Alpha and beta adrenergic receptors of canine lung tissue identification and characterization of alpha adrenergic receptors by two different ligands

Adrenergic receptors of canine peripheral lung tissues were measured by direct binding techniques using [³H]dihydroergocryptine ([³H]DHE), [³H]prazosin and [³H]dihydroalprenolol ([³H]DHA). All three ligands bound to canine lung tissue with saturability, stereospecificity and reversibility. Adrenergic agonists competed for binding of [³H]DHE and [³H]prazosin in the order: 1-epinephrine > 1-norepinephrine > d-epinephrine > d-norepinephrine > 1-isoproterenol. Adrenergic antagonists competed for binding of [³H]prazosin in the order: prazosin > phentolamine > yohimbine. Inhibition curves of [³H]DHE by prazosin or yohimbine were biphasic suggesting two subtypes of binding sites. Maximum binding capacities of [³H]DHE ranged from 30.6 to 42.7 fmol/mg protein. [³H]prazosin from 18.3 to 26.9 fmol/mg protein and [³H]DHA from 135.2 to 359.4 fmol/mg protein. When both [³H]DHE and [³H]prazosin were used in the same membrane preparation, specific binding of [³H]DHE was always more than that of [³H]prazosin. Since [³H]prazosin is considered to bind to alpha₁ adrenergic receptors specifically and [³H]DHE is considered to bind alpha₂ adrenergic receptors nonselectively, the difference between the numbers of the specific binding sites of these two ligands should represent alpha₂ adrenergic receptors. Alpha₂ adrenergic receptor density ranged from 9.5 to 21.1 fmol/mg protein. Our results suggest the existence of both alpha₁ and alpha₂ adrenergic receptors in canine peripheral lung tissue. Approximately 40% of alpha adrenergic receptors were alpha₂. The ratio of alpha/beta adrenrgic receptors ranged from 1:3.3 to 1:10.4. The ratio of alpha₁/be ta adrenergic receptors ranged from 1:6.7 to 1:21.1.     

Alpha-adrenergic receptors in liver membranes: delineation with subtype selective radioligands

Alpha₁ and alpha₂ adrenergic receptors have previously been demonstrated in rat liver membranes by competition curves of [³H]dihydroergocryptine ([³H]DHE) with the alpha₁ selective antagonist prazosin (B.B. Hoffman, D. Mullikin-Kilpatrick and R.J. Lefkowitz, J. Biol. Chem. $\text{255}$:4645–4652, 1980). The present studies have utilized the radioligands [³H]prazosin and [³H]yohimbine to further define alpha receptors in rat liver membranes. [³H]Prazosin was found to label alpha₁ receptors whereas [³H]yohimbine labelled alpha₂ receptors. The proportion of alpha₁ and alpha₂ receptors determined directly with these radioligands (79% and 20% respectively) was in good agreement with the proportions determined previously with [³H]DHE. Guanine nucleotides were found to reduce the affinity of the agonist epinephrine at the alpha₂ sites labelled by [³H]yohimbine but not at the alpha₁ sites labelled by [³H]prazosin. These results have implications for the interpretation of agonist interactions with alpha receptors in liver membranes.     

alpha 1-Adrenergic receptors in guinea pig myocardium: identification by binding of a new radioligand, (3H)-prazosin.

Binding of (³H)-prazosin to adrenoceptors in guinea pig myocardial membranes was rapid, readily reversible, stereospecific and saturable. By Scatchard analysis (n = 6) Bmax was 58 fmol of (³H)-prazosin bound/mg protein and the K_D was 0.58 nm. The Hill number was 1.05. Adrenergic agonists competed with (³H)-prazosin as follows: (?)adrenaline > (?)noradrenaline > (?)phenylephrine ? ($\text{+}$)isoprenaline > (+)noradrenaline; antagonists competed in the order: non-radioactive prazosin > phentolamine ? piperoxan > yohimbine > sulpiride > propranolol. The K_D for beta-adrenoceptors assessed by (?³H)-dihydroalprenolol was 0.86 nM and the Bmax (96 fmol/mg protein) was almost twice that of alpha-adrenoceptors. (³H)-prazosin appears to be a useful radioligand for the study of post-synaptic (alpha₁) adrenoceptors in myocardial tissue.     

