Regulation Of Phospholipase A2 Research Articles

Secretory Phospholipase A2 (sPLA2) Isozymes as Potential Targets in Tobacco Condensate- induced Colon Damage.

To find out the role of secretory phospholipase A2 (sPLA2) isozymes as potential targets in tobacco condensate-induced colon damage. The effects of cigarette smoke condensate (CSC) and the molecular mechanisms involved in the regulation of phospholipase A2 (PLA2) and its isozymes in colon cells, which are still unclear and emerging, are studied. The study aimed to check the effect of CSC on cell viability and reactive oxygen species (ROS) and superoxide. Also, the effect of CSC on gene expression of different secretory phospholipase A2 (sPLA2) was evaluated. Moreover, the impact of inhibition of sPLA2 on various cell properties i.e. cell viability, cell proliferation, membrane damage and free radicals' generation is also studied. CSC-induced changes were evaluated in cell viability by MTT assay, followed by the evaluation of membrane modulation by flow cytometry, free radical generation by fluorescent dyes, PLA2 isoforms gene expression patterns and their suppression by small interfering RNA (siRNA) studied in HCT-15 male and HT-29 female colon cells. Our results demonstrate that HCT-15 and HT-29 cells treated with CSC significantly reduced the cell viability by 50% within 48 h and significantly enhanced the total reactive oxygen species (ROS) by 2 to 10-fold, and mitochondrial ROS (mtROS) and superoxide radicals (SOR) by 2-fold each. Treatment with CSC significantly unregulated secretory phospholipase A2 (sPLA2) IID group and down-regulated IB and cytosolic phospholipase (cPLA2) IVA groups in HCT-15 cells without affecting them in HT-29 cells. Silencing the sPLA2 IID group results in an increase in cell viability and a decrease in ROS. Silencing the PLA2 IVA gene in the HCT-15 cells showed a reduced expression which had no impact on the CSC-induced cell proliferation, membrane damage and free radicals (ROS, mtROS, and SOR) generation. Therefore, identifying cell-specific sPLA2 isozymes seems to play a key role in controlling the ROSinduced damage by CSC and helps develop specific therapeutic strategies.

Development and Application of a New Platform for Measuring Phospholipase A2 Activity in Cells Using Mass Spectrometry

Members of the phospholipase A2 (PLA2) superfamily hydrolyze fatty acids from the sn‐2 position of membrane phospholipids. PLA2‐mediated release of n‐3 and n‐6 polyunsaturated fatty acids (PUFAs) is a highly regulated and critical step in producing eicosanoids and docosanoids. These signaling molecules play key roles in a variety of important processes including inflammation, onset of premature labor, and neural degenerative diseases. Thus, a better understanding of how PLA2 functions in cells may uncover new therapeutic avenues and lead to potent pharmacological agents. Though a rapid in vitro assay of PLA2 function has been recently published, one barrier to progress in this field is the lack of methods for determining PLA2 activity in cells. To address this issue, we have developed a new platform for measuring PLA2 activity ex vivo. Enriching cells with natural or deuterated PUFAs followed by mass spectrometry allows for tracking of hydrolysis at the sn‐2 position by both the disappearance of substrate phospholipids and the appearance of product PUFAs. An optimized cell culture procedure and lipid analysis workflow allow for significantly improved throughput. We anticipate that this platform will not only lead to a better understanding of PLA2 regulation and function in cells, but also allow for rapid screening of inhibitor candidates.Support or Funding InformationSupported by NIH grant GM20501

Metabolomic profiling in liver of adiponectin-knockout mice uncovers lysophospholipid metabolism as an important target of adiponectin action.

