David Hughes Edwards

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

We have investigated the role of cAMP in NO- and prostanoid-independent 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 ≈2-fold. Relaxation was associated with transient ≈1.5-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 ≈2-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.

Evidence that cyclic guanosine monophosphate (cGMP) mediates endothelium-dependent relaxation.

The mechanism of action of endothelium-derived relaxant factor (EDRF) was studied using aortic strip preparations of the rabbit and a bioassay system of a rabbit coronary artery perfused in series with an intact aorta. Methylene blue (an inhibitor of guanylate cyclase) inhibited, and 2-O-propoxyphenyl-8-azapurine-6-one (MB22948, an inhibitor of cGMP phosphodiesterase) potentiated the vascular effects of EDRF whether these were due to its basal or to stimulated release. Infusion of these agents at different sites in the bioassay indicated that they act pharmacologically at the smooth muscle level and not on release of EDRF or by chemical interaction with EDRF. The data are consistent with the hypothesis that EDRF-induced relaxation is mediated by elevation of smooth muscle cGMP levels.

Isolated perfused rabbit coronary artery and aortic strip preparations: the role of endothelium‐derived relaxant factor.

Isolated perfused coronary arteries and aortic ring preparations of rabbits were studied, both with intact endothelium and with endothelium removed by K‐rich solution and friction respectively. Constrictor dose‐responses to histamine, acetylcholine, phenylephrine and 5‐hydroxytryptamine (5‐HT) were measured. They were greatly depressed by the presence of endothelium in coronary preparations. In aortic preparations endothelium affected dose‐responses relatively little, depressing the response to acetylcholine but apparently increasing the responses to the other three agents. Acetylcholine relaxed pre‐constricted coronary or aortic preparations but only when endothelium was present. This relaxation was inhibited by quinacrine or hydroquinone. Aortic preparations had resting tone which could be increased by hydroquinone if endothelium was present, suggesting continual release of endothelium‐derived relaxant factor (EDRF) at rest. When allowance was made for basal EDRF activity in aortic preparations, the maximal constrictor response to acetylcholine remained lower in the presence of endothelium, consistent with acetylcholine stimulation of EDRF, but maximal constrictor responses to the other three agents were the same with and without endothelium, suggesting that the direct constrictor response overrides EDRF activity.

Connexin‐mimetic peptides dissociate electrotonic EDHF‐type signalling via myoendothelial and smooth muscle gap junctions in the rabbit iliac artery

1 Synthetic peptides corresponding to the Gap 26 and Gap 27 domains of the first and second extracellular loops of the major vascular connexins (Cx37, Cx40 and Cx43), designated as 43Gap 26, 40Gap 27, 37,40Gap 26 and 37,43Gap 27 according to Cx homology, were used to investigate the role of gap junctions in the spread of endothelial hyperpolarizations evoked by cyclopiazonic acid (CPA) through the wall of the rabbit iliac artery. 2 Immunostaining and confocal microscopy demonstrated that gap junction plaques constructed from Cx37 and Cx40 were abundant in the endothelium, whereas Cx43 was the dominant Cx visualized in the media. 3 None of the Cx‐mimetic peptides affected endothelial hyperpolarizations evoked by CPA directly. 4 When administered individually, 40Gap 27, 37,40Gap 26 and 37,43Gap 27, but not 43Gap 26, attenuated endothelium‐dependent subintimal smooth muscle hyperpolarization. By contrast, only 43Gap 26 and 37,43Gap 27 reduced the spread of subintimal hyperpolarization through the media of the rabbit iliac artery. The site of action of the peptides therefore correlated closely with the expression of their target Cxs in detectable gap junction plaques. 5 The findings provide further evidence that the EDHF phenomenon is electrotonic in nature, and highlight the contribution of myoendothelial and homocellular smooth muscle communication via gap junctions to arterial function.

Minimal model of arterial chaos generated by coupled intracellular and membrane Ca2+ oscillators.

We have developed a mathematical model of arterial vasomotion in which irregular rhythmic activity is generated by the nonlinear interaction of intracellular and membrane oscillators that depend on cyclic release of Ca2+ from internal stores and cyclic influx of extracellular Ca2+, respectively. Four key control variables were selected on the basis of the pharmacological characteristics of histamine-induced vasomotion in rabbit ear arteries: Ca2+ concentration in the cytosol, Ca2+ concentration in ryanodine-sensitive stores, cell membrane potential, and the open state probability of Ca2+-activated K+ channels. Although not represented by independent dynamic variables, the model also incorporates Na+/Ca2+exchange, the Na+-K+-ATPase, Cl- fluxes, and Ca2+ efflux via the extrusion ATPase. Simulations reproduce a wide spectrum of experimental observations, including 1) the effects of interventions that modulate the functionality of Ca2+ stores and membrane ion channels, 2) paradoxes such as the apparently unpredictable dual action of Ca2+ antagonists and low extracellular Na+ concentration, which can abolish vasomotion or promote the appearance of large-amplitude oscillations, and 3) period-doubling, quasiperiodic, and intermittent routes to chaos. Nonlinearity is essential to explain these diverse patterns of experimental vascular response.

