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1.
B. N. Manukhin L. A. Nesterova 《Biochemistry (Moscow) Supplemental Series A: Membrane and Cell Biology》2011,5(4):350-354
Activation and inhibition of muscarinic cholinoceptors by atropine and carbachol are shown to exert allosteric effects on
the binding of specific nonselective α2-adrenoceptor antagonist [3H]RX821002 in rat brain cortex membranes. The ligand-receptor interaction for α2-adrenoceptors corresponded to the model suggesting the presence of one homogeneous pool of receptors with two specific binding
sites. The parameters of the [3H]RX821002 binding were as follows: [3H]RX821002 -K
d = 1.94 ± 0.08 nM, B
max = 13.4 ± 1.8 fmol/mg protein, n = 2. The inhibition of muscarinic cholinoceptors by atropine induced an increase of affinity (K
d = 1.36 ± 0.12 nM) and a decrease of the α2-adrenoceptor density (B
max = 10.18 ± 0.48 fmol/mg protein). The muscarinic cholinoceptor agonist carbachol induced an increase of the affinity (K
d = 1.56 ± 0.05 nM) and quantity of binding sites (B
max = 16.61 ± 0.29 fmol/mg protein). As a result, under the influence of atropine and carbachol, the efficiency of binding (E = B
max/2K
d) increased from 3.50 ± 0.40 to 5.60 ± 0.79 and 6.86 ± 0.20 fmol/mg protein/nM, respectively. The data suggest that α2-adrenoceptors exist in rat brain cortex as homodimers. 相似文献
2.
Iñaki Ibarrola Kepa Ogiza Aida Marino José M. Macarulla Miguel Trueba 《Journal of bioenergetics and biomembranes》1991,23(6):919-926
A high-affinity and low-capacity corticosterone specific binding was detected in the purified plasma membrane preparation from rat kidney using anin vitro steroid hormone binding assay. The specific-bound hormone was efficiently distinguished from the irreversible-bound hormone with 10 µM corticosterone. Under standardized conditions of pH 7.4 at 2°C and 30 min incubation time, the binding was saturable and showedK
d=13±3 nM andB
max=616±34 fmol/mg of protein. Competitive binding studies with analogue steroids indicated that corticosterone binding to kidney plasma membrane is hormone-specific. Results indicated that the possible nongenomic effects of steroids could be mediated by their interaction with plasma membrane. 相似文献
3.
L. Felipe Barros J. Carlos Bustamante David L. Yudilevich Simon M. Jarvis 《The Journal of membrane biology》1991,119(2):151-161
Summary The nucleoside transport activity of human placental syncytiotrophoblast brush-border and basal membrane vesicles was compared. Adenosine and uridine were taken up into an osmotically active space. Adenosine was rapidly metabolized to inosine, metabolism was blocked by preincubating vesicles with 2-deoxycoformycin, and subsequent adenosine uptake studies were performed in the presence of 2-deoxycoformycin. Adenosine influx by brush-border membrane vesicles was fitted to a two-component system consisting of a saturable system with apparent Michaelis-Menten kinetics (apparentK
m
approx. 150 m) and a linear component. Adenosine uptake by the saturable system was blocked by nitrobenzylthioinosine (NBMPR), dilazep, dipyridamole and other nucleosides. Inhibition by NBMPR was associated with high-affinity binding of NBMPR to the brush-border membrane vesicles (apparentK
d
0.98±0.21nm). Binding of NBMPR to these sites was blocked by adenosine, inosine, uridine, thymidine, dilazep and dipyridamole, and the respective apparentK
i
values were 0.23±0.012, 0.36±0.035, 0.78±0.1, 0.70±0.12 (mm), and 0.12 and 4.2±1.4 (nm). In contrast, adenosine influx by basal membrane vesicles was low (less than 10% of the rate observed with brush-border membrane vesicles under similar conditions), and hence no quantitative studies of adenosine uptake could be performed with these vesicles. Nevertheless, high-affinity NBMPR binding sites were demonstrated in basal membrane vesicles with similar properties to those in brushborder membrane vesicles (apparentK
d
1.05±0.13nM and apparentK
i
values for adenosine, inosine, uridine, thymidine, dilazep and dipyridamole of 0.14±0.045, 0.54±0.046, 1.26±0.20, 1.09±0.18mm and 0.14 and 3.7±0.5nm, respectively). Exposure of both membrane vesicles to UV light in the presence of [3H]NBMPR resulted in covalent labeling of a membrane protein(s) with a broad apparentM
r
on SDS gel electropherograms of 77,000–45,000, similar to that previously reported for many other tissues, including human erythrocytes. We conclude that the maternal (brush-border) and fetal (basal) surface of the human placental syncytiotrophoblast posses broad-specificity, facilitated-diffusion, NBMPR-sensitive nucleoside transporters. 相似文献
4.
