P2X7受体与炎性细胞因子

P2X7受体是嘌呤受体中功能独特的一个亚型,为ATP控制的离子通道,在单核细胞、巨噬细胞、中性粒细胞中高表达,被ATP激活后导致K＋外流和Ca^2＋内流、非选择性膜孔形成,启动一系列信号途径如炎症小体NALP3的活化,丝裂原蛋白激酶途径激活NF-κB增强炎性细胞因子转录,ROS和氮介质的产生,介导IL-1β、IL-6、IL-18、TNF-α、MIP-2、CCL2、HMGB1等多种炎性细胞因子的释放,参与炎症的发生发展,与真菌感染及阿尔茨海默病、类风湿性关节炎、哮喘等炎症性疾病密切相关.     

中性粒细胞CXCR1、CXCR2活化及其信号传导

中性粒细胞属非特异性免疫细胞,其表面可表达CXCR1和CXCR2.IL-8是其共同配体,它们彼此结合激活后续级联信号传导,产生一系列生物学效应,在介导炎症反应、促进血管新生、维持中性粒细胞稳态等起重要作用.Reparixin是非竞争变构的CXCR1和CXCR2阻滞剂,可抑制中性粒细胞过度趋化、迁移介导的炎症反应.     

CuCl2胁迫对烟草BY-2悬浮细胞死亡的诱导

本文以不含叶绿体的烟草BY-2悬浮细胞系为实验材料,研究了CuCl2胁迫对BY-2细胞呼吸速率、呼吸途径以及细胞内H2O2和ATP含量的影响。结果表明：0.5mmol·L-1CuCl2胁迫明显导致了烟草BY-2细胞的死亡,引起了胞内H2O2爆发和ATP含量下降,严重抑制了BY-2细胞的总呼吸和交替氧化酶途径（alternative oxidase pathway,AOX）。此外,CuCl2对BY-2细胞的线粒体电子传递具有即时的抑制作用。加入外源腺苷（ATP合成的底物）显著抑制了CuCl2胁迫引起的ATP损耗,并阻止了细胞死亡。上述结果表明CuCl2胁迫导致的ATP损耗在CuCl2诱导烟草BY-2细胞死亡过程中起重要的作用。     

CuCl2胁迫对烟草BY-2悬浮细胞死亡的诱导

本文以不含叶绿体的烟草BY-2悬浮细胞系为实验材料,研究了CuCl2胁迫对BY-2细胞呼吸速率、呼吸途径以及细胞内H2O2和ATP含量的影响。结果表明:0.5mmol·L-1CuCl2胁迫明显导致了烟草BY-2细胞的死亡,引起了胞内H2O2爆发和ATP含量下降,严重抑制了BY-2细胞的总呼吸和交替氧化酶途径(alternative oxidase pathway,AOX)。此外,CuCl2对BY-2细胞的线粒体电子传递具有即时的抑制作用。加入外源腺苷(ATP合成的底物)显著抑制了CuCl2胁迫引起的ATP损耗,并阻止了细胞死亡。上述结果表明CuCl2胁迫导致的ATP损耗在CuCl2诱导烟草BY-2细胞死亡过程中起重要的作用。     

MAN2C1基因在病毒感染中的作用

目的 通过观察MAN2C1转基因小鼠对H5N1高致病性禽流感病毒的易感性,以了解此转基因小鼠的免疫应答情况和MAN2C1基因在病毒感染中的作用。方法 以H5N1高致病性禽流感病毒滴鼻感染MAN2C1转基因小鼠,HE染色观察小鼠肺组织病理变化;RT-PCR和免疫组织化学方法检测小鼠肺组织H5N1病毒载量;间接ELISA方法 检测小鼠血清抗体滴度变化。结果 与对照组小鼠比较,MAN2C1转基因小鼠表现为更为严重的间质性肺炎,肺组织病毒载量增加,外周血中性粒细胞数目降低,淋巴细胞数量增加,血清IgG抗体滴度降低。结论 MAN2C1基因抑制了小鼠的体液免疫。     

