Characterization of 3H-labelled platelet activating factor receptor complex solubilized from rabbit platelet membranes

Rabbit platelet membranes, preincubated with 3H-labeled platelet activating factor ([3H]PAF), were solubilized with 2% digitonin. Sedimentation of the detergent extract in a sucrose density gradient revealed a major labeled component with a sedimentation coefficient (s20,omega) of 10.5 S, which was substantially diminished when an excess of unlabeled PAF or L-652,731, (trans-2,5-bis(3,4,5-trimethoxyphenyl)tetrahydrofuran), (PAF antagonist) was present in the preincubation mixture, suggesting that the 10.5 S component is a specific receptor-bound [3H]PAF complex. Gel filtration of the [3H]PAF-receptor complex on Sephacryl S-300 revealed a single radiolabeled fraction with an apparent Stokes' radius of 4.9 nm. The apparent molecular weight and the frictional ratio of the agonist-receptor complex were computed to be 220,000 and 1.13, respectively. Dissociation of [3H]PAF from the radioligand-receptor complex was facilitated by Na+ and Li+, whereas K+ and Cs+ were ineffective. The guanine nucleotide, GTP, was also found to promote the dissociation in a manner that is additive with the effect of Na+, suggestive of the coupling of a guanine nucleotide binding protein to the solubilized PAF-receptor complex.     

Solubilization and Characterization of the A2-Adenosine Receptor

Abstract

Binding of [³H]CGS 21680, an agonist radioligand selective for A₂-adenosine receptors (A₂AR), to membranes and solubilized preparations from bovine brain striatum revealed labelling of a single high affinity binding state. In membranes, guanine nucleotides per se were ineffective in modulating agonist binding whereas cations, Na⁺ and Mg⁺⁺, had distinct effects. The addition of NaCl (200 mM) as well as the Mg⁺⁺-free preparation of membranes led to a significant decrease in binding affinity and the number of binding sites. Moreover, the presence of Na⁺ was required for the demonstration of a guanine nucleotide effect, i.e. a decrease in maximal binding. Following solubilization, agonist-A AR interactions were sensitive to guanine nucleotides even in the absence of Na⁺²; guanine nucleotides and Na⁺ had additive effects in reducing the number of binding sites. Moreover, the effect of GTP was reversible, i.e. binding returned to control levels upon removal of the nucleotide. This suggests the A₂AR and its G protein (presumably G_S) are solubilized as a functional unit and may not dissociate even in the presence of GTP following solubilization. We, therefore, believe that a “tight” association exists between receptor and G protein (G_S), and that guanine nucleotides and sodium act at different sites on the R–G complex. Drawing an analogy with similar observations on the avian β-adrenergic receptor (Hertel et al, J.Biol.Chem. 265:17988–94, 1990; Parker & Ross, J.Biol.Chem. 266:9987–96, 1991) we postulate that the regulatory features of the A₂AR can be attributed to a distinct receptor domain that interacts with cellular regulatory elements.     

ADP- and epinephrine-elicited release of [3H]guanylylimododiphosphate from platelet membranes: Implications for receptor-Ni stoichiometry

Epinephrine-promoted release of [³H]guanylylimidodiphosphate ([³H]Gpp(NH)p) from human platelet membranes has been used to probe the interactions between alpha₂-adrenergic recpetors and N_i, the guanine nucleotide binding protein that couples those receptors to an inhibition of adenylate cyclase activity. We show here that ADP, which also acts through specific platelet receptors to inhibit adenylate cyclase activity, also promotes the release of [³H]Gpp(NH). The amount of [³H]Gpp(NH)-release elicited by epinephrine and by ADP together is equal to the sum of the amounts released by the two agents acting individually. Furthermore the maximal amounts of [³H]Gpp(NH)-release elicited by each of the two agents approximates the numbers of receptors for ADP and epinephrine present in the platelet membranes. These results suggest that the two receptor types interact with distinct portions of the pool of N_i molecules and that each receptor initiates guanine-nucleotide exchange on a single molecule of N_i.     

