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 study the effect of polydatin on phospholipase A(2) in lung tissues in rats with endotoxic shock.

METHODSThirty-two healthy male Wistar rats were employed in this study. A total of 8 rats received normal saline intravenously (control group), 8 rats received 10 mg/kg of endotoxin (endotoxic shock group), 8 rats received 1 mg/kg of polydatin after endotoxin injection (polydatin treatment group), and 8 rats received 1 mg/kg of polydatin (polydatin prevention group) 30 minutes before endotoxin injection. Mean arterial pressure was measured once half an hour. Lung tissues were collected 6 hours later. Phospholipase A(2) activity was measured with acid titration. The gene expression of secretory phospholipase A(2) type IIA was detected with reverse transcription polymerase chain reaction. Meanwhile, the histological changes of the lungs among four groups were compared through microscopic examination.

RESULTSPhospholipase A(2) activity and the gene expression of secretory phospholipase A(2) type IIA increased after endotoxin injection, but polydatin could inhibit these effects of endotoxin. Obvious morphological evidence could be found in the lung pathological sections and the protective effect of polydatin was most significant in the polydatin prevention group.

CONCLUSIONSPolydatin has prophylactic and therapeutic effects (the former is more distinct than the latter) on acutely injured lungs in rats with endotoxic shock and which suggests that polydatin may be a phospholipase A(2) inhibitor.

Analysis of Variance ; Animals ; Glucosides ; pharmacology ; Lung ; metabolism ; Male ; Phospholipases A ; drug effects ; metabolism ; Phospholipases A2 ; Rats ; Rats, Wistar ; Reverse Transcriptase Polymerase Chain Reaction ; Shock, Septic ; metabolism ; Stilbenes ; pharmacology

OBJECTIVETo estimate the effect of aluminum on hippocampal intracellular Ca²+ concentration and expression of phospholipase C (PLC) and NMDA receptor α (NMDARα) genes in hippocampus as well as the neural behaviors in weaning rats through subchronic exposure in order to explore the mechanism which aluminum impaired the ability of learning and memory of central nervous system development.

METHODSWeaning Wistar rats were randomly divided into four groups based on their body weight. Aluminium chloride was administered by water at the doses of 0.2%, 0.4% and 0.6% (m/v) for 90 days. Platform experiment was used to detect the activity of learning and memory. Fura-2/AM calcium ions fluorescence indicator was used to measure Ca²+ concentration in hippocampal neurons. Western blot method was used to detect the expressions of PLC and NMDARα genes.

RESULTSThe incubation of rats in platform experiment [(232.20 ± 57.45), (35.00 ± 9.37), (16.10 ± 5.57) s] shortened while increase of mistake times (1.10 ± 0.74, 2.20 ± 0.92, 3.40 ± 1.51) was significantly associated with the dose of aluminum (P < 0.01). The Ca(2+) concentration decreased significantly in the rats of aluminum exposed groups (P < 0.01). The expression of PLC and NMDARα in aluminum exposed groups (0.30 ± 0.06, 0.18 ± 0.04, 0.16 ± 0.03; 0.38 ± 0.03, 0.32 ± 0.02, 0.25 ± 0.02) decreased significantly compared with that in the control group (0.47 ± 0.07, 0.48 ± 0.04) (P < 0.01) and there was a dose-effect relationship in the NMDARα expression.

CONCLUSIONSubchronic exposure of aluminium could impair the ability of learning and memory in rats during development, inhibit the expression of NMDARα and PLC and reduce Ca²+ concentration, suggesting that the disorder of Ca²+ signaling system might be one of mechanisms of aluminium damaging the ability of learning and memory.

Aluminum ; toxicity ; Animals ; Calcium ; metabolism ; Hippocampus ; metabolism ; Learning ; drug effects ; Male ; Memory ; drug effects ; Rats ; Rats, Wistar ; Receptors, N-Methyl-D-Aspartate ; metabolism ; Type C Phospholipases ; metabolism

