Increased Phospholipase D Activity Research Articles

Phospholipase D1 activation through Src and Ras is involved in basic fibroblast growth factor‐induced neurite outgrowth of H19‐7 cells

Phospholipase D (PLD) is implicated in a variety of physiological processes that reveal it to be a member of the signal transducing phospholipases. We found that PLD1 is activated when basic fibroblast growth factor (bFGF) stimulates neurite outgrowth of an immortalized hippocampal cell line (H19-7). Overexpression of PLD1 in H19-7 cells dramatically elongated bFGF-induced neurite outgrowth and increased PLD activity. Transfection of DN-rPLD1 blocked bFGF-induced PLD activation and completely inhibited neurite outgrowth induced by bFGF, suggesting that PLD1 activation is important in bFGF-induced neurite outgrowth of H19-7 cells. PLD activation and neurite outgrowth induced by bFGF was dependent on phospholipase C gamma (PLC-gamma) and Ca2+, but not protein kinase C (PKC). Furthermore, inhibition of Src and Ras partially blocked bFGF-induced PLD activation and neurite outgrowth, respectively. Coinhibition of Src and Ras completely blocked bFGF-induced PLD activation, suggesting that Src and Ras independently regulate PLD1 activation. Interestingly, bFGF-induced PLD activation and neurite outgrowth did not require ERK1/2 activated by Ras. Taken together, this study demonstrates that bFGF activates PLD1 through PLC-gamma activation, which leads to neurite outgrowth in H19-7 cells. Furthermore, our results show that PLD1 activation by bFGF is regulated by Src and Ras independently.

Phospholipase D Couples Survival and Migration Signals in Stress Response of Human Cancer Cells

MDA-MB-231 human breast cancer cells belong to a highly invasive metastatic cell line that depends on phospholipase D (PLD) activity for survival when deprived of serum growth factors. In response to the stress of serum withdrawal, there is a rapid and dramatic increase in PLD activity. Concomitant with increased PLD activity, there was an increase in the ability of MDA-MB-231 cells to both migrate and invade Matrigel. The ability of MDA-MB-231 cells to both migrate and invade Matrigel was dependent on both PLD and mTOR, a downstream target of PLD signals. Serum withdrawal also led to a PLD-dependent increase in the expression of the stress factor, hypoxia-inducible factor-1alpha. These data reveal that PLD survival signals not only prevent apoptosis but also stimulate cell migration and invasion, linking the ability to suppress apoptosis with the ability to metastasize.

Copper excess triggers phospholipase D activity in wheat roots

Wheat seedlings ( Triticum durum Desf.) were incubated in 100 μM Cu 2+ for different periods of time ranging from 1 min up to 16 h. Following metal addition a rapid intake of copper ions into the roots was observed. Cu 2+ induced an accumulation of both phosphatidic acid and phosphatidylbutanol within 1 min of incubation, the latter indicating a very rapid induction of phospholipase D (PLD) activity. The highest PLD stimulation was detected after 2 h from copper addition and decreased almost to the initial value at increasing times. Cycloheximide treatment of roots lowered phosphatidylbutanol accumulation because of a reduced PLD activity. The expression profile of a T. durum putative PLD-encoding gene showed a peak after 1 h of treatment as well, indicating that enhanced gene expression contributed to the increase in PLD activity. In the absence of copper ions, roots treated with the G protein activator mastoparan showed increases in phosphatidic acid and phosphatidylbutanol similar to those detected with the metal. PLD activity was also stimulated by cholera toxin. Two putatively G protein α subunit encoding sequences were isolated and no significant differences in transcription activity following Cu 2+ addition were observed. In copper-treated roots an early production of superoxide generated both by total and membrane-bound NADPH oxidase occurred. The G protein inhibitor suramin as well as the PLD antagonist 1-butanol abolished copper-induced superoxide production.

