Characterization of alpha 1-adrenergic receptor subtypes in rat brain: a reevaluation of [3H]WB4104 and [3H]prazosin binding

[3H]Prazosin and [3H]WB4101 [2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4 benzodioxane] have both been proposed to label alpha 1-adrenergic receptors in the rat central nervous system. As many discrepancies between the binding of these two ligands have arisen, we conducted these studies in order to reevaluate their binding characteristics and resolve the similarities and differences in the pharmacological characteristics of their respective binding sites. [3H]Prazosin binding is characterized by a monophasic saturation isotherm. Prazosin, indoramine, and dihydroergocryptine competitions with [3H]prazosin are steep and monophasic, and model best to a single binding site. In contrast, phentolamine and WB4101 competition curves are shallow in rat cortex, exhibiting Hill coefficients significantly less than 1.0, and model to two binding sites of approximately equal proportions. The higher and lower affinity components are defined as alpha 1A and alpha 1B, respectively. [3H]WB4101 also labels two binding sites in rat cortex and hippocampus with picomolar and nanomolar affinity, respectively. However, the nanomolar binding site is serotonergic and not adrenergic. The picomolar site (KD = 150 pm) has characteristics of an alpha 1-receptor binding site: prazosin, WB4101, and phentolamine affinities for this [3H]WB4101 binding site correlate with their affinities for the highest affinity component (alpha 1A) of [3H]prazosin binding. In addition, the Bmax of this [3H] WB4101-labeled site is equal to one-half of the total [3H]prazosin Bmax. Agonist competitions with [3H]prazosin binding are multiphasic with pseudo-Hill slopes less than 1.0 and with a rank order of affinity of epinephrine greater than norepinephrine greater than phenylephrine. When binding to the alpha 1A component is blocked by a 30 nM phentolamine mask, the same rank order of agonist affinities is preserved. Although the affinities of epinephrine and norepinephrine at the two subtypes are identical, phenylephrine is weaker at the alpha 1B site. The ratio of the potency of phentolamine versus prazosin is about 4 at the alpha 1A component but about 80 at the alpha 1B binding site. We discuss these data in relation to the reported potencies of these antagonists in blocking alpha 1-receptor-mediated responses which may correlate with our designation of alpha 1A or alpha 1B binding sites.     

Selective blockade of alpha 1-adrenoceptors of porcine vascular smooth muscle cells by bunazosin

The characteristics of bunazosin binding to alpha-adrenoceptors in the porcine aortic membranes were investigated using [3H]prazosin and [3H]yohimbine binding assays to identify alpha 1- and alpha 2-adrenoceptors, respectively. The extent of the inhibition (Ki values) of [3H]prazosin binding to alpha 1-adrenoceptors induced by bunazosin was 0.29 nmol/l and about the same as that induced by prazosin (Ki = 0.10 nmol/l). The Ki value of bunazosin inhibition of [3H]yohimbine binding to alpha 2-adrenoceptors was 350 nmol/l. There was over a 1000-fold difference in Ki value for bunazosin between alpha 1-and alpha 2-adrenoceptors. Thus, bunazosin is a highly alpha 1 selective agent in vascular smooth muscle.     

5-Methylurapidil may discriminate between alpha 1-adrenoceptors with a high affinity for WB4101 in rat lung.

The alpha 1-adrenoceptors of rat lung with a high affinity for [3H]-prazosin were subdivided into two populations (high and low affinity sites) by WB4101 and 5-methylurapidil but the proportions were different between both drugs. After pretreatment with chlorethylclonidine, WB4101 recognized only high affinity sites, while 5-methylurapidil still detected high and low affinity sites. These results indicate that alpha 1-adrenoceptors with a high affinity for WB4101 are not homogeneous in the rat lung, suggesting the possible existence of a new alpha 1-adrenoceptor subtype in addition to alpha 1A and alpha 1B subtypes.     

Pharmacological characterization of alpha 1-adrenoceptor subtypes in rat heart: a binding study.

