1d Adrenergic Receptor Research Articles

Combined vaccines against angiotensin II receptor type 1 and alpha 1D-adrenergic receptor for hypertension.

Compared with monotherapy, combination therapy with multiple antihypertensive drugs has demonstrated superior efficacy in the management of hypertension. The aim of this study was to explore the efficacy of multitarget combined vaccines in achieving simultaneous antihypertensive and target organ protection effects. Our team has developed ATRQβ-001 and ADRQβ-004 vaccines targeting Ang II type 1 receptor (AT1R) and α1D-adrenergic receptor (α1D-AR), respectively. In NG-nitroarginine methyl ester ( l -NAME) + abilities spontaneously hypertensive rats (SHRs) model, SHRs were simultaneously inoculated with ATRQβ-001 and ADRQβ-004 vaccines. Histological and biochemical analyses were performed to evaluate the antihypertensive effects and target organ protection of the ATRQβ-001 and ADRQβ-004 combined vaccines in comparison with those of the single vaccine. Both ATRQβ-001 and ADRQβ-004 vaccines induced robust antibody production, resulting in persistent high antibody titers in rats. Notably, the combined administration of both vaccines significantly decreased SBP in SHRs compared with treatment with a single vaccine, both before and after l -NAME administration. Furthermore, the combined vaccine regimen demonstrated superior efficacy in protecting against vascular remodeling, myocardial hypertrophy and fibrosis, and kidney injury in SHRs. Mechanistically, the combined vaccines exhibited significantly downregulated the expression of angiotensin II type 1 receptor (AT1R) and α1D-adrenergic receptor (α1D-AR). Importantly, no apparent immune-related adverse effects were observed in animals immunized with the combined vaccines. Preliminary findings from this investigation suggest that co-administration of the novel ATRQβ-001 and ADRQβ-004 vaccines holds potential as a groundbreaking therapeutic strategy for managing hypertension.

Vaccine Targeting Alpha 1D-Adrenergic Receptor Improved Metabolic Syndrome in Mice

PurposeMetabolic syndrome (MetS) is a complex chronic disease that includes obesity and hypertension, with rising evidence demonstrating that sympathetic nervous system (SNS) activation plays a key role. Our team designed a therapeutic vaccine called ADRQβ-004 targeting the α1D-adrenergic receptor (α1D-AR). This study was performed to investigate whether the ADRQβ-004 vaccine improves MetS by modulating SNS activity.MethodsC57BL/6N mice were fed a high-fat diet (HFD) and Nω-nitro-L-arginine methyl ester (L-NAME) combination diet for 18 weeks to elicit MetS. The MetS mice were subcutaneously immunized with the ADRQβ-004 vaccine four times to evaluate the therapeutic efficacy in obesity and hypertension and other associated abnormalities related to MetS by conducting echocardiographic, histological, and biochemical analyses.ResultsThe ADRQβ-004 vaccine induced strong antibody production and maintained a high anti-ADR-004 antibody titer in MetS mice. The ADRQβ-004 vaccine improved obesity (P < 0.001) and decreased systolic blood pressure (P < 0.001). Improvements in dysregulated glucose homeostasis and dyslipidemia resulting from the ADRQβ-004 vaccine were also confirmed. Furthermore, the ADRQβ-004 vaccine attenuated cardiovascular functional (P = 0.015) and structural changes (P < 0.001), decreased fat accumulation (P = 0.012) and inflammation (P = 0.050) in the epididymal white adipose tissue, and alleviated hepatic steatosis (P = 0.043) involved in MetS. Moreover, the ADRQβ-004 vaccine improved systematic and visceral organs SNS activities in the MetS.ConclusionThis study demonstrated for the first time that the ADRQβ-004 vaccine targeting α1D-AR improved obesity, hypertension, dyslipidemia, and dysglycemia, and further reduced end-organ damage, which may provide new motivation for MetS research.

