共查询到20条相似文献,搜索用时 13 毫秒
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We demonstrate that cells derived from primary cultures of rabbit proximal tubules (RPTC), human embryonic kidney (HEK293) and human kidney carcinomas (Caki-1) express microsomal Ca(2+)-independent phospholipase A(2) (iPLA(2)gamma) and cytosolic Ca(2+)-independent phospholipase A(2) (iPLA(2)beta). Inhibition of iPLA(2) activity in these cells using the iPLA(2) inhibitor bromoenol lactone (BEL) (0-5.0microM) for 24h did not induce cell death as determined by annexin V and propidium iodide (PI) staining. However, BEL treatment prior to cisplatin (50muM) or vincristine (2microM) exposure reduced apoptosis 30-50% in all cells tested (RPTC, HEK293 and Caki-1 cells). To identify the phospholipids altered during cell death electrospray ionization-mass spectrometry and lipidomic analysis of HEK293 and Caki-1 cells was performed. Cisplatin treatment reduced 14:0-16:0 and 16:0-16:0 phosphatidylcholine (PtdCho) 50% and 35%, respectively, in both cell lines, 16:0-18:2 PtdCho in Caki-1 cells and increased 16:1-22:6 plasmenylcholine (PlsCho). BEL treatment prior to cisplatin exposure further decreased 14:0-16:0 PtdCho, 16:0-16:1 PlsCho and 16:0-18:1 PlsCho in HEK293 cells, and inhibited cisplatin-induced increases in 16:1-22:6 PlsCho in Caki-1 cells. Treatment of cells with BEL prior to cisplatin exposure also increased the levels of several arachidonic containing phospholipids including 16:0-20:4, 18:1-20:4, and 18:0-20:4 PtdCho, compared to cisplatin only treated cells. These data demonstrate that inhibition of iPLA(2) protects against chemotherapeutic-induced cell death in multiple human renal cell models, identifies specific phospholipids whose levels are altered during cell death, and demonstrates that alterations in these phospholipids correlate to the protection against cell death in the presence of iPLA(2) inhibitors. 相似文献
3.
The present study investigates whether endothelin-1 (ET-1), like noradrenaline (NA), stimulates the release of arachidonic acid (AA) via cytosolic phospholipase A2 (cPLA2) in rat tail artery. In tail artery segments labelled with [3H]AA, ET-1-induced AA release in a concentration-dependent manner with an EC50 of 1.3 nM. The effect of ET-1 was inhibited by bosentan and was insensitive to BQ788, suggesting the involvement of ETA receptor. The stimulation of AA release induced by ET-1 was prevented by arachydonyl trifluoromethyl ketone (AACOCF3), a selective inhibitor of cPLA2 and not by RHC80267, a diacylglycerol lipase inhibitor. Furthermore, PD98059, inhibitor of mitogen-activated protein kinase kinase (MEK) cascade and calphostin C, a protein kinase C (PKC) inhibitor, prevented the stimulation of AA release induced by ET-1 and NA. Immunoblotting of the cytosolic fraction of rat tail arteries stimulated with ET-1 or NA showed an increase in extracellular signal-regulated kinases (ERKs) phosphorylation and this effect was abolished by calphostin C treatment. These findings show that in rat tail artery ET-1 and NA induce a sequential activation of protein kinase C and extracellular signal-regulated kinases that results in stimulation of AA release via cPLA2 activation. This may represent a general pathway by which G-proteins coupled receptors stimulate AA release and its metabolites in vascular smooth muscle. 相似文献
4.
