Identification of a second putative receptor of platelet-activating factor from human polymorphonuclear leukocytes

Due to multiple molecular species of platelet-activating factor (PAF) and the existence of high affinity binding sites in a variety of cells and tissues, possible existence of PAF receptor subtypes has been suggested. This report shows differences between specific PAF receptors in human leukocytes and platelets. Human polymorphonuclear leukocyte membranes showed high affinity binding sites for PAF with an equilibrium dissociation constant (KD) of 4.4 (+/- 0.3) x 10(-10) M. We compared the relative potencies of several PAF agonists and receptor antagonists between human platelet and human leukocyte membranes. One receptor antagonist (Ono-6240) was found to be 6-10 times less potent in inhibiting the specific [3H]PAF receptor binding, PAF-induced GTPase activity, as well as the PAF-induced aggregation in human leukocytes than in human platelets. Mg2+, Ca2+, and K+ ions potentiated the specific [3H]PAF binding in both systems. Na+ and Li+ ions inhibited the specific [3H]PAF binding to human platelets but showed no effects in human leukocytes. K+ ions decreased the Mg2+-potentiated [3H]PAF binding in human leukocytes but showed no effects in human platelets. PAF stimulates the hydrolysis of [gamma-32P] GTP with an ED50 of about 1 nM, whereas the biological inactive enantiomer shows no activity even at 10 microM in both human platelets and human leukocytes. The PAF-stimulated GTPase in human leukocytes can be abolished by the pretreatment of membranes with pertussis toxin and cholera toxin. However, the PAF-stimulated activity of GTPase in human platelets is insensitive to pertussis toxin and cholera toxin. These results suggest that there exists a second type of PAF receptor in human polymorphonuclear leukocytes, which is structurally different from the one characterized in human platelets, and that the guanine nucleotide-binding protein coupled to PAF receptors in human leukocytes is also different from the one in human platelets.     

Ionic and GTP regulation of binding of platelet-activating factor to receptors and platelet-activating factor-induced activation of GTPase in rabbit platelet membranes

Specific binding of 3H-labeled platelet-activating factor (PAF) to rabbit platelet membranes was found to be regulated by monovalent and divalent cations and GTP. At 0 degrees C, inhibition of [3H]PAF binding by sodium is specific, with an ED50 of 6 mM, while Li+ is 25-fold less effective. On the contrary, K+, Cs+, and Rb+ enhance the binding. The divalent cations, Mg2+, Ca2+, and Mn2+ enhance the specific binding 8-10-fold. From both Scatchard and Klotz analyses, the inhibitory effect of Na+ is apparently due to an increase in the equilibrium dissociation constant (KD) of PAF binding to its receptors. However, the Mg2+-induced enhancement of the PAF specific binding may be attributed to an increased affinity of the receptor and an increased availability of the receptor sites. In the presence of Na+, PAF receptor affinity decreased with increasing temperature with a 100-fold sharp discontinuous decrease in receptor affinity at 24 degrees C. In contrast, the Mg2+-induced increase is independent of temperature suggesting that the Mg2+ regulatory site is different from Na+ regulatory site. [3H]PAF binding is also specifically inhibited by GTP; other nucleotides have little effect. PAF also stimulates hydrolysis of [gamma-32P]GTP with an ED50 of 0.7 nM, whereas 3-O-hexadecyl-2-O-acetyl-sn-glyceryl-1-phosphorylcholine showed no activity even at 10 microM. Moreover, such stimulatory effect of PAF is dependent on Na+ and can be abolished by the PAF-specific receptor antagonist, kadsurenone, but not by an inactive analog, kadsurin B. These results suggest that the PAF receptor may be coupled with the adenylate cyclase system via an inhibitory guanine nucleotide regulatory protein.     

