Biochemical alterations in rat Thiry-Vella fistulas.

In order to obtain baseline information on the secretory function of normal rat bowel for our work on intestinal graft ischemia, we studied several biochemical parameters in rat Thiry-Vella fistulas (TVF). TVFs were created in 200-g male Lewis rats (n = 11) using the 25-cm segment of jejunum normally used as a graft in our intestinal transplant model. The stomas were matured primarily and the animals were allowed to recover. The TVFs were flushed at 0, 6, and 24 h and then daily for up to 21 days with 12 mL normal saline solution. The effluent was collected and analyzed for total protein (TP), secretory phospholipase A2 (sPLA2), intestinal fatty acid binding protein (I-FABP), lactate dehydrogenase (LDH), and N-acetylglucosamine (NAGA). TP content was 0.12 +/- 0.01 mg/mL up to 48 h, then gradually increased and stabilized at 0.39 +/- 0.05 mg/mL at day 21. By sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), one major protein band was identified in the low-molecular-mass range (15 kD), consistent with I-FABP and sPLA2. Secretory PLA2 levels decreased over the first 4 days to a low of 115 +/- 24.8% hydrolysis/min/fraction, then gradually rose to a plateau at approximately 529.76 +/- 88.36% hydrolysis/min/fraction by day 18. I-FABP levels rose rapidly from 0 ng/mL at 2 h to 900 +/- 250.0 ng/mL at 6 h and approximately 3000 +/- 304.9 ng/mL by day 14. LDH levels at 2 h and 48 h did not differ, with 0.03 +/- 0.004 and 0.03 +/- 0.005 optical density units (OD)/min/mL, respectively. NAGA levels were 0.07 +/- 0.05 OD/h/mL at 2 h and rose to 0.14 +/- 0.04 OD/h/mL at 48 h. These data suggest that after an early equilibration period, biochemical secretion into the lumen of normal rat bowel reaches a state of equilibrium, and therefore appears to reflect the baseline biochemical status of the bowel. Some of these levels are not negligible as one would expect in "normal" bowel. This information should prove extremely helpful as a baseline study of abnormal conditions of the intestine, such as ischemia or rejection.

Modification of disaccharidase activity measurement in rat bowel.

This study describes a modification of the existing disaccharidase assay in rat small bowel in which whole bowel, rather than mucosa, is utilized. In addition, the use of total vs. specific activity as a more accurate unit of measurement of disaccharidase activity is discussed.

Diphenyleneiodium chloride blocks inflammatory cytokine-induced up-regulation of group IIA phospholipase A(2) in rat mesangial cells.

Inflammatory cytokines, interleukin 1beta and tumor necrosis factor-alpha, potently stimulate rat mesangial cells to express and secrete group IIA phospholipase A(2) (PLA(2)). Cytokine-induced up-regulation of PLA(2) has been blocked by inhibitors (antioxidants) of the transcription factor, nuclear factor-kappaB (NF-kappaB), suggesting a role for NF-kappaB in the regulation of group IIA PLA(2) expression. Reactive oxygen species such as H(2)O(2), which are elevated in mesangial cells after cytokine activation, can mimic cytokine-induced NF-kappaB activation. However, the source of reactive oxygen species generation in mesangial cells, produced by cytokine stimulation, has yet to be clarified. Recently, tumor necrosis factor-alpha has been demonstrated to increase superoxide radical generation in mesangial cells. Therefore, we hypothesized that a selective NADPH oxidase inhibitor, diphenyleneiodium chloride (DPI), could block cytokine-induced group IIA PLA(2) up-regulation by attenuating NF-kappaB binding. To test this hypothesis, we isolated rat mesangial cells and characterized them by ultrastructural and immunochemical methods. This homogeneous mesangial cell population was responsive to cytokine as evidenced by an increase in steady-state levels of group IIA PLA(2) mRNA and extracellular enzymatic activity over time. DPI (0.02-20 microM), added 90 min before cytokine activation, inhibited both group IIA PLA(2) mRNA and enzymatic activity in a concentration-dependent manner. By electrophoretic mobility shift analysis, cytokine activation also increased specific NF-kappaB binding to one of two NF-kappaB consensus elements in the rat group IIA PLA(2) promoter and also was suppressed by DPI pretreatment. Antibodies to NF-kappaB p65 (Rel A) and p50 (but not normal rabbit IgG) supershifted this retardation signal and verified the type of NF-kappaB species as the classical p50/p65 heterodimer.

