G-protein beta gamma-subunits activate the cardiac muscarinic K+-channel via phospholipase A2

Muscarinic receptors of cardiac pacemaker and atrial cells are linked to a potassium channel (IK.ACh) by a pertussis toxin-sensitive GTP-binding protein. The dissociation of G-proteins leads to the generation of two potential transducing elements, alpha-GTP and beta gamma. IK.ACh is activated by G-protein alpha- and beta gamma-subunits applied to the intracellular surface of inside-out patches of membrane. beta gamma has been shown to activate the membrane-bound enzyme phospholipase A2 in retinal rods. Arachidonic acid, which is produced from the action of phospholipase A2 on phospholipids, is metabolized to compounds which may act as second messengers regulating ion channels in Aplysia. Muscarinic receptor activation leads to the generation of arachidonic acid in some cell lines. We therefore tested the hypothesis that beta gamma activates IK.ACh by stimulation of phospholipase A2. When patches were first incubated with antibody that blocks phospholipase A2 activity, or with the lipoxygenase inhibitor, nordihydroguaiaretic acid, beta gamma failed to activate IK.ACh. Arachidonic acid and several of its metabolites derived from the 5-lipoxygenase pathway, activated the channel. Blockade of the cyclooxygenase pathway did not inhibit arachidonic acid-induced channel activation. We conclude that the beta gamma-subunit of G-proteins activates IK.ACh by stimulating the production of lipoxygenase-derived second messengers.     

Potentiation of NMDA receptor currents by arachidonic acid.

Arachidonic acid is released by phospholipase A2 when activation of N-methyl-D-aspartate (NMDA) receptors by neurotransmitter glutamate raises the calcium concentration in neurons, for example during the initiation of long-term potentiation and during brain anoxia. Here we investigate the effect of arachidonic acid on glutamate-gated ion channels by whole-cell clamping isolated cerebellar granule cells. Arachidonic acid potentiates, and makes more transient, the current through NMDA receptor channels, and slightly reduces the current through non-NMDA receptor channels. Potentiation of the NMDA receptor current results from an increase in channel open probability, with no change in open channel current. We observe potentiation even with saturating levels of agonist at the glutamate- and glycine-binding sites on these channels; it does not result from conversion of arachidonic acid to lipoxygenase or cyclooxygenase derivatives, or from activation of protein kinase C. Arachidonic acid may act by binding to a site on the NMDA receptor, or by modifying the receptor's lipid environment. Our results suggest that arachidonic acid released by activation of NMDA (or other) receptors will potentiate NMDA receptor currents, and thus amplify increases in intracellular calcium concentration caused by glutamate. This may explain why inhibition of phospholipase A2 blocks the induction of long-term potentiation.     

Dopamine activation of the arachidonic acid cascade as a basis for D1/D2 receptor synergism

Understanding the actions of the neurotransmitter dopamine in the brain is important in view of its roles in neuropsychiatric illnesses. Dopamine D1 receptors, which stimulate both adenylyl cyclase and phospholipase C, and D2 receptors, which inhibit them, can nevertheless act synergistically to produce many electrophysiological and behavioral responses. Because this functional synergism can occur at the level of single neurons, another, as yet unidentified, signalling pathway activated by dopamine has been hypothesized. We report here that in Chinese hamster ovary (CHO) cells transfected with the D2 receptor complementary DNA, D2 agonists potently enhanced arachidonic acid release, provided that such release has been initiated by stimulating constitutive purinergic receptors or by increasing intracellular Ca2+. In CHO cells expressed D1 receptors, D1 agonists exert no such effect. When D1 and D2 receptors are coexpressed, however, activation of both subtypes results in a marked synergistic potentiation of arachidonic acid release. The numerous actions of arachidonic acid and its metabolites in neuronal signal transduction suggest that facilitation of its release may be implicated in dopaminergic responses, such as feedback inhibition mediated by D2 autoreceptors, and may constitute a molecular basis for D1/D2 receptor synergism.     

Arachidonic acid induces a prolonged inhibition of glutamate uptake into glial cells

Activation of NMDA (N-methyl-D-aspartate) receptors by neurotransmitter glutamate stimulates phospholipase A2 to release arachidonic acid. This second messenger facilitates long-term potentiation of glutamatergic synapses in the hippocampus, possibly by blocking glutamate uptake. We have studied the effect of arachidonic acid on glutamate uptake into glial cells using the whole-cell patch-clamp technique to monitor the uptake electrically. Micromolar levels of arachidonic acid inhibit glutamate uptake, mainly by reducing the maximum uptake rate with only small effects on the affinity for external glutamate and sodium. On removal of arachidonic acid a rapid (5 minutes) phase of partial recovery is followed by a maintained suppression of uptake lasting at least 20 minutes. Surprisingly, the action of arachidonic acid is unaffected by cyclo-oxygenase or lipoxygenase inhibitors suggesting that it inhibits uptake directly, possibly by increasing membrane fluidity. As blockade of phospholipase A2 prevents the induction of long-term potentiation (LTP), inhibition of glutamate uptake by arachidonic acid may contribute to the increase of synaptic gain that occurs in LTP. During anoxia, release of arachidonic acid could severely compromise glutamate uptake and thus contribute to neuronal death.     

