The tropolonic alkaloid colchicine significantly reduces the behavioural, electroencephalographic and histological damage seen after a 6-min occlusion of the two common carotid arteries of the Mongolian gerbil if the compound is administered at 2 or 4 mg/kg i.p. immediately upon reperfusion. A 45% increase in high-frequency ECoG activity and significant reduction of 80% in the hypermotility of the gerbils, with 63% less faults in a passive avoidance paradigm, were observed in conjunction with considerable protection of the hippocampus, after a single dose of 4 mg/kg colchicine. No adverse effects of colchicine treatment on animal movement and body weight were observable. Colchicine's possible mode of action, via inhibition of cellular transport systems, is discussed.

[3H]52770 RP, a PAF antagonist, was found to bind with high affinity and in a reversible manner on specific and saturable binding sites in guinea pig cortical membranes. Scatchard analysis revealed the presence of one class of binding sites with an equilibrium dissociation constant of 0.38 nM and a Bmax of 1190 fmol/mg protein. However, these binding sites did not correspond to the PAF receptors described with this ligand in platelet plasma membranes; indeed, PAF and its analogs were unable to displace [3H]52770 RP binding in guinea pig brain. Therefore, one may conclude that [3H]52770 RP in guinea pig brain labels acceptor or recognition sites, rather than true receptor sites.

Serotonin-S2 receptor sites were first described in studies in which 3H-spiperone was used in frontal cortical membrane preparations (Leysen et al. 1978; Peroutka and Snyder 1979). The receptor binding sites were further characterized and could be better distinguished by using the more selective serotonin antagonist 3H-ke-tanserin (Leysen et al. 1982). Serotonin-S2 receptors were shown to have a role in serotonin agonist-induced behavioral excitation and discriminative stimulus effects in rodents, and also to mediate serotonin-induced vasoconstriction and platelet function. Recently it has been shown that inositol phospholipid turnover forms part of the signal transducing system coupled to serotonin-S2 receptor sites (for review see Leysen et al. 1984).

AbstractChemical compounds synthesized at Janssen Pharmaceutica are commonly used to identify and characterize neurotransmitter receptors in the brain and in the peripheral system through in vitro or in vivo binding techniques. In this regard, they have played a prominent role in advancing our knowledge about dopamine and serotonin receptors; indeed the identification in vitro or in vivo of these receptors, their solubilization, and their purification were made possible by the availability of3H‐haloperidol,3H‐spiperone, and3H‐ketanserin. Other ligands such as3H‐dexetimide and3H‐lofentanil were used to demonstrate, for the first time, axonal transport of muscarinic and opiate receptors. Finally some of these ligands (spiperone, carfentanil, ketanserin) allowed human brain receptors to be visualized through PET scanner.

Several [3H] radioligands have been proposed for In vitro biochemical characterization of serotonin-S2 receptors. Among the most widely used are [3H]spiperone and [3H]ketanserin. Recently, a derivative of ketanserin, [3H]7-aminoketanserin has been Introduced as a high affinity selective serotonin-S2 receptor ligand with a very low non-specific binding [1]. A major drawback of all these ligands, however. Is the low specific radioactivity especially if one wants to study tissues with low receptor contents. [125I]-Labelled compounds have much greater sensitivity than tritiated probes. The available iodinated serotonin-S2 receptor ligands [125I]LSD [2] and [125I]MIL (a methylated derivative of [125I]LSD) [3] show, however, the major disadvantage of interacting with other serotonin binding sites at nanomolar ligand concentrations [4]. It therefore seemed desirable to develop a [125I]-labelled compound, based on a selective ligand for the serotonin-S2 receptor subtype, such as 7-aminoketanserin.

[3H]-7-Aminoketanserin (7-amino-3-[2-[4-(2-tritio-4-fluorobenzoyl)-1- piperidinyl]ethyl]-2,4-(1H,3H)-quinazolinedione), an amino derivative of the selective serotonin-S2 antagonist ketanserin, was synthesized and tested for in vitro labeling of serotonin-S2 receptors. The compound showed a very high affinity for both membrane-bound and detergent-solubilized serotonin-S2 receptors with KD values of 0.35 and 2.03 nM, respectively. At nanomolar concentrations, binding to serotonin-S1 sites was totally absent. Serotonin-S2 receptor binding was characterized by a slow dissociation and a very low nonspecific binding. In rat frontal cortex preparations, binding could be displaced by nanomolar concentrations of different serotonin antagonists and micromolar concentrations of serotonin agonists. Compounds with other pharmacological profiles were poorly or not active. Introduction of an amino function in this new radioligand led to a decreased lipophilicity. Therefore, besides being a valuable radioligand for routine binding studies, [3H]-7-aminoketanserin will probably be a good ligand for labeling serotonin-S2 receptors on intact cells.

