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Objective To establish mouse model of dementia by intracranial injection of A?25-35 and small amount of ibotenic acid(IBO) and to explore whether the effects of catalpol can affect the brain M receptor density and the short term memory. Methods The mice were randomly divided into three groups: control group,model group,treat group which were given orally for 2 months with 50 mg?kg-1?d-1 of catalpol.Dementia model was developed by single unilateral injection of 0.3 ?L of a solution of A?(1?L normal saline containing 4 ?g of A?25-35 and 1 ?g of ibotenic acid) into right basal ganglion region according the atlas of mouse brain with the aid of a stereotaxic equipment.The track of injection was observed by HE staining.The learning/memory ability was measured by Y-maze perfor-mance.The brain muscarinic receptor density was analyzed with single-site binding assay using 3H-quinuclidinyl benzilae(QNB).Results Two months after model development,the learning ability as well as the density of muscarinic receptor in brain were significantly decreased in model mice compared with those in control mice.Parallel models treated with daily oral administration of Catalpol for two months improved the learning ability and increased the brain muscarinic receptor density when compared with model mice.The correlation coefficient between total M receptor densities and the learning/memory ability was significant when examined with linear regresion.Conclusion A dementia model was established in mice.Dementia model was developed by single unilateral injection of 0.3 ?L of a solution of A?(1 ?L normal saline containing 4 ?g of A?25-35 and 1 ?g of ibotenic acid) into right basal ganglion region was established in mice.Catalpol can significantly improve the learning and increase the brain muscarinic receptor density of the model.
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Aim To observe the effect of three steroidal saponins on the M-cholinoceptor density of cultured rat myocardiac cells. Methods The time course of M-cholinoceptor density was observed and diosgenin (DIO),timosaponin aglycone (ZMS,S-configuration) and XMS,a stereoisomeric compound of ZMS in C-25 methyl group,R-configuration) were added to the culture medium from the 12th day of culture at three final concentration of 10~(-7),10~(-6) and 10~(-5) mol?L~(-1),and the M-cholinoceptor density was measured on the 16th day with radioligand binding assay. Results The density of M-cholinoceptor increased gradually at the beginning of culture,reached a plateau at 4～10 days,and then dropped gradually. On the 16th day of culture,the M-cholinoceptor density was about 60% of the plateau value.The up-regulation effect of ZMS on the density of cultured rat myocardiac cells on the 16 th day was only significant at a final concentration of 10~(-5) mol?L~(-1). On the contrary,XMS was effective even when its final concentration was as low as 10~(-7) mol?L~(-1). DIO showed no effect on the M-cholinoceptor density at any of its three concentration. Conclusion The above results indicate that XMS with lower concentration showed similar effect on the M-cholinoceptor density of cultured mylcardiac cells as that of ZMS with more higher concentration.
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Aim To study the effect of tetramethylpyrazine(TMP) on binding of 125I-VEGF to VEGF receptor. Methods The mice sera were collected after peritoneal injection with big-dose TMP,low-dose TMP,protamine and NS. A reversed-phase high performance liquid chromatography(RP-HPLC) method was used to determine the TMP in mice serum. The culture medium of ECV304 was treated with the mice sera in different groups. Radioligand binding assay(RBA) of receptor and Scatchard pot were performed to observe the changes of the maximum binding capacity(B_ max) and dissociation constant(K_d).Results The sera of big-dose TMP inhibited 125I-VEGF binding to its receptor, K_d=343.30?36.64 pmol?L-1,B_ max=46.26?5.85 fmol/2?10~5 cells(P0.05),but B_ max decreased(P
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Objective:To observe the change and clinical significance of glucocorticoid receptor on peripher alleukocytes frompatients with Graves disease.Methods:By radioligand binding assay,GR contents of peripher alleukocytes were determinated in 61 patients with Graves disease,20 healthy volunteers as controls.Results:The GR contents in Graves disease group were significantly lower than that of the normal control group(t = 7.389,P = 0.000 1).In 61 patients with graves disease,GR contents gradually decreased(F = 7.389,P = 0.000 1)with increased Graves disease severity.GR contents were negatively correlated with FT3,FT4 and TSH,correlation coefficients were 0.741(t = 5.643,P = 0.013),0.689(t = 5.119,P=0.025)and 0.755(t =6.391,P=0.009),respectively.GR contents were decreased(t = 4.274,P= 0.000)while patients complicated bythyrotoxic exophthalmos,but GR contents were not statisticallysignificantlydeclined while patients complicated by hyperthyroid heart disease(t = 0.273,P = 0.786) or thyrotoxic periodic paralysis were(t =0.961,P = 0.336).Conclusion:The binding capacity of GR on peripheral leukocytes in the Graves disease patients is lower than normal.This change may play roles in the pathogenic process of Graves disease and probably relate to the disease severity,thyrotoxic exophthalmos complicated.
