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.     

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.     

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.     

Identification of alpha adrenoceptor subtypes in dog arteries by (3H) yohimbine and (3H) prazosin

Binding of the alpha adrenergic antagonists (³H) prazosin and (³H) yohimbine to membranes of dog arteries exhibit the characteristics expected of alpha adrenoceptors. Binding of both ligands is saturable with dissociation constants of 0.19nM and 1.15nM for (³H) prazosin and (³H) yohimbine respectively. A series of catecholamines inhibit binding of both ligands with a potency in the order $\text{epinephrine}\text{>}\text{norepinephrine}\text{a}\text{?}\text{isoproterenol}$, corresponding with the activity of these agents at alpha adrenoceptors in blood vessels. Competition for binding in both instances is stereoselective. ?-Phenylephrine has similar potencies in inhibiting (³H) prazosin and (³H) yohimbine specific binding whilst the imidazoline related partial alpha adrenergic agonists clonidine and guanfacine are more potent in inhibiting (³H) yohimbine specific binding. The affinity of prazosin for the (³H) yohimbine binding site is approximately 2500 times less than for the (³H) prazosin site whilst yohimbine is approximately 150 times more potent in inhibiting (³H) yohimbine than (³H) prazosin specific binding. Non-selective alpha adrenergic antagonists have similar affinities for both binding sites. The concentrations of (³H) yohimbine binding sites in different arteries vary about two fold whilst for (³H) prazosin the variation was about three fold. These results indicate that there are two discrete noradrenergic binding sites in the major arteries of dog which have binding properties expected of alpha₁ and alpha₂ adrenoceptors.     

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.     

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.     

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.     

Competition by Estrogens for Catecholamine Receptor Binding In Vitro

Abstract: We have examined the ability of various steroids to compete for high-affinity binding of ³H-labeled ligands to catecholamine receptors in membranes prepared from rat cerebral cortex, striatum, and anterior pituitary. Ligands employed were: [³H]WB4101, [³H]prazosin, [³H]yohimbine, and [³H]clonidine (alpha-noradrenergic); [³H]dihydroalprenolol (beta-noradrenergic); [³H]spiperone and [³H]ADTN (dopaminergic). Only the 17β estrogens were effective and only binding of [³H]spiperone and [³H]ADTN in striatum and [³H]WB4101 and [³H]prazosin in cerebral cortex was reduced. Thus putative dopaminergic and alpha₁-noradrenergic sites alone appear to recognize estrogens. A slight competitive effect on [³H]spiperone binding to anterior pituitary membranes was also observed. Among the 17β estrogens tested, the most effective in all cases was the catechol estrogen 2-hydroxyestradiol (2-OHE₂). The ability of 2-OHE₂ (IC₅₀= 20–30 μM) to inhibit ligand binding to alpha₁ receptors was comparable to that of norepinephrine (IC₅₀= 10–20 μM), whereas for dopamine receptors in striatum and pituitary 2-OHE₂ was an order of magnitude less effective than dopamine (IC₃₀= 12 μM) in reducing binding of ³H ligands. Estradiol-17β and 2-hydroxyestrone were also able to inhibit binding, but the order of steroid potency was different for alpha₁ and dopaminergic receptors. Progesterone, testosterone, and corticosterone were without effect in all cases. These results show that there is specificity of steroid interactions with catecholamine receptors in the brain, both in terms of steroid structure and receptor type. The possible relevance of these interactions to neuroendocrine function is discussed.     

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.     

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.     

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.     

Behavior of rat hypothalamic and extrahypothalamic immuno-reactive TRF in thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC)

[³H] quinuclidinyl benzilate (QNB), a specific muscarinic antagonist, was utilized to identify muscarinic cholinergic receptors on dispersed anterior pituitary cells. Scatchard analysis of [³H] QNB binding to receptors departs from linearity with upward concavity. A high affinity binding site having a dissociation constant (K_d) of 1.5 nM was observed when the [³H] QNB concentration was varied from 0.15 to 20 nM. A low affinity binding site (K_d 20 nM) was observed when [³H] QNB concentration was above 20 nM. Using 10 nM [³H] QNB for binding, the second order association rate constant (k₁) of 0.064 nM^?1 min^?1 and first order dissociation rate constant (k₂) of 0.078 min^?1$\text{(}\text{T}\text{1}\text{2}\text{8}\text{min}\text{)}$ were observed. k₂/k₁ = K_d of 1.22 nM is in good agreement with K_d = 1.5 nM from equilibrium data. Muscarinic cholinergic receptor antagonists, atropine and scopolamine, and agonist oxtoremorine potently competed with [³H] QNB binding. A nicotinic cholinergic receptor agonist was 50 times less potent as a competitor of [³H] QNB binding than the muscarinic agonist.     

Demonstration of [3H] diazepam binding to benzodiazepine receptors in vivo.