The presence of high concentrations of both dopamine and cholecystokinin (CCK) in the striatum and in various limbic structures suggests that the CCK may not only influence dopaminergic transmission, but it also may be relevant to the psychopathology of schizophrenia and to the therapeutic effects of neuroleptics. By using a synaptosomal fraction isolated from the mouse cerebral cortex and [propionyl-3H]CCK8-sulphate ([3H]CCK8S) as a ligand, a single binding site for [3H]CCK8 with aK
d
value of 1.04 nM and aB
max value of 42.9 fmol/mg protein was identified. The competitive inhibition of [3H]CCK8S binding by related peptides produced an order of potency of CCK8-sulphated (IC50=5.4 nM)>CCK8-unsulfated (IC50=40 nM) and >CCK4 (IC50=125 nM). The regional distribution of [3H]CCK8S binding in the mouse brain was highest in the olfactory bulb (34.3±5.6 fmol/mg protein) > cerebral cortex > cerebellum > olfactory tubercle > striatum > pons-medulla > mid brain > hippocampus > hypothalamus (12.4±2.1 fmol/mg protein). The repeated administration of haloperidol (2.5 mg/kg/tid) increased the binding of [3H]CCK8S in cerebral cortex from 31.8±1.7 to 38.9±5.2 fmol/mg protein. The varied distribution of CCK8S receptors may signify nonuniform functions for the octapeptide in the brain. 相似文献
5.
Summary The sensitivity of nucleoside transport by rat erythrocytes to inhibition by nitrobenzylthioinosine (NBMPR) and the slowly permeating organomercurial,p-chloromercuriphenyl sulfonate (pCMBS), was investigated. The dose response curve for the inhibition of uridine transport (100 M) by NBMPR was biphasic –35% of the transport activity was inhibited with an IC50 value of 0.25 nM, but 65% of the activity remained insensitive to concentrations as high as 1 M. These two components of uridine transport are defined as NBMPR-sensitive and NBMPR-insensitive, respectively. Uridine influx by both components was saturable and conformed to simple Michaelis-Menten kinetics, and was inhibited by other nucleosides. The uridine affinity of the NBMPR-sensitive transport component was threefold higher than for the NBMPR-insensitive transport mechanism (apparentK
m for uridine 50±18 and 163±28 M, respectively). The two transport systems also differed in their sensitivity topCMBS. NBMPR-insensitive uridine transport was inhibited bypCMBS with an IC50 of 25M, while 1 mMpCMBS had little effect on NBMPR-sensitive transport by intact cells.pCMBS inhibition was reduced in the presence of uridine and adenosine and reversed by the addition by -mercaptoethanol, suggesting that thepCMBS-sensitive thiol group is located on the exterior surface of the erythrocyte membrane within the nucleoside binding site of the transport system. Inhibition of uridine transport by NBMPR was associated with high-affinity [3H]NBMPR binding to the cell membrane (apparentK
d46±25 pM). Binding of inhibitor to these sites was competitively blocked by uridine and inhibited by adenosine, thymidine, dipyridamole, dilazep and nitrobenzylthioguanosine. Assuming that each NBMPR-sensitive transport site binds a single molecule of NBMPR, the calculated translocation capacity of each site is 25±6 molecules/site per sec at 22°C.pCMBS had no effect on [3H]NBMPR binding to intact cells but markedly inhibited binding to disrupted membranes indicating that the NBMPR-sensitive nucleoside transporter probably has a thiol group located on the inner surface of the membrane. Exposure of rat erythrocyte membranes to UV light in the presence of [3H]NBMPR resulted in covalent radiolabeling of a membrane protein(s) (apparent Mr on SDS gel electropherograms of 62,000). Labeling of this protein was abolished in the presence of nitrobenzylthioguanosine. We conclude that nucleoside transport by rat erythrocytes occurs by two facilitated-diffusion systems which differ in their sensitivity to inhibition by both NBMPR andpCMBS. 相似文献
6.
