exercise (r = -0.66, P = 0.003) independently of insulin, HbA(1c), lipids, BMI, and blood pressure. CONCLUSIONS: The increase in LBF during exercise is substantially attenuated in type 2 diabetic compared with matched control subjects. Impaired endothelium-dependent vasodilation secondary to elevated plasma glucose may underlie this observation. This mechanism may be of importance in determining the leg ischemic threshold in diabetic individuals with peripheral vascular disease.     

Angiotensin-converting enzyme inhibition improves venous endothelial dysfunction in chronic smokers

OBJECTIVE: In arteries and veins smoking is associated with impaired nitric oxide-mediated relaxation to endothelium-dependent agonists such as bradykinin. We investigated whether acute local angiotensin-converting enzyme (ACE) inhibition, achieved by enalaprilat, could influence bradykinin-induced vasodilation in veins of smokers. METHODS: We studied 7 smokers and 7 nonsmokers with the hand vein technique. After preconstriction with phenylephrine was performed, endothelium-dependent and independent relaxations were assessed by infusing bradykinin (1 to 278 ng/min) and sodium nitroprusside (0.0001 to 3166 ng/min), respectively. Dose-response curves were constructed before and during enalaprilat coinfusion (1 microg/min for 40 minutes). RESULTS: Smokers had impaired venodilation to bradykinin compared with nonsmokers (P < .01). Apparent maximal relaxation induced by bradykinin was 78%+/-9% in the control group and 48%+/-9% in smokers (mean +/- SD). ACE inhibition shifted the bradykinin dose-response curve to the left in both groups (P < .001) and was associated with a minimal increase in apparent maximal venodilation in nonsmokers (78%+/-9% to 83%+/-18%). In contrast, in smokers ACE inhibition augmented the magnitude of apparent maximal venodilation to values comparable to those observed in the control group (48%+/-9% to 102%+/-21%). In both groups the response to sodium nitroprusside was not affected by enalaprilat. CONCLUSIONS: This study shows that acute local ACE inhibition restores bradykinin-induced relaxation in smokers to values found in nonsmokers. This observation suggests that increased vascular metabolism of bradykinin exists in veins of smokers and that the vascular renin-angiotensin system may play a key role in smoking-induced endothelial dysfunction.     

Basal nitric oxide production is impaired in offspring of patients with essential hypertension.

There is considerable evidence that endothelium-dependent nitric oxide (NO)-mediated vasodilatation in response to acetylcholine is impaired in essential hypertension, whereas the endothelium-independent response to sodium nitroprusside is normal. More limited data have suggested that there is also reduced vasoconstriction in response to N(G)-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of basal NO release. As it is not known whether endothelial dysfunction in hypertension, if indeed present, is a cause or consequence of the condition, we have studied the normotensive offspring of parents with essential hypertension. Both basal and stimulated vascular responses were examined in 12 normotensive offspring [mean age (+/-S.E.M.) 26.1+/-1.4 years] of parents with essential hypertension and compared with those in 12 age-matched offspring (mean age 25.6+/-1.1 years) of normotensive subjects. Forearm blood flow was measured simultaneously in both arms by venous occlusion plethysmography, both at baseline and during intra-arterial brachial infusion of increasing doses of acetylcholine, sodium nitroprusside, noradrenaline and L-NMMA. There were no significant differences between the groups in the responses to acetylcholine, sodium nitroprusside or noradrenaline. In contrast, the vasoconstrictor response to L-NMMA was significantly blunted in the offspring of hypertensive parents compared with that in the offspring of normotensive parents (P=0.005). Thus endothelial dysfunction, as demonstrated by impaired basal production of NO, is present in subjects at high risk of essential hypertension, and does not occur simply as a consequence of the condition.     

