Identification and characterization of beta1-adrenergic receptors in rat parotid membranes

• Beta-adrenergic receptor identification and properties are probed in rat parotid membranes utilizing the high affinity β-adrenergic antagonist(−)-[³H]dihydroalprenolol.

• The binding of (−)-[³H]dihydroalprenolol to membrane preparations of parotid is rapid, equilibrium being reached in 5 min. Strict stereospecificity is observed, (−)-propanolol being 100 times more potent than (+)-propranolol in competing with (−)-[³H]dihydroalprenolol for binding sites. Beta-adrenergic agonists compete for the binding sites with (−)-[³H]dihydroalprenolol with the same characteristics, i.e., much higher concentrations of the (+)-stereoisomers than the (−)-stereoisomers are required to produce 50% inhibition, the range varies from 14-fold for epinephrine to 300-fold for isoproterenol. Among the (−)-stereoisomers, the relative potency of inhibitory action is (−)-propranolol > (−)-isoproterenol > (−)-epinephrine ≡ (−)-norepinephrine. (−)-Isoproterenol is about 20 times as potent as norepinephrine, the least potent agonist among all the catecholamine (−)-stereoisomers.

• The binding of (−)-[³H]dihydroalprenolol is saturable, with a maximum number of binding sites equalling 450 fmol/mg protein and a dissociation constant of 7.9 nM. The Scatchard plots show no significant curvilinear character. Hill plots consistently give a Hill coefficient close to unity (0.92–1.05). Both pieces of evidence suggest a single-component system with no significant cooperativity.

• Dissociation kinetics study after the method of De Metys et al. (1973) Biochem. Biophys. Res. Commun. 155, 154, indicates a lack of site-to-site interactions among the binding sites. The rate of dissociation of bound (−)-[³H]dihydroalprenolol is the same in the presence and absence of 1 · 10⁻⁵ M (±)-alprenolol.

• Based on the binding of (−)-[³H]dihydroalprenolol, it is concluded that the beta-adrenergic receptors can be identified in rat parotid and that these binding sites display β₁ character. Results of the study indicate a one-component system with no observable site-to-site interactions.

Beta-adrenergic receptors and adenylate cyclase in hypertrophic and hyperplastic rat salivary glands

Isoproterenol induces both the secretion of protein and the stimulation of DNA synthesis and growth in rat salivary glands.The specific binding of the labelled beta-adrenergic antagonist [³H]dihydroalprenolol has been used to measure the number of beta-adrenergic receptors in rat parotid glands during isoproterenol-induced growth. Isoproterenol-enlarged glands display no change in the specific binding capacity per gland for [³H]-dihydroalprenolol compared with normal tissue.Catecholamine sensitive adenylate cyclase activity varies independently of the number of specific [³H]dihydroalprenolol binding sites during isoproterenol-induced growth.Previously-described differences in optimal isoproterenol doses which produce protein secretion and stimulation of DNA synthesis may reflect different responses to various rates of receptor occupancy, or may be due to the presence of more than one type of beta-adrenergic receptor.     

Beta-adrenergic activity of (+/-)-hydroxybenzylisoproterenol in isolated rat fat cells and hepatocytes

The pharmacology of (+/-)-hydroxybenzylisoproterenol with respect to stimulation of cyclic AMP accumulation by isolated rat fat cells and liver cells was examined. (+/-)-Hydroxybenzylisoproterenol was found to be a full agonist and twice as potent as (-)-isoproterenol in liver cells, and equipotent to (-)-isoproterenol in fat cells with regard to stimulating cyclic AMP accumulation. A study of the ability of this catecholamine to stimulate adenylate cyclase activity of broken-cell preparations revealed that (+/-)-hydroxybenzylisoproterenol was equipotent to (-)-isoproterenol in liver cell homogenates, while 3- to 4-fold more potent than (-)-isoproterenol in fat cell ghost membranes. (+/-)-Hydroxybenzylisoproterenol was also found to be as potent as (-)-isoproterenol in stimulating cyclase activity of S49 mouse lymphoma cell membranes. Competition studies of specific [125I]iodohydroxybenzylpindolol binding to liver cell membranes revealed a Kd of 10 nM for (+/-)-hydroxybenzylisoproterenol and 25 nM for (-)-isoproterenol binding to the liver beta-adrenergic receptor. Competition studies of specific (-)-[3H]dihydroalprenolol binding to fat cell membranes indicated a similar affinity of these sites for both (+/-)-hydroxybenzylisoproterenol and (-)-isoproterenol. The guanyl nucleotide Gpp(NH)p induced a shift in the curve for competition of (-)-[3H]dihydroalprenolol binding by (-)-isoproterenol to the right, but failed to do so when (+/-)-hydroxybenzylisoproterenol was the competing agonist. Properties of (+/-)-[3H]hydroxybenzylisoproterenol binding to fat cell or liver cell membranes were inconsistent with those expected of adenylate cyclase coupled beta-adrenergic receptors.     

