Gonadotropin releasing-hormone receptors: photoaffinity labeling with an antagonist

A photoaffinity antagonist of gonadotropin releasing hormone (GnRH), D pGlu-D-Phe-D-Trp-Ser-D-Lys6(N epsilon-azidobenzoyl)-Leu-Arg-Pro-Gly-NH2 (photoaffinity antagonist) was prepared by reacting [D-pGlu1, D-Phe2, D-Trp3, D-Lys6]GnRH with the N-hydroxysuccinimide ester of 4-azidobenzoic acid. The analog appeared homogeneous when analyzed by thin-layer chromatography and its photoreactivity was demonstrated by spectral changes when exposed to light. The photoaffinity antagonist retained high affinity binding to the GnRH receptor of pituitary membrane preparations and exhibited antagonistic activity when assayed in vitro in whole pituitaries. Pituitary membrane preparations were incubated with the radioactive photoaffinity GnRH antagonist and irradiated with light. Sodium dodecyl sulfate gel electrophoresis after solubilization and reduction showed the specific labeling of a single specific protein with an apparent molecular weight of 60,000 daltons. These results indicate that GnRH agonists and antagonists bind to the same receptor.     

Solubilization of phencyclidine receptors from rat cerebral cortex in an active ligand binding state

A phencyclidine (PCP) receptor binding site has been solubilized in an active ligand-binding state from rat cerebral cortical membranes with sodium deoxycholate. Optimal receptor solubilization occurs at a detergent/protein ratio of 0.5 (w/w); for 5 mg protein/ml solubilized with 0.25% sodium deoxycholate, about 60% of the protein and 25% of the receptor is solubilized. Specific binding of either [3H]-N-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) or [3H]MK-801 is measurable by filtration through Sephadex G-50 columns or glass fiber filters; more than 60% of the binding activity is stable after 48 h at 4 degrees C. In the presence of detergent, [3H]TCP binding exhibits a Kd of 250 nM, a Bmax of 0.56 pmol/mg protein, and a pharmacological profile consistent with that of the membrane-bound PCP receptor, although most drugs bind with affinities 2 to 8 fold lower than in membranes. Upon reduction of detergent concentration, binding parameters approximate those for the membrane-bound receptor ([3H]TCP binding: Kd = 48 nM, Bmax = 1.13 pmol/mg protein).     

Evidence for an endogenous peptide ligand for the phencyclidine receptor

Porcine brain contained an active factor that competed with [3H]-phencyclidine (PCP) for binding to rat brain membranes. On reverse phase high pressure liquid chromatography, the active material eluted between 38-42% acetonitrile. Gel filtration chromatography of the factor predicted a molecular weight of approximately 3000 daltons. The endogenous substance appeared to be selective for PCP receptors as it did not interact with either benzodiazepine, neurotensin, nor with mu, delta, or kappa opioid receptors. The active material showed a heterogenous distribution in brain, with highest concentrations found in hippocampus and cortex. It is likely to be a small peptide since various proteases eliminated or markedly reduced the potency of the compound in a [3H]-PCP binding assay. The material also possessed PCP-like activity in two bioassays. Like PCP, it induced contralateral rotational behavior after unilateral intranigral injection and depressed spontaneous cell activity after iontophoretic micropressure application in hippocampus and cerebral cortex. Thus, this small peptide is likely to be an endogenous ligand for the PCP receptor.     

Interaction of phencyclidine and its new adamantyl derivatives with muscarinic receptors

The antimuscarinic potency of new derivatives of phencyclidine containing the adamantyl moiety was evaluated in the guinea pig ileum test. The drugs tested competitively inhibited the acetylcholine-induced contractions. K_d values were calculated and compared to those obtained by direct binding to the muscarinic receptors from mouse brain. The new derivative, |1-(2-phenyl-2-adamantyl) piperidine| was found to be the most potent muscarinic antagonist among the many phencyclidine derivatives known to date. The results are discussed in terms of the acetylcholine-like molecular arrangement of these derivatives that gives rise to a characteristic muscarinic interaction.     

