Phospholipase D activity and phosphatidylethanol formation in stimulated HeLa cells expressing the human m1 muscarinic acetylcholine receptor gene

The human m1 and m2 muscarinic acetylcholine receptor (AChR) genes were subcloned, permanently expressed in HeLa cells and analyzed for their pharmacological and biochemical profiles. Both subtypes displayed saturable, high affinity binding of [3H]-quinuclidinyl benzilate (QNB) which was displaced by muscarinic agonists and antagonists. Stimulation of intact HeLa cells expressing the human m1 AChR gene by the muscarinic agonist oxotremorine-M, in the presence of ethanol, resulted in the activation of phospholipase D (PLD) and the formation of phosphatidylethanol (PEt). In contrast, oxotremorine-M did not activate PLD in the HeLa cells expressing the human m2 AChR subtype. These data suggest that the human m1 AChR is linked to the signal transduction mechanism of PLD activation, whereas the human m2 AChR interacts with a different guanine nucleotide regulatory binding protein (G-protein) which does not cause the activation of PLD or the formation of PEt.     

gamma-tocopherol decreases ox-LDL-mediated activation of nuclear factor-kappaB and apoptosis in human coronary artery endothelial cells.

gamma-Tocopherol, produced by many plants, is the major form of tocopherol in the United States diet. It is an effecient protector of lipids against peroxidative damage. Epidemiologic studies show that supplementation of diet with gamma-tocopherol is inversely related to the risk of death from cardiovascular disease. This study was conducted to examine the role of gamma-tocopherol in oxidized LDL (ox-LDL)-induced nuclear factor (NF)-kappaB activation and apoptosis in human coronary artery endothelial cells (HCAECs). Cultured HCAECs were treated with ox-LDL (10-40 microgram/ml). Incubation of HCAECs with ox-LDL resulted in apoptosis of HCAECs, as determined by TUNEL and DNA laddering. Ox-LDL degraded IkappaB protein and activated NF-kappaB in HCAECs (both P < 0.01 vs control), as determined by Western blot. Treatment of cells with gamma-tocopherol attenuated ox-LDL-mediated degradation of IkappaB and activation of NF-kappaB (both P < 0.01 vs ox-LDL alone). The presence of gamma-tocopherol also reduced ox-LDL-induced apoptosis (P < 0.01 vs ox-LDL alone). A high concentration of gamma-tocopherol (50 micromol/L) was more effective than the low concentration of gamma-tocopherol (10 micromol/L) in this process. These observations show that ox-LDL induces apoptosis of HCAECs at least partially by activation of NF-kappaB signal transduction pathway. gamma-Tocopherol significantly decreases ox-LDL-induced apoptosis of HCAECs by inhibiting the activation of NF-kappaB.     

Bacterial lipopolysaccharide activates nuclear factor-kappaB through interleukin-1 signaling mediators in cultured human dermal endothelial cells and mononuclear phagocytes

Bacterial lipopolysaccharide (LPS)-mediated immune responses, including activation of monocytes, macrophages, and endothelial cells, play an important role in the pathogenesis of Gram-negative bacteria-induced sepsis syndrome. Activation of NF-kappaB is thought to be required for cytokine release from LPS-responsive cells, a critical step for endotoxic effects. Here we investigated the role and involvement of interleukin-1 (IL-1) and tumor necrosis factor (TNF-alpha) signal transducer molecules in LPS signaling in human dermal microvessel endothelial cells (HDMEC) and THP-1 monocytic cells. LPS stimulation of HDMEC and THP-1 cells initiated an IL-1 receptor-like NF-kappaB signaling cascade. In transient cotransfection experiments, dominant negative mutants of the IL-1 signaling pathway, including MyD88, IRAK, IRAK2, and TRAF6 inhibited both IL-1- and LPS-induced NF-kappaB-luciferase activity. LPS-induced NF-kappaB activation was not inhibited by a dominant negative mutant of TRAF2 that is involved in TNF signaling. LPS-induced activation of NF-kappaB-responsive reporter gene was not inhibited by IL-1 receptor antagonist. TLR2 and TLR4 were expressed on the cell surface of HDMEC and THP-1 cells. These findings suggest that a signal transduction molecule in the LPS receptor complex may belong to the IL-1 receptor/toll-like receptor (TLR) super family, and the LPS signaling cascade uses an analogous molecular framework for signaling as IL-1 in mononuclear phagocytes and endothelial cells.     

