Cardiovascular, metabolic, and coronary dysfunction in high-fat-fed obesity-resistant/prone rats.

OBJECTIVE: Obesity is a global epidemic leading to several comorbidities including diabetes and cardiovascular disease. The hypothesis that the genetic background of the obesity-prone rat (OP) predisposes to physiologic, metabolic, and microvascular dysfunction which is exacerbated by a diet high in saturated fats was tested. METHODS: Male OP and obesity-resistant (OR) rats were fed either a diet containing 10% (chow) or 45% kcal fat (HF) for 42 weeks. RESULTS: Weight of OP rats was greater than OR rats by 8 weeks on both diets. Blood pressure was increased in OP rats on chow and further augmented by HF diet compared to OR rats on similar diets. In contrast to weight and blood pressure, glucose clearance was similar in OR and OP rats on chow and impaired in both models on HF diet. Relaxation to acetylcholine was attenuated in OP rats compared to OR rats by 8 weeks and remained reduced throughout the study. A longer period of time was required to observe vascular dysfunction in HF-fed OR rats. CONCLUSIONS: When compared to OR rats, OP rats are prone to develop not only greater obesity but also hypertension and vascular dysfunction on a normal diet which is further augmented with HF diet.

Characterization of diabetic neuropathy in the Zucker diabetic Sprague-Dawley rat: a new animal model for type 2 diabetes.

Recently a new rat model for type 2 diabetes the Zucker diabetic Sprague-Dawley (ZDSD/Pco) was created. In this study we sought to characterize the development of diabetic neuropathy in ZDSD rats using age-matched Sprague-Dawley rats as a control. Rats were examined at 34 weeks of age 12 weeks after the onset of hyperglycemia in ZDSD rats. At this time ZDSD rats were severely insulin resistant with slowing of both motor and sensory nerve conduction velocities. ZDSD rats also had fatty livers, elevated serum free fatty acids, triglycerides, and cholesterol, and elevated sciatic nerve nitrotyrosine levels. The corneas of ZDSD rats exhibited a decrease in subbasal epithelial corneal nerves and sensitivity. ZDSD rats were hypoalgesic but intraepidermal nerve fibers in the skin of the hindpaw were normal compared to Sprague-Dawley rats. However, the number of Langerhans cells was decreased. Vascular reactivity of epineurial arterioles, blood vessels that provide circulation to the sciatic nerve, to acetylcholine and calcitonin gene-related peptide was impaired in ZDSD rats. These data indicate that ZDSD rats develop many of the neural complications associated with type 2 diabetes and are a good animal model for preclinical investigations of drug development for diabetic neuropathy.

Progression and reversal of coronary and mesenteric vascular dysfunction associated with obesity.

OBJECTIVE: The purpose of this study was to examine progression and reversal of microvascular complications when rats were fed a high fat diet. METHODS: Sprague-Dawley rats 10 weeks of age were fed a diet containing 45% kcal fat for up to 32 weeks. Blood pressure and heart rate was measured by telemetry. Vascular reactivity of aorta and small coronary and mesenteric vessels was determined after 8, 16, 24, and 32 weeks on diet. RESULTS: There was a modest increase in weight and blood pressure in high fat fed rats. Sodium nitroprusside (SNP)-induced relaxation of coronary arteries was potentiated after 8 weeks on high fat diet, however, this enhanced response was not observed after 16, 24, or 32 weeks of diet. Acetylcholine (Ach) mediated relaxation was attenuated after 16, 24, and 32 weeks of high fat diet in coronary arteries; however, in aorta and mesenteric arteries, Ach-mediated response was not altered until 32 weeks on high fat diet. Reversing the high fat diet for 8 weeks resulted in partial recovery of metabolic parameters; however endothelial function in coronary arteries remained impaired. CONCLUSIONS: These studies indicate that high fat diet promotes progressive impairment of coronary vascular function that is difficult to reverse.

Dietary fat, fatty acid saturation and mitochondrial bioenergetics.

