cyclic-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

Exposure of cultured endothelial cells to shear stress resulting from well-defined fluid flow stimulates the production of nitric oxide (NO). We have established that an initial burst in production is followed by sustained steady-state NO production. The signal transduction events leading to this stimulation are not well understood. In the present study, we examined the role of regulatory guanine nucleotide binding proteins (G proteins) in shear stress-mediated NO production. In endothelial cells not exposed to shear stress, AIF4-, a general activator of G proteins, markedly elevated the production of guanosine 3',5'-cyclic monophosphate (cGMP). Pretreatment with NO synthase inhibitor N omega-nitro-L-arginine completely blocked this stimulation. Incubation with guanosine 5'-O-(2-thiodiphosphate) (GDP beta S), a general G protein inhibitor, blocked the flow-mediated burst in cGMP production in a dose-dependent manner. Likewise, GDP beta S inhibited NOx (NO2 + NO3) production for the 1st h. However, inhibition was not detectable between 1 and 3 h. Pertussis toxin (PTx) had no effect on the shear response at any time point. The burst in NO production caused by a change in shear stress appears to be dependent on a PTx-refractory G protein. Sustained shear-mediated production is independent of G protein activation.

Topics: Aluminum Compounds; Cells, Cultured; Cyclic GMP; Endothelium, Vascular; Fluorides; GTP-Binding Proteins; Guanosine Diphosphate; Humans; Nitric Oxide; Osmolar Concentration; Pertussis Toxin; Stress, Mechanical; Thionucleotides; Virulence Factors, Bordetella