The goal of this proposal is to elucidate the biology of crytpdin-induced channel formation in crypt epithelia. Cryptdins are intestinal (Paneth cell) specific alpha-defensins - cationic peptides first recognized as neutrophil products, which exhibit potent antimicrobial properties. Nineteen cryptdin isoforms have been described in the mouse and two, HD-5 and HD-6, in the human. The antimicrobial action of alpha-defensins is thought to reside in their ability to partition into biomembranes and produce anion conductive pores. Cryptdins, and HD-5 and HD-6 are known to be released from Paneth cells into the crypt lumen and thus other crypt cell types, the major one being the chloride secreting """"""""undifferentiated"""""""" crypt cell, are naturally exposed to these channel forming peptides. The PI and his collaborators have recently shown the cryptdins 2 and 3 induce a physiologic chloride secretory response in human intestinal T84 cells likely by forming anion conductive channels in the apical (lumenal) membrane. Studies proposed will test the hypothesis that Paneth cell-derived cryptdins act to regulate the physiology of the intestinal crypt by paracrine insertion (self-assembly) of chloride conducting pores into apical membranes of neighboring crypt epithelial cells and down regulation by endocytosis and apical membrane restitution. In vitro experiments will define the mechanism by which mouse cryptdins 2 and 3 induce chloride secretion. The PI and his collaborators will use matrilysin -/- mouse and CR2-tox176 mouse to elucidate the effects of cryptdins on small intestinal functions ex vivo and in vivo. The biophysical properties and structure function of cryptdin 3 and HD-5 and HD-6 will be examined in intact and semi-permeabilized T84 and renal HEK cell model systems and by single channel patch clamp. Finally studies are proposed to elucidate the cellular mechanism(s) of cryptdin 3-induced channel formation and down regulation. The significance of these studies is emphasized by accumulating evidence that the intestinal crypt plays a central role in intestinal epithelial cell ontogeny, physiology (solute and water transport), and specific and non-specific mucosal defense. Cryptdins act in paracrine fashion to regulate crypt cell function and appear to have a unique mechanism of action. Proposed studies will elucidate the biology of these novel alpha-defensins and may set the stage for future development of alpha-defensin inhibitors or for use of these (or related peptides) in treatment of disease at the mucosal surfaces of the intestine or respiratory tract.
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