Fat intake alters mitochondrial lipid composition which can affect function. We used novel methodology to assess bioenergetics, including simultaneous ATP and reactive oxygen species (ROS) production, in liver and heart mitochondria of C57BL/6 mice fed diets of variant fatty acid content and saturation. Our methodology allowed us to clamp ADP concentration and membrane potential (ΔΨ) at fixed levels. Mice received a control diet for 17­19 weeks, a high-fat (HF) diet (60% lard) for 17­19 weeks, or HF for 12 weeks followed by 6­7 weeks of HF with 50% of fat as menhaden oil (MO) which is rich in n-3 fatty acids. ATP production was determined as conversion of 2-deoxyglucose to 2-deoxyglucose phosphate by NMR spectroscopy. Respiration and ATP production were significantly reduced at all levels of ADP and resultant clamped ΔΨ in liver mitochondria from mice fed HF compared to controls. At given ΔΨ, ROS production per mg mitochondrial protein, per unit respiration, or per ATP generated were greater for liver mitochondria of HF-fed mice compared to control or MO-fed mice. Moreover, these ROS metrics began to increase at a lower ΔΨ threshold. Similar, but less marked, changes were observed in heart mitochondria of HF-fed mice compared to controls. No changes in mitochondrial bioenergetics were observed in studies of separate mice fed HF versus control for only 12 weeks. In summary, HF feeding of sufficient duration impairs mitochondrial bioenergetics and is associated with a greater ROS "cost" of ATP production compared to controls. These effects are, in part, mitigated by MO.

Role of the effect of inhibition of neutral endopeptidase on vascular and neural complications in streptozotocin-induced diabetic rats.

We have previously shown that treating streptozotocin-induced diabetic rats, an animal model of type 1 diabetes, with Ilepatril (an inhibitor of neutral endopeptidase and angiotensin converting enzyme (ACE)) improves vascular and neural function. In this study we sought to determine the individual effect of inhibition of neutral endopeptidase and ACE on diabetes-induced vascular and neural dysfunction. After 4 weeks of untreated diabetes, rats were treated for 12 weeks with Ilepatril, Enalapril (ACE inhibitor) or Candoxatril (neutral endopeptidase inhibitor) followed by analysis of neural and vascular function. Diabetes caused slowing of motor and sensory nerve conduction, thermal hypoalgesia, reduction in intraepidermal nerve fiber density in the hindpaw and impairment in vascular relaxation to acetylcholine and calcitonin gene-related peptide in epineural arterioles of the sciatic nerve and to atrial natriuretic peptide and calcitonin gene-related peptide in renal arteries. Inhibition of neutral endopeptidase or ACE improved neural function; however, dual inhibition of neutral endopeptidase and ACE with Ilepatril tended to have the greatest efficacy. Ilepatril and Candoxatril treatment of diabetic rats was more efficacious in improving vascular responsiveness in epineurial arterioles than treatment with Enalapril. Ilepatril, Enalapril or Candoxatril treatment of diabetic rats were all efficacious in renal arteries. These studies suggest that combination therapy may be the most effective approach for treatment of diabetic neural and vascular complications.

Diet-induced obesity in Sprague-Dawley rats causes microvascular and neural dysfunction.

BACKGROUND: The objective of this study was to determine the effect of diet-induced obesity (DIO) on microvascular and neural function. METHODS: Rats were fed a standard or high fat diet for up to 32 weeks. The following measurements were carried out: vasodilation in epineurial arterioles using videomicroscopy, endoneurial blood flow using hydrogen clearance, nerve conduction velocity using electrical stimulation, size-frequency distribution of myelinated fibres of the sciatic nerve, intraepidermal nerve fibre density using confocal microscopy and thermal nociception using the Hargreaves method. RESULTS: Rats fed a high fat diet for 32 weeks developed sensory neuropathy, as indicated by slowing of sensory nerve conduction velocity and thermal hypoalgesia. Motor nerve conduction velocity and endoneurial blood flow were not impaired. Mean axonal diameter of myelinated fibres of the sciatic nerve was unchanged in high fat-fed rats compared with that in control. Intraepidermal nerve fibre density was significantly reduced in high fat-fed rats. Vascular relaxation to acetylcholine and calcitonin gene-related peptide was decreased and expression of neutral endopeptidase (NEP) increased in epineurial arterioles of rats fed a high fat diet. In contrast, insulin-mediated vascular relaxation was increased in epineurial arterioles. NEP activity was significantly increased in the skin of the hindpaw. Markers of oxidative stress were increased in the aorta and serum of high fat-fed rats but not in epineurial arterioles. CONCLUSION: Chronic obesity causes microvascular and neural dysfunction. This is associated with increased expression of NEP but not oxidative stress in epineurial arterioles. NEP degrades vasoactive peptides, which may explain the decrease in microvascular function.