Carol E Cass Norbert Kolassa Yoshimasa Uehara Ewa Dahlig-Harley Eric R Harley Alan R.P Paterson 《生物化学与生物物理学报:生物膜》1981,649(3):769-777
Cells of an adenosine-resistant clone (AE1) of S49 mouse lymphoma cells were compared with cells of the parental line with respect to (a) characteristics of nucleoside transport, (b) high affinity binding of the inhibitor of nucleoside transport, nitrobenzylthionisine (NBMPR), and (c) the antiproliferative effects of the nucleoside antibiotics, tubercidin, arabinosyladenine and showdomycin. Rates of inward transport of uridine, thymidine, adenosine, 2′-deoxyadenosine, tubercidin, showdomycin, and arabinosyladenine in AE1 cells were less than 1% of those in cells of the parental S49 line. The inhibitor of nucleoside transport, NBMPR, reduced rates of inward nucleoside transport in S49 cells to levels comparable to those seen in the transport-defective mutant. S49 cells possessed high affinity sites that bound NBMPR (6.6 · 104 sites/cell, Kd 0.2 nM), whereas site-specific binding of NBMPR to AE1 cells was not demonstrable, indicating that loss of nucleoside transport activity in AE1 cells was accompanied by loss of the high affinity NBMPR binding sites. Relative to S49 cells, AE1 cells were resistant to the antiproliferative effects of tubercidin and showdomycin, but differences between the two cell lines in sensitivity toward arabinosyladenine were minor, suggesting that nucleoside transport activity was required for cytotoxicity of tubercidin and showdomycin, but not for that of arabinosyladenine. 相似文献
7.
E F Williams P H Barker A S Clanachan 《Canadian journal of physiology and pharmacology》1984,62(1):31-37
The site-specific binding of the potent and selective nucleoside transport inhibitor, [3H]nitrobenzylthioinosine (NBMPR), to the nucleoside transport system of cardiac membranes of several species was investigated. The affinity of [3H]NBMPR for these sites ranged from 0.03 nM in rat to 0.78 nM in dog. The maximal binding capacity of cardiac membranes for [3H]NBMPR was also species dependent and was greatest in bovine and guinea pig heart (2551 and 1700 fmol/mg protein, respectively) and least in rat (195 fmol/mg protein). The affinities of recognized nucleoside transport inhibitors and benzodiazepines for these transport inhibitory sites in guinea pig and rat heart were estimated by studying the inhibition of the site-specific binding of [3H]NBMPR in competition experiments. These values were compared with their inhibitory effects on the transporter-dependent accumulation of [3H]adenosine in guinea pig and rat cardiac muscle segments and with their ability to potentiate the negative inotropic action of adenosine in electrically driven guinea pig and rat left atria. In guinea pig heart, the recognized nucleoside transport inhibitors and benzodiazepines had an order of affinity (dilazep greater than hydroxynitrobenzylthioguanosine greater than dipyridamole greater than hexobendine much greater than lidoflazine much greater than flunitrazepam greater than diazepam greater than lorazepam greater than flurazepam) for the NBMPR site which was similar to those for the inhibition of [3H]adenosine accumulation and for potentiation of adenosine action. In contrast, in rat heart, where the maximal binding capacity of [3H]NBMPR was lower (eightfold), the nucleoside transporter dependent accumulation of [3H]adenosine was also lower (sixfold) and the negative inotropic action of adenosine was not significantly potentiated.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
8.
