Human cloned α1A-adrenoceptor isoforms display α1L-adrenoceptor pharmacology in functional studies

The recombinant α_1A-adrenoceptor displays a distinct pharmacological profile (‘classical α_1A-adrenoceptor') in homogenate binding assays, but displays the properties of the so-called α_1L-adrenoceptor in functional studies in whole cells at 37°C. As three splice variants of the human α_1A-adrenoceptor have been described previously (α_1A-1, α_1A-2 and α_1A-3), we have compared their functional pharmacological profiles, when expressed stably in Chinese hamster ovary (CHO-K1) cells (antagonist inhibition of noradrenaline-stimulated [³H]inositol phosphates accumulation). A fourth, novel isoform (α_1A-4) has also been studied: α_1A-4 mRNA predominates in several human tissues including prostate, liver, heart and bladder. In homogenate binding studies, all four isoforms displayed essentially identical affinity profiles, with prazosin (1-(4-amino-6,7-dimethoxy-2-quinazolinyl)-4-(2-furoyl)piperazine), tamsulosin (5-[2-[[2-(2-ethoxyphenoxy)ethyl]-amino]propyl]-2-methoxybenzenesulfonamide), RS-17053 (N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-α,α-dimethyl-1H-indole-3-ethanamine hydrochloride), WB 4101 ((2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane hydrochloride) and 5-Me-urapidil (5-methyl-6[[3-[4-(2-methoxyphenyl)-1-piperazinyl]propyl]amino]-1,3-dimethyuracil) all displaying subnanomolar affinities. In functional studies, noradrenaline accelerated [³H]inositol phosphates production with potencies (p[A]₅₀) of between 5.8 and 6.6. The affinities of prazosin, RS-17053, WB 4101 and 5-Me-urapidil, at antagonizing responses to noradrenaline, were reduced by approximately 10-fold (cf. binding data), while those for tamsulosin and indoramin (N-[1-[2-(1H-indol-3-yl)ethyl]-4-piperidinyl]benzamide) remained constant or increased, consistent with the previously described α_1L-adrenoceptor. Thus, all four human recombinant α_1A-adrenoceptor isoforms display the pharmacology of the α_1L-adrenoceptor when studied in functional assays, consistent with the hypothesis that the putative α_1L-adrenoceptor represents a functional phenotype of the α_1A-adrenoceptor.     

Identification of alpha 1L-adrenoceptor in mice and its abolition by alpha 1A-adrenoceptor gene knockout

Background and purpose:

The α_1L-adrenoceptor has pharmacological properties that distinguish it from three classical α₁-adrenoceptors (α_1A, α_1B and α_1D). The purpose of this was to identify α_1L-adrenoceptors in mice and to examine their relationship to classical α₁-adrenoceptors.

Experimental approach:

Radioligand binding and functional bioassay experiments were performed on the cerebral cortex, vas deferens and prostate of wild-type (WT) and α_1A-, α_1B- and α_1D-adrenoceptor gene knockout (AKO, BKO and DKO) mice.

Key results:

The radioligand [³H]-silodosin bound to intact segments of the cerebral cortex, vas deferens and prostate of WT, BKO and DKO but not of AKO mice. The binding sites were composed of two components with high and low affinities for prazosin or RS-17053, indicating the pharmacological profiles of α_1A-adrenoceptors and α_1L-adrenoceptors. In membrane preparations of WT mouse cortex, however, [³H]-silodosin bound to a single population of prazosin high-affinity sites, suggesting the presence of α_1A-adrenoceptors alone. In contrast, [³H]-prazosin bound to two components having α_1A-adrenoceptor and α_1B-adrenoceptor profiles in intact segments of WT and DKO mouse cortices, but AKO mice lacked α_1A-adrenoceptor profiles and BKO mice lacked α_1B-adrenoceptor profiles. Noradrenaline produced contractions through α_1L-adrenoceptors with low affinity for prazosin in the vas deferens and prostate of WT, BKO and DKO mice. However, the contractions were abolished or markedly attenuated in AKO mice.

