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1.
《Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids》2022,1867(12):159222
N-Acyl-phosphatidylethanolamines (NAPEs), a minor class of membrane glycerophospholipids, accumulate along with their bioactive metabolites, N-acylethanolamines (NAEs) during ischemia. NAPEs can be formed through N-acylation of phosphatidylethanolamine by cytosolic phospholipase A2ε (cPLA2ε, also known as PLA2G4E) or members of the phospholipase A and acyltransferase (PLAAT) family. However, the enzyme responsible for the NAPE production in brain ischemia has not yet been clarified. Here, we investigated a possible role of cPLA2ε using cPLA2ε-deficient (Pla2g4e?/?) mice. As analyzed with brain homogenates of wild-type mice, the age dependency of Ca2+-dependent NAPE-forming activity showed a bell-shape pattern being the highest at the first week of postnatal life, and the activity was completely abolished in Pla2g4e?/? mice. However, liquid chromatography-tandem mass spectrometry revealed that the NAPE levels of normal brain were similar between wild-type and Pla2g4e?/? mice. In contrast, post-mortal accumulations of NAPEs and most species of NAEs were only observed in decapitated brains of wild-type mice. These results suggested that cPLA2ε is responsible for Ca2+-dependent formation of NAPEs in the brain as well as the accumulation of NAPEs and NAEs during ischemia, while other enzyme(s) appeared to be involved in the maintenance of basal NAPE levels. 相似文献
2.
Hiroyuki Nakamura Shigeo Wakita Akiko Suganami Yutaka Tamura Kentaro Hanada Toshihiko Murayama 《Journal of lipid research》2010,51(4):720-728
We examined the effect of the cellular sphingolipid level on the release of arachidonic acid (AA) and activity of cytosolic phospholipase A2α (cPLA2α) using two Chinese hamster ovary (CHO)-K1-derived mutants deficient in sphingolipid synthesis: LY-B cells defective in the LCB1 subunit of serine palmitoyltransferase for de novo synthesis of sphingolipid species, and LY-A cells defective in the ceramide transfer protein CERT for SM synthesis. When LY-B and LY-A cells were cultured in Nutridoma medium and the sphingolipid level was reduced, the release of AA stimulated by the Ca2+ ionophore increased 2-fold and 1.7-fold, respectively, compared with that from control cells. The enhancement in LY-B cells was decreased by adding sphingosine and treatment with the cPLA2α inhibitor. When CHO cells were treated with an acid sphingomyelinase inhibitor to increase the cellular SM level, the release of AA induced by A23187 or PAF was decreased. In vitro studies were then conducted to test whether SM interacts directly with cPLA2α. Phosphatidylcholine vesicles containing SM reduced cPLA2α activity. Furthermore, SM disturbed the binding of cPLA2α to glycerophospholipids. These results suggest that SM at the biomembrane plays important roles in regulating the cPLA2α-dependent release of AA by inhibiting the binding of cPLA2α to glycerophospholipids. A23187相似文献
3.
It was recently suggested that the opening of neutrophil plasma membrane Ca2+ channels by chemotactic agents is mediated by a rise in free cytosolic Ca2+ concentration ([Ca2+]i). This hypothesis was tested in human cells monitoring [Ca2+]i with the indicator indo-1. In cells loaded with the Ca2+-chelating agent bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetate, transmembrane Ca2+ uptake could be stimulated by formyl-methionyl-leucyl-phenylalanine (fMLP) even when [Ca2+]i was at or below the resting level. In contrast, simply elevating [Ca2+]i in unstimulated cells failed to increase transmembrane uptake. It was concluded either that Ca2+ uptake across the plasma membrane is activated directly by the formation of the chemotactic factor-receptor complex or, more likely, that a transduction mechanism distinct from changes in [Ca2+]i is involved. 相似文献
4.
Noha SM Jazzar B Kuehnl S Rollinger JM Stuppner H Schaible AM Werz O Wolber G Schuster D 《Bioorganic & medicinal chemistry letters》2012,22(2):1202-1207
The release of arachidonic acid, a precursor in the production of prostaglandins and leukotrienes, is achieved by activity of the cytosolic phospholipase A(2)α (cPLA(2)α). Signaling mediated by this class of bioactive lipids, which are collectively referred to as eicosanoids, has numerous effects in physiological and pathological processes. Herein, we report the development of a ligand-based pharmacophore model and pharmacophore-based virtual screening of the National Cancer Institute (NCI) database, leading to the identification of 4-(hexadecyloxy)-3-(2-(hydroxyimino)-3-oxobutanamido)benzoic acid (NSC 119957) as cPLA(2)α inhibitor in cell-free and cell-based in vitro assays. 相似文献
5.
