ABSTRACT
In the 15 years since the identification and characterisation of the extracellular calcium-sensing receptor (CaR), it has become increasingly apparent that this cationic binding receptor is found in many tissues, not associated with the control of plasma calcium. One of these tissues is the pancreatic islet where insulin secretion provides the basis of energy regulation. It seems inherently unlikely that the islet responds to alterations in systemic calcium and a more plausible and intriguing possibility is that the CaR mediates cell-to-cell communication through local increases in the concentration of extracellular Ca(2+), co-released with insulin. This short article explores this possibility and suggests that this novel mechanism of cell communication, along with direct coupling via gap junctions and other local paracrine regulators helps explain why the glucose responsiveness of the intact islet is greater than the sum of the composite parts in isolation.
Subject(s)
Insulin/metabolism , Receptors, Calcium-Sensing/physiology , Animals , Calcium/metabolism , Cell Communication/physiology , Humans , Insulin Secretion , Islets of Langerhans/metabolism , Islets of Langerhans/physiology , Models, Biological , Receptors, Calcium-Sensing/metabolismABSTRACT
Phosphatidic acid, the product of phospholipase D catalysed phosphatidylcholine hydrolysis is an important signalling molecule that has been implicated in regulation of actin cytoskeleton remodelling and secretion from mast cells. We show that human PLD1b (hPLD1b) is an actin-binding protein and the N-terminus is predominantly involved in this interaction. Protein kinase C (PKC) is a major upstream regulator of PLD activity and PKC phosphorylation sites have been identified within the N-terminus of PLD1b at serine 2 and threonine 147. Over-expression of wild type hPLD1b in mast cells showed that antigen stimulation significantly enhanced co-localisation of PLD1b with actin structures. Mutation of serine 2 to alanine abolished antigen-induced co-localisation whereas mutation of threonine 147 had less dramatic effects on co-localisation. The absence of co-localisation of PLD1b (S2A) with actin coincides with a significant decrease in PLD activity in cells expressing the PLD1b (S2A) mutant. In resting RBL-2H3 cells, mutation of serine 2 to aspartate resulted in constitutive co-localisation of PLD with the actin cytoskeleton, coincident with restored PLD activity. These results reveal that serine 2 is an important regulatory site involved in controlling PLD enzyme activity and the interaction between PLD and actin.
Subject(s)
Actins/metabolism , Mast Cells/metabolism , Phospholipase D/metabolism , Animals , Antigens/physiology , Cell Line , Gene Expression Regulation , Humans , Mutation , Phospholipase D/genetics , Protein Binding , Protein Transport , Serine/chemistry , TransfectionABSTRACT
BACKGROUND: Efficient neuronal gene therapy is a goal for the long-term repair and regeneration of the injured central nervous system (CNS). We investigated whether targeting cDNA to neurons with cholera toxin b chain conjugated non-viral polyplexes led to increased efficiency of non-viral gene transfer in the CNS. Here, we illustrate the potential for this strategy by demonstrating enhanced transfection of a differentiated neuronal cell type, PC12. METHODS: In vitro transfection efficiency of a cholera toxin b chain-poly(D-lysine) molecular conjugate (CTb-K(100)) was compared by fluorescence-activated cell sorting (FACS) analysis of green fluorescent protein (GFP) expression and luminometric measurement of beta-galactosidase (beta-gal) expression, to untargeted poly(D-lysine) (K(100)) in undifferentiated and NGF-differentiated PC12 cells. RESULTS: Transfection of undifferentiated PC12 cells with CTb-K(100) polyplexes resulted in a 36-fold increase in levels of pCMV-DNA(LacZ) expression and a 20-fold increase in the frequency of transduction with pCMV-DNA(GFP), compared with untargeted K(100) polyplexes. Treatment of PC12 cells with 50 ng/ml/day of NGF for 14 days led to differentiation to a neuronal phenotype. Transfection of NGF-differentiated cells with CTb-K(100) polyplexes resulted in a 133-fold increase in levels of pCMV-DNA(LacZ) expression and a 11-fold increase in the percentage of cells transduced with pCMV-DNA(GFP), compared with untargeted K(100) polyplexes. Transfection was dependent on CTb, with CTb-K(100)-mediated transfections competitively inhibited with free CTb in both PC12 phenotypes. CONCLUSIONS: Non-viral systems for gene transfer in damaged CNS show superior toxicological profiles to most viruses but are limited by inefficient and non-selective gene expression in target tissue. Cholera toxin is known to interact preferentially with neuronal cells of the central and peripheral nervous systems, mediating binding through the b subunit, CTb, and the pentasaccharide moiety of the gangliosaccharide, GM1, which is present at high levels on the neuronal cell surface. Here, we show that a molecular conjugate of the CTb subunit, covalently linked to poly(D-lysine), is able to successfully target and significantly enhance transfection of a neuronal cell type, NGF-differentiated rat PC12 pheochromocytoma cells. This observation encourages the further development of non-viral strategies for the delivery of therapeutic genes to neurons.
