We propose to better understand the course of diarrheal diseases by studying the intracellular regulation of active intestinal electrolyte transport. We will concentrate on the role of Ca++ and calmodulin dependent phosphorylation in the regulation of active ileal electrolyte transport and will study:- 1. Protein kinase activity and endogenous protein substrates for phosphorylation in epithelial cells of rabbit ileum a. in whole homogenates and b. in purified membranes (microvillus and basolateral), intracellular organelles (endoplasmic reticulum and mitochondria) and cytosol. Each will be studied in the presence and absence of Ca++, calmodulin, cyclic AMP and combinations of the three. Each purified fraction will also be studied in the presence of cytosol to seek interactions between the cytosol kinases, phosphatases and substrates of the cytosol and the purified fractions. We will also study the effects of drugs which interfere with the action of Ca++ calmodulin, or cyclic AMP, or which are thought to interfere with transport or regulatory proteins directly e.g. trifluoperazine, W7, W5, dantrolene, TMB-8 and chloroquine. All these studies will be performed by incubation with Gamma32-P-ATP and analysis by SDS polyacrylamide gel electrophoresis, autoradiography and densitomery. 2. Protein phosphorylation and endogenous protein substrates in intact intestinal epithelial cells. After incorporation of 32PO4 into the nucleotide pools of the cells, the cells will be exposed to conditions and agents which affect the cytosol free Ca++ concentrations, cyclic AMP levels, Ca++ calmodulin, and electrolyte transport, e.g. ionophore A23187, carbachol, serotonin; calcium deprivation, EGTA (acutely), verapamil, dopamine; theophylline, 8-bromo cyclic AMP; and other drugs (as mentioned for the broken cell studies). Correlation of the phosphorylation of specific proteins with changes in intestinal ion transport may be possible as a result of these studies. This would contribute to the goal of understanding the mechanisms and control of absorption and secretion in intestine, and the ultimate goal of modifying active intestinal transport in such a way as to be useful in the therapy of diarrheal diseases.
