Specific Inhibitor Of Phospholipase Research Articles

Phospholipase D antagonist 1-butanol inhibited the mobilization of triacylglycerol during seed germination in Arabidopsis

Storage oil breakdown plays an important role in the life cycle of many plants by providing the carbon skeletons that support seedling growth immediately following germination. 1-Butanol, a specific inhibitor of phospholipase D (PLD)-dependent production of the signalling molecule phosphatidic acid (PA), inhibited Arabidopsis seed germination. N-Acylethanolamines (NAEs), which have been shown to inhibits PLDα1 activity, have no effect on seed germination. However, mobilization profile of triacylglycerols (TAG) that induced by each compound has not been reported. To gain deeper insights into the mode of mobilization of TAG during NAE 12:0 or 1-butanol treatment, we conducted a detailed comparative analysis of the effect of NAE 12:0, DMSO, 1-butanol and tert-butanol on Arabidopsis seed germination and fatty acid composition, tert-butanol and DMSO served as the corresponding controls treatment respectively. Our data show that 1-butanol, but not the inactive tert-butanol isomer, inhibited Arabidopsis seed germination, which is accompanied by a with retardation of the mobilization of triacylglycerols (TAG). In contrast, NAE 12:0 did not affect mobilization of TAG, nor did it significantly delay seed germination as monitored by radicle and cotyledon emergence. 1-Butanol induced RNA degradation in seeds and seedlings. We speculate that the large-scale degradation of RNA under the induction of 1-butanol may lead to abnormal gene expression in genes necessary for seed germination, including the genes needed for the mobilization of oil bodies, and thus cause a delay of seed germination. To the best of our knowledge, we report for the first time that 1-butanol delays the mobilization of TAG.

Stimulation of JNK Phosphorylation by the PTTH in Prothoracic Glands of the Silkworm, Bombyx mori.

In this study, phosphorylation of c-Jun N-terminal kinase (JNK) by the prothoracicotropic hormone (PTTH) was investigated in prothoracic glands (PGs) of the silkworm, Bombyx mori. Results showed that JNK phosphorylation was stimulated by the PTTH in time- and dose-dependent manners. In vitro activation of JNK phosphorylation in PGs by the PTTH was also confirmed in an in vivo experiment, in which a PTTH injection greatly increased JNK phosphorylation in PGs of day-6 last instar larvae. JNK phosphorylation caused by PTTH stimulation was greatly inhibited by U73122, a potent and specific inhibitor of phospholipase C (PLC) and an increase in JNK phosphorylation was also detected when PGs were treated with agents (either A23187 or thapsigargin) that directly elevated the intracellular Ca2+ concentration, thereby indicating involvement of PLC and Ca2+. Pretreatment with an inhibitor (U0126) of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) and an inhibitor (LY294002) of phosphoinositide 3-kinase (PI3K) failed to significantly inhibit PTTH-stimulated JNK phosphorylation, indicating that ERK and PI3K were not related to JNK. We further investigated the effect of modulation of the redox state on JNK phosphorylation. In the presence of either an antioxidant (N-acetylcysteine, NAC) or diphenylene iodonium (DPI), PTTH-stimulated JNK phosphorylation was blocked. The JNK kinase inhibitor, SP600125, markedly inhibited PTTH-stimulated JNK phosphorylation and ecdysteroid synthesis. The kinase assay of JNK in PGs confirmed its stimulation by PTTH and inhibition by SP600125. Moreover, PTTH treatment did not affect JNK or Jun mRNA expressions. Based on these findings, we concluded that PTTH stimulates JNK phosphorylation in Ca2+- and PLC-dependent manners and that the redox-regulated JNK signaling pathway is involved in PTTH-stimulated ecdysteroid synthesis in B. mori PGs.

CB1 receptors down-regulate a cAMP/Epac2/PLC pathway to silence the nerve terminals of cerebellar granule cells.