Binding of radiolabeled antagonist to platelet-activating-factor receptor in human lung membranes: shifting of agonist binding affinity states by cations and guanine nucleotide

Binding of the radiolabeled platelet-activating-factor (PAF) receptor antagonist RP52770, [( 3H]-N-(3-chlorophenyl)-3-(3-pyridinyl)-1H, 3H-pyrrolo- [1,2-c]thiazole-7-carboxamide) to receptors in human lung membranes was time- dependent, protein-dependent, reversible and saturable. The dissociation constant and maximal binding density were 14 +/- 2 nM and 2.1 +/- 0.6 pmol/mg protein, respectively. [3H]-RP52770 binding to the PAF receptor was competitively displaced by PAF and receptor antagonists. The rank order of the binding affinities were PAF greater than RP52770 (+) greater than RP52770 (-) greater than CV3988, equivalent to the PAF receptor specificities determined from functional studies. Binding of PAF to [3H]-RP52770 labeled receptors was regulated by sodium, guanylylimido- diphosphate (GppNHp) and divalent cations. In the presence of EDTA, Na+ and GppNHp, in combination, binding of PAF to the receptor was maximally shifted to the right. These results clearly demonstrate that cations and guanine nucleotide can regulate the affinity states of the PAF receptor in human lung membranes.     

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.     

The specific binding of the platelet-activating factor (PAF) receptor antagonist WEB 2086 and the benzodiazepine flunitrazepam to rat hepatocytes

Thieno-triazolodiazepines WEB 2086 and BN 50739 have been described as the potent PAF receptor antagonists. Binding of radiolabeled [³H]WEB 2086 has been widely employed to characterize PAF receptors in different cells. In a search for a PAF receptor in isolated rat hepatocytes, we discovered that the binding of [³H]WEB to rat hepatocytes was highly specific but had a relatively low affinity with a K_d of 113 nM and B_max of 0.65 pmol/10⁶ cells in freshly isolated cell suspension and K_d of 1.65 μM and B_max of 2.0 pmol/plate in cultured hepatocytes. No consistent specific binding of [³H]PAF itself was found in the same cell preparations. The binding of [³H]flunitrazepam in the presence of the peripheral type of benzodiazepine receptor antagonist Ro 5-4864 was saturated and exhibited a K_i of 3.8 nM and B_max of 3.5 pmol/plate. The central type of benzodiazepine receptor antagonist clonazepam also competed for the [³H]flunitrazepam binding, however with a much lower affinity. Various antagonists inhibited the binding of [³H]WEB 2086 with a rank order BN 50739⪢Ro 5-4864≥clonazepam. Interestingly, bicuculline, a specific antagonist of GABA(A) recognition sites, also significantly reduced the binding of [³H]WEB 2086. The binding of [³H]flunitrazepam was inhibited with a rank potency BN 50739⪢WEB 2086. Taken together, these findings suggest that the specific binding of PAF receptor antagonists WEB 2086 and BN 50739 in rat hepatocytes does not involve PAF receptors and occurs via peripheral benzodiazepine and, possibly GABA(A) receptor sites.     

Beta-adrenergic activity of (+/-)-hydroxybenzylisoproterenol in isolated rat fat cells and hepatocytes

The pharmacology of (+/-)-hydroxybenzylisoproterenol with respect to stimulation of cyclic AMP accumulation by isolated rat fat cells and liver cells was examined. (+/-)-Hydroxybenzylisoproterenol was found to be a full agonist and twice as potent as (-)-isoproterenol in liver cells, and equipotent to (-)-isoproterenol in fat cells with regard to stimulating cyclic AMP accumulation. A study of the ability of this catecholamine to stimulate adenylate cyclase activity of broken-cell preparations revealed that (+/-)-hydroxybenzylisoproterenol was equipotent to (-)-isoproterenol in liver cell homogenates, while 3- to 4-fold more potent than (-)-isoproterenol in fat cell ghost membranes. (+/-)-Hydroxybenzylisoproterenol was also found to be as potent as (-)-isoproterenol in stimulating cyclase activity of S49 mouse lymphoma cell membranes. Competition studies of specific [125I]iodohydroxybenzylpindolol binding to liver cell membranes revealed a Kd of 10 nM for (+/-)-hydroxybenzylisoproterenol and 25 nM for (-)-isoproterenol binding to the liver beta-adrenergic receptor. Competition studies of specific (-)-[3H]dihydroalprenolol binding to fat cell membranes indicated a similar affinity of these sites for both (+/-)-hydroxybenzylisoproterenol and (-)-isoproterenol. The guanyl nucleotide Gpp(NH)p induced a shift in the curve for competition of (-)-[3H]dihydroalprenolol binding by (-)-isoproterenol to the right, but failed to do so when (+/-)-hydroxybenzylisoproterenol was the competing agonist. Properties of (+/-)-[3H]hydroxybenzylisoproterenol binding to fat cell or liver cell membranes were inconsistent with those expected of adenylate cyclase coupled beta-adrenergic receptors.     

