Mechanism of dilation to reactive oxygen species in human coronary arterioles

We tested whether reactive oxygen species (ROS) generated from treatment with xanthine (XA) and xanthine oxidase (XO) alter vascular tone of human coronary arterioles (HCA). Fresh human coronary arterioles (HCA) from right atrial appendages were cannulated for video microscopy. ROS generated by XA (10(-4) M) + XO (10 mU/ml) dilated HCA (99 +/- 1%, 20 min after application of XA/XO). This dilation was not affected by denudation or superoxide dismutase (150 U/ml). Catalase (500 U/ml or 5,000 U/ml) attenuated the dilation early on, but a significant latent vasodilation appeared after 5 min peaking at 20 min (51 +/- 1%, 20 min after application of XA/XO + 500 U/ml catalase, P < 0.01 vs. control). KCl (40 mM) reduced the early and sustained vasodilation to XA/XO in the absence of catalase but 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 5 x 10(-5) M), diethyldithiocarbamate trihydrate (DDC, 10(-2) M), and deferoxamine (DFX, 10(-3) M) had no effect. In contrast, the catalase-resistant vasodilation was significantly attenuated by DDC, ODQ, and DFX as well as polyethylene-glycolated catalase (5,000 U/ml), but KCl had no effect. Confocal microscopy revealed that even in the presence of catalase, 2',7'-dichlorodihydrofluoresein diacetate fluorescence was observed in the vascular smooth muscle, but this was abolished by DDC. These data indicate that the exogenously generated superoxide anion (O2-*) by XA/XO is spontaneously converted to H2O2, which dilates HCA through vascular smooth muscle hyperpolarization. O2-* is also converted to H2O2 likely by superoxide dismustase within vascular cells and dilates HCA through a different pathway involving the activation of guanylate cyclase. These findings suggest that exogenously and endogenously produced H2O2 may elicit vasodilation by different mechanisms.     

Reactive oxygen species mediate arachidonic acid-induced dilation in porcine coronary microvessels

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.     

Preventing superoxide formation in epineurial arterioles of the sciatic nerve from diabetic rats restores endothelium-dependent vasodilation

We have previously reported that in streptozotocin-induced diabetic rats that increased formation of superoxide and peroxynitrite is associated with impairment in vascular relaxation in epineurial arterioles of the sciatic nerve. In this study we demonstrate that pretreating epineurial arterioles from diabetic rats in vitro with alpha-lipoic acid, dihydrolipoic acid, tempol or arginine restores acetylcholine-mediated vascular relaxation to near the reactivity observed in vessels from control rats. Suggesting that increased oxidative stress and reduction in nitric oxide availability is partially responsible for the impairment in endothelium-dependent vasodilation observed in epineurial arterioles from diabetic rats. In contrast, pretreating epineurial arterioles from diabetic rats with aminoguanidine or allopurinol had no effect. Studies designed to investigate the source of superoxide formation provided results suggesting that complex I of the mitochondrial electron transport chain and NAD(P)H oxidase are responsible for the increase in superoxide formation observed with epineurial arterioles from the sciatic nerve. Pretreating epineurial arterioles from diabetic rats with the protein kinase C inhibitor bisindolymaleimide I (GF 109203X) improved acetylcholine-mediated vascular relaxation but did not prevent the increase in superoxide formation suggesting that activation of protein kinase C by oxidative stress is downstream of superoxide formation. These studies imply that increased superoxide formation via the mitochondrial electron transport chain and perhaps NAD(P)H oxidase is partially responsible for reduced vascular reactivity observed in epineurial arterioles of the sciatic nerve from diabetic rats.     

Sensory Nerve Innervation of Epineurial Arterioles of the Sciatic Nerve Containing Calcitonin Gene–Related Peptide: Effect of Streptozotocin-Induced Diabetes

The authors have determined that epineurial arterioles of the sciatic nerve are innervated by nonadrenergic, noncholinergic nerves that contribute to the regulation of vasodilation. Using immunohistochemistry, the authors determined that nerves innervating epineurial arterioles contain the neuropeptide calcitonin gene–related peptide (CGRP). Using streptozotocin-induced diabetic rats, the authors demonstrated that CGRP content in sensory nerves innervating epineurial arterioles and vasodilation in response to exogenous CGRP was decreased. In summary, epineurial arterioles of the sciatic nerve are innervated by sensory nerves containing the neuropeptide CGRP. The diabetes-like condition induced by streptozotocin reduces the content of CGRP in these nerves and exogenous CGRPmediated vasodilation. CGRP is likely an important regulator of vascular tone and compromising its function could contribute to nerve ischemia and diabetic neuropathy.     

