Joseph Amaral

Excess membrane cholesterol alters human gallbladder muscle contractility and membrane fluidity

BACKGROUND & AIMS The relationship between muscle contractility, plasma membrane cholesterol, and fluidity was investigated in human gallbladders with gallstones. METHODS Isolated gallbladder muscle cells were used to measure contraction. Plasma membranes of gallbladder muscle were purified in a sucrose gradient and measured for cholesterol content and cholesterol/phospholipid mole ratio. Membrane fluidity was determined by using fluorescence polarization and was expressed as the reciprocal of anisotropy. RESULTS The maximal contraction induced by cholecystokinin octapeptide was significantly less in gallbladders with cholesterol stones than in those with pigment stones. The membrane cholesterol content and cholesterol/phospholipid mole ratio were significantly higher in gallbladders with cholesterol stones than in those with pigment stones. Membrane anisotropy was also higher than in gallbladders with pigment stones, reflecting lower membrane fluidity in gallbladders with cholesterol stones. After muscle cells from cholesterol stone gallbladders were incubated with cholesterol-free liposomes for 4 hours, cholecystokinin octapeptide-induced contraction, membrane cholesterol content and cholesterol/phospholipid ratio, and membrane fluidity returned to normal levels. CONCLUSIONS Gallbladder muscle from patients with cholesterol stones has increased membrane cholesterol/phospholipid mole ratio and decreased membrane fluidity resulting in impaired muscle contractility. These abnormalities are corrected by removing the excess cholesterol from the plasma membranes.

CCK receptor dysfunction in muscle membranes from human gallbladders with cholesterol stones

Human gallbladders with cholesterol stones exhibit impaired muscle contraction induced by agonists that act on transmembrane receptors, increased membrane cholesterol content, and abnormal cholesterol-to-phospholipid ratio compared with those with pigment stones. The present study was designed to investigate the functions of the CCK receptor of gallbladder muscle membranes by radioreceptor assay and cross-linking.125I-labeled CCK-8 binding was time-dependent, competitive, and specific. Scatchard analysis showed that the maximum specific binding (Bmax) was significantly decreased in cholesterol compared with pigment stone gallbladders (0.18 ± 0.07 vs. 0.38 ± 0.05 pmol/mg protein, P < 0.05). In contrast, the affinity for CCK was higher in cholesterol than pigment stone gallbladders (0.18 ± 0.06 vs. 1.2 ± 0.23 nM). Similar results were observed in binding studies with the CCK-A receptor antagonist [3H]L-364,718. Cross-linking and saturation binding studies also showed significantly less CCK binding in gallbladders with cholesterol stones. These abnormalities were reversible after incubation with cholesterol-free liposomes. The Bmax increased ( P < 0.01) and the dissociation constant decreased ( P < 0.001) after incubation with cholesterol-free liposomes. In conclusion, human gallbladders with cholesterol stones have impaired CCK receptor binding compared with those with pigment stones. These changes are reversed by removal of the excess membrane cholesterol. These receptor alterations may contribute to the defective contractility of the gallbladder muscle in patients with cholesterol stones.Human gallbladders with cholesterol stones exhibit impaired muscle contraction induced by agonists that act on transmembrane receptors, increased membrane cholesterol content, and abnormal cholesterol-to-phospholipid ratio compared with those with pigment stones. The present study was designed to investigate the functions of the CCK receptor of gallbladder muscle membranes by radioreceptor assay and cross-linking. 125I-labeled CCK-8 binding was time-dependent, competitive, and specific. Scatchard analysis showed that the maximum specific binding (Bmax) was significantly decreased in cholesterol compared with pigment stone gallbladders (0.18 +/- 0. 07 vs. 0.38 +/- 0.05 pmol/mg protein, P < 0.05). In contrast, the affinity for CCK was higher in cholesterol than pigment stone gallbladders (0.18 +/- 0.06 vs. 1.2 +/- 0.23 nM). Similar results were observed in binding studies with the CCK-A receptor antagonist [3H]L-364,718. Cross-linking and saturation binding studies also showed significantly less CCK binding in gallbladders with cholesterol stones. These abnormalities were reversible after incubation with cholesterol-free liposomes. The Bmax increased (P < 0.01) and the dissociation constant decreased (P < 0.001) after incubation with cholesterol-free liposomes. In conclusion, human gallbladders with cholesterol stones have impaired CCK receptor binding compared with those with pigment stones. These changes are reversed by removal of the excess membrane cholesterol. These receptor alterations may contribute to the defective contractility of the gallbladder muscle in patients with cholesterol stones.

