Estradiol binding components in intestinal mucosa of female rats.

The presence of estrogen binding components (EBC) in intestinal mucosa of female rats was investigated by competitive-binding assay using radiolabelled and nonlabelled estradiol 17 beta (E2). EBC were found exclusively in the nuclear fraction and were absent from the cytosolic and from the microsomal fractions. Two types of nuclear EBC with different binding characteristics and capacities were found: Kd1 = 4.8 +/- 0.8 nM, n1 = 18.4 +/- 4.2 fmol/mg protein (n1 = 83.4 +/- 12.5 fmol/mg DNA) and Kd2 = 31.1 +/- 6.8 nM, n2 = 91.1 +/- 18.5 fmol/mg protein (412.7 +/- 80.0 fmol/mg DNA). Type 1 component showed slightly greater affinity for estrogens as compared to progesterone and dexamethasone whereas type 2 component bound other competitors with even greater affinity than E2.     

Enhanced inhibition of estrogen receptor nuclear binding in the uterus of aged mice

We have studied why uteri from aging mice show a decrease nuclear concentration of estrogen receptors (UER). While 50-60% of available cytosolic UER from ovariectomized (OVX) mice aged 4-8 months, upon physiochemical activation, are able to bind either to DNA-cellulose or nuclear suspensions from young animals, only 20-30% of comparable concentrations of cytosolic UER from mice aged 15-18 months did so under identical experimental conditions. Nuclear dilutions with uterine cytosolic fractions from estrogen treated OVX mice prior to determination of [3H] UER binding sites in nuclear suspensions decreased the number of nuclear ER sites in both age groups. However, we observed that cytosols from aged animals showed a greater ability to prevent [3H]E2 binding to nuclear sites when compared to young ones (inhibition index: 0.286 +/- 0.013 (SE) vs. 0.137 +/- 0.025, P less than 0.05). These changes occur independently of protein concentration and result from dilution of a specific endogenous inhibitor of [3H]E2 binding to nuclear sites. The significance of these observed differences is discussed.     

Exchange assays using sodium thiocyanate to measure total nuclear content of estrogen receptors in the hypothalamic pituitary axis of mice

We studied the effect of sodium thiocyanate (NaSCN) in the determination of specific nuclear estrogen receptors from the hypothalamic-pituitary axis (HPA) of mice. We compared the results with those of the exchange assay using either suspensions of whole nuclei or KC1-nuclear extracts. Our findings demonstrated that 0.5 M NaSCN was more efficient than 0.5 M KC1 in extracting [³H] E₂ nuclear content from HPA (91% vs 79.3%). Nuclear fractions extracted with 0.5 M NaSCN revealed the presence of a single class of low-capacity-high affinity binding sites that sedimented in the 4.0 S area. Nuclear binding was T° dependent and reached maximum levels of 450 ± 156 (S.E.) fmol/mg DNA after an overnight incubation at 4°C. Such levels were comparable to those observed in whole nuclei suspensions after 1 hour incubation at 37°C (618 ± 71 fmol/mg DNA, p > 0.05) but two-fold higher (p <0.01) than the concentration of binding sites measured in KC1-extracted nuclear fractions under similar experimental conditions. We conclude that NaSCN extracted the total content of nuclear estrogen receptors in HPA of mature mice.     

Effects of chronic nicotine treatment on nicotinic receptors in the rabbit urinary bladder

Nicotine induced a phasic contraction in the rabbit urinary bladder. The response was abolished by hexamethonium and partially reduced by atropine and capsaicin. Simultaneous atropine and capsaicin treatment did not abolish the contraction. These findings suggest that the response to nicotine is due to acetylcholine, tachykinins, and unknown mediator release. In contrast, nicotine-induced contraction diminished following the chronic nicotine treatment without a change of its pharmacological properties. These results suggest the possibility that chronic nicotine treatment causes a decrease in nicotinic receptor numbers. Therefore, the binding properties of (-)-[3H]nicotine on rabbit urinary detrusor muscle membrane fractions were studied to evaluate the effects of chronic nicotine treatment on nicotinic receptors. Specific (-)-[3H]nicotine binding reached saturation and Scatchard plots were curvilinear, suggesting the existence of two different affinity sites for (-)-[3H]nicotine. Dissociation constants (KD) and maximum binding sites (Bmax) were KD1 = 4.91 +/- 1.88 nM, Bmax1 = 2.42 +/- 0.22 fmol/mg protein and KD2 = 263 +/- 56 nM, Bmax2 = 25.0 +/- 4.3 fmol/mg protein. In urinary bladder membrane fractions from chronic nicotine-treated rabbits, KD and Bmax values were KD1 = 3.96 +/- 0.38 nM, Bmax1 = 1.07 +/- 0.25 fmol/mg protein and KD2 = 249 +/- 12 nM, Bmax2 = 10.8 +/- 1.5 fmol/mg protein. Dissociation constants for both sites following chronic nicotine treatment did not change but maximum binding site numbers for both sites significantly decreased (p less than 0.05). These results suggest that the decrease in contractile response evoked by nicotine after chronic nicotine treatment in rabbit urinary bladder is due to a decrease in numbers of nicotinic receptors.     

