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1.  Maitotoxin-induced membrane blebbing and cell death in bovine aortic endothelial cells 
BMC Physiology  2001;1:2.
Maitotoxin, a potent cytolytic agent, causes an increase in cytosolic free Ca2+ concentration ([Ca2+]i) via activation of Ca2+-permeable, non-selective cation channels (CaNSC). Channel activation is followed by formation of large endogenous pores that allow ethidium and propidium-based vital dyes to enter the cell. Although activation of these cytolytic/oncotic pores, or COP, precedes release of lactate dehydrogenase, an indication of oncotic cell death, the relationship between CaNSC, COP, membrane lysis, and the associated changes in cell morphology has not been clearly defined. In the present study, the effect maitotoxin on [Ca2+]i, vital dye uptake, lactate dehydrogenase release, and membrane blebbing was examined in bovine aortic endothelial cells.
Maitotoxin produced a concentration-dependent increase in [Ca2+]i followed by a biphasic uptake of ethidium. Comparison of ethidium (Mw 314 Da), YO-PRO-1 (Mw 375 Da), and POPO-3 (Mw 715 Da) showed that the rate of dye uptake during the first phase was inversely proportional to molecular weight, whereas the second phase appeared to be all-or-nothing. The second phase of dye uptake correlated in time with the release of lactate dehydrogenase. Uptake of vital dyes at the single cell level, determined by time-lapse videomicroscopy, was also biphasic. The first phase was associated with formation of small membrane blebs, whereas the second phase was associated with dramatic bleb dilation.
These results suggest that maitotoxin-induced Ca2+ influx in bovine aortic endothelial cells is followed by activation of COP. COP formation is associated with controlled membrane blebbing which ultimately gives rise to uncontrolled bleb dilation, lactate dehydrogenase release, and oncotic cell death.
PMCID: PMC32181  PMID: 11231888
2.  The opening of maitotoxin-sensitive calcium channels induces the acrosome reaction in human spermatozoa: differences from the zona pellucida 
Asian Journal of Andrology  2010;13(1):159-165.
The acrosome reaction (AR), an absolute requirement for spermatozoa and egg fusion, requires the influx of Ca2+ into the spermatozoa through voltage-dependent Ca2+ channels and store-operated channels. Maitotoxin (MTx), a Ca2+-mobilizing agent, has been shown to be a potent inducer of the mouse sperm AR, with a pharmacology similar to that of the zona pellucida (ZP), possibly suggesting a common pathway for both inducers. Using recombinant human ZP3 (rhZP3), mouse ZP and two MTx channel blockers (U73122 and U73343), we investigated and compared the MTx- and ZP-induced ARs in human and mouse spermatozoa. Herein, we report that MTx induced AR and elevated intracellular Ca2+ ([Ca2+]i) in human spermatozoa, both of which were blocked by U73122 and U73343. These two compounds also inhibited the MTx-induced AR in mouse spermatozoa. In disagreement with our previous proposal, the AR triggered by rhZP3 or mouse ZP was not blocked by U73343, indicating that in human and mouse spermatozoa, the AR induction by the physiological ligands or by MTx occurred through distinct pathways. U73122, but not U73343 (inactive analogue), can block phospholipase C (PLC). Another PLC inhibitor, edelfosine, also blocked the rhZP3- and ZP-induced ARs. These findings confirmed the participation of a PLC-dependent signalling pathway in human and mouse zona protein-induced AR. Notably, edelfosine also inhibited the MTx-induced mouse sperm AR but not that of the human, suggesting that toxin-induced AR is PLC-dependent in mice and PLC-independent in humans.
PMCID: PMC3739404  PMID: 20835262
acrosome reaction; calcium channels; human sperm; maitotoxin; mouse sperm
3.  Phospholipse c inhibitor, u73122, stimulates release of hsp-70 stress protein from A431 human carcinoma cells 
Accumulating evidences suggest that Hsp 70, the inducible component of Hsp70 family, might release from a living cell. Here we show that a pharmacological inhibitor of phospholipase C activity U73122 caused a 2–4 fold reduction of an intracellular level of Hsp70 in A431 human carcinoma cells.
A depletion of Hsp70 under U73122 was a result of the protein release since it was detected in cell culture medium, as was established by immunoprecipitation and precipitation with ATP-agarose. The reduction of Hsp70 level was specifically attributed to the inhibition of PLC, since the non-active inhibitor, U73343, had no effect on Hsp70 level. The PLC-dependent decrease of Hsp70 intracellular level was accompanied by the enhanced sensitivity of A431 cells to the apoptogenic effect of hydrogen peroxide. Here for the first time we demonstrated one of the possibilities for a cell to export Hsp70 in PLC-dependent manner.
From our data we suggest that phospholipase C inhibition is one of the possible mechanisms of Hsp70 release from cells.
PMCID: PMC385244  PMID: 14989758
4.  Sphingosine-1-phosphate–induced oxygen free radical generation in smooth muscle cell migration requires Gα12/13 protein-mediated phospholipase C activation 
Sphingosine-1-phosphate (S-1-P) is a bioactive sphingolipid that stimulates the migration of vascular smooth muscle cell (VSMC) through G-protein coupled receptors; it has been shown to activate reduced nicotinamide dinucleotide phosphate hydrogen (NAD[P]H) oxidase. The role of phospholipase C (PLC) in oxygen free radical generation, and the regulation of VSMC migration in response to S-1-P, are poorly understood.
