Effects of connexin-mimetic peptides on gap junction functionality and connexin expression in cultured vascular cells

Abstract

1. We have investigated the effects of connexin-mimetic peptides homologous to the Gap 26 and Gap 27 domains of Cxs 37, 40 and 43 against gap junctional communication and connexin expression in rat aortic endothelial cells (RAECs) and A7r5 myocytes. 2. Immunostaining and Western blot analysis confirmed the presence of gap junction plaques containing Cx43, but not Cx40, in RAECs, whereas plaques containing Cxs 40 and 43 were evident in A7r5 cells. Expression of Cx37 was limited in RAECs and absent from A7r5 cells. 3. Under control conditions calcein-loaded RAECs transferred dye to approximately 70% of subjacent A7r5 cells after coculture for 4-5 h. Dye transfer was inhibited by a peptide targeted to Cxs 37 and 43 ((37,43)Gap 27), but minimally affected by peptides targeted to Cxs 37 and 40 ((37,40)Gap 26 and (40)Gap 27). These findings suggest that the myoendothelial gap junctions that couple RAECs and A7r5 cells are constructed principally from Cx43. 4. Inhibition of dye transfer from RAECs to A7r5 cells cocultured in the presence of (37,43)Gap 27 plus (37,40)Gap 26 for 5 h was fully reversible. 5. In A7r5 cells, endogenous expression of Cx40 and Cx43 was unaffected by incubation with (37,43)Gap 27, (37,40)Gap 26, either individually or in combination, and the peptide combination did not impair connexin trafficking or the de novo formation of gap plaques in A7r5 cells transfected to express Cx43-GFP. 6. Treatment of A7r5 cells with (37,43)Gap 27 plus (37,40)Gap 26 abolished synchronized oscillations in intracellular [Ca2+] induced by the alpha1-adrenoceptor agonist phenylephrine. 7. The reversibility and lack of effect of the peptides on plaque formation suggests that they may be considered ideal probes for functional studies of connexin-mediated communication in the vascular wall.

Figure 1

Connexin profiles in A7r5 cells and RAECs. (a, b) Costaining of A7r5 cells for Cx43 (green) and Cx40 (red) at two magnifications, with gap junction plaques containing both connexin subtypes identified in yellow. (c) Cx43 staining of RAECs. (d) Cx40 staining of RAECs. (e) Cx37 staining of RAECs. (f) von Willebrand factor staining of RAECs (green) with nuclei (red) identified by propidium iodide. Cells were viewed at a magnification of × 40 and in some cases a zoom of up to 1.4 was applied. The image size was calculated by the Biorad Lasersharp software. Bars=10 μM. Arrows indicate gap junction plaques at points of cell-to-cell contact.

Figure 2

Western blot analysis of connexin expression profiles in A7r5 and RAECs. NP, P1 and P2 denote nonphosphorylated and phosphorylated isoforms of Cx43, respectively.

Figure 3

Effects of gap junction blockade on dye transfer from endothelial to smooth muscle cells. A7r5 cells were labelled with PHK26 (red) and overlaid with calcein-loaded RAECs (green). (a) Percentage of RAECs donating dye to different numbers of A7r5 cells after 2, 3, 4 and 5 h under control conditions. (b) Effect of connexin-mimetic peptides on dye transfer at 600 μM each. (c) Recovery of dye transfer following washout of a combination of ^37,43Gap 27+^37,40Gap 26 peptides at 300 μM each. (d) Effect of 18α-GA (25 μM) on dye transfer followed by recovery. (e) Typical field of view of highly coupled cells under control conditions. (f) Typical field of view of cells cocultured for 5 h in the presence of ^37,43Gap 27+^37,40Gap 26 at 300 μM each. (g) Typical field of view following washout of this peptide combination for 1 h. Asterisks identify RAECs loaded with calcein. Bars=10 μM. Magnification × 40. ^*P<0.05, ^**P<0.001 compared to control.

Figure 4

Integrity of gap junction plaques in A7r5 cells fixed and stained for Cx43 (green) and Cx40 (red) following 4 h incubation with connexin-mimetic peptides. (a) Control, (b) ^37,43Gap 27 (600 μM), (c) ^37,40Gap 26 (600 μM), (d) ^37,43Gap 27+^37,40Gap 26 (300 μM each). (e) Histogram showing plaque integrity quantified by analysis of Cx43 and Cx40 fluorescence at the plasma membrane subtracted from background fluorescence following the various treatments. Results are given as mean relative fluorescence±s.e.m. Bars=10 μm. Magnification × 40.

Figure 5

Effect of ^37,43Gap 27 + ^37,40Gap 26 (300 μM each) on the assembly of gap junction plaques in A7r5 cells transfected with Cx43-GFP: (a) 6 h post-transfection, (b) 9 h post-transfection, (c) 18 h post-transfection, (d) 18 h post-transfection in the presence of ^37,43Gap 27+^37,40Gap 26 added 6 h after transfection, that is, the time point corresponding to (a). Bars=10 μm. Magnfication × 40. Arrows indicate gap junction plaques at points of cell-to-cell contact.

Figure 6

Effects of connexin-mimetic peptides on synchronized intracellular Ca²⁺ oscillations. Cells were loaded with Fura2 AM under control conditions or following incubation with connexin-mimetic peptides or 18α-GA for 90 min. They were then treated with phenylephrine and Fura2 fluorescence at 340 : 380 nm recorded for 5 min. (a) A7r5 cells, (b) RAECs, (c) A7r5 cells following treatment with ^37,43Gap 27+^37,40Gap 26 (300 μM each), (d) A7r5 cells following treatment with 25 μM 18α-GA.