Omega-3 and omega-6 fatty acids stimulate cell membrane expansion by acting on syntaxin 3

Growth of neurite processes from the cell body is the critical step in neuronal development and involves a large increase in cell membrane surface area. Arachidonic-acid-releasing phospholipases are highly enriched in nerve growth cones and have previously been implicated in neurite outgrowth. Cell membrane expansion is achieved through the fusion of transport organelles with the plasma membrane; however, the identity of the molecular target of arachidonic acid has remained elusive. Here we show that syntaxin 3 (STX3), a plasma membrane protein, has an important role in the growth of neurites, and also serves as a direct target for omega-6 arachidonic acid. By using syntaxin 3 in a screening assay, we determined that the dietary omega-3 linolenic and docosahexaenoic acids can efficiently substitute for arachidonic acid in activating syntaxin 3. Our findings provide a molecular basis for the previously established action of omega-3 and omega-6 polyunsaturated fatty acids in membrane expansion at the growth cones, and represent the first identification of a single effector molecule for these essential nutrients.     

Recombinant human lipocortin 1 inhibits thromboxane release from guinea-pig isolated perfused lung

The guinea-pig perfused isolated lung, used in conjunction with the cascade superfusion system to measure the release of thromboxane A2(TXA2), is a simple and convenient model for assessing the inhibition by glucocorticoids of eicosanoid formation. Dexamethasone inhibits the release of TXA2 from the lung when it is stimulated by agents such as RCS-RF2 of leukotrienes, but not when bradykinin or arachidonic acid are used. Using this model we have shown that the glucocorticoids suppress eicosanoid generation by cells through the induction of a family of phospholipase A2-inhibitory proteins now termed the 'lipocortins'. Recently the primary structure of one form of lipocortin has been elucidated and the human gene cloned. Lipocortin 1 is a polar monomeric protein with anti-phospholipase properties in vitro and we now report that when infused into guinea-pig lung preparations this protein has the same inhibitory profile as the glucocorticoids but with a more rapid onset of action. This is the first demonstration that eicosanoid formation can be inhibited by a recombinant phospholipase inhibitory protein applied extracellularly.     

Arachidonic acid metabolites as mediators of somatostatin-induced increase of neuronal M-current

The M-current (IM) is a time- and voltage-dependent K+ current that persists at slightly depolarized membrane potentials. IM is reduced by muscarinic cholinergic agonists and certain peptides, and is thought to be responsible in part for the slow and late slow excitatory postsynaptic potentials in sympathetic neurons. Recently, we reported that IM in hippocampal neurons was also augmented by somatostatin-14 and -28 suggesting that two different receptors reciprocally regulate one neuronal channel type. Muscarinic effects on IM may be mediated by various components of the phosphatidylinositol phosphate pathway. We now report the involvement of a different second messenger pathway, that generated by phospholipase A2, in the somatostatin-induced augmentation of IM in hippocampal cells. This pathway generates arachidonic acid from which leukotrienes can be produced by lipoxygenases. We find that the IM-augmenting effects of somatostatin are abolished by two substances that can inhibit phospholipase A2, quinacrine and 4-bromophenacyl bromide, and that both arachidonic acid and leukotriene C4 mimic the effects of somatostatin-14 on hippocampal pyramidal neurons in vitro. Arachidonic and somatostatin effects are blocked by a lipoxygenase inhibitor, implicating an arachidonic acid metabolite, perhaps a leukotriene, in the somatostatin effect.     

