Phospholipase D (PLD), one of the intracellular signal transduction enzymes, may play an important role in developing brain. However, the developmental regulation of PLD protein has not been determined. In the present study, we investigated the temporal and spatial expression of PLD isozyme, PLD1 in the developing rat forebrain using an affinity-purified peptide antibody against PLD1. Our data showed that immunoreactivity for PLD1 was first seen in the germinal zone of the lateral ventricle, differentiating neurons and their processes at embryonic day 18 (E18). At E20, clusters of immunoreactive cells were observed in the medial germinal zone of the lateral ventricle, restricted zones of the frontal and parietal cortex, the nuclei of the medial septum and the diagonal band. During the first postnatal week, there was an increase in the number and staining intensity of the immunoreactive neurons in the cerebral cortex, which peaked at postnatal day 7 (P7). During the second postnatal week, there was an abrupt decrease in the number of immunoreactive cortical pyramidal neurons. By P14, only a few of the pyramidal neurons in cerebral cortex layer V were immunoreactive. These results revealed that expression of PLD1 protein at various stages of development of the septum and cerebral cortex is differentially regulated. This suggests that PLD1 may regulate the developmental processes of some neuronal populations.
Animals ; Brain ; Cerebral Cortex ; Immunohistochemistry ; Lateral Ventricles ; Neurons ; Phospholipase D ; Phospholipases* ; Prosencephalon* ; Rabeprazole ; Rats* ; Signal Transduction

GTPrS-dependent phospholipase D activity in human neutrophils was investigated using exogenous phospholipid as a substrate. Both cytosolic and membrane- associated phospholipase D activities were identified. The previously described 50 kDa cytosolic activating factor was resolved chromatographically from the cytosolic phospholipase D. Using exogenous phospholipid as substrate along with chromatographically resolved 50 kDa factor and recombinant ADP-ribosylation factor 1, plasma membrane was required for activity, indicating that the activity which was previously seen using endogenous phospholipid substrate was due to a phospholipase D located in the plasma membrane. In addition, ADP-ribosylation factor and the 50 kDa factor activated synergistically. Using neutrophil plasma membranes, a third regulator of neutrophil membrane phospholipase D was identified from bovine brain cytosol. This factor was resolved from ADP-ribosylation factor and Rho A by successive column chromatographies. The brain factor showed a synergistic effect with the 50 kDa neutrophil activator but an additive effect with recombinant ADP- ribosylation factor. Whether or not ADP-ribosylation factor or the brain factor were present, high activities were seen only when the 50 kDa factor was present, indicating that the 50 kDa cytosolic factor is a major activating factor for the neutrophil plasma membrane phospholipase D.
ADP-Ribosylation Factor 1 ; ADP-Ribosylation Factors* ; Brain* ; Cell Membrane ; Chromatography ; Cytosol* ; Fibrinogen ; Humans ; Membranes ; Neutrophils* ; Phospholipase D* ; Phospholipases*

PURPOSE: Phospholipase D (PLD) catalyzes the hydrolytic cleavage of terminal phosphate diester bond of glycerophopholipids to produce phosphatidic acid (PA). PLD plays an important role in signal transduction and is known to be involved closely in cancer promotion, inflammation, and other cell responses. In order to evaluate radiation effect in tumor cells, various cells were screened for PLD activities and examined their radiation effects on PLD following gamma- ray irradiation. MATERIALS AND METHODS: PLD activities in 19 species of cell were measured by radioactive isotope method with 1,2 - di [1-14C] phosphatidylcholine in the presence of oleate. Among the cell lines examined, VERO 76, L 1210 and P 388 were selected and examined for their effects of metal ions and agonists on PLD activities before and after irradiation by Co-60 teletheraphy unit. RESULTS: The activities of oleate-PLD were observed in 11 species among 19 cell lines examined. VERO 76 and L 1210 cells showed that the PLD activity increased immediately after irradiation and reached to 150~200% of the control levels. The activation of PLD in response to gamma-ray was maximum at 20 Gy. In irradiated VERO 76, the stimulatory effect of Mg2+ was reduced and the activation of PLD by agonists in irradiated cells vary from those of the control cells. CONCLUSION: The activation effect of irradiation on PLD activity observed strongly implies that the PLD activity is closely related to the phenomena of cell necrosis. Therefore the cell lines examined here could provide a good source for the study of radiobiology that cover from cell death to cancer promotion.
Cell Death ; Cell Line* ; Inflammation ; Ions ; Necrosis ; Oleic Acid ; Phosphatidic Acids ; Phosphatidylcholines ; Phospholipase D ; Radiation Effects ; Radiobiology ; Signal Transduction

