The effect of sulphasalazine on neutrophil superoxide generation in rheumatoid arthritis

The production of superoxide by the peripheral blood neutrophils of 19 patients with active rheumatoid arthritis was measured during treatment with sulphasalazine (SASP). The response to drug treatment was determined by change in plasma viscosity, CRP, early morning stiffness and articular index over a 10-point scale. Of the 19 patients studied, eight were considered to have responded well to SASP and seven to have responded poorly or not at all. Over the treatment period, plateau levels of superoxide production fell in seven of the eight responders (P = 0.028) compared with a non-significant fall in 3/7 of the non- responder groups. The initial rate of superoxide production also fell in the responder group, but this was not statistically significant. Initial values in both the responder and non-responder groups were comparable with those seen for normal controls. Analysis of drug levels showed all patients to be compliant with drug treatment; however, drug levels and neutrophil activity were not correlated. Studies of the effect of SASP and sulphapyridine on superoxide production in vitro showed no difference between good and poor responders. These results suggest that there is no inherent difference between good and poor responders regarding the susceptibility of their neutrophils to SASP. SASP's action on neutrophils, therefore, appears not to be its main mechanism of disease-modifying activity in RA.      

Placental transfer of sulphasalazine and sulphapyridine and some of its metabolites

In eleven pregnant patients with ulcerative colitis or Crohn's disease who were treated with sulphasalazine (SASP) the serum concentrations of SASP and sulphapyridine (SP) were measured at delivery. The concentrations of SASP and SP were almost identical in the cord serum and the maternal serum. In seven of the women the same concentrations were measured at a later occasion when they were not pregnant. The serum SASP concentration remained the same, but the SP concentration was higher in the non-pregnant women. This probably reflects the different degree of protein binding of SASP and SP, respectively, and the change of distribution volume that occurs in pregnancy. In the newborn the concentrations of SASP and SP were 4.6 +/- 3.1 microgram/ml and 18.2 +/- 8.7 microgram/ml, respectively. Current studies have shown that neither SASP nor SP in these concentrations causes significant displacement of bilirubin from albumin. Thus, SASP can be given to the pregnant patient up to delivery without risks for the newborn full-term infant.     

Crystal-induced neutrophil activation. I. Initiation and modulation of calcium mobilization and superoxide production by microcrystals.

The effects of monosodium urate and calcium pyrophosphate dihydrate crystals on the levels of cytoplasmic free calcium and on the oxidative burst in normal human blood neutrophils were examined. The pattern of sensitivity to granulocyte-macrophage colony-stimulating factor, colchicine, cytochalasin B, pertussis toxin, diglyceride kinase, and protein kinase C inhibitors differentiated the mechanism(s) of neutrophil activation by the crystals from that involved in the responses to soluble chemotactic factors and indicated that individual crystals can use several activation pathways.     

Hormonal modulation of cytosolic free calcium

Many hormones are capable of increasing [Ca2+]i in many different tissues by mobilizing Ca2+ from internal stores, primarily the endoplasmic reticulum, and by promoting Ca2+ influx across the plasma membrane. Recent studies suggest that both processes involve G-proteins. In one case, a G-protein activates a PI 4,5-P2 specific phospholipase C, resulting in the formation of Ins 1,4,5-P3, which then releases Ca2+ from the endoplasmic reticulum. Ca2+ influx can be stimulated by low concentrations of agonist, which do not promote internal Ca2+ mobilization. This process appears to be stimulated directly by a G-protein.     

Plasma-mediated stimulation of neutrophil superoxide anion production during coronary artery bypass grafting: role of endothelin-1.

