Piero Biancani

Gallbladder Relaxation in Patients With Pigment and Cholesterol Stones

BACKGROUND & AIMSnGallbladders with cholesterol stones show a defective contraction in response to agonists. The aim of this study was to investigate the muscle relaxation of human gallbladders with cholesterol or black pigment gallstones.nnnMETHODSnGallbladder relaxation was measured in vitro using muscle strips and single muscle cells. Relaxation was expressed as percent inhibition of either basal active tension in strips or maximal cell contraction induced by diacylglycerol. The production of cyclic nucleotides was determined using a 125I-labeled radioimmunoassay kit.nnnRESULTSnFrequency-dependent relaxation evoked by electrical field stimulation was significantly lower in gallbladders with cholesterol stones than in gallbladders with pigment stones. Relaxation and adenosine 3,5-cyclic monophosphate (cAMP) production induced by isoproterenol, vasoactive intestinal peptide, and forskolin were also significantly decreased in gallbladders with cholesterol stones. However, the relaxation in response to 8-bromo-cAMP, nitric oxide (NO), and the NO donor S-nitroso-N-acetylpenicillamine (SNAP), which circumvent plasma membrane receptors and directly activate intracellular mechanisms, was similar in gallbladders with cholesterol and pigment stones. Guanosine 3,5-cyclic monophosphate production induced by NO and SNAP was also similar.nnnCONCLUSIONSnHuman gallbladder muscle from specimens with cholesterol stones show an impaired relaxation and lower cAMP production compared with specimens with pigment stones. The muscle defect(s) responsible for this impairment seem to be in the plasma membranes.

Effect of Vesical Outlet Obstruction on Detrusor Contractility and Passive Properties in Rabbits

Outlet obstruction was induced in 16 New Zealand rabbits by implanting a polyethylene tube (20 F) for a period of three months. The tube was slit longitudinally and placed around the bladder neck, between the ureters and vasa deferentia. It was left open to induce moderate obstruction and closed by a suture to induce severe obstruction. The animals were studied by cutting consecutive rings from each bladder, which were subjected to the following studies: morphology, contractility and mechanical properties. Morphology. Histology sections of the rings, studied by Hematoxylin and Eosin and Masson-Trichrome stains, demonstrated smooth muscle hypertrophy in moderate obstruction, while hyperplasia was the predominant response in severe obstruction. The average nuclear count per square millimeter of smooth muscle was 251 in controls, 87 in moderate obstruction and 705 in severe obstruction. Bladder wall thickness was significantly increased after both moderate and severe obstruction, as compared to controls. Contractility and mechanical properties. Each ring was tested in a muscle chamber under conditions of maximal electrical stimulation (100 V, 2 ms, 40 Hz). For each ring, full force-length relationships (active and passive) were obtained by stretching the rings in successive increments until length of maximal active force development was reached. Force was normalized per unit cross-sectional area (stress), and length according to a reference underformed state (per cent of unloaded length). The maximum active stress of the rings was taken as a measure of bladder contractility, and the rate of increase in passive force as a measure of detrusor stiffness. In all groups, the body of the detrusor exerted better contractility, as compared to a rigid, less contractile base. In the obstructed groups, detrusor contractility was significantly decreased at the body level, with increased stiffness, as compared to controls. The length at which maximum contractility was exerted, however, increased in moderate obstruction, and decreased in severe obstruction.

Excess membrane cholesterol alters human gallbladder muscle contractility and membrane fluidity

BACKGROUND & AIMSnThe relationship between muscle contractility, plasma membrane cholesterol, and fluidity was investigated in human gallbladders with gallstones.nnnMETHODSnIsolated 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.nnnRESULTSnThe 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.nnnCONCLUSIONSnGallbladder 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.