Differential contractile actions of reactive oxygen species on rat aorta: selective activation of ATP receptor by H2O2

This study aims to examine the effects of different reactive oxygen species (ROS) on the resting tension of endothelium-denuded rat aortic rings. In these preparations, H2O2 (30 microM) induced a fast and transient contraction, which could be abolished by pretreatment of catalase (800 U/ml), but not affected by superoxide anion scavenger, superoxide dismutase (SOD; 150 U/ml) or the hydroxyl free radical scavenger, DMSO/mannitol (each 3 mM). In contrast, pyrogallol, a putative superoxide anion donor, induced a biphasic contraction, which could be abolished by SOD, but not by catalase or DMSO/mannitol. Unlike H2O2 and pyrogallol, Vitamin C(VitC)/Fe2+ (each 100 microM), a commonly used hydroxyl radical-generating system, triggered a tonic contraction which could be prevented by DMSO/mannitol, but not by SOD or catalase. Interestingly, H2O2-induced contraction could be concentration-dependently (10-100 microM) inhibited by suramin and reactive blue-2 (RB-2), two widely used ATP receptor antagonists. On the other hand, suramin or RB-2, at concentration up to 100 microM, affected neither pyrogallol nor VitC/Fe2+-induced contraction. In conclusion, we showed for the first time that different ROS could contract rat aorta with different mechanisms of action, and H2O2 elicits a transient contraction probably as a result of the ATP receptor activation.     

Aging and estrogen alter endothelial reactivity to reactive oxygen species in coronary arterioles

Endothelium-dependent, nitric oxide (NO)-mediated vasodilation can be impaired by reactive oxygen species (ROS), and this deleterious effect of ROS on NO availability may increase with aging. Endothelial function declines rapidly after menopause, possibly because of loss of circulating estrogen and its antioxidant effects. The purpose of the current study was to determine the role of O(2)(-) and H(2)O(2) in regulating flow-induced dilation in coronary arterioles of young (6-mo) and aged (24-mo) intact, ovariectomized (OVX), or OVX + estrogen-treated (OVE) female Fischer 344 rats. Both aging and OVX reduced flow-induced NO production, whereas flow-induced H(2)O(2) production was not altered by age or estrogen status. Flow-induced vasodilation was evaluated before and after treatment with the superoxide dismutase (SOD) mimetic Tempol (100 μM) or the H(2)O(2) scavenger catalase (100 U/ml). Removal of H(2)O(2) with catalase reduced flow-induced dilation in all groups, whereas Tempol diminished vasodilation in intact and OVE, but not OVX, rats. Immunoblot analysis revealed elevated nitrotyrosine with aging and OVX. In young rats, OVX reduced SOD protein while OVE increased SOD in aged rats; catalase protein did not differ in any group. Collectively, these studies suggest that O(2)(-) and H(2)O(2) are critical components of flow-induced vasodilation in coronary arterioles from female rats; however, a chronic deficiency of O(2)(-) buffering by SOD contributes to impaired flow-induced dilation with aging and loss of estrogen. Furthermore, these data indicate that estrogen replacement restores O(2)(-) homeostasis and flow-induced dilation of coronary arterioles, even at an advanced age.     

Hypoxic constriction and reactive oxygen species in porcine distal pulmonary arteries

