Stimulation of phosphatidylcholine breakdown and diacylglycerol production by growth factors in Swiss-3T3 cells.

The effect of a number of growth factors on phosphatidylcholine (PtdCho) turnover in Swiss-3T3 cells was studied. Phorbol 12-myristate 13-acetate (PMA), bombesin, platelet-derived growth factor (PDGF) and vasopressin rapidly stimulated PtdCho hydrolysis, diacylglycerol (DAG) production, and PtdCho synthesis. Insulin and prostaglandin F2 alpha (PGF2 alpha) stimulated PtdCho synthesis, but not its breakdown, whereas epidermal growth factor (EGF) and bradykinin were without effect. Stimulation of PtdCho hydrolysis by the above ligands resulted in increased production of phosphocholine and DAG (due to phospholipase C activity) and significant amounts of choline, suggesting activation of a phospholipase D as well. CDP-choline and glycerophosphocholine levels were unchanged. Down-regulation of protein kinase C with PMA (400 nM, 40 h) abolished the stimulation of PtdCho hydrolysis and PtdCho synthesis by PMA, bombesin, PDGF and vasopressin, but not the stimulation of PtdCho synthesis by insulin and PGF2 alpha. PtdCho hydrolysis therefore occurs predominantly by activation of protein kinase C (either by PMA or PtdIns hydrolysis) leading to elevation of DAG levels derived from non-PtdIns(4,5)P2 sources. PtdCho synthesis occurs by both a protein kinase C-dependent pathway (stimulated by PMA, PDGF, bombesin and vasopressin) and a protein kinase C-independent pathway (stimulated by insulin and PGF2 alpha). DAG production from PtdCho hydrolysis is not the primary signal to activate protein kinase C, but may contribute to long-term activation of this kinase.     

De novo synthesis of diacylglycerol from glucose. A new pathway of signal transduction in human neutrophils stimulated during phagocytosis of beta-glucan particles.

The phagocytosis of beta-glucan particles by human neutrophils and the associated activation of NADPH O2- forming oxidase were accompanied by an increased hydrolysis of phosphoinositides by phospholipase C, hydrolysis of phosphatidylcholine by phospholipase D, accumulation of diglyceride (DG) mass, and [Ca2+]i rise. The reaction of phospholipid hydrolysis played a minor role in the formation of DG, which was mainly formed by de novo synthesis from glucose. The activation of this pathway was shown by the stimulation of the incorporation of [U-14C]glucose into DG, which occurred very rapidly after the challenge of neutrophils with beta-glucan particles. This DG derived from glucose was found almost completely as 1-acyl-2-acyl-glycerol (DAG). On the basis of the finding that phosphatidic acid was the precursor of DAG, an increase in the incorporation of [U-14C]acetate into DAG did not occur, and the [14C]radioactivity was in the glycerol backbone, the synthesis of DAG from [U-14C]glucose occurred very likely via dihydroxyacetone phosphate and glycerol 3-phosphate, stepwise acylation to phosphatidic acid, and dephosphorylation by phosphatidate phosphatase.     

v-Src increases diacylglycerol levels via a type D phospholipase-mediated hydrolysis of phosphatidylcholine.

Activating the protein-tyrosine kinase of v-Src in BALB/c 3T3 cells results in rapid increases in the intracellular second messenger, diacylglycerol (DAG). v-Src-induced increases in radiolabeled DAG were most readily detected when phospholipids were prelabeled with myristic acid, which is incorporated predominantly into phosphatidylcholine. Consistent with this observation, v-Src increased the level of intracellular choline. No increase in DAG was observed when cells were prelabeled with arachidonic acid, which is incorporated predominantly into phosphatidylinositol. Inhibiting phosphatidic acid (PA) phosphatase, which hydrolyzes PA to DAG, blocked v-Src-induced DAG production and enhanced PA production, implicating a type D phospholipase. Consistent with the involvement of a type D phospholipase, v-Src increased transphosphatidylation activity, which is characteristic of type D phospholipases. Thus, v-Src-induced increases in DAG most likely result from the activation of a type D phospholipase/PA phosphatase-mediated signaling pathway.     

