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D J Hanahan

Publications and source records attributed to D J Hanahan.

At least 19 recordsLinked to original sources
PubMed1994

Biochemical characterization of the interaction of lipid phosphoric acids with human platelets: comparison with platelet activating factor.

A series of lipid phosphoric acids, including 1-O-alkyl-2-lyso-glycerophosphoric acid, 1-O-acyl-2-lyso-glycerophosphoric acid, hexadecylpropanediolphosphoric acid, N-acyl-2-aminoethanolphosphoric acid, sphingosine phosphoric acid, and certain homologues and analogues, were synthesized and characterized by thin-layer chromatography, fast-atom bombardment mass spectrometry, and their ability to aggregate human platelets. The presence of a receptor for these lipid phosphoric acids that is distinct from the PAF receptor is strongly suggested from experiments involving a desensitization procedure, platelet-activating factor (PAF) receptor antagonists, and inhibitors of the lipid phosphoric acids. The unique features of the interaction of these lipid phosphoric acids with platelets include: (a) evidence for a separate receptor(s) for this diverse group of synthetic compounds, (b) no requirement for stereospecificity (i.e., no glycerol backbone), and (c) a structural requirement for a long-chain hydrocarbon residue covalently bound to a phosphoric acid residue. In the interaction of these compounds with the platelet, it is mandatory that extracellular Ca2+ and ADP be present for maximum biological activity. The potential involvement of a lipid phosphoric acid receptor, which could form a component of the activation pathway associated with various lysophospholipids and analogues, such as PAF, via a phospholipase D activation, is discussed.

Blood Platelets↗
PubMed1992

Protein tyrosine phosphorylation and regulation of the receptor for platelet-activating factor in rat Kupffer cells. Effect of sodium vanadate.

Platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, AGEPC) and sodium vanadate (a phosphotyrosine phosphatase inhibitor) induced a time- and concentration-dependent increase in phosphotyrosine in several proteins and stimulated prostaglandin (PG) E2 production in cultured rat Kupffer cells. In addition, vanadate induced a decrease in the surface expression of AGEPC receptors and, as a consequence, inhibited AGEPC-stimulated PGE2 production. The vanadate-induced decrease in the surface expression of AGEPC receptors was time- and concentration-dependent and was partially prevented by genistein, a putative tyrosine kinase inhibitor. Upon removal of vanadate from the culture medium and re-incubation of cells in vanadate-free medium, the surface AGEPC receptors were restored within 7 h after the removal of vanadate. Both AGEPC- and vanadate-stimulated PGE2 formation was attenuated by genistein. Thus the present investigation demonstrates that both AGEPC and sodium vanadate stimulate tyrosine phosphorylation of cellular proteins, and vanadate induces a decrease in the number of the surface AGEPC receptors. These results suggest that protein tyrosine phosphorylation may play a role, directly or indirectly, in the regulation of surface expression of AGEPC receptors as well as in PGE2 production in response to vanadate and AGEPC.

Animals↗
PubMed1992

Dual effects of oleic acid on Ca2+ mobilization and protein phosphorylation in human platelets in presence or absence of platelet activating factor.

