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.

α1 adrenergic receptors in serum and saliva of patients with oral squamous cell carcinoma.

BACKGROUND: Neurotransmitters released from the sympathetic nervous system attach to the adrenergic receptors on the surface of tumoral cells in response to stress, and alter the expression of genes programming cellular activity. This study aimed to assess the expression of α1 adrenergic receptors in the serum and saliva of patients with oral squamous cell carcinoma (OSCC) compared with healthy controls. MATERIALS AND METHODS: In this case-control study, serum and stimulated and unstimulated saliva samples were collected from 26 OSCC patients and 26 healthy controls. ELISA kits were used for measurement of the serum and salivary levels of α1 adrenergic receptors. RESULTS: The level of α1 adrenergic receptors was significantly higher in the stimulated and unstimulated saliva of OSCC patients than healthy controls (P = 0.000). However, their serum level was not significantly different between the two groups (P = 0.389). The serum level of α1 adrenergic receptors significantly increased by an increase in OSCC grade. No significant correlation was noted between the serum and salivary levels of α1 adrenergic receptors in OSCC patients. The salivary level of α1 adrenergic receptors was significantly higher in patients with tumors located in the gingiva, compared with other sites. CONCLUSION: Significantly higher salivary level of α1 adrenergic receptors in OSCC patients compared with healthy controls, and no significant difference in their serum level between the two groups may indirectly indicate the over-expression of these receptors in OSCC cells, compared with normal oral mucosa. Further studies and particularly histological analyses are required to confirm this finding.

INPHARMA-Based Determination of Ligand Binding Modes at α1 -Adrenergic Receptors Explains the Molecular Basis of Subtype Selectivity.

The structural poses of ligands that bind weakly to protein receptors are challenging to define. In this work we have studied ligand interactions with the adrenoreceptor (AR) subtypes, α1A -AR and α1B -AR, which belong to the G protein-coupled receptor (GPCR) superfamily, by employing the solution-based ligand-observed NMR method interligand NOEs for pharmacophore mapping (INPHARMA). A lack of receptor crystal structures and of subtype-selective drugs has hindered the definition of the physiological roles of each subtype and limited drug development. We determined the binding pose of the weakly binding α1A -AR-selective agonist A-61603 relative to an endogenous agonist, epinephrine, at both α1A -AR and α1B -AR. The NMR experimental data were quantitatively compared, by using SpINPHARMA, to the back-calculated spectra based on ligand poses obtained from all-atom molecular dynamics simulations. The results helped mechanistically explain the selectivity of (R)-A-61603 towards α1A -AR, thus demonstrating an approach for targeting subtype selectivity in ARs.

Analysis of Density Changes of Selected Brain Receptors After a 14-Day Supply of Chromium(III) and Evaluation of Chromium(III) Affinity to Selected Receptors and Transporters.

Chromium(III) is one of the most controversial biometals. Although, it is no longer on the list of minerals necessary for the proper functioning of the human body, and its pharmacological effect is still under discussion. One of the purposes of Cr(III) administration is to use it in patients with mood disorders and it is strictly related to its pharmacological, not dietary effect. This is because its high doses are necessary to obtain the results and additionally, no deficiencies in human population have been noted. In this study, the affinity of chromium(III) to selected receptors and transporters in the rat brain was evaluated, and the effect of the 14-day administration of this metal was assessed on the density of selected receptors. All analyses were performed in vitro using radioligand binding assays, and the results indicated lack of affinity to ß1 and α1 receptors and serotonin transporter (SERT), furthermore very weak affinity to the 5-HT1A receptor (30% inhibition at 10-4 and 10-5 M). Analysis of the α1 and ß1 adrenergic receptor density indicated lack of any adaptive effects after 14 days of Cr(III) administration through intraperitoneal injections (doses 6 and 12 mg/kg). The antidepressant activity of chromium(III) indicated in clinical trials concerned patients with atypical, seasonal, or dystonic symptoms. This effect, as it seems based on the presented results, does not depend on direct affinity to serotonin receptors and transporter nor is the result of adaptive changes in the adrenoreceptor system.

Distinct phosphorylation sites/clusters in the carboxyl terminus regulate α1D-adrenergic receptor subcellular localization and signaling.

