α1D Adrenoceptor Research Articles

Luteolin alleviates vascular dysfunctions in CLP-induced polymicrobial sepsis in mice.

Luteolin, a naturally occurring flavonoid, is thought to have health-promoting properties as a part of human diet and has been reported to possess a wide range of pharmacological activities. Therefore, the present study was undertaken to evaluate the effect of luteolin pre-treatment on vascular dysfunctions in sepsis induced by caecal ligation and puncture (CLP) in the mouse model. Mice were divided into four groups: sham, luteolin plus sham, CLP, and luteolin plus CLP. Luteolin was administered (0.2mg/kg body weight) intraperitoneally one hour (h) before CLP surgery in mice. 20 ± 2h post CLP surgery, the isolated thoracic aorta of mice was assessed for its vascular reactivity to noradrenaline (NA) and acetylcholine (ACh). To explore the underlying mechanism, aortic mRNA expressions of α1D adrenoceptors, eNOS and iNOS were investigated. In mice with CLP-induced sepsis luteolin pre-treatment markedly increased the survival time and attenuated serum lactate level. The CLP group manifested the reduced vascular reactivity to NA and this deficit was restored by luteolin pre-treatment. However, luteolin pre-treatment did not improve α1D adrenoceptors down-regulation observed in septic mice aorta. In the presence of 1400W, the NA contractile response was significantly restored in CLP mice aortic tissue in comparison with the respective control of septic mice and further enhanced in the presence of luteolin. Luteolin reduced the iNOS mRNA expression and iNOS-derived nitrite production. Pre-treatment with luteolin restored the endothelial dysfunction in septic mice aorta by improving eNOS mRNA expression and enhanced eNOS-derived nitric oxide (NO) production in septic mice aorta and aortic iNOS gene expression and inducible NO production. The present study suggests that the vasoplegic state to NA in aorta was restored through the iNOS pathway and endothelial dysfunction was reversed via eNOS and NO production pathway.

α1D–Adrenoceptor Mediated Components to Nerve‐Evoked Contractions in Mouse Vas Deferens

Contractions of the mouse vas deferens are mediated by α1‐adrenoceptors and P2X1‐purinoceptors (Brown et al., 1983). α1D‐Adrenoceptors are involved in the nerve‐evoked contraction to a single stimulus (Bexis et al., 2008). In this study, the role of these receptors in contractions to low and high frequency nerve stimulation have been investigated. Whole vas deferens from C57 mice were set up between electrodes in organ baths under 0.5 g tension in Krebs‐Henseleit solution, and stimulated electrically with 10 pulses at 1Hz or 40 pulses at 10 Hz using a Grass stimulator. The response to 1Hz stimulation consisted of discrete peaks of response corresponding to each stimulus. Threshold concentrations of the α1A‐adrenoceptor antagonist RS100329 (1nM) and the α1D‐adrenoceptor antagonist BMY7378 (100nM) significantly reduced contractions to the first pulse, and the non‐selective antagonist prazosin (10 nM) significantly reduced contractions to the first 4 pulses, at 1 Hz, but the combination of prazosin and the purinoceptor antagonist suramin produced marked inhibition of the response to all 10 pulses. The response to 10 Hz stimulation consisted of an early peak and later plateau of contraction. BMY7378 (100 nM) did not significantly affect the 10 Hz response but prazosin (10 nM) and RS100329 (1 nM) significantly reduced contractions, particularly the peak response, and higher concentrations markedly reduced responses. In the presence of the L‐type calcium channel blocker nifedipine, contractions to 10Hz stimulation were markedly reduced, particularly the later plateau component. In the presence of nifedipine, the peak response to 10 Hz stimulation was significantly reduced by prazosin (1nM) and by BMY7378 (100nM), demonstrating a small α1D‐adrenoceptor mediated component. In summary, contractions to 1 Hz stimulation have early α1A‐ and α1D‐adrenoceptor mediated components, but later responses are predominantly purinergic. Contractions even to 10 Hz stimulation have an early small α1D‐adrenoceptor component to the peak response, but both peak and plateau contractions largely involve α1A‐adrenoceptors and purinoceptors. The α1A‐adrenoceptor and purinoceptor components largely involve calcium entry through L‐type channels, but the α1D‐adrenoceptor mediated component involves T‐type calcium channels (Seto et al., 2010). In conclusion, the α1D‐adrenoceptor mediated component to both low and high frequency stimulation is very short‐lived, occuring in the first second of stimulation at low and high frequencies.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Synthesis and Pharmacological Evaluation of Novel Silodosin-Based Arylsulfonamide Derivatives as α1A/α1D-Adrenergic Receptor Antagonist with Potential Uroselective Profile.

