15-Deoxi-Δ-prostaglandin J2 is a tubulin-binding agent that destabilizes microtubules and induces mitotic arrest

15-Deoxi-Δ^12,14-prostaglandin J₂ (15d-PGJ₂) is known to play an important role in the pathophysiology of carcinogenesis, however, the molecular mechanisms underlying these effects are not yet fully understood. Recently, we have shown that 15d-PGJ₂ is a potent inducer of breast cancer cell death and that this effect is associated with a disruption of the microtubule cytoskeletal network. Here, we show that treatment of the MCF-7 breast cancer cell line with 15d-PGJ₂ induces an accumulation of cells in the G₂/M compartment of the cell cycle and a marked disruption of the microtubule network. 15d-PGJ₂ treatment causes mitotic abnormalities that consist of failure to form a stable metaphase plate, incapacity to progress through anaphase, and failure to complete cytokinesis. 15d-PGJ₂ binds to tubulin through the formation of a covalent adduct with at least four cysteine residues in α- and β-tubulin, as detected by hybrid triple-quadrupole mass spectrometry analysis. Overall, these results support the hypothesis that microtubule disruption and mitotic arrest, as a consequence of the binding of 15d-PGJ₂ to tubulin, can represent one important pathway leading to breast cancer cell death.     

The biphasic effects of cyclopentenone prostaglandins, prostaglandin J(2) and 15-deoxy-Delta(12,14)-prostaglandin J(2) on proliferation and apoptosis in rat basophilic leukemia (RBL-2H3) cells

Mast cells produce chemical mediators, including histamine and arachidonate metabolites such as prostaglandin D(2) (PGD(2)) after antigen stimulation. Cyclopentenone prostaglandins of the J series, prostaglandin J(2) (PGJ(2)) and 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), are thought to be derivatives of PGD(2). In this study, the biphasic effects of the PGJ(2) and 15d-PGJ(2) on proliferation and apoptosis in rat basophilic leukemia cells (RBL-2H3), a tumor analog of mast cells, were examined. At low concentrations, 1 or 3 microM PGJ(2) and 15d-PGJ(2) induced cell proliferation, respectively. At high concentrations (10-30 microM) both the inhibition of viability and decrease in histamine content in RBL-2H3 cells were dose dependent. These effects were independent of the nuclear hormone receptor, peroxisome proliferator-activated receptor gamma (PPARgamma), since troglitazone, an agonist of PPARgamma did not cause any effects in RBL-2H3 cells. Cell death induced by PGJ(2) and 15d-PGJ(2) was the result of apoptotic processes, since RBL-2H3 cells treated with 30 microM of the prostaglandins had condensed nuclei, DNA fragmentation and increase in activities of caspase-3 and -9. Moreover, PGJ(2) or 15d-PGJ(2)-induced apoptotic effects were prevented by the caspase inhibitor, z-VAD-fmk. In conclusion, the PGJ(2) or 15d-PGJ(2)-induced apoptosis in RBL-2H3 cells occurs mainly via mitochondrial pathways instead of by PPARgamma-dependent mechanisms.     

Ca2+ signals evoked by histamine H1 receptors are attenuated by activation of prostaglandin EP2 and EP4 receptors in human aortic smooth muscle cells

Background and Purpose

Histamine and prostaglandin E₂ (PGE₂), directly and via their effects on other cells, regulate the behaviour of vascular smooth muscle (VSM), but their effects on human VSM are incompletely resolved.

Experimental Approach

The effects of PGE₂ on histamine-evoked changes in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and adenylyl cyclase activity were measured in populations of cultured human aortic smooth muscle cells (ASMCs). Selective ligands of histamine and EP receptors were used to identify the receptors that mediate the responses.

Key Results

Histamine, via H₁ receptors, stimulates an increase in [Ca²⁺]_i that is entirely mediated by activation of inositol 1,4,5-trisphosphate receptors. Selective stimulation of EP₂ or EP₄ receptors attenuates histamine-evoked Ca²⁺ signals, but the effects of PGE₂ on both Ca²⁺ signals and AC activity are largely mediated by EP₂ receptors.

