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Background: NMDA receptor contributes to cause neuronal death in anoxic condition. It is not known how a part of NMDA receptors, NMDA-binding site and/or glycine-binding site, influence neuronal damage in rats' hippocampus in vitro., Methods: Rats' hippocampus, labeled with norepinephrine (3H-NE), was incubated in artificial cerebrospinal fluid (aCSF) and we measured 3H-NE in superfusion solution and remaining tissue. Glucose was eliminated from aCSF and 95% N2 + 5% CO2 produced the anoxic state., Results: The amount of 3H-NE release increased in anoxia with NMDA (NMDA-binding site agonist), while there was no influence on NMDA receptor in non-anoxic state even after D-serine (glycine-binding site agonist) has been administered. The 3H-NE was released more when D-serine (100 mu mM) and NMDA (100 mu mM) were administered together than when only D-serine (10 mu mM, 100 mu mM, 1000 mu mM) in anoxia or NMDA (10 mu mM, 100 mu mM, 1000 mu mM) in anoxia was administered., Conclusion: Glycine-binding site agonist alone does not act significantly but ion channels in NMDA receptor open more and become more effective when both glycine-binding site agonist and NMDA-binding site agonist exist, suggesting that there are interactions between NMDA-binding site and glycine-binding site in NMDA-receptor during anoxia.

MDR results from overexpression of P-glycoprotein (Pgp) and multidrug resistance protein (MRP or MRP1) that function as ATP-dependent efflux pumps. Lung resistance related protein (LRP) is also supposed to be involved in MDR. The human canalicular multispecific organic anion transporter (cMOAT) gene that is responsible for the defects in Dubin-Johnson syndrome was isolated. cMOAT is homologous to MRP1 and supposed to be involved in drug resistance. Human cMOAT cDNA transfected LLC-PK1 cells, LLC/cMOAT-1, have increased resistance to vincristine (VCR), 7-ethyl-10-hydroxycamptothecin (SN-38), and cisplatin. The multidrug resistance (MDR)-reversing agents, cyclosporin A (CsA) and PAK-104P, almost completely reversed the resistance to VCR, SN-38 and cisplatin of LLC/cMOAT-1 cells by interacting with the substrate binding site of cMOAT. Treatment of human colorectal carcinoma SW-620 cells with sodium butyrate(NaB) induced LRP in the cells and conferred resistance to Adrianycin(ADM), VCR, VP-16, gramicidin D and taxol. Two LRP-specific ribozymes inhibited the NaB-induced expression of LRP in SW-620 cells and almost completely abolished their acquisition of the MDR phenotype. The accumulation of ADM, VCR and taxol was not decreased in NaB-treated cells, suggesting that ATP-binding cassette transporters are not involved in the MDR of NaB-treated cells. ADM was mainly located in the nuclei of untreated and the cytoplasm of NaB-treated cells. The accumulation level of ADM in the nuclei isolated from untreated cells or those from treated cells in the presence of anti-LRP polyclonal antibody was higher than that from treated cells in the absence of the antibody. Efflux of ADM from nuclei isolated from NaB-treated cells was enhanced compared with those from untreated cells and NaB-treated cells transfected with a LRP-specific ribozyme. The polyclonal antibody against LRP inhibited the enhanced efflux of ADM from nuclei isolated from NaB-treated cells. These findings indicate that LRP is involved in resistance to ADM, VCR, VP-16, taxol and gramicidin D, and has an important role in the transport of ADM from the nucleus to the cytoplasm.

(-)-Epigallocatechin gallate (EGCG), the main constituent of green tea, inhibited a tumor promoting activity of okadaic acid in a two-stage carcinogenesis experiment on mouse skin. The group treated with a single application of 100 micrograms 7, 12-dimethylbenz (a) anthracene followed by repeated applications of 1 microgram okadaic acid resulted in 80% of tumor-bearing mice and 4.7 of average numbers of tumors per mouse in week 20. Repeated applications of 5 mg EGCG, prior to okadaic acid, completely inhibited the tumor formation in mice up to week 20. The inhibitory effects of EGCG with two different doses of each application, 1 mg and 5 mg, were dose-dependent. A topical application of 5 mg EGCG immediately reduced the specific binding of [3H]okadaic acid to a particulate fraction of mouse skin to as low as 30% of control. According to the Scatchard analysis, the reduction of specific [3H]okadaic acid binding was mainly due to the reduction of the binding sites, not due to the change of the affinity. The reduction of the specific binding was closely related to the inhibitory effct of EGCG on tumor promotion of okadaic acid. Since EGCG is a non-toxic compound, ingested in green tea in daily life in Japan, EGCG is one of the candidates for practical cancer chemopreventive agents.