Involvement of the Catecholamine Pathway in Glioblastoma Development

Glioblastoma (GBM) is the most aggressive tumor of the central nervous system (CNS). The standard of care improves the overall survival of patients only by a few months. Explorations of new therapeutic targets related to molecular properties of the tumor are under way. Even though neurotransmitters and their receptors normally function as mediators of interneuronal communication, growing data suggest that these molecules are also involved in modulating the development and growth of GBM by acting on neuronal and glioblastoma stem cells. In our previous DNA CpG methylation studies, gene ontology analyses revealed the involvement of the monoamine pathway in sequential GBM. In this follow-up study, we quantitated the expression levels of four selected catecholamine pathway markers (alpha 1D adrenergic receptor—ADRA1D; adrenergic beta receptor kinase 1 or G protein-coupled receptor kinase 2—ADRBK1/GRK2; dopamine receptor D2—DRD2; and synaptic vesicle monoamine transporter—SLC18A2) by immunohistochemistry, and compared the histological scores with the methylation levels within the promoters + genes of these markers in 21 pairs of sequential GBM and in controls. Subsequently, we also determined the promoter and gene methylation levels of the same markers in an independent database cohort of sequential GBM pairs. These analyses revealed partial inverse correlations between the catecholamine protein expression and promoter + gene methylation levels, when the tumor and control samples were compared. However, we found no differences in the promoter + gene methylation levels of these markers in either our own or in the database primary–recurrent GBM pairs, despite the higher protein expression of all markers in the primary samples. This observation suggests that regulation of catecholamine expression is only partially related to CpG methylation within the promoter + gene regions, and additional mechanisms may also influence the expression of these markers in progressive GBM. These analyses underscore the involvement of certain catecholamine pathway markers in GBM development and suggest that these molecules mediating or modulating tumor growth merit further exploration.

The effect of doxazosin and sildenafil citrate combination on bladder tissue contractility, alpha adrenergic receptor, and iNOS subtype expression in a male rat model of partially bladder outlet obstruction.

Our aim was to investigate the effects of doxazosin, sildenafil, and their combination on bladder tissue contractility and adrenergic receptor (AR) and inducible nitric oxide synthase (iNOS) expression utilizing a male rat model of partial urethral obstruction (PUO). Thirty male Sprague Dawley rats were divided into five groups. Except the SHAM group, all animals in remaining four groups underwent surgery for PUO. No further treatment was given to the first group (NT group). Remaining three groups received 6 weeks of treatment with 20 mg/kg doxazosin (D group), 20 mg/kg sildenafil (S group), 20 mg/kg doxazosin, and 20 mg/kg sildenafil combination (DS group), respectively via oral gavage. Then, bladder strips were harvested from each animal for isometric tension studies and for real time polymerase chain reaction studies of both AR subtypes and iNOS mRNA. Contractile responses to carbachol and electrical field stimulation at various concentrations/frequencies showed a significant increase after PUO. Any treatment helped to normalize these increased responses. Alpha 1a and 1d AR subtype expressions were found to be down- and up-regulated, respectively, in every group with PUO, compared to SHAM group. iNOS expression was similar in D and NT groups and significantly increased in S and DS groups. Contractile changes of rat bladder tissue due to PUO were prevented by sildenafil or doxazosin alone or in combination where combination treatment did not provide any additional advantage. Further studies are needed to clarify the role of phosphodiesterase inhibitors and combination treatment in the treatment of LUTS.

Effect of Chemical Stimulation of the Medial Frontal Lobe on the Micturition Reflex in Rats

We assessed the influence of the medial frontal lobe on micturition after chemical stimulation. We also examined the relation between the medial frontal lobe and the rostral pontine reticular formation, which has a strong inhibitory effect on micturition. A total of 35 female rats underwent continuous cystometry. Bladder activity changes were examined after physiological saline, glutamate, the glutamate receptor antagonist MK-801, noradrenaline or the adrenergic α-1 receptor antagonist naftopidil was injected in the medial frontal lobe. When glutamate was injected in the medial frontal lobe, MK-801 was also injected in the rostral pontine reticular formation. Glutamate injection in the medial frontal lobe prolonged the interval between bladder contractions while injection of the glutamate antagonist MK-801 shortened the interval. Glutamate injection in the medial frontal lobe just after MK-801 injection in the ipsilateral rostral pontine reticular formation also prolonged the interval between bladder contractions. However, after prior injection of MK-801 in the bilateral rostral pontine reticular formation glutamate injection in the medial frontal lobe did not influence cystometric parameters. Noradrenaline injection in the medial frontal lobe shortened the interval between bladder contractions while injection of its antagonist naftopidil prolonged the interval. Medial frontal lobe neurons excited by glutamate inhibited the micturition reflex via activation of the rostral pontine reticular formation by glutamatergic projection while medial frontal lobe neurons excited by noradrenaline facilitated the micturition reflex. Thus, the medial frontal lobe may be an important integration center for the initiation of micturition and urine storage mechanisms.