To determine whether reactive oxygen species (ROS) play an essential role in hypoxic pulmonary vasoconstriction (HPV) and the cellular locus of ROS production and action during HPV, we measured internal diameter (ID) at constant transmural pressure, lucigenin-derived chemiluminescence (LDCL), and electron paramagnetic resonance (EPR) spin adduct spectra in small distal porcine pulmonary arteries, and dichlorofluorescein (DCF) fluorescence in myocytes isolated from these arteries. Hypoxia (4% O2) decreased ID, increased DCF fluorescence, tended to increase LDCL, and in some preparations produced EPR spectra consistent with hydroxyl and alkyl radicals. Superoxide dismutase (SOD, 150 U/ml) or SOD + catalase (CAT, 200 U/ml) did not alter ID during normoxia but reduced or abolished the constriction induced by hypoxia. SOD also blocked HPV in endothelium-denuded arteries after restoration of the response by exposure to 10-10 M endothelin-1. Confocal fluorescence microscopy demonstrated that labeled SOD and CAT entered pulmonary arterial myocytes. SOD, SOD + CAT, and CAT blocked the increase in DCF fluorescence induced by hypoxia, but SOD + CAT and CAT also caused a stable increase in fluorescence during normoxia, suggesting that CAT diminished efflux of DCF from cells or oxidized the dye directly. We conclude that HPV required increased concentrations of ROS produced by and acting on pulmonary arterial smooth muscle rather than endothelium.     

Characterization of reactive oxygen species induced effects on human spermatozoa movement and energy metabolism

Reactive oxygen species (ROS) inhibit sperm movement and have been implicated in male infertility. In this study, we determined the effects of specific ROS produced by activated leukocytes on human spermatozoa and investigated their metabolic site of action. We used chemiluminescence and electron paramagnetic resonance (EPR) to characterize the ROS generated by both blood and seminal leukocytes. We also determined the effects of these ROS on sperm energy metabolism using biochemical analyses and flow cytometry. Both blood and seminal leukocytes produced the same characteristic ROS which were determined to be hydrogen peroxide (H2O2) and superoxide radicals (O2*-). EPR using the spin trapping technique indicated that superoxide radical-dependent hydroxyl radicals (HO.) were also generated. ROS generated by PMA-stimulated blood leukocytes (2-5 x 10(6)/ml) caused inhibition of sperm movement in 2 h (p < .01). Using the hypoxanthine/ xanthine oxidase (0.5 U/ml) system to generate ROS, we determined that spermatozoa ATP levels, after ROS treatment, were reduced approximately eight-fold in 30 min (0.10 x 10(10) moles/10(6) sperm cells) compared to control (0.84 X 10(-10) moles/10(6) sperm cells) (p < .01). Sperm ATP reduction paralleled the inhibition of sperm forward progression. Neither superoxide dismutase (100 U/ml) nor dimethyl sulfoxide (100 mM) reversed these effects; however, protection was observed with catalase (4 X 10(3) U/ml). Flow cytometric analyses of sperm treated with various doses of H2O2 (0.3 mM-20.0 mM) showed a dose-dependent decrease in sperm mitochondrial membrane potential (MMP); however, at low concentrations of H2O2, sperm MMP was not significantly inhibited. Also, sperm MMP uncoupling with CCClP had no effect on either sperm ATP levels or forward progression. These results indicate that H2O2 is the toxic ROS produced by activated leukocytes causing the inhibition of both sperm movement and ATP production. O2*- and HO. do not play a significant role in these processes. Low concentrations of H2O2 causing complete inhibition of sperm movement and ATP levels inhibit sperm energy metabolism at a site independent of mitochondrial oxidative phosphorylation.     

Reactive oxygen species promote tyrosine phosphorylation and capacitation in equine spermatozoa

The objective of this study was to examine the influence of reactive oxygen species (ROS) on equine sperm capacitation. Motile equine spermatozoa were separated on a discontinuous Percoll gradient, resuspended at 10 x 10(6)ml in Tyrode's medium supplemented with BSA (0.5%) and polyvinyl alcohol (0.5%) and incubated at 39 degrees C for 2h with or without the xanthine (X; 0.1mM)-xanthine oxidase (XO; 0.01 U/ml) system or NADPH (0.25 mM). The importance of hydrogen peroxide or superoxide for capacitation was determined by the addition of catalase (CAT; 150 U/ml) or superoxide dismutase (SOD; 150 U/ml), respectively. Following incubation, acrosomal exocytosis was induced by a 5 min incubation at 39 degrees C with progesterone (3.18 microM), and sperm viability and acrosomal integrity were then determined by staining with Hoechst 33258 and fluoroisothiocyanate-conjugated Pisum sativum agglutin. To examine tyrosine phosphorylation, treatments were subjected to sodium dodecyl sulfate-polyacrylaminde gel electrophoresis (SDS-PAGE) followed by Western blot analysis with the anti-phosphotyrosine antibody (alpha-PY; clone 4G10). Capacitation with the X-XO system or NADPH led to a significant (P<0.0001) increase in live acrosome-reacted spermatozoa compared to controls. The addition of CAT or SOD prevented the increase in live acrosome-reacted spermatozoa associated with X-XO treatment. Incubation with the X-XO system was also associated with a significant (P<0.005) increase in tyrosine phosphorylation when compared to controls, which could be prevented by the addition of CAT but not SOD. This study indicates that ROS can promote equine sperm capacitation and tyrosine phosphorylation, suggesting a physiological role for ROS generation by equine spermatozoa.     