Two subtypes (alpha 1A and alpha 1B) of alpha 1-adrenoceptors have been defined on the basis of radioligand binding studies with 2-(2,6-dimethoxyphenoxyethyl)-1,4-benzodioxane (WB4101). In contrast, functional studies with blood vessels have proposed another subclassification, where the alpha 1-adrenoceptors can be classified into putative two (alpha 1H and alpha 1L) or three (alpha 1H, alpha 1L and alpha 1N) subtypes. The present study was performed to elucidate the effects of repeated treatment with electroconvulsive shock (ECS) or imipramine on the subtypes of alpha 1-adrenoceptors. The density of the alpha 1H binding sites (including alpha 1A and alpha 1B binding sites) in the frontal cortex was increased by repeated treatment with ECS but decreased by imipramine. These treatments did not change the density of alpha 1L binding sites. These results indicated that repeated treatment with ECS and imipramine had different effects on the alpha 1H binding sites in the rat frontal cortex. In addition, the effects of antipsychotic drugs, antidepressant drugs and calcium channel antagonists on [3H]prazosin binding were studied. The order of inhibition of [3H]prazosin binding was: antipsychotic drugs (1.4-4.2 nM) > antidepressant drugs (69-116 nM) > calcium channel antagonists. The calcium channel antagonists, verapamil (720 nM), diltiazem (4.3 microM) and nicardipine (75 microM) were weakly bound. These results suggested that the antimanic effects of calcium channel antagonists were not associated with inhibition of alpha 1-adrenoceptors.

We investigated the effects of repeated administrations of methamphetamine on the dopamine uptake sites in the rat striatum, by using an increasing dose paradigm of methamphetamine (2.5, 5, 7.5, 10 mg/kg sc x 2, every other day for a week) which has been established to produce behavioral sensitization in methamphetamine-induced behavior. A significant decrease in the Bmax value of [3H] GBR12935 binding and in the Vmax value of [3H] dopamine uptake was demonstrated in rats sacrificed 7 days after the final methamphetamine administration, and it was also shown that the Bmax value of [3H] GBR12935 binding was reduced in the dose dependent manner. Moreover, repeated administrations of methamphetamine significantly reduced the [3H] GBR12935 binding sites in the rat striatum even 30 days after the drug discontinuation. These data suggest that repeated administrations of methamphetamine induces a long lasting decrease of the dopamine uptake sites in the rat striatum. For an understanding of neurochemical basis of these findings, we investigated the effects of pharmacological manipulations in the presynaptic dopaminergic system by the pretreatment of alpha-methyl-p-tyrosine, reserpine or amfonelic acid and the blockade of postsynaptic dopamine receptor by a pretreatment of SCH23390 or YM-09151-2, on decrease in the dopamine uptake sites following the repeated administration of methamphetamine in the rat striatum. While pretreatment of alpha-methyl-p-tyrosine or amfonelic acid prevented a reduction in the dopamine uptake sites, pretreatment of reserpine accelerated this reduction. These data suggest that presynaptic newly synthesised dopamine plays an important role in the methamphetamine-induced decrease in the dopamine uptake sites. Pretreatment of SCH23390 or YM-09151-2 protected the dopamine uptake sites from reductive effect of repeated methamphetamine administration. Because there was no presynaptic dopaminergic mechanism understanding this result, some other neronal networks, which communicate with dopaminergic system, might make a reasonable explanation of this result.