The human α1D-adrenergic receptor is a seven transmembrane-domain protein that mediates many of the physiological actions of adrenaline and noradrenaline and participates in the development of hypertension and benign prostatic hyperplasia. We recently reported that different phosphorylation patterns control α1D-adrenergic receptor desensitization. However, to our knowledge, there is no data regarding the role(s) of this receptor's specific phosphorylation residues in its subcellular localization and signaling. In order to address this issue, we mutated the identified phosphorylated residues located on the third intracellular loop and carboxyl tail. In this way, we experimentally confirmed α1D-AR phosphorylation sites and identified, in the carboxyl tail, two groups of residues in close proximity to each other, as well as two individual residues in the proximal (T442) and distal (S543) regions. Our results indicate that phosphorylation of the distal cluster (T507, S515, S516 and S518) favors α1D-AR localization at the plasma membrane, i. e., substitution of these residues for non-phosphorylatable amino acids results in the intracellular localization of the receptors, whereas phospho-mimetic substitution allows plasma membrane localization. Moreover, we found that T442 phosphorylation is necessary for agonist- and phorbol ester-induced receptor colocalization with ß-arrestins. Additionally, we observed that substitution of intracellular loop 3 phosphorylation sites for non-phosphorylatable amino acids resulted in sustained ERK1/2 activation; additional mutations in the phosphorylated residues in the carboxyl tail did not alter this pattern. In contrast, mobilization of intracellular calcium and receptor internalization appear to be controlled by the phosphorylation of both third-intracellular-loop and carboxyl terminus-domain residues. In summary, our data indicate that a) both the phosphorylation sites present in the third intracellular loop and in the carboxyl terminus participate in triggering calcium signaling and in turning-off α1D-AR-induced ERK activation; b) phosphorylation of the distal cluster appears to play a role in receptor's plasma membrane localization; and c) T442 appears to play a critical role in receptor phosphorylation and receptor-ß-arrestin colocalization.

The mechanism of botulinum A on Raynaud syndrome.

BACKGROUND: Botulinum neurotoxin type A (BoNT/A) is emerging as a treatment modality for Raynaud's phenomenon (RP). However, the mechanism of the role of BoNT/A in antagonizing the constriction of arteriola in RP remains unclear. MATERIALS AND METHODS: We tested the constriction of arteriole diameter and the distribution of adrenergic receptors on the rat cremaster modle. Moreover, we measured the secretion of norepinephrine (NE), protein level changes and related receptors on cultured rat superior cervical ganglia neurons(SCGNs), a model of sympathetic neuron. RESULTS: Based on our results, the inhibition of arteriole vasoconstriction was increased with increasing doses of BoNT/A. BoNT/A, prazosin, and BQ123 treatment can result in significant inhibition of arteriole vasoconstriction with the same electrical stimulation. The inhibition effect of prazosin was equivalent to BoNT/A, while BQ123 has a synergistic effect with BoNT/A. After treating SCGNs using BoNT/A for 30 min, the decrease in fluorescence intensity of FM1-43 slowed down which was correlated with the doses of BoNT/A. Furthermore, release of NE in the supernatant was significantly decreased as measured by enzyme-linked immunosorbent assay, 24 h after a high dose of BoNT/A (25 µ/mL). Cleaved-SNAP-25 was detected by Western blotting 24 h following BoNT/A (50 µ/mL) treatment. Moreover, receptor SV2C, GM1, and FGFR3 were detected on sympathetic neurons, similarly to cholinergic neurons. CONCLUSION: Our study showed that BoNT/A could significantly inhibit electrical stimulation-induced arteriole vasoconstriction through the sympathetic pathway. The mechanism was similar to the cholinergic one, in which the vesicle release of sympathetic neurons could be inhibited by cleavage of SNAP-25. The end result was blocked vesicle fusion with the presynaptic membrane after BoNT/A treatment, inhibiting the release of the NE.

Additive effect of oral LDD175 to tamsulosin and finasteride in a benign prostate hyperplasia rat model.

OBJECTIVE: We investigated the benefits of the BKCa agonist 4-chloro-7-trifluoromethyl-10H-benzo[4,5]furo[3,2-b]indole-1-carboxylic acid (LDD175) combined with tamsulosin and finasteride, in a benign prostatic hyperplasia (BPH) rat model. MATERIALS AND METHODS: Castration was performed by bilateral orchiectomy under ketamine anesthesia. A rat model of BPH was established by daily intramuscular administration of testosterone propionate plus 17ß-estradiol for 8 weeks. Model rats were administered combinations of 20 mg/kg LDD175, 0.01 mg/kg tamsulosin and 1 mg/kg finasteride once daily by oral gavage for 4 weeks from week 6 to 9 post-surgery. Intraurethral pressure induced by electrostimulation of the hypogastric nerve was measured at the end of administration. Body and genitourinary organ weights were recorded, serums were assayed for hormone concentrations, and tissues were subjected to histopathology, and analyses of α1-adrenoceptor mRNA and protein expression levels after treatment. RESULTS: Combined LDD175, tamsulosin, and finasteride significantly decreased prostatic index, serum hormone levels, epithelial thickness, and prostate expression of α1-adrenoceptors in BPH model rats. The 3-drug combination was more effective than any other combination or LDD175 alone. CONCLUSION: These results suggest that LDD175 addition to tamsulosin and finasteride may be beneficial for the treatment of BPH patients who do not respond to tamsulosin plus finasteride.

