pervanadate and erbstatin

Soluble immune complexes activate a rapid burst of reactive oxidant secretion from neutrophils that have previously been primed with GM-CSF. Binding of these complexes to the cell surface of unprimed neutrophils results in the generation of intracellular Ca2+ transients, but the NADPH oxidase fails to become activated. No phospholipase D activity was observed following the addition of soluble immune complexes to unprimed cells. Upon priming with GM-CSF, the intracellular Ca2+ signal generated following soluble complex binding was greatly extended and phospholipase D was activated: there was also increased phosphorylation of proteins on tyrosine residues and the NADPH oxidase was activated. When Ca2+ influx was prevented, this phospholipase D activity was not observed. This primed oxidase activity was completely inhibited by erbstatin. Treatment of unprimed neutrophils with pervanadate (to inhibit protein tyrosine phosphatases) mimicked the effects of priming in that pervanadate-treated neutrophils secreted reactive oxidants in response to soluble immune complexes. The data indicate that during priming a new signaling pathway is activated that involves Ca2+ influx, phosphorylation on tyrosine residues, phospholipase D activity, and NADPH oxidase activation.

Topics: Aniline Compounds; Antigen-Antibody Complex; Calcium; Enzyme Activation; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Hydroquinones; NADPH Oxidases; Neutrophils; Phospholipase D; Phosphorylation; Phosphotyrosine; Platelet Activating Factor; Protein-Tyrosine Kinases; Reactive Oxygen Species; Respiratory Burst; Signal Transduction; Vanadates; Xanthenes

The mediators involved in eosinophil accumulation in diseases such as allergy continue to be an area of interest, even though little is known regarding the signaling involved in the human cell type recruitment. In the present study, we demonstrate a novel modulatory role of tyrosine kinase and tyrosine phosphatase activities on normal human eosinophil chemotaxis induced by different groups of chemoattractant.. Purified eosinophils were obtained from normal healthy volunteers with the CD16-negative procedure. Chemotactic activities against platelet-activating factor (PAF), vasoactive intestinal peptide (VIP) and eotaxin were assessed using a 48-well microchemotaxis chamber assay. Purified eosinophils were pretreated with herbimycin A, erbastatin or pervanadate to examine the role of tyrosine kinase in chemoattractant signaling.. Pretreatment of eosinophils with the tyrosine kinase inhibitors herbimycin A and erbstatin significantly blocked chemotaxis induced by eotaxin whilst both inhibitors augmented chemotaxis induced by VIP; however, they had no effect on PAF-induced chemotaxis. On the other hand, pretreatment of eosinophils with the phosphotyrosine phosphatase inhibitor pervanadate resulted in augmentation of eotaxin-induced chemotaxis and inhibition of VIP-induced chemotaxis, but it had no effect on PAF-induced chemotaxis.. These results suggest that protein kinase plays a modulatory role in eosinophil chemotaxis induced by various chemoattractants.

Topics: Benzoquinones; Chemokine CCL11; Chemokines, CC; Chemotactic Factors, Eosinophil; Chemotaxis, Leukocyte; Cytokines; Enzyme Inhibitors; Eosinophils; Humans; Hydroquinones; In Vitro Techniques; Lactams, Macrocyclic; Platelet Activating Factor; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Quinones; Rifabutin; Vanadates; Vasoactive Intestinal Peptide

Cyclic nucleotide-gated (CNG) channels in vertebrate photoreceptors are crucial for transducing light-induced changes in cGMP concentration into electrical signals. In this study, we show that both native and exogenously expressed CNG channels from rods are modulated by tyrosine phosphorylation. The cGMP sensitivity of CNG channels, composed of rod alpha-subunits expressed in Xenopus oocytes, gradually increases after excision of inside-out patches from the oocyte membrane. This increase in sensitivity is inhibited by a protein tyrosine phosphatase (PTP) inhibitor and is unaffected by three different Ser/Thr phosphatase inhibitors. Moreover, it is suppressed or reversed by application of ATP but not by a nonhydrolyzable ATP analog. Application of protein tyrosine kinase (PTK) inhibitors causes an increase in cGMP sensitivity, but only in the presence of ATP. Taken together, these results suggest that CNG channels expressed in oocytes are associated with active PTK(s) and PTP(s) that regulate their cGMP sensitivity by changing phosphorylation state. The cGMP sensitivity of native CNG channels from salamander rod outer segments also increases and decreases after incubation with inhibitors of PTP(s) and PTK(s), respectively. These results suggest that rod CNG channels are modulated by tyrosine phosphorylation, which may function as a novel mechanism for regulating the sensitivity of rods to light.

Topics: Adenosine Triphosphate; Animals; Cyclic GMP; Enzyme Inhibitors; Eye Proteins; Hydroquinones; Ion Channel Gating; Marine Toxins; Microcystins; Okadaic Acid; Oocytes; Oxazoles; Patch-Clamp Techniques; Peptides, Cyclic; Phenols; Phosphorylation; Protein Processing, Post-Translational; Protein Tyrosine Phosphatases; Rod Cell Outer Segment; Staurosporine; Vanadates; Xenopus laevis

To investigate the role of tyrosine phosphorylation in polymorphonuclear leucocyte (PMN) activation we have examined the effect of the potent tyrosine phosphatase (PTPase) inhibitor, vanadyl hydroperoxide, on PMN function. Western blotting of vanadyl hydroperoxide-treated PMN showed that there was a rapid dose-dependent increase in tyrosine-phosphorylated proteins. Vanadyl hydroperoxide also induced superoxide production in PMN over the range 10-100 microM, similar to the concentrations that also induced tyrosine phosphorylation. The tyrosine kinase inhibitor erbstatin totally inhibited the respiratory burst induced by vandyl hydroperoxide, showing that tyrosine kinase activity was necessary for superoxide production. The protein kinase C (PKC) inhibitors chelerythrine and bisidolylmaleimide inhibited the vanadyl hydroperoxide-induced respiratory burst with an inhibitory concentration of 50% (IC50) close to that for PKC inhibition without affecting tyrosine phosphorylation. These results indicate a possible role for PKC in vanadyl hydroperoxide-mediated superoxide production, and that any PKC involvement is downstream of tyrosine phosphorylation. These results further demonstrate that inhibition of phosphotyrosine phosphatases results in the activation of a functional response, indicating a critical role for phosphotyrosine phosphatases in PMN stimulation.

Topics: Animals; Dose-Response Relationship, Drug; Hydroquinones; NADH, NADPH Oxidoreductases; NADPH Oxidases; Neutrophil Activation; Neutrophils; Phosphorylation; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Rats; Rats, Wistar; Respiratory Burst; Superoxides; Vanadates