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.     

Hormone action at the membrane level VIII. Adrenergic receptors in rat heart and adipocytes and their modulation by thyroxine

The regulation of adrenergic receptors in rat heart was measured in rats made hyperthyroid by injection with thyroxine and made hypothyroid by addition of propylthiouracil to the drinking water. Hyperthyroid rats displayed cardiac hypertrophy and a decrease in epididymal gat pad weight. The maximal beta-receptor level of ventricular membranes, as determined by (?)-[³H]dihydroalprenolol binding, was increased 60% by thyroxine treatment and decreased about 30% by propylthiouracil treatment. The affinity of the beta receptor was unchanged after thyroxine or propylthiouracil treatment. The maximal activity of the isoproterenol-stimulated adenylate cyclase (EC 4.6.1.1) varied with thyroid state in a manner parallel to the increase in beta-adrenergic binding sites. Thyroxine treatment also increases by 2-fold the beta receptors in isolated rat fat cells.Propylthiouracil treatment lowered the level of alpha receptors in heart by 30% as measured by [³H]dihydroergocryptine binding, but increased the affinity about 2.5 fold. The highest level of alpha receptors was seen in control hearts. These studies indicate that thyroxine may control the turnover of beta-adrenergic receptors in heart and fat cells and regulate physiological responses in these tissues via a hormone-hormone interplay system.Thyroxine treatment reduced the activity of the membrane-bound Mg²⁺-ATPase (EC 3.6.1.3) and 5′-mononucleotidase (EC 3.1.3.5) but appears to increase the activity of the (Na⁺ + K⁺)ATPase (EC 3.6.1.4).     

Characteristics of β-adrenergic-agonist binding to rat adipocyte membranes: Evidence that (±)-[3H]hydroxybenzylisoproterenol interacts selectively with the adipocyte β-adrenergic receptors

The binding characteristics of the β-adrenergic agonist $\text{(±)-[}{}^3\text{H]hydroxybenzylisoproterenol}$ to rat adipocyte membranes were studied. Binding was rapid, reaching equilibrium within 10 min at 37°C (second order rate constant k₁=1.37·10⁷·M^?1·min^?1). Dissociation of specific binding by 0.5 mM (?)-isoproterenol suggested dissociation from two different sites with respective dissociation rate constants k₂ of 0.106·min^?1 and 0.011·min^?1.[³H]Hydroxybenzylisoproterenol binding was saturable (B_max=690±107 fmol/mg protein), yielding curvilinear Scatchard plots. Computer modeling of these data were consistent with the existence of two classes of [³H]hydroxybenzylisoproterenol binding sites, one having high affinity (K_D=3.5±0.7 nM) but low binding capacity (10% of the total sites) and one haveing low affinity (K_D=101±20 nM) but high binding capacity (90% of the sites). Adrenergic ligands competed with [³H]hydroxybenzylisoproterenol binding with the following order of potency=(?)-propranolol>(?)-isoproterenol>(?)-norepinephrine≈ (?)-epinephrine>>(+)-isoproterenol=(+)-propranolo, which is consistent with binding to β₁-adrenergic receptors. Competition curves of [³H]hydroxybenzylisoproterenol binding by the β-agonist (?)-isoproterenol were shallow and modeled to two affinity states of binding, whereas, competition curves by β-antagonist (?)-propranolol were steeper with Hill number near to one. Gpp[NH]p severely reduced [³H]hydroxybenzyl-isoproterenol binding, an effect which apparently resulted from the reduction of the number of both the high and low affinity sites. In membranes which had been previously exposed to (?)-isoproterenol, then number of [³H]hydroxybenzylisoproterenol binding sites was reduced by 50%, an effect which apparently resulted from the loss of part of both the high and low affinity state binding sites. Finally, the ability of (?)-isoproterenol to stimulate adenylate cyclase correlate closely with the ability of (?)-isoproterenol to displace [³H]hydroxybenzylisoproterenol binding. Comparison of these findings with the binding characteristics of the β-antagonist [³H]dihydroalprenolol to rat adipocyte membranes, led to conclude that [³H]hydroxybenzylisoproterenol can be successfully used to label the β-adrenergic receptors of rat fat cells and suggests that it might be a better ligand than [³H]dihydroalprenolol in these cells.     