Subcellular compartmentalization of proximal Gαq-receptor signaling produces unique hypertrophic phenotypes in adult cardiac myocytes.

G protein-coupled receptors that signal through Gαq (Gq receptors), such as α1-adrenergic receptors (α1-ARs) or angiotensin receptors, share a common proximal signaling pathway that activates phospholipase Cß1 (PLCß1), which cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) to produce inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. Despite these common proximal signaling mechanisms, Gq receptors produce distinct physiological responses, yet the mechanistic basis for this remains unclear. In the heart, Gq receptors are thought to induce myocyte hypertrophy through a mechanism termed excitation-transcription coupling, which provides a mechanistic basis for compartmentalization of calcium required for contraction versus IP3-dependent intranuclear calcium required for hypertrophy. Here, we identified subcellular compartmentalization of Gq-receptor signaling as a mechanistic basis for unique Gq receptor-induced hypertrophic phenotypes in cardiac myocytes. We show that α1-ARs co-localize with PLCß1 and PIP2 at the nuclear membrane. Further, nuclear α1-ARs induced intranuclear PLCß1 activity, leading to histone deacetylase 5 (HDAC5) export and a robust transcriptional response (i.e. significant up- or down-regulation of 806 genes). Conversely, we found that angiotensin receptors localize to the sarcolemma and induce sarcolemmal PLCß1 activity, but fail to promote HDAC5 nuclear export, while producing a transcriptional response that is mostly a subset of α1-AR-induced transcription. In summary, these results link Gq-receptor compartmentalization in cardiac myocytes to unique hypertrophic transcription. They suggest a new model of excitation-transcription coupling in adult cardiac myocytes that accounts for differential Gq-receptor localization and better explains distinct physiological functions of Gq receptors.

Label-Free Dynamic Mass Redistribution Reveals Low-Density, Prosurvival α1B-Adrenergic Receptors in Human SW480 Colon Carcinoma Cells.

Small molecules that target the adrenergic family of G protein-coupled receptors (GPCRs) show promising therapeutic efficacy for the treatment of various cancers. In this study, we report that human colon cancer cell line SW480 expresses low-density functional α1B-adrenergic receptors (ARs) as revealed by label-free dynamic mass redistribution (DMR) signaling technology and confirmed by quantitative reverse-transcriptase polymerase chain reaction analysis. Remarkably, although endogenous α1B-ARs are not detectable via either [3H]-prazosin-binding analysis or phosphoinositol hydrolysis assays, their activation leads to robust DMR and enhanced cell viability. We provide pharmacological evidence that stimulation of α1B-ARs enhances SW480 cell viability without affecting proliferation, whereas stimulating ß-ARs diminishes both viability and proliferation of SW480 cells. Our study illustrates the power of label-free DMR technology for identifying and characterizing low-density GPCRs in cells and suggests that drugs targeting both α1B- and ß-ARs may represent valuable small-molecule therapeutics for the treatment of colon cancer.

Avaliação dos efeitos vasculares e expressão de mRNA de receptores de drogas vasoconstritoras em leito arterial mesentérico de ratos normotensos, diabéticos, hipertensos renais um-rim, um clip (1R-1C) e 1R-1C diabéticos / Vascular reactivity and vasoconstrictor drugs receptors RNAmexpression on mesenteric artery bed of normotensive, diabetic, 1K-1C hypertensive and 1K-1C hypertensive-diabetic rats

