A mechanism by which the osmotic laxative magnesium sulphate increases the intestinal aquaporin 3 expression in HT-29 cells

Abstract

Aims We have suggested that an osmotic laxative, magnesium sulphate (MgSO 4), may act as a cathartic in a very rational manner by increasing the aquaporin 3 (AQP3) expression level and by changing osmotic pressure in the colon. In this study, we examined the mechanism by which MgSO 4 increases the intestinal AQP3 expression level by using the human colon cancer HT-29 cell line. Main methods After the addition of MgSO 4 to HT-29 cells, the expression levels of AQP3 mRNA and protein were measured using real-time RT-PCR and western blotting, respectively. The intracellular Mg 2+ concentration, adenylate cyclase (AC) activity and protein kinase A (PKA) activity were also determined. The phosphorylated cAMP response element-binding protein (CREB) expression levels were determined by western blotting. Key findings The AQP3 mRNA expression level started to increase significantly at 1 h after MgSO 4 addition and peaked at 9 h, at a level 3 times as high as the control levels. The AQP3 protein expression level started to increase 6 h after the addition and reached a level almost twice as high as the control levels by hour 12. In the HT-29 cells treated with MgSO 4, there was a 1.4-fold increase in the intracellular Mg 2+ concentration, a 1.5-fold increase in AC activity, a 1.6-fold increase in PKA activity, and a significant increase in phosphorylation of the CREB. Significance These results suggest that the AC activation caused by an increase in the intracellular Mg 2+ concentration may trigger CREB phosphorylation through PKA activation and promote AQP3 gene transcription.