[3 H]Nitrobenzylthioinosine Binding to the Guinea Pig CNS Nucleoside Transport System: A Pharmacological Characterization 总被引:1,自引:0,他引:1
The binding of [3H]nitrobenzylthioinosine (NBMPR) to specific membrane sites in guinea pig brain was rapid, reversible, and saturable, and was dependent upon protein concentration, pH, and temperature. Mass law analysis of the binding data for cortical membranes indicated that NBMPR bound with high affinity to a single class of sites at which the equilibrium dissociation constant (KD) for NBMPR was 0.10-0.25 nM and which possessed a maximum binding capacity (Bmax) per mg of protein of 300 fmol of NBMPR. Kinetic analysis of the site-specific binding of NBMPR yielded an independent estimate of the KD of 0.16 nM. A relatively homogeneous subcellular distribution of the sites for NBMPR was found in cortical tissue. Recognized inhibitors of nucleoside transport were potent, competitive inhibitors of the binding of NBMPR in guinea pig CNS membranes whereas benzodiazepines and phenothiazines have low affinity for the sites. NBMPR sites in guinea pig cortical membranes have characteristics similar to those for NBMPR in human erythrocytes, the occupation of which is associated with inhibition of nucleoside transport. The comparable affinities for a range of agents for sites in human erythrocytes and guinea pig CNS membranes suggest that NBMPR also binds to transport inhibitory elements of the guinea pig CNS nucleoside transport system. It is proposed that the study of the binding of NBMPR provides an effective method by which to examine drug interactions with the membrane-located nucleoside transport system in CNS membranes. 相似文献
9.
L. A. Nesterova O. V. Boiko B. N. Manukhin 《Biochemistry (Moscow) Supplemental Series A: Membrane and Cell Biology》2016,10(4):278-286
The influence of β-adrenoceptor activation and inhibition by isoprenaline and propranolol on the specific binding of nonselective α1- and α2-adrenoceptor antagonists [3H]prazosin and [3H]RX821002 in rat cerebral cortex subcellular membrane fractions was studied. It was established that for the α1- and α2-adrenoceptors the ligand–receptor interaction corresponds to the model of one affinity pool of receptors and binding of two ligand molecules by one dimer receptor. The parameters of [3H]prazosin binding to α1-adrenoceptors were: K d = 1.85 ± 0.16 nM, B max = 31.14 ± 0.35 fmol/mg protein, n = 2. The parameters of [3H]RX821002 binding to α2-adrenoceptors were: K d = 1.57 ± 0.27 nM, B max = 7.2 ± 1.6 fmol/mg protein, n = 2. When β-adrenoceptors were activated by isoprenaline, the binding of radiolabelled ligands with α1- and α2-adrenoceptors occurred according to the same model. The affinity to [3H]prazosin and the concentration of active α1-adrenoceptors increased by 27% (K d = 1.36 ± 0.03 nM) and 84% (B max = 57.37 ± 0.28 fmol/mg protein), respectively. The affinity of α2-adrenoceptors to [3H]RX821002 decreased by 56% (K d = 3.55 ± 0.02 nM), and the concentration of active receptors increased by 69% (B max = 12.24 ± 0.06 fmol/mg protein). Propranolol alters the binding character of both ligands. For [3H]prazosin and [3H]RX821002, two pools of receptors were detected with the following parameters: K d1 = 1.13 ± 0.09, K d2 = 6.07 ± 1.06 nM, B m1 = 11.36 ± 1.77, Bm2 = 51.09 ± 0.41 fmol/mg protein, n = 2 and K d1 = 0.61 ± 0.02, K d2 = 3.41 ± 0.13 nM, B m1 = 1.88 ± 0.028, B m2 = 9.27 ± 0.08 fmol/mg protein, n = 2, respectively. The concentration of active receptors (B max) increased twofold for both ligands. It was suggested that α1- and α2-adrenoceptors in rat cerebral cortex subcellular membrane fractions exist as dimers. A modulating influence of isoprenaline and propranolol on the specific binding of the antagonists to α1- and α2- adrenoceptors was revealed, which was manifested in the activating effect on the [3H]prazosin binding parameters, in the inhibitory effect on the [3H]RX821002 binding parameters, and in a change of the general character of binding for both ligands. 相似文献
10.
