Cysteamine increases expression and activity of H+-K+-ATPase of gastric mucosal cells in weaning piglets

Abstract

Aim: To determine the in vivo and in vivo effects of cysteamine (CS) on expression and activity of H(+)-K(+)-ATPase of gastric mucosal cells in weaning piglets.

Methods: Eighteen litters of newborn Xinhuai piglets were employed in the in vivo experiment and allocated to control and treatment groups. From 12 d of age (D12), piglets in control group were fed basal diet, while the treatment group received basal diet supplemented with 120 mg/kg CS. Piglets were weaned on D35 in both groups. Six piglets from each group (n = 6) were slaughtered on D28 (one week before weaning), D35 (weaning), D36.5, D38, D42, and D45 (36 h, 72 h, one week and 10 d after weaning), respectively. Semi-quantitative RT-PCR was performed to determine the levels of H(+)-K(+)-ATPase mRNA in gastric mucosa. H(+)-K(+)-ATPase activity in gastric mucosa homogenate was also determined. Gastric mucosal epithelial cells from piglets through primary cultures were used to further elucidate the effect of CS on expression and activity of H(+)-K(+)-ATPase in vivo. Cells were treated for 20 h with 0.001, 0.01, and 0.1 mg/mL of CS (n = 4), respectively. The mRNA expression of H(+)-K(+)-ATPase and somatostatin (SS) as well as the H(+)-K(+)-ATPase activity were determined.

Results: in vivo, both mRNA expression and activity of H(+)-K(+)-ATPase in gastric mucosa of control group exhibited a trend to increase from D28 to D45, reaching a peak on D45, but did not show significant age differences. Furthermore, neither the mRNA expression nor the activity of H(+)-K(+)-ATPase was affected significantly by weaning. CS increased the mRNA expression of H(+)-K(+)-ATPase by 73%, 53%, 30% and 39% on D28 (P = 0.014), D35 (P = 0.017), D42 (P = 0.013) and D45 (P = 0.046), respectively. In accordance with the mRNA expression, H(+)-K(+)-ATPase activities were significantly higher in treatment group than in control group on D35 (P = 0.043) and D45 (P = 0.040). In vivo, CS exhibited a dose-dependent effect on mRNA expression and activity of H+-K+-ATPase. Both H(+)-K(+)-ATPase mRNA expression and activity in gastric mucosal epithelial cells were significantly elevated after 20 h of exposure to the moderate (H(+)-K(+)-ATPase expression: P=0.03; H(+)-K(+)-ATPase activity: P = 0.014) and high concentrations (H(+)-K(+)-ATPase expression: P=0.017; H(+)-K(+)-ATPase activity: P = 0.022) of CS. Significant increases in SS mRNA expression were observed to accompany the elevation of H(+)-K(+)-ATPase expression and activity induced by the moderate (P = 0.024) and high concentrations (P = 0.022) of CS. Low concentration of CS exerted no effects either on expression and activity of H(+)-K(+)-ATPase or on SS mRNA expression in cultured gastric mucosal epithelial cells.

Conclusion: No significant changes are observed in mRNA expression and activity of H(+)-K(+)-ATPase in gastric mucosa of piglets around weaning from D28 to D45. CS increases expression and activity of gastric H(+)-K(+)-ATPase in vivo and in vivo. SS is involved in mediating the effect of CS on gastric H(+)-K(+)-ATPase expression and activity in weaning piglets.

Figure 1

Expression (A_1-2) and activity (B) of H+-K+-ATPase in vivo. Lane M: PUC19 marker; lanes 1-6: piglets at 28, 35, 36.5, 38, 42, 45 d of age in control group, respectively; lanes 7-12: piglets at 28, 35, 36.5, 38, 42, 45 d of age in treatment, respectively. Bars without a common superscript representing significant differences between age groups (small letters a and b for control group and capital letters A and B for treatment group); ^aP<0.05 indicating differences between control and treatment groups at the same age.

Figure 2

Effect of CS on expression (A_1-2) and activity (B) of H+-K+-ATPase in vitro. Lane M: PUC19 marker, lanes 1-2: control; lanes 3-4: low concentration group; lanes 5-6: moderate concentration group; lanes 7-8: high concentration group. ^aP<0.05 vs 0 mg/mL of CS.

Figure 3

Effect of CS on SS mRNA expression in vitro. Lane M: PUC19 marker, lanes 1 and 2: control; lanes 3 and 4: low concentration group; lanes 5 and 6: moderate concentration group; lanes 7 and 8: high concentration group (A,B). ^aP<0.05 vs 0 mg/mL of CS.