OBJECTIVES: Systemic lupus erythematosus (SLE) is a kind of autoimmune inflammatory connective tissue disease which seriously endangers human health. Genetic factors play a key role in the pathogenesis of SLE. This study aims to investigate a novel phospholipase D2 (PLD2) mutation associated with familial SLE, and further explore the underlying mechanism of the mutation in SLE. METHODS: The blood samples from a SLE patient, the patient's parents, and 147 normal controls were collected and DNA was extracted. Whole genome high-throughput sequencing was performed in the patient and her parents and the results were further analyzed by various bioinformatics methods. The wild type (wt), mutant type (mu), and negative control PLD2 plasmids were further constructed and transfected into 293 cells. The expression level of HRAS protein in 293 cells was detected by Western blotting. RESULTS: In this SLE family, the female SLE patient and her mother, 1 in generation II and 1 in generation III had typical clinical manifestations of SLE, and all of them had lupus nephritis at early stage. The genetic characteristics are consistent with autosomal dominant inheritance. A novel PLD2 heterozygous mutation (c.2722C>T) was found in the patient and her mother, but not in her father and other normal controls. Compared with wtPLD2 plasmid and negative control PLD2 plasmid, the expression of HRAS in 293 cells transfected with muPLD2 plasmid was significantly up-regulated (both CONCLUSIONS PLD2 c.2722C>T mutation may be one of the pathogeny of SLE in this family.
Case-Control Studies ; Female ; High-Throughput Nucleotide Sequencing ; Humans ; Lupus Erythematosus, Systemic/genetics* ; Lupus Nephritis ; Mutation ; Phospholipase D

OBJECTIVETo investigate the fluctuations in arterial leukocyte phospholipase D (PLD) activity during the perioperative period of open heart surgery under cardiopulmonary bypass (CPB), and the relationship between PLD activity and systemic inflammatory response induced by CPB.

METHODSArterial blood was obtained from 26 patients undergoing open heart surgery at 8 different time points during the perioperative period, from which leukocytes were isolated for determination of PLD activity, CD11b expression and myeloperoxidase (MPO) activity. Plasma IL-6, IL-8 and C-reactive protein were also determined. The 26 cases were retrospectively divided into 3 groups according to perfusion time in order to detect the possible influences of CPB on PLD activity and IL-6 and IL-8 levels.

RESULTSWhen the ascending aorta was declamped, average arterial leukocyte PLD activity was 0.305 +/- 0.132 nmol choline.min(-1).mg(-1), 5.0 times higher of the pre-CPB value, and remained (5.4 times higher of the pre-CPB level) at 72 hours after CPB. Leukocyte CD11b expression and plasma IL-6 and IL-8 levels increased significantly at the end of CPB, while MPO activity and C-reactive protein concentration reached their peaks at 1 and 24 hours, respectively, after CPB. At the end of CPB, the arterial leukocyte PLD activity of patients whose CPB duration was longer than 90 minutes were 1.82- and 1.74-fold that of the other two groups with CPB lasting between 90 and 60 minutes and less than 60 minutes.

CONCLUSIONSArterial leukocyte PLD activity rises significantly in CPB and its elevation is earlier and more persistent than other inflammation-related indicators tested; longer CPB duration leads to higher leukocyte PLD activity at the end of CPB. These results imply that PLD could be a new target for prevention of systemic inflammatory response induced by CPB.

Adolescent ; Adult ; Cardiopulmonary Bypass ; Female ; Humans ; Interleukin-6 ; blood ; Interleukin-8 ; blood ; Leukocytes ; enzymology ; Male ; Middle Aged ; Phospholipase D ; blood ; Systemic Inflammatory Response Syndrome ; enzymology

Phospholipase D (PLD) hydrolyzes phosphocholine into choline and phosphatide acid, and these metabolites play an important role in regulating cell physiology and biochemistry. To study the biological function of phospholipase D3 (PLD3) during the insulin stimulation in C2C12 myoblasts, we constructed PLD3 over-expressed cell lines (C2C12/pPLD3) and investigated the phosphorylation of Akt. The results showed that the level of phosphorylated Akt (P-Akt) was significantly increased in control C2C12 cells when insulin concentration was elevated during cell treatment, whereas the level of P-Akt in C2C12/pPLD3 cells was not changed. When extending the time of insulin treatment, P-Akt level in C2C12/pPLD3 cells was increased around 2 folds, but the total level of P-Akt in C2C12/pPLD3 was still lower than that in control group. 1-Butanol, a PLD inhibitor, could completely block Akt phosphorylation in C2C12 cells that even stimulated by insulin. However, 1-Butanol did not inhibit the Akt phosphorylation in C2C12/pPLD3 cells, but increased the phosphorylation up to 6 folds higher than control cells. The level of Akt phosphorylation in control C2C12 cells was increased significantly when stimulated by phosphatidic acid (PA), while there was no change in C2C12/pPLD3 cells with the similar treatment. When cells simulated by both PA and insulin, P-Akt level in both C2C12/pPLD3 cells and C2C12 cells were down regulated. Our observations indicated that PLD3 over expression may inhibit Akt phosphorylation and further block the transduction of insulin signaling in C2C12 cells.
Cell Line ; Humans ; Insulin ; pharmacology ; Myoblasts ; cytology ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism ; Phospholipase D ; biosynthesis ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; chemistry ; drug effects ; Signal Transduction

