11,12-Epoxyeicosatrienoic acid induces vasodilator response in the rat perfused mesenteric vasculature.

Epoxyeicosatrienoic acids (EETs) are endogenous ligands that undergo hydrolysis by soluble epoxide hydrolase (sEH). The responses of 11, 12-EET in comparison with other vasodilator agonists including carbachol and sodium nitroprusside (SNP) were investigated. The effect of 1-cyclohexyl-3-dodecyl urea (CDU), a sEH, was tested on the vasodilator effect induced by 11, 12-EET in the perfused mesenteric beds isolated from normo-glycaemic and type-1 STZ-diabetic rats. In the perfused mesenteric beds of control and diabetic animals, 11, 12-EET produced vasodilation in a dose-dependent manner. The vasodilator response induced by 11, 12-EET was significantly decreased in tissues obtained from diabetic animals, but this was significantly corrected through inhibition of sEH. The effects of nitric oxide synthase inhibitor, cyclo-oxygenase inhibitor, specific potassium channel inhibitors, soluble guanylyl cyclase inhibitor and transient receptor potential channel V4 inhibitor, on vasodilator response to 11, 12-EET were investigated. In tissues isolated from control animals, vasodilator responses to 11, 12-EET were not inhibited by acute incubation with l-NAME, l-NAME with indomethacin, glibenclamide, iberiotoxin, charybdotoxin, apamin or ODQ. Incubation with the transient receptor potential channel V4 inhibitor ruthenium red caused significantly reduced vasodilator responses induced by 11, 12-EET. In conclusion, results from this study indicate that 11, 12-EET has a vasodilator effect in the perfused mesenteric bed, partly through activation of vanilloid receptor. A strategy to elevate the levels of EETs may have a significant impact in correcting microvascular abnormality associated with diabetes.

The role of tyrosine kinase-mediated pathways in diabetes-induced alterations in responsiveness of rat carotid artery.

1 G-protein-coupled receptor signalling, including transactivation of receptor tyrosine kinases (RTKs), has been implicated in vascular pathology. However, the role of specific RTKs in the development of diabetes-induced cardiovascular complications is not known. 2 We investigated the ability of a chronic administration of genistein, a broad-spectrum inhibitor of tyrosine kinases (TKs), AG1478, a specific inhibitor of epidermal growth factor receptor (EGFR) TK activity, and AG825, a specific inhibitor of Erb2, to modulate the altered vasoreactivity of isolated carotid artery ring segments to common vasoconstrictors and vasodilators in streptozotocin (STZ)-induced diabetes. 3 In diabetic carotid artery, the vasoconstrictor responses induced by noradrenaline (NE), endothelin-1 (ET-1), and angiotensin II (Ang II), were significantly increased whereas vasodilator responses to carbachol and histamine were significantly reduced. Inhibition of TKs, EGFR or Erb2 pathway did not affect the body weight or agonist-induced vasoconstrictor and vasodilator responses in the non-diabetic control animals. However, inhibition of TKs by genistein, EGFR TK by AG1478 or Erb2 by AG825 treatment produced a significant normalization of the altered agonist-induced vasoconstrictor responses without affecting blood glucose levels. Treatment with diadzein, an inactive analogue of genistein, did not affect the vasoconstrictor and vasodilator responses in the diabetic animals. 4 Treatment with genistein, AG1478 or AG825 resulted in a significant improvement in diabetes-induced impairment in endothelium-dependent relaxation to carbachol and histamine. 5 These data suggest that activation of TK-mediated pathways, including EGFR TK signalling and Erb2 pathway, are involved in the development of diabetic vascular dysfunction in the carotid artery.

Contribution of cytochrome P450 metabolites of arachidonic acid to hypertension and end-organ damage in spontaneously hypertensive rats treated with L-NAME.

1 The purpose of this study was to examine the effect of inhibition of the formation of cytochrome P450 metabolites of arachidonic acid with 1-aminobenzotriazole (ABT) on the development of hypertension and end-organ damage in spontaneously hypertensive rats (SHR) chronically treated with nitric oxide synthesis inhibitor L-NAME (SHR-L-NAME). 2 Administration of L-NAME in drinking water (80 mg l(-1)) to SHR for 3 weeks significantly elevated mean arterial blood pressure (MABP) (223 +/- 4 mmHg) as compared to SHR controls drinking regular water (165 +/- 3 mmHg). The administration of ABT (50 mg kg(-1) i.p. alt diem) for 6 days significantly attenuated elevation of blood pressure in SHR-L-NAME (204 +/- 4 mmHg). 3 L-NAME-induced increase in urine volume and protein was significantly lower in ABT-treated animals. 4 The impaired vascular responsiveness to noradrenaline and isoprenaline in the perfused mesenteric vascular bed of SHR-L-NAME-treated animals was significantly improved by ABT treatment. 5 Morphological studies of the kidneys and hearts showed that treatment with ABT minimized the extensive arterial fibrinoid necrosis, arterial thrombosis, significant narrowing of arterial lumen with marked arterial hyperplastic arterial changes that were observed in vehicle treated SHR-L-NAME. 6 In isolated perfused hearts, recovery of left ventricular function from 40 min of global ischaemia was significantly better in ABT-treated SHR-L-NAME. 7 These results suggest that in hypertensive individuals with endothelial dysfunction and chronic NO deficiency, inhibitors of 20-HETE synthesis may be able to attenuate development of high blood pressure and end-organ damage.