Secretory event in intestinal grafts during preservation ischemia.

BACKGROUND: Ischemia triggers secretion of proteins from the intestine, including type II secretory phospholipase A2 (sPLA2). This "secretory event" was studied in intestinal grafts during the first few hours of preservation by measuring total protein, sPLA2, and other enzymes in the UW preservation solution over time. The effect of PX-13, a PLA2 inhibitor, was also studied. MATERIALS AND METHODS: Twenty-five centimeter intestinal grafts were harvested from Lewis rats, flushed, and preserved in UW solution +/- PX-13 at 4 degrees C. UW samples from 0 to 48 h (n = 5 each) were analyzed for total protein, sPLA2, lactate dehydrogenase (LDH), N-acetylglucosamine (NAGA), and lysozyme. Nonpreserved grafts were homogenized in PBS as tissue controls. Standard biochemical methods were used for all assays. RESULTS: Total protein increased rapidly by 5 min, continued to rise more slowly until 30 min, and then stabilized. The most significant increase in sPLA2 activity occurred between 90 and 180 min. NAGA increased most markedly between 30 and 180 min, while LDH increased in the first 30 min, although the level of both enzymes was negligible compared to tissue enzyme. Lysozyme levels were minimal at all times. PX-13 decreased sPLA2 activity markedly at all time points. CONCLUSION: Total protein levels increased before sPLA2, suggesting that sPLA2 may be secreted in response to other proteins or enzymes released even earlier during preservation (e.g., cytokines). These elevations do not appear to be caused by cell death. Phospholipase A2 secretion may be blocked, and this may greatly improve the outcome of intestinal preservation.

Phospholipase A2 secretion during intestinal graft ischemia.

The time-dependent appearance of phospholipase A2 (PLA2) activity in the preservation media of ischemic rat intestinal grafts is described. In controls, Ca2+-dependent, secretory PLA2 activity accumulated rapidly during the first 6 hr of ischemia, followed by a linear increase for up to 48 hr. LDH levels, by contrast, increased linearly throughout the 48 hr of ischemia. Addition of inhibitors of PLA2, cyclooxygenase, and lipooxygenase blocked accumulation of PLA2, but not LDH. PX-13, a novel PLA2 inhibitor, was most effective: 40 microM inhibited release by 86%, while 25 microM indomethacin (cyclooxygenase blocker) or nordihydroguiaretic acid (lipooxygenase blocker) inhibited 41 and 36%, respectively. That appearance of PLA2 activity, but not LDH, is attenuated by inhibitors of the eicosanoid cascade suggests a secretory event rather than leakage from dying cells. The secreted PLA2 is most likely the proinflammatory sPLA2 that has been implicated as a stress-induced protein and priming agent in ischemia-reperfusion injury.

PX-52, A novel inhibitor of 14 kDa secretory and 85 kDa cytosolic phospholipases A2.

Previously we reported that PGBx, a prostaglandin oligomer with anti-inflammatory activity, inhibited 14 kDa phospholipase A2 (PLA2) activity and blocked arachidonic acid mobilization in prelabeled human neutrophils (Biochim. Biophys. Acta 1006:272-277, 278-286, 1989) This study describes a new inhibitor of phospholipase A2, PX-52, that also blocks agonist induced arachidonic acid mobilization in prelabeled cells. PX-52, a fatty acid polymer, inhibited hydrolysis of 14C-oleate labeled E.coli by a variety of 14 kDa PLA2s including human PMN, sperm, synovial fluid and disc, as well as porcine pancreas, N. naja, and bee venom in a dose-dependent manner with IC50s ranging from 1.0-3.7 uM. Inhibition of activity was comparable at different Ca2+ concentrations, but was relieved by increasing substrate concentration or by methylation of PX-52. Hydrolysis of [14C]-arachidonyl phosphatidylcholine by 85 kDa, cytosolic PLA2 from U937 cells was similarly inhibited by PX-52, the IC50 = 5 uM. Arachidonic acid mobilization induced by A23187 in prelabeled human PMNs was blocked by PX-52; IC50 = 10-15 uM while concentrations of up to 80 uM oleate had no effect. These results demonstrate that PX-52 inhibits the in vitro activity of secretory and cytosolic PLA2s and agonist-induced arachidonic acid release from human cells. Given its ability to block the arachidonic acid cascade, PX-52 may be useful in the control of inflammation.

