Functional and molecular characterization of NHE3 expression during ontogeny in rat jejunal epithelium

Abstract

Ontogenic changes occur in intestinal brush-border membrane vesicle (BBMV) Na+/H+exchange activity. The present studies were designed to investigate ontogenic changes in Na+/H+exchanger (NHE) isoform 3 in rat jejunum. pH-dependent Na+ uptake was assayed in four age groups of rats in the presence of 0, 50, or 800 μM HOE-694, a specific NHE inhibitor with differential sensitivities for NHE2 [inhibition constant ( K i) = 5 μM in PS120 fibroblasts] and NHE3 ( K i = 650 μM). Results showed that NHE2 and NHE3 contribute to basal BBMV uptake at all ages. Uptake levels were highest in 6-wk-old rats, lower in adult rats, and lowest in 2-wk-old (suckling) and 3-wk-old (weanling) rats. NHE3 contribution ranged from 92% at 6 wk of age to 59% at 2 and 3 wk of age. NHE3 inhibition by 800 μM HOE-694 was 38–45%. Statistical analysis showed that HOE-694 had a significant effect at both concentrations at all ages and that differences were present between all ages except 2- and 3-wk rats (at all HOE-694 concentrations). Northern blot analyses of jejunal mucosa showed lowest NHE3 mRNA levels in 2-wk animals and higher levels in all other age groups. Polyclonal antibodies were developed against an NHE3 COOH-terminal fusion protein, and antiserum was characterized with NHE3-transfected PS120 cells and by immunohistochemistry. Western blot analyses showed lowest protein levels in 2-wk animals and higher levels in the other ages. Suckling rats were subcutaneously injected with methylprednisone (MP) for 2 days and killed 1 day later. Northern blot analyses showed a twofold increase in NHE3 mRNA expression with MP treatment. Immunoblot analyses showed a 2.5-fold increase in NHE3 immunoreactive protein levels with MP injection. Overall, these data suggest that NHE3 is regulated during ontogeny and that ontogenic changes are most apparent around the time of weaning. Furthermore, the data suggest that NHE3 is regulated at transcriptional and posttranscriptional levels during mammalian intestinal development.