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.     

Soluble epoxide hydrolase contamination of specific catalase preparations inhibits epoxyeicosatrienoic acid vasodilation of rat renal arterioles

Cytochrome P-450 metabolites of arachidonic acid, the epoxyeicosatrienoic acids (EETs) and hydrogen peroxide (H(2)O(2)), are important signaling molecules in the kidney. In renal arteries, EETs cause vasodilation whereas H(2)O(2) causes vasoconstriction. To determine the physiological contribution of H(2)O(2), catalase is used to inactivate H(2)O(2). However, the consequence of catalase action on EET vascular activity has not been determined. In rat renal afferent arterioles, 14,15-EET caused concentration-related dilations that were inhibited by Sigma bovine liver (SBL) catalase (1,000 U/ml) but not Calbiochem bovine liver (CBL) catalase (1,000 U/ml). SBL catalase inhibition was reversed by the soluble epoxide hydrolase (sEH) inhibitor tAUCB (1 μM). In 14,15-EET incubations, SBL catalase caused a concentration-related increase in a polar metabolite. Using mass spectrometry, the metabolite was identified as 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), the inactive sEH metabolite. 14,15-EET hydrolysis was not altered by the catalase inhibitor 3-amino-1,2,4-triazole (3-ATZ; 10-50 mM), but was abolished by the sEH inhibitor BIRD-0826 (1-10 μM). SBL catalase EET hydrolysis showed a regioisomer preference with greatest hydrolysis of 14,15-EET followed by 11,12-, 8,9- and 5,6-EET (V(max) = 0.54 ± 0.07, 0.23 ± 0.06, 0.18 ± 0.01 and 0.08 ± 0.02 ng DHET·U catalase(-1)·min(-1), respectively). Of five different catalase preparations assayed, EET hydrolysis was observed with two Sigma liver catalases. These preparations had low specific catalase activity and positive sEH expression. Mass spectrometric analysis of the SBL catalase identified peptide fragments matching bovine sEH. Collectively, these data indicate that catalase does not affect EET-mediated dilation of renal arterioles. However, some commercial catalase preparations are contaminated with sEH, and these contaminated preparations diminish the biological activity of H(2)O(2) and EETs.     

Differential serotonin responses in the skeletal muscle microcirculation

Closed circuit television microscopy was used to quantitate in vivo responses of small vessels in the rat cremaster muscle to topically applied serotonin. Sprague-Dawley rats were anesthetized with a combination of urethane (800 mg/kg) and alpha-chloralose (60 mg/kg). The cremaster muscle with intact circulation and innervation was suspended in a bath which had controlled pH, pCO2, and pO2. Microvascular diameters of first order arterioles and venules and fourth-order arterioles were measured from the television monitor while serotonin (10(-9)M-10(-4)M) was added to the bath. Fourth-order arterioles (3-11 micron diameter) dilated to a maximum of 267% of their control value with a serotonin concentration of 10(-6)M. Serotonin (10(-4)M) constricted first-order arterioles (78-121 micron) to 61% of their control value. The threshold concentration (10(-8)M) for a serotonin-induced dilation of fourth-order arterioles was 1000 fold less than the threshold concentration (10(-5)M) for serotonin-induced constriction of first-order arterioles. Serotonin (10(-8)M - 10(-4)M) did not alter the diameter of first-order venules (115-195 micron) from the control value. The dose-dependent constriction of first-order arterioles and dose-dependent dilation of fourth-order arterioles by serotonin appear to be independent of each other. In addition, the lack of constriction of first-order venules suggests a heterogenous distribution of serotonin receptors and that the predominate control mechanisms are different at different levels of the arteriolar and venous microcirculation of rat skeletal muscle.     

