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.

Organic Cation Transporters are Involved in Fluoxetine Transport Across the Blood-Brain Barrier In Vivo and In Vitro.

BACKGROUND: The research and development of drugs for the treatment of central nervous system diseases faces many challenges at present. One of the most important questions to be answered is, how does the drug cross the blood-brain barrier to get to the target site for pharmacological action. Fluoxetine is widely used in clinical antidepressant therapy. However, the mechanism by which fluoxetine passes through the BBB also remains unclear. Under physiological pH conditions, fluoxetine is an organic cation with a relatively small molecular weight (<500), which is in line with the substrate characteristics of organic cation transporters (OCTs). Therefore, this study aimed to investigate the interaction of fluoxetine with OCTs at the BBB and BBB-associated efflux transporters. This is of great significance for fluoxetine to better treat depression. Moreover, it can provide a theoretical basis for clinical drug combination. METHODS: In vitro BBB model was developed using human brain microvascular endothelial cells (hCMEC/D3), and the cellular accumulation was tested in the presence or absence of transporter inhibitors. In addition, an in vivo trial was performed in rats to investigate the effect of OCTs on the distribution of fluoxetine in the brain tissue. Fluoxetine concentration was determined by a validated UPLC-MS/MS method. RESULTS: The results showed that amantadine (an OCT1/2 inhibitor) and prazosin (an OCT1/3 inhibitor) significantly decreased the cellular accumulation of fluoxetine (P <.001). Moreover, we found that N-methylnicotinamide (an OCT2 inhibitor) significantly inhibited the cellular uptake of 100 and 500 ng/mL fluoxetine (P <.01 and P <.05 respectively). In contrast, corticosterone (an OCT3 inhibitor) only significantly inhibited the cellular uptake of 1000 ng/mL fluoxetine (P <.05). The P-glycoprotein (P-gp) inhibitor, verapamil, and the multidrug resistance associated proteins (MRPs) inhibitor, MK571, significantly decreased the cellular uptake of fluoxetine. However, intracellular accumulation of fluoxetine was not significantly changed when fluoxetine was incubated with the breast cancer resistance protein (BCRP) inhibitor Ko143. Furthermore, in vivo experiments proved that corticosterone and prazosin significantly inhibited the brain-plasma ratio of fluoxetine at 5.5 h and 12 h, respectively. CONCLUSION: OCTs might play a significant role in the transport of fluoxetine across the BBB. In addition, P-gp, BCRP, and MRPs seemed not to mediate the efflux transport of fluoxetine.

Application of fluorescence correlation spectroscopy to study substrate binding in styrene maleic acid lipid copolymer encapsulated ABCG2.

ABCG2 is one of a trio of human ATP binding cassette transporters that have the ability to bind and transport a diverse array of chemical substrates out of cells. This so-called "multidrug" transport has numerous physiological consequences including effects on how drugs are absorbed into and eliminated from the body. Understanding how ABCG2 is able to interact with multiple drug substrates remains an important goal in transporter biology. Most drugs are believed to interact with ABCG2 through the hydrophobic lipid bilayer and experimental systems for ABCG2 study need to incorporate this. We have exploited styrene maleic acid to solubilise ABCG2 from HEK293T cells overexpressing the transporter, and confirmed by dynamic light scattering and fluorescence correlation spectroscopy (FCS) that this results in the extraction of SMA lipid copolymer (SMALP) particles that are uniform in size and contain a dimer of ABCG2, which is the predominant physiological state. FCS was further employed to measure the diffusion of a fluorescent ABCG2 substrate (BODIPY-prazosin) in the presence and absence of SMALP particles of purified ABCG2. Autocorrelation analysis of FCS traces enabled the mathematical separation of free BODIPY-prazosin from drug bound to ABCG2 and allowed us to show that combining SMALP extraction with FCS can be used to study specific drug: transporter interactions.

Elucidation of prazosin biodegradation by isolated Bacillus spp. from the tropical environment.

