PTX-sensitive G-protein Research Articles

Spinal β-adrenergic receptors’ activation increases the blood glucose level in mice

ABSTRACTWe examined the role of spinally located β-adrenergic receptors in the regulation of the blood glucose level. The intrathecal (i.t.) injections with dobutamine (β1-adrenergic receptor agonist) or terbutaline (β2-adrenergic receptor agonist) caused an elevation of the blood glucose level, whereas metoprolol (β1-adrenergic receptor antagonist) or butoxamine (β2-adrenergic receptor antagonist) did not. In addition, i.t. pretreatment with pertussis toxin (PTX) attenuated the hyperglycemic effect induced by dobutamine or terbutaline. Moreover, plasma insulin level was increased by dobutamine but not by terbutaline, and PTX reduced dobutamine-induced up-regulation of the plasma insulin level. Terbutaline significantly increased plasma corticosterone level, and PTX further enhanced terbutaline-induced corticosterone level. Furthermore, intraperitoneal (i.p.) pretreatment with hexamethonium- (a preganglionic blocker) attenuated dobutamine- and terbutaline-induced hyperglycemic effects. Our results suggest that activation of spinal β1- and β2-adrenergic receptors produces hyperglycemic effects in a different manner. Spinally located PTX-sensitive G-proteins appear to be involved in hyperglycemic effect induced by terbutaline. Furthermore, dobutamine- or terbutaline-induced hyperglycemia appears to be mediated through the spinal nerves.

The activation of α2-adrenergic receptor in the spinal cord lowers sepsis-induced mortality.

The effect of clonidine administered intrathecally (i.t.) on the mortality and the blood glucose level induced by sepsis was examined in mice. To produce sepsis, the mixture of D-galactosamine (GaLN; 0.6 g/10 ml)/lipopolysaccharide (LPS; 27 µg/27 µl) was treated intraperitoneally (i.p.). The i.t. pretreatment with clonidine (5 µg/5 µl) increased the blood glucose level and attenuated mortality induced by sepsis in a dose-dependent manner. The i.t. post-treatment with clonidine up to 3 h caused an elevation of the blood glucose level and protected sepsis-induced mortality, whereas clonidine post-treated at 6, 9, or 12 h did not affect. The pre-treatment with oral D-glucose for 30 min prior to i.t. post-treatment (6 h) with clonidine did not rescue sepsis-induced mortality. In addition, i.t. pretreatment with pertussis toxin (PTX) reduced clonidine-induced protection against mortality and clonidine-induced hyperglycemia, suggesting that protective effect against sepsis-induced mortality seems to be mediated via activating PTX-sensitive G-proteins in the spinal cord. Moreover, pretreatment with clonidine attenuated the plasma tumor necrosis factor α (TNF-α) induced by sepsis. Clonidine administered i.t. or i.p. increased p-AMPKα1 and p-AMPKα2, but decreased p-Tyk2 and p-mTOR levels in both control and sepsis groups, suggesting that the up-regulations of p-AMPKα1 and p-AMPKα2, or down-regulations of p-mTOR and p-Tyk2 may play critical roles for the protective effect of clonidine against sepsis-induced mortality.

Effect of D-glucose feeding on mortality induced by sepsis.

Sepsis is the life-threatening response to infection which can lead to tissue damage, organ failure, and death. In the current study, the effect of orally administered D-glucose on the mortality and the blood glucose level induced by D-Galactosamine (GaLN)/lipopolysaccharide (LPS)-induced sepsis was examined in ICR mice. After various amounts of D-glucose (from 1 to 8 g/kg) were orally fed, sepsis was induced by injecting intraperitoneally (i.p.) the mixture of GaLN /LPS. Oral pre-treatment with D-glucose dose-dependently increased the blood glucose level and caused a reduction of sepsis-induced mortality. The oral post-treatment with D-glucose (8 g/kg) up to 3 h caused an elevation of the blood glucose level and protected the mortality observed in sepsis model. However, D-glucose post-treated at 6, 9, or 12 h after sepsis induction did not affect the mortality and the blood glucose level induced by sepsis. Furthermore, the intrathecal (i.t.) pretreatment once with pertussis toxin (PTX; 0.1 µg/5 ml) for 6 days caused a reduction of D-glucose-induced protection of mortality and hyperglycemia. Furthermore, once the hypoglycemic state is continued up to 6 h after sepsis initiated, sepsis-induced mortality could not be reversed by D-glucose fed orally. Based on these findings, it is assumed that the hypoglycemic duration between 3 and 6 h after the sepsis induction may be a critical time of period for the survival. D-glucose-induced protective effect against sepsis-induced mortality appears to be mediated via activating PTX-sensitive G-proteins in the spinal cord. Finally, the production of hyperglycemic state may be critical for the survival against the sepsis-induced mortality.

