Effects Of Phospholipase A2 Inhibitors Research Articles

Effect of phospholipase A2 inhibitors during infection caused by Leishmania (Leishmania) amazonensis

BackgroundLipid metabolites play an important role in parasite differentiation and virulence. Studies have revealed that Leishmania sp. uses prostaglandins to evade innate barriers, thus enabling the parasites to survive inside immune cells. Despite the role of the enzyme Phospholipase A2 (PLA2) in prostaglandins production, few studies have investigated the role of parasite PLA2 during the interaction between L. (L.) amazonensis and the host (in vitro and in vivo) immune cells.MethodsIn the present work, the leishmanicidal effect of PLA2 inhibitors, methyl arachidonyl fluorophosphonate (MAFP), bromoenol lactone (BEL) and aristolochic acid (AA) were investigated in vitro (promastigote and intracellular amastigote forms of L. (L.) amazonensis) and during in vivo infection using BALB/c mice.ResultsThe aforementioned inhibitors were deleterious to promastigote and amastigote forms of the L. (L.) amazonensis and were non-toxic to peritoneal macrophages from BALB/c mice. L. (L.) amazonensis-infected BALB/c mice treated with the inhibitor BEL presented decreased lesion size and skin parasitism; however, BEL treatment induced hepatotoxicity in BALB/c mice.ConclusionsResults presented herein suggested that PLA2 inhibitors altered L. (L.) amazonensis viability. In spite of liver toxicity, treatment with BEL was the most selective compound in vitro, as well in vivo, resulting in lower skin parasitism in the infected mice. These findings corroborate the role of PLA2 in parasite virulence and maintenance in vertebrate hosts, and suggest that molecules structurally related to BEL should be considered when planning compounds against Leishmania sp.

Effects of Phospholipase A2 Inhibitors on Bilayer Lipid Membranes.

The work examines the effect of inhibitors of cytosolic Ca(2+)-dependent and Ca(2+)-independent phospholipases A2 on bilayer lipid membranes. It was established that trifluoroperazine (TFP) and, to a lesser extent, arachidonyl trifluoromethyl ketone (AACOCF3) and palmitoyl trifluoromethyl ketone (PACOCF3) were able to permeabilize artificial lipid membranes (BLM and liposomes). It was shown that AACOCF3 lowered the temperature of phase transition of DMPC liposomes, inducing disordering of the hydrophobic region of lipid bilayer. TFP disordered membranes both in the hydrophobic region and in the region of hydrophilic heads, this being accompanied by changes in the membrane permeability: appearance of a channel-like BLM activity and leakage of sulforhodamine B from liposomes. In contrast to AACOCF3 and TFP, PACOCF3 increased membrane orderliness in the hydrophobic region (heightened the temperature of phase transition of DMPC liposomes) and in the region of lipid heads. The effectiveness of AACOCF3 and PACOCF3 as inductors of BLM and liposome permeabilization was considerably lower comparatively to TFP. As revealed by dynamic light scattering, incorporation of TFP, AACOCF3 and PACOCF3 into the membrane of liposomes resulted in the increase of the average size of particles in the suspension, presumably due to their aggregation or fusion. The paper discusses possible mechanisms of the influence of phospholipase A2 inhibitors on bilayer lipid membranes.

Effect of Phospholipase A2 Inhibitors on the Release of Arachidonic Acid and Cell Viability in Cold-Stored Hepatocytes

We investigated the effect of phospholipase A2 (PLA2) inhibitors on PLA2 activity and cell viability in cold-stored rat hepatocytes. The cells were radiolabeled with [3H] arachidonic acid (AA) and cold stored in the University of Wisconsin (UW) solution containing various PLA2 inhibitors. PLA2 activity was determined by measuring the total free (cellular + supernatant) AA by thin-layer chromatography after inhibiting reacylation of free AA with inhibitors of energy production (carbonyl cyanide m-chlorophenylhydrazone + iodoacetate). Aristolochic acid, chlorpromazine, and quinacrine in the UW solution showed a significant inhibitory effect throughout 48 h cold storage but only at relatively high concentration. PLA2 activity was also suppressed (58% of control) by trifluoperazine (50 μM), but its effect was limited to only 24 h. In contrast, pretreatment of the cells prior to hypothermic preservation with trifluoperazine (10 to 100 μM) suppressed PLA2 activity during 48 h storage. Inclusion of calmodulin antagonist W-7 did not affect PLA2 activity. Thus, the inhibitory activity of these agents appears unrelated to Ca–calmodulin–phospholipid interaction but to have an inhibitory effect on PLA2 activity. To study the effects of PLA2 inhibitors on cell viability, lactate dehydrogenase (LDH) release was measured in the presence or absence of inhibitors upon rewarming cold-stored cells in Krebs–Henseleit buffer for 2 h at 37°C. None of the inhibitors tested improved cell viability after 48 h storage. Thus, although PLA2 inhibitors blocked PLA2 activity, there was no suppression of LDH release. PLA2 may play a minor role in preservation/reperfusion injury to cold-stored hepatocytes.