This laboratory demonstrated earlier that oleic acid inhibited platelet activating factor (PAF)-induced aggregation and serotonin release of rabbit platelets (M. Miwa, C. Hill, R. Kumar, J. Sugatani, M. S. Olson, and D. J. Hanahan, 1987, J. Biol. Chem. 262, 527-530). More recently, we reported that oleic acid caused a decrease in phosphatidylinositol-4-phosphate (PIP) and phosphatidylinositol-4,5-bisphosphate (PIP2), but did not affect the level of inositol-1,4,5-trisphosphate (IP3), in rabbit platelets (D. Nunez, J. Randon, C. Gandhi, A. Siafaka-Kapadai, M. S. Olson, and D. J. Hanahan, 1990, J. Biol. Chem. 265, 18330-18838). These results suggested that oleic acid did not stimulate phospholipase C. In contrast, PAF induced a decrease in PIP2 and an increase in PIP level and IP3. These effects were shown to be attenuated by oleic acid. In this current study, our experiments show that (a) oleic acid blocked PAF-induced rise in intracellular [Ca2+] (to provide a mechanism in agreement with our previous experiments which showed that oleic acid inhibited PAF-induced IP3 rise in platelets) and (b) oleic acid itself induced a gradual rise in [Ca2+]i, which would provide a mechanism for oleic acid-induced aggregation despite the fact that oleic acid did not cause the production of IP3 (Nunez et al., 1990). Oleic acid, in a dose-dependent manner, was shown to inhibit PAF-induced Ca2+ mobilization from intra- and extracellular sources. The inhibition was closely related to the suppressive effect of oleic acid on PAF-induced aggregation. Furthermore, oleic acid inhibited the PAF-stimulated phosphorylation of the 20- and 40-kDa proteins. At concentrations above 20 microM, oleic acid itself could induce platelet aggregation and Ca2+ mobilization, but the time sequence of these two responses in human platelets was significantly different from those obtained with PAF. Oleic acid alone, at 20 microM, caused a 1.4-fold increase in the cAMP level in platelets which was followed by a decline to a basal value at higher concentrations of this fatty acid. It seemed clear that elevation of adenylate cyclase activity was not associated with free fatty acid inhibition of platelet activation. Interestingly, both PAF and oleic acid added separately to human platelets induced protein-tyrosine phosphorylation, but oleic acid did not cause any inhibition of PAF-induced protein-tyrosine phosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)

Blood Platelets↗
PubMed1992

31P NMR spectroscopic studies on purified, native and cloned, expressed forms of NADPH-cytochrome P450 reductase.

31P NMR spectroscopy has been utilized in conjunction with site-directed mutagenesis and phospholipid analysis to determine structural aspects of the prosthetic flavins, FAD and FMN, of NADPH-cytochrome P450 reductase. Comparisons are made among detergent-solubilized and protease (steapsin)-solubilized preparations of porcine liver reductases, showing unequivocally that the 31P NMR signals at approximately 0.0 ppm in the detergent-solubilized, hydrophobic form are attributable to phospholipids. By extraction and TLC analysis, the phospholipid contents of detergent-solubilized rat liver reductase, both tissue-purified and Escherichia coli-expressed, have been determined to reflect the membranes from which the enzyme was extracted. In addition, the cloned, wild-type NADPH-cytochrome P450 reductase exhibits an additional pair of signals downfield of the normal FAD pyrophosphate resonances reported by Otvos et al. [(1986) Biochemistry 25, 7220-7228], but these signals are not observed with tissue-purified or mutant enzyme preparations. The Tyr140----Asp140 mutant, which exhibits only 20% of wild-type activity, displays no gross changes in 31P NMR spectra. However, the Tyr178----Asp178 mutant, which has no catalytic activity and does not bind FMN, exhibits no FMN 31P NMR signal and a normal, but low intensity, pair of signals for FAD. The latter experiments, taking advantage of mutations in residues putatively on either side of the FMN isoalloxazine ring, suggest subtle to severe changes in the binding of the flavin prosthetic groups and, perhaps, cooperative interactions of flavin binding to NADPH-cytochrome P450 reductase.

Animals↗
PubMed1992

Platelet-activating factor-stimulated protein tyrosine phosphorylation and eicosanoid synthesis in rat Kupffer cells. Evidence for calcium-dependent and protein kinase C-dependent and -independent pathways.