Subject(s)
Cholera Toxin/genetics , Gene Transfer Techniques , Neurons/physiology , Polylysine/genetics , Animals , Binding, Competitive , Cell Differentiation/drug effects , Cell Differentiation/genetics , Chemistry, Physical/methods , Cholera Toxin/metabolism , DNA, Complementary , G(M1) Ganglioside/metabolism , Gene Expression , Nerve Growth Factor/pharmacology , PC12 Cells , Rats , Transfection/methodsABSTRACT
Cell adhesion is fundamental to establishing and maintaining the discrete tissues in multicellular organisms. Adhesion must be sufficiently strong to preserve tissue architecture, whilst having the capacity to readily dissociate to permit fundamental processes, such as wound repair, to occur. However, very little is known about the signalling mechanisms involved in temporary down-regulation of cell adhesion to facilitate such processes. Cadherins are the principal mediators of cell-cell adhesion in a wide variety of tissues and species and form multi-protein complexes with cytosolic and cytoskeletal proteins to express their full adhesive capacity. In the present study we report that the p85 subunit of phosphoinositide 3-kinase (PI 3-kinase) is associated with the cadherin-based adhesion complex in human epithelial cells. The interaction of p85 with the complex is via beta-catenin. We also show that the interaction of p85 and beta-catenin is direct, involves the N-terminal Src homology domain 2 of p85 and is regulated by tyrosine phosphorylation. These data suggest that PI 3-kinase may play a role in the functional regulation of the cadherin-based adhesion complex.
Subject(s)
Cadherins/metabolism , Cytoskeletal Proteins/metabolism , Peptide Fragments/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Trans-Activators , Animals , Cadherins/isolation & purification , Catalytic Domain , Cell Adhesion , Cell Line , Chemical Precipitation , Cytoskeletal Proteins/genetics , Dogs , Epidermal Growth Factor/pharmacology , ErbB Receptors/metabolism , Glutathione Transferase/genetics , Humans , Keratinocytes/enzymology , Keratinocytes/metabolism , Macromolecular Substances , Peptide Fragments/isolation & purification , Phosphatidylinositol 3-Kinases/isolation & purification , Recombinant Fusion Proteins/metabolism , Two-Hybrid System Techniques , beta CateninABSTRACT
The signalling pathway leading, for example, to actin cytoskeletal reorganisation, secretion or superoxide generation involves phospholipase D (PLD)-catalysed hydrolysis of phosphatidylcholine to generate phosphatidic acid, which appears to mediate the messenger functions of this pathway. Two PLD genes (PLD1 and PLD2) with similar domain structures have been doned and progress has been made in identifying the protein regulators of PLD1 activation, for example Arf and Rho family members. The activities of both PLD isoforms are dependent on phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and our sequence analysis suggested the presence of a pleckstrin homology (PH) domain in PLD1, although its absence has also been daimed. Investigation of the inositide dependence showed that a bis-phosphorylated lipid with a vicinal pair of phosphates was required for PLD1 activity. Furthermore, PLD1 bound specifically and with high affinity to lipid surfaces containing PI(4,5)P2 independently of the substrate phosphatidylcholine, suggesting a key role for the PH domain in PLD function. Importantly, a glutathione-S-transferase (GST) fusion protein comprising GST and the PH domain of PLD1 (GST-PLD1-PH) also bound specifically to supported lipid monolayers containing PI(4,5)P2. Point mutations within the PLD1 PH domain inhibited enzyme activity, whereas deletion of the domain both inhibited enzyme activity and disrupted normal PLD1 localisation. Thus, the functional PH domain regulates PLD by mediating its interaction with polyphosphoinositide-containing membranes; this might also induce a conformational change, thereby regulating catalytic activity.