Cannabinoid receptors mediate short-term retrograde inhibition of neurotransmitter release, as well as long-term depression of synaptic transmission at excitatory synapses. The responses of individual nerve terminals in VGLUT1-pHluorin transfected cerebellar granule cells to cannabinoids have shown that prolonged activation of cannabinoid type 1 receptors (CB1Rs) silences a subpopulation of previously active synaptic boutons. Adopting a combined pharmacological and genetic approach to study the molecular mechanisms of CB1R-induced silencing, we found that adenylyl cyclase inhibition decreases cAMP levels while it increases the number of silent synaptic boutons and occludes the induction of further silencing by the cannabinoid agonist HU-210. Guanine nucleotide exchange proteins directly activated by cAMP (Epac proteins) mediate some of the presynaptic effects of cAMP in the potentiation of synaptic transmission. ESI05, a selective Epac2 inhibitor, and U-73122, the specific inhibitor of phospholipase C (PLC), both augment the number of silent synaptic boutons. Moreover, they abolish the capacity of the Epac activator, 8-(4-chlorophenylthio)-2'-O-methyladenosine 3',5'-cyclic monophosphate monosodium hydrate, to prevent HU-210-induced silencing consistent with PLC signaling lying downstream of Epac2 proteins. Furthermore, Rab3-interacting molecule (RIM)1α KO cells have many more basally silent synaptic boutons (12.9±3.5%) than wild-type cells (1.1±0.5%). HU-210 induced further silencing in these mutant cells, although 8-(4-chlorophenylthio)-2'-O-methyladenosine 3',5'-cyclic monophosphate monosodium hydrate only awoke the HU-210-induced silence and not the basally silent synaptic boutons. This behavior can be rescued by expressing RIM1α in RIM1α KO cells, these cells behaving very much like wild-type cells. These findings support the hypothesis that a cAMP/Epac/PLC signaling pathway targeting the release machinery appears to mediate cannabinoid-induced presynaptic silencing.

Signaling involved in PTTH-stimulated 4E-BP phosphorylation in prothoracic gland cells of Bombyx mori

Our previous studies showed that adenosine 5′-monophosphate-activated protein kinase (AMPK)/the target of rapamycin (TOR) signaling is involved in prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis in Bombyx mori prothoracic glands (PGs). In the present study, we further investigated the signaling involved in PTTH-stimulated phosphorylation of 4E-BP. We found that 4E-BP phosphorylation stimulated by PTTH was partially reduced in Ca2+-free medium, indicating the involvement of Ca2+. In addition, we found that a potent and specific inhibitor of phospholipase C (PLC), U73122, greatly inhibited 4E-BP phosphorylation. However, PTTH-stimulated 4E-BP phosphorylation was not attenuated by a protein kinase C (PKC) inhibitor (chelerythrine C). These results indicate that PLC, but not PKC, is involved in PTTH-stimulated 4E-BP phosphorylation. When PGs were treated with agents that directly elevate the intracellular Ca2+ concentration (either A23187 or thapsigargin), a great increase in 4E-BP phosphorylation was observed. A23187-stimulated phosphorylation of 4E-BP was blocked by a chemical activator of AMPK (5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside, AICAR) and a phosphoinositide 3-kinase (PI3K) inhibitor (LY294002), but not by U0126, indicating involvement of AMPK and PI3K. Determination of AMPK phosphorylation showed that treatment with either A23187 or thapsigargin inhibited AMPK phosphorylation. Moreover, PTTH appeared to inhibit AMPK phosphorylation in a Ca2+-dependent manner. Altogether, these results indicate interconnections among Ca2+ signaling, AMPK, and 4E-BP phosphorylation in PTTH-activated PGs of B. mori.

Regulation of histone H3 phosphorylation at serine 10 in PTTH-stimulated prothoracic glands of the silkworm, Bombyx mori

A complex signaling network appears to be involved in prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis in insect prothoracic glands (PGs). In the present study, we investigated the localization of phosphorylated extracellular signal-regulated kinase (ERK) in PTTH-stimulated PGs in Bombyx mori. The nuclear effect of PTTH was further studied by examining phosphorylation of histone H3 at serine 10. Results showed that in PTTH-stimulated PGs, higher phosphorylated ERK was detected in nuclear fraction compared to that in cytosolic fraction. PTTH treatment in vitro appears to rapidly enhance the transcriptional activation-associated histone H3 phosphorylation at serine 10. PTTH stimulated histone H3 phosphorylation in a time-dependent manner. Injection of PTTH into day-6 last instar larvae greatly increased histone H3 phosphorylation, verifying the in vitro effect. The stimulation of histone H3 phosphorylation by PTTH appears to be developmentally regulated. PTTH-stimulated histone H3 phosphorylation was greatly reduced in Ca2+-free saline or by pretreatment with a potent and specific inhibitor of phospholipase C (PLC), U73122. When PGs were treated with agents that directly elevate the intracellular Ca2+ concentration (either A23187 or thapsigargin), a greatly increase in histone H3 phosphorylation at serine 10 was observed, indicating Ca2+-dependency of histone H3 phosphorylation stimulated by PTTH. In addition, PTTH-stimulated histone H3 phosphorylation was partially reduced by U0126, a specific mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor, indicating the involvement of ERK. However, pretreatment with LY294002, a phosphoinositide 3-kinase (PI3K) inhibitor, did not inhibit PTTH-stimulated histone H3 phosphorylation, implying that PI3K signaling is not related to PTTH-stimulated histone H3 phosphorylation. Taken together, these results suggest that PTTH-stimulated histone H3 phosphorylation at serine 10 is mediated by Ca2+/ERK signaling in B. mori PGs.

