{'fraction_non_alphanumeric': 0.03246153846153846, 'fraction_numerical': 0.014615384615384615, 'mean_word_length': 5.43026706231454, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Binding characteristics of [3H]quinupramine to rat brain membrane fractions.

The binding characteristics of [3H]quinupramine to rat brain membrane fractions were studied. The specific binding of [3H]quinupramine to rat brain membrane fractions was stable, reversible and saturable. Scatchard analysis of the data from saturation experiments indicated that the specific binding was a single population with an affinity (KD) of 3.04 nM, a maximal binding site number (Bmax) of 714 fmol/mg protein, and a Hill coefficient (nH) of 1.08. Compounds known to inhibit muscarinic cholinergic receptors such as atropine and quinuclidinyl benzilate were the most potent competitors of [3H]quinupramine binding. When the drug potencies in inhibiting [3H]quinupramine binding were tested in the presence of 10 nM atropine, mianserin was the most potent competitor. Studies of the subcellular fractions showed that there was an enrichment of [3H]quinupramine binding sites in the synaptosome fraction. The regional distribution study revealed the highest densities of binding sites in the cerebral cortex and the lowest in the cerebellum. Thus, the specific binding of [3H]quinupramine observed here can be accounted for by both muscarinic cholinergic and serotonin S2 receptors.

The receptor binding assay has been considered as a useful technique for iden tifying the site of action for a variety of endobiotic and xenobiotic substances. For example, the discovery of high-affinity binding sites for psychoactive drugs (1-3) had opened up new approaches for the study of these drugs and their mechanisms of action. Furthermore, these procedures have been used to identify endogeneous ligands for some drug binding sites. Recently, some investigators reported the characteristics of the specific binding sites of antidepressant drugs (4)(5)(6)(7)(8)(9)(10)(11)(12)(13).
Quinupramine was a weak inhibitor of monoamine uptake systems (17), and it was found to possess decreased central serotonin S2 receptor binding without altered (S-adrenergic receptor binding after its repeated treatment (18). Through these observations, we have suggested that the pharmacological properties of quinupramine, chemically one of the typical tricyclic anti depressant drugs, are close to those of the atypical antidepressant drugs (18). In this paper, the studies were carried out to clarify the mechanisms of action of quinupramine by characterizing the specific binding sites in rat brain.  The rate of dissociation of [3H]quinupra mine binding was examined by adding 10 ,uM quinupramine at equilibrium (Fig. 2,  quinupramine was displaced by these com pounds (Fig. 3), the majority of the specific binding of [3H]quinupramine may be at tributed to the muscarinic cholinergic receptor binding sites.

Materials and Methods
Whether or not the binding sites labelled by [3H]quinupramine represent other neuro transmitter receptors was studied using various drugs (Fig. 4)  [3H]quinupramine to rat whole brain without cerebellum. The standard assay conditions were used. The specific binding was determined as the difference between the total and binding in the presence of the displacer, 10 pM quinupramine, and it was estimated to be about 3,500 dpm. Points shown are the mean±S.E.M. from three independent determina tions, each performed in duplicate.    Table 4. Subcellular distribution of the specific binding of [3H]quinupramine in rat whole brain without cerebellum The specific binding of [3H]quinupramine was determined in various subcellular fractions using the standard assay procedure. The data are presented in terms of specific binding per milligram of protein and as a percentage of the total binding activity recovered.
Values given are the mean±S.E.M. of three determinations, each performed in duplicate. carried out in the presence of atropine, with IC50 values of 2.7 and 98 nM, respectively. By contrast, the affinity of pyrilamine and phentolamine varied less than 7-fold between its affinities for [3H]quinupramine binding in the presence and absence of atropine. The differential effects of these drugs indicated that the binding sites labelled by [3H] quinupramine represented both muscarinic cholinergic and serotonin S2 receptors.
Regional distribution of the specific binding of [3H]quinupramine: The binding in the richest region, frontal cortex, was almost seven times greater than that in the poorest region, cerebellum (Table 3). Subcellular distribution of the specific binding of [3H]quinupramine: In the principal subcellular fractions of whole homogenates of rat whole brain without cerebellum, the greatest amounts and specific activity of [3H] quinupramine binding occurred in the crude mitochondrial (P2) fraction (  (25) binding. The KD-value derived from the equilibrium experi ments was very similar to the value derived from the kinetic experiment using the half time for the initial phase of dissociation. Hill analysis of the binding showed no coopera tivity (n"=1.08).
Many investigators reported the character istics of the specific binding sites of anti depressant drugs. In all cases, the binding sites had ultimately been found to be neuro transmitter receptors or transport sites. Mequitazine is a potent antihistamic drug with a potent antimuscarinic cholinergic activity (28), and it has a quinuclidine group as a moiety in the molecule. Though the chemical structure of mequitazine is very close to that of quinupramine, mequitazine has weak antiserotonergic activity (29) and no antidepressant activity. If the specific binding of [3H]quinupramine involves central neurotransmitter receptor binding sites, this binding should be dis tributed unevenly in the brain. The cerebral cortex had the highest regional distribution of [3H]quinupramine specific binding, while the cerebellum had the lowest. The highest specific binding of [3H]ketanserin, a ligand for serotonin S2 receptors, was determined in the frontal cortex in rat brain, and specific binding was virtually not detected in the hypothalamus and cerebellum (30). Similarly, the highest specific binding of [3H] quinuclidinyl benzilate, a ligand for muscarinic cholinergic receptors, was detected in the striatum and the lowest in the cerebellum (31). The data of the regional distribution of [3H]quinupramine binding, which labels both serotonin S2 and muscarinic cholinergic receptors, may be not contrary to these observations. Neurotransmitter receptors are generally assumed to involve postsynaptic membranes.