Nuclear translocation of cytosolic phospholipase A2 is induced by ATP depletion.

Phospholipase A(2) (PLA(2)) enzymes may play a role in cellular injury due to ATP depletion. Renal Madin-Darby canine kidney cells were subjected to ATP depletion to assess the effects of cellular energy metabolism on cytosolic PLA(2) (cPLA(2)) regulation. ATP depletion results in a decrease in soluble cPLA(2) activity and an increase in membrane-associated activity, which is reversed upon restoration of ATP levels by addition of dextrose. In ATP-depleted cells cPLA(2) mass shifts from cytosol to nuclear fractions. GFP-cPLA(2) is localized at the nuclear membrane of stably transfected ATP-depleted LLC-PK(1) cells under conditions where [Ca(2+)](i) is known to increase. cPLA(2) translocation does not occur if the increase in [Ca(2+)](i) increase is inhibited. If [Ca(2+)](i) is allowed to increase when ATP is depleted and the cells are then lysed, cPLA(2) remains associated with nuclear fractions even if the homogenate [Ca(2+)] is markedly reduced. In contrast, cPLA(2), which becomes associated with the nucleus when [Ca(2+)](i) is increased using ionophore, readily dissociates from the nuclear fractions of ATP-replete cells upon reduction of homogenate [Ca(2+)]. Okadaic acid inhibits the ATP depletion-induced association of cPLA(2) with nuclear fractions. Thus energy deprivation results in [Ca(2+)]-induced nuclear translocation, which is partially prevented by a phosphatase inhibitor.

Attenuation of hypoxic pulmonary vasoconstriction by endotoxemia requires 5-lipoxygenase in mice.

Sepsis and endotoxemia impair hypoxic pulmonary vasoconstriction (HPV), thereby reducing systemic oxygenation. To assess the role of leukotrienes (LTs) in the attenuation of HPV during endotoxemia, the increase in left lung pulmonary vascular resistance (LPVR) before and during left mainstem bronchus occlusion (LMBO) was measured in mice with and without a deletion of the gene encoding 5-lipoxygenase (5-LO). LMBO increased the LPVR equally in saline-challenged wild-type and 5-LO-deficient mice (96+/-20% and 94+/-19%, respectively). Twenty-two hours after challenge with Escherichia coli endotoxin, the ability of LMBO to increase LPVR was markedly impaired in wild-type mice (27+/-7%; P<0.05) but not in 5-LO-deficient mice (72+/-9%) or in wild-type mice pretreated with MK886, an inhibitor of 5-LO activity (76+/-10%). Compared with wild-type mice, endotoxin-induced disruption of lung structures and inflammatory cell influx in the lung were markedly attenuated in 5-LO-deficient mice. Administration of MK571, a selective cysteinyl LT(1) receptor antagonist, 1 hour before endotoxin challenge preserved HPV and attenuated pulmonary injury in wild-type mice but did not prevent the endotoxin-induced increase in pulmonary myeloperoxidase activity. Taken together, these findings demonstrate that a 5-LO product, most likely a cysteinyl LT, contributes to the attenuation of HPV and to pulmonary injury after challenge with endotoxin.

Interleukin-1beta-mediated induction of Cox-2 in the CNS contributes to inflammatory pain hypersensitivity.

Inflammation causes the induction of cyclooxygenase-2 (Cox-2), leading to the release of prostanoids, which sensitize peripheral nociceptor terminals and produce localized pain hypersensitivity. Peripheral inflammation also generates pain hypersensitivity in neighbouring uninjured tissue (secondary hyperalgesia), because of increased neuronal excitability in the spinal cord (central sensitization), and a syndrome comprising diffuse muscle and joint pain, fever, lethargy and anorexia. Here we show that Cox-2 may be involved in these central nervous system (CNS) responses, by finding a widespread induction of Cox-2 expression in spinal cord neurons and in other regions of the CNS, elevating prostaglandin E2 (PGE2) levels in the cerebrospinal fluid. The major inducer of central Cox-2 upregulation is interleukin-1beta in the CNS, and as basal phospholipase A2 activity in the CNS does not change with peripheral inflammation, Cox-2 levels must regulate central prostanoid production. Intraspinal administration of an interleukin-converting enzyme or Cox-2 inhibitor decreases inflammation-induced central PGE2 levels and mechanical hyperalgesia. Thus, preventing central prostanoid production by inhibiting the interleukin-1beta-mediated induction of Cox-2 in neurons or by inhibiting central Cox-2 activity reduces centrally generated inflammatory pain hypersensitivity.

Renal concentrating defect in mice lacking group IV cytosolic phospholipase A(2).

