Peripheral-type benzodiazepine receptors are highly concentrated in mitochondrial membranes of rat testicular interstitial cells

The binding of 3H-RO 5-4864 to the peripheral-type benzodiazepine receptors (PBZDR) in rat testicular interstitial cells (TIC) was characterized. The binding was saturable, reversible and showed a single high-affinity (Kd = 5.02 +/- 0.86 nM) class of binding sites. The maximal binding capacity (Bmax) in crude mitochondrial fractions (77.6 +/- 9.1 pmol/mg protein) represents the highest density of PBZDR in tissues thus far studied. In comparison with the crude mitochondrial fraction the subcellular fractionation of TIC revealed a 2-fold enrichment of 3H-RO 5-4864 binding sites to the purified mitochondria (Bmax = 140 +/- 23 pmol/mg protein). The ability of various drugs to displace 3H-RO 5-4864 from TIC binding sites was examined and the inhibition constants (Ki) for RO 5-4864, PK 11195, diazepam and flunitrazepam were 3.5, 4.4, 159, and 353 nM, respectively, whereas clonazepam and RO 15-1788 were inefficient in displacing 3H-RO 5-4864 (Ki greater than 10 microM). This pharmacological profile is characteristic of PBZDR described in other tissues. In conclusion, rat TIC possess a very high concentration of PBZDR primarily associated with mitochondrial membranes.     

The identification and characterization of 125I-labelled iodomelatonin-binding sites in the duck kidney.

The melatonin-binding sites in membrane preparations of duck kidney were demonstrated by utilizing 125I-labelled iodomelatonin as a radioligand. Binding at these sites was found to be reversible, saturable, specific and of high affinity. Scatchard analysis of the specific binding revealed an equilibrium binding constant (Kd) of 44.6 +/- 4.4 pmol/l (n = 6) and a total number of binding sites (Bmax) of 6.43 +/- 0.60 fmol/mg protein (n = 6) at the mid-point of the light period (mid-light). The Hill coefficient approached 1.0, suggesting a single class of 125I-labelled iodomelatonin-binding site in the duck kidney. Diurnal variation in 125I-labelled iodomelatonin binding showed that the Bmax was 53.4% higher at mid-light than at mid-point of the dark period (P < 0.05), with no significant variation in Kd. The Kd value determined from kinetic analysis was 22.5 pmol/l in birds at mid-light, which was comparable with values determined from equilibrium studies. The order of pharmacological affinity for 125I-labelled iodomelatonin-binding sites in the duck kidney membrane preparations was: 2-iodomelatonin > melatonin > 6-chloromelatonin > 6-hydroxymelatonin > N-acetylserotonin > 5-methoxytryptamine, 5-methoxytryptophol, 5-hydroxytryptamine, tryptamine, 1-acetylindole-3-carboxaldehyde, 5-hydroxyindole-3-acetic acid, L-tryptophan, 5-methoxyindole-3-acetic acid, 3-acetylindole, acetylcholine, epinephrine, norepinephrine and harmaline. The pharmacological characteristics indicated that 125I-labelled iodomelatonin-binding sites are highly specific for melatonin. Our finding of 125I-labelled iodomelatonin-binding sites in the kidney suggests that melatonin may regulate kidney function.(ABSTRACT TRUNCATED AT 250 WORDS)     

Age-related changes in glucocorticoid and androgen receptors of cultured human pubic skin fibroblasts

