Endothelins activate phospholipase A2 in brain capillary endothelial cells

Addition of endothelin-1 to cultured rat brain capillary endothelial cells induced a 2.7-fold activation of phospholipase A₂, as evidenced from the release of [³H]arachidonic acid from prelabelled cells. Half maximum activation by endothelin-1 was observed at 1 nM. The action of endothelin-1 was not mimicked by low concentrations of endothelin-3 and it was largely suppressed by BQ-123, suggesting the involvement of an ETA receptor subtype. It is suggested that the activation of phospholipase A₂ by endothelins plays a role in the development of delayed cerebral vasopasm following subarachnoid hemorrhage.     

A continuous fluorometric assay for phospholipase A2 activity in brain cytosol

Alterations in phospholipase A₂ (PLA₂) activity have been implicated in Alzheimer disease and other neurological disorders, although brain PLA₂ activity is currently measured using lengthy, non-continuous assays. We describe herein a rapid, continuous assay in which we measured PLA₂ activity in mouse brain cytosol (CB-57). Brains were homogenized in HEPES buffer (pH 7.5) and the cytosolic fraction was prepared by centrifugation at 25 000×g for 20 min, followed by centrifugation of the supernatant at 100 000×g for 60 min. Cytosolic protein content was determined using the Bradford assay. Pyrene labeled phosphatidylcholine was added to 50 μg of cytosolic protein in Tris buffer (pH 8.0) containing fatty acid free-bovine serum albumin for a final assay volume of 2 ml. Assay temperature was maintained at 30±1°C. The excitation wavelength was 345 nm and emission was measured at 377 nm. Fluorescence intensity was converted to molar concentrations using a standard curve. Under these conditions, bromoenol lactone inhibited up to 58% of the PLA₂ activity with an IC₅₀ of 0.5 μM. In a separate experiment, lack of appreciable alternative acylhydrolase activity was verified chromatographically. Using this method, brain PLA₂ activity can be measured in a continuous, rapid, and sensitive manner.     

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 x 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.     

Effect of bromophenacyl bromide, a phospholipase A2 inhibitor, on the induction and maintenance of LTP in hippocampal slices

The effect of bromophenacyl bromide (BPB), a phospholipase A₂ (PLA₂) inhibitor, on both the induction and the maintenance of long-term potentiation (LTP) was investigated in field CA₁ of the hippocampal slice preparation. One hour of BPB application (50 μM) caused a large reduction in the magnitude of LTP induced by a theta burst stimulation (TBS) paradigm. BPB had no significant effect on either the degree of paired-pulse facilitation or the amount of pre-established LTP. Furthermore, the facilitation of postsynaptic responses occuring during TBS and in the first minute following TBS was not reduced by the PLA₂ inhibitor. These results indicate that the inhibition of LTP produced by BPB is not due to an effect of the drug on a physiological event that triggers LTP. The data also suggest that PLA₂ activation plays a critical role in the expression of LTP, but is not required for the maintenance of the potentiation.     

Regulation of endogenous calcium-dependent synaptic membrane phospholipase A2

Synaptic plasma membrane preparations from brain tissue have endogenous Ca2+-dependent phospholipase A2 activity. Characterization of this activity revealed that it was maximally active at 10(-7)-10(-5) M Ca2+ and pH 7.0. The enzyme had a Km of 62.0 microM and a Vmax of 98.0 nmol/mg/h. Calmodulin and prostaglandin F2 alpha stimulated phospholipase A2 activity, whereas prostaglandin E2, cyclic AMP and ATP were inhibitory. Addition of exogenous phospholipase A2 to synaptic plasma membrane and synaptic vesicle preparations led to their disruption and/or lysis. We suggest that Ca2+-dependent regulation of phospholipase A2 activity may be required for synaptic vesicle and synaptic plasma membrane interaction.     

A light and electron microscopic study of cytoplasmic phospholipase A2 and cyclooxygenase-2 in the hippocampus after kainate lesions

The systemic administration of kainate (10 mg/ml) into adult Wistar rats produces seizures and neurodegeneration. We have studied the effect of kainate administration on cPLA₂ and COX-2 immunoreactivities after 3 days and 1, 2, 4 and 11 weeks. The cPLA₂ immunoreactivity was increased in hippocampal neurons at 1 and 3 days after kainate injection suggesting that PLA₂ may be involved in neurodegeneration. Increased cPLA₂ and COX-2 immunoreactivities in astrocytes at 1, 2, 4 and 11 weeks after kainate injection indicate an adaptive astrocytic response that may be associated with gliosis.     

