Na+/HCO3− cotransport is required for rapid repair of the gastric mucosa after injury

Abstract

Restitution is a protective mechanism essential to the gastrointestinal epithelium for repair of superficial wounds after injury. Of vital importance to restitution is the fact that epithelial cells migrate to close gaps in continuity between cells. Recently, it was shown that endothelial cells must alkalinize, or reduce intracellular pH (pHi), prior to migration; a process accomplished by activation of Na+/H + exchange. Thus, the present study was performed to determine whether the migration of gastric surface epithelial ceils after injury is dependent on transporters that regulate pH i. METHODS: Paired fundic mucosa from the bullfrog, Rana catesbeiana, were mounted in Ussing-type chambers. Tissues were injured with IM NaC1 for 10 min, washed, and allowedto repair for 4 hr. The following conditions were tested: (1) absence of Na + where Na + was replaced with coline; (2) amiloride (1 mM) to block Na+/H + exchange; (3) bafilomycin A t (25 nM) to block vacuolar-ATPase activity with or without amiloride (1 mM); (4) bumetanide (100 pM) to inhibit Na+-K+-2CI cotransport; (5) absence of C1where C1was substituted with gluconate; and (6) 4,4-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS-1 raM) to block Na+/HCO3 + cotransport and/or CI-/HCO 3exchange. The return of PD and resistance after injury (electrical restitution) was determined by established techniques. Tissue morphology (morphological restitution) was evaluated by light and electron microscopy. RESULTS: Cell migration after injury was dependent on Na +, as electrical and morphological restitution did not occur in the absence of Na +. Likewise, DIDS, blocking Na+/HCO3 cotransport and/or C1-/HCO 3exchange, completely inhibited electrical and morphological restitution. Amiloride, blocking Na+/H + exchange, did not influence restitution at neutral luminal pH (7.4) or acidic luminal pH (4.0). In fact, tissues incubated with amiloride at acidic luminal pH showed significantly better restitution (electrical) than did control tissues after injury. Since the epithelium had many vacuoles 4 hr after injury, we tested the hypothesis that cells might shuttle H + into vacuoles to regulate pH i. Bafilomycin A 1 was used to inhibit vacuole formation and alone or in combination with amiloride had no effect on restitution. Restitution occurred in the presence of bumetanide and in Cl--free solutions. CONCLUSIONS: Our results show that restitution of the frog gastric mucosa is dependent on Na + (as well as HCO 3as described by Svanes et al., Gastroenterology 1982;82:1409) and is DIDS-inhibitable. We suggest that restitution is dependent on Na+/HCO3 cotransport and not CI'/HCO 3exchange, since Cl'-free solutions did not block restitution. Since gastric surface cells seem to • use Na÷/H + exchange to regulate pH i (Seidler et al., Am. J. Physiol. 1989;256:G466) we suggest that Na÷/HCO3 cotransport influences restitution by some other means, perhaps by regulation of cell volume.