Hydrogen Peroxide Potentiates the EDHF Phenomenon by Promoting Endothelial Ca2+ Mobilization

Objective—The purpose of this study was to test the hypothesis that H2O2 contributes to the EDHF phenomenon by mobilizing endothelial Ca2+ stores. Methods and Results—Myograph studies with rabbit iliac arteries demonstrated that EDHF-type relaxations evoked by the SERCA inhibitor cyclopiazonic acid (CPA) required activation of KCa channels and were potentiated by exogenous H2O2 and the thiol oxidant thimerosal. Preincubation with a submaximal concentration of CPA unmasked an ability of exogenous H2O2 to stimulate an EDHF-type response that was sensitive to KCa channel blockade. Imaging of cytosolic and endoplasmic reticulum [Ca2+] in rabbit aortic valve endothelial cells with Fura-2 and Mag-fluo-4 demonstrated that H2O2 and thimerosal, which sensitizes the InsP3 receptor, both enhanced CPA-evoked Ca2+ release from stores, and that the potentiating effect of H2O2 was suppressed by the cell-permeant thiol reductant glutathione monoethylester. CPA-evoked relaxations were attenuated by exogenous catalase and potentiated by the catalase inhibitor 3-aminotriazole, and were abolished by the connexin-mimetic peptide 43Gap26, which interrupts intercellular communication via gap junctions constructed from connexin 43. Conclusions—H2O2 can enhance EDHF-type relaxations by potentiating Ca2+ release from endothelial stores, probably via redox modification of the InsP3 receptor, leading to the opening of hyperpolarizing endothelial KCa channels and an electrotonically-mediated relaxant response.

Distinct hyperpolarizing and relaxant roles for gap junctions and endothelium-derived H2O2 in NO-independent relaxations of rabbit arteries

We have compared the contributions of gap junctional communication and chemical signaling via H2O2 to NO-independent relaxations evoked by the Ca2+ ionophore A23187 and acetylcholine (ACh) in rabbit ilio-femoral arteries. Immunostaining confirmed the presence of connexins (Cxs) 37 and 40 in the endothelium and Cxs 40 and 43 in smooth muscle. Maximal endothelium-dependent subintimal smooth muscle hyperpolarizations evoked by A23187 and ACh were equivalent (≈20 mV) and almost abolished by an inhibitory peptide combination targeted against Cxs 37, 40, and 43. However, maximal NO-independent relaxations evoked by A23187 were unaffected by such peptides, whereas those evoked by ACh were depressed by ≈70%. By contrast, the enzyme catalase, which destroys H2O2, attenuated A23187-induced relaxations over a broad range of concentrations, but only minimally depressed the maximum response to ACh. Catalase did not affect A23187- or ACh-evoked hyperpolarizations. After loading with an H2O2-sensitive probe, A23187 caused a marked increase in endothelial fluorescence that correlated temporally with relaxation, whereas only a weak delayed increase was observed with ACh. In arteries without endothelium, the H2O2-generating system xanthine/xanthine oxidase induced a catalase-sensitive relaxation that mimicked the gap junction-independent response to A23187 as it was maximally equivalent to ≈80% of induced tone, but associated with a smooth muscle hyperpolarization <5 mV. We conclude that myoendothelial gap junctions underpin smooth muscle hyperpolarizations evoked by A23187 and ACh, but that A23187-induced relaxation is dominated by extracellular release of H2O2. Endothelium-derived H2O2 may thus be regarded as a relaxing factor, but not a hyperpolarizing factor, in rabbit arteries.

