Human cardiac beta-adrenergic receptors: subtype heterogeneity delineated by direct radioligand binding

Human myocardial beta-adrenergic receptors were directly identified and characterized using the high affinity antagonist radioligand [125I]iodocyanopindolol. Beta 1 and beta 2 adrenergic receptors were found to coexist in both the left ventricle and right atrium. The relative proportions of the two receptor subtypes were determined by the use of competition radioligand binding and computer modelling techniques employing the subtype selective agents atenolol (beta 1 selective) and zinterol (beta 2 selective). The left ventricle contains 86 +/- 1% beta 1 and 14 +/- 1% beta 2 adrenergic receptors while the right atrium contains 74 +/- 6% beta 1 and 26 +/- 6% beta 2 adrenergic receptors. The direct demonstration of beta 2 adrenergic receptors in the human heart, with a higher proportion in the right atrium agrees with pharmacologic data and supports the notion that chronotropic effects of adrenergic agonists in man may be mediated by both beta 1 and beta 2 adrenergic receptors.     

Endocrine aging in C57 BL mice--II. Dynamics of estrogen receptors in the hypothalamic-pituitary axis

Specific cytosolic and nuclear binding sites for estrogens were measured in the hypothalamic-pituitary axis (HPA) of young (4-8 months) and old (16-18 months) C57 BL mice in order to determine any age-related alteration in hormone-receptor interaction. Our results indicated no age differences in the affinity (KD = 0.89 +/- 0.03 (SEM) vs 1.09 +/- 0.2 X 10(-9) M), the specificity, the sedimentation profile (6 s) or in the number (98.9 +/- 4.9 vs 84.4 +/- 2.3 fmol/mg protein) of unoccupied estrogen binding sites in the cytosols. Estradiol administration to young mice induced a complete translocation of cytosolic estrogen receptors to the nucleus, and two types of nuclear binding sites were observed: Type I were specific for estrogens with high affinity (KD = 0.51 +/- 0.06 X 10(-9) M) and low binding capacity (115.1 +/- 22.7 fmol/mg DNA) and sedimented in the 4.0 s area, while Type II binding sites showed a much higher capacity and lower affinity for R2858. HPA nuclear suspensions of aged untreated mice showed undetectable (less than 50 fmol/mg DNA) levels of nuclear estrogen receptors and E2 pre-treatment resulted in a significant increase in both types of binding sites. While no significant changes in the physicochemical characteristics of these nuclear receptors were observed, when compared to young animals, aging was manifested by a translocation defect in the HPA of C57 BL mice. These results suggest aging changes in the endocrine regulating centers of the brain with defective activation of estrogen receptors.     

Identification of alpha-2 adrenergic receptors in rabbit adipocytes

Adrenaline, an alpha and beta adrenergic agonist don't modify theophylline induced lipolysis in perirenal and epididymal adipose tissue of the rabbit. Clonidine an alpha 2 adrenergic agonist inhibits theophylline stimulated lipolysis in the two tissue indicating the existence of alpha 2 adrenergic responsiveness. Direct identification of these receptors by radioligand binding studies shows that (H3) yohimbine an alpha 2 adrenergic antagonist binds to rabbit's fat cell membranes with high affinity: KD = 1.7 +/- 0.1 nmoles. The maximal number of binding sites at saturation is low 16 +/- 29 fmoles/mg protein.     

