Characterization of the μ and δ Opioid Receptors in the Brain of the C57BL/6 and DBA/2 Mice, Selected for Their Differences in Voluntary Ethanol Consumption

Genetically determined differences in the activity of the hypothalamic β-endorphin system have been demonstrated between the C57BL/6 (alcohol-preferring) and DBA/2 (alcohol-aversive) inbred strains of mice. The present studies examined the distribution and density of the μ and δ receptors in specific brain regions that may mediate the rewarding and reinforcing effects of ethanol, using quantitative auto-radiography and the specific μ agonist FK 33–824 and 6 agonist DPDPE, in their iodinated form. ¹²⁵I-FK 33–824 recognizes a high affinity binding site in brain membrane preparations from both the C57BL/6 (K_d= 1.37 ± 0.22 nM; B_max= 80 ± 12.3 fmol/mg protein) and DBA/2 mice (K_d= 1.02 ± 0.16 nM; B_max= 39.5 ± 9.6 fmol/mg protein), whereas ¹²⁵I-DPDPE binds to a high affinity binding site in brain membranes from both the C57BL/6 (K_d= 1.08 ± 0.34 nM; B_max= 24.4 ± 4.5 fmol/mg protein) and DBA/2 mice (K_d= 0.68 ± 0.24 nM; B_max= 15.3 ± 3.7 fmol/mg protein). The auto-radiographic studies demonstrated differences in the density of the μ opioid receptors between the two strains of mice in brain nuclei that are not directly related to the brain reward system. However, strain-related differences in the density of δ opioid receptors were observed in regions of the limbic system known to mediate the positive reinforcing effects of many drugs of abuse. The density of δ receptors was significantly higher in the ventral tegmental area and nucleus accumbens of the C57BL/6 mice. The results of the present study support the hypothesis that genetically determined differences exist in the density of opioid receptors in distinct regions of the brain between the C57BL/6 and DBA/2 inbred strains of mice, which may play a role in controlling their voluntary ethanol consumption.     

Binding of the calcium channel blocker nitrendipine to its receptor in purified sarcolemma from canine cardiac ventricle

High affinity binding of the 1,4-dihydropyridine calcium channel blocker [³H]nitrendipine was found in cardiac sarcolemma but not cardiac sarcoplasmic reticulum or mitochondria. Sarcolemmal binding of [³H]nitrendipine was saturable and reversible, with a maximum (B_max) of approximately 1 pmol/mg protein and a K_d of approximately 0.14 nm. Displacement of sarcolemma-bound [³H]nitrendipine by other nifedipine analogs was stereospecific. The K_d for nitrendipine binding was approximately three orders of magnitude lower than the IC₅₀ for the negative inotropic effect of this drug on isolated cat myocardium.     

Influence of α-Methyldopa Treatment on Adrenergic Receptor Binding in Rat Forebrain

We studied the influence of 6 days treatment with α₃-methyldopa (50 mg/kg s.c.) twice daily on radioligand binding to α₁ ((³H)prazosin), α₂ ((³H)clonidine) and β((³H)dihydroalprenolol (D₃HA)) receptors in rat forebrain. A 27% rise (p lt; 0.05) in the B_max for (³H)prazosin without change in Kd was found. Following α-methyldopa, the K_d for (³H)clonidine was increased from 1.78 ± 19 to 3.03 ± 0.30 nM and B_max fell from 197 ± 20 to 167 ± 19 fmoles/mg protein (p < 0.05). These findings suggest that chronic α-methyldopa therapy induces changes in α₁ and α₂ receptors which may modify the antihypertensive effect of α-methyldopa.     

Autoradiographic Localization of Dopamine D2-Like Receptors in the Rat Adrenal Gland