VIP receptors in mesenteric and coronary arteries: a radioligand binding study

Using a biologically active radioligand, [Tyr(125I)10]VIP, we have identified and characterized receptors for vasoactive intestinal peptide (VIP) on membranes prepared from the rat superior mesenteric artery and bovine coronary arteries. Binding was specific, saturable, reversible and dependent on time and temperature. Scatchard analysis suggested the presence of a high and a low affinity binding site in each arterial system with the following binding constants: the rat mesenteric artery, KD = 0.22 +/- 0.02 and 13.6 +/- 7.8 nM (corresponding maximum number of binding sites, RO = 606 +/- 44 fmol/mg protein and 2.1 +/- 0.2 pmol/mg protein); bovine circumflex coronary artery, KD = 0.10 +/- 0.01 and 37.8 +/- 16.1 nM (corresponding RO = 369 +/- 65 fmol/mg protein and 2.0 +/- 0.7 pmol/mg protein); bovine left and right descending coronary arteries, KD = 0.12 +/- 0.03 and 21.3 +/- 6.4 nM (corresponding RO = 472 +/- 7 fmol/mg protein and 2.2 +/- 0.3 pmol/mg protein). The arterial VIP receptors did not recognize secretin, glucagon, apamin or bovine parathyroid hormone, and had reduced affinity for PHI, PHM and growth hormone releasing factors (GRF). These recognition properties were, by and large, similar to those seen in the bovine cerebral arteries although a between-species heterogeneity of recognition function could be deduced from the differences in the competitive binding of rat and bovine vascular VIP receptors with the corresponding species-specific GRFs.     

Estrogen receptors in ovary and uterus of immature hamster and rat: effects of estrogens

Cytosolic and nuclear estrogen receptors in the ovary and uterus of immature rats and hamsters were determined to evaluate why exogenous estrogens were ineffective in stimulating follicular maturation in the hamster compared to the rat. Animals were injected sc with oil or single injection of 1 mg estradiol cyclopentylpropionate (ECP) on Day 23 or a daily injection of 2 mg diethylstilbestrol (DES) on Days 23-25 and killed on Day 26. Total binding sites for estrogen in ovarian cytosol of control hamsters were half the number in the rat ovary (28 fmole/mg protein) and about 50% of the receptors were occupied in the hamster. The apparent affinity of the estrogen-cytosol receptor complex was also lower in the hamster (Kd; 1.41 nM) than in the rat (Kd; 0.52 nM). After ECP treatment, there was a tendency for translocation in all 4 tissues examined even though some differences were not statistically significant. However, after DES treatment both cytosol and nuclear estrogen receptors decreased in both species. This discrepancy may be due to the difference in the time course of the nuclear translocation, the difference in metabolism and difference in the binding potencies of ECP and DES. The lack of ovarian responsiveness to estrogen in the hamster thus appears to be due to the reduced number of cytosol receptor sites which have a low affinity for estrogen and are already partially occupied.     

Heterogeneity of [3H]estradiol binding sites in the rat prostate: properties and distribution of type I and type II sites

In order to assess the rat prostate as a target tissue for receptor-mediated estrogen action, we have studied the properties and distributions of estrogen binding sites in the dorsolateral (DLP) and ventral (VP) prostate. Saturation analyses over a wide range of [3H]estradiol ([3H]E2) concentrations (0.5-100 nM) revealed two distinct types of binding sites in the cytosol and nuclear fractions of DLP of intact rats. The high affinity (type I) estrogen binding sites saturated at 2-4 nM of [3H]E2 and had a capacity of 170 fmol/mg DNA in the cytosol and 400 fmol/mg DNA in the nuclei. DLP type I sites had ligand specificity similar to that described for the classical estrogen receptors (ERs) found in female target tissues. The moderate affinity (type II) estrogen binding sites saturated at 15-30 nM of [3H]E2 and had a capacity of 850 fmol/mg DNA in the cytosol and 1600 fmol/mg DNA in the nuclei. DLP type II sites shared some characteristics of the type II ERs described for the rat uterus; they were estrogen specific, heat labile, and sensitive to reducing agents such as dithiothreitol. Saturation analyses on VP cytosols and nuclear fractions revealed only high affinity sites but no moderate affinity sites in the tissue preparations. Our finding that prostatic type II estrogen binding sites are present exclusively in the DLP supports the concept that basic biological differences exist between the two major prostatic lobes of the rat. Furthermore, our findings may help elucidate the observed differences in susceptibility between these two lobes to the hormonal induction of proliferative prostatic lesions.     