Mechanical Activation of mTOR Signaling Requires a Phospholipase D‐Mediated Increase in Phosphatidic Acid

Signaling by the mammalian target of rapamycin (mTOR) has been reported to be necessary for mechanical load-induced growth of skeletal muscle. The mechanisms involved in the mechanical activation of mTOR signaling are not known, but several studies indicate that an atypical (nutrient and PI3K-independent) mechanism is involved. Recently, a novel regulatory pathway for mTOR signaling involving phospholipase D (PLD) and the lipid second messenger phosphatidic acid (PA) was reported. In testing the role of the PLD /PA pathway in the mechanical activation of mTOR signaling, we demonstrated that both isoforms of PLD (PLD1 and PLD2) are localized to the z-band of skeletal muscle (the primary site of mechanical force transmission). Mechanically loading skeletal muscle with passive stretch, ex vivo, promoted an increase in PLD activity, PA accumulation and mTOR signaling. Incubation with a PLD / phospholipase C (PLC) inhibitor, neomycin, blocked the mechanical activation of mTOR signaling. Inhibitors of PLC, intracellular calcium and protein kinase C (U73122, BAPTA-AM and BIM, respectively) did not affect the mechanical activation of mTOR signaling. In contrast, inhibition of PLD with 1-Butanol blocked the mechanical activation of mTOR signaling. These results suggest that the effect of neomycin was attributable to the inhibition PLD, but not PLC. Furthermore, both neomycin and 1-Butanol blocked the mechanically induced increase in PA. Taken together, our results indicate that mechanical stimuli activate mTOR signaling through a PLD-mediated increase in PA. Funded By: NIH R01HL64382 to S.C. and F32AR052240 to T.A.H.

Disruption of Lipid Rafts Stimulates Phospholipase D Activity in Human Lymphocytes: Implication in the Regulation of Immune Function

Recent evidence suggests that phospholipase D (PLD) can be regulated through its association/dissociation to lipid rafts. We show here that modifying lipid rafts either by cholesterol depletion using methyl-beta-cyclodextrin and filipin or by conversion of sphingomyelin to ceramide with exogenous bacterial sphingomyelinase (bSMase) markedly activated the PLD of human PBMC. bSMase was the most potent PLD activator, giving maximal 6- to 7-fold increase in PLD activity. Triton X-100-treated lysates prepared from control PBMC and from bSMase-treated cells were fractionated by centrifugation on sucrose density gradient. We observed that bSMase treatment of the cells induced a larger ceramide increase in raft than in nonraft membranes and displaced both the Src kinase Lck and PLD1 out of the raft fractions. In addition, the three raft-modifying agents markedly inhibited the lymphoproliferative response to mitogenic lectin. To examine further the potential role of PLD activation in the control of lymphocyte responses, we transiently overexpressed either of the PLD1 and PLD2 isoforms in Jurkat cells and analyzed the phorbol ester plus ionomycin-induced expression of IL-2 mRNA, which is one of the early responses of lymphocyte to activation. We observed a 43% decrease of IL-2 mRNA level in Jurkat cells overexpressing PLD1 as compared with mock- or PLD2-transfected cells, which indicates that elevated PLD1, but not PLD2, activity impairs lymphocyte activation. Altogether, the present results support the hypothesis that PLD1 is activated by exclusion from lipid rafts and that this activation conveys antiproliferative signals in lymphoid cells.

Arf1-dependent PLD1 is localized to oleic acid-induced lipid droplets in NIH3T3 cells

Phospholipase D (PLD) is known to play a role in vesicle transport through the hydrolysis of phosphatidylcholine (PC) to produce the bioactive lipid, phosphatidic acid. Lipid droplets (LDs) are surrounded by a monolayer of phospholipids, including PC and its lyso derivative, and exhibit a number of signaling proteins. Our recent report suggests that the association of adipose differentiation-related protein (ADRP) to LDs is regulated by an ADP-ribosylation factor 1 (Arf1)-dependent mechanism. In the present study, we found an increase in PLD activity accompanied with LD formation in oleic acid-treated NIH3T3 cells. Brefeldin A, an inhibitor of ARF-GEFs, suppressed both PLD activation and LD formation in oleic acid-treated cells. PLD1, but not PLD2, was found to exist in LDs by immunocytochemical analysis. Furthermore, co-existence of PLD1, Arf1, and ADRP was observed in the LD-enriched subcellular fractions obtained from oleic acid-treated NIH3T3 cells by Western blot analysis. PLD1 activity in the LD-enriched fractions was stimulated by exogenously added Arf1. Although LDs were induced in either PLD1- or PLD2-overexpressing CHO cells by oleic acid treatment, the stimulation of PLD activity was observed only in PLD1-CHO cells. Taken together, the data suggest that the activation of Arf1-dependent PLD1 occurs in LDs and may be involved in their physiological function.