1. The alpha 1-adrenoceptor subtypes of rat heart were characterized in binding experiments performed with [3H]-prazosin as the radiolabel. The specific binding to the alpha 1-adrenoceptors was determined with 0.3 microM prazosin, because phentolamine (10 microM) was insufficient to inhibit completely the specific binding of high concentrations of [3H]-prazosin. 2. In saturation experiments, [3H]-prazosin bound to two distinct affinity sites (pKD = 10.39 and 8.19). The proportion of the low affinity sites was approximately 84% of total specific binding. Membranes pretreated with chloroethylclonidine (CEC, 10 microM) also showed two distinct affinity sites for [3H]-prazosin, although the maximum numbers of high and low affinity sites were reduced by 86 and 64%, respectively. 3. In competition experiments, [3H]-prazosin (100 pM) binding was inhibited by WB4101 (2-(2,6-dimethoxy-phenoxyethyl)aminomethyl-1,4-benzodioxane) and 5-methylurapidil. The inhibition curves displayed shallow slopes which could be subdivided into high and low affinity components (pKi = 10.43 and 8.36 for WB4101, 8.62 and 6.61 for 5-methylurapidil). However, unlabelled prazosin or HV723 (alpha-ethyl-3,4,5-trimethoxy-alpha-(3-((2-(2-methoxyphenoxy)-ethyl)amin o) propyl)benzeneacetonitrile fumarate) competed for [3H]-prazosin binding monophasically (pKi = 10.34 and 8.28, respectively). In CEC-pretreated membranes, prazosin, WB4101, 5-methylurapidil and HV723 antagonized the [3H]-prazosin (100 pM) binding monophasically (pKi = 9.70, 9.56, 8.60 and 8.82, for each antagonist). 4. On the other hand, 1000 pM [3H]-prazosin binding was inhibited by unlabelled prazosin biphasically (pKi = 10.49 and 8.49). HV723 did not discriminate both prazosin-high and low affinity sites (pKi = 8.18).(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for alpha 1-adrenergic receptor internalization in DDT1 MF-2 cells following exposure to agonists plus protein kinase C activators

Agonist-induced sequestration and internalization of alpha 1-adrenergic receptors were examined in DDT1 MF-2 cells. Pretreatment of cells with epinephrine or norepinephrine alone, but not with phorbol 12-myristate 13-acetate alone, resulted in a marked decrease in [3H]prazosin binding to intact cells at 4 degrees. These pretreatments resulted in little or no change in the fraction of alpha 1-adrenergic receptors exhibiting limited accessibility to the hydrophilic competing ligand epinephrine in short-time competition binding assays with intact cells and little or no change in the subcellular distribution of alpha 1-adrenergic receptors between the plasma membrane and light vesicle compartments as assessed by sucrose density gradient centrifugation assays. Pretreatment with a combination of agonist plus phorbol 12-myristate 13-acetate resulted in a greater decrease in [3H]prazosin binding at 4 degrees than was observed when cells were pretreated with agonist alone, induced the conversion of about half of cell surface alpha 1-adrenergic receptors to a form exhibiting limited accessibility to epinephrine in short-time assays, and induced a shift of about half of alpha 1-adrenergic receptors from the plasma membrane fraction to a light vesicle fraction on sucrose density gradients. A similar shift of alpha 1-adrenergic receptors was observed on sucrose density gradients after exposure to agonist plus either mezerein or beta-phorbol didecanoate, but not with agonist plus alpha-phorbol didecanoate, indicating involvement of protein kinase C. These results suggest that pretreatment with agonist alone induces the sequestration of alpha 1-adrenergic receptors into a compartment that is inaccessible to [3H]prazosin at 4 degrees but that is accessible to hydrophilic ligands at 37 degrees and remains associated with the plasma membrane. In contrast, alpha 1-adrenergic receptors are apparently internalized from the plasma membrane to a separate compartment, presumably an intracellular vesicle, when cells are pretreated simultaneously with a combination of agonist plus a protein kinase C activator.     