1-Butanol triggers programmed cell death in Populus euphratica cell cultures

1-Butanol, which is a specific inhibitor of phospholipase D, usually inhibits phosphatidic acid (PA) production and blocks the PA-dependent signaling pathway under stress conditions. However, the effects of 1-butanol on plant cells under non-stress condition are still unclear. In this study, we report that 1-butanol induced a dose dependent cell death in poplar (Populus euphratica) cell cultures. In contrast, the control 2-butanol and ethanol had no effects on cell viability. 1-Butanol-treated cells displayed hallmark features of programmed cell death (PCD), such as shrinkage of the cytoplasm, DNA fragmentation, condensed or stretched chromatin and the activation of caspase-3-like protease. Exogenous application of PA markedly inhibited the 1-butanol-induced PCD. 1-Butanol also caused a burst of mitochondrial H2O2 ([H2O2]mit) that was usually accompanied by a loss of mitochondrial membrane potential (∆Ψm). Supplement of PA, antioxidant enzyme (catalase) and antioxidant (ascorbic acid) reversed this effect. Moreover, a significant increase of nitric oxide (NO) was observed in 1-butanol-treated poplar cells. This NO burst was suppressed by PA or inhibitors of NO synthesis. Further pharmacological experiments indicate that the burst of NO contributed to the 1-butanol-induced inhibition of antioxidant enzymes and subsequent H2O2-dependent PCD. In conclusion, we propose that 1-butanol is a potent inducer of PCD in plants and this process is regulated by the PA, NO and H2O2.

Effects of Inositol 1,4,5-triphosphate on Osteoclast Differentiation in RANKL-induced Osteoclastogenesis

The receptor activator of NF-κB ligand (RANKL) signal is an activator of tumor necrosis factor receptor-associated factor 6 (TRAF6), which leads to the activation of NF-κB and other signal transduction pathways essential for osteoclastogenesis, such as Ca2+ signaling. However, the intracellular levels of inositol 1,4,5-trisphosphate (IP3) and IP3-mediated cellular function of RANKL during osteoclastogenesis are not known. In the present study, we determined the levels of IP3 and evaluated IP3-mediated osteoclast differentiation and osteoclast activity by RANKL treatment of mouse leukemic macrophage cells (RAW 264.7) and mouse bone marrow-derived monocyte/macrophage precursor cells (BMMs). During osteoclastogenesis, the expression levels of Ca2+ signaling proteins such as IP3 receptors (IP3Rs), plasma membrane Ca2+ ATPase, and sarco/endoplasmic reticulum Ca2+ ATPase type2 did not change by RANKL treatment for up to 6 days in both cell types. At 24 h after RANKL treatment, a higher steady-state level of IP3 was observed in RAW264.7 cells transfected with green fluorescent protein (GFP)-tagged pleckstrin homology (PH) domains of phospholipase C (PLC) δ, a probe specifically detecting intracellular IP3 levels. In BMMs, the inhibition of PLC with U73122 [a specific inhibitor of phospholipase C (PLC)] and of IP3Rs with 2-aminoethoxydiphenyl borate (2APB; a non-specific inhibitor of IP3Rs) inhibited the generation of RANKL-induced multinucleated cells and decreased the bone-resorption rate in dentin slice, respectively. These results suggest that intracellular IP3 levels and the IP3-mediated signaling pathway play an important role in RANKL-induced osteoclastogenesis.