The Effect of Phospholipase A2Inhibitors on Proliferation and Apoptosis of Murine Intestinal Cells

Background.Group II phospholipase A2(PLA2) enzymes, the rate controlling enzymes in arachidonic acid metabolism, have been well characterized and subdivided into secretory 14-kDa PLA2(sPLA2) and cytoplasmic 85-kDa PLA2(cPLA2). Previous research has demonstrated increased PLA2in colorectal tumors. The present study was performed to determine the effect of specific PLA2 inhibitors on the proliferation and induction of apoptosis of intestinal epithelial cells.Methods.A continuously proliferating rat small intestinal cell line (IEC-18) and a mouse colon cancer cell line (WB-2054-M4) were utilized for these experiments. The cells were placed in microwells with serum-free or serum-supplemented media. The effects of serum on proliferation were then evaluated in the presence of the cPLA2inhibitor, methylarachidonyl fluorophosphate (MAFP), or the sPLA2inhibitorp-bromophenacyl bromide (BPB). The sPLA2and cPLA2protein was estimated by Western blotting. Proliferation of intestinal cells was quantitated by incorporation of [3H]thymidine into DNA and PLA2activity was evaluated by quantitating arachidonic acid formation and prostaglandin E2production.Results.Western blotting of IEC-18 and WB-2054 cell protein demonstrated sPLA2and cPLA2enzyme in cells incubated in media containing 10% serum. Spontaneous DNA synthesis was present in both cell lines and serum consistently increased proliferation. In IEC-18 cells [3H]thymidine incorporation stimulated by serum was inhibited by MAFP and BPB, while in the malignant cell line, proliferation was inhibited only by BPB. BPB, but not MAFP, produced a dose-dependent increase in apoptotic ratios in both cell lines. Arachidonic acid and PGE2formation, stimulated by serum, was inhibited by MAFP and BPB.Conclusions.A differential effect on intestinal cell mitogenesis was seen with different PLA2inhibitors. The sPLA2inhibitor, but not the cPLA2inhibitor, significantly inhibited [3H]thymidine incorporation in the malignant cell line. This occurred with an induction of apoptosis. sPLA2inhibitors may be specific inhibitors of growth of malignant cells. The inhibition of arachidonic acid and PGE2production did not correlate with the inhibition of proliferation, suggesting that the two processes may be unrelated.

Effects of phospholipase A2 inhibitors on the antidepressant-induced axonal regeneration of noradrenergic locus coeruleus neurons.

In previous experiments, we have shown that antidepressants such as desipramine (DMI) can induce axonal regeneration of noradrenergic locus coeruleus neurons. In this article, we suggest that phospholipase A2 is involved in the molecular mechanism of the antidepressant-induced regeneration of brain noradrenergic axons. The effects of the PLA2 inhibitors, mepacrine (MEP) or 4-bromphenacyl bromide (BPB), upon the DMI-induced regeneration of noradrenergic axons in the rat cerebral cortex were assessed by either histofluorescence or immunohistochemistry using an antibody to dopamine-beta-hydroxylase. Symmetrical sites of the frontal cortex in both hemispheres were pretreated with 6-hydroxydopamine (6-OHDA). Then, the same cortical site of one hemisphere was infused with DMI by means of osmotic minipumps for more than 2 weeks, while the corresponding cortical site of the other hemisphere was infused with DMI plus MEP or BPB. It was found that the PLA2 inhibitors could attenuate the DMI-induced regeneration of noradrenergic axons. Thus, if axonal retraction or degeneration of brain noradrenergic neurons is involved in the pathogenesis of clinical depression, elucidating the malfunction of the PLA2 systems may provide substantial evidence to aid in our understanding of the cause of depression at the molecular level.

Effect of phospholipase A2 inhibitor ONO-RS-082 on substance P-induced histamine release from rat peritoneal mast cells.