Naftopidil for the treatment of urinary symptoms in patients with benign prostatic hyperplasia

Naftopidil, approved only in Japan, is an α1-adrenergic receptor antagonist (α1-blocker) used to treat lower urinary tract symptoms (LUTS) suggestive of benign prostatic hyperplasia (BPH). Different from tamsulosin hydrochloride and silodosin, in that it has higher and extremely higher affinity respectively, for the α1A-adrenergic receptor subtype than for the α1D type, naftopidil has distinct characteristics because it has a three times greater affinity for the α1D-adrenergic receptor subtype than for the α1A subtype. Although well-designed large-scale randomized controlled studies are lacking and the optimal dosage of naftopidil is not always completely determined, previous reports from Japan have shown that naftopidil has superior efficacy to a placebo and comparable efficacy to other α1-blockers such as tamsulosin. On the other hand, the incidences of ejaculatory disorders and intraoperative floppy iris syndrome induced by naftopidil may be lower than for tamsulosin and silodosin having high affinity for the α1A-adrenergic receptor subtype. However, it remains unknown if the efficacy and safety of naftopidil in Japanese is applicable to white, black and Hispanic men having LUTS/BPH in western countries.

Stress rapidly increases alpha 1d adrenergic receptor mRNA in the rat dentate gyrus

The hippocampal formation is a highly plastic brain region that is sensitive to stress. It receives extensive noradrenergic projections, and noradrenaline is released in the hippocampus in response to stressor exposure. The hippocampus expresses particularly high levels of the α 1D adrenergic receptor (ADR) and we have previously demonstrated that α 1d ADR mRNA expression in the rat hippocampus is modulated by corticosterone. One of the defining features of a stress response is activation of the hypothalamic pituitary adrenal (HPA) axis, resulting in the release of corticosterone from the adrenal glands. However, the effect of stress on hippocampal expression of α 1d ADR mRNA has not been determined. In this study, male rats were exposed to inescapable tail shock, loud noise or restraint, and the effect on α 1d ADR mRNA expression in the hippocampus was determined by semi-quantitative in situ hybridization. All three stressors resulted in a rapid upregulation of α 1d ADR mRNA in the dentate gyrus, with expression peaking at approximately 90 min after the start of the stressor. Physical activity has previously been reported to counteract some of the effects of stress that occur within the dentate gyrus. However, 6 weeks of voluntary wheel running in rats did not prevent the restraint stress-induced increase in α 1d ADR mRNA expression in the dentate gyrus. Although the function of the α 1D ADR in the dentate gyrus is not known, these data provide further evidence for a close interaction between stress and the noradrenergic system in the hippocampus.

Frontal analysis of cell-membrane chromatography for determination of drug-α 1D adrenergic receptor affinity

The aim of the present study was to determine drug-α 1D adrenergic receptor (AR) affinity by frontal analysis of cell-membrane chromatography (CMC). The cell-membrane stationary phase (CMSP) was prepared by immobilizing rat aorta cell membranes on porous silica, and the resulting CMSP was used to determine drug binding affinity to α 1D-AR by frontal analysis. The CMSP of rat aorta was stable and reproducible. Relative binding affinities (dissociation constant, K d) were determined by frontal chromatography for prazosin (166.13 ± 18.36 nmol), BMY7378 (537.40 ± 30.84 nmol), phentolamine (646.92 ± 23.17 nmol), 5-methylurapidil (725.66 ± 25.48 nmol), oxymetazoline (910.56 ± 40.62 nmol) and methoxamine (1299.27 ± 51.73 nmol). These results were consistent with the affinity rank order and showed a good correlation with the affinity of the same compounds for the cloned α 1D-AR subtype obtained from radioligand-binding assay. The study demonstrates that frontal analysis of CMC may be used for direct determination of drug–receptor binding interactions, and that CMC is an alternative reliable method to quantitatively study ligand–receptor interactions.

