Regulation of platelet-activating factor receptor and PAF receptor-mediated arachidonic acid release by protein kinase C activation in rat Kupffer cells

Phorbol 12-myristate 13-acetate (PMA), a potent protein kinase C activator, caused down-regulation of receptors for platelet-activating factor (AGEPC) on the plasma membrane of rat Kupffer cells (40-50% reduction) but had a relatively minor effect on the binding affinity of the receptors for AGEPC (Kd = 0.30 nM vs 0.56 nM) when incubated with the cells for a short period of time (30-60 min). As a consequence, the AGEPC receptor-mediated arachidonic acid release was attenuated. The PMA-induced down-regulation of AGEPC receptors was concentration-dependent, specific, and transient (the maximal effect was observed at about 1 h and the level of specific [3H]AGEPC binding gradually returned to the control level within 8.5 h and even higher than the control level at 24 h after addition of PMA). Upon removing PMA from the culture medium, more than half of the lost receptors were replaced within 1 h at 37 degrees C and the recovery process appeared to be independent of protein synthesis. The ability of PMA to down-regulate the AGEPC receptors was lost in cells "down-regulated" for protein kinase C, suggesting that the receptor-regulatory effect of PMA is protein kinase C-dependent. Protein kinase C appeared to be involved in the AGEPC-induced arachidonic acid release since 1-(5-isoquinolinesulfonyl)-2-methyl-piperazine dihydrochloride, a protein kinase C inhibitor, attenuated the stimulatory effect of AGEPC in this system. In addition, AGEPC-induced [3H]arachidonic acid release was inhibited significantly in cells down-regulated for protein kinase C. The present study thus demonstrates that protein kinase C has dual actions in the regulation of AGEPC-mediated events, i.e., a positive forward action, regulating AGEPC-stimulated arachidonic acid release, and a negative action, which inactivates or down-regulates AGEPC receptors.     

Regulation of platelet-activating factor receptors in rat Kupffer cells

Ligand binding studies indicate that 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC) down-regulates its own receptors on the plasma membrane of isolated rat Kupffer cells but has no significant effect on the binding affinity of the receptor for AGEPC. Exposure of isolated rat Kupffer cells to 10(-8) and 10(-6) M AGEPC resulted in a rapid, time-dependent reduction in the number of cell surface AGEPC receptors to a new steady state concentration (54.1 +/- 5.0% and 38.6 +/- 5.4% of control, respectively). During the observation period (6 h), the half-time of surface AGEPC receptors was about 60 and 45 min in the presence of 10(-8) and 10(-6) M AGEPC, respectively. Both the rate of loss and the maximal loss of the receptors were dependent upon the AGEPC concentration. With receptor synthesis inhibited by cycloheximide in the absence of AGEPC, the half-time of the surface AGEPC receptor was about 4 h, suggesting that AGEPC receptors are not recycled and that the loss of AGEPC receptors from the plasma membrane is accelerated by AGEPC binding. When incubated with Kupffer cells at 37 degrees C for 3 h, 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine (1.0 microM), an inactive metabolite of AGEPC, did not cause the loss of AGEPC receptors. Under the same conditions, AGEPC antagonists such as BN52021 (2 x 10(-5) M) or U66985 (2 x 10(-5) M) alone had no effect (97.0 +/- 3.9% of control for BN52021) or only a relatively slight effect (78.4 +/- 1.8% for U66985) on the number of surface AGEPC receptors. However, AGEPC antagonists inhibited the AGEPC-induced down-regulation of AGEPC receptors in a concentration-dependent manner, suggesting that the AGEPC-induced down-regulation of AGEPC receptors is a receptor-mediated process. The AGEPC-mediated decrease in receptor number on rat Kupffer cells is reversible. Upon removing AGEPC from the culture medium, about 67% of the lost receptors were replaced within 2 h. Cycloheximide, an inhibitor of protein synthesis, prevented the restoration of the AGEPC receptors. Similar results were obtained when Kupffer cells were incubated with Pronase followed by removing Pronase and reincubating the cells with or without cycloheximide. These observations suggest that the restored AGEPC receptor is newly synthesized rather than recycled. The present study demonstrates that under non-stimulatory (i.e. in the absence of AGEPC) conditions AGEPC receptors are lost from the plasma membrane and are reformed in the cells continuously.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of receptors for platelet-activating factor in rat Kupffer cells