We previously reported that zymosan-stimulated, protein kinase C (PKC)-dependent arachidonic acid liberation occurs with association of Ca2+-independent phospholipase A2 (iPLA2) with the membranes of macrophage-like P388D1 cells. In the present study, the possible involvement of PKC isoforms (alpha, beta, delta, and epsilon) on the increase in iPLA2 was examined. Stimulation of P388D1 cells with zymosan induced increases in iPLA2 activity and protein in the membranes and liberation of arachidonic acid. In the stimulated cells, PKCalpha, PKCdelta, and PKCepsilon, but not PKCbeta, were increased in the membranes. The zymosan-induced increase in iPLA2 activity was suppressed by pretreatment with 4beta-phorbol 12-myristate 13-acetate for 10 hr, by which PKCalpha and PKCdelta, but not PKCbeta and PKCepsilon, were depleted, and by G?6976, a PKCalpha inhibitor, but not rottlerin, a PKCdelta inhibitor. The zymosan-induced release of arachidonic acid was also reduced by the PKC depletion and G?6976. However, stimulation with 4beta-phorbol 12-myristate 13-acetate alone did not increase iPLA2 activity in the membranes. Furthermore, the depletion of intracellular Ca2+ also impaired the zymosan-induced increase in iPLA2 activity in the membranes. However, no increase in iPLA2 activity was observed upon stimulation with Ca2+-mobilizing agents (ionomycin or thapsigargin). Cytochalasin D, an inhibitor of actin polymerization, suppressed the zymosan-induced increases in iPLA2 activity and protein in the membranes and the release of arachidonic acid. These results suggest that zymosan stimulates an increase in iPLA2 in the membranes of P388D1 cells probably through activation of PKCalpha in concert with cytochalasin D-sensitive events. 相似文献
5.
We investigated the signal transduction pathway(s) of leukotriene D(4) (LTD(4)) in the human promonocytic U937 cells, a cell line known to constitutively express CysLT(1) receptors. Herein, we demonstrate that LTD(4) specifically acts on a CysLT(1) receptor to dose-dependently increase (three to five-fold over basal) RasGTP through a G(i/o) protein. In fact, while cytosolic Ca(2+) ([Ca(2+)](i)) increase was only partially sensitive to pertussis toxin (PTx), Ras activation was almost completely inhibited by the same toxin. Furthermore, the phospholipase C (PLC) inhibitor U73122 completely inhibited both [Ca(2+)](i) and RasGTP increase, suggesting that in these cells PLC is the point of convergence for both PTx insensitive and sensitive pathways leading to [Ca(2+)](i) release and Ras activation. Indeed, chelating intracellular Ca(2+) strongly (>70%) prevented LTD(4)-induced Ras activation, indicating that this ion plays an essential role for CysLT(1)-induced downstream signaling in differentiated U937 (dU937) cells. In addition, while Src did not appear to be substantially involved in CysLT(1)-induced signaling, genistein was able to partially inhibit LTD(4)-induced [Ca(2+)](i) transient ( approximately 34%) and almost completely prevented Ras activation (>90%), suggesting a potential role for other Ca(2+)-dependent tyrosine kinases in LTD(4)-induced signaling. Finally, agonist-induced CysLT(1) stimulation was followed by a specific extracellular regulated kinase (ERK) 1/2 phosphorylation, an event with a pharmacological profile similar to that of Ras activation, partially ( approximately 40%) sensitive to Clostridium sordellii lethal toxin and totally blocked by PTx. In conclusion, LTD(4)-induced CysLT(1) receptor activation in dU937 cells leads to Ras activation and ERK phosphorylation mostly through a PTx-sensitive G(i/o) protein, PLC, and Ca(2+)-dependent tyrosine kinase(s). 相似文献
6.
Egle Solito Celine Raguenes-Nicol Catherine de Coupade Anne Bisagni-Faure Franoise Russo-Marie 《British journal of pharmacology》1998,124(8):1675-1683
- Annexin 1 (An 1), a phospholipid and calcium binding protein, is strongly expressed in differentiated U 937 cells. In attempting to correlate the expression of An 1 with phospholipase A2 (PLA2) activity, U 937 cells were stably transfected both with a Sense and Antisense cDNA for An 1. PLA2 activity was measured by Flow cytometry analysis utilizing the bis-Bodipy-C11-PC fluorescent probe.