Prazosin (PRZ), a drug used to treat hypertensive patients, is an emergent contaminant in water systems. PRZ is an alpha-adrenergic receptor blocker used to treat anxiety, and is believed to reach the environment through human excretion, irresponsible disposal of unused medicine, and waste products from manufacturing plants. The purpose of this research was to isolate and characterize potential microbes for PRZ biodegradation and to identify the degradation pathway. After screening, isolated strain STP3 showed a capability for PRZ degradation and was chosen for further analysis. Resting cell assays with PRZ were conducted to identify the intermediate metabolites formed from biodegradation by Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) analysis. Two metabolites degraded from PRZ by STP3 were successfully found, and as these metabolites are derived from the main structure of PRZ, their presence proved PRZ degradation. Draft genome sequencing analysis of STP3 was performed to identify potential enzymes for PRZ biodegradation based on the metabolites found.

Reversible inhibition of efflux transporters by hydrogel microdevices.

Oral drug delivery is a preferred administration route due to its low cost, high patient compliance and fewer adverse events compared to intravenous administration. However, many pharmaceuticals suffer from poor solubility and low oral bioavailability. One major factor that contributes to low bioavailability are efflux transporters which prevent drug absorption through intestinal epithelial cells. P-glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP) are two important efflux transporters in the intestine functioning to prevent toxic materials from entering systemic circulation. However, due to its broad substrate specificity, P-gp limits the absorption of many therapeutics, including chemotherapeutics and antibacterial agents. Methods to inhibit P-gp with competitive inhibitors have not been clinically successful. Here, we show that micron scale devices (microdevices) made from a commonly used biomaterial, polyethylene glycol (PEG), inhibit P-gp through a biosimilar mucus in Caco-2 cells and that transporter function is restored when the microdevices are removed. Microdevices were shown to inhibit P-gp mediated transport of calcein AM, doxorubicin, and rhodamine 123 (R123) and BCRP mediated transport of BODIPY-FL-prazosin. When in contact with Caco-2 cells, microdevices decrease the cell surface amount of P-gp without affecting the passive transport. Moreover, there was an increase in mucosal to serosal transport of R123 with microdevices in an ex-vivo mouse model and increased absorption in vivo. This biomaterial-based approach to inhibit efflux transporters can be applied to a range of drug delivery systems and allows for a nonpharmacologic method to increase intestinal drug absorption while limiting toxic effects.

The pharmacology of α1-adrenoceptor subtypes.

This review examines the functions of α1-adrenoceptor subtypes, particularly in terms of contraction of smooth muscle. There are 3 subtypes of α1-adrenoceptor, α1A- α1B- and α1D-adrenoceptors. Evidence is presented that the postulated α1L-adrenoceptor is simply the native α1A-adrenoceptor at which prazosin has low potency. In most isolated tissue studies, smooth muscle contractions to exogenous agonists are mediated particularly by α1A-, with a lesser role for α1D-adrenoceptors, but α1B-adrenoceptors are clearly involved in contractions of some tissues, for example, the spleen. However, nerve-evoked responses are the most crucial physiologically, so that these studies of exogenous agonists may overestimate the importance of α1A-adrenoceptors. The major α1-adrenoceptors involved in blood pressure control by sympathetic nerves are the α1D- and the α1A-adrenoceptors, mediating peripheral vasoconstrictor actions. As noradrenaline has high potency at α1D-adrenceptors, these receptors mediate the fastest response and seem to be targets for neurally released noradrenaline especially to low frequency stimulation, with α1A-adrenoceptors being more important at high frequencies of stimulation. This is true in rodent vas deferens and may be true in vasopressor nerves controlling peripheral resistance and tissue blood flow. The αlA-adrenoceptor may act mainly through Ca2+ entry through L-type channels, whereas the α1D-adrenoceptor may act mainly through T-type channels and exhaustable Ca2+ stores. α1-Adrenoceptors may also act through non-G-protein linked second messenger systems. In many tissues, multiple subtypes of α-adrenoceptor are present, and this may be regarded as the norm rather than exception, although one receptor subtype is usually predominant.

