The central goal of this projet is the characterization of the epithelial location and transport mechanisms responsible for active K secretion and absorption across the mammalian colon as well as the mechanisms that regulate these processes. Mammalian colon absorbs and secretes fluid by processes dependent on Na absorption and Cl secretion, respectively. Active K secretion and absorption occur and could be associated with either Na absorbing cells or Cl secreting cells. The location of the K transport processes will be determined using conventional and ion-selective microelectrodes, electron microprobe analysis of tissue composition, and fluorescence mocroscopy of living tissue. Electrophysiologic techniques will be employed to detect changes in cell membrane electrical potential and intracellular ion activity consistent with K secretion and absorption. Electron microprobe analysis of intracellular Na, K and Cl concentration will confirm the morphologic location of active K secretion an dabsorption. The possibility that K absorption is coupled to acid secretion will be examined using fluorescent probes of pH in conjunction with direct visualization using differential interference contrast microscopy. Regulation of K transport will be examined using the patch clamp technique and morphologic techniques. Activation of K and Cl channels by stimulators of transport will be examined with single channel recording from isolated colonic epithelia and primary cultures of these cells. Stimulation of Cl secretion alters the morphology of the vacuolated, columnar cell type in the colonic epithelium. Direct visualization of the morphologic changes involved in stimulation of secretion will be accomplished with differential interference contrast microscopy. These studies will provide a complete characterization of the location of active K transport in the colonic epithelium and permit an understanding of the ion transport events associated with intestinal fluid absorption and secretion.
