Binding of some non-steroidal anti-inflammatory drugs to glucocorticoid receptors in vitro

The mechanism of action of non-steroidal anti-inflammatory drugs (NSAID) is felt to be via prostaglandin synthetase inhibition. Yet several clinical findings suggest these drugs may possess glucocorticoid agonist activity as well. To evaluate this possibility, a variety of non-steroidal antiinflammatory drugs was examined to determine whether they compete for rat kidney glucocorticoid receptor sites in vitro. Meclofenamic acid, MK 410 (an indomethacin analogue) and indomethacin were the most potent competitors requiring 7 × 10³-fold, 2 × 10⁴-fold and 3 × 10⁴-fold molar excess, respectively, to inhibit 50 per cent of the binding of 10 nM [³H]dexamethasone to kidney cytosol binding sites. The same drugs also inhibited the binding of [³H]dexamethasone in kidney slices requiring about 3-fold higher concentrations to achieve 50 per cent inhibition. At a 10⁵-fold molar ratio, sulfinpyrazone reduced cytosol binding to 60 per cent, phenylbutazone to 85 per cent and ibuprofen to 89 per cent of control levels; aspirin and naproxen did not compete even at this concentration. Similarly, prostaglandins failed to compete with [³H]dexamethasone for kidney receptors. Indomethacin inhibition of [³H]dexamethasone binding appeared competitive when analyzed by a double reciprocal plot. As expected, inhibition of [³H]dexamethasone cytosol binding by indomethacin prevented the appearance of the nuclear complex. Of the NSAID examined, none competed for plasma binding of [³H]corticosterone except indomethacin which reduced the binding to 50 per cent of control at a 10⁵-fold molar excess. In conclusion, these studies support the possibility that some NSAID may possess intrinsic glucocorticoid agonist activity. Furthermore, they indicate the glucocorticoid receptor site is not as specific as once thought and suggest that non-steroidal compounds may potentially serve as glucocorticoid agonists or antagonists.     

糖皮质激素及其受体与分娩启动的关系

目的 探讨糖皮质激素(GC)及其受体(GR)与分娩启动的关系.方法 40例是月剖宫产孕妇均分为临产后剖宫产(A)组及选择性剖宫产(B)组.孕妇均无妊娠合并症及并发症,孕期未使用GC.检测血清及羊水中GC水平.采用免疫组化和RT-PCR技术检测胎盘、胎膜组织GR-α和GR-β的表达.结果 A组血清GC水平明显高于B组(P＜0.05).两组胎盘组织几乎无GR表达;A组胎膜绀织中GR-β表达明显低于B组,GR-α/GR-β比例高于B组(P＜0.05).结论 临产后血清GC增加,胎膜上GR-β的表达降低,GR-α/GR-β增高,可能与分娩启动相关.     

孕三烯酮与兔子宫雌激素与孕激素受体的亲和力测定

孕三烯酮与兔子宫雌激素与孕激素受体的亲和力测定桂耀庭吴昌杰１吴熙瑞１（深圳市红十字会医院中西医结合研究所，深圳５１８０２９；１同济医科大学生殖医学中心，武汉４３００３０）孕三烯酮（ｇｅｓｔｒｉｎｏｎｅ，Ｒ２３２３）是一种１９—去甲基睾丸酮衍生物．８０...     

孕三烯酮与兔子宫雌激素和孕激素受体的亲和力测定

Radioligand receptor binding assay (RRBA) was used to determine the characteristics of gestrinone binding to estrogen and progestin receptors of the uterine cytosol in rabbits. The results showed that the binding of ［³H］ progesterone and ［³H］ estradiol to uterine cytosols was in a concentration-dependent saturation manner, the dissociation constants (K_d) were 0.82 and 0.18 nmol·L^-1 by the Scatchard analysis. If the affinities of progesterone and estradiol binding to progestin and estrogen receptors were taken to be 100%, the relative binding affinities of gestrinone were 54.87%, 0.74%, respectively. The results indicated that gestrinone showed a considerably high affinity with progestin receptor, but had only a very poor affinity with estrogen receptor.     

Binding of soman antidotes to acetylcholine receptors

It has been reported that several bis-quaternary compounds not necessarily having an oxime function can be used to treat soman poisoning in mice. The mechanism for this protection is not clear, but it has been proposed that such compounds may act by blocking muscarinic or nicotinic acetylcholine receptors. We have tested thirty-four compounds for muscarinic binding activity, using displacement of tritiated 3-quinuclidinyl benzilate (QNB) as a criterion, and thirty-two compounds for nicotinic binding based on inhibition of alpha-bungarotoxin (BGT) binding. Only sixteen of these compounds were able to displace QNB from rat brain membranes, and only ten of them affected BGT binding. With one exception, all of the effective compounds belonged to a series of bis-pyridinium compounds that are similar in structure to SAD-128. The binding affinities to muscarinic receptors of all these compounds were low compared to atropine. Some of the compounds bound to nicotinic receptors with affinities approaching that of d-tubocurarine. However, there was not a direct correlation between binding affinity and their reported efficacy against soman.     

