Characterization of benzodiazepine binding sites after short-wave photo-affinity labeling with flunitrazepam

Membranes prepared from rat cerebral cortex were irradiated with short-wave UV light in the presence of flunitrazepam (FZ). This photo-affinity labeling (PAL) drastically reduces the potency of FZ binding to these membranes, but the binding of 3H-beta-carboline-3-carboxylate ethyl ester (3H-BCCE) was found to be essentially unchanged. 3H-BCCE binding was therefore determined in the presence of an antagonist (BCCE itself), an agonist (FZ) and a compound reported to discriminate between multiple benzodiazepine sites (CL 218,872). The results with BCCE are consistent with a single population of sites, but FZ binds to some of the sites with a reduced affinity (KI = 30 nM) and to the remaining sites with a very low affinity (KI approximately equal to 1 microM). CL 218,872 shows a reduced affinity but appears to interact with all of the sites. Taken together, these results indicate that the binding domains for BCCE and FZ are not identical, and that CL 218,872 interacts more strongly with the antagonist domain.     

CL 218,872 Binding to Benzodiazepine Receptors in Rat Spinal Cord: Modulation by γ-Aminobutyric Acid and Evidence for Receptor Heterogeneity

The binding of the triazolopyridazine CL 218,872 to central benzodiazepine receptors identified with [3H]Ro 15-1788 was studied in extensively washed homogenates of rat spinal cord and cerebral cortex. CL 218,872 displacement curves were shallow in both spinal cord (nH = 0.67) and cortex (nH = 0.54), suggesting the presence of type 1 and type 2 benzodiazepine receptors in both tissues. CL 218,872 had lower affinity in spinal cord (IC50 = 825 nM) than cortex (IC50 = 152 nM), possibly reflecting the presence of fewer type 1 sites in the cord. Activating gamma-aminobutyric acid (GABA) receptors with 10 microM muscimol resulted in a two- to threefold increase in CL 218,872 affinity in both tissues without changes in the displacement curve slope. This indicates that GABA enhances CL 218,872 affinity for both type 1 and type 2 sites in both spinal cord and cerebral cortex.     

n-[3H]Butyl-β-Carboline-3-Carboxylate, a Putative Endogenous Ligand, Binds Preferentially to Subtype 1 of Central Benzodiazepine Receptors

Synthetic n-butyl beta-carboline-3-carboxylate, an endogenous central benzodiazepine receptor inhibitor found in brain, was tritium-labeled from the butenyl ester. Binding of this [3H]beta-carboline was concentrated particularly in the synaptosomal membrane fraction of the cerebral cortex; this fraction showed a single type of high-affinity site (KD = 2.7 +/- 0.1 nM) with a Bmax of 1.16 +/- 0.08 pmol/mg of protein. The number of sites labeled was about half of that obtained with [3H]flunitrazepam binding (Bmax = 2.36 +/- 0.06 pmol/mg of protein). On the other hand, in the cerebellum, both ligands bound to practically the same number of sites. When [3H]flunitrazepam binding was done in the presence of 10(-11)-10(-5) M butyl beta-carboline, the differences between the two brain regions were more apparent. In cerebellar membranes the data fitted a straight line in the Eadie-Hofstee plot; this finding and a Hill number near unity suggest a single type of binding site. In the cortical membranes the data of binding fitted a concave curve, and the Hill number was 0.6. These are characteristics of two types of binding sites with different affinities (KD1 = 0.6-1.5 nM and KD2 = 12-18 nM). The differentiation of a high- and low-affinity site in the cerebral cortex was corroborated by experiments in which [3H]butyl beta-carboline binding was displaced by the triazolopyridazine CL 218,872. These results demonstrate that in the cerebral cortex there are two subtypes of sites (1 and 2) of central benzodiazepine receptors and that CL 218,872 binds preferentially to subtype 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

A benzodiazepine antagonist action of CL 218,872

The effect of the Type I benzodiazepine (BDZ) receptor agonist, CL 218,872, on convulsions generated by low doses of methyl beta-carboline-3-carboxylic acid ( MBCC ), bicuculline, picrotoxin and pentylenetetrazole (PTZ) in mice was examined. Low doses of CL 218,872 enhanced the convulsions produced by all agents except PTZ. An anticonvulsant action of CL 218,872 was observed at higher doses. Since CL 218,872 exhibits proconvulsive effects at low doses, and a proconvulsant action is a characteristic of compounds classified as BDZ antagonists, it appears that CL 218,872 has some antagonist action.     