Secretory phospholipase A2 levels in rat small bowel.

Preliminary studies on ischemia/reperfusion injury in transplanted small bowel grafts showed that secretory phospholipase A2 (sPLA2) may play a substantial role by breaking down membrane phospholipids. This study sought to determine the normal values of sPLA2 in the rat small bowel as a function of site and length as a baseline for future studies. The entire small bowel of male Lewis rats (200 g) was flushed with normal saline to eliminate solid contents. In group 1, the entire small bowel was divided into 5-cm segments (numbered 1-9), which were snap frozen and processed the same day for sPLA2. In group 2, a 25-cm segment of bowel (corresponding to segments 2-6 in group 1) was harvested from each animal, snap frozen, and immediately processed for sPLA2. To assess the effect of bowel storage on enzyme content, group 3 and group 4 grafts were stored for 7 and 14 days, respectively, at -85 degrees C prior to processing. All samples were homogenized in buffer, extracted with H2SO4 and assayed for sPLA2 activity using [1-14C]oleate-labeled autoclaved Escherichia coli as substrate. Results were analyzed statistically by ANOVA. sPLA2 activity rose from 85.46 +/- 14.46% hydrolysis/min fraction-1 in segment 1, to 476.38 +/- 176.75% hydrolysis/min fraction-1 in segment 9. The increase was linear and statistically significant (p < .0001). There was no significant difference in enzymatic activity between groups 2, 3, and 4. Group 2 activity was 263.02 +/- 43.74% hydrolysis/min fraction-1. This value was not statistically different from the mathematically calculated mean of segments 2-6 in group 1 (237.75). The results show that (1) sPLA2 activity increases predictably with distance from the ligament of Treitz (2) storage at -85 degrees C does not affect sPLA2, activity, and (3) 25-cm grafts may be evaluated in toto with reproducible baseline enzyme activity. Given the variability of enzyme activity along the course of the rat small bowel, it is imperative that exact location be identified in any studies evaluating sPLA2 activity.

Haemodialysis activates phospholipase A2 enzyme.

BACKGROUND: Clinical and experimental evidence suggest that haemodialysis (HD) procedure is an inflammatory process. For the production of proinflammatory lipid mediators in many inflammatory reactions, the release of arachidonic acid by phospholipase A2 (PLA2) enzyme is a prerequisite. Therefore, the purpose of the present investigation was to establish whether the activity of PLA2 increases during HD and whether the increase depends on the type of dialyser used. METHODS: We performed dialysis in eight chronic HD patients. Blood samples entering and leaving the dialyser were obtained before and at 15, 60, 120 and 180 min after the dialysis was started, on one occasion using a cuprophane and on another occasion a cellulose triacetate dialyser. PLA2 activity was assessed in crude plasma and in plasma extract. RESULTS: PLA2 activity in plasma extract exhibited similar biochemical properties to that of inflammatory human synovial fluid PLA2 enzyme which is of group II PLA2. PLA2 activity in crude plasma represents a type of PLA2 other than the synovial type. In HD patients, baseline PLA2 activities in a crude plasma and plasma extract were significantly increased when compared to normal subjects. An increase in PLA2 activity was observed in crude plasma with a peak appearing at 15 min when the patients were dialysed with cuprophane and cellulose triacetate membranes. This increase was observed in both arterial and venous blood samples and was more pronounced when the patients were dialysed with cuprophane than with cellulose triacetate membranes. When PLA2 was assessed in plasma extract, the activity increased only with cuprophane but not with cellulose triacetate membranes. CONCLUSION: PLA2 activity in plasma is increased in HD patients and increases during the dialysis procedure to a greater extent with a less biocompatible membrane. Continuous activation of PLA2 might be relevant for long-term deleterious consequences of HD.

Development and use of ELISA to quantify type II phospholipase A2 in normal and uremic serum.

