A quantitative method of analyzing the interaction of slightly selective radioligands with multiple receptor subtypes

Subclasses of receptors exist for most neurotransmitters. Frequently, two subtypes of receptors coexist in the same tissue and, in some cases, they mediate the same physiological response. In tissues with two classes of binding sites for a given hormone, an estimate of the proportion of each class of binding sites is obtained by inhibiting the binding of a single concentration of a radioligand with a selective unlabeled ligand. Accurate estimates of the density of each class of receptors will only be obtained, however, if the radioligand is entirely nonselective. Selectivity of just 2- to 3-fold can markedly influence the results of subtype analysis. The conclusion that a radioligand is nonselective is usually based on the results of a saturation binding curve. If Scatchard analysis of such data results in a linear plot, then it is concluded that the radioligand is nonselective. However, Scatchard analysis cannot distinguish between a radioligand that is nonselective and one that is slightly selective. The use of a slightly selective radioligand can lead to errors of 50% or more, depending on the concentration of the radioligand relative to the Kd values of the two classes of sites. A new analytical method has been developed that can be used to quantitate 2- to 3-fold differences in the affinity of two distinct classes of binding sites for a radioligand. This new approach requires that a series of inhibition experiments with a selective unlabeled ligand be performed in the presence of increasing concentrations of the radioligand. Analysis of the resulting inhibition curves, utilizing the mathematical modeling program MLAB on the PROPHET system, yields accurate estimates of the density of each class of receptor as well as the affinity of each receptor for the labeled and unlabeled ligands. This approach was used to determine whether 125I-iodopindolol shows selectivity for beta 1- or beta 2-adrenergic receptors. A series of inhibition curves was generated with the unlabeled ligands ICI 89,406 (beta 1-selective) and ICI 118,551 (beta 2-selective), using membranes prepared from C6 glioma cells. These cells contain both beta 1- and beta 2-adrenergic receptors. 125I-Iodopindolol was determined to be 3-fold selective for beta 2-adrenergic receptors. Since the sensitivity of this approach is superior to that of Scatchard analysis, it is likely that other radioligands, previously thought to be nonselective, will be shown to be selective when analyzed by this method.     

Up-regulation of N-methyl-D-aspartate receptors on cultured cortical neurons after exposure to antagonists.

The density of N-methyl-D-aspartate (NMDA) receptors on membranes prepared from cultured cortical neurons was determined using binding assays with [125I]I-MK-801 after exposure of cultures to antagonists of the NMDA receptor complex. The density of binding sites for [125I]I-MK-801 was increased by 40-80% after exposure to D-2-amino-5-phosphonopentanoic acid (D-AP5), with no change in the number or viability of neurons. The effect of D-AP5 was concentration dependent, with an EC50 of 10 microM. Up-regulation of NMDA receptors was observed after 2-7 days but not after 1 day of exposure to 100 microM D-AP5. The density of NMDA receptors was also increased after exposure of cells to CGS 19755 and MK-801 but not after exposure to the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. The binding of [3H]AMPA was unaltered after exposure to D-AP5. These results demonstrate that the density of NMDA receptors on cultured neurons can be selectively up-regulated by exposure to NMDA receptor antagonists. Increases in the density of NMDA receptors occurring in vivo could complicate therapeutic approaches to the treatment of neurological disorders.     

Effects of mono-, di-, and triamines on the N-methyl-D-aspartate receptor complex: a model of the polyamine recognition site.