Benign prostatic hyperplasia (BPH) is the most common male clinical problem impacting the quality of life of older men. Clinical studies have indicated that the inhibition of α1A-/α1D adrenoceptors might offer effective therapy in lower urinary tract symptoms. Herein, a limited series of arylsulfonamide derivatives of (aryloxy)ethyl alicyclic amines was designed, synthesized, and biologically evaluated as potent α1-adrenoceptor antagonists with uroselective profile. Among them, compound 9 (3-chloro-2-fluoro-N-([1-(2-(2-(2,2,2-trifluoroethoxy)phenoxy]ethyl)piperidin-4-yl)methyl)benzenesulfonamide) behaved as an α1A-/α1D-adrenoceptor antagonist (Ki(α1) = 50 nM, EC50(α1A) = 0.8 nM, EC50(α1D) = 1.1 nM), displayed selectivity over α2-adrenoceptors (Ki(α2) = 858 nM), and a 5-fold functional preference over the α1B subtype. Compound 9 showed adequate metabolic stability in rat-liver microsome assay similar to the reference drug tamsulosin (Clint = 67 and 41 µL/min/mg, respectively). Compound 9 did not decrease systolic and diastolic blood pressure in normotensive anesthetized rats in the dose of 2 mg/kg, i.v. These data support development of uroselective agents in the group of arylsulfonamides of alicyclic amines with potential efficacy in the treatment of lower urinary tract symptoms associated to benign prostatic hyperplasia.

Identification of 3,4-dihydro-2H-thiochromene 1,1-dioxide derivatives with a phenoxyethylamine group as highly potent and selective α1D adrenoceptor antagonists

A series of phenoxyethylamine derivatives was designed and synthesized to discover potent and selective human α1D adrenoceptor (α1D adrenergic receptor; α1D–AR) antagonists. Compound 7 was taken from our internal compound collection as an attractive starting point and exhibited moderate binding affinity for α1D–AR and high selectivity against α1A– and α1B–ARs. We focused on modifying the 2-methylsulfonylbenzyl group of 7 to discover novel compounds structurally distinct from other reported α1–AR antagonists containing the phenoxyethylamine motif. Study of structure activity relationship guided by a targeted ligand-lipophilicity efficiency score led to the discovery of a novel scaffold of 3,4-dihydro-2H-thiochromene 1,1-dioxide for selective α1D–AR antagonists. Further optimization studies resulted in the identification of (4S)-N4-[2-(2,5-difluorophenoxy)ethyl]-N6-methyl-3,4-dihydro-2H-thiochromene-4,6-diamine 1,1-dioxide, (S)–41, as a novel, highly potent and selective α1D–AR antagonist. Herein, we provide details of the structure activity relationship of the phenoxyethylamine analog for the potency and selectivity.

Structure determination, synthesis, and biological evaluation of a metabolite of the selective α1D adrenoceptor antagonist TAK-259

5-Chloro-1-(5-chloro-2-(methylsulfonyl)benzyl)-2-imino-1,2-dihydropyridine-3-carboxamide hydrochloride (TAK-259) is a novel, selective, and orally active α1D adrenoceptor antagonist with anti-urinary frequency effect. A metabolite (2) of TAK-259 was identified from monkey urine samples. To elucidate the structure of 2, extraction and purification of the metabolite from TAK-259-treated monkey urine was conducted. Structural analysis of the purified compounds using NMR indicated the compound to be 2-amino-5-chloro-1-[5-chloro-2-(methylsulfonyl)benzyl]-4-oxo-1,4-dihydropyridine-3-carboxamide (2a). An authentic sample of compound 2a was synthesized via regioselective alkylation of 4-methoxypyridin-2-amine. The biological activity of metabolite 2a was also evaluated, and the compound found not to possess affinity toward any known α1 adrenoceptor subtype.

Discovery of 5-Chloro-1-(5-chloro-2-(methylsulfonyl)benzyl)-2-imino-1,2-dihydropyridine-3-carboxamide (TAK-259) as a Novel, Selective, and Orally Active α1D Adrenoceptor Antagonist with Antiurinary Frequency Effects: Reducing Human Ether-a-go-go-Related Gene (hERG) Liabilities.

A novel structural class of iminopyridine derivative 1 was identified as a potent and selective human α1D adrenoceptor (α1D adrenergic receptor; α1D-AR) antagonist against α1A- and α1B-AR through screening of an in-house compound library. From initial structure-activity relationship studies, we found lead compound 9m with hERG K(+) channel liability. To develop analogues with reduced hERG K(+) channel inhibition, a combination of site-directed mutagenesis and docking studies was employed. Further optimization led to the discovery of (R)-9s and 9u, which showed antagonistic activity by a bladder strip test in rats with bladder outlet obstruction, as well as ameliorated cystitis-induced urinary frequency in rats. Ultimately, 9u was selected as a clinical candidate. This is the first study to show the utility of iminopyridine derivatives as selective α1D-AR antagonists and evaluate their effects in vivo.

