Synthesis Of Endothelium-derived Hyperpolarizing Factor Research Articles

Abstract 1134: Crucial Role of Cu/Zn-SOD in the Synthesis of Endothelium-Derived Hyperpolarizing Factor (EDHF) during Reactive Hyperemia in Mouse Mesenteric Microcirculation in Vivo

Background: We have recently demonstrated that endothelial Cu/Zn-superoxide dismutase (SOD) plays an important role as an EDHF synthase. In this study, we examined the possible role of Cu/Zn-SOD in the synthesis of EDHF/H 2 O 2 during reactive hyperemia (RH) in mouse mesenteric microcirculation in vivo. Methods: Mesenteric arterioles from wild-type (wild) and Cu/Zn-SOD −/− mice (SOD-KO) (n=5 each, 22– 60 μm) were continuously observed with a pencil-typed intravital microscope (×600 magnification) during acetylcholine (ACh)-induced vasodilatation and RH (reperfusion after 60 sec of mesenteric artery occlusion) under cyclooxygenase blockade (indomethacin) under the following 3 conditions; control, NO synthase inhibitor (L-NMMA) and L-NMMA+catalase (a specific decomposer of H 2 O 2 ). Results: In mesenteric arterioles of wild-type mice, endothelium-dependent relaxations to ACh (34±4%) and vasodilatation during RH (40±5%) were resistant to L-NMMA (ACh 23±7% and RH 20±5%, both P<0.05), but were markedly inhibited by L-NMMA+catalase (ACh, 8±2% and RH, 4±3%, both P<0.01 vs. control). In mesenteric arterioles of SOD-KO mice, L-NMMA markedly inhibited the relaxations to ACh (30±4% to 8±2%, P<0.01) and vasodilatation during RH (40±5% to 5±2%, P<0.01), and the responses were not inhibited by the addition of catalase (ACh, 3±2% and RH, 4±2%, not significant vs. control). In the presence of L-NMMA, RH-induced increase in blood flow was 330±5% of baseline in the wild-type mice, whereas in the SOD-KO mice, the response was significantly reduced to 145±23% (P<0.01). In the mesenteric arterioles of SOD-KO mice, tempol, an SOD mimetic, significantly increased the ACh-induced vasodilation, which effect was abolished by catalase. Vascular H 2 O 2 production in response to ACh in mesenteric arterioles (fluorescent method) was significantly increased in wild-type mice but was markedly impaired in SOD-KO mice. By contrast, vascular NO production was significantly increased in wild-type mice and was unaltered in SOD-KO mice. Vascular level of BH 4 in the aorta was comparable between the two genotypes. Conclusions: These results indicate that endothelial Cu/Zn-SOD plays an important role as an EDHF synthase during RH in mouse mesenteric microcirculation in vivo.

Pulsatile stretch in coronary arteries elicits release of endothelium-derived hyperpolarizing factor: a modulator of arterial compliance.

To date, the release of the endothelium-derived hyperpolarizing factor (EDHF) has been demonstrated only in response to receptor-dependent Ca2+-elevating agonists. Since endothelial cells in situ are continuously subjected to rhythmic distension, we investigated the effect of rhythmic stretch on the release of EDHF from isolated porcine coronary arteries. In the combined presence of diclofenac and N(G)-nitro-L-arginine (L-NNA), sinusoidal pressure oscillations (from 40 to 50 mm Hg, 4 minutes, 1.5 Hz) led to simultaneous oscillations in the external diameter of coronary artery segments, the amplitude of which were decreased by iberiotoxin and apamin and also by endothelial denudation. In order to directly demonstrate the release of EDHF, the intraluminal solution from endothelium-intact coronary segments exposed to pulsatile stretch was applied to detector rat aortic smooth muscle cells, the membrane potential of which was continuously measured using the patch-clamp technique. The hyperpolarization of detector cells induced by the intraluminal solution was proportional to the amplitude of the pressure oscillations applied to the donor artery and was attenuated by either preincubation of donor arteries with 17-octadecynoic acid or application of either tetrabutylammonium or iberiotoxin to detector cells. In contrast to the bradykinin-induced release of EDHF, the EDHF synthesized in response to pulsatile stretch did not exhibit any tachyphylaxis. These findings demonstrate for the first time that the synthesis of EDHF in coronary arteries can be mechanically stimulated by rhythmic vessel wall distension and suggest that the continuous release of EDHF may contribute to the adjustment of an adequate vascular compliance and to the control of coronary blood flow.