Rat peritoneal mast cells purified on a Percoll gradient were challenged with substance P (SP) and the effect of phospholipase A2 inhibitor ONO-RS-082 on SP-induced histamine release from the cells was investigated. 10(-5) mol/l SP caused a significant histamine release and the amount of histamine release reached maximum at 1 min after the challenge. ONO-RS-082 inhibited the SP-induced histamine release in a concentration-dependent manner at the concentration from 10(-6) to 10(-4) mol/l, suggesting possible involvement of phospholipase A2 in SP-induced histamine release from rat peritoneal mast cells.

Group II phospholipase A2 inhibitors suppressed lysophosphatidylserine-dependent degranulation of rat peritoneal mast cells

Rat peritoneal mast cells were sensitized with IgE and challenged with the specific antigen in the presence of lysophosphatidylserine (lysoPS), an essential co-factor for rodent connective tissue mast cell degranulation, and the effects of phospholipase A2 inhibitors were examined. Mepacrine, a known inhibitor of phospholipase A2, at concentrations below 10 −5 M and anti-rat 14-kDa group II phospholipase A2 antibody inhibited histamine release, while they did not affect the prostaglandin generation. Like histamine release, prostaglandin generation in IgE- and antigen- challenged rat peritoneal mast cells was dependent on the presence of lysoPS. These results indicate that 14-kDa group II phospholipase A2 may play an essential role in IgE-, antigen-, and lysoPS-dependent degranulation process of rat peritoneal mast cells and that the mechanism whereby it participates may not be due to the production of lysoPS from PS in mast cell membranes.

The biphasic effect of phospholipase A2 inhibitors on axon elongation

We have analyzed changes in axon elongation rate in vitro induced by three different phospholipase A2 inhibitors, namely, bromophenacyl bromide (BPB), quinacrine and Sr2+. Spinal ganglia were obtained from 19.5-day-old rat fetuses and explanted onto polyornithine substrata. Axon length was measured at 1, 2 and 3 days in vitro (d.i.v.). We have previously shown that BPB added at 5 hr in vitro (h.i.v.) modifies the structure of growth cones and inhibits or promotes axon elongation according to its concentration. Now, we have observed that 0.5 × 10−6 M BPB also stimulates axon elongation when added at 1 d.i.v. Culture media with different Ca2+ concentrations were used to test Sr2+ added at 1 d.i.v. In 2.3 mM Ca2+ only an inhibitory effect was observed with 8 mM Sr2+. On the other hand, in 0.3 mM Ca2+, axon growth was stimulated by 0.6–1.2 mM Sr2+ but inhibited by 6 mM Sr2+. Quinacrine, added at 5 h.i.v. was inhibitory at 10−5 M and showed no effects at 10−6 M. However, after washing quinacrine, normal elongation rate was recovered by those previously treated with 10−5 M and axon growth was enhanced in those treated with 10−6 M. Since three different phospholipase A2 inhibitors, tested in different situations, produce a similar biphasic effect on axon elongation rate, we postulate that this enzymatic activity is involved in the motility of axon growth cones.

Effects of phospholipase A2 inhibitors on ruthenium red-induced Ca2+ release from mitochondria.

The pharmacologic agents verapamil, nifedipine, diltiazem, prenylamine, N-oleoylethanolamine, R 24571, trifluoperazine, dibucaine, and quinacrine are examined as potential inhibitors of rat liver mitochondrial phospholipase A2 acting on endogenous phospholipid. Their potency as inhibitors of the enzyme is compared to their activities as inhibitors of phospholipase A2-dependent swelling and ruthenium red-induced Ca2+ release in intact mitochondria. For verapamil, diltiazem, trifluoperazine, dibucaine, and quinacrine, there is complete agreement between the relative potencies as inhibitors of phospholipase A2 and the two other processes. Nifedipine and prenylamine, which are weak inhibitors of phospholipase A2, produce a permeable inner membrane, provided that the mitochondrial have accumulated Ca2+. R 24571, which strongly inhibits the enzyme, disrupts mitochondria by a Ca2+-independent mechanism. N-Oleoylethanolamine, which is an effective inhibitor of swelling, does not inhibit phospholipase A2 or ruthenium red-induced Ca2+ release. The results support a proposed scheme wherein ruthenium red-induced Ca2+ release is viewed as reverse activity of the Ca2+-uptake uniporter occurring subsequent to decline in the proton motive force. The latter effect is proposed to arise from a specific phospholipase A2-dependent increase in inner-membrane H+ conductance of mitochondrial subpopulations. It is further shown that mitochondrial membranes display cyclic oscillations in free fatty acid content which are not dependent on the presence of Ca2+ or on the capacity to generate acylcoenzyme A.