Development of Benzodiazepine and Picrotoxin (t-Butylbicyclophosphorothionate) Binding Sites in Rat Cerebellar Granule Cells in Culture

The specific bindings of [3H]flunitrazepam [( 3H]FLU), [3H]CGS 8216, and t-[35S]butylbicyclophosphorothionate [( 35S]TBPS) to sites on rat cerebellar granule cells all increase from 4 to 15 days in culture, although their time courses differ. Specific [3H]FLU binding doubles, [3H]CGS 8216 binding triples, and [35S]TBPS binding increases about fourfold from 4 to 15 days in culture. Displacement studies, using the type I-selective ligand CL 218,872, indicate that at 4 days the [3H]FLU binding sites are almost entirely "type II," judging from an IC50 value near 300 nM and a pseudo-Hill number near 1. By 10 days, approximately equal numbers of type I and type II binding sites are present in the cultured cells, and this ratio remains constant thereafter (12 and 15 days). At days 10-15, both the IC50 value for CL 218,872 (near 100 nM) and the pseudo-Hill number (near 0.7) remain constant and are significantly different from the values at culture day 4. The development of specific [35S]TBPS binding parallels that of [3H]CGS 8216 binding more closely than the development of [3H]FLU binding. The [3H]CGS 8216/[3H]FLU ratio increased by a factor of 1.6 from day 4 to day 15 (p less than 0.001). Taken together, our data suggest the existence of several gamma-aminobutyric acidA (GABAA) receptor subunits, the relative proportions of which change during development. The presence of the GABA-mimetic 4,5,6,7-tetrahydroisoxazolo[5,4c]pyridine-3-ol (THIP) in the culture medium had no apparent effect on any of the binding sites studied, although THIP was shown previously to induce low-affinity GABA binding sites.     

Synchronization of estrus in goats: The relationship between eCG binding in plasma,time of occurrence of estrus and fertility following artificial insemination

Radioimmunoassay (RIA) was used to measure plasma eCG binding in dairy goats (n = 524) at the beginning of a progestagen/eCG treatment and 25 d after eCG administration. The eCG binding was not dependent on the age of the females but increased with the number of treatments they had previously received (3.4 % ± 4.8, n = 47 vs 9.6 % ± 13.2, n = 249; mean ± SD; P < 0.01 for goats treated 0 and 1 time vs those treated 2 to 5 times, respectively). The synchronization treatment led to an increase in the binding of eCG (7.1 % ± 10.9 before vs 28.3 ± 24.5 after treatment; P < 0.01). When eCG binding before treatment was higher than 5 % the onset of estrus was delayed: 37.9 % of goats came into estrus more than 30 h after sponge removal vs 7.4 % when eCG binding was lower than 5 % (P < 0.01). Fertility was significantly decreased when eCG binding was higher than 10 %. These results show that the repetition of treatment with eCG to induce estrus in goats increases eCG binding. This could explain the lowered efficiency of the hormonal treatment to synchronize estrus and the associated decrease in fertility when goats are inseminated at a predetermined time.     

A single intrauterine infusion of sustained recombinant ovine interferon-tau extends corpus luteum lifespan in cyclic ewes

