We investigated the effect of alpha-adrenergic and cholinergic receptor agonists on Ca2+ current in adult rat trigeminal ganglion neurons using whole-cell patch clamp methods. The application of acetylcholine, carbachol, and oxotremorine (50 muM each) produced a rapid and reversible reduction of the Ca2+ current by 17+/-6%, 19+/-3% and 18+/-4%, respectively. Atropine, a muscarinic antagonist, blocked carbachol-induced Ca2+ current inhibition to 3 +/- 1%. Norepinephrine (50 muM) reduced Ca2+ current by 18 +/- 2%, while clonidine (50 muM), an alpha2-adrenergic receptor agonist, inhibited Ca2+ current by only 4 +/- 1%. Yohimbine, an alpha2-adrenergic receptor antagonist, did not block the inhibitory effect of norepinephrine on Ca2+ current, whereas prazosin, an alpha1-adrenergic receptor antagonist, attenuated the inhibitory effect of norepinephrine on Ca2+ current to 6 +/- 1%. This pharmacology contrasts with alpha2-adrenergic receptor modulation of Ca2+ channels in rat sympathetic neurons, which is sensitive to clonidine and blocked by yohimbine. Our data suggest that the modulation of voltage dependent Ca2+ channel by norepinephrine is mediated via an alpha1-adrenergic receptor. Pretreatment with pertussis toxin (250 ng/ml) for 16 h greatly reduced norepinephrine- and carbachol-induced Ca2+ current inhibition from 17 +/- 3% and 18 +/- 3% to 2 +/- 1% and 2 +/- 1%, respectively. These results demonstrate that norepinephrine, through an alpha1-adrenergic receptor, and carbachol, through a muscarinic receptor, inhibit Ca2+ currents in adult rat trigeminal ganglion neurons via pertussis toxin sensitive GTP-binding proteins.
Acetylcholine ; Adult ; Animals ; Atropine ; Carbachol ; Clonidine ; GTP-Binding Proteins ; Humans ; Neurons ; Norepinephrine ; Oxotremorine ; Pertussis Toxin ; Pharmacology ; Prazosin ; Rats ; Receptors, Muscarinic ; Trigeminal Ganglion ; Yohimbine

We investigated the mechanism of Cl- secretion by fluoroaluminate(AlF4-) and sodium orthovanadate(vanadate) using the human colonic T84 cell line. T84 cell monolayers grown on collagen-coated filters were mounted in Ussing chambers to measure short circuit current(ISC). Serosal addition of AlF4- or vanadate to T84 monolayers produced a sustained increase in ISC. Removal of Ca2+ from the serosal bathing solution partially inhibited AlF4-(-)and vanadate-induced ISC, and readministration of Ca2+ restored AlF4-(-)and vanadate-induced ISC. Carbachol application in the presence of forskolin, AlF4- or vanadate induced a synergistic increase of ISC. Forskolin and vanadate significantly increased cellular cAMP level, while carbachol and AlF4- did not. Carbachol, AlF4- and vanadate significantly increased [Ca2+]i. After Na+ in mucosal bathing solution was replaced with K+, and the mucosal membrane of T84 cell was permeabilized with amphotericin B, AlF4-, vanadate, and carbachol increased K+ conductance, but forskolin did not. After sodium chloride in serosal bathing solution was replaced with sodium gluconate and the serosal membrane was permeabilized with nystatin, forskolin, AlF4-, and vanadate increased Cl- conductance, but carbachol did not. AlF4-(-)induced ISC was remarkably inhibited by the pretreatment of pertussis toxin(2 micrograms/ml) for 2 hours. These results indicate that AlF4- and vanadate can increase Cl- secretion via simultaneous stimulation of Cl- channel and K+ channel in T84 cells. However, the AlF4- action is mostly attributed to stimulation of pertussis toxin-sensitive G-proteins, whereas the vanadate action mostly results from G protein-independent mechanisms.
Aluminum/*pharmacology ; Amphotericin B/pharmacology ; Carbachol/pharmacology ; Cell Polarity ; Cells, Cultured/drug effects ; Chloride Channels/drug effects/*physiology ; Chlorides/*physiology ; Colon ; Electrophysiology ; Fluorine/*pharmacology ; Forskolin/pharmacology ; GTP-Binding Proteins/physiology ; Human ; Pertussis Toxin ; Potassium/pharmacology ; Potassium Channels/drug effects/physiology ; Second Messenger Systems ; Signal Transduction ; Support, Non-U.S. Gov't ; Vanadates/*pharmacology ; Virulence Factors, Bordetella/pharmacology

