Involvement of de novo ceramide biosynthesis in macrophage death induced by activation of ATP-sensitive P2X7 receptor

Macrophage ionotropic P2X7 receptors regulate cell-death through ill-defined signaling pathways. Here, we investigated the role of ceramide, an apoptogenic sphingolipid and showed that ATP stimulated ceramide accumulation in macrophages. Benzoylbenzoyl-ATP, a potent P2X7 agonist, was able to mimic the effects of ATP on ceramide accumulation while oxidized ATP had the opposite effect. Ceramide accumulation was blocked by de novo ceramide biosynthesis inhibitors. Interestingly, ATP-induced caspase-3/7 activation was dependent on ceramide generation. Finally, we showed that de novo ceramide biosynthesis is involved in ATP-induced macrophage death in a caspase-dependent manner. Our results indicate a novel role of ceramide in P2X7-regulated cell-death.     

The Pro-451 to Leu polymorphism within the C-terminal tail of P2X7 receptor impairs cell death but not phospholipase D activation in murine thymocytes

The P2X family of ATP receptors (P2XR) are ligandgated channels that have been proposed to regulate cell death of immature thymocytes. However, the nature of the P2XR subtype involved has been controversial until recently. In agreement with previous studies, we found that extracellular ATP (ATPe) induces a caspase-dependent apoptosis of BALB/c thymocytes, as observed by DNA fragmentation. Additionally, ATPe induces a predominant caspase-independent thymocytes lysis characterized by plasma membrane disruption. Both responses to ATPe can be induced by a potent P2X7R agonist, benzoylbenzoyl-ATP, whereas P2X7R antagonists, oxidized ATP and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid, inhibited the effect of ATPe. These results are further supported by observations where disruption of the P2X7R gene (P2X7R(-/-) mice) completely abolishes thymocytes death induced by ATPe. Interestingly, the natural P451L mutation in the C-terminal tail of P2X7R present in C57BL/6 mice, which impairs ATPe-dependent pore formation in T lymphocytes, significantly reduces thymocytes death triggered by ATPe. Furthermore, we found that P2X7R from BW5147 thymoma cells also harbors this point mutation, accounting for their insensitivity to ATPe-induced cell death. Concentrations of ATPe effective in inducing cell death also increase phosphatidylcholine-hydrolyzing phospholipase D (PC-PLD) activity in BALB/c thymocytes through the stimulation of P2X7R. However, in contrast to ATPe-induced cell death, PC-PLD activation is totally Ca(2+)-dependent. Moreover, the stimulation of PC-PLD by ATPe is not affected by the P451L mutation present in C57BL/6 thymocytes and BW5147 cells, suggesting that cell death and PC-PLD activity are regulated through distinct domains of the P2X7R. Finally, the inhibition of ATPe-induced PC-PLD stimulation does not affect thymocytes death. Altogether, these data suggest that P2X7R-induced thymocytes death is independent of the stimulation of PC-PLD activity.     

Induction of apoptosis through B-cell receptor cross-linking occurs via de novo generated C16-ceramide and involves mitochondria

B-cells, triggered via their surface B-cell receptor (BcR), start an apoptotic program known as activation-induced cell death (AICD), and it is widely believed that this phenomenon plays a role in the restriction and focusing of the immune response. Although both ceramide and caspases have been proposed to be involved in AICD, the contribution of either and the exact molecular events through which AICD commences are still unknown. Here we show that in Ramos B-cells, BcR-triggered cell death is associated with an early rise of C16 ceramide that derives from activation of the de novo pathway, as demonstrated using a specific inhibitor of ceramide synthase, fumonisin B1 (FB1), and using pulse labeling with the metabolic sphingolipid precursor, palmitate. There was no evidence for activation of sphingomyelinases or hydrolysis of sphingomyelin. Importantly, FB1 inhibited several specific apoptotic hallmarks such as poly(A)DP-ribose polymerase cleavage and DNA fragmentation. Electron microscopy revealed morphological evidence of mitochondrial damage, suggesting the involvement of mitochondria in BcR-triggered apoptosis, and this was inhibited by FB1. Moreover, a loss of mitochondrial membrane potential was observed in Ramos cells after BcR cross-linking, which was inhibited by the addition of FB1. Interestingly, benzyloxycarbonyl-Val-Ala-dl-Asp, a broad spectrum caspase inhibitor did not inhibit BcR-induced mitochondrial membrane permeability transition but did block DNA fragmentation. These results suggest a crucial role for de novo generated C16 ceramide in the execution of AICD, and they further suggest an ordered and more specific sequence of biochemical events in which de novo generated C16 ceramide is involved in mitochondrial damage resulting in a downstream activation of caspases and apoptosis.     