The lipid mediator platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, AGEPC) has been shown to elicit several important biochemical signaling responses in mammalian cells, including polyphosphoinositide hydrolysis, arachidonic acid release/eicosanoid production, and protein tyrosine phosphorylation. In the present study, the roles of Ca2+ and protein kinase C (PKC), two signaling components of the phospholipase C pathway, in AGEPC-stimulated eicosanoid production and protein tyrosine phosphorylation, were investigated in cultured rat Kupffer cells. AGEPC at nanomolar concentrations induced an increase in intracellular calcium concentration ([Ca2+]i), stimulated membrane PKC activity, and resulted in protein tyrosine phosphorylation. The maximal increase in [Ca2+]i and membrane PKC activity in response to AGEPC were observed within 30-50 s, whereas the AGEPC-induced protein tyrosine phosphorylation reached maximal levels within 2-5 min. [Ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) but not 8-(N,N-diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), an inhibitor of calcium release from intracellular compartments, nearly abolished the AGEPC-induced increase in [Ca2+]i suggesting involvement of extracellular calcium influx in this event. Both EGTA and TMB-8 abolished or inhibited AGEPC-stimulated protein tyrosine phosphorylation and eicosanoid formation, respectively. The calcium ionophore A23187 alone stimulated eicosanoid production and protein tyrosine phosphorylation with an identical pattern to that of AGEPC. Phorbol myristate acetate (PMA), an activator of PKC, which did not affect [Ca2+]i, mimicked the actions of AGEPC, stimulating eicosanoid production and promoting tyrosine phosphorylation of a set of proteins similar to those phosphorylated following AGEPC stimulation. AGEPC-enhanced tyrosine phosphorylation of some of the protein substrates and eicosanoid production were inhibited in cells "down-regulated" for PKC. Furthermore, both PMA- and AGEPC-stimulated eicosanoid production and protein tyrosine phosphorylation were attenuated or abolished by at least one of the PKC inhibitors, staurosporine, and calphostin C. Taken together, these results are consistent with the conclusions that: (a) AGEPC stimulates the phospholipase-mediated arachidonic acid release/eicosanoid synthesis cascade and protein tyrosine phosphorylation through extracellular Ca(2+)-dependent and PKC-dependent and -independent mechanism(s) and (b) the Ca(2+)-PKC interaction determines the efficacy of the AGEPC-stimulated cellular events.

Animals↗
PubMed1992

The human platelet as a reproducible and sensitive cell for the detection and assay of platelet-activating factor.

A new procedure for the preparation of human platelets consistently sensitive to platelet-activating factor (PAF) in the low nanomolar range has been developed. Key to the success of this approach was the addition of adenosine during the isolation phase, providing an excellent recovery of stable cells, and the inclusion of ADP in the aggregation assay, providing increased sensitivity to PAF. Examination of the binding profile of tritium-labeled PAF to these platelets in the presence or absence of ADP revealed significant difference in the Kd values but not in the number of specific binding sites. Other reagents having an influence on the reactivity and stability of the human platelets, as regards its interaction with PAF, are described.

Adenosine↗
PubMed1991

Paracrine interactions between platelet-activating factor and prostaglandins in hormonally-treated human luteal phase endometrium in vitro.

