Proceedings of The Physiological Society
Newcastle University (2009) Proc Physiol Soc 16, PC19
The hydroxylase inhibitor DMOG attenuates colonic epithelial secretory function
J. B. Ward1, K. Lawler1, C. Taylor2, S. J. Keely1
1. Molecular Medicine, Royal College of Surgeons in Ireland, Dublin 9, Ireland. 2. School of Medicine and Medical Science, University College Dublin, Dublin, Ireland.
Gastrointestinal (GI) disorders, such as inflammatory bowel diseases and ischaemic colitis, cause inflammation of the GI tract and are commonly associated with hypoxia, altered epithelial transport and diarrhoea. In hypoxia, O2 sensors known as 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. Thus, the aim of the current studies was to investigate the role of hydroxylases in regulating intestinal epithelial secretory function. The pan specific inhibitor dimethyloxalylglycine (DMOG) was used to inhibit hydroxylase activity. Cl- secretion, the primary driving force for intestinal fluid secretion, was measured as changes in short circuit current (Isc) across voltage clamped monolayers of T84 cells. Results are expressed as mean ± SEM. Statistical analyses were made by one way ANOVA and Tukey multiple comparisons test. p values ≤0.05 were considered to be significant. Pre-treatment of T84 cells with DMOG (1mM, 24 hrs) significantly attenuated Cl- secretion in response to forskolin (FSK) and carbachol (CCh). 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. The effects of DMOG on secretory responses were apparent after 3 hours and maximal by 18 hours, which was concurrent with DMOG-induced stabilisation of HIF1-α. Pre-treatment of T84 cells with CoCl2 (500µM, 24hrs), which increases cellular HIF levels in a hydroxylase-independent manner, also attenuated Cl- secretory responses to CCh and FSK to 51.6% ± 8.1% (n=3; p≤0.05) and 53% ± 8.4% (n=4; p≤0.01) of those in control cells, respectively. DMOG did not alter the generation of prosecretory second messengers in response to CCh and FSK. Similarly, secretagogue-induced basolateral K+ and apical Cl- conductances were unaltered by DMOG. In contrast, Na+/K+ATPase activity was significantly reduced (to 27.6% ± 8.1%; n=7 p≤0.01) by DMOG. Analysis of transport protein expression revealed that DMOG significantly decreased both protein and mRNA expression of CFTR and NKCC1 but did not alter expression of the Na+/K+ATPase α subunit. Finally, cellular ATP levels were significantly reduced (to 67% ± 8.5% of controls; n=6; p≤0.01) by treatment with DMOG. These studies demonstrate a novel role for PHDs in regulating intestinal epithelial secretory function by a mechanism which appears to involve HIF, depletion of cellular ATP, and inhibition of transport protein function. Our data suggest that by virtue of their ability to alter epithelial transport, hydroxylases are likely to be important regulators of intestinal fluid transport in health and disease.
Where applicable, experiments conform with Society ethical requirements