The Role of Phosphorylated Cx43 on PKC Mediated Ser368 in Lung Injury Induced by Seawater Inhalation

Abstract

Seawater aspiration may result in acute lung injury/acute respiratory distress syndrome (ALI/ARDS), which is characterized by pulmonary inflammation and lung edema that closely related to pulmonary barrier dysfunction and intracellular communication. The aim of the present research was to explore the role of connexion 43 (Cx43) in seawater aspiration-induced ALI/ARDS. The results from in vivo experiments showed that seawater inhalation led to increased expression of p-PKC and phosphorylated Cx43 (p-Cx43), which were followed by protein rich fluid leakage and TNF-α and IL-1β secretion. Besides, the results from in vitro tests proved that the expression of p-PKC directly influenced phosphorylation state of Cx43 and its function, which could further affect the inflammatory factors secretion and intercellular communication. In conclusion, seawater aspiration causes p-Cx43 expression by PKC pathway, which is involved in the on come and development of pulmonary inflammation and lung edema.

Fig. 1

Pulmonary edema and microvascular permeability induced by seawater aspiration. Data are mean ± SD, n = 8. ^* P < 0.01 versus control; ^** P < 0.05 versus ^* P.

Fig. 2

Secretion of TNF-α and IL-1β in lung tissues (a and b). Data are mean ± SD, n = 8. ^* P < 0.05 versus control; ^* P < 0.05 versus **P. And TNF-α and IL-1β secretion in seawater stimulated A549 cells (c and d). ^# P < 0.05 versus ^* P.

Fig. 3

p-PKC expression in lungs stimulated by seawater. After protein quantitation, Western blot was performed and the ratios of p-PKC and PKC versus β-actin were obtained for each group to examine the p-PKC and PKC content. The expression of PKC kept stable after seawater inhalation, while p-PKC expression was significantly increased by seawater, ^* P < 0.05 versus control.

Fig. 4

Expression of phosphorylated Cx43 on p-Ser368 in lung stimulated by seawater. After protein quantitation, Western blot was performed and the ratios of phosphorylated Cx43 on p-Ser368 versus β-actin were obtained. The expression of p-Ser368 of Cx43 increased once seawater inhaled into lungs, ^* P < 0.05 versus control.

Fig. 5

The expression of p-PKC in seawater stimulated A549 cells. After protein quantitation, western blot was performed to evaluate p-PKC expression in A549 cells. Seawater, as well as PMA, markedly increased p-PKC expression ^* P < 0.05 versus control, while inhibitor of PKC (STS) inhibited the expression of p-PKC induced by seawater, ^# P < 0.05 versus ^* P.

Fig. 6

Expression of phosphorylated Cx43 on p-Ser368 in A549cells exposed to seawater. After protein quantitation, western blot was also performed to examine expression of phosphorylated Cx43 on p-Ser368. Seawater and PMA increased p-Ser368 of Cx43 expression^* P < 0.05 versus control, while inhibitor of PKC (STS) inhibited p-Ser368 of Cx43 expression stimulated by seawater, ^# P < 0.05 versus ^* P.

Fig. 7

Dye transfer in A549 cells. The panels showed regions of A549 cells on slides scrape-loaded with Lucifer yellow (LY): a Control; b seawater; c seawater + STS; d PMA. Green indicated LY, including donor cells initially loaded with LY and recipient cells linked together by gap junctional intercellular communication.

Fig. 8

Quantification of dye transfer in A549 cells. ^* P < 0.05. ^# P < 0.05 versus ^* P.