The alpha-adrenergic receptor: radiohistochemical analysis of functional characteristics and biochemical differences

The partial agonist [3H]para-aminoclonidine was used to label alpha 2-adrenergic binding sites in intact sections of the rat central nervous system using in vitro labeling receptor autoradiographic techniques. The distribution of alpha 2-agonist binding sites closely parallels the reported distribution of noradrenergic and adrenergic cell groups and their terminal fields, particularly the projections of the medullary catecholamine neurons. This distribution of alpha 2 binding sites confirms physiological studies which indicate that the anti-hypertensive actions of alpha 2-agonist compounds are mediated centrally in medullary and spinal centers involved in the control of parasympathetic and sympathetic outflow. Further, the high concentrations of alpha 2 binding sites in pontine and limbic areas such as the locus coeruleus, parabrachial nucleus, dorsal raphe, hypothalamus, amygdala, bed nucleus of the stria terminalis, septum and entorhinal cortex offer an anatomical basis for understanding the anxiolytic and antidepressant actions of drugs like clonidine. The antagonists [3H]prazosin and [3H]WB4101 were used to study the distribution of alpha 1-adrenergic binding sites in the rat forebrain and biochemical studies were performed to analyze the marked differences that were initially seen in the distribution of [3H]prazosin and [3H]WB4101 binding sites. Several pieces of evidence derived from both biochemical and autoradiographic studies suggest that [3H]prazosin and [3H]WB4101 act at distinctly different binding sites. However, both sites may represent components of an alpha 1-adrenergic receptor-effector complex since a high degree of overlap was seen in the binding site distribution of these two ligands and since kinetic interactions could be demonstrated in at least one region of the brain, the hippocampus. Differences noted in the relative displacements of [3H]prazosin and [3H]WB4101 binding in various forebrain regions could reflect differences in the coupling efficiency of the [3H]prazosin and [3H]WB4101 component of the hypothesized complex. Further, in some regions, [3H]WB4101 labeled a binding site that is different from the alpha 1-receptor. Thus, [3H]prazosin and [3H]WB4101 binding sites seen in forebrain regions such as lamina V of the cortex, thalamic nuclei and dorsal raphe probably represent alpha 1-adrenergic receptors and confirm electrophysiological and biochemical studies which demonstrate that adrenergic transmission in these regions can be mediated through an alpha 1-receptor.     

alpha 2-Adrenoceptors in the HT 29 human colon adenocarcinoma cell line: characterization with [3H]clonidine; effects on cyclic AMP accumulation

In the present work, [3H]clonidine was used to characterize alpha 2-adrenoceptors on the human adenocarcinoma cell line HT 29. The effects of alpha 2-adrenergic stimulation on cellular cyclic AMP levels were also investigated. The binding of [3H]clonidine on HT 29 cell membrane preparations was rapid and reversible. Scatchard analysis of the saturation curves indicated the existence of a single class of non-interacting sites with a KD of 1.29 +/- 0.07 nM and a Bmax of 114 +/- 7 fmol/mg of cell membrane protein. The binding sites for [3H]clonidine showed the required specificity for alpha 2-adrenoceptors. The potencies of alpha-adrenergic compounds to displace [3H]clonidine binding ranked as follows: yohimbine greater than phentolamine much greater than prazosin for antagonists and clonidine greater than epinephrine greater than norepinephrine greater than phenylephrine much greater than amidephrine for agonists. When tested on intact cells, epinephrine, norepinephrine and clonidine were found to counteract, in a dose-dependent manner, the increase of cyclic AMP triggered by vasoactive intestinal peptide (VIP). Such inhibitory effects were abolished by the addition of yohimbine but not of prazosin. The physiological amines were the most efficient agonists: both epinephrine and norepinephrine inhibited VIP-induced cyclic AMP accumulation by 50-55% with KD values of 50 nM and 300 nM respectively. Clonidine was a partial agonist only, provoking a weak (25-30%) inhibition of VIP-induced cyclic AMP accumulation even at high concentrations. These results indicate that, like normal colocytes, human colon adenocarcinoma cells HT 29 possess alpha 2-adrenoceptors, the stimulation of which is associated with an inhibition of cyclic AMP production.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological characterization of contractile responses induced by alpha 1-agonists, norepinephrine and clonidine, by selective antagonists of their subtypes in rabbit thoracic aorta.