Binding of the hydrophilic beta-adrenergic antagonist [3H]CGP-12177 to cardiac tissue slices: characterization and ontogenetic studies in dogs

A new technique was developed to characterize the binding of a hydrophilic beta-adrenergic antagonist, [3H]CGP-12177, to 1-mm thick slices of canine cardiac tissue. This technique was used to quantify the density (Bmax) and the affinity (Kd) of these receptors in the right ventricular conus (RVC) and the left ventricle (LV) at day 1 to 6 weeks of age, and in the adult. Binding was found to be reversible, saturable, stereospecific, of high affinity, and thermolabile. There was an increase in the density of beta-adrenergic receptors between day 1 (Bmax = 2.2 +/- 0.3 fmol/mg tissue in RVC and 2.9 +/- 0.8 fmol/mg tissue in the LV) and 2 weeks of age postnatally, after which it remained constant until 6 weeks of age (Bmax = 7.5 +/- 0.4 and 6.8 +/- 0.9 fmol/mg tissue in RVC and LV, respectively); however, by 6 weeks of age it had not reached adult levels (10.3 +/- 1.0 fmol/mg tissue). The affinity of these receptors did not change between early neonatal life (Kd = 1.3 +/- 0.4 nM) and adulthood (Kd = 1.4 +/- 0.2 nM). The density of beta-adrenergic receptors in the RVC was similar to that in the LV. This new method of quantifying beta-adrenergic receptors in cardiac tissue is simple and fast, and requires minimal tissue handling. It proved to be useful in studying the development of cardiac beta-adrenergic receptors with age.     

Comparison of muscarinic and beta adrenergic receptors between bovine peripheral lung and tracheal smooth muscles: a striking difference in the receptor concentration

This study was undertaken to compare the activity of muscarinic and beta adrenergic receptors in bovine peripheral lung to the corresponding receptor activity in tracheal smooth muscle. We used [3H] quinuclidinyl benzilate (QNB) and [3H]dihydroalprenolol (DHA) to measure muscarinic and beta receptor activity, respectively. Binding to QNB and DHA at 25 degrees C was rapid, reversible, saturable and of high affinity. The order of potency for cholinergic and adrenergic agents competing for binding was compatible with muscarinic and beta 2 adrenergic potencies. We found that the concentration of muscarinic receptor binding sites was 37-fold greater in the tracheal muscle preparation (2805 +/- 309 fmol/mg protein) than in the peripheral lung preparation (76 +/- 28 fmol/mg protein). Unlike muscarinic receptors, the lung contained 8-fold higher concentration of the beta adrenergic receptors than did the tracheal muscle (1588 +/- 417 vs. 199 +/- 42 fmol/mg protein). The dissociation constant or the agonist's inhibitory constant (Ki) for either receptor binding site, however, was not significantly different between the two tissues. Furthermore, in vitro contraction studies showed that the response of tracheal muscle strips to methacholine was markedly greater than the response of peripheral lung strips, a finding consistent with the QNB binding result. The muscle but not the peripheral lung strip exhibited a relaxing response to epinephrine. Our data indicate a striking quantitative difference in muscarinic and beta adrenergic receptors between lung tissue and tracheal muscle, and that each receptor in the lung is qualitatively similar to the corresponding receptor in the muscle.     

125I]iodopindolol: a new beta adrenergic receptor probe

When utilizing iodohydroxybenzylpindolol (IHYP) as an adrenergic receptor probe in muscle membrane systems, the data demonstrated an unacceptably high nonspecific binding component. Bearer et al. have reported that chloramine-T induced iodination of hydroxybenzylpindolol (HYP) results in the incorporation of iodine into the indole ring rather than into the phenolic moiety as noted previously by others. These results suggest that pindolol itself can also be iodinated. Therefore, the usefulness of carrier free 125I-labeled iodopindolol (IPIN) as an adrenergic receptor probe was investigated. Using between 0.01 nM and 0.1 nM [125I]IPIN in two different muscle membrane systems, we found the nonspecific binding component to be 10% or less of total binding. When [125I]IPIN was used with membranes prepared from rat skeletal muscle, we found it to interact with a single set of high affinity binding sites (KD = 0.13 +/- 0.01 nM) with the characteristics of beta adrenergic receptors and a density of 48.5 fmoles/mg protein. IPIN binding was also studied with purified dog cardiac sarcolemma. A single set of binding sites was detected having a KD of 1.64 +/- 0.5 nM; the density of these sites was 289 fmoles/mg membrane protein. [125I]IPIN may be a useful probe for the beta adrenergic receptor of tissues in which [125I]IHYP and other beta adrenergic receptor probes have a non-specific binding component which approaches that of the specific binding component.     