Delivery carriers were developed to permit sustained release of recombinant ovine tau-interferon (roIFN-tau) to increase corpus luteum (CL) lifespan in cyclic ewes following a single intrauterine administration on Day 10 post estrus. A single infusion with 1.7 mg roIFN-tau covalently bound to carboxymethyl biogel agarose (carbodiimide coupling) significantly increased the interestrus interval (P < 0.01) in treated (n = 4) versus control animals (n = 6), whereas liposomally encapsulated roIFN-tau administered to experimental ewes (n = 8) versus control ewes (n = 6) was less effective (P < 0.05). RoIFN-tau covalently bound to trisacryl (glutaraldehyde coupling) was also effective in cyclic ewes (n = 6), but covalent binding to Eupergit C through oxirane bonds yielded ineffective preparations. Ewes that were given 1.7 mg soluble roIFN-tau (n = 8) displayed slight extension of the CL lifespan compared with ewes that were given 1.7 mg soluble BSA (n = 6), but this extension lacked significance in the Mann-Whitney U-test (P > 0.05). These results are consistent with previous data from experiments performed with daily intrauterine infusion of soluble, native or recombinant oIFN-tau. In addition, because CL maintenance requires only a single administration, these methods are efficient and simple to use since they avoid animal catheterization and allow for reduced injection frequency. Moreover, they may permit the use of smaller amounts of IFN. It is concluded that the use of oIFN-tau sustained in some delivery systems may allow for the development of an experimental sheep pseudopregnancy model.     

Selective Increase of α2A-Adrenoceptor Agonist Binding Sites in Brains of Depressed Suicide Victims

Abstract: The α_2A- and α_2C-adrenoceptor subtypes were evaluated in postmortem brains from suicides with depression (n = 22), suicides with other diagnoses (n = 12), and controls (n = 26). Membrane assays with the antagonist [³H]RX821002 (2-[³H]methoxyidazoxan) suggested the presence of α_2A-adrenoceptors in the frontal cortex and both α_2C-adrenoceptors and α_2A-adrenoceptors in the caudate. The proportions in caudate were similar in controls (α_2A, 86%; α_2C, 14%), depressed suicides (α_2A, 91%; α_2C, 9%), and suicides with other diagnoses (α_2A, 88%; α_2C, 12%). Autoradiography of [³H]RX821002 binding under α_2B/C-adrenoceptor-masking conditions confirmed the similar densities of α_2A-adrenoceptors in the cortex, hippocampus, and striatum from controls and suicides. In the frontal cortex of depressed suicides, competition of [³H]RX821002 binding by (?)-adrenaline revealed a greater proportion (61 ± 9%) of α_2A-adrenoceptors in the high-affinity conformation for agonists than in controls (39 ± 5%). Simultaneous analysis with the agonists [³H]clonidine and [³H]UK14304 and the antagonist [³H]RX821002 in the same depressed suicides confirmed the enhanced α_2A-adrenoceptor density when evaluated by agonist, but not by antagonist, radioligands. The results indicate that depression is associated with a selective increase in the high-affinity conformation of the brain α_2A-adrenoceptors.     

Characterization of the Solubilized GABA and Benzodiazepine Receptors from Various Regions of Bovine Brain

GABA and benzodiazepine receptors were solubilized from bovine cerebral cortex, cerebellum, and hippocampus and then partially purified by gel filtration and characterized. The apparent molecular weights of all these receptors were determined to be 600,000-650,000 by gel filtration, the sedimentation coefficients being 11.0-11.3 S by sucrose density gradient centrifugation. [3H]Muscimol was bound to two classes of sites in fractions from all three regions, and [3H]flunitrazepam bound to one class of sites. A comparison of the ratios of Bmax for flunitrazepam binding to Bmax for muscimol binding revealed that the fractions from the hippocampus exhibited a much higher ratio of benzodiazepine binding sites than were detected in fractions from the cortex and cerebellum. GABA agonist and antagonist inhibited [3H]muscimol binding to the fractions from these regions, at similar concentrations. Benzodiazepine agonists and antagonists also inhibited [3H]flunitrazepam binding in these three fractions, with similar potency. CL 218,872, however, inhibited [3H]flunitrazepam binding in the cerebellar fraction with the lowest IC50 value and that in th hippocampal fraction with the highest IC50 value. Hill coefficients for CL 218,872 inhibition were 0.98, 0.64, and 0.58 for cerebellum, cortex, and hippocampus, respectively.     