Conclusions and Implications

Two important inflammatory mediators, histamine via H₁ receptors and PGE₂ acting largely via EP₂ receptors, exert opposing effects on [Ca²⁺]_i in human ASMCs.     

Synthetic peroxisome proliferator-activated receptor gamma agonists rosiglitazone and troglitazone suppress transcription by promoter 3 of the human thromboxane A2 receptor gene in human erythroleukemia cells

The human thromboxane (TX)A2 receptor (TP) gene encodes two TP isoforms, TPalpha and TPbeta, that are regulated by distinct promoters designated promoter Prm1 and Prm3, respectively. Previous studies established that 15d-Delta12,14-prostaglandin J2 (15d-PGJ2) selectively inhibits Prm3 activity and TPbeta expression through a peroxisome proliferator-activated receptor (PPAR)gamma mechanism without affecting Prm1 activity or TPalpha expression in human megakaryocytic erythroleukemia (HEL) 92.1.7 cells. Herein, we investigated the effect of synthetic thiazolidinedione (TZD) PPARgamma ligands rosiglitazone and troglitazone on TP gene expression in HEL cells. Like 15d-PGJ2, both TZDs suppressed Prm3 activity, TPbeta mRNA expression and TP-mediated calcium mobilization without affecting Prm1 or TPalpha mRNA expression. However, unlike 15d-PGJ2, both TZDs mediated their PPARgamma-dependent effects through trans-repression of an activator protein-1 (AP-1) element, a site previously found to be critical for basal Prm3 activity. These data provide further evidence for the role of PPARgamma in regulating the human TP gene; they highlight further differences in TPalpha and TPbeta expression/regulation and point to essential differences between natural and synthetic PPARgamma agonists in mediating those effects.     

Enzymatically modified low-density lipoprotein upregulates CD36 in low-differentiated monocytic cells in a peroxisome proliferator-activated receptor-gamma-dependent way

Peroxisome proliferator-activated receptor-gamma (PPARgamma) has been suggested to upregulate CD36. Since free oxidized polyunsaturated fatty acids are PPARgamma ligands, we studied the effects of LDL modified by the simultaneous action of sPLA2 and 15-lipoxygenase (15LO) on CD36 expression and PPARgamma activation in monocytic cells. Exposure of MM6 cells, which do not express CD36 or other scavenger receptors, to such enzymatically modified LDL (enzLDL) resulted in upregulation of CD36 surface protein and mRNA expression. Similar effects were observed with free 13-hydroperoxyoctadecadienoic acid but not its esterified counterpart. Less pronounced effects were observed with LDL modified by 15LO alone. Upregulation of CD36 was inversely correlated to the state of cell differentiation, as showed by lower response to enzLDL of the scavenger receptor-expressing MM6-sr and THP1 cells. Importantly, LDL modified by sPLA2 and 15LO did not efficiently induce upregulation CD36 in PPARgamma-deficient macrophage-differentiated embryonic stem cells confirming a role of PPARgamma in CD36 expression in cells stimulated with enzLDL. Our data show that LDL modified with physiologically relevant enzymes stimulates CD36 expression in non-differentiated monocytes and that this process involves PPARgamma activation. These effects of enzLDL can be considered pro-atherogenic in the context of early atherosclerosis.     

Co-operative signalling through DP(1) and DP(2) prostanoid receptors is required to enhance leukotriene C(4) synthesis induced by prostaglandin D(2) in eosinophils

BACKGROUND AND PURPOSE

Prostaglandin (PG) D₂ has emerged as a key mediator of allergic inflammatory pathologies and, particularly, PGD₂ induces leukotriene (LT) C₄ secretion from eosinophils. Here, we have characterized how PGD₂ signals to induce LTC₄ synthesis in eosinophils.

EXPERIMENTAL APPROACH

Antagonists and agonists of DP₁ and DP₂ prostanoid receptors were used in a model of PGD₂-induced eosinophilic inflammation in vivo and with PGD₂-stimulated human eosinophils in vitro, to identify PGD₂ receptor(s) mediating LTC₄ secretion. The signalling pathways involved were also investigated.

