Direct studies of beta-adrenergic receptors intact frog erythrocytes.

(?) [³H]Dihydroalprenolol, a potent competitive β-adrenergic antagonist can be used to directly study β-adrenergic receptors by ligand binding techniques in an intact cell system, the frog erythrocyte. At 37°, binding reached equilibrium within 1 minute. Upon addition of excess unlabeled propranolol, complete dissociation of receptor bound ligand occurred within 1 minute. The characteristics of (?) [³Hdihydroalprenolol binding to β-adrenergic receptors in intact cells were quite similar to those previously demonstrated with isolated membrane fractions. The equilibrium dissociation constant for (?) [³H]dihydroalprenolol was 1.5 nM. Order of potency of agonists and antagonists in competing for the binding sites was appropriate for the β-adrenergic receptor as was the stereospecificity of binding ((?) isomers more potent than (+) isomers). Saturation studies with these intact cells indicated 1700 binding sites/cell in excellent agreement with the number previously estimated from membrane studies. Preincubation of cells with 10^?5M isoproterenol produced a 36% fall in number of β-adrenergic receptors. It is concluded that (?) [³H]dihydroalprenolol can be used to directly study the properties and regulation of β-adrenergic receptors in intact cell as well as broken cell preparations.     

Antagonism of alpha- and beta-adrenergic-mediated accumulations of cyclic AMP in rat cerebral cortical slices by the beta-antagonist (-)alprenolol.

(?)Alprenolol, a compound reported to bind with a high degree of specificity and stereoselectivity to β-adrenergic receptors from rat cerebral cortex completely inhibited the accumulations of cyclic AMP elicited by maximally effective concentrations of norepinephrine and epinephrine at antagonist concentrations as low as 10^?5M. Other β-adrenergic antagonists such as (?)propranolol, (±)sotalol, and (+)alprenolol only partially antagonized accumulations of cyclic AMP elicited by these catecholamines even at 10-fold higher concentrations. α-Adrenergic antagonists such as phentolamine, phenoxybenzamine and clonidine only partially antagonized inhibited the accumulation of cyclic AMP elicited by methoxamine, a compound shown to stimulate the accumulation of cyclic AMP by interaction with α-adrenergic receptors. The results indicate that in brain tissue containing a mixed population of α- and β- adrenergic linked cyclic AMP generating systems, (?)alprenolol does not exhibit absolute specificity for β-receptors.     

Regulation of alpha- and beta-adrenergic receptor subclasses by gonadal steroids in human myometrium

Both alpha- and beta-adrenergic receptors have been identified in the human myometrium by radioligand binding. Both adrenergic receptor subclasses have been shown to mediate the contractile response of the uterus upon catecholamine stimulation: alpha-adrenergic receptors cause uterine contraction while beta-adrenergic receptors induce relaxation. We have identified alpha 1- and alpha 2-adrenergic receptors in myometrial membranes using the newly developed radiolabelled specific antagonists [3H]-prazosin and [3H]-rauwolscine. This enabled us to characterize both receptor subclasses individually. Beta adrenergic receptors were identified using the radiolabelled antagonist (-)-[3H]-dihydroalprenolol. Binding of radioligands to the myometrial membrane receptors was rapid, readily reversible, of high affinity and stereoselective. The total number of alpha 1-, alpha 2- and beta-receptors was determined by Scatchard analysis of radioligand saturation binding and the beta/beta 2-receptor ratio was determined by computer analysis of the beta 2-selective antagonist ICI 118 551) (-)-[3H]-dihydroalprenolol competition binding curves. This enabled us to study the regulation of both alpha- and beta-receptor subclasses under various physiological and pharmacological conditions in the human, i.e., during different phases of the menstrual cycle, in postmenopausal women and during depo-progestin (Medroxyprogesterone acetate) therapy. Only the alpha 2- and beta 1-adrenergic receptor concentrations were found to be subjected to gonadal steroid regulation. The number of alpha 2- and beta 1-adrenergic receptors increased concomitantly with circulating plasma oestradiol levels. This effect was counteracted by progesterone. The number of alpha 1- and beta 2-adrenergic receptors was unaffected by the gonadal steroid environment. These results are an example of the heteroregulation of membrane receptors by oestrogens and progesterone and cast new light on the regulatory mechanisms involved in uterine contractility in the human.     

Catecholamine receptors and cyclic AMP formation in the central nervous system: effects of tetrahydroisoquinoline derivatives.