BACKGROUND AND OBJECTIVES: The aim of this study was to determine the effect of ethanol on the regulation of vascular tone. MATERIAL AND METHODS: Using rat aorta ring, isometric contraction and 45Ca uptake were measured. Phorbol 12,13-dibutyrate (PDBu), phenylephrine, KCl were used for the regulation of smooth muscle tone. RESULTS: Ethanol induced transient contraction in rat aorta ring by dose-dependent manner. Ethanol suppressed the dose dependent contractile responses of vascular strip by phenylephrine, KCl and PDBu. Endothelium-dependent relaxation by acetylcholine was inhibited by ethanol. Ethanol depressed 45Ca uptake by high KCl but not by phenylephrine or PDBu in rat aorta. n-butanol selectively suppressed tonic contraction by high KCl, but t-butanol did not at the same concentration of butanol in rat aorta. PDBu-induced contraction was selectively suppressed by n-butanol but not by t-butanol. CONCLUSIONS: These findings suggest that the action of ethanol on phospholipase D is involved in the decreased response of rat aorta strip by vasoconstrictors.
1-Butanol ; Acetylcholine ; Animals ; Aorta* ; Ethanol* ; Isometric Contraction ; Muscle, Smooth* ; Phenylephrine ; Phorbol 12,13-Dibutyrate ; Phospholipase D ; Protein Kinase C ; Rats* ; Relaxation ; tert-Butyl Alcohol ; Vasoconstrictor Agents

To examine the involvement of phospholipase D (PLD)isozymes in postnatal testis development, the expression ofPLD1 and PLD2 was examined in the mouse testis atpostnatal weeks 1, 2, 4, and 8 using Western blot analysisand immunohistochemistry. The expression of both PLD1and PLD2 increased gradually with development frompostnatal week 1 to 8. Immunohistochemically, PLDimmunoreactivity was detected in some germ cells in thetestis and interstitial Leydig cells at postnatal week 1.PLD was mainly detected in the spermatocytes andresidual bodies of spermatids in the testis after 8 weeksafter birth. The intense immunostaining of PLD in Leydigcells remained unchanged by postnatal week 8. Thesefindings suggest that PLD isozymes are involved in thespermatogenesis of the mouse testis.
Animals ; Blotting, Western ; Female ; Immunohistochemistry ; Isoenzymes ; Male ; Mice ; Mice, Inbred BALB C ; Phospholipase D/biosynthesis/*metabolism ; Spermatogenesis/physiology ; Testis/*enzymology/growth & development

OBJECTIVETo investigate whether or not lipopolysaccharide (LPS) and ovalbumin (OA) prime rat peripheral leukocytes, the effect of sensitization on priming and the role of phospholipase D in priming.

METHODSThe peripheral leukocytes were separated and purified from sensitized or unsensitized rats. LPS or OA was used as a priming agent and formylmethionylphenylalanine (fMLP) as an activating agent. Degradation of leukocyte was determined by measurement of elastase release and myeloperoxidase (MPO) activity. Phospholipase D (PLD) activity was assayed by the generation of choline,which was measured by choline-oxidase-catalyzed formation of H(2)O(2) and Trinder reaction.

RESULTCompared with cells treated by fMLP alone,leukocytes from unsensitized rat challenged with fMLP after incubated with LPS released more elastase and MPO (P<0.05). But there was no significant difference between leukocytes challenged with fMLP after incubated with OA and fMLP treated alone. In sensitized rat,there was no difference between leukocytes challenged with fMLP after incubated with LPS and fMLP treated alone. But leukocytes challenged with fMLP after incubated with OA released significantly more elastase and MPO than fMLP treated alone (P<0.05). A significant correlation was obtained between the release of elastase and PLD activity (r(s)=0.51,P<0.01), and also between the release of MPO and PLD activity (r(s)=0.73,P<0.01) in unsensitized rat. In sensitized rat, it was 0.48 (P<0.01) and 0.37 (P<0.05) respectively.

CONCLUSION(1) LPS primes peripheral leukocytes from unsensitized rats; (2) OA primes peripheral leukocytes from actively sensitized rats; (3) PLD plays a role in priming of rat peripheral leukocytes.

Animals ; Leukocyte Elastase ; secretion ; Leukocytes ; drug effects ; enzymology ; Lipopolysaccharides ; pharmacology ; Male ; N-Formylmethionine Leucyl-Phenylalanine ; pharmacology ; Ovalbumin ; immunology ; Peroxidase ; blood ; Phospholipase D ; physiology ; Rats ; Rats, Sprague-Dawley

Animals ; Anti-Asthmatic Agents ; therapeutic use ; Asthma ; drug therapy ; etiology ; Humans ; Phosphodiesterase Inhibitors ; pharmacology ; Phospholipase D ; physiology ; Phosphoric Diester Hydrolases ; physiology