Breakdown Of Membrane Phospholipids Research Articles

Increased PLA2 activity in individuals at ultra-high risk for psychosis

Phospholipase A2 is the main enzyme in the metabolism of membrane phospholipids. It comprises a family of enzymes divided into iPLA2, cPLA2 and sPLA2. Studies have reported increased PLA2 activity in psychotic patients, which suggests an accelerated breakdown of membrane phospholipids. In the present study we investigated whether increased PLA2 activity is also present in individuals at ultra-high risk (UHR) for psychosis. One-hundred fifty adults were included in this study (85 UHR and 65 controls). UHR was assessed using the "structured interview for prodromal syndromes". PLA2 activity was determined in platelets by a radio-enzymatic assay. We found in UHR individuals increased activities of iPLA2 (p < 0.001) and cPLA2 (p = 0.012) as compared to controls. No correlations were found between socio-demographic and clinical parameters and PLA2 activity. Our findings suggest that increased PLA2 activities may be useful as a biological risk-marker for psychotic disorders.

S218. A Potential Therapeutic Target of Plasma Free Fatty Acids in First-Episode Antipsychotic-Naïve Patients With Schizophrenia

Previous findings indicated that schizophrenia (SZ) patients have increased breakdown of membrane phospholipids. The present study is thus to test whether the resulting plasma FFAs are altered at early course of disease development, and if so, whether such changes can be affected by treatment of atypical antipsychotic drugs (AAPD).

Lipid catabolism inhibition sensitizes prostate cancer cells to antiandrogen blockade.

Prostate cancer (PCa) is the most common malignancy among Western men and the second leading-cause of cancer related deaths. For men who develop metastatic castration resistant PCa (mCRPC), survival is limited, making the identification of novel therapies for mCRPC critical. We have found that deficient lipid oxidation via carnitine palmitoyltransferase (CPT1) results in decreased growth and invasion, underscoring the role of lipid oxidation to fuel PCa growth. Using immunohistochemistry we have found that the CPT1A isoform is abundant in PCa compared to benign tissue (n=39, p<0.001) especially in those with high-grade tumors. Since lipid oxidation is stimulated by androgens, we have evaluated the synergistic effects of combining CPT1A inhibition and anti-androgen therapy. Mechanistically, we have found that decreased CPT1A expression is associated with decreased AKT content and activation, likely driven by a breakdown of membrane phospholipids and activation of the INPP5K phosphatase. This results in increased androgen receptor (AR) action and increased sensitivity to the anti-androgen enzalutamide. To better understand the clinical implications of these findings, we have evaluated fat oxidation inhibitors (etomoxir, ranolazine and perhexiline) in combination with enzalutamide in PCa cell models. We have observed a robust growth inhibitory effect of the combinations, including in enzalutamide-resistant cells and mouse TRAMPC1 cells, a more neuroendocrine PCa model. Lastly, using a xenograft mouse model, we have observed decreased tumor growth with a systemic combination treatment of enzalutamide and ranolazine. In conclusion, our results show that improved anti-cancer efficacy can be achieved by co-targeting the AR axis and fat oxidation via CPT1A, which may have clinical implications, especially in the mCRPC setting.

Neuroprotective effect of 2-hydroxy arachidonic acid in a rat model of transient middle cerebral artery occlusion

Stroke modifies the composition of cell membranes by eliciting the breakdown of membrane phospholipids whose products, such as arachidonic acid (AA), are released in the cytosol. The action of enzymes such as cyclooxygenases on AA leads to inflammatory stimuli and increases the cell oxidative stress. We report here the neuroprotective effect of 2-hydroxyarachidonic acid (2OAA), a cyclooxygenase inhibitor derived from AA, as a promising neuroprotective therapy against stroke.The effect of a single dose of 2OAA, administered intragastrically 1h after the ischaemic insult, in a rat model of transient middle cerebral artery occlusion (tMCAO) was tested after 24h of reperfusion. Infarct volume was measured by TTC method to evaluate the neuroprotective effect. Levels of phospholipids and neutral lipids were measured by thin-layer chromatography. The expression of cPLA2 and sPLA2 phospholipases responsible for the cleavage of membrane phospholipids, as well as the expression of antioxidant enzymes, was measured by qPCR. Lipid peroxidation was measured as the concentration of malondialdehyde and 4-hydroxynonenal.The treatment with 2OAA reduced the infarct volume and prevented ischaemia-induced increases in transcription levels of free fatty acid (FFAs), as well as in both phospholipases A2 (cPLA2 and sPLA2). The lipid peroxidation and the transcription levels of antioxidant enzymes induced by ischaemia were also decreased by this treatment.We conclude that 2OAA treatment results in a strong neuroprotective effect that seems to rely on a decrease in PLA2 transcriptional activity. This would reduce their action on the membrane phospholipids reducing reactive oxygen and nitrogen species generated by FFAs. Based on the transcriptional activity of the antioxidant enzymes, we conclude that the treatment prevents oxidative stress rather than promoting the antioxidant response. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá.

