Proceedings of The Physiological Society

University College London (2011) Proc Physiol Soc 24, C15 and PC15

Oral Communications

The function of CFTR high expresser cells in the possum proximal colon.

S. Fan1, N. Harfoot1, R. C. Bartolo1, A. Butt1

1. Physiology, University of Otago, Dunedin, New Zealand.

Although the possum proximal colon expresses the cystic fibrosis transmembrane conductance regulator (CFTR) (4) and the NaK2Cl cotransporter (NKCC1) (2), secretagogues do not stimulate electrogenic Cl- secretion by this epithelium (3). Here we have investigated the function of CFTR in the possum proximal colon. CFTR was expressed at high levels in the proximal colon, but the CFTR immunoreactivity was restricted to a small population of randomly distributed cells in the surface epithelium and upper third of the crypts (n=3), reminiscent of the CFTR high expresser (CHE) cells seen in the intestine of eutherian mammals (1). Consistent with the absence of electrogenic Cl- secretion the CHE cells do not express NKCC1, and while forskolin (20µM) stimulated an increase in short circuit current when the tissues were mounted in the Ussing chamber (n=20), this was not inhibited by bumetanide (100µM), although it was inhibited by mucosal N-(2-Naphthalenyl)-((3,5-dibromo-2,4-dihydroxyphenyl)methylene)glycine hydrazide (GlyH101) (40µM) indicating it involved CFTR. Forskolin also stimulated a dose dependent (EC50=1µM, n=7) increase in transepithelial resistance (RT), which was dependent upon Cl- (n=16). Furthermore, mucosal GlyH101 (40µM, n=6) increased RT (ΔRT=45±5 Ω cm2, all values X±SEM) to a comparable extent as forskolin (20µM, ΔRT=48±10 Ω cm2), but the effects of forskolin and GlyH101 on RT were not additive. Serosal DIDS (1mM) had a similar effect on RT (ΔRT=42±13 Ω cm2, n= 6) and, as with mucosal GlyH101, the effects of serosal DIDS and forskolin were not additive. These data suggest that the CHE cells in the possum colon provide a transepithelial Cl- conductance, consisting of CFTR in the apical membrane and a DIDS-sensitive anion conductance in the basolateral membrane, and this conductance is inhibited by forskolin. Measurement of the Cl- current (ICl) in the presence of a mucosal to serosal gradient confirmed this proposal. When colonic epithelia were bathed in mucosal NaCl Ringer’s and serosal NaGluconate Ringer’s a ICl of 298±26µA cm-2 (n=8) developed, which was inhibited by forskolin with an IC50 (0.75µM, n=5) comparable to that for the increase in RT in the absence of a gradient. Furthermore, mucosal GlyH101 (40µM, n=8) and serosal DIDS (1mM, n=8) inhibited 82±4% and 89±4% of the ICl, respectively, whereas serosal GlyH101 and mucosal DIDS had no effect. Thus, in the possum proximal colon CHE cells provide a transepithelial Cl- conductance and, as absorption in this tissue is driven by electrogenic Na+ transport, it is likely that this is an important route for the passive absorption of Cl-. Inhibition of this conductance by forskolin would reduce Na+ absorption by restricting the movement of Cl-.

Where applicable, experiments conform with Society ethical requirements