Ontogeny of alpha 1- and beta-adrenergic receptors in rat lung

The binding characteristics of the alpha 1-selective adrenergic ligand [3H]-prazosin were determined in particulate membranes of rat lung from day 18 of gestation to adulthood. Specific binding was present at all ages studied, was reversible and inhibition of specific binding by agonists followed the order of potency: (-)-epinephrine = (-)-norepinephrine much greater than (-)-isoproterenol greater than (+)-norepinephrine. Inhibition by antagonists followed the order of potency: prazosin greater than WB4101, much greater than yohimbine. Binding capacity increased during the neonatal period from 52 +/- 9 fmoles x mg-1 protein in lung preparations on day 18 of a 21 day gestation increasing to 105 +/- 4 fmoles x mg-1 protein (mean +/- SE) by postnatal day 15. Binding activity decreased thereafter, reaching adult levels by 28 days of postnatal age, 62 +/- 3 fmoles x mg-1 protein. This pattern of alpha 1-adrenergic receptor density was distinct from that of beta-adrenergic receptors identified in rat lung membrane with the beta- adrenergic antagonist, (-)-[3H]dihydroalprenolol ((-)-[3H]DHA). (-)-[3H]DHA binding increased dramatically during this same time period, from 46 +/- 4 fmoles x mg-1 protein on day 18 of gestation to 496 +/- 44 fmoles x mg-1 protein in the adult lung. Affinity for [3H]-prazosin and (-)-[3H]DHA did not change with age. Pulmonary alpha 1-adrenergic receptors are present as early as 18 days of gestation in the rat and alpha 1-adrenergic receptor density is maximal by 15 days of postnatal age. The timing of the changes in alpha 1-adrenergic receptors correlates with the timing of increased sympathetic innervation of the developing rat lung and is distinct from that of beta-adrenergic receptor sites.     

Development of acetabulum after closed reduction in developmental hip dysplasia

The radiographic study of acetabulum development after closed reduction of 87 congenitally dislocated hips in 64 patients who were treated between 1980 and 1990 was conducted retrospectively. An average patient's age at the moment of closed hip reduction was 16 months (range between 6 to 24 months). On the average, patients were 16 months old (range between 6 to 24 months) at the moment of closed hip reduction. Development of bony acetabulum was followed for 8 years by measuring the AC angle. According to the age at the moment of closed reduction patients were sorted into three groups, the first group from 6 months to 12 months old (39 hips), the second group from 12 to 24 months old (28 hips) and the third group from 24 to 36 months old (20 hips). At the beginning of the treatment mean values of the AC angle in the first group were 37.2 degrees +/- 3.31 degrees; in the second group 39.7 degrees +/- 3.2 degrees; and in the third group 43.8 degrees +/- 2.7 degrees. The final values of the AC angle in the first group were 14.7 degrees +/- 3.21 degrees, in the second group 21 degrees +/- 3.02 degrees; and in the third group 24 degrees +/- 3.76 degrees. Normally and mildly dysplastic hips were achieved in 80.5% treated hips, while in 19.5% treated hips medium and serious dysplasia retarded. The acetabulum development in dysplastic hip after retained stable concentric reduction was the same as in the normal hip. The operative reconstruction of acetabulum should not be done before the third year of life.     

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.     

The effect of age on beta-adrenergic receptors and adenylate cyclase activity in rat erythrocytes.