Rat arterial VSMC were cultured in vitro. Oxygen free radical generation was measured by fluorescent redox indicator assays in response to S-1-P (0.1µM) in the presence and absence of the active PLC inhibitor (U73122; U7, 10nM) or its inactive analog U73343 (InactiveU7, 10nM). Activation of PLC was assessed by immunoprecipitation and Western blotting for the phosphorylated isozymes (β and γ). Small interfering (si) RNA to the G-proteins Gαi, Gαq, and Gα12/13 was used to downregulate specific proteins. Statistics were by one-way analysis of variance (n = 6).
S-1-P induced time-dependent activation of PLC-β and PLC-γ; PLC-β but not PLC-γ activation was blocked by U7 but not by InactiveU7. PLC-β activation was Gαi-independent (not blocked by pertussis toxin, a Gαi inhibitor, or Gαi2 and Gαi3 siRNA) and Gαq-independent (not blocked by glycoprotein [GP] 2A, a Gαq inhibitor, or Gαq siRNA). PLC-β activation and cell migration was blocked by siRNA to Gα12/13. Oxygen free radical generation induced by S-1-P, as measured by dihydroethidium staining, was significantly inhibited by U7 but not by InactiveU7. Inhibition of oxygen free radicals with the inhibitor diphenyleneiodonium resulted in decreased cell migration to S-1-P. VSMC mitogen-activated protein kinase activation and VSMC migration in response to S-1-P was inhibited by PLC- inhibition.
S-1-P induces oxygen free radical generation through a Gα12/13, PLC-β-mediated mechanism that facilitates VSMC migration. To our knowledge, this is the first description of PLC-mediated oxygen free radical generation as a mediator of S-1-P VSMC migration and illustrates the need for the definition of cell signaling to allow targeted strategies in molecular therapeutics for restenosis.
Clinical Relevance
Activation of vascular smooth muscle cells by growth factors leads to cell proliferation and migration, which are integral features of the healing response in a vessel that leads to the development of intimal hyperplasia after bypass grafting, angioplasty, and stenting. Sphingosine-1-phosphate (S-1-P) is a common phospholipid, released from activated platelets at sites of vessel injury. It is a G-protein–coupled receptor agonist that induces smooth muscle cell migration, a key event in the development of intimal hyperplasia. Mechanisms of cell migration are not well defined, and understanding the mechanisms of signal transduction is important in defining potential targets for therapeutic intervention. The present study shows that S-1-P induces oxygen free radical generation through a Gα12/13, PLC-β–mediated mechanism that facilitates smooth muscle cell migration. Targeting choke points in cell signaling, such as membrane G-proteins, is an attractive molecular target in developing therapeutic strategies to moderate restenosis.
PMCID: PMC2607047  PMID: 18155002
5.  Increased sensitivity to apoptosis induced by methotrexate is mediated by Jun N-terminal kinase 
Arthritis and rheumatism  2011;63(9):2606-2616.
Low-dose methotrexate [MTX] is an effective therapy for rheumatoid arthritis yet its mechanism of action is incompletely understood. Here, we explored induction of apoptosis by MTX.
We employed flow cytometry to assess changes in levels of intracellular proteins, reactive oxygen species [ROS], and apoptosis.Quantitative polymerase chain reaction was usedtoassess changes in transcript levels of select target genes in response to MTX.
MTX does not directly induce apoptosis but rather ‘primes’ cells for markedly increased sensitivity to apoptosis via either mitochondrial or death receptor pathways by a Jun N-terminal kinase [JNK]-dependent mechanism. Increased sensitivity to apoptosis is mediated, at least in part, by MTX-dependent production of reactive oxygen species, JNK activation and JNK-dependent induction of genes whose protein products promote apoptosis. Supplementation with tetrahydrobiopterin blocks these methotrexate-induced effects. Subjects with rheumatoid arthritis on low-dose MTX therapy express elevated levels of the JNK-target gene, JUN.
Our results support a model whereby methotrexate inhibits reduction of dihydrobiopterin to tetrahydrobiopterin resulting in increased production of ROS, increased JNK activity and increased sensitivity to apoptosis. The finding of increased JUN levels in subjects with RA taking low-dose MTX supports the notion that this pathway is activated by MTX, in vivo, and may contribute to efficacy of MTX in inflammatory disease.
PMCID: PMC3165146  PMID: 21618198
6.  WWOX suppresses autophagy for inducing apoptosis in methotrexate-treated human squamous cell carcinoma 
Cell Death & Disease  2013;4(9):e792-.
Squamous cell carcinoma (SCC) cells refractory to initial chemotherapy frequently develop disease relapse and distant metastasis. We show here that tumor suppressor WW domain-containing oxidoreductase (WWOX) (also named FOR or WOX1) regulates the susceptibility of SCC to methotrexate (MTX) in vitro and cure of SCC in MTX therapy. MTX increased WWOX expression, accompanied by caspase activation and apoptosis, in MTX-sensitive SCC cell lines and tumor biopsies. Suppression by a dominant-negative or small interfering RNA targeting WWOX blocked MTX-mediated cell death in sensitive SCC-15 cells that highly expressed WWOX. In stark contrast, SCC-9 cells expressed minimum amount of WWOX protein and resisted MTX-induced apoptosis. Transiently overexpressed WWOX sensitized SCC-9 cells to apoptosis by MTX. MTX significantly downregulated autophagy-related Beclin-1, Atg12–Atg5 and LC3-II protein expression and autophagosome formation in the sensitive SCC-15, whereas autophagy remained robust in the resistant SCC-9. Mechanistically, WWOX physically interacted with mammalian target of rapamycin (mTOR), which potentiated MTX-increased phosphorylation of mTOR and its downstream substrate p70 S6 kinase, along with dramatic downregulation of the aforementioned proteins in autophagy, in SCC-15. When WWOX was knocked down in SCC-15, MTX-induced mTOR signaling and autophagy inhibition were blocked. Thus, WWOX renders SCC cells susceptible to MTX-induced apoptosis by dampening autophagy, and the failure in inducing WWOX expression leads to chemotherapeutic drug resistance.