《Prostaglandins & other lipid mediators》2007,82(3-4):113-125
Cytosolic phospholipase A2-α (cPLA2) plays an important role in the release of arachidonic acid and in cell injury. Activation of cPLA2 is dependent on a rise in cytosolic Ca2+ concentration, membrane association via the Ca2+-dependent lipid binding (CaLB) domain, and phosphorylation. This study addresses the activation of cPLA2 via potential association with membrane phosphatidylinositol 4,5-bisphosphate (PIP2), including the role of a “pleckstrin homology (PH)-like” region of cPLA2 (amino acids 263-354). In cells incubated with complement, phorbol myristate acetate + the Ca2+ ionophore, A23187, or epidermal growth factor + A23187, expression of the PH domain of phospholipase C-δ1 (which sequesters membrane PIP2) attenuated cPLA2 activity. Stimulated cPLA2 activity was also attenuated by the expression of cPLA2 135-366, or cPLA2 2-366, and expression of a PIP2-specific 5′-phosphatase. However, in a yeast-based assay that tests the ability of proteins to bind to membrane lipids, including PIP2, with high affinity, only cPLA2 1-200 (CaLB domain) was able to interact with membrane lipids, whereas cPLA2s 135-366, 2-366, 201-648, and 1-648 were unable to do so. Therefore, cPLA2 activity can be modulated by sequestration or depletion of cellular PIP2, although the interaction of cPLA2 with membrane PIP2 appears to be indirect, or of weak affinity. 相似文献
6.
Mark Hess Alwine Schulze Elfringhoff 《Journal of enzyme inhibition and medicinal chemistry》2013,28(6):946-957
A series of 3-pyrrol-3-yl-3H-isobenzofuran-1-ones was synthesized and assessed for the ability to inhibit cytosolic phospholipase A2α (cPLA2α). Several of these compounds were found to be active in both a cell based assay and an isolated enzyme assay. The most potent inhibitor was the thiazolidine-2,4-dione substituted derivative 35. With IC50-values of 0.7 μM and 7.3 μM in the cellular and isolated enzyme assay, respectively, it possesses similar inhibitory potency as the known cPLA2α inhibitor arachidonyltrifluoromethyl ketone (AACOCF3). Structure–activity relationship studies revealed that the evaluated isobenzofuran-1-ones seem to exert their cellular activities not only by a direct interaction with the enzyme but also by other as yet unknown mechanisms. 相似文献
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The binding of Ca2+ antagonists to soluble proteins obtained by ammonium sulphate precipitation from cytosol fraction of rabbit skeletal muscles was studied. The KD values for 3H D-888 and 3H PN 200-110 binding to soluble proteins were 21.3 +/- 3.1 nmol.l-1 and 28.8 +/- 8.9 nmol.l-1 respectively. Photoaffinity labelling of the soluble proteins with the arylazide 1,4-dihydropyridine probe 3H azidopine resulted in labelling of the 85-95 K protein band as determined by SDS polyacrylamide gel electrophoresis. Partial purification of prelabelled soluble sample by gel filtration on Sephadex G-150 gave a more precise molecular weight of 90 +/- 2.5K. Polyclonal antibodies prepared against Ca2+ channel complex from rabbit muscle T-tubules inhibited the 3H PN 200-110 binding. Our results suggest that the soluble protein with Mr = 90K +/- 2.5K may be a precursor of the large subunit of the membrane bound L-type Ca2+ channel in rabbit skeletal muscle. 相似文献
10.
Masaya Shimizu Eiko Tada Tomohiko Makiyama Kana Yasufuku Yuta Moriyama Hiromichi Fujino Hiroyuki Nakamura Toshihiko Murayama 《Cellular signalling》2009,21(3):440-447
Ceramide and the metabolites including ceramide-1-phosphate (C1P) and sphingosine are reported to regulate the release of arachidonic acid (AA) and/or phospholipase A2 (PLA2) activity in many cell types including lymphocytes. Recent studies established that C1P, a product of ceramide kinase, interacts directly with Ca2+ binding regions in the C2 domain of α type cytosolic PLA2 (cPLA2α), leading to translocation of the enzyme from the cytosol to the perinuclear region in cells. However, a precise mechanism for C1P-induced activation of cPLA2α has not been well elucidated; such as the phosphorylation signal caused by the extracellular signal-regulated kinases (ERK1/2) pathway, a downstream of the protein kinase C activation with 4β-phorbol myristate acetate (PMA), is required or not. In the present study, we showed that the increase in intracellular ceramide levels (exogenously added cell permeable ceramides and an inhibition of ceramidase by (1S,2R)-D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol and the increase in C1P formation by transfection with the vector for human ceramide kinase significantly enhanced the Ca2+ ionophore (A23187) -induced release of AA via cPLA2α's activation in CHO cells. Ceramides did not show additional effects on the release from the cells treated with the inhibitor of ceramidase. Ceramides and C2-C1P neither had effect on the intracellular mobilization of Ca2+ nor the phosphorylation of cPLA2α in cells. A23187/PMA-induced release of AA was enhanced by ceramides and C2-C1P and by expression of ceramide kinase. Our findings suggest that C1P is a stimulatory factor on cPLA2α that is independent of the Ca2+ signal and the PKC-ERK-mediated phosphorylation signal. 相似文献
11.