Reconstitution into liposomes of the B°-like glutamine-neutral amino acid transporter from renal cell plasma membrane

Na⁺ dependent [³H]glutamine uptake was found in liposomes reconstituted with solubilized rat kidney brush border in the presence of intraliposomal K⁺. The reconstituted system was optimised with respect to the critical parameters of the cyclic detergent removal procedure, i.e., the detergent used for the solubilization, the protein concentration, the detergent/phospholipid ratio and the number of passages through a single Amberlite column. Time dependent [³H]glutamine accumulation in proteoliposomes occurred only in the presence of external Na⁺and internal K⁺. The transporter showed low if there is any tolerance towards the substitution of Na⁺ or K⁺ for other cations. Valinomycin strongly stimulated the transport indicating that it is electrogenic. Intraliposomal glutamine had no effect. From the dependence of the transport rate on the Na⁺ concentration cooperativity index close to 1 was derived, indicating that 1 Na⁺ should be involved in the cotransport with glutamine. The electrogenicity of the transport originated from the Na⁺ transport. Optimal rate of 0.1 mM [³H]glutamine uptake was found in the presence of 50 mM intraliposomal K-gluconate. At higher K-gluconate concentrations the transport rate decreased. The activity of the reconstituted transporter was pH dependent with optimal function in the range pH 6.5-7.0. [³H]glutamine (and [³H]leucine) uptake was inhibited by all the neutral but not by the positively or negatively charged amino acids. The sulfhydryl reagents HgCl₂, mersalyl, p-hydroxymercuribenzoate and the substrate analogue 2-aminobicyclo[2,2,1]heptane-2-carboxylate strongly inhibited the transporter, whereas the amino acid analogue α-(methylamino)isobutyrate had no effect. The inhibition by mersalyl was protected by the presence of the substrate. On the basis of the Na⁺ dependence, the electrogenic transport mode and the specificity towards the amino acids, the reconstituted transporter was classified as B°-like.     

Presence of Platelet-Activating Factor and Its Receptor in Baboon (Papio spp) Spermatozoa

Platelet-activating factor [1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine; PAF] is a novel potent signaling phospholipid which has unique pleiotropic biological properties in addition to platelet activation. PAF has been detected in the spermatozoa in several species. The concentration of PAF is inversely related to human spermatozoal quality. PAF is present in squirrel monkey (a seasonal breeder) spermatozoa and is significantly higher during the breeding season. PAFs mechanism of action is a receptor-mediated event. There is no report on the presence of PAF or the PAF-receptor in nonhuman Old World primate spermatozoa. Therefore, the primary objective of this study was to determine if PAF is present in the spermatozoa from baboons, which are year-round breeders. A secondary objective was to determine the presence and localization of the PAF-receptor in spermatozoa. We extracted endogenous lipids from mature hybrid baboon (Papio spp) epididymal spermatozoa and assayed them for the presence of PAF by [ ¹²⁵ I]-radioimmunoassay. We also exposed baboon spermatozoa to PAF-receptor antibody followed by FITC-conjugated antibody. PAF was in all samples assayed (mean: 2.29 (±0.63) pmol/10 ⁶ spermatozoa). Baboon spermatozoa possess PAF-receptors most prevalently along the neck and midpiece regions. The data demonstrates that PAF and its receptor are present in baboon spermatozoa. Additional studies will elucidate the role of PAF in spermatozoal function.     

Endogenous phosphorylation of platelet membrane proteins.

The characteristics of the phosphorylating activity of platelet membranes have been studied. Plasma membranes of human platelets isolated by the glycerol lysis technique were shown to incorporate significant amounts of [³²P]phosphate into specific membrane proteins. This activity was only partially cyclic 3′:5′-monophosphate (cyclic AMP)-dependent but had most of the other characteristics of protein kinases derived from other sources. Maximal stimulation of endogenous phosphorylation was obtained at 1 × 10^?7, m cyclic AMP and exceeded by approximately 30% the [³²P]phosphate incorporation in the absence of this cyclic nucleotide. The platelet membrane protein kinase was able to phosphorylate exogenous proteins, e.g., histone, fibrinogen etc., as well as endogenous membrane proteins. The latter solubilized by sodium dodecyl sulfate and separated by dodecyl sulfate-polyacrylamide gel electrophoresis incorporated [³²P]phosphate into three polypeptides of apparent molecular weights 52,000, 31,000, and 20,000. The phosphorylation of the polypeptide of molecular weight 52,000 was cyclic AMP-dependent.     