BACKGROUND: Activation of polymorphonuclear neutrophils (PMN) and subsequent release of free oxygen radicals, including the superoxide anion (O2-) has been shown to result in postischaemic myocardial dysfunction during coronary artery bypass grafting (CABG). Several neutrophil-oriented stimuli are known to be released from myocardium during ischaemia and reperfusion. Release of endothelin-1 has been documented during CABG. The aim of the current study was to evaluate plasma-mediated neutrophil stimulation and to verify whether endothelin-1, known to be a stimulus for PMN, is involved in plasma-mediated stimulation of PMN during coronary artery bypass grafting. METHODS: Plasma samples from peripheral artery, peripheral vein, and coronary sinus were obtained from 21 patients undergoing CABG before aortic clamping (global ischaemia), immediately after beginning reperfusion, and 30 min after reperfusion as well as from healthy controls. Plasma was incubated with PMN isolated from healthy donors preincubated in the presence of saline or specific endothelin-1 receptor antagonist (ET-A). PMN O2- production was measured spectrophotometrically. RESULTS: Plasma samples taken from the coronary sinus at the beginning of reperfusion were capable of higher stimulation of neutrophil superoxide anion production (24.2 +/- 2.0 nmol/5 x 10(6)PMN/30 min) than plasma obtained before reperfusion (15.6 +/- 1.5; p < 0.05) or plasma taken from peripheral artery (17.1 +/- 1.7; p < 0.05). Preincubation of PMN with endothelin-1 receptor antagonist decreased superoxide anion production by cells exposed to plasma taken from coronary sinus at the beginning of reperfusion (17.6 +/- 2.0, p < 0.05). CONCLUSIONS: Transcardiac release of soluble stimuli for PMN occurs as a result of myocardial ischaemia during CABG. Endothelin-1 may be involved in the plasma-mediated stimulation of neutrophil superoxide anion production.     

Effect of vasotocin on cytosolic free calcium concentrations in frog adrenocortical cells in primary culture.

In a previous report we have shown that vasotocin (AVT), the amphibian counterpart of vasopressin, is a potent stimulator of corticosterone and aldosterone secretion by frog adrenocortical cells. We have also observed that the stimulatory effect of AVT on corticosteroid secretion is mediated through activation of receptors positively coupled to phospholipase-C. In the present study we examined the effect of AVT on cytosolic Ca2+ concentrations ([Ca2+]i). Since the interrenal (adrenal) gland of the frog is composed of a mixed population of chromaffin and adrenocortical cells, cytochemical identification of cultured cells was performed by immunofluorescence, using antibodies to AVT or 11 beta-hydroxylase as markers of chromaffin cells or steroid-producing cells, respectively. Cultured interrenal cells were loaded with the fluorescent Ca2+ indicator indo-1, and variations in [Ca2+]i were studied using dual emission wavelength microfluorimetry. Exposure of adrenocortical cells to AVT induced elevation of [Ca2+]i. Prolonged infusion of AVT caused an immediate increase in [Ca2+]i, followed by a sustained response of adrenocortical cells. Repeated pulses of AVT resulted in a gradual decline in the [Ca2+]i increase, suggesting the existence of a desensitization phenomenon. The effect of AVT on calcium mobilization was totally blocked when the cells were incubated in the presence of the V2 antagonist [d(CH2)5,D-Phe2,Ile4,Ala9-NH2]AVP. In calcium-free medium, the AVT-evoked increase in [Ca2+]i was suppressed. In contrast, when Ca2+ was replaced by Mn2+ in the incubation medium, the early response of the cells (transient peak of [Ca2+]i) was preserved, while the plateau phase disappeared. Incubation of the cells with the dihydropyridine Ca2+ channel blocker nifedipine did not affect the AVT-induced [Ca2+]i rise. These results indicate that AVT exerts a dual action on [Ca2+]i in frog adrenocortical cells. The initial rise of [Ca2+]i can be ascribed to immediate mobilization of intracellular Ca2+ stores, probably mediated by inositol trisphosphategated channels, whereas the sustained increase in [Ca2+]i results from nifedipine-insensitive plasma membrane Ca2+ channels.     

Effect of prostaglandin F2 alpha on cytosolic free calcium ion concentrations in rat luteal cells.

Changes in cytosolic free calcium concentrations [( Ca2+]i) in response to prostaglandin F2 alpha (PGF2 alpha) were measured in single rat luteal cells, using the calcium-sensitive fluorescent dye fura-2. A total of 112 cells were studied in 20 experiments. The average resting [Ca2+]i was 113 +/- 6.4 nM. In 59 cells (53%), there was a 4.6 +/- 0.2-fold increase in [Ca2+]i within 29.3 +/- 1.0 sec of PGF2 alpha administration, and the cells recovered by 78.0 +/- 4.5 sec. The magnitude of the increase in [Ca2+]i in response to PGF2 alpha was not changed by an increase in the concentration of PGF2 alpha. Perifusion with low calcium buffer reduced, then eliminated, the [Ca2+]i response to PGF2 alpha. Perifusion of cells with PGF2 alpha resulted in a single transient [Ca2+]i response, similar to that after short term exposure to PGF2 alpha. Many (67%) of the cells that responded to PGF2 alpha also responded to GnRH. No additive increase in [Ca2+]i was seen when PGF2 alpha and GnRH were administered together. The source of the calcium appears to be intracellular stores that are shared by GnRH and PGF2 alpha.     

