Abstract: | Intestinal neutral NaCl absorption, which is made up ofbrush-border (BB)Na+/H+exchange linked to BBCl /HCO 3exchange, is up- and downregulated as part of digestion and diarrhealdiseases. Glucocorticoids stimulate ileal NaCl absorption and BBNa+/H+exchange. Intestinal BB contains twoNa+/H+exchanger isoforms, NHE2 and NHE3, but their relative roles in rabbitileal BBNa+/H+exchange has not been determined. A technique to separate the contribution of NHE2 and NHE3 to ileal BBNa+/H+exchange activity was standardized by using an amiloride-related compound, HOE-694. Under basal conditions, both NHE2 and NHE3 contribute ~50% to ilealNa+/H+exchange. Glucocorticoids (methylprednisolone) increase BBNa+/H+exchange (2.5 times) but increase only ileal NHE3 activity (4.1 times),without an effect on NHE2 activity. Thus ileal BBNa+/H+exchange in animals treated with glucocorticoids is 69% via NHE3. Aquantitative Western analysis for NHE3 was developed, using as aninternal standard a fusion protein of the COOH-terminal 85 amino acidsof NHE3 and maltose binding protein. Glucocorticoid treatment increasedthe amount of BB NHE3. The quantitative Western analysis showed thatNHE3 makes up 0.018% of ileal BB protein in control rabbits and0.042% (2.3 times as much) in methylprednisolone-treated rabbits.Methylprednisolone treatment did not alter the amount of ileal BB NHE2protein. NHE3 turnover number was estimated to be 458 cycles/s underbasal conditions and 708 cycles/s in glucocorticoid-treated ileum. Thusmethylprednisolone stimulates ileal BBNa+/H+exchange activity only by an effect on NHE3 and not on NHE2; it does soprimarily by increasing the amount of BB NHE3, although it alsoincreases the NHE3 turnover number. |