Previously we reported that uremic plasma contained eight times more phospholipase A2 (PLA2) activity than control plasma (Costello et al., Clin Chem 1990;36:198-200). That study, however, did not distinguish between various PLA2s that could contribute to the observed increase. Therefore, we developed a sandwich ELISA to specifically quantify serum type II PLA2. By ELISA, uremic sera contained significantly more type II PLA2 than control sera (median = 1025 micrograms/L, range = 52-3320 micrograms/L vs median = 9.2 micrograms/L, range = 4.6-17.5 micrograms/L; P = 0.002). When serum samples were incubated with 1-[14C]oleate-labeled autoclaved Escherichia coli, activity was increased 14.6-fold in uremic vs normal serum, with a median of 6.5 mumol/min per liter (range 1.1-16.3) vs a control median of 0.49 mumol/min per liter (range 0.32-0.60; P = 0.002). Thus, ELISA detects about eightfold more immunoreactive type II PLA2 in uremic serum than does enzymatic analysis. Evidently, the increase in PLA2 activity previously observed in uremic plasma is primarily due to increased concentrations of type II PLA2.

Human neutrophils store type II 14-kDa phospholipase A2 in granules and secrete active enzyme in response to soluble stimuli.

Although "secretory" type II 14-kDa phospholipase A2 (sPLA2) activity has been described in neutrophils, direct evidence of enzyme secretion has been elusive. We have used immunogold electron microscopy with polyclonal and monoclonal antibodies to sPLA2 to demonstrate localization of the enzyme to granules of resting human neutrophils and translocation to phagolysosomes. Soluble stimuli such as calcium ionophore A23187 stimulate loss of cell-associated enzymatic activity. Supernatant fluids from stimulated neutrophils lack measurable PLA2 but contain proteases which inactivate exogenous sPLA2. The use of alpha-1-antitrypsin as a protease inhibitor permitted this first demonstration of secretion of PLA2 activity from stimulated human neutrophils.

Cellular regulation of arachidonate mobilization and metabolism.

Synthesis of eicosanoids is initiated by signal transduction cascades which result in the hydrolysis of free arachidonic acid from membrane phospholipids. Both a cytosolic 85 kDa and a nonpancreatic 14 kDa PLA2 may contribute to cellular arachidonate mobilization. In many cells, agonist-stimulated fatty acid release is dependent upon increases in intracellular free calcium and is enhanced by pretreatment with agents such as phorbol esters and soluble diglycerides. The response is specific for arachidonate and structurally similar polyunsaturated fatty acids containing a cis 5, 6 double bond. DMSO-differentiation of U937 cells markedly enhances the A23187-stimulated release of [3H]arachidonate, which appears to be coupled to differentiation-induced enhancement of capacitance calcium entry. Although both phorbol esters and soluble diglycerides enhance subsequent fMLP or A23187-stimulated arachidonate release in human neutrophils, several lines of evidence indicate that the effects of oleoylacetylglycerol and 1,2-dioctanoylglycerol are protein kinase C-independent. Soluble diglycerides, but not phorbol esters, enhance the coupling of arachidonate mobilization to subsequent leukotriene B4 synthesis. Further studies will be required to elucidate the mechanisms which regulate activation of cellular phospholipases and subsequent synthesis.

Inhibitor of phospholipase A2 blocks eicosanoid and platelet activating factor biosynthesis and has topical anti-inflammatory activity.

The effect of a phospholipase A2 (PLA2) inhibitor on leukotriene, prostaglandin and platelet activating factor (PAF) biosynthesis in isolated cells and in vivo was determined. BMS-181162, [4(3'-carboxyphenyl)-3,7-dimethyl-9(2",6",6"-trimethyl-1"- cyclohexenyl)2Z,4E,6E,8E-nonatetraenoic acid], reversibly inhibited the 14-kdalton PLA2 purified from human synovial fluid with an IC50 of 8 microns. In A23187-stimulated human polymorphonuclear leukocytes (PMNs), BMS-181162 blocked arachidonic acid release with an IC50 of 10 microns. Leukotriene B4 and PAF biosynthesis in these cells was also inhibited. In a phorbol ester-induced chronic mouse skin inflammation model, topically applied BMS-181162 markedly lowered the tissue levels of leukotriene B4 and prostaglandin E2 and dose-dependently inhibited leukocyte infiltration (ED50 = 180 micrograms per ear). BMS-181162 is an inhibitor of PLA2 and may prove to be a useful tool in the delineation of the role of PLA2 in the inflammatory process.

