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 Isc is closely associated with epithelial Na+ channel (ENaC) but is not dependent on ENaC activity. Thus, inhibition of ENaC by 10 mu M amiloride or benzamil neither altered the direction nor its activity. Physiological studies suggested that this Cl- absorptive Isc 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.