O presente trabalho teve como objetivo avaliar e comparar a reatividade vascular de agentes vasoconstritores presentes nas soluções anestésicas locais (Adrenalina - vasoconstrição e vasodilatação; Felipressina - vasoconstrição), nas doses de 80, 160, 320, 640 e 1280ng (adrenalina) ou 0,25; 0,5; 1; 2 e 4 x10-3UI (felipressina), em leito arterial mesentérico deratos normotensos, diabéticos, hipertensos renais um-rim, um-clip (1R-1C) e hipertensos1R-1C-diabéticos. E correlacionar tal reatividadecom expressão de RNAm dos receptores 1A e 2- adrenérgicos, V1A para vasopressina e AT1A, AT1Be AT2 para angiotensina II visando verificar se a hipertensão arterial e o diabetes mellitus provocam alteração em modelo indutivo e isogênico. Ratos Wistar pesando 110-160g, foram anestesiados com mistura de quetamina e xilazina (50+10mg/ml/kg de peso), tiveram seu abdômen aberto e receberam um clip de prata com abertura 0,25mm na artéria renal esquerda, removendo-se cirurgicamente o rim direito (ratos 1R-1C). Após 14 dias, receberam injeção subcutânea de estreptozotocina (50 e 60mg/kg de peso) para indução do diabetes mellitus sendo a glicemia testada pela veia caudal previamente aos experimentos (diabéticos). Após 30-42 dias da implantação do clip, todos os grupos foram novamente anestesiados e implantou-se cânula de polietileno (PE-50) na artéria carótida esquerda para registro direto da pressão arterial. Após registro da pressão os animais tiveram a artéria principal mesentérica exposta e canulada. O leito arterial mesentérico foi então isolado e colocado em banho com solução nutritiva de Krebs a 37ºC. O cateter foi conectado ao sistema de registro computadorizado (PowerLab®) utilizando software específico (Chart 5Pro ®). Analisaram-se: a pressão máxima (vasoconstrição) e mínima (vasodilatação), o tempo necessário para atingir esse valor, duração total da resposta, integral e integral sobre a linha de base. Os dados foram submetidos à análise de variância de medidas repetidas (ANOVA), seguida do teste de Holm-Sidak (distribuição normal) ou de Mann-Whitney (nãoparamétrico), quando apropriado, nível de significância de 5%. Todas as respostas máximas de vasoconstrição apresentaram comportamento dose-dependente, contudo, para os quatro grupos estudados, a resposta vasoconstritora para adrenalina foi significativamente superior à felipressina (p<0,05). Diabetes e hipertensão reduziram a resposta vasoconstritora da adrenalina e da felipressina, valores de integral sobre a linha de base, respectivamente para grupo controle, diabético, hipertenso e hipertenso-diabético: 2462±465; 1511±236; 2542± 5456 e 3749±819mmHg.s (p<0,05) para adrenalina e 3749 ± 708; 746 ± 103; 1647 ± 422; 1359 ± 591 mmHg.s (p<0,05) para felipressina. Tanto o diabetes quanto a hipertensão, associadas ou não, aumentaram significativamente o tempo para atingir a pressão máxima de vasoconstrição e a duração (p<0,05). As artérias mesentéricas de ratos diabéticos, hipertensos e diabéticos-hipertensos apresentaram expressão significativamente aumentada dos receptores 1Aadrenérgico, AT1B e AT2 para angiotensina II (p<0,05), enquanto receptor AT1A estava com a expressão aumentada apenas nos grupos diabéticos. A expressão do receptor 1A-adrenérgico é discrepante com os achados funcionais, o que pode ser justificado pela fase crônica da doença em que a PCR foi realizada. É possível correlacionar os dados obtidos com a menor atividade vasoconstritora da felipressina observada clinicamente. A maior sensibilidade às moléculas vasoconstritoras pode explicar a maior tendência de pacientes diabéticos desenvolverem hipertensão. A partir dos dados obtidos pode-se concluir que a adrenalina é o vasoconstritor mais potente que a felipressina e ambas as moléculas tem seus efeitos reduzidos em pacientes hipertensos e diabéticos, o que reforça a indicação de se utilizar anestésicos locais associados a vasoconstritores nestas populações.(AU)