The pharmacological profile and the anatomical localization of dopamine D₂-like receptors were studied in sections of the rat adrenal gland using combined radioligand binding and autoradiographic techniques with [³H]-spiroperidol as a ligand. [³H]-Spiroperidol was bound to sections of the rat adrenal gland in a manner consistent with the labelling of dopamine D₂-like receptor sites. The binding was time-, temperature-and concentration-dependent and of high affinity with a dissociation constant (K_s) value of 1.6 ± 0.04 nM and a maximum density of binding sites (B_max) of 60 ± 3.6 fmol/mg tissue. Experiments on the pharmacological specificity of [³H]-spiroperidol binding to sections of the rat adrenal gland suggest the labelling of dopamine D₃ and/or D₄ receptors. The presence of dopamine D₃ and D₄ receptors in the rat adrenal gland was confirmed by the demonstration of a specific binding for the D₃ radioligand [³H]-7-hydroxy-N,N-di-n-propyl-2-aminotetralin (DPAT) and for the D₄ radioligand [³H]-clozapine. Light microscope autoradiography showed the highest accumulation of silver grains which correspond to [3H]-spiroperidol binding sites in the rat adrenal medulla. In the adrenal cortex, where density of silver grains is about 40% lower than in the medulla, the radioligand is accumulated primarily in the zona glomerulosa and to a lesser extent in the zona reticularis. These findings suggest that dopamine D2-like receptor sites in the rat adrenal gland cortex are primarily involved in the modulation of catecholamine secretion from the medulla and of aldosterone secretion from the cortex. The possible relevance of the occurrence of dopamine D3 and D4 receptor subtypes in the adrenal gland is discussed.     

TRH receptor binding in avian pituitary and brain

The pituitary gland of the domestic fowl binds [³H]-[3-methyl-His²]thyrotropin-releasing hormone ([³H]MeTRH) with properties very similar to those exhibited by mammalian TRH receptors, including affinity (K_D = 4.9 nM), density of binding sites (B_max = 5.1 pmol/g), and pharmacology for eight TRH analogs. Chicken brain appears to contain similar binding sites, but the level of binding was too low for detailed characterization.     

Mechanism of Action of Acamprosate. Part I. Characterization of Spermidine-Sensitive Acamprosate Binding Site in Rat Brain

It has been suggested that the anticraving drug, acamprosate, acts via the glutamatergic system, but the exact mechanism of action is still unknown. The aim of this study was to characterize [³H]acam-prosate binding and establish whether this showed any relation to sites on the NMDA receptor complex. We found saturable specific binding of [³H]acamprosate to rat brain membranes with a KD of 120 μM and a B_max of 450 pmol/mg of protein. This acamprosate binding site was sensitive to inhibition by spermidine (IC₅₀: 13.32 ± 1.1 μM; Hill coefficient = 1.04), and arcaine and glutamate both potentiated the inhibitory effect of spermidine. Acamprosate binding to the acamprosate binding site was also sensitive to inhibition by divalent cations (Ca2⁺, Mg2⁺, and Sr²⁺). Conversely, acamprosate displaced [¹⁴C]spermidine binding from rat brain membranes with an IC₅₀, of 645 μM and a Hill coefficient = 1.74. This inhibitory effect of acamprosate was not affected by arcaine, and was associated with a significant reduction in B_max and binding affinity for spermidine, suggesting an allosteric interaction between acamprosate and a spermidine binding site. These data are consistent with an effect of acamprosate on the NMDA receptor protein complex, and acamprosate was also found to alter binding of [³H]dizocilpine to rat brain membranes. When no agonists were present in vitro (minimal NMDA receptor activation), acamprosate markedly potentiated [³H]dizocilpine binding at concentrations in the 5 to 200 μ range. However, under conditions of maximal receptor activation (100 μM glutamate, 30 μM glycine), acamprosate only inhibited [³H]dizocilpine binding (at concentrations concentrations > 100 μM). When these binding studies were performed in the presence of 1 μM spermidine, the enhancing effects of acamprosate on [³H]dirocilpine binding were inhibited. The results show that acamprosate binds to a specific spermidine-sensitive site that modulates the NMDA receptor in a complex way. Together, with data from al Quatari et al. (see next paper), this work suggests that acamprosate acts as “partial co-agonist“ at the NMDA receptor, so that low concentrations enhance activation when receptor activity is low, whereas higher concentrations are inhibitory to high levels of receptor activation. This may be relevant to the clinical effects of acamprosate in alcohol-dependent patients during abstinence.     

Binding of [3H]ouabain to endothelial cells derived from various vascular beds

Summary Binding experiments were performed with [³H]ouabain on plasma membranes derived from several types of isolated and cultivated endothelial cells. Identical saturation curves for [³H]ouabain binding to endothelial cells form pig aorta, caval vein, and pulmonary artery were obtained with a dissociation constant (K_D) of 3.29±0.31 nmol/l and a binding capacity (B_max) of 5.22±0.12 pmol/mg protein. On guinea-pig coronary endothelial cells, saturation of [³H]ouabain revealed much lower affinity (K_D 95±15 nmol/l, B_max 2.08±0.09 pmol/mg protein). All Scatchard plots were linear, indicating a homogeneous class of binding sites. In competition experiments, cardiac glycosides and their aglycons displaced the radioligand with a structure-activity relationship typical for interaction with Na⁺/K⁺-ATPase (proscillaridin A>ouabain>digoxin>g-strophanthidin>digoxigenin>dihydrodigoxin); in particular, removal of the sugar moiety results in considerable reduction of affinity. Furthermore, K⁺ displayed a steep inhibition curve with a half-maximal inhibitory constant of 2 mmol/l. All these findings suggest the presence of endothelial ouabain receptors linked to Na⁺/K⁺-ATPase. However, direct measurement of this enzyme was not possible due to an extremely high Mg²⁺-ATPase activity.     