Modulation of cytosolic free calcium levels by extracellular phosphate and lanthanum.

The effects of extracellular phosphate and lanthanum on cytosolic free Ca2+ [( Ca2+]i) levels were studied in isolated rat pancreatic acini. In the presence of 1.28 mM Ca2+ and 1.0 mM phosphate, the mean resting [Ca2+]i level was 120 nM. Omission of phosphate from incubation medium significantly lowered this value to 94 nM. The gastrointestinal hormone cholecystokinin octapeptide (CCK-8) rapidly enhanced both [Ca2+]i levels and 45Ca2+ efflux, irrespective of the presence or absence of phosphate. Lanthanum (0.1 mM), a compound known to block transmembrane Ca2+ fluxes, attenuated both actions of CCK-8, but only in the absence of extracellular phosphate. There was a concomitant decrease in amylase secretion induced by 0.1 nM CCK-8 but not by 10 nM CCK-8, without a significant change in cellular ATP levels. The inhibitory actions of lanthanum on CCK-8-stimulated [Ca2+]i levels were very rapid and were mimicked only by prolonged incubation of acini in Ca2+-free medium supplemented with EGTA. Omission of phosphate from incubation medium also lowered basal [Ca2+]i levels in IM-9 lymphocytes. These findings suggest that extracellular phosphate may modulate resting [Ca2+]i levels in pancreatic acini and other cell types and that mobilization of intracellular Ca2+ may partly depend on the availability of a lanthanum-sensitive pool of cell-surface Ca2+ that is not readily removed by EGTA.     

Auranofin modulates human neutrophil superoxide production and protein phosphorylation

Summary The effect of auranofin (AF) was examined on human neutrophil superoxide production and protein phosphorylation stimulated by phorbol esters. Low concentrations of auranofin (0.5 M) enhanced while higher concentrations (0.5–10 M) inhibited superoxide release stimulated by a suboptimal concentration (0.005 M) of phorbol myristate acetate (PMA). The enhancing but not the inhibitory effect of AF was lost if a maximal stimulating dose (0.05 M) of PMA was used. In contrast AF had a biphasic effect on protein phosphorylation regardless of the stimulating concentration of PMA. Comparison of the dose-response curves for these effects of AF suggest that although changes in protein phosphorylation may be partly responsible for altered activity of the NADPH oxidase responsible for superoxide production, it is unlikely that they are mediated by a direct effect of AF on protein kinase C. Also, measurement of ³H-PDBu-binding to neutrophils showed that these actions of AF could not be attributed to altered binding of phorbol esters to their cellular receptor (protein kinase C).     

Cardiac energy metabolism homeostasis: role of cytosolic calcium

The heart is capable of dramatically altering its overall energy flux with minimal changes in the concentrations of metabolites that are associated with energy metabolism. This cardiac energy metabolism homeostasis is discussed with regard to the potential cytosolic control network responsible for controlling the major energy conversion pathway, oxidative phosphorylation in mitochondria. Several models for this cytosolic control network have been proposed, but a cytosolic Ca(2+) dependent parallel activation scheme for metabolism and work is consistent with most of the experimental results. That model proposes that cytosolic Ca(2+) regulates both the utilization of ATP by the work producing ATPases as well as the mitochondrial production of ATP. Recent studies have provided evidence supporting this role of cytosolic Ca(2+). These data include the demonstration that mitochondrial [Ca(2+)] can track cytosolic [Ca(2+)] and that the compartmentation of cytosolic [Ca(2+)] can facilitate this process. On the metabolic side, Ca(2+) has been shown to rapidly activate several steps in oxidative phosphorylation, including F(1)F(0)-ATPase ATP production as well as several dehydrogenases, which results in a homeostasis of mitochondrial metabolites similar to that observed in the cytosol. Numerous problems with the Ca(2+) parallel activation hypothesis remain including the lack of specific mechanisms of mitochondrial Ca(2+) transport and regulation of F(1)F(0)-ATPase, the time dependence of Ca(2+) activation of cytosolic ATPases as well as oxidative phosphorylation, and the role of cytosolic compartmentation. In addition, the lack of cytosolic or mitochondrial [Ca(2+)] measurements under in vivo conditions is problematic. Several lines of investigation to address these issues are suggested. A model of the cardiac energy metabolism control network is proposed that includes a Ca(2+) parallel activation component together with more classical elements including metabolite feedback and cytosolic compartmentation.     