Adiponectin mediates anti-diabetic effects via increasing hepatic insulin sensitivity and direct metabolic effects. In the present study, we conducted a comprehensive and unbiased metabolomic profiling of liver tissue from AdKO (adiponectin-knockout) mice, with and without adiponectin supplementation, fed on an HFD (high-fat diet) to derive insight into the mechanisms and consequences of insulin resistance. Hepatic lipid accumulation and insulin resistance induced by the HFD were reduced by adiponectin. The HFD significantly altered levels of 147 metabolites, and bioinformatic analysis indicated that one of the most striking changes was the profile of increased lysophospholipids. These changes were largely corrected by adiponectin, at least in part via direct regulation of PLA2 (phospholipase A2) as palmitate-induced PLA2 activation was attenuated by adiponectin in primary hepatocytes. Notable decreases in several glycerolipids after the HFD were reversed by adiponectin, which also corrected elevations in several diacyglycerol and ceramide species. Our data also indicate that stimulation of ω-oxidation of fatty acids by the HFD is enhanced by adiponectin. In conclusion, this metabolomic profiling approach in AdKO mice identified important targets of adiponectin action, including PLA2, to regulate lysophospholipid metabolism and ω-oxidation of fatty acids.

Evaluation of annexin II as a potential serum marker for hepatocellular carcinoma using a developed sandwich ELISA method

Annexin II (Annexin A2, ANXA2) is a 36 kDa calcium-dependent phospholipid-binding protein that is located on the surface of most eukaryotic cells. ANXA2 is involved in several biological processes, including anti-inflammatory effects, Ca27+-dependent exocytosis, immune responses, Ca2+ transport and phospholipase A2 regulation. In our previous study, ANXA2 was identified as an up-regulated gene in hepatocellular carcinoma (HCC) tissue by cDNA microarray. In the present study, we have evaluated ANXA2 as a tumor-associated marker of HCC. We determined the ANXA2 levels in human liver tissues with HCC using real-time RT-PCR and Western blot analysis. For quantitative analysis of the ANXA2 protein in body fluids, we developed a sandwich ELISA system in which a polyclonal antibody and a monoclonal antibody specific to ANXA2 were employed as a capture antibody and a probe antibody, respectively. We detected the ANXA2 concentration in human serum using our newly developed system and evaluated its usefulness as a tumor marker. Overexpression of ANXA2 in human liver tissue was confirmed by real-time RT-PCR and Western blot analysis. The sandwich ELISA system for ANXA2 was developed for the detection of ANXA2 in human samples. The dose-response relationship between ANXA2 and optical density was linear in the range of 0-10 microg/ml and the sensitivity was 0.02 microg/ml. We determined the ANXA2 concentration in serum specimens using the newly developed sandwich ELISA. The serum ANXA2 concentrations of the patients with HCC (53.38+/-36.23 microg/ml) were significantly elevated when compared with those of normal individuals (28.81+/-24.94 microg/ml). These results suggest that expression of ANXA2 may be increased in HCC patients and may play an important role in liver cancer progression. This new ELISA method can be used as a tool for the detection of ANXA2 in human serum, particularly for cancer diagnostics.

Release of arachidonic acid induced by tumor necrosis factor-α in the presence of caspase inhibition: Evidence for a cytosolic phospholipase A2α-independent pathway

Stimulation of L929 cells with tumor necrosis factor-α (TNFα) caused cell death accompanied by a release of arachidonic acid (AA). Although the inhibition of caspases has been shown to cause necrosis in TNFα-treated L929 cells, its role in the TNFα-induced release of AA has not been elucidated. The release of AA is tightly regulated by phospholipase A2 (PLA2). To find out the mechanisms underlying the TNFα-induced release of AA, we investigated the relationship between TNFα stimulation and PLA2 regulation with and without zVAD, an inhibitor of caspases. In the present study, we found that treatment with TNFα and zVAD stimulated release of AA and cell death in C12 cells (a variant of L929 cells lacking α type of cytosolic PLA2 (cPLA2α)). Stimulation with TNFα/zVAD also caused the release of AA from L929-cPLA2α-siRNA cells. Treatment with pyrrophenone (a selective inhibitor of cPLA2α) completely inhibited the TNFα-induced release of AA, but only partially inhibited the TNFα/zVAD-induced response in L929 cells. The TNFα/zVAD-induced release of AA from C12 and L929-cPLA2α-siRNA cells was pyrrophenone-insensitive, but inhibited by treatment with butylated hydroxyanisole (BHA, an antioxidant). Treatment with dithiothreitol, which inactivates secretory PLA2 activity, decreased the amount of AA released by TNFα/zVAD. TNFα/zVAD appears to stimulate release of AA from C12 cells in a cPLA2α-independent, BHA-sensitive manner. The possible roles of secretory PLA2 and reactive oxygen species from different pools in the release of AA and cell death were discussed.