- U 937 cells stably transfected with the sense or antisense vectors were differentiated for 24 h with phorbol 12-myristate 13-acetate (PMA, 6 ng ml−1). Both in undifferentiated and differentiated cells, the Antisense clone (36.4 AS) showed consistently higher PLA2 activity than the control Sense clone (15 S).
- Since the fluorescent probe measures the total PLA2 activity, we used two different stimuli, PMA: (100 ng ml−1) or lipopolysaccharide (LPS, 10 ng ml−1), and two different inhibitors, to discriminate the PLA2 involved (namely arachidonyl trifluoromethyl ketone or AACOCF3, which is specific for the cytosolic PLA2, and SB 203347 specific for the secretory PLA2).
- In the Antisense clone the inhibitory effect of AACOCF was stronger [68%, P<0.025] than in the Sense, which may reflect the lower endogenous level of An 1 present in the cells. On the contrary, the inhibitory effect of SB 203347 [60% of inhibition] was identical in both clones.
- Since cPLA2 activity is correlated with its phosphorylation, Western and shift blot analysis were performed. They did not show any significative difference between the phosphorylated and non phosphorylated form of the enzyme in both the differentiated or not, Sense and Antisense clones. Furthermore the tyrosine phosphorylation analysis of An 1 showed that less than 10% of An 1 was phosphorylated irrespective of PMA presence or absence.
- From the pattern of inhibition observed, we propose that the endogenous unphosphorylated form of An 1 may act intracellularly to block the activity of a cytosolic PLA2.
7.
Evangelia Pantazaka Emily J A Taylor William G Bernard Colin W Taylor 《British journal of pharmacology》2013,169(7):1624-1634
Background and Purpose
Histamine and prostaglandin E2 (PGE2), directly and via their effects on other cells, regulate the behaviour of vascular smooth muscle (VSM), but their effects on human VSM are incompletely resolved.Experimental Approach
The effects of PGE2 on histamine-evoked changes in intracellular free Ca2+ concentration ([Ca2+]i) and adenylyl cyclase activity were measured in populations of cultured human aortic smooth muscle cells (ASMCs). Selective ligands of histamine and EP receptors were used to identify the receptors that mediate the responses.Key Results
Histamine, via H1 receptors, stimulates an increase in [Ca2+]i that is entirely mediated by activation of inositol 1,4,5-trisphosphate receptors. Selective stimulation of EP2 or EP4 receptors attenuates histamine-evoked Ca2+ signals, but the effects of PGE2 on both Ca2+ signals and AC activity are largely mediated by EP2 receptors.Conclusions and Implications
Two important inflammatory mediators, histamine via H1 receptors and PGE2 acting largely via EP2 receptors, exert opposing effects on [Ca2+]i in human ASMCs. 相似文献8.
We recently demonstrated that endothelin-1 (ET-1) activates two types of Ca(2+)-permeable nonselective cation channels (NSCC-1 and NSCC-2) in C6 glioma cells. It is possible to discriminate between these channels by using the Ca(2+) channel blockers SK&F 96365 (1-[beta-(3-[4-methoxyphenyl]propoxy)-4-methoxyphenethyl]-1H-imidazole hydrochloride) and LOE 908 [(R,S)-(3,4-dihydro-6,7-dimethoxy-isoquinoline-1-yl)-2-phenyl-N,N-di-[2-(2,3,4-trimethoxyphenyl)ethyl]-acetamide]. LOE 908 is a blocker for NSCC-1 and NSCC-2, whereas SK&F 96365 is an inhibitor for NSCC-2. The purpose of the present study was to identify the G-proteins that are involved in ET-1-activated Ca(2+) channels in C6 glioma cells. ET-1 activated only NSCC-1 in C6 glioma cells preincubated with U73122 (1-[6-[((17beta)-3-methoxyestra-1,3,5[10]-trien-17-yl)amino]hexyl]-1H-pyrrole-2,5-dione), a phospholipase C (PLC) inhibitor. Microinjection of the dominant negative mutant of G(12)/G(13) (G(12)G228A/G(13)G225A) abolished activation of NSCC-1 and NSCC-2. In contrast, pertussis toxin did not affect any of the Ca(2+) channels in the ET-1-stimulated C6 glioma cells. These results indicate that G(12)/G(13) may couple with endothelin receptors and play an important role in the activation of NSCCs in C6 glioma cells. Moreover, the activation mechanisms of NSCC-1 and NSCC-2 by ET-1 were different. NSCC-1 activation depended upon a G(12)/G(13)-dependent cascade, whereas NSCC-2 activation depended upon both G(q)/PLC- and G(12)/G(13)-dependent cascades. 相似文献
9.