A specific acylating agent for the [3H]phencyclidine receptors in rat brain

A derivative of phencyclidine (PCP, 1 in fig. 1) bearing an isothiocyanate moiety on the meta position of the aromatic ring (Metaphit, 3 in fig. 1) has been synthesized and identified as a rapid and specific site-directed acylating agent of the [3H]phencyclidine binding site in rat brain homogenates. The percentage of sites irreversibly inactivated by Metaphit was found to be the same in the hippocampus and striatum and the remaining sites were unaffected by Metaphit treatment under any conditions, suggesting that at least two distinct binding sites are present. An isomeric isothiocyanate derivative did not irreversibly inhibit [3H]phencyclidine receptors, indicating structural specificity for Metaphit in the inhibition of these receptors. The availability of Metaphit should greatly facilitate study of the structure and function of the phencyclidine receptors.     

Regional heterogeneity of rat brain phencyclidine (PCP) receptors revealed by photoaffinity labeling with [3H] azido phencyclidine

Photoaffinity labeling of rat brain phencyclidine (PCP) receptors with [3H] azido phencyclidine ([3H]AZ-PCP) reveals the existence of five polypeptides which are specifically labeled by the affinity probe (Mr's 90,000, 62,000, 49,000, 40,000 and 33,000). These labeled components are unevenly distributed in rat brain. In the frontal cortex, thalamus and olfactory bulb, the major bands labeled are the Mr's 90 K and 62 K polypeptides; in the cerebellum most of the labeling is in the 90 K and 33 K bands; and in the hippocampus all but the Mr 40 K band are heavily labeled. Together with dexoxadrol/[3H]PCP competition binding data, which indicated the existence of high and low affinity dexoxadrol/PCP binding sites, these results suggest regional heterogeneity of PCP receptors. The regional distribution of the high affinity dexoxadrol binding sites correlates best with that of the Mr 90 K polypeptide.     

mCPP but not TFMPP is an antagonist at cardiac 5HT3 receptors.

The prototypic arylpiperazines, meta-chlorophenylpiperazine (mCPP), meta-trifluoromethylphenylpiperazine (TFMPP) and quipazine are widely studied serotonergic ligands with nonselective effects at 5HT1 and 5HT2 receptor subtypes. The present study was designed to compare the affinities of these arylipiperazines at 5HT3 receptors, and to determine agonist or antagonist activity at 5HT3 receptors. Quipazine showed high affinity at brain 5HT3 receptors (IC50 = 4.4 nM) and was a potent agonist of the von Bezold-Jarisch reflex in anesthetized rats, a response mediated by cardiac 5HT3 receptors. In concentrations that activated 5HT3 receptors, quipazine also antagonized serotonin-induced bradycardia in anesthetized rats. Taken together, these data suggest that quipazine is an agonist/antagonist with high affinity at 5HT3 receptors in both brain and cardiac tissue. Although mCPP also showed relatively high affinity at brain 5HT3 receptors (IC50 = 61.4 nM), it did not activate the von Bezold-Jarisch reflex; instead, mCPP potently antagonized serotonin-induced bradycardia. Thus, mCPP acts as an antagonist at 5HT3 receptors in the periphery. Although both quipazine and mCPP possessed relatively high affinity at brain 5HT3 receptors, TFMPP did not bind appreciably to 5HT3 receptors in brain (IC50 = 2373 nM) and neither activated nor inhibited cardiac 5HT3 receptors. That TFMPP did not interact with 5HT3 receptors, whereas quipazine and mCPP did, is in marked contrast to the similar effects of all three arylpiperazines at other serotonin receptors. The selectivity of TFMPP for 5HT1 and 5HT2 receptors (i.e., its minimal affinity for 5HT3 receptors) suggests that this arylpiperazine may be a preferred ligand relative to mCPP when studying 5HT1 or 5HT2 receptor mediated responses.     