KEY RESULTS

In vivo and in vitro assays with receptor antagonists showed that PGD₂-triggered cysteinyl-LT (cysLT) secretion depends on the activation of both DP₁ and DP₂ receptors. DP₁ and DP₂ receptor agonists elicited cysLTs production only after simultaneous activation of both receptors. In eosinophils, LTC₄ synthesis, but not LTC₄ transport/export, was activated by PGD₂ receptor stimulation, and lipid bodies (lipid droplets) were the intracellular compartments of DP₁/DP₂ receptor-driven LTC₄ synthesis. Although not sufficient to trigger LTC₄ synthesis by itself, DP₁ receptor activation, signalling through protein kinase A, did activate the biogenesis of eosinophil lipid bodies, a process crucial for PGD₂-induced LTC₄ synthesis. Similarly, concurrent DP₂ receptor activation used Pertussis toxin-sensitive and calcium-dependent signalling pathways to achieve effective PGD₂-induced LTC₄ synthesis.

CONCLUSIONS AND IMPLICATIONS

Based on pivotal roles of cysLTs in allergic inflammatory pathogenesis and the collaborative interaction between PGD₂ receptors described here, our data suggest that both DP₁ and DP₂ receptor antagonists might be attractive candidates for anti-allergic therapies.

LINKED ARTICLE

This article is commented on by Mackay and Stewart, pp. 1671–1673 of this issue. To view this commentary visit http://dx.doi.org/10.1111/j.1476-5381.2011.01236.x     

Blockade of calcium entry in smooth muscle cells by the antidepressant imipramine

The present study was designed to evaluate the effects of antidepressants on smooth muscle contractile activity. In rat aortic rings, the antidepressants imipramine, mianserin and sertraline provoked concentration-dependent inhibitions of the mechanical responses evoked by K+ (30 mM) depolarization. These myorelaxant effects were not modified by the presence of glibenclamide or 80 mM K+ in the bathing medium. Moreover, the vasodilator properties of imipramine were not affected by atropine, phentolamine and pyrilamine. Radioisotopic experiments indicated that imipramine failed to enhance 86Rb outflow from prelabelled and perifused aortic rings whilst counteracting the increase in 45Ca outflow provoked by a rise in the extracellular K+ concentration. Simultaneous measurements of contractile activity and fura-2 fluorescence revealed that, in aortic rings, imipramine reduced the mechanical and fluorimetric response to K+ challenge. In A7r5 smooth muscle cells, whole cell recordings further demonstrated that imipramine inhibited the inward Ca2+ current. Under different experimental conditions, the ionic and relaxation responses to the antidepressants were reminiscent of those mediated by the Ca2+ entry blocker verapamil. Lastly, it should be pointed out that imipramine exhibited a myorelaxant effect of similar amplitude on rat aorta and on rat distal colon. All together, these findings suggest that the myorelaxant properties of imipramine, and probably also setraline and mianserin, could result from their capacity to inhibit the voltage-sensitive Ca2+ channels.     

Stimulation of catecholamine biosynthesis via the protein kinase C pathway by endothelin-1 in PC12 rat pheochromocytoma cells

It has been reported that endothelins (ETs) stimulate catecholamine release from chromaffin cells. However, it is not known whether ETs also affect catecholamine biosynthesis. Thus, using a rat pheochromocytoma cell line, PC12, we examined the effects of ETs on catecholamine biosynthesis. The mRNA level and activity of tyrosine hydroxylase (TH), a rate-limiting enzyme in catecholamine biosynthesis, were increased significantly by endothelin-1 (ET-1) (100nM). These stimulatory effects were inhibited completely by a blocker for the A-type endothelin receptor, BQ-123 [cyclo(D-alpha-aspartyl-L-prolyl-D-valyl-L-leucyl-D-tryptophyl)] (1 microM), but not by a blocker for the B-type endothelin receptor, BQ-788 (N-cis 2,6-dimethylpiperidinocarbonyl-L-gamma-methylleucyl-D-1-methoxycarbonyltryptophanyl-D-norleucine (1 microM). Also, Ro-32-0432 (3-[8-[(dimethylamino)methyl]-6,7,8,9-tetrahydropyrido-[1,2-a]indol-10-yl]-4-(1-methyl-3-indolyl)-H-pyrrole-2,5-dione hydrochloride) (100nM), a protein kinase C inhibitor, completely inhibited ET-1-induced increases in TH activity and mRNA level. Furthermore, ET-1 (100nM) significantly stimulated protein kinase C activity, as well as inositol 1,4,5-triphosphate production; these stimulatory effects were abolished by BQ-123 but not by BQ-788. Moreover, ET-1 (100nM) significantly increased both the TH-protein level and the intracellular catecholamine content. By contrast to ET-1, endothelin-3 did not affect catecholamine synthesis. These results indicate that ET-1, but not ET-3, stimulates catecholamine synthesis through the PKC pathway in PC12 cells. Also, the use of selective ET receptor antagonists suggests that the effects of ET-1 on catecholamine biosynthesis are mediated through ET(A).     

