Serial Review: Redox-Regulated Phospholipase Signal Transduction
Serial Review Editors: Henry J. Forman, Viswanathan Natarajan
Phospholipase A2, reactive oxygen species, and lipid peroxidation in cerebral ischemia

https://doi.org/10.1016/j.freeradbiomed.2005.08.044Get rights and content

Abstract

Ischemic stroke is caused by obstruction of blood flow to the brain, resulting in energy failure that initiates a complex series of metabolic events, ultimately causing neuronal death. One such critical metabolic event is the activation of phospholipase A2 (PLA2), resulting in hydrolysis of membrane phospholipids and release of free fatty acids including arachidonic acid, a metabolic precursor for important cell-signaling eicosanoids. PLA2 enzymes have been classified as calcium-dependent cytosolic (cPLA2) and secretory (sPLA2) and calcium-independent (iPLA2) forms. Cardiolipin hydrolysis by mitochondrial sPLA2 disrupts the mitochondrial respiratory chain and increases production of reactive oxygen species (ROS). Oxidative metabolism of arachidonic acid also generates ROS. These two processes contribute to formation of lipid peroxides, which degrade to reactive aldehyde products (malondialdehyde, 4-hydroxynonenal, and acrolein) that covalently bind to proteins/nucleic acids, altering their function and causing cellular damage. Activation of PLA2 in cerebral ischemia has been shown while other studies have separately demonstrated increased lipid peroxidation. To the best of our knowledge no study has directly shown the role of PLA2 in lipid peroxidation in cerebral ischemia. To date, there are very limited data on PLA2 protein by Western blotting after cerebral ischemia, though some immunohistochemical studies (for cPLA2 and sPLA2) have been reported. Dissecting the contribution of PLA2 to lipid peroxidation in cerebral ischemia is challenging due to multiple forms of PLA2, cardiolipin hydrolysis, diverse sources of ROS arising from arachidonic acid metabolism, catecholamine autoxidation, xanthine oxidase activity, mitochondrial dysfunction, activated neutrophils coupled with NADPH oxidase activity, and lack of specific inhibitors. Although increased activity and expression of various PLA2 isoforms have been demonstrated in stroke, more studies are needed to clarify the cellular origin and localization of these isoforms in the brain, their responses in cerebral ischemic injury, and their role in oxidative stress.

Section snippets

Stroke is a major clinical problem

The leading cause of long-term disability and third leading cause of death, stroke, or “brain attack” continues to be a problem of vast clinical significance. It is estimated that more than 700,000 Americans suffer from stroke each year, and approximately 4.7 million Americans are stroke survivors. The economic impact of stroke amounts to more than $33 billion in direct health care costs annually, with an additional cost of $21 billion due to loss of productivity [1].

Ischemic stroke

Cerebral ischemia or stroke is characterized by an obstruction of blood flow to the brain. There are essentially two types of stroke: (1) global or total loss of blood flow to the brain caused by events such as cardiac arrest, or (2) focal (regional) arising from local interruption of blood flow to the brain due to an artery blockage. Animal models [2] have been developed to mimic the human condition. The global model involves the occlusion of bilateral common carotid and vertebral arteries,

Energy failure is the initial metabolic event in stroke

The energy needs of the brain are supplied by metabolism of glucose and oxygen for the phosphorylation of ADP to ATP. Most of the ATP generated is utilized in the brain in maintaining intracellular homeostasis and transmembrane ion gradients of sodium, potassium, and calcium. Energy failure results in rapid loss of ATP and uncontrolled leakage of ions across the cell membrane that results in membrane depolarization and release of the neurotransmitters such as glutamate and dopamine [3], [4].

Reactive oxygen species (ROS) contribute to stroke injury

ROS including hydrogen peroxide (H2O2) and superoxide radical (O2radical dot) are produced by a number of cellular oxidative metabolic processes involving xanthine oxidase, NAD(P)H oxidases, metabolism of arachidonic acid by cyclooxygenases and lipoxygenases, monoamine oxidases, and the mitochondrial respiratory chain. The involvement of phospholipase A2 (PLA2) in ROS formation is outlined in Fig. 1. ROS can also be formed nonenzymatically, for example, by autoxidation of catecholamines [3], [10].

ROS and reactive nitrogen species (RNS) initiate lipid peroxidation

The highly reactive hydroxyl radical (radical dotOH) is not produced as a by-product of any known enzymatic reaction, but is formed from H2O2 (in itself not highly reactive) in the presence of divalent metal ions, especially Fe2+ and Cu2+, via the Fenton reaction [14]. Once formed, radical dotOH reacts almost instantaneously with many cellular components, including polyunsaturated fatty acids of membrane lipids. The initial reaction of radical dotOH with polyunsaturated fatty acids produces an alkyl radical, which in turn

Lipid peroxidation products can alter cellular function

Peroxidation of lipids can disrupt the organization of the membrane, causing changes in fluidity and permeability, inhibition of metabolic processes, and alterations of ion transport [20]. Damage to mitochondria induced by lipid peroxidation can lead to further ROS generation [21]. In addition, lipid peroxides degrade to reactive aldehyde products, including malondialdehyde (MDA), 4-hydroxynonenal (HNE), and acrolein [22], [23], [24], [25]. These aldehydes in turn covalently bind to proteins