Evidence for the Interaction of A3 Adenosine Receptor Agonists at the Drug-Binding Site(s) of Human P-glycoprotein (ABCB1).

P-glycoprotein (P-gp) is a multidrug transporter that is expressed on the luminal surface of epithelial cells in the kidney, intestine, bile-canalicular membrane in the liver, blood-brain barrier, and adrenal gland. This transporter uses energy of ATP hydrolysis to efflux from cells a variety of structurally dissimilar hydrophobic and amphipathic compounds, including anticancer drugs. In this regard, understanding the interaction with P-gp of drug entities in development is important and highly recommended in current US Food and Drug Administration guidelines. Here we tested the P-gp interaction of some A3 adenosine receptor agonists that are being developed for the treatment of chronic diseases, including rheumatoid arthritis, psoriasis, chronic pain, and hepatocellular carcinoma. Biochemical assays of the ATPase activity of P-gp and by photolabeling P-gp with its transport substrate [125I]-iodoarylazidoprazosin led to the identification of rigidified (N)-methanocarba nucleosides (i.e., compound 3 as a stimulator and compound 8 as a partial inhibitor of P-gp ATPase activity). Compound 8 significantly inhibited boron-dipyrromethene (BODIPY)-verapamil transport mediated by human P-gp (IC50 2.4 ± 0.6 µM); however, the BODIPY-conjugated derivative of 8 (compound 24) was not transported by P-gp. In silico docking of compounds 3 and 8 was performed using the recently solved atomic structure of paclitaxel (Taxol)-bound human P-gp. Molecular modeling studies revealed that both compounds 3 and 8 bind in the same region of the drug-binding pocket as Taxol. Thus, this study indicates that nucleoside derivatives can exhibit varied modulatory effects on P-gp activity, depending on structural functionalization. SIGNIFICANCE STATEMENT: Certain A3 adenosine receptor agonists are being developed for the treatment of chronic diseases. The goal of this study was to test the interaction of these agonists with the human multidrug resistance-linked transporter P-glycoprotein (P-gp). ATPase and photolabeling assays demonstrated that compounds with rigidified (N)-methanocarba nucleosides inhibit the activity of P-gp; however, a fluorescent derivative of one of the compounds was not transported by P-gp. Furthermore, molecular docking studies revealed that the binding site for these compounds overlaps with the site for paclitaxel in the drug-binding pocket. These results suggest that nucleoside derivatives, depending on structural functionalization, can modulate the function of P-gp.

α1A-Adrenoceptors activate mTOR signalling and glucose uptake in cardiomyocytes.

The capacity of G protein-coupled receptors to modulate mechanistic target of rapamycin (mTOR) activity is a newly emerging paradigm with the potential to link cell surface receptors with cell survival. Cardiomyocyte viability is linked to signalling pathways involving Akt and mTOR, as well as increased glucose uptake and utilization. Our aim was to determine whether the α1A-adrenoceptor (AR) couples to these protective pathways, and increased glucose uptake. We characterised α1A-AR signalling in CHO-K1 cells co-expressing the human α1A-AR and GLUT4 (CHOα1AGLUT4myc) and in neonatal rat ventricular cardiomyocytes (NRVM), and measured glucose uptake, intracellular Ca2+ mobilization, and phosphorylation of mTOR, Akt, 5' adenosine monophosphate-activated kinase (AMPK) and S6 ribosomal protein (S6rp). In both systems, noradrenaline and the α1A-AR selective agonist A61603 stimulated glucose uptake by parallel pathways involving mTOR and AMPK, whereas another α1-AR agonist oxymetazoline increased glucose uptake predominantly by mTOR. All agonists promoted phosphorylation of mTOR at Ser2448 and Ser2481, indicating activation of both mTORC1 and mTORC2, but did not increase Akt phosphorylation. In CHOα1AGLUT4myc cells, siRNA directed against rictor but not raptor suppressed α1A-AR mediated glucose uptake. We have thus identified mTORC2 as a key component in glucose uptake stimulated by α1A-AR agonists. Our findings identify a novel link between the α1A-AR, mTORC2 and glucose uptake, that have been implicated separately in cardiomyocyte survival. Our studies provide an improved framework for examining the utility of α1A-AR selective agonists as tools in the treatment of cardiac dysfunction.

