Transepithelial resistance can be regulated by the intestinal brush-border NA⁺/H⁺ exchanger NHE3

Initiation of intestinal Na⁺-glucose cotransport results in transient cell swelling and sustained increases in tight junction permeability. Since Na⁺/H⁺ exchange has been implicated in volume regulation after physiological cell swelling, we hypothesized that Na⁺/H⁺ exchange might also be required for Na⁺-glucose cotransport-dependent tight junction regulation. In Caco-2 monolayers with active Na⁺-glucose cotransport, inhibition of Na⁺/H⁺ exchange with 200 μM 5-(N,N-dimethyl)amiloride induced 36 ± 2% increases in transepithelial resistance (TER). Evaluation using multiple Na⁺/H⁺ exchange inhibitors showed that inhibition of the Na⁺/H⁺ exchanger 3 (NHE3) isoform was most closely related to TER increases. TER increases due to NHE3 inhibition were related to cytoplasmic acidification because cytoplasmic alkalinization with 5 mM NH₄Cl prevented both cytoplasmic acidification and TER increases. However, NHE3 inhibition did not affect TER when Na⁺-glucose cotransport was inhibited. Myosin II regulatory light chain (MLC) phosphorylation decreased up to 43 ± 5% after inhibition of Na⁺/H⁺ exchange, similar to previous studies that associate decreased MLC phosphorylation with increased TER after inhibition of Na⁺-glucose cotransport. However, NHE3 inhibitors did not diminish Na⁺glucose cotransport. These data demonstrate that inhibition of NHE3 results in decreased MLC phosphorylation and increased TER and suggest that NHE3 may participate in the signaling pathway of Na⁺-glucose cotransport-dependent tight junction regulation.