Magga J Bart G Oker-Blom C Kukkonen JP Akerman KE Näsman J 《Biochemical pharmacology》2006,71(6):827-836
The G protein coupling characteristics of a flag epitope-tagged orexin receptor type 1 (OX1R) was investigated in HEK293 cells. Immunoprecipitation of the OX1R and immunoblotting revealed interactions with Gq/G11 proteins as well as with Gs and Gi proteins. Stimulation with orexin-A did not affect the ability of the OX1R to coprecipitate Gq/G11 proteins, but it robustly elevated the intracellular concentration of Ca2+, [Ca2+]i. No changes in cAMP levels could be detected upon receptor stimulation. To get further insight into the functional correlation of G protein activation and Ca2+ signalling, we used baculovirus transduction to express chimeric G proteins, containing the Galphas protein backbone with various Galpha donor sequences (Galphas/x) at the N and C termini, and measured cAMP as functional output. The Galphas/x chimeric proteins with Galpha11(Galphaq) and Galpha16 structure in the C terminus were stimulated by the OX1R. Concentration-response curves with Galphas/16 revealed an agonist potency correlation between G protein activation and the elevation of [Ca2+]i via discharge of intracellular Ca2+ stores, a feature also recognized for the muscarinic M3 receptor. However, in contrast to the M3 receptor, the OX1R elevated [Ca2+]i via influx from extracellular space at about 30-fold lower agonist concentration. The results suggest that the OX1R is linked to influx of Ca2+ through a signal pathway independent of Gq/G11 protein activation. 相似文献
10.
Wen-Hsin Liu Pei-Hsiu Kao Yi-Ling Chiou Shinne-ren Lin Ming-Jung Wu Long-Sen Chang 《Toxicology letters》2009
In view of the controversial role of catalytic activity on the cytotoxicity of phospholipase A2 (PLA2), the present study is conducted to explore whether PLA2 induces apoptotic process of human leukemia U937 cells through catalytic activity-independent pathway. Modification of His-48 (according to the sequence alignment with porcine pancreatic PLA2) with p-bromophenacyl bromide (BPB) caused over 99.9% drop in enzymatic activity Naja naja atra PLA2. It was found that BPB–PLA2-induced apoptotic death of U937 cells was associated with mitochondrial depolarization, modulation of Bcl-2 family members, cytochrome c release and activation of caspases 9 and 3. Upon exposure to BPB–PLA2, elevation of intracellular Ca2+ levels and p38 MAPK activation were observed in U937 cells. Pretreatment with BAPTA-AM (Ca2+ chelator) and nifedipine (L-type Ca2+ channel blocker) abrogated Ca2+ increase and p38 MAPK activation, and rescued viability of BPB–PLA2-treated U937 cells. BPB–PLA2-induced dissipation of mitochondrial membrane potential and down-regulation of Bcl-2 were suppressed by SB202190 (p38MAPK inhibitor). Although PLA2 mutants in which His-48 and Asp-49 were substituted by Ala and Lys, respectively, did not display detectable PLA2 activity, they induced death of U937 cells. The signaling pathway of PLA2 mutants in inducing cell death was indistinguishable from that of BPB–PLA2. Taken together, our data indicate that catalytic activity-independent pathway is involved in PLA2-induced apoptotic death of human leukemia U937 cells via mitochondria-mediated death pathway triggering by Ca2+-mediated p38 MAPK activation. 相似文献
11.