In order to evaluate age-related changes in glucocorticoid receptor and androgen receptor of cultured human pubic skin fibroblasts in young and aged men, we determined both [3H]dexamethasone binding and [3H]methyltrienolone (R1881, potent androgenic steroid) binding, using dispersed whole cell assay. Scatchard analyses of specific [3H]dexamethasone binding to the fibroblasts of young and aged men showed a single class of high-affinity binding sites with a mean (+/- SD) binding site concentration (Bmax) of 12.69 +/- 2.36 X 10(4) and 12.87 +/- 12.21 X 10(4) sites/cell, respectively, and mean (+/- SD) dissociation constant (Kd) of 5.60 +/- 0.41 and 7.36 +/- 2.17 nM, respectively. Scatchard analyses of specific [3H]R1881 binding to the same cultured skin fibroblasts of young and aged men showed a single class of high-affinity binding sites with a mean Bmax of 5.77 +/- 1.02 X 10(3) and 2.82 +/- 0.97 X 10(3) sites/cell, respectively, and a mean Kd of 0.56 +/- 0.23 and 0.50 +/- 0.28 nM, respectively. These findings indicate that there were no significant age-related changes in binding site and affinity of glucocorticoid receptor in cultured human pubic skin fibroblasts, whereas binding sites of androgen receptor significantly decreased in those of aged men as compared to young men, without significant change in affinity.     

Binding of oestradiol, progesterone and prolactin in rat lung

Binding of [3H]oestradiol and of [3H]progesterone in the cytosol from lungs of adult male rats was suppressible, dependent on incubation time and on protein concentration and was protein in nature. Suppressible binding of oestradiol consisted of a high affinity site (dissociation constant (Kd), 1 x 10(-9) +/- 0.2 x 10(-9) mol/l; maximum number of binding sites (Bmax), 0.7 +/- 0.2 pmol/mg protein) and a lower affinity site (Kd, 2.4 x 10(-8) +/- 0.6 x 10(-8) mol/l; Bmax, 6.3 +/- 0.4 pmol/mg protein) and showed evidence of positive co-operation. Suppressible binding of progesterone consisted of a single site with a Kd of 6 x 10(-9) +/- 1 x 10(-9) mol/l and Bmax of 44.5 +/- 8 fmol/mg protein. Binding of 125I-labelled ovine prolactin was found in homogenates of fetal lung (20 days of gestation) but not of adult lung (80 days of age). Treatment of adult rats with ovine prolactin was associated with an increase in the number of binding sites of high affinity for 125I-labelled ovine prolactin but these sites showed an altered specificity. This 'up-regulation' of the prolactin binding may provide a mechanism by which prolactin stimulates surfactant production in lung. These results, together with the known effects of these hormones on certain lung functions, provide further evidence that lung is a target organ for oestradiol, progesterone and prolactin.     

The identification of 125I-labelled iodomelatonin-binding sites in the testes and ovaries of the chicken (Gallus domesticus).

The existence of 125I-labelled iodomelatonin-binding sites in chicken ovaries and testes was investigated. The specific binding of 125I-labelled iodomelatonin to chicken ovarian and testicular tissue satisfies all the criteria for a binding site. It was rapid, stable, saturable, reversible, specific and of high affinity. Equilibrium studies showed that total and non-specific binding increased over a range of 5-150 pmol 125I-labelled iodomelatonin/l tested, with specific binding reaching saturation towards the middle range of radioligand concentrations. Scatchard analyses indicated a dissociation constant (Kd) of 36.5 +/- 5.3 pmol/l (means +/- S.E.M.) in the membrane preparations of chicken testes at the middle point in the period of light and a maximum number of binding sites (Bmax) of 0.93 +/- 0.40 fmol/mg protein (n = 6). In membrane preparations of chicken ovaries, the Kd was 102.2 +/- 27.3 pmol/l and the Bmax was 2.77 +/- 0.38 fmol/mg protein (n = 6). Equilibrium and kinetic dissociation constants in the picomolar range indicate high-affinity and physiologically relevant 125I-labelled iodomelatonin-binding sites. Competitive inhibition studies determined the following order of relative potency for inhibition of 125I-labelled iodomelatonin-binding to chicken gonadal membranes: 6-chloromelatonin greater than melatonin greater than N-acetylserotonin much much greater than 5-hydroxytryptamine, tryptamine, 5-methoxytryptophol, 1-acetylindole-3-acetic acid, 5-hydroxyindole-3-acetic acid and L-tryptophan. The presence of 125I-labelled iodomelatonin-binding sites suggests a direct pineal-gonadal connection in the chicken.     