PMCID: PMC3789168  PMID: 24008736
tumor suppressor; methotrexate; chemotherapy; autophagy; apoptosis
7.  Induction of the Gαq Signaling Cascade by the Human Immunodeficiency Virus Envelope Is Required for Virus Entry▿  
Journal of Virology  2008;82(18):9191-9205.
Binding of human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) with the primary receptor CD4 and one of two coreceptors, CXCR4 or CCR5, activates a signaling cascade resulting in Rac-1 GTPase activation and stimulation of actin cytoskeletal reorganizations critical for HIV-1-mediated membrane fusion. The mechanism by which HIV-1 Env induces Rac-1 activation and subsequent actin cytoskeleton rearrangement is unknown. In this study, we show that Env-mediated Rac-1 activation is dependent on the activation of Gαq and its downstream targets. Fusion and Rac-1 activation are mediated by Gαq and phospholipase C (PLC), as shown by attenuation of fusion and Rac-1 activation in cells either expressing small interfering RNA (siRNA) targeting Gαq or treated with the PLC inhibitor U73122. Rac-1 activation and fusion were also blocked by multiple protein kinase C inhibitors, by inhibitors of intracellular Ca2+ release, by Pyk2-targeted siRNA, and by the Ras inhibitor S-trans,trans-farnesylthiosalicylic acid (FTS). Fusion was blocked without altering cell viability or cell surface localization of CD4 and CCR5. Similar results were obtained when cell fusion was induced by Env expressed on viral and cellular membranes and when cell lines or primary cells were the target. Treatment with inhibitors and siRNA specific for Gαi or Gαs signaling mediators had no effect on Env-mediated Rac-1 activation or cell fusion, indicating that the Gαq pathway alone is responsible. These results could provide a new focus for therapeutic intervention with drugs targeting host signaling mediators rather than viral molecules, a strategy which is less likely to result in resistance.
PMCID: PMC2546909  PMID: 18632858
8.  The effect of an anti-membrane antibody-methotrexate conjugate on the human prostatic tumour line PC3. 
British Journal of Cancer  1990;61(5):702-708.
Methotrexate (MTX) was linked, via an active ester intermediate, to a purified IgG fraction of rabbit polyclonal antiserum raised against a cell membrane preparation from the human prostatic cell line PC3. The resulting conjugates contained an average of 0.044 mg of MTX per mg of antibody with acceptable losses in both the binding activity of the immunoglobulin (27.5%) and the enzyme inhibitory activity of the drug (32% at a MTX concentration of 3 x 10(-7) M). Using cultures of PC3 cells the antibody-MTX (Ab-MTX) conjugates were observed to be as effective as free drug in causing cell death and more effective than non-immune IgG-MTX (NIgG-MTX) conjugates. When athymic nude mice bearing PC3 tumours were administered with Ab-MTX conjugates, significant reductions in tumour growth rates were observed compared to animals given saline, MTX alone or NIgG-MTX conjugates (P less than 0.01 in all cases). Furthermore, the accumulation of radioactive MTX in the tumour tissue of animals injected with these Ab-MTX conjugates was 16-fold greater than those given free drug and 8.6-fold greater than those administered with NIgG-MTX conjugates. Uptake by the reticuloendothelial system, however, was not significantly different when animals from each treatment group were compared.
PMCID: PMC1971590  PMID: 2337507
9.  Trophoblast Adhesion of the Peri-implantation Mouse Blastocyst is Regulated by Integrin Signaling That Targets Phospholipase C 
Developmental biology  2006;302(1):143-153.
Integrin signaling modulates trophoblast adhesion to extracellular matrices during blastocyst implantation. Fibronectin (FN)-binding activity on the apical surface of trophoblast cells is strengthened after elevation of intracellular Ca2+ downstream of integrin ligation by FN. We report here that phosphoinositide-specific phospholipase C (PLC) mediates Ca2+ signaling in response to FN. Pharmacological agents used to antagonize PLC (U73122) or the inositol phosphate receptor (Xestospongin C) inhibited FN-induced elevation of intracellular Ca2+ and prevented the upregulation of FN-binding activity. In contrast, inhibitors of Ca2+ influx through either voltage-gated or non-voltage-gated Ca2+ channels were without effect. Inhibition of protein tyrosine kinase activity by genistein, but not G-protein inhibition by suramin, blocked FN-induced intracellular Ca2+ signaling and upregulation of adhesion, consistent with involvement of PLC-γ. Confocal immunofluorescence imaging of peri-implantation blastocysts demonstrated that PLC-γ2, but not PLC-γ1 nor PLC-β1, accumulated near the outer surface of the embryo. Phosphotyrosine site-directed antibodies revealed phosphorylation of PLC-γ2, but not PLC-γ1, upon integrin ligation by FN. These data suggest that integrin-mediated activation of PLC-γ to initiate phosphoinositide signaling and intracellular Ca2+ mobilization is required for blastocyst adhesion to FN. Signaling cascades regulating PLC-γ could, therefore, control a critical feature of trophoblast differentiation during peri-implantation development.