Eicosanoids regulate various cellular functions that are important in physiological and pathophysiological processes. Arachidonic acid is released from membranes by phospholipase A(2) (PLA(2)) activity. Activated macrophages derived from mice lacking the 85-kDa group IV cytosolic PLA(2) (cPLA(2)) have a markedly reduced release of prostaglandin E(2) and leukotrienes B(4) and C(4). Under basal conditions and after furosemide, urinary prostaglandin E(2) excretion is reduced in cPLA(2)-knockout (cPLA(2)(-/-)) mice. Serum creatinine, Na(+), K(+), and Ca(2+) concentrations, glomerular filtration rate, and fractional excretion of Na(+) and K(+) are not different in cPLA(2)(-/-) and cPLA(2)(+/+) mice. Maximal urinary concentration is lower in 48-h water-deprived cPLA(2)(-/-) mice compared with cPLA(2)(+/+) animals (1,934 +/- 324 vs. 3,541 +/- 251 mmol/kgH(2)O). Plasma osmolality is higher (337 +/- 5 vs. 319 +/- 3 mmol/kgH(2)O) in cPLA(2)(-/-) mice that lose a greater percentage of their body weight (20 +/- 2 vs. 13 +/- 1%) compared with cPLA(2)(+/+) mice after water deprivation. Vasopressin does not correct the concentrating defect. There is progressive reduction in urinary osmolality with age in cPLA(2)(-/-) mice. Membrane-associated aquaporin-1 (AQP1) expression, identified by immunocytochemical techniques, is reduced markedly in proximal tubules of older cPLA(2)(-/-) animals but is normal in thin descending limbs. However, Western blot analysis of kidney cortical samples revealed an equivalent AQP1 signal intensity in cPLA(2)(+/+) and cPLA(2)(-/-) animals. Young cPLA(2)(-/-) mice have normal proximal tubule AQP1 staining. Collecting duct AQP2, -3, and -4 were normally expressed in the cPLA(2)(-/-) mice. Thus mice lacking cPLA(2) develop an age-related defect in renal concentration that may be related to abnormal trafficking and/or folding of AQP1 in the proximal tubule, implicating cPLA(2) in these processes.

Specific physiological roles of cytosolic phospholipase A(2) as defined by gene knockouts.

The cytosolic 85 kDa phospholipase A(2) (cPLA(2)) is a unique member of the phospholipase A(2) (PLA(2)) superfamily. Because PLA(2) activity and eicosanoid production are important in normal and pathophysiological states we and the laboratory of Shimizu created a mouse deficient in cPLA(2) (cPLA(2)(-/-) mouse). cPLA(2)(-/-) mice develop normally but the females have severe reproductive defects. cPLA(2)(-/-) mice suffer smaller infarcts and fewer neurological deficits after transient occlusion of the middle cerebral artery and have less injury after administration of a dopaminergic selective neurotoxin. cPLA(2)(-/-) mice have a more rapid recovery from allergen-induced bronchoconstriction and have no airway hyperresponsiveness. Peritoneal macrophages from cPLA(2)(-/-) mice fail to produce prostaglandins, leukotriene B(4) and cysteinyl leukotrienes after stimulation. Bone marrow-derived mast cells from cPLA(2)(-/-) mice fail to produce eicosanoids in either immediate or delayed phase responses. Thus the cPLA(2) knockout mouse has revealed important roles of cPLA(2) in normal fertility, generation of eicosanoids from inflammatory cells, brain injuries and allergic responses. Furthermore the cPLA(2)(-/-) mouse reveals that the many other forms of PLA(2) cannot replace many functions of cPLA(2). The importance of cPLA(2) in inflammation and tissue injury suggests that pharmacological targeting of this enzyme may have important therapeutic benefits.

Phospholipases A2 in ischemic and toxic brain injury.

Phospholipases A2 (PLA2s) regulate hydrolysis of fatty acids, including arachidonic acid, from the sn-2 position of phospholipid membranes. PLA2 activity has been implicated in neurotoxicity and neurodegenerative processes secondary to ischemia and reperfusion and other oxidative stresses. The PLA2s constitute a superfamily whose members have diverse functions and patterns of expression. A large number of PLA2s have been identified within the central nervous systems of rodents and humans. We postulated that group IV large molecular weight, cytosolic phospholipase A2 (cPLA2) has a unique role in neurotoxicity associated with ischemic or toxin stress. We created mice deficient in cPLA2 and tested this hypothesis in two injury models, ischemia/reperfusion and MPTP neurotoxicity. In each model cPLA2 deficient mice are protected against neuronal injury when compared to their wild type littermate controls. These experiments support the hypothesis that cPLA2 is an important mediator of ischemic and oxidative injuries in the brain.

Cytosolic phospholipase A2 is essential for both the immediate and the delayed phases of eicosanoid generation in mouse bone marrow-derived mast cells.