sn-1,2-diacylglycerol and choline increase after fertilization in Xenopus laevis.

sn-1,2-Diacylglycerol (DAG) mass and translocation of protein kinase C alpha and beta to a membrane fraction increased approximately 7 min after insemination of Xenopus laevis eggs. The DAG mass increase of 48 pmol (from 62 to 110 pmol/cell) was greater than that for inositol 1,4,5-trisphosphate (IP3; an increase of approximately 170 fmol or approximately 280-fold smaller than the DAG increase), and DAG peaks approximately 5 min after IP3. Choline mass (a measure of phosphatidyl choline-specific phospholipase D) also peaked before DAG and the choline increase (134 pmol/cell) was greater than that of DAG. There was no detectable change in phosphocholine mass (a measure of phosphatidylcholine-specific phospholipase C). During first cleavage, DAG decreased, PKC translocation was low, and choline increased and peaked (whereas published work shows an increase in IP3 mass). Artificial elevation of intracellular calcium ([Ca2+]i) increased DAG levels but prevention of the [Ca2+]i increase after fertilization did not block DAG production. Thus, sperm stimulate production of DAG and choline through [Ca2+]i-independent and [Ca2+]i-dependent paths.     

Fluoride activates diradylglycerol and superoxide generation in human neutrophils via PLD/PA phosphohydrolase-dependent and -independent pathways

In contrast to the rapid, ethanol-inhibited superoxide generation by the receptor-linked agonist formyl-methionyl-leucyl-phenylalanine (fMLP), fluoride-activated superoxide generation occurs after a prolonged lag, and as shown herein is relatively ethanol-insensitive. We have investigated fluoride-activation of diradylglycerol generation and phospholipase D activity. Fluoride induces a very large increase in diradylglycerol mass (both 1,2-diacylglycerol (DAG) and 1-O-alkyl,2-acylglycerol (EAG)), with kinetics similar to superoxide generation. Unlike fMLP-activated diglyceride generation which is completely inhibited by ethanol, that produced by fluoride is only partially (30%) blocked. When the phosphatidylcholine pool is 3H-prelabeled, fluoride activates both [3H]phosphatidic acid (PA) and [3H]diglyceride generation with similar kinetics. Partial inhibition of the production of these species by ethanol was seen, coincident with the appearance of [3H]phosphatidylethanol, indicating phospholipase D-dependent transphosphatidylation had occurred. The data are consistent with the fluoride activation of PA and diglyceride generation by both phospholipase D-dependent and -independent (presumably phospholipase C) mechanisms.     

Amplification of Ca2+ signaling by diacylglycerol-mediated inositol 1,4,5-trisphosphate production

Stimulation of various cell surface receptors leads to the production of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG) through phospholipase C (PLC) activation, and the IP3 and DAG in turn trigger Ca2+ release through IP3 receptors and protein kinase C activation, respectively. The amount of IP(3) produced is particularly critical to determining the spatio-temporally coordinated Ca(2+)-signaling patterns. In this paper, we report a novel signal cross-talk between DAG and the IP3-mediated Ca(2+)-signaling pathway. We found that a DAG derivative, 1-oleoyl-2-acyl-sn-glycerol (OAG), induces Ca2+ oscillation in various types of cells independently of protein kinase C activity and extracellular Ca2+. The OAG-induced Ca2+ oscillation was completely abolished by depletion of Ca2+ stores or inhibition of PLC and IP3 receptors, indicating that OAG stimulates IP3 production through PLC activation and thereby induces IP3-induced Ca2+ release. Furthermore, intracellular accumulation of endogenous DAG by a DAG-lipase inhibitor greatly increased the number of cells responding to agonist stimulation at low doses. These results suggest a novel physiological function of DAG, i.e. amplification of Ca2+ signaling by enhancing IP3 production via its positive feedback effect on PLC activity.     