In humans, repeated use of methamphetamine produces hypersensitivity to the psychotogenic effects of methamphetamine that persists for months to years after the discontinuation of methamphetamine administration. Methamphetamine-induced psychosis has been thought to be a useful experimental model for schizophrenia. A possible involvement of the glutamate system in the hypersensitivity including behavioral sensitization or reverse tolerance is recognized in an animal model for methamphetamine psychosis. We investigated the effects of antagonists of N-methyl-D-aspartate (NMDA) receptor on methamphetamine-induced decrease in dopamine (DA) uptake sites in the rat striatum and on the behavioral sensitization. Repeated administrations of escalating doses of methamphetamine (2.5, 5, 7.5, 10mg/kg s. c. x 2, every other day for a week) decreased DA uptake sites to about 75% of the control in the striatum assayed by binding with [3H]GBR 12935. Co-administration of MK-801, a non-competitive antagonist of NMDA receptor, and methamphetamine significantly prevented the methamphetamine-induced decrease in striatal [3H]GBR 12935 binding in a dose dependent manner. Administration of MK-801 alone did not affect the [3H]GBR 12935 binding. Furthermore, co-administration of SDZ EAA494, a competitive antagonist of NMDA receptor, and methamphetamine also prevented the methamphetamine-induced decrease in the striatal [3H]GBR 12935 binding in a dose dependent manner. In methamphetamine-pretreated rats, the methamphetamine challenge (2.5mg/kg) after a 7-day-drug-free period produced an initial elevation in locomotion lasting for 10-30 min which was followed by a precipitous drop in the locomotion activity to very low levels for approximately 50-70 min. During the period of reduced locomotor activity, methamphetamine-pretreated rats showed intense focused stereotyped behavior. In contrast, animals treated with both MK-801 and methamphetamine showed neither the progressive enhancement of the locomotor activity nor the stereotyped behavior induced by the drug. Pretreatment with MK-801 blocked the development of the methamphetamine-induced behavioral sensitization. These results suggest an involvement of excitatory amino acids in neurochemical effects of methamphetamine on the dopamine system in the striatum.

Changes in rat and human testicular human chorionic gonadotropin (hCG) binding sites induced by hCG were estimated in vivo and in vitro. After a single administration of hCG, the specific hCG binding sites were significantly reduced for 7 and 5 days in rat and human testes, respectively. However, hCG binding sites had recovered to pretreatment values by the 14th day after the administration. Occupied hCG binding sites measured in both rat and human testes accounted for about half of the reduced binding sites on the day after administration of hCG, but thereafter the number of sites occupied was not correlated with the reduction of the binding induced a dose-related significant loss of the specific hCG binding sites for 7 and 5 days in rat and human testes, respectively. Thereafter, the number of binding sites gradually increased. These patterns of change in hCG binding sites in vitro were similar to those in vivo. These findings suggest that the reduction in hCG binding sites in rat and human testes by hCG is due not only to occupancy but also down-regulation of the binding sites. In conclusion, the present testicular organ culture method is useful to study hormonal regulation of testicular function, especially in human testes.

Estrogen priming increases uterine 8S macromolecule which binds progesterone specifically. Progesterone-8S complex in the cytoplasm enters into nucleus and is bound to chromatin finally. In this paper, the mode of nuclear translocation of steroid in exchange assay of receptor introduced by Anderson et al., and the mode of binding to chromatin were studied on the progesterone-receptor complex in the uterus of estrogen primed female rabbit. 1. After intravenous administration of 200 mug progesterone into the estrogen primed immature rabbit, uterine nuclei were prepared by the method in Table 1. These nuclei were incubated with 3H-progesterone and cold steroids at 4 degrees C for 30 minutes, and then washed with buffer A. The radioactivity of the nuclei was counted. This experiment was performed at 4 degrees C because progesterone receptor and chromatin were observed to be degraded at 37 degrees C for 20 minutes. The effect of cold steroids in vitro on the incorporation of 3H-progesterone into the uterine nuclei of rabbit pretreated with progesterone was found to be similar to their effect on progesterone-receptor binding in cytosol or chromatin (Fig. 1). 2. The effect of cold steroids on 3H-progesterone-receptor-chromatin triplex (Table 2 and Fig. 2) was examined. Once 3H-progesterone-receptor-chromatin triplex was formed, it was difficult to exchange 3H-progesterone to other steroids at 4 degrees C. These results (1 & 2) indicate that progesterone-receptor complex enters into nucleus and is bound to chromatin. Exchange of steroid may occur in the nuclear progesterone-receptor complex, which is free from the binding with chromatin. And thus exchange assay cannot represent quantitative data on receptor content. 3. 3H-progesterone-8S or 5S complexes were obtained by 5 approximately 20% sucrose linear gradient centrifugation (Fig. 3). The same molar concentration of these complexes from estrogen primed or castrated rabbit uterus were incubated with primed uterine chromatin for 30 minutes. Then the chromatin was washed with buffer A and the radioactivity was counted. It was shown in Fig. 4 that 3H-progesterone-8S complex was bound to chromatin much more tightly than 3H-progesterone 5-S complex in preparations obtained from both castrated or primed uterine cytosols. All these results indicate that 8S may be the biologically active form of the receptor. 4.3H-progesterone uptake into uterine nuclei was observed in very limited amount following the injection into uterine artery. The radioactivity in nuclei decreased easily by washing with buffer A as in Fig. 5. The small amount of residual radioactivity after washing, that is, very limited number of binding sites with high affinity is considered to be indicative of biologically active binding.