Evaluation of cytochrome P450 4 family as mediator of phospholipase D activation in aortic vascular smooth muscle cells

Norepinephrine (NE) stimulates phospholipase D (PLD) activity via phospholipase A 2-dependent arachidonic acid release in rabbit aortic vascular smooth muscle cells (VSMC). We have previously shown that exogenous 20-hydroxyeicosatetraenoic acid (20-HETE), an eicosanoid generated through the cytochrome P450 (CYP) 4A pathway in vivo, stimulates PLD activity. Whether endogenous CYP4-derived arachidonic acid metabolites act as intracellular mediators of NE-induced PLD activation in VSMC is not known. In rabbit aortic VSMC, prototypical hepatic/renal CYP4A inducers such as fenofibrate and Wy 14643 inhibited both basal and NE-induced PLD activity after 48 h of exposure. The level of CYP4F, and to a lesser extent CYP4A, was also decreased by these agents. The expression levels of rabbit aortic VSMC CYP4A and CYP4F isoforms were reduced by antisense oligonucleotides treatment for 48 hours as measured by RTQ-PCR or Western blotting. This reduction in CYP4A or CYP4F levels did not change NE-induced PLD activation. The corresponding CYP4A scrambled and CYP4F sense oligonucleotides did not alter CYP levels. PLD activity was increased by ~ 70% after 15 min of stimulation with NE, whereas lauric acid ω-hydroxylase activity, a measure of fatty acid ω-hydroxylation, was unchanged. Inhibition of ω-hydroxylation with DDMS and HET0016, selective ω-hydroxylase inhibitors, and 20-HEDE, an antagonist of 20-HETE, increased PLD activity in a concentration-dependent manner and did not alter NE-induced PLD activation. These data suggest that PLD activation by NE is independent of the CYP4A/4F enzymes in rabbit aortic VSMC.

Somatostatin Increases Phospholipase D Activity and Phosphatidylinositol 4,5-bisphosphate Synthesis in Clonal β Cells HIT-T15

In the presence of arginine vasopressin (AVP), somatostatin increases [Ca(2+)](i), leading to a transient increase in insulin release from clonal beta cells HIT-T15 via G(i/o) and phospholipase C (PLC) pathway (Cheng et al., 2002a). The present study was to elucidate the mechanisms underlying somatostatin-induced [Ca(2+)](i) increase in the presence of AVP. We found that the effect of somatostatin was mediated by betagamma subunits but not by the alpha subunit of G(i/o). Because somatostatin alone failed to increase [Ca(2+)](i), we hypothesized that somatostatin increases phosphatidylinositol 4,5-bisphosphate (PIP(2)) synthesis, providing extra substrate for preactivated PLC-beta to generate inositol 1,4,5-trisphosphate (IP(3)). Somatostatin alone did not increase IP(3) levels, but AVP + somatostatin did. Somatostatin increased PIP(2) levels but decreased phosphatidylinositol 4-phosphate levels. We further hypothesized that PLD mediates somatostatin-induced changes in PIP(2) levels. Both the phospholipase D (PLD) inhibitors and antibody versus PLD1 antagonized AVP-somatostatin-induced increases in [Ca(2+)](i). PLD inhibitor also antagonized somatostatin-induced increase in PIP(2) levels. In addition, somatostatin increased PLD activity. These results suggest that activation of somatostatin receptors that are coupled to the betagamma dimer of G(i/o) led to PLD1 activation, thus promoting the synthesis of phosphatidic acid. Phosphatidic acid activates PIP-5 kinase, which evokes an increase in PIP(2) synthesis. The PIP(2) generated by somatostatin administration increases substrate for preactivated phospholipase C-beta, which hydrolyzes PIP(2) to form IP(3), leading to an increase in [Ca(2+)](i). The regulation of PIP(2) synthesis by G(i/o)-coupled receptors via PLD activation represents a novel signaling mechanism for somatostatin and a novel concept in the cross-talk between G(q)- and G(i/o)-coupled receptors in beta cells.