Combination of direct infusion mass spectrometry and gas chromatography mass spectrometry for toxicometabolomic study of red blood cells and serum of mice Mus musculus after mercury exposure.

Although mercury (Hg) is an important environmental and occupational pollutant, its toxicological effects, especially in serum and red blood cells (RBCs), have been scarcely studied. A toxicometabolomics workflow based on high resolution mass spectrometry approaches has been applied to investigate the toxicological effects of Hg in Mus musculus mice after subcutaneous injection for 10 days, which produced inflammation and vacuolization, steatosis and karyolysis in the hepatic tissue. To this end, direct infusion mass spectrometry (DIMS) of polar and lipophilic extracts from serum and RBCs, using positive and negative mode of acquisition (ESI+/ESI-), and gas chromatography-mass spectrometry were used. A quantitative analysis of reversible oxidized thiols in serum proteins demonstrated a strong oxidative stress induction in the liver of Hg-exposed mice. Endogenous metabolites alterations were identified by partial least squares-discriminant analysis (PLS-DA). Mercury-exposed mice show perturbations in energy metabolism, amino acid metabolism, membrane phospholipid breakdown and oxidative stress-related metabolites in serum along the exposure. This work reports for the first time the effects of Hg-exposure on RBCs metabolic pathways, and reveals disturbances in glycolysis, membrane turnover, glutathione and ascorbate metabolisms.

Use of metallomics and metabolomics to assess metal pollution in Doñana National Park (SW Spain).

Monitoring organism exposure to heavy metals has acquired increased importance in the last decades. The mouse Mus spretus has been used to assess the biological response to contaminants in the relevant ecological area of Doñana National Park (DNP) and surrounding areas (SW Spain), where many migrating birds land for breeding and feeding every year. A metallomics approach, based on the characterization of metal biomolecules using size exclusion chromatography coupled with inductively coupled plasma-mass spectrometry (SEC-ICP-MS) and a metabolomics approach based on direct infusion to a mass spectrometer (DI-ESI-QTOF-MS) followed by a partial linear square-discriminant analysis (PLS-DA), were used to compare the biological responses of M. spretus living in three areas of DNP (the reference) and surrounding areas (El Partido and El Matochal). The activities of key antioxidant enzymes, such as Cu/Zn-SOD, Mn-SOD, CAT, GR, and guaiacol peroxidase, were also determined in connection with environmental contamination issues. The results show differences caused by the presence of metals in the ecosystem that affected to the levels of metals and metalloproteins, such as MT, Cu/Zn-SOD, or Mn-CA, the breakdown of membrane phospholipids, perturbations in metabolic pathways, related to energy metabolism, and oxidative stress.

A combination of metallomics and metabolomics studies to evaluate the effects of metal interactions in mammals. Application to Mus musculus mice under arsenic/cadmium exposure