PMCID: PMC1894903  PMID: 17027741
Blastocyst implantation; trophoblast; phospholipase C; Ca2+ signaling; signal transduction; integrins; fibronectin; extracellular matrix; cell adhesion; tyrosine phosphorylation
10.  Phospholipase C and myosin light chain kinase inhibition define a common step in actin regulation during cytokinesis 
BMC Cell Biology  2007;8:15.
Phosphatidylinositol 4,5-bisphosphate (PIP2) is required for successful completion of cytokinesis. In addition, both PIP2 and phosphoinositide-specific phospholipase C (PLC) have been localized to the cleavage furrow of dividing mammalian cells. PLC hydrolyzes PIP2 to yield diacylglycerol (DAG) and inositol trisphosphate (IP3), which in turn induces calcium (Ca2+) release from the ER. Several studies suggest PIP2 must be hydrolyzed continuously for continued cleavage furrow ingression. The majority of these studies employ the N-substituted maleimide U73122 as an inhibitor of PLC. However, the specificity of U73122 is unclear, as its active group closely resembles the non-specific alkylating agent N-ethylmaleimide (NEM). In addition, the pathway by which PIP2 regulates cytokinesis remains to be elucidated.
Here we compared the effects of U73122 and the structurally unrelated PLC inhibitor ET-18-OCH3 (edelfosine) on cytokinesis in crane-fly and Drosophila spermatocytes. Our data show that the effects of U73122 are indeed via PLC because U73122 and ET-18-OCH3 produced similar effects on cell morphology and actin cytoskeleton organization that were distinct from those caused by NEM. Furthermore, treatment with the myosin light chain kinase (MLCK) inhibitor ML-7 caused cleavage furrow regression and loss of both F-actin and phosphorylated myosin regulatory light chain from the contractile ring in a manner similar to treatment with U73122 and ET-18-OCH3.
We have used multiple inhibitors to examine the roles of PLC and MLCK, a predicted downstream target of PLC regulation, in cytokinesis. Our results are consistent with a model in which PIP2 hydrolysis acts via Ca2+ to activate myosin via MLCK and thereby control actin dynamics during constriction of the contractile ring.
PMCID: PMC1888687  PMID: 17509155
11.  Caribbean maitotoxin elevates [Ca2+]i and activates non-selective cation channels in HIT-T15 cells 
World Journal of Diabetes  2013;4(3):70-75.
AIM: To investigate the cytotoxic mechanism of caribbean maitotoxin (MTX-C) in mammalian cells.
METHODS: We used whole-cell patch-clamp techniques and fluorescence calcium imaging to determine the cellular toxic mechanisms of MTX-C in insulin secreting HIT-T15 cells, which is a system where the effects of MTX have been observed. HIT-T15 cells stably express L-type calcium current, making it a suitable model for this study. Using the fluorescence calcium indicator Indo-1 AM, we found that there is a profound increase in HIT-T15 intracellular free calcium 3 min after application of 200 nmol/L MTX-C.
RESULTS: About 3 min after perfusion of MTX-C, a gradual increase in free calcium concentration was observed. This elevation was sustained throughout the entire recording period. Application of MTX-C did not elicit the L-type calcium current, but large cationic currents appeared after applying MTX-C to the extracellular solution. The current-voltage relationship of the cation current is approximately linear within the voltage range from -60 to 50 mV, but flattened at voltages at -80 and -100 mV. These results indicate that MTX-C induces a non-voltage activated, inward current under normal physiological conditions, which by itself or through a secondary mechanism results in a large amount of cationic influx. The biophysical mechanism of MTX-C is different to its isoform, pacific maitotoxin (MTX-P), when the extracellular calcium is removed.
CONCLUSION: We conclude that MTX-C causes the opening of non-selective, non-voltage-activated ion channels, which elevates level of intracellular calcium concentration and leads to cellular toxicities.
PMCID: PMC3680626  PMID: 23772275
Maitotoxin; Calcium fluorescence; High voltage gated Ca2+ channels; Whole cell patch clamp; Insulin secreting cells
12.  Involvement of Calcium-Mediated Reactive Oxygen Species in Inductive GRP78 Expression by Geldanamycin in 9L Rat Brain Tumor Cells 
Treatment with geldanamycin (GA) leads to an increase in [Ca2+]c and the production of reactive oxygen species (ROS) in rat brain tumor 9L RBT cells. GA-exerted calcium signaling was blocked by BAPTA/AM and EGTA. The effect of GA on [Ca2+]c was significantly reduced in the presence of thapsigargin (TG) and ruthenium red (RR). GA-induced GRP78 expression is significantly decreased in the presence of BAPTA/AM, EGTA and RR, suggesting that the calcium influx from the extracellular space and intracellular calcium store oscillations are contributed to by the calcium mobilization and GRP78 expression induced by GA. The induced GRP78 expression is sensitive to added U73122 and Ro-31-8425, pinpointing the involvement of phospholipase C (PLC) and protein kinase C (PKC) in GA-induced endoplasmic reticulum (ER) stress. The antioxidants N-acetylcysteine (NAC), BAPTA/AM, EGTA and H7 also have significant inhibitory effects on ROS generation. Finally, neither H7 nor NAC was able to affect the calcium response elicited by GA. Our results suggest that the causal signaling cascade during GA-inducted GRP78 expression occurs via a pathway that connects PLC to cytoplasmic calcium increase, PKC activation and, then, finally, ROS generation. Our data provides new insights into the influence of GA on ER stress response in 9L RBT cells.