Systematic series of monoamines, diamines, and triamines were used to define the structural requirements for interaction at the polyamine recognition site of the N-methyl-D-aspartate receptor complex. Effects of amines on binding of [3H]MK-801 to washed synaptic plasma membranes were measured in the presence of L-glutamate and glycine (100 microM each), in the absence or presence of spermine (10 microM). Linear aliphatic monoamines of methylene chain length up to 12 (dodecylamine) did not interact with the polyamine recognition site. Nonspecific inhibition of binding was observed at high concentrations of the longer monoamines. alpha,omega-Diamines of methylene chain length 2 (1,2-diaminoethane, DA2) through 12 (1,12-diaminododecane, DA12) had varying actions, depending on chain length. The shortest diamines (DA2 and DA3) acted as weak partial agonists, enhancing the binding of [3H[MK-801. Intermediate-length diamines (DA4-DA7) were selective polyamine antagonists, having little or no effect on binding of [3H]MK-801 measured in the absence of spermine but inhibiting binding measured in the presence of spermine. The longest diamines tested (DA8-DA12) acted as inverse agonists; they inhibited binding in the absence or presence of spermine, and this inhibition was blocked by the selective polyamine antagonist diethylenetriamine. Computer modeling of conformations of the diamines quantitatively documented that 1) these molecules are flexible and 2) long diamines may easily adopt conformations with inter-nitrogen distances mimicking those of short diamines. The cis and trans isomers of 1,4-diaminocyclohexane are inflexible, conformationally restricted diamines with markedly different actions. The cis isomer was a partial agonist and the trans isomer was an antagonist at the polyamine recognition site. Triamines of general structure NH2(CH2)3NH(CH2)xNH2 (TRI[3,x]), in which x = 3-12, were synthesized and tested for activity at the polyamine recognition site. Despite the large range of size, TRI[3,3] through TRI[3,9] were all fully polyamine agonists of similar potency. TRI[3,10] was a partial agonist, whereas TRI[3,12] inhibited binding of [3H]MK-801. Diethylenetriamine did not attenuate the effect of TRI[3,12]. Based on the results of the radioligand binding studies and the computer analysis, a model of the polyamine recognition site is proposed.     

Regulation of the 5-hydroxytryptamine1B receptor in opossum kidney cells after exposure to agonists.

The density of 5-hydroxytryptamine (5-HT)1B receptors and their coupling to the inhibition of cAMP accumulation were investigated in opossum kidney cells maintained in culture. The density and properties of the receptor were determined using [125I] iodocyanopindolol as the radioligand. The pharmacological specificity of the binding site was consistent with that expected for a 5-HT1B receptor. Serotonin inhibited forskolin-stimulated cAMP accumulation with an EC50 of 4-8 nM. Compounds known to show selectivity at the 5-HT1B receptor, such as trifluoromethyl-phenylpiperazine and CGS-12066B, also inhibited forskolin-stimulated cAMP accumulation, acting as full agonists with efficacies comparable to that of serotonin. Other beta-adrenergic receptor antagonists, including (-)-pindolol and (-)-alprenolol, bound to the receptor with high affinity and acted as partial agonists capable of inhibiting forskolin-stimulated cAMP accumulation. Exposure of cells to 5-HT resulted in a time- and dose-dependent decrease in the density of 5-HT1B receptors that was not accompanied by a change in the Kd of the binding site for [125I] iodocyanopindolol. A maximum decrease of 60% in the number of 5-HT1B receptors was evident after a 16-hr treatment with 1 microM 5-HT. Concomitant with the observed decrease in the density of receptors was a marked increase in the EC50 for 5-HT-mediated inhibition of forskolin-stimulated cAMP accumulation. The EC50 was increased 4-5-fold after a 16-hr exposure to 1 microM 5-HT, and the maximal level of inhibition was markedly decreased. Whereas pretreatment with moderate concentrations of 5-HT (100-300 nM) for 16 hr produced significant decreases in the density of 5-HT1B receptors and increases in the EC50 for inhibition of forskolin-stimulated cAMP formation, there was little change in the maximal level of inhibition that could be attained. Such a combination of changes could be explained by the presence of "spare" 5-HT1B receptors on these cells.     