Subject(s)
Phosphatidylinositol 4,5-Diphosphate/metabolism , Phospholipase D/metabolism , Protein Isoforms/metabolism , Signal Transduction/physiology , Amino Acid Sequence , Animals , COS Cells , Catalysis , Cell Line , Chlorocebus aethiops , Consensus Sequence , Fibroblasts , Humans , Hydrolysis , Membrane Lipids/metabolism , Molecular Sequence Data , Phosphatidylcholines/metabolism , Phosphatidylserines/metabolism , Phospholipase D/chemistry , Phospholipase D/genetics , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Structure, Tertiary , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Surface Plasmon ResonanceABSTRACT
Fibroblast growth factor-mediated signalling was studied in porcine aortic endothelial cells expressing either wild-type fibroblast growth factor receptor-1 or a mutant receptor (Y766F) unable to bind phospholipase C-(&ggr;). Stimulation of cells expressing the wild-type receptor resulted in activation of phospholipases C, D and A(2) and increased phosphoinositide 3-kinase activity. Stimulation of the wild-type receptor also resulted in stress fibre formation and a cellular shape change. Cells expressing the Y766F mutant receptor failed to stimulate phospholipase C, D and A(2) as well as phosphoinositide 3-kinase. Furthermore, no stress fibre formation or shape change was observed. Both the wild-type and Y766F receptor mutant activated MAP kinase and elicited proliferative responses in the porcine aortic endothelial cells. Thus, fibroblast growth factor receptor-1 mediated activation of phospholipases C, D and A(2) and phosphoinositide 3-kinase was dependent on tyrosine 766. Furthermore, whilst tyrosine 766 was not required for a proliferative response, it was required for fibroblast growth factor receptor-1 mediated cytoskeletal reorganisation.
Subject(s)
Cytoskeleton/metabolism , Endothelium, Vascular/enzymology , Phosphatidylinositol 3-Kinases/metabolism , Phospholipases/metabolism , Receptor Protein-Tyrosine Kinases/genetics , Receptors, Fibroblast Growth Factor/genetics , Animals , Aorta/cytology , Cell Division/physiology , Cells, Cultured , Endothelium, Vascular/cytology , Mitogen-Activated Protein Kinases/metabolism , Mutagenesis/physiology , Phospholipase D/metabolism , Phospholipases A/metabolism , Phosphorylation , Receptor Protein-Tyrosine Kinases/metabolism , Receptor, Fibroblast Growth Factor, Type 1 , Receptors, Fibroblast Growth Factor/metabolism , Signal Transduction/physiology , Swine , Transfection , Type C Phospholipases/metabolism , Tyrosine/geneticsABSTRACT
Phospholipase D (PLD) activity has been shown to be GTP-dependent both in vivo and in vitro. One protein that confers GTP sensitivity to PLD activity in vitro is the low-molecular-mass G-protein ADP-ribosylation factor (Arf). However, members of the Rho family and protein kinase C (PKC) have also been reported to activate PLD in various cell systems. We have characterized the stimulation of PLD in HL60 cell membranes by these proteins. The results demonstrate that a considerable proportion of HL60 PLD activity is located in a detergent-insoluble fraction of the cell membrane that is unlikely to be a caveolae-like domain, but is probably cytoskeletal. This PLD activity required the presence of Arf1, a Rho-family member and PKC for efficient catalysis of the lipid substrate, suggesting that the activity represents PLD1. We show that recombinant human PLD1b is regulated in a similar manner to HL60-membrane PLD, and that PKCalpha and PKCdelta are equally effective PLD activators. Therefore maximum PLD activity requires Arf, a Rho-family member and PKC, emphasizing the high degree of regulation of this enzyme.