Solubilization of a thromboxane A2/prostaglandin H2 antagonist binding site from human platelets.

A binding site for 9,11-dimethylmethano-11,12-methano-16-(3-[125I]iodo-4-hydroxyph eny l)-13,14-dihydro-13-aza-15 alpha beta-omega-tetranorthromboxane A2 ([125I]-PTA-OH), a thromboxane A2/prostaglandin H2 antagonist, was solubilized into the 200,000 X g supernatant from human platelet membranes by using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Binding to the solubilized site was saturable, displaceable, and reversible. Displaceable binding was not affected by sodium, potassium, or phosphate concentrations up to 50 mM or by magnesium to 5 mM but was increased 14% (P less than 0.05) by 5 mM calcium. A pH optimum for displaceable binding occurred between pH 7.0 and 7.5. Scatchard analysis of [125I]-PTA-OH binding to the solubilized binding site revealed a single class of sites, having a dissociation constant (Kd) of 66 +/- 16 nM (n = 3) and a Bmax of 750 +/- 80 fmol/mg of protein. The Kd for the membranes prior to solubilization was 47 +/- 11 nM (n = 3) and the Bmax was 700 +/- 90 fmol sites per mg of protein. The association rate constant, k1, was 1.57 X 10(7) M-1 X min-1 and the dissociation rate constant, k-1, was 0.61 +/- 0.04 min-1 (n = 4), yielding a Kd (k-1/k1) of 39 nM. Several thromboxane A2/prostaglandin H2 agonists and antagonists displaced bound [125I]-PTA-OH at concentrations similar to those at which they affect platelet aggregation. Collectively, these observations suggest that the solubilized protein is the thromboxane A2/prostaglandin H2 binding site that mediates platelet aggregation.     

Binding of 125I-labelled thyrotrophin to transfected human thyrocytes (SGHTL-34 cells)

The binding of 125I-labelled bovine TSH to a human thyroid cell line (SGHTL-34) has been studied. Binding to hormonally responsive cells was time dependent, specific and reversible. Scatchard analysis of the binding data indicated the presence of a single binding site with high affinity (intrinsic dissociation constant (Kd) = 0.25 +/- 0.08 nmol/l; mean +/- S.E.M.; n = 4) and low capacity (maximum binding (Bmax) = 104 +/- 29 fmol/mg protein; mean +/- S.E.M.; n = 4). Hill plots confirmed the presence of a single site. Kinetic data demonstrated close agreement between the Kd and Bmax obtained from the competition data (Kd = 0.23 +/- 0.35 nmol/l; Bmax = 161 +/- 83 fmol/mg protein; n = 6).     

Oxytocin and vasopressin: distinct receptors in myometrium

The binding characteristics of [3H]oxytocin [( 3H]OT) and [3H]lysine vasopressin [( 3H]LVP) to nonpregnant human myometrium were investigated. Binding of both radioligands was saturable, time dependent, and reversible. Whereas [3H]OT was found to bind to a single class of sites with high affinity [Kd, 1.5 +/- 0.4 (+/- SEM) nM] and low capacity [maximum binding (Bmax), 34 +/- 6 fmol/mg protein], [3H]LVP bound to two classes of sites, one with high affinity (Kd, 2.2 +/- 0.1 nM) and low capacity (Bmax, 198 +/- 7 fmol/mg protein) and another with low affinity (Kd, 655 +/- 209 nM) and high capacity (Bmax, 5794 +/- 1616 fmol/mg protein). The binding of the labeled peptides also displayed a marked difference in sensitivity to Mg2+ and guanine nucleotides. These differences in binding characteristics as well as the differences in potency of analogs in competing for [3H]OT and [3H]LVP binding indicate the presence of distinct receptors for OT and vasopressin in human myometrium. Pharmacological characterization of the high affinity binding sites for [3H]LVP indicated that these are of the V1 subtype. Although, as suggested by others, vasopressin and OT can bind to the same sites, the presence of distinct receptors for both peptides provides an explanation for the previously reported difference in myometrial responsiveness to OT and vasopressin.     

