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PubMed1982

Isolation and preliminary characterisation of two toxic phospholipases A2 from the venom of the Malayan cobra (Naja naja sputatrix).

Two toxic phospholipases A have been isolated from the venom of the Malayan cobra (Naja naja sputatrix). The phospholipases A were purified by successive ion-exchange chromatography on SP-Sephadex C-25, Sephadex G-75 gel filtration chromatography and successive Bio-Rex 70 ion-exchange chromatography. The purified toxic phospholipases A were homogeneous electrophoretically. They were designated as sputatrix phospholipase A-I and sputatrix phospholipase A-II. Positional specificity studies showed that they belong to the A2-type phospholipase A. The medium lethal dose 50% (LD50) values of the two phospholipases A are 0.27 and 0.28 microgram/g, respectively, by intravenous injection and 1.05 and 1.00 microgram/g, respectively, by intraperitoneal injection. The molecular weights of the two enzymes are 14000 as determined by gel-filtration chromatography and SDS-polyacrylamide gel electrophoresis. Amino acid composition of sputatrix phospholipase A-I differs from sputatrix phospholipase A-II only by having one extra amino acid: a glutamic acid. Amino acid compositions of the two enzymes are also similar to those of other cobra venom phospholipases A.

Amino Acids↗

PubMed2004

Brain phospholipases A2: a perspective on the history.

The phospholipases A2 (PLA2) belong to a large family of enzymes involved in the generation of several second messengers that play an important role in signal transduction processes associated with normal brain function. The phospholipase A2 family includes secretory phospholipase A2, cytosolic phospholipase A2, calcium-independent phospholipase A2, plasmalogen-selective phospholipase A2 and many other enzymes with phospholipase A2 activity that have not been classified. Few attempts have been made purify and characterize the multiple forms of PLA2 and none have been fully characterized and cloned from brain tissue. A tight regulation of phospholipase A2 isozymes is necessary for maintaining physiological levels of free fatty acids including arachidonic acid and its metabolites in the various types of neural cells. Under normal conditions, phospholipase A2 isozymes may be involved in neurotransmitter release, long-term potentiation, growth and differentiation, and membrane repair. Under pathological conditions, high levels of lipid metabolites generated by phospholipase A2 are involved in neuroinflammation, oxidative stress, and neural cell injury.

Animals↗

PubMed1996

Relation between accumulation of phospholipase A2 reaction products and Ca2+ release in isolated liver mitochondria.

A Ca(2+)-dependent stimulation of mitochondrial phospholipase A2 is often assumed to play a role in mitochondrial Ca2+ release. We sought to clarify this relation by measuring Ca2+ transport and determining phospholipase A2 reaction products from the same sample of isolated, incubated rat liver mitochondria. When mitochondria had accumulated and spontaneously released again Ca2+, most probably by membrane permeability transition, there was no increase of phospholipase A2 reaction products. However, when the incubation was continued after Ca2+ release, significant increases of the content of lysophosphatidylcholine and unesterified fatty acids could be seen. Quinacrine, an inhibitor of phospholipase A2 activity, prevented Ca2+ release and p-hydroxymercuribenzoic acid, an inhibitor of lysophospholipid reesterification, induced a fast release of Ca2+ from isolated mitochondria. Such effects are usually taken as indirect evidence for a participation of phospholipase A2 in mitochondrial Ca2+ release, but analysis of the mitochondrial lipids revealed that no significant changes of the mass of phospholipase A2 reaction products had occurred. These experiments suggest that the accumulation of phospholipase A2 reaction products in mitochondria is the consequence rather than the cause of the membrane permeability transition. Exogenous phospholipase A2 products, lysophosphatidylcholine and arachidonic acid, induced mitochondrial Ca2+ release after a time lag, which decreased with aging of the mitochondrial preparation. The amount of lysophosphatidylcholine taken up by the mitochondria from the incubation medium during these experiments was measured and compared to the amount of lysophosphatidylcholine produced endogenously by mitochondrial phospholipase A2. From these data it appears likely that the amount of lysophosphatidylcholine generated in the mitochondria after the permeability transition is sufficient to sustain the permeable state. An accumulation of mitochondrially generated phospholipase A2 reaction products after the permeability transition could thus be a decisive factor for the limited reversibility of the membrane permeability transition.