Binding of antimorphine anti-idiotypic antibodies to opiate receptors

Antibodies that specifically recognize and bind opiate receptors were developed by the anti-idiotypic method. Antimorphine antibodies were generated in rabbits and showed selective binding to various opiate ligands. Guinea pigs were immunized with antimorphine antibodies to produce anti-idiotypic antibodies that cross-reacted with opiate receptors. Guinea pig antiserum inhibited binding of naloxone (0.6 nmol) to mouse brain homogenate. In isolated mouse vas deferens, contraction of electrically stimulated tissue was inhibited by the antiserum. The results indicate that anti-idiotypic antibodies which interact with opiate receptors were developed without isolating receptors.     

Differential binding in vitro to glucocorticoid receptors of deflazacort and prednisolone

Deflazacort, a new steroidal anti-inflammatory agent, has remarkable glucocorticoid activity in both animals, and humans. Its biologically active form, 21-desacetyl deflazacort, displaces [³H]dexamethasone from its cytosol receptor sites in rat kidney, thymus and liver in vitro. Although less active than prednisolone in its binding to glucocorticoid receptors, deflazacort ‘stabilizes’ the resulting steroid-receptor complex more effectively than the former in the kidney and thymus but not in the liver. This property might explain the greater activity of deflazacort than of prednisolone in the rat.     

Binding of buprenorphine to opiate receptors: Regulation by guanyl nucleotides and metal ions

The effects of guanosine-5'-triphosphate (GTP), sodium chloride and manganese chloride on the binding of buprenorphine to opiate receptors present in rat brain has been studied. Manganese chloride significantly decreased the affinity of binding of both [³H]buprenorphine and unlabelled buprenorphine to morphine and enkephalin receptors. Guanosine-5'-triphosphate increased the affinity of buprenorphine for morphine sites, but had no effect on binding of buprenorphine to enkephalin or benzomorphan sites, or binding of [³H]buprenorphine. Sodium chloride had no effect on binding of buprenorphine. Control studies indicated similar apparent affinities of buprenorphine for morphine (K_i = 0.30nM) and enkephalin (K_i=0.31nM) sites, and lower affinity for benzomorphan sites (K_i = 4.16nM). No evidence could be obtained for a differential effect of ions or guanosine-5'-triphosphate on binding of buprenorphine to opiate receptor subtypes.     

Binding analysis of nilvadipine to plasma lipoproteins by capillary electrophoresis-frontal analysis

Capillary electrophoresis coupled with frontal analysis (HPCE/FA) was applied to the ultramicro analysis of enantioselective binding of nilvadipine (NV), a calcium channel blocker, to plasma lipoproteins. The drug lipoprotein mixed solution was hydrodynamically introduced into a non-coated fused silica capillary for capillary electrophoresis. Since NV has no electric charge in the run buffer (pH 7.4), the unbound NV moved towards the cathodic end by electroosmotic flow, which was faster than the electrophoretic migrations of negatively charged lipoproteins and the bound NV. Once unbound NV migrated apart from lipoprotein, and bound NV was quickly released from the protein to maintain the binding equilibrium. Thus, NV migrated as a zone with a plateau region. The concentration of NV in this plateau region appearing on the electrophorogram was the same as the unbound NV concentration in the initial sample solution. It was found that the binding of NV to high-density lipoprotein (HDL), low-density lipoprotein (LDL) and oxidized LDL was non-specific and not enantioselective. Partition-like binding to the lipid part of these lipoproteins seemed to occur dominantly. The total binding affinities of NV to LDL were about seven times stronger than those to HDL, and the oxidation of LDL enhanced the binding affinity significantly.     

Ex vivo binding to glucocorticoid receptors in the thymus of the adrenalectomized rat

The interaction of glucocorticoids with thymic cytosol receptors in the adrenalectomized rat was studied by a method based on their capacity to deplete cytosol receptors when administered in vivo. Three h after a single oral administration of reference steroids at various dose levels, thymus cytosol aliquots were incubated with a saturating concentration of [³H]dexamethasome (30 nM) for 24 h at 0–2°C with and without 2 000 nM unlabeled dexamethasone. Bound radioactivity was determined by dextran-coated charcoal adsorption. The depletion of cytosol receptors was dose-dependent for each glucocorticoid, with the following ED₅₀ (mg/kg): fluocinolone acetonide 0.032, dexamethasone 17.0. A striking correlation (r 0.991) between ex vivo receptor binding and thymolytic effect was observed. When data from in vitro competition studies were compared with those obtained in ex vivo experiments, the latter were correlated more tightly with the biological response.     

