Glycerophosphrylocholine (GPC) is a renal medullary compatible organic osmolyte that is derived from choline via phosphatidylcholine, which is catalyzed in part by phospholipase A2 (PLA2) and its degradation by GPC: choline phosphodiesterase (GPC: choline PDE). We found that caffeine elevated intracellular free calcium ([Ca2+]i) and GPC level in cultured MDCK cells, canine kidney epithelial cells, and propose a possible biochemical mechanism. When MDCK cells were incubated for 3 h with 1 to 10 mM caffeine, cellular GPC was elevated in a dose-dependent manner, and this occurred independently of the extracellular osmolality. Caffeine stimulated the rate of [14C]choline incorporation into [14C]GPC and PLA2 activity. Whereas, GPC: choline PDE activity was accompanied by less of increase. These enzyme changes demonstrate the increased net synthesis of MDCK GPC. In order to identify what triggers the PLA2 activation, [Ca2+]i was measured by using a fluorescence dye, Fura-2. Caffeine (10 mM) resulted in a typical transient increase in MDCK [Ca2+]i concentration, and this increase was greatly inhibited by pretreatment of MDCK cells with 10 mM ryanodine for 5 min. Ryanodine (10 mM) also inhibited the caffeine-induced stimulation of PLA2 activity. These findings provide the first evidence that caffeine in MDCK cells causes a ryanodine-inhibitable increase of [Ca2+]i and PLA2 activity, resulting in cellular GPC accumulation.
Animal ; Caffeine/pharmacology* ; Calcium/metabolism* ; Carbon Radioisotopes ; Cell Line ; Choline/metabolism ; Dogs ; Glycerylphosphorylcholine/metabolism* ; Kidney/cytology ; Phospholipases A/metabolism* ; Phospholipases A/drug effects ; Phospholipases A/antagonists & inhibitors ; Phosphoric Diester Hydrolases/metabolism ; Phosphoric Diester Hydrolases/drug effects ; Ryanodine/pharmacology* ; Ryanodine/metabolism

OBJECTIVETo investigate the potential effects of angiogenic process by secretory phospholipase A2 (sPLA2) inhibitor-HyPE (linking N-derivatized phosphatidyl-ethanolamine to hyaluronic acid) on human bone marrow endothelial cell line (HBME-1).

METHODSIn order to examine the suppressing effects of HyPE on HBME-1 proliferation, migration, and capillary-like tube formation, HBME-1 were activated hy angiogenic factor, specifically by basic fibroblast growth factor (b-FGF), vascular endothelial growth factor (VEGF), and oncostatin M (OSM) (at a final concentration of 25, 20, and 2.5 ng/mL, respectively), then HBME-1 proliferation, migration, and tube formation were studied in the absence or presence of HyPE. HBME-1 tube formation was specially analyzed in fibrin gel.

RESULTSHyPE effectively inhibited HBME-1 proliferation and migration as a dose-dependent manner, whatever HBME-1 were grown in the control culture medium or stimulated with b-FGF, VEGF, or OSM. In fibrin, the formations of HBME-1 derived tube-like structures were enhanced by all angiogenic factors, but these were strongly suppressed by HyPE.

CONCLUSIONSThe results support the involvement of sPLA2 in angiogenesis. It is proposed that sPLA2 inhibitor introduces a novel approach in the control of cancer development.

Bone Marrow Cells ; cytology ; Capillaries ; drug effects ; Cell Division ; drug effects ; Cell Movement ; drug effects ; Cells, Cultured ; Endothelial Cells ; cytology ; Enzyme Inhibitors ; pharmacology ; Fibroblast Growth Factor 2 ; antagonists & inhibitors ; Humans ; Hyaluronic Acid ; pharmacology ; Neovascularization, Pathologic ; pathology ; Oncostatin M ; Peptides ; antagonists & inhibitors ; Phosphatidylethanolamines ; pharmacology ; Phospholipases A ; antagonists & inhibitors ; Phospholipases A2 ; Vascular Endothelial Growth Factor A ; antagonists & inhibitors