Separation of agonist-stimulated arachidonate mobilization from subsequent leukotriene B4 synthesis in human neutrophils: different effects of oleoylacetylglycerol and phorbol myristate acetate as priming agents.

Preincubation of human neutrophils with phorbol esters or soluble diglycerides enhances subsequent f-Met-Leu-Phe (fMLP)-stimulated arachidonate mobilization and leukotriene B4 (LTB4) synthesis. We have recently reported that 1,3-dioctanoylglycerol (1,3-diC8) is equipotent with 1,2-sn-dioctanoylglycerol (1,2-diC8) as priming agent, thus suggesting that the priming effects of diacylglycerols are protein kinase C (PKC) independent (Rosenthal et al., 1993, Biochim. Biophys. Acta 1177:79-86). In order to further investigate this question, the present study has directly compared the effects of oleoylacetylglycerol (OAG) and the PKC activator, phorbol 12-myristate 13-acetate (PMA), on agonist-stimulated lipid metabolism. The results indicate that both OAG and PMA dose dependently enhance f-Met-Leu-Phe (fMLP)-stimulated release of [3H]arachidonate. Optimal concentrations of OAG (5 microns) and PMA (10 nM) are equipotent in increasing fMLP-stimulated arachidonate mobilization as quantitated either with total radioactivity or by mass measurements of free arachidonate. By contrast OAG is sixfold more effective than PMA in enhancing synthesis of 5-lipoxygenase (5-LO) metabolites by mass and two to threefold more effective than PMA in enhancing synthesis of [3H]eicosanoids. Furthermore, OAG, but not PMA, enhances fMLP-stimulated synthesis of platelet-activating factor. By contrast, PMA directly stimulates [3H]arachidonate mobilization, while OAG (20 microM) does not; despite these differences, the combined effects of PMA + OAG on subsequent agonist-stimulated arachidonate release are not greater than those of PMA alone. In cells challenged with subthreshold concentrations (< 0.1 microM) of the calcium ionophore A23187, both OAG and PMA stimulate [3H]arachidonate release but not [3H]LTB4 synthesis. These findings suggest that OAG does not directly activate 5-LO, but instead couples arachidonate mobilization to leukotriene synthesis in a PKC-independent manner.

1,3-Dioctanoylglycerol modulates arachidonate mobilization in human neutrophils and its inhibition by PGBx: evidence of a protein-kinase-C-independent role for diacylglycerols in signal transduction.

Preincubation of human neutrophils with 1-oleoyl-2-acetylglycerol (OAG) enhances subsequent f-Met-Leu-Phe (fMLP)-stimulated arachidonate mobilization. We have recently demonstrated that preincubation of neutrophils with OAG also reverses inhibition of A23187 stimulated [3H]arachidonate mobilization by the phospholipase A2 inhibitors, PGBx and aristolochic acid. The present study has compared the effects of 1,2-sn-dioctanoylglycerol (1,2-diC8) and 1,3-dioctanoylglycerol (1,3-diC8) on these cellular events. Dose-dependent priming (ED50 < 2.5 microM) of fMLP-stimulated [3H]arachidonate mobilization is obtained with both 1,2-diC8 and 1,3-diC8. Both diC8s also enhance fMLP-stimulated synthesis of leukotriene B4, 5-hydroxyeicosatetraenoic acid and platelet-activating factor, and generation of superoxide. Furthermore, both 1,2-diC8 and 1,3-diC8 reverse the effects of PGBx on A23187-stimulated [3H]arachidonate mobilization and platelet-activating factor synthesis. By contrast, higher concentrations (5-10 microM) of 1,2-diC8, but not 1,3-diC8, directly stimulate both [3H] arachidonate mobilization and superoxide generation. Since 1,3-diC8 does not activate protein kinase C (PKC), these results suggest that PKC is involved in direct activation of neutrophils by diacylglycerols but not in priming. Furthermore, reversal of the inhibitory effects of PGBx by diacylglycerols also appears to involve a PKC-independent mechanism.