Synthesis, In Vitro Activities of (2-Cyclopropoxyphenyl)piperidine Derivatives for &amp;#945;1a and &amp;#945;1d Adrenergic Receptor Inhibitors

An alpha 1a- and alpha 1d-adrenergic receptor (AR) selective antagonist may be a more efficacious treatment for BPH/LUTS patients and may have fewer side effects than the existing pharmaceuticals. A facile synthesis for a series of (2-cyclopropoxyphenyl)piperidine derivatives has been developed, in which aryl vinyl ether formation and subsequent cyclopropyl formation provide efficient access to key intermediate N-Boc-4-(2-cyclopropoxyphenyl)piperidine. The synthesized (2-cyclopropoxyphenyl)piperidine derivatives display high affinity and selectivity for alpha1a-AR and alpha1d-AR compared to alpha1b-AR and D2 receptor, Ki values for alpha1a-AR are 0.91 nM to 79.0 nM and alpha1d-AR are 2.0 nM to 57 nM; Ki values for alpha1b-AR are 107 nM to 839.8 nM and D2 receptor are 66.2 nM to 187.1 nM. The selectivity ratios of Ki(alpha1b)/Ki(alpha1a) are 11 to 155 fold, Ki(alpha1b)/Ki(alpha1d) are 6 to 171 fold, Ki(D2)/Ki(alpha1a) are 2 to 158 fold, and Ki(D2)/Ki(alpha1d) are 1.2 to 89 fold. Compound 17a shows improved stability in human liver microsome test (t1/2 = 18 minutes).

Regulation of hippocampal α 1d adrenergic receptor mRNA by corticosterone in adrenalectomized rats

The hippocampal formation receives extensive noradrenergic projections and expresses high levels of mineralocorticoid (MR) and glucocorticoid (GR) receptors. Considerable evidence suggests that the noradrenergic system influences hippocampal corticosteroid receptors. However, there is relatively little data describing the influence of glucocorticoids on noradrenergic receptors in the hippocampal formation. α 1d adrenergic receptor (ADR) mRNA is expressed at high levels in the hippocampal formation, within cells that express MR or GR. In order to determine whether expression of α 1d ADR mRNA is influenced by circulating glucocorticoids, male rats underwent bilateral adrenalectomy (ADX) or sham surgery, and were killed after 1, 3, 7 or 14 days. Levels of α 1d ADR mRNA were profoundly decreased in hippocampal subfields CA1, CA2 and CA3 and the medial and lateral blades of the dentate gyrus, as early as 1day after ADX, as determined by in situ hybridization. The effect was specific for the hippocampal formation, with levels of α 1d mRNA unaltered by ADX in the lateral amygdala, reticular thalamic nucleus, retrosplenial cortex or primary somatosensory cortex. Additional rats underwent ADX or sham surgery and received a corticosterone pellet (10 or 50mg) or placebo for 7 days. Corticosterone replacement prevented the ADX-induced decrease in hippocampal α 1d ADR mRNA, with the magnitude of effect depending on corticosterone dose and hippocampal subregion. These data indicate that α 1d ADR mRNA expression in the hippocampal formation is highly sensitive to circulating levels of corticosterone, and provides further evidence for a close interaction between glucocorticoids and the noradrenergic system in the hippocampus.

EFEK 17β-ESTRADIOL TERHADAP DENSITAS RESEPTOR ADRENERGIK-αIDDAN KONTRAKTILITAS OTOT POLOS PEMBULUH DARAH TIKUS

The incidence of hypertension in menopausal and post menopausal women related to plasma estrogen leveland sympathetic nervous activity. Estrogen may have a specific effect to modulate adrenergic vasoconstriction by modulating adrenergic receptors. Blood vessels contractility is regulated mainly by the sympathetic nervous system, in aorta mediated particularly by  α1Dadrenergic receptor.We hypothesize that 17βestradiol decreases vascular smooth muscle contractility by decreasing  α1Dadrenergic receptor density. To prove the above mechanism in the present study we performed several interrelated assays, i.e bioassay using isolated organ and protein blotting. An isolated rat aortic ring without endothelium (2-3 month,150-200 gram) was incubated in 17β-estradiol (10-5M, 10-4M,and 10-3M ) for two hours prior to stimulation using phenylephrine as α1Dadrenergic receptor agonist. The response of the rat aortic ring to the stimulation of α1Dadrenergic receptor agonist was measured as the subsequent change on aortic contractility using bioassay, and the change of  α1Dadrenergic receptor density using Western and dotblot.

