Thyroid dependent maturation of β-adrenergic receptors in the rat lung

β-Adrenergic receptors were identified in membranes of fetal and postnatal rat lung with (?)-[³H]dihydroalprenolol, [³H]DHA. β-Receptor number (Bmax) increased 11-fold from day 18 of gestation to adult levels by day 28 of postnatal life. The increase of β-adrenergic receptors occurring between postnatal days 15 and 28 was dependent on thyroxine (T4) in propylthiouracil treated pups. β-Adrenergic receptors on day 28 were identical in euthyroid (PTU + T4) as compared to normal control pups (489±31 and 491±30 femtomoles·mg^?1) however receptors were markedly reduced in 28 day hypothyroid pups (PTU alone), Bmax = 294±21.5, m±S.E. p<0.01. Treatment of the hypothyroid pups with T4 for three days on postnatal day 25 increased β-adrenergic receptors approximately two-fold by day 28. This thyroid hormone dependent increase in lung β-adrenergic receptors occurs between postnatal days 15 and 28 coincident with the known increase in thyroid gland activity in the rat pup.     

Characteristics of an Adenylate Cyclase Coupled β-Adrenergic Receptor in a Smooth Muscle Tumor Cell Line

Abstract

We have shown that binding of ³H-dihydroalprenolol ([³H] DHA) to DDT₁ MF-2 cells and cell membranes was of high affinity, saturable, stereoselective and reversible. The [³H]DHA dissociation constants were 0.63 ± 0.15 nM (n=6) and 0.83 ± 0.04 nM (n=5) for intact cells and cell membranes, respectively, with a binding site concentration for cells of 27,300 ± 5,200 sites/ cell (n=6) and for membranes 468 ± 24 fmoles/mg protein (n=5). The order of agonist competition for the [³H]-DHA binding site of DDT₁ cell membranes was isoproterenol (K_i = 0.20 ± 0.07 μM) > epinephrine (K_i = 0.4 ± 0.2 μM) > norepinephrine (K_i = 66.5 ± 5.15 μM) consistent with a β₂-selective receptor interaction. Zinterol, a β₂-selective antagonist, (K_i = 0.05 ± 0.01 μM) was 18x more effective than metoprolol, a β₁-selective antagonist (K_i = 0.9 ± 0.1 μM), in competing for the DHA binding site. A nonlinear iterative curve fitting analysis of zinterol and metoprolol binding isotherms indicated that (p>0.05) DDT₁ cells possess a pure population of β₂-adrenergic receptors. Finally, we have shown that DDT₁ MF-2 cell β₂-adrenergic receptor is functionally coupled to adenylate cyclase via a G/F protein complex as demonstrated in part by a guanine nucleotide requirement for isoproterenol stimulation of adenylate cyclase activity. In addition, guanine nucleotide mediated a reduction in the affinities of isoproterenol and epinephrine for the [³H]DHA binding site.     

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.     

Kinetics of [3H]prazosin and [3H]dihydroalprenolol binding to α1- and β-adrenoreceptors in rat brain cortex membranes

The binding of nonselective α₁- and β-adrenoreceptor antagonists [³H]prazosin and [³H]dihydroalprenolol ([³H]DHA) to rat cerebral cortex synaptosomal membranes has been studied. It is found that ligand-receptor interactions of α₁-adrenoreceptors fit into a single receptor pool model, which assumes the binding of two ligand molecules to one receptor molecule. The parameters of [³H]prazosin binding to α₁-adrenoreceptors are as follows: K _d = 2.58 ± 0.20 nM; B _m = 2.95 ± 1.12 fmol/mg protein; Hill coefficient, n = 2. For β-adrenoreceptors, ligand-receptor interactions fit into a model assuming the presence of two receptor pools in the same effector system and binding of two ligand molecules to one receptor molecule. The corresponding parameters of the [³H]DHA binding to β-adrenoreceptors are as follows: K _d1 = 0.74 ± 0.09 nM; K _d2 = 7.63 ± 0.70 nM; B _m1 = 25 ± 2 fmol/mg, B _m2 = 48 ± 2 fmol/mg, n ₁ = 2; n ₂ = 2. We suggest that in rat cerebral cortex membranes α-and β-adrenoreceptors exist as dimers.     

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.     

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.     