The Protein Acyl Transferase ZDHHC21 Modulates α1 Adrenergic Receptor Function and Regulates Hemodynamics.

Palmitoylation, the reversible addition of the lipid palmitate to a cysteine, can alter protein localization, stability, and function. The ZDHHC family of protein acyl transferases catalyzes palmitoylation of numerous proteins. The role of ZDHHC enzymes in intact tissue and in vivo is largely unknown. Herein, we characterize vascular functions in a mouse that expresses a nonfunctional ZDHHC21 (F233Δ). Physiological studies of isolated aortae and mesenteric arteries from F233Δ mice revealed an unexpected defect in responsiveness to phenylephrine, an α1 adrenergic receptor agonist. In vivo, F233Δ mice displayed a blunted response to infusion of phenylephrine, and they were found to have elevated catecholamine levels and elevated vascular α1 adrenergic receptor gene expression. Telemetry studies showed that the F233Δ mice were tachycardic and hypotensive at baseline, consistent with diminished vascular tone. In biochemical studies, ZDHHC21 was shown to palmitoylate the α1D adrenoceptor and to interact with it in a molecular complex, thus suggesting a possible molecular mechanism by which the receptor can be regulated by ZDHHC21. Together, the data support a model in which ZDHHC21 F233Δ diminishes the function of vascular α1 adrenergic receptors, leading to reduced vascular tone, which manifests in vivo as hypotension and tachycardia. This is to our knowledge the first demonstration of a ZDHHC isoform affecting vascular function in vivo and identifies a novel molecular mode of regulation of vascular tone and blood pressure.

Erythropoietin Reverses Sepsis-Induced Vasoplegia to Norepinephrine Through Preservation of α1D-Adrenoceptor mRNA Expression and Inhibition of GRK2-Mediated Desensitization in Mouse Aorta.

We investigated the effect of erythropoietin (EPO) posttreatment on survival time and vascular functions in a mouse model of sepsis. Sepsis was induced by cecal ligation and puncture. After 20 ± 2 hours of sepsis, thoracic aorta was isolated for assessing its reactivity to norepinephrine (NE) and acetylcholine (ACh). We also measured the tissue nitric oxide (NO) level, inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), G protein-coupled receptor kinase 2 (GRK2), and α1D adrenoceptor messenger RNA (mRNA)/protein expression. In septic mice, EPO moderately improved the survival time from 19.68 ± 0.75 to 34.7 ± 3.2 hours. Sepsis significantly decreased the aortic contractile response to NE along with reduced α1D mRNA and protein expression. Erythropoietin significantly preserved the α1D receptor expression and restored NE-induced contractions to control levels in septic mice. Further, it attenuated the aortic α1D receptor desensitization in sepsis which was evident from reduced GRK2 mRNA expression. Accordingly, a selective GRK2 inhibitor markedly restored the contractile responses to NE in sepsis. Erythropoietin treatment attenuated iNOS mRNA expression and iNOS-induced overproduction of NO, but improved endothelium-dependent relaxation to ACh associated with increased eNOS mRNA expression. In conclusion, EPO seems to reverse sepsis-induced vasoplegia to NE through the preservation of α1D adrenoceptor mRNA/protein expression, inhibition of GRK2-mediated desensitization, and attenuation of NO overproduction in the mouse aorta.

Effects of Spinal and Peripheral Injection of α1A or α1D Adrenoceptor Antagonists on Bladder Activity in Rat Models with or without Bladder Outlet Obstruction