There is persuasive epidemiological and experimental evidence that dietary polyphenols have anti-inflammatory activity. Aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) have long been used to combat inflammation. Recently, cyclooxygenase (COX) inhibitors have been developed and recommended for treatment of rheumatoid arthritis (RA) and osteoarthritis (OA). However, two COX inhibitors have been withdrawn from the market due to unexpected side effects. Because conventional therapeutic and surgical approaches have not been able to fully control the incidence and outcome of many inflammatory diseases, there is an urgent need to find safer compounds and to develop mechanism-based approaches for the management of these diseases. Polyphenols are found in many dietary plant products, including fruits, vegetables, beverages, herbs, and spices. Several of these compounds have been found to inhibit the inflammation process as well as tumorigenesis in experimental animals; they can also exhibit potent biological properties. In addition, epidemiological studies have indicated that populations who consume foods rich in specific polyphenols have lower incidences of inflammatory disease. This paper provides an overview of the research approaches that can be used to unravel the biology and health effects of polyphenols. Polyphenols have diverse biological effects, however, this review will focus on some of the pivotal molecular targets that directly affect the inflammation process.
Phospholipases A/antagonists & inhibitors ; Phenols/*pharmacology ; Peroxisome Proliferator-Activated Receptors/drug effects/physiology ; NF-kappa B/metabolism ; Lipoxygenase Inhibitors/pharmacology ; Humans ; Flavonoids/*pharmacology ; Cytokines/biosynthesis ; Cyclooxygenase Inhibitors/pharmacology ; Arachidonic Acid/metabolism ; Anti-Inflammatory Agents/*pharmacology ; Animals

OBJECTIVE: To observe the effect of chloroquine on the apoptosis of intestinal mucosa epithelial cell and enterogenous bacteria-endotoxin translocation after total hepatic ischemia-reperfusion in rats. METHODS: The rat total hepatic ischemia-reperfusion model was built by blocking the hepatic portal, suprahepatic and infrahepatic vena cava for 20 minutes. Ninety Sprague-Dawley rats were assigned randomly into the sham operation group (Group A, n = 30), total hepatic ischemia-reperfusion treatment group (Group B, n = 30), and chloroquine administrated group (Group C, n = 30). Each group was subdivided randomly into 3 subgroups (n = 10) according to different experiment time phases as follows: after 20 minutes of total hepatic vascular exclusion (T0), 4 hours after reperfusion (T1), and the 48 hours of survival. Group A and Group B were intravenously injected with normal saline 1 mL/kg while Group C received chloroquine 10 mg/kg which dissolved in 1 mL/kg normal saline intravenously. The levels of portal blood D-lactate, TNF-alpha, endotoxin, and the intestinal mucosa MDA concentration were measured at T0 and T1; the portal blood, mesenteric lymph node, and spleen tissues were cultured for bacteria; and the apoptotic index of intestinal mucosa epithelial cells at T0 and T1 and the survival rate after 48 hour reperfusion were obtained. RESULTS: Compared with Group A, the levels of portal blood D-lactate, TNF-alpha, endotoxin and the intestinal mucosa MDA in Group B and Group C were significantly higher (P < 0.05 or P < 0.01). These indexes of Group C were lower than those of Group B (P < 0.05). The portal vein blood, mesenteric lymph node and spleen tissues existed the bacterium translocation both in Group B and Group C, and the positive rate in Group C was lower than that in Group B (P < 0.05). Apoptotic index of the intestinal mucosa epithelial cell increased significantly in Group B (P < 0.01) and Group C (P < 0.05), but the apoptotic index in Group C was lower than that in Group B (P < 0.05); the 48 hour survival rate of the rats in Group C was higher than that in group B (P < 0.05). CONCLUSION Chloroquine may decrease the intestinal mucosa epithelial cell apoptosis and the enterogenous bacteria-endotoxin translocation after total hepatic ischemia-reperfusion and increase the survival rate of the rats.
Animals ; Bacterial Translocation ; drug effects ; Chloroquine ; pharmacology ; Epithelial Cells ; pathology ; Escherichia coli ; physiology ; Female ; Intestinal Mucosa ; pathology ; Intestine, Small ; microbiology ; pathology ; Liver ; blood supply ; Male ; Phospholipases A ; antagonists & inhibitors ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; microbiology ; pathology