Elevated beta-adrenergic receptor number after chronic propranolol treatment.

Treatment of rats with the beta-adrenergic antagonist propranolol for two weeks leads to a 100% increase in the number of beta-adrenergic receptors ((?) [³H]dihydroalprenolol binding sites) in cardiac particulate fractions. No change in receptor affinity was observed. These findings may l) represent the converse of agonist-induced desensitization which is associated with decreases in beta-receptor number, 2) provide a potential explanation for the clinically observed “propranolol withdrawal syndrome”.     

The beta-adrenergic receptor of turkey erythrocyte membranes: conformational modification by beta-adrenergic agonists.

The β-adrenergic receptors of turkey erythrocyte membranes have been identified by the specific binding of the radiolabeled antagonist (?)-|³H|-dihydroalprenolol. Pretreatment of these membranes with either the alkylating agent N-ethylmaleimide or with β-adrenergic agonists does not affect (?)-|³H|-dihydroalprenolol binding to the receptor sites. However, the simultaneous presence of both types of products causes a 50% decline in the number of binding sites. A less pronounced decline occurs when the membranes are pretreated with N-ethylmaleimide in presence of the partial agonist (?)-phenylephrine, and no decline in the presence of the antagonist (?)-|³H|-dihydroalprenolol. β-adrenergic agonists thus appear to induce a conformational change of their receptor, with results in an increased susceptibility to inactivation by N-ethylmaleimide.     

Beta-adrenergic receptors in early human placenta characterization of [3H]-dihydroalprenolol binding

The binding characteristics of beta-adrenergic ligand [³H]-dihydroalprenolol (DHA) were determined in particulate membranes of early human placenta (8 – 12 weeks of gestation). [³H]-DHA binding to crude membrane fractions was rapid, reversible, saturable and linearly correlated with the membrane protein concentration. Scatchard analysis of saturation experiments showed a K_D of 2.80 ± 0.9 nM and a density of binding sites of 330.30 ± 93.5 fmol/mg protein. Agonist potency isoproterenol epinephrine norepinephrine indicated that early human placenta contains an adrenergic receptor of beta-2 subtype.     

A reliable assay for beta-adrenoceptors in intact isolated human fat cells with a hydrophilic radioligand, [3H]CGP-12177

The beta-adrenergic receptors of isolated human fat cells were identified using a new hydrophilic beta-adrenergic radioligand (+/-)[3H]CGP-12177. The results were compared with those from [3H]dihydroalprenolol binding to fat cells and membranes. [3H]CGP-12177 binding to isolated fat cells showed lower nonspecific binding (less than 15% of total binding) than the lipophilic [3H]dihydroalprenolol (40-60%) at 3 times the KD. At 37 degrees C, [3H]CGP-12177 binding was rapid, reversible, of high affinity (1.2 +/- 0.3 nM) and saturable. The total number of binding sites per cell in subcutaneous adipocytes was 25,000 +/- 6,000 and was equivalent to that found using membrane fractions. Displacement of [3H]CGP-12177 bound to adipocytes by propranolol was stereoselective, consistent with competition at a single site, and had the same characteristics as in membranes. The displacement curves of the beta 1-selective antagonists (atenolol and betaxolol) were biphasic, the high affinity displacement accounting for 70% of the total binding sites. Beta-adrenergic agonists also competed with [3H]CGP-12177 binding in the order of potency: (-) isoproterenol greater than (-) norepinephrine greater than (-) epinephrine, similar to that found in membranes and in in vitro studies on the lipolytic activity of isolated fat cells. This study demonstrates that the sites specifically labeled by [3H]CGP-12177 are the physiological beta-adrenoceptors and also shows that the ligand is better than [3H]dihydroalprenolol for the accurate identification of these receptors in intact human adipocytes. The methodology, which requires biopsies of less than 1 gram of adipose tissue, can be of potential interest for clinical studies investigating the status of fat cell beta-adrenoceptors in various pathophysiological situations.     