Growth of Pakistani children in relation to the 1990 growth standards

This study was designed to compare the growth of Pakistani schoolchildren in the UK with the 1990 UK growth standards. Measurements of height, weight, and sitting height were performed on 785 Pakistani schoolchildren aged 5-14 years with the mean values for each age and sex being plotted on the UK growth standards. The results were expressed as SD scores relative to the 1990 reference data. The mean height for the boys was only 0.2 SD scores below the mean for the new growth standards with the mean height for the girls being 0.4 SD scores below the mean. The mean values for weight and body mass index were 0.3 and 0.5 SD scores less than the mean for boys and girls respectively. This study demonstrates that the growth of Pakistani schoolchildren in the UK is comparable to the 1990 UK growth standards with only minor differences. It is not safe to assume that short stature or low body weight in a Pakistani child is due to his or her ethnic background.     

Hemorrhagic tumor necrosis during a pilot trial of tumor necrosis factor-alpha and anti-GD3 ganglioside monoclonal antibody in patients with metastatic melanoma

Hemorrhagic tumor necrosis is an inflammatory event that leads to selective destruction of malignant tissues, with both potentially toxic and beneficial consequences. A pilot clinical trial was undertaken combining tumor necrosis factor-alpha (TNF-alpha) with the monoclonal antibody R24 (MoAb R24) against GD3 ganglioside in patients with metastatic melanoma. Patients received MoAb R24 to recruit leukocytes to the tumor followed by low doses of recombinant TNF-alpha to activate leukocytes. Eight patients were treated and seven patients had mild toxicity. One patient with extensive metastatic melanoma developed tumor lysis syndrome within hours after treatment with almost complete necrosis of bulky tumors in multiple visceral sites. To our knowledge, this is the first documented case of hemorrhagic tumor necrosis in a patient with metastatic cancer in multiple visceral sites.     

Induction of immune tolerance in patients with hemophilia A and inhibitors treated with porcine VIIIC by home therapy

Home therapy with porcine factor VIIIC was safe and effective when administered to five hemophilic patients over periods of 8 1/2, 6, 4, 3 1/2, and 2 years. No significant transfusion reactions occurred. Before treatment with porcine factor VIIIC, all five had high-level, high- responding anti-human VIIIC inhibitors initially lacking anti-porcine factor VIIIC activity. Although specific anti-porcine VIIIC inhibitors arose in all patients, these were generally transient, and only one patient became refractory to treatment. We believe that porcine factor VIIIC is the treatment of choice in patients whose inhibitors do not cross-react. All five patients lost their original anti-human VIIIC inhibitors after starting treatment with porcine VIIIC, permitting the reintroduction of human VIIIC in three of them. There has been no recurrence of anti-human VIIIC inhibitor activity during 2 to 3 years of regular treatment with human VIIIC in these patients. This suggests that tolerance to human VIIIC has arisen as a result of treatment with porcine VIIIC. Porcine VIIIC may have a role in the desensitization of some factor VIIIC inhibitor patients.     

Factor V Quebec revisited

Factor V Quebec has been described as a bleeding disorder that exhibits an autosomal dominant inheritance pattern and presents severe bleeding after trauma. Two members of a fourth-generation (IV.13 and IV.15) Canadian family have been studied in detail and are the subject of this report. Their clinical presentations and histories have been described previously (Tracy et al: J Clin Invest 74:1221, 1984). Persistent abnormalities include mild thrombocytopenia and defective platelet factor V. Plasma factor V is present at near normal concentration and is fully functional. Thus, the bleeding diathesis appears to reflect the absence of platelet factor V activity. The recent report (Hayward et al: Blood 84:110a, 1994 [suppl, abstr]) of multimerin deficiency in these individuals led us to reevaluate these patients. Western blot analyses of platelet lysates developed with a variety of monoclonal antibodies show that the alpha-granule proteins, fibrinogen, von Willebrand factor, factor V and osteonectin are decreased in concentration and significantly degraded in the platelets of these patients. Thrombospondin, while not degraded, is substantially decreased. In contrast, platelet factor 4 and beta-thromboglobulin do not appear to be affected. These observations suggest that the alpha- granules are correctly assembled but the contents are subsequently subjected to proteolytic degradation. The results indicate that factor V Quebec disorder is probably associated with a generalized defect that leads to degradation of most proteins of the alpha-granules.