胞浆Ca~(2+)和某些激酶对NADPH氧化酶激活和肌动蛋白聚合的作用

 为澄清中性粒细胞胞浆 Ca2 +和某些 O-·2 产生相关激酶对 NADPH氧化酶激活和肌动蛋白聚合的作用 ,利用分化为中性粒细胞样的 HL- 60细胞研究了胞浆 Ca2 +螯合剂 BAPTA- AM和激酶抑制剂对这些激酶激活、NADPH氧化酶激活和肌动蛋白聚合的影响 .使用 1 0 μmol/L的 Ca2 +螯合剂 BAPTA- AM去除胞浆 Ca2 +后 ,趋化肽 f MLP诱导的 O-·2 产生明显减少 ,但不影响 f MLP诱导的肌动蛋白聚合 ;8μmol/L的 PKC激酶抑制物 GF1 0 92 0 3x几乎完全抑制 O-·2 产生 ;50 μmol/L的p38激酶抑制物 SB2 0 3580、50 μmol/L的 ERK激酶抑制物 PD0 980 59和 0 .1 μmol/L的 PI3激酶抑制物渥曼青霉素 (Wortmannin)使 f MLP诱导的 O-·2 产生大约减少一半 ;其中 Wortmannin还抑制 f MLP诱导的肌动蛋白聚合 ;f MLP刺激细胞后 ,PI3- K、p38和 ERK激酶迅速激活 ,但这些激酶的激活对 Ca2 +是非必需的 .这些结果说明 Ca2 +依赖途径 (PKC)和 Ca2 +非依赖途径 (PI3- K、p38和ERK)对 NADPH氧化酶激活都起着重要作用 ,而 Ca2 +非依赖途径中的 PI3- K激酶还参与中性粒细胞样 HL- 60细胞的肌动蛋白聚合 .     

胞浆Ca~(2 )和某些激酶对NADPH氧化酶激活和肌动蛋白聚合的作用

为澄清中性粒细胞胞浆 Ca2 和某些 O-·2 产生相关激酶对 NADPH氧化酶激活和肌动蛋白聚合的作用 ,利用分化为中性粒细胞样的 HL- 60细胞研究了胞浆 Ca2 螯合剂 BAPTA- AM和激酶抑制剂对这些激酶激活、NADPH氧化酶激活和肌动蛋白聚合的影响 .使用 1 0 μmol/L的 Ca2 螯合剂 BAPTA- AM去除胞浆 Ca2 后 ,趋化肽 f MLP诱导的 O-·2 产生明显减少 ,但不影响 f MLP诱导的肌动蛋白聚合 ;8μmol/L的 PKC激酶抑制物 GF1 0 92 0 3x几乎完全抑制 O-·2 产生 ;50 μmol/L的p38激酶抑制物 SB2 0 3580、50 μmol/L的 ERK激酶抑制物 PD0 980 59和 0 .1 μmol/L的 PI3激酶抑制物渥曼青霉素 (Wortmannin)使 f MLP诱导的 O-·2 产生大约减少一半 ;其中 Wortmannin还抑制 f MLP诱导的肌动蛋白聚合 ;f MLP刺激细胞后 ,PI3- K、p38和 ERK激酶迅速激活 ,但这些激酶的激活对 Ca2 是非必需的 .这些结果说明 Ca2 依赖途径 (PKC)和 Ca2 非依赖途径 (PI3- K、p38和ERK)对 NADPH氧化酶激活都起着重要作用 ,而 Ca2 非依赖途径中的 PI3- K激酶还参与中性粒细胞样 HL- 60细胞的肌动蛋白聚合 .     

P2z／P2x7嘌呤能受体的研究进展

P2受体是许多种类细胞共有的一类膜受体,能选择性地与胞外ATP(ATPe)结合,产生多种生物效应。P2受体不同于P1受体,因后者也是一类嘌呤能受体,但仅选择性识别腺嘌呤,现称为A1、A2受体。包括ATP在内的多种天然或合成的核苷酸对不同亚型的P2受体有着不同的激活能力,因此根据药理学性质将P2受体分为五类:P2x、P2y、P2u、P2z和P2t,其中前四种是ATP受体,而P2t则是血小板(PLT)表达的ADP受体。P2z受体有一个     

PMA诱导人中性粒细胞胞外诱捕网(NETS)形成的方法及其结构的研究

目的:建立佛波酯(PMA)诱导人中性粒细胞NETS形成的方法,并研究NETS的结构组成。方法:提取人中性粒细胞,使用10、30、90 n M的PMA分别刺激细胞2、3、4 h,采用核酸染料sytox green染色后,通过共聚焦显微镜检测和比较各组NETS的形成情况,并通过活性氧(ROS)探针对NETS进行荧光染色,对弹性蛋白酶(Elastase)、髓过氧化物酶(MPO)和组蛋白H3(Histone H3)进行免疫荧光染色。结果:PMA低于30 n M刺激细胞4 h都不会产生NETS,90 n M刺激3 h就会形成NETS,使用90 n M刺激中性粒细胞4 h后,其形成的NETS含量最高,显著高于30 n M刺激4 h及90 n M刺激3h(P0.05)。免疫荧光染色结果显示NETS结构上含有大量ROS和Elastase,含有少量MPO,几乎不含Histone H3。结论:90 n M PMA刺激中性粒细胞4 h可促进NETS形成,其含有大量ROS和Elastase。     

