Effects of cholesterol on CCK-1 receptors and caveolin-3 proteins recycling in human gallbladder muscle

• Published in: American journal of physiology: Gastrointestinal and liver physiology 2010-09, Vol.299 (3), p.G742-G750
• Main Authors: Cong, P, Pricolo, V, Biancani, P, Behar, J
• Format: Article
• Language: English
• Subjects: Animals; Antigens; Caveolin 3 - genetics; Caveolin 3 - metabolism; Cell Membrane; Cells; Cells, Cultured; Cholesterol; Cholesterol - pharmacology; Gallbladder; Gallbladder - anatomy & histology; Gene Expression Regulation; GTP-Binding Proteins - metabolism; Guinea Pigs; Humans; Liver and Biliary Tract; Male; Muscle Cells - cytology; Muscle Cells - drug effects; Muscle Cells - metabolism; Muscle, Smooth - drug effects; Muscle, Smooth - metabolism; Muscular system; Physiology; Proteins; Receptor, Cholecystokinin A - genetics; Receptor, Cholecystokinin A - metabolism; Rodents; Sincalide - pharmacology
• ISSN: 0193-1857; 1522-1547
• DOI: 10.1152/ajpgi.00064.2010

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.