Bumetanide increases Cl(-)-dependent short-circuit current in late distal colon: Evidence for the presence of active electrogenic Cl(-) absorption

Mammalian colonic epithelia consist of cells that are capable of both absorbing and secreting Cl(-). The present studies employing Ussing chamber technique identified two opposing short-circuit current (I(sc)) responses to basolateral bumetanide in rat distal colon. Apart from the transepithelial Cl(-)-secretory I(sc) in early distal colon that was inhibited by bumetanide, bumetanide also stimulated I(sc) in late distal colon that had not previously been identified. Since bumetanide inhibits basolateral Na(+)-K(+)-2Cl(-) cotransporter (NKCC) in crypt cells and basolateral K(+)-Cl(-) cotransporter (KCC) in surface epithelium, we proposed this stimulatory I(sc) could represent a KCC-mediated Cl(-) absorptive current. In support of this hypothesis, ion substitution experiments established Cl(-) dependency of this absorptive I(sc) and transport inhibitor studies demonstrated the involvement of an apical Cl(-) conductance. Current distribution and RNA sequencing analyses revealed that this Cl(-) absorptive I(sc) is closely associated with epithelial Na(+) channel (ENaC) but is not dependent on ENaC activity. Thus, inhibition of ENaC by 10 μM amiloride or benzamil neither altered the direction nor its activity. Physiological studies suggested that this Cl(-) absorptive I(sc) senses dietary Cl(-) content; thus when dietary Cl(-) was low, Cl(-) absorptive I(sc) was up-regulated. In contrast, when dietary Cl(-) was increased, Cl(-) absorptive I(sc) was down-regulated. We conclude that an active Cl(-) extrusion mechanism exists in ENaC-expressing late distal colon and likely operates in parallel with ENaC to facilitate NaCl absorption.