Biochemical characterization and autoradiographic localization of [125I]endothelin-1 binding sites on trophoblast and blood vessels of human placenta.

The presence of endothelin binding sites in the human placenta raises the question of the precise localization of these receptors on well defined placental constituents. In order to find an answer to this problem various approaches were used. Specific binding sites for [125I] endothelin-1 (ET-1) were identified on human term placenta, not only on membranes of smooth muscles stem villi vessels, but also on trophoblastic plasma membranes prepared from trophoblast in culture. Scatchard analysis of binding data revealed a single class of high affinity binding sites with Kd values of 26 +/- 4 pmol/L for stem villi vessels and 126 +/- 4 pmol/L for trophoblast in culture, with maximum binding capacities of 681 +/- 61 and 224 +/- 53 fmol/mg protein, respectively. The anatomical localization of these binding sites was determined by in vitro autoradiography. Autoradiograms obtained from placental sections incubated with [125I]ET-1 indicate that [125I]ET-1 high affinity binding sites exist on placental stem villi vessels and on the trophoblastic layer of the villi. The latter localization was also found on autoradiograms of trophoblast in culture. The human placental syncytiotrophoblast is a polarized epithelium with the microvillous membrane, facing maternal blood space and the basal plasma membrane, facing fetal circulation. [125I]ET-1 high affinity binding sites are present on both membranes but the number of binding sites is higher on the basal plasma membrane. These findings lead to the suggestion that ET-1 may be involved in the regulation of the feto-placental circulation and may subserve specific trophoblastic functions.

Radioligand binding of antagonists of platelet-activating factor to intact human platelets.

Two new antagonists of platelet-activating factor (PAF), the pyrrolothiazole derivative 52770 RP and the triazolodiazepine WEB 2086, have been studied as radioligands in intact human platelets. [3H]52770 RP and [3H]WEB 2086 bound specifically to high-affinity sites with dissociation constants (Kd) of 14.8 and 6.1 nM, respectively. The maximal number of sites for [3H]52770 RP binding was approx. 15-fold higher than for [3H]PAF and [3H]WEB 2086. In addition, C16-PAF, lyso-PAF, WEB 2086 and 52770 RP had Ki values which were nearly identical for both [3H]PAF and [3H]WEB 2086, whereas only 52770 RP competed for [3H]52770 RP-binding sites. These results demonstrate that in human platelets the sites of [3H]WEB 2086 binding are identical to [3H]PAF-binding sites, whereas those of [3H]52770 RP are not. [3H]WEB 2086 appears, therefore, to be a suitable antagonist radioligand for labelling PAF receptors.

PAF binding sites. Characterization by [3H]52770 RP, a pyrrolo[1,2-c]thiazole derivative, in rabbit platelets.

52770 RP, the N-(3-chlorophenyl)-3-(3-pyridinyl)-1H,3H-pyrrolo[1,2-c]thiazole -7-carboxamide, displaces in a potent, specific and competitive manner [3H]PAF from its binding sites on rabbit platelets. Since 52770 RP is not structurally related to PAF and has low liposolubility with respect to PAF, it was selected as a potential radioligand for PAF receptor sites. [3H]52770 RP displayed high-affinity, specificity, as well as saturable and displaceable binding to a single class of recognition sites in intact platelets and crude platelet membranes. In these preparations, the values of binding parameters were, respectively, 8.5 and 7.6 nM for Kd, 0.2 pmol/5 X 10(7) platelets and 3.66 pmol/mg protein for Bmax and 0.96 and 0.91 for nH. Inasmuch as the (+)-52770 RP was 300-fold more potent than the (-)-isomer at displacing [3H]52770 RP in intact platelets, the studied binding site manifested stereospecific discrimination. A variety of pharmacological agents including pro- and anti-aggregant compounds did not exhibit affinity for [3H]52770 RP binding sites. In contrast, PAF, some of its active analogues and several recognized PAF antagonists (BN 52021, brotizolam, L-652,731, triazolam), displaced the [3H]52770 RP binding. Studies carried out using [3H]PAF demonstrated that 52770 RP was approximately 4- and 200-fold more potent than L-652,731 and BN 52021 respectively, as a PAF-receptor antagonist. In washed rabbit platelets, the rank order of potency (Ki) for several analogues of 52770 RP, to displace [3H]PAF from its binding sites, was highly correlated (r = 0.96) to their ability to antagonize [3H]52770 RP binding. In functional studies, 52770 RP antagonized not only the PAF-induced aggregation in washed rabbit platelets but also the hypotension evoked by PAF in the anesthetized rat. In this respect, it was 26 and 2 times more potent than L-652,731, respectively. In conclusion, [3H]52770 RP might represent a novel interesting tool for furthering our understanding of the role of PAF binding sites in pathophysiological processes.

