A potentially critical role of phospholipases in central nervous system ischemic, traumatic, and neurodegenerative disorders.

Phospholipases are a diverse group of enzymes whose activation may be responsible for the development of injury following insult to the brain. Amongst the numerous isoforms of phospholipase proteins expressed in mammals are 19 different phospholipase A2's (PLA2s), classified functionally as either secretory, calcium dependent, or calcium independent, 11 isozymes belonging to three structural groups of PLC, and 3 PLD gene products. Many of these phospholipases have been identified in selected brain regions. Under normal conditions, these enzymes regulate the turnover of free fatty acids (FFAs) in membrane phospholipids affecting membrane stability, fluidity, and transport processes. The measurement of free fatty acids thus provides a convenient method to follow phospholipase activity and their regulation. Phospholipase activity is also responsible for the generation of an extensive list of intracellular messengers including arachidonic acid metabolites. Phospholipases are regulated by many factors including selective phosphorylation, intracellular calcium and pH. However, under abnormal conditions, excessive phospholipase activation, along with a decreased ability to resynthesize membrane phospholipids, can lead to the generation of free radicals, excitotoxicity, mitochondrial dysfunction, and apoptosis/necrosis. This review evaluates the critical contribution of the various phospholipases to brain injury following ischemia and trauma and in neurodegenerative diseases.

Effects of adenosine receptor antagonists on pial arteriolar dilation during carbon dioxide inhalation.

The role of adenosine in the cerebrovascular response to carbon dioxide inhalation was evaluated in two sets of experiments. The pial circulation was recorded by a Laser-Doppler flow probe placed over a closed cranial window in methoxyflurane anesthetized rats. Topical application of the nonselective adenosine receptor antagonist caffeine (1 mM), the selective A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX,1 microM), or the selective A2A receptor antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a]triazin-5-yl amino]ethyl) phenol (ZM 241385, 1 microM) all failed to affect mean arterial blood pressure, basal cerebral blood flow, or the carbon dioxide-evoked hyperemia. Systemically administered caffeine (20 mg/kg) also had no significant effects. However, following the systemic administration of the nonselective nitric oxide synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME, 20 mg/kg), the topical application of both caffeine and ZM 241385 (but not DPCPX) significantly reduced the carbon dioxide-evoked hyperemia. L-NAME (20 mg/kg) administered intravenously, evoked a significant increase in mean arterial blood pressure, a slow progressive decline in cerebral blood flow and, during brief (60-90 s) periods of 7.5% carbon dioxide inhalation, a significant decrease in arterial blood pressure. L-NAME failed to reduce the carbon dioxide-evoked increase in cerebral blood flow as measured by the area under the curve (AUC), although it did reduce the peak flow response. Topically applied L-NAME (1 mM) failed to alter mean arterial blood pressure, basal cerebral blood flow, or the carbon dioxide-evoked increases in cerebral blood flow. In a second series of experiments, we evaluated the ability of 10% carbon dioxide inhalation for 8 min to elicit a release of adenosine from the cerebral cortex. Adenosine levels in the cortical superfusates rose significantly during periods of carbon dioxide inhalation. The data suggest that following the removal of the confounding effects of nitric oxide, which are unlikely to be mediated locally, a significant contribution by adenosine A2A receptor activation to the carbon dioxide-evoked cortical hyperemia was evident.

The role of phospholipases, cyclooxygenases, and lipoxygenases in cerebral ischemic/traumatic injuries.

Free fatty acids (FFAs) are elevated in the brain following both ischemic and traumatic injury. Phospholipase activation, with the subsequent release of FFAs from membrane phospholipids, is the likely mechanism. In addition to phospholipases A1, B, C, and D, there are at least 19 groups of PLA2, including multiple cytosolic, calcium independent, and secretory isoforms. Phospholipase activity can be regulated by calcium, by phosphorylation, and by agonists binding to G-protein-coupled receptors. These enzymes normally function in the physiological remodeling of cellular membranes, whereby FFAs are removed by phospholipase activity and then reacylated with a different FFA. However, reductions in the cell's ability to maintain normal metabolic function and the resultant fall in ATP levels can cause the failure of reacylation of membrane phospholipids. Alterations to membrane phospholipids would be expected to compromise many cellular functions, including the ability to accumulate excitotoxic amino acids. This review presents evidence for a central role of phospholipases and their products in the etiology of damage following injury to the brain. Phospholipase expression and activity is increased in animal models of cerebral ischemia and trauma. FFA release from the in vivo rat brain is reduced following the application of selective phospholipase inhibitors, and this inhibition also decreases the severity of cortical damage following forebrain ischemia, focal (middle cerebral artery occlusion) ischemia, and cerebral trauma. Mice with knockouts of PLA2 have decreased infarct volumes. Human data demonstrate a correlation between the elevation of CSF FFAs and worsened outcome following stroke, traumatic brain injury, and subarachnoid hemorrhage. The released FFAs, especially arachidonic and docosahexaenoic acids, together with the production of lysophospholipids, can initiate a chain of events which may be responsible for the development of neuronal damage. Inhibitors of both cyclooxygenase and lipoxygenase pathways have been shown to reduce cerebral deficits following ischemia and trauma. These results suggest therapeutic strategies to reduce morbidity following cerebral injury using selective inhibitors of phospholipases, cyclooxygenases, and lipoxygenases, underlining the need for further investigation of their role in the development of cerebral damage.