BACKGROUND/AIMS: The major compounds of Cochinchina momordica seed extract (SK-MS10) include momordica saponins. We report that the gastroprotective effect of SK-MS10 in an ethanol-induced gastric damage rat model is mediated by suppressing proinflammatory cytokines and downregulating cytosolic phospholipase A2 (cPLA2), 5-lipoxygenase (5-LOX), and the activation of calcitonin gene-related peptide. In this study, we evaluated the gastroprotective effects of SK-MS10 in the nonsteroidal anti-inflammatory drug (NSAID)-induced gastric damage rat model. METHODS: The pretreatment effect of SK-MS10 was evaluated in the NSAID-induced gastric damage rat model using aspirin, indomethacin, and diclofenac in 7-week-old rats. Gastric damage was evaluated based on the gross ulcer index by gastroenterologists, and the damage area (%) was measured using the MetaMorph 7.0 video image analysis system. Myeloperoxidase (MPO) was measured by enzyme-linked immunosorbent assay, and Western blotting was used to analyze the levels of cyclooxygenase (COX)-1, COX-2, cPLA2, and 5-LOX. RESULTS: All NSAIDs induced gastric damage based on the gross ulcer index and damage area (p<0.05). Gastric damage was significantly attenuated by SK-MS10 pretreatment compared with NSAID treatment alone (p<0.05). The SK-MS10 pretreatment group exhibited lower MPO levels than the diclofenac group. The expression of cPLA2 and 5-LOX was decreased by SK-MS10 pretreatment in each of the three NSAID treatment groups. CONCLUSIONS: SK-MS10 exhibited a gastroprotective effect against NSAID-induced acute gastric damage in rats. However, its protective mechanism may be different across the three types of NSAID-induced gastric damage models in rats.
Animals ; Anti-Inflammatory Agents, Non-Steroidal/adverse effects ; Arachidonate 5-Lipoxygenase/drug effects ; Calcitonin Gene-Related Peptide/drug effects ; Cyclooxygenase 1/drug effects ; Cyclooxygenase 2/drug effects ; Disease Models, Animal ; Gastric Mucosa/chemistry/drug effects ; Group IV Phospholipases A2/drug effects ; Male ; Momordica/*chemistry ; Peroxidase/drug effects ; Plant Extracts/*pharmacology ; Rats ; Rats, Sprague-Dawley ; Seeds/*chemistry ; Stomach Ulcer/chemically induced/*prevention & control ; Treatment Outcome

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

OBJECTIVETo investigate the effects of magnesium isoglycyrrhizinate (MI) on the changes of phospholipase A2 (PLA2) induced during liver tissue injury following limb ischemia/reperfusion (I/R) in rats.

METHODTwenty-four healthy male Sprague-Dawley rats weighing (230+/-30) g were randomly divided into three groups (n = 8 each) as follows: control (Group C: anesthetization without any ischemia); I/R injury (Group I/R: 4 h ischemia induced by rubber band ligation of the left hind limb around the roots of the hind limb, followed by 6 h of reperfusion, with 1 mL normal saline given via tail vein prior to reperfusion); MI-treated group (Group MI: underwent ischemia and reperfusion, with 1 mL MI (30 mg/kg) infused prior to reperfusion). Levels of TNFa and PLA2 in plasma and liver tissue were measured by enzyme-linked immunosorbent assay (ELISA). Levels of plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), creatine kinase (CK), myeloperoxidase (MPO), and malondialdehyde (MDA), and activities of MPO and MDA in liver tissue were measured by colorimetry. Ultrastructural changes of liver tissue were observed by electron microscopy.

RESULTSThe MI group had significantly lower PLA2 and TNFa in liver homogenates and serum than the I/R group (both P less than 0.05). Serum ALT, AST, LDH, and CK were significantly lower in the MI group than in the I/R group (all P less than 0.05), as were the levels of MPO and MDA in liver homogenates and serum (all P less than 0.05). The I/R group showed significantly more liver tissue damage, which appeared to be attenuated in the MI group.

CONCLUSIONMI treatment can inhibit the I/R-induced TNFa, PLA2, and MDA in plasma and liver tissue, as well as decrease the I/R-induced MPO activity in rats. Thus, MI may have protective effects against liver tissue injury following limb ischemia/reperfusion.

Animals ; Extremities ; blood supply ; Liver ; drug effects ; injuries ; metabolism ; Male ; Malondialdehyde ; metabolism ; Phospholipases A2 ; metabolism ; Rats ; Rats, Sprague-Dawley ; Reperfusion Injury ; metabolism ; Saponins ; pharmacology ; Triterpenes ; pharmacology

