NO, via its target Cx37, modulates calcium signal propagation selectively at myoendothelial gap junctions

Abstract

Background: Gap junctional calcium signal propagation (transfer of calcium or a calcium releasing messenger via gap junctions) between vascular cells has been shown to be involved in the control of vascular tone. We have shown before that nitric oxide (NO) inhibits gap junctional communication in HeLa cells exclusively expressing connexin 37 (HeLa-Cx37) but not in HeLa-Cx40 or HeLa-Cx43. Here we studied the effect of NO on the gap junctional calcium signal propagation in endothelial cells which, in addition to Cx37, also express Cx40 and Cx43. Furthermore, we analyzed the impact of NO on intermuscle and on myoendothelial gap junction-dependent calcium signal propagation. Since specific effects of NO at one of these three junctional areas (interendothelial/ myoendothelial/ intermuscle) may depend on a differential membrane localization of the connexins, we also studied the distribution of the vascular connexins in small resistance arteries.

Results: In endothelial (HUVEC) or smooth muscle cells (HUVSMC) alone, NO did not affect gap junctional Ca2+ signal propagation as assessed by analyzing the spread of Ca2+ signals after mechanical stimulation of a single cell. In contrast, at myoendothelial junctions, it decreased Ca2+ signal propagation in both directions by about 60% (co-cultures of HUVEC and HUVSMC). This resulted in a longer maintenance of calcium elevation at the endothelial side and a faster calcium signal propagation at the smooth muscle side, respectively. Immunohistochemical stainings (confocal and two-photon-microscopy) of cells in co-cultures or of small arteries revealed that Cx37 expression was relatively higher in endothelial cells adjoining smooth muscle (culture) or in potential areas of myoendothelial junctions (arteries). Accordingly, Cx37 - in contrast to Cx40 - was not only expressed on the endothelial surface of small arteries but also in deeper layers (corresponding to the internal elastic lamina IEL). Holes of the IEL where myoendothelial contacts can only occur, stained significantly more frequently for Cx37 and Cx43 than for Cx40 (endothelium) or Cx45 (smooth muscle).

Conclusion: NO modulates the calcium signal propagation specifically between endothelial and smooth muscle cells. The effect is due to an augmented distribution of Cx37 towards myoendothelial contact areas and potentially counteracts endothelial Ca2+ signal loss from endothelial to smooth muscle cells. This targeted effect of NO may optimize calcium dependent endothelial vasomotor function.

Figure 1

NO reduced gap junctional Ca²⁺ signal propagation in HeLaCx37 cells. A depicts the fluorescence image (Fura2, excitation 380 nm), the stimulated cell (red), adjacent cells (green) and secondary adjacent cells (blue). All adjacent cells together are counted as neighbouring cells. B. The Ca²⁺ concentration (ratio) increased after mechanical stimulation of the red marked HeLaCx43 cell (0 s) and the signal propagated to some neighbouring cells (8 s, 12 s). C depicts the time course of the Ca_i²⁺ increase. The signal spread with a time delay of up to 7 s into adjacent (green, 1A green) but hardly to secondary adjacent (blue, 1A blue) cells. D. After stimulation, 30 ± 4% of the neighbouring HeLaCx37 cells showed a Ca²⁺_i increase (con). NO (15 min, 2 μM SNAP) reduced the number of cells responding with an elevated Ca²⁺_i signal to 16 ± 3% (p < 0.05, n = 15, w = C = 3) whereas it was virtually unchanged in HeLa-Cx43 cells (con: 38 ± 6%, NO: 37 ± 5%; n = 13, w = C = 3).

Figure 2

NO did not reduce gap junctional Ca²⁺ signal propagation in HUVEC. A depicts the fluorescence image (Fura2, excitation 380 nm), the stimulated cell (red), adjacent cells (green), and secondary adjacent cells (blue). All adjacent cells together are counted as neighbouring cells. B. The Ca²⁺ concentration (ratio) increased (0 s) after mechanical stimulation of the red marked HUVEC and the Ca²⁺_i signal propagated to most neighbouring cells within 6 s. The time course of the Ca²⁺_i increase in the marked cells (A) is shown in C. Ca²⁺_i increased in the stimulated cell (red) and the signal propagated with a time delay of up to 3 s to adjacent (green) and up to 10 s to secondary adjacent (blue) cells. In the HUVEC monolayer, incubation with NO (15 min, 2 μM SNAP) did not reduce the number of responding (D) but increased the time delay (E) of the Ca²⁺_i transfer to neighbouring endothelial cells. n = 39-66, w = 6, C = 4; *: p < 0.01, NG.

Figure 3

NO reduced gap junctional Ca²⁺ signal propagation in siRNA (against Cx43) treated HUVEC. A. Treatment with siRNA (24 h) against Cx43 decreased the amount of Cx43 protein in these cells. B. Depicts the time course of the Ca²⁺_i increase in the stimulated (red), adjacent (green) and secondary adjacent (blue) cells under control conditions (top) and after application of NO (bottom, 15 min 2 μM SNAP; representative experiment). The result of all experiments (n = 21, w = 6, C = 3; *: p < 0.05) is shown in C, demonstrating a decrease of responding cells after NO treatment.

Figure 4

NO inhibited myoendothelial signal propagation in co-cultured HUVEC and HUVSMC. A/B depicts the immunohistochemical staining of Cx37 (EC, red), CD31 (EC, blue) and α-smooth-muscle actin (SMC, red) in a co-culture of endothelial and smooth muscle cells (A, scale bar 30 μm) and an area with endothelial cells only within the same co-culture (B, scale bar 30 μm). C depicts the number of responding cells in co-cultures of HUVEC and HUVSMC. Treatment with NO (15 min, 2 μM SNAP) significantly reduced the Ca²⁺_i signal transfer from SMC to EC and also from EC to SMC whereas it did not affect the signal transfer from EC to EC and from SMC to SMC (n = 13-21, w = C = 5; p < 0.05). In the remaining responding cells, the time delay (D) was significantly increased from SMC to EC whereas the signal spread faster from SMC to SMC after exposure to NO. The amplitude of the Ca²⁺_i increase (in the remaining responding cells) was unchanged in all cells (n = 17-106, w = 14-21, C = 6; *: p < 0.05, con vs. NO, NG). E. The decrease of the mechanically induced calcium rise in the initial stimulated cells was reduced by incubation with SNAP (15 min, 2 μM).

Figure 5

Distribution of Cx37 within the internal elastic lamina. A. Representative 2-photon-image of a small resistance artery for visualization of the internal elastic lamina and the connexins. Cx37 (red) located in the small holes (dark dots) within the internal elastic lamina (autofluorescence, green). Arrows indicate some of the holes in which Cx37 could be detected; scale bar: 25 μm. B depicts the summary of n = 7-10 experiments (3 vessels each) revealing the percentage of holes in the internal elastic lamina that contain the different vascular Cx (*: p < 0.05 vs. Cx40; #: p < 0.05 vs. Cx45, NG).

Figure 6

Location of vascular Cx across the vessel wall. A. Confocal images of triple (Cx, α-actin, CD31) immunohistochemical stainings of Cx40, Cx45, Cx37, and Cx43 in small resistance arteries. Left panel: Overlay of a z-series in xy-direction, right panel: Cross section (slice) of the z-stack in yz-direction along the yellow lines in the z-stack. The arrows depict the Cx expression in ECL (yellow) or beyond EC and within SMC (SMCL, white), scale bars: 10 μm. B. Summary of the Cx distribution within the ECL and SMCL for all Cx (n = 4-8, at least 3 vessels each; *: p < 0.001).