An assay for beta-adrenergic receptors in isolated human fat cells

The beta-adrenergic receptors have been characterized in isolated human adipocytes using a potent beta-adrenergic antagonist (-)-[3H]dihydroalprenolol. Binding of (-)-[3H]dihydroalprenolol to isolated fat cells was stereospecific and saturable, the maximum number of binding sites calculated being 7.8 +/- 2.2 pmol of bound ligand/10(7) cells, corresponding to 450,000 binding sites/cell. The dissociation constant was estimated to be 2.7 +/- 1.1 nM. The results with competition-inhibition experiments using beta-adrenergic agonists and antagonists indicated that the binding sites in isolated adipocytes were predominantly of the beta1-subtype; about 80% of the receptors were of this type. With the present method, specific beta-adrenergic receptor number and affinity in isolated human adipocytes could be determined in about 1 g of human adipose tissue.     

Characteristics of beta-adrenergic receptors in isolated cells and in crude membranes of brown adipose tissue

In relation to decreased metabolic sensitivity to catecholamines observed, in vitro, in brown fat of cold-acclimated rats, beta-adrenergic receptors were studied in isolated cells and in a crude membrane preparation from rat interscapular brown adipose tissue. [3H] dihydroalprenolol binding had the same characteristics in both types of preparation; competition studies of [3H] dihydroalprenolol binding led to the characterization of beta 1 subtype adrenergic receptors with a lower affinity of beta-adrenergic agonists for [3H] dihydroalprenolol binding sites in membranes than that found in isolated cells. Cold acclimation produced, in isolated cells only, a decrease of 41% in the [3H] dihydroalprenolol binding sites and a beta-adrenergic agonist affinity increase. It is concluded that beta-adrenergic receptor decrease could be a factor, at the hormone receptor interaction level, in the regulation of the transmission of biological action responsible for the cold-induced decrease in catecholamine responsiveness in brown adipose tissue. For a study of the desensitization process in brown fat, isolated cells seem to offer certain advantages over a crude membrane preparation.     

Evidence for a second desensitized state of beta-adrenergic receptor with low affinity for beta-antagonists and normal reactivity towards beta-agonists in adipocyte membranes previously exposed to beta-antagonists.

When adipocyte membranes are successively exposed to (-)-propranolol or (+/- alprenolol at 25 or 4 degrees C, repeatedly washed and then assayed for (-)-[3H]dihydroalprenolol binding, the apparent number of beta-adrenergic binding sites is markedly decreased. Induction of this peculiar type of receptor desensitization does not require prolonged exposure of the membranes to the beta-adrenergic antagonists (half-time: 1 min), is stereospecific, concentration-dependent and almost complete with high concentrations of antagonists. p[NH]ppG, which reduces the affinity of fat cell beta-adrenergic receptors for agonists, does not prevent the antagonist-induced decrease in the receptor number. The magnitude of the desensitizating effect induced separately by (-)-isoproterenol and (-)-propranolol is not additive in membranes exposed to both drugs, suggesting that the receptors lost after exposure to agonists are the same sites as part of those lost after exposure to antagonists. However, contrary to the results found in membranes desensitized by agonists, adenylate cyclase activity remained fully responsive to catecholamines in membranes exposed to beta-antagonists. As shown by kinetic studies on (-)-[3H]dihydroalprenolol binding, this beta-antagonist-induced receptor desensitization is reversible after prolonged incubation. These data which have never yet been described in the other reported desensitizable beta-adrenergic systems, suggest that, when exposed to beta-antagonists, the fat cell beta-adrenergic receptors undergo a conformational change leading to a peculiar state which has low affinity for antagonists but behaves towards agonists as does the receptor in its resting state.     