Nitroglycerin-induced tolerance affects multiple sites in the organic nitrate bioconversion cascade

The effect of nitroglycerin (NTG)-induced tolerance on the spasmolytic effects of a series of vasodilators was determined to establish potential sites of tolerance. Concentration-effect curves to vasodilators were completed concurrently in U46619-contracted bovine isolated coronary artery rings pre-exposed to 100 microM NTG for 10 min (NTG-tolerant rings) and in control rings not pre-exposed to NTG. Compared to control rings, NTG-tolerant rings were markedly less responsive (P less than .01, n = 8-10) to the spasmolytic actions of NTG, isosorbide dinitrate, sodium nitroprusside (SNP) and 3-morpholinosydonimine (SIN-1), whereas the spasmolytic actions of S-nitroso-N-acetylpenicillamine and nitric oxide were only marginally attenuated in NTG-tolerant rings. On the other hand, no significant difference in the relaxant responses of NTG-tolerant and control coronary artery rings were observed to either the endothelium-dependent vasodilator, A23187 or the guanylate cyclase-independent vasodilator, theophylline. In additional cross-tolerance studies, relaxations to the organic nitrate vasodilator, NTG were significantly more attenuated (P less than .05, n = 5) by tolerance induced by NTG, than by either SNP or SIN-1, whereas the actions of the non-nitrate vasodilators, SNP and SIN-1 were attenuated more by SNP-and SIN-1-induced tolerance than by NTG-induced tolerance (P less than .05, n = 5). We conclude that, in this isolated coronary artery preparation, NTG-induced tolerance affects at least two major sites in the cascade of events between the initial site of NTG action and guanylate cyclase activation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of nitric oxide and noradrenergic activation in the posterior hypothalamus on arterial pressure tolerance to nitroglycerin in rats

The effects of nitric oxide (NO) and noradrenergic activation in the posterior hypothalamus on arterial pressure tolerance induced by subcutaneous injection of nitroglycerin (NTG) was investigated in anesthetized Sprague-Dawley rats. Intravenous injections of NTG (3, 10, and 30 microg/kg) and sodium nitroprusside (1, 3, and 10 microg/kg) produced dose-dependant decreases in arterial blood pressure. Tolerance to NTG was produced by subcutaneous administration of 4.0 mg of NTG as 4 separate hourly injections of 1.0 mg each, affecting the dose-dependent response of NTG IV injection. The 4 high-dose NTG pulse injections produced a marked shift in the dose-response curves for arterial pressure depression induced by intravenous injection of the challenge doses of NTG, but did not alter hypotensive responses to sodium nitroprusside. The tolerance responses to arterial pressure depression were enhanced by a bilateral microinjection of NTG (1 nmol) and by diethylamine NONOate (1 nmol), an NO donor, into the posterior hypothalamus. Bilateral microinjection of guanethidine (1.5 nmol), a noradrenergic blocker, into the posterior hypothalamus inhibits NTG tolerance in a period of time within 2 hours. We conclude that exogenous NO and noradrenergic activation in the posterior hypothalamus play an important role in arterial pressure tolerance to systemically administered NTG.     

The l-arginine — nitric oxide pathway in the canine femoral vascular bed: in vitro and in vivo experiments

Vascular endothelial cells synthesize nitric oxide from L-arginine, and this pathway can be inhibited by various analogues of L-arginine, including NG-nitro L-arginine methyl ester (L-NAME). To investigate the role of this pathway in the regulation of femoral arterial tone, the effect of L-NAME was studied in vitro in isolated canine femoral arteries suspended in organ chambers for isometric tension recording, and in vivo in conscious dogs chronically instrumented for the measurement of iliac blood flow and iliac artery diameter. In vitro, L-NAME induced an endothelium-dependent contraction, inhibited the endothelium-dependent relaxations to acetylcholine or bradykinin, and potentiated the relaxation evoked by the nitric oxide donor SIN-1. In vivo, locally administered L-NAME induced a decrease in iliac artery diameter and an increase in iliac resistance, potentiated the iliac responses to the organic nitrate nitroglycerin, but did not affect the iliac responses to the endothelium dependent vasodilator acetylcholine. Thus, in the canine femoral vascular bed: a) basal release of nitric oxide contributes in vivo to the maintenance of a permanent vasodilator tone at the level of both large conductance and small resistance vessels; b) the endothelium-dependent relaxations to acetylcholine and bradykinin in vitro are mostly mediated through the release of nitric oxide from L-arginine; c) the endothelium-dependent relaxations to acetylcholine in vivo are probably mediated by a relaxing factor distinct from nitric oxide, or by a nitric oxide-like molecule released from endothelial pools; and d) removal of the NO-mediated vasodilator tone by L-NAME leads to a supersensitivity to nitrovasodilators, both in vitro and in vivo.     

