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Pertussis toxin inhibits the N-formyl-MetLeu-Phe (fMet-Leu-Phe) mediated human neutrophil functions of enzyme release, superoxide generation, aggregation, and chemotaxis. As pertussis toxin modifies the GTP binding receptor-regulatory protein "N1," the association of the fMetLeu-Phe receptor with such a protein was further examined in purified neutrophil plasma membranes. Both fMet-Leu-Phemediated guanine nucleotide exchange and nucleotide-mediated regulation of the fMet-Leu-Phe receptor are inhibited by pertussis toxin. In addition, membrane pretreatment with pertussis toxin abolishes the fMet-Leu-Phe-mediated inhibition of adenylate cyclase. Actions of pertussis toxin are due to the ADP-ribosylation of a single subunit at 41 kDa in the neutrophil plasma membrane, which comigrates on NaDodSO4 gels with the N. GTP-binding protein in the platelet plasma membrane. Our results suggest that (i) the fMet-Leu-Phe receptor is associated with a N. GTP regulatory protein, and (it) a fMetLeu-Phe-N; complex is important in the control of several neutrophil functions, probably involving multiple transduction systems, including adenylate cyclase.

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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.

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To investigate the possible similarities between the reactive oxygen generating systems of the leukocyte and the spermatozoa, and to identify possible biochemical differences between fertile and infertile patients, the effect of various stimulants on the production of reactive oxygen in fertile, fertile with varicocele and infertile patients was examined. Generation of reactive oxygen species by human spermatozoa was examined in 2 patient groups: a fertile group (14), which included 7 patients with a palpable varicocele, and an infertile group (16) composed of 7 patients with a palpable varicocele and 9 who remained infertile 6 months to 3 years (mean 20 months) after internal spermatic vein ligation. Various known stimulants of reactive oxygen generation in leukocytes were used to assess spermatozoal reactive oxygen production. Reactive oxygen species generation was measured by the technique of chemiluminescence. In the infertile group reactive oxygen generation was markedly increased by the calcium ionophore A23187 and the chemoattractants N-formyl-methionyl-leucyl-phenylalanine and complement 5a (p < 0.05). In addition, fertile patients with varicoceles showed significantly enhanced chemoattractant stimulated reactive oxygen generation compared to fertile donors without varicoceles. This finding of a biochemical alteration in the sperm of infertile patients and patients with varicoceles may lend support to the argument for early varicocele repair.

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