σ-1 Receptor Inhibition of ASIC1a Channels is Dependent on a Pertussis Toxin-Sensitive G-Protein and an AKAP150/Calcineurin Complex.

ASIC1a channels play a major role in various pathophysiological conditions including depression, anxiety, epilepsy, and neurodegeneration following ischemic stroke. Sigma-1 (σ-1) receptor stimulation depresses the activity of ASIC1a channels in cortical neurons, but the mechanism(s) by which σ-1 receptors exert their influence on ASIC1a remains unknown. Experiments were undertaken to elucidate the signaling cascade linking σ-1 receptors to ASIC1a channels. Immunohistochemical studies showed that σ-1 receptors, ASIC1a and A-kinase anchoring peptide 150 colocalize in the plasma membrane of the cell body and processes of cortical neurons. Fluorometric Ca(2+) imaging experiments showed that disruption of the macromolecular complexes containing AKAP150 diminished the effects of the σ-1 on ASIC1a, as did application of the calcineurin inhibitors, cyclosporin A and FK-506. Moreover, whole-cell patch clamp experiments showed that σ-1 receptors were less effective at decreasing ASIC1a-mediated currents in the presence of the VIVIT peptide, which binds to calcineurin and prevents cellular effects dependent on AKAP150/calcineurin interaction. The coupling of σ-1 to ASIC1a was also disrupted by preincubation of the neurons in the G-protein inhibitor, pertussis toxin (PTX). Taken together, our data reveal that σ-1 receptor block of ASIC1a function is dependent on activation of a PTX-sensitive G-protein and stimulation of AKAP150 bound calcineurin.

β-Hydroxybutyrate Modulates N-Type Calcium Channels in Rat Sympathetic Neurons by Acting as an Agonist for the G-Protein-Coupled Receptor FFA3

Free fatty acids receptor 3 (FFA3, GPR41) and 2 (FFA2, GPR43), for which the short-chain fatty acids (SCFAs) acetate and propionate are agonist, have emerged as important G-protein-coupled receptors influenced by diet and gut flora composition. A recent study (Kimura et al., 2011) demonstrated functional expression of FFA3 in the rodent sympathetic nervous system (SNS) providing a potential link between nutritional status and autonomic function. However, little is known of the source of endogenous ligands, signaling pathways, or effectors in sympathetic neurons. In this study, we found that FFA3 and FFA2 are unevenly expressed in the rat SNS with higher transcript levels in prevertebral (e.g., celiac-superior mesenteric and major pelvic) versus paravertebral (e.g., superior cervical and stellate) ganglia. FFA3, whether heterologously or natively expressed, coupled via PTX-sensitive G-proteins to produce voltage-dependent inhibition of N-type Ca(2+) channels (Cav2.2) in sympathetic neurons. In addition to acetate and propionate, we show that β-hydroxybutyrate (BHB), a metabolite produced during ketogenic conditions, is also an FFA3 agonist. This contrasts with previous interpretations of BHB as an antagonist at FFA3. Together, these results indicate that endogenous BHB levels, especially when elevated under certain conditions, such as starvation, diabetic ketoacidosis, and ketogenic diets, play a potentially important role in regulating the activity of the SNS through FFA3.

EP3 receptor isoforms are differentially expressed in subpopulations of primate granulosa cells and couple to unique G-proteins