Improved Human Sperm Recovery Using Superoxide Dismutase and Catalase Supplementation in Semen Cryopreservation Procedure

The aim of this work was to evaluate the effects of ROS scavenger supplementation in human semen samples undergoing cryopreservation procedures.After screening out andrological pathologies, we selected 25 male partners of infertile couples with the following semen profile: volume >/= 2.0 ml, normal viscosity, sperm count >/=20 x 10(6)/ml, straight progressive motility (classes 1 and 2) >/= 40% (Mazzilli, Rossi, Delfino and Nofroni (1999) Andrologia 31: 187-194), atypical forms 相似文献   

Extracellular superoxide enhances 5-HT-induced murine pulmonary artery vasoconstriction

Accumulating evidence suggests that changes in both 5-hydroxytryptamine (5-HT) receptor activity and in the levels of reactive oxygen species (ROS) play an important role in regulating pulmonary artery (PA) vascular responsiveness, particularly in the setting of pulmonary hypertension. Therefore, we hypothesized that increased levels of superoxide enhance 5-HT-induced PA constriction. With the use of a small-vessel bioassay, 5-HT (0.01-10 microM) induced a concentration-dependent vasoconstriction in isolated wild-type murine intrapulmonary arteries (100-150 microm diameter) that was enhanced by both removal of the endothelium and by treatment with either N(G)-nitro-L-arginine methyl ester (30 microM) or xanthine (10 microM) + xanthine oxidase (0.005 U/ml). PA isolated from extracellular superoxide dismutase (EC-SOD) knockout mice also showed enhanced constriction. On the other hand, PA constriction to 5-HT was attenuated by either the addition of GR-127935 (0.1 microM, a selective inhibitor of 5-HT(1B/1D) receptor) or copper/zinc-containing superoxide dismutase (Cu/Zn SOD, 150 U/ml) and in PA isolated from transgenic mice overexpressing human EC-SOD. With the use of both oxidative fluorescent confocal microscopy and lucigenin-enhanced chemiluminescence, superoxide levels were increased significantly after 5-HT-induced PA vasoconstriction. This increase in superoxide levels could be blocked by the exogenous addition of Cu/Zn SOD (150 U/ml) or by apocynin (30 microM, an inhibitor of NADPH oxidase) but was not affected by gp91(phox) knockout mice. Overall, our results are consistent with 5-HT increasing vascular smooth muscle superoxide production via an NADPH oxidase pathway that is independent of gp91(phox), which leads to increases in extracellular superoxide levels, which in turn enhances 5-HT-induced murine pulmonary vasoconstriction.     

Arachidonic acid- and prostaglandin E2-induced cerebral vasodilation is mediated by carbon monoxide, independent of reactive oxygen species in piglets

Arachidonic acid (AA) and prostaglandin (PG) E(2) stimulate carbon monoxide (CO) production, and AA metabolism is known to be associated with the generation of reactive oxygen species (ROS). This study was conducted to address the hypothesis that CO and/or ROS mediate cerebrovascular dilation in newborn pigs. Experiments were performed on anesthetized newborn pigs with closed cranial windows. Different concentrations of AA (10(-8)-10(-6) M), PGE(2) (10(-8)-10(-6) M), iloprost (10(-8)-10(-6) M), and their vehicle (artificial cerebrospinal fluid) were given. Piglets with PGE(2) and iloprost received indomethacin (5 mg/kg iv) to inhibit cyclooxygenase. AA, PGE(2), and iloprost caused concentration-dependent increases in pial arteriolar diameter. The effects of both AA and PGE(2) in producing cerebral vascular dilation and associated CO production were blocked by the heme oxygenase inhibitor chromium mesoporphyrin (2 × 10(-5) M), but not by the prostacyclin analog, iloprost. ROS inhibitor tempol (SOD mimetic) (1 × 10(-5) M) and the H(2)O(2) scavenger catalase (1,000 U/ml) also do not block these vasodilator effects of AA and PGE(2). Heme-L-lysinate-induced cerebrovascular dilation and CO production was blocked by chromium mesoporphyrin. Hypoxanthine plus xanthine oxidase, a combination that is known to generate ROS, caused pial arteriolar dilation and CO production that was inhibited by tempol and catalase. These data suggest that AA- and PGE(2)-induced cerebral vascular dilation is mediated by CO, independent of ROS.     

