Studies on YM-12617: A selective and potent antagonist of postsynaptic α1-adrenoceptors

YM-12617, 5-[2-[[2-(2-ethoxyphenoxy)ethyl]-amino]propyl]-2 -methoxybenzenesulfonamide HCl is a structurally new type of extremely potent alpha 1-adrenoceptor antagonist. Its alpha-adrenoceptor blocking properties have been compared with those of prazosin, phentolamine and yohimbine using both pharmacological and 3H-ligand binding techniques in vitro and in vivo. In the isolated rabbit aorta, a tissue known to contain mainly alpha 1-adrenoceptors at postjunctional sites, YM-12617 competitively antagonized noradrenaline-induced contraction with a pA2 value of 10.11. Although YM-12617 was also a competitive antagonist toward clonidine at prejunctional alpha 2-adrenoceptors in the isolated rat vas deferens, its affinity for these receptors (pA2 = 6.41) was 5,000 times lower than that displayed for the postjunctional alpha 1-adrenoceptors in the isolated rabbit aorta. YM-12617 displaced both 3H-WB 4101 and 3H-clonidine binding to rat brain membranes; however, the affinity of YM-12617 for alpha 1-adrenoceptors (pKi = 9.64) was 3800 times higher than that for alpha 2-adrenoceptors (pKi = 6.06). Based on pA2 values obtained in the isolated tissues and pKi values in the binding assays, YM-12617 was 2-18, 36-117 and 1,740-5,750 times more potent than prazosin, phentolamine and yohimbine in antagonizing alpha 1-adrenoceptors, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Assessment of imiloxan as a selective alpha 2B-adrenoceptor antagonist

1. The alpha 2-adrenoceptor binding sites of rabbit spleen and rat kidney, labelled with [3H]-rauwolscine, were characterized using a range of subtype selective ligands. 2. In rabbit spleen, the alpha-2-adrenoceptor binding sites displayed high affinity for oxymetazoline and WB 4101 and low affinity for prazosin and chlorpromazine suggesting the presence of an alpha 2A subtype. 3. There was evidence for heterogeneity of the alpha 2-adrenoceptor binding sites present in rabbit spleen. The results obtained with oxymetazoline and WB 4101 indicated that at least 75% of the [3H]-rauwolscine binding sites in this preparation displayed a pharmacology consistent with the presence of an alpha 2A subtype. 4. In rat kidney, the alpha 2-adrenoceptor binding sites displayed high affinity for prazosin and chlorpromazine and low affinity for oxymetazoline and WB 4101 suggesting the presence of an alpha 2B subtype. 5. The inclusion of guanylylimidodiphosphate (Gpp(NH)p, 0.1 mM) did not modify the pharmacology of the alpha 2-adrenoceptor binding sites present in the two preparations. Furthermore, when the two membrane preparations were combined, the resultant pharmacology was still consistent with the presence of two receptors that retained the characteristics of the alpha 2A and alpha 2B subtypes. 6. Imiloxan was identified as a selective alpha 2B ligand while benoxathian displayed a high degree of selectivity for the alpha 2A-adrenoceptor binding site. The selectivity of imiloxan for the alpha 2B-adrenoceptor binding site, coupled with its specificity for alpha 2-adrenoceptors, should make it a valuable tool in the classification of alpha 2-adrenoceptor subtypes.     

Alpha 2-adrenoceptor and catecholamine-insensitive binding sites for [3H]rilmenidine in membranes from rat cerebral cortex.

