IMPAIRED GALLBLADDER CONTRACTION WITH GALLSTONES

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Gallbladder stasis due to a defective muscle contraction is thought to play a permissive role in gallstone formation and growth and may be a major contributing factor in the recurrence of gallstones. This defective gallbladder contraction in humans and prairie dogs is associated with excess bile cholesterol and is characterized by a reduced response to receptor-dependent ligands such as CCK-8 and acetylcholine. CCK-8 does not fully activate available intracellular pathways of defective muscle cells that appear to be functionally normal. The contraction of control muscle cells induced by low concentrations of CCK-8 is mediated by PKC, whereas that induced by high concentrations is calmodulin dependent. In contrast, the contraction of defective muscle cells induced by all CCK-8 concentrations can only utilize the PKC pathway. The magnitude of contraction of these defective muscle cells is normalized when membrane receptors are circumvented by directly activating G proteins with GTPgammas or by utilizing second messengers such as IP3 and DAG or by calmodulin. These data suggest that the defect may lie in the signal transduction across the plasma membrane possibly due to excessive incorporation of cholesterol which could affect its lipid milieu and transmembrane proteins. This hypothesis is supported by our preliminary findings: 1) that normal muscle cells incubated with cholesterol-rich liposomes increased their cholesterol:phospholipid (Ch:P1) ratio in the plasma membrane and develop a defective contraction in response to CCK which is normalized when treated with GTPgammas; and, 2) that the abnormal Ch:P1 ratio and defective contraction of muscle cells from gallbladders with cholesterol stones are normalized after incubation with cholesterol-free liposomes. We therefore propose to investigate in control and defective muscle cells from human and prairie dog gallbladders: 1) the direct role of cholesterol in the pathogenesis of the defective muscle contraction and relaxation; 2) the generation of second messengers that mediate contraction and relaxation induced by receptor-dependent ligands and by agonists that circumvent membrane receptors; 3) the changes of the plasma membrane of control and defective muscle cells before and after incubation with cholesterol-rich and cholesterol-free liposomes by measuring its cholesterol:phospholipid ratio and membrane fluidity; 4) the functional abnormalities of membrane receptors in these defective muscle cells by measuring ligand binding, receptor-G protein coupling and receptor activation of G proteins; and, 5) whether these cholesterol associated muscle abnormalities are reversible in vitro after incubation with cholesterol-free liposomes and in vivo after cholesterol-free or low cholesterol diets.