L. A. Nesterova B. N. Manukhin 《Biochemistry (Moscow) Supplemental Series A: Membrane and Cell Biology》2009,3(1):66-70
The binding of specific nonselective α1- and α2-adrenoceptor antagonists [3H]prazosine and [3H]RX821002 has been studied on rat cerebral cortex synaptosomal membranes. It is shown that for α1-adrenoceptors the ligand-receptor interaction corresponds to the model assuming the presence of one pool of receptors and binding of two ligand molecules to the receptor. The parameters of [3H]prazosine binding to α1-adrenoceptors were: K d= 1.56 ± 0.17 nM, B max = 30.25 ± 1.78 fmol/mg protein, n = 2. The parameters of [3H]RX821002 binding to α2-adrenoceptors were: K d = 1.94 ± 0.08 nM, B max = 12.77 ± 3.17 fmol/mg protein, n = 2. For α2 -adrenoceptors the ligand-receptor interaction corresponded to the same model. For α1 - and α2-adrenoceptor antagonists the dissociation constants (K d) are approximately equal (1.56 ± 0.17 and 1.94 ± 0.08 nM, respectively), but the concentration of α2-adrenoceptors is two times lower than that of α1-adrenoceptors ( 12.77 ± 3.17 and 30.25 ± 1.78 fmol/mg protein, respectively). The efficiency (E = B max/2K d) of the ligand binding to α1-adrenoceptors is 2.3 times higher than that to α2-adrenoceptors (7.46 ± 1.32 and 3.29 ± 0.68 fmol/mg protein/nM, respectively. The data suggest that α1- and α2 -adrenoceptors in rat cerebral cortex exist as dimers. 相似文献
11.
Edwin M. Meyer Sean O. Bryant Ruei-Hsin D. Wang R. Jason Watson 《Neurochemical research》1993,18(10):1067-1072
The binding of (1)-[3H]vesamicol was characterized in several subcellular fractions and brain regions of the rat. Binding to a lysed P2 fraction from the rat cerebral cortex reached equilibrium within 4 min at 37°C and was reversible (dissociation half-time 4.9 min). At least two binding affinities were found in P2 fractions from the cerebral cortex (Kd:21 nM and 980 nM), striatum (Kd:28 nM and 690 nM), and cerebellum (Kd:22 nM and 833 nM). High affinity Bmax values were highest in striatum (1.17 pmol/mg protein), followed by cerebellum (0.67 pmol/mg protein), and cerebral cortex (0.38 pmol/mg protein). Low affinity Bmax values were highest in cerebellum (5.2 pmol/mg protein), with similar values for cerebral cortex (3.7 pmol/mg protein) and striatum (3.8 pmol/mg protein). High affinity but not low affinity binding in each brain region was stereospecific. Another inhibitor of vesicular ACh-transport also displaced 1-vesamicol binding potently (IC50:17 nM) and efficaciously (over 90%). Both high affinity and low affinity Bmax values for [3H]vesamicol-binding were highest in a partially purified synaptic vesicle fraction, followed by puriffied synaptosomes, crude membranes and P2 fractions. Specific binding was not observed in a mitochondria-enriched fraction. Crude membrane preparations of primary, neuron-enriched whole brain cultures also exhibited high (64 nM) and low affinity (1062 nM) [3H]vesamicol binding. Isoosmotic replaement of 0.18 M KCl in the binding-buffer with NaCl had no effect on binding. These results suggest that at least some high affinity [3H]vesamicol binding in rat brain preparations may be associated with synaptic vesicles, some of which may not be cholinergic in origin. 相似文献
12.
Bradykinin (BK) and its analogs (1 nM-100 M) stimulated phosphoinositide (PI) turnover in murine fibrosarcoma (HSDM1C1) cells in a concentration-dependent manner. The relative potencies (EC50) were: BK=48±4 nM; Lys-BK=39±3 nM; Met-Lys-BK=158±33 nM; Des-Arg9-BK=2617±598 nM (means±SEM, n=3–14). All these analogs were full agonists and they produced up to 5.4±0.4-fold stimulation of PI turnover at the highest concentration (10–100 M) of the peptides. In contrast, the analogs [D-Arg0-HYP3-Thienyl5,8-D-Phe7]-BK (HYP3-antagonist), [D-Arg0-HYP3-Thienyl,5,8-D-Phe7]-BK (Thienyl antagonist) and Des-Arg9-Leu8-BK were inactive, as agonists, at 0.1 nM-1 M in this system. These data suggested that BK-induced PI turnover in these cells was mediated via B2-type of BK receptors. This was confirmed further by the fact that both the B2-selective Hyp3- and Thienyl-antagonists inhibited BK-induced PI turnover with KBS of 369±51 nM and 368±118 nM respectively while the B1-selective antagonist, Des-Arg9-Leu8-BK, was inactive at 1 M. [3H]BK receptor binding studies revealed two binding sites, one with high affinity (Kd=0.24±0.06 nM; Bmax=1.4±0.4 pmol/g tissue) and the other with low affinity (Kd=18.5±0.95 nM; Bmax=25.1±0.52 pmol/g tissue), on HSDM1C1 cell homogenates. The rank order of affinity of BK analogs at inhibiting specific [3H]BK binding was similar to that found for PI turnover. Taken together, these data have provided evidence for the presence of two B2-type BK binding sites on the HSDM1C1 cells. Based on the affinity parameters, the low-affinity component of [3H]BK binding in HSDM1C1 cells appears to be coupled to the phospholipase C-induced PI turnover mechanism. The high-affinity component has been previously shown to mediate the production of prostaglandins by activation of phospholipase A2. 相似文献
13.