Monocyte chemoattractant protein-1 (MCP1) plays a key role in monocyte/macrophage infiltration to the sub-endothelial space of the blood vessel wall, which is a critical initial step in atherosclerosis. In this study, we examined the intracellular signaling pathway of IL-1beta-induced MCP1 expression using various chemical inhibitors. The pretreatment of a phosphatidylcholine (PC)-specific PLC (PC-PLC) inhibitor (D609), PKC inhibitors, or an NF-kappaB inhibitor completely suppressed the IL-1beta-induced MCP1 expression through blocking NF-kappaB translocation to the nucleus. Pretreatment with inhibitors of tyrosine kinase or PLD partially suppressed MCP1 expression and failed to block nuclear NF-kappaB translocation. These results suggest that IL-1beta induces MCP1 expression through activation of NF-kappaB via the PC-PLC/PKC signaling pathway.
Active Transport, Cell Nucleus/drug effects ; Aorta/pathology ; Atherosclerosis/immunology/metabolism ; Bridged Compounds/pharmacology ; Cell Nucleus/*metabolism ; Cells, Cultured ; Chemokine CCL2/*biosynthesis ; Estrenes/pharmacology ; Genistein/pharmacology ; Humans ; Interleukin-1beta/metabolism ; Myocytes, Smooth Muscle/drug effects/immunology/*metabolism/pathology ; NF-kappa B/*metabolism ; Phospholipases/antagonists & inhibitors ; Protein-Tyrosine Kinases/antagonists & inhibitors ; Pyrrolidinones/pharmacology ; Recombinant Proteins/metabolism ; Signal Transduction/*drug effects ; Thiones/pharmacology

OBJECTIVETo explore the role of stromal-derived factor-1 (SDF-1) in the migration of mesenchymal stem cells (MSCs) and the underlying signal transduction mechanism.

METHODSRat bone marrow-derived MSCs were infected with 100 ml recombinant adenovirus containing human SDF-1alpha gene (Ad-hSDF-1alpha), and the cell migration changes were observed at 1, 2, and 3 days after the infection. Twelve hours after Ad-hSDF-1alpha transfection, the MSCs in separate cultures were treated with wortmannin (50 nmol/L), LY294002 (10 mmol/L), PD98059 (50 mmol/L), U73122 (10 mmol/L), AMD3100 (0.1 g/L), or verapamil (50 nmol/L), respectively, and the signal transduction pathways involved in MSC migration were analyzed.

RESULTSThe MSCs grew in colonies after transfection with Ad-hSDF-1alpha, but this growth pattern was substantially attenuated after treatment with wortmannin, LY294002, PD98059, U73122, AMD3100 and verapamil, among which U73122 and AMD3100 treatments resulted in the most conspicuous inhibitory effects.

CONCLUSIONThe effect of SDF-1 in promoting MSC migration is related to mitogen-activated protein kinase, phosphatidylinositol phospholipase C, and protein kinase signal pathways.

Adenoviridae ; genetics ; Animals ; Cell Movement ; genetics ; physiology ; Cells, Cultured ; Chemokine CXCL12 ; genetics ; physiology ; Enzyme Inhibitors ; pharmacology ; Genetic Vectors ; genetics ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; metabolism ; Rats ; Rats, Wistar ; Signal Transduction ; drug effects ; Transfection ; Type C Phospholipases ; antagonists & inhibitors ; metabolism

Inflammatory diseases are common medical conditions seen in disorders of human immune system. There is a great demand for anti-inflammatory drugs. There are major inflammatory mediators in arachidonic acid metabolic network. Several enzymes in this network have been used as key targets for the development of anti-inflammatory drugs. However, specific single-target inhibitors can not sufficiently control the network balance and may cause side effects at the same time. Most inflammation induced diseases come from the complicated coupling of inflammatory cascades involving multiple targets. In order to treat these complicated diseases, drugs that can intervene multi-targets at the same time attracted much attention. The goal of this review is mainly focused on the key enzymes in arachidonic acid metabolic network, such as phospholipase A2, cyclooxygenase, 5-lipoxygenase and eukotriene A4 hydrolase. Advance in single target and multi-targe inhibitors is summarized.
Animals ; Anti-Inflammatory Agents ; therapeutic use ; Arachidonate 5-Lipoxygenase ; metabolism ; therapeutic use ; Arachidonic Acid ; metabolism ; Cyclooxygenase Inhibitors ; therapeutic use ; Drug Delivery Systems ; methods ; Epoxide Hydrolases ; antagonists & inhibitors ; metabolism ; therapeutic use ; Humans ; Inflammation ; drug therapy ; Lipoxygenase Inhibitors ; Metabolic Networks and Pathways ; drug effects ; Phospholipase A2 Inhibitors ; Phospholipases A2 ; metabolism ; therapeutic use ; Prostaglandin-Endoperoxide Synthases ; metabolism