Endothelium-dependent relaxation in isolated renal arteries of diabetic rabbits.

1 In this study, we have investigated the vasodilator response to acetylcholine under diabetes conditions in isolated renal arteries of rabbits. We have also examined the contribution of endothelium-derived nitric oxide (EDNO) and endothelium-derived hyperpolarizing factor (EDHF) to the endothelium-dependent relaxation caused by acetylcholine in the renal arteries of alloxan-induced diabetic rabbits. 2 Acetylcholine (10(-10) - 10(-4) M) produced cumulative concentration-response curve in the renal arteries of both control and diabetic rabbits. The EC50 values and maximal responses to acetylcholine were not significantly different relative to diabetic conditions. In order to isolate the EDHF component of acetylcholine-induced vasodilator response, L-nitro-methyl arginine ester (L-NAME, 10(-4) M) and indomethacin (10(-6) M) were added to the Krebs' solution throughout the experiment. Under these conditions, acetylcholine induced vasodilatation in the isolated renal arteries from both control and diabetic rabbits. The vasodilator response to acetylcholine was not affected under diabetic conditions. 3 Sodium nitroprusside (SNP)-induced relaxation was increased in the diabetic rabbits compared with the control animals. 4 Tetrabutyl ammonium (TBA, 0.5 mM) produced a significant reduction in acetylcholine-induced vasodilatation in both preparations from control and diabetic animals, consistent with involvement of K+ channels in mediating this response. Glibenclamide (1 microM) attenuated acetylcholine-induced vasodilatation in preparations from control animals only, while iberiotoxin (0.05 microM) significantly reduced the vasodilator response to acetylcholine in preparations from both control and diabetic animals. 5 The role of EDNO in mediating acetylcholine-induced vasodilatation was examined. The vascular preparations were incubated with 20 mM K(+)-Krebs' solution to inhibit the EDHF contribution to acetylcholine-induced vasodilatation. Under this condition, acetylcholine induced a vasodilator response in both preparations from control and diabetic rats. Pretreatment with L-NAME (10(-4) M) attenuated acetylcholine-induced vasodilatation in both preparations, indicating an nitric oxide-mediated vasodilator response. 6 Our results indicated that acetylcholine-induced vasodilatation in the isolated renal arteries of alloxan-induced diabetic rabbits was not affected under diabetic conditions. Acetylcholine-induced vasodilatation is mediated by two vasodilator components; namely, EDHF and EDNO. The contribution of EDHF and EDNO to acetylcholine-induced vasodilatation was not affected under diabetic conditions and there was no indication of endothelial dysfunction associated with diabetes. EDHF component was found to act mainly through high conductance Ca(2+)-activated K+ channels under normal and diabetic conditions, while the adenosine triphosphate-dependent K+ channels were involved in mediating acetylcholine vasodilator response in the control preparations only.

Inhibition of calcium/calmodulin-dependent protein kinase II normalizes diabetes-induced abnormal vascular reactivity in the rat perfused mesenteric vascular bed.

1. Calcium/calmodulin-dependent protein kinase II (CaMKII) has an important function in mediating insulin release but its role in the development of diabetes-induced cardiovascular complications is not known. 2. We investigated the ability of a chronic administration of KN-93 (5 mg kg(-1) alt diem for 4 weeks), an inhibitor of CaMKII, to modulate the altered vasoreactivity of the perfused mesenteric bed to common vasoconstrictors and vasodilators in streptozotocin (STZ)-induced diabetes. 3. The vasoconstrictor responses induced by noradrenaline (NE), endothelin-1 (ET-1), and angiotensin II (Ang II), were significantly increased whereas, vasodilator responses to carbachol and histamine were significantly reduced in the perfused mesenteric bed of the STZ-diabetic rats as compared with non-diabetic controls. 4. Inhibition of CaMKII by KN-93 treatment did not affect blood glucose levels but produced a significant normalization of the altered agonist-induced vasoconstrictor and vasodilator responses. KN-93 did not affect agonist-induced responses in control animals. In addition, KN-93 significantly reduced weight loss in diabetic rats. 5. The present data suggest that CaMKII is an essential mediator in the development of diabetic vascular dysfunction and may also play an important role in signalling pathways leading to weight loss during diabetes.