Arsenic and cadmium are toxic metals of environmental significance with harmful effects on man. To study the toxicological and biochemical effects of arsenic/cadmium in mammals a combined metallomic and metabolomic approach has been developed, complemented with the measurement of biochemical parameters in blood and histopathological evaluation of liver injury in mice Mus musculus under exposure to both xenobiotics. Size-exclusion chromatography (SEC) was combined with affinity chromatography (AF) and ICP-MS detection using species unspecific isotopic dilution analysis (SUID) to characterize the biological effects of As/Cd on selenium containing proteins in the bloodstream of exposed mice. On the other hand, both direct infusion mass spectrometry (DIMS) and gas chromatography-mass spectrometry (GC-MS) provided information about changes in metabolites caused by metals. The results show that As/Cd exposure produces interactions in the distribution of both toxics between organs and plasma of mice and antagonistic interactions with selenium containing proteins in the bloodstream. Interplay with essential metabolic pathways, such as energy metabolism and breakdown of membrane phospholipids were observed, which are more pronounced under As/Cd exposure. In addition, heavy metal and metalloid causes differential liver injury, manifested by steatosis (non-alcoholic fatty liver disease, NAFLD) and infiltration of blood cells into the space of Disse. This work presents new contributions in the study of arsenic/cadmium interactions in mice Mus musculus under controlled exposure. With the combination of metallomic and metabolomic approaches the traffic of As and Cd from liver to kidney by means of blood was observed and excretion of As (as arsenic metabolites) or Cd (as MTCd) is inhibited with the simultaneous administration of As/Cd, and these toxic elements have important influence in the levels of seleno-proteins in the plasma. In addition, the metabolomic approach reveals inhibition of different metabolic cycles such as tricarboxylic acid and phospholipid degradation that causes membrane damage and apoptosis that is histopathologically confirmed. This article is part of a Special Issue entitled: Environmental and structural proteomics.

Signalling between G-protein-coupled receptors and TASK1 channels

This editorial refers to ‘Inhibition of the potassium channel TASK-1 in rat cardiac muscle by endothelin-1 is mediated by phospholipase C’ by J. Schiekel et al ., pp. 97–105, this issue. The superfamily of two pore-domain K+ (K2P) channels has a least 15 members,1 of which mainly TASK1 is expressed in cardiac myocytes. TASK1 gating is voltage independent, but the channel is acid sensitive, being activated by alkalotic conditions. Ion currents carried by TASK1 likely contribute to the cardiac action potential duration (APD), and pharmacological inhibition of TASK1 lengthens the APD2,3 while TASK1 knock-out animals display a long QT on the electrocardiogram.4,5 Interest in TASK1 channels has been mounting, because their activity in various cell types seems critical for several physiological processes while their changed expression and/or function may underlie many disease conditions. The channels are potential targets for diverse therapeutic actions, including antiarrhythmic, antihypertensive, antiepileptic, and antidepressive treatments as well as analgesia and inhalation anaesthesia.6 Therefore, identifying the signalling pathways involved in their regulation is important. Newly available investigation tools, such as those used in the study of Schiekel et al. 7 published in the current issue of Cardiovascular Research , allow one to investigate the signalling pathways for the regulation of TASK1 in intact cells while monitoring cell membrane lipid metabolism. A well-recognized regulation of TASK1 channels is their inhibition by receptors coupled to the G-protein Gp, which is linked to membrane phospholipid breakdown via the activation of phospholipase C (PLC). Neurotransmitters such as norepinephrine (acting on α–adrenergic receptors), hormones such angiotensin or endothelin-1, and other agonists (e.g. platelet-activating factor, PAF) reduce TASK1 ion currents.1 The signalling pathway linking Gp activation to TASK1 inhibition has been a source of controversy, given variable and inconsistent results …

Alterations of Brain Membranes in Schizophrenia: Impact of Phospholipase A2

Physiological enzymatic cleavage of membrane phospholipids by phospholipase A2 (PLA2) results in normal levels of phosphomonoester and phosphodiester, by which a normal dopamine neurotransmission is maintained. Data from postmortem tissue and in vivo imaging studies suggest that increased activity of intracellular calcium-independent PLA2 (iPLA2) in the brain of schizophrenic patients might accelerate the breakdown of membrane phospholipids and alter the properties of neuronal membranes, which in turn contributes to a hypodopaminergy. Alterations in PLA2 activity are probably genetically determined and represent a possible pharmacological target for Schizophrenia.

Insulin-resistant brain state (IRBS) changes membrane composition of fatty acids in temporal and entorhinal brain cortices of rats: relevance to sporadic Alzheimer’s disease?