NMDA receptors activate the arachidonic acid cascade system in striatal neurons

Receptors for excitatory amino-acid transmitters on nerve cells fall into two main categories associated with non-selective cationic channels, the NMDA (N-methyl-D-aspartate) and non-NMDA (kainate and quisqualate) receptors. Special properties of NMDA receptors such as their voltage-dependent blockade by Mg2+ (refs 3, 4) and their permeability to Na+, K+ as well as to Ca2+ (refs 5, 6), have led to the suggestion that these receptors are important in plasticity during development and learning. They have been implicated in long-term potentiation (LTP), a model for the study of the cellular mechanisms of learning. We report here that glutamate and NMDA, acting at typical NMDA receptors, stimulate the release of arachidonic acid (as well as 11- and 12-hydroxyeicosatetraenoic acids from striatal neurons probably by stimulation of a Ca2+-dependent phospholipase A2. Kainate and quisqualate, as well as K+-induced depolarization were ineffective. Our results provide direct evidence in favour of the hypothesis, that arachidonic acid derivatives, produced by activation of the postsynaptic cell, could be messengers that cross the synaptic cleft to modify the presynaptic functions known to be altered during LTP. In addition, we suggest that NMDA receptors are the postsynaptic receptors which trigger the synthesis of these putative transynaptic messengers.     

Arachidonic acid released from striatal neurons by joint stimulation of ionotropic and metabotropic quisqualate receptors

Associative stimulation of N-methyl-D-aspartate (NMDA) receptors and quisqualate ionotropic receptors (Qi) induces long-term potentiation at particular glutamatergic synapses. Release of arachidonic acid as a result of stimulation of NMDA receptors has been proposed to play a part in the establishment of long-term potentiation. But long-term plasticity events at some other glutamatergic synapses do not involve activation of NMDA receptors. Here we report that in mature striatal neurons in primary cultures, quisqualate can release arachidonic acid by associatively activating both quisqualate metabotropic receptors coupled to phospholipase C (Qp) and Qi receptors. Independent activation of these two receptor types with specific agonists did not stimulate arachidonic acid release. These results support a role for the associative activation of Qp and Qi receptors in synaptic plasticity events, including long-term potentiation at particular synapses.     

High-resolution (1.5 A) crystal structure of phospholipase C from Bacillus cereus

Both the phosphatidylinositol-hydrolysing and the phosphatidylcholine-hydrolysing phospholipases C have been implicated in the generation of second messengers in mammalian cells. The phosphatidylcholine-hydrolysing phospholipase C (PLC) from Bacillus cereus, a monomeric protein containing 245 amino-acid residues, is similar to some of the corresponding mammalian proteins. This, together with the fact that the bacterial enzyme can mimic the action of mammalian PLC in causing, for example, enhanced prostaglandin biosynthesis, suggests that B. cereus PLC can be used as a model for the hitherto poorly characterized mammalian PLCs. We report here the three-dimensional structure of B. cereus PLC at 1.5 A resolution. The enzyme is an all-helix protein belonging to a novel structural class and contains, at least in the crystalline state, three Zn2+ in the active site. We also present preliminary results from a study at 1.9 A resolution of the complex between PLC and inorganic phosphate (Pi) which indicate that the substrate binds directly to the metal ions.     

Go protein as signal transducer in the pertussis toxin-sensitive phosphatidylinositol pathway

Receptors stimulating phospholipase C do so through heterotrimeric GTP-binding proteins to produce two second messengers, inositol 1,4,5-trisphosphate (InsP3) and diacylglycerol. In spite of the detailed understanding of phospholipase C structure and phosphatidyl inositol signalling, the identity of the GTP-binding protein involved is so far unknown. To address this issue, we have used the Xenopus oocyte in which muscarinic receptors couple to phospholipase C through a pertussis toxin-sensitive GTP-binding protein. In this cell, InsP3 mobilizes intracellular Ca2+ to evoke a Cl- current. The magnitude of this Cl- current is proportional to the amount of InsP3 in the cell, and therefore can be used as an assay for InsP3 production. We report here that the activated alpha-subunit of the GTP-binding protein GO, when directly injected into oocytes, evokes a Cl- current by mobilizing Ca2+ from intracellular InsP3-sensitive stores. We also show that holo-GO, when injected into oocytes, can specifically enhance the muscarinic receptor-stimulated Cl- current. These data indicate that GO can serve as the signal transducer of the receptor-regulated phospholipase C in Xenopus oocytes.     

Positive feedback of glutamate exocytosis by metabotropic presynaptic receptor stimulation.