Endothelin-1 receptors in the human myometrium: evidence for different binding properties in post-menopausal as compared to premenopausal and pregnant women

OBJECTIVE We investigated the binding properties of the endothelin receptors in the human myometrium in clinical situations associated with different ovarian steroid levels. SUBJECTS AND METHODS Binding properties of the endothelin receptors were studied in myometrial membranes from post-menopausal women (n= 12), myomatous premenopausal women (n= 14) and pregnant women (n= 14), using ¹²⁵I-labelled endothelin-1. RESULTS The mean (SD) maximal receptor density (B_max) was significantly higher in samples from premenopausal and pregnant women than from post-menopausal women (983 ± 196, 1116 ± 201 and 490 ± 145 pmol/g protein, respectively). Receptor affinity (K_d) did not differ significantly between these groups. Among the pregnant women, mean B_max and K_d values were similar in those who electively underwent Caesarean section prior to the onset of labour and those operated on during the second stage of spontaneous labour. Binding properties of myometrial membranes of either pre or post-menopausal women were unaffected by the presence of high levels of beta-oestradiol or progesterone in the medium. Among samples of premenopausal women, no significant difference was found in binding properties between those operated on either during mid-follicular phase or during mid-luteal phase. CONCLUSIONS In clinical situations associated with relatively high levels of ovarian steroids, the density of endothelin receptors in the myometrium is higher than in situations associated with low ovarian steroid level. Ovarian steroids may exert their influence via the production of other mediators. Changes in density of the endothelin receptor, induced by change in ovarian steroids activity, might play a role in the regulation of myometrial contractility.     

In vitro studies on the relative potency of bronchodilator agents

This study describes a rapid in vitro assay for the order of potency of bronchodilator drugs using specific binding of (−)-[³H] dihydroalprenolol ([³H]DHA) to rat lung membranes. Under linear conditions with respect to tissue, specific binding of [³H]DHA showed saturability, rapid kinetics of association and dissociation of radioligand, and sterospecificity. Nanomolar (nM) concentrations for 50% inhibition (IC₅₀±SE) for the bronchodilator drugs examined were as follows: albuterol, 1485±170; isoproterenol, 136±53; procaterol, 162±28; terbutaline, 3310±934; and zinterol, 51±8.3. A comparison of binding studies using rat lung tissue membranes and similar preparations of rat heart and skeletal muscle demonstrated that lung tissue had 7 to 8 times more receptor sites (B_max) for [³H]DHA than heart or skeletal muscle. Adenyl cyclase activit of the rat lung membrane preparation almost doubled in the presence of (−)-isoproterenol. Displacement of specific (³H)DHA binding in membrane preparations may provide useful data for evaluating bronchodilator compounds.     

Effect of Acute and Chronic Alcohol Administration to Rats on the Expression of Interleukin-6 Cell-Surface Receptors of Hepatic Parenchymal and Nonparenchymal Cells

