The aim of this project is to clarify the role of the apical membrane large conductance K+ (BK) channels in colonic K+ secretion in health and disease. In infective diarrheas, the intestinal water secretion is driven by enhanced Cl- secretion, whereas in the common inflammatory bowel disease, ulcerative colitis (UC), the main transport defect contributing to diarrhea is a marked decrease or absence of Na+ absorption, which removes the driving force for water absorption. Historically, an increased colonic K+ secretion has been reported in patients with active UC, although its possible clinical implications and the mechanisms involved have not been studied. We have developed preliminary, but clear-cut, evidence showing increases in colonic K+ secretion, in the expression of apical BK channels (which mediates K+ exit from the cells) and in expression of the basolateral Na-K-2Cl co- transporter (NKCC1; which mediates K+ and water uptake into the cells) in both human UC and an experimental model of colitis. We propose a second novel mechanism in UC, that stimulation of colonic K+ secretion may provide a driving force for increased colonic water secretion in UC. Thus, Specific Aim 1 is to determine the relationship between BK channel-mediated colonic K+ secretion and water secretion in health and disease. We will accomplish this by using in vivo colonic perfusion, in vitro crypt-gland micro-perfusion, and Ussing chamber experiments, which will all be performed in a normal and experimentally induced UC in rat (DSS-colitis) and mouse (IL-10 knockout) colon, and a normal and UC human colon.
Specific Aim 2 will address the molecular mechanism of active K+ secretion within both a normal and an inflamed human colon (UC). This will allow us to identify the BK? splice variants and BK? (?1 and ?3) isoforms that distribute/express along the surface-crypt cell axis and establish which isoforms function in normal and inflamed (UC) rat and human colon. Additional studies will focus on basolateral K+ and water uptake mechanism(s) within a normal and inflamed human colon.
Specific Aim 3 will identify the pro-inflammatory mediator(s) that regulate BK channel expression transcriptionally and activation of BK channels in UC colon. In additional studies, the importance of BK channel for the synthesis and secretion of proinflammatory mediators and for the development of inflammation using BK? knockout, IL-10 knockout and BK?/IL-10 double-knockout mouse models.
The pathogenesis of diarrhea in ulcerative colitis (UC) is been attributed to decreased colonic Na+ absorption, rather than an increased Cl- secretion. While the transport proteins responsible for Cl- secretion are down- regulated, those responsible for active K+ secretion [i.e. apical BK channels, which mediate K+ exit into the lumen; and the basolateral Na-K-2Cl co-transporter 1 (NKCC1), which mediates cellular K+ and water uptake] are up-regulated in UC. Based on the clinical observation that active K+ secretion provides the driving force for diarrhea in patients with colonic pseudo-obstruction, this study is designed to establish the novel hypothesis that active K+ secretion is a significant driving force for water secretion and hence diarrhea in patients with UC.