Specific binding of [3H]dihydrokadsurenone to rabbit platelet membranes and its inhibition by the receptor agonists and antagonists of platelet-activating factor

Kadsurenone inhibits specifically and competitively the specific binding of 3H-labeled platelet-activating factor ([3H]PAF) to rabbit platelet membranes. Since the 5-propyl analog of kadsurenone (dihydrokadsurenone) retains roughly the same potency as kadsurenone, [3H]dihydrokadsurenone was therefore synthesized through tritiation of kadsurenone. Specific binding of [3H]dihydrokadsurenone in rabbit platelet membranes is saturable. Scatchard analysis of binding data reveals the presence of a single class of binding sites with an equilibrium dissociation constant (KD) of 16.81 ( +/- 0.57) nM. The total number (Bmax) of detectable binding sites is 2.27 ( +/- 0.09) pmol/mg protein. Both C16- and C18-PAF fully displace the specific binding of (3H]dihydrokadsurenone (5 nM) with an identical ED50 of 3.6 X 10(-9) M. Dihydrokadsurenone and kadsurenone also displace the specific binding with roughly the same potency (ED50 = 4.4 X 10(-8) M). Several other PAF analogs and PAF receptor antagonists tested show relative potencies roughly similar to those found in the [3H]PAF-specific binding assay. Other pharmacological agents with no PAF antagonistic activities did not inhibit the specific binding of [3H]dihydrokadsurenone. These results agree with our previous conclusion that kadsurenone is a specific and competitive receptor antagonist and strongly suggest that PAF and the PAF receptor antagonists tested may interact at a common binding site in the PAF receptor.     

Specific binding of platelet-activating factor (PAF) by human peripheral blood mononuclear leukocytes

Binding of platelet-activating factor (PAF) to human peripheral blood mononuclear leukocytes was time-dependent, reversible, and saturable. [3H]PAF binding to the cells was inhibited dose-dependently by unlabeled PAF and PAF receptor antagonists: L-659,989, triazolam, and alprazolam. Scatchard analysis of saturation binding data indicated one class of receptors for PAF with KD = 5.7 nM and Bmax = 18 fmol/10(6) cells (11,100 receptors/cell). PAF (10 nM) increased intracellular free calcium concentration in human lymphocytes and this effect was inhibited by L-659,989 dose-dependently. Our data suggest that human peripheral blood mononuclear leukocytes have specific receptors for PAF.     

The Binding Properties of the Particulate and Solubilized Sulfonylurea Receptor from Cerebral Cortex Are Modulated by the Mg2+ Complex of ATP

Glibenclamide closes an ATP-sensitive K+ channel (K-ATP channel) by interaction with the sulfonylurea receptor in the plasma membrane of pancreatic B cells and thereby initiates insulin release. Previous studies demonstrated that the Mg2+ complex of ATP decreases glibenclamide binding to the sulfonylurea receptor from pancreatic islets. The aim of the present study was to examine the effect of adenine and guanine nucleotides on binding of sulfonyl-ureas to the cerebral sulfonylurea receptor. For this purpose, binding properties of the particulate and solubilized site from rat or pig cerebral cortex were analyzed. Maximum recovery of receptors in detergent extracts amounted to 40-50%. Specific binding of [3H]glibenclamide to the solubilized receptors corresponded well to specific binding to microsomes. In microsomes and detergent extracts, the Mg2+ complexes of ATP, ADP, GTP, and GDP inhibited binding of [3H]glibenclamide. These effects were not observed in the absence of Mg2+. In detergent extracts, Mg-ATP (300 microM) reduced the number of high-affinity sites for [3H]-glibenclamide by 52% and increased the dissociation constant for [3H]glibenclamide by eightfold; Mg-ATP was half-maximally effective at 41 microM. Alkaline phosphatase accelerated the reversal of Mg-ATP-induced inhibition of [3H]glibenclamide binding. The data suggest similar control of the sulfonylurea receptor from brain and pancreatic islets by protein phosphorylation.     