We have used mice in which the gene for cytosolic phospholipase A2 (cPLA2) has been disrupted to demonstrate the absolute requirement for cPLA2 in both the immediate and the delayed phases of eicosanoid generation by bone marrow-derived mast cells. For the immediate phase, quantitative analysis of the products of the 5-lipoxygenase pathway showed that gene disruption of cPLA2 prevented the provision of arachidonic acid substrate for biosynthesis of proximal intermediates. By analogy, we conclude that arachidonic acid substrate was also not available to prostaglandin endoperoxide synthase 1 in the immediate phase of prostaglandin (PG) D2 generation. These defects occurred with two distinct stimuli, stem cell factor and IgE/antigen, which were, however, sufficient for signal transduction defined by exocytosis of beta-hexosaminidase. Whereas cPLA2 is essential for immediate eicosanoid generation by providing arachidonic acid, its role in delayed-phase PGD2 generation is more complex and involves the activation-dependent induction of prostaglandin endoperoxide synthase 2 and the supply of arachidonic acid for metabolism to PGD2.

Reduced fertility and postischaemic brain injury in mice deficient in cytosolic phospholipase A2.

Phospholipase A2 (PLA2) enzymes are critical regulators of prostaglandin and leukotriene synthesis and can directly modify the composition of cellular membranes. PLA2 enzymes release fatty acids and lysophospholipids, including the precursor of platelet-activating factor, PAF, from phospholipids. Free fatty acids, eicosanoids, lysophospholipids and PAF are potent regulators of inflammation, reproduction and neurotoxicity. The physiological roles of the various forms of PLA2 are not well defined. The cytosolic form, cPLA2, preferentially releases arachidonic acid from phospholipids and is regulated by changes in intracellular calcium concentration. We have now created 'knockout' (cPLA2-/-) mice that lack this enzyme, in order to evaluate its physiological importance. We find that cPLA2-/- mice develop normally, but that the females produce only small litters in which the pups are usually dead. Stimulated peritoneal macrophages from cPLA2-/- animals did not produce prostaglandin E2 or leukotriene B4 or C4. After transient middle cerebral artery occlusion, cPLA2-/- mice had smaller infarcts and developed less brain oedema and fewer neurological deficits. Thus cPLA2 is important for macrophage production of inflammatory mediators, fertility, and in the pathophysiology of neuronal death after transient focal cerebral ischaemia.

Cytosolic phospholipase A2 (PLA2), but not secretory PLA2, potentiates hydrogen peroxide cytotoxicity in kidney epithelial cells.

Phospholipase A2 (PLA2) and reactive oxygen species have been implicated both individually and synergistically in various forms of cellular injury. The form(s) of PLA2 important for cell injury and the implications of enhanced activity of the enzyme, however, have not been discerned. Previous studies reveal an increase in PLA2 activity associated with cell injury, but this association does not establish a causal relationship between the increase in activity and the injury. LLC-PK1 cell lines were created that express either the cytosolic PLA2 or a group II PLA2. The susceptibility of these cells to hydrogen peroxide toxicity was determined in order to evaluate the relative importance of these two forms of PLA2 in oxidant injury. Expression of cytosolic PLA2 in the LLC-cPLA2 cell line was associated with a 50-fold increase in PLA2 activity in the cytosolic fraction, an increase in agonist-stimulated arachidonate release, and immunodetection of the cytosolic PLA2 protein that was undetectable in control cells. Exposure to hydrogen peroxide or menadione, but not mercuric chloride, resulted in significantly greater lactate dehydrogenase release in LLC-cPLA2 cells when compared with control cells. Exogenous arachidonic acid (150 microM) did not enhance hydrogen peroxide-induced injury. The intracellular calcium chelator, 1,2-bis-(o-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid/tetra(acetoxymethyl) ester, protected the cells against injury, but the calcium ionophore, A23187, did not increase injury. Glycine conferred no protective effect against hydrogen peroxide toxicity. By contrast to these results with cytosolic PLA2-expressing cells, secretory PLA2 expression to very high levels did not increase susceptibility to hydrogen peroxide. Thus, cytosolic PLA2 may an be an important mediator of oxidant damage to renal epithelial cells.

Neuromuscular blocking agents in the management of respiratory failure. Indications and treatment guidelines.

Local custom continues to dictate the clinical use of NMBDs in critically ill patients with respiratory failure. The safety of long-term administration of NMBDs to critically ill patients remains of great concern. Studies that clearly delineate the cause of severe myopathies and neuropathies in critically ill paralyzed patients remain to be performed. Because it appears these disorders may be related to the administration of drugs with steroidal structure, we believe it is prudent to avoid such drugs, if possible, in critically ill patients. We therefore continue to use curare, a nonsteroidal drug with a long history of safety, for long-term paralysis in these patients. Similarly, we believe that it is prudent to monitor the efficacy of NMBDs via the routine use of ulnar nerve stimulation. Patient movement in the face of adequate neuromuscular blockade as assessed by ulnar nerve stimulation then can be treated by deepening the level of sedation rather than by continually increasing the dose of the NMBD.