Mitochondrial dysfunction is involved in P2X7 receptor-mediated neuronal cell death

J. Neurochem. (2012) 122, 1118-1128. ABSTRACT: P2X7 receptor (P2X7R) is known to be a 'death receptor' in immune cells, but its functional expression in non-immune cells such as neurons is controversial. Here, we examined the involvement of P2X7R activation and mitochondrial dysfunction in ATP-induced neuronal death in cultured cortical neurons. In P2X7R- and pannexin-1-expressing neuron cultures, 5 or more mM ATP or 0.1 or more mM BzATP induced neuronal death including apoptosis, and cell death was prevented by oxATP, P2X7R-selective antagonists. ATP-treated neurons exhibited Ca(2+) entry and YO-PRO-1 uptake, the former being inhibited by oxATP and A438079, and the latter by oxATP and carbenoxolone, while P2X7R antagonism with oxATP, but not pannexin-1 blocking with carbenoxolone, prevented the ATP-induced neuronal death. The ATP treatment induced reactive oxygen species generation through activation of NADPH oxidase and activated poly(ADP-ribose) polymerase, but both of them made no or negligible contribution to the neuronal death. Rhodamine123 efflux from neuronal mitochondria was increased by the ATP-treatment and was inhibited by oxATP, and a mitochondrial permeability transition pore inhibitor, cyclosporine A, significantly decreased the ATP-induced neuronal death. In ATP-treated neurons, the cleavage of pro-caspase-3 was increased, and caspase inhibitors, Q-VD-OPh and Z-DEVD-FMK, inhibited the neuronal death. The cleavage of apoptosis-inducing factor was increased, and calpain inhibitors, MDL28170 and PD151746, inhibited the neuronal death. These findings suggested that P2X7R was functionally expressed by cortical neuron cultures, and its activation-triggered Ca(2+) entry and mitochondrial dysfunction played important roles in the ATP-induced neuronal death.     

P2X7 receptor-dependent blebbing and the activation of Rho-effector kinases,caspases, and IL-1 beta release

In response to ATP binding, the P2X7R facilitates cation channel activation, nonspecific pore formation, rapid changes in plasma membrane morphology, and secretion of IL-1 beta from LPS-primed macrophages. To investigate the relationship between the P2X7R-dependent changes in plasma membrane organization and the release of IL-1 beta, we generated time-lapse movies of ATP-stimulated BAC1 murine macrophages in conjunction with biochemical analyses of IL-1 beta release. Similar image analyses in human embryonic kidney 293 cells expressing recombinant P2X7R (HEK-P2X7) permitted comparison of P2X7R-dependent effects in macrophage vs nonmacrophage backgrounds. Whereas HEK-P2X7 cells exhibit zeiotic blebbing within 5 min of ATP treatment, BAC1 macrophages initiated a distinct "tethered" blebbing 10 min after ATP addition. This blebbing was comparably induced by the P2X7R-selective agonist BzATP and was blocked by P2X7R inhibitors KN-62 and oxidized ATP. Blebbing was initiated at ATP concentrations > or = 3 mM, but optimal IL-1 beta release occurred at 1 mM ATP. P2X7R-dependent blebbing was abrogated in the presence of Rho-effector kinase inhibitors Fasudil and Y-27632, but ATP-induced IL-1 beta release was unaffected. ATP-induced activation of RhoA could be detected in both HEK-P2X7 cells and BAC1 murine macrophages. Thus, P2X7R activation signals distinct, novel membrane blebbing events (dependent on RhoA activation and Rho-effector kinase activity) and simultaneously initiates release of IL-1 beta. Our observations that blebbing and IL-1 beta release are dissociable suggest these events occur via parallel rather than convergent signaling pathways.     

