cAMP facilitates EDHF-type relaxations in conduit arteries by enhancing electrotonic conduction via gap junctions

Abstract

We have investigated the role of cAMP in NO-and prostanoidindependent relaxations that are widely attributed to an endothelium-derived hyperpolarizing factor (EDHF). Under control conditions EDHF-type relaxations evoked by acetylcholine (ACh) in rabbit iliac arteries were transient, but in the presence of the cAMP phosphodiesterase inhibitor isobutylmethylxanthine (IBMX) or the cell permeant cAMP analog 8-bromo-cAMP, relaxations became sustained with their maxima potentiated Ϸ2-fold. Relaxation was associated with transient Ϸ1.5-fold elevations in smooth muscle cAMP levels with both mechanical and nucleotide responses being abolished by interrupting gap junctional communication with the connexin-mimetic peptide Gap 27 and by endothelial denudation. However, IBMX induced a sustained endothelium-independent Ϸ2-fold rise in cAMP levels, which was not further amplified by ACh, suggesting that the contribution of cAMP to the EDHF phenomenon is permissive. After selective loading of the endothelium with calcein AM, direct transfer of dye from the endothelium to the media was enhanced by IBMX or 8-bromo-cAMP, but not by 8-bromo-cGMP, whereas Gap 27 promoted sequestration within the intima. ACh-induced hyperpolarizations of subintimal smooth muscle in arterial strips with intact endothelium were abolished by Gap 27 and the adenylyl cyclase inhibitor 2 ,5 -dideoxyadenosine but were unaffected by IBMX. By contrast, in strips partially denuded of endothelium, IBMX enhanced the transmission of hyperpolarization from the endothelium to remote smooth muscle cells. These findings support the hypothesis that endothelial hyperpolarization underpins the EDHF phenomenon, with cAMP governing subsequent electrotonic signaling via both myoendothelial and homocellular smooth muscle gap junctions.connexin ͉ acetylcholine ͉ IBMX ͉ 8-bromo-cAMP ͉ cGMP

Distinct hyperpolarizing and relaxant roles for gap junctions and endothelium-derived H 2 O 2 in NO-independent relaxations of rabbit arteries

“…Hemoglobin was obtained by lysis of red cells in HPLC grade water and purified on a Sephadex G25 column. In further experiments with endothelium-denuded rings in the presence of L-NAME and indomethacin, cumulative concentration-relaxation curves were constructed for xanthine in the presence of 100 milliunits͞ml of xanthine oxidase (XO) and for H 2 O 2 after incubation with catalase (2,000 units͞ml), superoxide dismutase (500 units͞ml; xanthine curve only), the K v channel blocker 4-aminopyridine (300 M), the SK Ca and IK Ca ͞BK Ca channel blockers apamin and charybdotoxin in combination (100 nM each), the K ATP channel blocker glibenclamide (10 M), the soluble guanylyl cyclase inhibitor 1H- [1,2,4]oxadiazolo [4,3-a]quinoxalin-1-one (ODQ, 10 M), the adenylyl cyclase inhibitor 2Ј,5Ј-dideoxyadenosine (2Ј,5Ј-ddA, 500 M), and the cell-permeant iron chelator 1,2 dimethyl-3-hydroxypyridin-4-one (deferiprone, 1 mM; xanthine curve only). All agents were obtained from Sigma, except 2Ј,5Ј-ddA (Affiniti, Nottingham, U.K.) and deferiprone (Calbiochem).…”

“…However, the mechanisms of relaxation activated by these agents may involve different pathways. In rabbit arteries, NO͞prostanoid-independent relaxations to ACh are attenuated by connexin (Cx)-mimetic peptides and glycyrrhetinic acid derivatives that interrupt gap junctional communication, whereas analogous responses to A23187 are insensitive to such inhibitors (1)(2)(3)(4)(5). By contrast, ''sandwich'' bioassay studies with closely apposed endothelium-intact and -denuded strips of rabbit superior mesenteric or ilio-femoral artery demonstrate that A23187, but not ACh, promotes the release of a diffusible factor that is capable of causing relaxation (2,5,6).…”

“…Relaxation was correlated with measurements of subintimal smooth muscle membrane potential, and the role of gap junctions was assessed with peptides homologous to the gap 26 and 27 domains of the first and second extracellular loops of Cxs 37, 40, and 43. Such peptides interrupt intercellular communication in a Cx-selective fashion without exerting nonspecific effects on endothelial or smooth muscle cells (15)(16)(17)(18)(19), and thereby attenuate the electrotonic spread of ACh-evoked endothelial hyperpolarization into the media via gap junctions (4). Endothelial H 2 O 2 production was assessed by sandwich bioassay and confocal imaging after loading of the endothelium with the H 2 O 2 -sensitive probe dihydrodichlorofluorescein (7,20).…”

Enhanced spontaneous Ca2+ events in endothelial cells reflect signalling through myoendothelial gap junctions in pressurized mesenteric arteries

“…The observation that calcein remained concentrated within the endothelial cells, including the endothelial cell projections which form gap junctions with the muscle, strongly suggests that the dye normally leaves the endothelium via gap junctions (and/or possibly connexin hemichannels) [42]. This technique has been used previously to show the effectiveness of another group of gap junction uncoupling agents, the connexin mimetic gap peptides [43].…”

Endothelium-dependent relaxation is resistant to inhibition of nitric oxide synthesis, but sensitive to blockade of calcium-activated potassium channels in essential hypertension

“…8 A recently published study by Taddei et al 9 has reported that ACh responses in the forearm of subjects with EH are sensitive to intra-brachial infusions of sulphaphenazole, an inhibitor of cytochrome P450 epoxygenase (CYP2C9). 9 On the basis of these data, these investigators suggest that a CYP2C9 product (11,12 epoxyeicosatrienoic acid (11,12 EET)) plays a key role in human EH as an endothelium-derived hyperpolarizing factor (EDHF) producing rapid compensatory vasorelaxation under conditions of decreased NO bioavailability. 9 The EDHF is a complex and multifaceted response, [10][11][12][13][14][15][16] playing a particularly prominent role in smaller resistance-sized vessels.…”