Monoclonal anti-idiotypic antibodies to platelet activating factor (PAF) and their interaction with PAF receptors.

Monoclonal anti-idiotypic antibodies (3C3F3E4 and 10D3F8H7) that interact with platelet activating factor (PAF) receptors were generated using an auto-anti-idiotypic approach by immunizing mice with an aldehydic analog of PAF coupled to bovine thyroglobulin. The resulting hybridomas were screened for anti-idiotypic antibody (anti-anti-PAF) with F(ab')2 fragments of affinity-purified polyclonal rabbit anti-PAF antibody. These antibodies displayed internal image properties of PAF and were considered as Ab2 beta according to the following criteria: (a) they bound to F(ab')2 fragments of the affinity-purified rabbit polyclonal anti-PAF antibody that had high affinity for PAF; (b) they inhibited [3H]PAF binding to rabbit polyclonal anti-PAF antibody and its F(ab')2 fragment in a concentration-dependent manner; (c) they displaced [3H]PAF from the anti-PAF antibody/[3H]PAF complex specifically; (d) they inhibited [3H]PAF binding to PAF receptors on rabbit platelet membranes dose dependently; (e) they displaced [3H]PAF from the [3H]PAF/PAF receptor complex specifically; and (f) they stimulated rabbit platelets to aggregate, and this aggregation could be inhibited or totally blocked by specific PAF receptor antagonists WEB 2086 and SRI 63-441. All of the above are consistent with the first successful production of monoclonal antibodies that mimic PAF and interact specifically with the PAF binding domain of PAF receptors on rabbit platelet membranes.     

A specific, photolabile and irreversible antagonist (L662,025) of the PAF-receptor

PAF (0.2 microM) induced maximal platelet aggregation in human PRP and [3H]-PAF (1-5 nM) binding to platelet membrane preparations had Kd value of 3.8 nM and Bmax of 200 fmoles/mg of protein. Without UV irradiation, a synthetic azido tetrahydrofuran derivative L662,025 was a reversible and competitive PAF-receptor antagonist with IC50 values of 5.6 +/- 0.3 microM (platelet aggregation) and 1.0 +/- 0.25 microM (receptor binding). Photolysis of L662,025 in the presence of PRP produced an irreversible inhibition of platelet aggregation and specific binding of [3H]-PAF (1 nM). L662,025 did not affect collagen- or ADP-induced human platelet aggregation before or after photolysis. It is a new probe that can be used to identify and characterize the PAF-receptor.     

Ciliary ganglion neurons in cell culture: high affinity choline uptake and autoradiographic choline labeling

Chick ciliary ganglion neurons grown in dissociated cell culture have a high affinity uptake mechanism for choline that has the properties expected for cholinergic neurons. The uptake has an apparent K_m of ca. 0.3 μM and is blocked by addition of 10 μM hemicholinium-3 or replacement of Na⁺ by Li⁺ in the uptake medium. When the choline uptake mechanism is used to label ciliary ganglion neuron-myotube cultures autoradiographically, over 99% of the neurons are labeled. A few cells with neuronal morphologies in such cultures (<1%) are labeled by γ-[³H]aminobutyric acid uptake. The number of [³H]choline-labeled neurons and the amount of Na⁺-dependent choline uptake is the same for ciliary ganglion neurons grown with and without skeletal myotubes. Rat superior cervical ganglion neurons, grown in cell culture under conditions that induce them to synthesize acetylcholine and form cholinergic synapses, are labeled by [³H]choline uptake, though not as heavily as ciliary ganglion neurons. In contrast, chick dorsal root ganglion neurons, a presumed population of noncholinergic neurons, are not labeled by [³H]choline uptake. Thus high affinity choline uptake can be used to label autoradiographically the cholinergic neurons tested, while at least one population of noncholinergic neurons remains unlabeled.     