The present study is to assess the prophylactic effect of quinacrine (QA) , an anti-malarial drug, against heatstroke in rats. Conscious rats were orally given equal volume normal saline or QA (dissolved in normal saline and final dosage for rats was 4.5, 9.0 and 18 mg x kg(-1)). An hour later rats were put into a warm water circulated hot chamber (41.0 +/- 0.5) degrees C. Rectal temperature (core temperature, T(co)) of rats in hot chamber was continuously monitored by a thermocouple. T(co) and survival time of rats showed that QA pre-treatment postponed the hyperthermia, and increased the survival time of rats in hot chamber. Primary striatum neurons' culture from new born rats was maintained with D-MEM and 10% FBS. After immuno-cytochemistry identification with antibodies against neural specific proteins, culture received 20 micromol x L(-1) QA only for 1 h and followed by 43.0 degrees C heat treatment for another hour, or 20 micromol x L(-1) QA for 1 h followed by 43.0 degrees C heat treatment for another hour. Control culture received heat treatment only. Cultures were labeled with the fluorescent indicator DPH and the relative membrane fluidity of neurons was measured with the help of fluorescent polarized spectrophotometer. [3H] Arachidonic acid (AA) labeled membrane of E. Coli cells was used as substrate to determine cPLA2 activity of neurons. Gas chromatography and mass spectrum were also employed to detect on the level of fatty acids level in rat striatum neurons. Results from cells indicated that inhibition of cPLA2, reduction the release of active fatty acids such as AA, and possibly, stabilization of the cell membrane which was disturbed by hot treatment, may contribute to the mechanism underlying heat protection and heatstroke preventive effects of quinacrine.
Animals ; Cells, Cultured ; Corpus Striatum ; drug effects ; pathology ; Fatty Acids ; metabolism ; Heat Stroke ; metabolism ; physiopathology ; prevention & control ; Hot Temperature ; adverse effects ; Male ; Membrane Fluidity ; drug effects ; Neurons ; enzymology ; metabolism ; physiology ; Phospholipases A2 ; metabolism ; Quinacrine ; pharmacology ; Rats ; Rats, Wistar

OBJECTIVETo isolate and purify a new phospholipase A2 (PLA2) homologue from Agkistrodon blomhoffii siniticus and investigate its effects on the gene expression profile of Hep3B cells.

METHODSThe PLA2 homologue was isolated and purified by reverse-phase high-performance liquid chromatography (HPLC) and its purity was determined also by HPLC. The relative molecular mass of the homologue was measured by electrospray ionization mass spectrum. The gene expression profile of Hep3B cells was detected with gene chip after exposure of the cells to 139 microg/ml PLA2 homologue for 12 h.

RESULTSThe purity of the PLA2 homologue was 97.2%, whose relative molecular mass was 13,900. After exposure of Hep3B cells to 139 microg/ml PLA2 homologue for 12 h, 19 genes were down-regulated and 20 up-regulated in the cells. The genes showing altered expressions in response to the exposure were mainly involved in cell cycle control and DNA damage repair, cell apoptosis and senescence, production of signal transduction molecules and transcription factors, cell adhesion, angiogenesis, and tumor invasion and metastasis.

CONCLUSIONSThe PLA2 homologue induces alterations in the expression of a wide variety of genes involved in the growth and metastasis of tumor cells. The results of this study provide clues for further study of the possible mechanism for the action of PLA2 homologue on Hep3B cells.

Agkistrodon ; Animals ; Carcinoma, Hepatocellular ; genetics ; Chromatography, High Pressure Liquid ; DNA Damage ; drug effects ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; drug effects ; Hyaluronan Receptors ; biosynthesis ; genetics ; Isoenzymes ; Liver Neoplasms ; genetics ; Phospholipases A ; isolation & purification ; pharmacology ; Phospholipases A2 ; Proto-Oncogene Proteins c-bcr ; biosynthesis ; genetics ; Snake Venoms ; enzymology ; Tumor Cells, Cultured

AIMTo determine the cellular distribution of secretory phospholipase A(2) (sPLA(2)) in dependence on the acrosomal state and under the action of elastase released under inflammatory processes from leukocytes.

METHODSAcrosome reaction of spermatozoa was triggered by calcimycin. Human leukocyte elastase was used to simulate inflammatory conditions. To visualize the distribution of sPLA(2) and to determine the acrosomal state, immunofluorescence techniques and lectin binding combined with confocal laser scanning fluorescence microscopy and flow cytometry were used.

RESULTSAlthough sPLA(2) was detected at the acrosome and tail regions in intact spermatozoa, it disappeared from the head region after triggering the acrosome reaction. This release of sPLA(2) was associated with enhanced binding of annexin V-fluoroscein isothiocyanate (FITC) to spermatozoa surfaces, intercalation of ethidium-homodimer I, and binding of FITC-labelled concanavalin A at the acrosomal region. Spermatozoa from healthy subjects treated with elastase were characterized by release of sPLA(2), disturbance of acrosome structure, and loss of vitality.