Basal activation of the P2X7 ATP receptor elevates mitochondrial calcium and potential, increases cellular ATP levels, and promotes serum-independent growth

P2X7 is a bifunctional receptor (P2X7R) for extracellular ATP that, depending on the level of activation, forms a cation-selective channel or a large conductance nonselective pore. The P2X7R has a strong proapoptotic activity but can also support growth. Here, we describe the mechanism involved in growth stimulation. Transfection of P2X7R increases resting mitochondrial potential (delta psi(mt)), basal mitochondrial Ca2+ ([Ca2+]mt), intracellular ATP content, and confers ability to grow in the absence of serum. These changes require a full pore-forming function, because they are abolished in cells transfected with a mutated P2X7R that retains channel activity but cannot form the nonselective pore, and depend on an autocrine/paracrine tonic stimulation by secreted ATP. On the other hand, sustained stimulation of P2X7R causes a delta psi(mt) drop, a large increase in [Ca2+]mt, mitochondrial fragmentation, and cell death. These findings reveal a hitherto undescribed mechanism for growth stimulation by a plasma membrane pore.     

P2X7 receptor‐induced death of motor neurons by a peroxynitrite/FAS‐dependent pathway

The P2X7 receptor/channel responds to extracellular ATP and is associated with neuronal death and neuroinflammation in spinal cord injury and amyotrophic lateral sclerosis. Whether activation of P2X7 directly causes motor neuron death is unknown. We found that cultured motor neurons isolated from embryonic rat spinal cord express P2X7 and underwent caspase‐dependent apoptosis when exposed to exceptionally low concentrations of the P2X7 agonist 2′(3′)‐O‐(4‐Benzoylbenzoyl)‐ATP. The P2X7 inhibitors BBG, oATP, and KN‐62 prevented 2′(3′)‐O‐(4‐Benzoylbenzoyl)‐ATP‐induced motor neuron death. The endogenous P2X7 agonist ATP induced motor neuron death at low concentrations (1‐100 μM). High concentrations of ATP (1 mM) paradoxically became protective due to degradation in the culture media to produce adenosine and activate adenosine receptors. P2X7‐induced motor neuron death was dependent on neuronal nitric oxide synthase‐mediated production of peroxynitrite, p38 activation, and autocrine FAS signaling. Taken together, our results indicate that motor neurons are highly sensitive to P2X7 activation, which triggers apoptosis by activation of the well‐established peroxynitrite/FAS death pathway in motor neurons.     

Neural progenitor cell death is induced by extracellular ATP via ligation of P2X7 receptor

Neural progenitor cells (NPCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes, and have been used to treat several animal models of CNS disorders. In the present study, we show that the P2X7 purinergic receptor (P2X7R) is present on NPCs. In NPCs, P2X7R activation by the agonists extracellular ATP or benzoyl ATP triggers opening of a non-selective cationic channel. Prolonged activation of P2X7R with these nucleotides leads to caspase independent death of NPCs. P2X7R ligation induces NPC lysis/necrosis demonstrated by cell membrane disruption accompanied with loss of mitochondrial membrane potential. In most cells that express P2X7R, sustained stimulation with ATP leads to the formation of a non-selective pore allowing the entry of solutes up to 900 Da, which are reportedly involved in P2X7R-mediated cell lysis. Surprisingly, activation of P2X7R in NPCs causes cell death in the absence of pore formation. Our data support the notion that high levels of extracellular ATP in inflammatory CNS lesions may delay the successful graft of NPCs used to replace cells and repair CNS damage.     