Virtual Screening against Phosphoglycerate Kinase 1 in Quest of Novel Apoptosis Inhibitors.

Inhibition of apoptosis is a potential therapy to treat human diseases such as neurodegenerative disorders (e.g., Parkinson's disease), stroke, and sepsis. Due to the lack of druggable targets, it remains a major challenge to discover apoptosis inhibitors. The recent repositioning of a marketed drug (i.e., terazosin) as an anti-apoptotic agent uncovered a novel target (i.e., human phosphoglycerate kinase 1 (hPgk1)). In this study, we developed a virtual screening (VS) pipeline based on the X-ray structure of Pgk1/terazosin complex and applied it to a screening campaign for potential anti-apoptotic agents. The hierarchical filters in the pipeline (i.e., similarity search, a pharmacophore model, a shape-based model, and molecular docking) rendered 13 potential hits from Specs chemical library. By using PC12 cells (exposed to rotenone) as a cell model for bioassay, we first identified that AK-918/42829299, AN-465/41520984, and AT-051/43421517 were able to protect PC12 cells from rotenone-induced cell death. Molecular docking suggested these hit compounds were likely to bind to hPgk1 in a similar mode to terazosin. In summary, we not only present a versatile VS pipeline for potential apoptosis inhibitors discovery, but also provide three novel-scaffold hit compounds that are worthy of further development and biological study.

Differential Interaction of Dantrolene, Glafenine, Nalidixic Acid, and Prazosin with Human Organic Anion Transporters 1 and 3.

In renal proximal tubule cells, the organic anion transporters 1 and 3 (OAT1 and OAT3) in the basolateral membrane and the multidrug resistance-associated protein 4 (MRP4) in the apical membrane share substrates and co-operate in renal drug secretion. We hypothesized that recently identified MRP4 inhibitors dantrolene, glafenine, nalidixic acid, and prazosin also interact with human OAT1 and/or OAT3 stably transfected in human embryonic kidney 293 cells. These four drugs were tested as possible inhibitors of p-[3H]aminohippurate (PAH) and [14C]glutarate uptake by OAT1, and of [3H]estrone-3-sulfate (ES) uptake by OAT3. In addition, we explored whether these drugs decrease the equilibrium distribution of radiolabeled PAH, glutarate, or ES, an approach intended to indirectly suggest drug/substrate exchange through OAT1 and OAT3. With OAT3, a dose-dependent inhibition of [3H]ES uptake and a downward shift in [3H]ES equilibrium were observed, indicating that all four drugs bind to OAT3 and may possibly be translocated. In contrast, the interaction with OAT1 was more complex. With [14C]glutarate as substrate, all four drugs inhibited uptake but only glafenine and nalidixic acid shifted glutarate equilibrium. Using [3H]PAH as a substrate of OAT1, nalidixic acid inhibited but dantrolene, glafenine, and prazosin stimulated uptake. Nalidixic acid decreased equilibrium content of [3H]PAH, suggesting that it may possibly be exchanged by OAT1. Taken together, OAT1 and OAT3 interact with the MRP4 inhibitors dantrolene, glafenine, nalidixic acid, and prazosin, indicating overlapping specificities. At OAT1, more than one binding site must be assumed to explain substrate and drug-dependent stimulation and inhibition of transport activity.