In the rabbit isolated thoracic aorta, WB 4101 and 5-methylurapidil dose-dependently shifted the concentration-response curves for norepinephrine to the right. Schild plots showed that the inhibition of responses for WB 4101 and 5-methylurapidil was biphasic, implying that norepinephrine acted through two receptor populations. Clonidine produced a concentration-dependent contraction in the isolated rabbit thoracic aorta. WB 4101 and 5-methylurapidil antagonized the contractions for clonidine, and the Schild plot to both antagonists against clonidine yielded a monophasic slope. Schild plots of the results obtained from the inhibition by WB 4101 and 5-methylurapidil for norepinephrine in strips pretreated with chloroethylclonidine yielded a straight line with a slope of unity. Specific binding of [3H]prazosin in the aortic membrane preparations was saturable. The Hill coefficient obtained from the inhibition curves for clonidine was significantly different from unity. Clonidine interacted with two binding sites labelled by [3H]prazosin, but the low affinity site was completely eliminated by pretreatment with 10 microM chloroethylclonidine. These results suggest that the subtype activated by norepinephrine is different from that activated by clonidine, and that norepinephrine-induced contraction through both alpha 1A- and alpha 1B-subtypes and clonidine through only the alpha 1A-subtype in the rabbit thoracic aorta.     

Subtypes of alpha 1-adrenoceptors in hippocampus of pigs, guinea-pigs, calves and humans: regional differences

Radioligand binding studies were performed with membranes of guinea-pig, pig, calf and human hippocampus using [125I]BE 2254 (also known as [125I]HEAT) as the radioligand. [125I]BE 2254 bound with similar high affinity to saturable populations of recognition sites in all four membrane preparations. Competition curves obtained with a variety of ligands (e.g., WB 4101, benoxathian, 5-methyl-urapidil) were biphasic and the profiles of the high- and low-affinity components of [125I]BE 2254 binding were similar in all four membrane preparations. The data suggest that [125I]BE 2254 labels two subtypes of alpha 1-adrenoceptors in the hippocampus of these species. [3H]WB 4101 was used to label alpha 1A recognition sites in pig hippocampus membranes. [3H]WB 4101 recognized with high affinity an apparently homogeneous class of sites, as suggested by monophasic saturation and competition experiments. The rank order of affinity of the compounds for the high-affinity component of [125I]BE 2254 binding was similar to the rank order of affinity of these drugs for [3H]WB 4101 sites. The pharmacological profile of the low-affinity component of [125I]BE 2254 binding was similar to that described recently for the alpha 1B-adrenoceptor cloned from DDT1 cells. In autoradiographic studies with human hippocampal slices, CEC (chloroethylclonidine), an alkylating agent described to show selectivity for alpha 1B-adrenoceptors, displaced preferentially [125I]BE 2254 binding from the molecular layer of the dentate gyrus. In contrast, WB 4101 an alpha 1A-adrenoceptor-selective ligand, displaced preferentially [125I]BE 2254 binding in the hilus and the CA3 region. The data show that 2 subtypes of alpha 1-adrenergic recognition sites can be identified in the hippocampus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective agonists reveal alpha(1A)- and alpha(1B)-adrenoceptor subtypes in caudal artery of the young rat

