Phospholipase D Family Member 4, a Transmembrane Glycoprotein with No Phospholipase D Activity, Expression in Spleen and Early Postnatal Microglia

BACKGROUND: Phospholipase D (PLD) catalyzes conversion of phosphatidylcholine into choline and phosphatidic acid, leading to a variety of intracellular signal transduction events. Two classical PLDs, PLD1 and PLD2, contain phosphatidylinositide-binding PX and PH domains and two conserved His-x-Lys-(x)(4)-Asp (HKD) motifs, which are critical for PLD activity. PLD4 officially belongs to the PLD family, because it possesses two HKD motifs. However, it lacks PX and PH domains and has a putative transmembrane domain instead. Nevertheless, little is known regarding expression, structure, and function of PLD4. METHODOLOGY/PRINCIPAL FINDINGS: PLD4 was analyzed in terms of expression, structure, and function. Expression was analyzed in developing mouse brains and non-neuronal tissues using microarray, in situ hybridization, immunohistochemistry, and immunocytochemistry. Structure was evaluated using bioinformatics analysis of protein domains, biochemical analyses of transmembrane property, and enzymatic deglycosylation. PLD activity was examined by choline release and transphosphatidylation assays. Results demonstrated low to modest, but characteristic, PLD4 mRNA expression in a subset of cells preferentially localized around white matter regions, including the corpus callosum and cerebellar white matter, during the first postnatal week. These PLD4 mRNA-expressing cells were identified as Iba1-positive microglia. In non-neuronal tissues, PLD4 mRNA expression was widespread, but predominantly distributed in the spleen. Intense PLD4 expression was detected around the marginal zone of the splenic red pulp, and splenic PLD4 protein recovered from subcellular membrane fractions was highly N-glycosylated. PLD4 was heterologously expressed in cell lines and localized in the endoplasmic reticulum and Golgi apparatus. Moreover, heterologously expressed PLD4 proteins did not exhibit PLD enzymatic activity. CONCLUSIONS/SIGNIFICANCE: Results showed that PLD4 is a non-PLD, HKD motif-carrying, transmembrane glycoprotein localized in the endoplasmic reticulum and Golgi apparatus. The spatiotemporally restricted expression patterns suggested that PLD4 might play a role in common function(s) among microglia during early postnatal brain development and splenic marginal zone cells

In thrombin stimulated human platelets Citalopram, Promethazine, Risperidone, and Ziprasidone, but not Diazepam, may exert their pharmacological effects also through intercalation in membrane phospholipids in a receptor-independent manner

Intercalation of drugs in the platelet membrane affects phospholipid-requiring enzymatic processes according to the drugs’ intercalation capability. We investigated effects of Promethazine, Citalopram, Ziprasidone, Risperidone, and Diazepam on phospholipase A2 (PLA2) and polyphosphoinositide (PPI) metabolism in thrombin-stimulated human platelets. We also examined effects of the drugs on monolayers of glycerophospholipids using the Langmuir technique. Diazepam did not influence PLA2 activity, had no effects on PPI cycle, and caused no change in mean molecular area of phospholipid monolayers. The remaining psychotropic drugs affected these parameters in different ways and levels of potency suggesting that they act by being intercalated between the molecules of adjacent membrane phospholipids, thus causing changes in substrate availability for phospholipid-hydrolyzing enzymes (PLA2 and Phospholipase C). We show that several psychotropic drugs can also have other cellular effects than receptor antagonism. These effects may be implicated in the psychotropic effects of the drugs and/or their side effects

Ca2+ store dynamics determines the pattern of activation of the store-operated Ca2+ current ICRAC in response to InsP3 in rat basophilic leukaemia cells

ICRAC and intracellular inositol 1,4,5-trisphosphate (InsP3) concentration is complex. In rat basophilic leukaemia (RBL-1) cells dialysed with high intracellular Ca2+ buffer, the relationship is supra-linear with a Hill coefficient of 12 and resembles an apparent ‘all-or-none’ phenomenon. The non-linearity seems to arise from InsP3 metabolism. However, it is not clear which InsP3-metabolising pathway engenders the non-linear behaviour nor whether ICRAC is always activated to its maximal extent by InsP3.Using the whole-cell patch clamp technique, we dialysed RBL-1 cells with different concentrations of the InsP3 analogue InsP3-F. InsP3-F is broken down by Ins(1,4,5)P3 5-phosphatase but is not a substrate for Ins(1,4,5)P3 3-kinase. The relationship between InsP3-F and ICRAC amplitude was supra-linear and very similar to that with InsP3 but was distinct from the graded relationship seen with the non-metabolisable analogue Ins2,4,5P3.In the presence of high intracellular Ca2+ buffer, InsP3-F activated ICRAC to its maximal extent. With moderate Ca2+ buffer, however, sub-maximal ICRAC could be obtained to a maximal InsP3-F concentration. Nevertheless, the relationship between the amplitude of ICRAC and InsP3-F concentration was still supra-linear.Submaximal ICRAC in response to InsP3-F in the presence of moderate Ca2+ buffer was due to partial depletion of the stores, because the size of the current could be increased by thapsigargin.The data suggest that first, Ins(1,4,5)P3 5-phosphatase is an important factor which contributes to the non-linear relationship between InsP3 concentration and the amplitude of ICRAC and second, InsP3 does not always activate ICRAC to its maximal extent. At moderate buffer strengths, submaximal ICRAC is evoked by maximal InsP3. However, the supra-linear relationship between InsP3 concentration and amplitude of the current still holds