An insulin-resistant brain state (IRBS) was induced by intracerebroventricular application of the diabetogenic substance streptozotocin (STZ) to investigate changes in cerebral fatty acids. Six weeks after the first STZ injection, increases were found in the concentrations of palmitic and stearic acid in temporal cortex; and of palmitic acid in entorhinal cortex indicative of membrane phospholipid breakdown, and damage of the membrane structure. The relevance of these changes is discussed in relation to sporadic Alzheimer's disease.

Calcium-independent phospholipase A2plays a key role in the endothelium-dependent contractions to acetylcholine in the aorta of the spontaneously hypertensive rat

Phospholipase A(2) (PLA(2)), a regulatory enzyme found in most mammalian cells, catalyzes the breakdown of membrane phospholipids to arachidonic acid. There are two major cytosolic types of the enzyme, calcium-dependent (cPLA(2)) and calcium-independent (iPLA(2)) PLA(2). The present study investigated whether or not iPLA(2) plays a role in the endothelium-dependent contractions of the aorta of the spontaneously hypertensive rat and its normotensive counterpart, the Wistar-Kyoto rat. The presence of iPLA(2) in the endothelial cells was identified by using immunochemistry and immunoblotting. Aortic rings with and without the endothelium were suspended in organ chambers for isometric tension recording. The production of prostanoids was measured by using enzyme immunoassay kits. iPLA(2) was densely distributed in endothelial cells of the aorta of both strains. At 3 x 10(-6) M, the selective iPLA(2) inhibitor, bromoenol lactone (BEL), abrogated endothelium-dependent contractions induced by acetylcholine but not those evoked by the calcium ionophore A-23187. The effects of BEL were similar in the aortae of Wistar-Kyoto and spontaneously hypertensive rats. The nonselective PLA(2) inhibitor quinacrine abolished the contractions triggered by both acetylcholine and A-23187, whereas the store-operated calcium channel inhibitor SKF-96365 prevented only the acetylcholine-induced contraction. The acetylcholine- but not the A-23187-induced release of 6-keto prostaglandin F(1alpha) was inhibited by BEL. The release of thromboxane B(2) by either acetylcholine or A-23187 was not affected by BEL. In conclusion, iPLA(2) plays a substantial role in the generation of endothelium-derived contracting factor evoked by acetylcholine.

Role of glycine receptors and glycine release for the neuroprotective activity of bilobalide

Bilobalide, a constituent of Ginkgo biloba, has neuroprotective properties. Its mechanism of action is unknown but it was recently found to interact with neuronal transmission mediated by glutamate, γ-aminobutyric acid (GABA) and glycine. The goal of this study was to test the interaction of bilobalide with glycine in assays of neuroprotection. In rat hippocampal slices exposed to N-methyl- d-aspartate (NMDA), release of choline indicates breakdown of membrane phospholipids. NMDA-induced choline release was almost completely blocked in the presence of bilobalide (10 µM). Glycine (10–100 µM) antagonized the inhibitory action of bilobalide in this assay. In a second assay of excitotoxicity, we measured tissue water content as an indicator of cytotoxic edema formation in hippocampal slices which were exposed to NMDA. In this assay, edema formation was suppressed by bilobalide but bilobalide's action was attenuated in the presence of glycine and of d-serine (100 µM each). To investigate bilobalide's interaction with glycine receptors directly, we determined 36chloride flux in rat cortico-hippocampal synaptoneurosomes. Glycine (100 µM) was inactive in this assay indicating an absence of functional glycine-A receptors in this preparation. [ 3H]Glycine was used to assess binding at the glycine binding site of the NMDA receptor but bilobalide was found to be inactive in this assay. Finally, [ 3H]glycine release was monitored in hippocampal slices exposed to oxygen-glucose deprivation. In this model, glycine release was induced by ischemia, an effect that was strongly reduced by bilobalide. We conclude that bilobalide does not interact with glycine receptors in neurochemical assays but it significantly reduces the release of glycine under ischemic conditions. This effect likely contributes to bilobalide's neuroprotective effects in assays of excitotoxicity and ischemia.