[3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) is thought to label a single population of serotonin (5-HT)1A receptor, but some reports implicate multiple binding sites exist. In addition, while 5-HT1A receptor activates or inhibits adenylate cyclase, 5-HT1A receptor high and low affinity states are not reported. In this experiment, we found that [3H]8-OH-DPAT had multiple binding sites, which contained 5-HT1A receptor high and low affinity states, in rat brain membranes. [3H]8-OH-DPAT saturation binding experiment revealed high and low affinity binding sites existed. High affinity binding site was dense in hippocampus and sparse in striatum. 5-HT agonist and antagonist biphasically displaced [3H]8-OH-DPAT binding in frontocortical, hippocampal, and striatal membranes. These drugs potently displaced high affinity [3H]8-OH-DPAT binding, but clomipramine (5-HT reuptake inhibitor) potently displaced low affinity binding. High affinity [3H]8-OH-DPAT binding site was decreased by guanosine triphosphate and Na+, but increased by divalent cations, implicating coupling with G protein(s). Low affinity [3H]8-OH-DPAT binding site was decreased by cations, especially by monovalent cations and Ca2+. After the destruction of 5-HT neuron by parachloroamphetamine, only the low affinity binding site decreased. These results indicate that [3H]8-OH-DPAT not only labels the 5-HT1A receptor high and low affinity states but has presynaptic binding site relating to 5-HT reuptake site.

