Quantitative contribution of NHE2 and NHE3 to rabbit ileal brush-border Na+/H+exchange.

Intestinal neutral NaCl absorption, which is made up of brush-border (BB) Na+/H+exchange linked to BB Cl-/[Formula: see text]exchange, is up- and downregulated as part of digestion and diarrheal diseases. Glucocorticoids stimulate ileal NaCl absorption and BB Na+/H+exchange. Intestinal BB contains two Na+/H+exchanger isoforms, NHE2 and NHE3, but their relative roles in rabbit ileal BB Na+/H+exchange has not been determined. A technique to separate the contribution of NHE2 and NHE3 to ileal BB Na+/H+exchange activity was standardized by using an amiloride-related compound, HOE-694. Under basal conditions, both NHE2 and NHE3 contribute ∼50% to ileal Na+/H+exchange. Glucocorticoids (methylprednisolone) increase BB Na+/H+exchange (2.5 times) but increase only ileal NHE3 activity (4.1 times), without an effect on NHE2 activity. Thus ileal BB Na+/H+exchange in animals treated with glucocorticoids is 69% via NHE3. A quantitative Western analysis for NHE3 was developed, using as an internal standard a fusion protein of the COOH-terminal 85 amino acids of NHE3 and maltose binding protein. Glucocorticoid treatment increased the amount of BB NHE3. The quantitative Western analysis showed that NHE3 makes up 0.018% of ileal BB protein in control rabbits and 0.042% (2.3 times as much) in methylprednisolone-treated rabbits. Methylprednisolone treatment did not alter the amount of ileal BB NHE2 protein. NHE3 turnover number was estimated to be 458 cycles/s under basal conditions and 708 cycles/s in glucocorticoid-treated ileum. Thus methylprednisolone stimulates ileal BB Na+/H+exchange activity only by an effect on NHE3 and not on NHE2; it does so primarily by increasing the amount of BB NHE3, although it also increases the NHE3 turnover number.