Effect of the purinergic receptor P2X7 on Chlamydia infection in cervical epithelial cells and vaginally infected mice

Ligation of the purinergic receptor, P2X7R, with its agonist ATP has been previously shown to inhibit intracellular infection by chlamydiae and mycobacteria in macrophages. The effect of P2X7R on chlamydial infection had never been investigated in the preferred target cells of chlamydiae, cervical epithelial cells, nor in vaginally infected mice. In this study, we show that treatment of epithelial cells with P2X7R agonists inhibits partially Chlamydia infection in epithelial cells. Chelation of ATP with magnesium or pretreatment with a P2X7R antagonist blocks the inhibitory effects of ATP. Similarly to previous results obtained with macrophages, ATP-mediated inhibition of infection in epithelial cells requires activation of host-cell phospholipase D. Vaginal infection was also more efficient in P2X7R-deficient mice, which also displayed a higher level of acute inflammation in the endocervix, oviduct, and mesosalpingeal tissues than in infected wild-type mice. However, secretion of IL-1beta, which requires P2X7R ligation during infection by other pathogens, was decreased mildly and only at short times of infection. Taken together, these results suggest that P2X7R affects Chlamydia infection by directly inhibiting infection in epithelial cells, rather than through the ability of P2X7R to modulate IL-1beta secretion.     

A role for mitogen-activated protein kinase(Erk1/2) activation and non-selective pore formation in P2X7 receptor-mediated thymocyte death

Extracellular ATP (ATPe) binds to P2X7 receptors (P2X7R) expressed on the surface of cells of hematopoietic lineage, including murine thymocytes. Activation of P2X7R by ATPe results in the opening of cation-specific channels, and prolonged ATPe exposure leads to the formation of non-selective pores enabling transmembrane passage of solutes up to 900 Da. In the presence of ATPe, P2X7R-mediated thymocyte death is due primarily to necrosis/lysis and not apoptosis, as measured by the release of lactate dehydrogenase indicative of a loss of plasma membrane integrity. The present study is focused on the identification of P2X7R signaling mediators in ATP-induced thymocyte necrosis/lysis. Thus, extracellular signal-regulated protein kinase 1/2 (Erk1/2) phosphorylation was found to be required for cell lysis, and both events were independent of ATP-induced calcium influx. P2X7R-dependent thymocyte death involved the chronological activation of Src family tyrosine kinase(s), phosphatidylinositol 3-kinase, the mitogen-activated protein (MAP) kinase(Erk1/2) module, and the proteasome. Although independent of this signaling cascade, non-selective pore formation may modulate ATP-mediated thymocyte death. These results therefore suggest a role for both activation of MAP kinase(Erk1/2) and non-selective pore opening in P2X7R-induced thymocyte death.     

Activation of the de novo biosynthesis of sphingolipids mediates angiotensin II type 2 receptor-induced apoptosis.

This study examines the role of sphingolipids in mediating the apoptosis of PC12W cells induced by the angiotensin II type 2 (AT2) receptor. PC12W cells express abundant AT2 receptor but not angiotensin II type 1 receptor and undergo apoptosis when stimulated by angiotensin II. AT2 receptor-induced ceramide accumulation preceded the onset of caspase 3 activation and DNA fragmentation. AT2 receptor-induced ceramide accumulation did not result from the degradation of complex sphingolipids (SL) such as sphingomyelin or glycosphingolipids, as no changes in neutral or acidic sphingomyelinase activities, sphingomyelin level, nor in cellular glycolipid composition were observed. AT2 receptor activated serine palmitoyltransferase with a maximum time of 24 h after angiotensin II stimulation. The AT2 receptor-induced accumulation of ceramide was blocked by inhibitors of the de novo pathway of SL synthesis, beta-chloro-L-alanine and fumonisin B1. Inhibition of the de novo biosynthesis of SLs by fumonisin B1 and beta-chloro-L-alanine completely abrogated the AT2 receptor-mediated apoptosis. Pertussis toxin and orthovanadate blocked AT2 receptor-mediated ceramide production. Taken together our data demonstrate that in PC12W cells the stimulation of AT2 receptor induces the activation of de novo pathway, and a metabolite of this pathway, possibly ceramide, mediates AT2 receptor-induced apoptosis.     