Iminodipropionitrile-Induced Dyskinesia in Mice: Striatal Calcium Channel Changes and Sensitivity to Calcium Channel Antagonists

Administration of 3,3'-iminodipropionitrile (IDPN) (1 g/kg, i.p. for 3 days) in mice leads to the development of a characteristic syndrome consisting of lateral and vertical head and neck movements, hyperactivity, random circling, increased locomotor activity, and increased startle response. Nifedipine, verapamil, and diltiazem (10 mg/kg) inhibited significantly the symptoms of IDPN-induced dyskinesia. However, there was no change in the affinity (KD) or the density of PN 200-110 binding sites (Bmax) in whole brains of IDPN-treated mice. Similarly, the K(+)-depolarization-dependent Ca2+ uptake in synaptosomes from whole brain, cortex, or striatum was not altered following IDPN treatment. However, IDPN caused a significant increase in the Bmax value (from 157 +/- 7 fmol/mg to 237 +/- 31 fmol/mg in control and treated groups, respectively) of PN 200-110 binding to the striatum without change of KD value (38 +/- 4.7 pM versus 33 +/- 1.6 pM). IDPN also caused a slight but significant decrease in the KD value (from 68 +/- 10.1 pM to 45 +/- 4.5 pM in control and treated groups, respectively), without significant change of Bmax value (563 +/- 51 fmol/mg versus 485 +/- 41 fmol/mg) of PN 200-110 binding to the cortex. IDPN did not alter omega-conotoxin binding in whole brain, striatum, or cortex. The behavioral effects of chronic IDPN treatment as inhibited by L-type calcium channel antagonists and this may be associated with the observed increase in striatal L-type calcium channels.     

Characterization of Opioid Receptors in Cultured Neurons

The appearance of mu-, delta-, and kappa-opioid receptors was examined in primary cultures of embryonic rat brain. Membranes prepared from striatal, hippocampal, and hypothalamic neurons grown in dissociated cell culture each exhibited high-affinity opioid binding sites as determined by equilibrium binding of the universal opioid ligand (-)-[3H]bremazocine. The highest density of binding sites (per mg of protein) was found in membranes prepared from cultured striatal neurons (Bmax = 210 +/- 40 fmol/mg protein); this density is approximately two-thirds that of adult striatal membranes. By contrast, membranes of cultured cerebellar neurons and cultured astrocytes were devoid of opioid binding sites. The opioid receptor types expressed in cultured striatal neurons were characterized by equilibrium binding of highly selective radioligands. Scatchard analysis of binding of the mu-specific ligand [3H]D-Ala2,N-Me-Phe4,Gly-ol5-enkephalin to embryonic striatal cell membranes revealed an apparent single class of sites with an affinity (KD) of 0.4 +/- 0.1 nM and a density (Bmax) of 160 +/- 20 fmol/mg of protein. Specific binding of (-)-[3H]bremazocine under conditions in which mu- and delta-receptor binding was suppressed (kappa-receptor labeling conditions) occurred to an apparent single class of sites (KD = 2 +/- 1 nM; Bmax = 40 +/- 15 fmol/mg of protein). There was no detectable binding of the selective delta-ligand [3H]D-Pen2,D-Pen5-enkephalin. Thus, cultured striatal neurons expressed mu- and kappa-receptor sites at densities comparable to those found in vivo for embryonic rat brain, but not delta-receptors.     

