Guanine nucleotides and alpha1 adrenergic receptors in the heart

Guanine nucleotides such as GTP and Gpp(NH)p are known to regulate the affinity of beta and alpha₂ adrenergic receptors for agonists as assessed by radioligand binding techniques. Recent studies in the rat heart using the radioligand [³H]WB4101, which reportedly labels alpha₁ adrenergic receptors, have suggested that the affinity of alpha₁ adrenergic receptors for epinephrine is altered by guanine nucleotides. To assess the possible role of guanine nucleotides in alpha₁ adrenergic function, we have constructed (?)epinephrine competition curves in the absence and presence of guanine nucleotides using the highly alpha₁ subtype selective agents [³H]prazosin and [¹²⁵I]BE2254 in rat cardiac membranes. Epinephrine competition curves in the absence of guanine nucleotides were steep and uniphasic in character, and the addition of Gpp(NH)p (10^?4 M) had no effect on the ability of epinephrine to compete for either [³H]prazosin or [¹²⁵I]BE2254 binding sites. In this same membrane preparation, the ability of (?)isoproterenol to compete with [³H]dihydroalprenolol for beta adrenergic receptors binding sites was decreased significantly by Gpp(NH)p. These findings demonstrate that, under conditions where guanine nucleotide regulation of agonist-receptor binding in these membranes can be observed, no nucleotide regulation of agonist-alpha₁ receptor interactions was evidenced using subtype selective radioligands. These results suggest that previous reports of agonist-alpha₁ receptor regulation by guanine nucleotides may represent a manifestation of the anomalous binding characteristics of [³H]WB4101 as compared with [³H]prazosin and [¹²⁵I]BE2254.     

3H]Dihydroergocryptine binding to alpha-adrenergic receptors of human platelets. A reassessment using the selective radioligands [3H]prazosin, [3H]yohimbine, and [3H]rauwolscine

Which subtype(s) of the alpha-adrenergic receptor occurs on human platelets? Studies of platelet responsiveness to adrenergic compounds and indirect radioligand binding studies addressing this question have yielded contradictory conclusions. These binding studies employed the ligand [³H]dihydroergocryptine ([³H]DHE), an alpha-adrenergic antagonist that does not select between alpha₁- and alpha₂-adrenergic receptors and that also binds to other receptor types in some tissues. To determine the subtype of the platelet alpha-adrenergic receptor, we have examined the binding to intact human platelets of [³H]prazosin (alpha₁-selective), [³H]yohimbine (alpha₂-selective), and [³H]rauwolscine (alpha₂-selective), and we have compared the binding of these selective radioligands with that of [³H]DHE. [³H]Yohimbine and [³H]rauwolscine both bound with high affinity (K_D = 2.7 and 4.6 nM, respectively) to an equal number and a single class (Hill coefficient ～1.0) of sites (～300 per platelet), but [³H]yohimbine yielded lower nonspecific binding than did [³H]rauwolscine. In paired experiments, [³H]DHE bound to 1.5 times as many (phentolamine-displaceable) sites as did [³H]yohimibine or [³H]rauwolscine. Unlabeled vohimbine and epinephrine competed for fewer [³H]DHE binding sites than did phentolamine. Thus, in addition to binding to the alpha₂-adrenergic receptors identified by [³H]yohimbine and [³H]rauwolscine, [³H]DHE seems to bind to other sites on human platelets. The nature of these sites is not clear. We found that [³H]prazosin did not identify alpha₁-adrenergic receptors on platelets, and that phenoxybenzamine only inhibited [³H]yohimbine and [³H]DHE binding at higher concentrations than usually observed for alpha₁-adrenergic receptors. We conclude that (1) all alpha-adrenergic sites on human platelets are of the alpha₂ subtype, (2) [³H]DHE may bind to additional, as yet ill-defined, sites in addition to those sites identified by [³H]yohimbine and [³H]rauwolscine, and (3) [³H]yohimbine is the preferred antagonist radioligand for studying the alpha₂-adrenergic receptors on human platelets.     