Regional distribution and pharmacological characterization of [125I]endothelin-1 binding sites in human fetal placental vessels.

High-affinity binding sites for [125I]endothelin(ET)-1 have been detected in purified membrane preparations of the fetal arteries and veins of the chorionic plate and the stem villi vessels of the human term placenta. Regardless of the vessel type, the apparent dissociation constant was found to be in the picomolar range (26----45 pM), and the Bmax value close to 600 fmol/mg protein. In stem villi vessels, ET-1, ET-2, sarafotoxin S6b and vasocontractor intestinal peptide (VIC) were approximately equipotent in their competitive displacement of [125I]ET-1 binding. The endothelin precursors, human and porcine big-endothelin, recognized ET-1 sites with low affinity (nM range), a finding which reflects their low potency as recognized vasocontractant agents. Interestingly, [125I]ET-1 binding parameters and pharmacological profiles were identical in fetal veins and arteries of the chorionic plate. Similarly, a study carried out in rat aortic membranes, revealed the presence of high affinity [125I]ET-1 binding sites with pharmacological characteristics close to those of the human stem villi vessels. In all vessels investigated, the binding pattern of ET-3 against [125I]ET-1 was of a non-competitive nature. Thus, these results demonstrate the presence of specific [125I]ET-1 binding sites along the vascular tree of the fetal side of the placenta and would support evidence currently available, favouring the existence of distinct ET-1 and ET-3 receptors. Finally, ET-1 in the human placenta may play an important physiological role as regulator of vascular resistance and/or be implicated as a pathological factor in certain pregnancy-related diseases.

Multiple high affinity binding sites for 5-hydroxytryptamine: a new class of sites distinct from 5-HT1 and S2.

Two different classes of binding sites probably related to serotonergic receptors have already been reported: 5-HT1 binding sites recognize [3H]5-hydroxytryptamine with a high affinity (Kd = 3 nM) and S2 binding sites recognize [3H]spiroperidol and [3H]ketanserine. An additional population of sites has been observed in crude membrane preparations or fractions enriched with synaptosomal membranes obtained from rat brain cortex. This population was observed as a single class of sites in a synaptosomal fraction (L fraction--according to Laduron (1977)). It corresponded to a dissociation constant Kd = 13-15 nM, and Bmax = 0.80 +/- 0.15 pmol/mg protein. Displacement experiments showed that it recognized preferentially the 5-HT structure (bufotenin, 5-MeO-tryptamine). Tryptamine was a weak displacer and 5,7-dihydroxytryptamine totally inefficient. Neither 8-OH-DPAT, nor quipazine had any effect. Methiothepin, cinanserin and cyproheptadine displaced 5-HT from these sites whereas ergot derivatives did not. Contrary to 5-HT1 binding, this recently observed binding was not altered by GTP; alpha-MSH reduced the corresponding Bmax whereas Leu-enkephalin did not. The degenerative lesion of the serotonergic fibers led to a slight increase in the Bmax of the binding without altering the Kd which means that corresponding sites are not located on serotonergic fibers and might be postsynaptically located.

5-Hydroxytryptamine receptors in neurones and glia.

Crude membranal fractions isolated from mammalian brain tissue contain two classes of recognition sites capable of binding [3H]5-HT with high affinity constants. These classes of sits are characteristics of the postsynaptosomal membrane fraction for the higher affinity and of a glial cell membrane fraction for the lowest. They are observed with similar properties in cultured neuronal and glial cell respectively. Two 5-HT stimulated adenylate cyclases are present in crude membrane fraction; they are also separable as neuronal and glial components. These observations correspond likely to the existence of two classes of receptors for 5-HT. Their mechanisms of regulation involve presumably structural conformation changes of the recognition site coupled to various states of the activity of the receptor.

[PAF-acether antagonistic pyrrolo[1,2-c]thiazoles: from RP 48740 to RP 59227]. / Pyrrolo[1,2-c]thiazoles antagonistes du PAF-acéther: du RP 48740 au RP 59227.

The optimisation of the PAF antagonistic activity of RP 48740 led to the discovery of still more potent molecules of the pyrrolo[1,2-c]thiazole family: RP 52629, RP 52770 and RP 59227. The latter compound, for which a stereo-specific synthesis was developed, exhibits a very high potency as a PAF receptor antagonist.