PurposeAntagonists of α1-adrenergic receptors (α1ARs) relax prostate smooth muscle and relieve voiding and storage symptoms. Recently, increased expression of α1ARs with change of its subtype expression has been proved in bladder outlet obstruction (BOO). To search for the evidence of changes in α1ARs subtype expression and activity in the peripheral and spinal routes, the effects of spinal and peripheral administration of tamsulosin (an α1A/D-selective AR), naftopidil (an α1A/D-selective AR), and doxazosin (non-selective AR) on bladder activity were investigated in a rat model with or without BOO.MethodsA total of 65 female Sprague-Dawley rats were divided into the BOO surgery group (n=47) and the sham surgery group (n=18). After 6 weeks, cystometry was assessed before and after intrathecal and intra-arterial administrations of tamsulosin, naftopidil, and doxazosin.ResultsAfter intra-arterial administrations of all three drugs, bladder capacity (BC) was increased and maximal intravesical pressure (Pmax) was decreased in both BOO and the sham rat models (P<0.05). After intrathecal administration of all three drugs, BC was increased and Pmax was decreased in only the BOO group. The episodes of involuntary contraction in the BOO rat models were decreased by intra-arterial administration (P=0.031). The increase of BC after intrathercal and intra-arterial administrations of α1ARs was significantly greater in the BOO group than in the sham group (P=0.023, P=0.041). In the BOO group, the increase of BC and decrease in Pmax were greater by intra-arterial administration than by intrathecal administration (P=0.035). There were no significant differences of the degrees of changes in the cystometric parameters among the three different α1ARs.ConclusionsUp-regulations of the α1ARs in BOO were observed by the greater increases of BC after α1AR antagonist administrations in the BOO group than in the sham group. However, there were no subtype differences of the α1ARs in functional parameters of bladder activity. In addition, α1ARs also act on the lumbosacral cord which implies that the sensitivity of α1ARs is increased in pathologic models such as BOO. Further evaluation including differential expression of α1ARs in BOO models are need.

Alpha 1d Adrenoceptor signaling is required for stimulus induced locomotor activity.

alpha 1 Adrenergic receptors mediate a variety of physiological responses and have been well studied in the cardiovascular and peripheral nervous system. However, their role in the central nervous system remains ill defined because of the lack of highly specific ligands to the alpha1 receptor subtypes. Here, we have employed gene targeting to elucidate the role of alpha 1d receptors in vivo. In addition to disrupting function, the insertion of the lacZ gene into the alpha 1d receptor locus enabled the specific identification of cells expressing the alpha 1d gene. These cells are localized in the cortex, hippocampus, olfactory bulb, dorsal geniculate and ventral posterolateral nuclei of the thalamus. Behaviorally, the alpha 1d(-/-) mice show normal locomotor activity during the subjective day, or resting phase of their cycle. However, during subjective night, or active phase, wheel-running activity is significantly reduced in mutant mice. Furthermore, these mice show a reduction in exploratory rearing behavior in a novel cage environment. Lastly, alpha 1d(-/-) mice show reduced hyperlocomotion after acute amphetamine administration. Together, these data reveal the functional importance of alpha 1d adrenoceptors in mediating a variety of stimulus-induced changes in locomotor behaviors. While the sensitivity of noradrenergic neurons to environmental stimuli has been well documented, our data demonstrate that at least some of these post-synaptic responses are mediated by alpha 1d adrenergic receptors.

Ligand design for α1 adrenoceptor subtype selective antagonists

α1 Adrenoceptors have three subtypes and drugs interacting selectively with these subtypes could be useful in the treatment of a variety of diseases. In order to gain an insight into the structural principles governing subtype selectivity, ligand based drug design (pharmacophore development) methods have been used to design a novel 1,2,3-thiadiazole ring d analogue of the aporphine system. Synthesis and testing of this compound as a ligand on cloned and expressed human α1 adrenoceptors is described. Low binding affinity was found, possibly due to an unfavourable electrostatic potential distribution. Pharmacophore models for antagonists at the three adrenoceptor sites (α1A, α1B, α1D) were generated from a number of different training sets and their value for the design of new selective antagonists discussed. The first preliminary antagonist pharmacophore model for the α1D adrenoceptor subtype is also reported.

Detection of alpha 1-adrenoceptor subtypes in human hypertrophied prostate by in situ hybridization.

Adrenergic stimulation induces contraction of hypertrophied prostatic tissue via the alpha 1 adrenoceptor, and the results of pharmacological studies suggested the existence of adrenoceptor subtypes. Recently three subtypes (alpha 1a, alpha 1b, and alpha 1d) were cloned. Using probes for these subtypes, we demonstrated their expression in the tissues of ten cases of benign prostatic hypertrophy, using in situ hybridization. To determine the ratio between these subtypes, an RNase protection assay was also performed in three cases. Expression of the alpha 1a and alpha 1d adrenoceptors was diffuse in the smooth muscles of the interstitium, but was absent in glandular epithelial cells. On the contrary, the alpha 1b adrenoceptor was hardly detectable. The RNase protection assay confirmed the absence of the alpha 1b adrenoceptor, the ratio of alpha 1a and alpha 1d being 4:1. These results supported the idea that the differences in prostatic contractile response to several adrenergic drugs are based on the affinities of these drugs for the different subtypes.

P-372 - Cloning, pharmacological characterization and tissue distribution of α1 d-adrenoceptor of rabbit.

P-372 - Cloning, pharmacological characterization and tissue distribution of α1 d-adrenoceptor of rabbit.