Dynamic Modulation of Interendothelial Gap Junctional Communication by 11,12-Epoxyeicosatrienoic Acid

Abstract

Abstract-Functional gap junctional communication between vascular cells has been implicated in ascending dilatation and the cytochrome P-450 (CYP) inhibitor-sensitive and NO-and prostacyclin-independent dilatation of many vascular beds. Here, we assessed the mechanisms by which the epoxyeicosatrienoic acids (EETs) generated by a CYP 2C enzyme control interendothelial gap junctional communication. In CYP 2C-expressing porcine coronary endothelial cells, bradykinin, which enhances EET formation, elicited a biphasic effect on the electrical coupling and transfer of Lucifer yellow between endothelial cells, consisting of a transient increase in coupling followed by a sustained uncoupling. The initial phase was sensitive to the CYP 2C9 inhibitor sulfaphenazole and the protein kinase A (PKA) inhibitors Rp-cAMPS and KT5720 and could be mimicked by forskolin and caged cAMP as well as by the PKA activators 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole 3Ј,5Ј-cyclic monophosphorothioate sodium salt and Sp-cAMPS. Gap junction uncoupling in bradykinin-stimulated porcine coronary endothelial cells was prevented by inhibiting the activation of extracellular signal-regulated kinase (ERK)1/2. In human endothelial cells, which express little CYP 2C, bradykinin elicited only an ERK1/2-mediated inhibition of intercellular communication. The CYP 2C9 product, 11,12-EET, also exerted a dual effect on the electrical and dye coupling of human endothelial cells, which was sensitive to PKA inhibition. These results demonstrate that an agonist-activated CYP-dependent pathway as well as 11,12-EET can positively regulate interendothelial gap junctional communication, most probably via the activation of PKA, an effect that is curtailed by the subsequent activation of ERK1/2. (Circ Res. 2002;90:800-806.)Key Words: connexin43 Ⅲ cytochrome P-450 2C Ⅲ cAMP Ⅲ endothelium-derived hyperpolarizing factors Ⅲ 11,12-epoxyeicosatrienoic acid I n large arteries and veins, gap junctions are known to be essential for the propagation of electrical signals between vascular smooth muscle cells, whereas in the microcirculation they appear to be involved in the phenomena of ascending dilation and endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation (see review 1 ).Myoendothelial gap junctions have been identified in numerous vascular beds, particularly in small arteries and terminal arterioles, 2 and it is assumed that the selective gating of such gap junctions plays an integral role in endotheliumdependent and in NO-and prostacyclin (PGI 2 )-independent relaxation. 3-6 However, there has been no convincing demonstration of a direct link between dynamic alterations in myoendothelial gap junctions and relaxation/vasodilatation.Interendothelial, rather than myoendothelial, cell communication has recently been proposed to be the pathway by which hyperpolarization and vasodilatation are conducted along agonist-stimulated resistance vessels. 7 In mice and hamsters, 8 -10 this phenomenon has been linked to a cytochrome P-450 (CYP)-dependent process t...

GPR40 is a low-affinity epoxyeicosatrienoic acid receptor in vascular cells

“…We also found that 11,12-EET (1 µM) increased Cx43 in the Triton-soluble fraction, and this increase was inhibited by pretreatment with U0126 ( Figure 10A) and GW1100 ( Figure 10B). There were several Cx43 bands detected indicating various levels of phosphorylation (12,53). These data indicate that 11,12-EET-induced phosphorylation and internalization of Cx43 involves GPR40-activated ERK phosphorylation.…”

“…Effect of 11,12-EET on Cx43 expression in HUVECs: 11,12-EET alters intercellular gap junction communication by activating Cx43 in ECs (12). Gap junctions are in a constant state of assembly and disassembly (53).…”

“…Additionally, EET regulation of Cx43 and COX-2 requires ERK phosphorylation (11,12). Activation of GPR40 also stimulates ERK phosphorylation in several cell types (38,51,52).…”

“…Additionally, 11,12-and 14,15-EETs inhibit smooth muscle cell migration and aromatase expression (8,9). On endothelial cells, 11,12-EET promotes cell growth, decrease apoptosis, migration, tube formation and angiogenesis, increases tissue plasminogen activator (tPA) release, increases cyclooxygenase-2 (COX-2) expression and alters connexin-43 (Cx43) expression and gap junctions (10)(11)(12)(13)(14)(15)(16)(17). EET isomers also inhibit platelet adhesion to the endothelium, and 11,12-and 8,9-EETs inhibit inflammation by decreasing leukocyte adhesion to the endothelium (18,19).…”

EDHF-mediated vasodilation involves different mechanisms in normotensive and hypertensive rat lungs

“…Although EETs may not be EDHF, several studies suggest that cytochrome P-450 (CYP450) activity is involved in the mechanism of EDHF-mediated vasodilation (6,22,43,45). For example, sulfaphenazole, an inhibitor of cytochrome 2C isozymes, inhibits EDHFmediated vasorelaxation in the porcine coronary artery (21) and 7-ethoxyresorufin, an inhibitor of CYP450 1A isozyme, inhibits the vasodilation in perfused rat mesenteric (1) and renal (2) vascular beds and the guinea pig cerebral artery (14).…”