Rats were treated with alcohol either acutely (continuous, 7-hr intravenous infusion; blood alcohol levels ～35 mM) or chronically (liquid diet, 12–14 weeks). Three hr before killing, the animals received Gram-negative bacterial lipopolysaccharide (LPS) or saline. Hepatocytes, Kupffer cells, and liver sinusoidal endothelial cells were isolated by liver collagenase perfusion and centrifugal elutriation, and used for measurements of recombinant human [¹²⁵I]interleukin-6 binding. Dissociation constant (K_d) and the amount of cell-surface receptors (B_max) were measured on whole cells, at 4°C. Two binding sites were detected on all three cell types: high-affinity (K_d1, from 20 to 125 PM) and low-affinity (K_d2, from 0.2 to 2 nM), with low B_max (B_max from 0.4 to 12 fmol/10⁶ cells) and high B_max (B_max2, from 10 to 210 fmol/10⁶ cells). Hepatocytes displayed an 8-fold higher binding capacity for high-affinity sites (B_max1) than the other two cell types. Acute ethanol treatment induced the following significant changes in the binding parameters: a decrease in K_d1 for hepatocytes and Kupffer cells, an increase in B_max2 for hepatocytes, and a decrease in B_max1 for Kupffer cells. Although the control (nonalcoholic) liquid diet per se completely suppressed the high-affinity binding sites, alcohol-containing diet induced only one change: a significant increase in K_d2 for hepatocytes. No changes in the binding parameters were seen after LPS administration to the chronically treated group. In the acute group, LPS mimicked alcohol action on hepatocyte binding parameters. Alcohol blunted LPS effects. No changes were observed in the cytokine binding to Kupffer cells after LPS injection. The results show that alcohol alters interleukin-6 cell-surface receptor properties and receptor amount on hepatocytes and Kupffer cells. By demonstrating the presence of interleukin-6 receptors on non-parenchymal liver cells, our data also suggest that these cells may be involved in an autocrine loop-like response, which could be a target for alcohol action on the liver.     

Characterization of the High Affinity [H]Nociceptin Binding Site in Membrane Preparations of Rat Heart: Correlations with the Non-opioid Dynorphin Binding Site

The binding parameters of [³H]nociceptin were examined in membrane preparations of rat heart and compared with those of [³H]dynorphin A-(1-13) ([³H]Dyn A-(1-13)). Scatchard analysis of [³H]nociceptin binding revealed the presence of two distinct sites: a high affinity (K_d: 583 nm) low capacity (B_max: 132 pmol/mg protein) site and a low affinity (K_d: 10 316 nm) high capacity (1552 pmol/mg protein) site. Dyn A and related peptides were potent competitors of the binding to the high affinity site with the following rank order of potencyα-neo-endorphin>Dyn A-(2-13)=Dyn A-(3-13)>Dyn A-(5-13)>Dyn A-(1-13)>Dyn A>Dyn B>Dyn A-(6-10)>>Dyn A-(1-8). Nociceptin was 6.7 times less potent than Dyn A with a K_iof 4.8μmas compared with 0.72μmfor Dyn A. The order of potency of the various peptides in inhibiting [³H]nociceptin binding correlated well (r=0.93) with their ability to compete with the binding of [³H]Dyn A-(1-13) (Dumont and Lemaire, 1993). In addition, the high affinity [³H]nociceptin and non-opioid [³H]Dyn A-(1-13) sites were both sensitive to NaCl (120 mm) and the phospholipase C (PLC) inhibitors, U-73122 and neomycin (100μm). The binding activities were less affected by the weak PLC inhibitor, U-73343, and no effect was observed with the non-hydrolysable GTP analogs, Gpp(NH)p and GTP-γ-S. Nociceptin (1–50μm) was also shown to inhibit the uptake of [³H]noradrenaline ( [³H]NA) by cardiac synaptosomal preparations. In spontaneously hypertensive rats (SHR), the potency of nociceptin in inhibiting [³H]NA uptake was increased by 1.6-fold as compared with Wistar Kyoto (WKY) control rats and such effect was accompanied by comparable increased levels of cardiac ORL₁mRNA and [³H]nociceptin high affinity sites. These changes correlated well with the previously observed increased levels of non-opioid cardiac [³H]Dyn A-(1-13) sites in SHR (1.3 times as compared with WKY) and increased potency of Dyn A-(1-13) in inhibiting [³H]NA uptake by cardiac synaptosomes in SHR (2.2-fold as compared with WKY) (Dumont and Lemaire, 1995). The results demonstrate that in rat heart the characteristics of the high affinity, low capacity [³H]nociceptin binding site are similar to those of the non-opioid Dyn binding site. The stimulation of this site by nociceptin, Dyn A or related peptides is more likely to produce a modulation of PLC activity and [³H]NA uptake and may participate to the pathophysiology of hypertension.     