Animals↗

PubMed1997

Suppression of cytokine synthesis, integrin expression and chronic inflammation by inhibitors of cytosolic phospholipase A2.

To define the isoform of phospholipases A2 active in inflammation we evaluated the effects of low-molecular-weight inhibitors of secretory and cytosolic phospholipases A2. We found that inhibitors of cytosolic phospholipase A2 had therapeutic efficacy in an in vivo model of chronic inflammation (rat adjuvant arthritis), whereas inhibitors of secretory phospholipase A2 had no beneficial effect. In vitro, inhibitors of cytosolic phospholipase A2 diminished surface expression of Mac-1 (CD11b/CD18) beta2-integrin on calcium ionophore-stimulated human blood granulocytes and suppressed synthesis of interleukin-1beta in lipopolysaccharide-stimulated human blood monocytes and U937 cells by reducing mRNA levels. Lipid mediators promote Mac-1 exocytosis and transcription of interleukin-1beta, which further enhances cytosolic phospholipase A2 activity and expression. Thus, superinduction of cytosolic phospholipase A2 may establish a positive feedback loop, converting acute inflammation into chronic inflammation. Consequently, inhibitors of cytosolic phospholipase A2 may prevent inflammation in vivo by interfering with cellular activation and infiltration. We conclude that cytosolic phospholipase A2 but not secretory phospholipase A2 is the predominant enzyme in inflammatory signalling.

Animals↗

PubMed1995

Type I skin reactivity to native and recombinant phospholipase A2 from honeybee venom is similar.

Phospholipase A2 is the major allergen in honeybee venom. Recombinant phospholipase A2 was produced in prokaryotes and tested for its biologic activity by intracutaneous skin testing with serial 10-fold dilutions in comparison with native and deglycosylated phospholipase A2 in patients allergic to bee venom. Linear regressions of the log of the wheal area versus the log of the allergen concentration were calculated for all allergens in each patient. The relative allergenic potency of the various preparations was analyzed by comparing the linear regressions. Native phospholipase A2 was about 10 times more potent than whole bee venom. None of 58 patients allergic to bee venom was missed by testing with native phospholipase A2 alone. This allergen and deglycosylated native phospholipase A2 resulted in similar skin reactions, indicating that the sugar residues were of little relevance for IgE-binding in the patients tested. Native phospholipase A2 also had relative potency similar to that of recombinant refolded phospholipase A2, whereas recombinant nonrefolded phospholipase A2 had almost no biologic activity in skin testing. These results demonstrate in vivo activity of the recombinant bee venom allergen phospholipase A2. Although correct refolding is a prerequisite for type I skin reactivity, glycosylation seems to be less important.

Adult↗

PubMed2002

Regulation of phospholipase C-gamma activity by glycosphingolipids.

Glycosphingolipid-enriched domains are hot spots for cell signaling within plasma membranes and are characterized by the enrichment of glycosphingolipids. A role for glucosylceramide-based glycosphingolipids in phospholipase C-mediated inositol 1,4,5-trisphosphate formation has been previously documented. These earlier studies utilized a first generation glucosylceramide synthase inhibitor to deplete cells of their glycosphingolipids. Recently, more active and specific glucosylceramide synthase inhibitors, including d-threo-ethylendioxyphenyl-2-palmitoylamino-3-pyrrolidinopropanol (d-t-EtDO-P4), have been designed. d-t-EtDO-P4 has the advantage of blocking glucosylceramide synthase at low nanomolar concentrations but does not cause secondary elevations in cell ceramide levels. In the present study, d-t-EtDO-P4 depleted cellular glucosylceramide and lactosylceramide in cultured ECV304 cells at nanomolar concentrations without obvious cellular toxicity. The expression of several signaling proteins was evaluated in glycosphingolipid-depleted ECV304 cells to study the role of glycosphingolipids in phospholipase C-mediated signaling. No difference was observed in the cellular expression of phospholipase C-gamma between controls and glycolipid-depleted cells. Western blot analysis, however, revealed that depletion of endogenous glycosphingolipids in cultured ECV304 cells with d-t-EtDO-P4 induced tyrosine phosphorylation of phospholipase C-gamma in a concentration-dependent manner with maximum induction at 100 nm. The phosphorylation of phospholipase C-gamma induced by d-t-EtDO-P4 was abolished by exogenously added glucosylceramide, consistent with a specific glycosphingolipid-phospholipase C-gamma interaction. The phospholipase C-gamma phosphorylation was maximally enhanced by bradykinin when cells were exposed to 100 nm d-t-EtDO-P4. The measurement of cellular activity of phospholipase C-gamma, by myo-inositol 1,4,5-trisphosphate radioreceptor assay, demonstrated that depletion of glucosylceramide-based glycosphingolipids in cultured ECV304 cells with d-t-EtDO-P4 resulted in significantly increased formation of inositol 1,4,5-trisphosphate above base line, and an increased sensitivity of phospholipase C-gamma to bradykinin stimulation. Thus, the activation of phospholipase C-gamma is negatively regulated by membrane glycosphingolipids in ECV304 cells.