Stromal cells and epithelial glands were separated after enzymic digestion of specimens obtained from 27 women at hysterectomy or endometrial biopsy during the luteal phase, and then cultured to confluence in vitro. PGE release into the culture medium (mean +/- s.e.m.: ng/mg protein/24 h) from gland cell cultures was not changed by oestradiol (17.6 +/- 1.3 for control and 25.5 +/- 2.8 for oestradiol, respectively). However, in the presence of oestradiol, PAF (5 ng/ml) significantly elevated PGE release to 44.2 +/- 5.8. No stimulation was observed in the presence of progesterone. Stromal cell medium had no effect on PGE release in gland cell cultures. PGE release was always much lower in stromal cell cultures than in glands (control: 4.7 +/- 0.6). PAF stimulated PGE release in the presence of oestradiol in these cells also; gland cell medium was without effect. In co-cultures of glandular and stromal cells, PGE release was more similar to that seen in gland cell cultures, with PAF being stimulatory under the influence of oestradiol. PGF release into the medium from the same gland cell cultures was significantly elevated by hormonal treatment, being greatest (62.0 +/- 11.3) with oestradiol alone, and was strongly inhibited in all wells by addition of PAF and stromal cell medium. In stromal cell cultures without hormonal addition, PGF levels (15.0 +/- 2.4) were similar to those seen in glands (18.1 +/- 3.1), and no stimulation was achieved by oestradiol (29.6 +/- 5.9). PAF was inhibitory on PGF release, while gland cell medium was without effect. Co-cultures gave PGF values generally similar to those of stromal cells; oestradiol was again stimulatory (55.0 +/- 9.3). PAF was significantly inhibitory in the presence of oestradiol. PAF (mean +/- s.e.m.: pmol/mg protein/24 h using a platelet serotonin release assay) in stromal cells was significantly increased from control [M199 alone] (0.31 +/- 0.12) by progesterone (1.00 +/- 0.17). Addition of PGE-2 (7.5 ng/ml) to progesterone-treated wells further increased PAF concentration (5.34 +/- 0.09), but was without effect in wells receiving oestradiol alone. Wells exposed to both hormones exhibited an intermediate response. Similar results were obtained with addition of gland cell culture medium, presumably due to its endogenous PGE content. In co-cultures, PAF concentrations were significantly elevated by progesterone alone (4.78 +/- 0.78) or when combined with oestradiol (2.38 +/- 0.51), but not by oestradiol alone. Treatment with PGE-2 caused no additional stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)

Cells, Cultured↗
PubMed1990

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.

Animals↗
PubMed1990

The inhibition of platelet-activating factor-induced platelet activation by oleic acid is associated with a decrease in polyphosphoinositide metabolism.

In an earlier study (Miwa, M., Hill, C., Kumar, R., Sugatani, J., Olson, M. S., and Hanahan, D. J. (1987) J. Biol. Chem. 262, 527-530) it was shown that an inhibitor of platelet-activating factor (PAF), a powerful endogenous mediator of platelet aggregation, was present in freeze-clamped perfused livers. Subsequently, we determined that this substance was a mixture of unsaturated free fatty acids (FFA). Among these FFA, oleic acid between 10 and 100 microM was found to be a potent inhibitor of PAF-induced platelet aggregation and serotonin secretion. Consequently, in order to understand the molecular mechanism of oleic acid action, we investigated the effects of this FFA on several biochemical events associated with platelet aggregation induced by PAF. The effect of oleic acid and/or PAF on the level of [32P]phosphatidylinositol 4-phosphate (PIP) and [32P]phosphatidylinositol 4,5-bisphosphate (PIP2) was examined by using platelets labeled with [32P]phosphate. Oleic acid induced a dose-dependent decrease in the levels of [32P]PIP and [32P]PIP2; a maximal decrease in [32P]PIP and [32P]PIP2 of approximately 50 and 25%, respectively, was observed within seconds after the addition of 20 microM oleic acid and persisted for at least 15 min. Oleic acid did not induce the formation of [3H]inositol phosphates in platelets prelabeled with [3H]inositol, suggesting that the decrease in [32P]PIP and [32P]PIP2 was not due to a stimulation of phospholipase C. In contrast to oleic acid, PAF induced a dose-dependent increase in the [32P]PIP level, reaching a maximum of approximately 200% 3 min after the addition of 1 nM PAF to the platelets. This increase in [32P]PIP was accompanied by platelet aggregation and secretion, and a close correlation was established between the [32P]PIP level and the degree of aggregation. Oleic acid and PAF, when added together to the platelets, interacted by affecting the level of [32P]PIP and [32P]PIP2 in an opposite way since the decrease in the level of [32P]PIP and [32P] PIP2 induced by oleic acid was partially reversed by an excess of PAF. The decrease in the levels of [32P] PIP and [32P]PIP2 caused by oleic acid was associated with an inhibition of platelet aggregation induced by PAF. Interestingly, oleic acid did not block [3H]PAF binding to platelets but inhibited the PAF-induced phosphorylation of platelet proteins of 20 kDa and 40 kDa. These results suggest that inhibition of the PAF response by oleic acid may be at one of the steps in the signal transduction.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Regulation of platelet-activating factor receptor and platelet-activating factor receptor-mediated biological responses by cAMP in rat Kupffer cells.

