LIGAND-BASED HISTOCHEMICAL-LOCALIZATION AND CAPTURE OF CELLS EXPRESSING HEAT-STABLE ENTEROTOXIN RECEPTORS

The heat stable enterotoxins (ST) of enterotoxigenic Escherichia coli (ETEC) cause diarrhoea by binding specific intestinal receptors. Preci se histochemical localization of ST receptors could provide more infor mation about the pathophysiology of secretory diarrhoea and the role o f ST receptors in normal biology. To accomplish this, we quantitativel y coupled biotin to the N-terminus of ST1b using biotin-X-X-N-hydroxys uccinimide ester. The derivatized toxin (BST) has an apparent K(d) of 11.7+/-10 nM for rat brush border receptors. We used BST in an affinit y panning cell-capture system, to validate its ability to discriminate between receptor-positive and receptor-negative cells. Cell lines exp ressing ST receptors (human colon carcinoma T84, and COS cells transfe cted with guanylyl cyclase-C (GC-C) ST receptor cDNA) were captured to streptavidin and anti-biotin-coated plates with high efficiency and s pecificity. This system provides a novel approach to screening cells f or the presence of unique ST-binding proteins. BST was then used with streptavidin-gold to demonstrate the cellular topography of ST recepto rs at the light microscopic level. Villus enterocytes were intensely s tained, but only a faint signal was observed in upper crypts of rat sm all intestine. Thus, a gradient of increasing receptor density was see n as upper crypt cells matured into villus enterocytes. Higher magnifi cation revealed that ST receptors are.concentrated at the apical aspec t of villus enterocytes. Recently, guanylin, a putative endogenous lig and for ST receptors, has been localized to Paneth cells, at the base of intestinal crypts. Thus, ST receptors are concentrated in villus en terocytes, while guanylin appears to be produced at the base of the cr ypts. This topographical arrangement suggests that there are autocrine or paracrine pathways by which ST receptors interact with endogenous ligands.