Sphingosine contributes to glucocorticoid-induced apoptosis of thymocytes independently of the mitochondrial pathway

During the selection process in the thymus, most thymocytes are eliminated by apoptosis through signaling via TCR or glucocorticoids. The involvement of ceramide (Cer) and sphingosine (SP), important apoptotic mediators, remains poorly defined in glucocorticoid-induced apoptosis. We report that, in mouse thymocytes, apoptosis triggered by 10(-6) M dexamethasone (DX) was preceded by a caspase-dependent Cer and SP generation, together with activation of acidic and neutral ceramidases. Apoptosis was drastically reduced by blocking either sphingolipid production (by acid sphingomyelinase inhibitor) or SP production (by ceramidase inhibitors), but not by inhibition of de novo Cer synthesis. Thus, SP generated through acid sphingomyelinase and ceramidase activity would contribute to the apoptotic effect of DX. Consistent with this hypothesis, SP addition or inhibition of SP kinase induced thymocyte apoptosis. DX induced a proteasome-dependent loss of mitochondrial membrane potential (Deltapsim) and caspase-8, -3, and -9 processing. Apoptosis was abolished by inhibition of Deltapsim loss or caspase-8 or -3, but not caspase-9. Deltapsim loss was independent of SP production and caspase-8, -3, and -9 activation. However, inhibition of SP production reduced caspase-8 and -3, but not caspase-9 processing. Proteasome inhibition impaired activation of the three caspases, whereas inhibition of Deltapsim loss solely blocked caspase-9 activation. These data indicate that DX-induced apoptosis is mediated in part by SP, which contributes, together with proteasome activity, to caspase-8-3 processing independently of mitochondria, and in part by the proteasome/mitochondria pathway, although independently of caspase-9 activation.     

Effects of protons on macroscopic and single-channel currents mediated by the human P2X7 receptor

Human P2X7 receptors (hP2X7Rs) belong to the P2X family, which opens an intrinsic cation channel when challenged by extracellular ATP. hP2X7Rs are expressed in cells of the inflammatory and immune system. During inflammation, ATP and protons are secreted into the interstitial fluid. Therefore, we investigated the effect of protons on the activation of hP2X7Rs. hP2X7Rs were expressed in Xenopus laevis oocytes and activated by the agonists ATP or benzoyl-benzoyl-ATP (BzATP) at different pH values. The protons reduced the hP2X7R-dependent cation current amplitude and slowed the current deactivation depending on the type and concentration of the agonist used. These effects can be explained by (i) the protonation of ATP, which reduces the effective concentration of the agonist ATP⁴⁻ at the high- and low-affinity ATP activation site of the hP2XR, and (ii) direct allosteric inhibition of the hP2X7R channel opening that follows ATP⁴⁻ binding to the low-affinity activation site. Due to the hampered activation via the low-affinity activation site, a low pH (as observed in inflamed tissues) leads to a relative increase in the contribution of the high-affinity activation site for hP2X7R channel opening.     

Trophic Activity of Human P2X7 Receptor Isoforms A and B in Osteosarcoma

The P2X7 receptor (P2X7R) is attracting increasing attention for its involvement in cancer. Several recent studies have shown a crucial role of P2X7R in tumour cell growth, angiogenesis and invasiveness. In this study, we investigated the role of the two known human P2X7R functional splice variants, the full length P2X7RA and the truncated P2X7RB, in osteosarcoma cell growth. Immunohistochemical analysis of a tissue array of human osteosarcomas showed that forty-four, of a total fifty-four tumours (81.4%), stained positive for both P2X7RA and B, thirty-one (57.4%) were positive using an anti-P2X7RA antibody, whereas fifteen of the total number (27.7%) expressed only P2X7RB. P2X7RB positive tumours showed increased cell density, at the expense of extracellular matrix. The human osteosarcoma cell line Te85, which lacks endogenous P2X7R expression, was stably transfected with either P2X7RA, P2X7RB, or both. Receptor expression was a powerful stimulus for cell growth, the most efficient growth-promoting isoform being P2X7RB alone. Growth stimulation was matched by increased Ca²⁺ mobilization and enhanced NFATc1 activity. Te85 P2X7RA+B cells presented pore formation as well as spontaneous extracellular ATP release. The ATP release was sustained in all clones by P2X7R agonist (BzATP) and reduced following P2X7R antagonist (A740003) application. BzATP also increased cell growth and activated NFATc1 levels. On the other hand cyclosporin A (CSA) affected both NFATc1 activation and cell growth, definitively linking P2X7R stimulation to NFATc1 and cell proliferation. All transfected clones also showed reduced RANK-L expression, and an overall decreased RANK-L/OPG ratio. Mineralization was increased in Te85 P2X7RA+B cells while it was significantly diminished in Te85 P2X7RB clones, in agreement with immunohistochemical results. In summary, our data show that the majority of human osteosarcomas express P2X7RA and B and suggest that expression of either isoform is differently coupled to cell growth or activity.     