Sphingosine 1-phosphate dynamically regulates myosin light chain phosphatase activity in human endothelial cells

Sphingosine 1-phosphate (S1P) is known to induce reorganization of the actin cytoskeleton through activation of the GTPase Rho. We have investigated the dynamic behavior of Rho/Rho kinase-regulated myosin light chain (MLC) phosphatase activity and MLC phosphorylation in Human Umbilical Vein Endothelial Cells (HUVEC) stimulated with S1P. Immediately (30-60 s) after S1P stimulation, MLC phosphatase activity dropped and MLC phosphorylation increased in a Rho/Rho kinase-dependent manner. Shortly thereafter (2 min), MLC phosphatase increased above baseline and MLC phosphorylation correspondingly decreased to near control values. At this time point, formation of actin ruffles and Rac activity assays indicated activation of Rac. Finally, between 5 and 15 min, MLC phosphatase dropped to a plateau below baseline. In parallel, MLC phosphorylation became constantly elevated above control values. These findings indicate that S1P is able to induce dynamic cycles of MLC phosphatase deactivation and activation. This novel feature of S1P could contribute to its chemotactic and angiogenic activity.     

Somatostatin regulates intracellular signaling in human carotid endothelial cells

Somatostatin (somatotropin release inhibitory factor; SRIF) is an endogenous peptide produced at sites of inflammation, making the SRIF a candidate in regulating vascular inflammation. We have used primary human coronary artery endothelial cells (hCAEC) as a model to study SRIF's vascular actions. RT-PCR analysis of hCAEC total mRNA demonstrated the presence of the sst(4) receptor subtype, providing a target for SRIF intracellular signaling. Western blotting with phospho-specific ERK1/2 antibodies showed that SRIF-14 acutely inhibited basal phosphorylation of the extracellular regulated kinases (ERK1/2) by 80%. In addition, SRIF-14 treated hCAEC cell lysates showed a 2.6-fold increase in phosphatase activity, which was inhibited by sodium vanadate. Furthermore, SRIF-14 appeared to be anti-inflammatory in hCAEC as IL-1beta-induced adhesion molecule expression was reduced by 50%. Together, these results show that the coronary artery endothelium is a direct target of SRIF action.     

Lymphocyte adhesion-dependent calcium signaling in human endothelial cells

Vascular endothelial cells (ECs) can undergo dramatic phenotypic and functional alterations in response to humoral and cellular stimuli. These changes promote endothelial participation in the inflammatory response through active recruitment of immune effector cells, increased vascular permeability, and alteration in vascular tone. In an attempt to define early events in lymphocyte-mediated EC signaling, we investigated cytosolic-free calcium (Ca2+) changes in single, Fluo-3- labeled human umbilical vein ECs (HUVECs), using an ACAS interactive laser cytometer. Of all lymphocyte subsets tested, allogeneic CD3-, CD56+ natural killer (NK) cells uniquely elicited oscillatory EC Ca2+ signals in cytokine (interleukin [IL]-1- or tumor necrosis factor [TNF])-treated ECs. The induction of these signals required avid intercellular adhesion, consisted of both Ca2+ mobilization and extracellular influx, and was associated with EC inositol phosphate (IP) generation. Simultaneous recording of NK and EC Ca2+ signals using two-color fluorescence detection revealed that, upon adhesion, NK cells flux prior to EC. Lymphocyte Ca2+ buffering with 1,2-bis-5-methyl-amino- phenoxylethane-N,N,N'-tetra-acetoxymethyl acetate (MAPTAM) demonstrated that lymphocyte fluxes are, in fact, prerequisites for the adhesion- dependent EC signals. mAb studies indicate that the beta 2 integrin- intercellular adhesion molecule (ICAM)-1 adhesion pathway is critically involved. However, ICAM-1 antisense oligonucleotide inhibition of IL-1- mediated ICAM-1 hyperinduction had no effect on EC Ca2+ signaling in lymphocyte-EC conjugates, indicating that additional cytokine-induced EC alteration is required. These experiments combine features of lymphocyte-endothelial interactions, intercellular adhesion, EC cytokine activation and transmembrane signaling. The results implicate the IP/Ca2+ second messenger pathway in EC outside-in signaling induced by cytotoxic lymphocytes, and suggest that these signals may play a role in EC alteration by lymphocyte adhesion.     