Role of phospholipase D1 in neurite outgrowth of neural stem cells

Employing neural stem cells from the brain cortex of E12 rat embryos, we investigated the possible role of phospholipase D (PLD) in the synaptogenesis and neurite formation of neural cells during differentiation. Expression level of PLD1 increased during neuronal differentiation of the neural stem cells, resulting in increased PLD activity. Expression level of synapsin I, a marker of synaptogenesis, also increased as the differentiation of neural stem cells progressed. To figure out the effect of PLD on synapsin I expression, we treated the neural stem cells with phorbol myristate acetate (PMA) to stimulate PLD activity. Increased PLD activity induced by PMA treatment resulted in elevated synapsin I expression and neurite outgrowth during neuronal differentiation. To further confirm the role of PLD in neurite outgrowth, we transfected the dominant-negative form of rat PLD1 cDNA (DN-rPLD1) into neural stem cells to downregulate PLD activity. Overexpression of DN-rPLD1 showed the complete inhibition of neurite outgrowth of neural stem cells under differentiation condition. While transfection of DN-rPLD1 did not affect the synapsin I expression, overexpression of rPLD1 resulted in increased synapsin I expression of the neural cells. These results suggest that PLD1 plays a critical role in neurite outgrowth during differentiation of the neural stem cells. In conclusion, this is the first evidence to show that PLD1 acts as an important regulator of neurite outgrowth in neural stem cell by promoting neuronal differentiation via increase of synapsin I expression.

Phospholipase D Is a Negative Regulator of Proline Biosynthesis in Arabidopsis thaliana

Accumulation of proline has been observed in a large number of plant species in response to drought and salt stresses, suggesting a key role of this amino acid in plant stress adaptation. Upstream components of the proline biosynthesis signal transduction pathways are still poorly defined. We provide experimental evidence that phospholipase D (PLD) is involved in the regulation of proline metabolism in Arabidopsis thaliana. The application of primary butyl alcohols, which divert part of PLD-derived phosphatidic acid by transphosphatidylation, stimulated proline biosynthesis even without hyperosmotic constraints. Moreover, application of primary butyl alcohols enhanced the proline responsiveness of seedlings to mild hyperosmotic stress. These data indicate that some PLDs are negative regulators of proline biosynthesis and that plants present a higher proline responsiveness to hyperosmotic stress when this regulator is abolished. We clearly demonstrate that PLD signaling for proline biosynthesis is similar to RD29A gene expression and different from the abscisic acid-dependent RAB18 gene expression. Our data reveal that PLDs play positive and negative roles in hyperosmotic stress signal transduction in plants, contributing to a precise regulation of ion homeostasis and plant salt tolerance.

Phospholipase D2 functions as a downstream signaling molecule of MAP kinase pathway in L1-stimulated neurite outgrowth of cerebellar granule neurons.

Stimulation of the neuronal cell adhesion molecule L1 in cerebellar granule neurons (CGNs) enhances neurite outgrowth and this response is inhibited by the primary alcohol ethanol. Because primary alcohols suppress the formation of the signaling lipid phosphatidic acid (PA) by phospholipase D (PLD), this observation prompted us to investigate whether PLD plays a role in the L1-mediated neurite outgrowth in CGNs. In the cerebellum of postnatal day 8 mice, PLD2 protein was abundantly expressed, while PLD1 expression was not detected. The L1-stimulated neurite outgrowth was inhibited by primary alcohols and by overexpression of lipase-deficient PLD2. Increases in cellular PA levels by direct PA application or overexpression of wild-type PLD2 mimicked the L1-dependent stimulation of neurite outgrowth. Furthermore, it was found that L1 stimulation in CGNs increased PLD activity concomitantly with phosphorylation of extracellular signal-regulated kinase (ERK), both of which were inhibited by the MAP kinase-ERK kinase (MEK) inhibitor. These results provide evidence that PLD2 functions as a downstream signaling molecule of ERK to mediate the L1-dependent neurite outgrowth of CGNs, a mechanism that may be related to alcohol-related neurodevelopmental disorders.

Overexpression of phospholipase D prevents actinomycin D-induced apoptosis through potentiation of phosphoinositide 3-kinase signalling pathways in Chinese-hamster ovary cells.