Alpha-adrenoceptor-mediated increase in cytosolic free calcium in isolated cardiac myocytes.

The effect of alpha-adrenoceptor stimulation on the concentration of cytosolic free calcium (Cai2+) was determined by measuring indo-l fluorescence in isolated ventricular cardiomyocytes from normal and streptozotocin-diabetic rat; 1.3 x 10(5) alpha 1-adrenoceptors per normal myocyte and an unaltered number of these receptors in cells from diabetic rats were detected using the alpha 1-selective ligand WB-4101. Under basal conditions, Cai2+ was found to be 154 +/- 4 nM (n = 34) reaching a value of 192 +/- 10 nM (n = 15) after stimulation of myocytes with a maximal dose of methoxamine for 5 min. Under the same conditions the leakage of dye produced a significantly smaller increase of basal values of 169 +/- 5 nM (n = 17). Indo-l loaded cells did not respond to beta-stimulation unless in the presence of KCl (50 mM), demonstrating the specificity of methoxamine action. Treatment of cells with nifedipine or chelation of extracellular calcium by EGTA did not modify the alpha-adrenergic response. Experiments with cardiomyocytes from streptozotocin-diabetic rats showed an unaltered modulation of Cai2+ by both alpha- and beta-receptor stimulation. It is concluded that signalling by alpha 1-adrenoceptors in ventricular cardiomyocytes results in mobilization of intracellular calcium stores.     

Effect of inhibition of Na, K-ATPase on cytosolic free sodium and calcium in platelets of spontaneously hypertensive rats.

Cytosolic free sodium concentrations ([Na+]i) in intact platelets of 18 spontaneously hypertensive rats (SHR) and of 18 age-matched normotensive Wistar-Kyoto rats (WKY) were measured using the sodium-sensitive fluorescent dye sodium-binding-benzofuran-isophthalate. In resting platelets [Na+]i tended to be higher in SHR compared to WKY (20.5 +/- 3.5 mmol/L v 15.1 +/- 1.9 mmol/L, mean +/- SEM), but the differences were not statistically significant. Stimulation of the Na-H-exchange by 1.0 U/mL thrombin increased [Na+]i in SHR by 22.9 +/- 4.3 mmol/L and in WKY by 35.0 +/- 5.6 mmol/L in a similar way. After inhibition of Na, K-ATPase by 1 mmol/L ouabain there was a significant rise of [Na+]i both in platelets of SHR to 38.0 +/- 5.1 mmol/L (P < .01 compared to resting platelets) and in platelets of WKY to 26.5 +/- 4.3 mmol/L (P < .01). However, no significant difference could be observed between these two groups. Using the calcium-sensitive dye fura-2, resting cytosolic free calcium concentrations ([Ca2+]i) were found to be significantly higher in platelets of SHR compared to WKY (171.9 +/- 21.5 nmol/L v 93.14 +/- 19.7 nmol/L, P < .05). After the addition of ouabain [Ca2+]i was significantly higher in SHR compared to WKY (245.5 +/- 32.6 nmol/L v 159.6 +/- 22.5 nmol/L, P < .05). The results do not support the hypothesis that altered sodium-calcium exchange causes elevated cytosolic free calcium in SHR.     

Activation of neutrophil superoxide production by concanavalin A can occur at low levels of intracellular ionized calcium

The effect of concanavalin A (Con A) on the concentration of ionized intracellular calcium [( Cai++]) in human granulocytes (PMN) was monitored using the fluorescent calcium indicator and chelator, Quin2. The addition of Con A to PMN resulted in a rise in [Cai++] that was markedly enhanced by the presence of Ca++ in the external buffer. The onset of the increment in [Cai++] preceded the onset of O2- production. The rise in [Cai++] induced by Con A is not transient, with a new, higher steady-state level of [Cai++] being attained within five minutes. The addition of alpha-methylmannoside (alpha-MM) one minute after Con A resulted in the return of [Cai++] to the original baseline level and the cessation of O2- production. The addition of a second stimulus (such as arachidonic acid) to these cells resulted in a second increment in [Cai++] and the return of O2- production. Thus the rise in [Cai++] induced by Con A is tightly coupled to the activation, inactivation, and reactivation of the O2- generating system by Con A. Further experiments were undertaken to assess the possible requirement for the rise in [Cai++] in the activation of PMN by Con A. PMN could be depleted of Cai++ by loading with Quin2 in the absence of extracellular Ca++. These Ca++- depleted PMN can be induced to produce O2- after treatment with PMA but not with Con A. The addition of Ca++ to Ca++ - depleted PMN results in a return of [Cai++] to the normal resting level of Ca++ -replete PMN. The time required to return to baseline is a function of the concentration of intracellular Quin2. The addition of Ca++ to Ca++ -depleted, Con A-treated PMN results in the elevation of [Cai++] and the production of O2-. Over a tenfold range of intracellular Quin2 concentration, the onset of O2- production always occurred at [Cai++] that were less than the normal resting level. Thus, activation of the O2- generating system by Con A can occur at [Cai++] which are much lower than the incremental level induced by Con A in Ca++ -replete PMN. Supporting this is the observation that only a very small increment in [Cai++] is induced by Con A in PMN cytoplasts, even though Con A could induce O2- production in the Quin2-loaded cytoplasts.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regulation of cytosolic free calcium in isolated rat osteoclasts by calcitonin.