Attenuation of vascular/neural dysfunction in Zucker rats treated with enalapril or rosuvastatin

Objective: Obese Zucker rats, animal model for the metabolic syndrome, develop a diabetes‐like neuropathy that is independent of hyperglycemia. The purpose of this study was to determine whether drugs used to treat cardiovascular dysfunction in metabolic syndrome also protect nerve function. Methods and Procedures: Obese Zucker rats at 20 weeks of age were treated for 12 weeks with enalapril or rosuvastatin. Lean rats were used as controls. Vasodilation in epineurial arterioles was measured by videomicroscopy. Endoneurial blood flow (EBF) was measured by hydrogen clearance and nerve conduction velocity was measured following electrical stimulation of motor or sensory nerves. Results: Enalapril treatment decreased serum angiotensin‐converting enzyme (ACE) activity and both drugs reduced serum cholesterol levels. In obese Zucker rats at 32 weeks of age superoxide levels were elevated in the aortas and epineurial arterioles, which were reduced by treatment with either drug. Nitrotyrosine levels were increased in epineurial arterioles and reduced with enalapril treatment. EBF was decreased and corrected by treatment with either drug. Motor nerve conduction velocity was decreased and significantly improved with enalapril treatment. Obese Zucker rats were hypoalgesic in response to a thermal stimulus and this was significantly improved with either treatment. Treatment with either enalapril or rosuvastatin significantly reversed the decrease in acetylcholine‐mediated vascular relaxation of epineurial arterioles in obese Zucker rats. Discussion: Even though obese Zucker rats have normal glycemia vascular and neural dysfunctions develop with age and can be improved by treatment with either enalapril or rosuvastatin.     

Effect of treatment of diabetic rats with dehydroepiandrosterone on vascular and neural function

Nutritional supplementation with dehydroepiandrosterone (DHEA) may be a candidate for treating diabetes-induced vascular and neural dysfunction. DHEA is a naturally occurring adrenal androgen that has antioxidant properties and is reportedly reduced in diabetes. Using a prevention protocol, we found that dietary supplementation of streptozotocin-induced diabetic rats with 0.1, 0.25, or 0.5% DHEA caused a concentration-dependent prevention in the development of motor nerve conduction velocity and endoneurial blood flow impairment, which are decreased in diabetes. At 0.25%, DHEA significantly prevented the diabetes-induced increase in serum thiobarbituric acid-reactive substances and sciatic nerve conjugated diene levels. This treatment also reduced the production of superoxide by epineurial arterioles of the sciatic nerve. DHEA treatment (0.25%) significantly improved vascular relaxation mediated by acetylcholine in epineurial vessels of diabetic rats. Sciatic nerve Na+-K+-ATPase activity and myoinositol content was also improved by DHEA treatment, whereas sorbitol and fructose content remained elevated. These studies suggest that DHEA, by preventing oxidative stress and perhaps improving sciatic nerve Na+-K+-ATPase activity, may improve vascular and neural dysfunction in diabetes.     

Chronic exposure to nicotine alters endothelium-dependent arteriolar dilatation: effect of superoxide dismutase.

The first goal of this study was to determine whether chronic injection of nicotine alters endothelium-dependent arteriolar dilatation. We measured the diameter of cheek pouch resistance arterioles (approximately 50 microm in diameter) in response to endothelium-dependent (acetylcholine and ADP) and -independent (nitroglycerin) agonists in control hamsters and hamsters treated with nicotine (2 microg. kg-1. day-1 for 2-3 wk). In control hamsters, acetylcholine (0.1 and 1.0 microM) dilated arterioles by 13 +/- 2 and 31 +/- 3%, respectively, and ADP (1.0 and 10 microM) dilated arterioles by 18 +/- 1 and 30 +/- 1%, respectively. In contrast, acetylcholine (0.1 and 1.0 microM) dilated arterioles by only 5 +/- 2 and 12 +/- 3%, respectively, and ADP (1.0 and 10 microM) dilated arterioles by only 7 +/- 2 and 13 +/- 3%, respectively, in animals treated with nicotine (P < 0.05 vs. response in control hamsters). Nitroglycerin produced similar dose-related dilatation of cheek pouch arterioles in control and nicotine-treated hamsters. Our second goal was to examine a possible mechanism for impaired endothelium-dependent arteriolar dilatation during chronic treatment with nicotine. We found that superfusion of the cheek pouch microcirculation with superoxide dismutase (150 U/ml) restored impaired endothelium-dependent, but did not alter endothelium-independent, arteriolar dilatation in hamsters treated with nicotine. Superfusion with superoxide dismutase did not alter endothelium-dependent or -independent arteriolar dilatation in control hamsters. We suggest that chronic exposure to nicotine produces selective impairment of endothelium-dependent arteriolar dilatation via a mechanism related to the synthesis/release of oxygen-derived free radicals.     