Subject(s)
GTP-Binding Proteins/metabolism , Phospholipase D/metabolism , Protein Kinase C/metabolism , ADP-Ribosylation Factor 1 , ADP-Ribosylation Factors , Cell Membrane/enzymology , Cloning, Molecular , Detergents , Enzyme Activation , HL-60 Cells , Humans , Phospholipase D/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , SolubilityABSTRACT
In eukaryotes, many receptor agonists use phospholipase-generated lipids as intracellular messengers. Receptor occupation stimulates the production of polyunsaturated 1,2-diacylglycerols by phosphatidylinositol-4,5-bisphosphate specific phospholipases C and/or of mono-unsaturated and saturated phosphatidates by phospholipase-D-catalysed phosphatidylcholine breakdown. The primary phospholipase products are rapidly metabolized: polyunsaturated 1,2-diacylglycerols are converted to polyunsaturated phosphatidates by diacylglycerol kinase; mono-unsaturated and saturated phosphatidates are dephosphorylated to give mono-unsaturated and saturated 1,2-diacylglycerols by phosphatidate phosphohydrolase. The phospholipase-generated polyunsaturated 1,2-diacylglycerols and mono-unsaturated and saturated phosphatidates appear to be intracellular messengers, whereas their immediate metabolites probably do not have signalling functions.
Subject(s)
Diglycerides/physiology , Phosphatidic Acids/physiology , Second Messenger Systems/physiology , Animals , Humans , Models, MolecularABSTRACT
p70(s6k) has a role in cell cycle progression in response to specific extracellular stimuli. The signal transduction pathway leading to activation of p70(s6k) by fibroblast growth factor receptor-1 (FGFR-1) was examined in FGF-2-treated rat L6 myoblasts. p70(s6k) was activated in a biphasic and rapamycin-sensitive manner. Although phosphatidylinositol 3'-kinase was not activated in the FGF-2 treated cells, as judged from in vitro and in vivo analyses, wortmannin and LY294002 treatment inhibited p70(s6k) activation. Inhibition of protein kinase C (PKC), by bisindolylmaleimide or by chronic phorbol ester treatment of the FGFR-1 cells, suppressed but did not block p70(s6k) activation. In cells expressing a point-mutated FGFR-1, Y766F, unable to mediate PKC activation, p70(s6k) was still activated, in a bisindolylmaleimide- and phorbol ester-resistant manner. The involvement of S6 kinase in FGFR-1-dependent biological responses was examined in murine brain endothelial cells. In response to FGF-2, these cells differentiate to form tube-like structures in collagen gel cultures and proliferate when cultured on fibronectin. p70(s6k) was not activated in endothelial cells on collagen, whereas activation was observed during proliferation on fibronectin. In agreement with this finding, rapamycin inhibited the proliferative but not the differentiation response. Our results indicate that FGFR-1 mediates p70(s6k) activation by a phosphatidylinositol 3'-kinase-independent mechanism that does not require PKC activation and, furthermore, proliferation, but not differentiation of endothelial cells in response to FGF-2, is associated with p70(s6k) activation.
Subject(s)
Endothelium, Vascular/growth & development , Fibroblast Growth Factor 2/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Protein Serine-Threonine Kinases/metabolism , Receptor Protein-Tyrosine Kinases , Receptors, Fibroblast Growth Factor/metabolism , Animals , Brain/blood supply , Cell Differentiation , Cell Division , Cells, Cultured , Dose-Response Relationship, Drug , Endothelium, Vascular/cytology , Enzyme Activation , Isoenzymes/metabolism , Mice , Muscles/cytology , Mutation , Phosphatidylinositol 3-Kinases , Phospholipase C gamma , Polyenes/pharmacology , Rats , Receptor, Fibroblast Growth Factor, Type 1 , Receptors, Fibroblast Growth Factor/genetics , Ribosomal Protein S6 Kinases , Signal Transduction/drug effects , Sirolimus , Type C Phospholipases/metabolismABSTRACT
1-Stearoyl-2-arachidonoylglycerol (SAG) kinase was identified in the particulate fraction of pig testes. This activity was enriched by hydroxyapatite and blue dye chromatography. The enzyme was selective for polyunsaturated diradylglycerol species and activity was not modulated by other diradylglycerol species or sphingomyelin metabolites. Further purification resulted in the isolation of 55 and 50 kDa proteins that corresponded with SAG kinase activity. These results support the view that the phosphorylation of polyunsaturated diradylglycerol is regulated by structural determinants in the molecule.