Deterioration of axotomy-induced neurodegeneration by group IIA secretory phospholipase A2

Phospholipase A₂ (PLA₂) is proposed to play a role in the repair of the ruptured membrane after axotomy. In neonatal rats, we examined the effect of Group IIA secretory PLA₂ (sPLA₂-IIA) on axotomy-induced cell death of motoneurons. sPLA₂-IIA significantly induced death of axotomized motoneurons. Indoxam, a specific inhibitor for sPLA₂-IIA, protected motoneurons from the sPLA₂-IIA-induced deterioration. The present study indicated that sPLA₂-IIA possessed neurotoxic effect rather than neuroprotective effect against facial nerve.     

Plasmalogens, phospholipases A2 and signal transduction

Several lines of evidence indicate that the breakdown of plasmalogens in neural membranes during neurodegenerative diseases is a receptor-mediated process catalyzed by a plasmalogen-selective phospholipase A₂. This enzyme has recently been purified from bovine brain. It does not require Ca²⁺ and is localized in cytosol. It has a molecular mass of 39 kDa and is strongly inhibited by glycosaminoglycans, with the pattern of inhibition being heparan sulfate0 > hyaluronic acid > chondroitin sulfate > heparin. This plasmalogen-selective phospholipase A₂ is also inhibited by gangliosides and sialoglycoproteins. Substrate specificity and the effects of metal ions, detergents and inhibitors suggest that this phospholipase A₂ is different from the well-known 85 kDa Ca²⁺-dependent cytosolic phospholipase A₂ that has recently been cloned and is not plasmalogen-selective. The plasmalogen-selective phospholipase A₂ may be regulated by glycosaminoglycans and sialoglycoconjugates and may be involved in the regulation of K⁺ channels. This enzyme, which plays a major role in the release of fatty acids during ischemic injury and reperfusion, shows promise as a major target for drug therapy.     

Role of phospholipase A2 on the variations of the choline signal intensity observed by H magnetic resonance spectroscopy in brain diseases

Phospholipase A₂ catalyzes the hydrolysis of membrane glycerophospholipids leading to the production of metabolites observable by both ¹H and ³¹P magnetic resonance spectroscopy. The signal of choline-containing compounds (Cho) observed by ¹H magnetic resonance spectroscopy is constituted of metabolites of phosphatidylcholine, especially phosphocholine (PCho) and glycerophosphocholine (GPCho). The phosphomonoester (PME) and phosphodiester (PDE) signals observed by ³¹P magnetic resonance spectroscopy are, respectively, precursors and catabolites of phospholipids. A large number of brain diseases have been reported to cause variations in the intensity of the Cho, PME and PDE signals. Changes in the activity of phospholipase A₂ have been measured in many brain diseases. In this review, the relationships between the results of ¹H and ³¹P magnetic resonance spectroscopy and the phospholipase A₂ assays are analyzed. In many brain diseases, the variation in the Cho signal intensity can be correlated with a stimulation or inhibition of the phospholipase A₂ activity.     

Effects of chronic administration of caffeine on adenosine A1 and A2 receptors in rat brain

Chronic administration of caffeine (75 mg/kg/day) to rats for 12 days increased [3H]R-PIA binding in the cerebral cortex and cerebellum and [3H]NECA binding to high affinity receptor sites in the striatum. The results indicate that both adenosine A1 and A2 receptor subtypes possess mechanisms of adaptation to chronic caffeine treatment. In addition, adenosine A1 receptor binding shows heterogenous neuroanatomical pattern indicating that the A1 response to caffeine treatment presents regional variation in the rat brain.     

Mechanism of phospholipase A2-induced conduction block in bullfrog

The effects of exogenously added phospholipase A₂ (PLA₂) and its hydrolytic products in isolated bullfrog sciatric nerve were investigated. Nerves were pretreated for 3 h with a dose of trypsin which did not affect conduction in order to enhance penetration of the added agents. Threatment of nerves with β-glucosidase, neuraminidase or chymotrypsin had no effect on conduction. Whereas incubatin of the nerves with normal Ringers for 2 h had no significant effect on conduction, incubation with PAL₂ in Ringers caused decrements in the height of the compound action potential in dose-related manner. In addition, incubation of the nerves with 10 mg/ml lysolecithin, arachidonic acid, or docosahexaenoic acid caused marked decrements in the height of the compound action potential. Electron microscopic analysis of nerves after each treatment which caused conduction block revealed varying levels of myelin damage. Although myelin was damaged at the paranodal and/or internodal region, depending on the agents used, the axonal membrane appeared to be intact at the ultrastructural level. It was concluded that the block in conduction resulting from PLA₂ was due to the formation of lysolecithin and long chain polyunsaturated fatty acids.     

