L- and N-current but not M-current inhibition by M1 muscarinic receptors requires DAG lipase activity

Stimulation of postsynaptic M(1) muscarinic receptors (M(1)Rs) increases firing rates of both sympathetic and central neurons that underlie increases in vasomotor tone, heart rate, and cognitive memory functioning. At the cellular level, M(1)R stimulation modulates currents through various voltage-gated ion channels, including KCNQ K+ channels (M-current) and both L- and N-type Ca2+ channels (L- and N-current) by a pertussis toxin-insensitive, slow signaling pathway. Depletion of phosphatidylinositol-4,5-bisphosphate (PIP2) during M(1)R stimulation suffices to inhibit M-current. We found previously that following PIP2 hydrolysis by phospholipase C, activation of phospholipase A2 and liberation of a lipid metabolite, most likely arachidonic acid (AA) are necessary for L- and N-current modulation. Here we examined the involvement of a third lipase, diacylglycerol lipase (DAGL), in the slow pathway. We documented the presence of DAGL in superior cervical ganglion neurons, and then tested the highly selective DAGL inhibitor, RHC-80267, for its capacity to antagonize M(1)R-mediated modulation of whole-cell Ca2+ currents. RHC-80267 significantly reduced L- and N-current inhibition by the muscarinic agonist oxotremorine-M (Oxo-M) but did not affect their inhibition by exogenous AA. Moreover, voltage-dependent inhibition of N-current by Oxo-M remained in the presence of RHC-80267, indicating selective action on the slow pathway. RHC also blocked inhibition of recombinant N-current. In contrast, RHC-80267 had no effect on native M-current inhibition. These data are consistent with a role for DAGL in mediating L- and N-current inhibition. These results extend our previous findings that the signaling pathway mediating L- and N-current inhibition diverges from the pathway initiating M-current inhibition.     

Interisland Mutation of a Novel Phospholipase A<Subscript>2</Subscript> from <Emphasis Type="Italic">Trimeresurus flavoviridis</Emphasis> Venom and Evolution of Crotalinae Group II Phospholipases A<Subscript>2</Subscript>

Trimeresurus flavoviridis (Crotalinae) snakes inhabit the southwestern islands of Japan: Amami-Oshima, Tokunoshima, and Okinawa. Affinity and conventional chromatographies of Amami-Oshima T. flavoviridis venom led to isolation of a novel phospholipase A₂ (PLA₂). This protein was highly homologous (91%) in sequence to trimucrotoxin, a neurotoxic PLA₂, which had been isolated from T. mucrosquamatus (Taiwan) venom, and exhibited weak neurotoxicity. This protein was named PLA-N. Its LD₅₀ for mice was 1.34 µg/g, which is comparable to that of trimucrotoxin. The cDNA encoding PLA-N was isolated from both the Amami-Oshima and the Tokunoshima T. flavoviridis venom-gland cDNA libraries. Screening of the Okinawa T. flavoviridis venom-gland cDNA library with PLA-N cDNA led to isolation of the cDNA encoding one amino acid-substituted PLA-N homologue, named PLA-N(O), suggesting that interisland mutation occurred and that Okinawa island was separated from a former island prior to dissociation of Amami-Oshima and Tokunoshima islands. Construction of a phylogenetic tree of Crotalinae venom group II PLA₂s based on the amino acid sequences revealed that neurotoxic PLA₂s including PLA-N and PLA-N(O) form an independent cluster which is distant from other PLA₂ groups such as PLA₂ type, basic [Asp⁴⁹]PLA₂ type, and [Lys⁴⁹]PLA₂ type. Comparison of the nucleotide sequence of PLA-N cDNA with those of the cDNAs encoding other T. flavoviridis venom PLA₂s showed that they have evolved in an accelerated manner. However, when comparison was made within the cDNAs encoding Crotalinae venom neurotoxic PLA₂s, their evolutionary rates appear to be reduced to a level between accelerated evolution and neutral evolution. It is likely that ancestral genes of neurotoxic PLA₂s evolved in an accelerated manner until they had acquired neurotoxic function and since then they have evolved with less frequent mutation, possibly for functional conservation. The nucleotide sequences reported in this paper are available from the GenBank/EMBL/DDBJ databases under accession numbers AB102728 and AB102729.     

