Age- and tissue-specific induction of NHE3 by glucocorticoids in the rat small intestine

Of the two known apical isoforms of theNa⁺/H⁺ exchanger (NHE) family, only the NHE3gene is regulated by glucocorticoids. The aim of these studies was toinvestigate the mechanisms underlying the effects of methylprednisolone(MP) on expression of NHE3 in the proximal and distal small intestineof suckling and adult rats. Immunoblots showed that the glucocorticoidresponsiveness in the proximal small intestine was greatest in sucklinganimals (NHE3/-actin: 0.43 ± 0.09 control vs. 1.57 ± 0.15 MP;P < 0.001), and responsiveness decreased with age with noeffect in adults (0.56 ± 0.14 vs. 0.64 ± 0.17). Distal smallintestine was responsive only in adult rats (0.49 ± 0.13 vs. 1.65 ± 0.09; P < 0.001). This pattern was confirmed at the mRNAlevel and by ²²Na⁺ uptake. Western blot and[³H]dexamethasone mesylate binding showed thatthe responsiveness of NHE3 to glucocorticoids is directly related tothe expression of glucocorticoid receptor (GR) in the small intestine.These studies suggest that loss and gain of glucocorticoidresponsiveness in the proximal and distal small intestine,respectively, are related to age- and segment-dependent expression of GR.

     

Cloning and characterization of a type III Na-dependent phosphate cotransporter from mouse intestine

Intestinal and renalabsorption of inorganic phosphate (P_i) is critical forphosphate homeostasis in mammals. We have isolated a cDNA that encodesa type III Na-dependent phosphate cotransporter from mouse smallintestine (mPit-2). The nucleotide sequence of mPit-2 predicts aprotein of 653 amino acids with at least 10 putative transmembranedomains. Kinetic studies, carried out in Xenopus oocytes,showed that mPit-2 cRNA induces significant Na-dependent P_iuptake with an apparent Michaelis constant (K_m)for phosphate of 38 µM. The transport of phosphate by mPit-2 isinhibited at high pH. Northern blot analysis demonstrated the presenceof mPit-2 mRNA in various tissues, including intestine, kidney, heart,liver, brain, testis, and skin. The highest expression of mPit-2 in the intestine was found in the jejunum. In situ hybridization revealed thatmPit-2 mRNA is expressed throughout the vertical crypt-villus axis ofthe intestinal epithelium. The presence of mPit-2 in the mouseintestine and its unique transport characteristics suggest thatmultiple Na-dependent cotransporters may contribute to phosphate absorption in the mammalian small intestine.

     

Effect of dietary polyamines and alpha-difluoromethylornithine on regulation of intestinal Na+/H+ exchanger

Polyamines are compounds required for initiation of rapid cellular growth and differentiation in many cell types. Ornithine decarboxylase is the rate limiting enzyme in polyamine synthesis. Fasting and refeeding regulates the activity of ornithine decarboxylase and polyamine content in the intestinal tract. We tested the hypothesis that polyamines regulate cell growth via the Na+/H+ exchanger which is believed to be intimately involved in cell growth. Ileal Na+/H+ activity was therefore examined in control, fasted, refed fasted, and in rats given the specific inhibitor of ornithine decarboxylase alpha-difluoromethylornithine. A well-validated ileal brush border membrane vesicles for the study of Na+/H+ exchange activity was utilized. Fasting markedly decreased while refeeding stimulated Na+/H+ exchange activity at all times studied (P less than 0.05-0.001). Maximal uptake of Na+ at 5 min was 3.12 +/- 0.05, 2.5 +/- 0.05 and 2.22 +/- 0.05 nmol/mg protein in refed, control and fasted rats respectively. Kinetics of amiloride sensitive Na+/H+ exchanger showed a Vmax of 17.1 +/- 3.5, 8.0 +/- 0.64 and 4.7 +/- 1.1 nmol/mg protein per 5 s in refed fasted, control and fasted rats respectively Km values were not significantly different between the groups studied. 2% alpha-difluoromethylornithine given in the drinking water abolished the stimulation in Na+/H+ exchange activity in refed fasted rats. These results suggest a close relationship between polyamines and Na+/H+ activity in the intestinal mucosa of rats.