Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, C33
Hydroxylases regulate epithelial Modulator of Na+/K+-ATPase expression: implications for intestinal fluid and electrolyte transport function.
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.
Cl- secretion, the primary driving force for intestinal fluid secretion, can become dysregulated in conditions of disease leading to the onset of diarrhoea. Our previous studies have shown that inhibition of hydroxylases, which are the primary intracellular sensors of O2 availability, exerts anti-secretory effects in vitro and in vivo through inhibition of Na+/K+-ATPase activity. Modulator of Na+/K+-ATPase (MONaKA) has been recently identified as an endogenous protein that regulates Na+/K+-ATPase activity in the nervous system by binding to the β subunits of the pump. Aim: Here we sought to investigate a potential role for MONaKA in mediating hydroxylase-induced attenuation of Na+/K+-ATPase activity in the colonic epithelium. Methods: Dimethyloxallyl glycine (DMOG) was used to inhibit hydroxylases. Ion transport was measured as changes in short-circuit current across voltage-clamped T84 cell monolayers. Protein expression and localisation was measured by Western blotting and confocal microscopic analysis. mRNA expression was measured by RT-PCR. Results: MONaKA is highly expressed in T84 colonic epithelial cells both at the mRNA and protein levels. Confocal imaging revealed that the protein is localised to the basolateral compartment of T84 cells and colonic crypts isolated from mice, rats, and humans. Treatment of T84 cells with DMOG (1 mM; 24hrs) increased expression of MONaKA to 190 ± 18.5% of that in control cells (n = 20, ***p ≤ 0.001) 24 hrs after treatment. DMOG also increased protein expression of the regulatory beta1 subunit of the Na+/K+-ATPase to 173.8 ± 17.3 % of controls with a time course coincident to that of its effects on MONaKA (n = 3, *p ≤ 0.05). MONaKA co-immunoprecipitated with both the catalytic α1 and regulatory beta1 subunits of the Na+/K+-ATPase. Finally, pre-treatment of cells with either actinomycin D (250ng/ml) or cycloheximide (5µM) did not alter DMOG-induced increases in MONaKA expression nor did they reverse the anti-secretory effects of DMOG in Ussing chambers. Conclusion: Hydroxylases regulate basolateral MONaKA expression in colonic epithelial cells by a postranslational mechanism. In turn, increased association of MONaKA with the regulatory β 1 subunit attenuates Na+/K+ ATPase pump activity, thereby inhibiting epithelial secretory function. Our data have important implications for our understanding of how epithelial fluid and electrolyte transport can be regulated under physiological and pathophysiological conditions and suggest that hydroxylases may be good targets for the development of new drugs to treat intestinal transport disorders.
Where applicable, experiments conform with Society ethical requirements