Acute effect of sidestream cigarette smoke extract on vascular endothelial function

Acute exposure to passive smoking adversely affects vascular function by promoting oxidative stress and endothelial dysfunction. However, it is not known whether tobacco sidestream (SS) smoke has a greater deleterious effect on the endothelium than non-tobacco SS smoke and whether these effects are related to nicotinic endothelial stimulation. To test these hypotheses, endothelial-dependent relaxation and superoxide anion production were assessed in isolated rat aortas incubated with tobacco SS smoke, non-tobacco SS smoke, or pure nicotine. Tobacco SS smoke decreased the maximal relaxation to acetylcholine (Ach) from 79 +/- 6% to 57 +/- 7.3% (% inhibition of phenylephrine-induced plateau, P < 0.001) and increased superoxide anion production from 31 +/- 9.7 to 116 +/- 24 count/10 sec/mg (P < 0.01, lucigenin-enhanced chemiluminescence technique). The non-tobacco SS smoke extract had no significant effect on the response to Ach but increased superoxide anion production in the aortic wall to 133 +/- 2 count/10 sec/mg (P < 0.001). Furthermore, concentration-response curves to Ach and superoxide production remained unaltered with nicotine (0.001, 0.01, or 0.1 mM). In conclusion, despite similar increases in vascular wall superoxide production with tobacco and non-tobacco SS smoke, only the tobacco SS smoke extracts affected endothelium-dependent vasorelaxation. Nicotine alone does not reproduce the effects seen with tobacco SS smoke, suggesting that the acute endothelial toxicity of passive smoking cannot simply be ascribed to a nicotine-dependent mechanism.     

(2R,3R)-2-(3',4'-dihydroxybenzyl)-3-(3',4'-dimethoxybenzyl)butyrolactone suppresses fMLP-induced superoxide production by inhibiting fMLP-receptor binding in human neutrophils

This study investigated the mechanism underlying the inhibiting effect of (2R,3R)-2-(3',4'-dihydroxybenzyl)-3-(3',4'-dimethoxybenzyl) butyrolactone (PP-6), a lignan from Piper philippinum, on superoxide anion production induced by the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (fMLP) in human neutrophils. Human neutrophils were stimulated with fMLP (1 microM), PMA (100 nM) or leukotriene B(4) (LTB(4); 1 microM) and induced superoxide anion release. PP-6 specifically inhibited fMLP-induced superoxide anion production in a concentration-dependent manner with an IC(50) value of 0.3+/-0.1 microM. Intracellular signaling caused by fMLP, PMA or LTB(4) were evaluated. PP-6 specifically inhibited fMLP-induced intracellular calcium mobilization and ERK (p42/p44), Akt and p38 phosphorylation. Moreover, PP-6 specifically inhibited fMLP-induced Mac-1 expression without affecting this caused by LTB(4) or PMA. PP-6 did not increase cAMP level in human neutrophils. PP-6 did not inhibit superoxide anion production by NaF (20 mM), a direct activator of G-protein, the target of the inhibitory action of PP-6 appears to be a component of the signal transduction pathway upstream of G-protein. PP-6 inhibited FITC-fMLP binding to neutrophils in a concentration-dependent manner with an IC(50) of 1.5+/-0.2 microM. PP-6 did not bring a parallel shift in the concentration response of fMLP-induced superoxide anion. Additionally, the inhibiting effect of PP-6 on fMLP-induced superoxide anion was reversed when PP-6 was washed out. These experimental results suggest that PP-6 exerts non-competitive and reversible antagonistic effect on fMLP receptor.     

Repeated pre-exposure to tobacco smoke potentiates subsequent locomotor responses to nicotine and tobacco smoke but not amphetamine in adult rats

