8-bromocyclic-gmp and tetrafluoroaluminate

Ca2+-sensitization of smooth muscle occurs through inhibition of myosin light chain phosphatase (MLCP) leading to an increase in the MLCK:MLCP activity ratio. MLCP is inhibited through phosphorylation of its regulatory subunit (MYPT-1) following activation of the RhoA/Rho kinase (ROK) pathway or through phosphorylation of the PP1c inhibitory protein, CPI-17, by PKC delta or ROK. Here, we explore the crosstalk between these two modes of MLCP inhibition in a smooth muscle of a natural CPI-17 knockout, chicken amnion. GTPgammaS elicited Ca2+-sensitized force which was relaxed by GDI or Y-27632, however, U46619, carbachol and phorbol ester failed to induce Ca2+-sensitized force, but were rescued by recombinant CPI-17, and were sensitive to Y-27632 inhibition. In the presence, but not absence, of CPI-17, U46619 also significantly increased GTP.RhoA. There was no affect on MYPT-1 phosphorylation at T695, however, T850 phosphorylation increased in response to GTPgammaS stimulation. Together, these data suggest a role for CPI-17 upstream of RhoA activation possibly through activation of another PP1 family member targeted by CPI-17.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Aluminum Compounds; Amnion; Animals; Bacterial Toxins; Calcium; Chickens; Cyclic GMP; Enzyme Activation; Enzyme Inhibitors; Fluorides; Guanosine 5'-O-(3-Thiotriphosphate); Guanosine Triphosphate; In Vitro Techniques; Muscle Contraction; Muscle Proteins; Muscle, Smooth; Myosin-Light-Chain Phosphatase; Phosphoprotein Phosphatases; Phosphoproteins; Protein Phosphatase 1; Protein Subunits; Receptors, G-Protein-Coupled; rhoA GTP-Binding Protein; Signal Transduction; Vasoconstrictor Agents