The kinetic and pharmacological characteristics of the binding of the oxazoline antihypertensive drug, [3H]rilmenidine, to membranes of rat cerebral cortex have been determined. Computerised resolution of curvi-linear, equilibrium binding isotherms was consistent with the existence of two distinct binding sites for [3H]rilmenidine: Kd 17.3 +/- 7.41 nM, Bmax 0.197 +/- 0.06 pmol/mg protein and Kd 254 +/- 48 nM, Bmax 1.59 +/- 0.08 pmol/mg protein. Moreover, the resolution of two association and dissociation rates also suggested the existence of two binding site populations. Drug inhibition studies revealed that specific binding of [3H]rilmenidine (2 nM) was only inhibited by a maximum of 50% by the catecholamines, adrenaline and noradrenaline, but was completely inhibited by some oxazolines, by guanabenz (a guanidino drug) and by several imidazoline compounds including naphazoline, oxymetazoline and clonidine. Binding isotherms for these drugs were also best fit by a two-site model. The relative Ki values at the high affinity site for [3H]rilmenidine and the number of these high affinity sites are consistent with this site being an alpha 2-adrenoceptor. The high affinity of oxymetazoline and low affinity of prazosin for high affinity [3H]rilmenidine binding sites together with the rank order of potency of oxymetazoline greater than phentolamine greater than SKF 104078 greater than ARC-239 greater than prazosin suggest that [3H]rilmenidine binds to the alpha 2A sub-type of adrenoceptor. Computer-resolved Ki values for drugs at the larger number of lower affinity binding sites were very similar to Ki values determined in the presence of 10 microM adrenaline (used to block alpha 2-adrenoceptor binding).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Subclassification of alpha 1-adrenoceptor recognition sites by urapidil derivatives and other selective antagonists

1. The affinities of urapidil derivatives and other antagonists for alpha 1-adrenoceptors labelled by [3H]-prazosin were determined on membranes of six different rat tissues. 2. Urapidil and its 5-acetyl-, 5-formyl- and 5-methyl-derivative displaced [3H]-prazosin from alpha 1-adrenoceptor binding sites in a concentration-dependent manner which varied with tissue. IC50 values were lower in vas deferens, hippocampus and cerebral cortex than in heart, liver and spleen. For 5-methyl-urapidil, binding to two distinct sites could be demonstrated with mean K1 values of about 0.6 and 45 nM. Saturation binding studies with [3H]-prazosin in the presence of 5-methyl-urapidil indicated a competitive type of interaction between 5-methyl-urapidil and [3H]-prazosin. 3. The proportion of [3H]-prazosin binding sites with high affinity for 5-methyl-urapidil was 58% in vas deferens, 69% in hippocampus, 41% in cerebral cortex and 23% in myocardium. In liver and spleen virtually no high affinity sites were found. These values were in good agreement with the percentages of binding sites with high affinities for WB-4101 and phentolamine, indicating that all these antagonists bind to the same subtype of alpha 1-recognition sites, whereas other alpha-antagonists like BE 2254, yohimbine and unlabelled prazosin did not discriminate between two binding sites. 4. Preincubating membranes of the cerebral cortex with chloroethylclonidine preferentially inactivated [3H]-prazosin binding sites with low affinity for 5-methyl-urapidil. 5. The antagonist potencies of 5-methyl-urapidil and WB-4101 against alpha 1- adrenoceptor-mediated contractile responses were higher in vas deferens than in myocardium. The alpha 1-mediated effects in vas deferens but not in the heart were highly susceptible to nitrendipine. 6. Using 5-methyl-urapidil, the existence of two distinct alpha 1-adrenoceptor recognition sites could be demonstrated which correspond to the proposed alpha 1A- and alpha 1B-subtypes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification of a single alpha 1-adrenoceptor corresponding to the alpha 1A-subtype in rat submaxillary gland.

1. The alpha 1-adrenoceptors present in membranes of rat liver, cortex and submaxillary gland were labelled with [3H]-prazosin and the affinity of 15 ligands for these receptors was determined. 2. In saturation studies, [3H]-prazosin bound with high affinity (Kd = 30-39 pM) to a single population of sites in all three preparations. 3. In competition studies using rat cortex, evidence for heterogeneity of the alpha 1-adrenoceptor binding sites was obtained. Displacement isotherms for amidephrine, benoxathian, oxymetazoline, phentolamine and WB 4101 were biphasic and were consistent with the presence of both alpha 1A- and alpha 1B-adrenoceptor subtypes as described by Morrow & Creese (1986) and Han et al. (1987). 4. The rat liver and submaxillary gland membrane preparations both possessed homogeneous populations of alpha 1-adrenoceptors. However, there were pharmacological differences between the receptors in these two preparations. Rat submaxillary gland alpha 1-adrenoceptors displayed high affinity for amidephrine, benoxathian, oxymetazoline, phentolamine and WB 4101 and therefore appeared to represent alpha 1A-adrenoceptors. Rat liver alpha 1-adrenoceptors possessed lower affinity for these ligands (6-65 fold) suggesting that these receptors were of the alpha 1B-subtype. 5. Spiperone exhibited 12.9 fold higher affinity for rat liver alpha 1B-adrenoceptors than for rat submaxillary gland alpha 1A-adrenoceptor and may therefore represent the first alpha 1B-adrenoceptor selective ligand.     