H-ras Inhibits RhoA/ROCK Leading to a Decrease in the Basal Tone in the Internal Anal Sphincter

The present studies evaluated the role of H-ras and its implications in the RhoA/Rho kinase (ROCK) pathway in regulating basal tone in the internal anal sphincter (IAS). Studies were performed in the IAS from the wild-type (H-ras(+/+)) and knock-out (H-ras(-/-)) mice. The basal tone of smooth muscle strips was measured by isometric force transducers. Length of smooth muscle cells (SMC) isolated from the IAS in the basal state was determined by phase contrast microscopy. Experiments were repeated in the presence of Y 27632, a ROCK inhibitor. Involvement of the RhoA/ROCK machinery was analyzed by reverse-transcription polymerase chain reaction, Western blot, and immunocytochemistry. Reversal of H-ras knock-out effect was evaluated by transfection of SMCs with the constitutively activated (G12V) mutant. Basal tone of the H-ras(-/-) IAS was significantly higher and resistant to relaxation by Y 27632, compared with the H-ras(+/+) IAS. Similarly, the length of SMCs from H-ras(-/-) IAS was significantly shorter. Y 27632 eliminated this difference. RhoA immunoreactivity shifted from cytoplasm to plasma membrane in H-ras(-/-) SMCs, a change typically associated with contraction. Further, SMCs from H-ras(-/-) mice exhibited higher levels of the contractile proteins ROCK II, phosphorylated-MYPT(1) and phosphorylated-MLC(20). Transfection with the G12V mutant increased the length of H-ras(-/-) cells. Conversely, the dominant negative H-ras (S17N) mutant decreased the length of H-ras(+/+) cells. H-ras negatively regulates basal tone in the IAS by inhibiting RhoA/Rho-kinase machinery. Studies may have significant relevance in the pathophysiology and therapy of certain anorectal motility disorders associated with the IAS dysfunction.

Cytosolic phospholipase A2 regulation in the hibernating thirteen-lined ground squirrel

Cytosolic calcium-dependent phospholipase A2 (cPLA2) has multiple roles including production of arachidonic acid (a key player in cellular signaling pathways) and membrane remodeling. Additionally, since catabolism of arachidonic acid generates free radicals, the enzyme is also implicated in ischemic injury to mammalian organs. Regulation of cPLA2 could be important in the suppression and prioritization of cellular pathways in animals that undergo reversible transitions into hypometabolic states. The present study examines the responses and regulation of cPLA2 in skeletal muscle and liver of hibernating thirteen-lined ground squirrels, Spermophilus tridecemlineatus. cPLA2 activity decreased significantly by 43% in liver during hibernation, compared with euthermic controls, and Km values for arachidonoyl thio-PC substrate fell in both organs during hibernation to 61% in liver and 28% in muscle of the corresponding euthermic value. To determine whether these responses were due to a change in the phosphorylation state of the enzyme, Western blotting was employed using antibodies recognizing phospho-Ser505 on α-cPLA2. The amount of phosphorylated α-cPLA2 in hibernator liver was just 38% of the value in euthermic liver. Furthermore, incubation of liver extracts under conditions that enhanced protein phosphatase action caused a greater reduction in the detectable amount of phospho-Ser505 enzyme content in euthermic, versus hibernator, extracts. The data are consistent with a suppression of cPLA2 function during torpor via enzyme dephosphorylation, an action that may contribute to the well-developed ischemia tolerance and lack of oxidative damage found in hibernating species over cycles of torpor and arousal.

