Treatment of rabbit neutrophils with phorbol esters results in increased ADP-ribosylation catalyzed by pertussis toxin and inhibition of the GTPase stimulated by fMet-Leu-Phe.
The effects of pretreatment of rabbit neutrophils with phorbol 12-myristate 13-acetate on the ability of pertussis toxin to catalyze ADP-ribosylation and of fMet-Leu-Phe to activate a high-affinity GTPase in these cell homogenates were examined. The addition of phorbol 12-myristate 13-acetate, but not 4 alpha-phorbol 12,13-didecanoate, to intact cells was found to stimulate by more than 100% the pertussis toxin-dependent ribosylation of a 41 kDa protein (either the alpha-subunit of the 'inhibitory' guanine nucleotide-binding protein N or a closely analogous protein) and to inhibit by more than 60% the activation by fMet-Leu-Phe of the GTPase of the neutrophil homogenates. The addition of fMet-Leu-Phe to intact cells increases the ADP-ribosylation catalyzed by pertussis toxin of the 41 kDa protein. On the other hand, the exposure of neutrophil homogenates to fMet-Leu-Phe results in a decreased level of ADP-ribosylation. This decreased ribosylation reflects a dissociation of the GTP-binding protein oligomer that is not followed by association, possibly because of the release of the alpha-subunit into the suspending media. The implications of these results for the understanding of the mechanism of inhibition of cell responsiveness by phorbol esters and the heterologous desensitization phenomenon are discussed. Prominent among these are the possibilities that (i) the rate of dissociation of the Ni oligomer is affected by the degree of its phosphorylation by protein kinase C, and/or (ii) the dissociated phosphorylated alpha-subunit (the 41 kDa protein) is functionally less active than its dephosphorylated couterpart.
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D020558:GTP Phosphohydrolases / Q000032:analysis* / Q000037:antagonists & inhibitors; D019204:GTP-Binding Proteins / Q000378:metabolism; D066298:In Vitro Techniques; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D009504:Neutrophils / Q000187:drug effects* / Q000378:metabolism; D009702:Nucleoside Diphosphate Sugars / Q000378:metabolism*; D037342:Pertussis Toxin; D010704:Phorbols / Q000494:pharmacology*; D010744:Phosphoric Monoester Hydrolases / Q000032:analysis*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000032:analysis; D011817:Rabbits; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D009702:Nucleoside Diphosphate Sugars; D010704:Phorbols; D010566:Virulence Factors, Bordetella; D000246:Adenosine Diphosphate Ribose; D009240:N-Formylmethionine Leucyl-Phenylalanine; D037342:Pertussis Toxin; D011493:Protein Kinase C; D010744:Phosphoric Monoester Hydrolases; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate