Inhibition of a Golgi complex lysophospholipid acyltransferase induces membrane tubule formation and retrograde trafficking

Recent studies have suggested that formation of Golgi membrane tubules involves the generation of membrane-associated lysophospholipids by a cytoplasmic Ca2+-independent phospholipase A2 (PLA2). Herein, we provide additional support for this idea by showing that inhibition of lysophospholipid reacylation by a novel Golgi-associated lysophosphatidylcholine acyltransferase (LPAT) induces the rapid tubulation of Golgi membranes, leading in their retrograde movement to the endoplasmic reticulum. Inhibition of the Golgi LPAT was achieved by 2,2-dimethyl-N-(2,4,6-trimethoxyphenyl)dodecanamide (CI-976), a previously characterized antagonist of acyl-CoA cholesterol acyltransferase. The effect of CI-976 was similar to that of brefeldin A, except that the coatomer subunit beta-COP remained on Golgi-derived membrane tubules. CI-976 also enhanced the cytosol-dependent formation of tubules from Golgi complexes in vitro and increased the levels of lysophosphatidylcholine in Golgi membranes. Moreover, preincubation of cells with PLA2 antagonists inhibited the ability of CI-976 to induce tubules. These results suggest that Golgi membrane tubule formation can result from increasing the content of lysophospholipids in membranes, either by stimulation of a PLA2 or by inhibition of an LPAT. These two opposing enzyme activities may help to coordinately regulate Golgi membrane shape and tubule formation.     

Membrane Tubule-mediated Reassembly and Maintenance of the Golgi Complex Is Disrupted by Phospholipase A2 Antagonists

Although membrane tubules can be found extending from, and associated with, the Golgi complex of eukaryotic cells, their physiological function has remained unclear. To gain insight into the biological significance of membrane tubules, we have developed methods for selectively preventing their formation. We show here that a broad range of phospholipase A2 (PLA2) antagonists not only arrest membrane tubule-mediated events that occur late in the assembly of the Golgi complex but also perturb its normal steady-state tubulovesicular architecture by inducing a reversible fragmentation into separate "mini-stacks." In addition, we show that these same compounds prevent the formation of membrane tubules from Golgi stacks in an in vitro reconstitution system. This in vitro assay was further used to demonstrate that the relevant PLA2 activity originates from the cytoplasm. Taken together, these results demonstrate that Golgi membrane tubules, sensitive to potent and selective PLA2 antagonists, mediate both late events in the reassembly of the Golgi complex and the dynamic maintenance of its steady-state architecture. In addition, they implicate a role for cytoplasmic PLA2 enzymes in mediating these membrane trafficking events.     

Inhibition of transferrin recycling and endosome tubulation by phospholipase A2 antagonists

We report here that a broad spectrum of phospholipase A(2) (PLA(2)) antagonists produce a concentration-dependent, differential block in the endocytic recycling pathway of transferrin (Tf) and Tf receptors (TfRs) but have no acute affect on Tf uptake from the cell surface. At low concentrations of antagonists (approximately 1 microm), Tf and TfR accumulated in centrally located recycling endosomes, whereas at higher concentrations (approximately 10 microm), Tf-TfR accumulated in peripheral sorting endosomes. Several independent lines of evidence suggest that this inhibition of recycling may result from the inhibition of tubule formation. First, BFA-stimulated endosome tubule formation was similarly inhibited by PLA(2) antagonists. Second, endocytosed tracers were found in larger spherical endosomes in the presence of PLA(2) antagonists. And third, endosome tubule formation in a cell-free, cytosol-dependent reconstitution system was equally sensitive PLA(2) antagonists. These results are consistent with the conclusion that endosome membrane tubules are formed by the action of a cytoplasmic PLA(2) and that PLA(2)-dependent tubules are involved in intracellular recycling of Tf and TfR. When taken together with previous studies on the Golgi complex, these results also indicate that an intracellular PLA(2) activity provides a novel molecular mechanism for inducing tubule formation from multiple organelles.     

Tubulation of Golgi membranes in vivo and in vitro in the absence of brefeldin A

Recent in vivo studies with the fungal metabolite, brefeldin A (BFA), have shown that in the absence of vesicle formation, membranes of the Golgi complex and the trans-Golgi network (TGN) are nevertheless able to extend long tubules which fuse with selected target organelles. We report here that the ability to form tubules (> 7 microns long) could be reproduced in vitro by treatment of isolated, intact Golgi membranes with BFA under certain conditions. Surprisingly, an even more impressive degree of tubulation could be achieved by incubating Golgi stacks with an ATP-reduced cytosolic fraction, without any BFA at all. Similarly, tubulation of Golgi membranes in vivo occurred after treatment of cells with intermediate levels of NaN3 and 2-deoxyglucose. The formation of tubules in vitro, either by BFA treatment or low-ATP cytosol, correlated precisely with a loss of the vesicle-associated coat protein beta-COP from Golgi membranes. After removal of BFA or addition of ATP, membrane tubules served as substrates for the rebinding of beta-COP and for the formation of vesicles in vitro. These results provide support for the idea that a reciprocal relationship exists between tubulation and vesiculation (Klausner, R. D., J. G. Donaldson, and J. Lippincott-Schwartz. 1992. J. Cell Biol. 116:1071- 1080). Moreover, they show that tubulation is an inherent property of Golgi membranes, since it occurs without the aid of microtubules or BFA treatment. Finally the results indicate the presence of cytosolic factors, independent of vesicle-associated coat proteins, that mediate the budding/tubulation of Golgi membranes.     

Intracellular Ca(2+) release triggers translocation of membrane marker FM1-43 from the extracellular leaflet of plasma membrane into endoplasmic reticulum in T lymphocytes

Stimulation of T cell receptor in lymphocytes enhances Ca(2+) signaling and accelerates membrane trafficking. The relationships between these processes are not well understood. We employed membrane-impermeable lipid marker FM1-43 to explore membrane trafficking upon mobilization of intracellular Ca(2+) in Jurkat T cells. We established that liberation of intracellular Ca(2+) with T cell receptor agonist phytohemagglutinin P or with Ca(2+)-mobilizing agents ionomycin or thapsigargin induced accumulation of FM1-43 within the lumen of the endoplasmic reticulum (ER), nuclear envelope (NE), and Golgi. FM1-43 loading into ER-NE and Golgi was not mediated via the cytosol because other organelles such as mitochondria and multivesicular bodies located in close proximity to the FM1-43-containing ER were free of dye. Intralumenal FM1-43 accumulation was observed even when Ca(2+) signaling in the cytosol was abolished by the removal of extracellular Ca(2+). Our findings strongly suggest that release of intracellular Ca(2+) may create continuity between the extracellular leaflet of the plasma membrane and the lumenal membrane leaflet of the ER by a mechanism that does not require global cytosolic Ca(2+) elevation.     

Purification and characterization of a membrane-associated 48-kilodalton phospholipase A(2) in leaves of broad bean

Several lines of evidence indicate that phospholipase A(2) (PLA(2)) plays a crucial role in plant cellular responses through production of linolenic acid, the precursor of jasmonic acid, from membrane phospholipids. Here we report the purification and characterization of a 48-kD PLA(2) from the membrane fractions of leaves of broad bean (Vicia faba). The plant PLA(2) was purified to near homogeneity by sequential column chromatographies from the membrane extracts. The purified 48-kD protein migrated as a single band on a SDS-PAGE gel and its density correlated with the PLA(2) activity. It was further confirmed that this 48-kD protein is the PLA(2) enzyme based on immunoprecipitating the activity with a monoclonal antibody against it and purifying the enzyme to homogeneity with the antibody affinity column. The purified plant PLA(2) preferred 2-linolenoyl-sn-glycerol-3-phosphocholine (GPC) to 2-linoleoyl-GPC, 2-palmitoyl-GPC and 2-arachidonyl-GPC as substrates with a pH optimum at pH 7.0 to 8.0. The plant PLA(2) was activated by calmodulin and inhibited by pretreatment of 5,8,11, 14-eicosatetraynoic acid known as an inhibitor of mammalian PLA(2)s. The enzyme was characterized as a Ca(2+)-independent PLA(2) different from mammalian PLA(2)s. This membrane-associated and Ca(2+)-independent PLA(2) is suggested to play an important role in the release of linolenic acid, the precursor of jasmonic acid, through a signal transduction pathway.     

1alpha,25(OH)2D3 and parathyroid hormone (PTH) signaling in rat intestinal cells: activation of cytosolic PLA2

In the current study, we have probed the role of cytosolic phospholipase A2 (cPLA2) activity in the cellular response to the calciotropic hormones, 1alpha,25,dihydroxy-vitamin D(3) [1alpha,25(OH)(2)D(3)] and PTH. Stimulation of rat enterocytes with either hormone, increased release of arachidonic acid (AA) 3H-AA] one-two fold in a concentration and time-dependent manner. The effect of either hormone on enterocytes was totally reduced by preincubation with the intracellular Ca(2+) chelator BAPTA-AM (5 microM), suggesting that the release of AA following cell exposure to the calciotropic hormones occurs mainly through a Ca(2+)-dependent mechanism involving activation of Ca(2+)-dependent cPLA2. Calciotropic homone stimulation of rat intestinal cells increases cPLA2 phosphorylation (three to four fold). This effect was decreased by PD 98059 (20 microM), a MAP kinase inhibitor, indicating that this action is, in part, mediated through activation of the MAP kinases ERK 1 and ERK2. Enterocytes exposure to 1alpha,25(OH)(2)D(3) (1nM) or PTH (10 nM) also resulted in P-cPLA2 translocation from cytosol to nuclei and membrane fractions, where phospholipase subtrates reside. Collectively, these data suggest that PTH and 1alpha,25(OH)(2)D(3) activate in duodenal cells, a Ca(2+)-dependent cytosolic PLA2 and attendant arachidonic acid release and that this activation requieres prior stimulation of intracellular ERK1/2. 1alpha,25(OH)(2)D(3) and PTH modulation of cPLA2 activity may change membrane fluidity and permeability and thereby affecting intestinal cell membrane function.     

A PLA1-2 punch regulates the Golgi complex

The mammalian Golgi complex, trans Golgi network (TGN) and ER-Golgi intermediate compartment (ERGIC) are comprised of membrane cisternae, coated vesicles and membrane tubules, all of which contribute to membrane trafficking and maintenance of their unique architectures. Recently, a new cast of players was discovered to regulate the Golgi and ERGIC: four unrelated cytoplasmic phospholipase A (PLA) enzymes, cPLA(2)α (GIVA cPLA(2)), PAFAH Ib (GVIII PLA(2)), iPLA(2)-β (GVIA-2 iPLA(2)) and iPLA(1)γ. These ubiquitously expressed enzymes regulate membrane trafficking from specific Golgi subcompartments, although there is evidence for some functional redundancy between PAFAH Ib and cPLA(2)α. Three of these enzymes, PAFAH Ib, cPLA(2)α and iPLA(2)-β, exert effects on Golgi structure and function by inducing the formation of membrane tubules. We review our current understanding of how PLA enzymes regulate Golgi and ERGIC morphology and function.     

Calcium-independent phospholipase A(2): structure and function

The classical Ca(2+)-independent phospholipase A(2) enzyme, now known as Group VIA PLA(2), was initially purified and characterized from the P388D(1) macrophage-like cell line. The corresponding cDNA was subsequently cloned from a variety of sources, and it is now known that multiple splice variants of the enzyme are expressed, some of which may act as negative regulators of the active enzyme. Group VIA PLA(2) has a consensus lipase motif (GTSTG) containing the catalytic serine, is 85-88 kDa, and exists in an aggregated form. The enzyme contains multiple ankyrin repeats, which may play a role in oligomerization. The Group VIA enzyme exhibits lysophospholipase activity as well as phospholipase A(2) activity, and it is capable of hydrolyzing a wide variety of phospholipid substrates. A major function of Group VIA PLA(2) is to mediate phospholipid remodeling, but the enzyme may play other roles as well. Other Ca(2+)-independent PLA(2) enzymes have more recently been identified, and it may be possible to discriminate between the various Ca(2+)-independent PLA(2) enzymes based on sequence or inhibitor-sensitivity. However, the physiological functions of the newly identified enzymes have yet to be elucidated.     

Human group IVC phospholipase A2 (cPLA2gamma). Roles in the membrane remodeling and activation induced by oxidative stress

To create the unique properties of a certain cellular membrane, both the composition and the metabolism of membrane phospholipids are key factors. Phospholipase A(2) (PLA(2)), with hydrolytic enzyme activities at the sn-2 position in glycerophospholipids, plays critical roles in maintaining the phospholipid composition as well as producing bioactive lipid mediators. In this study we examined the contribution of a Ca(2+)-independent group IVC PLA(2) isozyme (cPLA(2)gamma), a paralogue of cytosolic PLA(2)alpha (cPLA(2)alpha), to phospholipid remodeling. The enzyme was localized in the endoplasmic reticulum and Golgi apparatus, as seen using green fluorescence fusion proteins. Electrospray ionization mass spectrometric analysis of membrane extracts revealed that overexpression of cPLA(2)gamma increased the proportion of polyunsaturated fatty acids in phosphatidylethanolamine, suggesting that the enzyme modulates the phospholipid composition. We also found that H(2)O(2) and other hydroperoxides induced arachidonic acid release in cPLA(2)gamma-transfected human embryonic kidney 293 cells, possibly through the tyrosine phosphorylation pathway. Thus, we propose that cPLA(2)gamma is constitutively expressed in the endoplasmic reticulum and plays important roles in remodeling and maintaining membrane phospholipids under various conditions, including oxidative stress.     

The role of calcium in intracellular trafficking

The molecular mechanism of membrane fusion essential to vital cellular activities such as intracellular transport, hormone secretion, enzyme release, or neurotransmission, involve the assembly and disassembly of a specialized set of proteins in opposing bilayers. Recent evidences shed new light on the role Ca(2+) has in the regulation of this mechanism in which the Golgi apparatus works as a central station; from here, Ca(2+) ions are released into and recovered from the cytosol during the different steps of the cargo progression. In fact, transient cytosolic Ca(2+) fluctuations take a crucial role to recruit proteins and enzymes Ca(2+)-sensitive on Golgi membranes where they are involved in membranes remodelling which is fundamental process for the fusion events that allow protein trafficking. Here I provide an overview of the role Ca(2+) plays in intra-Golgi trafficking underlying some interesting aspects to clarify the mechanisms of cargo progression.     

Phospholipase A(2) antagonists inhibit nocodazole-induced Golgi ministack formation: evidence of an ER intermediate and constitutive cycling

Evidence has been presented both for and against obligate retrograde movement of resident Golgi proteins through the endoplasmic reticulum (ER) during nocodazole-induced Golgi ministack formation. Here, we studied the nocodazole-induced formation of ministacks using phospholipase A(2) (PLA(2)) antagonists, which have been shown previously to inhibit brefeldin A-stimulated Golgi-to-ER retrograde transport. Examination of clone 9 rat hepatocytes by immunofluorescence and immunoelectron microscopy revealed that a subset of PLA(2) antagonists prevented nocodazole-induced ministack formation by inhibiting two different trafficking pathways for resident Golgi enzymes; at 25 microM, retrograde Golgi-to-ER transport was inhibited, whereas at 5 microM, Golgi-to-ER trafficking was permitted, but resident Golgi enzymes accumulated in the ER. Moreover, resident Golgi enzymes gradually redistributed from the juxtanuclear Golgi or Golgi ministacks to the ER in cells treated with these PLA(2) antagonists alone. Not only was ER-to-Golgi transport of resident Golgi enzymes inhibited in cells treated with these PLA(2) antagonists, but transport of the vesicular stomatitis virus G protein out of the ER was also prevented. These results support a model of obligate retrograde recycling of Golgi resident enzymes during nocodazole-induced ministack formation and provide additional evidence that resident Golgi enzymes slowly and constitutively cycle between the Golgi and ER.     