Endothelium-derived relaxing factor (EDRF) and resistance vessels in an intact vascular bed: a microangiographic study of the rabbit isolated ear

1 Microradiographic techniques have been used to show that endothelium‐derived relaxing factor (EDRF), which is believed to be nitric oxide, influences vasomotor responses in small arteries and arterioles down to 25 μm in diameter in an isolated, intact, buffer‐perfused ear preparation of the rabbit. Arteries down to 75 μm in diameter, i.e. the central ear artery (G0) and its first three generations of branch vessels (G1, G2 and G3) were studied quantitatively. 2 Relative constrictor responses to 1 μm 5‐hydroxytryptamine (5‐HT) and the combination of 1 μm 5‐HT and 1 μm histamine diminished progressively from G0 to G3. Constrictor responses to 5‐HT were doubled in all generations by 1 μm haemoglobin which abolishes EDRF activity. 3 Relative dilator responses to acetylcholine or to substance P in preconstricted arteries were, in contrast, equal in the different generations. Mean —log (IC50) values calculated from diameter measurements were 7.63 ± 0.10 m and 9.80 ± 0.11 m, respectively. These dilator responses were abolished by 1 μm haemoglobin, implying that they were EDRF‐mediated. Spatial homogeneity of relative dilator responses was found also with glyceryl trinitrate (10 or 50 μm) whose activity is thought to depend on biotransformation to nitric oxide, in both the presence and the absence of haemoglobin. 4 This finding of spatial homogeneity of the diameter response to changes in EDRF activity (or to glyceryl trinitrate) implies that EDRF influences hydrodynamic resistance more in vessels where constrictor tone is high.

ASCORBIC ACID AND TETRAHYDROBIOPTERIN POTENTIATE THE EDHF PHENOMENON BY GENERATING HYDROGEN PEROXIDE

Aims Our objective was to investigate whether pro-oxidant properties of ascorbic acid (AA) and tetrahydrobiopterin (BH4) modulate endothelium-dependent, electrotonically mediated arterial relaxation. Methods and results In studies with rabbit iliac artery (RIA) rings, NO-independent, endothelium-derived hyperpolarizing factor (EDHF)-type relaxations evoked by the sarcoplasmic endoplasmic reticulum Ca2+-ATPase inhibitor cyclopiazonic acid and the G protein-coupled agonist acetylcholine (ACh) were enhanced by AA (1 mM) and BH4 (200 µM), which generated buffer concentrations of H2O2 in the range of 40–80 µM. Exogenous H2O2 potentiated cyclopiazonic acid (CPA)- and ACh-evoked relaxations with a threshold of 10–30 µM, and potentiation by AA and BH4 was abolished by catalase, which destroyed H2O2 generated by oxidation of these agents in the organ chamber. Adventitial application of H2O2 also enhanced EDHF-type dilator responses evoked by CPA and ACh in RIA segments perfused intraluminally with H2O2-free buffer, albeit with reduced efficacy. In RIA rings, both control relaxations and their potentiation by H2O2 were overcome by blockade of gap junctions by connexin-mimetic peptides (YDKSFPISHVR and SRPTEK) targeted to the first and second extracellular loops of the dominant vascular connexins expressed in the RIA. Superoxide dismutase attenuated the potentiation of EDHF-type relaxations by BH4, but not AA, consistent with findings demonstrating a differential role for superoxide anions in the generation of H2O2 by the two agents. Conclusion Pro-oxidant effects of AA and BH4 can enhance the EDHF phenomenon by generating H2O2, which has previously been shown to amplify electrotonic hyperpolarization-mediated relaxation by facilitating Ca2+ release from endothelial stores.

Angiographic contrast media relax isolated rabbit aorta through an endothelium-independent mechanism that may not depend on the presence of the iodine atom

Systemically administered iodinated angiographic contrast media evoke vasodilatation through mechanisms that are at present poorly understood. In the current investigation we have evaluated the role of the vascular endothelium in responses to an iso-osmolar formation of the non-ionic dimer iodixanol and a hyperosmolar formulation of the non-ionic monomer iopromide. Isolated rabbit aortic ring preparations with endothelium intact or removed by gentle abrasion were mounted in organ baths containing oxygenated Holmans solution, and cumulative concentration-response curves for relaxation to the contrast media were constructed after pre-constriction by phenylephrine (300 nM) in the presence of indomethacin to inhibit prostaglandin synthesis. Endothelial denudation did not influence the ability of either iodixanol or iopromide to relax the aortic ring preparations. Iopromide was significantly more potent than iodixanol when expressed in terms of iodine concentration (mg I ml-1), but both agents were equipotent when expressed in terms of molarity (mM). We conclude that relaxation of isolated rabbit aortic rings to iodixanol and iopromide under conditions where there is no fluid flow is endothelium-independent, and therefore not mediated by release of the potent endogeneous nitrovasodilator endothelium-derived relaxing factor (EDRF). Furthermore, their relaxant activity under the in vitro experimental conditions employed is attributable to a direct action on vascular smooth muscle by factors in addition to osmolality, and may depend on features that are not specifically associated with the presence of the iodine atom.