Na+,K+-ATPase levels were measured in different segments of rat sciatic nerves by in vitro binding of [3H]ouabain. Binding sites were found to accumulate on both sides of a ligature tied on the sciatic nerve, indicating an anterograde and retrograde axoplasmic transport of Na+,K+-ATPase. Accumulation of Na+,K+-ATPase at the ligature was time dependent and appeared to occur through fast axoplasmic transport mechanisms. This accumulation on both sides of the ligature was also visualized by autoradiographic studies in longitudinal section of sciatic nerves using [3H]ouabain.

Serotonin-S2 receptors from rat frontal cortex were solubilized using CHAPS/sodium chloride. Reconstitution of the solubilized receptors was achieved by dilution of the soluble preparation, followed by centrifugation to remove the detergent. The receptors were truly reconstituted as judged by sedimentation, increased thermostability and electron microscopy. The reconstituted preparation showed high-affinity binding of [3H]7-aminoketanserin. The binding characteristics resembled those obtained for membrane-bound receptors.

Axonal transport of receptors was studied in streptozocin-diabetic rats using two different binding models. Streptozocin-induced hyperglycemia caused a reduced accumulation of muscarinic receptors above a ligature placed on rat sciatic nerves when the binding assay was performed in vitro with [3H]QNB. In the vagus nerve, the retrograde axonal transport of receptor-bound opiate was strongly decreased in the streptozocin-treated rats when [3H]lofentanil was used in vivo to label opiate receptors. Insulin partly reversed the changes observed in the streptozocin-treated rats. These findings suggest that impaired axonal transport of receptors may explain part of the neurological disturbance which is seen in diabetic patients.

[3H]Fluspirilene, a neuroleptic molecule of the diphenylbutylpiperidine series, binds to skeletal muscle transverse tubule membranes with a high affinity corresponding to a Kd of 0.11 +/- 0.04 nM, A 1:1 stoichiometry was found between [3H]fluspirilene binding and the binding of (-)-[3H]desmethoxyverapamil [(-)[3H]D888], one of the most potent Ca2+ channel inhibitors. Ca2+ channel inhibitors such as D888, verapamil, gallopamil, bepridil, or diltiazem antagonize [3H]fluspirilene binding besides antagonizing (-)[3H]-D888 binding. Neuroleptics, especially those of the diphenylbutylpiperidine family, also antagonize both (-)[3H]D888 binding and [3H]fluspirilene binding. There is an excellent correlation between affinities found from [3H]fluspirilene binding experiments and those found from (-)[3H]D888 binding experiments. Analysis of the properties of these cross-inhibitions indicates that [3H]fluspirilene binds to a site that is not identical to that for phenylalkylamine derivatives (gallopamil, verapamil, diltiazem, and bepridil). Voltage-clamp experiments have shown that fluspirilene is an efficient inhibitor of the voltage dependent Ca2+ channel, achieving a half-maximal effect near 0.1-0.2 nM and nearly complete blockade at 1 nM. Fluspirilene blockade has little voltage dependence.

Opiate receptors measured in vivo with [3H]lofentanil in the rat vagus nerve were found to accumulate on both sides of a ligature. The time-course of accumulation was completely different in the proximal and the distal segments; the labelling was maximal 4 h after injection of [3H]lofentanil above the ligature but 16-24 h below the ligature. In unligated rats, a peak of radioactivity appeared in the nodose ganglion 16 h after injection; vagotomy, vinblastine or chronic treatment with capsaicin prevented the appearance of this delayed accumulation in the ganglion. These foregoing experiments suggest that opiate may act in the cell body of sensory neurones after being internalized at the nerve terminals and then transported retrogradely through fast axoplasmic mechanisms.

