Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC197
Prolyl hydroxylases regulate electrogenic sodium absorption across colonic epithelium
J. B. Ward1, C. T. Taylor2, S. J. Keely1
1. Molecular Medicine Laboratories, Royal College of Surgeons in Ireland, Dublin 9, Ireland. 2. School of Medicine & Medical Science, UCD, Dublin, Ireland.
Intestinal fluid movement is driven by osmotic gradients that are, in turn, established by active ion transport across the epithelium. Fluid absorption normally predominates and is promoted by Na+ absorption, while fluid secretion is driven by Cl- secretion. Epithelial transport function is dependent on cellular O2 and energy supply and our previous studies have shown that prolyl hydroxylases (PHDs), which function as cellular O2 sensors, are important regulators of intestinal fluid and electrolyte secretion in vitro and in vivo. However, little is known of the role of PHDs in regulating intestinal absorptive function. Here, we sought to investigate the role of PHDs in regulating epithelial sodium channel (ENaC) activity and expression. The pan-hydroxylase inhibitor dimethyloxallyl glycine (DMOG) was used to inhibit hydroxylases. ENaC activity was measured as amiloride-sensitive changes in short circuit current (Isc) across monolayers of T84 cells pretreated with 4-phenyl butyrate (4-PBA; 5 mM) to induce ENaC activity, or across muscle-stripped segments of rat rectum. Sodium glucose transporter-1 (SGLT1) activity was measured as phloridzin-sensitive changes in Isc across segments of mouse jejunum. Western blotting was used to assess protein expression. Treatment of T84 cell monolayers with 4-PBA (5 mM; 24 hrs) led to an induction of ENaC expression and an increase in basal Isc of 17.8 µA/cm2 that was wholly sensitive to the ENaC blocker, amiloride (10µM). Pretreatment of the cells with DMOG (1mM, 24 hrs) significantly reduced the 4-PBA-induced current by 29 ± 6.3 % (n=5, p < 0.01 by paired t-test). This reduction in activity was not due to altered abundance of the ENaCα subunit as shown by western blot analysis (n=3). Furthermore, treatment of rats by intraperitoneal injection of DMOG (40mg/kg) significantly reduced the amiloride-sensitive current across ex vivo rectal tissue to 64 ± 13.3% of that in control tissues when measured 24 hrs after treatment (n=6; p ≤ 0.05 by paired t-test). The effects of hydroxylase inhibition were specific for Na+ absorption through ENaC since treatment of mice with DMOG failed to alter glucose (25 mM)-stimulated SGLT1 activity in ex vivo sections of jejunum when measured 24 hrs after treatment (n=6). Conclusions: These studies demonstrate a novel role for PHDs in regulating intestinal absorptive function. PHD inhibitors specifically downregulate large intestinal ENaC activity while small intestinal SGLT-1 activity is unaltered. Mechanisms underlying DMOG inhibition of ENaC function remain to be elucidated but do not appear to involve alterations in expression of the ENaCα subunit of the protein. Our data suggest that PHDs may provide a new target for development of drugs to treat intestinal disorders associated with dysregulated epithelial absorptive function.
Where applicable, experiments conform with Society ethical requirements