The ability of a series of tetrahydroisoquinoline (THIQ) alkaloids to inhibit the binding of radioligands to catecholamine receptors in the CNS has been examined. $\text{(+)}$ THP was the most potent inhibitor of [³H] dihydroalprenolol binding to β-adrenergic receptors and of [³H] haloperidol to dopaminergic receptors and was the least potent inhibitor of [³H] WB-4101 binding to α-adrenergic receptors. Other THIQ alkaloids examined such as salsoline, salsolinol, and reticuline were less potent than $\text{(+)}$ THP in inhibiting radioligand binding to β-adrenergic and dopaminergic receptors, and more potent than $\text{(+)}$ THP in inhibiting radioligand to α-adrenergic receptors. The marked potency of $\text{(+)}$ THP in inhibiting radioligand binding to β-adrenergic receptors (IC₅₀ ～ 10^?7 M) was confirmed by the potency of this compound in inhibiting (?) isoproternol elicited accumulations of cyclic AMP in brain slice preparations. These data indicate that, if formed $\text{in}$$\text{vivo}$ during alcohol consumption, THIQ derivatives such as THP may affect catecholamine neurons in the CNS.     

Dissociation of beta-adrenergic receptors from hormone responsiveness during maturation of the rat reticulocyte

Intact rat erythrocytes and reticulocytes have been studied in relation to their concentration of β-adrenergic receptors and their responsiveness to β-adrenergic catecholamines. Characteristics of the β-receptor, as determined by binding of ¹²⁵I-labelled hydroxybenzylpindolol, were compared among control erythrocytes and reticulocytes. The dissociation constant (K_d=0.1?0.2 nM), association and dissociation kinetics, and stereospecificity for (?)-isomers of agonists and antagonists were similar in both cell types. The reticulocyte population contained four times more receptors per cell than the control erythrocytes. However, reticulocytes were 25 times more responsive than control cells to isoproterenol, as measured by the formation of cyclic AMP. After peak reticulocytosis, cells rapidly lost 95% of their maximum hormone responsiveness, but β-receptors declined much more slowly. The 4-fold decrease in β-receptors was associated with a 4-fold decrease in cell volume as the reticulocytes matured. The density of β-receptors was unchanged. However, responsiveness to isoproterenol in the reticulocytes when expressed on the basis of cell volume was still nine times greater than the control cells. Thus, maturation of reticulocytes is associated with an uncoupling of persistent β-receptors from catecholamine responsiveness.     

Beta-adrenergic receptors: stereospecificity and lack of affinity for catechols

Studies of adenylate cyclase activity in rat liver, heart and fat cell microsomal preparations and in turkey and rat erythrocyte ghosts indicate that β-adrenergic receptors exhibit very strict stereospecificity for (?)-catecholamines. (+)-Isomers of active catecholamines and inactive catechol compounds do not inhibit the β-adrenergic-mediated stimulation of adenylate cyclase and thus do not interact with specific receptors. However, very high concentrations (above 10^?4 M) of (?)- and (+)-isomers, as well as of biologically inactive non-catecholamine catechols (e.g., pyrocatechol, dihydroxymandelic acid), inhibit in a nonspecific manner the basal, hormone (catecholamine, glucagon)- and NaF-stimulated adenylate cyclase activity. Studies with propranolol suggest that the low activity (0.1 to 1%) of (+)-isomers of norepinephrine can be explained by contamination with the (?)-isomer. The activity of soterenol, a potent non-catechol β-adrenergic agonist, is uninfluenced by (+)-catecholamines or catechols. It is concluded that the binding of ³H-labeled catecholamines to a variety of cells, microsomes and membranes as described in various previous studies cannot represent specific receptor interactions. Binding to receptors must demonstrate strict stereospecificity and must not be affected by unrelated catechol substances.     

Beta-adrenergic receptors: evidence for negative cooperativity.

The specific binding of [³H] (?)alprenolol to sites in frog erythrocyte membranes provides a tool for directly assessing ligand binding to adenylatecyclase coupled β-adrenergic receptors. Hill Plots of such binding data yield slopes (n_H=“Hill Coefficients”) less than 1.0, suggesting that negatively cooperative interactions among the β-adrenergic receptors may occur. The existence of such negative cooperativity was confirmed by a direct kinetic method. The dissociation of receptor bound [³H] (?)alprenolol was studied under two conditions: 1) with dilution of the ligand-receptor complex sufficient to prevent rebinding of the dissociated tracer and 2) with this same dilution in the presence of excess unlabeled (?)alprenolol. If the sites are independent, the dissociation rates must be the same in both cases. However, the presence of (?)alprenolol increases the rate of [³H] (?)alprenolol dissociation, indicating that negatively cooperative interactions among the β-adrenergic receptor binding sites do occur.     