Agonists at PPAR-gamma suppress angiotensin II-induced production of plasminogen activator inhibitor-1 and extracellular matrix in rat cardiac fibroblasts

BACKGROUND AND PURPOSE: Peroxisome proliferator-activated receptor (PPAR)-gamma ligands have been shown to inhibit cardiac fibrosis. However, the underlying mechanisms are poorly understood. We investigated the regulation by PPAR-gamma ligands of angiotensin (Ang) II-induced plasminogen activator inhibitor (PAI)-1, extracellular matrix (ECM) production and cell growth in cardiac fibroblasts. EXPERIMENTAL APPROACH: The effects of PPAR-gamma ligands on Ang II-induced PAI-1, ECM expression and cell growth were assessed in primary-cultured rat cardiac fibroblasts; cardiac PAI-1 and ECM production was examined in Ang II-infused rats. KEY RESULTS: In growth-arrested cardiac fibroblasts, PPAR-gamma ligands rosiglitazone and 15-deoxy-Delta(12,14)-prostaglandin J2 (15d-PGJ2) dose-dependently attenuated Ang II-induced cell proliferation and expression of PAI-1, collagen type-I, collagen type-III and fibronectin. An accompanying increase in PPAR-gamma expression and activation was also observed. These suppressive effects were attenuated by the PPAR-gamma antagonists GW9662 and bisphenol A diglycidyl ether (BADGE). Moreover, rosiglitazone and 15d-PGJ2 inhibited in part the expression and phosphorylation of Ang II-induced transforming growth factor (TGF)-beta1, Smad2/3 and c-Jun NH(2)-terminal kinase (JNK). Ang II infusion in rats markedly increased left ventricular production of PAI-1, collagen and fibronectin, with a concurrent increase in the ratios of heart weight/body weight and left ventricle weight/body weight. Co-treatment with rosiglitazone significantly decreased these levels and upregulated PPAR-gamma expression. CONCLUSIONS AND IMPLICATIONS: Rosiglitazone and 15d-PGJ2 suppress Ang II-induced production of PAI-1 and ECM probably via interactions between PPAR-gamma and TGF-beta1/Smad2/3 and JNK signalling pathways. It is suggested that PPAR-gamma and its ligands may have potential applications in preventing cardiac fibrosis.     

A1 and A2 adenosine receptor modulation of contractility in the cauda epididymis of the guinea-pig