A 31-phosphorus neurospectroscopy study of ω-3 long-chain polyunsaturated fatty acid intervention with eicosapentaenoic acid and docosahexaenoic acid in patients with chronic refractory epilepsy

The aim of this study was to determine whether supplementation with the n-3 long-chain polyunsaturated fatty acids eicosapentaenoic acid and docosahexaenoic acid in patients with chronic refractory epilepsy is associated with beneficial changes in cerebral biochemistry. In a 3-month pilot randomized double-blind placebo-controlled study, three patients received eicosapentaenoic acid and docosahexaenoic acid daily and four received a placebo. 31-Phosphorus neurospectroscopy showed a decrease in phosphodiesters, an increase in γNTP and an increase in the broadband component in the active group over this period, while the opposite changes occurred in the placebo group. Therefore, in chronic refractory epilepsy, ω-3 supplementation may be associated with reduced membrane phospholipid breakdown in the brain, an improvement in brain energy metabolism, and an increased level of phospholipids in membranes and/or vesicle bilayers in cells in the brain. The unfavourable biochemical changes observed in the placebo group may be a feature of chronic intractable epilepsy.

Role of GABAergic antagonism in the neuroprotective effects of bilobalide

Bilobalide, a constituent of Ginkgo biloba, has neuroprotective properties. Its mechanism of action is unknown but it was recently found to block GABA A receptors. The goal of this study was to test the potential role of a GABAergic mechanism for the neuroprotective activity of bilobalide. In rat hippocampal slices exposed to NMDA, release of choline indicates breakdown of membrane phospholipids. NMDA-induced choline release was almost completely blocked in the presence of bilobalide (10 μM) and under low-chloride conditions. Bicuculline (100 μM), a competitive antagonist at GABA A receptors, reduced NMDA-induced choline release to a small extent (− 23%). GABA (100 μM) partially antagonized the inhibitory action of bilobalide. Exposure of hippocampal slices to NMDA also caused edema formation as measured by increases of tissue water content. NMDA-induced edema formation was suppressed by bilobalide and by low-chloride conditions. Bicuculline exerted partial protection (by 30%) while GABA reduced bilobalide's effect by about one third. To investigate bilobalide's interaction with GABA A receptors directly, we measured binding of [ 35S]-TBPS to rat cortical membranes. TBPS binding was competitively inhibited by bilobalide in the low micromolar range (IC 50 = 3.7 μM). As a functional test, we determined 36chloride flux in rat corticohippocampal synaptoneurosomes. GABA (100 μM) significantly increased 36chloride flux (+ 65%), and this increase was blocked by bilobalide, but with low potency (IC 50: 39 μM). We conclude that, while antagonism of GABA A receptors may contribute to bilobalide's neuroprotective effects, additional mechanisms must be postulated to fully explain bilobalide's actions.

Reduced phospholipid breakdown in Alzheimer’s brains: a 31P spectroscopy study

Abnormalities of membrane phospholipid metabolism have been described in Alzheimer's disease (AD). We investigated, with the aid of (31)P magnetic resonance spectroscopy, the in vivo intracerebral availability of phosphomonoesters (PME) and phosphodiesters (PDE) in patients with AD. Eighteen outpatients with mild or moderate probable AD and 16 nondemented elderly volunteers were assessed with the Cambridge Examination for Mental Disorders of the Elderly (CAMDEX) and its cognitive subscale of the CAMDEX schedule (CAMCOG). Scans were performed on a 1.5 T magnetic resonance imager addressing a 40-cm(3) voxel in the left prefrontal cortex. Main outcome measures were mean relative peak areas of PME and PDE, which provide an estimate of membrane phospholipid metabolism. PME resonance and the PME/PDE ratio were increased in AD patients as compared to controls (p<0.05). PME was negatively correlated with global cognitive performance as shown by the Mini-Mental State Examination (r(s)=-0.36, p=0.05) and CAMCOG scores (r(s)=-0.49, p=0.007), as well as with discrete neuropsychological functions, namely, memory (r(s)=-0.53, p=0.004), visual perception (r(s)=-0.54, p=0.003), orientation (r(s)=-0.36, p=0.05), and abstract thinking (r(s)=-0.48, p=0.01). We provide evidence of reduced membrane phospholipid breakdown in the prefrontal cortex of mild and moderately demented AD patients. These abnormalities correlate with neuropsychological deficits that are characteristic of AD.