PMCID: PMC3794827  PMID: 24051401
geldanamycin; calcium; PKC; ROS; GRP78
In this study, we report a phospholipase C (PLC)-mediated mechanism for the redistribution of interendothelial adherens junctions in response to melanoma cell contacts with the endothelium. We demonstrated that contact of melanoma cells to human umbilical vein endothelial cells (HUVEC) triggered rapid endothelial [Ca2+]i response through PLC-IP3 pathway. In addition, alternation of endothelial adherens junctions following contact of melanoma cells was evidenced by the changes in immunological staining patterns of vascular endothelial (VE)-cadherin. A PLC inhibitor, U73122 was shown to significantly diminish [Ca2+]i response and reduce the occurrence of melanoma cell–induced VE-cadherin reorganization. Moreover, inhibition of PLC attenuated melanoma cell transendothelial migration. However, melanoma cell-associated VE-cadherin breakdown was not sensitive to Ly294002, an inhibitor of phosphatidylinositol-3-kinase (PI3K), whereas inhibition of PI3K resulted in a reduction of melanoma cell transmigration. Taken together, our findings implicate that by inducing the PLC-Ca2+ signaling pathway, melanoma cells disrupt EC junctions to breach the endothelium and promote transvascular homing of tumor cells.
PMCID: PMC2782934  PMID: 15769649
Endothelial adherens junction; intracellular calcium; signaling; tumor cell migration; vascular endothelial-cadherin
14.  Involvement of phospholipase D and phosphatidic acid in the light-dependent up-regulation of sorghum leaf phosphoenolpyruvate carboxylase-kinase 
Journal of Experimental Botany  2010;61(10):2819-2827.
The photosynthetic phosphoenolpyruvate carboxylase (C4-PEPC) is regulated by phosphorylation by a phosphoenolpyruvate carboxylase kinase (PEPC-k). In Digitaria sanguinalis mesophyll protoplasts, this light-mediated transduction cascade principally requires a phosphoinositide-specific phospholipase C (PI-PLC) and a Ca2+-dependent step. The present study investigates the cascade components at the higher integrated level of Sorghum bicolor leaf discs and leaves. PEPC-k up-regulation required light and photosynthetic electron transport. However, the PI-PLC inhibitor U-73122 and inhibitors of calcium release from intracellular stores only partially blocked this process. Analysis of [32P]phosphate-labelled phospholipids showed a light-dependent increase in phospholipase D (PLD) activity. Treatment of leaf discs with n-butanol, which decreases the formation of phosphatidic acid (PA) by PLD, led to the partial inhibition of the C4-PEPC phosphorylation, suggesting the participation of PLD/PA in the signalling cascade. PPCK1 gene expression was strictly light-dependent. Addition of neomycin or n-butanol decreased, and a combination of both inhibitors markedly reduced PPCK1 expression and the concomitant rise in PEPC-k activity. The calcium/calmodulin antagonist W7 blocked the light-dependent up-regulation of PEPC-k, pointing to a Ca2+-dependent protein kinase (CDPK) integrating both second messengers, calcium and PA, which were shown to increase the activity of sorghum CDPK.
PMCID: PMC2882271  PMID: 20410319
Ca2+-dependent protein kinase; phosphatidic acid; phosphoenolpyruvate carboxylase; phosphoenolpyruvate carboxylase kinase; phospholipase C; phospholipase D; Sorghum bicolor
15.  Pharmacological Investigations of the Cellular Transduction Pathways Used by Cholecystokinin to Activate Nodose Neurons 
Cholecystokinin (CCK) directly activates vagal afferent neurons resulting in coordinated gastrointestinal functions and satiation. In vitro, the effects of CCK on dissociated vagal afferent neurons are mediated via activation of the vanilloid family of transient receptor potential (TRPV) cation channels leading to membrane depolarization and an increase in cytosolic calcium. However, the cellular transduction pathway(s) involved in this process between CCK receptors and channel opening have not been identified. To address this question, we monitored CCK-induced cytosolic calcium responses in dissociated nodose neurons from rat in the presence or absence of reagents that interact with various intracellular signaling pathways. We found that the phospholipase C (PLC) inhibitor U-73122 significantly attenuated CCK-induced responses, whereas the inactive analog U-73433 had no effect. Responses to CCK were also cross-desensitized by a brief pretreatment with m-3M3FBS, a PLC stimulator. Together these observations strongly support the participation of PLC in the effects of CCK on vagal afferent neurons. In contrast, pharmacological antagonism of phospholipase A2, protein kinase A, and phosphatidylinositol 3-kinase revealed that they are not critical in the CCK-induced calcium response in nodose neurons. Further investigations of the cellular pathways downstream of PLC showed that neither protein kinase C (PKC) nor generation of diacylglycerol (DAG) or release of calcium from intracellular stores participates in the response to CCK. These results suggest that alteration of membrane phosphatidylinositol 4,5-bisphosphate (PIP2) content by PLC activity mediates CCK-induced calcium response and that this pathway may underlie the vagally-mediated actions of CCK to induce satiation and alter gastrointestinal functions.