Glutamate is important in several forms of synaptic plasticity such as long-term potentiation, and in neuronal cell degeneration. Glutamate activates several types of receptors, including a metabotropic receptor that is sensitive to trans-1-amino-cyclopenthyl-1,3-dicarboxylate, coupled to G protein(s) and linked to inositol phospholipid metabolism. The activation of the metabotropic receptor in neurons generates inositol 1,4,5-trisphosphate, which causes the release of Ca2+ from intracellular stores and diacylglycerol, which activates protein kinase C. In nerve terminals, the activation of presynaptic protein kinase C with phorbol esters enhances glutamate release. But the presynaptic receptor involved in this protein kinase C-mediated increase in the release of glutamate has not yet been identified. Here we demonstrate the presence of a presynaptic glutamate receptor of the metabotropic type that mediates an enhancement of glutamate exocytosis in cerebrocortical nerve terminals. Interestingly, this potentiation of glutamate release is observed only in the presence of arachidonic acid, which may reflect that this positive feedback control of glutamate exocytosis operates in concert with other pre- or post-synaptic events of the glutamatergic neurotransmission that generate arachidonic acid. This presynaptic glutamate receptor may have a physiological role in the maintenance of long-term potentiation where there is an increase in glutamate release mediated by postsynaptically generated arachidonic acid.     

胞内型磷脂酶A2基因亚克隆与基因表达

目的 研究花生四稀酸在细胞中的释放及前列腺素的合成,构建与花生四稀酸释放相关的表达系统。即胞内型磷脂酶Ａ２（ｃｙｔｏｓｏｌｉｃ ｐｈｏｓｐｈｏｌｉｐａｓｅ ｃＰＬＡ２）ｃＤＮＡ－ｐＲＣ／ＣＭＶ。方法 从克隆载体ｐＭＴ２用Ｓａｌ１制备ｃＰＡＬ２ｃＤＮＡ;平末端连接ｃＰＡＬ２－ｐＲＣ／ＣＭＶ;转染鼠巨噬细胞及筛选正向阳性克隆;ｎｏｒｔｈｅｒｎ ｂｌｏｔ检测ｃＰＬＡ２ ｃＤＮＡ基因表达。结果 人ｃＰＬＡ     

Arachidonic acid metabolites as intracellular modulators of the G protein-gated cardiac K+ channel

Arachidonic acid is released from cell membranes in response to receptor-dependent as well as receptor-independent stimulation in various cells, including cardiac myocytes. Arachidonic acid is converted to prostaglandins by cyclooxygenase and to leukotrienes by 5-lipoxygenase, metabolites which are very biologically active and modulate cellular functions such as platelet aggregation, smooth muscle contraction and neural excitation. The molecular mechanisms underlying their modulations are, however, still badly understood. Here, we report that the 5-lipoxygenase metabolites of arachidonic acid activate the pertussis toxin-sensitive G protein-gated muscarinic K+ channel (IK.ACh): arachidonic acid activation of IK.ACh was prevented by the lipoxygenase inhibitors, nordihydroguaiaretic acid and AA-861; leukotriene A4 and C4 activated IK.ACh. The activation occurred in pertussis toxin-treated atrial cells and ceased when inside-out patches were formed but the patches were still susceptible to stimulation by GTP and to inhibition by GDP-beta-S. These results indicate that arachidonic acid metabolites may stimulate the G-protein in a receptor-independent way.     

Vitamin E modulates the lipoxygenation of arachidonic acid in leukocytes

The arachidonic acid released from cellular phospholipids of specifically stimulated platelets and leukocytes is oxygenated enzymatically by two major pathways. A complex cycloxygenase converts some of the free arachidonic acid to labile endoperoxides that are transformed to prostaglandins, thromboxanes and prostacyclin (PGI2). Lipoxygenases convert part of the arachidonic acid to unstable hydroperoxy-eicosatetraenoic acids (OOHETEs) that are transformed to monohydroxyeicosatetraenoic acids (HETEs), oligohydroxy-eicosatetraenoic or -eicostatrienoic acids such as di-HETEs and tri-HETEs, and, in some instances, more complex humoral mediators, including slow-reacting substances. Both the nature of the HETEs and the ratio of the HETEs to the cyclo-oxygenase products are specific characteristics of each type of cell. In human neutrophils, the sum of the lipoxygenase products 5-HETE, 11-HETE and 5,12-di-HETE substantially exceeds the total amount of PGE2 and other cyclo-oxygenase metabolites that are generated concurrently, and the endogenous lipoxygenase products regulate neutrophil function. The present data indicate that vitamin E (alpha-tocopherol) bidirectionally modulates the activity of the lipoxygenase pathway of human neutrophils in vitro. Normal plasma concentrations of alpha-tocopherol enhance the lipoxygenation of arachidonic acid, whereas higher concentrations of alpha-tocopherol exert a suppressive effect that is consistent with its role as a hydroperoxide scavenger.     