Volatile and intravenous anesthetics selectively attenuate the release of endothelium-derived hyperpolarizing factor elicited by bradykinin in the coronary microcirculation.

In addition to nitric oxide (NO) and prostacyclin (PGI2) another endothelium-derived factor, which hyperpolarizes vascular smooth muscle cell via activation of K+ channels, contributes to the vasorelaxant effect of bradykinin in different vascular beds. Preliminary findings suggest that this endothelium-derived hyperpolarizing factor (EDHF)-mediated vasodilatation is attenuated by both volatile and intravenous anesthetics. Since EDHF may play an important role in the coronary microcirculation, we investigated the effects of isoflurane (2 vol.% equivalent to approximately 250 microM), etomidate (30 and 100 microM), phenobarbital (100 microM) and thiopental (30 and 100 microM) on the EDHF-mediated dilator response to bradykinin and on the endothelium-independent dilatation evoked by sodium nitroprusside (SNP) in the isolated saline-perfused rat heart (Langendorff preparation). None of the anesthetics tested affected the dilator response to bradykinin or SNP under basal conditions. However, following inhibition of NO and PGI2 formation with NG-nitro-L-arginine (100 microM) and diclofenac (1 microM) respectively, isoflurane, etomidate and thiopental, but not phenobarbital, significantly attenuated the NO/PGI2-independent, i.e. EDHF-mediated dilator response to bradykinin, while the vasorelaxant effect of SNP remained unaffected. Isoflurane, etomidate and thiopental, but not phenobarbital, display cytochrome P450-inhibiting properties, suggesting that these anesthetics impair the cytochrome P450-dependent synthesis of EDHF in the coronary microcirculation.

Selective inhibition by barbiturates of the synthesis of endothelium-derived hyperpolarizing factor in the rabbit carotid artery.

1. Several lines of evidence suggest that both volatile and intravenous anaesthetics may interfere with the synthesis and release of endothelium-derived vasoactive factors. We have investigated the effects of three different barbiturates on the release of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF) in phenylephrine (1 microM)-preconstricted, endothelium-intact ring segments of the rabbit carotid artery. The segments were pretreated with the cyclo-oxygenase inhibitor, diclofenac (1 microM), to prevent the formation of vasoactive prostanoids, such as prostacyclin (PGI2). 2. Acetylcholine (ACh) elicited a concentration-dependent relaxation (EC50 0.15 microM) in control segments which was not significantly different from the relaxant responses of segments pretreated with methohexitone (0.03-0.3 mM), phenobarbitone (0.1-0.3 mM) or thiopentone (0.1-0.3 mM). 3. Inhibition of NO synthesis with NG-nitro-L-arginine (0.1 mM) significantly reduced the maximum relaxant response to ACh from 96 to 40%. This NO/PGI2-independent relaxation appeared to be mediated by the release of EDHF, since it was strongly diminished in the presence of the K+Ca inhibitors, tetrabutylammonium (1-3 mM) and charybdotoxin (10 nM), following preconstriction with potassium calcium (40 mM) or removal of the endothelium. Thiopentone or methohexitone markedly attenuated the EDHF-mediated relaxant response to ACh, while phenobarbitone had no effect. The endothelium-independent relaxation elicited by sodium nitroprusside (0.01-10 microM), on the other hand, was only marginally affected by these anaesthetics. 4. The cytochrome P450 inhibitor, clotrimazole (3-100 microM), mimicked the inhibitory effect of thiopentone and methohexitone on the NO/PGI2-independent relaxant response to ACh. Moreover the cytochrome P450-catalyzed O-dealkylation of 7-ethoxycoumarin by rabbit liver microsomes was inhibited in the presence of thiopentone or methohexitone (0.3-1 mM), while phenobarbitone was without effect.5. These findings suggest that thiopentone and methohexitone selectively attenuate the EDHF-mediated relaxant response to ACh in the rabbit carotid artery, presumably by interfering with its synthesis from arachidonic acid via the cytochrome P450 epoxygenase pathway.