Cholecystokinin-coupled intracellular signaling in human gallbladder muscle.

BACKGROUND/AIMS It has been shown that cholecystokinin (CCK) contracts the gallbladder muscle by utilizing intracellular calcium, but the intracellular pathways have not been elucidated. The present study was designed to characterize the signal transduction pathways that mediate CCK-induced contraction of human gallbladder muscle. METHODS Single muscle cells were isolated from human gallbladders by enzymatic digestion with collagenase. Permeable cells were obtained by incubation with saponin. Protein kinase C (PKC) activity was determined by measuring the phosphorylation of a specific substrate peptide from myelin basic protein, Ac-MBP(4-14). RESULTS The inositol-1,4,5-trisphosphate (IP3) antagonist heparin blocked the contractions induced by CCK. The PKC inhibitor H-7 blocked the contractions caused by low, but not high, concentrations of CCK and IP3. In contrast, the calmodulin inhibitor CGS9343B blocked the contractions induced by high, but not low, doses of CCK and IP3. Furthermore, exogenously activated calmodulin blocked the PKC-mediated contraction induced by diacylglycerol. Direct measurements of PKC activity showed that low, but not high, CCK concentrations caused PKC translocation. CONCLUSIONS CCK contracts the gallbladder muscle via IP3-mediated calcium release. CCK activates the PKC pathway at low concentrations, whereas it activates the calmodulin pathway at high concentrations, which in turn inhibits the activation of PKC.

Impaired muscle cytoprotection in human gallbladders with cholesterol stones

Background: The functional integrity of G protein coupling is a prerequisite for normal non-adrenergic, non-cholinergic (NANC) relaxation of the rabbit sphincter of Oddi (SO), a mechanism previously shown to be essentially nitrergic. To fulfil their biological function, however, G proteins must undergo a post-translational farnesylation process that enables them to associate with the membrane. Aim: We sought to establish whether the inhibition of farnesylation by either a dietary cholesterol overload or 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) inhibition influences the NANC relaxation of the rabbit SO. Methods: SO muscle rings from normal (N) or from hypercholesterolaemic (HC) animals, with or without treatment with lovastatin, a HMG-CoA reductase inhibitor (5 mg/kg/day orally during 5 days) were tested for changes in isometric tension in response to electrical field stimulation (FS :50 V, 0.1 ms, 20 Hz, 30 stimuli) in the presence of atropine (I /LM) and guanethidine (4 /LM). Additionally, both HC and lovastatin-treated animals underwent supplementary treatment with farnesol (30 /LM/kg i.v. twice a day during 3 days). Results FS relaxed the muscle rings, with an increase in tissue cyclic GMP content, as measured by radioimmunoassay. Preparations from HC rabbits (1.5% dietary cholesterol over 8 weeks, increasing the serum total cholesterollevel from 1.6~0.4 to 19.6~3.7 mmol/l) exhibited contractions with no change in cGMP content under the same conditions. The relaxation response was recaptured, with an increase in tissue cGMP content in preparations from HC rabbits treated with farnesol in the last 3 days of the dietary period. The treatment with lovastatin deteriorated the nitrergic relaxation of the SO from N animals, similarly to that produced by hypercholesterolemia. Conclusion: It has been demonstrated that treatment with lovastatin blunts the NANC relaxation phenomenon in preparations from normal animals, whereas farnesol supplementation improves the NANC relaxation function of the SO from animals with hypercholesterolemia, a condition which deteriorates NANC relaxation by itself. We therefore conclude that a deficiency in protein prenylation resulting from either the diet or HMG-CoA inhibition impairs the relaxation function of the rabbit SO. This work was supported by grant from the National Research Fund (OTKA No. F029398).