Conclusions and implications:

α_1L-Adrenoceptors were identified as binding and functional entities in WT, BKO and DKO mice but not in AKO mice, suggesting that the α_1L-adrenoceptor is one phenotype derived from the α_1A-adrenoceptor gene.     

Analysis of alpha 1L-adrenoceptor pharmacology in rat small mesenteric artery.

1. To illuminate the controversy on alpha 1A- or alpha 1L-adrenoceptor involvement in noradrenaline-mediated contractions of rat small mesenteric artery (SMA), we have studied the effects of subtype-selective alpha 1-adrenoceptor agonists and antagonists under different experimental conditions. 2. The agonist potency order in rat SMA was: A61603 > SKF89748-A > cirazoline > noradrenaline > ST-587 > methoxamine. Prazosin antagonized all agonists with a low potency (pA2: 8.29-8.80) indicating the involvement of alpha 1L-rather than alpha 1A-adrenoceptors. 3. The putative alpha 1L-adrenoceptor antagonist JTH-601, but not the alpha 1B-adrenoceptor antagonist chloroethylclonidine (10 microM) antagonized noradrenaline-induced contractions of SMA. The potency of the selective alpha 1D-adrenoceptor antagonist BMY 7378 against noradrenaline (pA2 = 6.16 +/- 0.13) and of the selective alpha 1A-adrenoceptor antagonist RS-17053 against noradrenaline (pKB = 8.35 +/- 0.10) and against the selective alpha 1A-adrenoceptor agonist A-61603 (pKB = 8.40 +/- 0.09) were too low to account for alpha 1D- and alpha 1A-adrenoceptor involvement. 4. The potency of RS-17053 (pKB/pA2's = 7.72-8.46) was not affected by lowering temperature, changing experimental protocol or inducing myogenic tone via KCl or U46619. 5. Selective protection of a putative alpha 1A-adrenoceptor population against the irreversible action of phenoxybenzamine also failed to increase the potency of RS-17053 (pA2 = 8.25 +/- 0.06 against A61603). 6. Combined concentration-ratio analysis demonstrated that tamsulosin, which does not discriminate between alpha 1A- and alpha 1L-adrenoceptors, and RS-17053 competed for binding at the same site in the SMA. 7. In summary, data obtained in our experiments in rat SMA indicate that the alpha 1-adrenoceptor mediating noradrenaline-induced contraction displays a distinct alpha 1L-adrenoceptor pharmacology. This study does not provide evidence for the hypothesis that alpha 1L-adrenoceptors represent an affinity state of the alpha 1A-adrenoceptor in functional assays. Furthermore, there is no co-existing alpha 1A-adrenoceptor in the SMA.     

High-affinity interactions between human alpha1A-adrenoceptor C-terminal splice variants produce homo- and heterodimers but do not generate the alpha1L-adrenoceptor

Using combinations of bioluminescence resonance energy transfer, time-resolved fluorescence resonance energy transfer and the functional complementation of pairs of inactive receptor-G protein fusion proteins, the human alpha(1A-1)-adrenoceptor was shown to form homodimeric/oligomeric complexes when expressed in human embryonic kidney (HEK) 293 cells. Saturation bioluminescence resonance energy transfer studies indicated the alpha(1A-1)-adrenoceptor homodimer interactions to be high affinity and some 75 times greater than interactions between the alpha(1A-1)-adrenoceptor and the delta opioid peptide receptor. Only a fraction of the alpha(1A-1)-adrenoceptors was at the plasma membrane of HEK293 cells at steady state. However, dimers of alpha(1A-1)-adrenoceptors were also present in intracellular membranes, and the dimer status of those delivered to the cell surface was unaffected by the presence of agonist. Splice variation can generate at least three forms of the human alpha(1A-1)-adrenoceptor with differences limited to the C-terminal tail. Each of the alpha(1A-1), alpha(1A-2a), and alpha(1A-3a)-adrenoceptor splice variants formed homodimers/oligomers, and all combinations of these splice variants were able to generate heterodimeric/oligomeric interactions. Despite the coexpression of these splice variants in human tissues that possess the pharmacologically defined alpha(1L)-adrenoceptor binding site, coexpression of any pair in HEK293 cells failed to generate ligand binding characteristic of the alpha(1L)-adrenoceptor.     