These studies investigated if pre-exposure to tobacco smoke affects the locomotor response to tobacco smoke, nicotine, and amphetamine in adult rats. The rats were habituated to an open field for 3-4 days and then exposed to tobacco smoke for 2 h/day for 13-14 days. The effect of exposure to tobacco smoke on locomotor activity was investigated after 1, 7, and 14 days of smoke exposure and after one 2-hour exposure session that followed a 3-week off period. The effects of tobacco smoke on the locomotor responses to nicotine (0.04 and 0.4 mg/kg, base) and amphetamine (0.1 and 0.5 mg/kg) were investigated on day 14, one day after the last smoke exposure session. The locomotor response to tobacco smoke was increased after 7 and 14 days of smoke exposure and after one exposure session after the 3-week off-period. The acute administration of the high dose of nicotine (0.4 mg/kg) led to a brief period of hypoactivity that was followed by a period of hyperactivity. Pre-exposure to tobacco smoke attenuated the nicotine-induced hypoactivity and potentiated the nicotine-induced hyperactivity. The low dose of nicotine (0.04 mg/kg) did not affect locomotor activity in the control rats but increased the total distance traveled in the tobacco smoke exposed rats. Exposure to tobacco smoke did not affect the locomotor response to amphetamine. These findings indicate that exposure to tobacco smoke leads to tolerance to the depressant effects of nicotine and potentiates the stimulant effects of nicotine and tobacco smoke.     

Effect of the lipopolysaccharide antagonist Planktothrix sp. FP1 cyanobacterial extract on human polymorphonuclear leukocytes

CyP is a lipopolysaccharide (LPS)-like molecule extracted from the freshwater cyanobacterium Oscillatoria planktothrix FP1, which has been reported to be a potent competitive inhibitor of bacterial LPS. In the present study the ability of CyP to affect human polymorphonuclear leukocyte (PMN) function was investigated. PMNs were isolated from venous blood by standard density-gradient centrifugation. Cell migration was measured by use of the Boyden chamber assay. Interleukin (IL)-8 and tumor necrosis factor (TNF)-α production was measured using a sandwich-type enzyme-linked immunosorbent assay. PMN intracellular reactive oxygen species (ROS) levels were assessed by the use of a fluorescent probe coupled to spectrophotometry. CyP 10-100 μg/ml was chemotactic for PMNs without affecting the chemotactic response to either E. coli LPS or N-formyl-Met-Leu-Phe (fMLP). CyP per se did not affect PMN production of either IL-8 or TNF-α, but concentration-dependently reduced LPS-induced production of both cytokines. On the contrary, CyP had no effect either on fMLP-induced production of IL-8 or on PMN oxidative burst (at rest and after stimulation with fMLP), a response which is known to be independent from LPS-operated pathways. In human PMNs CyP behaves as a selective and effective LPS antagonist. These findings support the therapeutic potential of CyP in endotoxin-dependent disease.     

Acute lung inflammation in rats induced by phorbol myristate acetate (PMA)

Intratracheal administration of PMA produces acute lung injury in part due to the generation of O2-derived free radicals. This study evaluated the role of the antioxidant enzyme superoxide dismutase (SOD) in PMA-induced lung injury in the rat. PMA was instilled into rats intratracheally (20-60 micrograms/kg), and the lungs were lavaged 4 hr later. Total number of cells recovered from lavage after PMA treatment was not different from the total number recovered from controls; lavagable PMNs increased in a dose-dependent manner. Albumin in lavage fluid (an index of lung vascular permeability) was significantly increased at 60 micrograms/kg PMA. SOD (10,000 U) + PMA (60 micrograms/kg) reduced the albumin level but significantly increased both total number of cells and number of PMNs recovered from lavage fluid. To investigate the possibility that SOD decreases the ability of PMNs to adhere, PMN aggregation was measured in vitro. The results indicated that 10,000 U SOD can inhibit PMA-induced aggregation by 50%. In contrast, aggregation to other stimuli (e.g., fMet-Leu-Phe, A23187) was unaffected by SOD. We conclude SOD prevents PMA-induced lung permeability and diminishes PMN adherence.     