Binding characteristics of the hepatic estrogen receptor of the spotted seatrout, Cynoscion nebulosus

Estrogen receptors were identified in cytosolic and nuclear extracts of livers of adult female spotted seatrout, Cynoscion nebulosus. A single class of high affinity binding sites was found, Kd = 1.26 +/- 0.55 nM (N = 55) for the cytosolic extract and Kd = 1.96 +/- 0.42 nM (N = 8) for the nuclear extract. The Kd did not differ between males and females or between vitellogenic and nonvitellogenic females. The binding in both the cytosolic and nuclear extracts was specific for estrogens (DES greater than E2 much greater than E1 = E3). Receptor concentrations in cytosolic extracts from late vitellogenic females (14.61 +/- 1.07 pmol/g liver, N = 40) were significantly higher than those from nonvitellogenic females (3.91 +/- 0.73 pmol/g liver, N = 7). The nuclear binding capacity of livers from midvitellogenic females (1.12 +/- 0.45 pmol/g liver, N = 10) was significantly higher than the binding capacity in livers from nonvitellogenic females (0.16 +/- 0.07 pmol/g liver, N = 26), but not that of late vitellogenic females (0.80 +/- 0.09 pmol/g liver, N = 77). The concentration of estradiol-binding sites was greatest in the liver (liver much greater than ovary greater than heart greater than spleen greater than muscle greater than brain). No interference from other steroid-binding proteins was detected using a simple dextran-coated charcoal method to separate bound from free hormone. Approximately 14% of the binding in the cytosolic extract had DNA-binding affinity. Estrogen receptor binding activity was maximally extracted from nuclei with buffer containing 0.6 M KCl. Nuclear receptors eluted from gel filtration columns with an apparent molecular weight of 95 kDa.     

Identification of serotonin 5HT2 receptors in bovine pineal gland

The concentration of serotonin in the pineal gland is extremely high, which prompted speculation that in addition to serving as a precursor of melatonin, serotonin may have an independent function of its own. By using [3H]-spiperone as a ligand, and ketanserine as a selective serotonin 5HT2 receptor antagonist, we have identified 5HT2 receptor in the bovine pineal gland, revealing a single population of binding sites with a dissociation equilibrium constant (Kd) value of 1.26 +/- 0.41 nM and a receptor density (Bmax) value of 193 +/- 38.85 fmol/mg protein. In displacement experiments, the concentrations of the drugs required to inhibit 50% of the specific binding of [3H]-spiperone in descending order of potency were methysergide greater than ritanserin greater than pirenperone greater than pipamperone greater than ketanserin greater than cyproheptadine greater than M-trifluoromethylphenyl-piperazine greater than prazosin greater than 5-methoxy-N-N-dimethyltryptamine hydrogen oxalate greater than 1-(3-chlorophenol) piperazine greater than serotonin. In the rat pineal gland, [3H]-spiperone revealed a low affinity serotonin binding site with a Kd value of 25.77 +/- 10.7 nM and a Bmax value of 1244 +/- 472 fmol/mg protein. The results of these studies are interpreted to indicate that the bovine pineal gland possess serotonin 5HT2 receptor. However, the rat pineal gland possess a serotoninergic binding site of unknown nature.     

