Chloride (Cl-) transport is important in a variety of cellular functions such as osmoregulation, neurotransmission, intracellular pH regulation, and transepithelial salt and fluid secretion. Pathological disturbances of chloride channels have been found to underlie anomalous salt and fluid secretion leading to secretory diarrhea and cystic fibrosis. Elevation of intracellular calcium [Ca++]i leads to cl- secretion in monolayers of the colonic epithelial cell line, T84. However, we have recently demonstrated that Cl- secretion becomes uncoupled from [Ca2+]i following maintained exposure to carbachol. Elevation of D-myo-inositol(3,4,5,6) (Ins(3,4,5,6)P4) may play a role in this uncoupling. 1) Ins(3,4,5,6)P4 levels are elevated at time points corresponding to inhibition of Cl- secretion. 2) A cell permeant analog of Ins(3,4,5,6)P4 partially uncouples short circuit current (Isc) from the thapsigargin stimulated rise in [Ca++]i. Very recently, we have found that the invasive bacteria, Salmonella, stimulates a specific and dramatic increase in DL- Ins(3,4,5,6)P4, suggesting that D or L Ins (3,4,5,6)Pr may also play a role in Salmonella entry or other response to bacterial invasion. The overall aim of this proposal is to ascertain the functional role of DL- Ins(3,4,5,6)Pr in colonic epithelia. Thus, we plan to study how Salmonella trigger a rise in InsP4 levels and test whether Ins(3,4,5,6)P4 elevation plays a role in bacterial entry or epithelial response to bacterial entry. Because of the specificity of the Ins(3,4,5,6)P4 response of T84 monolayers to Salmonella entry, it will enable us further test the role of Ins(3,4,5,6)P4 in the uncoupling of Cl- secretion from [Ca++]i. We plan to do this by identifying the metabolic pathways stimulated by carbachol which most closely correlate with the uncoupling. We study the activity of pertinent enzymes following stimulation to determine which enzyme mediates the rise in Ins(3,4,5,6)Pr. Preliminary data and findings by others indicate that the Ca2+-dependent Cl- secretion in confluent T84 cells is dependent on Ca2+- activated K+ channels, the probable target of Ins(3,4,5,6)P4 inhibition. We will test this hypothesis with experiments using cell permeant analogs of InsP4 and measure 86Rb+ and 125I efflux in response to Ca2+ elevation in T84 monolayers in Using chambers. These experiments will be complemented by patch clamp studies performed in collaboration with Dr. Greg Fitz. These studies will lead to a greater understanding of Cl- channels, especially mechanisms by which they are negatively regulated. Such information could lead to the development of therapeutic agents which could regulate pathological Cl- channel activity.
