A Concise and Useful Guide to Understand How Alpha1 Adrenoceptor Antagonists Work.

Adrenoceptors are the receptors for catecholamines, adrenaline, and noradrenaline. They are divided in α (α1 and α2) and ß (ß1, ß2 and ß3). α1-adrenoceptors are subdivided in α1A, α1B and α1D. Most tissues express mixtures of α1-adrenoceptors subtypes, which appear to coexist in different densities and ratios, and in most cases, their responses are probably due to the activation of more than one type. The three subtypes of α1-adrenoceptors are G-protein-coupled receptors (GPCR), specifically coupled to Gq/11. Additionally, the activation of these receptors may activate other signaling pathways or different components of these pathways, which leads to a great variety of possible cellular effects. The first clinically used α1 antagonist was Prazosin for Systemic Arterial Hypertension (SAH). It was followed by its congeners, Terazosin and Doxazosin. Nowadays, there are many classes of α-adrenergic antagonists with different selectivity profiles. In addition to SAH, the α1-adrenoceptors are used to treat Benign Prostatic Hyperplasia (BPH) and urolithiasis. This antagonism may be part of the mechanism of action of tricyclic antidepressants. Moreover, the activation of these receptors may lead to adverse effects such as orthostatic hypotension, similar to what happens with antidepressants and with some antipsychotics. Structure-activity relationships can explain, in part, how antagonists work and how selective they can be for each one of the subtypes. However, it is necessary to develop new molecules which antagonize the α1- adrenoceptors or make chemical modifications in these molecules to improve the selectivity and pharmacokinetic profile and/or reduce the adverse effects of known drugs.

Discovery of LASSBio-772, a 1,3-benzodioxole N-phenylpiperazine derivative with potent alpha 1A/D-adrenergic receptor blocking properties.

We described herein the discovery of 1-(2-(benzo[d] [1,3]dioxol-6-yl)ethyl)-4-(2-methoxyphenyl) piperazine (LASSBio-772), as a novel potent and selective alpha 1A/1D adrenoceptor (AR) antagonist selected after screening of functionalized N-phenylpiperazine derivatives in phenylephrine-induced vasoconstriction of rabbit aorta rings. The affinity of LASSBio-772 for alpha 1A and alpha 1B AR subtypes was determined through displacement of [(3)H]prazosin binding. We obtained Ki values of 0.14 nM for the alpha 1A-AR, similar to that displayed by tamsulosin (K(i) = 0.13 nM) and 5.55 nM for the alpha 1B-AR, representing a 40-fold higher affinity for alpha 1A-AR. LASSBio-772 also presented high affinity (K(B) = 0.025 nM) for the alpha 1D-AR subtype in the functional rat aorta assay, showing to be equipotent to tamsulosin (K(B) = 0.017 nM).

Paraventricular-coerulear interactions: role in hypertension induced by prenatal undernutrition in the rat.

Rats submitted to fetal growth retardation by in utero malnutrition develop hypertension when adult, showing increased hypothalamic mRNA expression for corticotropin-releasing hormone (CRH) and increased central noradrenergic activity. As hypothalamic CRH serves as an excitatory neurotransmitter within the locus coeruleus (LC) and coerulear norepinephrine plays a similar role within the paraventricular nucleus (PVN) of the hypothalamus, we studied, in both normal and prenatally undernourished 40-day-old anesthetized rats, the effects of intra-LC microinjection of CRH and intra-PVN microinjection of the alpha(1)-adrenoceptor antagonist prazosin on multiunit neuronal activity recorded simultaneously from the two nuclei, as well as the effects on systolic pressure. Undernutrition was induced during fetal life by restricting the diet of pregnant mothers to 10 g daily, whereas mothers of control rats received the same diet ad libitum. At day 40 of postnatal life: (i) undernourished rats showed increased neuronal activity in the PVN and LC, as well as increased systolic pressure; (ii) intra-LC CRH stimulated LC and PVN neurons and increased systolic pressure only in normal rats; (iii) intra-PVN prazosin decreased LC and PVN neuronal activity and systolic pressure only in undernourished rats; and (iv) in normal rats, prazosin prevented the stimulatory effect of CRH only in PVN activity; in undernourished rats, prazosin allowed CRH to regain its stimulatory effects. The results point to the existence of an excitatory PVN-LC closed loop, which seems to be hyperactive in prenatally undernourished rats as a consequence of fetal programming; this loop could be responsible, in part, for the hypertension developed by these animals.

Noradrenaline increases the firing rate of a subpopulation of rat subthalamic neurones through the activation of alpha 1-adrenoceptors.