Beta-adrenoceptor activity change after prolonged treatment with growth hormone and somatostatin

1. The effect of 10 days treatment with growth hormone (GH) (l mg/kg body wt/day) and somatostatin (SRIF) (0.25 mg/kg body wt/day) subcutaneously on the activity of β-adrenoceptors in rat hypothalamic, pituitary and cerebral cortical membrane fractions was studied using [³H]dihydroalprenolol ([³H]DHA) as radioligand.2. The administration of GH significantly increased the β-adrenoceptor binding affinity and the administration of SRIF decreased the β-adrenoceptor binding capacity in the hypothalamus.3. In the pituitary the β-adrenoceptor binding affinity was significantly decreased after both hormonal applications.4. In the cerebral cortex the β-adrenoceptor binding affinity was significantly decreased after the GH treatment and increased after the SRIF treatment.5. The present study provides direct evidence for GH and SRIF effects on the activity of rat β-adrenoceptors and supports the view about the involvement of β-adrenergic mechanisms in the neurotransmitter regulation of GH secretion in the rat.     

Pre- and Postnatal Ontogeny and Characterization of Dopaminergic D2, Serotonergic S2, and Spirodecanone Binding Sites in Rat Forebrain

Abstract: The ontogeny of binding sites for [³H] spiperone was studied in time-pregnant rats. Binding of [³H]spiperone to fresh homogenates of pre- and postnatal rat forebrain was characterized by Scatchard analysis and competition experiments with a number of dopaminergic and serotonergic agonists and antagonists and additional substances. A convenient discrimination of three high-affinity sites, i.e., the dopaminergic D₂, serotonergic S₂, and spirodecanone (Sd) sites, was obtained with l-(–)sulpiride and cis-flupenthixol. The analgesic R5573 was found not to be specific for the Sd site but to interact with all three sites. The three binding sites became detectable in sequential order. S₂ and D₂ binding sites were first found at embryonic days 15.75 and 17.75, respectively. The Sd site did not appear before postnatal day 8. All three binding sites reached adult values at approximately postnatal day 30. During the prenatal period, the increase in the number of D₂ binding sites paralleled the rise in forebrain dopamine concentrations. The kinetics of D₂ and S₂ sites were the same at embryonic day 19.75 and postnatal day 30. These observations provide evidence for the presence of the receptor substrate for actions of neuroleptics on dopaminergic and serotonergic systems during fetal life.     

Characterization of postsynaptic β-adrenergic receptors and presynaptic muscarinic cholinergic receptors in the rat spleen

The β-adrenergic and muscarinic cholinergic receptors in the splenic homogenates of control and 6-hydroxydopamine (6-OHDA) treated rats were characterized. The specific binding of [³H]dihydroalprenolol (DHA) and [³H]quinuclidinyl benzilate (QNB) in the rat spleen were saturable and of high affinity and showed pharmacological specificity of splenic β-adrenergic and muscarinic cholinergic receptors. Following 6-OHDA treatment, the Bmax value for specific [³H](-)DHA binding to the rat spleen was significantly increased by 26 percent and 22 percent compared to control at 2 and 3 weeks without a change in the Kd. In contrast, there was a 38 percent decrease in the Bmax for [³H](-)QNB in the 6-OHDA treated rat spleen at 2 and 3 weeks respectively without a change in the Kd. The Bmax value at 5 weeks was significantly greater than that at 2 or 3 weeks. The splenic norepinephrine (NE) concentration was markedly reduced by the 6-OHDA treatment at 1 to 3 weeks, while there was a significant recovery in the splenic NE concentration at 5 weeks. Thus, our results strongly suggest that we are biochemically localizing muscarinic cholinergic receptors on the sympathetic nerves of the rat spleen and that the β-adrenergic receptors of the spleen are localized postsynaptically.     

Identification and ontogeny of beta-adrenergic receptors in fetal rabbit lung

High affinity (Kd=0.2 nM), low capacity (48 fmoles per mg protein), stereospecific binding sites, with properties characteristic of the β₁-subtype of β-adrenergic receptors, have been detected in fetal rabbit lung membranes as early as the 22nd day of gestation. The concentration of the receptor did not change significantly between the 22nd and 26th day of gestation, but increased 3-fold between the 26th and 29th day, reaching a level of 198 fmoles per mg protein. A further increase (from 198 to 315 fmoles per mg protein) in receptor concentration was observed in adult female rabbits. The increase in the levels of pulmonary β-adrenergic receptors between the 26th and 29th day of gestation is temporally related to the increase in surfactant release into the alveolar spaces of the fetal lung. Thus β-adrenergic agonists may act directly on the fetal lung to regulate surfactant secretion.     