Prostaglandin E2 (PGE2) produced within the ovarian follicle is necessary for ovulation. PGE2 is recognized by four distinct G-protein-coupled receptors. Among them, PTGER3 (also known as EP3) is unique in that mRNA splicing generates multiple isoforms. Each isoform has a distinct amino acid composition in the C-terminal region, which is involved in G-protein coupling. To determine whether monkey EP3 isoforms couple to different G-proteins, each EP3 isoform was expressed in Chinese hamster ovary cells, and intracellular signals were examined after stimulation with the EP3 agonist sulprostone. Stimulation of EP3 isoform 5 (EP3-5) reduced cAMP in a pertussis toxin (PTX)-sensitive manner, indicating involvement of Gαi. Stimulation of EP3-9 increased cAMP, which was reduced by the general G-protein inhibitor GDP-β-S, and also increased intracellular calcium, which was reduced by PTX and GDP-β-S. So, EP3-9 likely couples to both Gαs and a PTX-sensitive G-protein to regulate intracellular signals. Stimulation of EP3-14 increased cAMP, which was further increased by PTX, so EP3-14 likely regulates cAMP via multiple G-proteins. Granulosa cell expression of all EP3 isoforms increased in response to an ovulatory dose of human chorionic gonadotropin. Two EP3 isoforms were differentially expressed in functional subpopulations of granulosa cells. EP3-5 was low in granulosa cells at the follicle apex while EP3-9 was high in cumulus granulosa cells. Differential expression of EP3 isoforms may yield different intracellular responses to PGE2 in granulosa cell subpopulations, contributing to the different roles played by granulosa cell subpopulations in the process of ovulation.

Effect of GABA Receptor Agonists or Antagonists Injected Spinally on the Blood Glucose Level in Mice

The possible roles of gamma-amino butyric acid (GABA) receptors located in the spinal cord for the regulation of the blood glucose level were studied in ICR mice. We found in the present study that intrathecal (i.t.) injection with baclofen (a GABAB receptor agonist; 1-10 μg/5 μl) or bicuculline (a GABAA receptor antagonist; 1-10 μg/5 μl) caused an elevation of the blood glucose level in a dose-dependent manner. The hyperglycemic effect induced by baclofen was more pronounced than that induced by bicuculline. However, muscimol (a GABAA receptor agonist; 1-5 μg/5 μl) or phaclofen (a GABAB receptor antagonist; 5-10 μg/5 μl) administered i.t. did not affect the blood glucose level. Baclofen-induced elevation of the blood glucose was dose-dependently attenuated by phaclofen. Furthermore, i.t. pretreatment with pertussis toxin (PTX; 0.05 or 0.1 μg/5 μl) for 6 days dose-dependently reduced the hyperglycemic effect induced by baclofen. Our results suggest that GABAB receptors located in the spinal cord play important roles for the elevation of the blood glucose level. Spinally located PTX-sensitive G-proteins appear to be involved in hyperglycemic effect induced by baclofen. Furthermore, inactivation of GABAA receptors located in the spinal cord appears to be responsible for tonic up-regulation of the blood glucose level.

Sphingosylphosphorylcholine Stimulates CCL2 Production from Human Umbilical Vein Endothelial Cells

Sphingosylphosphorylcholine (SPC) is a component of high-density lipoprotein particles. We investigated the functional role of SPC in HUVECs. SPC stimulation induced production of the CCL2 chemokine in a PTX-sensitive G-protein-dependent manner. SPC treatment caused the activation of NF-κB and AP-1, which are essential for SPC-induced CCL2 production, and induced the activation of three MAPKs, ERK, p38 MAPK, and JNK. Inhibition of p38 MAPK or JNK by specific inhibitors caused a dramatic decrease in SPC-induced CCL2 production. The Jak/STAT3 pathway was also activated upon SPC stimulation of HUVECs. Pretreatment with a Jak inhibitor blocked not only SPC-induced p38 MAPK and JNK activation, but also NF-κB and AP-1 activation. Our results suggest that SPC stimulates HUVECs, resulting in Jak/STAT3-, NF-κB-, and AP-1-mediated CCL2 production. We also observed that SPC stimulated expression of the adhesion molecule ICAM-1 in HUVECs. Our results suggest that SPC may contribute to atherosclerosis; therefore, SPC and its unidentified target receptor offer a starting point for the development of a treatment for atherosclerosis.

A farnesylated G-protein suppresses Akt phosphorylation in INS 832/13 cells and normal rat islets: Regulation by pertussis toxin and PGE 2