[3H]dipyridamole binding to nucleoside transporters from guinea-pig and rat lung. 总被引:1,自引:0,他引:1
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Membranes from guinea-pig lung exhibited high-affinity binding of [3H]dipyridamole, a potent inhibitor of nucleoside transport. Binding (apparent KD 2 nM) was inhibited by the nucleoside-transport inhibitors nitrobenzylthioinosine (NBMPR), dilazep and lidoflazine and by the transported nucleosides uridine and adenosine. In contrast, there was no detectable high-affinity binding of [3H]dipyridamole to lung membranes from the rat, a species whose nucleoside transporters exhibit a low sensitivity to dipyridamole inhibition. Bmax. values for high-affinity binding of [3H]dipyridamole and [3H]NBMPR to guinea-pig membranes were similar, suggesting that these structurally unrelated ligands bind to the NBMPR-sensitive nucleoside transporter with the same stoichiometry. 相似文献
14.
L. A. Nesterova B. N. Manukhin 《Biochemistry (Moscow) Supplemental Series A: Membrane and Cell Biology》2011,5(1):85-91
The influence of isoprenaline- and propranolole-induced activation and inhibition of β-adrenoreceptors on the specific nonselective
α2-antagonist [3H]RX821002 binding was studied on rat cerebral cortex subcellular membrane fractions. It was shown that the ligand-receptor
interaction for α2-adrenoreceptors corresponded to the model that assumed the presence of one receptor pool and binding of two ligand molecules
to a receptor dimer. The following parameters were determined for [3H]RX821002 binding to α2-adrenoreceptors: K
d1 = 1.57 ± 0.27 nM, B
max = 7.24 ± 1.63 fmol/mg of protein, n = 2. In the case of isoprenaline-induced activation of β-adrenoreceptors the binding of radiolabeled ligand to α2-adrenoreceptors was described by the same model. The affinity of α2-adrenoreceptors for [3H]RX821002 decreased more than twofold (K
d = 3.55 ± 0.02 nM) and the quantity of active receptors increased by 69% (B
max = 12.24 ± 0.06 fmol/mg of protein). Propranolole changed the model of ligand binding, and two pools of receptors were detected
with the following parameters: K
d1 = 0.61 ± 0.02 nM, K
d2 = 3.41 ± 0.13 nM, B
ml = 1.88 ± 0.028 fmol/mg of protein, B
m2 = 9.27 ± 0.08 fmol/mg of protein, n = 2. The data suggest that α2-adrenoreceptors in subcellular membrane fractions from rat cerebral cortex exist in dimeric form. Isoprenaline and propranolole
exhibit modulating effect on the specific antagonist binding to α2-adrenoreceptors, which results in the inhibition and alteration of [3H]RX821002 binding parameters. 相似文献
15.
Anna Siniscalchi Donata Rodi Stefania Gessi Franca Campi Pier A. Borea 《Neurochemistry international》1999,34(6):729
The effects of brain ischemia on the maximum binding capacity (Bmax) and affinity (Kd) of A1 receptors were studied in the rat cerebral cortex, with an in vitro approach. The results were correlated with changes in 3H-adenosine release, studied under identical experimental conditions. Fifteen minutes of in vitro ‘ischemia’ (hypoxic, glucose-free medium) induced a significant increase in both Bmax (2398±132 fmol/mg protein, 151% of the control, P<0.05) and in Kd (2.43±0.12 nM, 161% of the control, P<0.01). At the same time, an increase in tritium efflux from [3H]-adenosine labeled cerebral cortex slices to 324% of the control was observed. A trend toward normalization was evident 5–15 min after ‘reoxygenation’ (restoring normal medium), but the binding parameters were still altered after 60 min (Bmax 2110±82 fmol/mg protein, Kd 2.26±0.14 nM, P<0.01 vs the corresponding control) as was adenosine release (196% of the control). These findings suggest that the increased availability of adenosine and its receptors may be a defense mechanism against ischemic injury, while the reduced affinity of A1 receptors, possibly due to desensitization, may be a sign of ischemia-induced cellular damage. 相似文献
16.