Vascular and neural dysfunctions in obese Zucker rats: effect of AVE7688.

The purpose of this study was to determine whether AVE7688 a drug that inhibits both angiotensin converting enzyme and neutral endopeptidase activity protects vascular and nerve functions in an animal model of metabolic syndrome. Obese Zucker rats at 20 weeks of age were treated for 12 weeks with AVE7688. Vasodilation in epineurial arterioles was measured by videomicroscopy and nerve conduction velocity was measured following electrical stimulation. Treatment with AVE7688 improved vascular relaxation in response to acetylcholine and motor and sensory nerve conduction velocity. In obese Zucker rats superoxide levels and nitrotyrosine staining were elevated in the aorta and treatment corrected both conditions. Obese Zucker rats were hypoalgesic in response to a thermal stimulus and demonstrated signs of impaired tactile response and both conditions were significantly improved with treatment. Even though obese Zucker rats are normoglycemic vascular and neural dysfunctions develop with age and can be improved by treatment with AVE7688.

Treatment of cardiovascular dysfunction associated with the metabolic syndrome and type 2 diabetes.

UNLABELLED: Our previous studies have shown vascular dysfunction in small coronary and mesenteric arteries in Zucker obese rats, a model of the metabolic syndrome, and Zucker Diabetic Fatty (ZDF) rats, a model of type 2 diabetes. Because of their lipid lowering action and antioxidant activity, we predicted that treatment with Rosuvastatin, an HMG-CoA reductase inhibitor (statin) or Enalapril, an angiotensin converting enzyme (ACE) inhibitor would improve vascular dysfunction associated with the metabolic syndrome and type 2 diabetes. METHODS: 20-week-old Zucker obese and 16-week-old ZDF rats were treated with Rosuvastatin (25 mg/kg/day) or Enalapril (20 mg/kg/day) for 12 weeks. We examined metabolic parameters, indices of oxidative stress and vascular dysfunction in ventricular and mesenteric small arteries (75-175 microm intraluminal diameter) from lean, Zucker obese and ZDF rats (untreated and treated). RESULTS: Endothelial dependent responses were attenuated in coronary vessels from Zucker obese and ZDF rats compared to responses from lean rats. Both drugs improved metabolic parameters, oxidative stress, and vascular dysfunction in Zucker obese rats, however, only partial improvement was observed in ZDF rats, suggesting more aggressive treatment is needed when hyperglycemia is involved. CONCLUSION: Vascular dysfunction is improved when Zucker obese and, to a lesser degree, when ZDF rats were treated with Rosuvastatin or Enalapril.

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.

Role of nitrosative stress in early neuropathy and vascular dysfunction in streptozotocin-diabetic rats.

Evidence for important roles of the highly reactive oxidant peroxynitrite in diabetic complications is emerging. We evaluated the role of peroxynitrite in early peripheral neuropathy and vascular dysfunction in STZ-diabetic rats. In the first dose-finding study, control and STZ-diabetic rats were maintained with or without the potent peroxynitrite decomposition catalyst Fe(III)tetrakis-2-(N-triethylene glycol monomethyl ether) pyridyl porphyrin (FP15) at 3, 5, or 10 mg.kg(-1).day(-1) in the drinking water for 4 wk after an initial 2 wk without treatment for assessment of early neuropathy. In the second study with similar experimental design, control and STZ-diabetic rats were maintained with or without FP15, 5 mg.kg(-1).day(-1), for vascular studies. Rats with 6-wk duration of diabetes developed motor and sensory nerve conduction velocity deficits, mechanical hyperalgesia, and tactile allodynia in the absence of small sensory nerve fiber degeneration. They also had increased nitrotyrosine and poly(ADP-ribose) immunofluorescence in the sciatic nerve and dorsal root ganglia. All these variables were dose-dependently corrected by FP15, with minimal differences between the 5 and 10 mg.kg(-1).day(-1) doses. FP15, 5 mg.kg(-1).day(-1), also corrected endoneurial nutritive blood flow and nitrotyrosine, but not superoxide, fluorescence in aorta and epineurial arterioles. Diabetes-induced decreases in acetylcholine-mediated relaxation by epineurial arterioles and coronary and mesenteric arteries, as well as bradykinin-induced relaxation by coronary and mesenteric arteries, were alleviated by FP15 treatment. The findings reveal the important role of nitrosative stress in early neuropathy and vasculopathy and provide the rationale for further studies of peroxynitrite decomposition catalysts in long-term diabetic models.