Potentiation of nerve growth factor-induced neurite outgrowth by the ROCK inhibitor Y-27632: A possible role of IP 3 receptors

ROCK, a serine/threonine protein kinase that has been identified as a Rho GTP-binding protein, is a promising target for neuropsychiatric disorders. The selective ROCK inhibitor Y-27632 has been shown to induce neurite outgrowth in PC12 cells. However, the precise cellular and molecular mechanisms underlying ROCK inhibition-induced neurite outgrowth are not fully understood. In this study, we examined the roles of cellular signaling pathways in the potentiation of nerve growth factor (NGF)-induced neurite outgrowth by Y-27632. Y-27632 significantly potentiated NGF (2.5 ng/ml)-induced neurite outgrowth in PC12 cells, in a concentration-dependent manner. Furthermore, another ROCK inhibitor, H-1152, and the Rho inhibitor botulinum exoenzyme C3 also potentiated NGF (2.5 ng/ml)-induced neurite outgrowth. The effects by Y-27632 were antagonized by co-administration of inositol 1,4,5-trisphosphate (IP 3) receptor antagonists (xestospongin C or 2-aminoethoxydiphenylborate (2-APB)). Moreover, the potentiation by Y-27632 was blocked by co-administration of the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 or an Akt inhibitor. In contrast, the specific inhibitors of phospholipase C (PLC-γ), p38MAPK, c-Jun N-terminal kinase (JNK), and the Ras/Raf/mitogen-activated protein kinase (MAPK) signaling pathways did not affect the potentiation of NGF-induced neurite outgrowth by Y-27632. The results of double-staining immunocytochemistry suggested that both ROCK1 and type-1 IP 3 receptors may be co-localized in the cell body of PC12 cells. In conclusion, these findings suggest that IP 3 receptors and PI3K-Akt signaling pathways might be involved in the mechanisms of potentiation of NGF-induced neurite outgrowth by ROCK inhibitors.

The functional expression of calcium-sensing receptor in the differentiated THP-1 cells

The expression and function of calcium-sensing receptor (CaSR) in differentiated THP-1 (human acute monocytic leukemia cell line) cells are unknown currently. This study investigated above-mentioned issues using TRAP staining, immunofluorescence staining, Western blotting, ELISA, and Laser Confocal Scanning Microscopy techniques. We found that CaSR protein was expressed, and mainly located in the membrane and cytoplasm in differentiated THP-1 cells. Elevated extracellular calcium or GdCl(3) (an agonist of CaSR) raised intracellular calcium concentration. And this increase was inhibited or abolished by NPS2390 (an inhibitor of CaSR), U73122 (a specific inhibitor of phospholipase C, PLC) or thapsigargin (a Ca(2+)-ATPase inhibitor). The extracellular GdCl(3) elevation stimulated both of IL-1beta and TNFalpha release, and this effect of GdCl(3) was inhibited by NPS2390. In conclusion, CaSR is functionally expressed in differentiated THP-1 cells, and the activated CaSR contributes to intracellular calcium increment through Gq-PLC- inositol triphosphate (IP3) pathway and commits to cytokine secretion. These results suggest that CaSR might be involved in a variety of pathological processes mediated by activated monocyte-macrophages.

Oenocytoid cell lysis to release prophenoloxidase is induced by eicosanoid via protein kinase C

Eicosanoids mediate insect cellular immune responses, which depend largely on phenoloxidase (PO) activity. In plasma, PO is activated by the proteolytic cleavage of proPO, which is stored in oenocytoids, a specific hemocyte type, of the beet armyworm, Spodoptera exigua. Eicosanoids induce an acute cell lysis of oenocytoids, which releases proPO into the plasma. We investigated an intracellular signal pathway following a functional interaction of eicosanoid(s) to a putative membrane receptor. U-73122 (a specific inhibitor of phospholipase C) inhibited oenocytoid lysis of S. exigua significantly after bacterial infection. We concluded that oenocytoid lysis required a certain level of calcium ion because EGTA (a calcium chelator) treatment inhibited cell lysis. Two protein kinase C (PKC) inhibitors (staurosporine and calphostin C) significantly inhibited the oenocytoid lysis. Oenocytoid lysis was likely induced by Na + entry and subsequent osmotic shock because juvenile hormone analog, pyriproxyfen, which activates Na +-K + ATPase and induces subsequent cell shrinkage, antagonized the effect of eicosaniod on cell lysis. Furthermore, ouabain (a specific Na + pump inhibitor) significantly inhibited oenocytoid lysis. These results suggest that eicosanoid mediates oenocytoid lysis by activating the intracellular PKC pathway.