α 1-Adrenergic receptor stimulation induces the expression of receptor activator of nuclear factor κB ligand gene via protein kinase C and extracellular signal-regulated kinase pathways in MC3T3-E1 osteoblast-like cells

The receptor activator of nuclear factor κB ligand (RANKL) produced by bone marrow stromal/osteoblast cells is a crucial regulator of osteoclastgenesis and bone resorption. Osteoblastic cells have been demonstrated to express α 1-adrenergic receptors. Objective The purpose of this study was to test the hypothesis that α 1-adrenergic receptor stimulation induces the expression of RANKL gene via protein kinase C (PKC) and extracellular signal-regulated kinase (ERK) pathways in osteoblastic cells. Design The steady-state mRNA levels of RANKL and activation of ERK in mouse MC3T3-E1 osteoblast-like cells were analyzed by semi-quantitative RT-PCR and Western blotting, respectively. Results In three α 1-adrenergic receptor subtype mRNAs, α 1b- and α 1d-subtypes were expressed in MC3T3-E1 cells. The mRNA levels of RANKL were increased by phenylephrine (α 1-agonist) in time- and dose-dependent manners. Prazosin (α 1-antagonist) suppressed the phenylephrine-induced RANKL mRNA expression, but yohimbine (α 2-antagonist) and propranolol (β-antagonist) did not. Phorbol 12-myristate 13-acetate (PMA, PKC activator) increased RANKL mRNA expression and GF109203X (PKC inhibitor) suppressed the phenylephrine-induced RANKL mRNA expression. Both phenylephrine and PMA stimulated the phosphorylation of ERK, while both prazosin and GF109203X inhibited phenylephrine-induced ERK activation. Pretreatment with PD98059 (ERK kinase inhibitor) inhibited both the phosphorylation of ERK and the expression of RANKL gene induced by phenylephrine in MC3T3-E1 cells. Conclusion These results show that α 1b- and α 1d-adrenergic receptor subtype genes are expressed and the expression of RANKL mRNA may be regulated by α 1-adrenergic receptor stimulation in osteoblastic cells. The induction of RANKL mRNA by activating the α 1-adrenergic receptor is probably mediated via PKC and ERK signalling pathways in osteoblastic cells.

Inhibition of the α 1D-adrenergic receptor gene by RNA interference (RNAi) in rat vascular smooth muscle cells and its effects on other adrenergic receptors

Sympathetic-induced vasoconstriction is mediated by various adrenergic receptor (AR) subtypes located on membranes of vascular smooth muscle cells (VSMC) located on the arterial wall, but is mostly attributed to activation of the α 1D-AR. In order to study interaction and cross-talk among AR genes, we induced post-transcriptional silencing of the α 1D-AR gene in cultured VSMC using the RNAi technique. A pSEC neo expression plasmid vector containing a small interfering RNA (siRNA) sequence selected to bind to the targeted mRNA of the α 1D-AR gene was transfected into cultured VSMC from rat aorta. The RNA expression of all AR-subtype genes was assessed by Q-RT-PCR and the α 1D and α 2A-AR proteins quantified by Western blot. In siRNA-transfected cells, the α 1D-AR protein levels decreased by 55%, 69% and 75% at 24 h, 48 h and 72 h, respectively ( p < 0.03–0.01) with progressive increases in its gene expression by 50%–61% and concurrent increase in α 2A-AR protein peaking at 48 h. Decreases were noted in expression of the α 1A, α 2A, and β 3 AR genes. We conclude that post-transcriptional silencing of the α 1D-AR gene leads to significant decrease in receptor protein despite reactive increase in gene expression. However, suppression of one AR leads to reactive changes in other subtypes, indicating that cross-talk among related genes, whose products have overlapping functions, may partly offset anticipated effects in vivo.