Protein isoprenylation constitutes incorporation of either 15-carbon farnesyl or 20-carbon geranylgeranyl derivative of mevalonic acid onto the C-terminal cysteine, culminating in increased hydrophobicity of the modified proteins for optimal membrane anchoring and interaction with their respective effectors. Emerging evidence confirms the participatory role of prenylated proteins in pancreatic β-cell function including insulin secretion. Herein, we investigated the putative regulatory roles of protein farnesylation in cell survival signaling pathways in insulin-secreting INS 832/13 cells and normal rodent islets, specifically at the level of protein kinase-B/Akt phosphorylation induced by insulin-like growth factor [IGF-1]. Selective inhibitors of farnesylation [e.g., FTI-277 or FTI-2628] or knockdown of the β-subunit of farnesyl transferase by siRNA significantly increased Akt activation under basal and IGF-1-stimulated conditions. Consequentially, the relative abundance of phosphorylated FoxO1 and Bad were increased implicating inactivation of critical components of the cell death machinery. In addition, FTI-induced Akt activation was attenuated by the PI3-kinase inhibitor, LY294002. Exposure of INS 832/13 cells to pertussis toxin [PTx] markedly potentiated Akt phosphorylation suggesting involvement of a PTx-sensitive G-protein in this signaling axis. Furthermore, prostaglandin E 2, a known agonist of inhibitory G-proteins, significantly attenuated FTI-induced Akt phosphorylation. Taken together, our findings suggest expression of a farnesylated G-protein in INS 832/13 cells and normal rat islets, which appear to suppress Akt activation and subsequent cell survival signaling steps. Potential regulatory roles of the islet endogenous protein kinase-B inhibitory protein [Probin] in islet function are discussed.

Effects of Serum Amyloid A and Lysophosphatidylcholine on Intracellular Calcium Concentration in Human Coronary Artery Smooth Muscle Cells

Serum amyloid A (SAA), an acute-phase protein, and lysophosphatidylcholine (LPC), an oxidized LDL component, contribute to the physiological processes of atherosclerosis and cardiovascular disease. However, the effects of SAA/LPC on human coronary artery smooth muscle cells (hCASMCs) have not been fully investigated. Therefore, we examined the effects of SAA/LPC on Ca(2+)/Mg(2+) mobilization and its underlying mechanisms in hCASMCs. Intracellular Ca(2+)/Mg(2+) concentration ([Ca(2+)](i) / [Mg(2+)](i)) was measured with fura-2 AM/mag-fura-2 AM. Conventional RT-PCR analysis was also performed. Both SAA and LPC increased [Ca(2+)](i) by Ca(2+) entry. The SAA-induced Ca(2+) entry was inhibited by Gd(3+), SKF96365, and 2-aminoethoxydiphenyl borate (2-APB), a nonselective transient receptor potential (TRP) channel blocker, but not nifedipine. The LPC-induced Ca(2+) entry was blocked by Gd(3+), but not nifedipine, SKF96365 and 2-APB. U-73122 and PTX prevented the activation of SAA-, but not LPC-induced Ca(2+) influx. LPC, but not SAA, increased [Mg(2+)](i) as well as [Ca(2+)](i). The RT-PCR analysis revealed the expression of TRPC1/4, TRPV1/2/4, and TRPM7/8 mRNA. These results suggest that SAA/LPC activate Ca(2+) influx in hCASMCs; SAA activates it via PTX-sensitive G-protein, PLC and TRPC pathways, while LPC activates it independently of these pathways, where TRPM7 may be partly involved. Thus, TRP protein appears to be a target molecule of Ca(2+) signaling in hCASMCs elicited by SAA/LPC, which may play roles in coronary muscle dysfunction under pathophysiological and inflammatory conditions such as atherosclerosis.

Native Low-Density Lipoprotein-Dependent Interleukin-8 Production Through Pertussis Toxin-Sensitive G-Protein Coupled Receptors and Hydrogen Peroxide Generation Contributes to Migration of Human Aortic Smooth Muscle Cells

PurposeStimulation of human aortic smooth muscle cells (hAoSMCs) with native low-density lipoprotein (nLDL) induced the production of interleukin-8 (IL-8) that is involved in the pathogenesis of cardiovascular diseases. However, the process of signal transduction of nLDL was currently uncharacterized. Therefore, the aim of this study was to investigate the signal transduction pathway of nLDL-dependent IL-8 production and the effect of IL-8 on hAoSMCs migration.Materials and MethodsnLDL was prepared by ultracentrifugation with density-adjusted human serum of normocholesterolemia. In hAoSMCs, IL-8 secreted to medium was measured using ELISA assay, and Western blot analysis was performed to detect p38 MAPK activation as a key regulator of IL-8 production. nLDL-dependent H2O2 generation was determined by microscopic analysis using 2',7'-dichlorofluoroscein diacetate (DCF-DA). IL-8-induced migration of hAoSMCs was evaluated by counting the cell numbers moved to lower chamber using Transwell plates.ResultsnLDL-induced IL-8 production was completely blocked by preincubation of hAoSMCs with pertussis toxin (PTX), which inhibited nLDL-dependent p38 MAPK phosphorylation. PTX-sensitive G-protein coupled receptor was responsible for nLDL-dependent H2O2 generation that was abrogated with preincubation of the cells with of polyethylene glycol-conjugated catalase (PEG-Cat). Pretreatment of PEG-Cat prevented nLDL-induced p38 MAPK phosphorylation and IL-8 production, which was partly mimicked by treatment with exogenous H2O2. Finally, IL-8 increased hAoSMCs migration that was completely blocked by incubation with IL-8 neutralizing antibody.ConclusionPTX-sensitive G-protein coupled receptor-dependent H2O2 generation by nLDL plays a critical role in IL-8 production in hAoSMC, and IL-8 may contribute to atherogenesis through increased migration of hAoSMCs.