Vasopressin antisense peptide interactions with the V1 receptor

The molecular recognition hypothesis, that peptide ligands and their receptor binding sites are encoded by complementary nucleotide sequences, was tested for arginine vasopressin (AVP) and its V1 receptor. Binding of [125I] [d(CH2)5,Sar7]AVP (a selective V1 vasopressin antagonist radioligand) or [3H]AVP to rat liver plasma membranes was inhibited by peptides known to bind to V1 receptors but not by the AVP complementary peptide (Ser-Ser-Trp-Ala-Val-Leu-Glu-Val-Ala) (PVA). Rabbit anti-PVA antibodies were nonimmunoreactive with any protein in rat liver membranes or in a partially purified preparation from rat liver containing reconstitutable vasopressin binding activity. Furthermore, there was no suppression of the AVP pressor effect by PVA in vivo using a rat blood pressure bioassay. These findings do not support the hypothesis that the V1 receptor binding site is encoded by the antisense DNA strand to AVP.     

(+/-)-[3H]Epinephrine and (-)[3H]dihydroalprenolol binding to beta1- and beta2-noradrenergic receptors in brain, heart, and lung membranes.

(+/-)-[3H]Epinephrine binds to beta-receptors in calf cerebellar and rat lung membranes in the presence of 1.0 mM pyrocatechol and 1.0 microM phentolamine, with dissociation constants at 4 degrees C of 11 nM and 24 nM, respectively. (+/-)-[3H]Epinephrine associates to equilibrium within 20 min in both tissues, and over 50% of the binding is rapidly dissociable. Inhibition of binding by agonists and antagonists is highly stereoselective, and the structure-activity relationships of adrenergic agents in inhibiting (+/-)-[3H]epinephrine binding suggest an interaction with beta2 type noradrenergic receptors. (-)-Isoproterenol has an apparent Ki of 2 nM, (-)-epinephrine is 1.5 to 3 times weaker, and (-)-norepinephrine is 30 to 60 times weaker. Salbutamol and terbutaline, selective beta2-agonists, are potent inhibitors of binding, as are several nonspecific antagonists. Properties of the sites labeled by (+/-)-[3H]epinephrine in calf cerebellum and rat lung are closely similar. (-)-[3H]Dihydroalprenolol binding in calf cerebellum and rat lung also shows beta2 characteristics. Antagonists have similar potencies in inhibiting (-)-[3H]dihydroalprenolol and (+/-)-[3H]epinephrine binding in both tissues, but agonists are in general more potent inhibitors of (+/-)-[3H]epinephrine. Sodium and lithium selectively lower the affinity of (+/-)-[3H]epinephrine at its binding sites and the affinities of agonists, but not antagonists, at the (-)-[3H]dihydroalprenolol site. Specific (+/-)-[3H]epinephrine binding was not detectable in calf cortex and rat heart, where (-)-[3H]dihydroalprenolol binding suggests a beta1-receptor. A physiological significance of (+/-)-[3H]epinephrine binding is suggested by the strong correlation for agonists and antagonists between affinities in inhibiting binding, and in stimulating or inhibiting a beta-receptor-coupled adenylate cyclase in frog erythrocytes.     

Involvement of Ni protein in the functional coupling of the atrial natriuretic factor (ANF) receptor to adenylate cyclase in rat lung plasma membranes

In the presence of 1 microM atrial natriuretic factor (ANF) and low (0.1 mM) Mg2+ concentrations, the initial rate of binding of [3H]guanosine 5'-[beta, gamma-imido)triphosphate [( 3H]p[NH]ppG) to rat lung plasma membranes was increased twofold to threefold. ANF-dependent stimulation of the initial rate of [3H]p[NH]ppG binding was reduced at high (5 mM) Mg2+ concentrations. Preincubation of membranes with p[NH]ppG (5 min at 37 degrees C) eliminated the ANF-dependent effect on [3H]p[NH]ppG binding whereas ANF-dependent [3H]p[NH]ppG binding was unaffected by similar pretreatment with guanosine 5'-[beta-thio]diphosphate (GDP[beta S]). An increase in ANF concentration from 10 pM to 1 microM caused a 40% decrease in forskolin-stimulated or isoproterenol-stimulated adenylate cyclase activities (IC50 5 nM) in rat lung plasma membranes. GTP (100 microM) was obligatory for the ANF-dependent inhibition of adenylate cyclase, which could be completely overcome by the presence of 100 microM GDP[beta S] or the addition of 10 mM Mn2+. Reduction of Na2+ concentration from 120 mM to 20 mM had the same effect. Pertussis toxin eliminated ANF-dependent inhibition of adenylate cyclase by catalyzing ADP-ribosylation of membrane-bound Ni protein (41-kDa alpha subunit of the inhibitory guanyl-nucleotide-binding protein of adenylate cyclase). The data support the notion that one of the ANF receptors in rat lung plasma membranes is negatively coupled to a hormone-sensitive adenylate cyclase complex via the GTP-binding Ni protein.     