Impact of nicotine on myocardial neutrophil uptake

Excessive cigarette smoking is recognized as a major risk factor for ischemic heart disease. Although the mechanism by which smoking enhances this risk is not known, multiple lines of indirect evidence suggest that an adverse effect of nicotine on the interaction between neutrophils and the myocardium may play a central pathogenic role. Accordingly, this study employed an isolated rabbit heart preparation perfused at a constant flow rate with physiologic salt solution containing autologous neutrophils to test the hypotheses that nicotine promotes myocardial neutrophil uptake and that an augmented myocardial neutrophil burden intensifies the actions of stimulated neutrophils on the coronary circulation. Addition of 10(-7) M nicotine to the perfusion medium caused an abrupt and sustained sequestration of 111In-labeled neutrophils by isolated rabbit hearts. In contrast, preincubation of neutrophils in 10(-7) M nicotine without inclusion of the alkaloid in the perfusion medium failed to promote neutrophil uptake. Nicotine neither enhanced myocardial neutrophil sequestration induced by perfusion with hypoxic medium nor potentiated neutrophil chemotactic responses evoked by leukotriene B4, the putative mediator of hypoxia-induced myocardial neutrophil uptake. In addition, nicotine failed to influence either baseline levels or the hypoxia-induced accumulation of immunoreactive leukotriene B4 detected in myocardial biopsies. The inflammatory cell stimulant, formylmethionyl-leucyl-phenylalanine (fMLP), increased coronary vascular resistance in neutrophil-perfused hearts but not in hearts perfused with neutrophil-free medium. The magnitude of the fMLP-induced coronary response was augmented when the myocardial neutrophil burden was increased by the addition of nicotine to the perfusion medium or by perfusion with hypoxic medium. These observations suggest that nicotine promotes myocardial neutrophil uptake by mechanisms that do not relate to enhanced release and/or effects of endogenous leukotriene B4 and that sequestration of neutrophils intensifies their actions on the coronary circulation.     

Nicotine potentiates superoxide anion generation by human neutrophils

Cytotoxic neutrophil-derived oxygen radicals have been implicated in the pathogenesis of a variety of cardiovascular, pulmonary, and neoplastic disorders for which cigarette smoking is a prominent risk factor. Although nicotine alone failed to provoke neutrophil oxidative metabolism, the alkaloid caused dose-dependent (0.1 to 10 microM) potentiation of superoxide anion release induced by either phorbol myristate acetate or N-formyl-methionyl-leucyl-phenylalanine. The potentiating effect of nicotine was not attenuated by either atropine or hexamethonium nor was it mimicked by acetylcholine, suggesting involvement of noncholinergic receptors or a membrane-fluidizing effect of the alkaloid. Nicotine-induced exacerbation of neutrophil superoxide anion production may be involved with the enhanced risk of cardiovascular, pulmonary, or neoplastic disease in individuals who smoke.     

Comparison of environmental tobacco smoke (ETS) concentrations generated by an electrically heated cigarette smoking system and a conventional cigarette

Smoking conventional lit-end cigarettes results in exposure of nonsmokers to potentially harmful cigarette smoke constituents present in environmental tobacco smoke (ETS) generated by sidestream smoke emissions and exhaled mainstream smoke. ETS constituent concentrations generated by a conventional lit-end cigarette and a newly developed electrically heated cigarette smoking system (EHCSS) that produces only mainstream smoke and no sidestream smoke emissions were investigated in simulated "office" and "hospitality" environments with different levels of baseline indoor air quality. Smoking the EHCSS (International Organisation for Standardization yields: 5 mg tar, 0.3 mg nicotine, and 0.6 mg carbon monoxide) in simulated indoor environments resulted in significant reductions in ETS constituent concentrations compared to when smoking a representative lit-end cigarette (Marlboro: 6 mg tar, 0.5 mg nicotine, and 7 mg carbon monoxide). In direct comparisons, 24 of 29 measured smoke constituents (83%) showed mean reductions of greater than 90%, and 5 smoke constituents (17%) showed mean reductions between 80% and 90%. Gas-vapor phase ETS markers (nicotine and 3-ethenylpyridine) were reduced by an average of 97% (range 94-99%). Total respirable suspended particles, determined by online particle measurements and as gravimetric respirable suspended particles, were reduced by 90% (range 82-100%). The mean and standard deviation of the reduction of all constituents was 94 +/- 4%, indicating that smoking the new EHCSS in simulated "office" and "hospitality" indoor environments resulted in substantial reductions of ETS constituents in indoor air.     

Chronic administration of water soluble tobacco smoke extract or nicotine fails to influence porcine coronary artery reactivity

To determine if tobacco smoke components directly influence coronary vascular reactivity, we evaluated the contractile effects of prostaglandin (PG)F_2α and hypertonic KCl in porcine isolated left anterior descending coronary arteries derived from animals chronically treated (18 weeks) with either water soluble tobacco smoke extract, nicotine alkaloid, or saline. Dosing protocols for the extract and for nicotine were designed to achieve blood levels of nicotine approximating those attained by consumption of 2 packs/day of University of Kentucky 2Rl reference cigarettes. Histochemical evaluation of the arteries indicated that both the extract and nicotine caused expected increases in the collagen content of the tunica media. Neither the water soluble extract nor pure nicotine significantly altered coronary vascular sensitivity (as expressed by the ED₅₀) to KCl or PGF_2α. Similarly, neither treatment altered the maximum contractile responses (determined as the tension developed normalized to mg wet wt) evoked by KCl and PGF_2α. These results indicate that prolonged exposure to water soluble components to tobacco smoke, in doses sufficient to elevate the collagen content of the coronary vascular wall, do not have direct effects on coronary reactivity to selected vasoconstrictor stimuli.     