Rainbow trout somatostatin receptor subtypes SSTR1A, SSTR1B, and SSTR2 differentially activate the extracellular signal-regulated kinase and phosphatidylinositol 3-kinase signaling pathways in transfected cells

Previously, we reported that extracellular signal-regulated kinase (ERK) and protein kinase B (Akt), a downstream target of phosphatidylinositol 3-kinase (PI3K), mediated somatostatin (SS) inhibition of GH receptor, IGF1, and IGF1 receptor expression. In this study, we used Chinese hamster ovary-K1 cells that stably transfected individually with trout SS receptors (SSTR1A, SSTR1B, and SSTR2) to elucidate receptor-effector pathway linkages. SS induced ERK and Akt activation in a time- and concentration-related manner in all SSTR-expressing cells; however, the PI3K/Akt pathway was activated to a greater extent through SSTR1A than through either SSTR1B or SSTR2, whereas the ERK pathway was activated to a greater extent though SSTR2 than through either SSTR1A or SSTR1B. Although the ERK pathway inhibitor U0126 had no effect on Akt activation, the PI3K inhibitor LY294002 reduced ERK activation to near control levels in all SSTR-expressing cell lines, suggesting some cross talk between the pathways, possibly at the level of c-Raf, the phosphorylation of which also was induced by SS via each SSTR. Pertussis toxin (PTX) completely abolished SS-induced activation of ERK and Akt in SSTR1A-, SSTR1B-, and SSTR2-expressing cells, suggesting that these receptors link to the ERK and PI3K/Akt pathways via PTX-sensitive G-proteins. SS-induced activation of Elk1, Stat3, and C/EBPβ also was mediated by each of the trout SSTRs. These findings establish important receptor-effector pathway linkages for fish SSTRs and provide insight into the molecular mechanisms by which SSs may elicit diverse physiological effects in target cells.

A pertussis toxin sensitive G-protein-independent pathway is involved in serum amyloid A-induced formyl peptide receptor 2-mediated CCL2 production

Serum amyloid A (SAA) induced CCL2 production via a pertussis toxin (PTX)-insensitive pathway in human umbilical vein endothelial cells (HUVECs). SAA induced the activation of three MAPKs (ERK, p38 MAPK, and JNK), which were completely inhibited by knock-down of formyl peptide receptor 2 (FPR2). Inhibition of p38 MAPK and JNK by their specific inhibitors (SB203580 and SP600125), or inhibition by a dominant negative mutant of p38 MAPK dramatically decreased SAA-induced CCL2 production. Inactivation of G((i)) protein(s) by PTX inhibited the activation of SAA-induced ERK, but not p38 MAPK or JNK. The results indicate that SAA stimulates FPR2-mediated activation of p38 MAPK and JNK, which are independent of a PTX-sensitive G-protein and are essential for SAA-induced CCL2 production.