Specific 125I Neuropeptide Y Binding to Intact Cultured Bovine Adrenal Medulla Capillary Endothelial Cells

Objective: This study involved the pharmacological detection and characterization of binding sites for the neuromodulator neuropeptide Y (NPY) in an in vitro preparation of capillary endothelial cells derived from bovine adrenal medulla. Methods: Equilibrium binding assays were conducted on intact cells with ¹²⁵I Bolton-Hunter labeled NPY (¹²⁵I-BH-NPY). The specificity of the high-affinity binding site was evaluated in competition experiments with cold NPY, (Leu³¹, Pro³⁴)NPY (a Y₁ receptor ligand, Y₁RL), NPY_13–36 (a Y₂ receptor ligand, Y₂RL), and two other members of the pancreatic polypeptide-fold (PP-fold) family: peptide YY (PYY) and avian pancreatic polypeptide (APP). Forskolin-stimulated adenylate cyclase activity was assessed to detect the participation of this second messenger pathway in the neuromodulator action at the studied cell preparation. Results: Nonlinear regression analysis of the binding data indicated the existence of high-affinity binding sites with an equilibrium dissociation constant (K_d) value of 39.00 ± 12.84 nM and a maximal binding (B_max) of 489.89 ± 155.49 fmol/10⁶ cells (mean ± SE, n = 6). NPY, Y₁RL, and PYY displayed a concentration that inhibits the specific binding by 50% IC₅₀ (nM) values of 4.06 ± 1.66 (n = 4), 2.94 ± 0.75 (n = 5), and 18.36 ± 10.36 (n = 3), respectively. APP and Y₂RL were unable to compete with ¹²⁵I-NPY in the concentration range 0.001–1 μM. Further evaluation of second messenger pathways suggested that NPY binding sites in this model are coupled to the inhibition of adenylate cyclase. NPY significantly inhibited the forskolin-stimulated adenosine cyclic 3′,5′-(hydrogen phosphate) (cAMP) accumulation with a maximal effect of 37.03 ± 6.28%, n = 5 and an IC₅₀ of 5.96 ± 1.87 nM. The Y₁RL produced a comparable response (IC₅₀ = 5.35 ± 1.39 nM, n = 4; maximal inhibition of 61.05 ± 13.03%) and Y₂LR had no detectable effect at a similar concentration range. Conclusions: The results demonstrate the existence of a Y₁ receptor in the adrenal medulla capillary endothelial cells, which may be relevant to the postjunctional effect of NPY on this gland.     

Gonadal steroids as modulators of the function of the pineal gland

Sex steroids affect pineal function. Castration or the administration of estradiol, testosterone, or progesterone bring about changes in pineal melatonin synthesis, as well as in other aspects of pineal activity in rats. This paper deals with some of the mechanisms underlying steroid action on pineal metabolism. Estradiol is avidly taken up in vivo and in vitro by the pineal gland. A cytosol binding protein having a K_d of, (0.27 ± 0.08) × 10^?9M for estradiol and requiring SH groups for full expression of binding activity was detected in pineal homogenates. Estradiol uptake by the pineal and the uterus of mature rats attained their minima on the day of proestrus. A priming dose of estradiol increased high affinity binding sites for estradiol in pineal and uterine cytosol by about 150%. Testosterone is also readily taken up and retained in vivo and in vitro by the pineal of orchiectomized rats. Saturation kinetics analysis of androgen binding to bovine pineal cytosol indicated a K_d of 1.57 × 10^?9M for testosterone and 0.36 × 10^?9M for 5α-dihydrotestosterone. The cytoplasmic binder was shown to be a protein and to require SH groups for binding activity. Testosterone was metabolized by the pinealocytes into 5α-dihydrotestosterone, 5α-androstanediol, androstenedione and 5α-androstanedione; 5α-dihydrotestosterone was the major metabolite in the nuclei of pineal cells. Superior cervical ganglionectomy or exposure of rats to light increased androstenedione synthesis, whereas ganglionectomy or exposure to darkness decrease 5α-androstanedione synthesis. Progesterone is taken up by the pinealocytes in vitro up to a tissue concentration 18-fold that of the media. [³H]Progesterone is converted in the pinealocytes into 5α-pregnanedione and 3α-hydroxy-5α-pregnan-20-one. These data suggest that the early steps of sex steroid action on pineal cells resemble those of steroid target tissues.     

Tumor Necrosis Factor-α Cell-Surface Receptors of Liver Parenchymal and Nonparenchymal Cells during Acute and Chronic Alcohol Administration to Rats