Blotting, Western↗

PubMed1997

Fc epsilon RI-ligation induces association of tyrosine phosphorylated proteins with Src homology 2 domains of phospholipase C gamma 1 in RBL-2H3 rat basophilic leukemia cells.

Stimulation of the IgE receptors on mast cells and basophils activates protein tyrosine kinases and phospholipases leading to histamine release. However, the mechanism by which protein tyrosine kinases regulate the phospholipases is not clearly defined yet. In this study, we examined the possibility that phospholipase C gamma 1 associates with protein tyrosine kinases and tyrosine phosphorylated molecules as a result of activation of RBL-2H3 cells, and that this association involves the Src homology 2 domains of phospholipase C gamma 1. An increase in cytoplasmic Ca2+ level and tyrosine phosphorylations of proteins, including 72 and 40 kDa proteins, were observed after the cross-linking of the IgE receptors on RBL-2H3 rat basophilic cells by dinitrophenyl-specific IgE and dinitrophenyl-conjugated human serum albumin. Immunoprecipitation and coprecipitation experiments were performed to determine if the activation of protein tyrosine kinases is linked to the activation of phospholipase C gamma 1 via its SH2 domains. Tyrosine phosphorylation of phospholipase C gamma 1 was observed in 1 min following IgE receptor stimulation. several proteins (72, 50, 40, and 33 kDa) were identified to be tyrosine phosphorylated and specifically associated with phospholipase C gamma 1 by its Src homology 2 domains. In addition, the coprecipitated complex contains the tyrosine kinase activity which phosphorylates 72, 40, and 33 kDa proteins in the complex. In conclusion, these studies establish that tyrosine-phosphorylated proteins of 72, 40, and 33 kDa associate with phospholipase C gamma 1 via its SH2 domains following IgE receptor stimulation of RBL-2H3 basophilic cells, implying that protein tyrosine kinases may tyrosine-phosphorylate and recruit signaling proteins around the phospholipase C gamma 1 and that phospholipase C gamma 1 activation induces calcium mobilization, PKC activation and degranulation in mast cells or basophils.

Animals↗

PubMed2005

Role of nitric oxide in regulating secreted phospholipase A2 release from astrocytes.

Inflammatory stimuli such as lipopolysaccharide increase nitric oxide and secreted phospholipase A2 release from glial cells. However, the signaling mechanism(s) regulating secreted phospholipase A2 in glial cells is not known. Here, rat brain astrocytes treated with lipopolysaccharide generated nitrite and released secreted phospholipase A2, while microglia generated nitrite without releasing secreted phospholipase A2. Unexpectedly, attenuation of nitrite production by pretreatment with the nitric oxide synthase inhibitor N-Omega-nitro-L-arginine methyl ester greatly enhanced lipopolysaccharide-stimulated secreted phospholipase A2 release from astrocytes; postreatment with N-Omega-nitro-L-arginine methyl ester did not potentiate secreted phospholipase A2 release, and addition of an nitric oxide donor attenuated the secreted phospholipase A2 release. The regulation of secreted phospholipase A2 may act via the transcription factor nuclear factor-kappaB, as a nuclear factor-kappaB inhibitor attenuated lipopolysaccharide-stimulated secreted phospholipase A2 release. These results demonstrate the role of basal nitric oxide levels as a regulator of inflammatory secreted phospholipase A2 release from glial cells of the brain.

Analysis of Variance↗

PubMed1993

Differential effects of B2 receptor antagonists upon bradykinin-stimulated phospholipase C and D in guinea-pig cultured tracheal smooth muscle.