In the rat subthalamic nucleus, which plays a critical role in the control of motor behaviour, specific binding of [3H]-prazosin was detected by radioligand binding to homogenates and by autoradiography in slices. [3H]-Prazosin binding to homogenates (Bmax 71 +/- 5 fmol/mg protein; Kd 0.27 +/- 0.05 nM) was competed for by alpha1-antagonists. In subthalamic nucleus slices and in the presence of 10 mM LiCl, noradrenaline (100 microM) produced a modest, but consistent, stimulation of [3H]-inositol phosphate accumulation (146 +/- 6% of basal), reversed by the alpha1-antagonist prazosin (1 microM). Extracellular single-unit recordings in slices showed that in a subpopulation (61 out of 94 cells) of rat subthalamic neurones with regular, single-spike firing pattern, noradrenaline induced a concentration-dependent increase in the firing rate (EC50 2.5 +/- 0.2 microM, maximum effect 272 +/- 33% of basal). The action of noradrenaline was mimicked by the selective alpha1-agonist phenylephrine but not by selective alpha2- or beta-agonists, and was blocked by the alpha1-antagonist prazosin but not by alpha2- or beta-antagonists. The excitatory effect of noradrenaline was not prevented by perfusion with low Ca2+/high Mg2+ solution. In four out of 11 neurones perfusion with 3 microM noradrenaline resulted in a shift from bursting to regular firing. Taken together, our results indicate that rat subthalamic neurones express alpha1-adrenoceptors responsible for noradrenaline-induced stimulation of the firing rate of a subpopulation of neurones. By modulating the spontaneous activity of STN neurones, noradrenergic pathways might have a significant role in regulating basal ganglia function and thus motor activity.

Hormonal influence on expression and functionality of alpha1-adrenoceptor in rat submandibular gland.

In this work, we characterized alpha(1)-adrenoceptor expression and functionality in rat submandibular gland. Cumulative dose-response curve of methoxamine was constructed to determine the peroxidase secretion by glands from proestrous, estrous, metestrous and diestrous rats. They were compared to those from animals untreated or treated with sex hormones, estradiol and progesterone. The sensitivity of glands to an alpha(1)-adrenoceptor agonist varied depending on hormonal state, i.e. glands from proestrous and estrous were more sensitive to the stimulatory action of methoxamine than those from metestrous, diestrous and ovariectomised animals. The efficacy of the alpha(1) agonist was enhanced in glands from ovariectomised estrogen-treated rats but it was ineffective in glands from ovariectomised progesterone-treated rats. The functional studies correlated with 3H-prazosin binding assays in which estrogen increased alpha(1)-adrenoceptor density while progesterone decreased it. The results demonstrated that alpha(1)-adrenoceptor expression and functionality in rat submandibular glands are apparently under hormonal control and probably represent other examples of bidirectional interactions between neuronal and exocrine systems.

Alpha1-adrenoceptor subtypes in rat epididymis and the effects of sexual maturation.

We have characterized the expression of alpha1-adrenoceptor in epididymis from rats in different stages of sexual maturation: 40 (immature), 60 (young adult), and 120 (adult) days of age. Plasma testosterone levels were low in the immature animals but increased significantly in the 60- and 120-day-old animals. These changes were followed by a progressive increase in rat body weight and in caput and cauda epididymis relative weight. Reverse transcription polymerase chain reaction assay indicated that alpha1a-, alpha1b-, and alpha1d-adrenoceptor transcripts were present in both caput and cauda epididymis from adult rats. Ribonuclease protection assays further indicated that the expression of these alpha1-adrenoceptor transcripts differed with age and epididymal region analyzed. Prazosin (nonselective alpha1 antagonist), 5-methyl urapidil (alpha1A-selective), and BMY 7378 (alpha1D-selective) displaced [3H]prazosin binding curves in caput and cauda epididymis from 40- and 120-day-old rats. The potency order for these antagonists, as calculated from the negative logarithm of the inhibition constant (pK(i)) values for the high-affinity sites, indicated a predominant population of alpha1A-adrenoceptor subtype in caput and cauda epididymis from adult animals. Differences in pK(i) values in caput and cauda epididymis from immature and adult animals also suggested that the relative amount of alpha1-adrenoceptors, at the protein level, is modulated by sexual maturation. Taken together, the changes in alpha1-adrenoceptor expression during sexual maturation may suggest specific roles for these receptors in epididymal function.

Molecular cloning and functional expression of the guinea pig alpha(1a)-adrenoceptor.