Regulation of high molecular weight bovine brain neutral protease by phospholipids in vitro

The activity of the heat stable, glycosylated high molecular weight bovine brain neutral protease (HMW protease) is differentially regulated by phospholipids. While phosphatidylcholine (PC), phosphatidylserine (PS) and phosphatidic acid (PA) had only marginal stimulatory effect (40–75%) on the activity of HMW protease, lysophoshatidylcholine (lysoPC) and lysophosphatidic acid (lysoPA) activated the enzyme by more than two-fold. Both lysoPC and lysoPA exhibited concentration-dependent saturation kinetics for the activation of HMW protease. Surprisingly, phosphoinositides (phosphatidylinositol, PI; phosphatidylinositol 4-phosphate, PIP; and phosphatidylinositol 4,5-bisphosphate, PIP₂) modulated the activity of protease differently: activation of the enzyme was higher with PIP (90%) as compared to PI (21%), whereas PIP₂ inhibited the enzyme (16%). The inhibition of the protease by PIP₂ was concentration-dependent. During receptor-coupled cell activation, phospholipase A₂ (PLA₂) converts PC and PA to lysoPC and lysoPA, respectively; PI is converted to PIP₂ by successive enzymatic phosphorylation by PI 4-kinase and PIP 5-kinase; and phospholipase C (PLC) degrades PIP₂ to diacylglycerol and inositol 1,4,5-trisphosphate. Therefore, the data suggest that HMW protease may be coupled to cell signal transduction where PLA₂, PI 4-kinase, PIP 5-kinase and PLC are involved.     

Organelle motility regulated by the cell's environment: dissection of signaling pathways regulating movements of peroxisomes

Summary Chloroplasts and pigment granules are known to be intracellularly translocated upon discrete extracellular stimuli. The machineries transducing these signals inside cells are yet not understood. In studies investigating the motility of peroxisomes, we were able to identify both extracellular and intracellular signaling steps regulating movements of these organelles. Following simultaneous stimulation of CHO cells with both extracellular ATP and lysophosphatidic acid, an arrest of peroxisomes was observed. This block of motility was shown to be dependent on signaling cascades involving heterotrimeric G proteins of the class G_i/G_o, phospholipase C, calcium influx, and activation of protein kinase C as well as of mitogen-activated protein kinase. Cytosolic phospholipase A₂ is a point of convergence for these pathways, resulting in the release of arachidonic acid. This signaling pathway is specific for peroxisomes and does not influence motility of mitochondria, lysosomes, or endosomes. However, since the cytoskeleton and its associated proteins including the motor proteins play an important role in mediating motility of all cell organelles, it may well be that variant signaling cascades exist ensuring specific regulation of each distinct compartment.Abbreviations AA arachidonic acid - ATPS adenosine-5-O-(3-thiotriphosphate) - cAMP cyclic adenosine monophosphate - CaM-PK calmodulin-dependent protein kinase - CLIP cytosolic linker protein - DAG diacylglycerol - DiC₈ 1,2-dioctanoyl-sn-glycerol - GFP green-fluorescent protein - GTPS guanosine-5-O-(3-thiotriphosphate) - IP₃ inositol trisphosphate - LPA lysophosphatidic acid - MAPK mitogen-activated protein kinase - MEK MAPK kinase - PKA protein kinase A - PKC protein kinase C - cPKC classical PKC isoforms - PLA₂ phospholipase A₂ - PLAP PLA₂-activating proteinpeptide - PLC phospholipase C - PP2A protein phosphatase 2A     

Fluorescence changes reveal kinetic steps of muscarinic receptor–mediated modulation of phosphoinositides and Kv7.2/7.3 K+ channels