Role of alpha1A adrenoceptor subtype in production of the positive inotropic effect mediated via myocardial alpha,adrenoceptors in the rabbit papillary muscle: influence of selective alpha1A subtype antagonists WB 4101 and 5-methylurapidil

Summary In order to elucidate the contribution of alpha_1A subtype to the positive inotropic effect mediated by myocardial alpha, adrenoceptors, the influence of the alpha_1A selective antagonists WB 4101 and 5-methylurapidil on the alpha,-mediated positive inotropic effect (induced by phenylephrine in the presence of a beta adrenoceptor blocking agent bupranolol) was assessed in the isolated rabbit papillary muscle. WB 4101 (10^–9-10^–7mol/l) shifted the concentration-response curve of the alpha,-mediated positive inotropic effect to the right in parallel, but the slope of Schild plot did not meet the competitive antagonism: WB 4101 shifted the curve by log one unit at 10^–9 mol/1, whereas it did not cause further shift at higher concentrations of 10^–8 and 10^–7 mol/l. WB 4101 did not affect the beta adrenoceptor-mediated positive inotropic effect. 5-Methylurapidil (10^–9 to 10^–7 mol/l) shifted the curve of alpha₁-mediated positive inotropic effect to the right and downwards in a concentration-dependent manner; the slope of Schild plot calculated at the level of 20% of the maximum response to phenylephrine was close to unity. 5-Methylurapidil at 3 × 10^–7 mol/1 abolished the alpha₁-mediated positive inotropic effect. In addition, 5-methylurapidil inhibited the beta adrenoceptor-mediated positive inotropic effect in the same concentration range as it antagonized the alpha₁-mediated positive inotropic effect, indicating that 5-methylurapidil is not selective for myocardial alpha, adrenoceptors. In the membrane fraction derived from the rabbit ventricular muscle, 5-methylurapidil displaced the specific binding of [³H]CGP-12177 with high affinity, whereas WB 4101 did not affect the [³H]CGP-12177 binding in the concentration range that it antagonized the alpha,-mediated positive inotropic effect. The present results indicate that alpha_1A adrenoceptor subtype plays a role in production of the positive inotropic effect mediated by myocardial alpha, adrenoceptors, but the extent is less than that mediated by alpha_1B subtype in the rabbit ventricular myocardium. Send offprint requests to M. Endoh at the above address     

Interaction of the pesticide chlordimeform with adrenergic receptors in mouse brain: an in vitro study

Chlordimeform (N'(4-chloro-o-tolyl)-N, N-dimethylformamidine; CDM) is a formamidine insecticide/acaricide whose major active metabolite is its N-monomethyl analog, desmethylchlordimeform, (DCDM). While their pesticidal action in invertebrates appears to be related to activation of octopamine receptors, their mechanism of action in mammals has not been established. Because of similarities between octopamine and adrenergic receptors and suggestions of CDM and DCDM action on adrenoceptors, the in vitro interactions of CDM and DCDM with adrenoceptors were studied. In mouse brain membrane preparations CDM inhibited the binding of [³H]-clonidine to alpha₂- adrenoceptors and of [³H]-WB4101 to alpha₁-adrenoceptors with IC₅₀ values of 18.2 and 87 M, respectively. DCDM was a much more potent inhibitor, with IC₅₀ values toward alpha_2–, and alpha₁-adrenoceptors of 44 nM and 1 M, respectively. Both compounds were only weak inhibitors of the binding of [³H]-dihydroalprenolol to beta-adrenoceptors and of [³H]-quinuclidinyl benzilate to muscarinic receptors and were inactive toward benzodiazepines and gamma aminobutyric acid (GABA_A) receptors. Inhibition of [³H]-clonidine binding by both compounds was competitive, as indicated by a decreased receptor affinity without changes in receptor density. Interaction of CDM and DCDM with [³H]-WB4101 binding, on the other hand, was more complex, and not of the competitive type. These results show that CDM and its metabolite DCDM can interact directly in vitro with alpha-adrenergic receptors, suggesting that these receptors could mediate some of the effects of CDM and DCDM in vivo.     

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.     

Comparison of central and peripheral alpha1-adrenoceptors

Summary A series of alpha-adrenergic agonists and antagonists having diverse chemical structure was examined for both central and peripheral alpha₁-adrenoceptor activity. The agonists tested included several novel aminotetralin derivatives which were potent and selective alpha₁-agonists. Peripheral alpha₁-activity was determined in the isolated rabbit ear artery; central alpha₁-receptor affinity was measured as the ability to inhibit ³H-WB 4101 binding to rat brain homogenates. In the agonist series, an excellent correlation between peripheral alpha₁-activity and central alpha₁-affinity was obtained, providing that partial agonists were excluded. Likewise, the receptor dissociation constant for blockade of the peripheral alpha₁-adrenoceptor correlated well with affinity for the central receptor for all of the alphaantagonists. These data support the conclusion that central and peripheral alpha₁-adrenoceptors are similar or identical.     