Beta-adrenergic receptors and catecholamine-sensitive adenylate cyclase activity were studied in erythrocytes obtained from rats 6 weeks, 6 months, and 15 months of age. Intact erythrocytes from 6 week old rats contained significantly more beta receptors (411 ± 31 sites/cell) than 6 month (328 ± 21) or 15 month old rats (335 ± 16), as determined by binding of [¹²⁵I] iodohydroxybenzylpindolol. Erythrocytes from 6 week old rats also contained significantly greater isoproterenol-sensitive adenylate cyclase activity (95.0 ± 9.4pmoles/10⁹ cells) than erythrocytes from 6 month (27.9 ± 3.3) or 15 month old rats (23.7 ± 3.6). The erythrocyte population of 6 week old rats was bigger (mean corpuscular volume = 62 ± 2μ^3/cell) than the older rat erythrocytes (47 ± 1μ³ and 48 ± 1μ³). When the data were expressed relative to a unit of cell volume, there was no difference in the density of beta receptors among all three populations but a progressive and significant fall in hormone-sensitive adenylate cyclase activity. In the rat erythrocyte, the age-related loss of adenylate cyclase activity is not accompanied by changes in β-receptor density.     

BanAI a new isoschizomer of the type II restriction endonuclease HaeIII discovered in a Bacillus anthracis isolate from Amazon Basin

Steroid binding sites with high affinity for progesterone (Kd=40+/-14 nM determined by binding, and Kd=71+/-22 nM determined by displacement studies) and lower affinity for 21-hydroxyprogesterone and for testosterone, but no affinity for estradiol-17beta, onapristone and alpha-naphthoflavone were detected in the enriched plasma membrane fraction of the fungus Rhizopus nigricans. The amount of steroid binding sites is in accordance with the value of B(max)=744+/-151 fmol (mg protein)(-1). In the membrane fraction, progesterone induced about 30% activation of G proteins over basal level, as determined by GTPase activity (EC50=32+/-8 nM) and by the guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding rate (EC50=61+/-21 nM). The affinity of receptors for progesterone was substantially decreased in the presence of GTPgammaS and of cholera toxin. Our results suggest the existence of progesterone receptors in the membrane of Rhizopus nigricans and their coupling to G proteins.     

A point mutation in the seventh hydrophobic domain of the alpha 2 adrenergic receptor increases its affinity for a family of beta receptor antagonists.

Previous studies have shown that differences in subtype-specific ligand binding between alpha 2 and beta 2 adrenergic receptors are largely determined by the seventh hydrophobic domain. Here, we report that a single amino acid substitution (Phe412----Asn) in the seventh hydrophobic domain of the alpha 2 adrenergic receptor reduces affinity for the alpha 2 antagonist yohimbine by 350-fold and increases affinity for beta antagonist alprenolol by 3000-fold. The affinity of this mutant receptor alpha 2F----N for several alpha and beta adrenergic receptor agonists and antagonists was determined. Beta adrenergic receptor antagonists containing an oxygen atom linking the amino side chain with the aromatic ring bound to alpha 2F----N with high affinity, while the beta receptor antagonist sotalol, which lacks this oxygen, bound with low affinity. These data suggest that the Asn residue is involved in conferring specificity for binding to a specific class of beta receptor antagonists.     

Choline acetyltransferase activity and muscarinic binding in brain regions of aging fischer-344 rats

Muscarinic receptor binding and choline acetyltransferase (EC 2.3.1.6.) activity were assayed in three brain regions of 4-, 12- and 24-month-old Fischer-344 rats. Statistically significant age differences in cholinergic parameters were observed in each region. The affinity for [³H]quinuclidinyl benzilate increased in the cortex (24 vs 12 and 4 months), but B_max decreased in the cortex (24 vs 12 vs 4 months), striatum (24 vs 12 vs 4 months) and hippocampus (24 vs 12 and 24 vs 4). Assays of carbamylcholine inhibition of [³H]quinuclidinyl benzilate binding in the hippocampus showed that high affinity agonist binding increased with age (24 vs 12 and 4 months), and the percentage of muscarinic binding to high affinity agonist sites decreased (24 vs 12 vs 4 months). In addition, the affinity of the agonist oxotremorine for muscarinic binding sites also increased in the hippocampus (12 and 24 vs 4 months). Although the K_m of choline acetyltransferase for choline chloride did not change in any region tested, the K_m for acetyl coenzyme A decreased in the hippocampus (24 vs 12 months), but increased (4 vs 12 months) and then decreased (12 vs 24 months) in the striatum. Statistically significant age-related declines in V_max for choline acetyltransferase were noted in the striatum (24 < 12 < 4 months), but no age differences in this parameter were observed in the cortex or the hippocampus. Statistically significant positive correlations between V_max for choline acetyltransferase and B_max for [³H]quinuclidinyl benzilate binding were observed in each of the brain regions of 4-, 12- and 24-month-old rats.