Evidence for the existence and ionic modulation of platelet-activating factor receptors mediating degranulatory responses in human polymorphonuclear leukocytes.

High-affinity binding sites for platelet-activating-factor (PAF) have been identified in human polymorphonuclear leukocytes (PMNs). The aim of this investigation was to assess their functional relevance by characterizing PAF-induced elastase release, an enzyme present in azurophilic granules. At 37 degrees C, maximal release (measured with a spectrofluorimetric method) was achieved with 0.3 microM PAF. PAF analogs (e.g. lyso-PAF, 2-O-methyl-lyso-PAF) and enantio-PAF, which exhibit poor PAF-like activity in binding studies, were very weak releasers of elastase. The degranulation induced by PAF was fully and competitively inhibited by RP 59227, a potent, d-enantiomeric PAF antagonist. This effect was stereoselective and specific inasmuch as this compound was 500-fold more potent than its l-enantiomer and was devoid of effects on the degranulation promoted by N-formyl-l-methionyl-l-leucine-l-phenylalanine or [5S,12R]hydroxyeicosa-6,14-cis-8,10-trans-tetraenoic acid. The PAF-induced elastase extrusion was enhanced by physiological concentrations of CaCl2 and MgCl2. For instance, 1 mM Ca++ increased by 200% the release produced by 100 nM PAF. The mechanism of this effect is probably related to the capability of Ca++ and Mg++ to enhance the maximum number of [3H]PAF binding sites in whole or lysed PMNs without affecting the affinity (Kd) of the ligand. An additional possibility is that these ions intervene at the level of the signal transduction system leading to degranulation. For a series of 22 known PAF receptor antagonists, belonging to different chemical families, there was a strong correlation (r = 0.95) between their ability to displace [3H]PAF and to inhibit the PAF-evoked elastase release.(ABSTRACT TRUNCATED AT 250 WORDS)

[3H]52770 RP, a platelet-activating factor receptor antagonist, and tritiated platelet-activating factor label a common specific binding site in human polymorphonuclear leukocytes.

In human polymorphonuclear leukocytes (PMNs), the tritiated platelet activating factor ([3H]PAF) labels in a saturable manner a single class of binding sites with a Kd of 3.5 +/- 0.5 nM (n = 7) and a maximum binding capacity (Bmax) of 206 +/- 13 fmol/2.5 X 10(6) PMNs (n = 7). 52770 RP, a nonphospholipid antagonist of PAF receptors, fully and competitively displaced the [3H]PAF from its binding sites with a Ki of 7.0 +/- 0.7 nM (n = 4). The high potency and the low solubility in cellular membranes of this compound led us to prepare [3H]52770 RP. This ligand was characterized by a binding which was rapid, reversible, confined to a single site, saturable, specific and stereoselective. Its Kd and Bmax were 4.2 +/- 0.3 nM and 181 +/- 11 fmol/2.5 X 10(6) PMNs, respectively. The stereoselectivity of the binding was suggested by the 600- and 1050-fold higher potency of the d-enantiomer with respect to l-52770 RP in displacing [3H]52770 RP or [3H]PAF, respectively. Several PAF analogs (e.g., lyso-PAF, 2-O-methyl-lyso-PAF), which are poorly active as PAF receptor agonists in functional tests, were weak displacers of [3H]PAF and [3H]52770 RP. Furthermore, for a series of 14 known PAF receptor agonists or antagonists belonging to different chemical families, there was an excellent correlation (r = 0.98) between their ability to displace [3H]PAF and [3H]52770 RP. Thus, [3H]52770 RP and [3H]PAF appear to interact with the same binding site on human PMNs which is proposed to be the PAF receptor mediating functional responses.(ABSTRACT TRUNCATED AT 250 WORDS)

A method for cooperative or noncooperative binding studies using nonlinear regression analysis on a microcomputer.

Ligand-macromolecule interactions are studied by nonlinear regression analysis performed using a microcomputer (SYMAG-Micromachine 3000/Z). The basic phenomenon is described by the Clark equation B = (Formula: see text), where B and F represent bound and free ligand, respectively, Ni the total concentration of binding sites, and Ki their corresponding affinity constant. The programs of calculation have been extended also for Hill and for Adair equations using the Gauss algorithm described by E. E. Beaulieu and J. P. Raynaud [Eur. J. Biochem. 13, 293 (1970)]. A statistical test of F type is introduced to test the quality of the fit and compare the representations of the phenomenon using the different equations.