Characterization and visualization of rat and guinea pig brain kappa opioid receptors: evidence for kappa 1 and kappa 2 opioid receptors.

kappa opioid receptors (kappa receptors) have been characterized in homogenates of guinea pig and rat brain under in vitro binding conditions. kappa receptors were labeled by using the tritiated prototypic kappa opioid ethylketocyclazocine under conditions in which mu and delta opioid binding was suppressed. In the case of guinea pig brain membranes, a single population of high-affinity kappa opioid receptor sites (kappa sites; Kd = 0.66 nM, Bmax = 80 fmol/mg of protein) was observed. In contrast, in the case of rat brain, two populations of kappa sites were observed--high-affinity sites at low density (Kd = 1.0 nM, Bmax = 16 fmol/mg of protein) and low-affinity sites at high density (Kd = 13 nM, Bmax = 111 fmol/mg of protein). To test the hypothesis that the high- and low-affinity kappa sites represent two distinct kappa receptor subtypes, a series of opioids were tested for their abilities to compete for binding to the two sites. U-69,593 and Cambridge 20 selectively displaced the high-affinity kappa site in both guinea pig and rat tissue, but were inactive at the rat-brain low-affinity site. Other kappa opioid drugs, including U-50,488, ethylketocyclazocine, bremazocine, cyclazocine, and dynormphin (1-17), competed for binding to both sites, but with different rank orders of potency. Quantitative light microscopy in vitro autoradiography was used to visualize the neuroanatomical pattern of kappa receptors in rat and guinea pig brain. The distribution patterns of the two kappa receptor subtypes of rat brain were clearly different. The pattern of rat high-affinity kappa sites paralleled that of guinea pig in the caudate-putamen, mid-brain, central gray substance of cerebrum, and substantia nigra; interspecies differences were apparent throughout most of the rest of the brain. Collectively, these data provide direct evidence for the presence of two kappa receptor subtypes; the U-69,593-sensitive, high-affinity kappa 1 site predominates in guinea pig brain, and the U-69,593-insensitive, low-affinity kappa 2 site predominates in rat brain.     

Binding of sea anemone toxin to receptor sites associated with gating system of sodium channel in synaptic nerve endings in vitro.

Iodination of toxin II from the sea anemone Anemonia sulcata gives a labeled monoiododerivative that retains 80% of the original neurotoxicity. This derivative binds specifically to rat brain synaptosomes at 20 degrees C and pH 7.4 with a second-order rate constant of association ka = 4.6 x 10(4) M-1 sec-1 and a first-order rate constant of dissociation kd = 1.1 x 10(-2) sec-1. The binding occurs on the Na+ channel at a binding site distinct from that of other gating system toxins like batrachotoxin, veratridine, grayanotoxin, aconitine, and pyrethroids. The maximal binding capacity Bmax is 3.2 pmol/mg of protein (i.e., about two sea anemone toxin binding sites per tetrodotoxin binding site) and the Kd is 240 nM for the monoiododerivative and 150 nM for the native toxin. Corresponding binding parameters for the association of a 125I-labeled derivative of toxin II from the scorpion Androctonus australis Hector are Bmax = 0.3 pmol/mg of protein and Kd = 1 nM, whereas the Kd of the unmodified scorpion toxin is 0.6 nM. Competition experiments involving scorpion toxins, sea anemone toxins, and synaptosomes demonstrate that, although the sea anemone toxin is able to displace the scorpion toxin bound to synaptosomes, the scorpion toxin does not displace the sea anemone toxin. The sea anemone toxin but not the scorpion toxin binds to depolarized synaptosomes. Differences between binding properties of the two polypeptide toxins are analyzed in the discussion.     

Guanine nucleotide modulation of high affinity gonadotropin-releasing hormone receptors in rat mammary tumors.

