Proceedings of The Physiological Society

University College Dublin (2009) Proc Physiol Soc 15, C132

Oral Communications

Prolyl Hydroxylase Inhibition Attenuates Colonic Epithelial Secretory Function

J. B. Ward1, K. Lawler1, C. T. Taylor2, S. J. Keely1

1. Molecular Medicine, Royal College of Surgeons in Ireland, Dublin 9, Ireland. 2. UCD Conway Institute, UCD, Dublin, Ireland.

BACKGROUND: Inflammation of the gastrointestinal tract due to inflammatory bowel diseases and ischemic colitis is commonly associated with hypoxia and altered epithelial transport. In hypoxia, key O2 sensors called prolyl hydroxylases (PHDs) are inactivated, thereby allowing transcription factors such as hypoxia inducible factor (HIF) and nuclear factor κB (NFκB) to be activated. While recent studies have shown that inhibition of PHDs is protective in murine colitis, little is known of their role in regulating intestinal epithelial transport function. AIM: To investigate the role of PHDs in regulation of epithelial secretory function. METHODS: The pan specific inhibitor dimethyloxalylglycine (DMOG) was used to inhibit hydroxylase activity. Transepithelial Cl- ion secretion, the primary driving force for fluid secretion in the intestine, was measured as changes in short circuit current (Isc) across voltage clamped monolayers of T84 cells grown on permeable supports. Results are expressed as mean ± standard error of the mean for a series of n experiments. Statistical analyses were made by one way ANOVA using Tukey multiple comparisons test. p values ≤ 0.05 were considered to be significant. RESULTS: Pre-treatment of T84 cells with DMOG (1mM, 24 hrs) significantly attenuated Cl- secretion in response to the cAMP and Ca2+ dependent secretagogues, forskolin (FSK) and carbachol (CCh), respectively. Responses to CCh and FSK were 20.2% ± 2.6% (n = 16; p < 0.001) and 38.6% ± 6.7% (n = 11; p < 0.001) of those in control cells, respectively. Transepithelial resistance was not altered by DMOG treatment indicating that it did not exert toxic effects. The effects of DMOG on secretory responses were apparent after 3 hours and maximal at 18 hours, and this was concurrent with the cellular accumulation of HIF, which was apparent at 3 hours and sustained up to 24 hours. Secretagogue-induced basolateral K+ and apical Cl- conductances were not altered by DMOG. In contrast, Na+/K+ ATPase activity was significantly reduced to 27.6% ± 8.1% (n = 7; p ≤ 0.01) by DMOG. Western blot analysis of transport proteins revealed that DMOG also decreased expression of both the catalytic subunit of the Na+/K+ ATPase pump and the NKCC1 cotransporter. CONCLUSIONS: These studies demonstrate a novel role for PHDs in regulating intestinal epithelial secretory function. Our data suggest that by virtue of their ability to modulate transport protein expression, PHDs are likely to be important regulators of intestinal fluid and electrolyte transport in hypoxic conditions.

Where applicable, experiments conform with Society ethical requirements