卵巢癌患者化疗后骨髓抑制并发肺部感染病原菌特点及与外周血单核细胞Nrf2 mRNA和TLR7 mRNA的相关性

目的 观察卵巢癌患者化疗后骨髓抑制并发肺部感染病原菌特点,并分析其与外周血单核细胞（PBMC）Nrf2 mRNA、TLR7 mRNA表达的相关性。方法 选取我院30例化疗后骨髓抑制并发肺部感染的卵巢癌患者（并发肺部感染组）及同期30例化疗后骨髓抑制无肺部感染的卵巢癌患者（未并发肺部感染组）为研究对象,观察并发肺部感染患者病原菌特点,检测两组患者化疗后PBMC Nrf2 mRNA、TLR7 mRNA的表达水平,分析Nrf2 mRNA、TLR7 mRNA与骨髓抑制的关系及Nrf2 mRNA、TLR7 mRNA对骨髓抑制并发肺部感染的预测价值。结果共检出病原菌30株,其中革兰阳性菌12株（40.00%）,革兰阴性菌18株（60.00%）,且以铜绿假单胞菌为主（20.00%）。白细胞减少0～Ⅰ度患者、中性粒细胞减少0～Ⅰ度患者Nrf2 mRNA水平显著高于白细胞减少Ⅱ～Ⅲ度患者和中性粒细胞减少Ⅱ～Ⅲ度患者,而TLR7 mRNA水平显著低于白细胞减少Ⅱ～Ⅲ度患者和中性粒细胞减少Ⅱ～Ⅲ度患者（均P<0.05）。白细胞减少、中性粒细胞减少与Nrf2 mRNA表达水平呈负相关,与TLR7 mRN...     

Formyl peptide stimulates and ATP gamma S potentiates [3H]cytidine 5'-diphosphate diglyceride accumulation in human neutrophils.

Although it is evident that the chemotactic peptide FMLP activates O2-formation in neutrophils via the phosphoinositidase-mediated second messenger system, it is less clear how endogenous priming agents such as ATP and platelet activating factor potentiate FMLP action. In our study, we have examined the possible effects of the stable ATP analog adenosine 5'-O-[3-thiotriphosphate] (ATP gamma S) on cellular levels of inositol 1,4,5-trisphosphate, [Ca2+]i and diglyceride (DG), in resting and in FMLP-stimulated neutrophils. Although all three measures were increased in the presence of FMLP, only the increase in DG was enhanced by pretreatment (priming) with ATP gamma S. We also measured the accumulation of the phosphoinositide cycle intermediate cytidine 5'-diphosphate (CDP)-DG to assess possible effects of priming on phosphoinositide resynthesis. The addition of FMLP to [3H]cytidine-prelabeled neutrophils elicited an increase in the accumulation of [3H]CDP-DG that was maximally enhanced in cells that were pretreated with cytochalasin B. The stimulated accumulation of [3H]CDP-DG was completely reversed by the addition of myo-inositol. Treatment of [3H]cytidine-prelabeled neutrophils with ATP gamma S (10-100 microM) resulted in a dose-dependent synergistic increase in FMLP-stimulated [3H]CDP-DG accumulation, whereas ATP gamma S alone had no effect. The observed increases in DG and in [3H]CDP-DG, in contrast to inositol 1,4,5-trisphosphate and [Ca2+]i responses, correlates well with the ATP gamma S-priming of FMLP-induced O2-formation. A similar potentiation of FMLP-induced stimulation of CDP-DG formation was also observed with platelet-activating factor. It is proposed that the priming of FMLP responses in neutrophils proceeds via a mechanism that selectively enhances DG production through a mechanism that is independent of FMLP-induced breakdown of phosphatidylinositol bisphosphate.     