G protein–coupled receptors initiate signaling cascades. M₁ muscarinic receptor (M₁R) activation couples through Gα_q to stimulate phospholipase C (PLC), which cleaves phosphatidylinositol 4,5-bisphosphate (PIP₂). Depletion of PIP₂ closes PIP₂-requiring Kv7.2/7.3 potassium channels (M current), thereby increasing neuronal excitability. This modulation of M current is relatively slow (6.4 s to reach within 1/e of the steady-state value). To identify the rate-limiting steps, we investigated the kinetics of each step using pairwise optical interactions likely to represent fluorescence resonance energy transfer for M₁R activation, M₁R/Gβ interaction, Gα_q/Gβ separation, Gα_q/PLC interaction, and PIP₂ hydrolysis. Electrophysiology was used to monitor channel closure. Time constants for M₁R activation (<100 ms) and M₁R/Gβ interaction (200 ms) are both fast, suggesting that neither of them is rate limiting during muscarinic suppression of M current. Gα_q/Gβ separation and Gα_q/PLC interaction have intermediate 1/e times (2.9 and 1.7 s, respectively), and PIP₂ hydrolysis (6.7 s) occurs on the timescale of M current suppression. Overexpression of PLC accelerates the rate of M current suppression threefold (to 2.0 s) to become nearly contemporaneous with Gα_q/PLC interaction. Evidently, channel release of PIP₂ and closure are rapid, and the availability of active PLC limits the rate of M current suppression.     

The Cytotoxic Effect of Bromoenol Lactone,a Calcium-independent Phospholipase A<Subscript>2</Subscript> Inhibitor,on Rat Cortical Neurons in Culture

This study characterized the phospholipase A₂ (PLA₂) activity in cerebral cortex of fetal rat brain and investigated effects of chemical inhibition of Ca²⁺-independent PLA₂ (iPLA₂) on neurite outgrowth and cell development of cortical neurons in vitro. The PLA₂ activity in fetal brain was insensitive to a Ca²⁺-chelator EGTA and was significantly impaired by an iPLA₂ inhibitor, bromoenol lactone (BEL). Following treatment with BEL, cortical neurons showed acute loss of neurites and impaired cell body, which were clearly dose- and time-dependent. Nuclear staining revealed nuclear regression (shrinkage), but not fragmentation, in BEL-treated cells. The cytotoxic effect of BEL was additive with arachidonic acid (AA) and AA alone also induced neurite demise. BEL treatment resulted in increased production of prostaglandin E₂. Overall data suggest that iPLA₂, a primary PLA₂ isoform in cerebral cortex, displays a housekeeping role in development and neurite outgrowth in cortical neurons in vitro probably via maintaining phospholipid membrane remodeling rather than generating free fatty acids and lysophospholipids.     

Interisland Evolution of <Emphasis Type="Italic">Trimeresurus flavoviridis</Emphasis> Venom Phospholipase A<Subscript>2</Subscript> Isozymes

Abstract Trimeresurus flavoviridis snakes inhabit the southwestern islands of Japan. A phospholipase A₂ (PLA₂), named PL-Y, was isolated from Okinawa T. flavoviridis venom and its amino acid sequence was determined from both protein and cDNA. PL-Y was unable to induce edema. In contrast, PLA-B, a PLA₂ from Tokunoshima T. flavoviridis venom, which is different at only three positions from PL-Y, is known to induce edema. A new PLA₂, named PLA-B′, which is similar to PLA-B, was cloned from Amami-Oshima T. flavoviridis venom gland. Three T. flavoviridis venom basic [Asp⁴⁹]PLA₂ isozymes, PL-Y (Okinawa), PLA-B (Tokunoshima), and PLA-B′ (Amami-Oshima), are identical in the N-terminal half but have one to four amino acid substitutions in the β1-sheet and its vicinity. Such interisland sequence diversities among them are due to isolation in the different environments over 1 to 2 million years and appear to have been brought about by natural selection for point mutation in their genes. Otherwise, a major PLA₂, named PLA2, ubiquitously exists in the venoms of T. flavoviridis snakes from the three islands with one to three synonymous substitutions in their cDNAs. It is assumed that the PLA2 gene is a prototype among T. flavoviridis venom PLA₂ isozyme genes and has hardly undergone nonsynonymous mutation as a principal toxic component. Phylogenetic analysis based on the amino acid sequences revealed that T. flavoviridis PLA₂ isozymes are clearly separated into three groups, PLA2 type, basic [Asp⁴⁹]PLA₂ type, and [Lys⁴⁹]PLA₂ type. Basic [Asp⁴⁹]PLA₂-type isozymes may manifest their own particular toxic functions different from those of the isozymes of the PLA2 type and [Lys⁴⁹]PLA₂ type.     