Three distinct binding sites for [3H]-prazosin in the rat cerebral cortex.

1. The putative alpha 1-adrenoceptor subtypes of rat cerebral cortex membranes were characterized in binding. 2. Specific binding of [3H]-prazosin was saturable between 20-5000 pm. Scatchard plots of the binding data were non-linear, indicating the presence of two distinct affinity sites for prazosin (pKD, high = 10.18, Rhigh = 308 fmol mg-1 protein; pKD, low = 8.96, Rlow = 221 fmol mg-1 protein). 3. In the membranes pretreated with chlorethylclonidine (CEC) two affinity sites for prazosin were also observed: the affinities were similar to those without CEC pretreatment, but the maximum numbers of binding sites were reduced by CEC pretreatment to 23 and 62% for prazosin-high (Rhigh) and low affinity sites (Rlow), respectively. 4. The prazosin-high affinity sites were further subdivided into two subclasses by WB4101(2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane) and phentolamine; the low affinity sites for WB4101 and phentolamine were more potently inactivated by CEC as compared with the high affinity sites. On the other hand, prazosin, HV723 (alpha-ethyl-3,4,5-trimethoxy-alpha-(3-((2-(2-methoxyphenoxy)ethyl)- amino )-propyl)benzeneacetonitrile fumarate) and yohimbine inhibited [3H]-prazosin binding to prazosin-high affinity sites monophasically. 5. In addition to the high affinity sites, the prazosin-low affinity sites were labelled at high concentrations of [3H]-prazosin. Thus, prazosin and WB4101 showed shallow displacement curves. On the other hand, HV723 and yohimbine did not discriminate between prazosin-high and low affinity sites. 6. Two distinct alpha 1-adrenoceptor subclassifications have been recently proposed (alpha 1A, alpha 1B subtypes and alpha 1H, alpha 1L, alpha 1N subtypes).(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

Alpha 1-adrenoceptor subtypes in the rat ventricular muscle.

Scatchard analyses of [3H]prazosin binding in rat ventricular muscle membranes showed biphasic curves, which identified alpha 1High- and alpha 1Low-affinity sites. The alpha 1High-affinity site was completely inhibited by 1 microM phenoxybenzamine. The displacement potencies of alpha 1-adrenergic antagonists were characterized by [3H]prazosin binding to alpha 1High- and alpha 1Low-affinity sites in the absence and presence of 1 microM phenoxybenzamine. The affinities of most chemicals for alpha 1Low-affinity sites were significantly lower than those for alpha 1High-affinity sites, but WB-4101 (2-(2,6-dimethoxy-phenoxyethyl)aminomethyl-1,4-benzodioxane), arotinolol, cinanserin, nifedipine, and p-aminoclonidine had the same affinities for both alpha 1Low- and alpha 1High-affinity sites. These results show that two alpha 1-adrenoceptor subtypes, alpha 1High- and alpha 1Low-affinity, are present in the rat heart, and that there are physical variations in alpha 1-adrenoceptor binding sites, based on their selectivity to antagonists.     

Labelling of human platelet alpha 2-adrenoceptors with the full agonist [3H](-)adrenaline