Comparison of Environmental Tobacco Smoke (ETS) Concentrations Generated by an Electrically Heated Cigarette Smoking System and a Conventional Cigarette

Smoking conventional lit-end cigarettes results in exposure of nonsmokers to potentially harmful cigarette smoke constituents present in environmental tobacco smoke (ETS) generated by sidestream smoke emissions and exhaled mainstream smoke. ETS constituent concentrations generated by a conventional lit-end cigarette and a newly developed electrically heated cigarette smoking system (EHCSS) that produces only mainstream smoke and no sidestream smoke emissions were investigated in simulated “office” and “hospitality” environments with different levels of baseline indoor air quality. Smoking the EHCSS (International Organisation for Standardization yields: 5 mg tar, 0.3 mg nicotine, and 0.6 mg carbon monoxide) in simulated indoor environments resulted in significant reductions in ETS constituent concentrations compared to when smoking a representative lit-end cigarette (Marlboro: 6 mg tar, 0.5 mg nicotine, and 7 mg carbon monoxide). In direct comparisons, 24 of 29 measured smoke constituents (83%) showed mean reductions of greater than 90%, and 5 smoke constituents (17%) showed mean reductions between 80% and 90%. Gas–vapor phase ETS markers (nicotine and 3-ethenylpyridine) were reduced by an average of 97% (range 94–99%). Total respirable suspended particles, determined by online particle measurements and as gravimetric respirable suspended particles, were reduced by 90% (range 82–100%). The mean and standard deviation of the reduction of all constituents was 94?±?4%, indicating that smoking the new EHCSS in simulated “office” and “hospitality” indoor environments resulted in substantial reductions of ETS constituents in indoor air.     

Differential effects of nicotine and tobacco smoke alkaloids on swimming endurance in the rat

The swimming endurance of rats in a water tub was measured until the animals submerged for two seconds under the water surface. The total alkaloid fraction extracted from cigarette smoke produced deterioration of performance in doses of 0.05 to 0.2 mg/kg, whereas pure nicotine (0.1 and 0.2 mg/kg), as well as nicotine pretreated analogously to the extraction process of the total alkaloids produced performance improvements.This study was made possible through the help of a research grant from the Swiss Association of Cigarette Manufacturers.     

Inhibitory effect of nicotine on chemiluminescence response of human polymorphonuclear leukocytes stimulated by opsonized zymosan in vitro

Effect of nicotine on chemiluminescence (CL) responses of human polymorphonuclear leukocytes (PMN) in vitro was investigated. Nicotine, ranging from 10(-5) to 5 X 10(-4)M, showed a concentration-dependent inhibition of the CL induced by 2.5 mg of opsonized zymosan. This inhibitory effect of nicotine (10(-4)M) on CL was not affected by atropine (10(-4)M), hexamethonium (10(-4)M) or acetyl beta-methylcholine (10(-4)M). These results indicate that nicotine directly acts to PMN with no relation of cholinergic affecting receptors. As to cytotoxic effects on PMN, assessed by the methods of trypan blue exclusion and of lactate dehydrogenase leakage from the cells, nicotine showed no cytotoxic effect on PMN in its range of 10(-5) to 5 X 10(-4)M. It is concluded that tobacco alkaloid nicotine could directly inhibit phagocytizing function of PMN. Furthermore, it is presumed that PMN circulating in the respiratory organs in tobacco smoking would be exposed to high concentrations of nicotine, and that the nicotine incorporated into the circulating system could suppress PMN functions resulting in harmful influences on the host defense mechanisms.     