Ligand binding studies demonstrated that isolated rat Kupffer cells possess high affinity binding sites for platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, AGEPC). AGEPC binding reached saturation within 10 min at 25 degrees C and was reversible. A Scatchard analysis revealed a single class of AGEPC receptors numbering about 10,600 sites/cell and possessing a dissociation constant of 0.45 nM. Similar values for the dissociation constant for AGEPC (0.12 and 0.34 nM) were obtained independently by kinetic analysis of specific AGEPC binding. AGEPC binding was stereospecific and was inhibited by Zn2+ and AGEPC receptor antagonists including BN52021 and U66985. The AGEPC receptor was functionally active since it was shown to mediate arachidonic acid release and eicosanoid production in Kupffer cells, and these events were inhibited by AGEPC receptor antagonist BN52021. The receptor-mediated arachidonic acid release was extracellular calcium-dependent and was abolished by calcium channel blocker prenylamine and by [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, indicating that calcium influx through a receptor-regulated calcium channel in the plasma membrane is involved in the AGEPC-induced arachidonic acid release. It is suggested that rat Kupffer cells have specific and functionally active AGEPC receptors which are involved in signaling mechanisms which govern the production of several other autacoid-type mediators in the liver.     

Dictyostelium cells produce platelet-activating factor in response to cAMP.

Evidence is provided that Dictyostelium discoideum cells produce 1-O-alkyl-2-delta-acetyl-O-sn-glycero-3-phosphocholine (platelet-activating factor, PAF). D. discoideum PAF has been characterized as being identical with mammalian platelet-activating factor, based on its stimulation of rabbit platelet aggregation, its physicochemical properties and mass spectrum. The basal activity of PAF increases after starvation and during aggregation and declines at the slug stage. PAF is not detected in the extracellular space. Cell treatment with cAMP pulses stimulates a transient accumulation of PAF, probably via activation of a cAMP-dependent acetyltransferase, suggesting a possible involvement of PAF in cAMP-regulated processes in Dictyostelium.     

RGS4 inhibits platelet-activating factor receptor phosphorylation and cellular responses

To define the role of regulators of G-protein signaling (RGS) in chemoattractant-mediated responses, RGS4 and the receptors for platelet-activating factor (PAFR), formylated peptides (FR), or interleukin-8 (CXCR1) were stably coexpressed in a rat basophilic leukemia (RBL-2H3) cell line. The data demonstrate that RGS4 inhibited responses by PAFR (i.e., phosphoinositide (PI) hydrolysis, Ca2+ mobilization) but not by FR or CXCR1. An N-terminal 33 amino acid deletion mutant of RGS4 (DeltaRGS4), deficient in GAP (GTPase activating protein) activity and plasma membrane localization, had no effect on either PAFR, FR, or CXCR1. RGS4, but not DeltaRGS4, also blocked phosphorylation of PAFR by platelet-activating factor (PAF) and, unexpectedly, by phorbol 12-myristate 13-acetate (PMA); it also blocked cross-phosphorylation by formylmethionylleucylphenylalanine (fMLP). A point mutant of RGS4 (N88S), deficient in GAP activity but not membrane localization, partially blocked PAFR phosphorylation but had no effect on PAFR-mediated PI hydrolysis and Ca2+ mobilization. Truncation of the cytoplasmic tail of PAFR (mPAFR) resulted in a loss of its susceptibility to inhibition by RGS4. Taken together, the data indicate that of the receptors studied, RGS4 selectively inhibited responses to PAFR, which preferentially couples to Gq. At the level of expression studied, RGS4 did not inhibit FR or CXCR1 which activates Gi to transduce cellular signals. Since the tail-deleted mutant of PAFR was not affected by RGS4, and RGS4 blocked homologous as well as heterologous phosphorylation of this receptor, it is possible that RGS4 interferes sterically with the cytoplasmic tail of PAFR. Thus, in addition to stimulating the GTPase activity of Galpha, RGS4 prevents G protein activation by PAFR and the homologous and heterologous phosphorylation of this receptor.     