Charge Shielding of PIP2 by Cations Regulates Enzyme Activity of Phospholipase C

Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) of the plasma membrane by phospholipase C (PLC) generates two critical second messengers, inositol-1,4,5-trisphosphate and diacylglycerol. For the enzymatic reaction, PIP₂ binds to positively charged amino acids in the pleckstrin homology domain of PLC. Here we tested the hypothesis that positively charged divalent and multivalent cations accumulate around the negatively charged PIP₂, a process called electrostatic charge shielding, and therefore inhibit electrostatic PIP₂-PLC interaction. This charge shielding of PIP₂ was measured quantitatively with an in vitro enzyme assay using WH-15, a PIP₂ analog, and various recombinant PLC proteins (β1, γ1, and δ1). Reduction of PLC activity by divalent cations, polyamines, and neomycin was well described by a theoretical model considering accumulation of cations around PIP₂ via their electrostatic interaction and chemical binding. Finally, the charge shielding of PIP₂ was also observed in live cells. Perfusion of the cations into cells via patch clamp pipette reduced PIP₂ hydrolysis by PLC as triggered by M₁ muscarinic receptors with a potency order of Mg²⁺ < spermine⁴⁺ < neomycin⁶⁺. Accumulation of divalent cations into cells through divalent-permeable TRPM7 channel had the same effect. Altogether our results suggest that Mg²⁺ and polyamines modulate the activity of PLCs by controlling the amount of free PIP₂ available for the enzymes and that highly charged biomolecules can be inactivated by counterions electrostatically.     

Phospholipase A2 induced effects on the structural organization and physical properties of pea chloroplast membranes

Phospholipase A₂ (PLA₂)-induced effects on the membrane organization, fluidity properties and surface charge density of pea chloroplasts were investigated. It was observed that lipolytic treatment with PLA₂ altered the chloroplast structure having as a result a swelling of thylakoids and a total destruction of normal granal structure. In spite of this, the thylakoid membranes remained in close contact. At the same time, a slight decrease of surface charge density was registered, thus explaining the adhesion of swelled membranes. Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) was measured during PLA₂ treatment. A pronounced decrease of DPH fluorescence polarization was found, indicating that phospholipase treatment resulted in considerable disordering and/or fluidization of the thylakoid membranes. The increased fluidity could be attributed to the destabilizing effect of the products of enzymatic hydrolysis of the phospholipids (free fatty acids, lysophospholipids) on the bilayer structure of thylakoids membranes.Abbreviations 9-AA 9-aminoacridine - BSA bovine serium albumin - DCMU 3-/3,4-dichlorophenyl-1,1-dimethyl/urea - DPH 1,6-diphenyl-1,3,5-hexatriene - EDTA ethylenediaminetetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - LHC light harvesting chlorophyll a/b-protein complex of PS II - MES 2/N-morpholine/ethanesulfonic acid - PLA₂ phospholipase A₂ - PS I, PS II photosystem I and photosystem II, respectively - S lipid structural order parameter - THF tetrahydrofuran - TRICINE N-/tris/hydroxymethyl/methyl/glicine     

Is the PLC pathway involved in the response to phenol treatment in tobacco hairy roots?

Phospholipids and phospholipases play important roles in several cellular processes and responses to adverse growth conditions. However, the mechanism of action of phosphatidylinositol-specific phospholipase C (PI-PLC), and its resulting minor lipid function in response to pollutant-induced stress, remains to be elucidated. In this work, we studied the effects of phenol treatment on the PLC pathway, using two lines of hairy root cultures (HRs) from Nicotiana tabacum as plant model systems: wild-type (WT) and double-transgenic (DT) HRs. We quantified several product formations such as PIP, PIP₂, DGPP, and PA, which are mainly synthesized by specific lipid kinases belonging to the PLC pathway. In both HRs, phenol treatment significantly increased the formation of these compounds in a differential manner. In WT HRs, PA formation was twofold higher than in control. PIP₂ and PIP levels were about onefold higher than those of the controls while DGPP levels increased by 50%. In DT HRs, PIP levels were onefold higher than those of the controls while PIP₂, DGPP, and PA levels increased by 120%. Phenol treatment also upregulated the PLC4 gene expression mainly in the first hours of exposure in both HRs, while the DGK1 gene expression was only upregulated in WT HRs after 24 h of treatment. These results show an active participation of the PLC pathway under phenol treatment suggesting that this signal pathway could be important in plant cell responses to phenol-induced stress.