Differing calcium requirements for regulatory effects of ATP, ATP gamma S and adenosine on O2.- responses of human neutrophils

In formyl peptide stimulated human neutrophils the enhancement of O2.- responses by ATP and ATP S requires extracellular calcium. In contrast, the inhibitory effects of adenosine are independent of a calcium requirement. Rates of O2.- generation are not affected by these adenine compounds. Rather, ATP and ATP S cause a sustained period of generation whereas adenosine causes an abrupt early termination of the O2- response. The differing calcium requirements for regulatory effects of adenine compounds on O2.- responses of stimulated neutrophils suggests that ATP (or ATP gamma S) and adenosine may exert their effects at different points in the pathway of signal transduction events.     

Dual regulation of arachidonic acid release by P2U purinergic receptors in dibutyryl cyclic AMP-differentiated HL60 cells.

ATP promoted biphasic effects on both basal and fMLP-stimulated arachidonic acid (AA) release in neutrophil-like HL60 cells: stimulation in the micromolar range (EC50 = 3.2 +/- 0.9 microM) and inhibition at higher concentrations (EC50 = 90 +/- 11 microM). ATP also inhibited UTP- and platelet activating factor-stimulated AA release. Only stimulatory effects of ATP on basal or fMLP-stimulated phospholipase C were observed. The inhibitory effect of ATP on AA release was not due to reacylation of released AA, chelation of extracellular Ca2+, cell permeabilization, or changes in the rise of [Ca2+]i induced by agonist. The inhibition was rapid, being detected within 5-15 s. The inhibitory effect of ATP on fMLP-stimulated AA release could be desensitized by pretreatment of the cells with 2 mM ATP, but not 20 microM ATP, the concentration that resulted in maximal release of AA and inositol phosphates. The inhibition by ATP was neither dependent on generation of adenosine by ATP hydrolysis nor the result of direct interaction of ATP with P1 purinergic receptors. Among other nucleotides tested (CTP, GTP, ITP, TTP, XTP, adenosine 5'-(beta,gamma-methylene)triphosphate (AMP-PCP), adenyl-5'-yl imidodiphosphate (AMP-P(NH)P), ADP, adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S), and UTP), only UTP and ATP gamma S displayed biphasic effects with potencies and efficacies almost identical to those of ATP. The other nucleotides only exhibited stimulatory effects (EC50 = 60-300 microM). The results are consistent with a model of dual regulation of AA release by two distinct subtypes of P2U receptors in HL60 cells.     

H2O2, but not menadione, provokes a decrease in the ATP and an increase in the inosine levels in Saccharomyces cerevisiae. An experimental and theoretical approach.

When Saccharomyces cerevisiae cells, grown in galactose, glucose or mannose, were treated with 1.5 mm hydrogen peroxide (H2O2) for 30 min, an important decrease in the ATP, and a less extensive decrease in the GTP, CTP, UTP and ADP-ribose levels was estimated. Concomitantly a net increase in the inosine levels was observed. Treatment with 83 mm menadione promoted the appearance of a compound similar to adenosine but no appreciable changes in the nucleotide content of yeast cells, grown either in glucose or galactose. Changes in the specific activities of the enzymes involved in the pathway from ATP to inosine, in yeast extracts from (un)treated cells, could not explain the effect of H2O2 on the levels of ATP and inosine. Application of a mathematical model of differential equations previously developed in this laboratory pointed to a potential inhibition of glycolysis as the main reason for that effect. This theoretical consideration was reinforced both by the lack of an appreciable effect of 1.5 mm (or even higher concentrations) H2O2 on yeast grown in the presence of ethanol or glycerol, and by the observed inhibition of the synthesis of ethanol promoted by H2O2. Normal values for the adenylic charge, ATP and inosine levels were reached at 5, 30 and 120 min, respectively, after removal of H2O2 from the culture medium. The strong decrease in the ATP level upon H2O2 treatment is an important factor to be considered for understanding the response of yeast, and probably other cell types, to oxidative stress.     