Inactivation of protease-activated receptor-1 by proteolytic removal of the ligand region in vascular endothelial cells 总被引:1,自引:0,他引:1
Nakayama T Hirano K Hirano M Nishimura J Kuga H Nakamura K Takahashi S Kanaide H 《Biochemical pharmacology》2004,68(1):23-32
Proteolysis plays an important role in inactivating protease-activated receptor-1 (PAR1). We aimed to determine the cleavage site(s) responsive for the proteolytic inactivation of PAR1 in human umbilical vein endothelial cells. Fura-2 fluorometry revealed that the preceding stimulation with trypsin abolished the subsequent [Ca(2+)](i) response to thrombin, while the responses to PAR1-activating peptides remained intact. On the other hand, thrombin had no effect on the subsequent response to trypsin. The immunostaining with antibodies against the residues 35-46 (SPAN12) and 51-64 (WEDE15) revealed the broad boundaries of cleavage. Trypsin removed both epitopes from the cell surface within 3 min, while thrombin removed the epitope of SPAN12. The longer incubation with thrombin removed the epitope of WEDE15. However, PAR1-activating peptides thereafter induced an attenuated but significant elevation of [Ca(2+)](i). Not only the receptor internalization as observed with a confocal microscope, but also an additional cleavage was thus suggested to contribute to the thrombin-induced removal of the epitope of WEDE15. The analyses of the PAR1 mutants identified three cleavage sites for trypsin; residues 41-42, 70-71 and 82-83. The cleavage at the latter two sites was suggested to dominate that at the former, and thus remove the ligand region (residues 42-47). The inactivation of PAR1 due to proteolytic removal of the ligand region may contribute not only to the inactivation of PAR1 by proteases such as trypsin, but also to the termination of the intracellular signaling initiated by thrombin in the vascular endothelial cells. 相似文献
12.
Y Ikeda-Matsuo H Tanji A Ota Y Hirayama S Uematsu S Akira Y Sasaki 《British journal of pharmacology》2010,160(4):847-859
Background and purpose:
Although microsomal prostaglandin E synthase (mPGES)-1 is known to contribute to stroke injury, the underlying mechanisms remain poorly understood. This study examines the hypothesis that EP3 receptors contribute to stroke injury as downstream effectors of mPGES-1 neurotoxicity through Rho kinase activation.Experimental approach:
We used a glutamate-induced excitotoxicity model in cultured rat and mouse hippocampal slices and a mouse middle cerebral artery occlusion–reperfusion model. Effects of an EP3 receptor antagonist on neuronal damage in mPGES-1 knockout (KO) mice was compared with that in wild-type (WT) mice.Key results:
In cultures of rat hippocampal slices, the mRNAs of EP1–4 receptors were constitutively expressed and only the EP3 receptor antagonist ONO-AE3-240 attenuated and only the EP3 receptor agonist ONO-AE-248 augmented glutamate-induced excitotoxicity in CA1 neurons. Hippocampal slices from mPGES-1 KO mice showed less excitotoxicity than those from WT mice and the EP3 receptor antagonist did not attenuate the excitotoxicity. In transient focal ischaemia models, injection (i.p.) of an EP3 antagonist reduced infarction, oedema and neurological dysfunction in WT mice, but not in mPGES-1 KO mice, which showed less injury than WT mice. EP3 receptor agonist-induced augmentation of excitotoxicity in vitro was ameliorated by the Rho kinase inhibitor Y-27632 and Pertussis toxin. The Rho kinase inhibitor HA-1077 also ameliorated stroke injury in vivo.Conclusion and implications:
Activity of mPGES-1 exacerbated stroke injury through EP3 receptors and activation of Rho kinase and/or Gi. Thus, mPGES-1 and EP3 receptors may be valuable therapeutic targets for treatment of human stroke.This article is commented on by Andreasson, pp. 844–846 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2010.00715.x 相似文献13.