Inhibition of brain protein synthesis suppresses the release of prostaglandin E2 in febrile rabbits

In rabbits the third cerebral ventricle was perfused using a push-pull cannula. Prostaglandin E2 concentration in the perfusate was measured by radioimmunoassay. Prostaglandin concentration rose during fever induced by an intraventricular injection of endogenous pyrogen. Both fever and the increased prostaglandin concentration were suppressed by the intraventricular injection of 100 micrograms of the protein synthesis inhibitor, anisomycin. A possible interpretation of the findings is that anisomycin inhibits the formation of phospholipase A2. If this is true, the implication is that phospholipase A2 has a rapid turnover in brain.     

Regulation of 5-HT1A and 5-HT1B receptor systems by phospholipid signaling cascades

The 5-HT_1A and 5-HT_1B receptor systems play central roles in the control of serotonergic neurotransmission and feature prominently in many behaviors and physiological functions. In addition, the regulation of these receptors and their effector mechanisms has been the focus of intense interest because of their potential importance in the therapeutic actions of anxiolytic and antidepressant drugs. Here we describe the regulation of 5-HT_1A and 5-HT_1B receptor-mediated inhibition of adenylyl cyclase activity by receptors which activate phospholipid signaling cascades. Although it might be expected that these two highly homologous Gi-coupled receptors would be regulated similarly by activation of phospholipase C (PLC) and phospholipase A₂ (PLA₂), we have found that the regulation differs markedly between these receptor systems. Further, our data suggest that the modulation of agonist efficacy at these receptor subtypes is dependent on the nature of receptor coupling to PLC and PLA₂ activation. Moreover, regulation at the level of the effector (e.g., adenylyl cyclase) appears to play a significant role in the regulation of both the 5-HT_1A and 5-HT_1B receptor systems by the PLA₂ signaling cascade. Such data illustrate multiple levels for control of biochemical signaling cascades within cells and demonstrate that although different receptors may couple to the same effector pathways, the ultimate cellular effects produced by these receptors may differ due to differential cross-talk regulation.     

Characterization of phospholipase A2 secretion from human platelets

Human platelets secreted phospholipase A₂ in a dose- and time-dependent manner when challenged with thrombin, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), or collagen. Enzyme release was maximal at concentrations of 0.1 units/ml of thrombin, 100 nM TPA, or 2 μg/ml of collagen; and complete by 2 min in platelets treated with thrombin or TPA. Cells challenged with collagen required up to 5 min for maximal secretion. Besides dose and time functions, phospholipase A₂ secretion was also dependent on platelet concentration and the levels of bovine serum albumin in the incubation medium. The secreted enzyme was soluble and exhibited substrate and Ca²⁺ requirements similar to a detergent-solubilized, partially purified phospholipase A₂ from whole platelets [Kramer et al., Biochim. Biophys. Acta (1988) 959, 269–279]. The pH optimum of the secreted enzyme, however, was 1–2 units lower than the pH optimum of the phospholipase A₂ from whole cells. Secreted phospholipase A₂ hydrolyzed phosphatidylethanolamine at 5–12 times the rate of phosphatidylcholine when the substrates were present in pure form. These apparent differences in activity were greatly diminished, though, when 1:1 molar mixtures of the two substrates were employed. Because phospholipase A₂ catalyzes a key reaction during the formation of bioactive arachidonate metabolites, the secretion of this enzyme from platelets may be important in the regulation of thrombosis.     

Dopamine denervation leads to an increase in the intramembrane interaction between adenosine A2 and dopamine D2 receptors in the neostriatum