Elevation of cytosolic free calcium by platelet-activating factor in cultured rat mesangial cells

Rat glomerular mesangial cell monolayers loaded with the fluorescent probe fura-2 responded to exogenous platelet-activating factor (PAF) with a rapid increase in cytosolic free calcium concentration ([Ca2+]i). PAF-induced [CA2+]i transients consisted of a dose-dependent phasic peak response followed by a sustained tonic phase of increased [Ca2+]i. Chelation of extracellular calcium with EGTA suppressed the tonic phase of increased [Ca2+]i but did not affect the phasic peak response. This suggests two mechanisms for the elevation of [Ca2+]i: a transient mobilization from intracellular stores and an enhanced calcium influx across the plasma membrane, possibly mediated by receptor-operated channels. Lyso-PAF had no effect on basal [Ca2+]i and the PAF-receptor antagonist L652,731 selectively inhibited responses to PAF. PAF-stimulated mesangial cells displayed homologous desensitization to reexposure to PAF while still being responsive to other calcium-mobilizing agonists. Preincubation of cells with the protein kinase C (PKC) activator phorbol myristate acetate diminished the PAF-induced [Ca2+]i transient, suggesting a regulatory role for PKC in PAF-activation of mesangial cells. An increase in [Ca2+]i, as a result of receptor-linked activation of phospholipase C, may mediate PAF-induced hemodynamic and inflammatory events in renal glomeruli.     

Two distinct pathways of platelet-activating factor-induced hydrolysis of phosphoinositides in primary cultures of rat Kupffer cells

Stimulation of rat Kupffer cells in primary culture with platelet-activating factor (PAF) caused a rapid hydrolysis of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate with a concomitant increase in the levels of myo-inositol 1,4,5-trisphosphate and myo-inositol 1,4-bisphosphate. This phospholipase C-mediated hydrolysis of polyphosphoinositides was independent of extracellular Ca2+ but was inhibited by the intracellular Ca2+ antagonist TMB-8. A second slower response to PAF was characterized by deacylation of PI leading to the accumulation of glycerophosphoinositol (GPI). PAF-induced GPI synthesis was not inhibited by TMB-8. These effects of PAF were accompanied by initial transient mobilization of Ca2+ from intracellular stores followed by a rather slow influx of Ca2+ from the extracellular medium. PAF-stimulated deacylation and phosphodiesteric hydrolysis of inositol lipids were differentially affected by cholera toxin and pertussis toxin. Pretreatment of the Kupffer cells with either of these toxins caused inhibition of phospholipase C activity. Pertussis toxin also inhibited PAF-stimulated deacylation. However, cholera toxin itself stimulated GPI release and addition of PAF to the cholera toxin-treated cells caused a further increase in GPI release. Phorbol ester inhibited PAF-induced phosphodiesteric hydrolysis of phosphoinositides, but not deacylation. PAF-induced metabolism of phosphoinositides was inhibited by the PAF antagonist, U66985. These results suggest that PAF-induced phosphodiesteric hydrolysis and deacylation of inositol phospholipids are regulated via distinct mechanisms involving activation of separate G-proteins in rat Kupffer cells. Also the regulation of phosphoinositide metabolism by Ca2+ mobilization from two separate Ca2+ pools is indicated by this study.     

血小板活化因子受体研究进展

血汴板活化因子是通过靶细胞膜上的受体而发挥其作用的,该受体属Ｇ联的受体家族,含３４２个氨基酸,有７个疏水的跨膜片段。其作用机制是通过激活磷酯酰肌醇、钙信使系统及相关蛋白激酶,使某些蛋白质发生磷酸化并产生相应的生物学效应。     

Regulation of the biosynthesis of acyl analogs of platelet-activating factor by purinergic agonist in endothlial cells

We have previously shown that platelet-activating factor (PAF)-dependent transacetylase (TA) contains three catalytic activities, namely PAF: lysophospholipid TA (TAL), PAF: sphingosine TA (TAs) and PAF acetylhydrolase. It serves as a modifier of PAF actions by producing different lipid signal molecules. The TAL activity is involved in the biosynthesis of acyl analogs of PAF (acyl-PAF, 1-acyl-2-acetyl-sn-glycero-3-phosphocholine, acylacetyl-GPC) in agonist-stimulated endothelial cells. In the present investigation, we have studied the mechanism(s) by which the TA activity is regulated in ATP-treated endothelial cells. We have demonstrated that ATP, and thiol-modifying agents with ATP, specifically regulate only the TAL part of the TA activities.     