3H]WB4101 labels the 5-HT1A serotonin receptor subtype in rat brain. Guanine nucleotide and divalent cation sensitivity

In the presence of a 30 nM prazosin mask, [3H]-2-(2,6-dimethoxyphenoxyethyl) aminomethyl-1,4-benzodioxane ([3H]WB4101) can selectively label 5-HT1 serotonin receptors. Serotonin exhibits high affinity (Ki = 2.5 nM) and monophasic competition for [3H] WB4101 binding in cerebral cortex. Furthermore, we have found a significant correlation (r = 0.96) between the affinities of a number of serotonergic and nonserotonergic compounds at [3H]WB4101-binding sites in the presence of 30 nM prazosin and [3H] lysergic acid diethylamide ([3H]LSD)-labeled 5-HT1 serotonin receptors in homogenates of rat cerebral cortex. Despite similar pharmacological profiles, distribution studies indicate that, in the presence of 5 mM MgSO4, the Bmax of [3H]WB4101 is significantly lower than the Bmax of [3H]LSD in various brain regions. WB4101 competition for [3H] LSD-labeled 5-HT1 receptors fits best to a computer-derived model assuming two binding sites, with the KH for WB4101 being similar to the KD of [3H]WB4101 binding derived from saturation experiments. This suggests that [3H]WB4101 labels only one of the subtypes of the 5-HT1 serotonin receptors labeled by [3H]LSD. Interestingly, the selective 5-HT1A serotonin receptor antagonist, spiperone, and the selective 5-HT1A agonist, 8-hydroxy-2-(di-n-propylamino) tetraline, exhibit high affinity and monophasic competition for [3H]WB4101 but compete for multiple [3H]LSD 5-HT1 binding sites. These data indicate that [3H]WB4101 selectively labels the 5-HT1A serotonin receptor, whereas [3H] LSD appears to label both the 5-HT1A and the 5-HT1B serotonin receptor subtypes. The divalent cations, Mn2+, Mg2+, and Ca2+ were found to markedly increase the affinity and Bmax of [3H]WB4101 binding in cerebral cortex. Conversely, the guanine nucleotides guanylylimidodiphosphate and GTP, but not the adenosine nucleotide ATP, markedly reduce the Bmax of [3H]WB4101 binding. These characteristics are typical of agonists interacting with receptors which modulate cellular function via a guanine nucleotide-regulatory subunit.     

Subtypes of α1-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 ligangs (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 α₁-adrenoceptors in the hippocampus of these species. [³H]WB 4101 was used to label α_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 α_1B-adrenoceptor cloned from DDT₁ cells. In autoradiographic studies with human hippocampal slices, CEC (chloroethylclonidine), an alkylating agent described to show selectivity for α_1B-adrenoceptors, displaced preferentially [¹²⁵I]BE 2254 binding from the molecular layer of the dentate gyrus. In contrast, WB 4101 an α_1A-adrenoceptor-selective ligand, displaced preferentially [¹²⁵I]BE 2254 binding in the hilus and the CA3 region. The data show that 2 subtypes of α₁-adrenergic recognition sites can be identified in the hippocampus. In the human hippocampus, α_1A sites are predominant in the hilus and the CA3 region, whereas α_1B sites are predominant in the molecular layer of the dentate gyrus. These subtypes show a similar pharmacological profile in man, calf and guinea-pig, and may have a different functional role in these two areas of the hippocampus.     