CONCLUSIONThe ability of spermatozoa to release secretory phospholipase A(2) is related to the acrosomal state. Premature destabilization of the acrosome and loss of sPLA(2) can occur during silent inflammations in the male genital tract. The distribution pattern of sPLA(2) in intact spermatozoa might be an additional parameter for evaluating sperm quality.

Acrosome ; drug effects ; physiology ; Acrosome Reaction ; drug effects ; Annexin A5 ; metabolism ; Anti-Bacterial Agents ; pharmacology ; Calcimycin ; pharmacology ; Ethidium ; Flow Cytometry ; Fluorescent Dyes ; Humans ; In Vitro Techniques ; Male ; Microscopy, Confocal ; Pancreatic Elastase ; metabolism ; Phosphatidylserines ; metabolism ; Phospholipases A2, Secretory ; metabolism ; Semen ; cytology ; drug effects ; Spermatozoa ; drug effects ; enzymology

OBJECTIVETo investigate the effect of Chaiqin Chengqi Decoction (,CQCQD) on cholecystokinin receptor 1 (CCKR1)-mediated signal transduction of pancreatic acinar cell in rats with acute necrotic pancreatitis (ANP).

METHODSTwenty-seven Sprague-Dawley rats were randomized into three groups: the control group, the ANP group, and the CQCQD group (9 in each group). ANP rats were induced by two intraperitoneal injections of 8% L-arginine (pH=7.0, 4.4 g/kg) over a 2-h period. Rats were treated with 1.5 mL/100 g body weight of CQCQD (CQCQD group) or physiological saline (control and ANP groups) at 2 h interval. And 6 h after induction, pancreatic tissues were collected for histopathological examination. Pancreatic acinar cells were isolated for determination of CCKR1 mRNA and protein expression, phospholipase C (PLC) and inositol-1,4,5-triphosphate (IP3), and determination of fluorescence intensity (FI) as a measure of intracellular calcium ion concentration [Ca(2+)]i.

RESULTSThe pancreatic histopathological score (6.2 ± 1.1) and the levels of PLC (1,187.2 ± 228.2 μg/mL) and IP3 (872.2 ± 88.4 μg/mL) of acinar cells in the ANP group were higher than those in the control (2.8 ± 0.4, 682.5 ± 121.8 μg/mL, 518.4 ± 115.8 μg/mL) and the CQCQD (3.8 ± 0.8, 905.3 ± 78.5 μg/mL, 611.0 ± 42.5 μg/mL) groups (P<0.05). [Ca(2+)]i FI for the ANP group (34.8±27.0) was higher than that in the control (5.1 ± 2.2) and CQCQD (12.6 ± 2.5) groups (P<0.05). The expression of pancreatic acinar cell CCKR1 mRNA in the ANP group was up-regulated (expression ratio=1.761; P=0.024) compared with the control group. The expression of pancreatic acinar cell CCKR1 mRNA in the CQCQD group was down-regulated (expression ratio=0.311; P=0.035) compared with the ANP group. The ratio of gray values of the CCKR1 and β-actin in the ANP group (1.43 ± 0.17) was higher than those in the control (0.70 ± 0.15) and CQCQD (0.79 ± 0.11) groups (P<0.05).

CONCLUSIONSPancreatic acinar cell calcium overload of ANP induced by L-arginine was related to the up-regulated expressions of pancreatic acinar cell CCKR1 mRNA and protein. CQCQD can down-regulate expressions of pancreatic acinar cell CCKR1 mRNA and protein to reduce the PLC and IP3 of pancreatic acinar cells, relieving the calcium overload and reducing the pathological changes in rats with ANP.

Acinar Cells ; drug effects ; metabolism ; Animals ; Blotting, Western ; Calcium ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Fluorescence ; Gene Expression Regulation ; drug effects ; Inositol 1,4,5-Trisphosphate ; metabolism ; Pancreas ; pathology ; Pancreatitis, Acute Necrotizing ; drug therapy ; pathology ; RNA, Messenger ; genetics ; metabolism ; Rats, Sprague-Dawley ; Receptors, Cholecystokinin ; genetics ; metabolism ; Signal Transduction ; drug effects ; Type C Phospholipases ; metabolism