The C-terminal flavin domain of gp91phox bound to plasma membranes of granulocyte-like X-CGD PLB-985 cells is sufficient to anchor cytosolic oxidase components and support NADPH oxidase-associated diaphorase activity independent of cytosolic phospholipase A2 regulation

We have previously established a model of cytosolic phospholipase A(2) (cPLA(2))-deficient PLB-985 cells and demonstrated that cPLA(2)-generated arachidonic acid (AA) is essential for reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and NADPH-dependent diaphorase activity. The present study focuses on the C-terminal cytoplasmic domain of gp91(phox) (residues 283-570), which contains the NADPH binding and flavin adenine dinucleotide-reducing center, to determine if this portion is regulated by AA. The gp91(phox) C-terminal reductase domain was expressed in X-CGD PLB-985 cells lacking normal gp91(phox) (X-CGD PLB 91CT cells) and was detected in the plasma membrane. It appears to be bound electrostatically to the plasma membrane, as it is eluted by high salt. Permeabilized, granulocyte-like X-CGD PLB 91CT cells lacking cPLA(2) protein and activity, as well as AA release after stimulation, supported NADPH-dependent diaphorase activity after stimulation, similar to granulocyte-like X-CGD PLB 91CT cells. Normal translocation of p47(phox) and p67(phox) to the membrane fractions of both stimulated cell types indicated that the gp91(phox) C-terminal region is sufficient to anchor the cytosolic oxidase components to the membranes. cPLA(2) translocated to membranes and bound the assembled oxidase in granulocyte-like X-CGD PLB 91CT cells after stimulation. Therefore, the assembled membrane-bound oxidase complex encompassing the flavin domain of gp91(phox) provides a docking site for cPLA(2) but is not the site of AA-based regulation of oxidase activity.

Adenosine Regulation of Cystic Fibrosis Transmembrane Conductance Regulator through Prostenoids in Airway Epithelia

Cystic fibrosis is caused by dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, leading to altered ion transport, chronic infection, and excessive inflammation. Here we investigated regulation of CFTR in airway cell monolayers by adenosine, adenosine receptors, and arachidonic acid. Our studies demonstrate that the A2B adenosine receptor is expressed at high levels relative to the other adenosine receptor subtypes, with a characteristic low-affinity profile for adenosine-stimulated CFTR Cl- currents in both Calu-3 cells and CFBE41o- airway cell monolayers stably transduced with wild-type CFTR. The levels of adenosine found in sputum from patients with cystic fibrosis with moderate to severe lung disease stimulated apical prostaglandin release in Calu-3 and CFBE41o- cells, implicating adenosine regulation of phospholipase A2 (PLA2) activity. A2B adenosine receptor and arachidonic acid stimulation produced CFTR-dependent currents in airway monolayers and increased cAMP levels that were sensitive to cyclooxygenase inhibition. Arachidonic acid demonstrated dual regulation of CFTR, stimulating CFTR and Cl- currents in intact airway monolayers, and potently inhibiting PKA-activated Cl- currents in excised membrane patches. Cl- currents produced by arachidonic acid were sensitive to inhibition of PKA, cyclooxygenase, and 5-lipoxygenase. Together, the results provide a converging mechanism to link regulation of CFTR and airway cell inflammation through adenosine and adenosine receptors.

Progesterone primes zona pellucida‐induced activation of phospholipase A2 during acrosomal exocytosis in guinea pig spermatozoa

We investigated, using guinea-pig spermatozoa as a model, whether phospholipase A2 (PLA2) is involved in progesterone or zona pellucida (ZP)-stimulated acrosomal exocytosis, if progesterone enhances ZP-induced activation of PLA2, and mechanisms underlying PLA2 regulation. Spermatozoa were capacitated and labeled in low Ca2+ medium with [14C]choline chloride or [14C]arachidonic acid, washed, and then exposed to millimolar Ca2+ and progesterone and/or ZP. Each agonist stimulated decrease of phosphatidylcholine (PC) and release of arachidonic acid and lysoPC, indicative of PLA2 activation. Aristolochic acid (a PLA2 inhibitor) abrogated lipid changes and exocytosis, indicating that these lipid changes are essential for exocytosis. Exposure of spermatozoa to submaximal concentrations of both progesterone and ZP resulted in a synergistic increase of arachidonic acid and lysoPC releases, and exocytosis, suggesting that, under natural conditions, both agonists interact to bring about acrosomal exocytosis. Progesterone-induced PLA2 activation appears to be mediated by a GABA(A)-like receptor, because bicuculline (a GABA(A) receptor antagonist) blocked arachidonic acid release and exocytosis. In agreement with this, GABA mimicked progesterone actions. ZP-induced activation of PLA2 seemed to be transduced via G(i) proteins because pertussis toxin blocked arachidonic acid release and acrosomal exocytosis. PLA2 may be regulated by PKC because progesterone- or ZP-induced release of arachidonic acid was blocked by the PKC inhibitors staurosporine or chelerythrine chloride. PLA2 could also be regulated by the cAMP-PKA pathway; inclusion of the PKA inhibitor 14-22 amide or H-89 led to a reduction in arachidonic acid release or exocytosis after progesterone or ZP. Taken together, these results suggest that PLA2 plays an essential role in progesterone or ZP-stimulated exocytosis with progesterone priming ZP action.

