Endothelial Connexin37 and Connexin40 participate in basal but not agonist-induced NO release

Abstract

Background: Connexin37 (Cx37) and Cx40 are crucial for endothelial cell-cell communication and homeostasis. Both connexins interact with endothelial nitric oxide synthase (eNOS). The exact contribution of these interactions to the regulation of vascular tone is unknown.

Results: Cx37 and Cx40 were expressed in close proximity to eNOS at cell-cell interfaces of mouse aortic endothelial cells. Absence of Cx37 did not affect expression of Cx40 and a 50 % reduction of Cx40 in Cx40(+/-) aortas did not affect the expression of Cx37. However, absence of Cx40 was associated with reduced expression of Cx37. Basal NO release and the sensitivity for ACh were decreased in Cx37(-/-) and Cx40(-/-) aortas but not in Cx40(+/-) aortas. Moreover, ACh-induced release of constricting cyclooxygenase products was present in WT, Cx40(-/-) and Cx40(+/-) aortas but not in Cx37(-/-) aortas. Finally, agonist-induced NO-dependent relaxations and the sensitivity for exogenous NO were not affected by genotype.

Conclusions: Cx37 is more markedly involved in basal NO release, release of cyclooxygenase products and the regulation of the sensitivity for ACh as compared to Cx40.

Fig. 1

Mouse aortic endothelial cells express Cx37 and Cx40 at cell-cell interfaces. a-c Representative images of en face confocal immunofluorescent stainings for Cx37 (a), Cx40 (b) or Cx43 (c) in wild-type (WT) mouse aortic endothelium, respectively. d-f Representative images of en face confocal immunofluorescent stainings for Cx37 (d), Cx40 (e) or Cx43 (f) in Cx37^−/− mouse aortic endothelium, respectively. g-i Representative images of en face confocal immunofluorescent stainings for Cx37 (g), Cx40 (h) or Cx43 (i) in Cx40^−/− mouse aortic endothelium, respectively. Scalebar equals 15 μM

Fig. 2

Cx37-deficiency does not affect expression of Cx40 while Cx40-deficiency reduces expression of Cx37. a-b Total mRNA was isolated from aortas obtained from WT, Cx37^−/−, Cx40^−/− or Cx40^+/− mice (N = 3). Thereafter, the expression of Gja4 (A, the gene coding for Cx37) or Gja5 (B, the gene coding for Cx40) was assessed by real-time PCR. Cx40 is expressed at WT levels in the Cx37^−/− aortas while Cx37 showed large variation and tended to be reduced in the Cx40^−/− aortas (p = 0.08). Moreover, expression of Cx37 is not reduced in Cx40^+/− aortas. c Representative image of Cx37 and Cx40 protein expression in scraped mouse aortic endothelial cells as assessed by western blot. Tubulin and von Willebrand factor (vWf) were assessed for equal protein loading. d Cx37 protein quantification (N = 6–8). Similar to the expression of Cx37 at the level of RNA, Cx37 expression was reduced by half in Cx40^−/− aortic endothelial cells whereas Cx37 expression was not significantly reduced in Cx40^+/− endothelium. e Cx40 protein quantification (N = 6–8). Cx40 was diminished by half in Cx40^+/− endothelial cells and was not altered in Cx37^−/− endothelium. Values are expressed as mean ± SEM. *, ** or *** p < 0.05, 0.01 or 0.001 vs WT control, respectively

Fig. 3

Interactions between connexins and eNOS are present at cell-cell interfaces between mouse aortic endothelial cells. Representative images of proximity ligation assays performed with antibodies targeting Cx37 and eNOS or Cx40 and eNOS. a Close proximity of Cx37 and eNOS in WT mouse aortic endothelium. b Cx40 staining to highlight the intercellular gap junctions. c Merge of (a) and (b) highlighting close proximity of eNOS and Cx37 at the gap junctions between endothelial cells. d Close proximity of Cx40 and eNOS in WT endothelium. e Cx37 staining to highlight the intercellular gap junctions. f Merge of (d) and (e) highlighting close proximity of eNOS and Cx40 at the gap junctions between endothelial cells. g-i Control assays revealed that the intense red staining observed in a-c was no longer observed after omitting the eNOS antibody from the proximity ligation assays (g-i) even though cell-cell junctions could clearly be visualized (h-i). j-l: Similar control stainings, which revealed similar results, were performed with the Cx40 antibodies, but not the Cx37 antibodies present during the proximity ligation assays. Scalebar equals 20 μM

Fig. 4

Cx37 is implicated in basal NO release. Aortic segments obtained from WT, Cx37^−/−, Cx40^−/− or Cx40^+/− mice were mounted in a wire-myograph and concentration-response curves for PHE were generated in presence or absence of L-NAME (100 μM). a Inhibition of NO synthesis by L-NAME increased the contractile amplitude for PHE in WT aortas indicating presence of basal NO release. This effect of L-NAME was not observed in aortas isolated from Cx37^−/− (b) or Cx40^−/− (c) mice. In contrast (d), in aortas obtained from Cx40^+/− mice L-NAME did increase the contractile amplitude of PHE. Values are expressed as mean ± SEM. N = 6–10. **, *** p < 0.01, 0.001 vs control, respectively. e Representative image and quantification of eNOS protein expression in protein samples from scraped mouse aortic endothelial cells as assessed by western blot. Values are expressed as mean ± SEM. N = 6–8. * p < 0.05 vs WT

Fig. 5

Cx37 may be implicated in the release of COX products but not in activated NO release. Concentration-response curves for the endothelium-specific agonist ACh were generated in PHE pre-contracted aortas obtained from WT, Cx37^−/−, Cx40^−/− or Cx40^+/− mice. a WT aortas displayed relaxations in response to ACh (black) which were i) increased in presence of INDO (blue) and ii) inhibited in presence of L-NAME (red) or in presence of L-NAME and INDO (green). b Cx37^−/− aortas displayed relaxation responses to ACh (black) which were i) unaffected by presence of INDO (blue), ii) inhibited in presence of L-NAME (red) or in presence of L-NAME and INDO (green). c Aortas obtained from Cx40^−/− mice (c) displayed relaxations in response to ACh (black) which were i) slightly, but significantly, increased in presence of INDO (blue) and ii) inhibited in presence of L-NAME (red) or in presence of L-NAME and INDO (green). d Aortas obtained from Cx40^+/− mice displayed relaxations in response to ACh (black) which were i) increased in presence of INDO (blue) and ii) inhibited in presence of L-NAME (red) or in presence of L-NAME and INDO (green). Values are expressed as mean ± SEM. N = 6–10. *, ** or *** p < 0.05, 0.01 or 0.001 vs. control, respectively