Distribution of Major and Minor Alkaloids in Tobacco, Mainstream and Sidestream Smoke of Popular Indian Smoking Products

Various Indian smoking products—cigarette, bidi, chutta and a brand of US cigarette—were analysed by gas chromatography–flame ionization detection (GC–FID) for the levels of nicotine and minor tobacco alkaloids in tobacco, mainstream smoke (MS) and sidestream smoke (SS) employing modified smoking standards, namely two puffs/min. The analysis clearly demonstrated relatively higher levels of nicotine and minor tobacco alkaloids in tobacco from bidi (37.7 mg/g) and chutta (34.5 mg/g) when compared with Indian and US cigarettes (14–16 mg/g) studied. Relatively lower levels (SS/MS) of nicotine in SS from bidi and chutta compared with Indian/US cigarettes, suggest that the contribution of nicotine in SS from a single bidi/chutta to environmental tobacco smoke (ETS) is very much less than that of a single Indian/US cigarette. Reduced levels of nicotine in SS of bidi/chutta result in relatively higher deliveries of nicotine in MS as reflected by higher MS/SS values. The observed differences are likely to be due to difference in tobacco processing, burning rate/temperature and design of the smoking product.     

Comparison of the behavioral effects of cigarette smoke and pure nicotine in rats

Animal models of tobacco dependence typically rely on parenteral administration of pure nicotine. Models using cigarette smoke inhalation might more accurately simulate nicotine exposure in smokers. The primary goal of this study was to validate methods for administering cigarette smoke to rats using exposure conditions that were clinically relevant and also produced brain nicotine levels similar to those produced by behaviorally active doses of pure nicotine. A secondary goal was to begin examining the behavioral effects of smoke. Nose-only exposure (NOE) to smoke for 10-45 min or whole-body exposure (WBE) to smoke for 1-4 h produced serum nicotine concentrations similar to those in smokers (14-55 ng/ml), without excessive carbon monoxide exposure. Daily nicotine (0.1 mg/kg, s.c.) induced locomotor sensitization whereas 45-min NOE producing brain nicotine levels within the same range did not. Nicotine 0.125 mg/kg s.c. reversed withdrawal from a chronic nicotine infusion as measured by elevations in intracranial self-stimulation thresholds whereas 4-h WBE producing similar brain nicotine levels did not. These data demonstrate the feasibility of delivering cigarette smoke to rats at clinically relevant doses, and provide preliminary evidence that the behavioral effects of nicotine delivered in smoke may differ from those of pure nicotine.     

Inhibition of formyl-methionyl-leucyl-phenylalanine-stimulated respiratory burst in human neutrophils by adrenaline: inhibition of Phospholipase A2 activity but not p47phox phosphorylation and translocation

The polymorphonuclear neutrophil (PMN)-respiratory burst plays a key role in host defense and inflammatory reactions. Modulation of this key neutrophil function by endogenous agents and the mechanisms involved are poorly understood. This study was designed to analyze the mechanisms involved in the effect of adrenaline on neutrophil superoxide anions production. Using the superoxide dismutase (SOD)-inhibitable cytochrome c reduction assay, we report here that the beta-adrenergic agonist, adrenaline at physiologic concentrations (5-100 nM) inhibited formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated but not phorbol-myristate-acetate (PMA)-stimulated PMN superoxide anion production. The inhibitory effect of adrenaline runs in parallel with an increase in intracellular levels of cAMP which was reversed by the protein kinase A (PKA) inhibitor H-89, suggesting a role for PKA in mediating the inhibitory effect of adrenaline on fMLP-induced superoxide production. Adrenaline at physiological concentrations did not inhibit the fMLP-stimulated membrane translocation of the NADPH oxidase components p47phox and p67phox, nor the fMLP-stimulated phosphorylation of p47phox. However, adrenaline strongly depressed the activity of the cytosolic isoform of Phospholipase A(2) (cPLA(2)). We suggest that adrenaline inhibits fMLP induced superoxide production upstream of the NADPH oxidase via a mechanism involving PKA and cPLA(2).     

In vitro and in vivo effects of gold salts on chemotaxis and random migration of rat polymorphonuclear leucocytes

The effect produced by three gold salts (sodium aurothiomalate, allochrysine, auranofin) on chemotaxis and random migration of rat polymorphonuclear leucocytes (PMN) was investigated under various experimental conditions. The drug activity was examined after incubation in vitro or after administration in vivo. PMNs were recruited after the induction of two acute inflammatory reactions (pleurisies induced by isologous serum or a suspension of calcium pyrophosphate (CaPP) crystals). The three gold salts administered in vivo and in vitro inhibited the chemotactic responses of the two cell types. This action was dose-dependent. Auranofin was the most effective substance while sodium aurothiomalate was the least. The random migration was not always significantly depressed especially for CaPP-elicited cells. Reduction in neutrophil chemotaxis might be an important additional mechanism in the action of gold salts and their activity on inflammatory PMNs recruited at inflammatory foci might be beneficial in the treatment in rheumatic diseases in which PMN migration would be implicated.     