Multiple alpha(1)-adrenoceptors were evaluated in caudal artery of the young Wistar rat using selective agonists and antagonists. Arteries were exposed to the selective alpha(1A)-adrenoceptor agonist, A-61603 (N-[5-(4,5-dihydro-1H-imidazol-2-yl)-2-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl] methanesulfonamide) or to phenylephrine and to prazosin (alpha(1)-adrenoceptor antagonist), or the selective alpha(1A)-adrenoceptor antagonists 5-methylurapidil, RS 100329 (5-methyl-3-[3-[4-[2-(2,2,2,-trifluoroethoxy)phenyl]-1-piperazinyl]propyl]-2,4-(1H)-pyrimidinedione), RS 17053 (N-[2(2-cyclopropylmethoxy) ethyl]-5-chloro-alpha, alpha-dimethyl-1H-indole-3-ethanamide), and the selective alpha(1D)-adrenoceptor antagonist BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5] decane-7,9-dione). Results showed a 100-fold higher potency of A-61603 for the alpha(1)-adrenoceptor present in the artery, compared with phenylephrine. Prazosin displaced both agonists with high affinity, whereas 5-methylurapidil, RS 100329 and RS 17053 displaced A-61603 with high affinity, indicating the presence of alpha(1A)-adrenoceptors. The selective alpha(1A)-adrenoceptor antagonists blocked phenylephrine responses with low affinity, suggesting that phenylephrine activated a second receptor population in caudal artery. BMY 7378 antagonized with low affinity both A-61603 and phenylephrine-induced contractions, indicating absence of alpha(1D)-adrenoceptors in the vessel. The results suggest that functional alpha(1B)-adrenoceptors are present in caudal arteries of the young Wistar rat.     

Heterogeneity of mammalian alpha 2-adrenoceptors delineated by [3H]yohimbine binding

[3H]Yohimbine binding to membrane preparations of human colon, cerebral cortex, kidney, spleen and platelets was compared with binding to preparations of animal tissues (rabbit spleen, kidney and cerebral cortex; rat cerebral cortex; guinea-pig and cat spleen). Specific binding to all preparations was saturable and indicative of binding to a uniform population of sites. The equilibrium dissociation constants (KD) of [3H]yohimbine ranged from 1.6 to 2.6 nM for human tissue and from 5.1 to 9.4 nM for the animal tissues. Binding to all tissues was displaced by drugs with an order of potency yohimbine greater than phentolamine greater than prazosin, indicating an alpha 2-adrenoceptor classification of the labelled sites. Whilst certain drugs (phentolamine, corynanthine) possessed similar affinities for all alpha 2-adrenoceptors, others (prazosin, idazoxan, WY 26392) exhibited differential potencies for alpha 2-adrenoceptors in certain species. The pharmacological characteristics of human alpha 2-adrenoceptors were conserved within the tissues examined. These results suggest that human alpha 2-adrenoceptors differ in a number of ways from those present in tissues from the other mammalian species examined. The possible existence of a spectrum of alpha 2-adrenoceptors is discussed in the light of these findings.     

Different alpha-adrenoceptors modulate the release of 5-hydroxytryptamine and noradrenaline in rat cortex.

1 The potassium-evoked release of [3H]-noradrenaline from slices of rat occipital cortex and the potassium-evoked release of [3H]-5-hydroxytryptamine from slices of rat frontal cortex were measured using a superfusion system. 2 The rank order of potency for a number of alpha-adrenoceptor agonists was different for the two neuronal systems, clonidine and azepexole being the most potent inhibitors of noradrenaline release and methoxamine and phenylephrine being the most potent against 5-hydroxytryptamine release. 3 The rank order of potency for a series of alpha-adrenoceptor antagonists in reversing the inhibition of noradrenaline release produced by clonidine was: phentolamine greater than rauwolscine = yohimbine = corynanthine much greater than WB4101, whereas against methoxamine-inhibition of 5-hydroxytryptamine release the rank order of potency was: WB4101 greater than phentolamine greater than corynanthine greater than yohimbine greater than rauwolscine. 4 The results suggest that the alpha-adrenoceptors which modulate potassium-evoked 5-hydroxytryptamine release are not identical with the alpha 2-adrenoceptors which modulate potassium-evoked 5-hydroxytryptamine release are not identical with the alpha 2-adrenoceptors located on noradrenergic nerve terminals and may more closely resemble alpha 1-than alpha 2-adrenoceptors.     