Calcium-dependent and calcium-independent contractions in smooth muscles

This review examines signal transduction pathways mediating agonist-induced contraction of circular muscle in the body of the esophagus and in the lower esophageal sphincter (LES). In the LES, circular muscle agonists activate a well-defined contractile pathway, involving calcium (Ca(2+))-induced activation of calmodulin and myosin kinase, causing phosphorylation of 20-kDa myosin light chains (MLCs) and contraction. In this pathway, phosphorylation and contraction may be modulated by other factors, resulting, for instance, in inhibition of phosphatase activity, which may potentiate MLC phosphorylation. The agonist-activated contractile pathway of circular muscle from the esophageal body is not as well defined, and it is different from the LES contractile pathway, as it depends on activation of a Ca(2+)-independent protein kinase C (PKC), PKC-epsilon. In this pathway, agonist-induced Ca(2+) influx and/or release activate phospholipases to produce second messengers, such as diacylglycerol and arachidonic acid. The second messengers, however, activate a PKC-epsilon and a contractile pathway, which is Ca(2+) independent. This contractile pathway depends on activation of the mitogen-activated protein (MAP) kinases ERK1 and ERK2 and of p38 MAP kinase. These kinases are, in turn, linked to the small heat-shock protein HSP27, to integrin-linked kinase, and perhaps to other Ca(2+)-independent kinases, such as zipper kinase capable of producing MLC phosphorylation and contraction.

Signal transduction pathways in esophageal and lower esophageal sphincter circular muscle.

Esophageal reflux is a common condition that affects children and 1 in 10 adults, and if untreated may result in chronic esophagitis, aspiration pneumonia, esophageal strictures, and Barretts esophagus, a premalignant condition. Although esophagitis is a multifactorial disease that may depend on transient lower esophageal sphincter (LES) relaxation, speed of esophageal clearance, mucosal resistance, and other factors, impairment of LES pressure is a common finding in patients complaining of chronic heartburn. Our data suggest that esophageal and LES circular muscle utilize distinct Ca2+ sources, phospholipid pools, and signal transduction pathways to contract in response to acetylcholine (ACh): (1) In esophageal muscle ACh-induced contraction requires influx of extracellular Ca2+ and may be linked to phosphatidylcholine metabolism, production of diacylglycerol (DAG) and arachidonic acid, and activation of a protein kinase C (PKC)-dependent pathway. (2) In LES muscle ACh-induced contraction utilizes intracellular Ca2+ release arising from metabolism of phosphatidylinositol (PI), and a calmodulin-myosin light chain kinase-dependent pathway. Resting LES tone, on the other hand, may be due to relatively low basal PI hydrolysis resulting in submaximal levels of inositol triphosphate (IP3)-induced calcium release and interaction with DAG to activate PKC. (3) After induction of experimental esophagitis, basal levels of PI hydrolysis and intracellular calcium stores are substantially reduced, resulting in a reduction of resting tone. In addition the signal transduction pathway responsible for LES contraction in response to ACh changes from one that depends on IP3 production, calcium release, and calmodulin activation to one that relies on influx of extracellular calcium and activation of PKC.

Signal transduction pathways mediating CCK-induced gallbladder muscle contraction

The signal transduction that mediates CCK-induced contraction of gallbladder muscle was investigated in the cat. Contraction was measured by scanning micrometry in single muscle cells isolated enzymatically with collagenase. Production ofd- myo-inositol 1,4,5-trisphosphate (IP3) and sn-1,2-diacylglycerol (DAG) was quantitated using HPLC and TLC, respectively. Protein kinase C (PKC) activity was determined by measuring the phosphorylation of a specific substrate peptide from myelin basic protein, Ac-MBP-(4-14). CCK-induced contraction was blocked by incubation in strontium medium, pertussis toxin (PTx), and antibodies against Giα3or βγ-subunits but was not blocked by Ca2+-free medium or by antibodies against Gq/11α, Giα1-2, or Goα. The contraction induced by CCK was inhibited by the phospholipase C (PLC) inhibitor U-73122, anti-PLC-β3 antibody, and the IP3 receptor antagonist heparin but was not inhibited by the the phospholipase D inhibitor propranolol or antibodies against PLC-β1 or PLC-β2. Western blot analysis of gallbladder muscle revealed the presence of PLC-β2 and PLC-β3 but not PLC-β1. CCK caused a 94% increase in IP3 generation and an 86% increase in DAG generation. A low dose of CCK caused PKC translocation, and CCK-induced contraction was blocked by the PKC inhibitor H-7. A high dose of CCK, however, caused no PKC translocation, and its contraction was blocked by the calmodulin antagonist CGS9343B. In conclusion, CCK contracts cat gallbladder muscle by stimulating PTx-sensitive Gi 3 protein coupled with PLC-β3, producing IP3 and DAG. Low doses activate PKC, whereas high doses activate calmodulin.