Reactivation of nuclear RNA polymerase activity in growth stimulated epithelial cells

The activation of endogenous RNA polymerases has been studied in mouse kidney epithelial cells which have been induced to grow in culture. Slide cultures were assayed for RNA polymerase, in situ, after brief fixation. Enzyme activity was detected by autoradiography. Polymerases I (nucleolar) and II (nucleoplasmic) were distinguished by localization and by the activities in the presence of different ions as well as sensitivity to α-amanitin.Kidney epithelial cells resuming their growth activity when inoculated in vitro show an early and rapid increase in nuclear size (nuclear protein content) which precedes cell multiplication. Both nucleolar and nucleoplasmic polymerase activities appear at an early phase of growth activation and increase during the whole period of growth activation in proportion to increase in nuclear size. RNA polymerases may also be activated in cells which have not undergone an increase in nuclear size if assayed in the presence of a high salt concentration (0.4 M ammonium sulphate). The role of modification of chromatin structure for genetic reactivation is discussed.     

Phospholipase D in platelets and megakaryocytic cells

Phospholipase D (PLD) is stimulated in platelets by various agents. Phosphatidylcholine is the major substrate for PLD. This enzymatic pathway generates phosphatidic acid selectively. Guanine nucleotides also stimulate PLD in platelet membranes. Furthermore, tyrosine kinase may also be involved in platelet PLD regulation. It appears that multiple signals acting sequentially or in parallel converge on PLD. Among others, PLD has been proposed to play a role in platelet secretion and PLA₂ regulation. PLD is also present in platelet percursor megakaryoctric cells and can be activated by platelet agonists. In these cells both PKC and G-proteins (e.g. Rho) may regulate PLD activity. The significance of PLD in megakaryocytes awaits investigation. These recent developments offer new avenues of research to further elucidate the biochemistry of platelet and megakaryocyte function.     

Phospholipase D2 activity suppresses hydrogen peroxide-induced apoptosis in PC12 cells

Phospholipase D (PLD) plays an important role as an effector in the membrane lipid-mediated signal transduction. However, the precise physiological functions of PLD are not yet well understood. In this study, we examined the role of PLD activity in hydrogen peroxide (H(2)O(2))-induced apoptosis in rat pheochromocytoma (PC12) cells. Treatment of PC12 cells with H(2)O(2) resulted in induction of apoptosis in these cells, which is accompanied by the activation of PLD. This H(2)O(2)-induced apoptosis was enhanced remarkably when phosphatidic acid production by PLD was selectively inhibited by pretreating the PC12 cells with 1-butanol. Expression of PLD2, but not of PLD1, correlated with increased H(2)O(2)-induced PLD activity in a concentration- and time-dependent manner. Concomitant with PLD activation, the PLD2 activity suppressed H(2)O(2)-induced apoptosis in PC12 cells. Expression of PLD2 lipase-inactive mutant (K758R) had no effect on either PLD activity or apoptosis. PLD2 activity also suppressed H(2)O(2)-induced cleavage and activation of caspase-3. Taken together, the results suggest that PLD2 activity is specifically up-regulated by H(2)O(2) in PC12 cells and that it plays a suppressive role in H(2)O(2)-induced apoptosis.     