Phytosphingosine-1-phosphate (PhS1P) was found to stimulate an intracellular calcium increase via phospholipase C but not pertussis toxin (PTX)- sensitive G-proteins in L2071 mouse fibroblasts. PhS1P also activated ERK and p38 kinase, and these activations by PhS1P were inhibited by PTX. Moreover, PhS1P stimulated the chemotactic migration of L2071 cells via PTX-sensitive Gi protein(s). In addition, the PhS1P-induced chemotactic migration of L2071 cells was also dramatically inhibited by LY294002 and SB203580 (inhibitors of phosphoinositide 3-kinase and p38 kinase, respectively). L2071 cells are known to express four S1P receptors, i.e., S1P1, S1P2, S1P3, and S1P4, and pretreatment with an S1P1 and S1P3 antagonist (VPC 23019) did not affect on PhS1P-induced chemotaxis. This study demonstrates that PhS1P stimulates at least two different signaling cascades, one is a PTX-insensitive but phospholipase C dependent intracellular calcium increase, and the other is a PTX-sensitive chemotactic migration mediated by phosphoinositide 3-kinase and p38 kinase.
1-Phosphatidylinositol 3-Kinase/metabolism ; Animals ; Calcium Signaling/drug effects ; Chemotaxis/*drug effects ; Estrenes/pharmacology ; Extracellular Signal-Regulated MAP Kinases/metabolism ; Fibroblasts/*cytology/*drug effects ; GTP-Binding Proteins/*metabolism ; Gene Expression Regulation/drug effects ; Humans ; Mice ; Pertussis Toxin/*pharmacology ; Phosphorylation/drug effects ; Pyrrolidinones/pharmacology ; RNA, Messenger/genetics/metabolism ; Receptors, Lysosphingolipid/genetics/metabolism ; Sphingosine/*analogs & derivatives/pharmacology ; p38 Mitogen-Activated Protein Kinases/metabolism

His-Phe-Tyr-Leu-Pro-Met (HFYLPM) is a synthetic peptide that stimulates Jurkat T cells resulting in intracellular calcium ([Ca2+]i) increase in a pertussis toxin (PTX)-sensitive manner. We have examined the physiological role of the peptide in T cell activity by comparative investigation of intracellular signaling pathways accompanied with HFYLPM-induced T cell chemotaxis with a well-known chemokine, stromal cell-derived factor-1 (SDF-1)-induced signalings. Wortmannin and genistein inhibited both of HFYLPM- and SDF-1-induced Jurkat T cell chemotaxis indicating that phosphoinositide-3-kinase and tyrosine kinase activity were required for the processes. However, U-73122 and BAPTA/AM preferentially blocked HFYLPM- but not SDF-1-induced T cell chemotaxis. It indicates that phospholipase C/calcium signaling is necessary for only chemotaxis by HFYLPM. One of the well-known cellular molecules involving chemotaxis, extracellular signal-regulated protein kinase (ERK), was activated by SDF-1 but not by HFYLPM ruling out a possible role of ERK on the peptide-mediated chemotaxis. These results indicate that the synthetic peptide, HFYLPM, stimulates T cell chemotaxis showing unique signaling and provide a useful tool for the study of T cell activation mechanism.
1-Phosphatidylinositol 3-Kinase/metabolism ; Androstadienes/pharmacology ; Calcium/metabolism ; Cell Line ; Chemokines, CXC/*pharmacology ; Chemotaxis, Leukocyte/drug effects/*physiology ; Dose-Response Relationship, Drug ; Genistein/pharmacology ; Human ; Jurkat Cells ; Oligopeptides ; Peptide Fragments/chemical synthesis/metabolism/*physiology ; Pertussis Toxin ; Phospholipase C/metabolism ; Protein-Tyrosine Kinase/metabolism ; Signal Transduction/drug effects ; T-Lymphocytes/*drug effects ; Virulence Factors, Bordetella/pharmacology

His-Phe-Tyr-Leu-Pro-Met (HFYLPM) is a synthetic peptide that stimulates Jurkat T cells resulting in intracellular calcium ([Ca2+]i) increase in a pertussis toxin (PTX)-sensitive manner. We have examined the physiological role of the peptide in T cell activity by comparative investigation of intracellular signaling pathways accompanied with HFYLPM-induced T cell chemotaxis with a well-known chemokine, stromal cell-derived factor-1 (SDF-1)-induced signalings. Wortmannin and genistein inhibited both of HFYLPM- and SDF-1-induced Jurkat T cell chemotaxis indicating that phosphoinositide-3-kinase and tyrosine kinase activity were required for the processes. However, U-73122 and BAPTA/AM preferentially blocked HFYLPM- but not SDF-1-induced T cell chemotaxis. It indicates that phospholipase C/calcium signaling is necessary for only chemotaxis by HFYLPM. One of the well-known cellular molecules involving chemotaxis, extracellular signal-regulated protein kinase (ERK), was activated by SDF-1 but not by HFYLPM ruling out a possible role of ERK on the peptide-mediated chemotaxis. These results indicate that the synthetic peptide, HFYLPM, stimulates T cell chemotaxis showing unique signaling and provide a useful tool for the study of T cell activation mechanism.
1-Phosphatidylinositol 3-Kinase/metabolism ; Androstadienes/pharmacology ; Calcium/metabolism ; Cell Line ; Chemokines, CXC/*pharmacology ; Chemotaxis, Leukocyte/drug effects/*physiology ; Dose-Response Relationship, Drug ; Genistein/pharmacology ; Human ; Jurkat Cells ; Oligopeptides ; Peptide Fragments/chemical synthesis/metabolism/*physiology ; Pertussis Toxin ; Phospholipase C/metabolism ; Protein-Tyrosine Kinase/metabolism ; Signal Transduction/drug effects ; T-Lymphocytes/*drug effects ; Virulence Factors, Bordetella/pharmacology