In vitro alpha1-adrenoceptor pharmacology of Ro 70-0004 and RS-100329, novel alpha1A-adrenoceptor selective antagonists.

It has been hypothesized that in patients with benign prostatic hyperplasia, selective antagonism of the alpha1A-adrenoceptor-mediated contraction of lower urinary tract tissues may, via a selective relief of outlet obstruction, lead to an improvement in symptoms. The present study describes the alpha1-adrenoceptor (alpha1-AR) subtype selectivities of two novel alpha1-AR antagonists, Ro 70-0004 (aka RS-100975) and a structurally-related compound RS-100329, and compares them with those of prazosin and tamsulosin. Radioligand binding and second-messenger studies in intact CHO-K1 cells expressing human cloned alpha1A-, alpha1B- and alpha1D-AR showed nanomolar affinity and significant alpha1A-AR subtype selectivity for both Ro 70-0004 (pKi 8.9: 60 and 50 fold selectivity) and RS-100329 (pKi 9.6: 126 and 50 fold selectivity) over the alpha1B- and alpha1D-AR subtypes respectively. In contrast, prazosin and tamsulosin showed little subtype selectivity. Noradrenaline-induced contractions of human lower urinary tract (LUT) tissues or rabbit bladder neck were competitively antagonized by Ro 70-0004 (pA2 8.8 and 8.9), RS-100329 (pA2 9.2 and 9.2), tamsulosin (pA2 10.4 and 9.8) and prazosin (pA2 8.7 and 8.3 respectively). Affinity estimates for tamsulosin and prazosin in antagonizing alpha1-AR-mediated contractions of human renal artery (HRA) and rat aorta (RA) were similar to those observed in LUT tissues, whereas Ro 70-0004 and RS-100329 were approximately 100 fold less potent (pA2 values of 6.8/6.8 and 7.3/7.9 in HRA/RA respectively). The alpha1A-AR subtype selectivity of Ro 70-0004 and RS-100329, demonstrated in both cloned and native systems, should allow for an evaluation of the clinical utility of a 'uroselective' agent for the treatment of symptoms associated with benign prostatic hyperplasia.     

Pharmacological properties of the cloned alpha 1A/D-adrenoceptor subtype are consistent with the alpha 1A-adrenoceptor characterized in rat cerebral cortex and vas deferens.

1. The pharmacological characteristics of cloned mammalian alpha 1A/D-, alpha 1B- and alpha 1C-adrenoceptor subtypes expressed in rat 1 fibroblasts were determined in comparison to the binding and functional properties of these subtypes in rat tissues. 2. Analysis of [3H]-prazosin binding to membrane homogenates from rat 1 fibroblast cells expressing each of the alpha 1-subtypes indicated high affinity binding to a single population of binding sites. Binding affinities were similar for alpha 1A/D-, alpha 1B- and alpha 1C-subtypes (Kds: 0.13, 0.10 and 0.15 nM respectively) although a higher density of alpha 1B- and alpha 1C-receptors (Bmax: 4068 and 10,323 fmol mg-1 protein respectively) were expressed in comparison to alpha 1A/D (838 fmol mg-1). 3. Displacement of [3H]-prazosin from membranes expressing cloned alpha 1-adrenoceptor subtypes revealed that 5-methyl-urapidil, WB 4101, benoxathian and phentolamine displayed high affinity and selectivity for alpha 1A/D- over alpha 1B-subtypes. These compounds also had high affinity and selectivity for alpha 1C- over alpha 1B-subtypes. 5-Methyl-urapidil showed selectivity for alpha 1C (Ki 0.60 +/- 0.16 nM) over both alpha 1A/D (Ki, 9.8 +/- 2.8 nM) and alpha 1B (Ki 57.2 +/- 12 nM) subtypes. Prazosin and doxazosin were not subtype selective. 4. In comparison to [3H]-prazosin a similar pharmacological profile was obtained with [125I]-HEAT using cloned alpha 1A/D-, alpha 1B- and alpha 1C-adrenoceptors expressed in rat 1 fibroblasts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Age-related change of the role of alpha1L-adrenoceptor in canine urethral smooth muscle.