Global cerebral ischemia

The gerbil model of transient forebrain ischemia is characterized by delayed hippocampal CA1 neuronal death. Neurodegeneration is evident by 3 days reperfusion and neuronal death culminates by 6 days [26], [27]. Five minutes of forebrain ischemia and 6 h reperfusion in gerbil resulted in significantly increasing the levels of MDA, HNE, and lipid hydroperoxides in the cortex, striatum, and hippocampus and thus preceded the onset of neuronal death [28]. These increases persisted over 4 days

PLA2 enzymes occur in multiple forms

PLA2 enzymes cleave fatty acids at the sn-2 position of glycerophospholipids to give free fatty acids and lysophospholipids. The reaction is of particular importance when the fatty acid released from the sn-2 position is arachidonic acid, since it can be metabolized by cyclooxygenases and lipoxygenases to various bioactive eicosanoids, including prostaglandins, thromboxanes, and leukotrienes. The other PLA2 products, lysophospholipids, are also biologically active and are important in cell

In vitro

One in vitro model of transient cerebral ischemia uses organotypic cultures of hippocampal slices. Ischemia is mimicked by incubation in an oxygen-depleted atmosphere in culture media free of glucose (oxygen glucose deprivation, or OGD). PLA2 activity increased by 2-fold as compared to controls in the hippocampal pyramidal cell layer immediately after 35 min of OGD and remained elevated at 24 h reoxygenation. PLA2 activity in the dentate gyrus granule cell layer was twice as high as in the

Forebrain ischemia

PLA2 activities significantly increased in cytosolic, mitochondrial, and microsomal fractions after 10 min of global cerebral ischemia in gerbils and 10 min of reperfusion. The specific PLA2 isoforms which increased after ischemia/reperfusion were not characterized; however, the majority of PLA2 activity was calcium dependent. Separation of the cytosolic fraction by gel-filtration chromatography gave two peaks of PLA2 activity at 14 and 60 kDa; mitochondrial and microsomal fractions gave a

PLA2 and lipid peroxidation

Some of the sources of ROS in cerebral ischemia are summarized in Fig. 1. During energy failure in ischemia, AMP increases and is metabolized to hypoxanthine via the purine salvage pathway. With reoxygenation of brain tissue, hypoxanthine is metabolized to xanthine and uric acid by xanthine oxidase, with the generation of O2radical dot [102]. The action of PLA2 contributes indirectly to the generation of ROS and lipid peroxides by releasing arachidonic acid, which is then oxidatively metabolized by

PLA2 contributes to ischemic injury

The PLA2 inhibitor quinacrine attenuated neurological deficits and decreased infarction following 2 h of transient focal cerebral ischemia [98]. Quinacrine also attenuated CA1 neuronal death in transient forebrain ischemia in gerbil [97]. These studies provide evidence that PLA2 contributes to ischemic injury; however, since quinacrine is a general PLA2 inhibitor, these results do not indicate which PLA2 form was involved in the ischemic injury [98]. Indirect evidence has been provided by

Conclusions

The published findings, so far, reveal increases in cPLA2 (activity, mRNA expression, and immunoreactivity) and sPLA2 (activity and mRNA expression) in stroke. The contribution of cPLA2 to ischemic injury has been demonstrated in gene knockout studies [95], [96]. However, several areas are in need of further studies. While iPLA2s are generally regarded as housekeeping enzymes in phospholipid remodeling [54], their role in cerebral ischemia needs to be investigated. A neuronal sPLA2 receptor has

Acknowledgments

This study was supported in part by grants to RMA from NIH/NINDS (NS42008), UW-Medical School, and UW-graduate school, and laboratory resources were provided by William S. Middleton VA Hospital. We thank Prof. R. Dempsey for the support and encouragement, Drs. F. Tsao and E. Larsen and Mr. X. Chen for assistance in preparation of this review.

Dr. Rao Adibhatla received his Ph.D. in 1984 from Indian Institute of Science, Bangalore, India. He did postdoctoral training at CCMB (India), University of Iowa, University of Missouri, and University of Minnesota and was recipient of an Alexander von Humboldt fellowship (1987–1988). He was research faculty at University of Kentucky (1994–1995) and later moved to University of Wisconsin in 1995 where he is currently a research faculty in the Medical School. Research areas of interest include

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    Dr. Rao Adibhatla received his Ph.D. in 1984 from Indian Institute of Science, Bangalore, India. He did postdoctoral training at CCMB (India), University of Iowa, University of Missouri, and University of Minnesota and was recipient of an Alexander von Humboldt fellowship (1987–1988). He was research faculty at University of Kentucky (1994–1995) and later moved to University of Wisconsin in 1995 where he is currently a research faculty in the Medical School. Research areas of interest include (1) lipid metabolism and phospholipid homeostasis in stroke, (2) signal transduction mechanisms in CNS trauma. He has been an invited guest speaker at MIT, NIH, and Ferrer Grupo, Barcelona, Spain/Elder Pharmaceuticals, India, on “CDP-choline mechanisms and efficacy in stroke. Publications include 60 peer-reviewed papers. Awarded an NIH RO1 grant for his research on CDP-choline in stroke. Ad hoc reviewer on more than 15 neurochemistry/neuroscience journals. Member of the National Brain Committee, American Heart Association peer review study section.

    Mr. James Hatcher obtained his B.Sc. in chemistry from Iowa State University, Ames, Iowa, in 1973. After more than 20 years in cancer research, he joined Dr. Adibhatla's lab in 1997. He is coauthor on 48 peer-reviewed papers including 23 since joining Dr. Adibhatla's lab.

    This article is part of a series of reviews on “Redox-Regulated Phospholipase Signal Transduction.” The full list of papers may be found on the home page of the journal.

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