Age and growth of stargazer (Uranoscopus scaber L., 1758) in the southeastern Black Sea

A sample of 346 Uranoscopus scaber (L., 1758) was collected from the southeastern Black Sea between January 2002 and May 2005 in order to provide information on age, growth, length–weight relationship, and stomach contents of this species. Total length and total weight of sampled fish ranged from 5.2 to 21.9 cm and from 2.0 to 182.5 g, respectively. The sex ratio (1 : 1.98) was biased toward females (P < 0.05). Isometric growth was determined in the population. Length–weight relationships for all individuals were described by the parameters: a = 0.0167, b = 3.00, with the r² = 0.99. The population was composed of five age‐classes (I–V years). The von Bertalanffy growth parameters (±SE) and confidence limit (CL, 95%) for the entire population were: L_∞ = 26.31 ± 0.838 cm (CL: 24.66–27.96), k = 0.339 ± 0.023 1/year (CL: 0.294–0.385), and t_o = 0.087 ± 0.0346 year (CL: 0.019–0.155) (r² = 0.96). Growth performance index (Φ) was 2.37. The diet was composed of Gobius sp. (2.2%),Trachurus trachurus (2.1%), Merlangius merlangus euxinus (4.3%) and unidentified fish species (17.4%); 39.1% were unidentified remains.     

Potent interaction of histamine and polyamines at microsomal cytochrome P450, nuclei,and chromatin from rat hepatocytes

Histamine and polyamines have been implicated in the mediation of cell proliferation. Our previous work linked the growth-modulatory effects of histamine with its binding to intracellular sites in microsomes and nuclei of various tissues. In this study, we identify cytochrome P450 enzymes as a major component of microsomal intracellular sites in hepatocytes and demonstrate that polyamines compete with high affinity for histamine binding to them. Spectral measurement of histamine binding to P450 in liver microsomes resolved high and intermediate affinity binding sites (K_s1 = 2.4 ± 1.6 μM; K_s2 = 90 ± 17 μM) that corresponded to microsomal binding sites (K_d1 = 1.0 ± 0.9 μM; K_d2 = 57 ± 13 μM) resolved by ³H-histamine binding; additional low affinity (K_d3 ∼ 3 mM), and probably physiologically irrelevant, sites were resolved only by ³H-histamine radioligand studies. As determined spectrally, treatment of microsomes with NADPH/carbon monoxide decreased histamine binding to P450 by about 90% and, as determined by ³H-histamine binding, abolished the high affinity sites and reduced by 85% the number of intermediate sites. Spermine competed potently for ³H-histamine binding: in microsomes, K_i = 9.8 ± 5.8 μM; in nuclei, K_i = 13.7 ± 3.1 μM; in chromatin, K_i = 46 ± 33 nM. Polyamines inhibited the P450/histamine absorbance complex with the rank order of potency: spermine > spermidine ≫ putrescine. In contrast, histamine did not compete for ³H- spermidine binding in nuclei or microsomes, suggesting that polyamines modulate histamine binding allosterically. We propose that certain P450 isozymes that modulate gene function by controlling the level of oxygenated lipids, represent at least one common intracellular target of growth-regulatory endogenous bioamines and, as shown previously, of exogenous growth-modulatory drugs including antiestrogens, antiandrogens, and certain antidepressants and antihistamines. J. Cell. Biochem. 69:233–243, 1998. © 1998 Wiley-Liss, Inc.     

Effects of hemicastration and unilateral vasectomy on the remaining gonad and on the FSH,LH and testosterone blood concentration in bulls

The effects of unilateral castration and vasectomy on the weight and microscopic appearance of the contralateral testis and on the blood levels of testosterone, LH and FSH, were studied in German Fleckvieh bulls. Testicular weights were higher in hemicastrated bulls (P < 0.01) and unilaterally vasectomized bulls (P < 0.05) when compared to controls, 377 ± 45g (x ± s, N = 4 and 281 ± 12g, N = 4 vs 226 ± 38g, N = 3, respectively.Testosterone concentrations were higher during the weeks 14 to 22 after surgery in both treated groups. LH levels were not different from controls, but FSH levels increased significantly (P < 0.01) two weeks after hemicastration and unilateral vasectomy.Different factors appear to regulate the LH and the FSH concentrations in bulls. The increase of FSH after hemicastration may indicate a reduced production of inhibin or an inhibin-like substance from the testes, and a similar increase after unilateral vasectomy suggests that this substance may be resorbed distal to the testes.     