1. Guinea-pig tracheal smooth muscle cells were isolated and maintained in culture for 14-21 days prior to the study of the effect of a selective bradykinin B1 agonist and B2 antagonists upon bradykinin-stimulated phospholipase C and D activities. 2. Bradykinin-stimulated phospholipase C activity was determined by mass measurement of inositol (1,4,5)trisphosphate (Ins(1,4,5)P3) in unlabelled cells, whereas phospholipase D activity was assayed by the accumulation of [3H]-phosphatidylbutanol ([3H]-PtdBut) in [3H]-palmitate-labelled cells, which were stimulated in the presence of butan-1-o1 (0.3%, v/v). 3. Bradykinin elicited the rapid and transient formation of Ins(1,4,5)P3, in a concentration-dependent manner (log EC50 = -7.55 +/- 0.1 M, N = 3). Bradykinin also rapidly activated the concentration-dependent (log EC50 = -8.3 +/- 0.4 M, n = 3) phospholipase D-catalysed accumulation of [3H]-PtdBut; the accumulation of [3H]-PtdBut was sustained. These effects were not inhibited by pretreatment of the cells with indomethacin (1 microM). 4. The bradykinin B1 agonist, desArg9-bradykinin (1 microM) was without effect upon phospholipase C or phospholipase D activity. Bradykinin-stimulated (10 nM, EC40) Ins(1,4,5)P3 formation was inhibited by B2 receptor antagonists, D-Arg-[Hyp3,D-Phe7]-bradykinin (NPC 567) and D-Arg-[Hyp3,Thi5,8,D-Phe7]-bradykinin (NPC 349), with log IC50 values of -6.3 +/- 0.5 M and -6.3 +/- 0.4 M, respectively. However, bradykinin-stimulated (10 nM, EC100) [3H]-PtdBut accumulation was poorly inhibited and with low potency by each B2 receptor antagonist and bradykinin-stimulated phospholipase D activity persisted at concentrations of antagonist that completely blocked bradykinin-stimulated Ins(1,4,5)P3 formation (30 microM). 5. These observations suggest that the activation of phospholipase C by bradykinin may be mediated through a bradykinin B2 receptor population, whereas bradykinin-stimulated phospholipase D may be activated via a distinct population of bradykinin receptors that do not appear to be either B1 or B2 receptor types, based upon pharmacological specificity. The mechanism of the activation of phospholipase D by bradykinin and the role of the putative B3 bradykinin receptor are discussed.

Animals↗

PubMed1992

Raised serum activity of phospholipase A2 immunochemically related to group II enzyme in inflammatory bowel disease: its correlation with disease activity of Crohn's disease and ulcerative colitis.

Calcium dependent phospholipase A2 activity in the mixed micelles of 1-palmitoyl-2-oleoyl-phosphatidylglycerol and cholate was measured in sera of 39 patients with Crohn's disease, 40 patients with ulcerative colitis, and 40 healthy controls. The phospholipase A2 activity was significantly raised in those sera of the patients with active Crohn's disease and those with moderate and severe ulcerative colitis. The major phospholipase A2 activity derived from the sera was separated into two peaks by reverse phase high performance liquid chromatography. The phospholipase A2 active fractions were immunochemically characterised using specific antibody directed against human group II phospholipase A2 purified from rheumatoid synovial fluid. The results suggest that raised serum phospholipase A2 activity in patients with Crohn's disease and ulcerative colitis was mainly attributed to the two forms of phospholipase A2 immunochemically related to group II enzyme. In patients with Crohn's disease, serum phospholipase A2 activity decreased in parallel with clinical improvement, and correlated with serum C-reactive protein and erythrocyte sedimentation rate. The results suggest that serum phospholipase A2 activity may serve as an additional indicator of disease activity. Serum phospholipase A2 activity in patients with ulcerative colitis tends to increase in relation with endoscopic severity, and may be a more sensitive laboratory index than serum C-reactive protein and erythrocyte sedimentation rate to evaluate disease activity.

Acute Disease↗

PubMed1994

Sequential activation of phospholipase-C and -D in agonist-stimulated gonadotrophs.