1. The effects of adenosine receptor agonists upon phenylephrine-stimulated contractility and [³H]-cyclic adenosine monophosphate ([³H]-cyclic AMP) accumulation in the cauda epididymis of the guinea-pig were investigated. The α₁-adrenoceptor agonist, phenylephrine elicited concentration dependent contractile responses from preparations of epididymis. In the absence or presence of the L-type Ca²⁺ channel blocker, nifedipine (10 μM) the non-selective adenosine receptor agonist, 5′-N-ethylcarboxamido-adenosine (NECA, 1 μM) shifted phenylephrine concentration-response curves to the left (4 and 5 fold respectively). Following the incubation of preparations with pertussis toxin (200 ng ml⁻¹ 24 h) NECA shifted phenylephrine concentration-response curves to the right (5.7±0.9 fold).
2. In the presence of phenylephrine (1 μM), NECA and the A₁ adenosine receptor selective agonists, N⁶-cyclopentyladenosine (CPA) and (2S)-N⁶-[2-endo-norbornyl]adenosine ((S)-ENBA) elicited concentration-responses dependent contractions from preparations of epididymis (pEC₅₀ values 8.18±0.19, 7.79±0.29 and 8.15±0.43 respectively). The A₃ adenosine receptor agonists N⁶-iodobenzyl-5′-N-methyl-carboxamido adenosine (IBMECA) and N⁶-2-(4-aminophenyl) ethyladenosine (APNEA) mimicked this effect (but only at concentrations greater than 10 μM). In the presence of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 30 nM) CPA concentration-response curves were shifted, in parallel to the right (apparent pK_B 8.75±0.88) and the maximal response to NECA was reduced.
3. In the presence of DPCPX (100 nM) the adenosine agonist NECA and the A_2A adenosine receptor selective agonist, CGS 21680 (2-p-(2-carboxyethyl)-phenethylamino-N-ethylcarboxamido adenosine), but not CPA, inhibited phenylephrine (20 μM) stimulated contractions (pIC₅₀ 7.15±0.48). This effect of NECA was blocked by xanthine amine congener (XAC, 1 μM) and the A_2A adenosine receptor-selective antagonist 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385; 30 nM).
4. (S)-ENBA (in the absence and presence of ZM 241385, 100 nM), but not NECA or CPA inhibited the forskolin (30 μM)-stimulated accumulation of [³H]-cyclic AMP in preparations of the epididymis of the guinea-pig (by 17±6% of control). In the presence of DPCPX (100 nM) NECA and CGS 21680, but not (S)-ENBA, increased the accumulation of [³H]-cyclic AMP in preparations of epididymis (pEC₅₀ values 5.35±0.35 and 6.42±0.40 respectively), the NECA-induced elevation of [³H]-cyclic AMP was antagonised by XAC (apparent pK_B 6.88±0.88) and also by the A_2A adenosine receptor antagonist, ZM 241385 (apparent pK_B 8.60± 0.76).
5. These studies are consistent with the action of stable adenosine analogues at post-junctional A₁ and A₂ adenosine receptors in the epididymis of the guinea-pig. A₁ Adenosine receptors potentiate α₁-adrenoceptor contractility, an effect blocked by pertussis toxin, but which may not be dependent upon an inhibition of adenylyl cyclase. The epididymis of the guinea-pig also contains A₂ adenosine receptors, possibly of the A_2A subtype, which both inhibit contractility and also stimulate adenylyl cyclase.

     

Oxidative stress and alpha1-adrenoceptor-mediated stimulation of the Cl-/HCO3- exchanger in immortalized SHR proximal tubular epithelial cells

BACKGROUND AND PURPOSE: This study evaluated the signalling coupled to the alpha1-adrenoceptor-induced stimulation of the Cl-/HCO3- exchanger in hypertension. EXPERIMENTAL APPROACH: The Na+ -independent HCO3- transport system activity was assayed as the initial rate of pHi recovery after an alkaline load (CO2/HCO3 removal) in immortalized renal proximal tubular epithelial cells from spontaneously hypertensive rat (SHR) and their normotensive control (Wistar Kyoto rat; WKY). KEY RESULTS: Noradrenaline increased Cl-/HCO3- exchanger activity with EC50 values of 0.6 and 5.3 microM in SHR and WKY cells, respectively. These effects were abolished by prazosin, but not by yohimbine. Phenylephrine increased Cl-/HCO3- exchanger activity in SHR and WKY cells (EC50 of 2.6 and 4.9 microM, respectively). Phenylephrine-mediated increase in Cl-/HCO3- exchanger activity in WKY and SHR cells was inhibited by protein kinase C (PKC), MAPK/ERK kinase (MEK) and p38 mitogen-activated protein kinase (p38 MAPK) inhibitors. The expression of alpha1A- and alpha1B-adrenoceptors was identical in WKY and SHR cells. SHR cells generated more H2O2 than WKY cells. In SHR cells, the NADPH oxidase inhibitor apocynin reduced their increased ability to generate H2O2 and abolished their hypersensitivity to phenylephrine, but failed to affect basal Cl-/HCO3- exchanger activity. H2O2-dependent stimulation of Cl-/HCO3- exchange activity was significantly higher in SHR than in WKY cells. CONCLUSIONS AND IMPLICATIONS: Differences between WKY and SHR cells on their sensitivity to alpha1-adrenoceptor stimulation did not correlate with the abundance of alpha1A- and alpha1B-adrenoceptors and may be related to the increased generation of H2O2, which may amplify the response downstream of alpha1-adrenoceptor activation.     