Expression and regulation of phospholipase A2 in venom gland of the chinese honeybee,Apis cerana cerana

Phospholipase A(2) (PLA(2)) is one of the components of bee venom with a wide range of pharmacological functions. It operates as a major allergen working with other venom components to defend the colony from intruder. In the present study, the cDNA sequence of the Ac-pla(2) gene from cDNA library of the venom gland of Apis cerana was compared with the amplified corresponding region of genomic DNA. The result showed that the Ac-pla(2) gene consisted of four exons and three introns. Southern blot showed that the Ac-pla(2) gene was a single copy per haploid genome. The most active transcription period was during the first 8 days of adults, which correspondingly was the period of sharp increase of PLA(2) protein. ELISA analysis revealed that the PLA(2) was undetectable in pupal stage and the newly eclosed adult, but increased sharply to a maximum of 10-12 mug per honeybee by 8-10 days of adult life, followed by a gradual decrease to 8 mug for the rest of adult life. Transcriptional or post transcriptional regulation is the key step for Ac-pla(2) expression. The early secreted Ac-PLA(2) showed a low degree of post-translational modification; with increasing age, glycosylation was detected by Western blot and glycoprotein staining analysis. Different post-translational modifications were found among different individuals in A. cerana when compared to A. mellifera.

Regulation of Phospholipase A2 by Interleulin-1 in Human Airway Smooth Muscle

Regulation of Phospholipase A2 by Interleulin-1 in Human Airway Smooth Muscle

Regulation of Phospholipase A2 by Interleulin-1 in Human Airway Smooth Muscle

Treatment of human airway smooth muscle (HASM) with interleukin (IL)-1β alone or in combination with serum or growth factors is known to induce cyclooxygenase (COX)-2 and prostaglandin E2 synthesis, an effect presumably underlying IL-1β–mediated β2 adrenergic receptor and epoprostanoid receptor desensitization and inhibition of mitogen-stimulated cell growth in HASM. Although COX-2 induction is clearly required and COX-2 activity is believed to be limiting in IL-1β-stimulated prostaglandin E2 synthesis, the role of phospholipase A2 (PLA2) in this process in unclear. We therefore assessed potential alterations in the following: (1) cytosolic PLA2 (cPLA2) protein levels, (2) cPLA2 phosphorylation state, and (3) PLA2 activity in growth-arrested HASM cultures treated with IL-1β or growth factors. Treatment with IL-1β induced a time-dependent increase in cPLA2 protein levels (assessed by immunoblotting) with a twofold increase observed at 18 h. Epidermal growth factor had only a minimal effect on cPLA2 induction, but cotreatment with IL-1β plus endothelial growth factor was synergistic, resulting in a greater than fourfold induction. cPLA2 induction was partially inhibited by the p38 inhibitor SB203580 and the classical protein kinase C inhibitor Bis I. IL-1β induced a rapid phosphorylation of cPLA2, which peaked at approximately 1 h and then waned toward basal levels by 18 h. Lastly, PLA2 activity in cultured HASM, characterized by cleavage of a phospholipid analog conjugated to a BODIPY fluorophore (D3803; Molecular Probes; Eugene, OR), was regulated by IL-1β but occurred against a background of high basal PLA2 activity in cell culture medium, suggesting an important role for secretory PLA2 in HASM arachidonate metabolism. These data suggest that regulation of HASM PLA2 activity may be important in cytokine- and growth factor-mediated eicosanoid synthesis and associated functional consequences.

P38 MAPK regulates group IIa phospholipase A2 expression in interleukin-1beta -stimulated rat neonatal cardiomyocytes.