Thiol reactivity and the molecular individuality of α- and β-adrenoreceptors in rat liver plasma membranes

Whether or not α- and β-adrenoreceptors are non-identical binding sites on the same protein is still an open question. We investigated the effects of sulfhydryl reagents and dithiothreitol on the binding of [³H]dihydroalprenolol and [³H]dihydroergocryptine to β- and α-adrenoreceptors of rat liver plasma membranes. Dithiothreitol inhibited the binding of [³H]dihydroalprenolol to the β-adrenoreceptor, whereas it had no effect on the specific binding of [³H]dihydroergocryptine to the α-adrenoreceptor. In contrast, mersalyl, a mercurial SH reagent, readily blocked the α-adrenoreceptor and, although to a lesser extent, the β-adrenoreceptor. The interaction of mersalyl with the α-adrenoreceptors was almost instantaneous. In contrast, under the same experimental conditions, the inactivation of the β-adrenoreceptors was much slower ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{:7}\text{min}$). Finally, a marked difference in the accessibility of the SH groups to mersalyl was observed between the α- and β-adrenoceptors. The presence of 15 μM (?)-epinephrine or 1.5 μM phentolamine was sufficient to prevent the blockade of the α-adrenoreceptor by mersalyl, but inactivation of the β-adrenoreceptor by mersalyl was not modified by 500 μM (?)-epinephrine and was only slightly decreased by 50 μM (?)-propranolol. Thus, the α- and β-adrenoreceptors from rat liver plasma membranes exhibited biochemical differences which may be interpreted in favor of their molecular individuality.     

Beta-adrenergic receptors and adenylate cyclase in hypertrophic and hyperplastic rat salivary glands.

Isoproterenol induces both the secretion of protein and the stimulation of DNA synthesis and growth in rat salivary glands. The specific binding of the labelled beta-adrenergic antagonist [3H]dihydroalprenolol has been used to measure the number of beta-adrenergic receptors in rat parotid glands during isoproterenol-induced growth. Isoproterenol-enlarged glands display no change in the specific binding capacity per gland for [3H]-dihydroalprenolol compared with normal tissue. Catecholamine sensitive adenylate cyclase activity varies independently of the number of specific [3H]dihydroalprenolol binding sites during isoproterenol-induced growth. Previously-described di-ferences in optimal isoproterenol doses which produce protein secretion and stimulation of DNA synthesis may reflect different responses to various rates of receptor occupancy, or may be due to the presence of more than one type of beta-adrenergic receptor.     

Characterization of beta-adrenergic receptor and adenylate cyclase in canine cerebellum.

Binding of (?)-[${}^3\text{H}$]dihydroalprenolol to the synaptic membrane fractions of canine cerebellum was rapid and reversible with rate constants of 1.62 × 10⁸m^?1 min^?1 and 0.189 min^?1 for the forward and reverse reactions, respectively. The binding was of high affinity and saturable with an equilibrium dissociation constant (K_D) of 5 to 7 nm. Bound (?)-[${}^3\text{H}$]-dihydroalprenolol was displaceable with β-adrenergic agonists and antagonists, but not with a variety of other neuroactive substances such as acetylcholine, histamine, serotonin, dopamine, tyramine, (?)-phenylephrine, γ-aminobutyric acid, glycine, and glutamic acid. Adenylate cyclase of the membranes was stimulated at most three times by β-adrenergic agonists, but not significantly by the other neuroactive substances. Guanine nucleotides such as GTP and guanyl-5′-yl imidodiphosphate (Gpp(NH)p) were strictly required for β-adrenergic stimulation of adenylate cyclase with their optimum concentrations of 50 μm, although the nucleotides alone elevated virtually no basal activity. The affinities of β-adrenergic ligands including some stereoisomers for (?)-[${}^3\text{H}$]dihydroalprenolol binding sites were very similar to those for adenylate cyclase in the presence of GTP. Binding of β-adrenergic agonists to the membranes exhibited an apparent negative cooperativity as determined by displacement of (?)-[${}^3\text{H}$]dihydroalprenolol in the absence of purine nucleotides. This negative cooperativity was entirely abolished by addition of either GTP or Gpp(NH)p at 50 μm. Both (?)-isoproterenol-stimulated adenylate cyclase activity and binding of (?)-[${}^3\text{H}$]dihydroalprenolol were not affected by β₁-selective antagonists, (±)-atenolol, and (±)-practolol, at concentrations which completely inhibit peripheral β₁-responses in vitro, whereas β₂-selective agonists such as YM-08316 (BD-40A) and (±)-salbutamol not only stimulated adenylate cyclase but also competitively inhibited binding of (?)-[${}^3\text{H}$]dihydroalprenolol. These results indicate that canine cerebellar adenylate cyclase may be coupled specifically with β₂-adrenergic receptor.     