Inhibition of acetylcholine-induced EDHF response by elevated glucose in rat mesenteric artery

The effects of high glucose on endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxations of isolated rat mesenteric artery and the possible involvement of reactive oxygen species in these responses were investigated. After precontraction with phenylephrine (3 x 10(-8)-10(-7) M), acetylcholine (10(-8)-3 x 10(-6) M) and A 23187 (10(-8)-3 x 10(-6) M), a calcium ionophore, induced concentration-dependent relaxations in the presence of N(W)-nitro-l-arginine methyl ester (L-NAME) (10(-4) M) and indomethacin (10(-5) M). These relaxations were abolished in the presence of charybdotoxin (2 x 10(-7) M) plus apamin (10(-7) M) and were assumed to be mediated by EDHF. Effects of elevated glucose were examined by incubating the arterial rings for 6 h in Krebs-Henseleit solution containing 22.2 mM glucose. Under these conditions relaxation to acetylcholine was significantly attenuated but was unchanged when the tissues were incubated for 6 h in solution containing 11.1 mM mannitol used as hyperosmotic control. Addition of superoxide dismutase (SOD) (75 U/ml) and combination of SOD with catalase (200 U/ml) during incubation with high glucose significantly preserved the impairment of EDHF-mediated relaxations to acetylcholine. A 23187-induced endothelium-dependent relaxation was not affected by high glucose. Similarly, relaxations to pinacidil (10(-10)-10(-5) M) and to sodium nitroprusside (SNP) (10(-10)-3 x 10(-7) M) were also unchanged in the rings exposed to high glucose. These results suggest that in rat mesenteric arteries exposed to elevated glucose receptor-dependent EDHF-mediated relaxations (acetylcholine-induced) are impaired whereas receptor-independent ones (A 23187-induced) and responses to smooth muscle relaxants that exert their effects through mechanisms independent of endothelium are unaffected. Our findings lead us to propose that reactive oxygen species like superoxide ((.)O(2)(-)) and hydrogen peroxide (H(2)O(2)) do seem to play a role in the impairment of EDHF-mediated relaxations in the presence of elevated glucose.     

Analysis of purine- and pyrimidine-induced vascular responses in the isolated rat cerebral arteriole

Effects of extraluminal UTP were studied and compared with vascular responses to ATP and its analogs in rat cerebral-penetrating arterioles. UTP, UDP, 2-methylthio-ATP, and alpha,beta-methylene-ATP dilated arterioles at the lowest concentration and constricted them at high concentrations. Low concentrations of ATP dilated the vessels; high concentrations caused a biphasic response, with transient constriction followed by dilation. Endothelial impairment inhibited ATP- and UTP-mediated dilation and potentiated constriction to UTP but not to ATP. ATP- and 2-methylthio-ATP- but not UTP-mediated constrictions were inhibited by desensitization with 10(-6) M alpha,beta-methylene-ATP or 3 x 10(-6) M pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). PPADS at 10(-4) M abolished the UTP-mediated constriction and induced vasodilation in a dose-dependent manner but did not affect the dilation to ATP. These results suggest that in rat cerebral microvessels 1) ATP and 2-methylthio-ATP induce transient constriction via smooth muscle P(2X1) receptors in the cerebral arteriole, 2) UTP stimulates two different classes of P(2Y) receptors, resulting in constriction (smooth muscle P(2Y4)) and dilation (possibly endothelial P(2Y2)), and 3) ATP and UTP produce dilation by stimulation of a single receptor (P(2Y2)).