Tumor necrosis factor-α (TNF-α) has been shown to contribute to the alcohol [ethanol (ETOH)]-induced alteration of hepatic function. Therefore we tested the hypothesis that the hepatic action of TNF-α could be due, at least in part, to alterations in TNF-α cell-surface receptors of hepatic parenchymal (hepatocytes) and nonparenchymal (Kupffer and sinusoidal endothelial) cells. Rats were either acutely treated with ETOH by a primed, continuous 7-hr intravenous infusion of 20% (w/v) ETOH (30 mg/100 g body weight/h) or chronically fed an ETOH-containing liquid diet (5.2% ETOH, w/v, with ETOH as 36% of total calories) for 14 weeks. Control rats in the acute group were infused with sterile saline, whereas control rats in the chronic group were fed liquid diet containing dextrin to replace ETOH in isocaloric amounts. Three hr before killing, the rats were injected intravenously with Gram-negative bacterial lipopolysaccharide [(LPS) 100 /μg/100 g body weight] or saline. Hepatocytes, Kupffer cells, and sinusoidal endothelial cells were isolated after liver perfusion with collagenase (without pronase), separated by centrifugal elutriation, and used to determine the affinity (K_d) and capacity (B_max) of binding sites, using recombinant human-[¹²⁵l]TNF-α as the ligand. Two binding sites were detected on Kupffer cells and sinusoidal endothelial cells isolated from control animals: a high-affinity (K_d1, in the range of 150–200 PM), low-capacity (B_max1, in the range of 2–3 fmol/10⁶ cells) binding site and a low-affinity (K_d2, in the range of 2–9 nm), high-capacity (B_max2, in the range of 3–15 fmol/10⁶ cells) binding site. One binding site was detected on hepatocytes isolated from control rats (K_d1= 1.0 nm and a B_max= 95 fmol/10⁶ cells). Acute ETOH administration caused an increase in K_d1 on both Kupffer and sinusoidal endothelial cells; a decrease in K_d1 on hepatocytes; and an increase in K_d2, B_max1, and B_max2 on endothelial cells. A second binding site on hepatocytes (K_d2= 5.8 nm, S_max2= 186 fmol/10⁶ cells) was observed only in the ETOH-treated group after LPS administration. Chronic alcohol exposure markedly elevated K_d1 and S_max1 on hepatocytes. Overall, LPS-induced changes mimicked those induced by alcohol, except for a decrease in B_max1 on Kupffer cells of rats in the acute treatment group. Also, the liquid diet containing alcohol or dextrin abrogated the high-affinity, low-capacity binding sites on both Kupffer cells and sinusoidal endothelial cells, and low-affinity, high-capacity binding sites on the hepatocyte. After LPS administration, the high-affinity binding sites on Kupffer and sinusoidal endothelial cells were reexpressed. These data show that: (1) ETOH induces changes in both the capacity (B_max) and affinity (K_d) of TNF-α cell-surface receptors of hepatocytes, Kupffer cells, and sinusoidal endothelial cells; and (2) ETOH-induced changes are consistent with an increased sensitivity of these cells to the action of TNF-α.     

Stability of [3H]MK-801 Binding Sites Following Chronic Ethanol Consumption

Previous work has demonstrated that short periods (1–2 weeks) of exposure to ethanol produce an upregulation of the N-methyl-D-aspartate (NMDA) receptor complex in hippocampus; an alteration that appears to be associated with the development of physical dependence, because a return to control levels occurs over a 24- to 48-hr abstinence period. Prolonged periods of chronic ethanol treatment (CET; 4–8 months of treatment) have been shown to produce severe and permanent alterations in the morphological and functional characteristics of hippocampal pyramidal neurons. Several lines of research have demonstrated that the NMDA receptor complex is involved in excitotoxic cell loss during certain pathological states. On the basis of this evidence, we hypothesized that prolonged ethanol exposure would be accompanied by an enduring increase in NMDA receptors and that NMDA receptor binding in cells surviving CET would be altered. To test this hypothesis, we measured the binding characteristics of the NMDA receptor complex in a variety of brain structures following CET. Animals were fed a nutritionally complete, ethanol-containing diet for 28 weeks and then allowed a 48-hr abstinence period. A control group was fed the same diet, except sucrose was isocalorically substituted for ethanol. We first examined the effect of CET on the binding properties of a noncompetitive antagonist to the NMDA receptor channel, [³H]diclozipene ([³H]MK-801). Next, as an indirect examination of NMDA receptor function, we measured the ability of glutamate to stimulate channel opening and thus [³H]MK-801 binding. In all brain structures examined, neither the K_d nor the B_max of [³H]MK-801 binding to the NMDA receptor was altered following CET. In addition, no effect of treatment was seen on the ability of glutamate to stimulate [³H]MK-801 binding.     