The main goal of this study wasto evaluate and compare vasoconstrictor agents present in local anesthetic solutions (Epinephrine - vasoconstriction and vasodilation, Felypressin - vasoconstriction) vascular reactivity on mesenteric artery bed of normotensive, diabetic, renal hypertensive one-kidney-one-clip (1K1C) and hypertensive 1K1C diabetic rats. Dosagesstudied were 80, 160, 320, 640 and 1280ng (epinephrine) or 0,25; 0,5;1; 2 and 4 x 10-3UI (felypressin). Also, we aimed to correlate artery response with RNAm expression of 1A and 2-adrenoceptors, V1A vasopressin receptor and AT1A, AT1B e AT2 angiotensin receptors, in order to verify if arterial hypertension and diabetes can lead to alterations on a inductive and isogenic model. Wistar male rats weighing 110-160g were anaesthetized with a mixture of ketamine and xylazine (50+10mg/ml/kg), had their abdominal cavity opened and a silver clipwith 0.25-mm gap was implanted in the main left kidney artery, the right kidney was surgically removed (1K1C-rats). After 14 days, they received a subcutaneous injection of streptozotocin (50 and 60 mg/ml/kg) for inducing diabetes, whereas the glycemia was tested via the tail vein prior to surgery (diabetic rats). Around 30-42 after the clip was implanted, all the groups were anaesthetized again and a polyethylene (PE-50) cannula was implanted on the left carotid artery for direct arterial pressure register. After registering the pressure, the animals had their main mesenteric artery exposed and cannulated. The mesenteric artery bed was then isolated and transferred to a bath with Krebs nutritive solution at 37ºC. The catheter was connected to the computer register system (PowerLab®) using a specific software (Chart 5Pro ®). The following parameters were analyzed: maximum (vasoconstriction) and minimal pressure (vasodilating), the amount of time necessary to achieve this number, total duration of the reaction, integral and integral over baseline. The data was submitted to analysis of variance of repeated measures (ANOVA), followed by a Holm-Sidak (normal distribution) test or Mann Whitney (parametrics) test when suitable, with a significance level of 5%. All maximum vasoconstriction results presented dosage-dependant behavior, however, for the four groups tested, the vasoconstrictive result for epinephrine was significantly superior to felypressin (p<0,05). Diabetes and hypertension significantly reducedepinephrine and felypressin vasoconstrictor responses, integral above baseline, respectively, for control, diabetic, hypertensive and hypertensive-diabetic groups:2462±465; 1511±236; 2542± 5456 e 3749±819 mmHg.s (p<0.05, epinephrine) and 3749 ± 708; 746 ± 103; 1647 ± 422; 1359 ± 591 mmHg.s (p<0.05, felypressin). Both diabetes and hypertension, associated or not, significantly increased time necessary to achieve maximum vasoconstrictor response and its duration (p<0,05). Diabetic, hypertensive and hypertensive-diabetic mesenteric arteries presented 1A-adrenoceptor, AT1B and AT2 angiotensin II-receptor gene expression significantly increased when compared with control group (p<0,05), while AT1Areceptor presented this pattern only in diabetic groups.1A-adrenoceptor gene expression did not confirm functional data, probably due to chronic disease state in wich PCR was performed. A partir dos dados obtidos pode-se concluir que a adrenalina é o vasoconstritor mais potente que a felipressina e ambas as moléculas tem seus efeitos reduzidos em ratos hipertensos e diabéticos não tratados, o que reforça a indicação de se utilizar anestésicos locais associados a vasoconstritores nestas populações.Its possible to correlate our datawith reducedvasoconstrictor activity of felypressinin clinical use. Increased sensibility and receptor population for vasoconstrictor endogenous molecules could explain diabetic populations tendency to develop arterial hypertension. Our results suggest that epinephrine is more potent than felypressin and both vasoconstrictors presents reduced effects on diabetic and hypertensive patients, what reinforces vasoconstrictor associated with local anesthetic use in this population.(AU)

Sympathetic innervation of the suprasesamoidean region of the deep digital flexor tendon in the forelimbs of horses.

The purpose of this study was to delineate the pattern of sympathetic innervation in the suprasesamoidean region of the deep digital flexor tendon (DDFT) in horses using immunohistochemical staining (IHC) for tyrosine hydroxylase (TH) and alpha-1 adrenergic receptor (α1-AR). Fourteen forelimbs were collected from 10 horses. Longitudinal sections of the suprasesamoidean region of healthy DDFTs were harvested. Most of the sympathetic innervation was found to be in the walls of blood vessels. The tendon tissue proper was sparsely innervated, with a lesser degree of innervation within the dorsal fibrocartilage. Increased α1-AR immunostaining was also detected in walls of blood vessels and in spindle cells of fibrocartilage. Both α1-AR and TH immunostaining were detected in tenocytes. These findings support the presence of autocrine/paracrine catecholaminergic signalling in equine tendon tissue.

Chronic stress enhances progression of periodontitis via α1-adrenergic signaling: a potential target for periodontal disease therapy.

This study assessed the roles of chronic stress (CS) in the stimulation of the sympathetic nervous system and explored the underlying mechanisms of periodontitis. Using an animal model of periodontitis and CS, the expression of tyrosine hydroxylase (TH) and the protein levels of the α1-adrenergic receptor (α1-AR) and ß2-adrenergic receptor (ß2-AR) were assessed. Furthermore, human periodontal ligament fibroblasts (HPDLFs) were stimulated with lipopolysaccharide (LPS) to mimic the process of inflammation. The proliferation of the HPDLFs and the expression of α1-AR and ß2-AR were assessed. The inflammatory-related cytokines interleukin (IL)-1ß, IL-6 and IL-8 were detected after pretreatment with the α1/ß2-AR blockers phentolamine/propranolol, both in vitro and in vivo. Results show that periodontitis under CS conditions enhanced the expression of TH, α1-AR and ß2-AR. Phentolamine significantly reduced the inflammatory cytokine levels. Furthermore, we observed a marked decrease in HPDLF proliferation and the increased expression of α1-ARfollowing LPS pretreatment. Pretreatment with phentolamine dramatically ameliorated LPS-inhibited cell proliferation. In addition, the blocking of α1-ARsignaling also hindered the upregulation of the inflammatory-related cytokines IL-1ß, IL-6 and IL-8. These results suggest that CS can significantly enhance the pathological progression of periodontitis by an α1-adrenergic signaling-mediated inflammatory response. We have identified a potential therapeutic target for the treatment of periodontal disease, particularly in those patients suffering from concurrent CS.