     

Differences between cellular mechanisms of ATP-and noradrenaline-induced inhibition of visceral smooth muscles under conditions of selective and combined activation of M<Subscript>2</Subscript> or M<Subscript>3</Subscript> cholinoreceptors

Our studies of the role of phospholipase C in inhibitory synaptic action upon visceral smooth muscles demonstrated that, under conditions of carbachol (CCh)-induced pre-activation of cholinoreceptors, ATP-or noradrenaline (NA)-evoked relaxation of these muscles is mediated by the phospholipase C-independent pathway, while the phospholipase C-dependent pathway does not manifest itself as a mechanism that determines the inhibitory effect of the above transmitters. Under conditions of pre-activation of muscarinic cholinoreceptors, ATP-and NA-induced relaxation is continued due to activation of inositol trisphosphate (InsP₃)-sensitive receptors despite the fact that the pathway of inhibition is phospholipase C-independent. This is confirmed by complete depression of the inhibitory effects of ATP and NA against the background of CCh-induced contraction after pre-incubation of the studied preparations together with 100 μM 2-APB, a blocker of InsP₃ receptors. Selective blockings of either M₂ or M₃ cholinoreceptors are accompanied by a complete loss of the ability of the above blocker of InsP₃ receptors (2-APB) to suppress ATP-and NA-induced contraction of smooth muscles in the state of CCh-induced contraction. It can be hypothesized that, under conditions of selective pre-activation of M₂ or M₃ cholinoreceptors, the mechanisms of intracellular signalling mediating the inhibition events are modified. The InsP₃-dependent pathway that determines both adrenergic and purinergic inhibition of smooth muscles is switched off, and the inhibitory action of neurotransmitters is realized under such conditions through the InsP₃-independent pathway. Therefore, in our study we first found differences between cellular mechanisms underlying ATP-and NA-induced inhibition of smooth muscles under conditions of selective activation of either M₂ or M₃ cholinoreceptors and the mechanisms underlying the relaxing action of inhibitory neurotransmitters under conditions of combined synergistic activation of cholinoreceptors of both the above-mentioned subtypes. Neirofiziologiya/Neurophysiology, Vol. 39, No. 1, pp. 22–31, January–February, 2007.     

Mechanism of thrombin-induced arachidonic acid release in osteoblast-like cells

In a previous study, we have reported that thrombin stimulates phosphatidylcholine hydrolysis by phospholipase (PL) D, but has little effect on phosphoinositide hydrolysis by PLC in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the mechanism of the thrombin-induced arachidonic acid (AA) release in MC3T3-E1 cells. Thrombin stimulated AA release dose dependently in the range between 0.1 and 1 U/ml. Quinacrine, a PLA₂ inhibitor, suppressed the thrombin-induced AA release. In addition, quinacrine also suppressed the thrombin-induced prostaglandin E₂ synthesis in these cells. On the other hand, propranolol, which is known to inhibit phosphatidic acid phosphohydrolase, did not affect the thrombin-induced AA release. 1(6-((17β-3-Methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-d |ione (U-73122), a PLC inhibitor, had no effect on the AA release by thrombin. In addition, 1,6-bis-(cyclohexyloximinocarbonylamino)-hexane (RHC-80267), a selective inhibitor of diacylglycerol lipase, had little effect on the thrombin-induced AA release. Neither propranolol, U-73122 nor RHC-80267 affect the thrombin-induced prostaglandin E₂ synthesis. These results strongly suggest that thrombin induces AA release not by phosphatidylcholine hydrolysis by PLD nor phosphoinositide hydrolysis by PLC but mainly by PLA₂ in osteoblast-like cells.     