Comparison of two classes of non-peptide drugs as antagonists of neutrophil receptors for f-Met-Leu-Phe. Pyrazolons and iodinated radiographic contrast agents.

The radiographic contrast agent sodium diatrizoate (DTR) reportedly inhibits f-Met-Leu-Phe-induced chemotaxis in human neutrophils. DTR is also an ingredient of Ficoll-Paque, a density centrifugation medium widely used to purify human polymorphonuclear leukocytes (PMNs). Exposure of PMNs to DTR during preparation had no detrimental effect on subsequent binding characteristics of tritiated f-Met-Leu-Phe, probably owing to a rapid dissociation of DTR from the PMN receptors. DTR competed directly with f-Met-Leu-Phe for receptor binding, but was 160- and 640-fold less potent than phenylbutazone and 1,2-diphenyl-4-[3-(1-naphthyl)-propyl]-3,5-pyrazolidinedione (DPN; an analog of phenylbutazone), respectively. Iohexol and the methylamide of DTR did not compete with [3H]f-Met-Leu-Phe in receptor binding, supporting the existence of a definite interaction between iodinated aromatic molecules and the f-Met-Leu-Phe receptor. DTR did not inhibit prostaglandin synthesis, as did DPN. Both drugs inhibited chemotactic peptide-induced release of superoxide anion in a concentration-dependent manner, and were relatively selective for f-Met-Leu-Phe, as opposed to C5a. Both drugs at 10 microM interfered non-selectively with chemotactic peptide-induced beta-glucuronidase release from PMNs. Available non-peptide antagonists of f-Met-Leu-Phe exhibited other pharmacodynamic properties that could make them unsuitable for future in vivo studies designed to probe the physiological role of the receptor.     

Secondhand smoke and nicotine exposure: a brief review

Secondhand tobacco smoke exposure is linked to a number of adverse health outcomes. This paper reviews published studies examining nicotine levels related to exposure to secondhand tobacco smoke. Twenty-two field studies measuring biological levels of nicotine associated with secondhand tobacco smoke exposure were evaluated. Positive associations between self-reported and/or objective measures of secondhand tobacco smoke exposure and concentrations of nicotine and/or biomarkers of nicotine in the body were frequently reported. Two studies indicated that nicotine exposure from secondhand tobacco smoke can engender plasma nicotine concentrations that are equivalent to levels produced by tobacco smoking and that are associated with nicotine-induced changes in behavior. Future research should examine whether nicotine exposure from secondhand tobacco smoke has functional effects on neurobiological and behavioral processes associated with tobacco use.     

Effect of mammalian lignans on fMLP-induced oxidative bursts in human polymorphonuclear leucocytes.

We examined the effects of mammalian lignans, enterolactone, prestegane B and 2,3-dibenzylbutane-1,4-diol (DBB) on superoxide production and luminol-dependent chemiluminescence (LCL) response in human polymorphonuclear leucocytes (PMNs). The three lignans had no direct effect on the responses of human PMNs. DBB and prestegane B enhanced the superoxide production and LCL response induced by formylmethionyl-leucyl-phenylalanine (fMLP), but enterolactone inhibited fMLP-induced effects. The effects of DBB were stronger than those of prestegane B and the effects of DBB were inhibited by bromophenacyl bromide, mepacrine, N-(6-aminophenyl)-5-chloro-1-naphthalene, sulphonamide and trifluoroperazine, but not by gossypol, nordihydroguaretic acid, indomethacin, staurosporine, 1-(5-isoquinolinesulphonyl)-2-methylpiperazine dihydrochloride or (R,S)-2-methoxy-3-(octadecyl-carbamoyloxy)-propyl-2-(2-thiazoli o)-ethylphosphate. These results suggest that DBB primes the responses of human PMNs, and the priming effect is caused by the activation of phospholipase A2--and Ca(2+)-calmodulin-pathways, but not by the activation of lipoxygenase, cyclo-oxygenase and protein kinase C or by the release of platelet activating factor.