Leukotriene C4 receptors in cultured smooth muscle cells from bovine anterior cerebral arteries and microcerebrovasculatures.

Specific receptors for leukotriene C4 (LTC4) were identified on smooth muscle cells isolated from bovine anterior cerebral arteries (BACASMC) and bovine microcerebrovasculatures (BMSMC). [3H]LTC4 specific bindings to both cells at a fixed input reached the maxima at 60 min and 20 min, respectively. With incremental inputs of radioligand and a constant cell number, [3H]LTC4 specific bindings reached a plateau indicative of a saturable binding site. Analysis of Scatchard plots demonstrated a single population of high-affinity binding sites in both cells. The dissociation constant (Kd) for BACASMC was 39.2 +/- 1.3 nmol.L-1 and its Bmax was 19.3 +/- 2.1 pmol/10(6) cells. For BMSMC, Kd = 2.0 +/- 0.4 nmol.L-1, Bmax = 157 +/- 13 fmol/10(6) cells. The specific [3H]LTC4 bindings was inhibited by unlabeled LTC4, LTD4 and FPL-55712 (an SRS-A antagonist). The inhibitory rates for BACASMC were 70.4% and 35.3% by LTC4 and FPL-55712 at 1 mumol.L-1, respectively. For BMSMC the inhibitory rates were 96.9%, 73.9%, and 44.9% by LTC4, LTD4, and FPL-55712 at 10 mumol.L-1, respectively.     

PAF binding sites. Characterization by [3H]52770 RP, a pyrrolo[1,2-c]thiazole derivative, in rabbit platelets

52770 RP, the N-(3-chlorophenyl)-3-(3-pyridinyl)-1H,3H-pyrrolo[1,2-c]thiazole -7-carboxamide, displaces in a potent, specific and competitive manner [3H]PAF from its binding sites on rabbit platelets. Since 52770 RP is not structurally related to PAF and has low liposolubility with respect to PAF, it was selected as a potential radioligand for PAF receptor sites. [3H]52770 RP displayed high-affinity, specificity, as well as saturable and displaceable binding to a single class of recognition sites in intact platelets and crude platelet membranes. In these preparations, the values of binding parameters were, respectively, 8.5 and 7.6 nM for Kd, 0.2 pmol/5 X 10(7) platelets and 3.66 pmol/mg protein for Bmax and 0.96 and 0.91 for nH. Inasmuch as the (+)-52770 RP was 300-fold more potent than the (-)-isomer at displacing [3H]52770 RP in intact platelets, the studied binding site manifested stereospecific discrimination. A variety of pharmacological agents including pro- and anti-aggregant compounds did not exhibit affinity for [3H]52770 RP binding sites. In contrast, PAF, some of its active analogues and several recognized PAF antagonists (BN 52021, brotizolam, L-652,731, triazolam), displaced the [3H]52770 RP binding. Studies carried out using [3H]PAF demonstrated that 52770 RP was approximately 4- and 200-fold more potent than L-652,731 and BN 52021 respectively, as a PAF-receptor antagonist. In washed rabbit platelets, the rank order of potency (Ki) for several analogues of 52770 RP, to displace [3H]PAF from its binding sites, was highly correlated (r = 0.96) to their ability to antagonize [3H]52770 RP binding. In functional studies, 52770 RP antagonized not only the PAF-induced aggregation in washed rabbit platelets but also the hypotension evoked by PAF in the anesthetized rat. In this respect, it was 26 and 2 times more potent than L-652,731, respectively. In conclusion, [3H]52770 RP might represent a novel interesting tool for furthering our understanding of the role of PAF binding sites in pathophysiological processes.     

新抗胆碱药[3H]三环哌酯对人脑M受体的作用

用放射配体受体结合试验法,研究了新化合物三环哌酯与人大脑皮质M受体的结合特性,并与QNB作了比较。饱和实验结果显示,[³H]三环哌酯的结合参数与[³H]QNB相近,两种配体的作用均符合单位点模型。竞争性抑制实验结果表明二者作用强度相当。[³H]三环哌酯的结合和解离速率常数均较[³H]QNB大,且其与皮质M受体的解离受季铵酚的变构调节,结果提示,两种配体与M受体有一些不同的结合特性,在M受体研究中,[³H]三环哌酯可以作为[³H]QNB的补充工具。     

Characterization of the binding of [3H]L-689,560, an antagonist for the glycine site on the N-methyl-D-aspartate receptor, to rat brain membranes.

