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.     

Biochemical Characterization of α-Adrenergic Receptors in Human Brain and Changes in Alzheimer-Type Dementia

Abstract Using ligand binding techniques, we studied α-adrenergic receptors in brains obtained at autopsy from seven histologically normal controls and seven patients with histopathologically verified Alzheimer-type dementia (ATD). Binding of the α-adrenergic antagonists [³H]prazosin and [³H]yohimbine to membranes of human brains exhibited characteristics compatible with α₁- and α₂-adrenergic receptors, respectively. Binding of both ligands was saturable and reversible, with dissociation constants of 0.15 nM for [³H]prazosin and 5.5 nM for [³H]yohimbine. [³H]Prazosin binding was highest in the hippocampus and frontal cortex and lowest in the caudate and putamen in the control brains. [³H]Yohimbine binding was highest in the nucleus basalis of Meynert (NbM) and frontal cortex and lowest in the caudate and cerebellar hemisphere in the control brains. Compared with values for the controls, [³H]prazosin binding sites were significantly reduced in number in the hippocampus and cerebellar hemisphere, and [³H]yohimbine binding sites were significantly reduced in number in the NbM in the ATD brains. These results suggest that α₁ and α₂-adrenergic receptors are present in the human brain and that there are significant changes in numbers of both receptors in selected regions in patients with ATD.     

Down regulation of enkephalin (δ) receptors Demonstration in membrane-bound and solubilized receptors

The pentapeptide leucine enkephalin induced down-regulation of enkephalin receptors in neuroblastoma-glioma NG108-15 hybrid cells in a reversible fashion, whereas the stable enkephalin analogue, d-Ala²-Met-enkephalinamide (AMEA), and the potent opiate alkaloid, etorphine, had a prolonged effect. The opiate alkaloid, morphine, which has low affinity to δ-type enkephalin receptors of these cells did not induce down-regulation, whereas AMEA decreased the binding of both opiate agonists and antagonists but had no effect on the binding of the α₂-adrenergic ligand, [³H]yohimbine. From several experiments that were designed to remove the tightly bound AMEA, and from experiments with solubilized receptor we ruled out the possibility that the decreased binding capacity of enkephalin-treated cells reflects only receptor masking. The study suggests that down-regulation of enkephalin receptors that may also occur in vivo can account for some of the abnormal physiological responses of subjects treated chromically with opiates. However, since opiates from the morphine type can induce opiate tolerance in vivo, but not down-regulation of enkephalin receptors in the cultured cells, we suggest that down-regulation of δ-type opiate receptors may not be prerequisite for the development of the physiological tolerance/dependence on these alkaloids.     

3H]Para-amino-clonidine: a novel ligand which binds with high affinity to alpha-adrenergic receptors.

Para-amino-clonidine (PAC) is an α-adrenergic agonist with extraordinarily high potency in some peripheral tissues. We have demonstrated the labeling of α-adrenergic binding sites in central and peripheral tissues with [³H]PAC and compared properties of this binding to those of [³H]clonidine. [³H]PAC binds saturably with a dissociation constant (K_D) of about 0.9 nM to rat cerebral cortex membranes. It has about 2–3 times the affinity of [³H]clonidine for α-receptor binding sites. The greater affinity is attributable mainly to a slower dissociation of [³H]PAC than [³H]clonidine from binding sites. The relative and absolute potencies of various adrenergic agonists and antagonists in competing for [³H]PAC and [³H]clonidine binding are essentially the same. [³H]PAC can also be utilized to label α-adrenergic binding sites in the kidney and spleen where the relative potencies of PAC and clonidine are the same as in the brain.     

Characterization of binding of [3H]PD 128907, A selective Dopamine D3 receptor agonist ligand,to CHO-K1 cells

PD 128907 [4a R, 10 b R-(+)-trans- 3, 4, 4a, 10 b - tetrahydro - 4- n-propy12 H,5H-[1] benzopyrano[4,3-b]1,4-oxazin-9-ol.], a selective dopamine (DA) D3 receptor agonist ligand exhibits about a 1000-fold selectivity for human D3 receptors (K_i, 1 nM) versus human D₂ receptors (K_i, 1183 nM) and a 10000-fold selectivity versus human D₄ receptors (K_i, 7000 nM) using [³H]spiperone as the radioligand in CHO-K1-cells. Studies with [³H]PD 128907, showed saturable, high affinity binding to human D₃ receptors expressed in CHO-K1 cells (CHO-K1-D₃) with an equilibrium dissociation constant (K_d) of 0.99 nM and a binding density (B_max) of 475 fmol/mg protein. Under the same conditions, there was no significant specific binding in CHO-K1-cells expressing human D₂ receptors (CHO-K1-D₂). The rank order of potency for inhibition of [³H]PD 128907 binding with reference DA agents was consistent with reported values for D₃ receptors. These results indicate that [³H]PD 128907 is a new, highly selective D₃ receptor ligand with high specific activity, high specific binding and low non-specific binding and therefore should be useful for further characterizing the DA D₃ receptors.     