1-Arylpiperazinyl-4-cyclohexylamine derived isoindole-1,3-diones as potent and selective α-1a/1d adrenergic receptor ligands

Subtype-selective α-1a and/or α-1d adrenergic receptor antagonists may be useful for the treatment of benign prostatic hyperplasia (BPH) and lower urinary tract symptoms (LUTS) with fewer adverse effects than non-selective drugs. A series of 1-arylpiperazinyl-4-cyclohexylamine derived isoindole-1,3-diones has been synthesized, displaying in vitro α 1a and α 1d binding affinity K i values in the range of 0.09–38 nM with K i(α 1b)/ K i(α 1a) and K i(α 1b)/ K i(α 1d) selectivity ratios up to 3607-fold.

Signal transduction and regulation: Are all α 1-adrenergic receptor subtypes created equal?

The current manuscript reviews the evidence whether and how subtypes of α 1-adrenergic receptors, i.e. α 1A-, α 1B- and α 1D-adrenergic receptors, differentially couple to signal transduction pathways and exhibit differential susceptibility to regulation. In both regards studies in tissues or cells natively expressing the subtypes are hampered because the relative expression of the subtypes is poorly controlled and the observed effects may be cell-type specific. An alternative approach, i.e. transfection of multiple subtypes into the same host cell line overcomes this limitation, but it often remains unclear whether results in such artificial systems are representative for the physiological situation. The overall evidence suggests that indeed subtype-intrinsic and cell type-specific factors interact to direct α 1-adrenergic receptor signaling and regulation. This may explain why so many apparently controversial findings have been reported from various tissues and cells. One of the few consistent themes is that α 1D-adrenergic receptors signal less effectively upon agonist stimulation than the other subtypes, most likely because they exhibit spontaneous internalization.

DIETARY GLYCINE INHIBITS BLADDER ACTIVITY IN NORMAL RATS AND RATS WITH SPINAL CORD INJURY

The influence of dietary glycine on bladder activity, and on glutamate and glycine levels in the serum and lumbosacral cord was examined in rats with or without spinal cord injury (SCI). A total of 84 female rats were divided into an intact and an SCI group. Each group of rats was divided into 7 subgroups. Two intact and 2 SCI subgroups were fed a standard diet and the remaining subgroups were fed diets containing 0.1% to 3% glycine. After 4 weeks isovolumetric cystometry was performed with rats under urethane anesthesia. Following cystometry glutamate and glycine levels in the serum and lumbosacral cord were measured as well as the glycine receptor alpha1 mRNA level in the lumbosacral cord. Dietary glycine (1% to 3%) prolonged the interval between bladder contractions in intact rats but did not change the amplitude of contractions. On the other hand, dietary glycine (1% to 3%) prolonged the interval and decreased the amplitude of bladder contractions in SCI rats. The glycine levels in the serum and lumbosacral cord of SCI rats on the standard diet were respectively 43% and 45% lower than those in intact rats on the standard diet. Dietary glycine (1% to 3%) increased the serum glycine level in intact and SCI rats but the glycine receptor alpha1 mRNA level in the lumbosacral cord was unchanged. Dietary glycine crosses the blood-brain barrier and inhibits the micturition reflex pathway in the lumbosacral cord but SCI and/or dietary glycine do not influence glycine receptor expression.

Synthesis and biological evaluation of bicyclic and tricyclic substituted nortropane derivatives: discovery of a novel selective α 1D-adrenergic receptor ligand

A range of 3,6,6-trisubstituted nortropane derivatives based upon 6 β-phenyltropane-3 β,6 α-diol have been synthesised from 6 β-hydroxytropinone, including some novel related tricyclic hemi-ketal and tricyclic ketal compounds. Derivatives were assessed for pharmacological affinity and selectivity at α 1-adrenergic receptors, and 6 β-phenyl-8-azabicyclo[3.2.1]octan-3-spiro-2′-(1′,3′-dioxolane)-6-ol, a novel lead compound selective for the alpha 1D-adrenergic receptor, is reported.