Regulation of human neutrophil functions by adenine nucleotides

Previous work has shown that platelet-derived adenine nucleotides modulate neutrophil superoxide anion (O2-) generation. Additional studies were undertaken to characterize the effects of authentic adenosine (ADO) and its nucleotide derivatives on the inflammatory functions of human neutrophils. Stimulus-specific inhibition of neutrophil O2- generation by ADO in response to FMLP was verified. In addition, the ability of ATP, ADP, and AMP to limit neutrophil O2- generation induced by FMLP (0.2 to 0.5 microM) was demonstrated. The concentration producing 50% inhibition for nucleotide inhibition of neutrophil O2- generation was in the rank order of ADO (0.1 microM) less than AMP (0.5 microM) less than ADP less than or equal to ATP (5 microM). Guanine and inosine nucleotides (0.01 to 100 microM) did not inhibit FMLP-stimulated neutrophil O2- generation. Neutrophil degranulation in response to FMLP was only modestly inhibited by adenine nucleotides and ADO. Adenosine and ADP failed to affect chemotaxis of neutrophils stimulated with FMLP. The inability of non-metabolizable analogs to mimic the inhibitory effects of authentic ATP or ADP on the neutrophil O2- response suggested that metabolism of added nucleotides is necessary for their effectiveness. Both TLC and HPLC confirmed that ATP and ADP were converted to AMP and ADO after their incubation with unstimulated or FMLP-activated neutrophils. The addition of adenosine deaminase to neutrophil reaction mixtures in which conversion of added nucleotides was apparent removed detectable ADO but failed to completely abrogate the inhibition of neutrophil O2- generation by accumulated AMP. The kinetics of inhibition of FMLP-induced neutrophil O2- generation by ATP and ADP also indicated that conversion of these nucleotides to ADO and/or AMP may be essential for their ability to reduce neutrophil responses.     

Inhibition of neutrophil apoptosis by ATP is mediated by the P2Y11 receptor

Neutrophils undergo rapid constitutive apoptosis that is delayed by a range of pathogen- and host-derived inflammatory mediators. We have investigated the ability of the nucleotide ATP, to which neutrophils are exposed both in the circulation and at sites of inflammation, to modulate the lifespan of human neutrophils. We found that physiologically relevant concentrations of ATP cause a concentration-dependent delay of neutrophil apoptosis (assessed by morphology, annexin V/To-Pro3 staining, and mitochondrial membrane permeabilization). We found that even brief exposure to ATP (10 min) was sufficient to cause a long-lasting delay of apoptosis and showed that the effects were not mediated by ATP breakdown to adenosine. The P2 receptor mediating the antiapoptotic actions of ATP was identified using a combination of more selective ATP analogs, receptor expression studies, and study of downstream signaling pathways. Neutrophils were shown to express the P2Y11 receptor and inhibition of P2Y11 signaling using the antagonist NF157 abrogated the ATP-mediated delay of neutrophil apoptosis, as did inhibition of type I cAMP-dependent protein kinases activated downstream of P2Y11, without effects on constitutive apoptosis. Specific targeting of P2Y11 could retain key immune functions of neutrophils but reduce the injurious effects of increased neutrophil longevity during inflammation.     

Extracellular ATP-induced NO production and its dependence on membrane Ca2+ flux in Salvia miltiorrhiza hairy roots

Extracellular ATP (eATP) is a novel signalling agent, and nitric oxide (NO) is a well-established signal molecule with diverse functions in plant growth and development. This study characterizes NO production induced by exogenous ATP and examines its relationship with other important signalling agents, Ca(2+) and H(2)O(2) in Salvia miltiorrhiza hairy root culture. Exogenous ATP was applied at 10-500 microM to the hairy root cultures and stimulated NO production was detectable within 30 min. The NO level increased with ATP dose from 10-100 microM but decreased from 100-200 muM or higher. The ATP-induced NO production was mimicked by a non-hydrolysable ATP analogue ATPgammaS, but only weakly by ADP, AMP or adenosine. The ATP-induced NO production was blocked by Ca(2+) antagonists, but not affected by a protein kinase inhibitor. ATP also induced H(2)O(2) production, which was dependent on both Ca(2+) and protein kinases, and also on NO biosynthesis. On the other hand, ATP induced a rapid increase in the intracellular Ca(2+) level, which was dependent on NO but not H(2)O(2). The results suggest that NO is implicated in ATP-induced responses and signal transduction in plant cells, and ATP signalling is closely related to Ca(2+) and ROS signalling.     