The Mitogen-activated protein kinase p38 links Shiga Toxin-dependent signaling and trafficking

Shiga toxin (Stx) binds to the cell, and it is transported via endosomes and the Golgi apparatus to the endoplasmic reticulum and cytosol, where it exerts its toxic effect. We have recently shown that Stx activates the tyrosine kinase Syk, which in turn induces clathrin phosphorylation and up-regulates Stx uptake. Here, we show that toxin-induced signaling can also regulate another step in intracellular Stx transport. We demonstrate that transport of Stx to the Golgi apparatus is dependent on the mitogen-activated protein kinase p38. Treatment of cells with chemical inhibitors or small interfering RNA targeting p38 inhibited Stx transport to the Golgi and reduced Stx toxicity. This p38 dependence is specific to Stx, because transport of the related toxin ricin was not affected by p38 inhibition. Stx rapidly activated p38, and recruited it to early endosomes in a Ca(2+)-dependent manner. Furthermore, agonist-induced oscillations in cytosolic Ca(2+) levels were inhibited upon Stx stimulation, possibly reflecting Stx-dependent local alterations in cytosolic Ca(2+) levels. Intracellular transport of Stx is Ca(2+) dependent, and we provide evidence that Stx activates a signaling cascade involving cross talk between Ca(2+) and p38, to regulate its trafficking to the Golgi apparatus.     

A role for calmodulin in organelle membrane tubulation.

Membrane tubules of uniform diameter (60-80 nm) and variable lengths have been seen to extend from the main bodies of the Golgi complex, trans Golgi network (TGN), and endosomes. In the case of endosomes, these tubules appear to mediate membrane and receptor recycling events. Brefeldin A (BFA) is a potent drug that completely blocks coated vesicle formation from the Golgi complex and TGN, but at the same time causes the enhanced formation of membrane tubules from these same organelles. Recently, experiments have shown that calmodulin antagonists inhibit the transport of receptors out of endosomes, perhaps by inhibiting the formation of recycling tubules. Using the potent calmodulin-specific antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide (W-13), and N-(4-aminobutyl)-5-chloro-1-naphthalenesulfonamide (C-1), we found that the recycling of transferrin from endosomes to the cell surface was significantly inhibited, resulting in the formation of enlarged endosomal vacuoles. In addition, these same calmodulin antagonists also potently inhibited the formation of BFA-stimulated membrane tubules from the Golgi complex, TGN, and endosomes. In the case of the Golgi complex, failure to form tubules resulted in the inhibition of BFA-stimulated retrograde transport to the endoplasmic reticulum. These results suggest that calmodulin is a general regulator of membrane tubulation and is capable of influencing the morphology of several organelles.     

A frontal variant of Alzheimer's disease exhibits decreased calcium-independent phospholipase A2 activity in the prefrontal cortex