P2X7 purinoceptors contribute to the death of Schwann cells transplanted into the spinal cord

The potential to use Schwann cells (SCs) in neural repair for patients suffering from neurotrauma and neurodegenerative diseases is well recognized. However, significant cell death after transplantation hinders the clinical translation of SC-based therapies. Various factors may contribute to the death of transplanted cells. It is known that prolonged activation of P2X7 purinoceptors (P2X7R) can lead to death of certain types of cells. In this study, we show that rat SCs express P2X7R and exposure of cultured SCs to high concentrations of ATP (3–5 mM) or a P2X7R agonist, 2′(3′)-O-(4-benzoylbenzoyl)ATP (BzATP) induced significant cell death rapidly. High concentrations of ATP and BzATP increased ethidium uptake by SCs, indicating increased membrane permeability to large molecules, a typical feature of prolonged P2X7R activation. SC death, as well as ethidium uptake, induced by ATP was blocked by an irreversible P2X7R antagonist oxidized ATP (oxATP) or a reversible P2X7R antagonist A438079. oxATP also significantly inhibits the increase of intracellular free calcium induced by minimolar ATP concentrations. Furthermore, ATP did not cause death of SCs isolated from P2X7R-knockout mice. All these results suggest that P2X7R is responsible for ATP-induced SC death in vitro. When rat SCs were treated with oxATP before transplantation into uninjured rat spinal cord, 35% more SCs survived than untreated SCs 1 week after transplantation. Moreover, 58% more SCs isolated from P2X7R-knockout mice survived after being transplanted into rat spinal cord than SCs from wild-type mice. This further confirms that P2X7R is involved in the death of transplanted SCs. These results indicate that targeting P2X7R on SCs could be a potential strategy to improve the survival of transplanted cells. As many other types of cells, including neural stem cells, also express P2X7R, deactivating P2X7R may improve the survival of other types of transplanted cells.     

P2X7 receptor as sensitive flow sensor for ERK activation in osteoblasts

The involvement of the P2 receptor in the activation of ERK induced by a short transient fluid flow stimulation in MC3T3-E1 osteoblasts was examined in the current study. The ERK activation induced by this transient fluid flow stimulation was followed by an increase in c-fos mRNA expression. Suramin, a non-selective P2 receptor antagonist, and two different P2X7 receptor (P2X7R) antagonists, ATP analogue (oxidized ATP) and dye (Brilliant blue G), inhibited fluid flow-induced ERK activation. However, the P2Y receptor pathway inhibitor U73122 did not abolish this ERK activation. The P2X7R agonist 2′,3′-O-(4-benzoylbenzoyl)-ATP (BzATP) significantly increased ERK activation and this activation could be completely inhibited by oxidized ATP and Brilliant blue G. Our results suggest that P2X7R is a highly sensitive P2 receptor for fluid flow-induced ERK activation in osteoblasts.     

FAS activation induces dephosphorylation of SR proteins; dependence on the de novo generation of ceramide and activation of protein phosphatase 1

