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-/HCO3- exchange, is up- and downre gulated as Part of digestion and diarrheal diseases. Glucocorticoids s timulate ileal NaCl absorption and BE Na+/H+ exchange. Intestinal BE c ontains two Na+/H+ exchanger isoforms, NHE2 and NHE3, but their relati ve 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 N a+/H+ exchange activity was standardized by using an amiloride-related compound, HOE-694. Under basal conditions, both NHE2 and NHE3 contrib ute similar to 50% to ileal Na+/H+ exchange. Glucocorticoids (methylpr ednisolone) increase BB Na+/H+ exchange (2.5 times)but increase only i leal NHE3 activity (4.1 times), without an effect on NHE2 activity. Th us 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 8 5 amino acids of NHE3 and maltose binding protein. Glucocorticoid trea tment increased the amount of BN NHE3. The quantitative Western analys is showed that NHE3 makes up 0.018% of ileal BB protein in control rab bits and 0.042% (2.3 times as much) in methylprednisolone-treated rabb its. Methylprednisolone treatment did not alter the amount of ileal BB NHE2 protein. NHE3 turnover number was estimated to be 458 cycles/s u nder 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 in creasing the amount of BB NHE3, although it also increases the NHE3 tu rnover number.