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Objective To compare the binding characteristics of insulin receptors (IR) on ovary,adrenal and hepatic plasma membrane from rats. Methods The number and affinity of IR was detected by radioligand binding assay. Results Scatchard plot analyses showed that the receptor numbers for high affinity sites are 7. 359?10/mg protein, 8. 029? 10/mg protein and 6. 440?10/mg protein of liver,ovarian and adrenal plasma membrane with KD of 6.147?107 M-1, 1. 528?10, M-1, and 1. 010?107 M-1 that. The receptor numbers for low affinity sites are 2. 403?10/mg protein,2. 212?10/mg protein,and 2. 257?10/mg protein respectively with KD of 2. 920?10M-1,2. 008?10 M-1 and 0. 433?10sM-1. Conclusion There are abundent IR on ovary and adrenal just as many as those on liver, although the KD are smaller. It suggests that insulin may play an important regulatory role in ovary and adrenal.
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Aim To observe the changes of endothelin receptors and their subtypes of left ventricules in normal SD rats and dilated cardiomyopathy rats. Methods To establish the best conditions of the binding experiment, different protein concentrations, incubation temperature ?and?incubating?time?were?tested? with 125 I-ET-1 ligand respectively. With the selected conditions, saturation binding experiments were performed to determine the amount of endothelin receptor and its subtypes in normal SD rats and in dilated cardiomyopathy ones. Results (1) The optimal incubating temperature was 37 ℃. Under this condition, the binding amount of 125 I-ET-1 increased rapidly in 0～30 minutes, and reached to saturation point at 60 minutes, and there was a linear correlation between 125 I-ET-1 binding amount and cell membrane protein concentration. (2) Endothelin-1, bosentan,BQ123,BQ788 etc. could competitively suppress the bound of 125 I-ET-1 to endothelin receptors. (3) The amount of endothelin receptor in left ventricle of dilated cardiomyopathy rats was ( 92.21? 34.34) nmol?kg -1 protein, which was significantly low than that in normal SD ones. There was no change on the ratio of endothelin receptor subtypes A and B. Conclusion 125I-ET-1 can be used to determine the amount of endothelin receptor and its subtypes in varied tissues specifically. The amount of endothelin receptor in left ventricle of dilated cardiomyopathy rats is down regulated, but the ratio of endothelin receptor subtype A vs B remains to be 21.
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The N-methyl-D-aspartate (NMDA) receptor-mediated glutamatergic neurotransmission is involved in synaptic plasticity, developmental processes, learning and memory and many neuropathological disorders including age-related diseases. In the present study, regulation of the NMDA receptor properties by various ligands was investigated using (3H)MK-801 binding studies in the synaptic membranes of young and aged rat forebrains. The binding in the presence of glutamate and glycine increased dramatically with growth between 1 and 6 weeks old, and thereafter declined gradually with aging. Glutamate, glycine or spermine respectively increased the binding with growth. Glutamate maintained the binding during aging, while glycine or spermine significantly decreased the binding in the aged brain. The maximum stimulation by glycine varied depending on the ages of brains. Greater sensitivity to glycine was observed at 1 week and 3 months and the sensitivity was significantly reduced in the aged brain. In contrast, spermine showed similar stimulation patterns in young and aged rats. These results indicated that the functional properties of the NMDA receptor-ion channel complex in young and aged rat forebrains are differentially regulated by agonists, and the reduction of the receptor function with normal aging may be, in some degree, due to the reduction of the receptor sensitivity to glycine.
Subject(s)
Animals , Rats , Aging , Brain , Dizocilpine Maleate , Glutamic Acid , Glycine , Learning , Ligands , Memory , N-Methylaspartate , Plastics , Prosencephalon , Spermine , Synaptic Membranes , Synaptic TransmissionABSTRACT
The action of BTHP on a-adrenoceptor was studied in anococcy-geus muscles, vas deferens and cell membrane of cerebral cortex of rats and aortic strips, saphenous vein of rabbit. BTHP produced parallel shifts to the right of dose-response curves for phenylephrine in rat anococcygeus muscle and rabbit aortic strips without change in the maximal responses. Their pA2 values were 5.8 and 5.86 respectively. BTHP was also a competitive antagonist for presynaptic and postsynaptic ?2-adrenoceptors on prostatic portion of rat vas deferens( pA2 against clonidine=5.2 ) and on rabbit saphenous vein ( pA2 against B-HT920=5.3 ) . BTHP decreased in dose-dependent manner 〔8H〕 prazosin and 〔3H〕 clonidine specific binding to ?1- and ?2- adrenoceptors on cell membrane of cerebral cortex in rats with IC50 of4 and 9.5 ?mol/L and with Ki ( apparent dissociation constant ) of 3.5 and 8.1?mol/L respectively, suggeating that BTHP has affinity for both ?1 and ?2- adrenoceptors of rat cerebrance.