To examine the roles of PLD (phospholipase D) in the regulation of the apoptotic process, PLD1 and PLD2 were stably overexpressed in S1P3-CHO cells [CHO (Chinese-hamster ovary) cells expressing the S1P (sphingosine 1-phosphate) receptor S1P3]. Treatment of S1P3-CHO cells with ActD (actinomycin D) induced apoptosis, as shown by the occurrence of nuclear fragmentation and the caspase-dependent proteolytic cleavage of PARP [poly(ADP-ribose) polymerase] and protein kinase Cd. Overexpression of either PLD1 or PLD2 protected S1P3-CHO cells from ActD-induced apoptosis, as demonstrated by an increased number of viable cells and inhibition of PARP and protein kinase Cd cleavage. However, in the early phase of apoptosis, ActD induced an increase in PLD activity and activation of key factors in the cell-survival signalling pathways, such as PI3K (phosphoinositide 3-kinase), Akt, p70S6K (p70 S6 kinase) and ERK (extracellular-signal-regulated kinase). Furthermore, the ActD-induced activation of these survival signalling enzymes was potentiated by overexpression of either PLD1 or PLD2. The PI3K inhibitor LY294002 inhibited the ActD-induced activation of Akt and p70S6K, and completely abolished the effects of PLD1 or PLD2, whereas inhibition of ERK activity by the MEK inhibitor U0126 had a milder effect. The ActD-induced activation of p70S6K and ERKs was blocked by 1-butanol, but not by t-butanol; similar to S1P, exogenous PLD suppressed the ActD-induced events in the apoptosis signalling pathways. These results show that, in S1P3-CHO cells, increased expression of PLDs prevents ActD-induced apoptosis by enhanced activation of the PI3K signalling pathways.

High glucose-induced membrane translocation of PKC betaI is associated with Arf6 in glomerular mesangial cells.

Protein kinase C (PKC)-induced changes in glomerular mesangial cell (MC) phenotypic behavior has been implicated in diabetes. The activity of diacylglycerol-sensitive PKC isoforms in MCs is altered by ambient changes in glucose, but the regulation of PKC activity and subsequent intracellular signaling events are not yet clearly defined. Small GTP-binding proteins of the ADP-ribosylation factor (Arfs) family, may regulate protein kinase membrane recruitment and hence its activity in signaling events of non-polarized cells. Members of the ARF family may coordinate membrane dynamics and other cellular functions through their interaction with PKC. We studied the activation of Arf, PKC betaI and phospholipase D (PLD) in MCs cultured under normal or high glucose conditions. MCs cultured in high glucose medium exhibited predominantly cytosolic localization of PKC betaI, Arf3 and Arf6. However, phorbol ester (PMA) stimulation of cells cultured in high glucose significantly enhanced membrane association of PKC betaI and Arf6, but not Arf3. Using [3H]choline chloride to prelabel MCs and measuring [3H]choline-containing metabolite release as PLD activity, PMA stimulated a significant increase of PLD activity under high glucose condition. Our data suggest that Arf6 plays a specific role in activation of PKC betaI and PLD under high glucose condition, and may be a significant intracellular event in the change of the mesangial cell phenotype associated with diabetic nephropathy.

Retinoic acid-induced differentiation into astrocytes and glutamatergic neurons is associated with expression of functional and activable phospholipase D

Phospholipase D (PLD) activity in mammalian cells has been associated with cell proliferation and differentiation. Here, we investigated the expression of PLD during differentiation of pluripotent embryonal carcinoma cells (P19) into astrocytes and neurons. Retinoic acid (RA)-induced differentiation increased PLD1 and PLD2 mRNA levels and PLD activity that was responsive to phorbol myristate acetate. Various agonists of membrane receptors activated PLD in RA-differentiated cells. Glutamate was a potent activator of PLD in neurons but not in astrocytes, whereas noradrenaline and carbachol increased PLD activity only in astrocytes. P19 neurons but not astrocytes released glutamate in response to a depolarizing stimulus, confirming the glutamatergic phenotype of these neurons. These results indicate upregulation of PLD gene expression associated with RA-induced neural differentiation.