Estrogen and androgen receptors in the liver of cynomolgus monkeys (Macaca fascicularis)

Estrogen receptors (ER) and androgen receptors (AR) were evaluated in the hepatic cytosol from cynomolgus macaques to determine if there were differences associated with gender and endogenous hormone secretion. Saturable, high affinity binding (Kd = 0.2-0.8 nM) was demonstrated for both ER and AR from either male or female monkeys. Displacement of tritiated estradiol from the ER was estrogen specific (including ethinyl estradiol). Both androgens and the synthetic progestins (levonorgestrel and norethindrone) displaced tritiated mibolerone from the AR. Both 8S and 4S molecular forms of ER and AR were demonstrated on 5-20% sucrose density gradients. The ER levels were higher in females in the follicular phase of the menstrual cycle (40.5 +/- 1.9 fmol/mg protein) than levels in males (26.4 +/- 4.8 fmol/mg protein; P less than 0.01) or levels in luteal phase females (31.8 +/- 2.4 fmol/mg protein; P less than 0.05). AR levels were not different between females during different phases of the menstrual cycle (65.8 +/- 4.6 and 69.5 +/- 4.3 fmol/mg protein, follicular and luteal, respectively), but there was a tendency (P less than 0.10) for the levels in males (54.4 +/- 6.6 fmol/mg protein) to be lower than female levels. The demonstration of saturable, high affinity binding of androgens and estrogens in liver tissue of these primates, along with differences associated with gender and the stage of the menstrual cycle, suggests that hepatic receptors are functional and may play an important role in hepatic protein secretion.     

Identification of α2-Adrenergic Receptors in Chicken Pineal Gland Using [3H]Rauwolscine

The norepinephrine-induced inhibition of avian pineal N-acetyltransferase activity appears to be mediated by alpha 2-adrenergic receptors. In this study, alpha 2-adrenergic receptors in the chicken pineal gland were directly identified by radioligand binding. Membrane preparations of pineal glands from chickens from 1 to 6 weeks of age were examined using [3H]rauwolscine, a selective alpha 2-adrenergic receptor antagonist, to characterize the binding sites. The results indicate no ontological change in either the affinity (KD) or density of receptor binding sites (Bmax) during the time span examined. The binding was saturable and of high affinity with a mean KD of 0.27 +/- 0.01 nM and a mean Bmax of 242 +/- 12 fmol/mg protein. Further characterization of these binding sites indicated that the alpha 2-adrenergic receptor is of the alpha 2A subtype, since prazosin and ARC-239 bound with low affinities and oxymetazoline bound with high affinity.     

Absence of specific luteinizing hormone-releasing hormone (LHRH) receptors in the ovaries of Djungarian hamsters (Phodopus sungorus)

High affinity binding sites for luteinizing hormone-releasing hormone (LHRH) were characterized in Djungarian hamsters. Scatchard analysis was used to demonstrate specific LHRH-binding in hamster and, serving as controls, rat pituitaries (dissociation constant, KD = 0.6 nM, binding capacity, BM = 2.5 +/- 0.7 fmol/mg tissue; KD = 0.6 nM, BM = 6.9 +/- 1.9 fmol/mg tissue, respectively). In contrast to results obtained with rat ovaries (KD = 0.9 nM, BM = 3.0 +/- 0.9 fmol/mg tissue), no specific LHRH-binding was detected in hamster ovaries. Thus, it seems that direct gonadal action of LHRH in the Djungarian hamster is not involved in ovarian regulation.     

Characterization of estrogen receptor in estrogen-dependent transplantable rat pituitary tumor MtT/F84

Properties of nuclear and cytosolic estrogen receptors (ERs) were examined in a new transplantable rat pituitary tumor designated as MtT/F84, of which growth is stimulated by estrogen. The optimal incubation conditions of both nuclear and cytosolic exchange were found to be at 37 degrees C for 15 min and at 25 degrees C for 2 hr, respectively. Molybdate increased a specific binding of estradiol (E2) as determined by [3H]E2-binding assay. Sucrose density gradient analyses of crude cytosol revealed specific peaks of radioactivity in both 4-5S and 8-10S areas. However, only a single 5S peak was present in 0.4M KCl-extractable nuclear ER. Molybdate also enhanced the stability of cytosolic 8-10S receptor in density gradient sedimentation behavior. Scatchard plot analysis for nuclear ER yielded a single class of binding sites with a dissociation constant (Kd) of 0.317 nM and the maximum number of binding sites (NBSmax) of 25.4 fmol/mg protein. Saturation analysis of [3H]estrogen binding to cytosolic ER also yielded a straight line with a Kd of 0.146 nM and NBSmax of 58.5 fmol/mg protein. The effect of E2 administration on the intracellular distribution of ER was also examined. A marked disappearance in the ER binding in cytosol with a concomitant increase in binding in nuclear fraction was found after the administration of the unlabeled E2 in vivo, whereas the total number of ER did not change. Thus, it is concluded that properties of ER in the MtT/F84 were very similar to those in other target organs such as uterus and pituitary gland.     