To examine age-related alteration of the role of alpha1L-adrenoceptor in the urethra, young non-parous and aged parous female dogs were used. In a functional study, we evaluated phenylephrine-induced contraction and antagonistic effects of JTH-601, a newly synthesized alpha1-adrenoceptor antagonist, and prazosin; in a localization survey using autoradiographic technique, we investigated specific [3H]JTH-601 and [3H]tamsulosin binding. Concentration-response curves were obtained for phenylephrine (pD2 = 5.0-5.3). JTH-601 and prazosin antagonized this contraction with pA2 values of 8.2-8.3 and 8.0-8.1, respectively. Specific binding of both [3H]JTH-601 and [3H]tamsulosin were observed in the bladder neck and proximal section of urethra. There were no significant differences of the pD2, pA2, and radio ligand binding between young non-parous and aged parous dogs.     

Identification of the alpha1L-adrenoceptor in rat cerebral cortex and possible relationship between alpha1L- and alpha1A-adrenoceptors

BACKGROUND AND PURPOSE: In addition to alpha1A, alpha1B and alpha1D-adrenoceptors (ARs), putative alpha1L-ARs with a low affinity for prazosin have been proposed. The purpose of the present study was to identify the alpha1A-AR and clarify its pharmacological profile using a radioligand binding assay. EXPERIMENTAL APPROACH: Binding experiments with [3H]-silodosin and [3H]-prazosin were performed in intact tissue segments and crude membrane preparations of rat cerebral cortex. Intact tissue binding assays were also conducted in rat tail artery. KEY RESULTS: [3H]-silodosin at subnanomolar concentrations specifically bound to intact tissue segments and membrane preparations of rat cerebral cortex at the same density (approximately 150 fmol mg(-1) total tissue protein). The binding sites in intact segments consisted of alpha1A and alpha1L-ARs that had different affinities for prazosin, while the binding sites in membranes showed an alpha1A-AR-like profile having single high affinity for prazosin. [3H]-prazosin also bound at subnanomolar concentrations to alpha1A and alpha1B-ARs but not alpha1L-ARs in cerebral cortex; the binding densities being approximately 200 and 290 fmol mg(-1) protein in the segments and the membranes, respectively. In the segments of tail artery, [3H]-silodosin only recognized alpha1A-ARs, whereas [3H]-prazosin bound to alpha1A and alpha1B-ARs. CONCLUSIONS AND IMPLICATIONS: The present study clearly reveals the presence of alpha1L-ARs as a pharmacologically distinct entity from alpha1A and alpha1B-ARs in intact tissue segments of rat cerebral cortex but not tail artery. However, the alpha1L-ARs disappeared after tissue homogenization, suggesting their decomposition and/or their pharmacological profile changes to that of alpha1A-ARs.     

一种新的α1A肾上腺素受体选择性拮抗剂—Sertindole

本工作分别在稳定表达α_1A,α_1B和α_1D肾上腺素受体(adrenoceptor,AR)的人胚胎肾脏细胞( human embryonic kidney 293,HEK 293)和大鼠离体血管上,用放射配体结合实验和离体血管收缩功能实验方法以确定sertindole对α₁-AR亚型的选择性拮抗作用。结果显示sertindole与克隆α_1A-AR的亲和性分别是与克隆α_1B-AR和克隆α_1D-AR的69倍和132倍。Sertindole拮抗去甲肾上腺素引起的主动脉和肾动脉收缩反应的pA₂值分别与其对α_1D和α_1A亚型的pKI值相符。分别稳定表达3种亚型受体的HEK293细胞膜标本经与sertindole预温育30min后,受体与¹²⁵IBE2254结合的B_max值显著降低,KD值无显著变化;而在 sertindole 存在条件下,α₁-AR3种亚型与¹²⁵IBE2254 结合的KD值显著增大,但B_max值无显著改变。上述结果表明sertindole为不可逆性竞争性α₁-AR拮抗剂,并有α_1A亚型选择性。     

Ex vivo and in vivo alpha1-adrenoceptor binding characteristics of a novel alpha1L-adrenoceptor antagonist, JTH-601, in rat tissues.