The Janus Face of Cyclooxygenase-2 in Ischemic Stroke

The rate-limiting enzyme for prostanoid synthesis cyclooxygenase-2 (COX-2) has been implicated in the basic mechanisms of several brain diseases, including stroke, multiple sclerosis and neurodegenerative diseases.1 The approval by the Food and Drug Administration (FDA) of highly selective COX-2 inhibitors for the treatment of pain and rheumatoid arthritis (RA) raised the possibility that these agents could also be used in the treatment of neurological diseases including stroke. However, the occurrence of serious cardiovascular complications in patients receiving COX-2 inhibitors has led to the recent withdrawal from the market of a popular COX-2 inhibitor and has called for a re-evaluation of the therapeutic potential of these drugs. In this article, we briefly review the role of COX-2 in ischemic brain injury and re-examine the validity of the COX-2 pathway as a therapeutic target for ischemic stroke. COX enzymes catalyze the conversion of arachidonic acid into prostaglandin H2 (PGH2; Figure).2 Arachidonic acid, produced by the breakdown of membrane phospholipids, is metabolized by COX into PGH2 in a 2-step reaction in which the free radical superoxide is also produced.2 Three isoforms of COX have been described. COX-1 is present in most cells and is involved in normal cellular physiology, such as gastric secretion and platelet function.2 COX-2 is expressed constitutively in some organs, such as brain, but is markedly upregulated by a wide variety of stimuli, most notably inflammatory mediators.2 COX-3, a splice variant of COX-1, is highly sensitive to inhibition by acetaminophen and is most abundant in heart and brain.3 The COX reaction product PGH2 is converted into 5 different prostanoids by a corresponding number of isomerases (Figure), the distribution of which is cell and organ specific.2 Thus, PGH2 can give rise to different prostanoids depending on the …

Glycerophosphocholine is elevated in cerebrospinal fluid of Alzheimer patients

Experimental and clinical studies give evidence for breakdown of membrane phospholipids during neurodegeneration. In the present study, we measured the levels of glycerophosphocholine (GPCh), phosphocholine (PCh), and choline, that is, water-soluble metabolites of phosphatidylcholine (PtdCho), in human cerebrospinal fluid (CSF). Among 30 cognitively normal patients the average CSF levels of GPCh, phosphocholine and choline were 3.64, 1.28, and 1.93 μM, respectively; metabolite levels did not change with increasing age. When compared with age-matched controls, patients with Alzheimer’s disease had elevated levels of all choline metabolites: GPCh was significantly increased by 76% ( P<0.01), phosphocholine by 52% ( P<0.05), and free choline (Ch) by 39%. Six patients with vascular dementia had lower choline and elevated phosphocholine levels, when compared to controls, but normal levels of GPCh. These data demonstrate that Alzheimer’s disease is accompanied by an increased PtdCho hydrolysis in the brain. PtdCho breakdown seems to be mediated by phospholipase A 2 and leads to significantly elevated levels of GPCh in CSF.

Current progress in the understanding of and therapeutic strategies for ischemia and reperfusion injury of the liver.

Ischemia and reperfusion (IR) injury of the liver consists of two distinct phases. The first phase is caused by acute cellular injury at 3 to 6 h postreperfusion, which may be mainly induced by the increased production of oxygen radical species. The secondary, subacute, phase results from inflammatory responses at 18 to 24 h, leading to the progression of liver damage. The inflammatory response observed here is caused by proinflammatory cytokines and accumulating neutrophils which secrete oxidants, proteases, and so on. The production of proinflammatory cytokines, including chemokines and adhesion molecules, is regulated by transcriptional factors nuclear factor kappa B (NFKappaB), and AP-1. The progression of the injury is generated by the recruiting leucocytes which release oxidants and proteases. Recruitment and adhesion of neutrophils to the liver are accomplished by multiple steps in which many chemoattractants and adhesion molecules participate. Recent investigations suggest that calcium-dependent proteases, among various kinds of proteases, also play important roles in the aggravation of IR injury. Based on the mechanisms stated above, numerous strategies have been proposed as for prophylaxis and treatment. Most of these therapeutic strategies are derived from the inhibition of the production of oxygen radicals, inflammatory cytokines, and adhesion molecules; inhibition of leucocyte infiltration and elastase production; and inhibition of microcirculatory impairment, apoptosis-related molecules, and the breakdown of membrane phospholipids; and so on. Moreover, recent studies clarified that short periods of ischemia and subsequent reperfusion, termed ischemic preconditioning, exert a preventive effect against IR injuries in various organs, including the liver. Based on clarification of the candidates responsible for this phenomenon, pharmacological ischemic preconditioning has been proposed. In this review article, the authors outline the current progress in the understanding of and therapeutic strategies for hepatic IR injury.