In vitro and in vivo receptor-binding properties of the new serotonin antagonist, ritanserin, are reported. In in vitro binding assays, ritanserin shows high affinity binding to serotonin-S2 sites in rat frontal cortex tissue: IC50 = 0.9 nM without drug preincubation and 0.3 nM with 30-min drug preincubation; IC50 values for histamine-H1, dopamine-D2, and adrenergic-alpha 1 and -alpha 2 sites were 39-, 77-, 107-, and 166-fold higher, and at up to 1 microM, the drug did not bind to serotonin-S1 sites. In in vitro assays, ritanserin dissociated very slowly from serotonin-S2 (t1/2 = 160 min) and histamine-H1 sites (t1/2 = 77 min) and rapidly from dopamine-D2 sites (t1/2 = 11 min). Half-times of dissociation from adrenergic-alpha 1 and -alpha 2 sites were 18 and 26 min. The inhibition by ritanserin of [3H]ketanserin binding was found to be partially noncompetitive and the inhibitory potency increased with drug preincubation. Due to the slow dissociation of ritanserin from the serotonin-S2 sites, the drug cannot be displaced completely by [3H]ketanserin. In contrast, inhibition by ritanserin of [3H]haloperidol binding to dopamine-D2 sites in rat striatum was fully competitive, in agreement with the rapid dissociation of the drug from the latter sites. In ex vivo binding assays using brain areas of rats and guinea pigs treated subcutaneously with ritanserin, occupation of serotonin-S2 sites was observed at very low dosage (50% occupation at 0.08-0.1 mg/kg) and sites remained occupied during a prolonged time period (greater than 70% occupation up to 48 hr after 2.5 mg/kg ritanserin). Histamine-H1 receptor sites in guinea pig cerebellum became occupied at dosages 25-fold higher than the dosage producing occupation of frontal cortical serotonin-S2 sites. Dopamine-D2 sites in rat striatum and cortical adrenergic-alpha 1 sites became only slightly occupied (less than 20%) at higher dosages and the effect was not dose-dependent. Adrenergic-alpha 2 sites were not occupied up to doses of 160 mg/kg given subcutaneously. In vivo binding assays using [3H]spiperone confirmed the occupation of frontal cortical serotonin-S2 sites following low dosage of ritanserin and a minor occupation of striatal dopamine-D2 sites. Levels of dopamine and serotonin and their metabolites remained unchanged in brain areas of rats orally treated with ritanserin up to dosages of 40 mg/kg. At 160 mg/kg, there seemed to be a slight reduction in dopamine and serotonin content.(ABSTRACT TRUNCATED AT 400 WORDS)

Na+ channels levels were measured in different segments of rat vagus and sciatic nerves by in vitro binding using a tritiated ethylene-diamine tetrodotoxin derivative ([3H]en-TTX). Binding sites were found to accumulate on both sides of a ligature tied on the sciatic nerve indicating an anterograde and retrograde axoplasmic transport of Na+ channels. Accumulation of Na+ channels at the ligature was time-dependent and appeared to occur through fast axoplasmic transport mechanisms. This accumulation on both sides of a ligature was also visualized by autoradiographic studies in longitudinal sections of sciatic nerves using [3H]en-TTX.

Binding on/in whole cells seems to be a more appropriate approach for studying receptor sites in physiological conditions. However, certain difficulties encountered throughout the characterization of [3H]spiperone binding in human lymphocytes led us to reconsider this problem. The IC50 values of [3H]spiperone binding to human lymphocytes did not correlate with those found in rat striatum; domperidone was inactive in lymphocytes whereas it is one of the most potent dopamine antagonists in rat striatal preparations in vitro. In contrast, chloroquine, a lysosomotropic drug, displaced [3H]spiperone at low concentration in intact lymphocytes but did not in the striatum. [3H]Spiperone binding was not displaceable in the membrane preparation of lymphocytes. Similar results were obtained with other intact cells, fibroblasts, hepatocytes and neuroblastoma cells using [3H]spiperone and other ligands, such as [3H]haloperidol, [3H]pyrilamine and [3H]ketanserin. Here again, displaceable binding was only present in intact cells but not in membrane fractions. Such a 'displaceable' binding was not related to receptor sites but may be regarded as non-specific binding which should correspond to a trapping phenomenon presumably in the lysosomes. Binding studies on intact cells need more caution than when performed on membrane preparations; indeed, permeation or trapping of ligands in the nanomolar range represents a serious drawback which, sometimes, can give the illusion of specific binding.