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

Date Published: February 2, 2017

Publisher: Public Library of Science

Author(s): Lieqi Tang, Xiefan Fang, Steven P. Winesett, Catherine Y. Cheng, Henry J. Binder, Scott A. Rivkees, Sam X. Cheng, Diego Alvarez de la Rosa.


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 (Isc) responses to basolateral bumetanide in rat distal colon. Apart from the transepithelial Cl–secretory Isc in early distal colon that was inhibited by bumetanide, bumetanide also stimulated Isc 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 Isc could represent a KCC-mediated Cl- absorptive current. In support of this hypothesis, ion substitution experiments established Cl- dependency of this absorptive Isc 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 μM 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 Isc was up-regulated. In contrast, when dietary Cl- was increased, Cl- absorptive Isc 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.

Partial Text

Sodium chloride transport in mammalian colon occurs in two opposing directions: secretion and absorption. The secretory process is primarily localized to crypt cells, whereas the absorptive process is mainly present in surface cells [1, 2]. During secretion, basolateral bumetanide-sensitive Na+-K+-2Cl–cotransporter (NKCC1) moves Cl- uphill into the cell using the Na+ electrochemical gradient generated by Na+,K+-ATPase (NKA); Cl- is then transported out of the cell into the lumen via apical Cl- channels such as cystic fibrosis transmembrane conductor regulator (CFTR), and Na+ flows through a paracellular shunt [2]. In this secretory process, the transport of Cl- anion is active and can be measured in an Ussing chamber as a decrease in bumetanide-sensitive short-circuit current (Isc), and the transport of the counter ion Na+ is passive, i.e., it only follows the transepithelial voltage, VT, set up by CFTR-mediated Cl- secretion, and is therefore not electrogenic and will not be reflected in Isc measurement [2].

Unlike other extracellular milieu, the lumen of the colon is home to hundreds of trillions of bacteria. There, as high as 120 mM of short-chain fatty acids (SCFA) are normally present due to bacterial fermentation of undigested carbohydrates [3, 4]. Accordingly, in the lumen of the colon, the major anions are SCFA; other anions, such as Cl-, are low [5]. For passive Cl- absorption to occur at low [Cl-]L, apical membrane potential (Ψa) must depolarize markedly to maintain the driving force for Cl- entry into the cell across the apical membrane. Alternatively, intracellular Cl- concentration ([Cl-]i) must fall appreciably. In the distal colon, while Ψa depolarizes due to ENaC-mediated electrogenic Na+ absorption, it also hyperpolarizes due to spontaneous electrogenic K+ secretion. Furthermore, unlike exclusive absorptive epithelia (e.g., sweat duct), colonic epithelia have a more complex task of providing both absorption and secretion of Cl-, and [Cl-]i may or may not fall in this tissue due to the presence of Cl- entry into the cell from the basolateral NKCC1 [2] or Cl-/ HCO3- exchanger [13]. Thus, it is not entirely clear whether the changes in Ψa and [Cl-]i are sufficient to maintain the driving force for Cl- influx from the lumen, requisite for passive Cl- absorption to occur in a low [Cl-]L situation. It is likely that an alternative active mechanism for Cl- absorption is present in the distal colon, particularly in the late distal colon where, due to reabsorption, [Cl-]L is the lowest [5].




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