The Research: The Principal Investigator's LONG TERM RESEARCH OBJECTIVE is to elucidate the intracellular events that regulate passive flow through the paracellular space of intestinal epithelia in states of both health and disease. The zonula occludens (ZO) is the rate-limiting barrier to passive flow through the paracellular space. It has been determined recently that a large portion of meal-related nutrients are harvested via the paracellular space. In addition, it appears that alterations in ZO permeability also play a role in disease states. Clostridium difficile toxin A, which is responsible for producing the pathologic changes associated with pseudomembranous colitis, has been shown to induce a morphologically undetectable but functionally significant alteration in the ZO barrier function of cultured intestinal epithelial cells. Although the intracellular events that regulate flow across this space have not been elucidated, it appears that second messengers (including protein kinase C, PKC) and the cytoskeleton are involved. Preliminary data indicates that the phorbol ester 12-0- tetradecanoyl-phorbol-13-acetate (TPA) and C. difficile toxins A and B will serve as useful agents for probing the intracellular mediators that regulate ZO permeability. For these studies we will use a cultured intestinal epithelial cell line, T84.
The SPECIFIC AIMS of this proposal are: 1) To correlate TPA-induced cytoskeletal changes with alterations in T84 barrier function; to determine if such effects are mediated by PKC and if so, to determine if phosphorylation of key cytoskeletal or ZO- associated proteins is responsible for the effects; 2) To investigate the role of PKC and other intracellular mediators in C. difficile toxin A and B-induced effects on T84 barrier function and cytoskeleton; and 3) To determine whether actin assembly-dissembly is required for TPA, toxin A and toxin B to exert their effects on ZO barrier function. The Environment: The University of Illinois at Chicago College of Medicine provides a rich environment for research in both clinical and basic science areas. The Section of Gastroenterology consists of 7 full time faculty members. 5 of which are committed to basic science research. The Section alone has 2,500 sq. ft. of research space. There are strong ties with many basic science faculty members from various departments, in particular Physiology. Dr. Rao, the applicant's sponsor, is an established investigator in the Department of Physiology and Biophysics. The applicant also utilizes other members in the Department of Physiology on a consultative basis regarding electrophysiologic issues (Dr. Randall Hudson) and regulation of actomyosin contraction (Dr. Primal deLanerolle).
