title: Roles of human peripheral blood leukocyte protein kinase C and G proteins in inflammatory mediator release by isogenic Escherichia coli strains.
abstract: The signal transduction pathway (protein kinase C [PKC], calcium influx, and G protein involvement) was studied with isogenic Escherichia coli strains expressing different types of adhesins (MSH+/- MS-Fim+/-, P-MRH+/- P-Fim+/-, and S-MRH+/- S-Fim+/-) or varying only in the expression of E. coli alpha-hemolysin. As target cells, human polymorphonuclear granulocytes (PMN) and a lymphocyte-monocyte-basophil (LMB) cell suspension were used. The alpha-hemolysin-producing (Hly+) strain E. coli K-12(pANN5211) induced calcium influx in a dose-dependent manner in both cell types. No calcium influx was detected after stimulation with the hemolysin-negative (Hly-) E. coli bacteria independent of the type of fimbriae. With Hly+ bacteria, a dose-dependent activation of PKC was observed in both cell types. The Hly- E. coli K-12 induced PKC to a lesser degree, expressing kinetics different from those of E. coli K-12(pANN5211) (Hly+). E. coli MSH+ MS-Fim+ was the most potent activator for PKC. Membrane preparations from leukocytes stimulated with Hly+ E. coli K-12(pANN5211) showed increased binding of [3H]guanylylimidodiphosphate, a nonhydrolyzable GTP analog, and increased GTPase activity compared with leukocytes stimulated with Hly- E. coli K-12. The amounts of GTPase activation and [3H]guanylylimidodiphosphate binding were similar for all Hly- E. coli bacteria in human PMN as well as in human LMB; no activation was obtained for E. coli bacteria without any type of fimbriae. GTP-gamma-S, a nonhydrolyzable GTP analog, inhibited the leukotriene B4 (LTB4) generation from human PMN by Hly- bacteria, unlike E. coli K-12(pANN5211). However, in the presence of NaF, a predominant activator of Gi, LTB4 generation by Hly+ and by Hly- bacteria was significantly enhanced. For LMBs only LTB4 generation by Hly+ bacteria was increased in the presence of GTP-gamma-S. NaF decreased the chemiluminescence induced by all E. coli strains. Our results thus indicate that (i) Hly+ and Hly- bacteria induce the activation of distinct G proteins, e.g., Gi, to different degrees, (ii) LTB4 generation and chemiluminescence response are differently regulated, and (iii) in comparison with PMN, a different signal transduction pathway is activated by E. coli bacteria in LMBs.
authors.affiliation: Lehrstuhl für Medizinische Mikrobiologie und Immunologie, Arbeitsgruppe für Infektabwehrmechanismen, Ruhr-Universität Bochum, Germany.
authors.forename: B
authors.initials: B
authors.lastname: König
authors.forename: W
authors.initials: W
authors.lastname: König
mesh_terms: D002118:Calcium / Q000378:metabolism; D004789:Enzyme Activation; D004926:Escherichia coli / Q000378:metabolism* / Q000472:pathogenicity; D019204:GTP-Binding Proteins / Q000502:physiology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D007962:Leukocytes / Q000378:metabolism*; D007975:Leukotriene B4 / Q000096:biosynthesis*; D008163:Luminescent Measurements; D009504:Neutrophils / Q000378:metabolism; D011493:Protein Kinase C / Q000502:physiology*; D015398:Signal Transduction; D012969:Sodium Fluoride / Q000494:pharmacology
chemical_list: D007975:Leukotriene B4; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D012969:Sodium Fluoride; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; D002118:Calcium