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. 2019 May 28;9:7932. doi: 10.1038/s41598-019-44333-w

Figure 7.

Figure 7

The CGRP-mediated increase in ethidium uptake induced by capsaicin is initiated by Panx-1-formed channel opening in endothelial cells. (A) Representative images of the Lucifer Yellow uptake (green) induced by perfusion of 1 µM capsaicin and 100 nM CGRP in mesenteric resistance arteries during the stimulation period (10 min) or 1 h after. (B) Representative images of Lucifer Yellow uptake attained after 20 min stimulation of isolated mesenteric resistance arteries with 1 µM capsaicin. In these experiments, Lucifer Yellow and capsaicin were applied in the bath solution. Note that the Lucifer Yellow fluorescent signal is only observed at the inner side of the internal elastic lamina (IEL), confirming that capsaicin activated the uptake of this dye exclusively in the endothelium. (C) Time course of the ethidium (Et) uptake achieved in control conditions and after the stimulation with 1 µM capsaicin or 100 nM CGRP in primary cultures of mesenteric endothelial cells. The horizontal bar indicates the period of stimulation. (D) Analysis of the Et uptake rate observed during the stimulation with capsaicin (Caps) or CGRP in control conditions and in the presence of 1 µM CGRP8–37 or 1 mM probenecid (Prob). The rate of ethidium uptake was assessed by calculating the slope of the increase in fluorescence intensity along the time in basal conditions and during the stimulation period. (E) Time course of the ethidium uptake induced by CGRP in the absence and presence of a combination of the connexin blocking peptides 37,43GAP27 (200 µM) plus 40GAP27 (200 µM) or the Panx-1 blocking peptide 10panx (60 µM). Note that, after treatment with GAP 27 peptides, the ethidium uptake curve shows two components: an initial increase similar to control that starts to decline after 6 min. Changes in ethidium-fluorescence signal are expressed in arbitrary units (AU). Numbers inside the bars indicate the n value. Values are means ± SEM. *P < 0.05 vs control by one-way ANOVA plus Dunnett post hoc test. †P < 0.01 vs basal by Student’s paired t-test. #P < 0.05 vs control by two-way ANOVA plus Fisher´s LSD post hoc test.