title: Effects of cholesterol on CCK-1 receptors and caveolin-3 proteins recycling in human gallbladder muscle.
abstract: The contraction of gallbladders (GBs) with cholesterol stones is impaired due to high cholesterol concentrations in caveolae compared with GBs with pigment stones. The reduced contraction is caused by a lower cholecystokinin (CCK)-8 binding to CCK-1 receptors (CCK-1R) due to caveolar sequestration of receptors. We aimed to examine the mechanism of cholesterol-induced sequestration of receptors. Muscle cells from human and guinea pig GBs were studied. Antibodies were used to examine CCK-1R, antigens of early and recycling endosomes, and total (CAV-3) and phosphorylated caveolar-3 protein (pCAV-3) by Western blots. Contraction was measured in muscle cells transfected with CAV3 mRNA or clathrin heavy-chain small-interfering RNA (siRNA). CCK-1R returned back to the bulk plasma membrane (PM) 30 min after CCK-8 recycled by endosomes, peaking at 5 min in early endosomes and at 20 min in recycling endosomes. Pretreatment with cholesterol-rich liposomes inhibited the transfer of CCK-1R and of CAV-3 in the endosomes by blocking CAV-3 phosphorylation. 4-Amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (inhibitor of tyrosine kinase) reproduced these effects by blocking pCAV-3 formation, increasing CAV-3 and CCK-1R sequestration in the caveolae and impairing CCK-8-induced contraction. CAV-3 siRNA reduced CAV-3 protein expression, decreased CCK-8-induced contraction, and accumulated CCK-1R in the caveolae. Abnormal concentrations of caveolar cholesterol had no effect on met-enkephalin that stimulates a delta-opioid receptor that internalizes through clathrin. We found that impaired muscle contraction in GBs with cholesterol stones is due to high caveolar levels of cholesterol that inhibits pCAV-3 generation. Caveolar cholesterol increases the caveolar sequestration of CAV-3 and CCK-1R caused by their reduced recycling to the PM.
authors.affiliation: Department of Medicine, Rhode Island Hospital, Brown University Medical School, Providence, USA.
authors.forename: P
authors.initials: P
authors.lastname: Cong
authors.forename: V
authors.initials: V
authors.lastname: Pricolo
authors.forename: P
authors.initials: P
authors.lastname: Biancani
authors.forename: J
authors.initials: J
authors.lastname: Behar
mesh_terms: D000818:Animals; D051244:Caveolin 3 / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane; D002478:Cells, Cultured; D002784:Cholesterol / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000378:metabolism; D005704:Gallbladder / Q000033:anatomy & histology*; D005786:Gene Expression Regulation; D006168:Guinea Pigs; D006801:Humans; D008297:Male; D032342:Muscle Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009130:Muscle, Smooth / Q000187:drug effects / Q000378:metabolism*; D043886:Receptor, Cholecystokinin A / Q000235:genetics / Q000378:metabolism*; D012844:Sincalide / Q000494:pharmacology
chemical_list: D051244:Caveolin 3; D043886:Receptor, Cholecystokinin A; D002784:Cholesterol; D019204:GTP-Binding Proteins; D012844:Sincalide