A novel mechanism for acetylcholine to generate diacylglycerol in brain

The classical scheme involving inositol phospholipid breakdown by phospholipase C as the sole source of diacylglycerol (DAG) has recently been challenged by evidence that phosphatidylcholine (PC) is an alternative source. In synaptic membranes of canine cerebral cortex, cholinergic agonists caused rapid accumulation of [3H]phosphatidic acid (PA) from [3H]PC within 15 s, whereas [3H]DAG formation showed a transient lag period before becoming elevated and then exceeding the amount of [3H]PA. Additional evidence shows that DAG is produced from PC by the action of phospholipase D to yield PA, which is further dephosphorylated to DAG by PA phosphatase. Our results indicate that this muscarinic acetylcholine receptor-regulated PC phospholipase D-PA phosphatase pathway may be a novel mechanism in cell signal transduction processes for activation of protein kinase C in brain.     

Muscarinic receptor activation of phosphatidylcholine hydrolysis. Relationship to phosphoinositide hydrolysis and diacylglycerol metabolism

We examined the relationship between phosphatidylcholine (PC) hydrolysis, phosphoinositide hydrolysis, and diacylglycerol (DAG) formation in response to muscarinic acetylcholine receptor (mAChR) stimulation in 1321N1 astrocytoma cells. Carbachol increases the release of [3H]choline and [3H]phosphorylcholine ([3H]Pchol) from cells containing [3H]choline-labeled PC. The production of Pchol is rapid and transient, while choline production continues for at least 30 min. mAChR-stimulated release of Pchol is reduced in cells that have been depleted of intracellular Ca2+ stores by ionomycin pretreatment, whereas choline release is unaffected by this pretreatment. Phorbol 12-myristate 13-acetate (PMA) increases the release of choline, but not Pchol, from 1321N1 cells, and down-regulation of protein kinase C blocks the ability of carbachol to stimulate choline production. Taken together, these results suggest that Ca2+ mobilization is involved in mAChR-mediated hydrolysis of PC by a phospholipase C, whereas protein kinase C activation is required for mAChR-stimulated hydrolysis of PC by a phospholipase D. Both carbachol and PMA rapidly increase the formation of [3H]phosphatidic acid ([3H]PA) in cells containing [3H]myristate-labeled PC. [3H]Diacylglycerol ([3H]DAG) levels increase more slowly, suggesting that the predominant pathway for PC hydrolysis is via phospholipase D. When cells are labeled with [3H]myristate and [14C]arachidonate such that there is a much greater 3H/14C ratio in PC compared with the phosphoinositides, the 3H/14C ratio in DAG and PA increases with PMA treatment but decreases in response to carbachol. By analyzing the increase in 3H versus 14C in DAG, we estimate that the DAG that is formed in response to PMA arises largely from PC. Muscarinic receptor activation also causes formation of DAG from PC, but approximately 20% of carbachol-stimulated DAG appears to arise from hydrolysis of the phosphoinositides.     

Phosphatidylethanolamine and phosphatidylcholine are sources of diacylglycerol in ras-transformed NIH 3T3 fibroblasts

Ras-transformation of cells is accompanied by an increase of the level of diacylglycerol (DAG), which participates in the signal transduction pathways. DAG could be generated from phospholipids either by activation of phospholipase C or by a more complex pathway involving phospholipase D and phosphatidate phosphohydrolase. To clarify which phospholipids produce DAG and which pathways are involved, we examined the DAG generating enzyme activities, using phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) as substrates. The study showed that the breakdown of PC and more markedly of PE by phospholipases C and D was stimulated in membranes from ras-transformed cells. Phosphatidate phosphohydrolase activity was also elevated in oncogene-expressing cells. The increase in glycerol uptake was most pronounced in cells given PE, followed by PC. The fatty acid analysis revealed apparent similarities between the acyl chains of PE and DAG only in the transformed cells. These findings suggest that PE is a source of DAG in ras-fibroblasts but does not rule out the role of PC in DAG production, due to the activation of the PC-specific phospholipases C and D.     