Treatment of streptozotocin-induced diabetic rats with AVE7688, a vasopeptidase inhibitor: effect on vascular and neural disease.

In epineurial arterioles, acetylcholine-mediated vascular relaxation is mediated by nitric oxide and endothelium-derived hyperpolarizing factor (EDHF), and both mechanisms are impaired by diabetes. The mediator responsible for the effect of EDHF is unknown. In epineurial arterioles, C-type natriuretic peptide (CNP) has properties consistent with EDHF-like activity. Epineurial arterioles express CNP, and exogenous CNP causes a concentration-dependent vascular relaxation. In streptozotocin-induced diabetic rats, CNP-mediated vascular relaxation in epineurial arterioles is decreased. Since CNP may be a regulator of vascular function, a vasopeptidase inhibitor may be an effective treatment for diabetes-induced vascular and neural disease. Vasopeptidase inhibitors inhibit ACE activity and neutral endopeptidase, which degrades natriuretic peptides. Streptozotocin-induced diabetic rats were treated with AVE7688 (450 mg/kg in the diet), a vasopeptidase inhibitor, for 8-10 weeks after 4 weeks of untreated diabetes. Treatment of diabetic rats corrected the diabetes-induced decrease in endoneurial blood flow, significantly improved motor and sensory nerve conduction velocity, prevented the development of hypoalgesia in the hind paw, and reduced superoxide and nitrotyrosine levels in epineurial arterioles. The diabetes-induced decrease in acetylcholine-mediated vascular relaxation by epineurial arterioles was significantly improved with treatment. These studies suggest that vasopeptidase inhibitors may be an effective approach for the treatment of diabetic vascular and neural dysfunction.

Progression of coronary and mesenteric vascular dysfunction in Zucker obese and Zucker diabetic fatty rats.

We investigated the progression of vascular dysfunction associated with the metabolic syndrome with and without hyperglycemia in lean, Zucker obese, and Zucker diabetic fatty (ZDF) rats. Responses of aorta and small coronary and mesenteric arteries were measured to endothelium-dependent and -independent vasodilators. Indices of oxidative stress were increased in serum from ZDF rats throughout the study, whereas values were increased in Zucker obese rats later in the study [thiobarbituric acid reactive substances: 0.45 +/- 0.02, 0.59 +/- 0.03 (P < 0.05), and 0.58 +/- 0.03 (P < 0.05) mug/ml in serum from 28- to 40-wk-old lean, Zucker obese, and ZDF rats, respectively]. Acetylcholine (ACh)-induced relaxation was not altered in vessels from lean animals from 8-40 wk. ACh-induced relaxation was nearly abolished in coronary arteries from 28- to 36-wk-old Zucker obese rats and by 16-36 wk in ZDF rats and was attenuated in aorta and mesenteric vessels from ZDF rats [%relaxation to 10 muM ACh: 72.2 +/- 7.1, 17.9 +/- 5.9 (P < 0.05), and 23.0 +/- 4.5 (P < 0.05) in coronary vessels; and 67.9 +/- 9.2, 50.1 +/- 5.5, and 42.3 +/- 4.7 (P < 0.05) in mesenteric vessels from 28- to 40-wk-old lean, Zucker obese, and ZDF rats, respectively]. The attenuated ACh-induced relaxation was improved when vessels were incubated with tiron, suggesting superoxide as a mechanism of endothelial dysfunction. Sodium nitroprusside-induced relaxation was not altered in aorta or coronary arteries and was potentiated in mesenteric arteries from Zucker obese rats. Our data suggest that diabetes enhances the progression of vascular dysfunction. Increases in indices of oxidative stress precede the development of dysfunction and may serve as a marker of endothelial damage.

ACE inhibitor or angiotensin II receptor antagonist attenuates diabetic neuropathy in streptozotocin-induced diabetic rats.