White fat cell alpha-adrenergic receptors and responsiveness in altered thyroid status

[³H]-Dihydroergocryptine was used to identify α-adrenergic receptors in a crude adipocyte membrane fraction obtained from hyperthyroid, hypothyroid and euthyroid hamsters. Hyperthyroidism produced a 35–45 % decrease in the number of both the high and the low affinity [³H]-dihydroergocryptine binding sites but failed to affect the respective affinities of both these sites. On the other hand, binding of [³H]-dihydroergocryptine was unaltered in membranes of hypothyroid animals. Hamster adipocyte α-adrenergic responsiveness, reflected by the increment of epinephrine-stimulated cyclic AMP synthesis induced by phentolamine, was 50 % reduced by hyperthyroidism but unchanged by hypothyroidism. These results which demonstrate that thyroid hormones can regulate the density of adipocyte α-adrenergic receptors, suggest that in human fat cells where catecholamines produce opposite α- and β-adrenergic effects on lipolysis, the increased α-adrenergic responsiveness found in hyperthyroidism could be due, at least in part, to a reduction in the number of α-adrenergic receptors.     

Are α-adrenergic antagonists potent tocolytics? In vivo experiments on postpartum rats

The aim of this study was to investigate the effects of α-adrenergic antagonists on the motor activity of the postpartum uterus of the rat in vivo. Intrauterine pressure was assessed by means of a Millar catheter fitted with a latex microballoon. Some of the tested compounds (urapidil, yohimbine, phentolamine, benoxathian and prazosin) decreased the uterine activity to a significant extent (57.4–67.4%). However, none of the investigated α-receptor blockers exerted the same effect as β-adrenergic agonists. Our results suggest that α-adrenergic antagonists could possibly be used as an alternative to β-adrenergic agonists in clinical tocolysis after an appropriate clinical evaluation.     

The Mammalian β-Adrenergic Receptor: Structural and Functional Characterization of the Carbohydrate Moiety

Abstract

Mammalian β-adrenergic receptors are glycoproteins consisting of a single polypeptide chain of M_r ～64,000. Treatment of purified [¹²⁵I]-labeled hamster lung β-adrenergic receptor with α-mannosi-dase reveals two discrete populations of receptor consistent with previous studies using membrane bound photoaffinity-labeled receptor. Treatment of the [¹²⁵I]-labeled receptor with endo-glycosidase F results initially in the formation of a M_r ～57,000 peptide which is further converted to M_r ～49,000 suggesting that there are two N-linked carbohydrate chains per receptor polypeptide. Exoglycosidase treatments and lectin chromatography of the [¹²⁵I]-labeled receptor reveals the presence of two complex type carbohydrate chains (～10% of which are fucosylated) on ～45% of the receptors. The remaining ～55% of the receptors appear to contain a mixture of carbohydrate chains (possibly high mannose, hybrid and complex type chains). Deglycosylation of the receptor by endoglycosidase F does not appear to alter the binding affinity of the receptor for a variety of β-adrenergic agonists and antagonists. Moreover, the ability of control, α-mannosidase sensitive or insensitive (fractionated on immobilized wheat germ agglutinin) and neuraminidase, α-mannosidase or endoglycosidase F treated receptors to interact with the stimulatory guanine nucleo-tide regulatory protein in a reconstituted system were virtually identical. The deglycosylated receptor was also unaltered in its heat lability as well as its susceptibility to a variety of proteases. These findings demonstrate that the carbohydrate portion of the β-receptor does not contribute to determining either its specificity of ligand binding or coupling to the adenylate cyclase system.     

Stereospecific (3H)(minus)-alprenolol binding sites, beta-adrenergic receptors and adenylate cyclase

The binding of [³H](?)-alprenolol (a potent β-adrenergic antagonist) to sites in frog erythrocyte membranes has been studied by a centrifugal assay. The specificity of the binding sites is strikingly similar to what might be expected of the β-adrenergic receptor binding sites which mediate stimulation of adenylate cyclase by catecholamines in these membranes. The sites bind β-adrenergic antagonists and agonists with affinities which are directly related to their antagonist or agonist potency on the frog erythrocyte membrane adenylate cyclase. Binding shows strict stereospecificity with (?)-isomers exhibiting two orders of magnitude higher affinities than (+)-isomers. Dissociation constants for potent β-adrenergic antagonists are in the range of 10^?9 – 10^?8M whereas those for β-adrenergic agonists are about two orders of magnitude higher (≥ 10^?6M).     