Affinity of the enantiomers of alpha- and beta-cyclazocine for binding to the phencyclidine and mu opioid receptors

The enantiomers in the alpha and beta series of cyclazocine were evaluated for their ability to bind to phencyclidine (PCP) and mu-opioid receptors in order to determine their receptor selectivity. The affinity of (-)-beta-cyclazocine for the PCP receptor was 1.5 greater than PCP itself. In contrast, (-)-alpha-cyclazocine, (+)-alpha-cyclazocine, and (+)-beta-cyclazocine were 3-, 5- and 138-fold less potent than PCP, respectively. Scatchard analysis of saturable binding of [3H]Tyr-D-Ala-Gly-N-MePhe-Gly-ol (DAMGO) also exhibited a homogeneous population of binding sites with an apparent KD of 1.9 nM and an estimated Bmax of 117 pM. [3H]Tyr-D-Ala-Gly-N-MePhe-Gly-ol (DAMGO) binding studies revealed that (-)-alpha-cyclazocine (KD = 0.48 nM) was 31-, 1020- and 12,600-fold more potent than (-)-beta-cyclazocine, (+)-alpha-cyclazocine and (+)-beta-cyclazocine, respectively, for binding to the mu-opioid receptor. These data show that, although (-)-beta-cyclazocine is a potent PCP receptor ligand consistent with its potent PCP-like discriminative stimulus effects, it shows little selectivity for PCP receptors since it also potently displaces mu-opioid binding. However, these cyclazocine isomers, due to their extraordinary degree of stereoselectivity, may be useful in characterizing the structural requirements for benzomorphans having activity at the PCP receptor.     

An odorant derivative as an antagonist for an olfactory receptor

Different odorants are recognized by different combinations of G protein-coupled olfactory receptors, and thereby, odor identity is determined by a combinatorial receptor code for each odorant. We recently demonstrated that odorants appeared to compete for receptor sites to act as an agonist or an antagonist. Therefore, in natural circumstances where we always perceive a mixture of various odorants, olfactory receptor antagonism between odorants may result in a receptor code for the mixture that cannot be predicted from the codes for its individual components. Here we show that stored isoeugenol has an antagonistic effect on a mouse olfactory receptor, mOR-EG. However, freshly purified isoeugenol did not have an inhibitory effect. Instead, an isoeugenol derivative produced during storage turned out to be a potent competitive antagonist of mOR-EG. Structural analysis revealed that this derivative is an oxidatively dimerized isoeugenol that naturally occurs by oxidative reaction. The current study indicates that as odorants age, they decompose or react with other odorants, which in turn affects responsiveness of an olfactory receptor(s).     

Binding studies and photoaffinity labeling identify two classes of phencyclidine receptors in rat brain

Binding and photoaffinity labeling experiments were employed in order to differentiate 1-(1-phenylcyclohexyl)piperidine (PCP) receptor sites in rat brain. Two classes of PCP receptors were characterized and localized: one class binds [3H]-N-[1-(2-thienyl)cyclohexyl]piperidine [( 3H]TCP) with high affinity (Kd = 10-15 nM) and the other binds the ligand with a relatively low affinity (Kd = 80-100 nM). The two classes of sites have different patterns of distribution. Forebrain regions are characterized by high-affinity sites (hippocampus greater than frontal cortex greater than thalamus greater than olfactory bulb greater than hypothalamus), but some parts (e.g., hippocampus, hypothalamus) contain low-affinity sites as well. In the cerebellum only low-affinity sites were detected. Binding sites for [3H]PCP and for its photolabile analogue [3H]azido-PCP showed a regional distribution similar to that of the [3H]TCP sites. The neuroleptic drug haloperidol did not block binding to either the high- or the low-affinity [3H]TCP sites, whereas Ca2+ inhibited binding to both. Photoaffinity labeling of the PCP receptors with [3H]AZ-PCP indicated that five specifically labeled polypeptides of these receptors (Mr 90,000, 62,000, 49,000, 40,000, and 33,000) are unevenly distributed in the rat brain. Two of the stereoselectively labeled polypeptides (Mr 90,000 and 33,000) appear to be associated with the high- and low-affinity [3H]TCP-binding sites; the density of the Mr 90,000 polypeptide in various brain regions correlates well with the localization of the high-affinity sites, whereas the density of the Mr 33,000 polypeptide correlates best with the distribution of the low-affinity sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Xanthine oxidase inhibition by 1,3-dipropyl-8-sulfophenylxanthine (DPSPX), an antagonist of adenosine receptors