[3H](-)Adrenaline has been used to characterize alpha 2-adrenoceptors on human platelets. Although (-)adrenaline is a good substrate for the platelet enzyme MAO-B, enzymatic inhibition was not a prerequisite to quantify the specific binding of the radioligand to platelet membranes. At 25 degrees C the binding was rapid (t1/2 of association: 10.3 min), reversible (t1/2 of dissociation: 4.0 min) and linearly dependent on the amount of protein present in the assay. The binding sites for [3H](-)adrenaline showed the specificity required for an alpha 2-adrenoceptor. The rank order of potency of inhibitors of [3H](-)adrenaline binding was oxymetazoline greater than idazoxan congruent to phentolamine congruent to clonidine congruent to (-)adrenaline greater than (-)noradrenaline greater than yohimbine much much greater than phenylephrine much greater than prazosin greater than (+)propranolol. Moreover, the nucleotide guanosine triphosphate (GTP) inhibited in a concentration-dependent manner (10(-9)-10(-4) M) the specific binding of [3H](-)adrenaline, suggesting that the radioligand preferentially labelled the high affinity state of the alpha 2-adrenoceptor. Linear (Scatchard) and non-linear analyses of [3H](-)adrenaline binding indicated the existence of a single population of non-interacting sites (KD = 2.5-2.7 nM; Bmax = 49-53 fmol/mg protein). The binding characteristics for [3H](-)adrenaline were not affected by age and sex of the donors or by freezing of platelet-rich plasma. In the same subjects alpha 2-adrenoceptor density (Bmax) for the full agonist [3H](-)adrenaline was 2.9-fold lower than that quantitated by the selective antagonist [3H]yohimbine. The inhibition constants (Ki) of adrenergic drugs and of various antidepressant drugs in competing with [3H](-)adrenaline were correlated with the inhibition constants of these drugs in competing with [3H]clonidine (r = 0.96; P less than 0.001) which suggests that both radioligands labelled the same alpha 2-adrenoceptor on the human platelet. The binding of the full agonist [3H](-)adrenaline to human platelet membranes might be a useful tool for the study of dysfunctions related to the high affinity state of the alpha 2-adrenoceptor.     

Effect of postganglionic denervation and pretreatment with reserpine on alpha-adrenoceptors of the guinea-pig vas deferens

The radiolabeled alpha-1-adrenoceptor antagonist [3H]WB-4101 was used to determine whether supersensitivity of the guinea pig vas deferens that develops after denervation or reserpine pretreatment is associated with a change in the characteristics of the alpha-1-adrenoceptor. Specific binding of [3H]WB-4101 to membrane fractions of vasa deferentia was saturable and reversible. The dissociation constant (KD) was similar when obtained by equilibrium or kinetic studies. As determined by Scatchard analysis, neither chronic (5 days) pretreatment with reserpine (1.0 mg/kg/day) nor postganglionic denervation (7 days) altered the number or affinity of [3H] WB-4101 binding sites. It is concluded that changes in alpha-1-receptor characteristics are not responsible for postjunctional supersensitivity in this tissue.     

3H]-RS-15385-197, a selective and high affinity radioligand for alpha 2-adrenoceptors: implications for receptor classification.

1. RS-15385-197 is the most potent and selective alpha 2-adrenoceptor antagonist available. We have used [3H]-RS-15385-197 to define alpha 2-adrenoceptor subtypes. The binding of [3H]-RS-15385-197 to membranes of rat cerebral cortex, rat neonatal lung and human platelets was reversible, saturable and of high affinity. Saturation experiments indicated that [3H]-RS-15385-197 bound to a single population of sites in all 3 tissues with high affinity (0.08-0.14 nM). The density of sites labelled by [3H]-RS-15385-197 was greater in the cortex (275 fmol mg-1 protein) than in the neonate lung (174 fmol mg-1 protein) and human platelet (170 fmol mg-1 protein). The density of sites labelled with [3H]-RS-15385-197 in the cortex was significantly greater than that labelled with [3H]-yohimbine (121 fmol mg-1 protein). 2. The selective alpha 2-adrenoceptor antagonists, idazoxan, yohimbine, rauwolscine and WY 26703 displaced [3H]-RS-15385-197 binding to rat cerebral cortex in a simple manner with Hill slopes close to unity. The affinities derived for these antagonists against [3H]-RS-15385-197 were similar to the values obtained for the displacement of [3H]-yohimbine indicating the alpha 2-adrenoceptor nature of the binding site. 3. alpha 2A-Adrenoceptor selective compounds, oxymetazoline and BRL 44409, showed high affinity for [3H]-RS-15385-197 binding in the human platelet and lower affinity in the neonate lung, while the alpha 2B-selective compounds, prazosin and imiloxan, showed high affinity for [3H]-RS-15385-197 binding in the neonate lung.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of electroconvulsive shock or imipramine on subtypes of alpha 1-adrenoceptors in the frontal cortex of the rat.

