sincalide and aluminum-fluoride

This study was designed to elucidate the mechanism of action of progesterone on gallbladder smooth muscle in guinea pigs. Adult male guinea pigs were treated with either progesterone (2 mg.kg-1.day-1) or saline for 7 days. Gallbladder muscle cells were isolated by enzymatic digestion with collagenase. Contractile responses to agonists were expressed as percent shortening from control cell length. [35S]guanosine 5'-O-(3-thiotriphosphate) ([35S]GTP gamma S)-binding properties of G proteins were assessed in crude membranes of gallbladder muscle with or without cholecystokinin octapeptide (CCK-8) stimulation. Gallbladder muscle cells from progesterone-treated guinea pigs exhibited an impaired contractile response to CCK-8, GTP gamma S, or aluminum fluoride but a normal response to potassium chloride or D-myo-inositol 1,4,5-trisphosphate compared with controls. Western blot analysis of gallbladder muscle revealed the presence of Gi1-2, Gi3, Gq/11, and Gs proteins. The maximal contraction induced by CCK-8 was blocked by pertussis toxin and Gi alpha 3-specific antibodies, but not by Gi alpha 1-2 or Gq/11 alpha antibodies. CCK-8 caused a significant increase in [35S]GTP gamma S binding to Gi alpha 3, but not to Gq/11 alpha or Gi alpha 1-2. The stimulation of Gi alpha 3 binding, however, was significantly reduced in gallbladder muscle membranes from progesterone-treated guinea pigs compared with that in control animals. In conclusion, progesterone might cause gallbladder hypomotility by downregulating Gi3 proteins.

Topics: Aluminum Compounds; Animals; Cholecystokinin; Fluorides; Gallbladder; GTP-Binding Proteins; Guinea Pigs; Inositol 1,4,5-Trisphosphate; Male; Muscle Contraction; Muscle, Smooth; Pertussis Toxin; Progesterone; Signal Transduction; Sincalide; Virulence Factors, Bordetella

Human gallbladders were used to investigate the mechanisms of the impaired contraction induced by cholecystokinin (CCK) associated with cholesterol stones. Single muscle cells were isolated enzymatically with collagenase. Inositol 1,4,5-trisphosphate was measured by high-performance liquid chromatography. Diacylglycerol was assayed by thin-layer chromatography. CCK stimulation showed decreased muscle contraction and production of inositol 1,4,5-trisphosphate and diacylglycerol in gallbladders with cholesterol stones compared with those with pigment stones. Exogenous calmodulin induced maximal contraction of 22.4 +/- 0.5 and 21.0 +/- 0.6% in gallbladders with cholesterol and pigment stones, respectively. Similar findings were observed with a synthetic diacylglycerol analogue. Two G protein activators, aluminum fluoride and guanosine 5'-O-(3-thiotriphosphate), evoked similar responses in these two types of gallbladders, with maximal contractions of 21.3 +/- 0.4 and 23.3 +/- 0.5%, respectively, in those with cholesterol stones and 20.9 +/- 0.8 and 22.6 +/- 0.4%, respectively, in those with pigment stones. These results suggest that receptor-dependent ligands like CCK cannot fully activate the intracellular pathways, which, however, can be fully stimulated by circumventing receptors with G protein activators or second messengers. After G protein activation, the pathways appear to be functionally intact. The defect might then reside in the receptor or in the interaction between receptors and G proteins.

Topics: Aluminum Compounds; Calmodulin; Cholecystokinin; Cholelithiasis; Cholesterol; Diglycerides; Dose-Response Relationship, Drug; Female; Fluorides; Gallbladder; GTP-Binding Proteins; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Inositol 1,4,5-Trisphosphate; Male; Middle Aged; Muscle Contraction; Signal Transduction; Sincalide