Characterization of cortisol binding sites in chicken liver plasma membrane

1. The presence of sites specifically binding [3H]cortisol in plasma membrane isolated from chicken liver has been determined. The kinetic parameters of this binding are: Kd = 4.5 nM and Bmax = 2225 fmol/mg protein in presence of 10(-6) M progesterone. 2. The affinities of several natural and synthetic steroids for the membrane binding site respect to the binding of 4 nM [3H]cortisol without competitor increased in the following order: Testosterone less than pregnenone less than dexamethasone less than progesterone less than prednisolone less than corticosterone less than deoxycorticosterone. 3. Other steroids such as estradiol, ouabain and triamcinolone acetonide does not bind to the plasma membrane. 4. Metal ions such as Ca2+ and Mg2+ did not modify the binding of [3H]cortisol. 5. Neither propranolol nor phentolamine, beta- and alpha-adrenergic antagonists affected [3H]cortisol binding to the plasma membranes. 6. The result suggest that the binding site detected is more specific for glucocorticoids and it is different of nuclear glucocorticoid receptor and progesterone receptor.     

Pharmacologic characterization of the rabbit neutrophil receptor for platelet-activating factor

The characteristics of receptors for platelet-activating factor (PAF) on rabbit neutrophils are investigated in this report. The presence of PAF-specific binding to rabbit neutrophils was confirmed using radiolabeled ligand binding assays and a rabbit peritoneal neutrophil membrane preparation. Binding of PAF to the neutrophil membranes was reversible and reached equilibrium within 30 min. Scatchard analysis of PAF-specific binding to the rabbit neutrophil membranes revealed a dissociation constant (Kd) for PAF of 0.41 +/- 0.045 nM and a Bmax of 0.32 +/- 0.11 pmol of PAF receptor/mg of protein. The order of potencies of PAF receptor antagonists to inhibit the binding of 3H-PAF to rabbit peritoneal neutrophil membranes was determined. For the competition assays, 100 micrograms of neutrophil or platelet membrane protein, 0.18 nM 3H-PAF, and varying amounts of PAF antagonist were incubated at room temperature for 1 hr. PAF receptor antagonists tested were ONO-6240, brotizolam, kadsurenone, WEB-2086, L-652-731, BN-52021, CV-3988, triazolam, alprazolam, and verapamil. The orders of potencies of these PAF receptor antagonists were similar for inhibition of 3H-PAF binding to rabbit peritoneal neutrophil and platelet membranes (correlation coefficient, r = 0.97). PAF had a significantly higher affinity for rabbit neutrophil membranes (Kd = 0.41 +/- 0.045 nM), as compared with its affinity for rabbit platelet membranes (Kd = 0.87 +/- 0.092 nM). In addition, sodium was found to inhibit 3H-PAF specific binding to rabbit platelet membranes and not to affect 3H-PAF binding to neutrophil membranes. These data indicate that, although PAF receptors on rabbit platelets and neutrophils exhibit similar orders of potencies of PAF receptor antagonists to inhibit the binding of 3H-PAF, the disparity in Kd of PAF for the receptors and the effect of NaCl on the binding of 3H-PAF reveal subtle differences between the cell types.     

Solubilization and partial purification of 3-((+-)-2-carboxypiperazine-4-yl)-[1,2-3H]propyl-1-phosphonic acid recognition proteins from rat brain synaptic membranes