Synthesis, screening, and molecular modeling of new potent and selective antagonists at the alpha 1d adrenergic receptor.

In the present study, more than 75 compounds structurally related to BMY 7378 have been designed and synthesized. Structural variations of each part of the reference molecule have been introduced, obtaining highly selective ligands for the alpha(1d) adrenergic receptor. The molecular determinants for selectivity at this receptor are essentially held by the phenyl substituent in the phenylpiperazine moiety. The integration of an extensive SAR analysis with docking simulations using the rhodopsin-based models of the three alpha(1)-AR subtypes and of the 5-HT(1A) receptor provides significant insights into the characterization of the receptor binding sites as well as into the molecular determinants of ligand selectivity at the alpha(1d)-AR and the 5-HT(1A) receptors. The results of multiple copies simultaneous search (MCSS) on the substituted phenylpiperazines together with those of manual docking of compounds BMY 7378 and 69 into the putative binding sites of the alpha(1a)-AR, alpha(1b)-AR, alpha(1d)-AR, and the 5-HT(1A) receptors suggest that the phenylpiperazine moiety would dock into a site formed by amino acids in helices 3, 4, 5, 6 and extracellular loop 2 (E2), whereas the spirocyclic ring of the ligand docks into a site formed by amino acids of helices 1, 2, 3, and 7. This docking mode is consistent with the SAR data produced in this work. Furthermore, the binding site of the imide moiety does not allow for the simultaneous involvement of the two carbonyl oxygen atoms in H-bonding interactions, consistent with the SAR data, in particular with the results obtained with the lactam derivative 128. The results of docking simulations also suggest that the second and third extracellular loops may act as selectivity filters for the substituted phenylpiperazines. The most potent and selective compounds for alpha(1d) adrenergic receptor, i.e., 69 (Rec 26D/038) and 128 (Rec 26D/073), are characterized by the presence of the 2,5-dichlorophenylpiperazine moiety.

Characteristics of behavioral abnormalities in α 1d-adrenoceptors deficient mice

To investigate the functional role of α 1d-adrenergic receptor (α 1d-AR) in the CNS, we have generated mutant mice lacking the α 1d-AR using a gene targeting approach and examined in detail the effects of α 1d-AR knockout mice on motor function, sensory function, and learning and memory. α 1d-AR knockout mice showed better motor coordination at the highest rotating speed of the rotarod performance and stronger muscle tone using the traction meter, but their locomotor activity and swimming ability in the water maze were not affected. In the water maze requiring reference memory, α 1d-AR knockout mice showed normal spatial learning. In the Y-maze task requiring working memory or attention, α 1d-AR knockout mice displayed an impaired spontaneous alternation performance. The α 1d-AR knockout mice tended to display lower levels of acoustic startle responses than the wild-type group at lower pulse intensities, although the acoustic prepulse inhibition was not impaired in the α 1d-AR knockout mice. Furthermore, the NMDA receptor antagonist, MK-801-induced deficits of acoustic prepulse inhibition were not observed in the α 1d-AR knockout mice. These results clearly demonstrate that the α 1d-AR receptor plays an important role in the process of auditory sensory function, attention or working memory rather than reference memory, and the sensorimotor gating deficits induced by the NMDA receptor antagonist.

The α 1D-adrenergic receptor modulates cardiovascular and drinking responses to central salt loading in mice

To characterize the involvement of specific α 1-adrenergic receptor (α 1-AR) subtypes in hypertension, parameters related to central salt- or angiotensin II (ANG II)-induced hypertension were investigated in α 1D-AR-deficient mice (knockout). Baseline daily water intake and food intake were larger in α 1D −/− mice than in α 1D +/+ mice. Intracerebroventricular (i.c.v.) administration of NaCl (0.67 M NaCl, 1 μl) elicited smaller increases in mean arterial blood pressure (MABP), heart rate, and water intake in α 1D −/− mice than it did in α 1D +/+ mice. I.c.v. administration of ANG II (10 pmol) resulted in increases in MABP and water intake that were similar in α 1D −/− mice and α 1D +/+ mice. These results suggest that α 1D-AR is, at least in part, involved in central salt-induced but not ANG II-induced hypertension and water intake.