Role of alpha-adrenergic receptors in denervation supersensitivity of rat vas deferens

Contractile responses of rat vas deferens were studied with particular attention directed to the role of receptors and neuronal control. Marked contraction of the vas deferens was observed with alpha-adrenergic agonists, depending on their concentrations. This tissue had a low sensitivity to ACh. Four days after denervation, this tissue showed a supersensitivity to alpha-adrenergic agonists and a high K+ concentration, but not to ACh. The increase in sensitivity to alpha-agonists resulted in an enhancement of the maximal response and a shift of the concentration response curve to lower concentrations of these reagents. Alterations were seen in the alpha-adrenergic receptors in the rat vas deferens, assayed by measuring the binding of [3H]WB4101. The maximal binding sites decreased significantly to 86 from 142 fmoles per mg protein. The affinity of the receptors for alpha-agonist, determined by measuring the ability of agonists to displace bound [3H] WB4101, increased significantly, while the affinity to alpha-antagonists remained unchanged. Studies on [3H]QNB binding indicated no significant change in muscarinic ACh receptors after denervation. Thus, supersensitivity of the alpha-adrenergic mechanism mediated by a specific change in affinity of alpha-receptors occurs after denervation of rat vas deferens. These changes in sensitivity and in receptors are discussed in relation to the characteristics and roles of alpha-receptors in the rat vas deferens.     

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.     

Two distinct α1-adrenergic receptor sites in rat liver: Differential binding of (−)-[3H]norepinephrine, [3H]prazosin and [3H]dihydroergocryptine: Effects of guanine nucleotides and proteolysis; implications for a two-site model of α-receptor regulation

(?)-[³H]Norepinephrine, [³H]prazosin and [³H]dihydroergocryptine bind to rat liver plasma membranes in a manner indicating a selective interaction with α₁-adrenergic receptors. All three ligands display monophasic saturation with a single component on Scatchard analysis. The binding capacities of (?)-[³H]norepinephrine, [³H]prazosin and [³H]dihydroergocryptine are 340 ± 70 fmol/mg of protein 760 ± 40 fmol/mg of protein and 1200 ± 300 fmol/mg of protein, respectively. Differential drug potencies in competing for (?)-[³H]norepinephrine and [³H]prazosin binding sites suggest that these two ligands label two distinct binding sites at the α₁-adrenergic receptor, while [³H]dihydroergocryptine labels both sites. Guanine nucleotides lower the apparent affinity of (?)-[³H]norepinephrine for its binding site, without affecting the number of sites or the binding of [³H]prazosin and [³H]dihydroergocryptine. Incubation of membranes with α-chymotrypsin slightly reduces the binding of [³H]prazosin and [³H]dihydroergocryptine, but causes a 2-fold increase in (?)-[³H]norepinephrine binding. Both the number of (?)-[³H]norepinephrine binding sites and the affinity are increased. Following proteolysis, (?)-[³H]norepinephrine binding still occurs to a single class of sites, but is no longer affected by guanine nucleotides. The effect of α-chymotrypsin is abolished by pretreatment of membranes with the irreversible α-adrenergic antagonist phenoxybenzamine. We propose that in rat liver, (?)-[³H]norepinephrine labels the physiologically active form of the α-adrenergic receptor while [³H]prazosin binds to a precursor form of the active receptor, and/or to an α₁-adrenergic binding site not coupled to an effector system.     

Characterization of alpha- and beta-adrenergic receptors in membranes prepared from the rabbit iris before and after development of supersensitivity.

α-Adrenergic and β-adrenergic receptors were studied by measuring the binding of [³H]dihydroergocryptine and [³H]dihydroalprenolol, respectively, to membranes prepared from homo-genized rabbit irides. The binding of [³H]dihydroergocryptine appears to be specific for α-adrenergic receptors as adrenergic agents displace this radioligand with the following order of potency: phentolamine > epinephrine ? norepinephrine ? isoproterenol = propranolol. The binding of [³H]dihydroalprenolol appears to be specific for β-adrenergic receptors as adrenergic agents displace this radioligand with the following order of potency: propranolol ? isoproterenol ? epinephrine > norepinephrine ? phentolamine. Several weeks after removal of the superior cervical ganglion, when all the adrenergic nerves to the tissue have degenerated, membranes prepared from denervated irides have an increased density of β-adrenergic receptors with no increase in the density of α-adrenergic receptors. A small decrease in the total number of α-adrenergic receptors probably occurs, which is due to the loss of pre-junctional receptors. The affinities of the receptors do not change. These findings suggest that unlike skeletal muscle, the supersensitivity that occurs in smooth muscle is not due to an increase in the population of receptors governing contraction. However, the change in population of β-adrenergic receptors is consistent with the hypothesis that, as in other tissues, the level of cyclic AMP modulates the density of the β-adrenergic receptor.     