Solubilization and Characterization of Rat Brain α2-Adrenergic Receptor

alpha 2-Adrenergic receptors labelled by [3H]-clonidine (alpha 2-agonist) can be solubilized from the rat brain in a form sensitive to guanine nucleotides with a zwitterionic detergent, 3-[3-(cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS). About 40% of the original [3H]CLO binding sites in the membranes were solubilized with 6 mM CHAPS. Separation of the soluble [3H]CLO-bound complex was performed by the vacuum filtration method using polyethylenimine-treated GF/B filters. Solubilized [3H]CLO binding sites retained the same pharmacological characteristics of membrane-bound alpha 2-adrenergic receptors. Scatchard plots of [3H]CLO binding to solubilized alpha 2-receptors were curvilinear, indicating the existence of the two distinct binding components. Solubilized receptors were eluted as a single peak from Bio-Gel A-1.5 m column with a Stokes radius of 6.6 nm. The isoelectric point was 5.6-5.8. Regulations of the receptor binding by guanine nucleotides, monovalent cations, and sulfhydryl-reactive agents were maintained intact in the soluble state, whereas those by divalent cations were lost. The apparent retention of receptors and guanine nucleotide binding regulatory component(s) in the soluble state may allow a investigation of the regulation mechanisms of the brain alpha 2-adrenergic receptor system at the molecular level.     

Guanine nucleotide regulation of [3H]vasopressin binding to liver plasma membranes and solubilized receptors. Evidence for the involvement of a guanine nucleotide regulatory protein.

A guanine nucleotide regulatory protein may be involved in vasopressin-receptor-mediated polyphosphoinositide breakdown in rat liver. Therefore we examined the effects of the non-hydrolysable guanine nucleotide guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) on [3H]vasopressin ([3H]AVP) binding to hepatic plasma membranes and detergent extracts. [3H]AVP bound to a single set of high-affinity binding sites in membranes. Addition of p[NH]ppG decreased the affinity of receptor binding without altering the maximal binding capacity. The rate of dissociation of [3H]AVP from membrane-bound receptors was also enhanced by p[NH]ppG. Solubilization of [3H]AVP-prelabelled membranes with dodecyl beta-D-maltoside resulted in a [3H]AVP-receptor complex that was unstable in solution. Incubation of these extracts for 5 min at 30 degrees C resulted in a 40% loss of bound [3H]AVP, whereas in the presence of p[NH]ppG there was a 54% loss. However, when membranes were prelabelled with [3H]AVP and p[NH]ppG and then solubilized, the resulting hormone-receptor complex was still temperature-labile but insensitive to the further addition of p[NH]ppG. The molecular size of soluble vasopressin receptors was estimated by gel filtration. The [3H]AVP-receptor complex was eluted as a single peak with an apparent molecular size of 258 kDa. However, no peak was detected when solubilized extract was made from membranes prelabelled with [3H]AVP and p[NH]ppG, suggesting that this receptor complex had dissociated during chromatography. It is possible therefore that the high-Mr complex contains the hormone, its receptor and a guanine nucleotide binding protein.     

Characterization of a Digitonin-Solubilized Bovine Brain H3 Histamine Receptor Coupled to a Guanine Nucleotide-Binding Protein

The H3 receptor is a high-affinity histamine receptor that inhibits release of several neurotransmitters, including histamine. We have characterized H3 receptor binding in bovine brain and developed conditions for its solubilization. Particulate [3H]histamine binding showed an apparently single class of sites (KD = 4.6 nM; Bmax = 78 fmol/mg of protein). Of the detergents tested, digitonin at a detergent/protein ratio of 1:1 (wt/wt) yielded the greatest amount of solubilized receptors, typically 15-30% of particulate binding. Neither equilibrium binding of [3H]histamine to receptors (KD = 6.1 nM; Bmax = 92 fmol/mg of protein) nor the inhibitor profile was substantially altered by digitonin solubilization. However, solubilization did increase the rate of [3H]histamine association with and dissociation from the receptor. Size-exclusion chromatography indicated an apparent molecular weight of 220,000 for the solubilized receptor, and peak binding from this column retained its guanine nucleotide sensitivity. These last two observations are consistent with the solubilized receptor occurring in complex with a guanine nucleotide-binding protein.     