Potentiation of Nerve Growth Factor-Induced Neurite Outgrowth by Fluvoxamine: Role of Sigma-1 Receptors, IP3 Receptors and Cellular Signaling Pathways

BackgroundSelective serotonin reuptake inhibitors (SSRIs) have been widely used and are a major therapeutic advance in psychopharmacology. However, their pharmacology is quite heterogeneous. The SSRI fluvoxamine, with sigma-1 receptor agonism, is shown to potentiate nerve-growth factor (NGF)-induced neurite outgrowth in PC 12 cells. However, the precise cellular and molecular mechanisms underlying potentiation by fluvoxamine are not fully understood. In this study, we examined the roles of cellular signaling pathways in the potentiation of NGF-induced neurite outgrowth by fluvoxamine and sigma-1 receptor agonists.Methods and FindingsThe effects of three SSRIs (fluvoxamine, sertraline, paroxetine) and three sigma-1 receptor agonists (SA4503, 4-phenyl-1-(4-phenylbutyl) piperidine (PPBP), and dehydroepiandrosterone (DHEA)-sulfate) on NGF-induced neurite outgrowth in PC12 cells were examined. Also examined were the effects of the sigma-1 receptor antagonist NE-100, inositol 1,4,5-triphosphate (IP3) receptor antagonist, and specific inhibitors of signaling pathways in the potentiation of NGF-induced neurite outgrowth by selective sigma-1 receptor agonist SA4503. Fluvoxamine (but not sertraline or paroxetine) and the sigma-1 receptor agonists SA4503, PPBP, and DHEA-sulfate significantly potentiated NGF-induced neurite outgrowth in PC12 cells in a concentration-dependent manner. The potentiation by fluvoxamine and the three sigma-1 receptor agonists was blocked by co-administration of the selective sigma-1 receptor antagonist NE-100, suggesting that sigma-1 receptors play a role in blocking the enhancement of NGF-induced neurite outgrowth. Moreover, the potentiation by SA4503 was blocked by co-administration of the IP3 receptor antagonist xestospongin C. In addition, the specific inhibitors of phospholipase C (PLC-γ), phosphatidylinositol 3-kinase (PI3K), p38MAPK, c-Jun N-terminal kinase (JNK), and the Ras/Raf/mitogen-activated protein kinase (MAPK) signaling pathways blocked the potentiation of NGF-induced neurite outgrowth by SA4503.ConclusionThese findings suggest that stimulation of sigma-1 receptors and subsequent interaction with IP3 receptors, PLC-γ, PI3K, p38MAPK, JNK, and the Ras/Raf/MAPK signaling pathways are involved in the mechanisms of action of sigma-1 receptor agonists such as fluvoxamine and SA4503.

Fast action of estrogen on intracellular calcium in dormant mouse blastocyst and its possible mechanism

To study the fast action of estrogen on intracellular calcium of dormant mouse blastocysts and the possible mechanism. Controlled, prospective study. Academic research unit. Forty adult Kun-ming mice. A laser scanning confocal microscope was used to detect the dynamic change of intracellular calcium labeled by Fluo-3/AM, a fluorescent probe of calcium, which was caused by 17-beta-estradiol (17beta-E(2)) in dormant mouse blastocysts. A fluorescent microscope was used to check out the alteration of [Ca(2+)](i) induced by E(2)-BSA, a large molecule of estrogen coupling with bovine serum albumin; by 17beta-E(2) with Ca(2+)-free M2 medium; by 17beta-E(2) with tamoxifen, a blocking agent of traditional estrogen receptor (ER); and by 17beta-E(2) with U73122, a specific inhibitor of phospholipase C. Immunocytochemistry was used to detect the change of intracellular phosphorylated phospholipase C (p-PLC) induced by 17beta-E(2). Intracellular calcium and intracellular p-PLC in dormant mouse blastocysts. Both 17beta-E(2) and E(2)-BSA could increase the intracellular calcium concentration ([Ca(2+)](i)) of blastocysts rapidly, and this increase of [Ca(2+)](i) was independent of either estrogen getting into the cells or the extracellular calcium in the incubation medium. However, this action was possibly blocked by a specific inhibitor of phospholipase C but not by the traditional blocking agent of ER. Moreover, the intracellular p-PLC increased after estrogen acting on blastocysts. Estrogen may induce the increase of intracellular calcium in dormant mouse blastocysts by its action on the composition of the cell membrane to cause the release of Ca(2+) from the endoplasmic reticulum through the transmembrane signal transduction mediated by PLC.