In vitro, ex vivo and in vivo alpha1-adrenoceptor binding of JTH-601 (3-[N-[2-(4-hydroxy-2-isopropyl-5-methylphenoxy)ethyl]-N-methylaminometh yl]-4-methoxy-2,5,6-trimethyl-phenol hemifumarate), a novel alpha1L-adrenoceptor antagonist, in rat tissues was investigated. JTH-601 competed in a concentration-dependent manner with [3H]prazosin for binding sites in the prostate, submaxillary gland and spleen of rats in vitro, and the inhibitory effect was not largely different among these tissues, as shown by the Ki values of 2-3 nM. At 0.25, 0.5 and 3 h after oral administration of JTH-601 (6.5 micromol/kg) in rats, there was a significant (57, 64 and 28%, respectively) increase in the apparent dissociation constant (Kd) for prostatic [3H]prazosin binding, compared to the control value. The administration of a higher dose (21.8 micromol/kg) of this agent produced greater (67-99%) increases in Kd values for prostatic [3H]prazosin binding at 0.5-12 h later. Similar significant increases in Kd values, as with the prostate, were seen in the submaxillary gland and heart 0.25-12 h after the oral administration of JTH-601 (6.5 and 21.8 micromol/kg), but significant increases in the spleen and cerebral cortex were seen only at 0.25-3 h and 0.5 h, respectively. At 10 min of i.v. injection of [3H]JTH-601 in rats, in vivo specific binding was observed in the prostate, cerebral cortex, submaxillary gland, spleen and heart but not in the aorta. The binding in the prostate, submaxillary gland and heart, but not in the cerebral cortex and spleen, lasted until 120 min. It is concluded that JTH-601 may exert a considerably sustained blockade of alpha1-adrenoceptors in the prostate of rats. This finding may be important in characterizing the therapeutic effect of JTH-601 for bladder outlet obstruction in patients with benign prostatic hyperplasia.     

Recent progress in alpha 1-adrenoceptor pharmacology

The adrenoceptors (ARs) play a key role in the modulation of sympathetic nervous system activity and are a site of action for many clinically important therapeutic agents. The alpha1-adrenoceptor subtypes (alpha1A-, alpha1B-, and alpha1D-AR) play a prominent role in regulating vascular tone and hypertrophic growth of smooth muscle and cardiac cells. Their functional characteristics with respect to ligand binding and second messenger utilization have been well described. Here, we review recent progress on subtype-specific subcellular localization, participation in signaling cascades, and the pivotal function of alpha1-ARs, as delineated through studies on genetically engineered animals. Together, these findings will provide new insights into the physiological and pathophysiological roles of the alpha1-ARs.     

Renal excretion mechanism of NS-49, a phenethylamine class alpha 1A-adrenoceptor agonist.

Renal handling of NS-49, which is an organic cation and a chiral compound, was investigated in rats, rabbits and dogs. Renal clearance (Cl(re)) of NS-49 was 3.4-fold the glomerular filtration rate (GFR) in the rat in vivo study. The clearance ratio (Cl(re)/GFR) approached unity during cimetidine infusion. Change in the urine flow rate or urinary pH did not affect the Cl(re) of NS-49. The stop-flow patterns of NS-49 in the rabbits and dogs showed a secretion peak in the proximal tubules. On concomitant administration of cimetidine, the secretion peak disappeared, the stop-flow pattern showing neither a secretion nor reabsorption peak. These findings indicate that in these species NS-49 undergoes glomerular filtration and extensive proximal tubular secretion, but little reabsorption. A transport mechanism study of NS-49 in brush-border membrane vesicles (BBMVs) isolated from rat kidney cortex showed that it is transported via the carrier-mediated H(+)/organic cation antiport system. In the rat renal clearance studies (in vivo) tubular secretion of NS-49 was significantly inhibited by quinine (p<0.01) but not by quinidine. Transport studies done with rat BBMVs (in vitro) also showed quinine to be more potent than quinidine in inhibiting NS-49 uptake. These results indicate that stereoselective interaction occurs in active renal tubular secretion.     

