SIGNAL-TRANSDUCTION PATHWAYS MEDIATING CCK-INDUCED GALLBLADDER MUSCLE-CONTRACTION

The signal transduction that mediates CCK-induced contraction of gallb ladder muscle was investigated in the cat. Contraction was measured by scanning micrometry in single muscle cells isolated enzymatically wit h collagenase. Production of D-myo-inositol 1,4,5-trisphosphate (IP3) and sn.-1,2-diacylglycerol (DAG) was quantitated using HPLC and TLC, r espectively. Protein kinase C (PKC) activity was determined by measuri ng the phosphorylation of a specific substrate peptide from myelin bas ic protein, Ac-MBP-(4-14). CCK-induced contraction was blocked by incu bation in strontium medium, pertussis toxin (PTx), and antibodies agai nst G(i)alpha(3) or beta gamma-subunits but was not blocked by Ca2+-fr ee medium or by antibodies against G(q/11)alpha, G(i)alpha(1-2), or G( 0)alpha. The contraction induced by CCK was inhibited by the phospholi pase C (PLC) inhibitor U-73122, anti-PLC-beta 3 antibody, and the IP3 receptor antagonist heparin but was not inhibited by the the phospholi pase D inhibitor propranolol or antibodies against PLC-beta 1 or PLC-b eta 2. Western blot analysis of gallbladder muscle revealed the presen ce of PLC-beta 2 and PLC-beta 3 but not PLC-beta 1. CCK caused a 94% i ncrease 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 calmodul in antagonist CCS9343B. In conclusion, CCK contracts cat gallbladder m uscle by stimulating PTx-sensitive G(i3)protein coupled with PLC-beta 3, producing IP3 and DAG. Low doses activate PKC, whereas high doses a ctivate calmodulin.