Mechanism of cAMP-induced Ca2+ influx in Dictyostelium: role of phospholipase A2.
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ABSTRACT:
SUBMITTER: Schaloske R
PROVIDER: S-EPMC1218785 | biostudies-other | 1997 Oct
REPOSITORIES: biostudies-other
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Project description:Chemotaxis toward different cyclic adenosine monophosphate (cAMP) concentrations was tested in Dictyostelium discoideum cell lines with deletion of specific genes together with drugs to inhibit one or all combinations of the second-messenger systems PI3-kinase, phospholipase C (PLC), phospholipase A2 (PLA2), and cytosolic Ca(2+). The results show that inhibition of either PI3-kinase or PLA2 inhibits chemotaxis in shallow cAMP gradients, whereas both enzymes must be inhibited to prevent chemotaxis in steep cAMP gradients, suggesting that PI3-kinase and PLA2 are two redundant mediators of chemotaxis. Mutant cells lacking PLC activity have normal chemotaxis; however, additional inhibition of PLA2 completely blocks chemotaxis, whereas inhibition of PI3-kinase has no effect, suggesting that all chemotaxis in plc-null cells is mediated by PLA2. Cells with deletion of the IP(3) receptor have the opposite phenotype: chemotaxis is completely dependent on PI3-kinase and insensitive to PLA2 inhibitors. This suggest that PI3-kinase-mediated chemotaxis is regulated by PLC, probably through controlling PIP(2) levels and phosphatase and tensin homologue (PTEN) activity, whereas chemotaxis mediated by PLA2 appears to be controlled by intracellular Ca(2+).
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Project description:We previously reported that INS-1 cells expressing the intracellular II-III loop of the L-type Ca(2+) channel Cav1.2 (Cav1.2/II-III cells) are deficient in Ca(2+)-induced Ca(2+) release (CICR). Here we show that glucose-stimulated ERK 1/2 phosphorylation (GSEP) is slowed and reduced in Cav1.2/II-III cells compared to INS-1 cells. This parallels a decrease in glucose-stimulated cAMP accumulation (GS-cAMP) in Cav1.2/II-III cells. Influx of Ca(2+) via L-type Ca(2+) channels and CICR play roles in both GSEP and GS-cAMP in INS-1 cells since both are inhibited by nicardipine or ryanodine. Further, the Epac1-selective inhibitor CE3F4 abolishes glucose-stimulated ERK activation in INS-1 cells, as measured using the FRET-based sensor EKAR. The non-selective Epac antagonist ESI-09 but not the Epac2-selective antagonist ESI-05 nor the PKA antagonist Rp-cAMPs inhibits GSEP in both INS-1 and Cav1.2/II-III cells. We conclude that L-type Ca(2+) channel-dependent cAMP accumulation, that's amplified by CICR, activates Epac1 and drives GSEP in INS-1 cells.
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Project description: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)? specific) and (S)-bromoenol lactone (S-BEL, iPLA(2)? specific). Immunohistochemical analysis revealed intense staining for both iPLA(2)? and ? isoforms throughout the TM, juxtacanalicular tissue, and Schlemm's canal of human eye. Inhibition of iPLA(2)? by R-BEL or small interfering RNA-mediated silencing of iPLA(2)? 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)? 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)? on TM contractile status appear to involve arachidonic acid and Rho GTPase signaling pathways.
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Project description:Phospholipidosis, the excessive accumulation of phospholipids within lysosomes, is a pathological response observed following exposure to many drugs across multiple therapeutic groups. A clear mechanistic understanding of the causes and implications of this form of drug toxicity has remained elusive. We previously reported the discovery and characterization of a lysosome-specific phospholipase A2 (PLA2G15) and later reported that amiodarone, a known cause of drug-induced phospholipidosis, inhibits this enzyme. Here, we assayed a library of 163 drugs for inhibition of PLA2G15 to determine whether this phospholipase was the cellular target for therapeutics other than amiodarone that cause phospholipidosis. We observed that 144 compounds inhibited PLA2G15 activity. Thirty-six compounds not previously reported to cause phospholipidosis inhibited PLA2G15 with IC50 values less than 1 mM and were confirmed to cause phospholipidosis in an in vitro assay. Within this group, fosinopril was the most potent inhibitor (IC50 0.18 μM). Additional characterization of the inhibition of PLA2G15 by fosinopril was consistent with interference of PLA2G15 binding to liposomes. PLA2G15 inhibition was more accurate in predicting phospholipidosis compared with in silico models based on pKa and ClogP, measures of protonation, and transport-independent distribution in the lysosome, respectively. In summary, PLA2G15 is a primary target for cationic amphiphilic drugs that cause phospholipidosis, and PLA2G15 inhibition by cationic amphiphilic compounds provides a potentially robust screening platform for potential toxicity during drug development.