The receptors on neuronal membranes for N-methyl-D-aspartate (NMDA), an analog of L-glutamic acid, are the focus of intensive study because of their importance in many neurophysiological and neuropathological states. Since there is very little knowledge of the molecular characteristics of the NMDA receptors, we undertook the development of methods for the solubilization and purification of proteins that form the receptor complex. Optimal conditions for solubilization of NMDA receptors from isolated synaptic plasma membranes involved the use of the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS) together with NH4SCN, 10% glycerol, and the nonionic detergent polyoxyethylene 10 tridecyl ether. The presence of NMDA receptors was monitored as the binding activity for the specific NMDA receptor ligand 3-((+-)-2-carboxypiperazine-4-yl)-[1,2-3H]propyl-1-phosphonic acid ([3H]CPP). Approximately 50% of membrane proteins were solubilized, and an equal quantitative recovery of [3H]CPP-binding proteins was achieved. The selectivity of [3H] CPP-binding proteins for excitatory amino acid agonists and aminophosphonocarboxylic acid antagonists remained essentially unchanged following solubilization. The effect of the NMDA receptor modulator, glycine, and of the ion channel-blocking cation Mg2+ on [3H]CPP-binding proteins was drastically altered by solubilization. Both became activators of [3H]CPP-binding sites. The NMDA receptor agonist ibotenic acid was used to develop an affinity matrix for the isolation of the NMDA receptor complex. The [3H]CPP-binding proteins were selectively eluted by the introduction of 2 mM Mg2+ in the elution buffers. This fraction was highly enriched in CPP-binding entities and in a protein of 58-60-kDa molecular size. The CPP binding activity of the proteins in this fraction was enriched by a factor of approximately 20,000 over that of brain homogenate. There was no L-[3H]glutamate binding activity associated with this fraction. Proteins interacting with glutamate, NMDA, and ibotenate were recovered in the 1 M KCl-eluted fraction. We propose that the 58-60-kDa protein is the aminophosphonocarboxylic acid antagonist-binding subunit of the NMDA receptor complex.     

[3H]MK-801 Labels a Site on the N-Methyl-D-Aspartate Receptor Channel Complex in Rat Brain Membranes

The potent noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist [3H]MK-801 bound with nanomolar affinity to rat brain membranes in a reversible, saturable, and stereospecific manner. The affinity of [3H]MK-801 was considerably higher in 5 mM Tris-HCl (pH 7.4) than in previous studies using Krebs-Henseleit buffer. [3H]MK-801 labels a homogeneous population of sites in rat cerebral cortical membranes with KD of 6.3 nM and Bmax of 2.37 pmol/mg of protein. This binding was unevenly distributed among brain regions, with hippocampus greater than cortex greater than olfactory bulb = striatum greater than medulla-pons, and the cerebellum failing to show significant binding. Detailed pharmacological characterization indicated [3H]MK-801 binding to a site which was competitively and potently inhibited by known noncompetitive NMDA receptor antagonists, such as phencyclidine, thienylcyclohexylpiperidine (TCP), ketamine, N-allylnormetazocine (SKF 10,047), cyclazocine, and etoxadrol, a specificity similar to sites labelled by [3H]TCP. These sites were distinct from the high-affinity sites labelled by the sigma receptor ligand (+)-[3H]SKF 10,047. [3H]MK-801 binding was allosterically modulated by the endogenous NMDA receptor antagonist Mg2+ and by other active divalent cations. These data suggest that [3H]MK-801 labels a high-affinity site on the NMDA receptor channel complex, distinct from the NMDA recognition site, which is responsible for the blocking action of MK-801 and other noncompetitive NMDA receptor antagonists.     

Inhibition of hepatic alpha 1-adrenergic effects and binding by phorbol myristate acetate

Treatment of isolated hepatocytes with the tumor-promoting agent, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) produced a time- and dose-dependent, non-competitive inhibition of alpha 1-adrenergic responses, including the activation of phosphorylase, increase in Ca2+ efflux, increase in free cytosolic Ca2+, and release of myo-inositol-1,4,5-P3. The actions of [8-arginine] vasopressin (AVP) on liver cells were also inhibited by PMA, but the inhibition could be overcome by high AVP concentrations. No significant inhibition of beta-adrenergic and glucagon-mediated activation of phosphorylase was induced by PMA and no inhibitory or synergistic effects of PMA were observed on the dose-dependent activation of phosphorylase by the Ca2+ ionophore A23187. In radioligand binding studies, PMA did not directly interfere with [3H]prazosin specific binding, the displacement of [3H]prazosin by (-)-norepinephrine nor with [3H]AVP specific binding to purified liver plasma membranes. Plasma membranes prepared from livers perfused with PMA exhibited a 30-44% reduction in [3H]prazosin binding capacity. Under identical conditions [3H]AVP binding was unchanged. The alpha 1-receptors remaining in membranes from PMA-treated livers had equivalent affinities for [3H]prazosin and (-)-norepinephrine, and were unaffected in terms of coupling to guanine nucleotide-regulating proteins as indicated by the ability of guanosine 5'-(beta, gamma-imido)triphosphate to promote the conversion of the remaining alpha 1-receptors into a low affinity state. These data indicate that tumor promoters are potent antagonists of alpha 1-adrenergic and vasopressin (low dose) responses in liver. It is proposed that PMA acting via protein kinase C (which presumably mediates the action of PMA) exerts its inhibitory action on alpha 1-adrenergic responses at the alpha 1-adrenergic receptor itself and also at a site close to or before myo-inositol-1,4,5-P3 release.     