Differential role for phospholipase D1 and phospholipase D2 in 12- O-tetradecanoyl-13-phorbol acetate-stimulated MAPK activation, Cox-2 and IL-8 expression

Phospholipase D (PLD) is expressed in many tissues and stimulated by growth factors and cytokines. However, the role of PLD in signal transduction is still not well-understood. Human embryonic kidney (HEK-293) cells exhibit low levels of both PLD1 and PLD2 mRNA, however, only PLD1 protein was detected by Western blot. When either isoform of PLD was stably expressed in HEK-293 cells, we observed an increased PLD activity in a cell-free system and a 12- O-tetradecanoyl-13-phorbol acetate (TPA)-stimulated increase in PLD activity in intact cells. This system was then used to elucidate the effects of PLD activity on TPA-stimulated signaling pathways. Two such pathways, the mitogen-activated protein kinases (MAPK), extracellular regulated protein kinase (ERK) and p38 are activated by growth factors and cellular stress, respectively. We found that TPA stimulated ERK phosphorylation regardless of the expression status of PLD. In contrast to ERK kinase, HEK-293 cells were unable to induce p38 phosphorylation by TPA stimulation. When HEK-293 cells expressed either PLD1 or PLD2, we observed elevated p38 phosphorylation in response to TPA stimulation. The ERK and p38 MAPKs can also stimulate the expression of both cyclooxygenase-2 (Cox-2) and interleukin-8 (IL-8). We used this system to differentiate the effect of PLD1 or PLD2 activity on the expression of Cox-2 and IL-8. Increased Cox-2 and IL-8 expression was found only in HEK-293 cells expressing PLD1. These data identify a novel role for the PLD1 isoform in the induction of gene expression and provide new insight into the differential role of PLD1 and PLD2 in cells.

Effects of Norepinephrine and Cardiotrophin-1 on Phospholipase D Activity and Incorporation of Myristic Acid Into Phosphatidylcholine in Rat Heart

The present study is part of a project on phospholipase D (PLD) in cardiac hypertrophy and analyzed effects on PLD activity of two growth stimuli, norepinephrine (NE) and cardiotrophin-1 (CT-1), in incubated rat heart. Phosphatidylcholine (PC) was labeled by 3H-myristic acid. PLD produced 3H-phosphatidylethanol (3H-PEth) from 3H-PC in the presence of ethanol and maintained a basal formation of 3H-PEth. Short-term and long-term exposure to NE for 2 or 13 h, respectively, enhanced the formation of 3H-PEth, which was blocked by prazosin. Long-term pretreatment with NE or CT-1 increased the incorporation of 3H-myristic acid into PC, which was blocked by atenolol. When the 3H-PEth formation was expressed as a fraction of 3H-PC, PLD activity seemingly was unchanged (NE) or markedly reduced (CT-1); the true effects, namely, stimulation by NE and nonresponsiveness towards CT-1, were unraveled by atenolol (NE) or when PLD activity was expressed as 3H-PEth per ng protein. In conclusion, α-adrenoceptor activation increased PLD activity. Long-term treatment with NE (via β-receptors) or CT-1 enhanced the 3H-myristic acid incorporation into a PC compartment, that was not available for the α-receptor-mediated PLD activation. These results were discussed in regard to cellular mechanisms of cardiac hypertrophy and to the transphosphatidylation assay of PLD.

Role of direct RhoA-phospholipase D1 interaction in mediating adenosine-induced protection from cardiac ischemia.