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Project description:We previously reported in atrial myocytes that inhibition of cAMP-dependent protein kinase (PKA) by laminin (LMN)-integrin signaling activates ?2-adrenergic receptor (?2-AR) stimulation of cytosolic phospholipase A2 (cPLA2). The present study sought to determine the signaling mechanisms by which inhibition of PKA activates ?2-AR stimulation of cPLA2. We therefore determined the effects of zinterol (0.1 ?M; zint-?2-AR) to stimulate ICa,L in atrial myocytes in the absence (+PKA) and presence (-PKA) of the PKA inhibitor (1 ?M) KT5720 and compared these results with atrial myocytes attached to laminin (+LMN). Inhibition of Raf-1 (10 ?M GW5074), phospholipase C (PLC; 0.5 ?M edelfosine), PKC (4 ?M chelerythrine) or IP3 receptor (IP3R) signaling (2 ?M 2-APB) significantly inhibited zint-?2-AR stimulation of ICa,L in-PKA but not +PKA myocytes. Western blots showed that zint-?2-AR stimulation increased ERK1/2 phosphorylation in-PKA compared to +PKA myocytes. Adenoviral (Adv) expression of dominant negative (dn) -PKC?, dn-Raf-1 or an IP3 affinity trap, each inhibited zint-?2-AR stimulation of ICa,L in + LMN myocytes compared to control +LMN myocytes infected with Adv-?gal. In +LMN myocytes, zint-?2-AR stimulation of ICa,L was enhanced by adenoviral overexpression of wild-type cPLA2 and inhibited by double dn-cPLA2S505A/S515A mutant compared to control +LMN myocytes infected with Adv-?gal. In-PKA myocytes depletion of intracellular Ca2+ stores by 5 ?M thapsigargin failed to inhibit zint-?2-AR stimulation of ICa,L via cPLA2. However, disruption of caveolae formation by 10 mM methyl-?-cyclodextrin inhibited zint-?2-AR stimulation of ICa,L in-PKA myocytes significantly more than in +PKA myocytes. We conclude that inhibition of PKA removes inhibition of Raf-1 and thereby allows ?2-AR stimulation to act via PKC?/Raf-1/MEK/ERK1/2 and IP3-mediated Ca2+ signaling to stimulate cPLA2 signaling within caveolae. These findings may be relevant to the remodeling of ?-AR signaling in failing and/or aging heart, both of which exhibit decreases in adenylate cyclase activity.
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Project description:BackgroundThe combined effects of glyphosate and hard water on chronic kidney disease of unknown etiology (CDKu) have attracted much interest, but the mechanisms remain unknown. Cytoplasmic phospholipase A2 (cPLA2) plays a key role in the acute and chronic inflammatory reactions. This study explored the effect of glyphosate combined with hard water on renal tubules and the possible targets and mechanisms involved.MethodsIn vivo experiments were conducted to investigate the synergistic effects and potential mechanisms of glyphosate and hard water on renal tubular injury in mice.ResultsAdministration of glyphosate in mice resulted in elevated levels of β2-microglobulin (β2-MG), albumin (ALB), and serum creatinine (SCr) compared to control mice. This increase was more pronounce when glyphosate was combined with hard water. In the glyphosate-treated mice, small areas of the kidney revealed fibroblast proliferation and vacuolar degeneration, particularly at the higher dose of 400 mg/kg glyphosate. However, the combination of glyphosate and hard water induced an even greater degree of pathological changes in the kidney. Immunofluorescence and western blot analyses showed that glyphosate and hard water had a coordinated effect on calcium ions (Ca2+)-activated phospholipase A2 and the activation may play a key role in inflammation and renal tubular injury. Exposure to glyphosate alone or glyphosate plus hard water increased the levels of oxidative stress markers and inflammatory biomarkers, namely, thromboxane A2 (TX-A2), leukotriene B4 (LTB4), prostaglandin E2 (PGE2), nitric oxide synthase (NOS), and nitric oxide (NO). Parameters of oxidative stress, including the levels of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were decreased. Further analysis showed that the levels of these biomarkers were significantly different between the mice treated with glyphosate plus hard water and the mice treated with glyphosate alone.ConclusionsThese findings suggested that hard water combined with glyphosate can induce renal tubular injury in mice, and this may involve mitogen-activated protein kinases (MAPK)/cytosolic phospholipase A2 (cPLA2)/arachidonic acid (AA) and its downstream factors.