We have previously found, in striatal membrane preparations from young (2 months old) rats, that stimulation of adenosine A₂ receptors (with the selective adenosine A₂ agonist CGS 21680) increases the dissociation constants of high- (K_h) and low-affinity (K_l) dopamine D₂ binding sites (labelled with the selective dopamine D₂ antagonist [³H]raclopride) without changing the proportion of high affinity binding sites (R_h). In the present study in striatal preparations from adult (6 months old) rats, it was found that in addition to the increase in both K_h and K_l values, stimulation of adenosine A₂ receptors is associated with an increase in R_h. These result suggest that, in the adult rat, adenosine A₂ stimulation may inhibit the behavioural effects induced by dopamine D₂ stimulation both by decreasing the affinity and the transduction of dopamine D₂ receptors. We have also studied the intramembrane A₂-D₂ receptor interaction in an experimental model of Parkinson's disease, namely in rats with a unilateral 6-OH-dopamine-induced lesion of the nigro-striatal dopamine pathway. It was found that a unilateral dopamine denervation is associated with a higher density of striatal dopamine D₂ receptors in the order of 20%, without any change in their affinity compared with the unlesioned neostriatum. Furthermore, the density (B_max values) of dopamine D₂ receptors in the contralateral neostriatum was significantly higher (about 20%) than in the striatum from native animals. This finding suggests that an unilateral dopamine denervation also induces compensatory long-lasting changes of dopamine D₂ receptors in the contralateral neostriatum. In addition to the hightened sensitivity to dopamine agonists, it is known that the dopamine denervated striatum is more sensitive to adenosine antagonists like methylxanthines. If the adenosine A₂-dopamine D₂ interaction is the main mechanism of action mediating the central effects of methylxanthines, the dopamine denervation might also potentiate this interaction, i.e., dopamine D₂ receptors could be not only more sensitive to dopamine but also to adenosine A₂ receptor activation. Our results support this hypothesis, since membrane preparations from the denervated neostriatum are more sensitive to the effect of CGS 21680 on dopamine D₂ receptors. Thus a low dose of CGS 21680 (3 nM), which is not effective in membrane preparations from the neostriatum of naive animals, is still effective in membranes from the denervated neostriatum. These results underline the potential antiparkinsonian activity of adenosine A₂ antagonists.     

Release of choline from rat brain under hypoxia: contribution from phospholipase A2 but not from phospholipase D

Moderate hypoxia induced in rats by inhalation of 10% oxygen led to an increase of the concentration of free choline in the brain and caused a large net-release of choline from the brain into the venous blood as determined by the measurement of the arterio-venous difference. In hippocampal slices from rat brain, hypoxia increased the release of choline into the superfusion medium. The activity of phospholipase D, as measured by the formation of phosphatidylpropanol in the presence of propanol, was not stimulated under these conditions. However, the mobilization of choline was completely depressed by lowering extracellular calcium and by 0.1 mM mepacrine. We conclude that hypoxia leads to a selective activation of phospholipase A₂ in the brain and, consequently, to a net loss of choline-containing phospholipids and membrane structures.     

Investigation of the Relocation of Cytosolic Phospholipase A and Annexin V in Activated Platelets

Cytosolic phospholipase A₂ is a Ca²⁺-dependent enzyme that acts on membrane phospholipids to release arachidonic acid, which in platelets is converted to thromboxane A₂. Annexin V is a Ca²⁺-dependent, phospholipid-binding protein, which is proposed to regulate inflammation by inhibiting cytosolic phospholipase A₂. Here, we have studied the association of cytosolic phospholipase A₂ and annexin V with platelet membranes after thrombin stimulation. In a time-dependent manner, an exact correlation was found between the membrane association of cytosolic phospholipase A₂ and annexin V. Calcium from the intracellular stores was sufficient for the relocation of intracellular annexin V and cytosolic phospholipase A₂ to platelet membranes. Activation in the presence of arginyl-glycyl-aspartyl-serine (RGDS), which inhibits binding of fibrinogen to its adhesive ligand, does not alter the amount of cytosolic phospholipase A₂ or annexin V that binds to membranes. When activation-induced actin polymerisation was prevented by cytochalasin E, the recovery of both annexin V and cytosolic phospholipase A₂ remained unchanged. However, complete depolymerisation of the cytoskeleton with DNase I almost abolished the association of cytosolic phospholipase A₂ with the membranes, and it completely abolished the relocation of annexin V to platelet membranes. Finally, we show that cytosolic phospholipase A₂ can be specifically purified from platelet membranes by affinity chromatography on GST-annexin V and that immunoprecipitation using antibodies against cytosolic phospholipase A₂ copurify annexin V and cytosolic phospholipase A₂ from activated platelets. These findings suggest that following platelet activation with thrombin, both cytosolic phospholipase A₂ and annexin V, relocate to platelet membranes where they interact. An intact cytoskeleton seems to be a prerequisite for the interaction of cytosolic phospholipase A₂ and annexin V with platelet membranes. The incorporation of cytosolic phospholipase A₂ into the membrane fraction of thrombin-activated platelets parallels that of annexin V, which suggests an interaction between the two proteins.