α-1 Adrenergic receptors are localized on presynaptic elements in the nucleus accumbens and regulate mesolimbic dopamine transmission.

Brainstem noradrenergic neurons innervate the mesocorticolimbic reward pathway both directly and indirectly, with norepinephrine facilitating dopamine (DA) neurotransmission via α1-adrenergic receptors (α1ARs). Although α1AR signaling in the prefrontal cortex (PFC) promotes mesolimbic transmission and drug-induced behaviors, the potential contribution of α1ARs in other parts of the pathway, such as the ventral tegmental area (VTA) and nucleus accumbens (NAc), has not been investigated before. We found that local blockade of α1ARs in the medial NAc shell, but not the VTA, attenuates cocaine- and morphine-induced locomotion. To determine the neuronal substrates that could mediate these effects, we analyzed the cellular, subcellular, and subsynaptic localization of α1ARs and characterized the chemical phenotypes of α1AR-containing elements within the mesocorticolimbic system using single and double immunocytochemical methods at the electron microscopic (EM) level. We found that α1ARs are found mainly extra-synaptically in axons and axon terminals in the NAc and are enriched in glutamatergic and dopaminergic elements. α1ARs are also abundant in glutamatergic terminals in the PFC, and in GABA-positive terminals in the VTA. In line with these observations, microdialysis experiments revealed that local blockade of α1ARs attenuated the increase in extracellular DA in the medial NAc shell following administration of cocaine. These data indicate that local α1ARs control DA transmission in the medial NAc shell and behavioral responses to drugs of abuse.

Nuclear localization drives α1-adrenergic receptor oligomerization and signaling in cardiac myocytes.

Conventional models of G-protein coupled receptor (GPCR) signaling describe cell surface receptors binding to external ligands, such as hormones or circulating peptides, to induce intracellular signaling and a physiologic response. However, recent studies identify new paradigms indicating that GPCRs localize to and signal at the nucleus and that GPCR oligomers can influence receptor function. Previously, we reported that endogenous α1-adrenergic receptors (α1-ARs) localize to and signal at the nuclei in adult cardiac myocytes. In this study, we examined the mechanisms behind α1-AR nuclear localization and how nuclear localization impacted receptor function. We verified that endogenous α1-ARs localized to the nuclear membrane of intact nuclei isolated from wild-type adult cardiac myocytes. Next, we identified and disrupted putative nuclear localization sequences in both the α1A- and α1B-adrenergic receptors, which led to mis-localization of α1-ARs in cultured adult cardiac myocytes. Using these mutants, we demonstrated that nuclear localization was required for α1-signaling in adult cardiac myocytes. We also found that the nuclear export inhibitor leptomycin B inhibited α1-AR signaling, indicating α1-AR signaling must arise in the nucleus in adult cardiac myocytes. Finally, we found that co-localization of the α1-subtypes at the nuclei in adult cardiac myocytes facilitated the formation of receptor oligomers that could affect receptor signaling. In summary, our data indicate that α1-AR nuclear localization can drive the formation of receptor oligomers and regulate signaling in adult cardiac myocytes.

Visualization and tissue distribution of alpha1L-adrenoceptor in human prostate by the fluorescently labeled ligand Alexa-488-silodosin.

PURPOSE: Although alpha(1L)-adrenoceptor is recognized as a target of alpha(1) antagonist therapy for benign prostatic hyperplasia, the most common techniques, such as immunohistochemistry and in situ hybridization, are not applicable to examine alpha(1L)-AR vs alpha(1A)-AR tissue distribution because alpha(1L)-AR is now considered another phenotype sharing the alpha(1A)-AR gene and protein molecule. We labeled the alpha(1A) and alpha(1L)-adrenoceptor selective antagonist silodosin (Kissei Pharmaceutical, Matsumoto, Japan) with the fluorophore Alexa Fluor(R) 488 (Alexa-488-silodosin) to visualize alpha(1L)-AR expression. MATERIALS AND METHODS: Radioligand binding and functional bioassay experiments were done to assess alpha(1)-AR expression in Chinese hamster ovary cells and human prostate tissues. Confocal imaging was subsequently performed. RESULTS: Although Alexa-488-silodosin had about 10 times lower affinity for all alpha(1)-AR subtypes than silodosin in binding and functional studies, it had high selectivity to alpha(1A) and alpha(1L)-ARs. Confocal imaging revealed clear localization of fluorescence on the membrane of Chinese hamster ovary cells expressing alpha(1A)-AR but not alpha(1B)-and alpha(1D)-ARs, and in the muscle layer of the human prostate. The fluorescent signal in Chinese hamster ovary cells disappeared in the presence of 3 nM prazosin but fluorescence was observed in the human prostate even in the presence of 100 nM prazosin. CONCLUSIONS: Alexa-488-silodosin is a powerful fluorescent probe with high selectivity to alpha(1A) and alpha(1L)-ARs. Thus, Alexa-488-silodosin successfully visualizes the site of alpha(1L)-ARs in the muscle layer of the human prostate without losing its distinct pharmacological profile.