It is now established that calcium is a second messenger mediating the action of calcitonin on the osteoclast. We have demonstrated that an increase in the concentration of intracellular free calcium ([Ca2+]i) is associated with (and possibly mediates) the functional effects of calcitonin, including an acute reduction of cell spread area (the R effect) and, in the longer term, a reduction in enzyme release. The present study addresses questions relating to mechanisms of calcitonin action on osteoclast [Ca2+]i. We have used asusuberic(1-7) eel and human calcitonin as agonists, and an indo-1-based dual-emission microspectrofluorimetric method for the measurement of [Ca2+]i in single osteoclasts. Whilst asusuberic(1-7) eel calcitonin caused a biphasic increase in [Ca2+]i, human calcitonin produced only a monophasic [Ca2+]i response of a much lower magnitude. Each biphasic response consisted of a rapid initial transient increase, occurring within seconds of exposure, followed by a sustained increase in [Ca2+]i. The magnitude of the latter response was more variable, but was consistently below the peak value of [Ca2+]i. The sustained phase of the calcitonin effect was abolished in extracellular Ca(2+)-free medium. This phase is therefore dependent on extracellular [Ca2+] ([Ca2+]e) whilst the rapid transient increase appeared to be dependent on Ca2+i redistribution. The effects of calcitonin on [Ca2+]i were concentration-dependent, with neither latency nor oscillations. Repetitive 30-s exposures to calcitonin failed to produce subsequent responses. There was a marked concentration-dependent correlation between changes in osteoclast [Ca2+]i and the magnitude of the R effect. Thus the likely components of the biphasic [Ca2-]i response are a rapid redistribution followed by the transmembrane flux of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rhythmic oscillations of cytosolic free calcium in rat C-cells

The relationship between stimulation of single C-cells (rMTC-6-23 cell line) with extracellular calcium, glucagon or 8-bromo-cAMP and fluctuations of intracellular free calcium concentration was studied. After pretreatment of rMTC cells with either 1 microM glucagon (30-60 min) or 1 mM 8-bromo-cAMP (5 min) [Ca2+]i started to oscillate when extracellular calcium was raised to 3 mM. These fluctuations in [Ca2+]i could be stopped by chelating the external calcium with EGTA or by adding calcium channel blockers. The voltage-dependent calcium channels in the plasma membrane seem to play a major role in maintaining the oscillations of [Ca2+]i.     

Effect of ethanol on adenosine triphosphate,cytosolic free calcium,and cell injury in rat hepatocytes

The events implicated in the early phases of acute ethanol-induced hepatocyte injury and their relation with the nutritional status of the liver are not clearly defined. We aimed to determine the effect of ethanol on ATP and cytosolic free Ca²⁺ in hepatocytes isolated from fed or fasted rats. Cell injury was assessed by LDH release and trypan blue uptake, ATP by [³¹P]NMR spectroscopy, and cytosolic free Ca²⁺ with aequorin. In control conditions, cells from fasted animals had a lower ATP level (–52%) and a higher cytosolic free Ca²⁺ (+101%) than did those isolated from fed animals. Ethanol caused a dose-dependent cell injury in both groups. At all ethanol doses, greater damage occurred when using hepatocytes isolated from fasted rats. In both groups, a dose-dependent decrease in ATP content and a rise in cytosolic free Ca²⁺ were seen. The magnitude of these changes were significantly greater in the fasted group. In conclusion, these data showed that fasting affects the energy status and cytosolic free calcium level in hepatocytes; ethanol causes a dose-dependent cell injury that occurs in association with a fall in ATP and a rise in cytosolic free Ca²⁺ levels. The nutritional status of an animals is an important determinant of the severity of ethanol-induced damage to liver cells.     

