Central role of heterocellular gap junctional communication in endothelium-dependent relaxations of rabbit arteries

Abstract

1. The contribution of gap junctions to endothelium-dependent relaxation was investigated in isolated rabbit conduit artery preparations pre-constricted by 10 microM phenylephrine (PhE). 2. Acetylcholine (ACh) relaxed the thoracic aorta by approximately 60 % and the superior mesenteric artery (SMA) by approximately 90 %. A peptide possessing sequence homology with extracellular loop 2 of connexin 43 (Gap 27, 300 microM) inhibited relaxation by approximately 40 % in both artery types. Gap 27 also attenuated the endothelium-dependent component of the relaxation induced by ATP in thoracic aorta but did not modify force development in response to PhE. 3. NG-nitro-L-arginine methyl ester (L-NAME, 300 microM), an inhibitor of NO synthase, attenuated ACh-induced relaxation by approximately 90 % in the aorta but only by approximately 40 % in SMA (P < 0.05). Residual L-NAME-insensitive relaxations were almost abolished by 300 microM Gap 27 in aorta and inhibited in a concentration-dependent fashion in SMA (approximately 50 % at 100 microM and approximately 80 % at 10 mM). Gap 27 similarly attenuated the endothelium-dependent component of L-NAME-insensitive relaxations to ATP in aorta. 4. Responses to cyclopiazonic acid, which stimulates endothelium-dependent relaxation through a receptor-independent mechanism, were also attenuated by Gap 27, whereas this peptide exerted no effect on the NO-mediated relaxation induced by sodium nitroprusside in preparations denuded of endothelium. 5. ACh-induced relaxation of 'sandwich' mounts of aorta or SMA were unaffected by Gap 27 but completely abolished by L-NAME. 6. We conclude that direct heterocellular communication between the endothelium and smooth muscle contributes to endothelium-dependent relaxations evoked by both receptor-dependent and -independent mechanisms. The inhibitory effects of Gap 27 peptide do not involve homocellular communication within the vessel wall or modulation of NO release or action.

Figure 1. Original traces demonstrating reversible inhibition of ACh-induced relaxation by gap junction peptide 27

A, Gap 27 (300 μm) markedly inhibited the relaxant response to ACh, an effect that was fully reversible on washout for ≈60 min. B, in matched experiments relaxations to ACh showed no significant attenuation over time.

Figure 2. Concentration-relaxation curves showing the effects of gap junction peptide 27 against ACh and ATP in thoracic aorta in the presence and absence of l-NAME

A, Gap 27 (300 μm, ▴) inhibited the relaxation induced by ACh by ≈40% whereas l-NAME (300 μm, ▿) inhibited relaxation by ≈90%. ○, control responses; •, time-matched controls. Gap 27 further attenuated the residual relaxation observed in the presence of l-NAME (♦). B, analogous effects were found with ATP, symbols denoting the same experimental protocols as in A. In endothelium-denuded rings ATP evoked relaxation only at concentrations ≥ 300 μm (□).

Figure 3. Concentration-relaxation curves showing the effects of gap junction peptide 27 against CPA and SNP in the thoracic aorta

A, Gap 27 (300 μm, ▴) inhibited the relaxation to CPA by ≈30%, whereas relaxation was attenuated by ≈80% in the presence of l-NAME (300 μm, ▿). The residual relaxation observed in the presence of l-NAME was further reduced by Gap 27 (♦). CPA itself caused < 5% relaxation in endothelium-denuded preparations (□). B, SNP concentration-relaxation curves obtained in endothelium-denuded rings were not affected by Gap 27 (300 μm, ▴). In both panels: ○, control responses; •, time-matched controls.

Figure 4. Concentration-relaxation curves showing the effects of heptanol against ACh- (A), CPA- (B) and SNP- (C) induced relaxation in the thoracic aorta

Heptanol (3 mm, ▴) significantly reduced relaxation to all three agents. ○ and •, control and time-matched relaxations to each agent, respectively. SNP concentration-relaxation curves were obtained in endothelium-denuded rings.

Figure 5. Concentration-relaxation curves showing the effects of gap junction peptide 27 against ACh in superior mesenteric artery in the presence and absence of l-NAME

A, maximal control ACh-induced relaxation (○) was greater in the superior mesenteric artery than in the aorta, although Gap 27 (300 μm) again resulted in ≈40% inhibition (▴). In contrast to the aorta, however, l-NAME (300 μm) inhibited the maximum response to ACh by only ≈40% (▿). These inhibitory effects of Gap 27 and l-NAME were synergistic (♦). B, concentration-dependent augmentation of the inhibitory effects of l-NAME (300 μm) by Gap 27 over the range 100 μm to 10 mm. ▿, maximum relaxation induced by ACh in the presence of l-NAME; ♦, maximum relaxations to ACh in the additional presence of Gap 27.

Figure 6. Concentration-relaxation curves showing no effect of gap junction peptide 20 (3 mm, ▵) against responses to ACh in superior mesenteric artery

○ and •, control and time-matched responses to ACh, respectively.

Figure 7. Concentration-relaxation curves for ACh in superior mesenteric artery in the presence and absence of indomethacin

Indomethacin (10 μm, •) did not modulate control ACh-induced relaxations (○). The inhibition observed with Gap 27 (300 μm, ▵) or l-NAME (300 μm, ▿) was not amplified by indomethacin (▴ and ▾, respectively). Inhibition by the combination of Gap 27 and l-NAME (□) was similarly unaffected by indomethacin (▪).

Figure 8. Original traces showing effects of Gap 27 peptide and l-NAME on phenylephrine-induced contraction

Force development was not enhanced by Gap 27 (300 μm, A) and rises in tension induced by l-NAME (300 μm) were similar in the presence (C) or absence (B) of this peptide.

Figure 9. Original traces showing ACh-induced relaxation in sandwich mounts of endothelium-intact and -denuded strips of thoracic aorta in the presence and absence of gap junction peptide 27

A, control relaxation following constriction by phenylephrine (PhE). B, repeat protocol in the presence of Gap 27 (3 mm) showing no loss of relaxation. Gap 27 was similarly without effect on responses to ACh in sandwich mounts of mesenteric artery (not shown).

Figure 10. Concentration-relaxation curves for ACh in sandwich preparations from thoracic aorta (A) and superior mesenteric artery (B)

In contrast to the findings with intact rings Gap 27 (3 mm, ▴) was completely without effect, whereas l-NAME (300 μm, ▿) abolished responses to ACh in both artery types. ○ and •, control and time-matched controls, respectively, in both panels.

Figure 11. Responses of sandwich preparations to substance P

Original traces (A) and histograms (B) showing that Gap 27 did not affect responses to substance P (10 nm) in sandwich mounts from thoracic aorta, whereas l-NAME (300 μm) abolished relaxation completely.