Subject(s)
Diglycerides/metabolism , Phosphotransferases (Alcohol Group Acceptor)/isolation & purification , Testis/enzymology , Animals , Chromatography, Thin Layer , Diglycerides/pharmacology , Male , Phosphorylation , Phosphotransferases (Alcohol Group Acceptor)/drug effects , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Silver Staining , Subcellular Fractions/enzymology , Substrate Specificity , SwineABSTRACT
PLD is regulated by the small GTP binding proteins Rho and Arf, though predominantly by the latter. The PA product of PLD activation is an activator of Rho-regulated actin stress fibre formation and in invasive cells of MMP-9 synthesis and activation. Together this may explain the increased invasion of cells in response to PA.
Subject(s)
GTP-Binding Proteins/metabolism , Phospholipase D/metabolism , ADP-Ribosylation Factors , Actins/metabolism , Blotting, Western , Cell Membrane/enzymology , Cell Movement/physiology , Edetic Acid/pharmacology , Enzyme Activation , GTP-Binding Proteins/pharmacology , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , HL-60 Cells , Humans , Lipids/analysis , Metalloendopeptidases/metabolism , Phosphatidic Acids/metabolism , Phosphatidic Acids/pharmacology , rhoA GTP-Binding ProteinABSTRACT
Growth factors and certain oncogenes activate a range of phospholipid-mediated signal transduction pathways resulting in cell proliferation. Demethoxyviridin (DMV), a structural analogue of wortmannin and recently reported as a potent inhibitor of phosphoinositide-3-kinase, inhibited bombesin plus insulin-stimulated increase in cell number in Swiss 3T3 cells, a model of cell proliferation. The drug produced cytostatic effects at concentrations below 1 microM and cytotoxic effects at 10 microM. In intact Swiss 3T3 cells DMV inhibited insulin-stimulated PI 3- and 4-kinases and bombesin-stimulated phospholipases C, D and A2 in the nanomolar range. DMV also inhibited bombesin-stimulated tyrosine phosphorylation of a range of proteins at nM concentrations. This study shows that DMV inhibited multiple stimulated signalling pathways which lead to increased Swiss 3T3 cell proliferation. A stable analogue of DMV may have chemotherapeutic potential.
Subject(s)
Androstenes/pharmacology , Cell Division/drug effects , Phosphoinositide-3 Kinase Inhibitors , Phospholipids/physiology , Signal Transduction/drug effects , 3T3 Cells , Androstadienes , Androstenes/toxicity , Animals , Bombesin/pharmacology , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/toxicity , Insulin/pharmacology , Mice , Phosphatidylinositols/metabolism , Phospholipases/antagonists & inhibitors , Phospholipases/metabolism , Phosphorylation , Receptor Protein-Tyrosine Kinases/metabolism , Tyrosine/metabolism , WortmanninSubject(s)
Cell Nucleus/enzymology , GTP-Binding Proteins/metabolism , HL-60 Cells/enzymology , Nuclear Envelope/ultrastructure , Phospholipase D/metabolism , ADP-Ribosylation Factors , Cell Fractionation/methods , Cell Membrane/enzymology , Cell Nucleus/ultrastructure , Cytosol/physiology , Endoplasmic Reticulum/enzymology , Golgi Apparatus/enzymology , Humans , Intracellular Membranes/enzymology , Lysosomes/enzymology , Recombinant Proteins/pharmacologyABSTRACT
Membrane-associated phospholipase D (PLD) in HL60 cells can be activated by the small GTP-binding proteins Arf and RhoA, but polyphosphorylated inositol lipids were required for maximum activity. The intact lipid was required because neither inositol 1,4, 5-trisphosphate nor stearoyl-arachidonyl glycerol could substitute for phosphatidylinositol 4,5-bisphosphate (PIP2). Arf-stimulated but not Rho-stimulated PLD activity was increased by the inclusion of Mg2+ and ATP. ATP-dependent PLD activation occurred when phosphatidylinositol 4-phosphate (PIP), PIP2, or phosphatidylinositol 3,4,5-trisphosphate (PIP3) were included, but PIP2 formation was only detected with PIP; no PIP3 production was detected under any conditions. Therefore, the ATP-dependent increase in PLD activity cannot be explained by PIP2 or PIP3 formation. Association of endogenous Arf and RhoA with membranes was increased by incubation with GTPgammaS. This treatment increased membrane PLD and PIP kinase activities in the absence of exogenous p21 proteins. Reduction of Arf translocation suppressed the increase in PLD and PIP kinase activities, whereas complete removal of Rho but not Arf from membranes with RhoGDI was without effect on PLD activity but increased PIP kinase activity. Therefore, although recombinant Arf and Rho can activate PLD and PIP kinase in HL60 cells, it is the endogenous Arf but not Rho that regulates PLD, and thus a role for Rho in the physiological regulation of PLD in HL60 cells is unlikely.