Two different modes of inhibition of the rat brain Na+,K+-ATPase by triterpene glycosides,psolusosides A and B from the holothurian Psolus fabricii

Effects of two triterpene glycosides, isolated from the holothurian Psolus fabricii, on rat brain Na⁺,K⁺-ATPase (Na,K-pump; EC 3.6.1.3) were investigated. Psolusosides A and B (PsA and PsB) inhibited rat brain Na⁺,K⁺-ATPase with I₅₀ values of 1×10⁻⁴ M and 3×10⁻⁴ M, respectively. PsA significantly stimulated [³H]ATP binding to Na⁺,K⁺-ATPase, weakly increased [³H]ouabain binding to the enzyme, and inhibited K⁺-phosphatase activity to a smaller degree than the total reaction of ATP hydrolysis. In contrast, PsB decreased [³H]ATP binding to Na⁺,K⁺-ATPase, and had no effect on [³H]ouabain binding to the enzyme. K⁺-Phosphatase activity was inhibited by PsB in parallel with Na⁺,K⁺-ATPase activity. The fluorescence intensity of tryptophanyl residues of Na⁺,K⁺-ATPase was increased by PsA and decreased by PsB in a dose-dependent manner. The excimer formation of pyrene, a hydrophobic fluorescent probe, was decreased by PsA only. The different characteristics of inhibition mode for these substances were explained by peculiarities of their chemical structures and distinctive affinity to membrane cholesterol.     

Ca2+-Guanine Nucleotide Interactions in Brain Membranes. II. Characteristics of [3H]Guanosine Triphosphate and [3H]β, γ-Imidoguanosine 5'-Triphosphate Binding and Catabolism in the Rat Hippocampus and Striatum

Abstract: With [³H]guanosine triphosphate ([³H]GTP) and [³H]β, γ -imidoguanosine 5′-triphosphate ([³H]GppNHp) as the labelled substrates, both the binding and the catabolism of guanine nucleotides have been studied in various brain membrane preparations. Both labelled nucleotides bound to a single class of noninteracting sites (K_D= 0.1-0.5 μm ) in membranes from various brain regions (hippocampus, striatum, cerebral cortex). Unlabelled GTP, GppNHp, and guanosine diphosphate (GDP) but not guanosine monophosphate (GMP) and guanosine competitively inhibited the specific binding of [³H]guanine nucleotides. Calcium (0.1–5 mm ) partially prevented the binding of [³H]GTP and [³H]GppNHp to hippocampal and striatal membranes. This resulted from both an increased catabolism of [³H]GTP (into [³H]guanosine) and the likely formation of Ca-guanine nucleotide^2- complexes. The blockade of guanine nucleotide catabolism was responsible for the enhanced binding of [³H]GTP to hippocampal membranes in the presence of 0.1 mm -ATP or 0.1 mm -GMP. Striatal lesions with kainic acid produced both a 50% reduction of the number of specific guanine nucleotide binding sites and an acceleration of [³H]GTP and [³H]GppNHp catabolism (into [³H]guanosine) in membranes from the lesioned striatum. This suggests that guanine nucleotide binding sites were associated (at least in part) with intrinsic neurones whereas the catabolising enzyme(s) would be (mainly) located to glial cells (which proliferate after kainic acid lesion). The characteristics of the [³H]guanine nucleotide binding sites strongly suggest that they may correspond to the GTP subunits regulating neurotransmitter receptors including those labelled with [³H]5-hydroxytryptamine ([³H]5-HT) in the rat brain.     