The binding characteristics of [3H]L-689,560 [(+/-)-4-(trans)-2-carboxy-5,7-dichloro-4-phenylaminocarbonylamino -1,2,3,4- tetrahydroquinoline], a selective antagonist for the glycine site on the N-methyl-D-aspartate receptor, have been evaluated using rat cortex/hippocampus P2 membranes. Specific [3H]L-689,560 binding was saturable, having a Kd of 2.97 nM and a Bmax of 4.15 pmol/mg of protein. The Bmax value was not significantly different from that obtained for [3H]glycine in the same membrane preparation, and L-689,560 and glycine were found to be mutually competitive. The specific binding of [3H]L-689,560 (1 nM) represented 96 +/- 0.02% (four experiments) of total binding. Association experiments at 4 degrees revealed that [3H]L-689,560 reached equilibrium in 120 min, with a t1/2 of 40 min. The dissociation of [3H]L-689,560 was slow at 4 degrees (t1/2 = 118 min), allowing the use of filtration to separate free from bound radioactivity. Both association and dissociation curves were best fitted by a double-exponential function, suggesting the presence of two components. Comparison of IC50 values obtained using [3H]glycine and [3H]L-689,560 binding for 21 glycine site ligands (including agonists, partial agonists, and antagonists, with affinities spanning 5 orders of magnitude) showed a 1:1 correlation, with a correlation coefficient of 0.97. This suggests that efficacy does not have a large influence on the affinity of glycine site ligands when an agonist or antagonist radioligand is used. Ligands for other amino acid recognition sites did not directly inhibit [3H]L-689,560 binding. [3H]L-689,560 is an improved radioligand for the glycine site that will facilitate further investigations of its properties.     

小鼠脾脏淋巴细胞上存在μ阿片受体的证据

AIM: Using ohmefentanyl (Ohm), a high affinity mu opioid receptor agonist, to find whether mu opioid receptor exists on mouse spleen lymphocytes. METHODS: The proliferation rates of T-lymphocytes and B-lymphocytes were determined under various concentrations of Ohm with or without naloxone(Nal) in vitro. Binding characteristics of [3H] Ohm with mice spleen lymphocytes were studied by radioligand assays. ReSULTS: Ohm 0.1 pmol.L(-1)-1 nmol.L-1 enhanced Con A-induced spleen T-cell proliferation in vitro. Nal 50 mumol.L-1, which per se enhanced the T-cell proliferation, blocked the enhancing effects of Ohm. However, Ohm had no effect on B-cell proliferation. Furthermore, a satisfactory saturable, specific, and reversible binding was demonstrated with Kd of (6.9 +/- 0.6) nmol.L-1, Bmax of (74 +/- 6) fmol/10(7) cells. The binding of [3H] Ohm was blocked by unlabeled Ohm or Nal. CONCLUSION: Stimulating effects of Ohm on lymphocytes were mediated by opioid receptors. Mouse spleen lymphocytes present mu opioid receptors.     

八肽胆囊收缩素对[~3H]埃托啡与大鼠脑阿片受体结合的抑制作用

本实验观察了CCK—8对[~3H]Et与大鼠脑阿片受体结合的影响.含硫CCK—8能抑制[~3H]Et与高亲和位点的结合,使K_D值增大(P<0.001),B_(max)减小(P<0.01),在10 fmol/L～Lμmol/L范围内呈量效关系.但对低亲和位点的结合没有影响.无硫CCK—8仅对[~3H]Et高亲和位点的K_D值有较小程度的增大(P<0.05),不影响B_(max)值.CCK—8(10 nmol/L)抑制[~3H]Et与阿片受体结合的作用能被CCK受体拮抗剂谷丙酰胺(1μmol/L)所阻断.结果提示,CCK—8可能通过激活CCK受体发挥对阿片受体的抑制作用。     