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AIM To establish radioligand binding assay of PAF (platelet activating factor) receptors in mouse peritoneal macrophages and observe the characteristics of PAF receptors. METHODS PAF receptor radioligand binding was studied in intact adherent mouse peritoneal macrophages by -PAF. The radioactivity was counted with an LS6500 scintillation system. RESULTS The PAF receptor binding was shown to be saturable with an equilibrium K D of 3.2 nmol?L -1 and a B max of 100.2 fmol?1?10 6 cells -1. The competitive analysis showed that such specific binding could be inhibited by BN52021. CONCLUSION Utilizing the adherent character of macrophages, the binding ligands could be separated from non-binding ligands without negative pressure filtration, then cells could reserve fine activity, and PAF receptors could be near to physiological properties for screening of PAF antagonist.
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AIM To establish radioligand binding assay of gastrin receptor in rat gastric parietal cell. METHODS Using 125 I labeled [Leu 15 ] gastrin 17 I as the radioligand, rat gastric parietal cells as the receptor preparations. RESULTS In the range of the study, a Scatchard plot of the binding was made by Ligand program with an equilibrium K d of approximately 1 249?10 -10 mol?L -1 and a maximum binding capacity of 4 4604?10 -12 mol?L -1 . The amount of gastrin binding was strongly associated with the cell concentration of rat gastric parietal cell preparations ranged from 0 25?10 9 to 2?10 9 cells?L -1 . The variant coefficent of the binding assay was 7 04% within the same sample. The competitive inhibiting analyses showed that 125 I gastrin was bound to gastric parietal cell preparations with a high specificity. CONCLUSION The radioligand binding assay of gastrin receptor in rat gastric parietal cell preparations meets the criteria for establishing receptor binding assay.
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AIM: To investigate the binding characteristics of cholecystokinin receptors in rat pulmonary interstitial macrophages (PIMs). METHODS: The PIMs were isolated from rat lung tissues, purified using the collagenase digestion method combined with alveolar lavage and pulmonary vessel perfusion. The PIM membrane was obtained by supercentrifuge. Receptors for CCK in PIMs were examined using [~3H] labeled CCK-8S as ligand. The specificity of [~3H]-CCK-8S binding to PIMs membrane and the subtypes of CCK receptors were determined by competitive inhibition experiments with CCK-8S, CCK-A and CCK-B receptors selective antagonists (CR1409 and CR2945). The effects of time and incubation temperature on the specific binding were also observed. RESULTS: The specific binding of [~3H]-CCK-8S was not detected in normal rat PIMs, but was detected in the rat administrated with LPS for 48 h. The capacity of ligand-receptor binding was dependent on the incubation temperature and time. Scatchard analysis of the saturation curves suggested that the presence of CCK receptors with high affinity [Kd=(0.68?0.28)mmol/L] and low binding capacity [Bmax=(32.50?2.70) pmol?g~(-1) protein] in PIMs. By means of competitive inhibition studies, the specific binding of [~3H]-CCK-8S to rat PIMs was inhibited by unlabelled CCK-8S [IC_(50)=(3.20?1.13) nmol/L], CCK-AR specific antagonist CR 1 409 [IC_(50)=(0.19?0.06)?mol/L] and CCK-BR specific antagonist CR 2945[IC_(50)=(2.30?0.80)nmol/L]. CONCLUSION: These results suggest the presence of two subtypes of CCK-AR and CCK-BR and provide a structural basis for CCK to play a pivotal role in PIMs.
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The number and affinity of ?-adrenoceptors on rat uterine smooth muscle membranes were determined by radioligand binding technique. The number of ?-adrenoceptors (shown by maximum binding capacity RT) of uterus in proestrus and estrus stage (126.84?54.69 fmol/mg preicin) was significantly higher than those in diestrus and metestrus stage (77.98 + 30.66 fmol/mg protein) (p
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Thyroid hormones are known to influence the activities of sympathetic nerve by modifying the P-adrenoceptor numbers in several peripheral organs. In order to find out whether similar changes exist in central nervous system ( CNS ) , the changes of ?-adrenergic re-cetors in mouse brain during experimental hyperthyroidism and the effects of some Yin Tonice( Rehmannia ) on that were investigated by method of radioligand binding. The results were as follows: 1 . The maximum Binding capacity (Bmax ) value of hyperthyroid mouse brain was 29% higher than the control's. No significant difference in KD value between the control and hyperthyroid animals was found . 2. By administration of rehmannia, the Bmax of hyperthyroid mouse was reduced to control value.
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The properties of the corticosterone receptors and the effects of para-chlorophenylalanine(pCPA) on the binding capacity of the corticosterone receptors of the hippocampal cytosol in adrenalectomized rats were investigated with receptor radioligand binding assay(RLA) using (3H)-corticosterone as the ligand. It was fonud that the corticosterone receptors possessed the properties of satu-rability, limited binding capacity, high affinity and high specificity. RLA demonstrated a linear Scatchard plot, which implies that there is only one receptor or one group of receptors with similar Kd values. Four days after intracerebroven-tricular injection of 1 mg of pCPA, the hippocampal concentration of 5-HT was markedly decreased, and the binding capacity of the cytosol receptors was increased, which implies that the functional activity of the serotoninergic system in the hippocampus is an important factor in the control of the number of corticosterone receptors in rats.