The contributions of phospholipase-C and -D to diacylglycerol (DG) formation during agonist-induced cell signaling were investigated in rat pituitary cells and alpha T3-1 gonadotrophs. In both cell types, GnRH caused a biphasic increase in DG formation, with an initial spike within 60 sec, followed by a larger and sustained rise to reach a second peak after 15 min of stimulation. Both phases of DG production were temporally correlated with inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] formation, consistent with the dependence of DG formation on phospholipase-C-mediated phosphoinositide hydrolysis. However, the ability of GnRH to stimulate phosphatidylethanol (PEt) in the presence of ethanol suggested that phospholipase-D may also participate in DG formation. Two inhibitors of phospholipase-C-dependent phosphoinositide hydrolysis, U73122 and neomycin sulfate, reduced the PEt as well as the Ins(1,4,5)P3 response to GnRH, indicating that phospholipase-D is activated during phospholipase-C-dependent signaling in pituitary gonadotrophs. The production of both DG and PEt was increased by treatment with the active phorbol ester phorbol 12-myristate 13-acetate (PMA), but not with inactive 4 alpha-phorbol 13-didecanoate, indicating that stimulation of protein kinase-C leads to activation of phospholipase-D. In accord with this, GnRH- and PMA-induced elevations of DG and PEt production were attenuated or abolished in protein kinase-C-depleted cells. In contrast, short and long term stimulation with PMA had no effect on basal inositol phosphate production. Also, GnRH-induced inositol phosphate production was not affected by protein kinase-C depletion. Finally, U73122 and neomycin sulfate did not inhibit PMA-induced PEt formation. These data indicate that GnRH activates a dual phospholipase pathway in a sequential and synchronized manner; phospholipase-C initiates the biphasic increase in Ins(1,4,5)P3 and DG formation, and protein kinase-C mediates the integration of phospholipase-D into the signaling response during the sustained phase of agonist stimulation.

Animals↗

PubMed2003

CaM kinase IIalpha mediates norepinephrine-induced translocation of cytosolic phospholipase A2 to the nuclear envelope.

Several growth factors, hormones and neurotransmitters, including norepinephrine, increase cellular calcium levels, promoting the translocation of cytosolic phospholipase A(2) to the nuclear envelope. This study was conducted to investigate the contributions of the calcium-binding protein calmodulin and of calcium-calmodulin-dependent protein kinase II to cytosolic phospholipase A(2) translocation to the nuclear envelope elicited by norepinephrine in rabbit aortic smooth-muscle cells. Norepinephrine caused cytosolic phospholipase A(2) accumulation around the nuclear envelope as determined from its immunofluorescence; cytosolic phospholipase A(2) translocation was blocked by inhibitors of calmodulin and calcium-calmodulin-dependent protein kinase II or calcium-calmodulin-dependent protein kinase IIalpha antisense oligonucleotide. Calmodulin and calcium-calmodulin-dependent protein kinase II inhibitors did not prevent cytosolic calcium increase but attenuated cytosolic phospholipase A(2) phosphorylation caused by norepinephrine or ionomycin. In vascular smooth-muscle cells reversibly permeabilized with beta-escin and treated with alkaline phosphatase, norepinephrine failed to cause cytosolic phospholipase A(2) phosphorylation and translocation to the nuclear envelope; these effects of norepinephrine were minimized by the phosphatase inhibitor okadaic acid. Recombinant cytosolic phospholipase A(2) phosphorylated by purified calcium-calmodulin-dependent protein kinase II, but not unphosphorylated or dephosphorylated cytosolic phospholipase A(2), introduced into permeabilized vascular smooth-muscle cells in the absence of calcium accumulated around the nuclear envelope. These data suggest that norepinephrine-induced translocation of cytosolic phospholipase A(2) to the nuclear envelope is mediated by its phosphorylation by calcium-calmodulin-dependent protein kinase II and that calcium alone is insufficient for cytosolic phospholipase A(2) translocation to the nuclear envelope in rabbit vascular smooth-muscle cells.

Active Transport, Cell Nucleus↗

PubMed1995

Serum phospholipases A2 after aortobifemoral reconstruction.