Formation of diacylglycerol by a phospholipase D-phosphatidate phosphatase pathway specific for phosphatidylcholine in endothelial cells

The conversion of phosphatidylcholine (PC) to diacylglycerol (DAG) was studied in sonicated endothelial cells and in subcellular fractions in the presence of 0.05% Triton X-100 and 2 mM EDTA. DAG formation occurred predominantly in an organelle fraction that sedimented at 15,000 x g. In parallel reactions with exogenous 1-oleoyl-2-[3H]oleoyl-PC (sn-2-[3H]DOPC) and phosphatidyl[3H]choline ([choline-3H]PC), [3H]DAG was formed by a reaction pathway in which [3H]choline was the only product derived from [choline-3H]PC. [3H]Choline was not formed secondarily from [3H]glycerophosphocholine or [3H]phosphocholine. Small amounts of [3H]phosphatidate ([3H]PA) were isolated from reactions with sn-2-[3H]DOPC at short incubation times, and substantial PA phosphatase activity was demonstrated. These data, taken together, supported a phospholipase D-PA phosphatase pathway of DAG formation. Kinetic data established that the low ratio of [3H]PA/[3H]DAG formed in reactions with sn-2-[3H]DOPC was due to a 15-fold higher Vmax and 7-fold lower apparent Km of the PA phosphatase. The [3H]PA/[3H]DAG product ratio was increased by addition of unlabeled PA or by selective extraction of phospholipase D with Triton X-100. The characteristics of the phospholipase D indicated a unique enzyme. Activity was optimal in the presence of EDTA and was almost totally dependent upon Triton X-100. The pH profile displayed a peak at 7.0. Of particular significance was the stringent substrate specificity. Phosphatidylinositol was not hydrolyzed, and activities towards phosphatidylethanolamine and sphingomyelin were at most 30- to 50-fold lower than those towards PC. Phospholipase D and PA phosphatase were identified in a number of rat tissues and other cells. The highest activities of phospholipase D were present in lung and endothelial cells. Phospholipase D was partially purified from rat lung by Triton X-100 extraction and anion exchange chromatography. When linked with PA phosphatase, the phospholipase D could initiate a pathway of DAG formation that is highly specific for PC.     

A phosphatidylcholine-specific phospholipase C regulates activation of p42/44 mitogen-activated protein kinases in lipopolysaccharide-stimulated human alveolar macrophages

This study uses human alveolar macrophages to determine whether activation of a phosphatidylcholine (PC)-specific phospholipase C (PC-PLC) is linked to activation of the p42/44 (ERK) kinases by LPS. LPS-induced ERK kinase activation was inhibited by tricyclodecan-9-yl xanthogenate (D609), a relatively specific inhibitor of PC-PLC. LPS also increased amounts of diacylglycerol (DAG), and this increase in DAG was inhibited by D609. LPS induction of DAG was, at least in part, derived from PC hydrolysis. Ceramide was also increased in LPS-treated alveolar macrophages, and this increase in ceramide was inhibited by D609. Addition of exogenous C2 ceramide or bacterial-derived sphingomyelinase to alveolar macrophages increased ERK kinase activity. LPS also activated PKC zeta, and this activation was inhibited by D609. LPS-activated PKC zeta phosphorylated MAP kinase kinase, the kinase directly upstream of the ERK kinases. LPS-induced cytokine production (RNA and protein) was also inhibited by D609. As an aggregate, these studies support the hypothesis that one way by which LPS activates the ERK kinases is via activation of PC-PLC and that activation of a PC-PLC is an important component of macrophage activation by LPS.     

An investigation of the transverse topology of bilirubin UDP-glucuronosyltransferase in rat hepatic endoplasmic reticulum.

Neutrophils stimulated with formylmethionyl-leucylphenylalanine (fMet-Leu-Phe) in the presence of butanol and ethanol formed phosphatidyl alcohols through a phospholipase D mechanism. The alcohols inhibited phosphatidic acid and diradylglycerol (DRG) formation, but did not block inositol 1, 4, 5-trisphosphate release. fMet-Leu-Phe-stimulated superoxide production was inhibited by alcohol concentrations which blocked DRG formation, whereas opsonized-zymosan-stimulated superoxide production was only partially decreased. These results suggest that phospholipase D activation is functionally linked to superoxide production in the human neutrophil.     

ADH-dependent phosphoinositide signalling system and prostaglandin E production in the frog urinary bladder.