Human adenosine A1 receptor and P2Y2-purinoceptor-mediated activation of the mitogen-activated protein kinase cascade in transfected CHO cells

1. The mitogen-activated protein (MAP) kinase signalling pathway can be activated by a variety of heterotrimeric G_i/G_o protein-coupled and G_q/G₁₁ protein-coupled receptors. The aims of the current study were: (i) to investigate whether the G_i/G_o protein-coupled adenosine A₁ receptor activates the MAP kinase pathway in transfected Chinese hamster ovary cells (CHO-A1) and (ii) to determine whether adenosine A1 receptor activation would modulate the MAP kinase response elicited by the endogenous P2Y2 purinoceptor.
2. The selective adenosine A₁ receptor agonist N⁶-cyclopentyladenosine (CPA) stimulated time and concentration-dependent increases in MAP kinase activity in CHO-A1 cells (EC50 7.1±0.4 nM). CPA-mediated increases in MAP kinase activity were blocked by PD 98059 (50 μM; 89±4% inhibition), an inhibitor of MAP kinase kinase 1 (MEKI) activation, and by pre-treating cells with pertussis toxin (to block G_i/G_o-dependent pathways).
3. Adenosine A₁ receptor-mediated activation of MAP kinase was abolished by pre-treatment with the protein tyrosine inhibitor, genistein (100 μM; 6±10% of control). In contrast, daidzein (100 μM), the inactive analogue of genistein had no significant effect (96±12 of control). MAP kinase responses to CPA (1 μM) were also sensitive to the phosphatidylinositol 3-kinase inhibitors wortmannin (100 nM; 55±8% inhibition) and LY 294002 (30 μM; 40±5% inhibition) but not to the protein kinase C (PKC) inhibitor Ro 31-8220 (10 μM).
4. Activation of the endogenous P2Y₂ purinoceptor with UTP also stimulated time and concentration-dependent increases in MAP kinase activity in CHO-A1 cells (EC₅₀=1.6±0.3 μM). The MAP kinase response to UTP was partially blocked by pertussis toxin (67±3% inhibition) and by the PKC inhibitor Ro 31-8220 (10 μM; 45±5% inhibition), indicating the possible involvement of both G_i/G_o protein and G_q protein-dependent pathways in the overall response to UTP.
5. CPA and UTP stimulated concentration-dependent increases in the phosphorylation state of the 42 kDa and 44 kDa forms of MAP kinase as demonstrated by Western blotting.
6. Co-activation of CHO-A1 cells with CPA (10 nM) and UTP (1 μM) produced synergistic increases in MAP kinase activity which were not blocked by the PKC inhibitor Ro 31-8220 (10 μM).
7. Adenosine A1 and P2Y2 purinoceptor activation increased the expression of luciferase in CHO cells transfected with a luciferase reporter gene containing the c-fos promoter. However, co-activating these two receptors produced only additive increases in luciferase expression.
8. In conclusion, our studies have shown that the transfected adenosine A₁ receptor and the endogenous P2Y₂ purinoceptor couple to the MAP kinase signalling pathway in CHO-A1 cells. Furthermore, co-stimulation of the adenosine A1 receptor and the P2Y₂ purinoceptor produced synergistic increases in MAP kinase activity but not c-fos mediated luciferase expression.