Pauli M Turunen Marie E Ekholm Pentti Somerharju Jyrki P Kukkonen 《British journal of pharmacology》2010,159(1):212-221
Background and purpose:
We have previously shown that lipid mediators, produced by phospholipase D and C, are generated in OX1 orexin receptor signalling with high potency, and presumably mediate some of the physiological responses to orexin. In this study, we investigated whether the ubiquitous phospholipase A2 (PLA2) signalling system is also involved in orexin receptor signalling.Experimental approach:
Recombinant Chinese hamster ovary-K1 cells, expressing human OX1 receptors, were used as a model system. Arachidonic acid (AA) release was measured from 3H-AA-labelled cells. Ca2+ signalling was assessed using single-cell imaging.Key results:
Orexins strongly stimulated [3H]-AA release (maximally 4.4-fold). Orexin-A was somewhat more potent than orexin-B (pEC50= 8.90 and 8.38 respectively). The concentration–response curves appeared biphasic. The release was fully inhibited by the potent cPLA2 and iPLA2 inhibitor, methyl arachidonyl fluorophosphonate, whereas the iPLA2 inhibitors, R- and S-bromoenol lactone, caused only a partial inhibition. The response was also fully dependent on Ca2+ influx, and the inhibitor studies suggested involvement of the receptor-operated influx pathway. The receptor-operated pathway, on the other hand, was partially dependent on PLA2 activity. The extracellular signal-regulated kinase, but not protein kinase C, were involved in the PLA2 activation at low orexin concentrations.Conclusions and implications:
Activation of OX1 orexin receptors induced a strong, high-potency AA release, possibly via multiple PLA2 species, and this response may be important for the receptor-operated Ca2+ influx. The response coincided with other high-potency lipid messenger responses, and may interact with these signals. 相似文献14.
We recently demonstrated that endothelin-1 (ET-1) activates two types of Ca(2+)-permeable nonselective cation channel (designated NSCC-1 and NSCC-2) and a store-operated Ca(2+) channel (SOCC) in rabbit basilar artery (BA) vascular smooth muscle cells (VSMCs). In this study, we investigated the effects of phosphoinositide 3-kinase (PI3K) on ET-1-induced activation of these channels and BA contraction by using PI3K inhibitors, wortmannin and LY 249002. To determine which Ca(2+) channels are activated via PI3K, monitoring of intracellular Ca(2+) concentration was performed. Role of PI3K in ET-1-induced vasoconstriction was examined by tension study using rabbit BA rings. Only NSCC-1 was activated by ET-1 in wortmannin- or LY 294002-pretreated VSMCs. In contrast, addition of these drugs after ET-1 stimulation did not suppress Ca(2+) influx. Wortmannin inhibited the ET-1-induced contraction of rabbit BA rings that depends on the Ca(2+) influx through NSCC-2 and SOCC. The IC(50) values of wortmannin for the ET-1-induced Ca(2+) influx and vasoconstriction were similar to those for the ET-1-induced PI3K activation. These results indicate that (1) NSCC-2 and SOCC are stimulated by ET-1 via PI3K-dependent cascade, whereas NSCC-1 is stimulated via PI3K-independent cascade; (2) PI3K is required for the activation of the Ca(2+) entry, but not for its maintenance; and (3) PI3K is involved in the ET-1-induced contraction of rabbit BA rings that depends on the extracellular Ca(2+) influx through SOCC and NSCC-2. 相似文献
15.