A role for G-proteins in the epidermal growth factor stimulation of phospholipase A2 in rat kidney mesangial cells

We have previously demonstrated phospholipase C (PLC) independent activation of phospholipase A₂(PLA₂) by epidermal growth factor (EGF) in glomerular mesangial cells in culture. In the current study using glass beads to permeabilize [³H]- or [¹⁴C]-arachidonate labelled mesangial cells we demonstrate that guanine nucleotides modulate the EGF-mediated stimulation of arachidonic acid release (75% inhibition with 100 M GDPS and 108% augmentation with 100 M GTPS). GTPS alone stimulated both the release of free arachidonic acid and production of diacylglycerol (DAG), while EGF itself neither stimulated DAG nor augmented the DAG response to GTPS. These findings suggest the intermediacy of a G-protein in PLC-independent stimulation of PLA₂ by a growth factor, and provide a model system for determining the relationship between G-protein intermediacy and the intrinsic tyrosine kinase activity of the growth factor receptor.Abbreviations EGF Epidermal Growth Factor - PLC phospholipase C - PLA₂ phospholipase A₂ - DAG Diacylglycerol - NEFA non-esterified fatty acid - GTPS guanosine-5-0-[3-thio]triphosphate - GDP\S guanosine-5-0-[2-thio]diphosphate     

Interactions of selected indole derivatives with phospholipase A<Subscript>2</Subscript>: <Emphasis Type="Italic">in silico and in vitro</Emphasis> analysis

Phospholipase A2 (PLA₂) is one of the key enzymes involved in the formation of inflammatory mediators. Inhibition of PLA₂ is considered to be one of the efficient methods to control inflammation. In silico docking studies of 160 selected indole derivatives performed against porcine pancreatic PLA₂ (ppsPLA₂) suggested that, CID2324681, CID8617 (indolebutyric acid or IBA), CID22097771 and CID802 (indoleacetic acid or IAA) exhibited highest binding energies. In silico analysis was carried out to predict some of the ADME properties. The binding potential of these compounds with human non pancreatic secretory PLA₂ (hnpsPLA₂) was determined using molecular docking studies. In order to corroborate the in silico results, enzyme kinetics and isothermal titration calorimetric analysis of the two selected compounds, IAA and IBA were performed against ppsPLA₂. From the analysis, it was concluded that IAA and IBA can act as competitive inhibitors to the enzyme and may be used as anti inflammatory agents.

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Figure Inhibitory activity of IAA and IBA against PLA₂

     

Polyphosphoinositide phospholipase C and evidence for inositol-phosphate-hydrolysing activities in the plasma-membrane fraction from light-grown wheat (Triticum aestivum L.) leaves

The phospholipase C (PLC; EC 3.1.4.3) activity in isolated plasma membranes of light-grown wheat (Triticum aestivum L. cv. Prelude) leaves was investigated. The activity against the polyphosphoinositides was strongly dependent on Ca²⁺ and was affected by the anionic detergent deoxycholate (DOC). In the presence of 20 M Ca²⁺ the PLC activity preferred phosphatidylinositol 4,5-bisphosphate (PIP₂) over phosphatidylinositol 4-monophosphate (PIP) as a substrate. Instead, with 1 mM Ca²⁺ the enzyme clearly favoured PIP. In addition, the PIP₂-PLC activity was increased by Mg²⁺ and in the presence of GTP, guanosine 5-(-thio)-triphosphate as well as ATP, CTP, guanosine 5-diphosphate and guanosine 5-(-thio)-diphosphate. Further analysis showed that a molybdate-sensitive phosphatase activity catalysing the dephosphorylation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P₃) is also associated with the plasma-membrane vesicles. Dephosphorylation of Ins(1,4,5)P₃ was reduced in the presence of GTP or by inclusion of the unspecific phosphatase inhibitor molybdate. The results indicate the presence of a PIP₂-PLC activity and the presence of a molybdate-sensitive phosphatase activity in wheat plasma-membrane vesicles.Abbreviations DOC deoxycholate - IDPase inosine 5-diphosphatase - InsP_s inositol phosphates, the numbering at the end indicates the number of phosphate residues and when their positions on the inositol ring are known they are indicated in parentheses, i.e. - Ins(1,4,5)P₃ inositol 1,4,5-trisphosphate - PIP phosphatidylinositol 4-monophosphate - PIP₂ phosphatidylinositol 4,5-bisphosphate - PLC phospholipase C This work was financially supported by grant from the Deutsche Forschungsgemeinschaft (DFG). M. C. Arz gratefully acknowledges the support of a Graduiertenstipendium des Landes Nordrhein-Westfalen (Germany). We wish to thank S. Laden and G.E. Grambow for assistance.     