     

Mechanisms for prostaglandin E2 formation caused by proteinase-activated receptor-1 activation in rat gastric mucosal epithelial cells

Proteinase-activated receptor-1 (PAR1), a thrombin receptor, plays a protective role in gastric mucosa via prostanoid formation. Thus, we studied effects of PAR1 stimulation on prostaglandin E(2) (PGE(2)) formation in rat normal gastric mucosal epithelial RGM1 cells and analyzed the underlying signal transduction mechanisms. The PAR1-activating peptide (PAR1-AP) and thrombin increased PGE(2) release from RGM1 cells for 18h, an effect being suppressed by inhibitors of COX-1, COX-2, MEK, p38 MAP kinase (p38 MAPK), protein kinase C (PKC), Src and EGF receptor-tyrosine kinase (EGFR-TK), but not JNK and matrix metalloproteinase (MMP)/a disintegrin and metalloproteinases (ADAMs). PAR1-AP caused persistent (6h or more) and transient (5min) phosphorylation of ERK and p38 MAPK, respectively, followed by delayed reinforcement at 18h. PAR1-AP up-regulated COX-2 in a manner dependent on MEK and EGFR-TK, but not p38 MAPK. The PAR1-mediated persistent ERK phosphorylation was reduced by inhibitors of Src and EGFR-TK. PAR1-AP actually phosphorylated EGF receptors and up-regulated mRNA for heparin-binding-EGF (HB-EGF), the latter effect being blocked by inhibitors of Src, EGFR-TK and MEK. Heparin, an inhibitor for HB-EGF, suppressed PAR1-mediated PGE(2) formation and persistent ERK phosphorylation. These results suggest that PAR1 up-regulates COX-2 via persistent activation of MEK/ERK that is dependent on EGFR-TK activation following induction of HB-EGF, leading to PGE(2) formation. In addition, our data also indicate involvement of COX-1, PKC and p38 MAPK in PAR1-triggered PGE(2) formation. PAR1, thus stimulates complex multiple signaling pathways responsible for PGE(2) formation in RGM1 cells.     

Modulation of HDL metabolism by the niacin receptor GPR109A in mouse hepatocytes

The niacin receptor GPR109A is a G_i-protein-coupled receptor which mediates the effects of niacin on inhibiting intracellular triglyceride lipolysis in adipocytes. However, the role of GPR109A in mediating the effects of niacin on high density lipoprotein (HDL) metabolism is unclear. We found niacin has no effect on HDL-C in GPR109A knockout mice. Furthermore, niacin lowered intracellular cAMP in primary hepatocytes mediated by GPR109A. We used an adeno-associated viral (AAV) serotype 8 vector encoding GPR109A under the control of the hepatic-specific thyroxine-binding globulin promoter to specifically overexpress GPR109A in mouse liver. Plasma HDL-C, hepatic ABCA1 and the HDL cholesterol production rate were significantly reduced in mice overexpressing GPR109A. Overexpression of GPR109A reduced primary hepatocyte free cholesterol efflux to apoA-I; conversely, GPR109A deficient hepatocytes had increased ABCA1-mediated cholesterol efflux. These data support the concept that the HDL-C lowering effect of niacin in wild-type mice is mediated through stimulation of GPR109A in hepatocytes; such an effect then leads to reduced hepatocyte ABCA1 expression and activity, decreased cholesterol efflux to nascent apoA-I, and reduced HDL-C levels. These results indicate that niacin-mediated activation of GP109A in liver lowers ABCA1 expression leading to reduced hepatic cholesterol efflux to HDL.     

MLCK and PKC Involvements via Gi and Rho A Protein in Contraction by the Electrical Field Stimulation in Feline Esophageal Smooth Muscle