Comparative molecular field analysis of fenoterol derivatives interacting with an agonist-stabilized form of the β2-adrenergic receptor

The β₂-adrenergic receptor (β₂-AR) agonist [³H]-(R,R′)-methoxyfenoterol was employed as the marker ligand in displacement studies measuring the binding affinities (K_i values) of the stereoisomers of a series of 4′-methoxyfenoterol analogs in which the length of the alkyl substituent at α′ position was varied from 0 to 3 carbon atoms. The binding affinities of the compounds were additionally determined using the inverse agonist [³H]-CGP-12177 as the marker ligand and the ability of the compounds to stimulate cAMP accumulation, measured as EC₅₀ values, were determined in HEK293 cells expressing the β₂-AR. The data indicate that the highest binding affinities and functional activities were produced by methyl and ethyl substituents at the α′ position. The results also indicate that the K_i values obtained using [³H]-(R,R′)-methoxyfenoterol as the marker ligand modeled the EC₅₀ values obtained from cAMP stimulation better than the data obtained using [³H]-CGP-12177 as the marker ligand. The data from this study was combined with data from previous studies and processed using the Comparative Molecular Field Analysis approach to produce a CoMFA model reflecting the binding to the β₂-AR conformation probed by [³H]-(R,R′)-4′-methoxyfenoterol. The CoMFA model of the agonist-stabilized β₂-AR suggests that the binding of the fenoterol analogs to an agonist-stabilized conformation of the β₂-AR is governed to a greater extend by steric effects than binding to the [³H]-CGP-12177-stabilized conformation(s) in which electrostatic interactions play a more predominate role.     

Central neurotransmitter receptors in hypertensive rats

Muscarinic cholinergic ([³H]QNB), α₁ ? ([³H]WB-4101), and α₂ ? ([³H]clonidine) adrenergic ligand binding was measured in various regions of the brains of adult normotensive, spontaneously hypertensive, and DOCA-salt hypertensive rats. There was a 66% increase in the number of α₁-adrenergic receptors in hypothalamus of the spontaneously hypertensive rats as compared to normotensive controls, with no change in the K_d value. There were no other differences in the spontaneously hypertensive rats and none in the DOCA-salt model. α₁-Adrenergic binding was elevated in hypothalamus of spontaneously hypertensive rats 4–20 weeks of age even though blood pressure in the 4-week old animals was not at hypertensive levels (i.e., <150 mmHg). Treatment of adult spontaneously hypertensive rats with clonidine HCl significantly reduced blood pressure but failed to alter the binding of [³H]WB-4101 in hypothalamus. Thus, it appears that the enhanced number of α₁-adrenergic receptors in hypothalamus of spontaneously hypertensive rats is neither a consequence of the increased blood pressure, nor a phenomenon common to all models of hypertension.     

Alpha-noradrenergic receptor binding in mammalian brain: differential labeling of agonist and antagonist states.

[³H]Clonidine, a α-noradrenergic agonist, and [³H]WB-4101, a benzodioxan derivative α-antagonist, bind with high affinity and selectivity to membranes of rat brain in a fashion indicating that they label postsynaptic α-noradrenergic receptors. Binding for both ligands is saturable with K_D values of 5 nM and 0.6 nM respectively for clonidine and WB-4101. The relative affinities of a series of phenylethylamines for binding sites corresponds well with their relative influences at α-receptors. Binding of both [³H]-ligands is stereoselective with about a 50 fold preference for (-)-norepinephrine. Of a series of ergot alkaloids, only those with known α-receptor activity have high affinities for the binding sites. Binding does not involve pre-synaptic norepinephrine nerve endings, because after an 80% depletion of endogenous norepinephrine by treatment with 6-hydroxydopamine, no decrease can be detected in [³H]clonidine and [³H]WB-4101 binding. α-Agonists have much higher affinities for [³H]clonidine than [³H]WB-4101 sites, while the reverse holds true for α-antagonists. Mixed agonist-antagonist ergots have similar affinities for binding of the two [³H]ligands. These data suggest that [³H]clonidine and [³H]WB-4101 respectively label distinct agonist and antagonist states of the α-receptor.     

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.     