A frontal variant of Alzheimer's disease (AD) has recently been identified on neuropathological and neuropsychological grounds (Johnson, J.K., Head, E., Kim, R., Starr, A., Cotman, C.W., 1999. Clinical and pathological evidence for a frontal variant of Alzheimer Disease. Arch. Neurol. 56, 1233-1239). Frontal AD differs strikingly from typical AD by the occurrence of neurofibrillary tangle densities in the frontal cortex as high or higher than in the entorhinal cortex. Since cerebrocortical membranes are commonly abnormal in Alzheimer's disease (AD), we assayed frontal AD cases for enzymes regulating membrane phospholipid composition. We specifically measured activity of phospholipase A2s (PLA2s) in dorsolateral prefrontal and lateral temporal cortices of frontal AD cases (n=12), which have respectively high and low densities of neurofibrillary tangles. In neither cortical area was Ca(2+)-dependent PLA2 activity abnormal compared to controls (n=12). In contrast, a significant 42% decrease in Ca(2+)-independent PLA2 activity was found in the dorsolateral prefrontal, but not the lateral temporal, cortex of the frontal AD cases. Similarly, the dorsolateral prefrontal cortex, but not the lateral temporal cortex of the frontal AD cases suffered a 42% decrease in total free fatty acid content, though neither that decrease nor those in any one species of free fatty acid was significant. The observed biochemical changes probably occurred in neurons given (a) our finding that PLA2 activity of cultured human NT2 neurons is virtually all Ca(2+)-independent and (b) the finding of others that nearly all Ca(2+)-independent PLA2 in brain gray matter is neuronal. The decrease in Ca(2+)-independent PLA2 activity is not readily attributable to Group VI or VIII iPLA2s since neither NT2N neurons nor our brain homogenates were greatly inhibited by drugs potently suppressing those iPLA2s. Decreased Ca(2+)-independent PLA2 activity in frontal AD may reflect a compensatory response to pathologically accelerated phospholipid metabolism early in the disorder. That could cause an early elevation of prefrontal free fatty acids, which can stimulate polymerization of tau and thus promote the prefrontal neurofibrillary tangle formation characteristic of frontal AD.     

The cytosolic phospholipase A2 catalytic domain modulates association and residence time at Golgi membranes

Cytosolic phospholipase A2 (cPLA2) catalyzes release of arachidonic acid from membranes following translocation to Golgi and endoplasmic reticulum. In response to an intracellular calcium concentration ([Ca2+]i) increase, the C2 domain binds Ca2+ and brings the catalytic domain into proximity with its phospholipid substrate. Because membrane residence is important in the regulation of cPLA2 activity, we explored the contributions of the C2 and catalytic domains in mediating membrane residence using an imaging approach in live cells with fluorescent protein chimeras of cPLA2. The isolated cPLA2 C2 domain associated with Golgi membranes rapidly in proportion to the [Ca2+]i, allowing for its use as a [Ca2+]i indicator. cPLA2 association with Golgi was slower than the isolated C2 domain in response to a [Ca2+]i increase. After [Ca2+]i decrease, cPLA2 remained associated with membrane in a Ca(2+)-independent fashion whereas C2 domain rapidly dissociated. Ca(2+)-independent membrane association was greatly reduced by mutation of Trp464, located at the membrane-exposed face of the catalytic domain, to Gly or Ala. Mutation of Trp464 to Phe supported Ca(2+)-independent association similar to wild type. These results demonstrate a role for the cPLA2 catalytic domain in regulating membrane association and membrane residence time.     

Phospholipase A2-independent Ca2+ entry and subsequent apoptosis induced by melittin in human MG63 osteosarcoma cells