{alpha}1-Adrenergic receptor subtypes in nonfailing and failing human myocardium.

BACKGROUND: alpha1-adrenergic receptors (alpha1-ARs) play adaptive roles in the heart and protect against the development of heart failure. The 3 alpha1-AR subtypes, alpha1A, alpha1B, and alpha1D, have distinct physiological roles in mouse heart, but very little is known about alpha1 subtypes in human heart. Here, we test the hypothesis that the alpha1A and alpha1B subtypes are present in human myocardium, similar to the mouse, and are not downregulated in heart failure. METHODS AND RESULTS: Hearts from transplant recipients and unused donors were failing (n=12; mean ejection fraction, 24%) or nonfailing (n=9; mean ejection fraction, 59%) and similar in age ( approximately 44 years) and sex ( approximately 70% male). We measured the alpha1-AR subtypes in multiple regions of both ventricles by quantitative real-time reverse-transcription polymerase chain reaction and radioligand binding. All 3 alpha1-AR subtype mRNAs were present, and alpha1A mRNA was most abundant ( approximately 65% of total alpha1-AR mRNA). However, only alpha1A and alpha1B binding were present, and the alpha1B was most abundant (60% of total). In failing hearts, alpha1A and alpha1B binding was not downregulated, in contrast with beta1-ARs. CONCLUSIONS: Our data show for the first time that the alpha1A and alpha1B subtypes are both present in human myocardium, but alpha1D binding is not, and the alpha1 subtypes are not downregulated in heart failure. Because alpha1 subtypes in the human heart are similar to those in the mouse, where adaptive and protective effects of alpha1 subtypes are most convincing, it might become feasible to treat heart failure with a drug targeting the alpha1A and/or alpha1B.

Ten commercial antibodies for alpha-1-adrenergic receptor subtypes are nonspecific.

Commercial antibodies are used widely to quantify and localize the alpha1-adrenergic receptor (AR) subtypes, alpha1A, alpha1B, and alpha1D. We tested ten antibodies, from abcam and Santa Cruz, using western blot with heart and brain tissue from wild-type (WT) mice and mice with systemic knockout (KO) of one or all three subtypes. We found that none of the antibodies detected a band in WT that was absent in the appropriate KO or in the KO that was null for all alpha1-ARs (ABDKO). We conclude that the antibodies we tested are not specific for alpha1-ARs. These results raise caution with prior studies using these reagents. For now, competition radioligand binding is the only reliable approach to quantify the alpha1-AR subtype proteins. Receptor protein localization remains a challenge.

Native profiles of alpha(1A)-adrenoceptor phenotypes in rabbit prostate.

BACKGROUND AND PURPOSE: alpha(1)-Adrenoceptors in the rabbit prostate have been studied because of their controversial pharmacological profiles in functional and radioligand binding studies. The purpose of the present study is to determine the native profiles of alpha(1)-adrenoceptor phenotypes and to clarify their relationship. EXPERIMENTAL APPROACH: Binding experiments with [3H]-silodosin and [3H]-prazosin were performed using intact tissue segments and crude membrane preparations of rabbit prostate and the results were compared with alpha(1)-adrenoceptor-mediated prostate contraction. KEY RESULTS: [3H]-Silodosin at subnanomolar concentrations bound specifically to intact tissue segments of rabbit prostate. However, [3H]-prazosin at the same range of concentrations failed to bind to alpha(1)-adrenoceptors of intact segments. Binding sites of [3H]-silodosin in intact segments were composed of alpha(1L) phenotype with low affinities for prazosin (pKi=7.1), 5-methyurapidil and N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-alpha,alpha-dimethyl-1H-indole-3-ethamine hydrochloride (RS-17053), and alpha(1A)-like phenotype with moderate affinity for prazosin (pKi=8.8) but high affinity for 5-methyurapidil and RS-17053. In contrast, both radioligands bound to a single population of alpha(1)-adrenoceptors in the membrane preparations at the same density with a subnanomolar affinity, showing a typical profile of 'classical' alpha(1A)-adrenoceptors (pKi for prazosin=9.8). The pharmacological profile of alpha(1)-adrenoceptor-mediated prostate contraction was in accord with the alpha(1L) phenotype observed by intact segment binding approach. CONCLUSIONS AND IMPLICATIONS: Three distinct phenotypes (alpha(1L) and alpha(1A)-like phenotypes in the intact segments and a classical alpha(1A) phenotype in the membranes) with different affinities for prazosin were detected in rabbit prostate. It appears that the three phenotypes are phenotypic subtypes of alpha(1A)-adrenoceptors, but are not genetically different subtypes.