Cholecystokinin-coupled intracellular signaling in human gallbladder muscle.

BACKGROUND/AIMSnIt 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.nnnMETHODSnSingle 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).nnnRESULTSnThe 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.nnnCONCLUSIONSnCCK 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.

Effects of bile acids on the muscle functions of guinea pig gallbladder

Hydrophobic bile acids impair gallbladder emptying in vivo and inhibit gallbladder muscle contraction in response to CCK-8 in vitro. This study was aimed at determining the mechanisms of muscle cell dysfunction caused by bile acids in guinea pig gallbladders. Muscle cells were obtained by enzymatic digestion. Taurochenodeoxycholic acid (TCDC), a hydrophobic bile acid, caused a contraction of up to 15% and blocked CCK-induced contraction. Indomethacin abolished the TCDC-induced contraction. Hydrophilic bile acid tauroursodeoxycholic acid (TUDC) had no effect on muscle contraction but prevented the TCDC-induced contraction and its inhibition on CCK-induced contraction. Pretreatment with NADPH oxidase inhibitor PH2I, xanthine oxidase inhibitor allopurinol, and free-radical scavenger catalase also prevented TCDC-induced contraction and its inhibition of the CCK-induced contraction. TCDC caused H2O2 production, lipid peroxidation, and increased PGE2 synthesis and activities of catalase and SOD. These changes were significantly inhibited by pretreatment of PH2I or allopurinol. Inhibitors of cytosolic phospholipase A2 (cPLA2), protein kinase C (PKC), and mitogen-activating protein kinase (MAPK) also blocked the TCDC-induced contraction. It is concluded that hydrophobic bile acids cause muscle cell dysfunction by stimulating the formation of H2O2 via activation of NADPH and xanthine oxidase. H2O2 causes lipid peroxidation and activates cPLA2 to increase PGE2 production, which, in turn, stimulates the synthesis of free-radical scavengers through the PKC-MAPK pathway.

Mechanisms of gallbladder hypomotility in pregnant guinea pigs

BACKGROUND & AIMSnGallbladder muscle contraction becomes impaired during pregnancy. This study was designed to investigate the mechanisms of gallbladder hypomotility induced by pregnancy in guinea pigs.nnnMETHODSnGallbladder muscle cells were obtained by enzymatic digestion. Cell contraction was expressed as percent shortening of initial control cell length.nnnRESULTSnContraction induced by cholecystokinin (CCK)-8 or guanosine 5-O-(3-thiotriphosphate) (GTPgammaS) was reduced in muscle cells from pregnant guinea pigs. The response to KCl or D-myo-inositol 1,4, 5-trisphosphate was not different between controls and pregnant animals. These findings suggest that impaired contraction in pregnancy might be caused by defective G protein activation. The function and content of G proteins were examined by using [35S]GTPgammaS binding and G protein subunit quantitation. In female controls, CCK-8 at 1 micromol/L caused increased [35S]GTPgammaS binding to Galphai3 but not to Galphaq/11, Galphai1-2, or Galphas. GTPgammaS binding to Galphai3 induced by CCK-8 was reduced in gallbladder muscle from pregnant guinea pigs. Measurements of basal G proteins showed that the content of Galphai3 was significantly lower and the Galphas content was higher in muscles from pregnant guinea pigs than in controls.nnnCONCLUSIONSnPregnancy may cause down-regulation of contractile G proteins such as Galphai3 and up-regulation of Galphas that mediates relaxation, resulting in impaired gallbladder muscle contraction.