A pertussis toxin-sensitive GTP-binding protein in the human neutrophil regulates multiple receptors, calcium mobilization, and lectin-induced capping.

Abstract

Human neutrophils treated with pertussis toxin had decreased functional responses to several agents including zymosan-treated serum, heat-aggregated immunoglobulin, platelet-activating factor, and fMet-Leu-Phe. Responses affected include superoxide generation and release of lysozyme. The degree and type of inhibition was dependent on the individual receptor and the cellular response studied. Measurement of intracellular calcium levels with quin-2 showed that both fMet-Leu-Phe-and platelet-activating factormediated increases in quin-2 fluorescence were diminished as a result of pertussis toxin treatment. fMet-Leu-Phe-mediated calcium uptake was also inhibited. However, under conditions where fMet-Leu-Phe-mediated effects on cell function were completely abolished, only a partial inhibition of 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8) sensitive calcium uptake was observed. A study of the linked reactions of chemotaxis, capping, and shape change revealed that (i) chemotaxis was inhibited regardless of the chemoattractant utilized (zymosan-treated serum, fMet-Leu-Phe, and platelet-activating factor) and (ii) the associated reactions of Con A capping and fMet-Leu-Phe-or Con A-mediated shape change were reduced in pertussis toxin-treated cells. Our results suggest that multiple mediators of inflammation act through a pertussis toxin-sensitive GTP-binding protein that regulates the mobilization of internal calcium as well as calcium uptake and is, in addition, a key control element of shape change, capping, and chemotaxis.The human neutrophil carries out several complex cellular host-defense and inflammation reactions in response to diverse receptor-mediated signals (1-4). We have shown that the fMet-Leu-Phe (FMLP) receptor acts through Ni, a GTP-binding protein sensitive to modification by pertussis toxin (PT) (5). As a result of the modification of Ni, guanine nucleotide regulation of the receptor and multiple responses associated with this receptor are inhibited (5). By contrast, the prostaglandin E1 and f-adrenergic receptors act through a cholera toxin-sensitive GTP-binding protein, Ns (6), to modulate the fMet-Leu-Phe-initiated reactions in a manner that is specific for the receptor occupied and the level of cyclic AMP generated (7-9).We have explored whether a PT-sensitive substrate may participate in the regulation of human neutrophils and have investigated specifically its role in the action of several mediators of inflammation, calcium-related transduction mechanisms (10-13), and the-linked cellular reactions of locomotion, capping, and shape change (14, 15). Our results indicate that the PT-sensitive Ni protein is central to the regulation of multiple reactions of the human neutrophil.

Granulocyte/macrophage colony-stimulating factor affects myo-inositol metabolism in a novel manner. Implications for its priming action on human neutrophils

“…2) of how GM-CSF acts to prime neutrophils is also totally consistent with the observation that its priming capability is inhibited by pertussis toxin [10][11][12]. A pertussis toxin-sensitive G-protein has been demonstrated to be intimately involved in the coupling of Ca2+-mobilizing receptors to PIC in neutrophils [13][14][15]. Thus, according to the postulate derived from the present study, one would accurately predict that GM-CSF's priming action would be pertussis toxin-sensitive.…”

“…Furthermore, the observation that the priming ability of GM-CSF can be significantly reduced in pertussis toxin-treated neutrophils [10][11][12] implies an essential role for a G-protein in the cytokine's priming action. It is also noteworthy that the responses of neutrophils to various Ca2+-mobilizing agonists are also acutely sensitive to pertussis toxin pretreatment [10,[13][14][15]. Taken together, these data suggest that GM-CSF and the Ca2+-mobilizing agonists share the same G-protein to mediate their physiological functions.…”

The Coupling of 5-Oxo-Eicosanoid Receptors to Heterotrimeric G Proteins

“…This led to total blockage of Eo random motility and migration responses, effects that cannot be related simply to GP inhibition. Second, the efficacy of PT in PMN (32,33) and Eo (34 -36) sometimes depends on CF dosage, with high concentrations of CF typically overcoming the PT inhibition, even if total, that occurs at a lower CF concentration. Note that we observed PT completely blocked optimal doses of 5-oxoETE at levels only weakly blocking even suboptimal levels of other CF.…”

Polymorphonuclear leukocyte locomotion is insensitive to lowered cytoplasmic calcium levels

“…Treatment of cells with isletactivating protein (IAP) of pertussis toxin, which ADPribosylates a 41 kD protein of PMNs, blocks peptidestimulated locomotion [Bokoch and Gilman, 1984;Goldman et al, 1982;Lad et al, 1985;Okajima et al, 19851. IAP also inhibits the peptide-induced increase in cytoplasmic calcium [Goldman et al, 1985;Molski et al, 1984;White et al, 19831.…”