Group IIa phospholipase A(2) (GIIa PLA(2)) is released by some cells in response to interleukin-1beta. The purpose of this study was to determine whether interleukin-1beta would stimulate the synthesis and release of GIIa PLA(2) from cardiomyocytes, and to define the role of p38 MAPK and cytosolic PLA(2) in the regulation of this process. Whereas GIIa PLA(2) mRNA was not identified in untreated cells, exposure to interleukin-1beta resulted in the sustained expression of GIIa PLA(2) mRNA. Interleukin-1beta also stimulated a progressive increase in cellular and extracellular GIIa PLA(2) protein levels and increased extracellular PLA(2) activity 70-fold. In addition, interleukin-1beta stimulated the p38 MAPK-dependent activation of the downstream MAPK-activated protein kinase, MAPKAP-K2. Treatment with the p38 MAPK inhibitor, SB202190, decreased interleukin-1beta stimulated MAPKAP-K2 activity, GIIa PLA(2) mRNA expression, GIIa PLA(2) protein synthesis, and the release of extracellular PLA(2) activity. Infection with an adenovirus encoding a constitutively active form of MKK6, MKK6(Glu), which selectively phosphorylates p38 MAPK, induced cellular GIIa PLA(2) protein synthesis and the release of GIIa PLA(2) and increased extracellular PLA(2) activity 3-fold. In contrast, infection with an adenovirus encoding a phosphorylation-resistant MKK6, MKK6(A), did not result in GIIa PLA(2) protein synthesis or release by unstimulated cardiomyocytes. In addition, infection with an adenovirus encoding MKK6(A) abrogated GIIa PLA(2) protein synthesis and release by interleukin-1beta-stimulated cells. These results provide direct evidence that p38 MAPK activation was necessary for interleukin-1beta-induced synthesis and release of GIIa PLA(2) by cardiomyocytes.

Differential Regulation of Phospholipase A2(PLA2)-dependent Ca2+ Signaling in Smooth Muscle by cAMP- and cGMP-dependent Protein Kinases: INHIBITORY PHOSPHORYLATION OF PLA2 BY CYCLIC NUCLEOTIDE-DEPENDENT PROTEIN KINASES

Both cAMP- and cGMP-dependent protein kinases inhibit agonist-stimulated phospholipase C-beta (PLC-beta) activity and inositol 1,4,5-trisphosphate-dependent Ca2+ release in vascular and visceral smooth muscle. In smooth muscle of the intestinal longitudinal layer, however, the initial steps in Ca2+ mobilization involve activation of cytosolic PLA2 (cPLA2) and arachidonic acid (AA)-dependent stimulation of Ca2+ influx. The present study examined whether cAMP- and cGMP-dependent protein kinases are capable of regulating these processes also. Agents that activated cAMP-dependent protein kinase (5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole 3',5'-cyclic monophosphothioate (Sp-isomer) and isoproterenol), cGMP-dependent protein kinase (8-(4-chlorophenylthio)-guanosine 3',5'-cyclic monophosphate and Na nitroprusside), or both kinases (vasoactive intestinal peptide and isoproterenol >1 microM) induced phosphorylation of cPLA2 and inhibition of agonist-stimulated cPLA2 activity. Phosphorylation and inhibition of cPLA2 activity by cAMP- and cGMP-dependent protein kinases were blocked by the corresponding selective inhibitors (cAMP-dependent protein kinase, N-[2(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide hydrochloride (H-89) and myristoylated protein kinase inhibitor () amide; cGMP-dependent protein kinase, (8R,9S, 11S)-(-)-9-methoxy-carbamyl-8-methyl-2,3,9,10-tetrahydro-8, 11-epoxy-1H,8H,11H,-2,7b,11a-trizadizobenzo(a,g)cycloocta(c, d, e)-trinden-1-one (KT-5823)). In contrast, AA-stimulated Ca2+ influx was inhibited by agents that activated cGMP-dependent protein kinase only; the inhibition was selectively blocked by KT-5823. The study provides the first evidence of inhibitory phosphorylation of cPLA2 in vivo by cAMP- and cGMP-dependent protein kinases. Inhibition of cPLA2 activity and AA-induced Ca2+ influx partly account for the ability of cAMP-dependent protein kinase and/or cGMP-dependent protein kinase to cause relaxation. Their importance resides in their location at the inception of the Ca2+ signaling cascade.