Characterization of α2-adrenergic receptors in human platelets by binding of a radioactive ligand [3H]yohimbine

[³H]Yohimbine, a potent α₂-adrenergic antagonist, was used to label the α₂-adrenergic receptors in membranes isolated from human platelets. Binding of [³H]yohimbine to platelet membranes appears to have all the characteristics of binding to α₂-adrenergic receptors. Binding reached a steady state in 2–3 min at 37°C and was completely reversible upon the addition of excess phentolamine or yohimbine (both at 10^?5 M;$\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 2.37}\text{min}$). [³H]Yohimbine bound to a single class of noncooperative sites with a dissociation constant of 1.74 nM. At saturation, the total number of binding sites was calculated to be 191 fmol/mg protein. [³H]Yohimbine binding was stereo-specifically inhibited by epinephrine: the (?) isomer was 11-times more potent than the (+) isomer. Cathecholamine agonists competed for the occupancy of the [³H]yohimbine-binding sites with an order of potency: clonidine > (?)-epinephrine > (?)-norepinephrine >> (?)-isoproterenol. The potent α₂-adrenergic antagonist, phentolamine, competed for the sites whereas the β-antagonist, (±)-propanolol, was a very weak inhibitor. 0.1 mM GTP reduced the bindng affinity of the agonists, while producing no change in antagonist-binding affinity. Dopamine and serotonine competed only at very high concentrations. Similarly, muscarinic cholinergic ligands were also poor inhibitors of [³H]yohimbine binding. These results suggest tht [³H]yohimbine binding to human platelet membranes is specific, rapid, saturable, reversible and, therefore, can be successfully used to label α₂-adrenergic receptors.     

The characteristics of adrenoceptors in homogenates of human cerebral cortex labelled by (3H)-rauwolscine

The alpha-2-adrenoceptor antagonist (³H)-rauwolscine has been used to label adrenoreptors in membranes from human cerebral cortex. The radioligand binds with high affinity (K_D 2.08 nM) to a single population of sites with a density of 135 fmoles/mg protein. Adrenoceptor antagonists displaced binding in a simple monomolecular fashion with an order of affinity rauwolscine > yohimbine > phentolamine > corynanthine > prazosin indicating binding to alpha-2-adrenoceptors. Agonists displaced with an order of affinity clonidine > (-) adrenaline > (-) noradrenaline > dopamine > (-) isoprenaline but all displayed apparent Hill coefficients less than unity indicating heterogeneity of binding. The relatively high affinity of the alpha-1 antagonist prazosin for (³H)-rauwolscine binding sites in rat cerebral cortex was not observed in the human tissue which had pharmacological properties similar to those described previously in human platelet.     

Alpha-adrenergic receptor subtypes: quantitative assessment by ligand binding.

We describe a method for quantitatively determining the alpha- adrenergic receptor subtypes in membrane fractions by studying the displacement of [³H] dihydroergocryptine by selective alpha antagonists and analyzing this data by a computer modeling technique. Alpha₁ receptors are those with a higher affinity for prazosin than for yohimbine; alpha₂ receptors have a higher affinity for yohimbine than for prazosin. Phentolamine does not discriminate between the two receptor subtypes present in rabbit uterus. The alpha receptor population of rabbit uterus was found to be 37% alpha₁ receptors and 63% alpha₂ receptors. The human platelet and rat liver alpha receptors were determined to be exclusively alpha₂ and alpha₁, respectively. In the uterus, prazosin had a 8800 fold greater affinity for alpha₁ than alpha₂ receptors while yohimbine had a 510 fold greater affinity for alpha₂ than alpha₁ receptors. The use of [³H] dihydroergocryptine displacement curves generated with selective alpha receptor antagonists coupled with subsequent computer modeling provides a precise and powerful method for quantifying the alpha receptor population of a tissue; this technique should be of value in studying the detailed regulation of alpha receptors in tissues which have both alpha₁ and alpha₂ receptors.     

Lack of specific (3H) prazosin binding sites in dog and rabbit cerebral arteries

In order to explore the characteristics of alpha adrenergic receptors on cerebrovascular smooth muscle, specific binding sites for the alpha 1 adrenergic ligand, (3H) prazosin, were studied in blood vessel homogenates. No specific (3H) prazosin binding was found in either rabbit or dog cerebral arteries, but specific binding was demonstrated in the rabbit saphenous and ear arteries. In the ear artery 3H-prazosin binding was saturable with a Kd of 0.51 +/- 0.20 nM and a Bmax of 89 +/- 29 fmoles/mg protein. To confirm the adequacy of our membrane preparation, homogenates of both dog and rabbit cerebral arteries showed saturable specific binding with two different ligands: one for muscarinic receptors, [3H](-) quinuclidinyl benzilate (QNB) and one for alpha 2 adrenergic receptors, (3H) yohimbine. The results of these studies demonstrate a lack of alpha 1 adrenergic receptors on cerebral blood vessels, confirming functional studies showing only a weak contractile response to norepinephrine.     