Beta-adrenoceptors of human lymphocytes: decrease in affinity of agonist for (-)3h-dihydroalprenolol binding sites by cytoplasmic fractions

The beta-adrenoceptors of intact human lymphocytes were investigated by binding assays with a radiolabeled β-adrenergic antagonists, (?)³H-dihydroalprenolol (DHA). Results shown are 1.48 ± 0.57 × 10⁴ binding sites for the ligand per cell. Binding of (?)³H-DHA to the membrane fractions of human lymphocytes was studied for comparison. The affinity of (?)³H-DHA for membrane was similar to that for whole lymphocytes, but the binding to the membranes was inhibited more strongly by β-adrenergic agonist. Moreover, the cytoplasmic fraction of human lymphocytes decreased the inhibition by (?)-isoproterenol of (?)³H-DHA binding to the membrane fraction. The results imply that the cytoplasmic fraction of human lymphocytes lowered the affinity of (?)-isoproterenol to (?)³H-DHA binding sites and left no effect on the binding of the β-adrenergic antagonist to the sites.     

Quantitation of Beta Adrenergic Receptors in Rat Liver: Confounding Effect of Displaceable But Nonstereospecific Antagonist Binding

Abstract

In rat liver membranes three types of ligand binding were seen using [³H]-dihydroalprenolol (DHA) and [¹²⁵I]-hydroxybenzylpindolol (HYP): binding stereospecifically displaced by β-adrenergic agonists and antagonists, binding nonstereospecifically displaced by β-adrenergic antagonists, and binding which was not displaced by β-adrenergic agonists or antagonists.

The magnitude of the nonstereospecific displaceable binding varied with the physiological state of the animal. It was sufficient to prevent the quantitation of the stereospecific displaceable binding in some preparations from young rats but in all preparations of rats greater than 150 g or more than about 6 weeks of age. In adrenalectomized weanling rats 10–30% of the total binding was of nonstereospecific displaceable type while in control rats it comprised up to 60% of the total binding. Addition of 5 X 10^-6 M phentolamine to the assay eliminated a large proportion of the nonstereospecific displaceable binding. When phentolamine was included in the assay, liver membranes from weanling rats stereo-specifically bound 30–35% of total binding; membranes from adrenalectomized rats showed stereospecific binding of up to 50 to 80%.

Because the amount of displaceable, nonstereospecific binding varied greatly depending on the physiologic state of the animals, stereospecific displacement should be monitored for every type of liver membrane preparation. Furthermore, animal age is an important variable. Using the published antagonist binding methodology (DHA or HYP) in liver membranes, it is not presently possible to quantitate liver β-adrenergic receptors in normal rats that have reached maturity.     

β-Adrenergic receptors in innervated and denervated skeletal muscle

In order to determine if the development of β-adrenergic receptors may explain the catecholamine evoked contracture of denervated mammalian skeletal muscle, the binding capacities and dissociation constants of β-adrenergic receptors of innervated and denervated rat skeletal muscle membrane preparations were determined by using [³H] dihydroalprenolol. The dissociation constants of [³H] dihydroalprenolol binding to innervated and denervated muscle microsomal suspensions were similar. The maximal number of binding sites increased from 27 pmol/g protein to 85 pmol/g protein following 25 days denervation. These results suggest that motor nerve may be involved in part, in the regulation of β-adrenergic receptors in skeletal muscle membrane preparations.     

Properties of β-adrenergic receptors in untreated and butyrate-threated HeLa cells

HeLa cells contain receptors on their surface which are β-adrenergic in nature. The binding of (?)-[³H]dihydroalprenolol is rapid, reversible, stereo-specific and of relatively high affinity. The HeLa cells also contain an adenylate cyclase which is activated by (?)-isoproterenol > (?)-epinephrine > (?)-norepinephrine. The adenylate cyclase of HeLa is also activated by guanyl-5′-yl-imidodophosphate (Gpp(NH)p), a nonhydrolyzable analogue of GTP. Inclusion of both (?)-isoproterenol and Gpp(NH)p leads to approximately additive rathen than synergistic activation of adenylate cyclase. After treatment of HeLa cells with 5 mM sodium butyrate there is an increase in the number of β-adrenergic receptors, but not in their affinity, which is reflected in an increased ability of (?)-isoproterenol to activate adenylate cyclase. Other properties of the β-adrenergic receptor including association and dissociation rates, temperature optimum of adenylate cyclase and response to Gpp(NH)p are relatively unaffected by butyrate pretreatment of the cells.     