Endothelin-1 enhances proliferation of lung cancer cells by increasing intracellular free Ca 2+

Endothelin-1 (ET-1), the most potent vasoconstrictor, has been shown to be mitogenic in many tumor cells as well as in vascular cells. It was previously reported that the mRNA of ET-1 and endothelin receptors (ETRs) are expressed in lung cancer cells. However, their biological role in lung cancer remains to be explored. The purpose of this study was to determine whether ET-1 stimulates proliferation of the human lung adenocarcinoma cell SPC-A1 and probe its cellular mechanism. Reverse-transcription polymerase chain reaction and Western blot analysis showed that both the mRNA and protein of ET-1, ET AR and ET BR are expressed in SPC-A1 cells. Application of ET-1 at 10 − 15 –10 − 8 M caused a dose-dependent cell proliferation and an increase in intracellular free Ca 2+ concentration ([Ca 2+] i). This ET-1-induced cell proliferation and [Ca 2+] i increase were completely abolished by BQ123, a selective ET AR antagonist, but not by BQ788, a selective ET BR antagonist. Furthermore, it was significantly reduced by U73122, a specific inhibitor of phospholipase C (PLC), but not by U73433, the structural isomer of U73122. Chelating extracellular Ca 2+ or blocking voltage dependent calcium channels by nifedipine also significantly reduced the mitogenic effect of ET-1 and [Ca 2+] i increase in SPC-A1 cells. These results indicate that ET-1 acts as an autocrine growth factor and enhances proliferation of SPC-A1 cells via activation of ET AR. The phosphoinositol/Ca 2+ pathway and Ca 2+ influx through voltage dependent Ca 2+ channels activated by ET AR contribute to this process.

Calcium Signaling of Dioleoyl Phosphatidic Acid via Endogenous LPA Receptors: A Study Using HCT116 and HT29 Human Colon Cancer Cell Lines

In the present study, we have tested the effect of dioleoyl phosphatidic acid (PA) on intracellular <TEX>$Ca_{2+}$</TEX> concentration (<TEX>$[Ca^{2+}]_{i}$</TEX>) in two human colon cancer cell lines (HCT116 and HT29). PA and lysophosphatidic acid (LPA), a bioactive lysolipid, increased <TEX>$[Ca^{2+}]_{i}$</TEX> in both HCT116 and HT29 cell lines. Increases of <TEX>$[Ca^{2+}]_{i}$</TEX> by PA and LPA were more robust in HCT116 cells than in HT29 cells. A specific inhibitor of phospholipase C (U73122), however, was not inhibitory to the cell responses. Pertussis toxin, a specific inhibitor of <TEX>$G_{i/o}$</TEX> type G proteins, however, had an inhibitory effect on the responses except for an LPA-induced one in HT29 cells. Ruthenium red, an inhibitor of the ryanodine receptor, was not inhibitory on the responses, however, 2-APB, a specific inhibitor of inositol 1,4,5-trisphosphate receptor, completely inhibited both lipid-induced <TEX>$Ca^{2+}$</TEX> increases in both cell types. Furthermore, by using Ki16425 and VPC32183, two structurally dissimilar specific antagonists for <TEX>$LPA_{1}/LPA_{3}$</TEX> receptors, an involvement of endogenous LPA receptors in the <TEX>$Ca^{2+}$</TEX> responses was observed. Ki16425 completely inhibited the responses but the susceptibility to VPC32183 was different to PA and LPA in the two cell types. Expression levels of five LPA receptors in the HCT116 and HT29 cells were also assessed. Our data support the notion that PA could increase <TEX>$[Ca^{2+}]_{i}$</TEX> in human colon cancer cells, probably via endogenous LPA receptors, G proteins and <TEX>$IP_{3}$</TEX> receptors, thereby suggesting a role of PA as an intercellular lipid mediator.

Prostaglandin I2 Production and cAMP Accumulation in Response to Acidic Extracellular pH through OGR1 in Human Aortic Smooth Muscle Cells

Ovarian cancer G-protein-coupled receptor 1 (OGR1) and GPR4 have recently been identified as proton-sensing or extracellular pH-responsive G-protein-coupled receptors stimulating inositol phosphate production and cAMP accumulation, respectively. In the present study, we found that OGR1 and GPR4 mRNAs were expressed in human aortic smooth muscle cells (AoSMCs). Acidic extracellular pH induced inositol phosphate production, a transient increase in intracellular Ca(2+) concentration ([Ca(2+)](i)), and cAMP accumulation in these cells. When small interfering RNAs (siRNAs) targeted for OGR1 and GPR4 were transfected to the cells, the acid-induced inositol phosphate production and [Ca(2+)](i) increase were markedly inhibited by the OGR1 siRNA but not by the GPR4 siRNA. Unexpectedly, the acid-induced cAMP accumulation was also largely inhibited by OGR1 siRNA but only slightly by GPR4 siRNA. Acidic extracellular pH also stimulated prostaglandin I2 (PGI(2)) production, which was again inhibited by OGR1 siRNA. The specific inhibitors for extracellular signal-regulated kinase kinase and cyclooxygenase attenuated the acid-induced PGI(2) production and cAMP accumulation without changes in the inositol phosphate production. A specific inhibitor of phospholipase C also inhibited the acid-induced cAMP accumulation. In conclusion, OGR1 is a major receptor involved in the extracellular acid-induced stimulation of PGI(2) production and cAMP accumulation in AoSMCs. The cAMP accumulation may occur through OGR1-mediated stimulation of the phospholipase C/cyclooxygenase/PGI(2) pathway.