Thermodynamic Study of Iodocyanopindolol β-Adrenoceptors Interactions with Rat Lung and Cerebral Cortex

Abstract

(±)¹²⁵ I-cyanopindolol (±) I CYP) was used to characterize β-adrenoceptors on rat lung and cerebral cortex membranes. The affinity of (±) ICYP was higher for lung (K^d = 64.3 pM) at 37°C. The association reaction of (±) ICYP was faster with lung (k₊₁ = 1.52 × 10⁹ M^?1.min^?1) than with cerebral cortex β-adrenoceptors (k₊₁ = 1.75 × 10⁸ M^?1.min^?1). In both tissues, the dissociation reaction followed a biphasic process with a fast (t ½ = 15.4 min and 5.6 min for lung and cerebral cortex respectively) and a slow component (t ½ = 474 min and 255 min for lung and cerebral cortex respectively). The thermodynamic parameters for (±) ICYP - β-adrenoceptors binding have been determined from kinetics and equilibrium studies, for the two tissues, at several temperatures between 0° and 44° C. For lung and cerebral cortex, Arrhenius plots were linear with different energies of activation. Van't Hoff plot was not linear for lung and the standard enthalpy and entropy changes of (±) ICYP - β-adrenoceptors interaction decreased linearly with temperature : the binding occured with a negative heat capacity change (ΔCp° = -368.9 cal. moles^?1. K^?1) at 25° C. Thermodynamic and kinetic results show that binding of (±) ICYP to lung β-adrenoceptors could involve two successive equilibria with a conformational change of the β-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.     

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.     

Blockade of central beta-adrenergic receptors by tazolol (1-isopropylamino-3-(2-thiazoloxy)-2-propanol).

Tazolol, a β₁-adrenergic agonist in heart, had no intrinsic β-adrenergic agonist activity with respect to cyclic AMP-generating systems in rat cerebral cortical slices or with respect to firing of rat cerebellar Purkinje cells. Instead, tazolol proved to be a relatively potent and specific β-adrenergic antagonist. The IC₅₀ for (±) tazolol in antagonizing (-) isoproterenol-elicited accumulation of cyclic AMP in rat cortical slices was 7 × 10^?7M. The IC₅₀ in antagonizing [³H] dihydroalprenolol-binding in rat cortical homogenates was 2.9 × 10^?7 M. Tazolol was about 10 fold more potent in both cases than the β-antagonist, (±) sotalol. Tazolol antagonized the inhibitory, β-adrenergically mediated effects of iontophoretically applied norepinephrine on firing of cerebellar Purkinje cells. The inhibitory effects of γ-aminobutyric acid on firing of Purkinje cells were not altered by tazolol. Tazolol appeared to lack significant local anesthetic activity as evidenced by its lack of effect on spike height in spontaneous firing Purkinje cells.     

Interaction of synthetic peptide octarphin with human blood lymphocytes

The synthetic peptide octarphin (TPLVTLFK, fragment 12–19 of β-endorphin), a selective agonist of nonopioid β-endorphin receptor, was prepared with specific activity 28 Ci/mmol. The binding of [³H]octarphin to T and B lymphocytes isolated from the blood of donors was studied. It was found that [³H]octarphin binds both to T and B cells with high affinity: K _d = 3.0 ± 0.2 and 3.2 ± 0.3 nM, respectively. The specific binding of [³H]octarphin to T and B lymphocytes was competitively inhibited by unlabeled β-endorphin (K _i = 1.9 ± 0.2 and 2.2 ± 0.3 nM, respectively) and was not inhibited by unlabeled naloxone, [Met⁵]enkephalin, [Leu⁵]enkephalin, α-endorphin, and γ-endorphin. Thus, T and B lymphocytes of human blood possess a nonopioid β-endorphin receptor whose binding is provided by the fragment 12–19 (the octarphin sequence).     

Binding of β-endorphin and its fragments to the nonopiod receptor of murine peritoneal macrophages

The tritium-labeled selective agonist of the nonopioid β-endorphin receptor the decapeptide immunorphin ([³H]SLTCLVKGFY) with a specific activity of 24 Ci/mmol was prepared. It was shown that [³H]immunorphin binds with a high affinity to the non-opioid β-endorphin receptor of mouse peritoneal macrophages (K _d 2.4 ± 0.1 nM). The specific binding of [³H]immunorphin to macrophages was inhibited by unlabeled β-endorphin (K _i of the [³H]immunorphin-receptor complex 2.9 ± 0.2 nM) and was not inhibited by unlabeled naloxone, α-endorphin, γ-endorphin, and [Met⁵]enkephalin (K _i > 10 μM). Thirty fragments of β-endorphin were synthesized, and their ability to inhibit the specific binding of [³H]immunorphin to macrophages was studied. It was found that the shortest peptide having practically the same inhibitory activity as β-endorphin is its fragment 12–19 (K _i 3.1 ± 0.3 nM).     

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.