Further characterization of the myocardial alpha-adrenoceptors mediating positive inotropic effects in the rabbit myocardium

[3H]Prazosin bound with high affinity to the membrane fraction derived from the rabbit ventricular myocardium. Oxymetazoline displaced [3H]prazosin from its binding site, did not elicit a positive inotropic effect but antagonized the positive inotropic effect of phenylephrine mediated by alpha-adrenoceptors in the presence of a beta-antagonist. Naphazoline was more potent in displacing [3H]prazosin and behaved as a weak partial agonist. YM-12617 (5-[2-[[2-(2-ethoxyphenoxy)ethyl]amino]propyl]-2- methoxybenzenesulfonamide HCl), a potent selective alpha 1-antagonist, displaced [3H]prazosin and antagonized the alpha-mediated positive inotropic effect with equal potency. Thus, a good correlation was found between the potency of alpha-antagonists to displace [3H]prazosin and their ability to antagonize the alpha-mediated positive inotropic effect. On the other hand, there was no significant correlation between the Ki and the pD2 value of the alpha-agonists (norepinephrine, epinephrine, phenylephrine and naphazoline), indicating that there is a non-linear relationship between agonist binding to myocardial alpha 1-adrenoceptors and subsequent functional changes. Myocardial alpha 1-adrenoceptors showed some pharmacological characteristics which appear to be different from those in smooth muscle tissues.     

Characterisation of α1B-adrenoceptors linked to inositol phosphate formation and calcium mobilisation in human astrocytoma U373 MG cells

The human U373 MG astrocytoma cell line has been widely used as a model system for the investigation of astrocyte function. The aim of this study was to establish which alpha1-adrenoceptors are present on these cells. The specific binding of [3H]prazosin to membranes of U373 MG cells (Bmax 32+/-3 fmol mg(-1) protein, Kd 0.27+/-0.03 nM) was inhibited in a monophasic manner by alpha1-antagonists that have different affinities for alpha1A-, alpha1B- and alpha1D-adrenoceptors. Estimates for pKi values were: prazosin 9.69+/-0.06, 5-methylurapidil 7.10+/-0.21; (+)-niguldipine 7.06+/-0.26; WB 4101 8.26+/-0.16; and BMY 7378 6.60+/-0.21. The specific binding of [3H]prazosin was reduced to low levels by pretreatment of cells with 10 microM chloroethylclonidine for 15 min. In the presence of 30 mM LiCl, 100 microM noradrenaline stimulated [3H]inositol phosphate accumulation by 2.1+/-0.1-fold of basal after 30-min incubation. The EC50 for the accumulation of [3H]IP1, the major product detected (85+/-2% of total [3H]IP1 + [3H]IP2 + [3H]IP3), was 0.38+/-0.05 microM. Noradrenaline-induced [3H]IP1 accumulation was also inhibited by alpha1-antagonists. Estimates for pKi values were: 5-methylurapidil 6.95+/-0.01; WB 4101 8.31+/-0.07; and BMY 7378 6.71+/-0.28. The accumulation of [3H]IP1 in response to 100 microM noradrenaline was not significantly affected by raising the extracellular Ca2+ concentration from 1.3 to 4 mM. Noradrenaline (100 microM) also produced an increase in intracellular Ca2+ (mean peak 86+/-5 nM above basal). Pretreatment with chloroethylclonidine (10 microM, 15 min) abolished noradrenaline-induced [3H]IP1 accumulation and Ca2+ mobilisation. Activation of the alpha1B-adrenoceptors by 10 microM phenylephrine increased [3H]thymidine uptake to 140+/-5% of control uptake. Taken together, these results indicate that U373 MG cells express a single class of alpha1-adrenoceptors, the alpha1B-subtype, which are coupled to phosphoinositide hydrolysis and calcium mobilisation, and which mediate a mitogenic response to alpha1-agonists.     