The 5-HT1A Receptor PET Radioligand 11C-CUMI-101 Has Significant Binding to α1-Adrenoceptors in Human Cerebellum, Limiting Its Use as a Reference Region.

Prazosin, a potent and selective α1-adrenoceptor antagonist, displaces 25% of 11C-CUMI-101 ([O-methyl-11C]2-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)-4-methyl-1,2,4-triazine-3,5(2H,4H)dione) binding in monkey cerebellum. We sought to estimate the percentage contamination of 11C-CUMI-101 binding to α1-adrenoceptors in human cerebellum under in vivo conditions. In vitro receptor-binding techniques were used to measure α1-adrenoceptor density and the affinity of CUMI-101 for these receptors in human, monkey, and rat cerebellum. METHODS: Binding potential (maximum number of binding sites × affinity [(1/dissociation constant]) was determined using in vitro homogenate binding assays in human, monkey, and rat cerebellum. 3H-prazosin was used to determine the maximum number of binding sites, as well as the dissociation constant of 3H-prazosin and the inhibition constant of CUMI-101. RESULTS: α1-adrenoceptor density and the affinity of CUMI-101 for these receptors were similar across species. Cerebellar binding potentials were 3.7 for humans, 2.3 for monkeys, and 3.4 for rats. CONCLUSION: Reasoning by analogy, 25% of 11C-CUMI-101 uptake in human cerebellum reflects binding to α1-adrenoceptors, suggesting that the cerebellum is of limited usefulness as a reference tissue for quantification in human studies.

The cytotoxicity of the α1-adrenoceptor antagonist prazosin is linked to an endocytotic mechanism equivalent to transport-P.

Since the α1-adrenergic antagonist prazosin (PRZ) was introduced into medicine as a treatment for hypertension and benign prostate hyperplasia, several studies have shown that PRZ induces apoptosis in various cell types and interferes with endocytotic trafficking. Because PRZ is also able to induce apoptosis in malignant cells, its cytotoxicity is a focus of interest in cancer research. Besides inducing apoptosis, PRZ was shown to serve as a substrate for an amine uptake mechanism originally discovered in neurones called transport-P. In line with our hypothesis that transport-P is an endocytotic mechanism also present in non-neuronal tissue and linked to the cytotoxicity of PRZ, we tested the uptake of QAPB, a fluorescent derivative of PRZ, in cancer cell lines in the presence of inhibitors of transport-P and endocytosis. Early endosomes and lysosomes were visualised by expression of RAB5-RFP and LAMP1-RFP, respectively; growth and viability of cells in the presence of PRZ and uptake inhibitors were also tested. Cancer cells showed co-localisation of QAPB with RAB5 and LAMP1 positive vesicles as well as tubulation of lysosomes. The uptake of QAPB was sensitive to transport-P inhibitors bafilomycin A1 (inhibits v-ATPase) and the antidepressant desipramine. Endocytosis inhibitors pitstop(®) 2 (general inhibitor of endocytosis), dynasore (dynamin inhibitor) and methyl-ß-cyclodextrin (cholesterol chelator) inhibited the uptake of QAPB. Bafilomycin A1 and methyl-ß-cyclodextrin but not desipramine were able to preserve growth and viability of cells in the presence of PRZ. In summary, we confirmed the hypothesis that the cellular uptake of QAPB/PRZ represents an endocytotic mechanism equivalent to transport-P. Endocytosis of QAPB/PRZ depends on a proton gradient, dynamin and cholesterol, and results in reorganisation of the LAMP1 positive endolysosomal system. Finally, the link seen between the cellular uptake of PRZ and cell death implies a still unknown pro-apoptotic membrane protein with affinity towards PRZ.

Molecular exploration of the α(1A)-adrenoceptor orthosteric site: binding site definition for epinephrine, HEAT and prazosin.