The search for potential targets for ceramide action led to the identification of ceramide-activated protein phosphatases (CAPP). To date, two serine/threonine protein phosphatases, protein phosphatase 2A (PP2A) and protein phosphatase 1 (PP1), have been demonstrated to function as ceramide-activated protein phosphatases. In this study, we show that treatment with either anti-FAS IgM (CH-11) (150 ng/ml) or exogenous d-(e)-C(6-)ceramide (20 microm) induces the dephosphorylation of the PP1 substrates, serine/arginine-rich (SR) proteins, in Jurkat acute leukemia T-cells. The serine/threonine protein phosphatase inhibitor, calyculin A, but not the PP2A-specific inhibitor, okadaic acid, inhibited both FAS- and ceramide-induced dephosphorylation of SR proteins. Anti-FAS IgM treatment of Jurkat cells led to a significant increase in levels of endogenous ceramide beginning at 2 h with a maximal increase of 10-fold after 7 h. A 2-h pretreatment of Jurkat cells with fumonisin B(1) (100 microm), a specific inhibitor of CoA-dependent ceramide synthase, blocked 80% of the ceramide generated and completely inhibited the dephosphorylation of SR proteins in response to anti-FAS IgM. Moreover, pretreatment of Jurkat cells with myriocin, a specific inhibitor of serine-palmitoyl transferase (the first step in de novo synthesis of ceramide), also blocked FAS-induced SR protein dephosphorylation, thus demonstrating a role for de novo ceramide. These results were further supported using A549 lung adenocarcinoma cells treated with d-(e)-C(6-)ceramide. Dephosphorylation of SR proteins was inhibited by fumonisin B(1) and by overexpression of glucosylceramide synthase; again implicating endogenous ceramide generated de novo in regulating the dephosphorylation of SR proteins in response to FAS activation. These results establish a specific intracellular pathway involving both de novo ceramide generation and activation of PP1 to mediate the effects of FAS activation on SR proteins.     

Regulation of P2X(7) nucleotide receptor function in human monocytes by extracellular ions and receptor density

P2X receptors function as ATP-gated cation channels. The P2X(7) receptor subtype is distinguished from other P2X family members by a very low affinity for extracellular ATP (millimolar EC50) and its ability to trigger induction of nonselective pores on repeated or prolonged stimulation. Previous studies have indicated that certain P2X(7) receptor-positive cell types, such as human blood monocytes and murine thymocytes, lack this pore-forming response. In the present study we compared pore formation in response to P2X(7) receptor activation in human blood monocytes with that in macrophages derived from these monocytes by in vitro tissue culture. ATP induced nonselective pores in macrophages but not in freshly isolated monocytes when both cell types were identically stimulated in standard NaCl-based salines. However, ion substitution studies revealed that replacement of extracellular Na+ and Cl- with K+ and nonhalide anions strongly facilitated ATP-dependent pore formation in monocytes. These ionic conditions also resulted in increased agonist affinity, such that 30-100 microM ATP was sufficient for activation of nonselective pores by P2X(7) receptors. Comparison of P2X(7) receptor expression in blood monocytes with that in macrophages indicated no differences in steady-state receptor mRNA levels but significant increases (up to 10-fold) in the amount of immunoreactive P2X(7) receptor protein at the cell surface of macrophages. Thus ability of ATP to activate nonselective pores in cells that natively express P2X(7) receptors can be modulated by receptor subunit density at the cell surface and ambient levels of extracellular Na+ and Cl-. These mechanisms may prevent adventitious P2X(7) receptor activation in monocytes until these proinflammatory leukocytes migrate to extravascular sites of tissue damage.     

Regulation of phospholipase D by P2X7 receptors in submandibular ductal cells

ATP (1 mM) increased the phospholipase D (PLD) activity of rat submandibular gland (RSMG) ductal cells in a concentration-dependent and calcium-sensitive manner. The response to ATP was reproduced by benzoylbenzoyl-ATP (Bz-ATP, 100 microM) and also partly by adenosine 5'-(gamma-thio)triphosphate (ATPgammaS, 1 mM). A similar stimulation was observed in control mice (P2X7R+/+ mice) but not in mice lacking the P2X7 receptors (P2X7R-/- mice). Oxidized ATP and Coomassie blue or the addition of magnesium or nickel to the incubation medium inhibited the response to ATP. The stimulation of PLD by purinergic agonist was inhibited by about 50% by calphostin C and chelerythrine, two protein kinase C (PKC) inhibitors. The stimulation of PLD by Bz-ATP and by o-tetradecanoylphorbol 13-acetate (TPA), a phorbol ester which activates PKC, were not additive.From these results we can conclude that the activation of P2X7 receptors in RSMG ductal cells is coupled to the activation of a PLD. This activation is partly mediated by protein kinase C.