Cross Talk Between Substance P and Melittin-Activated Cellular Signaling Pathways in Rat Lactotroph-Enriched Cell Cultures

Abstract: We have investigated the possible interaction (cross talk) between the phospholipase A₂ (PLA₂) and inositol 1,4,5-trisphosphate/protein kinase C (PKC) signaling pathways in rat lactotroph-enriched cell cultures. Melittin, a bee venom peptide, stimulated release of [³H]-arachidonic acid ([³H]AA) from [³H]AA-labeled enriched lactotrophs in a dose-dependent manner. Moreover, melittin and exogenous AA induced a redistribution of PKC catalytic activity and PKCα and β immunoreactivity from the soluble to the particulate fraction in resting and substance P (SP)-stimulated cells. Mellitin had no effect on phospholipase C (PLC) activity. Pretreatment of cell cultures with the PLA₂ inhibitors quinacrine and aristolochic acid resulted in a dose-dependent inhibition of melittin-stimulated PKC isozyme translocation as did the inhibitor of lipoxygenase, nordihydroguaiaretic acid, whereas the cyclooxygenase inhibitor indomethacin had no effect. SP and the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) dose-dependently increased levels of [³H]AA released from cells. Pretreatment of cell cultures with quinacrine reduced the effect of SP on [³H]AA formation. After long-term treatment (24 h) of cells with TPA, the effect of TPA on [³H]AA production was not different from control, whereas SP still displayed [³H]AA-releasing abilities although not at full scale. Pretreatment of cells with thapsigargin, U 73122, methoxyverapamil, and RHC 80267, an inhibitor of diacylglycerol lipase, all resulted in reduced SP-stimulated [³H]AA liberation. Treatment of cell cultures with pertussis toxin (PTX) reduced the release of [³H]AA induced by SP, whereas PTX had no effect on SP-stimulated generation of ³H-inositol phosphates. On the basis of these results, it is concluded that (1) the PLA₂ pathways interfere with the phosphoinositide-PLC signaling system at the level of PKC isozymes α and β, the product responsible for this interaction being either AA or a metabolite produced by the action of lipoxygenase; (2) SP and TPA are able to activate the PLA₂ pathway at a level at or beyond PLA₂, and this effect is mediated, in part, through PKCα and β species and (for SP) intracellular Ca²⁺ recruited from internal stores as well as from external sources; and (3) SP also activates PLA₂ through a PTX-sensitive pathway distinct from the one coupled to phosphoinositide-PLC, which is PTX insensitive.     

Biochemical characteristics of immune-associated phospholipase A<Subscript>2</Subscript> and its inhibition by an entomopathogenic bacterium, <Emphasis Type="Italic">Xenorhabdus nematophila</Emphasis>

An entomopathogenic bacterium, Xenorhabdus nematophila, induces an immunosuppression of target insects by inhibiting phospholipase A₂ (PLA₂) activity. Recently, an immune-associated PLA₂ gene was identified from the red flour beetle, Tribolium castaneum. This study cloned this PLA₂ gene in a bacterial expression vector to produce a recombinant enzyme. The recombinant T. castaneum PLA₂ (TcPLA₂) exhibited its characteristic enzyme activity with substrate concentration, pH, and ambient temperature. Its biochemical characteristics matched to a secretory type of PLA₂ (sPLA₂) because its activity was inhibited by dithiothreitol (a reducing agent of disulfide bond) and bromophenacyl bromide (a specific sPLA₂ inhibitor) but not by methylarachidonyl fluorophosphonate (a specific cytosolic type of PLA₂). The X. nematophila culture broth contained PLA₂ inhibitory factor(s), which was most abundant in the media obtained at a stationary bacterial growth phase. The PLA₂ inhibitory factor(s) was heat-resistant and extracted in both aqueous and organic fractions. Effect of a PLA₂-inhibitory fraction on the immunosuppression of T. castaneum was equally comparable with that resulted from inhibition of the TcPLA₂ gene expression by RNA interference.     