ACE inhibition and/or blocking of the angiotensin II receptor are recognized as first-line treatment for nephropathy and cardiovascular disease in diabetic patients. However, little information is available about the potential benefits of these drugs on diabetic neuropathy. We examined vascular and neural activity in streptozotocin-induced diabetic rats that were treated for 12 weeks with enalapril, an ACE inhibitor, or L-158809, an angiotensin II receptor blocker. A prevention protocol (group 1) as well as three intervention protocols (treatment was initiated after 4, 8, or 12 weeks of diabetes [groups 2, 3, and 4, respectively]) were used. Endoneurial blood flow and motor nerve conduction velocity (MNCV) were impaired in all groups of untreated diabetic rats. In group 1, treatment of diabetic rats with enalapril or L-158809 partially prevented the diabetes-induced decrease in endoneurial blood flow and MNCV. In groups 2-4, intervention with enalapril was more effective in reversing the diabetes-induced impairment in endoneurial blood flow and MNCV than L-158809. The superoxide level in the aorta and epineurial arterioles of diabetic rats was increased. Treatment of diabetic rats with enalapril or L-158809 reduced the superoxide level in the aorta in all groups but was less effective in epineurial arterioles. Acetylcholine and calcitonin gene-related peptide (CGRP) cause vasodilation in epineurial arterioles of the sciatic nerve, which was impaired by diabetes. Treatment of diabetic rats (all groups) with enalapril or L-158809 completely prevented/reversed the diabetes-induced impairment in CGRP-mediated vascular relaxation. Treatment with enalapril or L-158809 was also effective in improving impaired acetylcholine-mediated vasodilation, but the efficacy was diminished from groups 1 to 4. These studies suggest that ACE inhibitors and/or angiotensin II receptor blockers may be effective treatments for diabetes and vascular and neural dysfunction. However, the efficacy of these treatments may be dependent on when the treatment is initiated.

Progression of vascular and neural dysfunction in sciatic nerves of Zucker diabetic fatty and Zucker rats.

We have examined the progression of vascular and neural deficits in Zucker rats, Zucker diabetic fatty (ZDF) diabetic rats, and age-matched lean ZDF rats from 8 to 40 wk of age. Both the ZDF diabetic and Zucker rats were glucose intolerant at 8 wk of age. The Zucker rats did not become hyperglycemic but were hyperinsulinemic through 32 wk of age. All ZDF diabetic rats became hyperglycemic by 8 wk of age. Through their life span, serum free fatty acids and triglycerides levels were significantly higher in Zucker and ZDF diabetic rats compared with age-matched lean ZDF rats. After 24 and 28 wk of age, endoneurial blood flow was significantly decreased in ZDF diabetic and Zucker rats. Motor nerve conduction velocity was significantly decreased after 12-14 wk of age in ZDF diabetic rats and at 32 wk of age in Zucker rats. ACh-mediated vascular relaxation of epineurial arterioles of the sciatic nerve was impaired after 8-10 wk of age in ZDF diabetic rats and after approximately 16 wk of age in Zucker rats. In contrast, vascular relaxation mediated by calcitonin gene-related peptide was impaired significantly after 28 wk of age in ZDF diabetic rats but not impaired in Zucker rats up to 40 wk of age. Markers of oxidative stress were differentially elevated in ZDF diabetic rats and Zucker rats. These data indicate that vascular and neural dysfunction develops in both Zucker and ZDF diabetic rats but at different rates, which may be the result of hyperglycemia.

20-hydroxyeicosatetraenoic acid (20-HETE) metabolism in coronary endothelial cells.

We have investigated the role of endothelial cells in the metabolism of 20-hydroxyeicosatetraenoic acid (20-HETE), a vasoactive mediator synthesized from arachidonic acid by cytochrome P450 omega-oxidases. Porcine coronary artery endothelial cells (PCEC) incorporated 20-[(3)H]HETE primarily into the sn-2 position of phospholipids through a coenzyme A-dependent process. The incorporation was reduced by equimolar amounts of arachidonic, eicosapentaenoic or 8,9-epoxyeicosatrienoic acids, but some uptake persisted even when a 10-fold excess of arachidonic acid was available. The retention of 20-[(3)H]HETE increased substantially when methyl arachidonoyl fluorophosphonate, but not bromoenol lactone, was added, suggesting that a Ca(2+)-dependent cytosolic phospholipase A(2) released the 20-HETE contained in PCEC phospholipids. Addition of calcium ionophore A23187 produced a rapid release of 20-[(3)H]HETE from the PCEC, a finding that also is consistent with a Ca(2+)-dependent mobilization process. PCEC also converted 20-[(3)H]HETE to 20-carboxy-arachidonic acid (20-COOH-AA) and 18-, 16-, and 14-carbon beta-oxidation products. 20-COOH-AA produced vasodilation in porcine coronary arterioles, but 20-HETE was inactive. These results suggest that the incorporation of 20-HETE and its subsequent conversion to 20-COOH-AA in the endothelium may be important in modulating coronary vascular function.