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Project description:Recent reports demonstrated an association of human parvovirus B19 with inflammatory cardiomyopathy (iCMP), which is accompanied by endothelial dysfunction. As intracellular Ca(2+) activity is a key regulator of cell function and participates in mechanisms leading to endothelial dysfunction, the present experiments explored the effects of the B19 capsid proteins VP1 and VP2. A secreted phospholipase A2 (PLA2)-like activity has been located in the VP1 unique region of the B19 minor capsid protein. As PLA2 has recently been shown to activate the store-operated or capacitative Ca(2+) channel I(CRAC), we analyzed the impact of the viral PLA2 motif on Ca(2+) entry. We cloned the VP1 and VP2 genes isolated from a patient suffering from fatal B19 iCMP into eukaryotic expression vectors. We also generated a B19 replication-competent plasmid to demonstrate PLA2 activity under the control of the complete B19 genome. After the transfection of human endothelial cells (HMEC-1), cytosolic Ca(2+) activity was determined by utilizing Fura-2 fluorescence. VP1 and VP2 expression did not significantly modify basal cytosolic Ca(2+) activity or the decline of cytosolic Ca(2+) activity following the removal of extracellular Ca(2+). However, expression of VP1 and of the full-length B19 clone, but not of VP2, significantly accelerated the increase of cytosolic Ca(2+) activity following the readdition of extracellular Ca(2+) in the presence of thapsigargin, indicating an activation of I(CRAC.) The effect of VP1 was mimicked by the PLA2 product lysophosphatidylcholine and abolished by an inactivating mutation of the PLA2-encoding region of the VP1 gene. Our observations point to the activation of Ca(2+) entry by VP1 PLA2 activity, an effect likely participating in the pathophysiology of B19 infection.
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Project description:Although alkaline pH is known to trigger Ca(2+) influx in diverse cells, no pH-sensitive Ca(2+) channel has been identified. Here, we report that extracellular alkalinization induces opening of connexin 43 hemichannels (Cx43 HCs). Increasing extracellular pH from 7.4 to 8.5, in the presence of physiological Ca(2+)/Mg(2+) concentrations, rapidly increased the ethidium uptake rate and open probability of HCs in Cx43 and Cx43EGFP HeLa transfectants (HeLa-Cx3 and HeLa-Cx43EGFP, respectively) but not in parental HeLa cells (HeLa-parental) lacking Cx43 HCs. The increase in ethidium uptake induced by pH 8.5 was not affected by raising the extracellular Ca(2+) concentration from 1.8 to 10 mM but was inhibited by a connexin HC inhibitor (La(3+)). Probenecid, a pannexin HC blocker, had no effect. Extracellular alkalinization increased the intracellular Ca(2+) levels only in cells expressing HCs. The above changes induced by extracellular alkalinization did not change the cellular distribution of Cx43, suggesting that HC activation occurs through a gating mechanism. Experiments on cells expressing a COOH-terminal truncated Cx43 mutant indicated that the effects of alkalinization on intracellular Ca(2+) and ethidium uptake did not depend on the Cx43 C terminus. Moreover, purified dephosphorylated Cx43 HCs reconstituted in liposomes were Ca(2+) permeable, suggesting that Ca(2+) influx through Cx43 HCs could account for the elevation in intracellular Ca(2+) elicited by extracellular alkalinization. These studies identify a membrane pathway for Ca(2+) influx and provide a potential explanation for the activation of cellular events induced by extracellular alkalinization.
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Project description:The involvement of the phospholipase C (PLC) pathway in the non-genomic regulation of duodenal cell Ca2+ concentration by 17beta-oestradiol was investigated. The PLC inhibitors neomycin (0.5 mM) and U-73122 (2 microM) suppressed the stimulatory effect of 0.1 nM 17beta-oestradiol on the 45Ca2+ influx into enterocytes isolated from rat duodenum. The hormone (1 pM to 10 nM) increased the formation of 1,2-diacylglycerol in a biphasic pattern, characterized by an early peak at 45 s (+82%) and a later peak at 5 min (+46%). Both PLC inhibitors suppressed the first peak but were unable to block the 17beta-oestradiol effect at 5 min. 17beta-Oestradiol also increased the generation of inositol 1,4,5-trisphosphate within 15 s, with maximal stimulation at 30 s. 17beta-Oestradiol induced a rapid (30 s) and sustained (up to 5 min) increase in the intracellular Ca2+ concentration ([Ca2+]i) of fura 2-loaded enterocytes. The fast rise in [Ca2+]i was specific because other sex steroid hormones were without effect and could be blocked to a great extent by U-73122 (by 86% at 1 min). The effects of 17beta-oestradiol on enterocyte [Ca2+]i were decreased significantly (by 75%) in a Ca2+-free extracellular medium but a pronounced increase in [Ca2+]i was obtained after readmission of Ca2+ to the medium. The latter change was suppressed by 10 microM La3+, whereas nitrendipine (1 microM) and verapamil (10 microM) separately were without effect. The permeability of the 17beta-oestradiol-induced Ca2+ influx pathway to Mn2+ was increased 2.8-fold by treatment with oestrogen. These results suggest the operation of a PLC-dependent store-operated Ca2+ channel mechanism in 17beta-oestradiol regulation of enterocyte extracellular Ca2+ influx.
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