Activation of adenosine A1 or A3 receptors protects heart cells from ischemia-induced injury. The A3 receptor signals via RhoA and phospholipase D (PLD) to induce cardioprotection. The objective of the study was to investigate how RhoA activates PLD to achieve the anti-ischemic effect of adenosine A3 receptors. In an established cardiac myocyte model of preconditioning using the cultured chick embryo heart cells, overexpression of the RhoA-noninteracting PLD1 mutant I870R selectively blocked the A3 agonist (Cl-IBMECA, 10 nM)-induced cardioprotection. I870R caused a significantly higher percentage of cardiac cells killed in A3 agonist-treated than in A1 agonist (CCPA, 10 nM)-treated myocytes (ANOVA and posttest comparison, P<0.01). Consistent with its inhibitory effect on the PLD activity, I870R attenuated the Cl-IBMECA-mediated PLD activation. Cl-IBMECA caused a 41 +/- 15% increase in PLD activity in mock-transfected myocytes (P<0.01, paired t test) while having only a slight stimulatory effect on the PLD activity in I870R-transfected cells. To further test the anti-ischemic role of a direct RhoA-PLD1 interaction, atrial cardiac myocytes were rendered null for native adenosine receptors by treatment with irreversible A1 antagonist m-DITC-XAC and were selectively transfected with the human adenosine A1 or A3 receptor cDNA individually or they were cotransfected with cDNAs encoding either receptor plus I870R. I870R preferentially inhibited the human A3 receptor-mediated protection from ischemia. The RhoA-noninteracting PLD1 mutant caused a significantly higher percentage of cardiac cells killed in myocytes cotransfected with the human A3 receptor than in those cells expressing the human A1 receptor (ANOVA and posttest comparison, P<0.01). The present data provided the first demonstration of a novel physiological role for the direct RhoA-PLD1 interaction, that of potent protection from cardiac ischemia. The study further supported the concept that a divergent signaling mechanism mediates the anti-ischemic effect of adenosine A1 and A3 receptors.

Human cardiac phospholipase D activity is tightly controlled by phosphatidylinositol 4,5-bisphosphate

Phospholipase D (PLD) plays a central role in receptor-mediated breakdown of choline phospholipids and formation of phosphatidic acid (PA), an important regulator of cardiac function. However, specific mechanisms that regulate myocardial PLD activity remain largely unknown, particularly in the human heart. We hypothesized that phosphatidylinositol 4,5-bisphosphate (PIP2), best known as substrate for phospholipase C (PLC) isozymes, plays a critical role in regulating myocardial PLD activity. We examined the effect of PIP2 on human myocardial PLD activity in vitro by utilizing a fluorescence HPLC assay. PIP2 increased 10-fold the maximal activity of a partially solubilized PLD from human atrial myocardium. PIP2-stimulated PLD activity was accompanied by a consecutive increase in diacylglycerol, indicating dephosphorylation of PA by PA phosphohydrolase. Likewise, phosphatidylinositol 3,4,5-trisphosphate, which is produced from PIP2 by phosphatidylinositol 3-kinase, increased PLD activity with about the same potency but with somewhat lower efficacy. In contrast, other phospholipids were ineffective, indicating that the action of PIP2 on PLD is highly specific. Neomycin, a high-affinity ligand of PIP2, inhibited PLD activity in human atrial myocardium, but had no effect on the activity of partially solubilized enzyme. The addition of PIP2 restored the sensitivity of solubilized PLD to neomycin inhibition, indicating that neomycin inhibits PLD activity by binding to endogenous PIP2. Our results demonstrate a critical role for PIP2 in human cardiac PLD activity and suggest that PIP2 synthesis (by phosphatidylinositol 4-phosphate 5-kinase) and hydrolysis (by PIP2-specific PLC) could be important determinants in regulating PLD signal transduction in the human heart.