Coronary vascular dysfunction associated with direct current shock injury.

OBJECTIVE: To determine if cardiac injury following DC shocks includes impairment of coronary vascular reactivity. METHODS: 36 dogs (18-32 kg) were anesthetized and a thoracotomy was performed. Either antioxidant enzymes, superoxide dismutase (SOD, 15,000 U/kg) plus catalase (55,000 U/kg) or the NO synthase inhibitor N(G)-nitro-L-arginine (L-NNA, 5 mg/kg) was administered IV prior to sham (no shocks) or DC shock treatment, and the results were compared to dogs which did not receive SOD/catalase or L-NNA. In sham dogs, electrodes cradled the heart, but no shocks were delivered. In shock dogs, three 20 Joule DC shocks were delivered to the epicardium using hand-held paddles. Other dogs were allowed a 3-hour recovery period after the shocks. Epicardial microvessels and conduit rings were studied in vitro. Antagonists were not added to the bath of the study vessel. Internal diameter was measured in microvessels after constriction with endothelin. Tension of conduit arteries was measured after constriction with PGF(2alpha). Responses to acetylcholine (Ach, 10(-10)-10(-4) M), bradykinin (10(-14)-10(-6) M), the calcium ionophore A23187 (A23187, 10(-12)-10(-4) M) or nitroprusside (SNP, 10(-10)-10(-4) M) were measured. RESULTS: Bradykinin, A23187 and SNP dependent dilation was not different between vessels from sham and shocked animals. Dilation to Ach was attenuated in vessels from shocked dogs. Superoxide production probably contributed to the impaired dilation to Ach since treatment with SOD/catalase improved dilation. Treatment with L-NNA also improved vascular function after DC shock. CONCLUSION: DC shocks cause endothelial dysfunction, as demonstrated by impaired dilation to acetylcholine, in both canine coronary microvascular and conduit arteries. Since pretreatment with either SOD/catalase or L-NNA protects against this damage, a free radical mechanism, possibly involving eNOS, may contribute to endothelial dysfunction.DC shocks for cardioversion and defibrillation cause myocardial injury that may be free radical mediated.

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.

Human coronary endothelial cells convert 14,15-EET to a biologically active chain-shortened epoxide.

Cytochrome P-450 epoxygenase-derived epoxyeicosatrienoic acids (EETs) play an important role in the regulation of vascular reactivity and function. Conversion to the corresponding dihydroxyeicosatrienoic acids (DHETs) by soluble epoxide hydrolases is thought to be the major pathway of EET metabolism in mammalian vascular cells. However, when human coronary artery endothelial cells (HCEC) were incubated with (3)H-labeled 14,15-EET, chain-shortened epoxy fatty acids, rather than DHET, were the most abundant metabolites. After 4 h of incubation, 23% of the total radioactivity remaining in the medium was converted to 10,11-epoxy-hexadecadienoic acid (16:2), a product formed from 14,15-EET by two cycles of beta-oxidation, whereas only 15% was present as 14,15-DHET. Although abundantly present in the medium, 10,11-epoxy-16:2 was not detected in the cell lipids. Exogenously applied (3)H-labeled 10,11-epoxy-16:2 was neither metabolized nor retained in the cells, suggesting that 10,11-epoxy-16:2 is a major product of 14,15-EET metabolism in HCEC. 10,11-Epoxy-16:2 produced potent dilation in coronary microvessels. 10,11-Epoxy-16:2 also potently inhibited tumor necrosis factor-alpha-induced production of IL-8, a proinflammatory cytokine, by HCEC. These findings implicate beta-oxidation as a major pathway of 14,15-EET metabolism in HCEC and provide the first evidence that EET-derived chain-shortened epoxy fatty acids are biologically active.