We previously suggested that gonadotropin-releasing-hormone (GnRH) analogues activate the phosphoinositide pathway in rat mammary tumor membranes. In the present study we analyzed the binding of GnRH analogues to these membranes and assessed its modulation by guanine nucleotides. [125I]Buserelin (a GnRH superagonist) binding is specific because it is displaced only by GnRH analogues. Scatchard plot analysis reveals high affinity binding sites (Kd = 2.5 +/- 0.8 nM, Bmax = 250 +/- 120 fmol/mg membrane protein) and low affinity binding sites (Kd 1.1 +/- 0.3 microM, Bmax = 200 +/- 105 pmol/mg membrane protein). Guanine nucleotides increased the ED50 of [125I]buserelin displacement, and almost completely eliminated the high affinity binding. Similar results were obtained with [125I]D-Trp6-GnRH--another GnRH superagonist. The inhibition of buserelin binding by guanine nucleotides was specific for nucleotides that interact with G-binding proteins and was dose-dependent with a maximal effect at 10 microM GTP gamma S. Kinetic analysis of buserelin binding revealed that the dissociation rate increased at least 4-fold in the presence of 10 microM GTP gamma S. These results support the hypothesis that GnRH analogues interact directly with mammary tumors and activate a G-protein-dependent transducing mechanism.     

Localization of neuropeptide Y binding sites in the zona glomerulosa of the bovine adrenal gland

We studied neuropeptide Y (NPY) binding sites in the bovine adrenal gland by incubating tissue sections with [125I]-Bolton Hunter NPY, and then by measuring the number and affinity of binding sites in the tissue with quantitative autoradiography. Specific NPY binding sites were localized exclusively in the zona glomerulosa. These binding sites have an apparent dissociation constant (Kd) of 0.45 +/- 0.06 nM and a binding capacity (Bmax of 134 +/- 15 fmol mg-1 protein. Our results suggest that NPY may directly affect the release of aldosterone in the zona glomerulosa of the adrenal gland.     

Characteristics of 2-[125I]iodomelatonin binding sites in the pigeon spleen and modulation of binding by guanine nucleotides

Abstract: 2-[¹²⁵]Iodomelatonin binding sites in membrane preparations of pigeon spleen have been characterized. The binding was stable, saturable, reversible, and of high affinity. Rosenthal and Hill analyses showed that the radioligand-receptor interaction involved a single class of binding sites. Analysis of the binding results of spleens collected during mid-light revealed an equilibrium dissociation constant (Kd) of 36.6 ± 4.8 pmol/1 (mean ± sem, n = 10) and a maximum density (Bmax) of 2.3 ± 0.2 fmol/mg protein. There was no significant difference in the Kd (46.9 ± 5.0 pmol/1) or the Bmax values (2.4 ± 0.3 fmol/mg protein) for spleens collected during mid-dark (n = 9), although the mid-dark serum and pineal melatonin levels were significantly higher ( P < 0.05) than the corresponding mid-light values. Kinetic analysis showed a Kd of 8.6 ± 2.0 pmol/1 (n ± 4), in agreement with that derived from the saturation studies. Except for inhibition by 2-iodomelatonin, melatonin, 6-chloromelatonin, 6-hydroxymelatonin and N-acetylserotonin, the other indoles or neurotransmitters tested have little inhibition on the binding. In addition, guanosine 5'-O-(3-thiophosphate) (GTPγS), a nonhydrolysable analog of GTP, was found to inhibit the binding in a dose-dependent manner. Saturation studies revealed that this is due to a decrease in both the affinity and density of the binding sites. These data suggest that a single type of melatonin receptor is found in the pigeon spleen and that the site is coupled to a guinine nucleotide binding protein (G-protein). Our findings support a direct pineal melatonin action on the immune system.     

Interaction of amiodarone and desethylamiodarone with the cardiac muscarinic receptor in vitro

