Effect Of Cysteamine On Expression And Activity Of H～+-K～+-ATPase In Gastric Mucosa Of Pre-and Post-weaning Piglets And Possible Mechanisms Involved

The effects of cysteamine on mRNA expression and activity of H⁺-K⁺-ATPase in gastric mucosa of pre- and post-weaning piglets and possible mechanisms involved were investigated both in vivo and in vitro. The piglets were fed diet supplemented with 120mg.kg^-1 cysteamine and a semi-quantitative RT-PCR was applied to determine the levels of H⁺-K⁺-ATPase mRNA in gastric mucosa, while H⁺-K⁺-ATPase activity in gastric mucosa homogenate was also determined. In addition, cultured epithelial cells isolated from gastric mucosa of piglets were treated with cysteamine. The SS expression, mRNA expression and activity of H⁺-K⁺-ATPase were determined. Furthermore, parietal cells were purified to elucidate the possible direct action of cysteamine on H⁺-K⁺-ATPase activity.1. Effects of cysteamine on mRNA expression and activity of H⁺-K⁺-ATPase in gastric mucosa of pre- and post-weaning piglets.18 litters of newborn piglets were divided randomly to control and treatment groups. From 12 days of age, piglets in treatment group were fed starter diet supplemented with 120mg.kg^-1 cysteamine, while the control group received basal diet. Piglets were weaned at 35 days of age (D35) in both groups. 6 piglets from each group were slaughtered on D28 (one week before weaning), D35 (weaning day), D36.5, D38, D42 and D45 (36 hrs, 72hrs, one week and 10 days after weaning), respectively. A semi-quantitative RT-PCR was applied to determine the levels of H⁺-K⁺-ATPase mRNA in gastric mucosa. H⁺-K⁺-ATPase activity in gastric mucosa homogenate was also determined. The results indicated that: (1) H⁺-K⁺-ATPase mRNA expression and H⁺-K⁺-ATPase activity in control and treatment group reached a peak on D45, but did not show significant age differences from D28 to D45; (2) Cysteamine increased the level of H⁺-K⁺-ATPase mRNA expression and the differences were significant on D28, D35, D42 and D45, respectively. The H⁺-K⁺-ATPase activity in treatment group markedly increased by 32.3% on D35 and 18.3% on D45, respectively, compared with that of control; (3) No significant correlation was observedbetween H⁺-K⁺-ATPase mRNA expression and H⁺-K⁺-ATPase activity throughout the period of investigation. In present study, no clear developmental pattern was observed both for gastric expression of H⁺-K⁺-ATPase mRNA and H⁺-K⁺-ATPase activity in piglets pre-and post-weaning. Furthermore, neither the expression of H⁺-K⁺-ATPase mRNA nor the H⁺-K⁺-ATPase activity was affected significantly by weaning. However, dietary supplementation of cysteamine improved markedly both the mRNA expression and the activity of H⁺-K⁺-ATPase in piglets pre- and post-weaning.2. Possible mechanisms involved in the effect of cysteamine on mRNA expression and activity of H⁺-K⁺-ATPaseMucosal epithelial cells isolated from gastric tissue of piglets were divided into 4 groups and then cultured in DMEM medium (high glucose) supplemented with 10% fetal calf serum. After 24h, cysteamine hydrochloride was added into freshly replaced medium at the concentration of 0, 0.001, 0.01, and 0.1mg/mL for the control, low, moderate and high concentration groups, respectively. After 20h of treatment, cells were collected and the relative abundances of somatostatin and H⁺-K⁺-ATPase mRNA were determined by semi-quantitative RT-PCR. The H⁺-K⁺-ATPase activity was also analyzed. Then the isolated parietal cells were cultured for 24h and exposed to cysteamine for 20h. The H⁺-K⁺-ATPase activity was analyzed. The results showed that: (1) Low concentration of cysteamine had no influence on SS mRNA expression, whereas the moderate and high concentrations of cysteamine significantly increased SS mRNA expression by 56%and 64%, respectively; (2) Low concentration of cysteamine exhibited no effect on both mRNA expression and activity of H+-K+-ATPase, while moderate and high concentrations of cysteamine markedly increased both H+-K+-ATPase mRNA expression by 61% and 65%, and H+-K+-ATPase activity by 48% and 50%, respectively. (3) Cysteamine exhibited no significant effect on H+-K+-ATPa...