In the present paper, the cloning and expression of the guinea pig alpha(1A)-adrenoceptor is presented. The nucleotide sequence had an open reading frame of 1401 bp that encoded a 466 amino-acid protein with an estimated molecular mass of approximately 51.5 kDa. When the clone was expressed in Cos-1 cells, specific high-affinity binding of [(3)H]prazosin and [(3)H]tamsulosin was observed. Chloroethylclonidine treatment of membranes slightly decreased the total binding with both radioligands. Binding competition experiments using [(3)H]tamsulosin showed the following potency order: (a) for agonists: oxymetazoline >>epinephrine>norepinephrine>methoxamine, and (b) for antagonists: prazosin> or 5-methyl-urapidil=benoxathian>phentolamine>>BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4,5]decane-7,9-dione). Photoaffinity labeling using [(125)I-aryl]azido-prazosin revealed a major broad band with a molecular mass between 70 and 80 kDa. The receptor was functional, as evidenced by an epinephrine-increased production of [(3)H]inositol phosphates that was blocked by prazosin.

Protein kinase C-mediated phosphorylation and desensitization of human alpha(1b)-adrenoceptors.

Human alpha(1b)-adrenoceptors stably expressed (B(max) approximately 800 fmol/mg membrane protein) in mouse fibroblasts were able to increase intracellular Ca(2+) and inositol phosphate production in response to noradrenaline. Activation of protein kinase C desensitized the alpha(1b)-adrenergic-mediated actions but did not block the ability of the cells to respond to lysophosphatidic acid. Inhibition or downregulation of protein kinase C also blocked the action of the tumor promoter on the adrenergic effects. Photolabeling experiments indicated that the receptor has an apparent molecular weight of approximately 80 kDa. The receptors were phosphorylated in the basal state and such phosphorylation was increased when the cells were incubated with phorbol myristate acetate or noradrenaline. Incubation of the cells with phorbol myristate acetate or noradrenaline blocked noradrenaline-promoted [35S]GTP-gamma-S binding to membranes, suggesting receptor-G protein uncoupling. The results indicate that activation of protein kinase C blocked/desensitized human alpha(1b)-adrenoceptors and that such effect was associated to receptor phosphorylation.

Patterns of adrenoceptor change during liver regeneration of the wistar kyoto rat: A bunding study

Background. Adrenoceptors have been involved in the regulation of hepatocyte proliferation after partial hepatectomy, as well as in primary culture. This report characterized Ó1 and ß-adrenoceptor change during the time-course of liver regeneration in adult Wistar Kyoto rats. Methods. Saturation binding assays with [3H]prazosin or [3H]dihydroalprenolol (for Ó1- and ß-adrenoceptors, respectively) were done in liver plasma membranes from 6-monthold rats subjected to 70 percent hepatectomy followed by hepatic regeneration. Results. [3H]Prazosin and [3H]dihydroalprenolol binding gave control Bmax values of 101 ñ 10 and 12 ñ 1 fmol/mg protein and Kd of 0.50 ñ 0.10 and 4.1 ñ 0.4 nM for Ó1- and ß-adrenoceptors, respectively. Ó1-Adrenoceptor number and Kd increades at 24 and 48 h and returned to control values at 72 and 96 h after surgery, whereas ß-adrenoceptors augmented at 48 and 72 h, with a Kd change at 24 and 48 h postherapetectomy. Conclusions.These results suggest that dual control of Ó1- and ß-adrenoceptor membrane expression could be involved in different steps during hepatocyte proliferation, and that Wistar Kyoto rats have a different adrenoceptor pattern expression from other rat strains

Long-term treatment with fluoxetine associates with peripheral effects on rat vas deferens contractility.

The aim of this work was to study whether long-term treatment with fluoxetine could induce peripheral effects by modifying vas deferens contractile activity. For this purpose the contractile response to NE, and 5-HT of vas deferens isolated from male Wistar rats that received fluoxetine 10 mg/kg/day i.p., during 21 days, was studied using the isolated organ bath technique. Results show that vas deferens of treated rats presented spontaneous activity, an effect that was abolished by prazosin and isoproterenol and that was not affected by nitroprusside or indomethacin. In addition, fluoxetine did not modify the response to calcium suggesting that spontaneous activity was not a consequence of an abnormal calcium movement. Fluoxetine induced a significant increase in the response of vas deferens to 5-HT and to low NE concentrations while NE maximal effect was unaffected. Fluoxetine treatment did not modify the binding parameters of [3H]-prazosin to vas deferens. It is concluded that long-term treatment with fluoxetine modifies vas deferens contractile activity. This effect could be the result of an alteration of adrenergic neurotransmission and could account for some of the untoward effects observed during clinical course with fluoxetine.