The actions of intracellular pH (pH i ) on Ca2+dependent Cl? channels were studied in secretory epithelial cells derived from human colon carcinoma (T84) and in isolated rat parotid acinar cells. Channel currents were measured with the whole cell voltage clamp technique with pipette solutions of different pH. Ca2+dependent Cl? channels were activated by superfusing ionomycin to increase the intracellular calcium concentration ([Ca2+] i ) or by using pipette solutions with buffered Ca2+ levels. Large currents were activated in T84 and parotid cells by both methods with pH i levels of 7.3 or 8.3. Little or no Cl? channel current was activated with pH i at 6.4. We used on-cell patch clamp methods to investigate the actions of low pH i on single Cl? channel current amplitude in T84 cells. Lowering the pH i had little or no effect on the current amplitude of a 8 pS Cl? channel, but did reduce channel activity. These results suggest that cytosolic acidification may be able to modulate stimulus-secretion coupling in fluid-secreting epithelia by inhibiting the activation of Ca2+-activated Cl? channels. 相似文献
12.
Ziechner U Schönherr R Born AK Gavrilova-Ruch O Glaser RW Malesevic M Küllertz G Heinemann SH 《The FEBS journal》2006,273(5):1074-1086
Human ether à go-go potassium channels (hEAG1) open in response to membrane depolarization and they are inhibited by Ca2+/calmodulin (CaM), presumably binding to the C-terminal domain of the channel subunits. Deletion of the cytosolic N-terminal domain resulted in complete abolition of Ca2+/CaM sensitivity suggesting the existence of further CaM binding sites. A peptide array-based screen of the entire cytosolic protein of hEAG1 identified three putative CaM-binding domains, two in the C-terminus (BD-C1: 674-683, BD-C2: 711-721) and one in the N-terminus (BD-N: 151-165). Binding of GST-fusion proteins to Ca2+/CaM was assayed with fluorescence correlation spectroscopy, surface plasmon resonance spectroscopy and precipitation assays. In the presence of Ca2+, BD-N and BD-C2 provided dissociation constants in the nanomolar range, BD-C1 bound with lower affinity. Mutations in the binding domains reduced inhibition of the functional channels by Ca2+/CaM. Employment of CaM-EF-hand mutants showed that CaM binding to the N- and C-terminus are primarily dependent on EF-hand motifs 3 and 4. Hence, closure of EAG channels presumably requires the binding of multiple CaM molecules in a manner more complex than previously assumed. 相似文献
13.
Shigeki Kobayashi Takehisa Susa Hironori Ishiguchi Takeki Myoren Wakako Murakami Takayoshi Kato Masakazu Fukuda Akihiro Hino Takeshi Suetomi Makoto Ono Hitoshi Uchinoumi Hiroki Tateishi Mamoru Mochizuki Tetsuro Oda Shinichi Okuda Masahiro Doi Takeshi Yamamoto Masafumi Yano 《PloS one》2015,10(1)
Objectives
The purpose of this study was to investigate whether adding a low-dose β1-blocker to milrinone improves cardiac function in failing cardiomyocytes and the underlying cardioprotective mechanism.Background
The molecular mechanism underlying how the combination of low-dose β1-blocker and milrinone affects intracellular Ca2+ handling in heart failure remains unclear.Methods
We investigated the effect of milrinone plus landiolol on intracellular Ca2+ transient (CaT), cell shortening (CS), the frequency of diastolic Ca2+ sparks (CaSF), and sarcoplasmic reticulum Ca2+ concentration ({Ca2+}SR) in normal and failing canine cardiomyocytes and used immunoblotting to determine the phosphorylation level of ryanodine receptor (RyR2) and phospholamban (PLB).Results
In failing cardiomyocytes, CaSF significantly increased, and peak CaT and CS markedly decreased compared with normal myocytes. Administration of milrinone alone slightly increased peak CaT and CS, while CaSF greatly increased with a slight increase in {Ca2+}SR. Co-administration of β1-blocker landiolol to failing cardiomyocytes at a dose that does not inhibit cardiomyocyte function significantly decreased CaSF with a further increase in {Ca2+}SR, and peak CaT and CS improved compared with milrinone alone. Landiolol suppressed the hyperphosphorylation of RyR2 (Ser2808) in failing cardiomyocytes but had no effect on levels of phosphorylated PLB (Ser16 and Thr17). Low-dose landiolol significantly inhibited the alternans of CaT and CS under a fixed pacing rate (0.5 Hz) in failing cardiomyocytes.Conclusion
A low-dose β1-blocker in combination with milrinone improved cardiac function in failing cardiomyocytes, apparently by inhibiting the phosphorylation of RyR2, not PLB, and subsequent diastolic Ca2+ leak. 相似文献14.