Direct binding of 3H-lisuride to adrenergic and serotonergic receptors

The characteristics of the specific binding of ³H-lisuride hydrogen maleate (³H-LHM) to homogenates of rat striatum and bovine frontal cortex tissue were investigated. In rat striatum 50% of ³H-LHM binding was inhibited potently by spiperone and haloperidol indicating a component of ³H-LHM binding to D₂ dopamine receptors. Specific ³H-LHM binding was detected in rat striatum after selective blockade of the D₂ dopamine component indicating specific ³H-LHM binding to other striatal sites. In bovine frontal cortex clonidine and serotonin competition curves for specific ³H-LHM binding included high affinity phases indicating alpha₂ adrenergic and high affinity serotonergic components of binding. Blockade of the adrenergic component by 10^?7M clonidine resulted in the specific ³H-LHM binding exhibiting distinctly serotonergic characteristics. Conversely, blockade of the serotonergic component by 2 × 10^?7M serotonin resulted in the specific ³H-LHM binding exhibiting distinct alpha₂ receptor characteristics. These data demonstrate the specific binding of ³H-LHM to alpha₂ adrenergic receptors, to a high affinity serotonin site, and to D₂ dopamine receptors.     

Alpha1 Adrenergic Receptors in the Porcine Pituitary Neurointermediate Lobe: Detection with [3H]Ketanserin

Abstract

[³H]Ketanserin, a serotonin receptor antagonist, labelled high affinity, saturable sites in homogenates of porcine neurointermediate lobe tissue. Cinanserin, a potent and selective serotonin receptor antagonist, inhibited the specific binding of 5 × 10^-10M [³H]ketanserin with a high affinity component representing 20% of the total binding. Prazosin, a potent and selective alpha₁ adrenergic antagonist, inhibited [³H]ketanserin binding with a high affinity component representing 60% of total binding. The prazosin-specific component was demonstrated to be distinct from the cinanserin-specific component. 10^-7M cinanserin was co-incubated with [³H]ketanserin to eliminate the serotonergic component of the binding and allow pharmacological characterization of the remaining prazosin-specific component. The prazosin-specific binding of [³H]ketanserin binding closely resembled the results of experiments using [³H]prazosin to label alpha₁ receptors in neurointermediate lobe tissue homogenates. Ketanserin was found to be sevenfold more potent in inhibiting [³H]prazosin binding to alpha₁ adrenergic receptors in the neurointermediate lobe tissue than in brain tissue. This observation explains why low concentrations of [³H]ketanserin can selectively label serotonin receptors in the brain but will label both adrenergic and serotonin receptors in the neurointermediate lobe.     

[3H]Clonidine binding to rat hippocampal membranes

Alpha adrenergic receptor subtypes in rat hippocampal membranes were studied, using [³H]clonidine as the radioactive ligand. On the basis of competitive binding studies, using the selective antagonist-prazosin, WB-4101, and yohimbine, [³H] clonidine appeared to bind to a population of presynaptic sites that are pharmacologically similar to receptors previously classified as alpha₂. A computerized model that linearized and produced the best possible fit to the experimental data points indicated that [³H]clonidine binds to a single population of receptors possessing equal affinity for the ligand. Binding data also indicated that rat hippocampus contains significantly fewer [³H]clonidine binding sites than rat cortex.     

Simultaneous loss and reappearance of α1-adrenergic responses and [3H]prazosin binding sites in rat liver after irreversible blockade by phenoxybenzamine

The relative influences of the in vivo administration of phenoxybenzamine on in vitro binding to α₁-adrenergic receptors and α₁-receptor-mediated responses were studied. Phenoxybenzamine treatment reduced maximal specific binding of the α₁-selective antagonist [³H]prazosin to liver cell membranes. This response was rapid (< 90 min) and half-maximal following a phenoxybenzamine dose of approx. 10 mg/kg. A similar decrease in the ability of phenylephrine to stimulate glucose release and ⁴⁵Ca²⁺ efflux from liver slices was also noted after phenoxybenzamine treatment. During the recovery period following administration of 30 mg/kg phenoxybenzamine, [³H]prazosin specific binding and phenylephrine-stimulated glucose release and ⁴⁵Ca²⁺ efflux returned to their respective control levels with $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ values of 42, 49 and 38 h, respectively. At all times studied during the recovery period, α₁-binding and both of the α₁-responses were similar fractions of their respective control values. These observations indicate that a close relationship exists between the density of [³H]prazosin binding sites and the ability of rat liver to respond to α₁-stimulation. We suggest that the binding sites identified in studies using the antagonist [³H]prazosin and those through which the agonist phenylephrine stimulates glucose release and ⁴⁵Ca²⁺ efflux are either identical or in equilibrium with each other.     