Mineralocorticoid and glucocorticoid receptors in the amygdala regulate distinct responses to colorectal distension

Previously we found that exposure of the amygdala to elevated levels of corticosterone (CORT) induces anxiety-like behavior coupled to colonic hypersensitivity to distension, however, the specific corticoid receptor mediating the CORT responses remains controversial. In this study we investigated, through the use of selective antagonists, the relative role of amygdaloid mineralocorticoid (MR) versus glucocorticoid receptors (GR) in CORT-mediated spinal and cardiovascular pseudoaffective reflex responses to colorectal distension (CRD). Micropellets containing, CORT and a selective MR antagonist (spironolactone) or GR antagonist (mifepristone) were implanted stereotaxically onto the dorsal margin of the amygdala in rats. On day 7 post-implantation in response to graded CRD we measured: (i) changes in the electrical activity of dorsal horn neurons in the L6-S1 spinal cord and (ii) the cardiovascular depressor responses. Exposure of the amygdala to CORT-releasing micropellets increased the proportion of spinal neurons showing high-threshold and/or long-lasting responses and potentiated the magnitude of excitation. Elevated levels of amygdala CORT also increased the magnitude of the cardiovascular depressor response to CRD. MR but not GR antagonism prevented the increase in spinal cord neuronal excitation, whereas either the MR or GR antagonist decreased the magnitude of the depressor cardiovascular response to CRD. We conclude that MR in the amygdala trigger descending pathways facilitating viscero-nociceptive processing in the spinal cord, whereas MR and GR have a non-redundant role in CORT-induced potentiation of the autonomic pseudoaffective responses to colorectal stimuli.     

Binding profile of trazodone and dapiprazole to some brain receptors

Trazodone and dapiprazole displace ligands binding to rat brain alpha-1 adrenoceptors. The displacement of 3H-ligands to alpha-2, serotonin1 (5-HT1), dopamine, beta and opiate receptors is either absent or takes place at relatively higher concentrations. Trazodone, unlike dapiprazole, also inhibits binding to serotonin2 (5-HT2) receptors. Some pharmacological effects show a satisfactory correlation with these data. The psychopharmacological effects of trazodone and dapiprazole are similar, whereas the binding inhibition to 5-HT2 receptors is different, which would indicate that the psychopharmacological effects do not primarily depend on these receptors. The displacement of the binding to alpha-1 adrenoceptors by dapiprazole has a time course similar to that of its brain concentrations as well as to that of sedative and alpha-blocking effects. Dapiprazole and trazodone have antinociceptive effects and inhibit the binding to opiate receptors, although at relatively high concentrations.     

Binding of semirigid nicotinic agonists to nicotinic and muscarinic receptors

Isoarecolone methiodide (1-methyl-4-acetyl-1,2,3,6-tetrahydropyridine methiodide) was previously shown to be among the most potent agonists tested at the frog neuromuscular junction. Because nicotinic receptors from different sources vary in their selectivities, isoarecolone methiodide as well as 19 additional congeners, most of which were also previously tested at the frog neuromuscular junction, were studied in binding assays. Torpedo nobiliana was the tissue source for nicotinic receptors. Two types of experiments were conducted. The first evaluated the affinities of the agonists (including acetylcholine and carbamylcholine) for the recognition site by allowing the agonists to compete for that site with 125I-alpha-bungarotoxin. The inhibition potencies obtained correlated strongly (Spearman's correlation coefficient,-0.91) with the potency obtained at the frog neuromuscular junction. The second type of experiment evaluated the agonists for their ability to activate the receptor. The binding of [3H]perhydrohistrionicotoxin, which was employed as an indicator of the activation of the receptor, was measured in the presence of each of the agonists. Isoarecolone methiodide was the most potent of all. A few of the agonists (partial agonists) were incapable of fully enhancing this binding. For the full agonists, the concentration that produced half of the maximum binding of [3H]perhydrohistrionicotoxin was defined as the EC50. The correlation coefficient (Spearman's) for EC50 versus potency at the frog neuromuscular junction was -0.73, indicating innate differences between Torpedo and frog receptors. In addition, these compounds were tested for their affinity at muscarinic receptors from rat brain. Competition experiments were carried out using [3H]N-methylscopolamine. The affinity of isoarecolone methiodide was only about 7-fold lower than that of acetylcholine and less than 2-fold lower than that of carbamylcholine. In contrast, 1-methyl-4-acetylpiperazine methiodide was much more selective for nicotinic receptors. Its activity was similar to isoarecolone methiodide at the nicotinic receptor, but it was among the weakest compounds in its affinity for the muscarinic receptor.     

Metabotropic receptors for ATP and UTP: exploring the correspondence between native and recombinant nucleotide receptors

In the past five years, an extended series (P2Y_1−n) of metabotropic nucleotide (P2) receptors has been cloned from vertebrate tissues; these receptors are activated by either ATP or UTP, or both nucleotides. While certain cloned P2Y receptors appear to correspond functionally to particular native P2 receptor phenotypes, such pharmacological phenotypes could be explained by either a combination of several members of the P2Y_1−n series being coexpressed in the same tissue or the existence of novel, uncloned P2Y subtypes. Here, Brian King, Andrea Townsend-Nicholson and Geoffrey Burnstock review recent findings on the matter of pharmacological relationships between native P2 and cloned P2Y receptors.