PMCID: PMC3167007  PMID: 21664195
CCK; CCK1 Receptor; PLC; Signal Transduction; Vagal Afferent
16.  Interleukin-6 regulates anti-arthritic effect of methotrexate via reduction of SLC19A1 expression in a mouse arthritis model 
Methotrexate (MTX) enters cells via the reduced folate carrier SLC19A1, suggesting that SLC19A1 is associated with the efficacy of MTX. We here examined the relationship between the efficacy of MTX and the expression of SLC19A1 in glucose 6-phosphate isomerase (GPI)-induced arthritis. We found that interleukin-6 (IL-6) regulated the expression of SLC19A1, so we studied the effect of a combination of MTX and anti-mouse IL-6 receptor antibody (MR16-1).
GPI-induced arthritis was induced by intradermal immunization with recombinant GPI. MTX was given from the first day of immunization. Mice were injected once with MR16-1 10 days after immunization. The levels of SLC19A1 mRNA in whole hind limbs and immune cells were measured. Synovial cells from arthritic mice were cultured with cytokines, and cell proliferation and gene expressions were measured.
MTX inhibited the development of GPI-induced arthritis; however, the efficacy of MTX gradually diminished. SLC19A1 expression in immunized mice with arthritis was lower than in intact mice; moreover, SLC19A1 expression in arthritic mice was further decreased when they were treated with MTX. IL-6 was highly expressed in whole hind limbs of arthritic mice. In an in vitro study using synovial cells from arthritic mice, IL-6 + soluble IL-6 receptor (sIL-6R) weakened the anti-proliferative effect of MTX and reduced SLC19A1 expression. Finally, although MR16-1 did not improve arthritis at all when administered on day 10, MTX in combination with MR16-1 more potently reduced the development of arthritis than did MTX alone. When used in combination with MTX, MR16-1 apparently reversed the decrease in SLC19A1 induced by MTX alone.
In the present study, we demonstrated for the first time that IL-6 reduced the efficacy of MTX by decreasing the expression of SLC19A1, which is important for MTX uptake into cells.
PMCID: PMC3446470  PMID: 22546471
17.  Phospholipase C in Living Cells 
The Journal of General Physiology  2005;126(3):243-262.
We have further tested the hypothesis that receptor-mediated modulation of KCNQ channels involves depletion of phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphoinositide-specific phospholipase C (PLC). We used four parallel assays to characterize the agonist-induced PLC response of cells (tsA or CHO cells) expressing M1 muscarinic receptors: translocation of two fluorescent probes for membrane lipids, release of calcium from intracellular stores, and chemical measurement of acidic lipids. Occupation of M1 receptors activates PLC and consumes cellular PIP2 in less than a minute and also partially depletes mono- and unphosphorylated phosphoinositides. KCNQ current is simultaneously suppressed. Two inhibitors of PLC, U73122 and edelfosine (ET-18-OCH3), can block the muscarinic actions completely, including suppression of KCNQ current. However, U73122 also had many side effects that were attributable to alkylation of various proteins. These were mimicked or occluded by prior reaction with the alkylating agent N-ethylmaleimide and included block of pertussis toxin–sensitive G proteins and effects that resembled a weak activation of PLC or an inhibition of lipid kinases. By our functional criteria, the putative PLC activator m-3M3FBS did stimulate PLC, but with a delay and an irregular time course. It also suppressed KCNQ current. The M1 receptor–mediated activation of PLC and suppression of KCNQ current were stopped by lowering intracellular calcium well below resting levels and were slowed by not allowing intracellular calcium to rise in response to PLC activation. Thus calcium release induced by PLC activation feeds back immediately on PLC, accelerating it during muscarinic stimulation in strong positive feedback. These experiments clarify important properties of receptor-coupled PLC responses and their inhibition in the context of the living cell. In each test, the suppression of KCNQ current closely paralleled the expected fall of PIP2. The results are described by a kinetic model.
PMCID: PMC2266577  PMID: 16129772
18.  OXER1, a G protein-coupled oxoeicosatetraenoid receptor, mediates the survival-promoting effects of arachidonate 5-lipoxygenase in prostate cancer cells 
Cancer letters  2013;336(1):185-195.
Inhibition of 5-Lox induces apoptosis in prostate cancer cells by inactivating PKCε which is prevented by 5-oxoETE, and activators of PKCε prevent 5-Lox inhibition-induced apoptosis, suggesting that 5-Lox metabolites exert survival signaling via PKCε. However, mechanisms by which 5-Lox metabolites activate PKCε are not understood yet. We found that prostate cancer cells express high levels of OXER1, a G protein-coupled 5-oxoETE receptor, which delivers signal by generating diacyl-glycerol through phospholipase C-beta. Interestingly, we found that U73122, an inhibitor of PLC-beta, interrupts the apoptosis-preventing effect of 5-oxoETE, and exogenous diacyl-glycerol effectively prevents 5-Lox inhibition-induced apoptosis, suggesting that 5-oxoETE signals via OXER1 to promote prostate cancer cell survival.
PMCID: PMC3892773  PMID: 23643940
5-Lipoxygenase; Apoptosis; 5-OxoETE; OXER1; PLC-beta; PKC-epsilon
19.  Distinct Phospholipase C-β Isozymes Mediate Lysophosphatidic Acid Receptor 1 Effects on Intestinal Epithelial Homeostasis and Wound Closure 
Molecular and Cellular Biology  2013;33(10):2016-2028.