Different sedimentation properties of agonist- and antagonist-labelled platelet alpha2 adrenergic receptors

Alpha₂ adrenergic receptors were solubilized from human platelet particulate preparations with digitonin. The solubilized alpha₂ receptors retained the essential binding specificity characteristics of the membrane-bound receptors. The alpha₂ receptors could be labelled in platelet membranes with either agonist ([³H]epinephrine) or antagonist ([³H]yohimbine) radioligands. When these membranes were solubilized with digitonin and centrifuged on sucrose density gradients, the sedimentation coefficient of the agonist-labelled receptor (14.6S) was greater than that of the antagonist-labelled receptor (12.9S). This observation may provide insight into the mechanism of adenylate cyclase inhibition by alpha₂ adrenergic receptors.     

Pharmacologic identification of muscarinic receptors in the pylorus of the cat by binding of [3H]-quinuclidinyl benzilate

Muscarinic receptors in the smooth muscle of the cat pylorus (pyloric sphincter) were identified by binding of the ligand (±) [³H]-quinuclidinyl benzilate ([³H]-QNB). Receptor related binding of [³H]-QNB reached steady-state in thirty minutes at 37°C, was saturable, showed pharmacologic specificity and was stereoselective. An apparent equilibrium dissociation constant, K_D, of 1.9 ± 0.3 nM and maximum receptor concentration of 122 ± 13 femtomoles per mg of protein (means ± S.E.M.) were determined from Scatchard plots of [³H]-QNB binding. Hill coefficients of 0.99 and 1.01 indicated the absence of cooperative interactions. The muscarinic antagonists atropine and propantheline inhibited binding with IC₅₀ values in the nanomolar range, whereas bethanechol was over four orders of magnitude less potent. Noncholinergic agents had little or no effect on [³H]-QNB binding. The $\text{levo}$ isomer of QNB was about seventy times more effective at inhibiting binding than its $\text{dextro}$ isomer while $\text{dextro}$ benzetimide was greater than two thousand fold more active than $\text{levo}$ benzetimide. The isomers of another anticholinergic compound, tropicamide, also competed for [³H]-QNB binding sites in a stereoselective manner, the $\text{levo}$ isomer being eighty-five times more potent than the $\text{dextro}$ isomer.     

Temperature-sensitive high affinity [3H]serotonin binding: Characterization and effects of antidepressant treatment

Characterization of temperature-sensitive [³H]serotonin (5-HT) binding sites (1 and 4 nM Kd sites) revealed complex inhibition by neuroleptics and serotonin antagonists. There was no simple correlation with affinities for S₁ and S₂ receptors. $\text{In vivo}$ pretreatment (48 h before) with mianserin did not alter Bmax or Kd for the 1 nM Kd [³H]5-HT site, although [³H]ketanserin (S₂) densities were decreased by 50%. This suggested that possible S₂ components of [³H]5-HT binding must be negligeable, even though ketanserin competed with high affinity (IC₅₀ = 3 nM) for a portion of the 1 nM Kd [³H]5-HT site. Low concentrations of mianserin inhibited the 1 nM Kd [³H]5-HT site in a non-competitive manner, as shown by a decrease in Bmax with no change in Kd after $\text{in vitro}$ incubation. The complex inhibition data may therefore represent indirect interactions through another site.     

[3H]norepinephrine binding by rat glial cells in culture lack of correlation between binding and adenylate cyclase activation

Subcellular fractions prepared from rat glial cells in culture (clonal line C₆) were used in an attempt to characterize the adrenergic receptor involved in adenylate cyclase activation. Both [³H]norepinephrine binding and enzyme activation were measured under identical experimental conditions.Binding sites for norepinephrine could be detected; their main characteristics were: apparent K_m : 4 · 10⁻⁶ M, maximal capacity: 20 pmol/mg protein.Their stereospecificity towards structually related drugs was found to be different from the stereospecificity of the receptor involved in adenylate cyclase activation. Thus, 3-methoxydopamine (a competitive inhibitor of norepinephrine for adenylate cyclase activation), phenylephrine (a partial adrenergic agonist) and the blocking agent propranolol were unable to compete with [³H]norepinephrine for binding. On the other hand, several molecules like dopa bearing a catechol group and which are unable to interact with the adenylate cyclase as agonists or competitive inhibitors strongly inhibited [³H]norepinephrine binding.As in several other systems so far studied, the presence on the glial cell's membrane of a large number of “catechol-binding sites” makes it difficult to characterize the β-adrenergic receptor.     