Muscarinic Receptor Binding of Imidafenacin in the Human Bladder Mucosa and Detrusor and Parotid Gland

Objectives: The current study aimed to characterize comparatively the binding of imidafenacin to muscarinic receptors in the human bladder mucosa and detrusor muscle and parotid gland. Methods: The muscarinic receptor in homogenates of human tissues (bladder mucosa and detrusor muscle and parotid gland) was measured using a radioligand binding assay with [N‐methyl‐³H]scopolamine methyl chloride ([³H]NMS). Results: Imidafenacin competed with [³H]NMS for binding sites in the bladder mucosa and detrusor muscle and parotid gland, and its affinity was significantly (2.6–8.7 times) higher than that of oxybutynin. Also, the affinity of imidafenacin for muscarinic receptors was approximately two‐fold higher in the parotid gland than bladder tissue. The affinity of imidafenacin in the mucosa was similar to that in the detrusor muscle, suggesting that this agent exhibits therapeutic effects by blocking muscarinic receptors in the mucosa as well as detrusor muscle. Scatchard analysis revealed that imidafenacin increased significantly (approximately four‐fold) K_d values for [³H]NMS binding in the human detrusor muscle and parotid gland, with little effect on B_max values. This observation indicates that imidafenacin binds to the muscarinic receptors in human tissues in a competitive and reversible manner. Conclusion: Imidafenacin binds to muscarinic receptors in the human bladder mucosa and detrusor muscle and parotid gland with high affinity. This agent was considered to exhibit therapeutic effects on the lower urinary tract symptoms due to an overactive bladder by blocking muscarinic receptors in the urothelium as well as detrusor muscle.     

Characterization of Binding of an RGD Mimetic, [3H]-SC-52012, to Platelet GPIIb/IIIa

In order to compare binding of small peptide mimetics on activated vs resting platelets and with fibrinogen (fgn) on activated platelets, the binding of [³H]-SC-52012, a low molecular weight (483) mimetic of the RGDF sequence present in fgn, was evaluated. This compound is a potent inhibitor of fgn binding to activated platelets, IC, 9.0 ± 0.6 nM (mean ± SEM), and inhibits ADP induced human platelet aggregation (IC, 44 ± 5 nM). The dissociation constant (Kd) of [³H]-SC-52012 was 21.6 ± 4.7 nM (n = 13) in ADP-induced human washed platelets while the Kd for resting platelets was 156 ± 8.3 nM (n = 3). The maximum number of binding sites on ADP-activated and resting platelets were 60846 ± 7158 and 59464 ± 5898 molecules/platelet, respectively. By comparison, results with [¹²⁵I]-fgn binding to activated platelets gave values of 363 ± 73 nM and 58046 ± 6386 molecules/platelet (n = 8) for the Kd and receptor number, respectively. These data suggest that the small molecule binds regardless of activation state of the platelet with only a change in affinity. [³H]-SC-52012 could be displaced by unlabelled SC-52012 with an IC₅₀ of 135 ± 20 nM.     

Expression of functional Y1 receptors for neuropeptide Y in human Ewing's sarcoma cell lines

Summary In the human Ewing's sarcoma cell line WE-68, saturation analysis using³H-labelled neuropeptide Y ([³H]NPY) as the radioligand disclosed a homogeneous population of binding sites with a dissociation constant (K _d ) of 4.5 nM and maximal binding capacity (B _max) of 712 fmol/mg cell protein. Besides the WE-68 cell line, ten other human Ewing's sarcoma cell lines (FM-62, HS-80, HT-78, HT-M1-78, NT-68, RM-82, RS-63, VH-64, WE-M1-68, WE-M2-68) were also found to display NPY receptors withK _d varying from 3.5 nM to 10.7 nM andB _max=247–3744 fmol/mg cell protein. NPY, its natural analogues and the Y₁-receptor-specific peptide ligand [Leu³¹, Pro³⁴]NPY inhibited [³H]NPY binding in the potency order: [Leu³¹,Pro³⁴]NPYhuman NPYpeptide YY (PYY)>> salmon pancreatic polypeptide (PP) > human PP>porcine NPY_13–36NPY_22–36. In the Ewing's sarcoma cell lines NPY provoked inhibition of forskolin-stimulated cyclic AMP formation by up to 98%. Pertussis toxin alleviated the cyclic-AMP-inhibitory response to NPY. In isolated Ewing's sarcoma plasma membranes pertussis toxin [³²P]ADP-ribosylated a 41-kDa protein. The ability of NPY and analogues to inhibit cyclic AMP accumulation paralleled their potencies in displacing radioligand binding. By contrast, a cell line derived from an atypical form of Ewing's sarcoma did not express specific and functional NPY receptors. These results demonstrate that conventional Ewing's sarcoma cells possess G_i-potein-coupled NPY receptors of the Y₁ type, which upon interaction with NPY, PYY, and PP mediate inhibition of cyclic AMP generation.Abbreviations NPY neuropeptide Y - PP pancreatic polypeptide - PYY peptide YY - VIP vasoactive intestinal peptide     