Xanthine oxidase (XO), an enzyme involved in purine metabolism, is a source of either oxidants (superoxide radical) or antioxidants (uric acid). Interference with XO activity can lead to oxidative stress, thus contributing to the pathogenesis of cardiovascular diseases. The adenosine receptors antagonist, 1,3-dipropyl-8-sulfophenylxanthine (DPSPX), induces hypertension and cardiovascular injury in rats. Since DPSPX is a xanthine, we aimed at evaluating DPSPX's influence on XO activity to ascertain its contribution to DPSPX-induced hypertension. The activity of isolated XO in the presence of DPSPX was evaluated spectrophotometrically. Serum and urinary uric acid levels of DPSPX-treated rats were measured using a commercial kit. DPSPX inhibited XO activity in a concentration-dependent manner and reduced rat serum and urinary uric acid levels. It can be concluded that: DPSPX is an inhibitor of XO; decreased generation of uric acid may lead to oxidative stress, thus contributing to endothelial dysfunction and vascular morphological changes in DPSPX-treated rats.     

Alantrypinone and its derivatives: Synthesis and antagonist activity toward insect GABA receptors

The γ-aminobutyric acid (GABA) receptor bears important sites of action for insecticides. Alantrypinone is an insecticidal alkaloid that acts as a selective antagonist for housefly (vs rat) GABA receptors, and is considered to be a lead compound for the development of a safer insecticide. In an attempt to obtain compounds with greater activity, a series of racemic alantrypinone derivatives were systematically synthesized using hetero Diels–Alder reactions, and a total of 34 compounds were examined for their ability to inhibit the specific binding of [³H]4′-ethynyl-4-n-propylbicycloorthobenzoate, a high-affinity non-competitive antagonist, to housefly-head membranes. The assay results showed that (1) there is no significant difference between the potencies of natural (+)-alantrypinone and its synthetic racemate; (2) the amide NHs at the 2- and 18-positions are important for high activity; (3) there is a considerable drop in potency for compounds without an aromatic ring at the 16-position; and (4) a large substituent at the 3-position is detrimental to high activity.     

Radioimmunoassay for phencyclidine: Application to kinetic analysis in the rat

We report the development of a radioimmunoassay for phencyclidine (PCP) that is simple, rapid and sensitive to 0.5 ng/ml. Antibodies were raised in rabbits against the hapten, N-succinyl-3-aminophencyclidine. These antibodies proved to be very specific for PCP and exhibited less than 4% cross reactivity with the drug's two major metabolites. The assay was used for kinetic analysis of PCP in the rat following subcutaneous injection of 5 mg/kg of the drug. Serum and brain tissues were analyzed for PCP and the respective half lives were calculated to be 36 and 29 min for the α phase and 130 and 121 min for the β phase. The accuracy of the method was verified by concomitant assay of a number of kinetic samples by gas chromatography employing a nitrogen-phosphorus detector.     