We studied the interaction of amiodarone hydrochloride (Cordarone) and its major metabolite desethylamiodarone with the muscarinic receptor in purified canine cardiac sarcolemmal vesicles by measuring equilibrium binding of the muscarinic antagonist [3H]quinuclidinyl benzilate (QNB) and carbachol displacement of [3H]-QNB. At a [3H]-QNB concentration of 0.02 nM, equilibrium binding was inhibited by amiodarone and desethylamiodarone with an IC50 of 6.86 x 10(-6) M and 2.25 x 10(-6) M, respectively. The presence of increasing concentrations of [3H]-QNB in the incubation medium was able to reverse the inhibition seen with 1 x 10(-6) M amiodarone. Scatchard analysis of [3H]-QNB saturation isotherms (37 degrees C, pH 7.4) in the presence of 1 x 10(-6) M amiodarone showed an apparent increase in equilibrium dissociation constant (Kd) over control from 0.045 +/- 0.002 nM to 0.084 +/- 0.001 nM while maximal binding capacity (Bmax) was unaffected: 10.8 +/- 1.14 and 10.5 +/- 1.48 pmol/mg (means +/- S.E.M., n = 3), respectively. The inhibitory effect of amiodarone on equilibrium binding was highly dependent on the drug:membrane phospholipid mole ratio with effects beginning at a ratio of less than 0.1:1. Hill plot analysis was consistent with the interaction of [3H]-QNB at a single site in the presence or absence of amiodarone. Amiodarone (3 x 10(-6) M) decreased the pseudo-first order forward rate constant of [3H]-QNB (0.02 nM) with the muscarinic receptor (kobs = 4.05 +/- 0.61 x 10(-4)/s under control conditions and 2.36 +/- 0.15 x 10(-4)/s in the presence of amiodarone).(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of flunitrazepam and beta-carboline high affinity binding in bovine pineal gland

High affinity binding of 3H-flunitrazepam (FNZP) to crude membrane preparations of bovine pineal membranes was examined by a rapid filtration procedure through Whatman GFB paper. At 0 degrees C binding reached equilibrium in about 20 min. Scatchard analysis of data at equilibrium revealed a single population of binding sites with dissociation constant (Kd) = 3.14 +/- 0.45 nM and binding site concentration (Bmax) = 55.6 +/- 5.6 fmol/mg protein. Kinetic analysis of the association and dissociation curves indicated a kinetic Kd = 1.13 nM, in reasonable agreement to that obtained at equilibrium. When various benzodiazepine (BZP) analogues were tested for their ability to inhibit 3H-FNZP binding, the following Ki (nM) were obtained: clonazepam (0.22), Ro 15-1788 (0.48), FNZP (0.95), Ro 5-4864 (greater than 10,000). When the beta-carboline derivative 3H-ethyl-beta-carboline-3-carboxylate ester (E beta CEE) was used as a radioligand, Kd at equilibrium (0.98 nM), kinetic Kd (1.69 nM) and affinity order for analogues were in close agreement to those found for FNZP binding; however, Bmax was about 60% that observed for 3H-FNZP binding. Addition of GABA or pentobarbital (100 microM) to pineal membranes increased 3H-FNZP binding by 55 and 72%. These results suggest the existence of a mixed population of type 1 and type 2 central BZP receptor subclass in bovine pineal gland.     

Putative melatonin receptors in the male guinea pig kidney

Abstract: The direct action of pineal melatonin on the renal system is supported by our demonstration of 2-[¹²⁵I]iodomelatonin binding sites in the male guinea pig kidney. Scatchard analyses and Hill coefficients revealed a single type of binding site with an equilibrium dissociation constant (Kd) of 22.3 ±1.6 pmol/1 and a maximum binding density (Bmax) of 0.99 ±0.03 fmol/mg protein (n = 7) at mid-light. There was no significant difference in the Kd and Bmax values between kidney tissues collected at the middle of light and dark periods. The pharmacological profile of these 2-[¹²⁵I]iodomelatonin binding sites indicated high specificity for melatonin, 2-iodomelatonin and 6-chloromelatonin while kinetic studies generated a Kd value of 28.4 ±7.3 pmol/1 (n = 5) which was comparable to that determined from Scatchard transformations. Our results suggest that these binding sites are stable, reversible, saturable, specific, and of high affinity. Regional distribution study showed that specific binding of 2-[¹²⁵I]iodomelatonin was 8-fold higher in the cortical region than that in the medullary region. Studies of subcellular distribution showed that 59.3% of binding sites were localized in crude nuclear fractions followed by crude mitochondrial fractions (22.3%) and crude microsomal fractions (18.3%) with no detectable binding in cytosolic fractions. Our present findings suggest the presence of putative melatonin receptors in the guinea pig kidney, which support the hypotheses of melatonin-regulated renin secretion together with renal excretory functions via melatonin receptors.     