The characteristic binding of catechol estrogens to estrogen receptors in 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors

The binding of catechol estrogens (2-hydroxyestrone, 4-hydroxyestrone, 2-hydroxyestradiol, and 4-hydroxyestradiol) to estrogen receptors in 7,12-dimethylbenz(a)anthracene (DMBA)-induced rat mammary tumor cytosols was investigated. Cytosol estrogen receptors exhibited high affinities (Ka = 1.12-1.88 X 10(8) M-1) for all catechol estrogens as well as estradiol. The receptor level of catechol estrogens (46.1-97.5 fmol/mg protein) was 1.6-3.0 times higher than that of estradiol; especially the binding of 4-hydroxyestrone to estrogen receptors was the highest of all catechol estrogens and estradiol. In judging the receptor level of more than 20 fmol/mg protein to be positive, the binding of catechol estrogens to estrogen receptors was approximately correlated with that of estradiol. The positive receptor level of catechol estrogens was found in a half of tumor cytosols which showed the negative receptor level of estradiol. These results suggested that characteristic estrogen receptors indicating high affinities for catechol estrogens might be present in rat mammary tumor cytosols.     

3H]mepyramine binding to histamine H1 receptors in bovine retina

The promethazine-sensitive [3H]mepyramine binding was used to determine the presence of histamine H1 receptors in membranes from bovine retina. Specific mepyramine binding to retinal membranes was reversible, saturable and of high affinity. The apparent dissociation constant (KD = 2.2 +/- 0.4 nM) and the density of binding sites (Bmax = 60.9 +/- 5.1 fmol/mg protein), obtained in equilibrium studies, were similar to those found in bovine brain cortex. Binding was stereospecific and the inhibitory potencies of H1 and H2 antagonists indicated that [3H] mepyramine binding sites in the retina have characteristics of H1 receptors.     

Preponderance of alpha 2- over beta 1-adrenergic receptor sites in human fat cells is not predictive of the lipolytic effect of physiological catecholamines

Adrenergic control of human fat cell lipolysis is mediated by two kinds of receptor sites that are simultaneously stimulated by physiological amines. To establish a correlation between the binding characteristics of the receptor and biological functions, the ability of physiological amines to stimulate or inhibit isolated fat cell lipolysis in vitro was compared to the beta- and alpha 2-adrenoceptor properties of the same fat cell batch. The beta-selective antagonist (-)[3H]dihydroalprenolol ([3H]DHA) and the alpha 2-selective antagonists [3H]yohimbine ([3H]YOH) and [3H]rauwolscine ([3H]RAU) were used to identify and characterize the two receptor sites. Binding of each ligand was rapid, saturable, and specific. The results demonstrate 1) the weaker lipolytic effect of epinephrine compared with norepinephrine. This can be explained by the equipotency of the amines at the beta 1-sites and the higher affinity of epinephrine for alpha 2-adrenergic receptors. 2) The preponderance of alpha 2-adrenergic receptor sites labeled by [3H]YOH (Bmax, 586 +/- 95 fmol/mg protein; KD, 2.7 +/- 0.2 nM) or [3H]RAU (Bmax, 580 +/- 100 fmol/mg protein; KD, 3.7 +/- 0.1 nM). These two ligands can be successfully used to label alpha 2-adrenergic receptor sites. 3) The beta 1-adrenergic receptor population labeled by [3H]DHA(Bmax, 234 +/- 37 fmol/mg protein; KD, 1.8 +/- 0.4 nM), although a third as numerous as the alpha 2-adrenergic population, is responsible for the lipolytic effect of physiological amines and is weakly counteracted by simultaneous alpha 2-adrenergic receptor stimulation under our experimental conditions. It is concluded that, in human fat cells, the characterization of beta 1- and alpha 2-adrenergic receptors by saturation studies or kinetic analysis to determine affinity (KD) and maximal number of binding sites (Bmax) is not sufficient for an accurate characterization of the functional adrenergic receptors involved in the observed biological effect.     