Platelet activation--a role for a 40K anti-phospholipase A2 protein indistinguishable from lipocortin

Stimulus-response (S-R) coupling in platelets requires an intermediary other than an elevation in cytosolic free calcium ([Ca2+]i). While an increase in [Ca2+]i is essential in S-R coupling, effecting phosphorylation of myosin of relative molecular mass (Mr) 20,000 (20 K), platelet activation is also associated with phosphorylation of a 40K protein, which can occur in the absence of changes in [Ca2+]i. The 40K protein is the substrate for protein kinase C (PKC). Mounting evidence suggests that activation of PKC by diacylglycerol is the other signal involved in S-R coupling. Although phosphorylation of the 40K protein is associated with certain platelet functional responses, no precise role has been accredited to it. Recently, we and others have described several proteins (collectively known as lipocortin) which inhibit phospholipase A2 (PLA2). One of the most conspicuous proteins of this group is a 40K peptide whose inhibitory activity can be suppressed by prior phosphorylation. We hypothesized that the 40K protein described in platelets may possess anti-PLA2 activity and that phosphorylation by PKC, suppressing its inhibitory activity, may represent the mechanism underlying mobilization of arachidonic acid, the precursor of prostaglandins. The results of the present study strongly support this hypothesis.     

Second messenger role for Mg2+ revealed by human T-cell immunodeficiency

The magnesium ion, Mg(2+), is essential for all life as a cofactor for ATP, polyphosphates such as DNA and RNA, and metabolic enzymes, but whether it plays a part in intracellular signalling (as Ca(2+) does) is unknown. Here we identify mutations in the magnesium transporter gene, MAGT1, in a novel X-linked human immunodeficiency characterized by CD4 lymphopenia, severe chronic viral infections, and defective T-lymphocyte activation. We demonstrate that a rapid transient Mg(2+) influx is induced by antigen receptor stimulation in normal T cells and by growth factor stimulation in non-lymphoid cells. MAGT1 deficiency abrogates the Mg(2+) influx, leading to impaired responses to antigen receptor engagement, including defective activation of phospholipase Cγ1 and a markedly impaired Ca(2+) influx in T cells but not B cells. These observations reveal a role for Mg(2+) as an intracellular second messenger coupling cell-surface receptor activation to intracellular effectors and identify MAGT1 as a possible target for novel therapeutics.     

PPAR-γ is a major driver of the accumulation and phenotype of adipose tissue Treg cells

Obesity and type-2 diabetes have increased markedly over the past few decades, in parallel. One of the major links between these two disorders is chronic, low-grade inflammation. Prolonged nutrient excess promotes the accumulation and activation of leukocytes in visceral adipose tissue (VAT) and ultimately other tissues, leading to metabolic abnormalities such as insulin resistance, type-2 diabetes and fatty-liver disease. Although invasion of VAT by pro-inflammatory macrophages is considered to be a key event driving adipose-tissue inflammation and insulin resistance, little is known about the roles of other immune system cell types in these processes. A unique population of VAT-resident regulatory T (Treg) cells was recently implicated in control of the inflammatory state of adipose tissue and, thereby, insulin sensitivity. Here we identify peroxisome proliferator-activated receptor (PPAR)-γ, the 'master regulator' of adipocyte differentiation, as a crucial molecular orchestrator of VAT Treg cell accumulation, phenotype and function. Unexpectedly, PPAR-γ expression by VAT Treg cells was necessary for complete restoration of insulin sensitivity in obese mice by the thiazolidinedione drug pioglitazone. These findings suggest a previously unknown cellular mechanism for this important class of thiazolidinedione drugs, and provide proof-of-principle that discrete populations of Treg cells with unique functions can be precisely targeted to therapeutic ends.     

Prostaglandins and the synergism between VIP and noradrenaline in the cerebral cortex

We have previously shown that vasoactive intestinal peptide (VIP) and noradrenaline (NA) interact synergistically to increase cyclic AMP levels in mouse cerebral cortical slices. The pharmacological mechanism of this synergism is the potentiation by NA, through alpha 1 adrenergic receptors, of the stimulatory effect of VIP on cAMP formation. A similar interaction has been confirmed in guinea pig cerebral cortex and in discrete nuclei of the rat hypothalamus. Furthermore VIP and NA interact synergistically to depress the spontaneous activity of identified neurons in rat neocortex. At the cellular level, this synergistic interaction suggests that VIP- and NA-containing neuronal systems may converge, at least in part, on the same target cells to increase cAMP levels in the cerebral cortex. At the molecular level, the interaction may occur at various steps in signal transduction, between receptors, intramembrane transduction processes or intracellular effector mechanisms. Here we report that the alpha 1-adrenergic potentiation of the increases in cAMP elicited by VIP involves the formation of arachidonic acid metabolites and is mimicked by prostglandins F2 alpha and E2.