Free fatty acids in cerebrospinal fluids from patients with traumatic brain injury.

Free fatty acid (FFA) concentrations in cerebrospinal fluid (CSF) are recognized as markers of brain damage in animal studies. There is, however, relatively little information regarding FFA concentrations in human CSF in normal and pathological conditions. The present study examined FFA concentrations in CSF from 15 patients with traumatic brain injury (TBI) and compared the data with values obtained from 73 contemporary controls. Concentrations of specific FFAs from TBI patients, obtained within 48 h of the insult were significantly greater than those in the control group (arachidonic, docosahexaenoic and myristic, P<0.001; oleic, palmitic, P<0.01; linoleic, P<0.05). Higher concentrations of total polyunsaturated fatty acids (P<0.001) and of arachidonic, myristic and palmitic acids measured individually in CSF (P<0.01) obtained 1 week after the insult were associated with a worse outcome at the time of hospital discharge using the Glasgow Outcome Scale. This preliminary investigation suggests that CSF FFA concentrations may be useful as a predictive marker of outcome following TBI.

Free fatty acids in human cerebrospinal fluid following subarachnoid hemorrhage and their potential role in vasospasm: a preliminary observation.

OBJECT: The mechanisms leading to vasospasm following subarachnoid hemorrhage (SAH) remain unclear. Accumulation in cerebrospinal fluid (CSF) of free fatty acids (FFAs) may play a role in the development of vasospasm; however, in no previous study have concentrations of FFAs in CSF been examined after SAH. METHODS: We collected samples of CSF from 20 patients with SAH (18 cases of aneurysmal SAH and two cases of spontaneous cryptogenic SAH) and used a high-performance liquid chromatography assay to determine the FFA concentrations in these samples. We then compared these findings with FFA concentrations in the CSF of control patients. All FFA concentrations measured 24 hours after SAH were significantly greater than control concentrations (p < 0.01 for palmitic acid and < 0.001 for all other FFAs). All measured FFAs remained elevated for the first 48 hours after SAH (p < 0.05 for linoleic acid, p < 0.01 for palmitic acid, and p < 0.001 for the other FFAs). After 7 days, a second elevation in all FFAs was observed (p < 0.05 for linoleic acid, p < 0.01 for palmitic acid, and p < 0.001 for the other FFAs). Samples of CSF collected within 48 hours after SAH from patients in whom angiography and clinical examination confirmed the development of vasospasm after SAH were found to have significantly higher concentrations of arachidonic, linoleic, and palmitic acids than samples collected from patients in whom vasospasm did not develop (p < 0.05). CONCLUSIONS: Following SAH, all FFAs are initially elevated. A secondary elevation occurs between 8 and 10 days after SAH. This study provides preliminary evidence of FFA elevation following SAH and of a potential role for FFAs in SAH-induced vasospasm. A prospective study is warranted to determine if CSF concentrations of FFAs are predictive of vasospasm.

Inhibition of mitochondrial Na(+)/Ca(2+) exchange by 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one attenuates free fatty acid efflux in rat cerebral cortex during ischemia-reperfusion injury.

We evaluated the effects of 7-chloro-5-(2-chlorophenyl)-1,5-dihydro-4,1-benzothiazepin-2(3H)-one (CGP-37157) (50 muM), a specific inhibitor of mitochondrial Na(+)/Ca(2+) exchange, applied topically onto rat cerebral cortex during ischemia-reperfusion injury. Free fatty acid (FFA) levels in cortical superfusates, withdrawn at 10 min intervals from bilateral cortical windows, were analyzed by high performance liquid chromatography. During a 20 min period of ischemia in control animals, there were significant increases in all FFAs. Following reperfusion, FFA levels remained significantly elevated. Application of CGP-37157 significantly inhibited effluxes of all FFAs during both ischemia and reperfusion. These data indicate that inhibition of mitochondrial Na(+)/Ca(2+) exchange likely prevented the activation of phospholipases that usually occurs following an ischemic insult as evidenced by its attenuation of FFA efflux.