Adenylate cyclase coupled beta adrenergic receptors of Rauscher erythroleukemia cells

Rauscher murine erythroleukemia cells undergo erythroid differentiation in response to the hormonal inducer erythropoietin and to synthetic inducers. Incubation of these cells with (?) isoproterenol or (?) epinephrine results in a 2–3-fold increase in cyclic AMP levels. This effect is completely blocked by propranolol, identifying the response as due to a β-adrenergic receptor·adenylate cyclase system. Experiments with [¹²⁵I]iodohydroxybenzylpindolol demonstrate 1700 β-adrenergic receptors/cell with a K_D of 320 pM. These results form the basis for further studies of the β-adrenergic receptor·adenylate cyclase complex during erythropoiesis.     

Endogenous and Exogenous Regulation of Human α- and β-Adrenergic Receptors

Abstract

Regulation of human β2-adrenergic receptors in lymphocytes (determined by (±)-125 iodocyanopindolol (ICYP) binding) and α₂-adrenergic receptors in platelets (determined by ³H-yohimbine binding) was studied. While α₂-adrenergic receptor number did not change with age, a significant negative correlation between the number of α₂-adrenergic receptors and age was found; plasma catecholamines, on the contrary, were elevated in the elderly.In healthy women during normal menstrual cycle the number of α₂-adrenergic receptors decreased with increasing plasma estradiol levels.Incubation of lymphocyte membranes with isoprenaline (100 μM) and of platelet membranes with clonidine (1-100 μM) led to a reduction of the number of β₂- and α₂-receptors, respectively, without changes in the K_D-values. Treatment of hypertensive patients with clonidine (3x150 μg/die) for 7 days reduced the number of α₂-adrenergic receptors in platelets. In platelet membranes from such treated patients inhibition of ³H-yohimbine binding by clonidine and adrenaline was not affected by 10-⁴MGTP. It is concluded, that human α- and β-adrenergic receptors undergo regulatory mechanisms similar to those recently described for adrenergic receptors in a variety of animal models.     

Alpha-adrenergic receptors on human platelets.

[³H] dihydroergocyrptine, an α-adrenergic antagonist, binds specifically to sites on human platelet membranes. Prostaglandin E₁ (PGE₁) stimulates the production of cyclic AMP (cAMP) in human platelets. Alpha-adrenergic agonists, 1-epinephrine and 1-norepinephrine, and antagonists, phentolamine, phenoxybenzamine, and dihydroergocyrptine inhibit the binding of [³H] dihydroergocryptine. The α-adrenergic agonists inhibit PGE₁-stimulated cAMP production and the α-adrenergic antagonists phentolamine and dihydroergocryptine reverse this inhibition. The β-adrenergic agonist 1-isoproterenol and the β-adrenergic antagonists d1-propranolol and 1-alprenolol do not significantly alter binding or PGE₁-stimulated cAMP production. Clonidine, dopamine, and serotonin inhibit binding, but clonidine and dopamine are weak inhibitors of PGE₁-stimulated cAMP production, and serotonin is without effect. Tyramine, an amine without direct adrenergic activity fails to inhibit binding. Alpha-adrenergic agonists decrease the apparent affinity of a PGE₁-receptor activating cAMP production. The inhibition of PGE₁-stimulated cAMP production is a physiological measure of α-adrenergic agonist binding to the α-receptor.     

Characteristics of β-adrenergic receptors in longitudinal muscle membranes of rat uterus: Changes in kinetic properties of the receptor during gestation

Binding characteristics of β-adrenergic receptors of longitudinal muscle membranes isolated from different stages of pregnant rat myometrium were investigated using [³H]dihydroalprenolol. Between Days 15 and 21 of gestation, the ratio of β₁- and β₂-adrenergic receptors of longitudinal membranes was constant. The membranes were found to be predominant in β₂-adrenergic receptors. The concentration of longitudinal muscle β-adrenergic receptors increased significantly during the last 7 days of gestation. Kinetic binding studies implied that the affinity of the membrane β-adrenergic receptors decreased through a slight decrease in the association rate and a large increase in the dissociation rate with progression of pregnancy. A Scatchard plot indicated that longitudinal muscle in β-adrenergic receptors on Days 15 and 18 constitute a single class of independent sites. By contrast, the dissociation kinetics, the convex downward curvature in a Scatchard plot and a Hill coefficient (h) of less than 1.00 of [³H] DHA binding to β-receptors of muscle on Day 21 suggested the existence of negatively cooperative multiple binding sites for β-adrenergic ligand. These results suggest that changes in the dynamics of uterus β-adrenergic receptors play an important role in the onset of labor.