Levi R Seyedi N Schaefer U Estephan R Mackins CJ Tyler E Silver RB 《Biochemical pharmacology》2007,73(8):1146-1156
We hypothesized that the histamine H(3)-receptor (H(3)R)-mediated attenuation of norepinephrine (NE) exocytosis from cardiac sympathetic nerves results not only from a Galpha(i)-mediated inhibition of the adenylyl cyclase-cAMP-PKA pathway, but also from a Gbetagamma(i)-mediated activation of the MAPK-PLA(2) cascade, culminating in the formation of an arachidonate metabolite with anti-exocytotic characteristics (e.g., PGE(2)). We report that in Langendorff-perfused guinea-pig hearts and isolated sympathetic nerve endings (cardiac synaptosomes), H(3)R-mediated attenuation of K(+)-induced NE exocytosis was prevented by MAPK and PLA(2) inhibitors, and by cyclooxygenase and EP(3)-receptor (EP(3)R) antagonists. Moreover, H(3)R activation resulted in MAPK phosphorylation in H(3)R-transfected SH-SY5Y neuroblastoma cells, and in PLA(2) activation and PGE(2) production in cardiac synaptosomes; H(3)R-induced MAPK phosphorylation was prevented by an anti-betagamma peptide. Synergism between H(3)R and EP(3)R agonists (i.e., imetit and sulprostone, respectively) suggested that PGE(2) may be a downstream effector of the anti-exocytotic effect of H(3)R activation. Furthermore, the anti-exocytotic effect of imetit and sulprostone was potentiated by the N-type Ca(2+)-channel antagonist omega-conotoxin GVIA, and prevented by an anti-Gbetagamma peptide. Our findings imply that an EP(3)R Gbetagamma(i)-induced decrease in Ca(2+) influx through N-type Ca(2+)-channels is involved in the PGE(2)/EP(3)R-mediated attenuation of NE exocytosis elicited by H(3)R activation. Conceivably, activation of the Gbetagamma(i) subunit of H(3)R and EP(3)R may also inhibit Ca(2+) entry directly, independent of MAPK intervention. As heart failure, myocardial ischemia and arrhythmic dysfunction are associated with excessive local NE release, attenuation of NE release by H(3)R activation is cardioprotective. Accordingly, this novel H(3)R signaling pathway may ultimately bear therapeutic significance in hyper-adrenergic states. 相似文献
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Serum amyloid A (SAA) and Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm) have been reported as formyl peptide receptor-like 1 (FPRL1) ligands. WKYMVm but not SAA stimulated superoxide generation by human neutrophils. In terms of the downstream signalings triggered by WKYMVm and SAA, both agonists stimulated cytosolic phospholipase A2-mediated arachidonic acid release, a precursor of leukotriene B4 (LTB4) and prostaglandin E2 (PGE2). WKYMVm also strongly stimulated LTB4 production in human neutrophils without affecting PGE2 production, whereas SAA strongly stimulates cyclooxygenase-2 (COX-2) expression and PGE2 production but not LTB4 production. In terms of the receptors responsible for the differential actions of these two agonists, we found that FPRL1 is involved in the production of LTB4 by WKYMVm and PGE2 production by SAA. This study demonstrates that the chemoattractant receptor, FPRL1, can be differentially regulated by distinct ligands to generate different lipid mediators, and thus, different immune responses. 相似文献
18.
In this study, we have used a combination of immunocytochemical and Ca(2+) imaging techniques to determine the functional localisation of insulin receptors as well as the potential role for insulin in modulating hippocampal synaptic activity. Comparison of insulin receptor and MAP2 labelling demonstrated extensive insulin receptor immunoreactivity on the soma and dendrites of cultured hippocampal neurones. Dual labelling with synapsin 1 also showed punctate insulin receptor labelling associated with synapses. In functional studies, insulin inhibited spontaneous Ca(2+) oscillations evoked in cultured hippocampal neurones following Mg(2+) removal. This action of insulin was mimicked by the ATP-sensitive K(+) (K(ATP)) channel opener diazoxide or the large conductance Ca(2+)-activated K(+) (BK) channel activator NS-1619. Furthermore, application of the K(ATP) channel blocker glybenclamide or the BK channel inhibitors iberiotoxin or charybdotoxin attenuated the actions of insulin, whereas prior incubation with a combination of glybenclamide and iberiotoxin completely blocked insulin action. The ability of insulin to modulate the Ca(2+) oscillations was reduced by the inhibitors of MAPK activation PD 98059 and U0126, but not by the PI 3-kinase inhibitors LY 294002 or wortmannin, indicating that a MAPK-driven process underlies insulin action. In conclusion, insulin inhibits spontaneous Ca(2+) oscillations via a process involving MAPK-driven activation of BK and K(ATP) channels. This process may be a useful therapeutic target for the treatment of epilepsy and certain neurodegenerative diseases. 相似文献
19.