Nongenomic vascular action of aldosterone in the glomerular microcirculation.

Aldosterone (Aldo) accelerates hypertension, proteinuria, and glomerulosclerosis in animal models of malignant hypertension or chronic renal failure. Aldo may exert these deleterious renal effects by elevating renal vascular resistance and glomerular capillary pressure. To test this possibility, directly examined were the action of Aldo on the afferent (Af) and efferent (Ef) arterioles (Arts). Examined were the effect of Aldo added to both the bath and lumen on the intraluminal diameter (measured at the most responsive point) of rabbits. Aldo caused dose-dependent constriction in both arterioles with a higher sensitivity in Ef-Arts. Vasoconstrictor action of Aldo was not affected by a mineralocorticoid receptor antagonist spironolactone and was reproduced by membrane-impermeable albumin-conjugated Aldo, suggesting that the vasoconstrictor actions are nongenomic. Pretreatment with neomycin (a specific inhibitor of phospholipase C) abolished the vasoconstrictor action of Aldo in both arterioles. In addition, the vasoconstrictor action of Aldo on Af-Arts was inhibited by both nifedipine and efonidipine, whereas that on Ef-Arts was inhibited by efonidipine but not nifedipine. The results demonstrate that Aldo causes nongenomic vasoconstriction by activating phospholipase C with a subsequent calcium mobilization thorough L- or T-type voltage-dependent calcium channels in Af- or Ef-Arts, respectively. These vasoconstrictor actions on the glomerular microcirculation may play an important role in the pathophysiology and progression of renal diseases by elevating renal vascular resistance and glomerular capillary pressure.

Plasma Glucose-Lowering Effect of β-Endorphin in Streptozotocin-Induced Diabetic Rats

The effect of beta-endorphin on plasma glucose levels was investigated in streptozotocin-induced diabetic rats (STZ-diabetic rats). A dose-dependent lowering of plasma glucose was observed in the fasting STZ-diabetic rat fifteen minutes after intravenous injection of beta-endorphin. The plasma glucose-lowering effect of beta-endorphin was abolished by pretreatment with naloxone or naloxonazine at doses sufficient to block opioid mu-receptors. Also, unlike wild-type diabetic mice, beta-endorphin failed to induce its plasma glucose-lowering effect in the opioid mu-receptor knock-out diabetic mice. In isolated soleus muscle, beta-endorphin enhanced the uptake of radioactive glucose in a concentration-dependent manner. Stimulatory effects of beta-endorphin on glycogen synthesis were also seen in hepatocytes isolated from STZ-diabetic rats. The blockade of these actions by naloxone and naloxonazine indicated the mediation of opioid mu-receptors. In the presence of U73312, the specific inhibitor of phospholipase C (PLC), the uptake of radioactive glucose into isolated soleus muscle induced by beta-endorphin was reduced in a concentration-dependent manner, but it was not affected by U73343, the negative control of U73312. Moreover, chelerythrine and GF 109203X diminished the stimulatory action of beta-endorphin on the uptake of radioactive glucose at a concentration sufficient to inhibit protein kinase C (PKC). The data obtained suggest that activating opioid mu-receptors by beta-endorphin may increase glucose utilization in peripheral tissues via the PLC-PKC pathway to lower plasma glucose in diabetic rats lacking insulin.

Indomethacin Causes Prostaglandin D2-like and Eotaxin-like Selective Responses in Eosinophils and Basophils

We investigated the actions of a panel of nonsteroidal anti-inflammatory drugs on eosinophils, basophils, neutrophils, and monocytes. Indomethacin alone was a potent and selective inducer of eosinophil and basophil shape change. In eosinophils, indomethacin induced chemotaxis, CD11b up-regulation, respiratory burst, and L-selectin shedding but did not cause up-regulation of CD63 expression. Pretreatment of eosinophils with indomethacin also enhanced subsequent eosinophil shape change induced by eotaxin, although treatment with higher concentrations of indomethacin resulted in a decrease in the expression of the major eosinophil chemokine receptor, CCR3. Indomethacin activities and cell selectivity closely resembled those of prostaglandin D(2) (PGD(2)). Eosinophil shape change in response to eotaxin was inhibited by pertussis toxin, but indomethacin- and PGD(2)-induced shape change responses were not. Treatment of eosinophils with specific inhibitors of phospholipase C (U-73122), phosphatidylinositol 3-kinase (LY-294002), and p38 mitogen-activated protein kinase (SB-202190) revealed roles for these pathways in indomethacin signaling. Indomethacin and its analogues may therefore provide a structural basis from which selective PGD(2) receptor small molecule antagonists may be designed and which may have utility in the treatment of allergic inflammatory disease.