Subject(s)
GTP-Binding Proteins/metabolism , Guanine Nucleotide Dissociation Inhibitors , Phospholipase D/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , ADP-Ribosylation Factors , Cell Membrane/enzymology , Enzyme Activation , Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology , HL-60 Cells , Humans , Kinetics , Phosphatidylinositol 4,5-Diphosphate , Phosphatidylinositol Phosphates/pharmacology , Recombinant Fusion Proteins/metabolism , Recombinant Proteins/metabolism , rho Guanine Nucleotide Dissociation Inhibitor alpha , rho-Specific Guanine Nucleotide Dissociation Inhibitors , rhoA GTP-Binding ProteinABSTRACT
BACKGROUND: Agonist-stimulated phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine, generating the putative messenger phosphatidate (PA). Proposed functions for PA, and hence for PLD, include kinase activation, the regulation of small molecular weight GTP-binding proteins, actin polymerization and secretion. It has not been possible to define a physiological function for PLD activation as it is generally stimulated together with other signalling pathways, such as those involving phospholipases A2 and C, phosphatidylinositide (PI) 3-kinase and the p21(ras)/mitogen-activated protein (MAP) kinase cascade. RESULTS: We report that, in porcine aortic endothelial (PAE) cells, lysophosphatidic acid (LPA) stimulated PLD activity and rapidly generated PA in the absence of other phospholipase, PI 3-kinase or MAP kinase activities. PLD activation was controlled by a tyrosine kinase-regulated pathway. LPA also stimulated actin stress fibre formation, but was inhibited by butan-1-ol; the alcohol also reduced the accumulation of PA. The addition of PA to cells did not stimulate PLD activity, but did cause stress fibre formation in a manner that was insensitive to butan-1-ol. Stimulation of stress fibre formation by LPA and PA was sensitive to genistein, and was inhibited by micro-injection of the Rho-inhibiting C3 exotoxin into PAE cells. CONCLUSIONS: This study provides the first clear demonstration of a physiological role for PLD activity. In PAE cells, the stimulation of actin stress fibre formation was a consequence of PA generation and, therefore, PLD activation. The results suggest that PA generation is upstream of Rho activation, and imply a role for PLD in the regulation of Rho-mediated pathways.
Subject(s)
Actins/metabolism , Phospholipase D/metabolism , Animals , Cells, Cultured , Endothelium, Vascular/drug effects , Endothelium, Vascular/enzymology , Enzyme Activation , GTP-Binding Proteins/metabolism , Lysophospholipids/pharmacology , Signal Transduction , SwineABSTRACT
Wortmannin and its structural analogue demethoxyviridin (DMV) have been reported to be specific inhibitors of phosphatidylinositol 3-kinase activity. Here we report that these compounds are not as selective as assumed and demonstrate inhibition of bombesin-stimulated phospholipase A2 activity by both wortmannin and DMV with an IC50 (2 nM) which is slightly more potent than the inhibition of insulin-stimulated phosphatidylinositol 3,4,5-trisphosphate generation in these cells (approximately 10nM). While it has not been possible to fully block in vitro phospholipase A2 activity with wortmannin, inhibition cannot be a consequence of inhibition of PI 3-kinase activity since bombesin fails to generate 3-phosphorylated lipids in the intact cell. Therefore, while wortmannin is indeed a PI 3-kinase inhibitor, it is not as specific as previously reported, and experimental conclusions based solely on its use should be treated with caution.