KINETICS AND REGULATION OF A POLARIZED NA+-H+ EXCHANGER FROM CACO-2 CELLS, A HUMAN INTESTINAL-CELL LINE

The kinetics and regulation of Na+-H+ exchange were studied using BCECF to measure pH(i) in Caco-2 cells grown on membrane filters. Na+-H+ exchange was defined as a Na+- dependent H+ efflux in response to an acid load imposed by an NH4Cl prepulse in the absence of added CO2. Na+-H+ exchange was present exclusively on the basolateral membrane, had a K(t) (Na+) of 21 +/- 2 mM, and an ID50 for amiloride dependent on medium [Na+] with an apparent K(i) for amiloride of 3-mu-M. Na+-H+ exchange rates had a greater than first-order dependence on intracellular [H+], suggesting the presence of an internal proton modifier site. Results also suggest that Na+-H+ exchange is kinetically inactivated at resting pH(i) (7.35 +/- 0.02), since neither removal of Na+ nor addition of amiloride affected resting pH(i), although monensin alkalinized cells to pH(i) 7.6. To evaluate regulation of Na+-H+ exchange, cells were exposed to either forskolin, 1,9-dideoxyforskolin (a noncyclase-activating forskolin derivative), 8-BrcAMP, E. coli ST(a) toxin, ionomycin, phorbol dibutyrate, or cellular shrinkage in hypertonic medium. Only forskolin and 1,9-dideoxyforskolin caused a significant change (inhibition) in Na+-H+ exchange rate. Experiments performed with the Ussing chamber-voltage clamp technique verified that forskolin, 8-BrcAMP, E. coli ST(a) toxin, ionomycin, and phorbol dibutyrate increased transepithelial I(sc), verifying that all the regulatory pathways tested were functional and responsive to agonists. Results suggested that the I(sc) was due to Cl- secretion, since no net transcellular Na+ or Cl- flux was detected in basal conditions, and the I(sc) response to forskolin was abolished by omission of serosal Cl-. Because forskolin, but not 1,9-dideoxyforskolin, increased both cellular cAMP and I(sc), the inhibition of Na+-H+ exchange by forskolin derivatives was mediated by a mechanism not involving activation of adenylyl cyclase. In conclusion, Caco-2 cells use a basolateral Na+-H+ exchanger to regulate pH(i), but this exchanger is not affected by cell shrinkage or second messenger pathways that regulate Na+-H+ exchangers in other cell systems.