Benzodiazepine receptors were labeled with [³H] diazepam following intravenous injection in rats. Binding of [³H] diazepam $\text{in}$$\text{vivo}$ to rat forebrain membranes was displaceable by co-injection of clonazepam or the pharmacologically active enantiomers of two benzodiazepines, B9 and B10, but was not displaced by equal doses of the pharmacologically in-active enantiomers. Binding of [³H] diazepam $\text{in}$$\text{vivo}$ was bserved in kidney, liver, and abdominal muscle, but was not stereospecifically diplaced in any peripheral tissue studied. The regional distribution of benzodiazepine receptors in brain was uneven, with specific [³H] diazepam binding being highest in the cerebral cortex and lowest in the ponsmedulla. Preliminary studies of the subcellular distribution of [³H] diazepam binding demonstrated highest specific binding to synaptosomal membranes. These data demonstrate the feasibility of labeling benzodiazepine receptors in rat brain $\text{in}$$\text{vivo}$.     

[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.     

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.     

Isolation and partial characterization of a mucin-type glycoprotein from plasma membranes of human melanoma cells

Plasma membranes were isolated from HM7 melanoma cells grown in the presence of [³H]glucosamine and Na₂³⁵SO₄ or [³H]mannose and [¹⁴C]glucosamine. The labelled glucoconjugates were solubilized with 0.6 M lithium diiodosalicylate/0.5% Triton X-100. Fractionation of glycoconjugates by repeated chromatography on columns of Sepharose CL-6B and DEAE-Sepharose and by affinity chromatography on WGA-Sepharose yielded three radiochemically homogenous glycoproteins. One of these having an apparent molecular weight of 100 000 was found to contain clusters of (AcNeu)_{1 or in2} å [Gal å GalNAc] linked $\text{O-}\text{glycosidically}$ to the protein. One other glycoprotein contained both $\text{O-}\text{glycosidically}$ and $\text{N-}\text{glycosidically-linked}$ oligosaccharides, and the third contained only $\text{N-}\text{glycosidically-linked}$ carbohydrates. Preliminary results indicate that the 100 000 molecular weight mucin-type glycoprotein is present in significantly reduced quantities in cultured human fetal uveal melanocytes. Further, the bulk of the glycoproteins from the melanocytes were of lower molecular size compared to those from the melanoma cells.     

Prostaglandin F2α receptors in bovine corpus luteum cell membranes. Effect of enzymes and protein reagents

Various enzymes and proteins reagents inhibited [³H]prostaglandin F₂α binding to bovine corpus luteum cell membranes. Studies were undertaken (a) to explore further on the dose response relationships with the above agents, (b) to investigate the mechanism of inhibition of binding with respect to receptor affinities and number and (c) to assess whether decreased binding reflected changes in receptors and/or other membrane components.Preincubation of membranes with phoshpolipase A, trypsin, pronase, lipase, tetranitromethane, dinitrofluorobenzene, acetic anhydride and $\text{N-}\text{ethylmaleimide}$ resulted in moderate to drastic inhibitions of [³H]prostaglandin F₂α binding. The dose-dependent inhibition of binding by enzymes, but not by protein reagents (except for $\text{N-}\text{ethylmaleimide}$), exhibited a biphasic pattern: at lower concentrations, the loss of binding was low and relatively plateaued, but at higher concentrations, the losses were dramatic. The drastic reduction in binding by trypsin was due to destruction rather than solubilization of receptors from membranes. Phospholipase A was intrinsically more effective than phospholipases C and Ca²⁺ was not required for its inhibition of [³H]prostaglandin F₂α binding. Protein reagents inhibition of binding was differently influenced by added Ca²⁺ i.e., loss of binding increased with some ($\text{N-}\text{ethylmaleimide}$), decreased with others (tetranitromethane, dinitrofluorobenzene and azobenzene sulfenylbromide). These results are interpreted to indicate that Ca²⁺ induced conformational changes in membranes which may result in exposure of new groups and burying of already exposed modifiable groups.Treatment of membranes wiht trypsin and $\text{N-}\text{ethylmaleimide}$ selectively abolished high affinity prostaglandin F₂α receptors. The low affinity receptors were present but their numbers as well as their affinity were decreased. Lipase, phospholipase A, acetic anhydride, dinitrofluorobenzen and tetranitromethane appear to decrease binding by totally abolishing all prostaglandin F₂α receptors or by severely reducing their affinities.The occupancy of receptors by prostaglandin F₂α afforded considerable protection against trypsin, phospholipase A, lipase and dinitrofluorobenzene. These data indicated that the inhibition of binding by the above agents, at least in part, can be attributable to changes in receptor sites alone.     