The findings have implications for use of the Fischer-344 male rat as an animal model of aging and age-related disorders of the human brain, including dementia of the Alzheimer type.     

Interleukin 1 receptors on rabbit articular chondrocytes: relationship between biological activity and receptor binding kinetics

This study gives an account of the biologic and kinetic binding properties of interleukin 1 alpha (IL 1 alpha), interleukin 1 beta (IL 1 beta), and Glu-4 (an NH2-terminal mutant of IL 1 beta) to interleukin 1 (IL 1) receptors in rabbit articular chondrocytes. All three IL 1's demonstrated full agonist properties in their ability to stimulate prostaglandin E2 (PGE2) synthesis. IL 1 alpha was 23-fold more biologically active than IL 1 beta, which was around 110-fold more active than Glu-4 based on the concentration of IL 1 required for half-maximal stimulation of PGE2. The binding of all three ligands was concentration-dependent and saturable at 4 degrees C. Scatchard analysis of receptor binding data showed that the dissociation constant (KD) of IL 1 alpha was 46 +/- 12 pM, and the receptor density was 3120 sites/cell. The association of IL 1 alpha at 4 degrees C did not attain equilibrium until after 10 h at 100 pM of 125I-labeled IL 1 alpha. The dissociation of bound IL 1 alpha was very slow, t1/2 of 21 h, although only one class of high-affinity receptors was detected. The KD of IL 1 beta binding was 72 +/- 3 pM with a receptor density of 800 +/- 40 sites/cell. Dissociation of bound 125I-labeled IL 1 beta at 4 degrees C appeared to indicate the presence of two receptor subsets, a fast and a slower component with a t1/2 of 2 min and 5 h, respectively. The receptor binding affinity of Glu-4 was 324 +/- 3 pM, in line with its reduced biologic activity. Both IL 1 alpha and IL 1 beta are rapidly internalized in chondrocytes in a time- and temperature-dependent manner.     

Adrenergic receptors in the liver parenchyma in children with chronic hepatitis]

In the present study adrenergic receptors have been investigated in liver parenchyma, obtained at the resection of extrahepatic portal hypertension children without parenchymal affection (control group, n-7) and the resection of children in parenchymal affection (group of chronic hepatitis children, n-6). It has been shown, that the binding of beta-adrenergic radioligand 3H-dihydroalprenolol (3H-DHA) in liver parenchyma membranes of both control and chronic hepatitis groups was saturable and showed high affinity. The Scatchard analysis of the binding data indicated that the binding site was characterized by Kd and Bmax of 1.2 +/- 0.5 nM, 261.2 +/- 50 fmol/mg, respectively, for the control group; and 0.9 +/- 0.15 nM, 68.5 +/- 18.8 fmol/mg, respectively, for the group of chronic hepatitis patients; (mean+SEM). The binding of alpha 1-adrenergic antagonist 3H-prazosin (3H-PRZ) in liver parenchyma was also saturable and showed high affinity. The binding site is characterized by Kd = 0.6 +/- 0.12 nM, Bmax = 92.8 +/- 8.0 fmol/mg, for the control group; and Kd = 0.8 +/- 0.15 nM, Bmax = 195.0 +/- 22.0 fmol/mg, for the group of chronic hepatitis. It has been found that the number of binding sites of 3H-DHA significantly decreased and the number of binding sites of 3H-PRZ did not change in chronic hepatitis liver parenchyma in comparison with the control group. The results obtained suggest the important role of beta-adrenergic receptors in the pathogenesis of chronic hepatitis and in liver regeneration in children.     