Binding of [3H]idazoxan and of its methoxy derivative [3H] RX821002 in human fat cells: [3H]idazoxan but not [3H] RX821002 labels additional non-alpha 2-adrenergic binding sites

Binding studies were carried out in human fat cell membranes with two alpha 2-adrenergic antagonists, [3H]idazoxan and its methoxy derivative [3H]RX821002. Inhibition studies with epinephrine enantiomers indicate that [3H]RX821002 only binds to alpha 2-adrenoceptors, whereas [3H]idazoxan labels alpha 2-adrenoceptors and additional nonadrenergic sites (NAIBS). NAIBS and alpha 2-adrenoceptors display different affinities towards drugs from various chemical families. Imidazoline and some guanidine derivatives exhibit a high affinity for NAIBS. Pharmacological studies of human NAIBS indicate that they are slightly different from those previously reported in the rabbit, suggesting the existence of several subtypes of NAIBS. Furthermore, NAIBS are different from the previously described "imidazoline-preferring sites." [3H]idazoxan and [3H]RX821002 saturation analyses were performed in human adipocytes from different anatomical locations, in order to compare the number of NAIBS and alpha 2-adrenoceptors. Although there was an important variation in NAIBS and alpha 2-adrenoceptor numbers in the studied samples, a very poor correlation was obtained between the Bmax values of the two sites. Moreover, alkylation of alpha 2-adrenoceptors by phenoxybenzamine produces a 90% reduction in accessible [3H]RX821002 binding sites, without modification of [3H]idazoxan binding. These data show that NAIBS are not closely related to the alpha 2-adrenergic molecule. In addition, benextramine appears to be a reversible competitor at NAIBS. [3H]idazoxan binding, but not [3H]RX821002 binding, is sensitive to K+, suggesting that the domains involved in the ligand-NAIBS interaction are different from those involved in the ligand-alpha 2-adrenoceptor interaction.     

Alpha 1-adrenoceptor subtypes in aorta (alpha 1A) and liver (alpha 1B).

The effect of several alpha 1 adrenoceptor antagonists on the alpha 1-adrenoceptor-mediated stimulation of phosphatidylinositol labeling was studied comparatively in rat hepatocytes and rabbit aorta. It was observed that 5-methyl urapidil and WB 4101 were much more potent in rabbit aorta than in hepatocytes. The orders of potency were prazosin much greater than 5-methyl urapidil greater than or equal to WB 4101 in liver cells and WB 4101 greater than or equal to 5 methyl urapidil = prazosin in aorta. Treatment with chlorethylclonidine inhibited 70-80% of the stimulation of labeling induced by epinephrine in rat liver, but only 30-40% of that in aorta. Our data suggest the existence of two pharmacologically distinct receptors in these tissues i.e.m alpha 1A-adrenoceptors in aorta and alpha 1B in liver cells.     

Alpha-adrenoceptor antagonism by apoyohimbine and some observations on the pharmacology of alpha-adrenoceptors in the rat anococcygeus and vas deferens

alpha-Adrenoceptor antagonism of several test drugs was assessed against adrenergic contractile responses to field stimulation in rat vas deferens and anococcygeus, the prejunctional inhibitory effect of xylazine in vas deferens and the contractile effects of alpha-adrenoceptor agonists in anococcygeus. Against the adrenergic nerve-induced contraction in vas deferens, the potency series was WB 4101 greater than prazosin greater than apoyohimbine greater than corynanthine greater than yohimbine greater than rauwolscine. Against the inhibitory effect of xylazine in vas deferens the potency series was apoyohimbine greater than rauwolscine = yohimbine greater than WB 4101 much greater than prazosin and corynanthine. In anococcygeus, against the contractile responses to adrenergic nerve stimulation or to the agonists amidephrine, noradrenaline and xylazine, the potency series was apoyohimbine greater than corynanthine greater than rauwolscine. These results show that apoyohimbine is more potent than the yohimbine sterioisomers as an antagonist at alpha 1- and alpha 2-adrenoceptors but is not more selective. The assay methods employed confirm the current classification of 'alpha'-receptors and drugs.     