Skupin A Kettenmann H Winkler U Wartenberg M Sauer H Tovey SC Taylor CW Falcke M 《Biophysical journal》2008,94(6):2404-2411
Ca2+ oscillations have been considered to obey deterministic dynamics for almost two decades. We show for four cell types that Ca2+ oscillations are instead a sequence of random spikes. The standard deviation of the interspike intervals (ISIs) of individual spike trains is similar to the average ISI; it increases approximately linearly with the average ISI; and consecutive ISIs are uncorrelated. Decreasing the effective diffusion coefficient of free Ca2+ using Ca2+ buffers increases the average ISI and the standard deviation in agreement with the idea that individual spikes are caused by random wave nucleation. Array-enhanced coherence resonance leads to regular Ca2+ oscillations with small standard deviation of ISIs. 相似文献
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16.
Pattabiraman PP Lih FB Tomer KB Rao PV 《American journal of physiology. Cell physiology》2012,302(7):C979-C991
The contractile and relaxation characteristics of trabecular meshwork (TM) are presumed to influence aqueous humor (AH) drainage and intraocular pressure. The mechanisms underlying regulation of TM cell contractile properties, however, are not well understood. This study investigates the role of calcium-independent phospholipase A(2) (iPLA(2)), which controls eicosanoid synthesis, in regulation of TM cell contraction and AH outflow using mechanism-based isoform specific inhibitors (R)-bromoenol lactone (R-BEL, iPLA(2)γ specific) and (S)-bromoenol lactone (S-BEL, iPLA(2)β specific). Immunohistochemical analysis revealed intense staining for both iPLA(2)β and γ isoforms throughout the TM, juxtacanalicular tissue, and Schlemm's canal of human eye. Inhibition of iPLA(2)γ by R-BEL or small interfering RNA-mediated silencing of iPLA(2)γ expression induced dramatic changes in TM cell morphology, and decreased actin stress fibers, focal adhesions, and myosin light-chain (MLC) phosphorylation. AH outflow facility increased progressively and significantly in enucleated porcine eyes perfused with R-BEL. This response was associated with a significant decrease in TM tissue MLC phosphorylation and alterations in the morphology of aqueous plexi in R-BEL-perfused eyes. In contrast, S-BEL did not affect either of these parameters. Additionally, R-BEL-induced cellular relaxation of the TM was associated with a significant decrease in the levels of active Rho GTPase, phospho-MLC phosphatase, phospho-CPI-17, and arachidonic acid. Taken together, these observations demonstrate that iPLA(2)γ plays a significant and isoform-specific role in regulation of AH outflow facility by altering the contractile characteristics of the TM. The effects of iPLA(2)γ on TM contractile status appear to involve arachidonic acid and Rho GTPase signaling pathways. 相似文献
17.
A. Chorvatova A. Guyot C. Ojeda O. Rougier A. Bilbaut 《The Journal of membrane biology》1998,162(1):39-50
The effects of angiotensin II (100 nm) on the electrical membrane properties of zona fasciculata cells isolated from calf adrenal gland were studied using the
whole cell patch recording method. In current-clamp condition, angiotension II induced a biphasic membrane response which
began by a transient hyperpolarization followed by a depolarization more positive than the control resting potential. These
effects were abolished by Losartan (10−5
m), an antagonist of angiotensin receptors of type 1. The angiotensin II-induced transient hyperpolarization was characterized
in voltage-clamp condition from a holding potential of −10 mV. Using either the perforated or the standard recording method,
a transient outward current accompanied by an increase of the membrane conductance was observed in response to the hormonal
stimulation. This outward current consisted of an initial fast peak followed by an oscillating or a slowly decaying plateau
current. In Cl−-free solution, the outward current reversed at −78.5 mV, a value close to E
K. It was blocked by external TEA (20 mm) and by apamin (50 nm). In K+-free solution, the transient outward current, sensitive to Cl− channel blocker DPC (400 μm), reversed at −52 mV, a more positive potential than E
Cl. Its magnitude changed in the same direction as the driving force for Cl−. The hormone-induced transient outward current was never observed when EGTA (5 mm) was added to the pipette solution. The plateau current was suppressed in nominally Ca2+-free solution (47% of cells) and was reversibly blocked by Cd2+ (300 μm) but not by nisoldipine (0.5–1 μm) which inhibited voltage-gated Ca2+ currents identified in this cell type. The present experiments show that the transient hyperpolarization induced by angiotensin
II is due to Ca2+-dependent K+ and Cl− currents. These two membrane currents are co-activated in response to an internal increase of [Ca2+]
i
originating from intra- and extracellular stores.