Some in vivo and in vitro effects of ethacrynic acid on renal Na+,K+ -ATPase

Binding of [¹⁴C]ethaerynic acid [EA]at concentrations of EA from 10^?4m to 10^?2m to a membrane preparation containing Na⁺,K⁺-ATPase activity in vitro occurred in a nonsaturable manner; binding was stimulated by Na⁺ or K⁺, but was not affected by Mg²⁺ and/or ATP. [¹⁴C]EA significantly bound to a microsomal preparation with low Na⁺,K⁺-ATPase activity as well as to a heat-denatured enzyme; this binding reaction was not stimulated by Na⁺. These observations suggest that EA binds non-specifically or to nonspecific sites on membrane preparations. Nonselective binding of [¹⁴C]EA to subcellular particles after fractionation of slices also suggested the presence of nonspecific EA binding sites in vivo. In vitro [³H]ouabain binding to medullary and cortical Na⁺,K⁺-ATPase preparations was partially reduced by pretreatment with EA. On the other hand, [¹⁴C]EA binding to Na⁺,K⁺-ATPase was not affected by pretreatment of the preparation with ouabain (10^?6m to 5 × 10^?4m). EA reduced the sensitivity of [³H]ouabain binding to the enzyme preparation to Na⁴ and K⁺.EA was infused (0.1, 1.0, and 10 mg/min) into one renal artery of hydropenic dogs. A prompt natriuresis in the infused kidney occurred. Similar changes were observed in the contralateral kidney 20 min after starting the infusion. Both kidneys were removed 30 min after the beginning of the infusion, and Na⁺,K⁺-ATPase was isolated from the cortex and the medulla. Enzyme activity from cortex and medulla of either kidney was not significantly different from enzyme activity from cortex and medulla of control, uninfused dogs, regardless of dose of EA or method of enzyme isolation. Furthermore, in vitro binding of [³H]ouabain to Na⁺,K⁺-ATPase membrane preparations from cortex and medulla was the same for experimental and control kidneys. In vitro incubation of 2 × 10^?3m EA with a membrane preparation caused the same inhibition of ATPase activity when the enzyme was isolated either from control or EA-infused dogs. The inhibition could not be reversed by recentrifugation or rehomogenization of the enzyme. Our results do not support the concept that Na⁺,K⁺-ATPase is a pharmacological receptor for ethacrynic acid.     

Lack of specificity in cation effects on solubilized benzodiazepine receptor

Receptors for benzodiazepines (BZ) and -carboline-carboxylic acid ethyl ester (-CCE) has been solubilized with decanoly-N-methylglucamide (DMG), a new kind of nonionic detergent. The apparent dissociation constants of diazepam and -CCE for solubilized receptor were similar to those for synaptic membranes. Sucrose density gradient centrifugation of the solubilized receptor protein revealed that the binding profile of [³H]-CCE essentially parallels that of [³H]diazepam and that both sedimentation coefficients were 10.5S. Co²⁺ and Ni²⁺, which increase [³H]diazepam binding and decrease [³H]-CCE binding to synaptic membranes, remarkably increased the binding of both to the solubilized receptor. Mg²⁺ and Ca²⁺, which had no effect on membrane receptor binding, also enhanced [³H]diazepam and [³H]-CCE binding to the solubilized receptor. The increase in binding in the presence of these divalent cations was due to a change in the apparent number of binding sites, with no change in binding affinities. The relative lack of specificity in divalent cation effects on solubilized BZ receptor may be caused by separation or destruction of the cation recognition site or channel of the BZ receptor complex by solubilization of the synaptic membrane with DMG.     

Stoichiometry for guanine nucleotide binding to tubulin under polymerizing and nonpolymerizing conditions

The maximal stoichiometry for [³H]GTP binding to depolymerized tubulin with saturating amounts of added [³H]GTP is 0.4 mol/110,000 g protein. In contrast, 1 mol of radioactive nucleotide is incorporated into microtubules as a result of polymerization with [³H]GTP. The different stoichiometries result from a difference in the nucleotide binding properties of ring protein under polymerizing and nonpolymerizing conditions: ring protein at 0 °C is devoid of binding activity but binds added radioactive guanine nucleotide during microtubule assembly. The radioactive nucleotide which is incorporated into rings during microtubule assembly is not displaced by excess GDP, although it is at a site which is distinct from the N site.     