Despite the physiological and pharmacological importance of the α1A-adrenoreceptor, the mode of interactions of classical agonists and radioactive ligands with this receptor is not yet clearly defined. Here, we used mutagenesis studies and binding experiments to evaluate the importance of 11 receptor sites for the binding of (125)I-HEAT, (3)H-prazosin and epinephrine. Only one residue (F312) commonly interacts with the three molecules, and, surprisingly, D106 interacts only with epinephrine in a moderate way. Our docking model shows that prazosin and HEAT are almost superimposed into the orthosteric pocket with their tetralone and quinazoline rings close to the phenyl ring of the agonist.

Molecular and pharmacological characteristics of the gerbil α(1a)-adrenergic receptor.

The spiral modiolar artery supplies blood and essential nutrients to the cochlea. Our previous functional study indicates the α(1A)-adrenergic receptor subtype mediates vasoconstriction of the gerbil spiral modiolar artery. Although the gerbil cochlea is often used as a model in hearing research, the molecular and pharmacological characteristics of the cloned gerbil α(1a)-adrenergic receptor have not been determined. Thus we cloned, expressed and characterized the gerbil α(1a)-adrenergic receptor and then compared its molecular and pharmacological properties to those of other mammalian α(1a)-adrenergic receptors. The cDNA clone contained 1404 nucleotides, which encoded a 467 amino acid peptide with a deduced sequence having 96.8, 96.4 and 91.6% identity to rat, mouse and human α(1a)-receptors, respectively. We transiently transfected the α(1a)-adrenergic receptor into COS-1 cells and determined its pharmacological characteristics by [(3)H]prazosin binding. Unlabeled prazosin had a K(i) of 0.89±0.1nM. The α(1A)-adrenergic receptor-selective antagonists, 5-methylurapidil and WB-4101, bound with high affinity and had K(i) values of 4.9±1 and 1.0±0.1nM, respectively. BMY-7378, an α(1D)-adrenergic receptor-selective antagonist, bound with low affinity (260±60nM). The 91.6% amino acid sequence identity and K(i)s of the cloned gerbil α(1a)-adrenergic receptor are similar to those of the human α(1a)-adrenergic receptor clone. These results show that the gerbil α(1a)-adrenergic receptor is representative of the human α(1a)-adrenergic receptor, lending validity to the use of the gerbil spiral modiolar artery as a model in studies of vascular disorders of the cochlea.

Effect of amitriptyline on adrenergic receptor number and second messenger function in rat brain.

Radioligand binding studies were done to investigate the effect of chronic administration of Amitriptyline on alpha1-adrenoceptor (alpha1-AR) receptor mediated response to inositol triphosphate (IP3) in rat brain. Our studies revealed a significant decrease in the densities of alpha1-ARs in cortex and cerebellum of rat brain after chronic administration of Amitriptyline (10 mg kg-1 b.wt.). However, there was no significant change in the affinity of [3H]prazosin to alpha1-ARs. Displacement studies showed that Amitriptyline has higher affinity for alpha1-AR with a Ki value of 182+/-16 nM. Significant change was observed in basal IP3 activity in cortex and cerebellum after Amitriptyline exposure. In cortex and cerebellum of experimental rats the NE (Norepinephrine) stimulated IP3 activity was significantly decreased (1460+/-102 DPM/g tissue; p<0.0001; 1188+/-112 DPM/g tissue; p<0.0001), when compared to NE stimulated IP3 activity (4152+/-286 and 3952+/-245 DPM/g tissue, respectively) in control rats. The decrease in NE stimulated IP3 activity in both regions may be due to the significant downregulation of alpha1-ARs in cortex after Amitriptyline exposure as these sites are positively coupled to IP3. The observed significant decrease in alphal-ARs with concomitant decrease in NE stimulated IP3 activity, after Amitriptyline treatment, suggests that Amitriptyline which has high affinity for these sites, acts by modulating the alpha1-AR receptor mediated response in brain.

Functional analysis of a novel antagonistic antibody against the short epitope of the α1A-adrenergic receptor.