Characteristics of corpus luteum formed from the first wave dominant follicle following hCG in cattle

Two experiments were conducted to characterize the development and function of corpus luteum (CL) induced by hCG. In Experiment 1, cows (n = 18) were randomly assigned either to serve as controls (CONT, n = 6) or to receive hCG on Day 7 with (hCG-LUT, n = 6), or without (hCG-CONT, n = 6) surgical removal of the spontaneous CL on Day 12. The diameters of the hCG-induced and spontaneous CL of similar age did not differ (P > 0.05) between Days 1 and 4. At Day 5, the CONT (spontaneous) CL diameter (29.3 +/- 1.4 mm) was larger (P < 0.05) than that of the hCG-LUT (24.5 +/- 1.5 mm) or the hCG-CONT (24.6 +/- 1.7 mm) induced CL. Similarly, induced CL diameter for hCG-LUT and hCG-CONT groups was smaller (P < 0.01) than the CONT (spontaneous) CL between Days 10 to 14. Plasma progesterone (P(4)) levels were not different (P > 0.05) among treatment groups until Day 12. On Day 14, the P(4) concentration of hCG-LUT cows decreased (P < 0.01) to 1.1 +/- 0.9 ng/ml, then increased to 3.1 +/- 0.9 ng/ml by Day 18. Comparative values for hCG-CONT and CONT cows were 5.8 +/- 0.8 and 4.2 +/- 0.8; 4.5 +/- 0.8 and 5.5 +/- 0.8 ng/ml, respectively. The onset of regression of CL as well as estrous cycle length were similar (P > 0.05) for all treatment groups. In Experiment 2, the effects of intrauterine infusion of indomethacin on the diameter, function and life span of hCG-induced CL were examined. A slight, albeit not significant, suppression of PGFM levels was observed in indomethacin-infused cows (n = 4) compared with the controls (n = 4) in blood samples obtained once a day during the infusion period. However, in 2 cows from which blood samples were collected every 6 h, the control cow showed several pulses of PGFM while the indomethacin-treated cow exhibited none. Induced CL diameter and lifespan were not affected by indomethacin infusion. However, mean P(4) levels were higher (P < 0.05) between Days 16 and 20 in the indomethacin-infused group. In conclusion, the results suggest that 1) hCG-induced CL are functional but appear to be smaller and secrete less P(4) than spontaneous CL of similar age, and 2) the small size and reduced secretary function observed is not necessarily due to PGF(2alpha) secreted by the uterine endometrium but, probably, to inherent characteristics.     

Effect of dialysis on oxidative stress in uraemia

Abstract

It has been postulated that dialysis of patients with chronic renal failure (CRF) is associated with increased lipid peroxidation which may contribute to vascular and other complications of the syndrome. In the present study, a specific and precise technique [ferrous oxidation in xylenol orange (FOX) assay] was used to measure plasma lipid hydroperoxides (ROOHs) in three groups of uraemic patients. Patients were either studied before starting dialysis (n= 12) or on continuous ambulatory peritoneal dialysis (CAPD, n= 12) or haemodialysis (HD, n= 36) and compared to healthy controls (n=20). Plasma ROOHs were markedly elevated in HD patients compared with the controls (7.01±2.9 µM versus 4.25±2.05 µM; P < 0.005, Mann-Whitney test). Plasma ROOH concentrations in the CAPD patients were increased but not significantly higher than controls (5.36±3.56 µM versus 4.25±2.05 µM). By contrast, no differences in ROOH levels were found between controls and predialysis patients. There was no difference in plasma thiobarbituric acid reactive substances (TBARS)between control and the three CRF groups. Absolute and cholesterol standardised plasma α-tocopherol levels were lower in the patients (whether they were on dialysis or not) than in the controls (18.62±6.88 µM versus 22.73±5.33 µM; P < 0.01 and 1.99±1.88 µM/mM versus 5.25±1.0 µM/mM; P < 0.0005, respectively). This study provides direct evidence that enhanced oxidative stress in CRF patients is related to the dialysis treatment rather than the disease itself. Further studies will be necessary to establish the relationships between plasma measures of oxidative stress and cardiovascular complications in CRF patients under dialysis and whether treatment with antioxidants may reduce oxidative stress or reverse adverse effects associated with dialysis.     