O2‐ Interacts with Pertussis Toxin‐sensitive G‐proteins to Disrupt α2 Adrenergic Receptor Function in Sympathetic Nerves Supplying Mesenteric Arteries in DOCA‐salt Hypertension

Norepinephrine (NE) is released from sympathetic nerves supplying arteries and veins. We studied differences in NE release mechanisms in vitro in mesenteric arteries (MA) from normotensive (sham) and DOCA‐salt hypertensive rats using amperometry with a microelectrode. NE oxidation currents were larger in DOCA‐salt MA compared to sham MA. Idazoxan (1 µM, blocks α2 adrenergic receptors, α2ARs) increased NE current in sham but not DOCA‐salt MA indicating impaired function of α2ARs in DOCA‐salt rats. α2ARs on sympathetic nerve terminals couple to inhibition of NE release via pertussis toxin inhibited G‐proteins. After 2h incubation in pertussis toxin (2 µg/ml), NE currents increased in sham MA but not DOCA‐salt MA indicating that pertussis toxin sensitive G‐proteins were inactive in DOCA‐salt MA. O2− levels are elevated in MA of DOCA‐salt rats. Therefore, we treated DOCA‐salt rats with the NADPH oxidase inhibitor, apocynin (1.5 mM) in the drinking water for 4 weeks. NE currents were similar in sham MA and apocynin‐treated DOCA‐salt MA and α2AR function was restored in MA from apocynin‐treated DOCA‐salt rats. We conclude that α2AR receptor function is impaired in sympathetic nerves supplying MA in DOCA‐salt rats. This effect is mediated by O2− induced inhibition of PTX sensitive G‐proteins linking α2ARs to NE release. These data support a new mechanism for the beneficial effect of antioxidant treatment in hypertension.

Identification of novel synthetic peptide showing angiogenic activity in human endothelial cells

A novel synthetic hexapeptide (SFKLRY-NH 2) that displays angiogenic activity has been identified by positional scanning of a synthetic peptide combinatorial library (PS-SPCL). The peptide induced proliferation, migration, and capillary-like tube formation in primary cultured HUVECs, and augmented vessel sprouting ex vivo while attenuated by the treatment with pertussis toxin (PTX) or U73122 (PLC-inhibitor) suggesting the influence of PTX-sensitive G-proteins and PLC. In addition, SFKLRY-NH 2 up-regulated the expression of VEGF-A in HUVECs and the neutralizing antibody against VEGF suppressed SFKLRY-NH 2-induced tube formation activity. Taken together, these results suggest that SFKLRY-NH 2 may induce blood vessel formation by PTX-sensitive G protein-coupled receptor-PLC-Ca 2+ signaling cascade leading into VEGF-A expression in HUVECs.

Collagen-binding motif peptide, a cleavage product of osteopontin, stimulates human neutrophil chemotaxis via pertussis toxin-sensitive G protein-mediated signaling

The collagen-binding motif (CBM) peptide, a cleavage product of osteopontin (OPN), stimulated intracellular calcium increase in human neutrophils. CBM peptide-stimulated calcium was inhibited by pertussis toxin (PTX), suggesting the influence of PTX-sensitive G-proteins. In addition CBM peptide stimulated the chemotactic migration of human neutrophils and human monocytes. CBM peptide-induced neutrophil chemotaxis was completely inhibited by PTX, once again indicating the influence of Gi proteins. CBM peptide was also found to induce mitogen activated protein kinase activation. CBM peptide-induced neutrophil chemotaxis was mediated by p38 kinase as well as an extracellular signal-regulated protein kinase. Taken together, the results suggest that a cleavage product of OPN, CBM peptide, initiates immune responses by inducing neutrophil trafficking via certain PTX-sensitive cell surface receptors.

Lysophosphatidylglycerol stimulates chemotactic migration in human natural killer cells

We observed that lysophosphatidylglycerol (LPG) stimulates chemotactic migration in human natural killer (NK) cells. The LPG-induced chemotactic migration of NK cells was completely inhibited by pertussis toxin (PTX). LPG also stimulated the extracellular signal-regulated kinase (ERK) and Akt activities in NK cells. LPG-stimulated ERK activity was inhibited by PTX, indicating the involvement of PTX-sensitive G-proteins. The preincubation of NK cells with an ERK inhibitor (PD98059) or phosphoinositide-3-kinase (PI3K) inhibitors (wortmannin and LY294002) completely inhibited LPG-induced chemotactic migration, suggesting the essential role of ERK and PI3K in the process. Moreover, LPG-induced chemotactic migration in NK cell was inhibited by Ki16425, an LPA 1/3 receptor-selective antagonist, suggesting the involvement of the Ki16425-sensitive G-protein coupled receptor (GPCR) in the process. Taken together, the results indicate that LPG stimulates chemotactic migration in NK cells through GPCR, suggesting a new function of LPG as a modulator of NK cell functioning.