The alpha1A-adrenoceptor gene is required for the alpha1L-adrenoceptor-mediated response in isolated preparations of the mouse prostate

Background and purpose:This study investigated whether deletion of the alpha(1A)-adrenoceptor gene influences contractile responses of mouse prostate to noradrenaline. Responses of mouse prostate to noradrenaline are known to be mediated by alpha(1L)-adrenoceptors, which are thought to be a functional phenotype of alpha(1A)-adrenoceptor.Experimental approach:Prostate tissues from alpha(1A)-adrenoceptor knockout mice which were homozygous (alpha(1A)-/-) and heterozygous (alpha(1A)+/-) for the disrupted alpha(1A)-adrenoceptor gene, as well as wild-type (alpha(1A)+/+) littermates were mounted in glass-isolated organ baths. Electrical field stimulation of nerves and exogenous application of noradrenaline were used to investigate the effects of alpha(1A)-adrenoceptor disruption on prostate contractility.Key results:Frequency-response curves to electrical field stimulation (0.5 ms pulse duration, 60 V, 0.1-20 Hz) yielded frequency-dependent contractions. At frequencies of 10 and 20 Hz, prostates from alpha(1A)-/- mice elicited an approximately 30% decreased response compared with prostates from alpha(1A)+/+ mice. Prazosin (0.3 muM) attenuated responses to electrical field stimulation in prostates from alpha(1A)+/+ and alpha(1A)+/- mice but not from alpha(1A)-/- mice. Increasing concentrations of exogenously administered noradrenaline (10 nM-1 mM) produced mean concentration-response curves in prostates from alpha(1A)+/+ and alpha(1A)+/- mice, which were not different. Maximum responses to noradrenaline were decreased by approximately 80% in prostates from alpha(1A)-/- mice compared with alpha(1A)+/+ mice. Prazosin attenuated responses to noradrenaline in all genotypes.Conclusions and implications:alpha(1L)-Adrenoceptor-mediated responses in mouse prostate are abolished in alpha(1A)-/- mice, demonstrating that the alpha(1A)-adrenoceptor gene is essential to the manifestation of the prostatic alpha(1L)-adrenoceptor phenotype. This implies that alpha(1L)-adrenoceptors are indeed a functional phenotype of alpha(1A)-adrenoceptor.British Journal of Pharmacology (2008) 155, 103-109; doi:10.1038/bjp.2008.245; published online 16 June 2008.     

Evidence that an alpha 2A-adrenoceptor subtype mediates antinociception in mice.

In the hot-plate test in mice, the antinociceptive action of the alpha 2-adrenoceptor agonist, UK 14,304, was abolished by the alpha 2-adrenoceptor antagonist, idazoxan, the potent alpha 2A-adrenoceptor antagonist, RX 821002 and the preferential alpha 2A-adrenoceptor antagonist, BRL 44408. In contrast, the preferential alpha 2B- (and alpha 2C)-adrenoceptor ligands ('antagonists'), ARC-239, BRL 41992 and prazosin were inactive. The preferential alpha 2A-adrenoceptor partial agonist, guanfacine, partially inhibited UK 14,304-induced antinociception. Further, guanfacine BRL 44408 reversibly elicited submaximal antinociception. It is concluded that alpha 2A-adrenoceptors mediate antinociception in mice.     