Comparison of orocaecal transit times assessed by the lactulose/breath hydrogen and the sulphasalazine/sulphapyridine methods.

The lactulose/breath hydrogen and the sulphasalazine/sulphapyridine methods of assessing orocaecal transit time have been compared. In a two part crossover study in healthy normal subjects the median orocaecal transit time by the SLZ/SP method was 4.84 hours but only 2.92 hours by the lactulose/breath hydrogen method. Coadministration of lactulose and sulphazalazine to nine subjects with assessment of orocaecal transit time by hydrogen breath determination and plasma sulphapyridine assay gave orocaecal transit times of 2.33 and 2.25 hours respectively suggesting that the lactulose reduces transit time and that the lactulose/breath hydrogen method, which is so convenient to use, gives artificially low transit times. A third experiment was undertaken to compare the orocaecal transit times after 1.5 and 3.0 g sulphazalazine. The orocaecal transit times after the two doses were not statistically different.     

Platelet cytosolic proton and free calcium concentrations in essential hypertension

Alterations in the metabolism of intracellular messengers, such as calcium and cyclic adenosine 5'-phosphate (cAMP), have been reported in essential hypertension. Since intracellular pH (pHi) participates in the control of fundamental cell functions, we looked for changes in platelet cytosolic H+ concentration [( H+]i) in hypertension and investigated whether or not its impaired metabolism is linked to the calcium handling abnormalities. The fluorescent pH indicator BCECF has been used to evaluate intracellular H+ concentration in platelets, unstimulated ex vivo, from normotensive (n = 20) and hypertensive patients (n = 20). Cytosolic [H+] was 20% lower in hypertensive than in normotensive subjects (49.5 +/- 3.4 and 61.8 +/- 2.2 nmol/l cells, respectively, P less than 0.005; mean pHi values were 7.21 and 7.33, respectively). Platelet cytosolic H+ and free Ca2+ concentrations ([Ca2+]i) were determined in parallel in 15 normotensive and 15 hypertensive patients. [Ca2+]i was found to be 19% higher (P less than 0.01), and [H+]i 22% lower (P less than 0.02), in the hypertensive patients compared with the normotensive subjects. Platelet pHi and [Ca2+]i were increased simultaneously in some hypertensive patients. These results are compatible with the hypothesis of an in vivo activation of platelets in hypertension. If a similar alkalinization exists in smooth muscle cells, it may participate in cell proliferation and in an enhanced sensitivity to agonists, two parameters thought to be involved in blood pressure elevation.     

The regulation of neutrophil phospholipase A2 by granulocyte-macrophage colony-stimulating factor and its role in priming superoxide production

Experiments were performed to investigate the relative role of phospholipase A₂(PLA₂) in the activation and cytokine-mediated priming of neutrophil superoxide production. PLA₂ activity was measured with a radiometric assay which discriminates between PLA₂ and the downstream enzyme, 5-lipoxygenase. In cells that had not been primed by prior incubation with granulocyte-macrophage collony stimulating factor (GM-CSF), PLA₂ and NADPH oxidase were differentially stimulated by the chemotactic peptide N-formyl-met-leu-phe (FMLP), calcium ionophore, or phorbol ester. In addition, inhibition of PLA₂ by mepacrine (0–100 μmol/l) did not concomitantly inhibit FMLP-stimulated superoxide production. These findings suggest that the activity of PLA₂ and NADPH oxidase may be uncoupled in the unprimed cell. In cells preincubated with GM-CSF, time- and dose-dependent priming of FMLP-stimulated PLA₂ responses were observed and inhibition of PLA₂ by mepacrine was accompanied by the inhibition of FMLP-stimulated superoxide production down to the level of unprimed cells. The effect of mepacrine was not due to inhibition of FMLP receptor expression. These data suggest that a mepacrine-sensitive PLA₂ may have a role in the GM-CSF mediated priming of superoxide production. Using ionophore-stimulated PLA₂ activity as a model, we showed that Bordatella pertussis toxin did not inhibit GM-CSF mediated priming, demonstrating that a pertussis-sensitive GTP-binding protein does not mediate signal transduction from the GM-CSF receptor to PLA₂. The tyrosine kinase inhibitor, genestein, selectively inhibited GM-CSF primed but not unprimed PLA₂ activity, demonstrating that GM-CSF-mediated priming requires tyrosine kinase activity.