Cytochrome P-450 metabolites but not NO, PGI₂, and H₂O₂ contribute to ACh-induced hyperpolarization of pressurized canine coronary microvessels

Abstract

. Cytochrome P-450 metabolites but not NO, PGI2, and H2O2 contribute to ACh-induced hyperpolarization of pressurized canine coronary microvessels. Am J Physiol Heart Circ Physiol 285: H1939-H1948, 2003. First published July 24, 2003 10.1152/ajpheart.00190.2003.-The endothelium-dependent hyperpolarization of cells has a crucial role in regulating vascular tone, especially in microvessels. Nitric oxide (NO) and prostacyclin (PGI 2), in addition to endothelium-derived hyperpolarizing factor (EDHF), have been reported to hyperpolarize vascular smooth muscle in several organs. Studies have reported the hyperpolarizing effects of these factors are increased by a stretch in large coronary arteries. EDHF has not yet been identified and cytochrome P-450 metabolites and H2O2 are candidates for EDHF. With the use of the membrane potential-sensitive fluorescent dye bis-(1,3-dibutylbarbituric acid)trimethione oxonol [DiBAC4(3)], we examined whether NO, PGI2, cytochrome P-450 metabolites, and H2O2 contribute to AChinduced hyperpolarization in pressurized coronary microvessels. Canine coronary arterial microvessels (60-356 m internal diameter) were cannulated and pressurized at 60 cmH2O in a vessel chamber perfused with physiological salt solution containing DiBAC4(3). Fluorescence intensity and diameter were measured on a computer. There was a linear correlation between changes in the fluorescence intensity and membrane potential. ACh significantly decreased the fluorescence intensity (hyperpolarization) of the microvessels without any inhibitors. Endothelial damage caused by air perfusion abolished the ACh-induced decrease in fluorescence intensity. The inhibitors of NO synthase and cyclooxygenase did not affect the ACh-induced decreases in the fluorescence intensity. The addition of 17-octadecynoic acid, a cytochrome P-450 monooxygenase inhibitor, to those inhibitors significantly attenuated the ACh-induced decreases in fluorescence intensity, whereas catalase, an enzyme that dismutates H 2O2 to form water and oxygen, did not. Furthermore, catalase did not affect the vasodilation produced by ACh. These results indicate that NO and PGI 2 do not contribute to the AChinduced hyperpolarization and that the cytochrome P-450 metabolites but not H 2O2 are involved in EDHF-mediated hyperpolarization in canine coronary arterial microvessels. microcirculation; endothelium-derived hyperpolarizing factor; endothelium THE ENDOTHELIUM PLAYS an important role in regulating the vascular tone by releasing vasodilator substances such as prostacyclin (PGI 2 ), nitric oxide (NO), and endothelium-derived hyperpolarizing factor (EDHF) (37, 50). Part of the endothelium-dependent relaxation is accompanied by endothelium-dependent hyperpolarization of the vascular smooth muscle cells (10) and the contribution of the hyperpolarizing mechanism to endothelium-dependent relaxation increases as the vessel size decreases, whereas the contribution of NO decreases (24,29,36,48).It has been shown that NO and PGI 2 , in addition to EDHF, hyperpolarize the m...

Eradication of Pseudomonas aeruginosa biofilms and persister cells using an electrochemical scaffold and enhanced antibiotic susceptibility

“…39 Membrane potential sensitive dye 3,3′-dipropylthiacarbocyanine iodide enters only depolarized cells, where it binds reversibly to lipid-rich intracellular components. 40 The increased fluorescence intensity of this dye in e-scaffold treated cells (Fig. 3c) indicates that the e-scaffold disrupted the cytoplasmic membrane and that this induced depolarization of the plasma membrane potential.…”

EDHF and Gap Junctions: Important Regulators of Vascular Tone Within the Microcirculation

“…However, there is ample evidence that EDHF is an important mediator in the microcirculation because inhibition of NO-synthase and prostaglandin synthesis does not alter or only attenuates dilations in response to endothelial stimulation in kidney arterioles [131][132][133], the cremaster microcirculation of mice [127,134], rats [135] or hamsters [136,137], the cheek pouch preparation in hamsters [138,139], and other microcirculatory preparations [117,140] which allow the direct observation of arterioles in the range of 20 to 80 m. Similarly, in isolated perfused organs where the responses rely on the behavior of the resistance vessels EDHF-type dilations are present in response to stimulation with acetylcholine or bradykinin, e.g. the isolated perfused heart [141], mesenteric circulation [142], hindquarter [143], and in numerous preparations using isolated small arteries or arterioles [56,[144][145][146][147][148][149][150][151]. EDHF-type dilations have likewise been documented in humans using isolated arterioles stimulated with arachidonic acid [152] or bradykinin [153], but also in vivo in the human forearm microcirculation assessed by plethysmography [154].…”

“…However, other blockers of CYP 450 that do not inhibit K + -channels (e.g. ODYA, sulfaphenazole) attenuated EDHF-type dilations in a similar way in the coronary, skeletal muscle, and mesenteric microcirculation in various species [136,142,151,196] and were also effective in human coronary arterioles [152] or small arteries from subcutaneous tissue studied in vitro [179]. In the murine coronary microcirculation acetylcholine-induced dilations are mediated mainly by NO and prostaglandins [202] and only a different endothelial stimulator (bradykinin) uncovers a weak role of CYP 450 products, which is accentuated in mice deficient for endothelial NO synthase (eNOS) [203].…”

Arachidonic Acid Epoxygenase Metabolites Stimulate Endothelial Cell Growth and Angiogenesis via Mitogen-Activated Protein Kinase and Phosphatidylinositol 3-Kinase/Akt Signaling Pathways

“…EETs activate vascular smooth muscle cell large conductance Ca 2ϩ -activated K ϩ channels leading to hyperpolarization of the resting membrane potential and resulting in vasorelaxation and lowering of blood pressure. Indeed, 11,12-EET has been proposed to be identical to endothelium-derived hyperpolarizing factor (Bauersachs et al, 1997;Bolz et al, 2000;Matoba et al, 2000Matoba et al, , 2002Hamilton et al, 2001;Lacza et al, 2002;Miura et al, 2003;Morikawa et al, 2003;Tanaka et al, 2003;Yada et al, 2003). Exogenous application of EETs inhibits vascular smooth muscle cell migration, platelet aggregation, nuclear factor-B activation, and vascular cell adhesion molecule-1 expression (Node et al, 1999;Fleming et al, 2001;Sun et al, 2002;Krotz et al, 2004), suggesting an overall beneficial role for EETs within the vasculature and a protective role in the development of atherosclerosis.…”