Phospholipase D1 regulates secretagogue-stimulated insulin release in pancreatic beta-cells

Phospholipase D (PLD) has been strongly implicated in the regulation of Golgi trafficking as well as endocytosis and exocytosis. Our aim was to investigate the role of PLD in regulating the biphasic exocytosis of insulin from pancreatic beta-cells that is essential for mammalian glucose homeostasis. We observed that PLD activity in MIN6 pancreatic beta-cells is closely coupled to secretion. Cellular PLD activity was increased in response to a variety of secretagogues including the nutrient glucose and the cholinergic receptor agonist carbamoylcholine. Conversely, pharmacological or hormonal inhibition of stimulated secretion reduced PLD activity. Most importantly, blockade of PLD-catalyzed phosphatidic acid formation using butan-1-ol inhibited insulin secretion in both MIN6 cells and isolated pancreatic islets. It was further established that PLD activity was required for both the first and the second phase of glucose-stimulated insulin release, suggesting a role in the very distal steps of exocytosis, beyond granule recruitment into a readily releasable pool. Visualization of granules using green fluorescent protein-phogrin confirmed a requirement for PLD prior to granule fusion with the plasma membrane. PLD1 was shown to be the predominant isoform in MIN6 cells, and it was located at least partially on insulin granules. Overexpression of wild-type or a dominant negative catalytically inactive mutant of PLD1 augmented or inhibited secretagogue-stimulated secretion, respectively. The results suggest that phosphatidic acid formation on the granule membrane by PLD1 is essential for the regulated secretion of insulin from pancreatic beta-cells.     

Diethylstilbestrol inhibits phospholipase D activity and degranulation by stimulated human neutrophils

In the present study the effects of diethylstilbestrol on phospholipase D activity and degranulation by human neutrophils were examined. Diethylstilbestrol is a synthetic estrogen and has structural similarity to resveratrol. Resveratrol is a natural polyphenolic antioxidant and has been shown to inhibit the activity of phospholipase D in stimulated neutrophils. Phospholipase D catalyzes the hydrolysis of phosphatidylcholine to yield phosphatidic acid and choline. It also catalyzes the transfer of the phosphatidyl group to ethanol forming phosphatidylethanol at the expense of phosphatidic acid. Phospholipase D activation is associated with degranulation by neutrophils stimulated with chemotactic peptide, formyl-methionyl-leucyl-phenylalanine. The results show that diethylstilbestrol at 100 microM induced a complete inhibition of phosphatidic acid formation in neutrophils, the latter activated by chemotactic peptide. In the presence of ethanol, diethylstilbestrol dose dependently reduced phosphatidylethanol formation induced by chemotactic peptide or by phorbol 12-myristate 13-acetate, indicative of diethylstilbestyrol inhibition of phospholipase D activity. The results also demonstrate that diethylstilbestrol inhibited degranulation by chemotactic peptide-stimulated neutrophils. In comparison to resveratrol, diethylstilbestrol exhibits a stronger inhibition on PA formation, phospholipase D activity and degranulation. These findings suggest that diethylstilbestrol-like resveratrol, may have anti-inflammatory effect in vitro.     

Phospholipase D activity of cytochrome P450 in human liver endoplasmic reticulum.

Phospholipase D (PLD) activity in mammalian liver endoplasmic reticulum (ER) has not been characterized. Purified human liver microsomal cytochromes P450 (P450)-P450 1A2 and P450 2E1-were shown to have appreciable PLD activity, hydrolyzing phosphatidylcholine but not other phospholipids, generating PA and choline. The activity was confirmed using recombinant and mutated human P450s expressed in bacteria. In human liver microsomes, immunoinhibition of PLD activity was observed with anti-P450 1A2 > anti-P450 2C > anti-P450 2E1. Thus, P450 may act as a significant PLD in human liver ER and exert its biological effects by several mechanisms, including signaling functions and change of membrane properties.