Characterization of oxytocin receptors and serotonin transporters in mast cells

Oxytocin (OT) inhibits the uptake of serotonin (5HT) into uterine mast cells. This may modulate 5HT bioavailability in the myometrium. Because 5HT is an important endogenous uterotonic compound, it has been postulated that this effect of OT may contribute to its potency as a labor inducer. This also predicts the presence of oxytocin receptors (OTRs) and transducing signals that will interact with 5HT transporters (SERT) in mast cells. In this study, OTR and SERT were characterized in murine peritoneal mast cells by radioligandbinding studies. Saturation assays for OTR showed no changes in K _d along the estrous cycle (6.95±2.76 nM in estrus and 4.07±1.73 nM in diestrus) but an increase in B _max in estrus (0.71±0.08 pmol/10⁶ cells and 0.37±0.05 pmol/10⁶ cells in estrus and diestrus, respectively). B _max and K _d for SERT were not affected along the estrous cycle. The signaling between the OTR and the SERT was analyzed by measuring the extent of inhibition of OT and PMA (activator of protein kinase C on 5HT uptake and the capability of Ro318220 (specific inhibitor of PKC) to increase 5HT uptake and block the effect of the above compounds in mast cells. The results showed that in murine peritoneal mast cells in vitro (1) ovarian hormones modulate OTR but not SERT expression, (2) the magnitude of OT action on 5HT uptake depends on the number of OTRs expressed in mast cells, and (3) the signaling between OTR and the SERT is mediated through the activation of protein kinase C. It is concluded that the ovarian hormones have a modulatory action on 5HT uptake which involves OT-mediated mechanism.     

Expression of Tumor Necrosis Factor-α and Interleukin-6 Cell-Surface Receptors of the Alveolar Macrophage in Alcohol-Treated Rats

The hypothesis was tested that alcohol may modulate alveolar macrophage cytokine receptors, thus interfering in lung immune defense mechanisms. Male rats were treated with alcohol either acutely (7 hr continuous intravenous alcohol infusion at a rate of 30 mg/100 g body weight/hr after a priming dose of 175 mg/100 g body weight) or chronically (feeding an alcohol-containing liquid diet for 12–14 weeks). Three hr before killing, the rats received an intravenous injection of Gram-negative bacterial lipopolysaccharide (LPS; Escherichia coll, O26:B6, 100 μg/100 g body weight). After anesthesia with sodium pentobarbital, the trachea was cannulated, and the lungs excised and lavaged to obtain alveolar macrophages. The recovered cells were used to measure the binding of recombinant human [¹²⁵I]tumor necrosis factor-α (TNF-α) and [¹²⁵I]interleukin-6 (IL-6). K_d and B_max, were determined at 4°C, thus reflecting only the cell-surface binding sites and their affinity. Two binding sites were detected for both cytokines: high-affinity (K_d1 in the range of 20–110 pM), low-capacity (B_max, in the range of 1–13 fmol/10⁵ cells), and low-affinity (K_d2 in the range of 0.6–1.3 nM), high-capacity (B_max2 in the range of 34–100 fmol/10⁵ cells). Acute alcohol treatment significantly decreased B_max1 (39%) and B_max (79%) for TNF-α, whereas chronic alcohol feeding abrogated the B_max1 (B_max1= 0), without affecting B_max2. In the acute group, LPS had an effect similar to that of alcohol. Alcohol administration did not modify the LPS effects. The following changes were monitored for IL-6 binding. Acute alcohol treatment markedly reduced (86%) B_max2. LPS administration to saline-infused group increased K_d1 (122%) and B_max1 (197%), and significantly diminished (72%) B_max2 These effects of LPS were not altered by alcohol administration, with the exception of K_d1 which was decreased (43%) by LPS administration to acutely, alcohol-intoxicated rats. In the chronic treatment group, alcohol feeding increased B_max2 (426%), an effect that was not modified by LPS injection. These data suggest that: (1) both acute and chronic alcohol administration to rats differentially affects the alveolar macrophage cell-surface receptors for TNF-α and IL-6, and (2) alcohol-induced alterations in these two cytokine receptors may in part mediate alcohol effects on lung immune defense.