Regulation of platelet-activating factor synthesis in human neutrophils by MAP kinases

Human neutrophils (PMN) are potentially a major source of platelet-activating factor (PAF) produced during inflammatory responses. The stimulated synthesis of PAF in PMN is carried out by a phospholipid remodeling pathway involving three enzymes: acetyl-CoA:lyso-PAF acetyltransferase (acetyltransferase), type IV phospholipase A(2) (cPLA(2)) and CoA-independent transacylase (CoA-IT). However, the coordinated actions and the regulatory mechanisms of these enzymes in PAF synthesis are poorly defined. A23187 has been widely used to activate the remodeling pathway, but it has not been shown how closely its actions mimic those of physiological stimuli. Here we address this important problem and compare responses of the three remodeling enzymes and PAF synthesis by intact cells. In both A23187- and N-formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated PMN, acetyltransferase activation is blocked by SB 203580, a p38 MAP kinase inhibitor, but not by PD 98059, which blocks activation of the ERKs. In contrast, either agent attenuated cPLA(2) activation. Correlating with these results, SB 203580 decreased stimulated PAF formation by 60%, whereas PD 98059 had little effect. However, the combination of both inhibitors decreased PAF formation to control levels. Although a role for CoA-IT in PAF synthesis is recognized, we did not detect activation of the enzyme in stimulated PMN. CoA-IT thus appears to exhibit full activity in resting as well as stimulated cells. We conclude that the calcium ionophore A23187 and the receptor agonist fMLP both act through common pathways to stimulate PAF synthesis, with p38 MAP kinase regulating acetyltransferase and supplementing ERK activation of cPLA(2).     

Receptor-independent metabolism of platelet-activating factor by myelogenous cells

Human neutrophils incorporate and metabolize platelet-activating factor (PAF). We dissociated these events from PAF binding to its receptors. Cells were pretreated with either pronase, a PAF antagonist (L652731), or excess PAF. This reduced PAF receptor numbers by 70 to almost 100% but had no comparable effect upon the neutrophil's ability to metabolize PAF. Furthermore, HL-60 cells efficiently metabolized, but did not specifically bind, PAF. Thus, PAF receptor availability did not correlate with PAF metabolic capacity and we conclude that myelogenous tissues can process this bioactive ligand by a receptor-independent pathway.     

Biochemical and functional responses stimulated by platelet-activating factor in murine peritoneal macrophages

Platelet-activating factor (PAF) is a potent stimulant of leukocytes, including macrophages. To analyze the mechanisms of its effects upon macrophages, we determined whether macrophages bear specific surface receptors for PAF. By competitive radioactive binding assays, we determined two classes of specific receptors to be present on purified membranes derived from murine peritoneal macrophages (one having a Kd of approximately 1 X 10(-10) M and one a Kd of approximately 2 X 10(-9) M). When the macrophages were incubated with PAF, rapid formation of several inositol phosphates including inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate were observed. PAF also elevated intracellular levels of calcium to 290 +/- 27% of basal levels which were 82.7 +/- 12 nM. Increases in calcium were observed first in submembranous areas of the macrophages. PAF also led to increases of 1,2-diacylglycerol of approximately 200 pmol/10(7) cells. A characteristic pattern of enhanced protein phosphorylation, similar to that initiated by both phorbol 12,13-myristate and lipopolysaccharide, was observed and involved enhanced phosphorylation of proteins of 28, 33, 67, and 103 kD. The half-maximal dose of PAF for initiating all the above effects was approximately 5 X 10(-9) M. PAF also initiated significant chemotaxis of the cells; the half-maximal dose for this effect was approximately 1 X 10(-11) M. Taken together, these observations suggest that murine mononuclear phagocytes bear specific membrane receptors for PAF and that addition of PAF leads to generation of break-down products of polyphosphoinositides, subsequent changes in intracellular calcium and protein phosphorylation, and chemotaxis.     

Pressor responses to platelet-activating factor and thromboxane are mediated by Rho-kinase

Platelet-activating factor (PAF) contracts smooth muscle of airways and vessels primarily via release of thromboxane. Contraction of smooth muscle is thought to be mediated either by calcium and inositol trisphosphate (IP(3))-dependent activation of the myosin light chain kinase or, alternatively, via the recently discovered Rho-kinase pathway. Here we investigated the contribution of these two pathways to PAF and thromboxane receptor-mediated broncho- and vasoconstriction in two different rat models: the isolated perfused lung (IPL) and precision-cut lung slices. Inhibition of the IP(3) receptor (1-10 microM xestospongin C) or inhibition of phosphatidylinositol-specific PLC (30 microM L-108) did not affect bronchoconstriction but attenuated the sustained vasoconstriction by PAF. Inhibition of myosin light chain kinase (35 microM ML-7) or of calmodulin kinase kinase (26 microM STO609), which regulates the phosphorylation of the myosin light chain, had only a small effect on PAF- or thromboxane-induced pressor responses. Similarly, calmidazolium (10 microM), which inhibits calmodulin-dependent proteins, only weakly reduced the airway responses. In contrast, Y-27632 (10 microM), a Rho-kinase inhibitor, attenuated the thromboxane release triggered by PAF and provided partial or complete inhibition against PAF- and thromboxane-induced pressor responses, respectively. Together, our data indicate that PAF- and thus thromboxane receptor-mediated smooth muscle contraction depends largely on the Rho-kinase pathway.     