The effects of repeated treatment (14 days) with electroconvulsive shock (ECS) or imipramine on binding sites on alpha 1-adrenoceptors in the rat were studied. The binding of [3H]prazosin studied with WB4101 and phentolamine, as binding inhibitors, showed the existence of two subtypes of alpha 1-adrenoceptor (alpha 1A and alpha 1B). Proportions of the alpha 1A and alpha 1B binding sites were about 3:7 in the frontal cortex and 9:1 in the hippocampus. Pretreatment of the membranes with chlorethylclonidine (CEC) almost abolished the alpha 1B binding sites. Inhibition of the binding of [3H]prazosin studied with antidepressants (imipramine, desipramine, maprotiline and mianserin) showed that these drugs bound to alpha 1-adrenoceptors with low affinity, in an apparent monophasic manner. The characteristics of the alpha 1A and alpha 1B binding sites were studied by the binding assay with [3H]prazosin, in the presence of a small concentration (2 nM) of WB4101 to mask the alpha 1A binding sites, as well as the assay without WB4101, for the total alpha 1-adrenoceptor (alpha 1A and alpha 1B) binding. Repeated treatment with electroconvulsive shock increased but that with imipramine decreased, the density of the alpha 1B binding sites in the frontal cortex, without change of the affinity. Neither treatment affected the alpha 1A binding sites in the frontal cortex. The alpha 1-adrenoceptors (alpha 1A and alpha 1B) in the hippocampus were not affected at all by these repeated treatments. The electroconvulsive shock-induced increase in the alpha 1B binding sites in the frontal cortex of the rat could contribute to differences in clinical effects between electroconvulsive shock and antidepressant drugs.     

Heterogeneity of alpha 2-adrenoceptors in rat cortex but not human platelets can be defined by 8-OH-DPAT, RU 24969 and methysergide.

1. Saturation experiments indicated that [3H]-yohimbine binding was specific, saturable and labelled a single population of sites in rat cerebral cortex (Kd 5.3 +/- 0.9 nM, Bmax 121 +/- 10 fmol mg-1 protein) and human platelets (Kd 0.7 +/- 0.1 nM, Bmax 152 +/- 10 fmol mg-1 protein). 2. The alpha 2-adrenoceptor antagonists, yohimbine, rauwolscine, WY 26703, idazoxan and BDF 6143 displaced [3H]-yohimbine binding to each tissue in a simple manner, with high affinity and Hill slopes close to unity. 3. The alpha 1-adrenoceptor agonist, oxymetazoline and the antagonist prazosin inhibited the binding of [3H]-yohimbine to rat in a complex manner consistent with an interaction at more than one site. However, indoramin and WB 4101 only appeared to interact with one site. In contrast, in human platelets, all antagonists gave rise to monophasic displacement curves with Hill slopes close to unity suggesting a single site of interaction. 4. The 5-hydroxytryptamine (5-HT) receptor ligands, 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT), RU 24969, and methysergide inhibited the binding of [3H]-yohimbine to rat cortex with high and low affinity, consistent with an interaction with two populations of binding sites. However, inhibition of [3H]-yohimbine binding to human platelets suggested a single site of interaction. The low affinity of 5-HT, 5-carboxyamidotryptamine (5-CT) and dipropyl-5-CT indicated that [3H]-yohimbine was not labelling a 5-HT1-like site in rat cortex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of agonist and antagonist interactions at kidney alpha 1-adrenoceptors by nucleotides and metal ions

In order to characterize putative high- and low-affinity states of the renal alpha 1-adrenoceptor, binding sites for the selective antagonist radioligand [3H]prazosin were examined in washed membranes prepared from rat renal cortex and medulla. Norepinephrine competition curves at [3H]prazosin sites were biphasic and were best fit by a two-site model. Na+ and GTP selectively decreased the proportion of sites exhibiting a high affinity for norepinephrine. In contrast, Mg2+ facilitated high-affinity interactions of norepinephrine at the renal alpha 1-receptor. Guanine nucleotides and Na+ increased the affinity of some antagonists [( 3H]prazosin, WB-4101), but not others (phentolamine). Mg2+ again had opposite effects. The effects of ions and nucleotides on both agonist and antagonist interactions were concentration-dependent. The order of potencies for monovalent cations (Na+ greater than Li+ much greater than K+), divalent cations (Mn2+ greater than Mg2+) and nucleotides (Gpp (NH)p, GTP much greater than GMP, ATP) were similar to those reported for cyclase-coupled receptor systems. However, unlike other divalent cations Ca2+ decreased both agonist and antagonist binding, possibly due to a Ca2+-sensitive proteinase. Receptor binding properties were similar in renal cortex and medulla. Renal alpha 1-receptor sites appear to display high- and low-affinity states with respect to agonists, and the equilibrium between these states may be modulated by guanine nucleotides and mono- and divalent metal ions. Some antagonists appear to bind preferentially to sites with low agonist affinity, and this effect is probably independent of retained endogenous catecholamines.     