Functional interaction between nicotinic cholinergic receptors and Na, K-ATPase in the skeletal muscles

Acetylcholine (ACh) hyperpolarized the rat diaphragm muscle fibers by 4.5 +/- 0.8 mV (K0.5 = = 36 +/- 6 nmol/l). The AC-induced hyperpolarization was blocked by d-tubocurarine and ouabain in nanomolar concentrations. This effect of ACh was not observed in cultured C2C12 muscle cells and in Xenopus oocytes with expressed embryonic mouse muscle nicotinic acetylcholine receptors (nAChR) or with neuronal alpha 4 beta 2 nAChR. In membrane preparations from the Torpedo californica electric organ, containing both nAChR and Na, K-ATPase, 10 nmol/l ouabain modulated the binding kinetics of the cholinergic ligand dansyl-C6-choline to the nAChR. These results suggest that in-sensitive alpha 2 isoform) and nAChR in a state with high affinity to Ach and d-tubocurarine may form a functional complex in which binding of ACh to nAchR is coupled to activation of the Na, K-ATPase.     

Agonist-Induced Regulation of Adrenoceptor Subtypes in Cerebral Cortical Slices

Cerebral cortical slices from rat brain were incubated at 37 degrees C for 2 h in the presence of isoproterenol, noradrenaline, or adrenaline, and binding affinities and densities of adrenoceptor subtypes were subsequently examined in homogenized tissue. The density of alpha 2- and total beta-adrenoceptors was estimated using the radioligands [3H]rauwolscine and [3H]dihydroalprenolol (DHA), respectively. The percentages of beta 1- and beta 2-adrenoceptors were defined by inhibiting the binding of [3H]DHA with the beta 1-selective antagonist metoprolol. Exposure of slices to noradrenaline and adrenaline significantly decreased the maximal number of binding sites (Bmax) of alpha 2-adrenoceptors (48 and 37% respectively) without significantly affecting affinity; isoproterenol had no effect. Exposure to isoproterenol, noradrenaline, and adrenaline significantly decreased the Bmax of beta-adrenoceptors (by 60, 34, and 24%, respectively) but did not affect the affinity. Isoproterenol and adrenaline significantly decreased the density of beta 1-adrenoceptors by 75 and 24% and beta 2-adrenoceptors by 23 and 28%, respectively. Noradrenaline significantly decreased the density of beta 1-adrenoceptors by 42% without affecting the number of beta 2-adrenoceptors. These findings indicate that subtypes of adrenoceptors in rat cerebral cortex are differentially regulated by adrenergic agonists.     

Emergence of beta adrenergic-responsive hepatic glycogenolysis in male rats during post-maturational aging

Adrenergic-stimulated glycogenolysis (estimated as glucose output) was determined in hepatocytes from 7, 14, 20, and 24 mo old male Fischer 344 rats. Glucose output in response to the beta adrenergic agonist isoproterenol was minimal at 7 mo but increased progressively with increasing age. At all ages the isoproterenol response was concentration dependent and was inhibited by the beta adrenergic antagonist propranolol. Stimulation of glucose output by the mixed alpha-beta agonist epinephrine also increased between 7 and 24 mo. Glycogenolytic responses to alpha agonist (assessed in the presence of epinephrine and excess beta antagonist), glucagon, and forskolin did not increase substantially with age and at 24 mo were less than the response to beta agonist. In hepatocyte homogenates adenylate cyclase activation by beta agonist but not glucagon and forskolin increased between 7 and 24 mo. These results suggest that adrenergic stimulation of glycogenolysis, which in young adult male rats is generally attributed to alpha adrenergic-mediated processes, becomes mediated predominantly by beta adrenergic-responsive adenylate cyclase during post-maturational aging.