Antidiuretic hormone (ADH) stimulated formation of inositol 1,4,5-trisphosphate (IP3), 1,2-diaclyglycerol (DAG) and an increase of phosphatidylinositol 4,5-biphosphate (PIP2) breakdown in the frog urinary bladder 20 s after addition. ADH also increased the prostaglandin E (PGE) secretion into serosal medium 3.5-fold and the release of arachidonic acid (AA) from 1,2-DAG, which was intensified in the presence of DAG kinase inhibitor R59022. Neomycin sulphate (10(-5) M) from the serosal side blocked ADH-stimulated PIP2 hydrolysis, IP3 production and increased the hydro-osmotic response to ADH. It also inhibited the ADH-stimulated PGE production (55%) and release of AA from 1,2-DAG. This data suggest that PIP2 breakdown is involved in the mechanism of feedback regulation of ADH action and is associated with PGE production via (i) the increase of AA release from PIP2-generated 1,2-DAG and (ii) possible activation of phospholipase A2 by IP3-induced elevation of cytosol Ca2+.     

Signal transduction in esophageal and LES circular muscle contraction

Contraction of normal esophageal circular muscle (ESO) in response to acetylcholine (ACh) is linked to M2 muscarinic receptors activating at least three intracellular phospholipases, i.e., phosphatidylcholine-specific phospholipase C (PC-PLC), phospholipase D (PLD), and the high molecular weight (85 kDa) cytosolic phospholipase A2 (cPLA2) to induce phosphatidylcholine (PC) metabolism, production of diacylglycerol (DAG) and arachidonic acid (AA), resulting in activation of a protein kinase C (PKC)-dependent pathway. In contrast, lower esophageal sphincter (LES) contraction induced by maximally effective doses of ACh is mediated by muscarinic M3 receptors, linked to pertussis toxin-insensitive GTP-binding proteins of the G(q/11) type. They activate phospholipase C, which hydrolyzes phosphatidylinositol bisphosphate (PIP2), producing inositol 1,4,5-trisphosphate (IP3) and DAG. IP3 causes release of intracellular Ca++ and formation of a Ca++-calmodulin complex, resulting in activation of myosin light chain kinase and contraction through a calmodulin-dependent pathway. Signal transduction pathways responsible for maintenance of LES tone are quite distinct from those activated during contraction in response to maximally effective doses of agonists (e.g., ACh). Resting LES tone is associated with activity of a low molecular weight (approximately 14 kDa) pancreatic-like (group 1) secreted phospholipase A2 (sPLA2) and production of arachidonic acid (AA), which is metabolized to prostaglandins and thromboxanes. These AA metabolites act on receptors linked to G-proteins to induce activation of PI- and PC-specific phospholipases, and production of second messengers. Resting LES tone is associated with submaximal PI hydrolysis resulting in submaximal levels of inositol trisphosphate (IP3-induced Ca++ release, and interaction with DAG to activate PKC. In an animal model of acute esophagitis, acid-induced inflammation alters the contractile pathway of ESO and LES. In LES circular muscle, after induction of experimental esophagitis, basal levels of PI hydrolysis are substantially reduced and intracellular Ca++ stores are functionally damaged, resulting in a reduction of resting tone. The reduction in intracellular Ca++ release causes a switch in the signal transduction pathway mediating contraction in response to ACh. In the normal LES, ACh causes release of Ca++ from intracellular stores and activation of a calmodulin-dependent pathway. After esophagitis, ACh-induced contraction depends on influx of extracellular Ca++, which is insufficient to activate calmodulin, and contraction is mediated by a PKC-dependent pathway. These changes are reproduced in normal LES cells by thapsigargin-induced depletion of Ca++ stores, suggesting that the amount of Ca++ available for release from intracellular stores defines the signal transduction pathway activated by a maximally effective dose of ACh.     

Cyclic AMP-elevating agents block chemoattractant activation of diradylglycerol generation by inhibiting phospholipase D activation.