Many studies demonstrated the presence of cytosolic thyroid hormone binding proteins (CTHBPs) in various tissues, but the physiologic significance of these CTHBPs is not clear, partially because of the lack of information about the physicochemical properties of CTHBPs as purified forms. Since the difficulty in isolating these CTHBPs is considered to be due to instability during the various procedures for their isolation, studies on the stability of CTHBPs of rat liver were performed using a charcoal binding method to separate bound and free hormones. Binding characteristics of CTHBPs of rat liver were also determined. Specific triiodothyronine (T3) binding sites of cytosolic T3 binding protein (CT3BP) of rat liver were destroyed as the time progressed in homogenate at 0 degrees C, and Aprotinin (500 U/ml) had little effect in protecting these binding sites. T3 binding sites were stable in the form of cytosol at -20 degrees C up to 10 weeks. Dithiothreitol (DTT) had no effect on T3 binding to cytosol. T3 binding to CT3BP was pH-dependent with maximum specific binding at pH 7.4. T3 binding to CT3BP was stable at 4 degrees C overnight but was destroyed rapidly at 37 degrees C. Interestingly, specific T3 binding sites of CT3BP were completely abolished by dialysis, and Ca2+ or Mg2+ had no effect on retaining the specific binding sites. Thus, CT3BP was supposed to require some dialysable small molecule(s) to maintain specific T3 binding sites. Scatchard plot of T3 binding to crude cytosol revealed a high affinity, limited capacity T3 binding site with affinity constant (Ka) of 5.9 X 10(7) M-1 and maximum binding capacity (MBC) of 118 ng/g. liver. Relative affinities of T3 analogues for CT3BP were determined by comparing the molar concentrations of T3 analogues required for 50% inhibition of tracer 125I-T3 binding. If the affinity of L-T3 was assigned 100, D-T3 would have a value of 66.1; L-T4, 22.3; D-T4, 16.5; Triac, 6.2; and both Tetrac and reverse T3 were less than 1. Thus, the binding characteristics of CT3BP were fundamentally different from those of nuclear T3 receptor. Cytosolic thyroxine (T4) binding protein (CT4BP) of rat liver was relatively stable compared with CT3BP in homogenate at 0 degrees C. CT4BP was also stable in the form of cytosol at -20 degrees C for 10 weeks. CT4BP was pH-dependent with maximum specific binding at pH 7.4. It was stable at 4 degrees C overnight but destroyed rapidly at 37 degrees C. Specific T4 binding was decreased by dialysis but not abolished completely.(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of subchronic administration of various psychotropic agents on the density of 5HT 2 receptor binding sites in rat cerebral cortex were investigated. In addition to antidepressant agents, some neuroleptic drugs including chlorpromazine (CPZ), spiperon, and cisflupentixol reduced the numbers of 5HT-2 receptor binding sites after 21 days treatment. The more selective D-2 antagonist haloperidol and sulpiride were totally ineffective in this regard. Anxiolytic agents, benzodiazepine derivatives were also ineffective. Central 5, 7-DHT-induced lesion of 5HT neurons demonstrated that intact 5HT neurons were not required for the reduction of 5HT-2 receptors by desipramine (DMI). Co-administration of DMI and alpha-2 antagonist yohimbine (YOH) produced down-regulation of 5HT-2 receptors within 3 days, whereas each agent alone did not produce such effect. The effect of 3-day treatment with mianserin (MIA, alpha-2 and 5HT-2 antagonist) alone and DMI plus YOH producing 5HT-2 down-regulation, were not prevented by the pretreatment with DSP-4 which selectively destroyed NE neurons. These results suggest that the synaptic availability of 5HT may not be required for DMI-induced down regulation of 5HT-2 receptor binding sites, and the mechanisms mediated through postsynaptic alpha-2 and 5HT-2 receptors are important factors in the regulation of 5HT-2 receptor density. Evidence suggests that the 5HT-1 receptor site is functionally linked to adenylate cyclase in the brain, but a biochemical effector system which is linked to the 5HT-2 receptor site has not been found. The metabolism of inositol phospholipids in response to 5HT was, therefore, investigated in human platelets using sensitive radioisotopic method of Berridge (1983). In platelets prelabeled with 3H-myo-inositol, in Ca++ free HEPES buffer containing 10 mM LiCl, 5HT caused a dose-dependent accumulation of inositol-1-phosphate (IP1) during 15 min incubation. A maximal increase in IP1 formation was observed at 30 microM of 5HT and the EC50 value was 4 microM. Ketanserin, a selective 5HT-2 receptor antagonist was a potent inhibitor of 5HT-stimulated IP1 accumulation, with a Ki value of 12 nM, but a selective 5HT-1 antagonist, (-)-propranolol (1 microM) failed to block the 5HT response. These results indicate that 5HT is activating 5HT-2 receptors, but not 5HT-1 in human platelets. CPZ and imipramine inhibited 5HT-stimulated IP1 accumulation, with Ki values of 124 nM and 2.56 microM, respectively.

The effects of tricyclic antidepressant drugs on cardiac receptor binding sites and Ca2+ binding to cardiac sarcoplasmic reticulum were investigated in rats. 1) Quinupramine was found to possess higher affinity for muscarinic cholinergic receptor binding sites in brain and heart, compared with imipramine. 2) Repeated quinupramine treatment induced a significant increase in the Bmax value in cardiac muscarinic cholinergic receptors. 3) In imipramine treated heart, isoproterenol-induced stimulation of ornithine decarboxylase activity was significantly decreased compared with that in the vehicle control. 4) Repeated quinupramine and imipramine treatment caused a significant reduction of Bmax of 45Ca2+ binding in cardiac sarcoplasmic reticulum fractions. 5) After single quinupramine treatment, its concentrations in heart were higher than those in brain and plasma. 6) After the repeated quinupramine treatment, its concentration in heart was 2.4 times higher than that after single treatment. These results demonstrate that repeated tricyclic antidepressant drug treatment caused a functional impairment in Ca2+ binding sites of cardiac sarcoplasmic reticulum and adaptive changes of receptor binding sites.