Binding and functional characterization of alpha1-adrenoceptor subtypes in the rat prostate

The alpha1-adrenoceptor subtypes of rat prostate were characterized in binding and functional experiments. In binding experiments, [3H]tamsulosin bound to a single class of binding sites with an affinity (pKD) of 10.79+/-0.04 and Bmax of 87+/-2 fmol mg(-1) protein. This binding was inhibited by prazosin, 2-(2,6-dimethoxy-phenoxyethyl)-aminomethyl-1,4-benzodioxane hydrochloride (WB4101), 5-methylurapidil, alpha-ethyl-3,4,5,-trimethoxy-alpha-(3-((2-(2-methoxyphenoxy)ethyl)-amin o)-propyl)benzeneacetonitrile fumarate (HV723) and oxymetazoline with high efficacy, resulting in a good correlation with the binding characteristics of cloned alpha1a but not alpha1b and alpha1d-adrenoceptor subtypes. In functional studies, noradrenaline and oxymetazoline produced concentration-dependent contractions. These contractions were antagonized by tamsulosin, prazosin, WB4101 and 5-methylurapidil with an efficacy lower than that exhibited by these agents for inhibition of [3H]tamsulosin binding. The relationship between receptor occupancy and contractile amplitude revealed the presence of receptor reserve for noradrenaline, but the contraction induced by oxymetazoline was not in parallel with receptor occupation and developed after predicted receptor saturation. From these results, it is suggested that alpha1A-adrenoceptors are the dominant subtype in the rat prostate which can be detected with [3H]tamsulosin, but that the functional subtype mediating adrenergic contractions has the characteristics of the alpha1L-adrenoceptor subtype, having a lower affinity for prazosin and some other drugs than the alpha1A-adrenoceptor subtype.     

Effect of detergent solubilization on the affinity of some quinazoline derivatives for the alpha 1-adrenoceptor

[3H]Prazosin bound to a single class of high-affinity sites in both bovine aortic and rat hepatic membranes. The absolute affinity values of displacing ligands (prazosin greater than doxazosin much greater than trimazosin greater than yohimbine) were the same for both tissues. After solubilization of the alpha 1-adrenoceptors with digitonin and 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate, an identical rank order potency was observed. However, solubilization significantly reduced ligand affinity. In both tissues the affinity of prazosin was reduced 10- to 13-fold, whereas the affinities of doxazosin, trimazosin and yohimbine were reduced two- to six-fold. There appeared to be no relationship between the lipophilicities of the ligands and the degree to which affinity is affected by solubilization. The results suggest that the reductions in affinity are the consequence of a conformational change in the alpha 1-adrenoceptor and appear to support the hypothesis that the alpha 1-adrenoceptor is so constructed that the spatial configuration of the binding site can change in response to an alteration in its microenvironment.     

RX781094 a potent and selective alpha 2-adrenergic antagonist. Effects in adipocytes and hepatocytes

The potency and selectivity of RX781094, 2-(2-(1,4-benzodioxanyl]-2-imidazoline HCl, as alpha 1- and alpha 2-adrenergic antagonist was studied using rat hepatocytes and hamster adipocytes. The alpha 1-adrenergic stimulation of ureogenesis produced by epinephrine in rat hepatocytes was slightly diminished by 10(-4) M RX781094. On the contrary the alpha 2-adrenergic effect of epinephrine in hamster adipocytes (inhibition of adenylate cyclase) was antagonized dose-dependently by RX781094. This agent was approximately 10-fold more potent than yohimbine. Radioligand binding studies also showed that RX781094 was more potent and selective than yohimbine at alpha 2-adrenergic sites. It is concluded that RX781094 is a potent and selective alpha 2-adrenergic antagonist.