Involvement of the beta gamma subunits of inhibitory GTP-binding protein in chemoattractant receptor-mediated potentiation of cyclic AMP formation in guinea pig neutrophils.
Cellular cyclic AMP formation in response to prostaglandin (PG) E1 was markedly potentiated by the chemoattractant formyl-Met-Leu-Phe (fMLP) in guinea pig neutrophils. This potentiation by fMLP was abolished by prior treatment of the cells with pertussis toxin, but not by the prevention of an fMLP-induced intracellular Ca2+ increase in the cells, indicating the direct involvement of the inhibitory GTP-binding protein (Gj), but not Ca2+, in the fMLP-induced potentiation of cyclic AMP formation. Cyclic AMP formation in the neutrophils was also unique in response to forskolin; the diterpene inhibited cyclic AMP formation stimulated by PGE1 plus fMLP at low concentrations, but it slightly stimulated the basal and fMLP-induced cyclic AMP formation at high concentrations. Such a forskolin-induced inhibition was also observed in the adenylyl cyclase of the cell membranes and detergent extract therefrom only when the cyclase was activated by GTP or its nonhydrolyzable analogue (GTP gamma S). The forskolin-inhibitable activity could be affinity-purified from the GTP gamma S-treated cell membranes with a forskolin-agarose column. The cyclase appeared to be purified as a complex with the GTP gamma S-bound alpha subunit of the stimulatory GTP-binding protein (Gs alpha), but not with the beta gamma subunits, as judged from immunoblot analysis with specific antisera. The GTP gamma S-bound Gs alpha-stimulated cyclase activity was further enhanced by beta gamma, and this enhancement was again inhibited by forskolin. These results suggest that the GTP-bound Gs alpha produced by PGE1 receptor stimulation and the beta gamma subunits released from Gj by fMLP receptor stimulation were acting synergistically in the cyclic AMP formation of intact neutrophils.
Suzuki
T
T
Department of Physiological Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
Hazeki
O
O
Hazeki
K
K
Ui
M
M
Katada
T
T
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000527:Alprostadil / Q000502:physiology; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000378:metabolism*; D004789:Enzyme Activation; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000502:physiology*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006168:Guinea Pigs; D046911:Macromolecular Substances; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000378:metabolism*; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000502:physiology*; D018000:Receptors, Peptide / Q000502:physiology*; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000378:metabolism
D046911:Macromolecular Substances; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D018000:Receptors, Peptide; D005576:Colforsin; D009240:N-Formylmethionine Leucyl-Phenylalanine; D006160:Guanosine Triphosphate; D000242:Cyclic AMP; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D000527:Alprostadil; D002118:Calcium