In vitro studies on L-771,688 (SNAP 6383), a new potent and selective alpha1A-adrenoceptor antagonist

L-771,688 (SNAP 6383, methyl(4S)-4-(3, 4-difluorophenyl)-6-[(methyloxy)methyl]-2-oxo-3-[(?3-[4-(2-pyridin yl)-1-piperidinyl]propyl?amino)carbonyl]-1,2,3, 4-tetrahydro-5-pyrimidine carboxylate) had high affinity (Ki less than or = 1 nM) for [3H]prazosin binding to cloned human, rat and dog alpha1A-adrenoceptors and high selectivity (>500-fold) over alpha1B and alpha1D-adrenoceptors. [3H]Prazosin / (+/-)-beta-[125I]-4-hydroxy-phenyl)-ethyl-aminomethylteralone ([125I]HEAT) binding studies in human and animal tissues known to contain alpha1A and non-alpha1A-adrenoceptors further demonstrated the potency and alpha1A-subtype selectivity of L-771,688. [3H]L-771,688 binding studies at the cloned human alpha1A-adrenoceptors and in rat tissues indicated that specific [3H]L-771,688 binding was saturable and of high affinity (Kd=43-90 pM) and represented binding to the pharmacologically relevant alpha1A-adrenoceptors. L-771,688 antagonized norepinephrine-induced inositol-phosphate responses in cloned human alpha1A-adrenoceptors, as well as phenylephrine or A-61603 (N-[5-4,5-dihydro-1H-imidazol-2yl)-2-hydroxy-5,6,7, 8-terahydro-naphthlen-1-yl] methanesulfonamide hydrobromide) induced contraction in isolated rat, dog and human prostate, human and monkey bladder neck and rat caudal artery with apparent Kb values of 0.02-0.28 nM. In contrast, the contraction of rat aorta induced by norepinephrine was resistant to L-771,688. These data indicate that L-771,688 is a highly selective alpha1A-adrenoceptor antagonist.     

Altered glucose homeostasis in alpha2A-adrenoceptor knockout mice

To elucidate the functions of alpha2-adrenoceptor subtypes in metabolic regulation, we determined plasma glucose and insulin levels and tissue uptake of the glucose analogue 2-[18F]fluoro-2-deoxy-d-glucose ([18F]FDG) in C57Bl/6J wild-type (WT) and alpha2A-adrenoceptor knockout (alpha2A-KO) mice at baseline and following alpha2-adrenoceptor agonist ((+)-4-(S)-[1-(2,3-dimethylphenyl)ethyl]-1H-imidazole (dexmedetomidine)) and antagonist (4-[2-ethyl-2,3-dihydro-1H-inden-2-yl]-1H-imidazole (atipamezole)) administration. Basal glucose levels were 30% lower in alpha2A-KO mice than in WT mice. In WT mice, dexmedetomidine lowered insulin and elevated glucose levels, and atipamezole reduced glucose levels. In alpha2A-KO mice, neither drug affected the glucose or insulin levels. [18F]FDG uptake was investigated in plasma, heart, liver, kidney, pancreas, lung, fat, and skeletal muscle. Cardiac [18F]FDG uptake was a sensitive indicator of sympathetic function. Liver [18F]FDG uptake conformed to the plasma glucose levels. In alpha2A-KO mice, drug effects on [18F]FDG tissue uptake were absent. Thus, the alpha2A-adrenoceptor is the alpha2-adrenoceptor subtype primarily involved in the regulation of blood glucose homeostasis in vivo.     

The effect of site-directed mutagenesis of two transmembrane serine residues on agonist-specific coupling of a cloned human alpha2A-adrenoceptor to adenylyl cyclase.