E.C.K. Liu A. Hedberg H.J. Goldenberg D.N. Harris M.L. Webb 《Prostaglandins & other lipid mediators》1992,44(2)
DuP 753 is a potent, selective angiotensin II type 1 (AT1) receptor antagonist. The possibility was investigated that DuP 753 may crossreact with thromboxane A2/prostaglandin H2 (TP) receptors. DuP 753 inhibited the specific binding of the TP receptor antagonist [3H]SQ 29,548 (5 nM) in human platelets with kd/slope factor values of 9.6±1.4 μM/1.1±0.02. The AT2-selective angiotensin receptor ligand, PD 123,177 was a very weak inhibitor of specific [3H]SQ 29,548 binding in platelets (Kd/slope factor:200 μM/0.86). [3H]SQ 29,548 saturation binding in the absence and presence of DuP 753 resulted in an increase in equilibrium affinity constant (Kd: 9.3, 22, 33 nM, respectively) without a concentration-dependent reduction in binding site maxima (Bmax: 3597, 4597, 3109 fmol/mg protein, respectively). Platelet aggregation induced by the TP receptor agonist U 46,619 was concentration-dependently inhibited by DuP 753 (IC50=46 μM). These data indicate for the first time that DuP 753 is a weak but competitive antagonist at human platelet TP receptors. 相似文献
17.
Specific binding of [3H]nitrendipine to membranes from coronary arteries and heart in relation to pharmacological effects. Paradoxical stimulation by diltiazem 总被引:13,自引:0,他引:13
A DePover M A Matlib S W Lee G P Dubé I L Grupp G Grupp A Schwartz 《Biochemical and biophysical research communications》1982,108(1):110-117
High affinity binding sites for the calcium channel inhibitor [3H]nitrendipine have been identified in microsomes from pig coronary arteries (KD=1.6 nM; Bmax=35 fmol/mg) and in purified sarcolemma from dog heart (KD=0.11 nM; Bmax=230 fmol/mg). [3H]nitrendipine binding to coronary artery microsomes was completely inhibited by nifedipine, partially by verapamil and D600 and, surprisingly, was stimulated by d-cis-diltiazem but not by 1-cis-diltiazem, a less active isomer. Half-maximal relaxation of KCl-depolarized coronary rings occurred in a slow process at 1 nM nitrendipine or 100 nM d-cis-diltiazem. In dog trabecular strips, nitrendipine caused a negative inotropic response (ED50=1μM). These results suggest that there may be multiple binding sites for different “subclasses” of calcium channel inhibitors, and that drug binding sites may be different molecular entities from the putative calcium channels. 相似文献
18.
Leonor Mendoza-Vargas Hctor Solís-Chagoyn Gloria Benítez-King Beatriz Fuentes-Pardo 《Comparative biochemistry and physiology. Part A, Molecular & integrative physiology》2009,154(4):486-492
Retinular photoreceptors are structures involved in the expression and synchronization of the circadian rhythm of sensitivity to light in crayfish. To determine whether melatonin possesses a differential effect upon the receptor potential (RP) amplitude of retinular photoreceptors circadian time (CT)-dependent, we conducted experiments by means of applying melatonin every 2 h during a 24-hour cycle. Melatonin with 100 nM increased RP amplitude during subjective day to a greater degree than during subjective night. To determine whether MT2 melatonin receptors regulate the melatonin-produced effect, we carried out two experiments, circadian times (CTs) 6 and 18, which included the following: (1) application of the MT2 receptor selective agonist 8-M-PDOT and antagonist DH97, and (2) the specific binding of [125I]-melatonin in eyestalk membranes. The amount of 10 nM of 8-M-PDOT increased RP amplitude in a similar manner to melatonin, and 1 nM DH97 abolished the increase produced by melatonin and 8-M-PDOT. Binding of [125I]-melatonin was saturable and specific. Scatchard analysis revealed an affinity constant (Kd) of 1.1 nM and a total number of binding sites (Bmax) of 6 fmol/mg protein at CT 6, and a Kd of 1.46 nM and Bmax of 7 fmol/mg protein at CT 18. Our results indicate that melatonin increased RP amplitude of crayfish retinular photoreceptors through MT2-like melatonin receptors. These data support the idea that melatonin is a signal of darkness for the circadian system in crayfish retinular cells. 相似文献
19.