Melittin, a peptide from bee venom, is thought to be a phospholipase A(2) activator and Ca(2+) influx inducer that can evoke cell death in different cell types. However, the effect of melittin on cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and viability has not been explored in human osteoblast-like cells. This study examined whether melittin altered [Ca(2+)](i) and killed cells in MG63 human osteosarcoma cells. [Ca(2+)](i) changes and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Melittin at concentrations above 0.075 microM increased [Ca(2+)](i) in a concentration-dependent manner. The Ca(2+) signal was abolished by removing extracellular Ca(2+). Melittin-induced Ca(2+) entry was confirmed by Mn(2+) quenching of fura-2 fluorescence at 360 nm excitation wavelength which was Ca(2+)-insensitive. The melittin-induced Ca(2+) influx was unchanged by modulation of protein kinase-C activity with phorbol 12-myristate 13-acetate (PMA) and GF 109203X, or inhibition of phospholipase A(2) with AACOCF(3) and aristolochic acid; but was substantially inhibited by blocking L-type Ca(2+) channels. At concentrations of 0.5 microM and 1 microM, melittin killed 33% and 45% of cells, respectively, via inducing apoptosis. Lower concentrations of melittin failed to kill cells. The cytotoxic effect of 1 microM melittin was completely reversed by pre-chelating cytosolic Ca(2+) with BAPTA. Taken together, these data showed that in MG63 cells, melittin induced a [Ca(2+)](i) increase by causing Ca(2+) entry through L-type Ca(2+) channels in a manner independent of protein kinase-C and phospholipase A(2) activity; and this [Ca(2+)](i) increase subsequently caused apoptosis.     

Phospholipase A2 in the central nervous system: implications for neurodegenerative diseases

Phospholipase A2 (PLA2) belongs to a family of enzymes that catalyze the cleavage of fatty acids from the sn-2 position of phospholipids. There are more than 19 different isoforms of PLA2 in the mammalian system, but recent studies have focused on three major groups, namely, the group IV cytosolic PLA2, the group II secretory PLA2 (sPLA2), and the group VI Ca(2+)-independent PLA2. These PLA2s are involved in a complex network of signaling pathways that link receptor agonists, oxidative agents, and proinflammatory cytokines to the release of arachidonic acid (AA) and the synthesis of eicosanoids. PLA2s acting on membrane phospholipids have been implicated in intracellular membrane trafficking, differentiation, proliferation, and apoptotic processes. All major groups of PLA2 are present in the central nervous system (CNS). Therefore, this review is focused on PLA2 and AA release in neural cells, especially in astrocytes and neurons. In addition, because many neurodegenerative diseases are associated with increased oxidative and inflammatory responses, an attempt was made to include studies on PLA2 in cerebral ischemia, Alzheimer's disease, and neuronal injury due to excitotoxic agents. Information from these studies has provided clear evidence for the important role of PLA2 in regulating physiological and pathological functions in the CNS.     

Atrial natriuretic peptide attenuates elevations in Ca2+ and protects hepatocytes by stimulating net plasma membrane Ca2+ efflux

Elevations in intracellular Ca(2+) concentration and calpain activity are common early events in cellular injury, including that of hepatocytes. Atrial natriuretic peptide is a circulating hormone that has been shown to be hepatoprotective. The aim of this study was to examine the effects of atrial natriuretic peptide on potentially harmful elevations in cytosolic free Ca(2+) and calpain activity induced by extracellular ATP in rat hepatocytes. We show that atrial natriuretic peptide, through protein kinase G, attenuated both the amplitude and duration of ATP-induced cytosolic Ca(2+) rises in single hepatocytes. Atrial natriuretic peptide also prevented stimulation of calpain activity by ATP, taurolithocholate, or Ca(2+) mobilization by thapsigargin and ionomycin. We therefore investigated the cellular Ca(2+) handling mechanisms through which ANP attenuates this sustained elevation in cytosolic Ca(2+). We show that atrial natriuretic peptide does not modulate the release from or re-uptake of Ca(2+) into intracellular stores but, through protein kinase G, both stimulates plasma membrane Ca(2+) efflux from and inhibits ATP-stimulated Ca(2+) influx into hepatocytes. These findings suggest that stimulation of net plasma membrane Ca(2+) efflux (to which both Ca(2+) efflux stimulation and Ca(2+) influx inhibition contribute) is the key process through which atrial natriuretic peptide attenuates elevations in cytosolic Ca(2+) and calpain activity. Moreover we propose that plasma membrane Ca(2+) efflux is a valuable, previously undiscovered, mechanism through which atrial natriuretic peptide protects rat hepatocytes, and perhaps other cell types, against Ca(2+)-dependent injury.