Received: 29 May 1997/Revised: 4 November 1997 相似文献
18.
Steven J. Kirincich Jason Xiang Neal Green Steve Tam Hui Y. Yang Jaechul Shim Marina W.H. Shen James D. Clark John C. McKew 《Bioorganic & medicinal chemistry》2009,17(13):4383-4405
The synthesis and optimization of a class of trisubstituted quinazoline-2,4(1H,3H)-dione cPLA2α inhibitors are described. Utilizing pharmacophores that were found to be important in our indole series, we discovered inhibitors with reduced lipophilicity and improved aqueous solubility. These compounds are active in whole blood assays, and cell-based assay results indicate that prevention of arachidonic acid release arises from selective cPLA2α inhibition. 相似文献
19.
Ca2+-dependent Inactivation of CaV1.2 Channels Prevents Gd3+ Block: Does Ca2+ Block the Pore of Inactivated Channels?
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Lanthanide gadolinium (Gd(3+)) blocks Ca(V)1.2 channels at the selectivity filter. Here we investigated whether Gd(3+) block interferes with Ca(2+)-dependent inactivation, which requires Ca(2+) entry through the same site. Using brief pulses to 200 mV that relieve Gd(3+) block but not inactivation, we monitored how the proportions of open and open-blocked channels change during inactivation. We found that blocked channels inactivate much less. This is expected for Gd(3+) block of the Ca(2+) influx that enhances inactivation. However, we also found that the extent of Gd(3+) block did not change when inactivation was reduced by abolition of Ca(2+)/calmodulin interaction, showing that Gd(3+) does not block the inactivated channel. Thus, Gd(3+) block and inactivation are mutually exclusive, suggesting action at a common site. These observations suggest that inactivation causes a change at the selectivity filter that either hides the Gd(3+) site or reduces its affinity, or that Ca(2+) occupies the binding site at the selectivity filter in inactivated channels. The latter possibility is supported by previous findings that the EEQE mutation of the selectivity EEEE locus is void of Ca(2+)-dependent inactivation (Zong Z.Q., J.Y. Zhou, and T. Tanabe. 1994. Biochem. Biophys. Res. Commun. 201:1117-11123), and that Ca(2+)-inactivated channels conduct Na(+) when Ca(2+) is removed from the extracellular medium (Babich O., D. Isaev, and R. Shirokov. 2005. J. Physiol. 565:709-717). Based on these results, we propose that inactivation increases affinity of the selectivity filter for Ca(2+) so that Ca(2+) ion blocks the pore. A minimal model, in which the inactivation "gate" is an increase in affinity of the selectivity filter for permeating ions, successfully simulates the characteristic U-shaped voltage dependence of inactivation in Ca(2+). 相似文献
20.
Christopher M. R. Bax Vijai S. Shankar A. S. M. Towhidul Alam Bridget E. Bax Baljit S. Moonga Christopher L. -H. Huang Mone Zaidi Barry R. Rifkin 《Bioscience reports》1993,13(3):169-174
We report the effects of tetracycline analogues on cytosolic Ca2+ transients resulting from application of ionic nickel (Ni2+), a potent surrogate agonist of the osteoclast Ca2+ receptor. Preincubation with minocycline (1 mg/l) or a chemically modified tetracycline, 4-dedimethyl-aminotetracycline (CMT-1) (1 or 10 mg/l), resulted in a significant attenuation of the magnitude of the cytosolic [Ca2+] response to an application of 5 mM-[Ni2+]. Preincubation with doxycycline (1 or 10 mg/l) failed to produce similar results. In addition, application of minocycline alone (0.1–100 mg/l) resulted in a 3.5-fold elevation of cytosolic [Ca2+]. The results suggest a novel action of tetracyclines on the osteoclast Ca2+ receptor. 相似文献