A radioiodinated linear vasopressin antagonist: a ligand with high affinity and specificity for V1a receptors

A linear vasopressin antagonist, Phaa-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH2 (Linear AVP Antag) (Phaa = Phenylacetyl), was monoiodinated at the phenyl moiety of the tyrosylamide residue at position 9. This antagonist appeared to be a highly potent anti-vasopressor peptide with a pA2 value in vivo of 8.94. It was demonstrated to bind to rat liver membrane preparations with a very high affinity (Kd = 0.06 nM). The affinity for the rat uterus oxytocin receptor was lower (Ki = 2.1 nM), and affinities for the rat kidney- and adenohypophysis-vasopressin receptors were much lower (Ki = 47 nM and 92 nM, respectively), resulting in a highly specific vasopressin V1a receptor ligand. Autoradiographical studies using rat brain slices showed that this ligand is a good tool for studies on vasopressin receptor localization and characterization.     

Agonist and antagonist binding to tachykinin peptide NK-2 receptors

The binding of tachykinin peptides and fragments to NK-2 receptor sites in hamster urinary bladder membranes was examined and compared to binding to NK-1 receptor sites in rat submandibular gland. Neurokinin A (NKA) and its C-terminal fragments bound with highest NK-2 affinity and selectivity. N-terminal fragments of NKA did not bind to either type of receptor. Kassinin and eledoisin were NK-2 selective while physalaemin, phyllomedusin, and uperolein were NK-1 selective. Of fifteen tachykinin antagonists examined, none exhibited appreciable affinity or selectivity (relative to agonists) for NK-1, NK-2, or rat cerebral cortical NK-3 receptor sites. NKA binding to NK-2 sites was stimulated by Mn++ greater than Mg++ greater than Ca++. At the optimal concentration, the Mn++ stimulation was due to both an increased Bmax and increased affinity. The nonhydrolyzable guanine nucleotide, GppNHp, reduced agonist binding but not antagonist binding to NK-2 receptor sites. The nucleotide effect was due to a reduction in both Bmax and affinity and was potentiated by Mn++. The results indicate that tachykinin NK-2 receptor sites possess distinct structural requirements for agonists and are linked to a G-protein coupling system.     

Novel selective antagonist radioligands for the pharmacological study of A2B adenosine receptors

The adenosine A_2B receptor is the least well characterized of the four adenosine subtypes due to the lack of potent and selective agonists and antagonists. Despite the widespread distribution of A_2B receptor mRNA, little information is available with regard to their function. The characterization of A_2B receptors, through radioligand binding studies, has been performed, until now, by using low-affinity and non-selective antagonists like 1,3-dipropyl-8-cyclopentylxanthine ([³H]DPCPX),(4-(2-[7-amino-2-(2-furyl)-[1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-ylamino]ethyl)-phenol ([³H]ZM 241385) and 3-(3,4-aminobenzyl)-8-(4-oxyacetate)phenyl-1-propyl-xanthine ([¹²⁵I]ABOPX). Recently, high-affinity radioligands for A_2B receptors, [N-(4-cyanophenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)-phenoxy]acetamide ([³H]MRS 1754), N-(2-(2-Phenyl-6-[4-(2,2,3,3-tetratritrio-3-phenylpropyl)-piperazine-1-carbonyl]-7H-pyrrolo[2,3-d]pyrimidin-4-ylamino)-ethyl)-acetamide ([³H]OSIP339391) and N-benzo[1,3]dioxol-5-yl-2-[5-(1,3-dipropyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-3-yloxy]-acetamide] ([³H]MRE 2029F20), have been introduced. This minireview offers an overview of these recently developed radioligands and the most important applications of drugs towards A_2B receptors.     

Topological similarity of antagonist binding sites in biogenic amine receptors

The influence of physical and chemical properties of some sites of transmembrane receptor domains on the receptors ability to interact with nonspecific antagonists was investigated mathematically. The properties of sites located in 3rd and 7th transmembrane domains are most likely to explain pharmacological characteristics of the receptors. The possibility of receptor blocking by nonspecific antagonists not by competing with agonists but by influencing the receptor conformation is discussed.