calyculin-a and 1-2-dioctanoylglycerol

Neutrophils contain a 21-kDa phosphoprotein that undergoes rapid dephosphorylation upon stimulation of these cells with the chemoattractant N-fMet-Leu-Phe (fMLP), activators of protein kinase C [e.g., 4 beta-phorbol 12-myristate 13-acetate (PMA)] or the calcium ionophore A23187. This phosphoprotein was identified as the non-muscle form of cofilin by peptide sequencing and immunoblotting with specific antibodies. Evidence is presented that in neutrophils cofilin is regulated by a continual cycle of phosphorylation and dephosphorylation, and that the phosphatase undergoes activation during cell stimulation. Experiments with a wide variety of antagonists further suggested that the protein kinase that participates in these reactions may be a novel enzyme. The kinetics of cofilin dephosphorylation in neutrophils stimulated with fMLP or PMA were very similar to those observed for superoxide (O2-) release. Immunofluorescent studies revealed that cofilin was present throughout the cytosol of resting neutrophils and underwent rapid translocation to the F-actin-rich, ruffled membranes of stimulated cells. Cytochemical analysis further revealed that the ruffled membranes also contained large amounts of hydrogen peroxide (H2O2), a product of the O2-/H2O2-generating activity of stimulated neutrophils (NADPH oxidase). Cofilin is therefore well placed to participate in the continual polymerization and depolymerization of F-actin that is thought to give rise to the oscillatory pattern of H2O2 production observed under certain conditions.

Topics: Actin Depolymerizing Factors; Actins; Amino Acid Sequence; Animals; Calcimycin; Cell Membrane; Diglycerides; Diterpenes; Guinea Pigs; Humans; Hydrogen Peroxide; Immunoblotting; Marine Toxins; Microfilament Proteins; Molecular Sequence Data; N-Formylmethionine Leucyl-Phenylalanine; NADPH Oxidases; Neutrophil Activation; Neutrophils; Okadaic Acid; Oxazoles; Oxides; Phosphorylation; Staurosporine; Terpenes; Tetradecanoylphorbol Acetate

The goal was to investigate the role of protein kinases in modulating taurine transporter activity in Xenopus laevis oocytes expressing the mouse retinal Na+/C-/taurine transporter. The currents generated by the taurine transporter were studied with a two-electrode voltage clamp and we recorded the maximal current (Imax), presteady-state charge transfer Q, and membrane capacitance Cm. 8-Br-cAMP, a membrane-permeable activator of the cAMP-dependent protein kinase (PKA), decreased Imax (41%), Q (41%) and Cm (10%). Similarly, 1 microM sn-1,2-dioctanoylglycerol (DOG), an activator of the Ca2+/diacylglycerol-dependent protein kinase (PKC), decreased Imax (56%), Q (37%), and Cm (9%). Calyculin A, a specific inhibitor of protein phosphatases 1 and 2A, also produced effects similar to those of 8-Br-cAMP and DOG, and decreased Imax (64 %), Q (38%), and Cm (10%). We conclude that the taurine transporter is regulated by activators of PKA and PKC, and regulation occurs largely by changes in the number of transporters in the plasma membrane.

Topics: 8-Bromo Cyclic Adenosine Monophosphate; Animals; Biological Transport; Carrier Proteins; Chlorides; Cyclic AMP-Dependent Protein Kinases; Diglycerides; Electrophysiology; Endocytosis; Enzyme Activation; Exocytosis; Female; Gene Expression Regulation; Marine Toxins; Membrane Glycoproteins; Membrane Transport Proteins; Mice; Oocytes; Oxazoles; Phosphoprotein Phosphatases; Protein Kinase C; Recombinant Proteins; Retina; Sodium; Xenopus laevis