Specific binding sites for platelet activating factor in human lung tissues

Specific and saturable binding of [3H]-labeled 1-0-alkyl-2-0-acetyl-sn-glycero-3-phosphocholine (PAF) to membrane preparations of human lung tissues is demonstrated. The equilibrium dissociation constant (KD) was determined by Scatchard analysis to be 4.9 (+/- 1.7) X 10(-10)M and the maximal number of binding sites was estimated to be 140 (+/- 37) fmole/mg protein. The binding site is PAF specific and its selectivity toward PAF analogs is very similar to that in rabbit platelets. Two PAF receptor antagonists, kadsurenone and ginkgolide B, previously characterized in platelet systems, also displace the binding of [3H]-PAF to human lung homogenates. These data indicate that human lung tissues contain PAF specific receptors, and binding of PAF to these receptor sites may be the first step to initiate PAF-induced lung pathophysiology.     

Identification and characterization of leukotriene D4 receptors and signal transduction processes in rat basophilic leukemia cells

The leukotriene D4 (LTD4) receptor on rat basophilic leukemia (RBL-1) cell membranes was characterized using a radioligand binding assay. [3H]LTD4 binding to RBL-1 membrane receptors was stereoselective, specific, and saturable. The binding affinity and maximum binding density of [3H]LTD4 to RBL-1 membrane receptors were 0.9 +/- 0.2 nM and 800 +/- 125 fmol/mg protein, respectively. Binding of [3H]LTD4 to the receptors was enhanced by divalent cations (Ca2+, Mg2+, and Mn2+) and inhibited by guanine nucleotides and sodium ions, specifically, indicating that a guanine nucleotide-binding protein may regulate the agonist-receptor interaction. LTD4, LTE4 agonist and antagonist analogs competed with the radioligand in binding to the RBL-1 LTD4 receptors. The binding affinities of these analogs correlated with (a) those determined from the guinea pig lung LTD4 receptors and (b) the pharmacological activities in smooth muscle contraction. LTD4 and related agonists also induced time- and concentration-dependent phosphatidylinositol hydrolysis in RBL-1 cells. The LTD4 induction of inositol 1-phosphate was potent, stereoselective, specific, and was blocked by LTD4 receptor antagonists. The rank order potency of agonist-induced inositol 1-phosphate formation in RBL-1 cells was equivalent to the receptor binding affinity determined using either RBL-1 cell or guinea pig lung membranes. These studies have demonstrated the G protein coupled LTD4 receptors on RBL-1 cell membranes. Binding of agonists to the receptor may activate the G protein-regulated phospholipase C to induce hydrolysis of phosphatidylinositol. The hydrolytic products of phosphatidylinositol, possibly inositol trisphosphate and diacylglycerol, may be the intracellular messengers for LTD4 receptors in RBL-1 cells.     

Characterization of specific binding sites for corticosterone in mouse liver plasma membrane

The specific binding of [3H]corticosterone to mouse liver purified plasma membrane fractions is a saturable, reversible, and temperature-dependent process. Only one type of independent and equivalent binding sites has been determined in plasma membrane (Kd = 4.1 nM and Bmax = 3368 fmol/mg). As can be deduced from displacement data obtained in plasma membrane, the high-affinity binding site is different from nuclear glucocorticoid, nuclear progesterone, and Na+, K(+)-ATPase digitalis receptors. Probably this corticosterone binding site or receptor is the same one determined previously for [3H]cortisol in mouse liver plasma membrane. Such beta- and alpha-adrenergic antagonists as propranolol and phentolamine did not affect [3H]corticosterone binding to plasma membranes; therefore, this binding site is independent of these receptors. The binding sites in plasma membranes are not exclusive for corticosterone, but other steroids are also bound with very different affinities.