Helicobacter pylori (H. pylori) is an important risk factor for chronic gastritis, peptic ulcer, and gastric cancer. Proteinase-activated receptor 2 (PAR2), subgroup of G-protein coupled receptor family, is highly expressed in gastric cancer, and chronic expression of cyclooxygenase-2 (COX-2) plays an important role in H. pylori-associated gastric carcinogenesis and inflammation. We previously demonstrated that H. pylori induced the expression of PAR2 and COX-2 in gastric epithelial cells. Present study aims to investigate whether COX-2 expression induced by H. pylori in Korean isolates is mediated by PAR2 via activation of Gi protein and Src kinase in gastric epithelial AGS cells. Results showed that H. pylori-induced COX-2 expression was inhibited in the cells transfected with antisense oligonucleotide for PAR2 or treated with Gi protein blocker pertussis toxin, Src kinase inhibitor herbimycin A and soybean trypsin inbitor, indicating that COX-2 expression is mediated by PAR2 through activation of Gi protein and Src kinase in gastric epithelial cells infected with H. pylori in Korean isolates. Thus, targeting the activation of PAR2 may be beneficial for prevention or treatment of gastric inflammation and carcinogenesis associated with H. pylori infection.
Benzoquinones/*pharmacology ; Cell Line, Tumor ; Cyclooxygenase 2/genetics/*metabolism ; Epithelial Cells/enzymology/metabolism/microbiology ; GTP-Binding Protein alpha Subunits, Gi-Go/metabolism ; Gastric Mucosa/enzymology/metabolism/*microbiology ; *Helicobacter pylori ; Humans ; Lactams, Macrocyclic/*pharmacology ; Oligonucleotides, Antisense ; Pertussis Toxin/*pharmacology ; RNA, Messenger/metabolism ; Receptor, PAR-2/*physiology ; src-Family Kinases/metabolism

PURPOSE: Stimulation 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 METHODS: nLDL 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. RESULTS: nLDL-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. CONCLUSION: PTX-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.
Cell Movement/*physiology ; Cells, Cultured ; Humans ; Hydrogen Peroxide/*metabolism ; Interleukin-8/*biosynthesis ; Lipoproteins, LDL/*pharmacology ; Muscle, Smooth, Vascular/cytology/*metabolism ; Myocytes, Smooth Muscle/cytology/*metabolism ; Pertussis Toxin/pharmacology ; Phosphorylation/drug effects ; Reactive Oxygen Species/metabolism ; Receptors, G-Protein-Coupled/*physiology ; Signal Transduction ; p38 Mitogen-Activated Protein Kinases/metabolism

OBJECTIVETo observe the role of G protein in the dual regulation of opioid receptor agonist on the delayed rectified potassium channels.

METHODSUsing whole-cell patch-clamp techniques applied to NG108-15 cells, investigate the effect of opioid receptor agonist on the delayed rectified potassium channels by administration of Guanosine-5'-0'-2-thiociphosphate (GDP beta S), Pertusis Toxin (PTX), Tetroacetic acid nueleoside diphosphate kinase (NDPK) and Adenosine-3' 5' cyclic monophosphate cAMP in the pipette solution.

RESULTS(1) GDP beta S could block the changes induced by both high and low concentration of (D-Pen2.5)-enkephalin (DPDPE) (P < 0.05). (2) PTX could inhibit the excitative regulation on K+ channel by high concentration of DPDPE (P < 0.05). But CTX had no effect on K+ channel caused by DPDPE. (3) UDP could block the excitative effect of K+ channel by high concentration of NDPK, while have no changes on the inhibitory effect caused by low concentration of opioid agonists. (4) cAMP took part in the regulation in high concentration of agonist administration (P < 0.05), while no changes for low concentration of agonists.