Evidence for the AT1 subtype of the angiotensin II receptor in the rat submandibular gland

We have characterized angiotensin II (Ang II) receptor subtypes on rat submandibular gland membranes using a radioligand binding assay. [3H]Ang II binding to the membrane fractions exhibited both high (Kd =0.08 nm, Bmax =2.19 fmol/mg protein) and low (Kd =4.19 nm, Bmax = 13.7 fmol/mg protein) affinity. Ang 11, Ang III and saralasin completely displaced the [3H]Ang II binding, whereas CV-11974, an AT1 receptor antagonist and PD123319, an AT2 receptor antagonist maximally displaced up to approximately 87 and 13% of the total binding, respectively. [3H]DuP753 binding to the membrane fractions exhibited a single population of binding site with a Kd of 4.22 nM and Bmax of 3.77 pmol/mg protein. Ang II, Ang III and CV-11974 completely displaced the [3H]DuP753 binding with slope factors near unity, but PD123319 did not. These findings suggest that rat submandibular gland membranes contain predominantly the AT1 receptor subtype.     

血小板激活因子刺激血小板在脑微血管内皮细胞上粘附及药物的阻断作用(英文)

目的:研究血小板激活因子(PAF)刺激脑微血管内皮细胞导致血小板在内皮细胞上粘附及WEB,DMPP和粉防己碱的抑制作用. 方法:用[~3H]腺嘌呤标记血小板探讨PAF导致血小板在脑微血管内皮细胞上粘附和药物的抑制作用. 结果:PAF 10—100 nmol L~(-1)显著增加血小板与脑微血管内皮细胞的粘附率,WEB,DMPP和粉防己碱抑制由PAF刺激而导致的血小板在脑微血管内皮细胞上的粘附. 结论:DMPP和粉防己碱能够抑制PAF对脑血管的损害作用.     

Characterization of [3H]estradiol-17 beta-(beta-D-glucuronide) binding sites in basolateral and canalicular liver plasma membranes

The specific binding of [3H]estradiol-17 beta-(beta-D-glucuronide) ([3H]E217G) was examined in isolated basolateral (bLPM) and canalicular (cLPM) liver plasma membranes. Two distinct binding sites were identified in each membrane fraction by competition and saturation experiments. Binding parameters obtained from competition studies were: Kd1 = 26 nM, Bmax1 = 0.26 pmol/mg protein; Kd2 = 2.6 microM, Bmax2 = 27 pmol/mg protein for bLPM; and Kd1 = 81 nM, Bmax1 = 0.61 pmol/mg protein; Kd2 = 6.7 microM, Bmax2 = 79 pmol/mg protein for cLPM. Binding parameters obtained from saturation experiments were not significantly different. There was no Na+ requirement for binding. Kinetic dissociation experiments showed that binding was reversible and revealed two components. The dissociation rate constants did not vary with the method of dilution of radioligand, i.e. by "infinite" volume, or excess unlabeled ligand, thus ruling out the possibility of cooperativity. The ability of a series of compounds to inhibit the binding of [3H]E217G was also examined. In bLPM, taurocholate (TC), estrone sulfate (E1SO4) and bromosulfophthalein (BSP) were able to compete with both binding sites, whereas estriol-17 beta-(beta-D-glucuronide) (E317G), estriol-16 alpha-(beta-D-glucuronide) (E316G), testosterone glucuronide (TG), estradiol-3-(beta-D-glucuronide) (E23G), estriol-3-(beta-D-glucuronide) (E(3)3G), cholate and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) were able to inhibit binding to only the low-affinity site. In cLPM, only the cholestatic steroid D-ring glucuronides (E(3)17G, E(3)16G and TG) and TC were able to compete with both sites, whereas the non-cholestatic steroid A-ring glucuronides (E(2)3G and E(3)3G), BSP and DIDS competed for only the low-affinity site. Based on the observed substrate specificities, the low-affinity sites in bLPM and cLPM are postulated to represent multispecific organic anion carriers. The high-affinity site in cLPM may play a role in mediating steroid D-ring glucuronide-induced cholestasis.