호중구에서 phospholipase D의 활성에 대한 protein kinase G의 영향

Phosphipase D(PLD)는 호중구의 활성에서 중요한 신호전달 인자로 작용한다. 본 연구에서는 호중구에서 PLD의 활성화에 대한 nitric oxide(NO)와 cGMP의 영향을 조사하였다. 세포 내 NO의 생성을 증가시키는 물질인 sodium nitroprusside (SNP)를 단독으로 처리하였을 때 SNP를 처리하지 않은 세포에 비교하여 PLD 활성은 0.5 mM 농도에서 2배 이상 증가하였다. 세포 내 cAMP의 농도를 증가시키는 물질인 dibutyryl-cAMP를 처리하였을 때 formyl-Met-Leu-Phe(fMLP)에 의한 PLD활성은 억제되었으나 cGMP를 증가시키는 물질인 8-bromo-cGMP(300 <TEX>$\mu$</TEX>M)를 단독으로나 fMLP와 같이 처리하였을 때 PLD의 활성은 큰 영향이 없었다. NO에 의한 PLD의 활성은 cGMP-의존형 인산화 효소인 protein kinase G(PKG)의 억제제인 KT 5823에 의하여 억제되지 않았는데 이러한 결과는 PKG 이외의 경로를 통하여 일어남을 제시한다. NO를 처리한 호중구에서 p38 mitogen activated protein kinase(MAPK)가 활성화되어 인산화된 p38 MAPK가 Western blot에서 증가되었다. NO에 의한 p38 MAPK의 인산화는 KT 5823에 의하여 억제되지 않았고 PLD 억제제인 n-butanol에 의하여도 영향을 받지 않았다. PLD 활성의 인자인 RhoA는 fMLP나 phorbol myristate acetate(PMA)의 자극에 의하여 세포질로부터 세포막으로 전이가 되었으나 cGMP의 전처리에 의하여 fMLP에 의한 RhoA의 전이는 억제되었으나 PMA에 의한 전이는 영향을 받지 않았다. 이들 결과들은 호중구 내 증가된 cGMP가 RhoA를 억제하였으나 세포 내 증가된 NO는 cGMP 이외의 인자를 통하여 PLD의 활성화를 일으킨다는 것을 제시하고 있다. Phospholipase D (PLD) plays an important role as a signaling molecule in the activation of neutrophils. In this study, effect of nitric oxide (NO) and cGMP on the activation of PLD in human neutrophils was investigated. Sodium nitroprusside (SNP), an agent to produce NO spontaneously in cells, alone increased PLD activity and the maximal activation was obtained with 0.5 mM SNP. Dibutyryl-cAMP, an agent to increase an intracellular cAMP concentration inhibited formyl-Met-Leu-Phe (fMLP)-stimulated PLD activity but 8-bromo-cGMP (300 <TEX>$\mu$</TEX>M), an agent to increase an intracellular cGMP concentration did not affect basal and fMLP-stimulated PLD activity. NO-induced activation of PLD was not blocked by KT 5823, an inhibitor of cGMP-dependent protein kinase (PKG), suggesting that NO-induced PLD activation is not mediated by cGMP. NO also stimulated p38 mitogen activated protein kinase (MAPK) in human neutrophils, indicated by increased phosphorylation of p38 MAPK in Western blotting. NO-induced phosphorylation of p38 MAPK was not inhibited by KT 5823 or n-butanol. RhoA, an regulatory factor of PLD activation was trans-located from cytosolic fraction to plasma membranes by fMLP or phorbol ester, and fMLP-stimulated but not phorbol ester-stimulated translocation of RhoA was inhibited by cGMP. These results suggest that NO stimulates PLD activity through other unidentified facto.(s) than cGMP even though cGMP inhibits the artivation of RhoA.

Prostacyclin production in rat aortic smooth muscle cells: role of protein kinase C, phospholipase D and cyclooxygenase-2 expression

The present study was designed to investigate the role of protein kinase C (PKC) and phospholipase D (PLD) in angiotensin II (AngII)- and phorbol ester (PMA)-induced cyclooxygenase-2 (COX-2) expression and prostacyclin (PGI(2)) production in rat aortic smooth muscle cells (VSMC). Prostacyclin production in cultured VSMC was determined by radioimmunoassay. PKC activity was examined by measuring the transfer of 32P from (gamma-32P)ATP to histone III-S. COX-2 expression was determined by Western blotting. To measure PLD activity, thin layer chromatography was used. AngII (50 nM) and PMA (100 nM) promoted the translocation of PKC activity from the cytosol to the membranes within 30 min, followed by a strong increase in PLD activity as well as COX-2 expression and PGI(2) production. After 48 h exposure to PMA, PKC was downregulated resulting in a complete suppression of its activity. PKC-downregulation and the PKC inhibitor CGP41251 abolished PMA- and AngII-induced PLD activation, suppressed the stimulatory effect of PMA on COX-2 expression and PGI(2) production and strongly inhibited that of AngII. Furthermore, AngII- and PMA-induced PGI(2) production depended on protein synthesis and COX-2 but not COX-1 activity. Inhibition of PLD-mediated phosphatidic acid (PA) formation by 1% 1-butanol abolished AngII-induced COX-2 expression and PGI(2) secretion, while dioctanoyl PA increased COX-2 expression and PGI(2) production in a time- and concentration-dependent manner. Our results indicate that in VSMC, AngII promotes PGI(2) production to a large extent through a rise in COX-2 expression which is mediated by PA generated from increased PKC-dependent PLD activity.