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.     

Sodium modulation of 3H-agonist and 3H-antagonist binding to alpha 2-adrenoceptor subtypes.

1. The alpha 2-adrenoceptors on human platelets and neonatal rat lung were characterized with the agonist and antagonist ligands [3H]-adrenaline and [3H]-RS-15385-197 respectively. A correlation of affinities for 3H-antagonist binding showed the receptors to be of the alpha 2A-(platelet) and alpha 2B-(neonatal rat lung) adrenoceptor subtypes, whereas a correlation of affinities for 3H-agonist binding showed the receptors to have similar characteristics (r = 0.88). 2. NaCl (100 mM) had no effect on [3H]-RS-15385-197 binding in the human platelet, but increased the density of sites labelled with [3H]-RS-15385-197 in neonatal rat lung by 52%. NaCl increased the density of sites labelled by [3H]-adrenaline in neonatal rat lung, but there was a consequent 3.5 fold decrease in affinity. In the human platelet, no specific [3H]-adrenaline binding was observed in the presence of 100 mM NaCl. 3. In the neonatal rat lung, NaCl had no significant effect on the affinity of prazosin for [3H]-RS-15385-197 binding; however, imiloxan affinity was increased 13 fold. The affinity of the catecholamines, adrenaline and noradrenaline was significantly decreased, whereas the imidazolines, oxymetazoline and UK-14,304 were much less affected. The affinity of prazosin and imiloxan for [3H]-adrenaline binding was significantly increased in the presence of 10 and 100 mM NaCl. Conversely, the affinity of adrenaline and noradrenaline was decreased in the presence of NaCl, and there was no change in the affinity of the imidazoline agonists.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological characterization and regional distribution of alpha-noradrenergic binding sites of rat spinal cord

In order better to interpret their physiological role in rat spinal cord, we characterized binding sites of [3H]WB-4101 and [3H]p-aminoclonidine ( [3H] PAC), and determined their regional distribution. These binding sites have characteristics required for, respectively, alpha 1 and alpha 2 receptors of norepinephrine. Binding to these sites is saturable, with Kd values of 0.38 nM and 35 nM for high and low affinity binding sites respectively of [3H]WB-4101; and 1.7 nM, for a single binding site of [3H]PAC. For whole cord, Bmax values are 52 and 320 (high and low affinity sites respectively); and 21 fmol/mg protein. Catecholamines compete stereoselectively for these sites, while selected noradrenergic agents compete with an order of potency corresponding to their relative activity at the alpha 1 and alpha 2 receptors. We conclude that spinal alpha 1 and alpha 2 binding sites have the same pharmacologic properties as corresponding peripheral sites. The alpha 2 and, to a lesser degree, the alpha 1 binding sites vary in concentration with region. Our results support the contention that alpha 2 binding sites subserve neuronal function in the spinal cord.     

Binding of adrenergic ligands ([3H]clonidine and [3H]WB-4101) to multiple sites in human brain

In order to identify alpha-adrenoceptors in post-mortem human brain and to detect the possible existence of multiple types of binding sites for adrenergic [³H]ligands, we studied the binding of [³H]clonidine and [³H]WB-4101 to human brain cerebral cortex, hippocampus, hypothalamus and striatum. Frontal cortex revealed two binding sites for [³H]clonidine (with K_D values of approximately 1 and 8 nM), as indicated by the biphasic Scatchard plot, the biphasic pattern of dissociation kinetics, and the biphasic inhibition by phentolamine on the binding of [³H]clonidine; the high-affinity site was heat-labile. Two high-affinity binding sites for [³H]WB-4101 were also detected in the human frontal cortex (with K_D values of about 0.09 and 1.5 nM), as revealed by a biphasic pattern of dissociation. A third site with low affinity binding for [³H]WB-4101 was detected by the biphasic inhibition by phentolamine (as well as by WB-4101 and prazosin) on the binding of [³H]WB-4101. The three other brain regions revealed very similar patterns exhibited by the frontal cortex, except that the density of the [³H]clonidine sites (of either high or low affinity) was highest in the hypothalamus, whereas the density of [³H]WB-4101 sites was highest in the hippocampus.