Insulin modulation of an endothelial nitric oxide component present in the alpha2- and beta-adrenergic responses in human forearm.

We explored in 51 normal subjects, distributed in various series of experiments, whether endothelium nitric oxide may play a role in insulin modulation of alpha2- and beta-adrenergic- evoked vascular responses. In particular, we examined the forearm blood flow response (FBF, ml.min-1.dl-1) to intrabrachial infusion of BHT-933 (0.5, 1, and 2 microg.min-1.dl-1) or isoproterenol (1, 3, and 6 ng. min-1.dl-1) in control conditions, during intrabrachial infusion of insulin alone (0.05 mU.kg-1.min-1) and associated with l-N-monomethylarginine (L-NMMA) (0.05 microg.min-1.dl-1), a nitric oxide synthase inhibitor. In control conditions both BHT-933 and isoproterenol induced a dose-dependent vascular response. Local hyperinsulinemia (deep venous plasma insulin 68.5+/-4 microU/ml) did not change basal FBF whereas attenuated BHT-933 vasoconstriction and enhanced isoproterenol vasodilation. L-NMMA reduced basal FBF and abolished the insulin effect on BHT-933 and isoproterenol response. To clarify whether a nitric oxide component is included in alpha2- and beta-adrenergic response and may be responsible for insulin vascular effect, we further examined BHT-933 and isoproterenol responses during nitric oxide inhibition. Interestingly, L-NMMA potentiated the BHT-933 vasoconstriction and attenuated the isoproterenol vasodilation and, in these conditions, insulin was no more able to exhibit its vascular effects. Finally, to rule out the possibility that the conteracting effect of L-NMMA may not be specifically related to insulin action, dose-response curves to phenylephrine (0.5, 1, and 2 microg.min-1.dl-1) or sodium nitroprusside (1, 2, and 4 microg.min-1.dl-1) were also performed. Both insulin and L-NMMA were unable to alter the phenylephrine-induced vasoconstriction and the sodium nitroprusside vasodilation. In conclusion, our data demonstrate an endothelial nitric oxide component in the alpha2- and beta-adrenergic vascular responses which is the target of the insulin vascular action.     

Heterogeneity of endothelium-dependent vasodilation in pressurized cerebral and small mesenteric resistance arteries of the rat.

We compared endothelial responses to calcium-mobilizing agents in mesenteric and cerebral resistance arteries of the rat. Middle cerebral and small mesenteric arteries were mounted in a pressure myograph, and myogenic responses were recorded. The effects of acetylcholine (ACh), bradykinin, substance P, histamine, A23187, cyclopiazonic acid (CPA), and sodium nitroprusside were investigated in both arteries with myogenic tone in the absence and presence of nitric oxide synthase and cyclooxygenase inhibitors. The effects of raised potassium, K(+) channel blockers, and arachidonic metabolism inhibition were examined on the nitric oxide (NO) synthase/cyclooxygenase inhibitor-resistant dilation induced by ACh and CPA. Cerebral arteries display a high level of myogenic reactivity compared with mesenteric arteries. In cerebral arteries, only bradykinin and substance P induced endothelium-dependent dilation. The observed dilation was solely related to the activation of the NO pathway. However, in mesenteric arteries, all of the vasoactive agents induced endothelium-dependent dilation. A combination of NO, cyclooxygenase-derived prostanoids, and a factor with endothelium-derived hyperpolarizing factor-like properties was responsible for the observed vasodilation. NO and cyclooxygenase derivatives were able to compensate for each other in the CPA-induced endothelium-dependent vasodilation when one of the two pathways was blocked. Moreover, small Ca(2+)-activated K(+) channels and a combination of both large and small Ca(2+)-activated K(+) channels were implicated in the endothelium-derived hyperpolarizing factor-like component of dilation to ACh and CPA, respectively. Finally, the results suggest that the pathway by which agonists raise intracellular calcium concentration may determine the nature of the endothelial secretory product.