Treatment of cultured Kupffer cells with the beta-adrenergic agonist isoproterenol (10 microM) for a short period of time (30 min) attenuated the subsequent platelet-activating factor (PAF)-induced arachidonic acid release and cyclooxygenase-derived eicosanoid (e.g. thromboxane B2 and prostaglandin E2) production. This effect of isoproterenol was highly specific since the alpha-adrenergic agonist phenylephrine and the beta-adrenergic antagonist propranolol had no effect on the stimulatory effect of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC). The inhibitory effect of isoproterenol on the AGEPC-induced arachidonic acid release was demonstrated through the use of a specific beta-adrenergic subtype agonist and antagonist to be mediated by beta 2-adrenergic receptors on Kupffer cells. These inhibitory effects of isoproterenol can be mimicked by dibutyryl cAMP but not by dibutyryl cGMP, suggesting that a cAMP-dependent mechanism is likely involved in the regulatory action of isoproterenol. Ligand binding studies indicated that short term (i.e. 30 min) treatment of the cultured Kupffer cells with either isoproterenol or dibutyryl cAMP had no effect on the specific [3H]PAF binding. However, long term incubation (9-24 h) with dibutyryl cAMP caused down-regulation of the PAF receptors in rat Kupffer cells. Forskolin (0.1 mM), an adenylyl cyclase activator, down-regulated the surface expression of the AGEPC receptors more rapidly, decreasing the specific [3H]AGEPC binding by approximately 40% within 2 h. The receptor regulatory effect of dibutyryl cAMP and forskolin was time- and concentration-dependent. These observations suggest that a cAMP-dependent mechanism coupled with beta 2-adrenergic receptors may have important regulatory effects on the PAF receptor and post-receptor signal transducing mechanisms for PAF in hepatic Kupffer cells.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1990

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.

Animals↗
PubMed1990

Isolation of platelet-activating factor and purification by thin-layer chromatography.

Isolation and purification of platelet-activating factor can be accomplished with reasonable ease and effectiveness given the precautions and suggestions outlined above. These experimental approaches represent a composite of those practiced in the author's laboratory combined with selected observations reported in the literature.

Chromatography, Thin Layer↗
PubMed1990

Novel route to preparation of high purity lysoplasmenylethanolamine.

A rapid and simple method has been developed for the preparation of highly purified lysoplasmenylethanolamine. The starting material, a phosphatidylethanolamine (PE) sample that contained a mixture of the 1, 2 diacyl- and 1-O-alkenyl-2-acyl forms was subjected to mild alkaline methanolysis for 20 min at room temperature. Addition of chloroform and water with vigorous mixing, but without acidification at this point, led to a preferential retention of the lysoplasmenylethanolamine in the alkaline aqueous phase and complete separation of the methyl esters into the chloroform phase. Neutralization of the alkaline phase with dilute acetic acid, followed by addition of chloroform, allowed recovery of the lysoplasmenylethanolamine in the chloroform phase in very high yields (75-80% based on vinyl ether content of starting material). On the other hand, a preparation of cholineglycerophospholipids enriched in plasmenylcholine, treated in exactly the same manner, gave a lysoplasmenylcholine that was not retained in the alkaline phase, but partitioned primarily into the chloroform-rich phase together with the methyl esters. Characterization of the purified lysoplasmenylethanolamine was achieved by thin-layer chromatography and compositional analysis. In addition, fast atom bombardment mass spectral analysis of the intact lysoplasmenylethanolamine together with gas chromatography-mass spectrometry of the dimethyl acetals derived from the 1-O-alkenyl chains allowed further proof of the structure and an assessment of the purity of this compound.