Muscarinic acetylcholine receptor activation induces Ca2+ mobilization and Na+/K+-ATPase activity inhibition in eel enterocytes.

The effect of carbachol (Cch) on intracellular calcium concentration ([Ca2+]i) in eel enterocytes was examined using the fluorescent Ca2+ indicator fura-2. Cch caused a biphasic increase in [Ca2+]i, with an initial spike followed by a progressively decreasing level (over 6 min) to the initial, pre-stimulated, level. The effect of Cch was dose-dependent with a 7.5-fold increase in [Ca2+]i over basal level induced by the maximal dose of Cch (100 microM). In Ca2+-free/EGTA buffer the effect of Cch was less pronounced and the [Ca2+]i returned rapidly to basal levels. The increment of [Ca2+]i was dose-dependently attenuated in cells pre-treated with U73122, a specific inhibitor of phospholipase C, suggesting that the Cch-stimulated increment of [Ca2+]i required inositol triphosphate formation. In the presence of extracellular Ca2+, thapsigargin (TG), a specific microsomal Ca2+-ATPase inhibitor, caused a sustained rise in [Ca2+]i whereas in Ca2+-free medium the increase in [Ca2+]i was transient; in both cases, subsequent addition of Cch was without effect. When 2 mM CaCl2 were added to the cells stimulated with TG or with Cch in Ca2+-free medium, a rapid increase in [Ca2+]i was detected, corresponding to the capacitative Ca2+ entry. Thus, both TG and Cch depleted intracellular Ca2+ stores and stimulated influx of extracellular Ca2+ consistent with capacitative Ca2+ entry. K+ depolarization obtained with increasing concentrations of KCl in the extracellular medium induced a dose-related increase in [Ca2+]i which was blocked by 2 microM nifedipine, a non-specific L-type Ca2+ channel blocker. Nifedipine also changed significantly the height of the Ca2+ transient, and the rate of decrement to the pre-stimulated [Ca2+]i level, indicating that Ca2+ entry into enterocytes also occurs through an L-type voltage-dependent calcium channel pathway. We also show that isolated enterocytes stimulated with increasing Cch concentrations (0.1-1000 microM) showed a dose-dependent inhibition of the Na+/K+-ATPase activity. The threshold decrease was at 1 microM Cch; it reached a maximum at 100 microM (50.5% inhibition) and did not decrease further with the use of higher dose. The effect of Cch on Na+/K+-ATPase activity was dependent on both protein kinase C (PKC) and protein phosphatase calcineurin activation since the PKC inhibitor calphostin C abolished Cch effects, while the calcineurin inhibitor FK506 augmented Cch effect. Collectively, these data establish a functional pathway by which Cch can modulate the activity of the Na+/K+-ATPase through a PKC-dependent (calphostin C-sensitive) pathway and a calcineurin-dependent (FK506-sensitive) pathway.

An Antibody to BP 180 kDa Antigen is Able to Induce an Increase of Intracellular Ca 2+ Concentration in DJM-1 (Human Squamous Cell Carcinoma) Cells

To elucidate the induction of transmembrane signal transduction of keratinocytes by bullous pemphigoid (BP) antibodies, we assessed the intracellular Ca 2+ concentration ([Ca 2+ ]i) in DJM-1 cells (a squamous cell carcinoma cell line). The time course of fluo-3-fluorescence intensity was investigated, and transient increase of fluorescence intensity in DJM-1 cells was observed when the DJM-1 cells were incubated with rabbit IgG anti-human BP 180 kDa antigen (BP 180). No increase of fluorescence intensity was observed when the cells were incubated with rabbit IgG anti-human BP 230 kDa antigen (BP 230). This transient increase of fluorescence intensity was inhibited by U-73122, a specific inhibitor of phospholipase C. These results indicate the possibility that an antibody to BP 180 induces transmembrane signal transduction in keratinocytes.