Solubilization and characterization of high-affinity [3H]serotonin binding sites from bovine cortical membranes.

High-affinity [3H]serotonin binding activity has been solubilized from bovine cerebral cortical membranes by using Triton X-100, Tween-80, and octyl-beta-D-glucopyranoside. This mixture of detergents solubilizes the high-affinity [3H]serotonin binding activity present in crude membrane preparations with retention of 75-90% specific binding. The detergent mixture was chosen because it can easily be removed from the solubilized fraction by dialysis and polystyrene bead adsorption, thus permitting further purification and isolation of the binding sites. Saturation analysis reveals multiple components of high-affinity [3H]serotonin binding. In crude bovine cortical membranes, at least two binding components are present. A higher-affinity binding component, as defined from curvilinear Scatchard plots, has a Kd for [3H]serotonin of 1-3 nM, whereas a lower-affinity component has a Kd of 10-20 nM. In the solubilized preparation, only a single class of binding sites is apparent, with a Kd of 50-100 nM. Removal of detergents by dialysis and polystyrene bead adsorption results in restoration of the curvilinear Scatchard plot with apparent Kds similar to those observed in crude membrane preparations and with increased Bmax values for each component. [3H]Serotonin binding activity in the solubilized preparation is stable to Sephacryl S-300 column chromatography and to glycerol gradient sedimentation. Saturation analysis of the peak binding fractions from both these procedures once again yields curvilinear Scatchard plots, indicating that the multiple high-affinity binding components are preserved and migrate together. The molecular weight, Stokes radius, and frictional coefficient of the binding site(s) have been calculated. After detergent removal the solubilized material shows many of the characteristics usually attributed to S1 receptors, such as high affinity for [3H]serotonin and its analogs and low affinity for serotonin antagonists.     

Identification of alpha-adrenergic receptor subtypes in the steer stalk-median eminence

The presence of alpha 1- and alpha 2-receptor subtypes were studied in steer stalk-median eminence (SME) using a selective alpha 1-ligand, [3H]-prazosin, and a selective alpha 2-ligand, [3H]-clonidine. Scatchard analysis of the equilibrium isotherms of specific [3H]-prazosin binding indicated a single class of high affinity (Kd = 0.36 +/- 0.05 nM) and low capacity (Bmax = 111 +/- 19 fmol/mg protein) binding sites, which accounted for approximately 26% of the total sites identified by the non-subtype-selective-alpha-antagonist, dihydroergocryptine ([3H]-DHE). Similarly, computer modeling of the biphasic competition curve of prazosin displacing [3H]-DHE also revealed that there are approximately 26% high affinity sites, presumably alpha 1-, and approximately 74% low affinity sites, presumably alpha 2-receptors in the steer SME. A scatchard plot of specific [3H]-clonidine binding was biphasic and was resolved into a high (Kd = 1.60 +/- 0.34 nM) and low (Kd = 34.5 +/- 7.85 nM) affinity site with the binding capacities of 43 +/- 5 and 200 +/- 37 fmol/mg protein, respectively. The total number of alpha 2-sites identified by [3H]-clonidine accounted for approximately 58% of the total sites labeled by [3H]-DHE, which was in good agreement with that calculated from the competition of clonidine for [3H]-DHE binding (approximately 65% alpha 2 and approximately 35% alpha 1). The rank orders of potency of alpha-adrenergic agents to compete for sites labeled by [3H]-prazosin and [3H]-clonidine were consistent with their labeling alpha 1- and alpha 2-subtypes, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)