Maintenance of the epithelial barrier in the intestinal tract is necessary to protect the host from the hostile luminal environment. Phospholipase C-β (PLC-β) has been implicated to control myriad signaling cascades. However, the biological effects of selective PLC-β isozymes are poorly understood. We describe novel findings that lysophosphatidic acid (LPA) regulates PLC-β1 and PLC-β2 via two distinct pathways to enhance intestinal epithelial cell (IEC) proliferation and migration that facilitate wound closure and recovery of the intestinal epithelial barrier. LPA acting on the LPA1 receptor promotes IEC migration by facilitating the interaction of Gαq with PLC-β2. LPA-induced cell proliferation is PLC-β1 dependent and involves translocation of Gαq to the nucleus, where it interacts with PLC-β1 to induce cell cycle progression. An in vivo study using LPA1-deficient mice (Lpar1−/−) shows a decreased number of proliferating IECs and migration along the crypt-luminal axis. Additionally, LPA enhances migration and proliferation of IECs in an LPA1-dependent manner, and Lpar1−/− mice display defective mucosal wound repair that requires cell proliferation and migration. These findings delineate novel LPA1-dependent lipid signaling that facilitates mucosal wound repair via spatial targeting of distinct PLC-βs within the cell.
PMCID: PMC3647975  PMID: 23478264
20.  Pasteurella haemolytica leukotoxin induces bovine leukocytes to undergo morphologic changes consistent with apoptosis in vitro. 
Infection and Immunity  1996;64(7):2687-2694.
Infection of the bovine lung with Pasteurella haemolytica results in an acute respiratory disorder known as pneumonic pasteurellosis. One of the key virulence determinants used by this bacterium is secretion of an exotoxin that is specific for ruminant leukocytes (leukotoxin). At low concentrations, the leukotoxin can activate ruminant leukocytes, whereas at higher concentrations, it inhibits leukocyte functions and is cytolytic, presumably as a result of pore formation and subsequent membrane permeabilization. We have investigated the possibility that the activation-inhibition paradox is explained in part by leukotoxin-mediated apoptosis (i.e., activation-induced cell death) of bovine leukocytes. Incubation of bovine leukocytes with P. haemolytica leukotoxin caused marked cytoplasmic membrane blebbing (zeiosis) and chromatin condensation and margination, both of which are hallmarks of apoptosis. The observed morphologic changes in bovine leukocytes were leukotoxin dependent, because they were significantly diminished in the presence of an anti-leukotoxin monoclonal antibody. In addition, bovine leukocytes incubated with culture supernatant from a mutant strain of P. haemolytica that does not produce any detectable leukotoxin failed to exhibit the morphologic changes characteristic of cells undergoing apoptosis. These observations may represent an important mechanism by which P. haemolytica overwhelms host defenses, contributing to the fibrinous pleuropneumonia characteristic of bovine pasteurellosis.
PMCID: PMC174127  PMID: 8698496
21.  Mitogenic signaling from the egf receptor is attenuated by a phospholipase C-gamma/protein kinase C feedback mechanism. 
Molecular Biology of the Cell  1996;7(6):871-881.
We recently demonstrated that epidermal growth factor receptor (EGFR)-mediated signaling of cell motility and mitogenesis diverge at the immediate post-receptor level. How these two mutually exclusive cell responses cross-communicate is not known. We investigated a possible role for a phospholipase C (PLC)-dependent feedback mechanism that attenuates EGF-induced mitogenesis. Inhibition of PLC gamma activation by U73122 (1 microM) augmented the EGF-induced [3H]thymidine incorporation by 23-55% in two transduced NR6 fibroblast lines expressing motility-responsive EGFR; increased cell division and mitosis was observed in parallel. The time dependence of this increase revealed that it was due to an increase in maximal incorporation and not a foreshortened cell cycle. Motility-responsive cell lines expressing a dominant-negative PLC gamma fragment (PLCz) also demonstrated augmented mitogenic responses by 25-68% when compared with control cells. PLCz- or U73122-augmented mitogenesis was not observed in three non-PLC gamma activating, nonmotility-responsive EGFR-expressing cell lines. Protein kinase C (PKC), which may be activated by PLC-generated second messengers, has been proposed as mediating feedback attenuation due to its capacity to phosphorylate EGFR and inhibit the receptor's tyrosine kinase activity. Inhibition of PKC by Calphostin C (0.05 microM) resulted in a 57% augmentation in the fold of EGF-induced thymidine incorporation. To further establish PKC's role in this feedback attenuation mechanism, an EGFR point mutation, in which the PKC target threonine654 was replaced by alanine, was expressed. Cells expressing these PKC-resistant EGFR constructs demonstrated EGF-induced motility comparable to cells expressing the threonine-containing EGFR. However, when these cells were treated with U73122 or Calphostin C, the mitogenic responses are not enhanced. These findings suggest a model in which PKC activation subsequent to triggering of motility-associated PLC gamma activity attenuates the EGFR mitogenic response.
PMCID: PMC275939  PMID: 8816994
22.  Monoclonal antibody targeting of methotrexate (MTX) against MTX-resistant tumour cell lines. 
British Journal of Cancer  1992;65(6):838-844.