Involvement of B-50 (GAP-43) phosphorylation in the modulation of transmitter release by protein kinase C.

1. Protein kinase C (PKC) is a family of enzymes that is activated by diacylglycerol (DAG) following phospholipase (PL) C activation. Protein kinase C may also be activated by metabolites and arachidonic acid generated by breakdown of membrane phospholipids by PLD and PLA2, respectively. Subsequent to PKC activation, key protein substrates are phosphorylated, resulting in the facilitation of transmitter release. 2. Phorbol esters are compounds that mimic the actions of DAG on PKC and have been shown to facilitate stimulation-induced (S-I) transmitter release in rat brain. However, some phorbol esters that have a high affinity for PKC have no effect on transmitter release, whereas others with a lower affinity for PKC markedly elevate S-I transmitter release. 3. The structure and, more importantly, the lipophilicity of the phorbol esters determines their ability to access and activate the intraneuronal pools of PKC that are involved with transmitter release. In studies in which cell membranes were intact, phorbol esters did not display the characteristics expected based on their affinities for PKC in contrast with studies in disrupted synaptosomes. This supports the hypothesis that the membrane plays a critical role in determining the effects of phorbol esters on PKC. 4. B-50, a PKC substrate thought to be involved in transmitter release, also appears to be differentially phosphorylated by various phorbol esters. The effects on B-50 phosphorylation in intact synaptosomes, but not disrupted synaptosomes, are well correlated with the effects of phorbol esters on S-I transmitter release. 5. B-50 is colocalized with actin, which has also been suggested to play an important role in facilitating the movement of reserve pools of transmitter vesicles to the readily releasable state. Therefore, it is possible that the phosphorylation status of B-50 directly influences the organization of actin filaments, thereby allowing transmitter output to be sustained under high levels of stimulation.

Attenuation of ischemia and reperfusion injury of canine livers by inhibition of type II phospholipase A2 with LY329722.

Membrane phospholipid breakdown, caused by ischemia and reperfusion (I/R) of the liver, releases free fatty acids including arachidonic acids and lysophospholipids, which serve as precursors of various inflammatory lipid derivatives. Phospholipase A2 (PLA2) is a key enzyme that initiates this reaction. In this study, we tested our hypothesis that a type II PLA2 inhibitor, LY329722, could attenuate hepatic I/R injury caused by a 2-hr total hepatic vascular exclusion (THVE) in dogs. Eighteen beagle dogs, subjected to a 2-hr THVE, were divided into three groups. Group 1 (n=6) was untreated and served as a control group. LY329722 was administered to animals in group 2 (n=6) intravenously (0.2 mg x kg(-1) x hr(-1)) for 60 min before ischemia, and to animals in group 3 (n=6) for 60 min starting 15 min before reperfusion (0.2 mg x kg(-1) x hr(-1)). Animal survival, systemic and splanchnic hemodynamics, hepatic tissue blood flow, liver functions, energy metabolism, hepatic venous thromboxane B2 and endothelin-1 levels, phospholipid levels and tumor necrosis factor-a mRNA expression in liver tissue, and histopathologic findings were evaluated. Two-week animal survival was 33% (two of six) in group 1, and 100% (six of six) in groups 2 and 3. LY329722 improved systemic and splanchnic hemodynamics, hepatic tissue blood flow, and energy metabolism, reduced liver enzyme, thromboxane B2, and endothelin-1 release, prevented hepatic phospholipid degradation and tumor necrosis factor-alpha mRNA expression, and lessened histopathologic damage and the number of neutrophil infiltrating into the liver tissue. The present study demonstrated that a type II PLA2 inhibitor, LY329722, attenuated hepatic I/R injury caused by a 2-hr THVE model in dogs.