Swine as a model of skin inflammation. Phospholipase A2-induced inflammation.

A predictive animal model of skin inflammation is needed for the development of potential therapeutic agents. The existing models of inflammation rely on animals whose skin physiology or biochemistry differs significantly from human. The objective of this investigation was to evaluate the swine as a potential model of inflammation, because its skin has been recognized to exhibit morphologic and functional similarities to human skin. In the swine, an inflammatory response was produced following intradermal injection of snake venom phospholipase A2 (PLA2). This response was characterized by transient erythema (2-3 h) and microscopic changes of cell infiltration, epidermal hyperplasia, and dermal damage, which were apparent two days after PLA2 and peaked by day 7. In general, these microscopic changes persisted up to 21 days. Treatment with the antiinflammatory steroid, betamethasone dipropionate (Diprolene), gave a significant reduction of the inflammatory responses. Heat-inactivated PLA2, ovalbumin, or saline did not provoke this reaction, although PLA2 inactivated by bromophenacyl bromide alkylation did produce an inflammatory response. The alkylated PLA2 was also able to provoke an inflammatory response in the mouse paw edema assay. These results demonstrate that PLA2 can stimulate an inflammatory response in the swine skin, but that phospholipid hydrolytic activity is not required.

Role of human sperm phospholipase A2 in fertilization: effects of a novel inhibitor of phospholipase A2 activity on membrane perturbations and oocyte penetration.

Phospholipase A2 was isolated from human sperm and its potential role in the membrane fusion events of fertilization was examined. Highly purified enzyme hydrolyzed the phospholipids of [1-14C]oleate-labeled Escherichia coli optimally at neutral to alkaline pH with 5 mM CaCl2 and 150 mM NaCl (specific activity = 20 mumol/min/mg). Activity was inhibited in a dose-dependent manner by an oligomer of prostaglandin B1 (IC50 = 1.5 microM) reported to inhibit human phospholipases A2 in vitro and in situ. Sperm phospholipase A2 injected into mouse foot pad induced a dose-dependent edema that was inhibited by oral administration of prostaglandin Bx (IC50 < or = 10 mg/kg) or by pretreatment of the enzyme with 4-bromophenacyl bromide. Human sperm phospholipase A2 (10 micrograms) induced fusion of phosphatidylserine vesicles in the presence of 1 mM calcium chloride by approximately 80% (+/- 10%) as determined by monitoring turbidity (O.D.400) and efficiency of fluorescence resonance energy transfer. This enzyme-induced fusion was accompanied by phospholipid hydrolysis, and both fusion and phospholipid degradation were inhibited by more than 60% when enzyme was preincubated with 5 microM prostaglandin Bx. Sperm penetration of zona pellucida-free hamster oocytes was inhibited in a dose-dependent fashion when sperm were incubated with prostaglandin Bx (IC50 approximately 15 microM) during capacitation; sperm motility was not affected by this treatment. Capacitation in the presence of prostaglandin Bx had little to no effect on the in vitro acrosome reaction. These results suggest that sperm phospholipase A2 and its modulators may contribute to membrane fusion events in mammalian fertilization.

Novel inhibitor of phospholipase A2 with topical anti-inflammatory activity.

Activation of a phospholipase A2 (PLA2) is a key step in the production of precursors for the biosynthesis of lipid mediators of inflammation. Inhibition of this enzyme could result in the suppression of three important classes of inflammatory lipids, prostaglandins, leukotrienes and platelet activating factor (PAF), and offers an attractive therapeutic approach to design novel agents for the treatment of inflammation and tissue injury. In this report we describe a novel compound, BMS-181162 4(3'-carboxyphenyl)-3,7-dimethyl-9(2",6"6"-trimethyl-1"-cyclohexenyl),++ +2Z,4E,6E, 8E-nonatetraenoic acid which specifically inhibits a 14 kD human PLA2 and effectively blocks phorbol ester induced skin inflammation in mice. BMS-181162 is the first reported specific inhibitor of PLA2 and its specificity may make useful tool in the dissection of the role of PLA2 in the inflammatory process.