Several Methotrexate (MTX)-resistant sublines of the osteogenic sarcoma cell line 791T were derived by continuous selection in the presence of MTX and 12-O-tetradecanoylphorbol-13-acetate (TPA). Studies including assays of the uptake and binding of [3H]MTX and fluoresceinated-MTX, determined that these sublines showed diminished MTX transport, and that none of them appeared to overproduce the MTX-target enzyme dihydrofolate reductase. Conjugates of the anti-791T monoclonal antibody 791T/36 linked to MTX via human serum albumin (HSA) were prepared by Dr M.C. Garnett. These were cytotoxic selectively for cells bearing the 791T/36-defined antigen (gp72), and were found to be as cytotoxic to most of the MTX-resistant 791T sublines as they were to parental 791T cells. Furthermore, an anti-MTX/anti-gp72 bispecific antibody 516 augmented the cytotoxicity of HSA-MTX conjugate to the MTX-resistant 791T variant R120 apparently as efficiently as for parental 791T cells. It is suggested that acquired drug resistance caused by deficient transport mechanisms may be partially or wholly overcome by targeting the drug to a readily-internalised cell surface antigen.
PMCID: PMC1977768  PMID: 1616855
23.  In the Ventral Tegmental Area, Progestogens’ Membrane-Mediated Actions for Lordosis of Rats Involve the Second Messenger Phospholipase C 
Brain research  2008;1230C:218-223.
Steroid hormones have pervasive functional effects. Although steroids are generally known to have actions via binding to their cognate steroid receptors, it is becoming more clear that steroids can have non-traditional actions that do not require activation of cognate steroid receptors. We have found that progestogen-facilitated lordosis of rodents is enhanced by activation of dopamine type 1 (D1) or GABAA receptors and their downstream effectors, such as second messengers, in the ventral tegmental area (VTA). The role of phospholipase C in these effects is not clear. If progestins’ actions through D1 and GABAA receptors in the VTA are mediated through PLC, then inhibiting PLC formation in the VTA, via infusions of U73122 (400 nM/side), should reduce progestin (5α-pregnan-3α -ol-20-one; 3α,5α -THP; 100 or 200 ng/side)-facilitated lordosis and its enhancement by D1 (SKF38393; 100 ng/side) or GABAA (muscimol; 100 ng/side) receptor agonists in ovariectomized, estradiol-primed rats. We found that 3α, 5α -THP-, SKF38393-, and muscimol-facilitated lordosis was attenuated by infusions of the PLC inhibitor, U73122, but not vehicle, to the VTA. Thus, progestogens’ non-traditional actions in the VTA to enhance lordosis through D1 and/or GABAA include activity of PLC.
PMCID: PMC2572825  PMID: 18671954
neurosteroids; GABAA; dopamine type 1 receptors; cAMP; protein kinase C; 5α-pregnan-3α-ol-20-one
24.  Bile Acid Reflux Contributes To Development of Esophageal Adenocarcinoma Via Activation Of Phosphatidylinositol-Specific Phospholipase Cγ2 And NADPH Oxidase NOX5-S 
Cancer research  2010;70(3):1247-1255.
Gastroesophageal reflux disease complicated by Barrett’s esophagus (BE) is a major risk factor for esophageal adenocarcinoma (EA). However, the mechanisms of the progression from BE to EA are not fully understood. Besides acid reflux, bile acid reflux may also play an important role in the progression from BE to EA. In this study we examined the role of phosphatidylinositol-specific phospholipase C (PI-PLC) and a novel NADPH oxidase NOX5-S in bile acid-induced in cell proliferation. We found that taurodeoxycholic acid (TDCA) significantly increased NOX5-S expression, H2O2 production and cell proliferation in EA cells. The TDCA-induced increase in cell proliferation was significantly reduced by U73122, an inhibiter of PI-PLC. PI-PLCβ1, β3, β4, γ1 and γ2, but not β2 and δ1 were detectable in FLO cells by Western blot analysis. Knockdown of PI-PLCγ2 or ERK-2 MAP kinase with siRNAs significantly decreased TDCA-induced increase in NOX5-S expression, H2O2 production and cell proliferation. In contrast, knockdown of PI-PLC β1, β3, β4, γ1 or ERK-1 MAP kinase had no significant effect. TDCA significantly increased ERK-2 phosphorylation, an increase which was reduced by U73122 or PI-PLCγ2 siRNA. We conclude that TDCA-induced increase in NOX5-S expression and cell proliferation may depend on sequential activation of PI-PLCγ2 and ERK-2 MAP kinase in EA cells. It is possible that bile acid reflux present in patients with Barrett’s esophagus may increase ROS production and cell proliferation via activation of PI-PLCγ2, ERK-2 MAP kinase and NADPH oxidase NOX5-S, thereby contributing to the development of EA.
PMCID: PMC3057572  PMID: 20086178
phosphatidylinositol-specific phospholipase C; NOX5; Barrett’s esophagus; esophageal adenocarcinoma; bile acid
25.  Biochemical Characterization, Action on Macrophages, and Superoxide Anion Production of Four Basic Phospholipases A2 from Panamanian Bothrops asper Snake Venom 
BioMed Research International  2012;2013:789689.
Bothrops asper (Squamata: Viperidae) is the most important venomous snake in Central America, being responsible for the majority of snakebite accidents. Four basic PLA2s (pMTX-I to -IV) were purified from crude venom by a single-step chromatography using a CM-Sepharose ion-exchange column (1.5 × 15 cm). Analysis of the N-terminal sequence demonstrated that pMTX-I and III belong to the catalytically active Asp49 phospholipase A2 subclass, whereas pMTX-II and IV belong to the enzymatically inactive Lys49 PLA2s-like subclass. The PLA2s isolated from Panama Bothrops asper venom (pMTX-I, II, III, and IV) are able to induce myotoxic activity, inflammatory reaction mainly leukocyte migration to the muscle, and induce J774A.1 macrophages activation to start phagocytic activity and superoxide production.
PMCID: PMC3591126  PMID: 23509779

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