Heterogeneous effects of rapid eye movement sleep deprivation on binding to alpha- and beta-adrenergic receptor subtypes in rat brain.

Quantitative receptor autoradiography was used to map alterations in binding to alpha1-, alpha2-, beta1- and beta2-adrenergic receptors throughout the brain of rats deprived of rapid eye movement sleep for 96 h. Binding of [3H]prazosin to alpha1 sites, while not significantly different in any of 46 brain regions examined, showed a clear overall tendency towards decreased values after sleep deprivation. [3H]UK-14,314-labeled alpha2 binding sites were not significantly affected by sleep deprivation in any of 91 brain regions analysed, despite a trend towards increased values. In contrast, beta-adrenergic binding was significantly reduced throughout the brain. Binding to beta1 sites labeled by [125I]iodopindolol in the presence of ICI-11855 was significantly reduced in 13 of 69 brain areas examined; binding to beta2 sites labeled by [125I]iodopindolol in the presence of CGP-20712A was likewise reduced throughout the brain and significantly so in 25 of the 72 brain areas analysed. Rank ordering of the binding changes indicated that reductions in beta1 vs beta2 binding were maximal in different brain areas. This pattern of results may reflect a particular configuration of effects specifically associated with sleep loss stress. The results are consistent with evidence of persisting noradrenergic cell activity during sleep deprivation. The observed heterogeneity of effects suggests that not all norepinephrine receptors are equally affected by rapid eye movement sleep deprivation.

Tratamento da hipertensäo arterial: monoterapia da hipertensäo leve a moderada com dose única diária de prazosina cápsulas de liberaçäo gradual (SR) / Treatment of essential hypertension: monoterapy of mild and moderate forms with single daily doses of sustained-release prazosin

A eficácia e a tolerabilidade de uma nova formulaçäo de liberaçäo gradual do bloqueador do receptor pós-dináptico alfa, prazosina, foram investigadas em um estudo clínico multicêntrico aberto de pacientes ambulatoriais portadores de hipertensäo essencial leve e moderada. Com a posologia de dose única diária, as pressöes arteriais sistólica e diastólica foram significativamente reduzidas logo ao início do tratamento e durante as 12 semanas da duraçäo do estudo. Em média, a freqüência cardíaca permaneceu constante inicialmente e durante os três meses do período de tratamento. A dose inicial de prazosina de liberaçäo gradual (1mg) proporcionou uma reduçäo suave e regular da pressäo arterial, com mínimos efeito colaaterais. Além disso, a eficácia da droga permaneceu inalterada após os três meses de tratamento

Binding of adrenergic ligands to liver plasma membrane preparations from the axolotl, Ambystoma mexicanum; the toad, Xenopus laevis; and the Australian lungfish, Neoceratodus forsteri.

The beta-adrenergic ligand iodocyanopindolol (ICP) bound specifically to hepatic plasma membrane preparations from the axolotl, Ambystoma mexicanum (Bmax, 40 fmol/mg protein (P) at free concentration above 140 pM; KD, 42 pM); the toad, Xenopus laevis (Bmax, 200 fmol/mg P at 1 nM; KD, 300 pM); and the Australian lungfish, Neoceratodus forsteri (Bmax, 100 fmol/mg P at 5 nM). For the lungfish, the Scatchard plot was curved showing two classes of binding site with KD's of 20 and 500 pM. Neither the alpha 1-adrenergic ligand prazosin nor the alpha 2-adrenergic ligand yohimbine bound specifically to hepatic membrane preparations from any of the three species. Several adrenergic ligands displaced ICP from hepatic membrane preparations of all three species with KD's of Axolotl--propranolol, 50 nM; isoprenaline, 600 nM; adrenaline, 10 microM; phenylephrine, 20 microM; noradrenaline, 40 microM; and phentolamine, greater than 100 microM; X. laevis--propranolol, 30 nM; isoprenaline, 100 microM; adrenaline, 200 microM; noradrenaline, 300 microM; phenylephrine, 1 mM; and phentolamine, greater than 1 mM; N. forsteri,--propranolol, 25 nM; isoprenaline, 1 microM; adrenaline, 20 microM; phenylephrine, 35 microM; noradrenaline, 600 microM; and phentolamine, 400 microM. These findings suggest that alpha-adrenergic receptors are not present in hepatic plasma membrane preparations from these three species and that the hepatic actions of catecholamines are mediated via beta-adrenergic receptors. The order of binding of the beta-adrenergic ligands suggests that the receptors are of the beta 2 type.