Impaired G protein function in gallbladder muscle from progesterone-treated guinea pigs

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Abstract
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]GTPγS)-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, GTPγS, or aluminum fluoride but a normal response to potassium chloride ord- myo-inositol 1,4,5-trisphosphate compared with controls. Western blot analysis of gallbladder muscle revealed the presence of Gi 1–2, Gi 3, Gq/11, and Gsproteins. The maximal contraction induced by CCK-8 was blocked by pertussis toxin and Giα3-specific antibodies, but not by Giα1–2or Gq/11α antibodies. CCK-8 caused a significant increase in [35S]GTPγS binding to Giα3, but not to Gq/11α or Giα1–2. The stimulation of Giα3binding, 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 Gi 3proteins.