Subcellular distribution of α1-adrenergic receptors in rat liver

The distribution of α₁-adrenergic receptors in rat liver subcellular fractions was studied using the α₁-adrenergic receptor ligand [³H]prazosin. The highest number of [³H]prazosin binding sites was found in a plasma membrane fraction followed by 2 Golgi and a residual microsomal fraction, the numbers of binding sites were 1145, 845, 629 and 223 fmol/mg protein, respectively. When the binding in these fractions was compared with the activity of plasma membrane ‘marker’ enzymes in the same fractions a relative enrichment of [³H]prazosin binding sites was found in the residual microsomes and one of the Golgi fractions. Photoaffinity labelling with ¹²⁵I-arylazidoprazosin in combination with SDS-polyacrylamide gel electrophoresis revealed the specific binding to 40 and 23 kDa entities in a Golgi fraction, while in plasma membranes the binders had an apparent molecular mass of 36 and 23 kDa. When [³H]prazosin was injected in vivo into rat portal blood followed by subcellular fractionation of liver, a pattern of an initial rapid decline and thereafter a slow decline of radioactivity was noted in all fractions. Additionally, in the two Golgi fractions a transient accumulation of radioactivity occurred between 5 and 10 min after the injection. The ED₅₀ values for displacement of [³H]prazosin with adrenaline was lowest in the plasma membrane fraction, followed by the residual microsomes and Golgi fractions, the values were 10⁻⁶, 10⁻⁵ and 10⁻⁴ mol/l, respectively. On the basis of lack of correlation between distribution of α₁-adrenergic antagonist binding and adenylate cyclase activity, differences in the molecular mass of α₁-adrenergic antagonist binders, differences in the kinetics of in vivo binding and accumulation of [³H]prazosin and also differences in agonist affinity between plasma membrane and Golgi fractions, it is concluded that α₁-adrenergic receptors are localized to low-density intracellular membranes involved in receptor biosynthesis and endocytosis.     

Interaction of amiloride with alpha-adrenoreceptors: evidence from radioligand binding studies

We investigated the effect of amiloride on alpha-adrenoreceptors (alpha 1 and alpha 2) using radioligand binding techniques. Amiloride inhibited [3H]yohimbine and [3H]prazosin binding to alpha 2- and alpha 1-adrenoreceptors, respectively, from various tissues in a concentration-dependent manner. Amiloride was approximately 9-12 times more potent in inhibiting [3H]yohimbine binding to alpha 2-adrenoreceptors from rat tissues than from other mammalian tissues. However, it had almost the same potency in inhibiting [3H]prazosin binding to alpha 1-adrenoreceptors from rat as well as other mammalian tissues. Further, in rat tissues, amiloride was approximately 10 times more potent in inhibiting [3H]yohimbine than [3H]prazosin binding. Amiloride inhibited [3H]yohimbine binding noncompetitively and [3H]prazosin binding competitively. The inhibition of [3H]yohimbine and [3H]prazosin binding by amiloride was reversible. Since amiloride has been shown to be an inhibitor of Na+-H+ exchanger protein, we believe that it regulates the alpha 2-adrenoreceptors by binding to Na+ -H+ exchanger protein. Triamterene, a compound similar to amiloride in regard to diuretic effect, had very little effect on [3H]yohimbine and [3H]prazosin binding to rat kidney membranes, suggesting that the alpha-adrenoreceptor antagonistic properties of amiloride are not related to its antikaliuretic effect. The results of the present study suggest that some of the pharmacological actions of amiloride (antihypertensive and diuretic effects) can be explained in part by its regulatory effect on both alpha 1- and alpha 2-adrenoreceptors.     

Binding of the imidazoline UK-14, 304, a putative full α2-adrenoceptor agonist,to rat cerebral cortex membranes

The aryl imidazoline compound UK-14, 304 (5-bromo-6-[2-imidazolin-2-yl-amino]-quinoxaline) is a potent and selective α₂-adrenoceptor agonist with full intrinsic activity, unlike other imidazolines. We examined the characteristics of high specific activity (84 Ci/mmol) [³H] UK-14, 304 binding to rat cerebral cortex membranes. [³H] UK-14, 304 specific binding was enhanced by Mn²⁺ ion, and associated and dissociated moderately rapidly at 25°C. Norepinephrine-displaceable binding was saturable and monophasic, with a K_D of 1.4 nM, in agreement with rate and competition experiments, and a B_max of 200 fmol/mg protein. Competition studies revealed that binding was α₂-adrenoceptor-specific, with yohimbine being 12 times more potent than prazosin. [³H] UK-14, 304 appeared to label predominantly the R(H) state of the brain α₂-adrenoceptor, as judged by the high affinity of catecholamine and imidazoline agonists (IC₅₀, 1–13 nM), and the relatively low affinity of yohimbine and rauwolscine (IC₅₀, 100–300 nM), at the binding site. [³H] UK-14,304 compares favorably with other α₂-adrenoceptor ligands because of its high affinity and specific activity.