CONCLUSIONSDual changes were observed on delayed rectifier potassium channel by agonist treatment on NG108-15 cells. The excitative effect was Gi/o coupled in high concentration of agonist incubation, related to cAMP. While the inhibitory effect was possibly induced by G protein beta gamma subunit directly.

Animals ; Enkephalin, D-Penicillamine (2,5)- ; pharmacology ; GTP-Binding Proteins ; physiology ; Glioma ; metabolism ; pathology ; Guanosine Monophosphate ; analogs & derivatives ; pharmacokinetics ; Hybrid Cells ; metabolism ; pathology ; Mice ; Neuroblastoma ; metabolism ; pathology ; Patch-Clamp Techniques ; Pertussis Toxin ; pharmacology ; Potassium Channels, Inwardly Rectifying ; metabolism ; Rats ; Receptors, Opioid ; agonists ; Thionucleotides ; pharmacokinetics

This study investigated the production of stromal cell-derived factor-1 (SDF-1) and the expression of CXCR4 in human bone marrow endothelial cells (BMECs). Human BMEC cell line BMEC-1 cells expressed SDF-1 mRNA, and conditioned medium induced chemoattraction of CD34+ cells. Migration was not inhibited by pretreating the input cells with pertussis toxin, indicating that the chemoattractive activity was not dependent on SDF-1. Three-day culture of BMEC-1 and primary human BMEC cells produced 1,710+/-204 and 1,050+/-153 pg/mL SDF-1alpha, respectively, which was much less than primary human BM stromal cells (29,536+/-532 pg/ mL). By immuno-histochemistry, CXCR4 was detected in the endothelial cells lining sinusoids, arterioles, and venules in the bone marrow. However, cultured BMECs and BMEC-1 cells did not express CXCR4 on their surfaces. These results indicate that BMECs produce and release small amounts of SDF-1 and express CXCR4 in vivo only.
Antigens, CD34/biosynthesis ; Bone Marrow Cells/*metabolism ; Cell Movement ; Cells, Cultured ; Chemokines, CXC/*biosynthesis ; Chemotaxis ; Culture Media, Conditioned/pharmacology ; Endothelial Cells/*metabolism ; Enzyme-Linked Immunosorbent Assay ; Flow Cytometry ; Hematopoietic Stem Cells/metabolism ; Human ; Immunohistochemistry ; Pertussis Toxin/pharmacology ; RNA, Messenger/metabolism ; Receptors, CXCR4/*biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction ; Support, Non-U.S. Gov't ; Time Factors ; Umbilical Veins/cytology

Lysophosphatidic acid (LPA) is a bioactive phospholipids and involves in various cellular events, including tumor cell migration. In the present study, we investigated LPA receptor and its transactivation to EGFR for cyclooxygenase-2 (COX-2) expression and cell migration in CAOV-3 ovarian cancer cells. LPA induced COX-2 expression in a dose-dependent manner, and pretreatment of the cells with pharmacological inhibitors of Gi (pertussis toxin), Src (PP2), EGF receptor (EGFR) (AG1478), ERK (PD98059) significantly inhibited LPA- induced COX-2 expression. Consistent to these results, transfection of the cells with selective Src siRNA attenuated COX-2 expression by LPA. LPA stimulated CAOV-3 cell migration that was abrogated by pharmacological inhibitors and antibody of EP2. Higher expression of LPA2 mRNA was observed in CAOV-3 cells, and transfection of the cells with a selective LPA2 siRNA significantly inhibited LPA-induced activation of EGFR and ERK, as well as COX-2 expression. Importantly, LPA2 siRNA also blocked LPA-induced ovarian cancer cell migration. Collectively, our results clearly show the significance of LPA2 and Gi/Src pathway for LPA-induced COX-2 expression and cell migration that could be a promising drug target for ovarian cancer cell metastasis.
Butadienes/pharmacology ; Cell Line, Tumor ; Cell Movement/drug effects/*physiology ; Cyclooxygenase 2/*biosynthesis ; Extracellular Signal-Regulated MAP Kinases/antagonists & inhibitors/metabolism ; Female ; Flavonoids/pharmacology ; GTP-Binding Protein alpha Subunits, Gi-Go/antagonists & inhibitors/*metabolism ; Humans ; Lysophospholipids/pharmacology ; Nitriles/pharmacology ; Ovarian Neoplasms/metabolism/*pathology ; Pertussis Toxin/pharmacology ; Protein-Tyrosine Kinases/antagonists & inhibitors/*metabolism ; Proto-Oncogene Proteins/antagonists & inhibitors/*metabolism ; Pyrimidines/pharmacology ; Receptor, Epidermal Growth Factor/antagonists & inhibitors/metabolism ; Receptors, Lysophosphatidic Acid/*metabolism ; Receptors, Prostaglandin E/metabolism ; Signal Transduction ; Transcriptional Activation ; Tyrphostins/pharmacology