We have complelling preliminary data suggesting that genes associated with the murine Major Histocompatibility Complex (MHC,H-2) control the degree of small intestinal fluid accumulation (FA) in SAM (Sealed Adult Mice) challenged orally with cholera enterotoxin (CT). Various strains of inbred mice carrying the H-2b haplotype are sensitive (S) to CT H-2k haplotypes are resistant (R). Assays in 3 different strains (B10, C3H, and BALB) of congenic and congenic/recombinant mice differing genetically only in H-2 haplotype, corroborated the original observation. The objectives of the proposed research are to unequivocally confirm the H-2 associated linkage, map the responsible genes, and elucidate the mechanisms involved in their phenotypic expression. To meet these objectives we will: (1) determine the segregation patterns of S/R and haplotype in F1, F2, and backcross progeny from SxR matings, (ii) localize the genes encoding for S/R in appropriate congenic/recombinants, (iii) look for soluble intra-luminal inhibitors of CT in intestine of S/R strains, (iv) determine CT receptor densities on S/R enterocytes and extra-intestinal (EI) cells grown in vitro, (v) compare adenyl-cyclase/phosphodiesterase/NADase activities and intracellular c-AMP/NAD concentrations in S/R membranes and cells, (vi) quantitate cytosolic factors required for CT-stimulated cyclase activity, measure CT-catalysed ADP-rebosylation of membrane and soluble proteins, and assay c-AMP-stimulated phosphorylation of proteins in S/R enterocytes and EI cells, (vii) analyse S/R membranes for functional/compositional differences (e.g. gangliosides, glycoproteins, and their biosynthetic enzymes) involved in interactions with CT and its A and B subunits, (viii) explore the possibility that nucleotide-independent mechanisms are completely or partially involved in some CT-activated events. These findings may be applicable to the human MHC system, HLA, and """"""""natural"""""""" immunity to enterotoxin incited diarrhea and thus could form the basis for immunity testing and rational vaccination programs.
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