Heterogeneity of Guinea-Pig Lung Muscarinic Receptors Revealed by [3H]4-DAMP-binding

Summary: Muscarinic receptors present in guinea-pig lung were characterized using the M₃ selective radioligand [³H]4-diphenylacetoxy-N-methyl-piperidine methiodide ([³H]4-DAMP). In saturation studies, [³H]4-DAMP identified two populations of binding sites with approximately 4% of the sites displaying high affinity (K_d=0.21 nM and B_max= 10 fmol/mg prot.) while the remaining sites were low affinity ones (K_d=18.11 nM and B_max=269 fmol/mg prot.). In competition studies with [³H]4-DAMP (0.35 nM), methoctramine and hexahydro-siladifenidol (HHSiD) identified 50 and 70% of high affinity binding sites displaying the pharmacological profile of the M₂ and the M₃ receptors, respectively. No evidence was found for high affinity [³H]pirenzepine binding sites in guinea-pig lung. However, pirenzepine/[³H]4-DAMP competition experiments suggested that pirenzepine recognized an equal proportion of [³H]4-DAMP binding sites with intermediate and low affinity binding constants. The intermediate affinity binding constant was inconsistent with the presence of M₁ receptors and reflected more the presence of M₄ or a mixture of M₃ and M₄ receptors. The low affinity pirenzepine binding sites may represent M₂ receptors. These results provide further evidence for the occurrence of M₂ and M₃ receptors and suggest the presence of the M₄ muscarinic receptor subtype in guinea-pig lung.     

Binding studies with the 5-HT1B receptor agonist [3H]CP-96,501 in brain tissues

Binding studies in rat brain membranes have shown that [³H]CP-96,501 is a selective, high affinity radioligand for 5-HT_1B receptors. Additional studies with [³H]CP-96,501 in brain tissues of several other species were undertaken to investigate its utility in detecting this receptor subtype. The presence of 5-HT_1B receptors in brain membranes of the hamster and gerbil was demonstrated with [³H]CP-96,501. This finding was confirmed by [³H]5-HT binding experiments that also indicated the presence of other 5-HT₁ subtypes (possibly 5-HT_1D, 5-HT_1E) in these species as well as the rat. Negligible specific binding of [³H]CP-96,501 was found in membranes of guinea pig brain, dog hypothalamus, bovine caudate, pig choroid plexus, and several human brain tissues, consistent with the reported absence of the 5-HT_1B receptor subtype in these species. Autoradiographic examination of rat brain sections labeled by [³H]CP-96,501 showed a dense localization in areas known to be enriched in 5-HT_1B receptors (e.g., globus pallidus, substantia nigra, superior colliculus, subiculum). On the other hand, brain sections of dog and guinea pig appeared to show no specific binding of [³H]CP-96,501 by autoradiography, in agreement with the lack of brain 5-HT_1B receptors in these tissues. © 1992 Wiley-Liss, Inc.     

Antagonism by antidepressants of serotonin S1 and S2 receptors of normal human brain in vitro

Using radioligand binding techniques and human frontal cortex, we determined the equilibrium dissociation constants (KDs) of 25 antidepressants at the serotonin S1 (probably the S1A subtype) and serotonin S2 receptors using [3H]WB4101 and [3H]ketanserin, respectively. At the serotonin S1 receptor, the most and least potent antidepressants were trazodone (KD = 60 nM) and bupropion (KD = 170 microM), respectively. At the serotonin S2 receptor, the most and least potent antidepressants were amoxapine (KD = 0.6 nM) and bupropion (KD = 90 microM), respectively. Analysis of the data revealed a relationship between structure and serotonin S1 affinity for some tricyclic antidepressants. Buspirone, a new anxiolytic agent, possessed high affinity for the serotonin S1 receptor (KD = 3.8 nM).     

Characterization of alpha-adrenergic receptors in guinea pig cerebral cortex: Effect of chronic antidepressant treatments

Alpha-adrenergic receptor sites in cerebral cortex membranes of the guinea pig brain have been characterized by the specific binding of ³H-WB4101, an alpha-adrenergic antagonist. The binding was rapid, reversible, saturable, and stereoselective. The maximal binding site (B _max) and disociation constant (k _d) for ³H-WB4101 were calculated to be 251 fmol/mg protein and 1.23 nM, respectively. Ligand displacement experiments suggest that ³H-WB4101 binds to alpha₁-adrenergic receptors. Chronic treatment of guinea pigs with desipramine, amitriptyline, phenelzine, or electroconvulsive shock failed to show any statistically significant change in alpha-adrenergic receptor sensitivity as measured by the binding of ³H-WB4101.     