α1- and α2-adrenergic receptors in rat corpus striatum

Rat corpus striatum contained α₂-adrenergic receptor which were labelled with [³H]clonidine (95 ± 6 fmol/mg protein). The affinity of these receptors (K_d = 1.3 to 3.6 nM) was similar to that found in cerebral cortex. Five days after kainic lesions, the number of α₂-adrenergic receptors had dropped by half, suggesting that their location might be neuronal. One month after lesions, the number of α₂-adrenergic receptors had risen to that of the controls and was higher after two months. This increase would suggest a glial localization of the α₂-adrenergic receptor. We have previously described the presence of α₂-adrenergic receptors in primary astrocyte cultures (Ebersolt et al., 1981). Rat corpus striatum contained less α₁-adrenergic receptors than α₂-adrenergic receptors. They were labelled with [³H]prazosin (28 ± 1.9 fmol/mg protein) and were only slightly altered 5 days after kainic acid lesions (?20%). In addition to these classical α₁-adrenergic receptors, rat corpus striatum also contained [³H]WB4101 binding sites having high affinity for WB4101 (2–5 nM) and norepinephrine (1 μM) but a very low affinity for prazosin (4.4 μM). The exact nature of these sites remains unknown.     

Correlation Between Beta- and Alpha-Adrenergic Receptor Concentrations in Human Placenta

Abstract

α₂- and β-adrenergic receptors in human placental membranes have been investigated using the radioligands [³H]-RX 821002 and [³H]-dihydroalprenolol, respectively. The specific binding of the α₂-adrenoceptor antagonist RX 821002 confirms the presence of an α₂-adrenoceptor in the human placenta, which has been characterized previously with [³H]-rauwolscine. The major finding presented here is a correlation between the α₂- and β-adrenergic receptor concentrations (r=0.765) in the human placenta at term. It is suggested that the α₂/β adrenoceptor balance may play an important role in regulation of the vascular bed of the placenta. Determination of the α₂/β ratio may help towards an understanding of the contractility of the placental vascular muscles.     

Independent and Coordinate Regulation of β1- and β2-Adrenergic Receptors in Rat C6 Glioma Cells

Abstract

Rat C6 glioma cells have both β₁- and β₂-adrenergic receptors in ～ 7:3 ratio. When the cells were exposed to the β-adrenergic agonist isoproterenol, there was a rapid sequestration of up to 50% of the surface receptor population over a 30-min period as measured by the loss of binding of the hydrophilic ligand [³H] CGP-12177 to intact cells. Using the β₂-selective antagonist CGP 20712A to quantify the proportion of the two subtypes, it was found that although both β₁ and β₂ receptors were sequestered, the latter were sequestered initially twice as fast as the former. More prolonged agonist exposure led to a down-regulation of ～ 90% of the total receptor population by 6 h as measured by the loss of binding of the more hydrophobic ligand [¹²⁵I] iodocyanopindolol to cell lysates. The two subtypes, however, underwent down-regulation with similar kinetics. Treatment of the cells with agents that raise cyclic AMP levels such as cholera toxin and forskolin resulted in a slower, but still coordinated down-regulation of both subtypes. Thus, there appears to be both independent and coordinate regulation of endogenous β₁-and β₂-adrenergic receptors in the same cell line.     

[3H]Mesulergine,a selective ligand for serotonin-2 receptors

[3H]Mesulergine binds with high affinity to rat cerebral cortex membranes. (K_D = 1.9 nM, B_max = 11.3 pM/g tissue). The binding of this ligand is selective for serotonin-2 receptors: Its next highest affinity, which is for dopamine receptors labelled by neuroleptics, is about 50 times weaker than its affinity for serotonin-2 receptors. No significant affinity for serotonin-1, α₁-adrenergic or histamine H1 receptors was observed.Specific [3H]mesulergine binding was diminished in the presence of low concentrations of lithium ions.     

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.     

Effects of local anesthetics of guanyl nucleotide modulation of the catecholamine-sensitive adenylate cyclase system and on β-adrenergic receptors

Tetracaine and other local anesthetics exert multiple actions on the catecholamine-sensitive adenylate cyclase system of frog erythrocyte membranes. Tetracaine (0.2–2.0 mM) reduces the responsiveness of adenylate cyclose to (a) guanyl-5′-yl-imidodiphosphate and (b) isoproterenol in the presence of GTP or guanyl-5′-yl-imidodiphosphate. Local anesthetics did not affect (a) basal enzyme activity, and (b) enzyme responsiveness to NaF. Tetracaine inhibited stimulation of adenylate cyclase by guanyl-5′-yl-imidodiphosphate over the whole range of nucleotide concentrations. By contrast, inhibition by tetracaine of isoproterenol activity in the presence of GTP was significant only if GTP concentrations exceeded 10^?7 M.Tetracaine also competitively inhibited binding of both the antagonist [³H]-dihydroalprenolol and the agonist [³H]hydroxybenzylisoproterenol to β-adrenergic receptors. However, it was twice as potent in inhibiting [³H]-hydroxybenzylisoproterenol as [³H]dihydroalprenolol binding. The greater potency for inhibition of agonist binding was due to the ability of the anesthetics to promote dissociation of the high-affinity nucleotide sensitive state of the β-adrenergic receptor induced by agonists.Other local anesthetics mimicked the effects of tetracaine on adenylate     

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.     