Innervation and receptor profiles of the human apocrine (epitrichial) sweat gland: routes for intervention in bromhidrosis.

BACKGROUND: Human apocrine (epitrichial) sweat glands secrete in response to local or systemic administration of catecholamines and cholinergic agonists. As the process of secretion in human apocrine glands is not fully understood and no literature detailing the expression of adrenergic, cholinergic and purinergic receptors is available, there is a need to know the receptor types. Such data could provide new approaches for the treatment of axillary bromhidrosis. OBJECTIVES: To investigate the localization of nerve fibres, adrenergic, cholinergic and purinergic receptors in human axillary apocrine sweat glands by immunohistochemistry. METHODS: Human axillary apocrine sweat glands were investigated by serial sectioning of paraffin wax-embedded skin samples from volunteers. Sections were examined by light microscopy and immunohistochemistry, using antibodies against neurofilament, alpha- and beta-adrenoceptors, P2Y(1), P2Y(2) and P2Y(4) purinoceptors, and M(3) cholinoceptors. RESULTS: Neurofilaments were found near the eccrine but not the apocrine gland. Apocrine glands demonstrated the presence of beta-2 and beta-3 adrenoceptors in the secretory coil of the gland, but not alpha-1, beta-1 or M(3) receptors. Glandular purinergic staining (P2Y(1), P2Y(2) and P2Y(4)) was found in what looked like myoepithelial cells, while P2Y(1) and P2Y(2) staining was found on apical membranes and diffusely throughout secretory cells. Eccrine gland staining acted as internal positive controls. CONCLUSIONS: No nerve fibres were found near the apocrine gland, suggesting that any catecholamine influence is through humoral effects and that glands could be influenced by beta-adrenoceptor subtypes and purinoceptors. Blockage of both these types of receptors offers a route to controlling apocrine secretion from axillary glands and reducing the opportunity for the development of bromhidrosis.

Comparison of the binding affinity of CGP-12177A at recombinant rat alpha(1D)-adrenoceptors expressed in BHK-21 cell membranes and alpha(1)-adrenoceptors present in rat cerebral cortex membranes.

Recent in vitro studies, performed in rat aorta, mesenteric and intrapulmonary arteries, and human pulmonary artery, demonstrated that the beta-adrenoceptor ligand CGP-12177A (4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one) is also provided with antagonist or partial agonist properties at alpha(1)-adrenoceptors. These observations were supported by estimates of CGP-12177A binding affinity at alpha(1)-adrenoceptors, which have been always performed in rat cerebral cortex membranes, as a surrogate of vascular tissue. Since alpha(1D)-adrenoceptors are predominant in both rat aorta and mesenteric artery, in the present study, we measured, for the first time, the binding affinity of CGP-12177A at recombinant rat alpha(1D)-adrenoceptors expressed in BHK-21 cell membranes. CGP-12177A binding affinity was also determined in rat cerebral cortex membranes, where various alpha(1)-adrenoceptor subtypes are present. By means of [(3)H]prazosin binding competition experiments, we found that CGP-12177A bound to alpha(1D)-adrenoceptor-expressing BHK-21 cell membranes, with a binding affinity (pK(i)=5.39+/-0.27) almost identical to that measured in cerebral membranes (pK(i)=5.44+/-0.07), indicating that it is a non-subtype selective alpha(1)-adrenoceptor ligand. Moreover, CGP-12177A binding affinity was very close to its functional affinity evaluated in rat aorta in terms of antagonist potency against phenylephrine-induced contraction (pK(B)=5.65+/-0.07). In conclusion, our results demonstrate that, in order to evaluate CGP-12177A binding affinity at aorta and mesenteric artery alpha(1)-adrenoceptors, estimates in rat cerebral membranes are as reliable as those in recombinant rat alpha(1D)-adrenoceptors, since both values are very close to CGP-12177A functional affinities in isolated vessels.