Balance of purines may determine life or death of retinal ganglion cells as A3 adenosine receptors prevent loss following P2X7 receptor stimulation

The purines ATP and adenosine can act as a coordinated team of transmitters. As extracellular adenosine is frequently derived from the enzymatic dephosphorylation of released ATP, the distinct actions of the two purines can be synchronized. In retinal ganglion cells (RGCs), stimulation of the P2X7 receptor for ATP leads to increased intracellular Ca2+ and death. Here we define the contrasting effects of adenosine and identify protective actions mediated by the A3 receptor. Adenosine attenuated the rise in Ca2+ produced by the P2X7 agonist 3'-O-(4-benzoylbenzoyl)ATP (BzATP). Adenosine was also neuroprotective, increasing the survival of ganglion cells exposed to BzATP. The A3 adenosine receptor agonist 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronimide (Cl-IB-MECA) mimicked the inhibition of the Ca2+ rise, whereas the A3 antagonist 3-Ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS-1191) reduced the protective effects of adenosine. Both Cl-IB-MECA and a second A3 receptor agonist IB-MECA reduced the cell loss triggered by BzATP. The actions of BzATP were mimicked by ATPgammaS, but not by ATP. In summary, adenosine can stop the rise in Ca2+ and cell death resulting from stimulation of the P2X7 receptor on RGCs, with the A3 adenosine receptor contributing to this protection. Hydrolysis of ATP into adenosine and perhaps inosine shifts the balance of purinergic action from that of death to the preservation of life.     

Metabolism of round spermatids: gossypol induces uncoupling of respiratory chain and oxidative phosphorylation

The effect of gossypol on energy metabolism of round spermatids of rats was examined. When spermatids were treated with various concentrations of gossypol for 30 min at 32 degrees C, a biphasic response (stimulation at low concentrations and inhibition at high concentrations) was seen in pyruvate and CO2 production from lactate. At the early period of incubation, gossypol at even high concentrations stimulated CO2 production to an extent similar to that stimulated by 2,4-dinitrophenol (DNP). At longer periods of incubation, however, the rates of CO2 production from lactate dropped to those seen in the rotenone-treated cells. The rates of oxygen consumption were not increased further by DNP when cells were pretreated with gossypol. The adenosine triphosphate (ATP) content in spermatids was reduced markedly, although lactate oxidation to CO2 and mitochondrial respiration were stimulated by gossypol. These results suggest that gossypol probably exerts its effect on spermatids by uncoupling respiratory chain and oxidative phosphorylation.     

The receptors for ATP and fMetLeuPhe are independently coupled to phospholipases C and A2 via G-protein(s). Relationship between phospholipase C and A2 activation and exocytosis in HL60 cells and human neutrophils.

The relationship between phospholipase A2 and C activation and secretion was investigated in intact human neutrophils and differentiated HL60 cells. Activation by either ATP or fMetLeuPhe leads to [3H]arachidonic acid release into the external medium from prelabelled cells. This response was inhibited when the cells were pretreated with pertussis toxin. When the [3H]arachidonic acid-labelled cells were stimulated with fMetLeuPhe, ATP or Ca2+ ionophore A23187, and the lipids analysed by t.l.c., the increase in free fatty acid was accompanied by decreases in label from phosphatidylinositol and phosphatidylcholine. Moreover, incorporation of label into triacylglycerol and to a lesser extent phosphatidylethanolamine was evident. Activation of secretion was evident with ATP and fMetLeuPhe but not with A23187. The pharmacological specificity of the ATP receptor in HL60 cells was investigated by measuring secretion of beta-glucuronidase, formation of inositol phosphatases and release of [3H]arachidonic acid. External addition of ATP, UTP, ITP, adenosine 5'-[gamma-thio]triphosphate (ATP[S]), adenosine 5'-[beta gamma-imido]triphosphate (App[NH]p), XTP, CTP, GTP, 8-bromo-ATP and guanosine 5'-[gamma-thio]triphosphate (GTP[S]) to intact HL60 cells stimulated inositol phosphate production, but only the first five nucleotides were effective at stimulating secretion or [3H]arachidonic acid release. In human neutrophils, addition of ATP, ITP, UTP and ATP[S] also stimulated secretion from specific and azurophilic granules, and this was accompanied by increases in cytosolic Ca2+ and in [3H]arachidonic acid release. The addition of phorbol 12-myristate 13-acetate (PMA; 1 nM) prior to the addition of either fMetLeuPhe or ATP led to inhibition of phospholipase C activity. In contrast, this had no effect on phospholipase A2 activation, whilst secretion was potentiated. Phospholipase A2 activation by either agonist was dependent on an intact cell metabolism, as was secretion. It is concluded that (1) activation of phospholipase C does not always lead to activation of phospholipase A2, (2) phospholipase A2 is coupled to the receptor independently of phospholipase C via a pertussis-toxin-sensitive G-protein and (3) for secretion to take place, the receptor has to activate both phospholipases C and A2.