Ze-bing Huang Hai-yan Wang Na-na Sun Jing-ke Wang Meng-qin Zhao Jian-xin Shen Ming Gao Ronald P Hammer Jr Xue-gong Fan Jie Wu 《Acta pharmacologica Sinica》2014,35(12):1514-1520
Aim:
Congo red, a secondary diazo dye, is usually used as an indicator for the presence of amyloid fibrils. Recent studies show that congo red exerts neuroprotective effects in a variety of models of neurodegenerative diseases. However, its pharmacological profile remains unknown. In this study, we investigated the effects of congo red on ACh-induced Ca2+ oscillations in mouse pancreatic acinar cells in vitro.Methods:
Acutely dissociated pancreatic acinar cells of mice were prepared. A U-tube drug application system was used to deliver drugs into the bath. Intracellular Ca2+ oscillations were monitored by whole-cell recording of Ca2+-activated Cl− currents and by using confocal Ca2+ imaging. For intracellular drug application, the drug was added in pipette solution and diffused into cell after the whole-cell configuration was established.Results:
Bath application of ACh (10 nmol/L) induced typical Ca2+ oscillations in dissociated pancreatic acinar cells. Addition of congo red (1, 10, 100 μmol/L) dose-dependently enhanced Ach-induced Ca2+ oscillations, but congo red alone did not induce any detectable response. Furthermore, this enhancement depended on the concentrations of ACh: congo red markedly enhanced the Ca2+ oscillations induced by ACh (10–30 nmol/L), but did not alter the Ca2+ oscillations induced by ACh (100–10000 nmol/L). Congo red also enhanced the Ca2+ oscillations induced by bath application of IP3 (30 μmol/L). Intracellular application of congo red failed to alter ACh-induced Ca2+ oscillations.Conclusion:
Congo red significantly modulates intracellular Ca2+ signaling in pancreatic acinar cells, and this pharmacological effect should be fully considered when developing congo red as a novel therapeutic drug. 相似文献20.
The present study was designed to evaluate the effects of antidepressants on smooth muscle contractile activity. In rat aortic rings, the antidepressants imipramine, mianserin and sertraline provoked concentration-dependent inhibitions of the mechanical responses evoked by K+ (30 mM) depolarization. These myorelaxant effects were not modified by the presence of glibenclamide or 80 mM K+ in the bathing medium. Moreover, the vasodilator properties of imipramine were not affected by atropine, phentolamine and pyrilamine. Radioisotopic experiments indicated that imipramine failed to enhance 86Rb outflow from prelabelled and perifused aortic rings whilst counteracting the increase in 45Ca outflow provoked by a rise in the extracellular K+ concentration. Simultaneous measurements of contractile activity and fura-2 fluorescence revealed that, in aortic rings, imipramine reduced the mechanical and fluorimetric response to K+ challenge. In A7r5 smooth muscle cells, whole cell recordings further demonstrated that imipramine inhibited the inward Ca2+ current. Under different experimental conditions, the ionic and relaxation responses to the antidepressants were reminiscent of those mediated by the Ca2+ entry blocker verapamil. Lastly, it should be pointed out that imipramine exhibited a myorelaxant effect of similar amplitude on rat aorta and on rat distal colon. All together, these findings suggest that the myorelaxant properties of imipramine, and probably also setraline and mianserin, could result from their capacity to inhibit the voltage-sensitive Ca2+ channels. 相似文献