Retinoylation Reactions are Inversely Related to the Cardiolipin Level in Testes Mitochondria from Hypothyroid Rats

The effect of hypothyroidism, induced by 6-n-propyl-2-thiouracil (PTU) administration to rats, on the retinoylation reaction and oxidative status was investigated in rat-testes mitochondria. In hypothyroid mitochondria, when compared to euthyroid controls, we found a noticeable increase in the amount of all-trans-retinoic acid (atRA) bound to mitochondrial proteins by an acylation process (34.2 ± 1.9 pmoles atRA/mg protein/360 min and 22.2 ± 1.7 pmoles atRA/mg protein/360 min, respectively). This increase, which was time- and temperature-dependent, was accompanied by a strong reduction in the cardiolipin (CL) amount in the mitochondrial membranes of hypothyroid (2.6 ± 0.2%) as compared to euthyroid rats (4.5 ± 0.5%) Conversely, a decreased retinoylation reaction was observed when CL liposomes were added to mitochondria or mitoplasts from both euthyroid and hypothyroid rats, thus confirming a role of CL in the retinoylation process. In mitochondria from the latter animals an increase of the level of oxidized CL occurred. The ATP level, which was reduced in hypothyroid mitochondria (27.3 ± 4.1 pmoles ATP/mg protein versus 67.1 ± 8.3 pmoles ATP/mg protein of euthyroid animals), was surprisingly increased in mitochondria by the retinoylation reaction in the presence of 100 nM atRA (481.5 ± 19.3 pmoles ATP/mg protein of hypothyroid animals versus 84.7 ± 7.7 pmoles ATP/mg protein of euthyroid animals). Overall, in hypothyroid rat-testes mitochondria the increase in retinoylation activity correlates with a significant depletion of the CL level, due to a peroxidation of this lipid. In addition, an enhanced production of reactive oxygen species was observed.     

Differential ontogeny of type 1 and type 2 benzodiazepine receptors

The postnatal development of Type 1 and Type 2 benzodiazepine receptors in rat cerebral cortex was studied using CL 218,872, a novel triazolopyridazine. On postnatal day 1 most ³H-flunitrazepam binding sites appeared to be Type 2 receptors, which increased rapidly during the first week of life and reached adult levels by 3–4 weeks of age. Type 1 receptors, on the other hand, represented only a small percentage of the binding sites on postnatal day 1 and did not begin to increase in number until approximately 7–16 days of age. These results demonstrate a differential postnatal development of two sub-populations of benzodiazepine receptors.     

Autoradiographic Localization of [3H]Zolpidem Binding Sites in the Rat CNS: Comparison with the Distribution of [3H]Flunitrazepam Binding Sites

