Biphasic increase of gap junction coupling induced by dipyridamole in the rat aortic A-10 vascular smooth muscle cell line

Abstract

The rat aortic smooth muscle cell line A-10 was used to investigate the effect of dipyridamole on the gap junction coupling of smooth muscle cells. The scrape loading/dye transfer (SL/DT) technique revealed that dipyridamole concentrations between 5 μM and 100 μM significantly increased gap junction coupling. The adenosine receptor antagonist MRS 1754, as well as the PKA inhibitors Rp-cAMPS and H-89 were able to inhibit the dipyridamole-related increase in coupling, while forskolin and Br-cAMP also induced an enhancement of the gap junction coupling. Regarding the time-dependent behaviour of dipyridamole, a short-term effect characterised by an oscillatory reaction was observed for application times of less than 5 h, while applications times of at least 6 h resulted in a long-term effect, characterised by a constant increase of gap junction coupling to its maximum levels. This increase was not altered by prolonged presence of dipyridamole. In parallel, a short application of dipyridamole for at least 15 min was found to be sufficient to evoke the long-term effect measured 6 h after drug washout. We propose that in both the short-term and long-term effect, cAMP-related pathways are activated. The short-term phase could be related to an oscillatory cAMP effect, which might directly affect connexin trafficking, assembly and/or gap junction gating. The long-term effect is most likely related to the new expression and synthesis of connexins. With previous data from a bovine aortic endothelial cell line, the present results show that gap junction coupling of vascular cells is a target for dipyridamole.

Fig. 1

Dipyridamole affects gap junction coupling of rat aortic A-10 smooth muscle cells. Cells grown to a monolayer were treated with dipyridamole (dip.) for 24 h before SL/DT experiments. a The fluorescence micrographs show the diffusion of LY (green) in cells cultured under control conditions (eth.) and incubated with 50 μM dip. The cellular nuclei were stained with DAPI (blue). The scale bar corresponds to 100 μm. b Quantitative evaluation of the dye diffusion distance in relationship to dipyridamole concentration. Dipyridamole induced a significant increase of the dye diffusion distance compared with 0.5% ethanol, which alone did not affect the dye diffusion distance compared to untreated cells (unt.). A significant increase in gap junction coupling was observed at dipyridamole concentrations of at least 5 μM

Fig. 2

a The antagonist of the adenosine receptor A_2B MRS 1754 as well as (b) the PKA inhibitors Rp-cAMPS (200 μM) and H-89 (15 μM) significantly (# for p < 0.05) inhibited the dipyridamole- (dip., 50 μM) induced enhancement of gap junction coupling. c In contrast, forskolin (for., 100 μM) and 8-Br-cAMP (1 mM) significantly increased gap junction coupling after 6 h of incubation time similar to dipyridamole

Fig. 3

Time-dependent effect of dipyridamole on gap junction coupling. The figure shows the change of the diffusion distance induced by 10 μM (●) or 50 μM (▲) dipyridamole (7) after an application time of 0.25 h, 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 9 h or 24 h compared with control cells (0.5% ethanol (■)). After the respective time period, SL/DT experiments were performed (see material and methods). The analysis of dye diffusion revealed that dipyridamole induced a change in the gap junction coupling of A-10 smooth muscle cell line in a biphasic manner, which could be separated into short-term (0.25 h to 5 h) and long-term effects (over 6 h). Short-term behaviour was characterised by an oscillatory change of coupling by high dose dipyridamole (for more detail see table 1). The long-term effect was observed after at least 6 h of incubation and reached a constant level for both low (10 μM) and high concentrations (50 μM) of dipyridamole. The further presence of dipyridamole sustained the enhanced gap junction coupling, while a washout of the drug correlated with a decline of gap junction coupling to a basal level within the next 24 h (dashed line for 50 μM and dotted line for 10 μM)

Fig. 4

A short period of dipyridamole application for 15 min induced a long-term effect on gap junction coupling that lasted for 18 h. Cells were incubated for 15 min with 10 μM (●) or 50 μM (▲) dipyridamole and were compared with control cells (■). After drug washout, the gap junction coupling was analysed using SL/DT experiments as described above (material and methods) at the following time points: 0 h, 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 12 h, 18 h and 24 h. The figure shows that both doses of dipyridamole evoked a significant long-term increase of gap junction coupling that was first detected 5–6 h after drug washout