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.     

Alpha- and beta-adrenergic receptors of the rat salivary gland. Elevation after chemical sympathectomy.

Binding of [3H]dihydroergokryptine and [3H]dihydroalprenolol to membrane preparations from rat submaxillary gland was measured to characterize the alpha- and beta-adrenergic receptors, respectively. Kinetic analysis of the data revealed a high affinity binding site for each radioligand. Inhibition of binding at each site was stereospecific for the active isomer of the catecholamine used. The greater ability of a beta1 than beta2 specific beta-adrenergic antagonist to displace [3H]dihydroalprenolol binding indicated that this binding site was of the beta1 type. Chemical sympathectomy with reserpine or 6-hydroxydopamine resulted in a significant increase in both [3H]dihydroalprenolol and [3H]dihydroergokryptine binding in the rat submaxillary gland. 3scatchard analysis of the data indicated that these increases in binding were due to a change in total number of binding sites for [3H]dihydroergokryptine and [3H]dihydroalprenolol with little change in apparent affinities. This suggests that changes in alpha- and beta-adrenergic receptor density may be important in the development of supersensitivity in salivary glands after reserpine and 6-hydroxydopamine treatment.     

Potent beta-adrenergic antagonist possessing chemically reactive group

A highly potent beta-adrenergic antagonist based on the structure of alprenolol has been prepared by replacement of the isopropylamine residue of alprenolol by 1, 8-diamino-p-menthane (AlpM). The resulting mixture of isomers (AlpM) competes for occupancy of beta-adrenergic receptors in frog erythrocycte membranes with an apparent K_D of 210 pM. The bromoacetylated derivative of AlpM (BrAlpM) leads to an irreversible inactivation of the [³H]dihydroalprenolol ([³H]DHA) binding sites. Various adrenergic agonists and antagonists afford specific and stereoselective protection of the receptor against inactivation by BrAlpM. Tritiation of AlpM followed by bromoacetylation and chromatographic separation yielded two isomers, Br-1-AlpM and Br-8-AlpM of high specific radioactivity (～ 40 Ci/mmol). Both radiolabelled isomers interacted specifically and with high affinity with the beta-adrenergic receptor, but only a small amount of the ligands could be covalently incorporated into the receptor subunit. This agent provides a powerful new probe for studies of beta-adrenergic receptors in analogy with bungarotoxin for the nicotinic cholinergic receptor.     

The (--)[3H]dihydroalprenolol binding to rat adipocyte membranes: an explanation of curvilinear Scatchard plots and implications for quantitation of beta-adrenergic sites

In rat adipocyte membranes, both beta-adrenergic agonists and beta-adrenergic antagonists competed with (--)[3H]dihydroalprenolol for high affinity (KD 2-4 nM) and low capacity binding sites. The antagonists but not the agonists competed with (--)[3H]dihydroalprenolol for lower affinity and higher capacity sites. The present studies were performed in order to characterize the adipocyte beta-adrenergic receptor and distinguish it from low affinity, higher capacity sites which were heat-labile and not stereoselective. When isoproterenol was used to define the nonspecific binding, saturation studies showed a single binding site with a capacity of approximately 100 fmol/mg membrane protein (corresponding to approximately 50,000 sites/adipocyte). Binding was saturated by 10 nM (--)[3H]dihydroalprenolol. Approximate KD's of 204 nM were observed. Kinetic analysis of (--)[3H]dihydroalprenolol binding provided an independent measurement of KD between 0.75 and 1.1 nM. This binding site had the characteristics of a beta 1-adrenergic receptor with the potency of isoproterenol greater than norepinephrine greater than or equal to epinephrine as competitors of binding. Furthermore, the KD of inhibition of (--)[3H]dihydroalprenolol binding correlated with the Ki of inhibition by antagonists or Ka of activation by agonists of glycerol release in isolated adipocytes (r = 0.968, P less than 0.001). These results suggest that beta-adrenergic agonists compete with (--)[3H]dihydroalprenolol for the high affinity binding site which represents the physiological site. Furthermore, the use of antagonists (propranolol, alprenolol) to define specific beta-binding includes nonspecific site(s) as well as the beta-adrenergic site. Previous characterization and quantitation of beta receptors in rat fat cell membranes may have been in error by incorporating both types of binding in their measurement.