Agents which elevate cellular cAMP (prostaglandin E2, theophylline, and forskolin) or mimic cAMP action (dibutyryl cAMP) are known to inhibit human neutrophil activation (superoxide generation and secretion) by receptor-linked agonists such as formyl-methionyl-leucyl-phenylalanine (fMLP). Herein, we show that these agents also markedly inhibit fMLP-stimulated diradylglycerol generation (assayed by mass methods). The magnitude of inhibition correlated with the ability of a given agent or combination of agents to elevate cAMP. Both 1,2-diacylglycerol and 1-O-alkyl,2-acyl glycerol generation were affected. Effects on the latter species, as well as a lack of effect on fMLP-stimulated inositol phosphate release, implied that cAMP affected diradylglycerol generation from a source other than phospholipase C-dependent phosphoinositide hydrolysis, since phosphatidylinositols do not contain appreciable quantities of the 1-O-alkyl linkage. In cells in which the phosphatidylcholine pool was prelabeled using 1-O-[3H]octadecyl-2-lyso-sn-glycero-3-phosphocholine, prostaglandin E2 plus theophylline inhibited the fMLP-activated rapid generation of [3H]phosphatidic acid and its subsequent conversion to [3H]diradylglycerol, implying an effect at the level of phospholipase D. In the presence of ethanol, the fMLP-activated transphosphatidylation of [3H]phosphatidylcholine to generate [3H]phosphatidylethanol (a phospholipase D-dependent reaction) was also markedly inhibited. In contrast, when phorbol 12-myristate 13-acetate was used to activate cells, cAMP-related agents had no effect on phospholipase D activity, diradylglycerol generation, or superoxide generation. The data indicate an inhibitory effect of cyclic AMP on receptor-mediated phospholipase D activation at a site proximal to phospholipase D (e.g., the receptor or G protein). These studies provide a new example of "cross-talk" among signal transduction systems.     

Platelet-activating factor mediates phosphatidylcholine hydrolysis by phospholipase D in human endometrium.

Human preimplantation embryos and endometrium secrete platelet-activating factor (PAF). The mechanism of phosphatidylcholine (PC) degradation stimulated by PAF was investigated in endometrial explants prelabeled with [methyl-3H]choline or preincubated with [3H]butan-1-ol. Analysis of the water-soluble metabolites of PAF-induced PC hydrolysis in secretory endometrium demonstrated that the stimulated generation of [3H]choline ([3H]Cho) precedes that of [3H]choline phosphate ([3H]ChoP) and [3H]glycerophosphocholine ([3H]GPC). Within 30 sec there was a rapid rise in PAF-induced [3H]Cho generation and by 2 min this had increased to 59.9% +/- 10.6% (p less than 0.02), with no effect upon [3H]ChoP and [3H]GPC during this period. Both [3H]GPC and [3H]ChoP, however, were increased at a later time point. The slower [3H]ChoP generation may suggest that PC-specific phospholipase C activation as well as delayed [3H]GPC rise may be due to PC-specific phospholipase A2 and lysophospholipase activation. Phospholipase D activity was confirmed by the incorporation of high-specific-activity [3H]butan-1-ol into [3H]phosphatidylbutanol ([3H]PBut). The rapid generation of [3H]PBut, which paralleled the rise in intracellular [3H]Cho, strongly suggests that PC breakdown is catalyzed by the phospholipase D pathway. It is proposed that PAF induces PC hydrolysis as a consequence of an early phospholipase D-catalyzed breakdown of PC in human secretory endometrium. This may be an alternative source for prostaglandin synthesis and an important pathway essential for long-term activation of local cellular events at the time of implantation.     

Regulation of diacylglycerol levels in carbachol-stimulated pancreatic acinar cells: relationship to the breakdown of phosphatidylcholine and metabolism to phosphatidic acid.