Phospholipase A2 has been implicated in the pathogenesis of local and distant tissue injury after ischaemia and reperfusion. A common operation inducing ischaemia and reperfusion is aortobifemoral reconstruction, during which the aorta is clamped and the blood supply via the inferior mesenteric artery and iliac arteries is interrupted. The purpose of the present work was to study the catalytic activity concentration of phospholipase A2 and the mass concentrations of group I and group II phospholipases A2 in the sera of patients undergoing aortobifemoral reconstruction. Both the catalytic activity concentration of phospholipase A2 and the mass concentrations of group I and group II phospholipases A2 increased in serum samples after the operation. The catalytic activity concentration of phospholipase A2 correlated well with group II phospholipase A2 mass values (r = 0.81, p < 0.001), whereas no correlation was found between the catalytic activity concentration of phospholipase A2 and group I phospholipase A2 mass values (r = 0.12, p = 0.54). The results emphasize the role of group II phospholipase A2 in tissue injury after ischaemia and reperfusion.

Aged↗

PubMed1989

[Effect of diglyceride on phospholipase A2 activities in plasma membrane of guinea pig peritoneal macrophases].

The AA release from phospholipids is believed to be controlled by the activation of phospholipase A2 which are probably localized in the plasma membranes. And the considerable interest has been focused on the study of cells of the immune system including macrophages which metabolize arachidonic acid (AA) to prostaglandins, thromboxanes and leukotriens. Recently, it has been reported that phosphatidylinositol (PI) turnover plays an important role in the response to external stimuli in these cells. PI turnover has been accepted as being initiated by the hydrolysis of PI which is catalyzed by the phospholipase C and produced AA. Recently, we demonstrated that PI-specific phospholipase A2 in macrophage plasma membrane directly acted for AA release from PI. On the other hand, diglyceride, which is produced from PI by phospholipase C activation, is known to regulate the several cell functions with a protein kinase C activation. In this report, we examined the effect of diglyceride on phospholipase A2 activities in macrophage plasma membrane. PI-phospholipase A2 and PC-phospholipase A2 activities were stimulated by the addition of diglyceride. In contrast, PE-phospholipase A2 was not affected by diglyceride addition. The results suggested that phospholipase A2 activation was occurred with coupling the diglyceride production via the phospholipase C pathway.

Arachidonic Acid↗

PubMed1997

Serum phospholipase A2, amylase, lipase, and urinary amylase activities in relation to the severity of acute pancreatitis.

OBJECTIVE: To compare serum phospholipase A2 activity with measurements of conventional enzymes as an indicator of the severity of acute pancreatitis. DESIGN: Prospective study. SETTING: University hospital, Finland. SUBJECTS: 80 Consecutive patients with acute pancreatitis. INTERVENTIONS: Serum and urine samples were taken daily for a week after admission. MAIN OUTCOME MEASURES: Serum phospholipase A2, amylase, lipase, and urinary amylase activities. RESULTS: On admission, the serum amylase and lipase activities increased in parallel in all patients. However, the mean serum phospholipase A2 activity was three times higher in the patients with acute fulminant pancreatitis than in those with milder disease. The phospholipase A2 activity remained high during the course of the severe disease, whereas the other enzyme activities decreased appreciably during the first week. In contrast to the other enzyme activities that of serum phospholipase A2 correlated well with the severity of the acute pancreatitis. Heating at 60 degrees C for 45 minutes to inactivate the non-pancreatic thermolabile phospholipase A2 reduced the total serum phospholipase A2 activity more than the enzyme activity in the homogenates of pancreatic tissue, which suggests that extrapancreatic phospholipase A2 is present in serum. The receiver operating characteristic (ROC) curves confirmed the high sensitivity and specificity of serum phospholipase A2 activity with a mean (SEM) area under the curve up to 0.870 (0.062) compared with the other enzyme activities of which the highest area under the curve was 0.52 (0.089). CONCLUSIONS: In contrast to amylase and lipase activities, measurement of serum phospholipase A2 activity is important in the assessment of the severity of acute pancreatitis so that optimal treatment may be given.

Acute Disease↗

PubMed1985

Phospholipase-A1 and -A2 in experimental acute pancreatitis in rats.