Metabolism of 4-hydroxynonenal by rat Kupffer cells

Kupffer cells are known to participate in the early events of liver injury involving lipid peroxidation. 4-Hydroxy-2,3-(E)-nonenal (4-HNE), a major aldehydic product of lipid peroxidation, has been shown to modulate numerous cellular systems and is implicated in the pathogenesis of chemically induced liver damage. The purpose of this study was to characterize the metabolic ability of Kupffer cells to detoxify 4-HNE through oxidative (aldehyde dehydrogenase; ALDH), reductive (alcohol dehydrogenase; ADH), and conjugative (glutathione S-transferase; GST) pathways. Aldehyde dehydrogenase and GST activity was observed, while ADH activity was not detectable in isolated Kupffer cells. Additionally, immunoblots demonstrated that Kupffer cells contain ALDH 1 and ALDH 2 isoforms as well as GST A4-4, P1-1, Ya, and Yb. The cytotoxicity of 4-HNE on Kupffer cells was assessed and the TD50 value of 32.5+/-2.2 microM for 4-HNE was determined. HPLC measurement of 4-HNE metabolism using suspensions of Kupffer cells incubated with 25 microLM 4-HNE indicated a loss of 4-HNE over the 30-min time period. Subsequent production of 4-hydroxy-2-nonenoic acid (HNA) suggested the involvement of the ALDH enzyme system and formation of the 4-HNE-glutathione conjugate implicated GST-mediated catalysis. The basal level of glutathione in Kupffer cells (1.33+/-0.3 nmol of glutathione per 10(6) cells) decreased significantly during incubation with 4-HNE concurrent with formation of the 4-HNE-glutathione conjugate. These data demonstrate that oxidative and conjugative pathways are primarily responsible for the metabolism of 4-HNE in Kupffer cells. However, this cell type is characterized by a relatively low capacity to metabolize 4-HNE in comparison to other liver cell types. Collectively, these data suggest that Kupffer cells are potentially vulnerable to the increased concentrations of 4-HNE occurring during oxidative stress.     

Regulation of AMPA receptor-mediated synaptic transmission by clathrin-dependent receptor internalization

Redistribution of postsynaptic AMPA- (alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid-) subtype glutamate receptors may regulate synaptic strength at glutamatergic synapses, but the mediation of the redistribution is poorly understood. We show that AMPA receptors underwent clathrin-dependent endocytosis, which was accelerated by insulin in a GluR2 subunit-dependent manner. Insulin-stimulated endocytosis rapidly decreased AMPA receptor numbers in the plasma membrane, resulting in long-term depression (LTD) of AMPA receptor-mediated synaptic transmission in hippocampal CA1 neurons. Moreover, insulin-induced LTD and low-frequency stimulation-(LFS-) induced homosynaptic CA1 LTD were found to be mutually occlusive and were both blocked by inhibiting postsynaptic clathrin-mediated endocytosis. Thus, controlling postsynaptic receptor numbers through endocytosis may be an important mechanism underlying synaptic plasticity in the mammalian CNS.     

Superoxide release by zymosan-stimulated rat Kupffer cells in vitro

Kupffer cells were isolated from pronase-perfused rat livers and were maintained as a monolayer culture in a state of high purity and viability. Immediately after contact with zymosan particles, O2 uptake of the Kupffer cells increased fivefold; about 50% of the net oxygen consumed was accounted for as superoxide released into the medium. Concomitantly, a transient burst of luminol-dependent chemiluminescence, an increased activity of NAD(P)H oxidase and a stimulation of the flow of glucose through the hexose monophosphate shunt were observed. Chemiluminescence and O2- production were almost completely inhibited by superoxide dismutase and iodoacetate. Zymosan-induced chemiluminescence was not inhibited in the presence of the non-penetrating thiol reagents, 5,5'-dithio-bis-2-nitrobenzoate and iodoacetyl-sepharose. Iodoacetate acted on the cytosolic glucose-6-phosphate dehydrogenase rather than on NAD(P)H oxidase of the cell membrane.