Gap junctions regulate vessel diameter in chick chorioallantoic membrane vasculature by both tone-dependent and structural mechanisms

Abstract

Objective: In this study, we examined the impact of gap junction blockade on chick chorioallantoic membrane microvessels.

Methods: Expression of Cx37, Cx40/42, and Cx43 in chick chorioallantoic membrane tissue was studied by PCR, Western blot, and confocal immunofluorescence microscopy. Vessel diameter changes occurring under gap junction blockade with carbenoxolone (175 µmol/L), palmitoleic acid (100 µmol/L), ⁴³ GAP27 (1 mmol/L) were analyzed by intravital microscopy. To analyze vascular tone, chick chorioallantoic membrane vessels were exposed to a vasodilator cocktail consisting of acetylcholine (10 μmol/L), adenosine (100 μmol/L), papaverine (200 μmol/L), and sodium nitroprusside (10 μmol/L).

Results: In chick chorioallantoic membrane lysates, Western blot analysis revealed the expression of Cx40 and Cx43. Immunofluorescence in intact chick chorioallantoic membrane vasculature showed only Cx43, limited to arterial vessel walls. Upon gap junction blockade (3 hours) arterial and venous diameters decreased to 0.50 ± 0.03 and 0.36 ± 0.06 (carbenoxolone), 0.72 ± 0.08 and 0.63 ± 0.15 (palmitoleic acid) and 0.77 ± 0.004 and 0.58 ± 0.05 (GAP27), relative to initial values. Initially, diameter decrease was dominated by increasing vascular tone. After 6 hours, however, vessel tone was reduced, suggesting structural network remodeling.

Conclusions: Our findings suggest a major role for connexins in mediating acute and chronic diameter changes in developing vascular networks.

Keywords: CAM; carbenoxolone; chick chorioallantoic membrane; connexin 43; gap junctions; vascular remodeling.