Rat pancreatic acinar cells prelabeled with [14C]palmitic acid and then exposed to carbachol (CCh) exhibited a time-dependent increase in 1,2-[14C]diacylglycerol ([14C]DAG) levels, which was first detected at 2 min and then continued to rise in a linear manner. There was a concomitant increase in [14C]phosphatidic acid, which plateaued after 2 min and then remained at steady-state levels. CCh also promoted the release of phosphocholine, but not choline, within 60 s and caused a decrease in [14C]phosphatidylcholine in cells prelabeled with [14C]glycerol after 15 min. The inability to detect a rise in [14C]phosphatidylethanol accumulation and a fall in [14C]phosphatidate levels in [14C]palmitate prelabeled cells after exposure to CCh plus ethanol documented the absence of a phospholipase D-mediated pathway. The rapid phosphorylation of diglyceride in homogenates from unstimulated and carbachol-treated cells increased with increasing concentrations of exogenous substrate, thereby affirming that carbachol stimulates the phosphorylation of DAG by promoting the accumulation of the diglyceride. These collective findings provide evidence for the existence of an integrative control mechanism for regulating endogenous DAG levels during pancreatic acinar cell activation involving phosphatidylcholine-specific phospholipase C and DAG kinase.     

Effect of staurosporine on fMet-Leu-Phe-stimulated human neutrophils: dissociated release of inositol 1,4,5-trisphosphate, diacylglycerol and intracellular calcium.

Staurosporine, a microbial alkaloid, enhances inositol 1,4,5-trisphosphate (IP3) and 1,2-diacylglycerol (DG) production rapidly and dose-dependently in fMet-Leu-Phe (FMLP)-stimulated human neutrophils showing maximal effects at 1 microM concentration. The IP3 increase was specific for staurosporine as three other putative protein kinase C (PKC) inhibitors, H7, sphingosine and palmitoylcarnitine were unable to enhance the IP3 generation in FMLP-stimulated human neutrophils. Staurosporine, at concentrations 0.3-1.0 microM, did not affect the initial mobilization of FMLP-induced intracellular Ca2+ (Ca2+i), although a sustained elevation of cytosolic Ca2+ level was observed within 5 min. This effect could not be suppressed, even by 1 microM phorbol-myristate 12,13-acetate (PMA). Whereas lower concentrations of staurosporine (less than or equal to 100 nM) were unable to affect FMLP-induced IP3 production, DG accumulation and Ca2+i, the PMA-inhibited initial Ca2+i signal and IP3 formation triggered by FMLP were almost completely restored. At higher concentrations (greater than or equal to 300 nM) staurosporine reversed the inhibitory effect of other protein kinases, distinct from the PMA-inducible one, which may be responsible for the phosphatidyl inositol 4,5-bisphosphate (PIP2) breakdown, thus causing accumulation of IP3 and DG and an elevation of C2+i level. Whereas IP3 declined to basal level within 5 min, the DG level remained elevated during the same period. This phenomenon is attributed to phospholipase D (PLD) stimulation by staurosporine, which augments the DG synthesis, in part through PA degradation via phosphatidic acid (PA) phosphohydrolase.     

The chymotrypsin inhibitor carbobenzyloxy-leucine-tyrosine-chloromethylketone interferes with phospholipase D activation induced by formyl-methionyl-leucyl-phenylalanine in human neutrophils

The effects of carbobenzyloxy-leucine-tyrosine-chloromethylketone (zLYCK), an inhibitor of chymotrypsin-like proteases, on signal transduction in human neutrophils triggered by the chemoattractant formyl-methionyl-leucyl-phenylalanine (fMLP) were investigated. zLYCK (10 microM) inhibited the fMLP-induced respiratory burst in neutrophils treated with cytochalasin B. In the presence of zLYCK (10 microM), the activation of phospholipase D in response to fMLP addition was inhibited. zLYCK did not inhibit the binding of [3H] fMLP to its receptor or the enzymic activity of phospholipase D because the response to ionomycin was unaffected. The effect of zLYCK on phospholipase D correlated well with its effects on the accumulation of diglycerides, which was also inhibited in the presence of zLYCK. In electropermeabilized neutrophils, too, zLYCK caused an inhibition of the fMLP-induced respiratory burst and the fMLP-induced activation of phospholipase D. Interestingly, this inhibition could be bypassed by guanosine 5'-O-(thiotriphosphate). We conclude that the inhibition of the respiratory burst in human neutrophils by zLYCK is caused by the selective inhibition of signal transduction leading to activation of phospholipase D and that zLYCK might be a useful probe to study the role of phospholipase D in neutrophil activation.