We have shown that myosin light chain kinase (MLCK) was required for the off-contraction in response to the electrical field stimulation (EFS) of feline esophageal smooth muscle. In this study, we investigated whether protein kinase C (PKC) may require the on-contraction in response to EFS using feline esophageal smooth muscle. The contractions were recorded using an isometric force transducer. On-contraction occurred in the presence of N^G-nitro-L-arginine methyl ester (L-NAME), suggesting that nitric oxide acts as an inhibitory mediator in smooth muscle. The excitatory composition of both contractions was cholinergic dependent which was blocked by tetrodotoxin or atropine. The on-contraction was abolished in Ca²⁺-free buffer but reappeared in normal Ca²⁺-containing buffer indicating that the contraction was Ca²⁺ dependent. 4-aminopyridine (4-AP), voltage-dependent K⁺ channel blocker, significantly enhanced on-contraction. Aluminum fluoride (a G-protein activator) increased on-contraction. Pertussis toxin (a G_i inactivator) and C3 exoenzyme (a rhoA inactivator) significantly decreased on-contraction suggesting that Gi or rhoA protein may be related with Ca²⁺ and K⁺ channel. ML-9, a MLCK inhibitor, significantly inhibited on-contraction, and chelerythrine (PKC inhibitor) affected on the contraction. These results suggest that endogenous cholinergic contractions activated directly by low-frequency EFS may be mediated by Ca²⁺, and G proteins, such as Gi and rhoA, which resulted in the activation of MLCK, and PKC to produce the contraction in feline distal esophageal smooth muscle.     

Agonist potency differentiates G protein activation and Ca2+ signalling by the orexin receptor type 1

The G protein coupling characteristics of a flag epitope-tagged orexin receptor type 1 (OX1R) was investigated in HEK293 cells. Immunoprecipitation of the OX1R and immunoblotting revealed interactions with Gq/G11 proteins as well as with Gs and Gi proteins. Stimulation with orexin-A did not affect the ability of the OX1R to coprecipitate Gq/G11 proteins, but it robustly elevated the intracellular concentration of Ca2+, [Ca2+]i. No changes in cAMP levels could be detected upon receptor stimulation. To get further insight into the functional correlation of G protein activation and Ca2+ signalling, we used baculovirus transduction to express chimeric G proteins, containing the Galphas protein backbone with various Galpha donor sequences (Galphas/x) at the N and C termini, and measured cAMP as functional output. The Galphas/x chimeric proteins with Galpha11(Galphaq) and Galpha16 structure in the C terminus were stimulated by the OX1R. Concentration-response curves with Galphas/16 revealed an agonist potency correlation between G protein activation and the elevation of [Ca2+]i via discharge of intracellular Ca2+ stores, a feature also recognized for the muscarinic M3 receptor. However, in contrast to the M3 receptor, the OX1R elevated [Ca2+]i via influx from extracellular space at about 30-fold lower agonist concentration. The results suggest that the OX1R is linked to influx of Ca2+ through a signal pathway independent of Gq/G11 protein activation.     

Synergistic effect of PGD2 via prostanoid DP receptor on TNF-alpha-induced production of MCP-1 and IL-8 in human monocytic THP-1 cells

Prostaglandin (PG) D₂, a major cyclooxygenase metabolite generated predominantly from immunologically stimulated mast cells, is thought to contribute to the pathogenesis of allergic diseases via the two PGD₂ receptors, prostanoid DP receptor and chemoattractant receptor homologous molecule expressed on Th2 cells (CRTH2). Monocytes are known to express the prostanoid DP receptor, however, the role of it in inflammatory responses is still unclear. In the present study, to clarify the functional roles of prostanoid DP receptor on monocytes, we examined the effect of PGD₂ on the production of monocyte chemoattractant protein (MCP)-1 and interleukin (IL)-8 from a human monocytic cell line, THP-1. Single activation of prostanoid DP receptor hardly produced any cytokines or chemokines. However, activation with PGD₂ in the presence of tumor necrosis factor (TNF)-α mediated significant production of MCP-1 and IL-8, but not the other cytokines and chemokines, in comparison to single stimulation with TNF-α. In addition, the selective prostanoid DP receptor antagonist, pinagladin ((Z)-7-[(1R,2R,3S,5S)-2-(benzothiophen-3-ylcarbonylamide)-10-norpinan-3-yl]hept-5-enoic acid) inhibited the production of MCP-1 and IL-8 upon combined stimulation with PGD₂ and TNF-α. The synergistic production of MCP-1 and IL-8 by PGD₂ was mimicked by dibutyryl cAMP (db-cAMP) and was inhibited by a protein kinase A (PKA) inhibitor. Our findings suggest that activation of the prostanoid DP receptor on THP-1 cells enhances TNF-α-induced MCP-1 and IL-8 production via the cAMP/PKA signaling pathway.