Binding of 125I-labelled human chorionic gonadotropin to cell membrane receptors in rabbit ovarian slices

The binding of 125I-labelled human chorionic gonadotropin (HCG) was studied using thick slices (300 micron) of rabbit ovarian tissue. Binding was saturable, reversible, stereospecific, and of high affinity. The amount of binding was proportional to the number of slices used and could be destroyed by boiling. Ovarian slices from eight individual rabbits were found to have two binding sites for 125I-labelled HCG with KD values of 272 +/- 64 and 1263 +/- 274 pM and Bmax values of 25.7 +/- 5.3 and 94.1 +/- 18.8 fmol/mg protein, respectively. In a comparative study the KD and Bmax values were 351 +/- 151 pM and 25.3 +/- 11.1 fmol/mg protein with slices from one ovary and 134 +/- 24 pM and 109 +/- 32 fmol/mg protein with membranes from the contralateral ovary. These data suggest that the binding of HCG can be determined in live tissue.     

Thyroid hormone regulation of beta-adrenergic receptor number.

The effects of exogenous thyroid hormones (thyroxine and triiodothyronine) on beta-adrenergic receptors in the rat myocardium were investigated. The potent beta-adrenergic antagonist, (-)-[3H]dihydroalprenolol, was used to directly estimate the number and affinity of beta-adrenergic receptors in rat heart membranes from control and hyperthyroid rats. Cardiac membranes from hyperthyroid rats contained 196 +/- 7 fmol of (-)-[3H]dihydroalprenolol binding sites/mg of protein which was significantly (p less than 0.005) greater than the number of binding sites (89 +/- 5 fmol/mg of protein) present in control membranes. The equilibrium dissociation constant (KD) for the interaction of receptors with dihydroalprenolol was the same (2 to 15 nM) in membranes from control and hyperthyroid rats. Similarly, there was no significant difference between the control and hyperthyroid membranes in the affinity of the beta-adrenergic receptor binding sites for the beta-adrenergic agonist isoproterenol. The results of this study demonstrate that thyroid hormones can regulate the number of cardiac beta-adrenergic receptors. The increased numbers of receptors may be responsible, at least in part, for the enhanced catecholamine sensitivity of beta-adrenergic-coupled cardiac responses in the hyperthyroid state.     

Decreased nuclear binding of estrogen receptors in the pituitary of mice: correlation with biological effects throughout aging

We have determined the biological impact of our previously reported decreased activation of pituitary E2 receptors (PER) in mice. Young (4-8 months), middle-aged (10-14 months) and aged (15-18 months) female mice were studied either intact (metestrous) or gonadectomized (ovx) for 2 weeks prior to decapitation. Recombination studies using heat-treated cytosolic PER and enriched nuclear fractions of various age groups showed a markedly reduced ability for nuclear binding with advancing age, despite unchanged affinity of activated PER for nuclear acceptor sites. Cross incubation studies of heat-activated cytosolic PER with nuclei from mice of various age groups suggested that the activation defect followed the onset of anestrous whereas a reduced nuclear ability to support receptor binding preceded this manifestation. In parallel with these endocrine changes we observed a decrease in baseline cytosolic progesterone receptor (RcP) concentration and in the activity of the enzyme G6PDH in intact aging mice. Ovx induced a further decrease of both markers in young and middle-aged, but not in old mice, while E2 administration induced a decreased pituitary response in RcP but not in G6PDH in aged mice. Our results suggest functional changes in cytosolic nuclear interactions in the pituitary of aging female mice.     

Identification and characterization of melatonin binding sites in the gastrointestinal tract of ducks.

Utilizing 2-[125I]iodomelatonin as the radioligand, melatonin binding sites were identified and characterized in the jejunum of ducks. These sites were found to be reversible, saturable, specific and exhibited high affinity for melatonin. Scatchard analyses have established the equilibrium dissociation constant (Kd) for tissues collected during mid-photophase to be 40.9 +/- 7 pM and the maximum quantity of binding sites (Bmax) to be 2.0 +/- 0.4 fmol/mg protein while Kd of samples collected during mid-scotophase was found to be 54.1 +/- 10 pM with a corresponding Bmax of 1.5 +/- 0.3 fmol/mg protein. These Kd values are in good proximity to the kinetically derived equilibrium dissociation constant of 47.3 +/- 20 pM. No significant difference in Kd or Bmax was detected between the mid-light and mid-dark samples. Pharmacological profile of these binding sites, developed by their interactions with other indoles and compounds, indicated that these binding sites are highly specific for melatonin. Subcellularly, different densities of binding sites were localized to various fractions in the following order: nuclear greater than microsomal greater than mitochondrial greater than cytosolic. These binding sites in the jejunum might be the receptors accountable for promoting paracrine activities for the locally synthesized gastrointestinal melatonin and/or responsible for eliciting hormonal actions via interactions with melatonin of pineal origin.