AIMS: The alpha1A-adrenergic receptor (α1A-AR) regulates various vascular functions and participates in the pathogenesis of primary hypertension. However, highly specific and subtype-selective antagonists of α1A-AR have not been developed. METHODS AND RESULTS: Two novel antibodies against the short peptides CP-7, which is located in the second extracellular loop of α1A-AR, and CPE-8, which is located in the third extracellular loop of α1A-AR, were prepared. The two antibodies specifically bound to α1A-AR. However, neither antibody prevented [(3)H]-epinephrine or [(3)H]-prazosin from binding to the receptor. In vitro, the anti-CP-7 antibody inhibited Ca(2+)-dependent signal transduction processes including protein kinase C translocation and extracellular signal-regulated kinase (ERK1/2) phosphorylation induced by phenylephrine (PHE). The anti-CP-7 antibody decreased the beating rates of neonatal rat cardiomyocytes with or without PHE stimulation and reduced the blood pressure of spontaneously hypertensive rats that were immunized with CP-7-keyhole limpet haemocyanin. CONCLUSION: The anti-CP-7 antibody specifically inhibited the activation of α1A-AR both in vitro and in vivo. No competition binding was found between anti-CP-7 and [(3)H]-epinephrine or [(3)H]-prazosin. An antibody that specifically inhibits a receptor could be useful in research on G-protein-coupled receptors that lack specific antagonists. The antibody against the epitope CP-7 might have potential in a therapeutic application for treating primary hypertension.

Synthesis of a quinazoline derivative: a new α1-adrenoceptor ligand for conjugation to quantum dots to study α1-adrenoceptors in living cells.

Quantum dots (QDs) that are conjugated to small molecule derivatives of drugs and endogenous ligands may be useful tools to study the distribution and dynamic of membrane bound receptors, ion channels and transporters in live cells. In order to use these tools, it is necessary to functionalize QDs with bioactive ligands. In this paper, we successfully synthesized a ligand of α(1)-adrenoceptor that could be conjugated to QDs. In addition, the conjugation of the ligands to QDs and their biological activity were evaluated through binding assay with 30 nM QD conjugates in living human embryonic kidney 293 cells.

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).

Pharmacological characterization of zinc and copper interaction with the human alpha(1A)-adrenoceptor.

Metal ions have a major role in human health, and interact with many classes of receptors including the G-protein coupled receptors. In the peripheral system, zinc mainly accumulates in the soft prostate organ and, with copper, influences prostate disease progression, from normal to hypertrophic or cancerous states. The development of these pathologies may be influenced by the α(1A)-adrenoceptor, the principal regulator of prostate tonicity. There is currently no information on possible interactions between metals and the α(1A)-adrenoceptor. We therefore studied the effects of several mono- and divalent ions on this receptor subtype using binding and functional experiments performed on expressed cloned human α(1A)-adrenoceptor. Regardless of the counter anion used, Zn(2+) and Cu(2+) interact with α(1A)-adrenoceptor with apparent affinities in the low micromolar range. In addition, using specific binding experiments, we established that these ions acted as negative allosteric ligands on prazosin/α(1A)-adrenoceptor interaction, but in a different manner from the allosteric modulator 5-(N-ethyl-N-isopropyl)-amiloride, suggesting distinct mode of interaction. In addition, the presence of Cu(2+) weakly decreased epinephrine affinity, whereas the addition of Zn(2+) shifted to the left the epinephrine binding curve, revealing a positive allosteric effect but only on half of the binding site. Finally, cell-based functional experiments demonstrated that Zn(2+) and Cu(2+) antagonized epinephrine activation in an insurmountable manner, by reducing agonist efficacy without any shift in the epinephrine activation curves. This study shows the interactions between metal ions and the α(1A)-adrenoceptor with affinities compatible with physiological concentrations and suggests that zinc and copper may have a biological role in prostate function.