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.     

Characterization and quantitation of alpha-adrenergic receptor subtypes in rat hypothalamus

The binding of [³H]dihydroergocryptine ([³H]-DHE) to rat hypothalmic membranes was examined. Displacement of [³H]-DHE by 100 nM phentolamine, prazosin and clonidine can be used to assay for total α-, α₁- and α₂-adrenergic receptor sites respectively. The α₁-adrenergic receptor content of the hypothalmus is 4.1 pmoles/g tissue whereas the α₂-level is 6.5 pmoles/g tissue. [³H]-WB 4101 (α₁ selective) and [³H]clonidine (α₂ selective) binding yielded similar levels of 3.2 and 5.8 pmoles/g tissue. It is concluded that [³H]-DHE is a suitable ligand for the assay of α-adrenergic receptor subtypes under the conditions determined in this study.     

Selective visualization of rat brain 5-HT2A receptors by autoradiography with [3H]MDL 100,907

The recently developed 5-HT_2A receptor selective antagonist [³H]MDL100,907 ((+/–)2,3-dimethoxyphenyl-1-[2-(4-piperidine)-methanol]) has been characterized as a radioligand for the autoradiographic visualization of these receptors. [³H]MDL100,907 binding to rat brain tissue sections was saturable, had sub-nanomolar affinity (Kd=0.2–0.3nM), and presented a pharmacological profile consistent with its binding to 5-HT_2A receptors (rank order of affinity for [³H]MDL100,907-labelled receptors: MDL100,907 > spiperone > ketanserin > mesulergine). The distribution of receptors labelled by [³H]MDL100,907 was compared to the autoradiographical patterns obtained with [³H]Ketanserin, [³H]Mesulergine, and [³H]RP62203 (N-[3-[4-(4-fluorophenyl)-piperazin-1-y1]propyl]-1,8-naphtalenesultam) and to the distribution of 5-HT_2A receptor mRNA as determined by in situ hybridization. As opposed to the other radioligands, [³H]MDL100,907 labelled a single population of sites (5-HT_2A receptors) and presented extremely low levels of non-specific binding. The close similarity of the distributions of [³H]MDL100,907-labelled receptors and 5-HT_2A mRNA further supports the selectivity of this radioligand for 5-HT_2A receptors and suggests a predominant somatodendritic localization of these receptors. The present results point to [³H]MDL100,907 as the ligand of choice for the autoradiographic visualization of 5-HT_2A receptors. Received: 7 April / Accepted: 18 May 1997     

Characterization of alpha-adrenoceptors in myometrium of preparturient rats

We describe three methods for the quantitative analysis of the alpha-adrenoceptor subtypes in preparturient rat myometrial membrane fractions. A non-subtype-selective antagonist radioligand. [3H]dihydroergocryptine ([3H]DHE), was used to label all of the alpha-receptors. [3H]DHE bound to both alpha 1- and alpha 2-receptors with indistinguishable affinity. Computer modelling of competition curves of unlabeled selective antagonists or agonists was then required in order to determine reliably alpha 1 and alpha 2 affinities and proportions: the alpha 1-receptors represent 45% and the alpha 2-receptors 55% of the entire alpha-receptor population in rat uterus. The second approach involved the administration of phenoxybenzamine (POB) that irreversibly blocks the alpha 1-adrenoceptors. Myometrial membranes obtained from rats 1 h after the administration of varying amounts of POB showed a dose-dependent reduction in specific [3H]DHE binding. This reduction was accompanied by a progressive increase of the value of the dissociation constant. Our data indicate that a dose of 1 mg of POB left the alpha 2-receptors intact while entirely blocking the alpha 1-receptors in rat myometrium. The third approach utilized the selective radioligand antagonists [3H]prazosin ([3H]PRAZ) and [3H]rauwolscine ([3H]RAUW). The results obtained with these radioligands confirmed our observations on the alpha-adrenoceptor subtypes in experiments with [3H]DHE. The results obtained with the 3 methods are in good agreement. Each approach appears valid and applicable to the characterization of alpha 1- and alpha 2-adrenoceptor subtypes in rat uterus, but the method using [3H]PRAZ and [3H]RAUW demonstrates more directly the presence of the two receptor subtypes.