Regulation of voltage-gated Ca channels by lipids

Great skepticism has surrounded the question of whether modulation of voltage-gated Ca²⁺ channels (VGCCs) by the polyunsaturated free fatty acid arachidonic acid (AA) has any physiological basis. Here we synthesize findings from studies of both native and recombinant channels where micromolar concentrations of AA consistently inhibit both native and recombinant activity by stabilizing VGCCs in one or more closed states. Structural requirements for these inhibitory actions include a chain length of at least 18 carbons and multiple double bonds located near the fatty acid's carboxy terminus. Acting at a second site, AA increases the rate of VGCC activation kinetics, and in Ca_V2.2 channels, increases current amplitude. We present evidence that phosphatidylinositol 4,5-bisphosphate (PIP₂), a palmitoylated accessory subunit (β_2a) of VGCCs and AA appear to have overlapping sites of action giving rise to complex channel behavior. Their actions converge in a physiologically relevant manner during muscarinic modulation of VGCCs. We speculate that M₁ muscarinic receptors may stimulate multiple lipases to break down the PIP₂ associated with VGCCs and leave PIP₂'s freed fatty acid tails bound to the channels to confer modulation. This unexpectedly simple scheme gives rise to unanticipated predictions and redirects thinking about lipid regulation of VGCCs.     

Muscarinic Inhibition of Hippocampal and Striatal Adenylyl Cyclase is Mainly Due to the M<Subscript>4</Subscript> Receptor

The five muscarinic acetylcholine receptors (M₁–M₅) are differentially expressed in the brain. M₂ and M₄ are coupled to inhibition of stimulated adenylyl cyclase, while M₁, M₃ and M₅ are mainly coupled to the phosphoinositide pathway. We studied the muscarinic receptor regulation of adenylyl cyclase activity in the rat hippocampus, compared to the striatum and amygdala. Basal and forskolin-stimulated adenylyl cyclase activity was higher in the striatum but the muscarinic inhibition was much lower. Highly selective muscarinic toxins MT1 and MT2—affinity order M₁ ≥ M₄ >> others—and MT3—highly selective M₄ antagonist—did not show significant effects on basal or forskolin-stimulated cyclic AMP production but, like scopolamine, counteracted oxotremorine inhibition. Since MTs have negligible affinity for M₂, M₄ would be the main subtype responsible for muscarinic inhibition of forskolin-stimulated enzyme. Dopamine stimulated a small fraction of the enzyme (3.1% in striatum, 1.3% in the hippocampus). Since MT3 fully blocked muscarinic inhibition of dopamine-stimulated enzyme, M₄ receptor would be responsible for this regulation. Diana Jerusalinsky and Edgar Kornisiuk contributed equally to this paper.     

Lysosomotropic agents activate the capacity for calcium dependent pinocytosis in starvedAmoeba proteus: evidence for a mechanism involving phospholipase A2

Summary Pinocytosis induced by Na⁺ was assayed by phase contrast microscopy in 8–12 days starvedAmoeba proteus. These cultures were inactive with respect to calcium-dependent Na⁺-induced pinocytosis, but treatment with amino acid methyl and ethyl esters increased their capacity for pinocytosis. Besides promoting pinocytosis these compounds also stimulated calcium-sensitive secretion of lysosomal enzymes from normal, 2–3 days starved, cells. Only uncharged 1-forms of the amino acid esters were effective. Also other lysosomotropic compounds including monodansylcadaverine, glycine-phenylalanine-2-naphthylamide, NH₄Cl, and the ionophores monensin and A23187 activated starved cells. The effect of these agents (except A23187) was inhibited by the drug dantrolene suggesting that activation is a consequence of release of Ca²⁺ from intracellular stores. Several of the lysosomotropic agents also lost their activating effect in the presence of phospholipase A₂ (PLA₂) inhibitors. To investigate whether or not PLA₂ activity in the cell culture could imitate the effect of the lysosomotropic agents, we incubated starved cells with snake venom PLA₂s. These enzymes caused rapid, dantrolene-sensitive activation of the cells. Measurement of endogenous PLA₂in normal cells revealed significant cellular activity but no significant secretion of the enzyme into the culture medium was observed. Together the studies with enzyme inhibitors and dantrolene suggest that the process by which lysosomotropic agents affect pinocytosis involves activation of PLA₂ and release of Ca²⁺ from intracellular stores.Abbreviations AnBOMe amino-n-butyric acid methylester - Et ethylester - GPN glycine-1-phenylalanine-2-naphthylamide - MDC monodansylcadaverine - MDTC monodansylthiacadaverine - Me methylester - pBPB p-bromo phenacylbromide - PLA₂ phospholipase A₂