Chromatography, Thin Layer↗
PubMed1989

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)

Animals↗
PubMed1989

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.

Animals↗
PubMed1989

Inhibition of [3H]platelet activating factor (PAF) binding by Zn2+: a possible explanation for its specific PAF antiaggregating effects in human platelets.

Zinc ions in the micromolar range exhibited a strong inhibitory activity toward platelet activating factor (PAF)-induced human washed platelet activation, if added prior to this lipid chemical mediator. The concentration of Zn2+ required for 50% inhibition of aggregation (IC50) was inversely proportional to the concentration of PAF present. The IC50 values (in microM) for Zn2+ were 8.8 +/- 3.9, 27 +/- 5.8, and 34 +/- 1.7 against 2, 5, and 10 nM PAF, respectively (n = 3-6). Zn2+ exhibited comparable inhibitory effects on [3H]serotonin secretion and the IC50 values (in microM) were 10 +/- 1.2, 18 +/- 3.5, and 35 +/- 0.0 against 2, 5, and 10 nM PAF, respectively (n = 3). Under the same experimental conditions, aggregation and serotonin secretion induced by ADP (5 microM), arachidonic acid (3.3 microM), or thrombin (0.05 U/ml) were not inhibited. Introduction of Zn2+ within 0-2 min after PAF addition not only blocked further platelet aggregation and [3H]serotonin secretion but also caused reversal of aggregation. Analysis of [3H]PAF binding to platelets showed that Zn2+ as well as unlabeled PAF prevented the specific binding of [3H]PAF. The inhibition of [3H]PAF specific binding was proportional to the concentration of Zn2+ and the IC50 value was 18 +/- 2 microM against 1 nM [3H]PAF (n = 3). Other cations, such as Cd2+, Cu2+, and La3+, were ineffective as inhibitors of PAF at concentrations where Zn2+ showed its maximal effects. However, Cd2+ and Cu2+ at high concentrations exhibited a significant inhibition of the aggregation induced by 10 nM PAF with IC50 values being five- and sevenfold higher, respectively, than the IC50 for Zn2+, and with the IC50 values for inhibition of binding of 1 nM [3H]PAF being 5 and 19 times higher, respectively, than the IC50 for Zn2+. The specific inhibition of PAF-induced platelet activation and PAF binding to platelets suggested strongly that Zn2+ interacted with the functional receptor site of PAF or at a contiguous site.

Arachidonic Acid↗
PubMed1989

Beta 2-adrenergic agonist regulation of immune aggregate- and platelet-activating factor-stimulated hepatic metabolism.

Vasoconstriction and subsequent glycogenolysis stimulated by immune complex infusion into perfused rat livers was inhibited by prior infusion of isoproterenol. Similarly, isoproterenol inhibited the biosynthesis of bioactive lipid autacoids such as platelet-activating factor, prostaglandin E2, and thromboxane B2 which was stimulated by immune aggregates. The adrenergic receptor specificity of these effects was determined through the use of specific adrenergic subtype-specific agonists and antagonists to be mediated by beta 2-adrenergic receptors. Indirect evidence for the differential expression of hepatic sinusoidal and parenchymal beta-adrenergic receptors in the male rat during ontogeny suggested that inhibition of immune aggregate-stimulated autacoid biosynthesis, vasoconstriction, and glycogenolysis by isoproterenol occurs at a sinusoidal locus, most likely Kupffer cells. In contrast with the ability of beta 2-adrenergic agonists to inhibit immune aggregate- and platelet-activating factor-stimulated hepatic metabolism, dibutyryl cyclic AMP did not mimic these sinusoidal beta 2-adrenergic effects, despite stimulating hepatic parenchymal cell glycogenolysis as effectively as isoproterenol. These observations suggest a role for cyclic AMP-independent mechanisms in the regulation of heterologous stimulus-response coupling by hepatic sinusoidal beta 2-adrenergic receptors.

Animals↗