Antigen activation of mitogen-activated protein kinase in mast cells through protein kinase C-dependent and independent pathways.

We demonstrate discrete pathways for activation of mitogen-activated protein (MAP) kinase in cultured RBL-2H3 mast cells through protein kinase C (PKC), cytosolic calcium, and a third pathway that provides sustained signals for activation in Ag-stimulated cells. Thus, p42 MAP kinase was activated by increasing intracellular free Ca2+ with thapsigargin or by stimulating PKC with PMA. The latter stimulation was selectively blocked by the protein kinase C inhibitor, Ro31-7549. Stimulation of p42 MAP kinase by Ag resulted in relatively sustained activation of MAP kinase which was only partially suppressed by Ro31-7549. Kinetic studies revealed two components of the MAP kinase response to Ag: a rapid but transient component that was Ro31-7549 sensitive and presumably PKC dependent; and a more sustained component that was Ro31-7549 resistant and presumably PKC independent. Similarly, Ro31-7549 inhibited the early but not late release of arachidonic acid, a finding that was consistent with the known regulation of phospholipase A2 by MAP kinase. Early tyrosine phosphorylation events which were thought to be essential for Ag-induced activation of p42 MAP kinase and release of arachidonic acid were unaffected by Ro31-7549. The findings suggested that release of arachidonic acid was regulated primarily through MAP kinase but that PKC may transiently influence this release, either directly or indirectly through MAP kinase.

Dual regulation of PLA2 and PGI2 production by G proteins in bovine aortic endothelial cells.

NaF, a nonselective activator of heterotrimeric guanine nucleotide-binding proteins (G proteins), increased the release of arachidonic acid (AA) and prostacyclin (PGI2) production in bovine aortic endothelial cells (BAEC) at low concentrations (40-60 mM). On the other hand, higher concentrations (100 mM) inhibited phospholipase A2 (PLA2) compared with the basal activity. Intracellular Ca2+ levels did not rise after treatment with stimulatory concentrations of NaF, and, moreover, neither neomycin nor Ca(2+)-free medium affected the biphasic pattern of PGI2 synthesis in response to NaF. CGP-43187, an inhibitor of the 14-kDa secretory PLA2, did not affect NaF-induced AA release. However, AACOCF3, a specific inhibitor of the cytosolic 85-kDa PLA2 (cPLA2), abrogated AA release and PGI2 production in response to 60 mM NaF. A biphasic pattern of PGI2 production was also obtained with the guanosine 5'-triphosphate analogues guanosine 5'-O-(3-thiotriphosphate) and guanylylimidodiphosphate in permeabilized BAEC. Pretreatment of the cells with guanosine 5'-O-(2-thiodiphosphate) suppressed the inhibition and the stimulation of AA release induced by guanylylimidodiphosphate. In addition, phenylisopropyl adenosine inhibited the release of AA and PGI2, whereas ATP and bradykinin increased PGI2. Pertussis toxin not only inhibited ATP- and bradykinin-stimulated PGI2 release, it also reversed the inhibitory effect of phenylisopropyl adenosine, resulting in a significant stimulation. These findings strongly suggest that, in BAEC, cPLA2 is coupled with more than one G protein that are involved in inhibition and stimulation of cPLA2 activity.

The Regulation of Phospholipase-A2 (PLA-2) by Cytokines Expressing Hematopoietic Growth-Stimulating Properties