Cytochrome P450 metabolites of arachidonic acid play a role in the enhanced cardiac dysfunction in diabetic rats following ischaemic reperfusion injury.

1 This study examined the contribution of cytochrome P450 metabolites of arachidonic acid in mediating ischaemia/reperfusion (I/R)-induced cardiac dysfunction in normal and diabetic rats. 2 We first compared the metabolism of arachidonic acid in microsomes prepared from the hearts of control rats and rats treated with streptozotocin (55 mg kg(-1)) to induce diabetes. The production of dihydroxyeicosatrienoic acids and epoxyeicosatrienoic acids (EETs) were similar in microsomes prepared from the hearts of control and diabetic rats, but the production of 20-hydroxyeicosatetraenoic acid (20-HETE) was two-fold higher in diabetic hearts than in control animals. 3 We then compared the change in left ventricular pressure (P(max)), left ventricular end-diastolic pressure, coronary flow and coronary vascular resistance in isolated perfused hearts obtained from control and diabetic animals after 40 min of global ischaemia (I) followed by 30 min of reperfusion (R). The decline in cardiac function was three- to five-fold greater in the hearts obtained from diabetic vs. control animals. 4 Pretreatment of the hearts with N-hydroxy-N'-(4-butyl-2-methyl-phenyl)-formamidine (HET0016, 1 microm), a selective inhibitor of the synthesis of 20-HETE, for 30 min before I/R resulted in significant improvement in the recovery of cardiac function in the hearts obtained from diabetic but not in control rats. Perfusion with an inhibitor of soluble epoxide hydrolase, 1-cyclohexyl-3-dodecyl urea (CDU), before I/R improved the recovery of cardiac function in hearts obtained from both control and diabetic animals. Perfusion with both HET0016 and CDU resulted in significantly better recovery of cardiac function of diabetic hearts following I/R than that seen using either drug alone. Pretreatment of the hearts with glibenclamide (1 microm), an inhibitor of ATP-sensitive potassium channels, attenuated the cardioprotective effects of both CDU and HET0016. 5 This is the first study to suggest that acute blockade of the formation of 20-HETE and/or reduced inactivation of EETs could be an important strategy to reduce cardiac dysfunction following I/R events in diabetes.

Role of 20-hydroxyeicosatetraenoic acid in altering vascular reactivity in diabetes.

1 This study examined the role of 20-hydroxyeicosatetraenoic (20-HETE) in altering vascular function in streptozotocin (STZ)-induced diabetic rats. 2 The expression of CYP4A protein and the formation of 20-HETE were elevated in the kidney, but not in the renal or mesenteric vasculature, of diabetic animals. The vasoconstrictor responses to norepinephrine (NE), endothelin-1 (ET-1), and angiotensin II (Ang II) were significantly enhanced in the isolated perfused mesenteric vascular bed and renal artery segments of diabetic rats. Chronic treatment of the diabetic rats with 1-aminobenzotriazole (ABT, 50 mg kg(-1) alt(-1) diem) or N-hydroxy-N'-(4-butyl-2-methylphenyl) formamidine (HET0016, 2.5 mg kg(-1) day(-1)) attenuated the responses to these vasoconstrictors in both vascular beds. 3 The synthesis of 20-HETE in renal microsomes was reduced by >80% confirming that the doses of ABT and HET0016 were sufficient to achieve system blockade. Addition of HET0016 (1 microM) in vitro also normalized the enhanced vascular responsiveness of renal and mesenteric vessels obtained from diabetic animals to NE and inhibited the formation of 20-HETE by >90% while having no effect on the formation of epoxides. Vasodilator responses to carbachol and histamine were reduced in the mesenteric vasculature, but not in renal arteries, of diabetic rats. Treatment of the diabetic animals with HET0016 improved vasodilator responses in both vascular beds. Vascular sensitivity to exogenous 20-HETE was elevated in the mesenteric bed of diabetic animals compared to controls. 4 These results suggest that 20-HETE contributes to the elevation in vascular reactivity in diabetic animals. This effect is not due to increased vascular expression of CYP4A but may be related to either enhanced agonist-induced release of substrate (arachidonic acid) by the CaMKII/Ras-GTPase system and/or elevated vascular responsiveness to 20-HETE by the CaMKII/Ras-GTPase system and/or elevated vascular responsiveness to 20-HETE.