Using a standardized model of bile-induced acute pancreatitis, the reaction of phospholipase-A1 activity was investigated in parallel to that of phospholipase-A2, as well as their relationship to the pathomorphological spread of pancreatitis. While the measurement of the total free fatty acids (FFA) in serum as metabolites of phospholipase-A activity indicated variable reactions with the average remaining the same (0.186 +/- 0.15 to 0.192 +/- 0.153 mEq/l), phospholipase-A2 exhibited a highly significant increase from 9.6 +/- 2.2 to 18.2 +/- 5.4 nmol/ml/min (P less than 0.01). It was demonstrated that in parallel phospholipase-A1 also showed a highly significant increase from 2.5 +/- 1.2 to 6.7 +/- 3.1 nmol/ml/min (P less than 0.01). In relation to the pathomorphological score, both phospholipases showed a small increase at a low score (edema to focal necrosis), whereas in groups with a larger spread of the pancreatitis, score 5 or 6 (extended necrosis to retroperitoneal necrosis), the increase was substantially clearer. As a result of these findings, we conclude that phospholipase-A1 could possibly be used as phospholipase-A2 as an indicator of the severity of acute pancreatitis and that it should be taken into consideration in the very promising therapy with phospholipase-A-antagonists.

Acute Disease↗

PubMed1988

Solubilization and partial characterization of phospholipase A from rat heart sarcoplasmic reticulum.

Phospholipase A has been solubilized from the sarcoplasmic reticulum of rat heart by treatment with Tris buffer, potassium chloride, taurodeoxycholate or octyl glucoside. On HPLC gel permeation, two phospholipases were identified at the void volume of a TSK 3000 column and at an apparent molecular mass of 60 kDa. The two activity peaks exhibited a predominance of phospholipase A1 activity (83-91%) and a lesser phospholipase C activity (4-9%) using sonicated 1-palmitoyl-2[1-14C]oleoylphosphatidylcholine liposomes as substrate. The voiding phospholipase A peak, which represented the bulk of the recovered activity, exhibited a requirement for calcium ions in the 0.3-3 microM range. The heat stability and response to mercuric ions was studied and some similarities were noted between the solubilized sarcoplasmic reticulum phospholipases A and the cytosolic phospholipases A of rat heart. It is speculated that the cytosolic phospholipase A which we reported earlier may represent in part phospholipase A released from sarcoplasmic reticulum during isolation of the subcellular membrane fractions.

Animals↗

PubMed1996

Basic fibroblast growth factor-stimulated arachidonic acid release in rat pancreatic acini: sequential action of tyrosine kinase, phospholipase C, protein kinase C and diacylglycerol lipase.

This study was performed to evaluate the effect of human recombinant basic fibroblast growth factor on arachidonic acid release from rat pancreatic acini and to determine the cellular mechanism involved. From enzymatic assays, basic fibroblast growth factor did not significantly stimulate phospholipase A2 activity, whereas it significantly increased diacylglycerol lipase activity. Validity of phospholipase A2 or diacylglycerol lipase inhibitors was confirmed by their ability to inhibit phospholipase A2 or diacylglycerol lipase activities. Basic fibroblast growth factor increased intracellular accumulation and extracellular release of arachidonic acid from metabolically labelled acinar cells in a concentration- and time-dependent manner. This effect was maximal with 50 pM basic fibroblast growth factor and became significant after a 5-min incubation period. The protein tyrosine kinase inhibitor, 0.5 mM genistein, inhibited arachidonic acid release in basic fibroblast growth factor-stimulated acini, whereas 100 microM vanadate, a protein tyrosine phosphatase inhibitor, enhanced arachidonic acid release. Two phospholipase A2 inhibitors, mepacrine and aristolochic acid, failed to attenuate basic fibroblast growth factor-stimulated arachidonic acid release. A diacylglycerol lipase inhibitor RHC 80267 at 150 microM and 50 microM completely inhibited 50 pM basic fibroblast growth factor-induced intracellular accumulation and extracellular release of arachidonic acid, respectively. Furthermore, basic fibroblast growth factor stimulated arachidonic acid release was also inhibited by 10 microM U73122 and by 100 nM staurosporine, phospholipase C and protein kinase C respective inhibitors. Wortmannin, an inhibitor of basic fibroblast growth factor-stimulated phospholipase D, did not affect arachidonic acid release. 100 nM 4 beta-phorbol 12-myristate 13-acetate also increased arachidonic acid release, an effect also inhibited by staurosporine. Taken together, these data demonstrate activation of diacylglycerol lipase and arachidonic acid release in pancreatic acini upon stimulation by basic fibroblast growth factor, and strongly indicate that arachidonic acid release in response to basic fibroblast growth factor depends upon the sequential action of tyrosine kinase, phospholipase C, protein kinase C and diacylglycerol lipase but not from phospholipase A2 not phospholipase D activation.

Animals↗