The regional distribution of [3H]zolpidem, a novel imidazopyridine hypnotic possessing preferential affinity for the BZD1 (benzodiazepine subtype 1) receptor, has been studied autoradiographically in the rat CNS and compared with that of [3H]flunitrazepam. The binding of [3H]zolpidem to rat brain sections was saturable, specific, reversible, and of high affinity (KD = 6.4 nM). It occurred at a single population of sites whose pharmacological characteristics were similar to those of the benzodiazepine receptors labeled with [3H]flunitrazepam. However, ethyl-beta-carboline-3-carboxylate and CL 218,872 were more potent displacers of [3H]zolpidem than of [3H]flunitrazepam. The autoradiographic brain distribution of [3H]zolpidem binding sites was qualitatively similar to that previously reported for benzodiazepine receptors. The highest levels of [3H]-zolpidem binding sites occurred in the olfactory bulb (glomerular layer), inferior colliculus, ventral pallidum, nucleus of the diagonal band of Broca, cerebral cortex (layer IV), medial septum, islands of Calleja, subthalamic nucleus, and substantia nigra pars reticulata, whereas the lowest densities were found in parts of the thalamus, pons, and medulla. Comparative quantitative autoradiographic analysis of the binding of [3H]zolpidem and [3H]flunitrazepam [a mixed BZD1/BZD2 (benzodiazepine subtype 2) receptor agonist] in the CNS revealed that the relative density of both 3H-labeled ligands differed in several brain areas. Similar levels of binding for both ligands were found in brain regions enriched in BZD1 receptors, e.g., substantia nigra pars reticulata, inferior colliculus, cerebellum, and cerebral cortex lamina IV. The levels of [3H]zolpidem binding were five times lower than those of [3H]flunitrazepam binding in those brain regions enriched in BZD2 receptors, e.g., nucleus accumbens, dentate gyrus, and striatum. Moreover, [3H]zolpidem binding was undetectable in the spinal cord (which contains predominantly BZD2 receptors). Finally, like CL 218,872 and ethyl-beta-carboline-3-carboxylate, zolpidem was a more potent displacer of [3H]flunitrazepam binding in brain regions enriched in BZD1 receptors than in brain areas enriched in BZD2 receptors. The present data add further support to the view that zolpidem, although structurally unrelated to the benzodiazepines, binds to the benzodiazepine receptor and possesses selectivity for the BZD1 receptor subtype.     

Heterogeneity of γ-Aminobutyric Acid/Benzodiazepine/β-Carboline Receptor Complex in Rat Spinal Cord

The properties of muscimol, beta-carboline (BC), and benzodiazepine (BZD) binding to crude synaptic membranes were studied in the spinal cord and cerebellum of rats. In cerebellar membranes, the density of high-affinity [3H]muscimol and [3H]6,7-dimethoxy-4-ethyl-beta-carboline ([3H]BCCM) binding sites is almost identical to that of [3H]flunitrazepam ([3H]FLU) or [3H]flumazenil (Ro 15-1788; ethyl-8-fluoro-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a] [1-4]benzodiazepine-3-carboxylate). In contrast to the cerebellum, the number of muscimol and BC binding sites in rat spinal cord is approximately 20-25% of the number of FLU or flumazenil binding sites. Moreover, in spinal cord membranes, BC recognition site ligands displace [3H]-flumazenil bound to those sites, with low affinity and a Hill slope significantly less than 1; the potency of the different BCs in displacing [3H]flumazenil is 20-50-fold lower in the spinal cord than in the cerebellum. [3H]Flumazenil is not displaced from spinal cord membranes by the peripheral BZD ligand Ro 5-4864 (4'-chlorodiazepam), whereas it is displaced with low affinity and a Hill slope of less than 1 (nH = 0.4) by CL 218,872 (3-methyl-6-(3-trifluoromethylphenyl)-1,2,4-triazolol[4,3-b] pyridazine). These data suggest that a large number of BZD binding sites in spinal cord (approximately 80%) are of the central-type, BZD2 subclass, whereas the BZD binding sites in cerebellum are predominantly of the central-type, BZD1 subclass.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and Characterization of Specific Binding Sites for Somatostatin in Cytosol of Bovine Cystic Duct Mucosa

Abstract

Specific binding sites for somatostatin have been detected in cytosolic fraction of bovine cystic duct mucosa. At 37°C, the interaction of ¹²⁵I-Tyr¹¹-somatostatin with cytosolic fraction was rapid, reversible, specific and saturable. At equilibrium, the binding of tracer was competitively inhibited by native peptide in the 1 nM to 2 µ M range of concentrations. Scatchard analysis of binding data suggested the presence of two distinct classes of somatostatin binding sites: a class with a high affinity (K_d = 7.8 ± 0.3 nM) and a low capacity (1.3 ± 0.3 pmol somatostatin/mg protein) and a class with a low affinity (K_d = 129.1 ± 2.0 nM) and a high capacity (43.5 ± 6.7 pmol somatostatin/mg protein). The binding sites were shown to be highly specific for somatostatin since neuropeptides present in cystic duct such as Leu-enkephalin, neurotensin, substance P and vasoactive intestinal peptide did practically not show competition. These findings suggest that somatostatin could contribute to the regulation of the functions of the cystic duct mucosa in physiological and pathological conditions.