In vitro characterization of skeletal muscle β-adrenergic receptors coupled to adenylate cyclase

[³H]Dihydroalprenolol, a potent ß-adrenergic antagonist, was used to identify the adenylate cyclase-coupled ß-adrenoceptors in isolated membranes of rat skeletal muscle. The receptor sites, as revealed [³H]dihydroalprenolol binding, were predominantly localized in plasmalemmal fraction. That skeletal muscle fraction may also contain the plasmalemma of other intramuscular cells, especially that of blood vessels. Hence, the [³H]dihydroalprenolol binding observed in that fraction may be due partly to its binding to the plasmalemma of blood vessels. Small but consistent binding was also observed in sarcoplasmic reticulum and mitochondria. The level of [³H]dihydroalprenolol binding in different subcellular fractions closely correlated with the level of adenylate cyclase present in those fractions.The binding of [³H]dihydroalprenolol to plasmalemma exhibited saturation kinetics. The binding was rapid, reaching equilibrium within 5 min, and it was readily dissociable. From the kinetics of binding, association (K₁) and dissociation (K₂) rate constants of 2.21 · M^? · min^?1 and 3.21 · 10^?1, respectively, were obtained. The dissociation constant (K_d) of 15 nM for [³H]dihydroalprenolol obtained from saturation binding data closely agreed with the (K_d) derived from the ratio of dissociation and association rate constants (K₂/K₁).Several β-adrenergic agents known to be active on intact skeletal muscle also competed for [³H]dihydroalprenolol binding sites in isolated plasmalemma with essentially similar selectivity and stereospecificity. Catecholamines competed for [³H]dihydroalprenolol binding sites with a potency of isoproterenol > epinephrine > norepinephrine. A similar order of potency was noted for catecholamines in the activation of adenylate cyclase. Effects of catecholamines were stereospecific, (?)-isomers being more than potent than (+)-isomers. Phenylephrine, an α-adrenergic agonist, showed no effect either on [³H]dihydroalprenolol binding or on adenylate cyclase. Known ß-adrenergic antagonists, propranolol and alprenolol, stereospecifically inhibited the [³H]dihydroalprenolol binding and the isoproterenol-stimulated adenylate cyclase. The (K_i) values for the antagonists determined from inhibition of [³H]dihydroalprenolol binding agreed closely with the (K_i) values obtained from the inhibition of adenylate cyclase. The data suggest that the binding of [³H]dihydroalprenolol in skeletal muscle membranes possess the characteristics of a substance binding to the ß-adrenergic receptor.     

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.     

Evidence for heterogeneous distribution of α1, α2- and β-adrenergic binding sites on rat-liver cell surface

Fractionation of preparations of rat-liver membranes on linear sucrose gradients revealed different profiles for the binding of α₁-, α₂- and β-adrenergic radioligands. The peaks of binding activities of [³H]prazosin and [³H]epinephrine were clearly separated from those of [³H]yohimbine and [¹²⁵I]iodocyanopindolol which appeared at lower sucrose densities. Enzyme marker activities in the sucrose subfractions indicated the presence of plasma membranes in all of the subfractions. Furthermore, the binding peaks of the various adrenergic radioligands cannot be correlated with the presence of membranes derived from microsomes, lysosomes or Golgi apparatus. Pretreatment of rat livers with concanavalin A, in order to prevent the fragmentation of the plasma membranes during isolation, resulted in the shift of the binding of [³H]yohimbine and [¹²⁵I]iodocyanopindolol to sucrose-gradient subfractions of higher densities, clearly separate from fractions containing microsomes and Golgi apparatus. There was no distinct separation of the binding peaks of prazosin, yohimbine, and cyanopindolol in sucrose-gradient subfractions from concanavalin A-pretreated livers. These results are consistent with the hypothesis that α₁-, α₂-, and β-adrenergic binding sites are associated with plasma membranes, and are heterogeneously distributed on the rat-liver cell surface.     

Opiate binding to membrane preparations of neuroblastoma x glioma hybrid cells NG108-15: effects of ions and nucleotides.

Interaction of a number of opiate agonists with the opiate receptors in NG108-15 cell membranes is influenced by ions, as well as certain nucleotides. Steady state binding of [³H]leu-enkephalin is increased by Mg⁺⁺ and decreased by Na⁺, GMP-P(NH)P, GTP, GDP, ITP and IMP-P(NH)P. Half-maximal inhibition produced by GMP-P(NH)P occurred at 4.6 μM. The dissociation of [³H]leu- and [³H]met-enkephalin, as well as [³H]etorphine, from these opiate receptors was also shown to be altered by both ions and nucleotides.