Acetylcholine receptor-mediated sodium ion efflux after rapid hypoosmotic loading of radiotracer

Torpedo membrane vesicles (microsacs) enriched in acetylcholine receptors can be loaded with radiotracer permeants such as ²²Na⁺ and [³H]sucrose in only a few minutes. The fast loading technique employs exposure of vesicles to hypoosmotic solutions containing the radiotracers. Subsequent to loading of permeants, their efflux from the vesicles can be determined by a Millipore filtration technique. Efflux rates following hypoosmotic loading are comparable to those following the conventional loading technique which employs isotonic solutions. Hence, efflux measurements can be begun immediately after vesicle preparation. Furthermore, simultaneous loading of ²²Na⁺ and [³H]sucrose allows a normalization of ²²Na⁺ efflux with respect to vesicle volume, since ²²Na⁺, but not [³H]sucrose, is released specifically by cholinergic effectors. Such double-isotope measurements increase the reliability of the data derived.     

Ligation of the α2-macroglobulin signaling receptor on macrophages induces synthesis of platelet activating factor

The binding of receptor-recognized forms of α₂-macroglobulin (α₂M) to macrophage α₂M signaling receptors increases inositol-1,4,5-triphosphate synthesis and induces Ca²⁺ mobilization. In this report, we demonstrate that ligation of the macrophage α₂M signaling receptor is also associated with synthesis of platelet activating factor (PAF) by both the de novo and remodeling pathways. Both α₂M-methylamine and a cloned and expressed 20-kDa receptor binding fragment (RBF) from rat α₂M+, stimulated macrophage synthesis of PAF from [³H]acetate, [³H]methylcholine, and 1-O-[³H]alkyl lyso-PAF by two- to threefold. PAF levels reached a peak in 20 min after the cells were exposed to α₂M-methylamine or RBF; they remained elevated for about 1 h after ligand addition to the cells. When [³H]methylcholine was the substrate, pertussis toxin did not block PAF synthesis, but the protein kinase C inhibitor staurosporin reduced synthesis by 65–70%. Cycloheximide completely abolished the increase in synthesis of PAF by macrophages exposed to α₂M-methylamine. By contrast, when [³H]acetate was employed as a precursor, staurosporin or cycloheximide did not abolish the increase in PAF synthesis. These studies suggest that protein kinase C is necessary for the induction of the de novo pathway by α₂M-methylamine. Both α₂M-methylamine and RBF stimulated the activity of lyso-PAF acetyltransferase by about fourfold. Both ligands also stimulated the activity of PAF acetylhydrolase by about six- to sevenfold, indicating that ligation of the α₂M signaling receptor also regulates the degradation of PAF. The ability of receptor-recognized forms of α₂M to regulate levels of PAF suggests that α₂M-proteinase complexes not only regulate macrophage function by activating intracellular signaling but also may indirectly regulate the function of other cells that cannot bind α₂M-proteinase complexes. © 1996 Wiley-Liss, Inc.     

gamma-Aminobutyric acid receptors in cerebellar membranes of rat brain after a treatment with Triton X-100.

Abstract— The treatment of cerebellar membranes of rat brain with a low concentration of Triton X-100 followed by sufficient washing results in an increase of the Na⁺-independent binding of [³H]GABA and a total loss of the Na ⁺-dependent binding of [³H]GABA. The Na⁺-independent binding of [³H]GABA was more abundant in membranes of cerebellum than in membranes of other rat brain regions and mainly localized in the synaptic membrane fraction of a cerebellar homogenate. In the Triton-treated membranes, the Na⁺-independent binding of [³H]GABA was a saturable process, which could be resolved into two components, a high and a low affinity component with dissociation constants of 4.5 and 30 nm , respectively. The neurophysiological agonists, muscimol, GABA, and imidazole acetic acid, and the antagonist, bicuculline, inhibited the high affinity Na⁺-independent binding of [³H]GABA by 50% at 0.003, 0.012, 0.3 and 10 μm respectively. These data suggest that the Na⁺-independent binding of [³H]GABA in the Triton-treated cerebellar membranes represents the synaptic receptors of GABA. It is emphasized that extensive washing of the membranes after a Triton treatment is necessary in order to detect the high affinity Na⁺-independent binding of [³H]GABA.