Proceedings of The Physiological Society

University of Manchester (2010) Proc Physiol Soc 19, PC242

Poster Communications

Epidermal growth factor chronically upregulates colonic epithelial Cl- secretion through a mechanism involving protein kinase C-dependent upregulation of Ca2+-activated Cl- conductances.

M. S. Mroz1, F. O'Mahony1, S. J. Keely1

1. RCSI, Dublin, Ireland.


Cl- secretion, the primary driving force for intestinal fluid secretion, is dysregulated in many intestinal disorders, leading to the onset of diarrhoea. Growth factors are important regulators of epithelial transport and our previous work has shown that epidermal growth factor (EGF) chronically enhances colonic epithelial secretory function. Here we investigated the effects of EGF on activity and expression of apical Cl- channels, which constitute the exit pathways for Cl- in epithelial cells. T84 cell monolayers were mounted in Ussing chambers and Cl- secretion was measured as changes in short-circuit current. Cl- channel expression was measured by RT-PCR or western blotting. Statistical analyses were performed by one way ANOVA with the Newman-Keuls post-test or by Student t-tests as appropriate. p values < 0.05 were considered to be significant. As previously reported acute treatment with EGF (100 ng/ml; 15 min) enhanced Cl- secretory responses to the Ca2+ and cAMP-dependent agonists, carbachol (CCh; 100 μM) and forskolin (FSK; 10 μM) by 187 ± 15 % (n= 3; p<0.001) and 125 ± 4 % (n= 9; p<0.001) respectively, when measured 24 hrs later. The protein kinase C delta (PKCδ) inhibitor, rottlerin (20μM), abolished the effect of EGF on Ca2+ and cAMP induced Cl- secretion. Treatment with EGF increased mRNA levels for CFTR, the cAMP-dependent Cl- channel, to 270 ± 20 % of controls within 4 hrs of treatment (n = 3; p < 0.01). However, western blot analysis revealed no change in CFTR protein expression in response to EGF (80 ± 36 % of controls; n = 5) after 24 hrs. Furthermore, under experimental conditions that specifically isolate Cl- ion conductance through CFTR, EGF pretreatment did not alter responses to FSK which were 97 ± 9 % of those in controls (n = 5). In contrast, under conditions that specifically isolate Cl- conductances through Ca2+-dependent channels, EGF potentiated CCh-induced responses to 184 ± 31% of those in control cells (n = 17; p<0.005). This effect was reduced to 115 ± 51% (n = 4) by PKCδ inhibition with rottlerin. Furthermore, we found that mRNA expression of transmembrane protein 16A (TMEM16A), a recently identified protein associated with Ca2+-dependent Cl- channel activity, was increased by EGF to 256 ± 45% of control levels (n = 7; p < 0.01). Our data suggest that acute exposure to EGF chronically enhances colonic epithelial secretory capacity through a PKCδ-dependent upregulation of Ca2+-activated Cl- conductances. Our data further suggest that the identity of this upregulated conductance may be the novel Ca2+-activated channel, TMEM16A. These data provide new insights into the roles of EGF in regulating intestinal secretory function.

Where applicable, experiments conform with Society ethical requirements