Lysophosphatidylethanolamine stimulates chemotactic migration and cellular invasion in SK-OV3 human ovarian cancer cells: Involvement of pertussis toxin-sensitive G-protein coupled receptor

We investigated whether lysophosphatidylethanolamine (LPE) modulates cellular signaling in different cell types. SK-OV3 ovarian cancer cells and OVCAR-3 ovarian cancer cells were responsive to LPE. LPE-stimulated intracellular calcium concentration ([Ca 2+] i) increase was inhibited by U-73122, suggesting that LPE stimulates calcium signaling via phospholipase C activation. Moreover, pertussis toxin (PTX) almost completely inhibited [Ca 2+] i increase by LPE, indicating the involvement of PTX-sensitive G-proteins. Furthermore, we found that LPE stimulated chemotactic migration and cellular invasion in SK-OV3 ovarian cancer cells. We examined the role of lysophosphatidic acid receptors on LPE-stimulated cellular responses using HepG2 cells transfected with different LPA receptors, and found that LPE failed to stimulate nuclear factor kappa B-driven luciferase. We suggest that LPE stimulates a membrane bound receptor, different from well known LPA receptors, resulting in chemotactic migration and cellular invasion in SK-OV3 ovarian cancer cells.

GABA B receptor activation inhibits dopamine D1 receptor-mediated facilitation of [ 3H]GABA release in substantia nigra pars reticulata

GABA B receptors inhibit and dopamine D1 receptors stimulate the release of GABA from striatal terminals in the pars reticulata of the substantia nigra. Here we have studied the interaction between both classes of receptors by exploring the effect of GABA B receptors upon the stimulation of depolarization-induced [ 3H]GABA release induced by the activation of D1 receptors in slices of the pars reticulata of the rat substantia nigra. The activation of GABA B receptors with baclofen (100 μM) inhibited by 48 ± 8% the evoked [ 3H]GABA release in normal slices but did not modify the release in slices from reserpine-treated rats, indicating that the inhibition was dependent on endogenous dopamine. The inhibitory effect of baclofen was also abolished by the D1 receptor antagonist SCH 23390 (1 μM), indicating a D1 receptor-dependence of the baclofen inhibition. Baclofen dose-dependently inhibited (IC 50 = 3.6 μM) the stimulation of release induced by the D1 agonist SKF 38393 (1 μM). Baclofen also blocked the stimulation of release induced by forskolin but not that induced by 8-Br-cAMP, indicating that the inhibitory effect was exerted before cAMP synthesis. N-ethylmaleimide (NEM), a selective inactivator of PTX-sensitive G-proteins, abolished the baclofen inhibition of the SKF 38393-induced stimulation of the release without affecting the stimulation induced by the D1 agonist, suggesting that the baclofen effect was mediated by Gα i/o proteins. These results might have relevance in the control motor disorders associated with D1 receptor supersensitivity.

Serum amyloid A induces WISH cell apoptosis

Serum amyloid A (SAA) is an important mammalian acute reactant. Here, we aim to investigate the effect of SAA on apoptosis and its mechanism of action in human amniotic WISH cells. The expression of formyl peptide receptor (FPRL1), which is reported as a SAA receptor, was tested using RT-PCR and ligand binding assay with radio-labeled FPRL1 ligand. The effect of SAA on proliferating cell population was evaluated by thymidine incorporation assay. The protein phosphorylation levels and caspase-3 activity were detected by Western blot assay. SAA inhibits thymidine incorporation in human amniotic WISH cells. A SAA-induced decrease of proliferating cell population was accompanied with nuclear condensation and caspase-3 activation in WISH cells, suggesting that SAA induces WISH cell apoptosis. Since FPRL1 has been reported as a SAA receptor, we investigated the effects of several FRPL1 agonists on a proliferating cell population in WISH cells. Among the tested FPRL1 agonists, only SAA induced a decrease of proliferating cell population in WISH cells. On the downstream signaling of SAA, we found that SAA stimulated extracellular signal-regulated kinase and p38 kinase, which were not inhibited by pertussis toxin (PTX), ruling out the role of PTX-sensitive G-proteins. Furthermore a SAAinduced decrease of proliferating cell population was not affected by PTX, suggesting that SAA inhibits WISH cell apoptosis in a PTX-sensitive G-proteinindependent manner. A SAA-induced decrease of a proliferating cell population was completely blocked by PD98059 and SB203580, suggesting that mitogenactivated protein kinase activities are essentially required for the process. SAA is a novel inducer for WISH cell apoptosis, and the PTX-insensitive pathway is involved in the process.