Various growth factors released by macrophages and other cell types modulate normal hematopoiesis. The physiological mechanisms whereby these molecules interact with specific target cells are ill defined. Eicosanoids, the products of fatty acid metabolism, are known to regulate cell proliferation and differentiation. The release of membrane-bound phospholipid by phospholipase-A2 (PLA-2) is the first critical step in the initiation of membrane remodeling and eventually eicosanoid synthesis. We report here data that demonstrates how various cytokines exhibit a marked hydrolytic activity mediated through PLA-2 against both [1-14C] oleic acid- and [1-14C] arachidonic acid-labeled Escherichia coli (micelle) substrates. PLA-2 extracts were prepared from neutrophils elicited by injecting rats ip with 8% glycogen. The rate of hydrolysis of free fatty acids from the phospholipid substrate was found to be linear, rapid, and pH dependent and was calculated to be 30 nmoles of phospholipid/hr/mg protein lysate. Cytokines (i.e., interleukin-1 [IL-1, human and murine recombinant, alpha], mouse lung cell-derived colony-stimulating factor [L-CSF], granulocyte-macrophage colony-stimulating factor [murine recombinant GM-CSF], tumor necrosis factor [murine recombinant TNF-alpha], and granulocyte colony-stimulating factor [human recombinant, G-CSF] all induced PLA-2 activity with the release of free fatty acids above basal levels. In contrast, lipopolysaccharide (LPS), interleukin-2, (IL-2, human recombinant), and macrophage colony-stimulating factor (M-CSF) did not significantly activate PLA-2 hydrolysis. The activation of this membrane-bound enzyme-substrate complex by these growth factors may serve as a mechanism whereby the appropriate target cells expressing receptors respond through either direct or secondary signals leading to the formation of free fatty acids with the eventual synthesis of prostanoid or lipoxygenase products, resulting in cellular proliferation and differentiation.

Phospholipase A2 is secreted by murine keratinocytes after stimulation with IL-1 alpha and TNF-alpha.

Phospholipase A2 (PLA2)-catalysed liberation of arachidonic acid is the rate-limiting step in the generation of the lipid mediators prostaglandins and leukotrienes. PLA2 regulation thus represents a pivotal mechanism in the pathogenesis of inflammation. In this study we investigated the effects of TNF alpha and IL-1 alpha on PLA2 activity in cultured murine keratinocytes. Starting 18 h after stimulation, PLA2 activity increased significantly by about 250-320%) in the supernatants and in the cell pellets. This effect was completely inhibited either by preincubation of the cells with dexamethasone 48 h before stimulation or by coincubation with actinomycin D. PLA2 activity detected in the supernatants was blocked by reduction with dithiothreitol, whereas the PLA2 activity in the pellets was dithiothreitol-resistant. We conclude that in murine keratinocytes IL-1 alpha induce de novo synthesis and release of a secretory PLA2 and the induction of a different PLA2 activity in the cytosol. These findings indicate a crucial link between early cytokine effects and the initiation of the lipid mediator cascade in keratinocytes. The observation that PLA2 induction could be completely inhibited by preincubation with dexamethasone allows new insights into the mechanism of steroid effects on epidermal inflammation and renders PLA2 regulation an interesting therapeutic target.

SYMPOSIUM: Experimental Biology 1995 Role of Mesangial Cell Ion Transport in Glomerular Physiology and Disease: REGULATION OF RAT KIDNEY MESANGIAL CELL PHOSPHOLIPASE A2

1. The precursor of eicosanoids is arachidonic acid, which emanates from the cleavage of the sn-2 position of phospholipids by phospholipase A2 (PLA2). Eicosanoids have diverse physiological and pathophysiological effects in the kidney. The regulation of phospholipase A2 has important implications for kidney function. 2. In the current communication we focus our attention on mesangial cell cytosolic PLA2 (cPLA2) and its regulation at the post-translational and post-transcriptional level. 3. At the post-translational level, using site directed mutagenesis of cPLA2 and a dominant negative ras, we have demonstrated that cPLA2 can be phosphorylated by mitogen activated protein (MAP-2) kinase leading to increased cPLA2 enzymatic activity. 4. At the post-transcriptional level we show that the half-life of cPLA2 mRNA in mesangial cells is significantly increased when mesangial cells are stimulated by mitogens. We further demonstrate the presence of three ATTTA motifs in the 3' untranslated region (3' UTR) of the cPLA2 cDNA. 5. Using chimeric constructs bearing the 3' UTR from rat cPLA2 fused downstream of the luciferase reporter, we demonstrate that this region exerts a destabilizing effect on cPLA2. 6. We have isolated and mapped genomic DNA and polymorphic markers for cPLA2 in the human and rat.