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.     

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.     

Binding of [3H]methyltrienolone to androgen receptor in rat liver

The synthetic androgen methyltrienolone is superior to testosterone and androstenedione for the measurement of androgen receptor in tissues where the native ligands are metabolized into inactive derivatives. [³H]Methyltrienolone binds with a high affinity to androgen receptor in cytosol prepared from male rat livers, as the Scatchard analysis revealed that the K_d value was 3.3 · 10^?8 M and the number of binding sites was 35.5 fmol/mg protein. Since methyltrienolone also binds glucocorticoid receptor which exists in rat liver, the apparent binding of androgen receptor is faulty when measured in the presence of glucocorticoid receptor. The binding of methyltrienolone to glucocorticoid receptor can be blocked by the presence of a 100-fold molar excess of unlabeled synthetic glucocorticoid, triamcinolone acetonide, without interfering in its binding to androgen receptor, because triamcinolone does not bind to androgen receptor. Triamcinolone-blocked cytosol exhibited that the K_d value was 2.5 · 10^?8 M and the number of binding sites was 26.3 fmol/mg protein, indicating a reduction to $\text{3}\text{4}$ of that in the untreated cytosol. The profile of glycerol gradient centrifiguration indicated that [³H]methyltriemolone-bound receptor migrated in the 8–9 S region in both untreated and triamcinolone-blocked cytosols, but the 8–9 S peak in triamcinolone-blocked cytosol was reduced to about $\text{3}\text{4}$ of that of untreated cytosol.     

Role of alpha 1 adrenoceptors in the turnover of phosphatidylinositol and of alpha 2 adrenoceptors in the regulation of cyclic AMP accumulation in hamster adipocytes

The incorporation of radioactive phosphate into phosphatidylinositol was stimulated by epinephrine in hamster fat cells. This action was inhibited by alpha-adrenergic antagonists in the potency order: Prazosin?phentolamine>yohimbine. Methoxamine, but not clonidine, was able to mimic the effect of epinephrine. These data indicate that the phosphatidylinositol effect in fat cells is due to activation of alpha₁ adrenoceptors. On the other hand, the accumulation of cyclic AMP due to epinephrine was potentiated by alpha-adrenergic antagonists in the potency order phentolamine>yohimbine ?prazosin, in hamster fat cells. Clonidine significantly decreased the accumulation of cyclic AMP due to isoproterenol or ACTH in hamster fat cells, suggesting that the alpha-adrenergic modulation of cyclic AMP levels in hamster fat cells is mediated by alpha₂ adrenoceptors. Radioligand binding studies with plasma membranes from hamster adipocytes demonstrated the presence of both alpha₁ and alpha₂ adrenoceptors but about 90% of the binding sites were alpha₂. These data support the hypothesis that alpha₂ effects of catecholamines are due to inhibition of adenylate cyclase while the increases in phosphatidylinositol turnover that seem to be involved in the mobilization of calcium are linked exclusively to alpha₁ adrenoceptor activation.     

Autoradiographic localisation of [3H]ouabain bound to cultured epithelial cell monolayers of MDCK cells

[³H]Ouabain binding to intact MDCK (cultured monolayers of dog kidney) cells of 60 serial passages is dependent upon ouabain concentration, time and medium K⁺. By utilising high K⁺ incubations to estimate non-specific [³H]ouabain-binding, the concentration of ouabain giving half maximal specific binding was estimated to be 1.0 · 10^?7 M and the total maximum binding to be 2.33 · 10⁵ sites/cell. Ouabain inhibition of (Na⁺, K⁺)-pump function was monitored by the cellular uptake of ^B6Rb over 5 min. The larger fraction of ^B6Rb uptake was ouabain sensitive and the ouabain concentration giving half-maximal inhibition was 2 · 10^?7 M. The cellular distribution of the ($\text{Na}{}^{\mathrm{+}}\text{+}\text{K}{}^{\mathrm{+}}\text{)-}\text{ATPase}$ was investigated using [³H]ouabain autoradiography of intact freeze-dried epithelial monolayers of MDCK cells grown upon millipore filter supports. Binding of [³H]ouabain is localised over the lateral cellular membranes. Autoradiographic silver grain density is close to background levels over both the apical and basal (attachment) membranes.