Privitera PJ Beckstead RM Yates P Walgren R 《Cellular and molecular neurobiology》2003,23(4-5):805-815
1. The present study was undertaken to localize and characterize bradykinin (BK) binding sites in brains from Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR).2. Serial sections of brains were cut from adult WKY and SHR and specific [125I-Tyr0]bradykinin ([125I-Tyr0]BK) binding was determined using in vitro quantitative receptor autoradiographic techniques.3. Specific binding of [125I Tyr0]BK was localized in the medulla oblongata to the regions of the nucleus of the solitary tract (NTS), area postrema (AP), dorsal motor nucleus of the vagus (X), and caudal subnucleus of the spinal trigeminal nucleus in both strains of rat. The specific binding (85–90% of total binding) was of high affinity and saturable with K
D values in the range of 100 pM and a B
max of 0.75 fmol per mg tissue equivalent in the NTS–X–AP complex of both the WKY and SHR. In competition studies, the rank order of potencies was similar in both strains with BK = Lys-BK > icatibant >>> DesArg9-BK. The B2 receptor antagonist icatibant inhibited [125I-Tyr0]BK binding with a K
i value of 0.63 ± 0.19 nM in WKY and 0.91 ± 0.73 nM in SHR, while K
i values for the B>
1 receptor agonist DesArg9-BK were 1475 ± 1055 and 806 ± 362 nM in WKY and SHR, respectively.4. Our finding of specific high-affinity [125I-Tyr0]BK B2 binding sites in the NTS, AP, and the X of WKY and SHR is important because these brain areas are associated with central cardiovascular regulation. However, alterations in BK B2 receptors in the medulla that could contribute to the hypertensive state in the SHR were not detected. 相似文献
20.
L. A. Nesterova B. N. Manukhin 《Biochemistry (Moscow) Supplemental Series A: Membrane and Cell Biology》2013,7(1):45-51
The effects of activation and inhibition of serotonin receptors by serotonin (5-HT) and mianserin on the specific nonselective α1-antagonist [3H]prazosine binding in rat cerebral cortex membranes was studied. It was shown that the ligand-receptor interaction of α1-adrenoceptors corresponded to the model suggesting the presence of one pool of receptors and the binding of two ligand molecules to the receptor. The parameters of [3H]prazosine binding to α1-adrenoceptors were as follows: K d =1.85 ± 0.16 nM, B max = 31.1 ± 0.3 fmol/mg protein, n = 2. In case of activation of 5HT-receptors by serotonin, the character of ligand binding was different: two pools of receptors were detected with the parameters K d1 = 0.61 ± 0.04, K d2 = 3.82 ± 0.15 nM, B m1 = 6.6 ± 0.7, B m2 = 25.6 ± 0.4 fmol/mg protein, n = 2. The sensitivity of the high-affinity pool increased threefold and the sensitivity of the low-affinity pool decreased twofold as compared to the control. The value of maximal reaction (B max) did not change. In the case of inhibition of 5HT-receptors by mianserin, radioactive ligand is bound to α1-adrenoceptors according to the same model as in the control conditions. The affinity of α1-adrenoceptors to [3H]prazosine decreases twofold and the concentration increases (K d = 3.97 ± 0.12 nM, B max = 40.0 ± 0.5 fmol/mg protein). The data suggest that α1-adrenoceptors in rat cerebral cortex exist as a dimer. The modulatory effects of serotonin and mianserin on the specific binding of [3H]prazosine to α1-adrenoceptors was detected, manifesting itself as changes in the binding parameters and in the general character of ligand-receptor interactions. 相似文献