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

Abstract

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 (approximately 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 approximately 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 approximately 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.

Fig. 1.

Concentration-response curves for EDHF-type relaxations of rabbit iliac arteries evoked by A23187 and ACh and associated changes in subintimal smooth muscle membrane potential. (A) Cx-mimetic peptides targeting Cxs 37, 40, and 43 did not affect maximal relaxations to A23187, either singly or in combination, but caused a small rightward shift in the response curve when used as the triple combination ^37,43Gap27 + ⁴⁰Gap27 + ⁴³Gap26 (†, P < 0.05). Total peptide concentration was 900 μM in all experiments. (B and C) Catalase caused a concentration-dependent depression of relaxation to A23187, with residual responses observed in the presence of 2,000 units/ml catalase being abolished by ^37,43Gap27 + ⁴⁰Gap27 + ⁴³Gap26. By contrast, relaxations to ACh were inhibited by ^37,43Gap27 + ⁴⁰Gap27 + ⁴³Gap26, but minimally affected by catalase. ‡, P < 0.05 for specific ACh concentrations compared with control; ^*, P < 0.05; ^**, P < 0.01, and ^***, P < 0.001 for whole curves. (D) Representative traces showing that the triple peptide combination abolished smooth muscle hyperpolarizations evoked by A23187, whereas catalase was without effect. (E) Histograms summarizing maximal smooth muscle membrane potential changes. The triple peptide combination was a more effective inhibitor of smooth muscle hyperpolarizations evoked by 3 μM A23187 than ^37,43Gap27 or ^37,43Gap27 + ⁴⁰Gap27, and almost abolished the hyperpolarizing response to 3 μM ACh. Catalase (2,000 units/ml) did not affect the hyperpolarizing response to either A23187 or ACh. ^*, P < 0.05 and ^***, P < 0.001 compared with control.

Fig. 2.

(A) Transverse sections of rabbit iliac artery stained with primary antibodies to Cxs 37, 40, and 43 and secondary antibodies of goat anti-mouse-conjugated Alexa 488 or goat anti-rabbit-conjugated Alexa 546 as appropriate. Punctate fluorescence in the media and endothelium indicates the presence of gap junction plaques. Note autofluorescence of internal elastic lamina (IEL) in panel stained for Cx 43 (Bottom), but absence of endothelial Cx protein. L = lumen. (B) Time course of endothelial fluorescence induced by 3 μM A23187 in femoral arterial rings loaded with the H₂O₂-sensitive probe dihydrodichlorofluorescein. (C) Only a small and relatively delayed increase in fluorescence was observed after administration of 3 μM ACh. The ridged structure apparent in B and C reflects the intimal folds evident in A. (Magnifications: ×40 in A Top and Middle, B, and C; ×50 in A Bottom.)

Fig. 3.

(A) Representative traces from ring and sandwich preparations showing that neither preincubation with 20 μM hemoglobin nor the additional acute administration of 20 μM hemoglobin inhibited relaxation to A23187. (B) Representative traces from sandwich bioassay experiments confirming that A23187-evoked relaxations were sensitive to catalase, whereas ACh did not evoke detectable release of H₂O₂.

Fig. 4.

(A) Concentration-dependent relaxations of endothelium-denuded iliac artery rings evoked by enzymatically generated (xanthine plus XO) and authentic H₂O₂. Relaxations to both sources of H₂O₂ were effectively abolished by catalase. (B) Histograms showing that X/XO reduced smooth muscle membrane potential by <5 mV, and that significant hyperpolarization was observed only after administration of supraphysiological concentrations of authentic H₂O₂.