The long-term goal is to elucidate the molecular mechanisms by which the second messengers cAMP, cGMP and Ca regulate electrolyte and water transport in epithelia. Sodium- or Na/K-dependent mechanisms of Cl permeation into cells are important both in the vectorial transport of salt and water across epithelia and in volume regulation in a variety of tissues. Malfunctioning of ion transport processes, including cation-coupled Cl entry mechanisms, has been implicated in a number of diseases, including glaucoma, cystic fibrosis, diarrhea and essential hypertension. In epithelia, net fluid transport represents a balance between active absorption and active secretion and Na/Cl or Na/K/Cl cotransport mechanisms are necessary components of both processes. An increase in the intracellular content of either Ca, cAMP or, in some cases, cGMP, regulates Na/Cl transport in most epithelia, leading to a decrease in fluid absorption in absorptive epithelia and to an increase in fluid secretion in secretory epithelia. These second messengers probably act via specific protein kinases. This proposal aims to correlate Ca-, cAMP- and cGM-mediated changes in Na/K/Cl cotransport with phosphorylation of specific proteins in intact tissues and to characterize the transport-related phosphoproteins. The model system is the intestine of the winter flounder, where Na/K/Cl cotransport is important in osmoregulation. This simple epi thelium is relatively homogeneous, and possesses a well-characterized Na/K/Cl cotransport mechanism that can be regulated by all three second messengers. Changes in phosphorylation and cotransport wil be correlated on the basis of time, dose dependency and reversibility of effects. Specific phosphoprotein substrates will be identified by one- and two-dimensional gel electrophoresis followed by autoradiography, and transport will be assessed by radioisotope fluxes. Specific substrates and kinases will be characterized by various techniques, including subcellular fractionation, photoaffinity labelling, peptide mapping, and phosphoaminoacid analysis. The overall objective is to identify the molecular nature of ion transport components; second-messenger-specific phosphoproteins could be the transporter per se or modulaters thereof. These studies will provide an excellent model for examining similar transport mechanisms in more complex epithelia such as mammalian ileum and the thick ascending limb of Henle's loop in mammalian kidney.