1. The effects of substitution of the Ser200 and Ser204 residues with alanine on the signalling properties of the cloned human alpha2A-adrenoceptor, stably expressed in Chinese hamster ovary (CHO) cell lines, have been investigated using noradrenaline and the structural isomers of octopamine. 2. The Ser-->Ala200 or the Ser-->Ala204 mutant forms of the alpha2A-adrenoceptor, when expressed in cells in the absence of pertussis toxin pretreatment, are two orders of magnitude more sensitive to inhibition of cyclic AMP production by (+/-)-para-octopamine and (+/-)-meta-octopamine, respectively, than cells expressing the wild-type receptor. Binding studies indicate that the effects are not due to an increased agonist affinity for the mutant receptors and that they are likely to be due to agonist-mediated conformational changes in receptor structure. 3. After incubation with pertussis toxin, (+/-)-meta-octopamine (100 microM and above) produced a stimulation of cyclic AMP levels in cells expressing the Ser-->Ala204 mutant form of the alpha2A-adrenoceptor but showed no stimulation in cells expressing the Ser-->Ala200 mutant receptor. Under these conditions (+/-)-para-octopamine did not produce any increases in cyclic AMP production in cells expressing either of the mutant receptor forms or the wild-type receptor. 4. The results emphasise the importance of the Ser200 and Ser204 residues of the alpha2A-adrenoceptor in exerting an inhibitory influence on the ability of (+/-)-para-octopamine and (+/-)-meta-octopamine respectively, to induce a receptor-agonist conformation capable of inhibiting forskolin-stimulation of cyclic AMP levels. 5. It is clear that Ser204 also prevents meta-octopamine from generating a receptor-agonist conformation that can increase cyclic AMP levels, emphasising the importance of this residue in the agonist-specific coupling of this receptor to different second messenger systems.     

The effects of SB 216469, an antagonist which discriminates between the alpha 1A-adrenoceptor and the human prostatic alpha 1-adrenoceptor.

1. The affinity of the alpha 1-adrenoceptor antagonist SB 216469 (also known as REC 15/2739) has been determined at native and cloned alpha 1-adrenoceptor subtypes by radioligand binding and at functional alpha 1-adrenoceptor subtypes in isolated tissues. 2. In radioligand binding studies with [3H]-prazosin, SB 216469 had a high affinity at the alpha 1A-adrenoceptors of the rat cerebral cortex and kidney (9.5-9.8) but a lower affinity at the alpha 1B-adrenoceptors of the rat spleen and liver (7.7-8.2). 3. At cloned rat alpha 1-adrenoceptor subtypes transiently expressed in COS-1 cells and also at cloned human alpha 1-adrenoceptor subtypes stably transfected in Rat-1 cells, SB 216469 exhibited a high affinity at the alpha 1a-adrenoceptors (9.6-10.4) with a significantly lower affinity at the alpha 1b-adrenoceptor (8.0-8.4) and an intermediate affinity at the alpha 1d-adrenoceptor (8.7-9.2). 4. At functional alpha 1-adrenoceptors, SB 216469 had a similar pharmacological profile, with a high affinity at the alpha 1A-adrenoceptors of the rat vas deferens and anococcygeus muscle (pA2 = 9.5-10.0), a low affinity at the alpha 1B-adrenoceptors of the rat spleen (6.7) and guinea-pig aorta (8.0), and an intermediate affinity at the alpha 1D-adrenoceptors of the rat aorta (8.8). 5. Several recent studies have concluded that the alpha 1-adrenoceptor present in the human prostate has the pharmacological characteristics of the alpha 1A-adrenoceptor subtype. However, the affinity of SB 216469 at human prostatic alpha 1-adrenoceptors (pA2 = 8.1) determined in isolated tissue strips, was significantly lower than the values obtained at either the cloned alpha 1a-adrenoceptors (human, rat, bovine) or the native alpha 1A-adrenoceptors in radioligand binding and functional studies in the rat. 6. Our results with SB 216469, therefore, suggest that the alpha 1-adrenoceptor mediating contractile responses of the human prostate has properties which distinguish it from the cloned alpha 1a-adrenoceptor or native alpha 1A-adrenoceptor. Since it has previously been shown that the receptor is not the alpha 1B- or alpha 1D-adrenoceptor, the functional alpha 1-adrenoceptor of the human prostate may represent a novel receptor with properties which differ from any of the alpha 1-adrenoceptors currently defined by pharmacological means.