Alpha 1D-AR Research Articles

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 [ 3H]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).

Inhibition of vascular smooth muscle G protein-coupled receptor kinase 2 enhances alpha1D-adrenergic receptor constriction.

G protein-coupled receptor kinase 2 (GRK2) is a serine/theorinine kinase that phosphorylates and desensitizes agonist-bound G protein-coupled receptors. GRK2 is increased in expression and activity in lymphocytes and vascular smooth muscle (VSM) in human hypertension and animal models of the disease. Inhibition of GRK2 using the carboxyl-terminal portion of the protein (GRK2ct) has been an effective tool to restore compromised beta-adrenergic receptor (AR) function in heart failure and improve outcome. A well-characterized dysfunction in hypertension is attenuation of betaAR-mediated vasodilation. Therefore, we tested the role of inhibition of GRK2 using GRK2ct or VSM-selective GRK2 gene ablation in a renal artery stenosis model of elevated blood pressure (BP) [the two-kidney, one-clip (2K1C) model]. Use of the 2K1C model resulted in a 30% increase in conscious BP, a threefold increase in plasma norepinephrine levels, and a 50% increase in VSM GRK2 mRNA levels. BP remained increased despite VSM-specific GRK2 inhibition by either GRK2 knockout (GRK2KO) or peptide inhibition (GRK2ct). Although betaAR-mediated dilation in vivo and in situ was enhanced, alpha(1)AR-mediated vasoconstriction was also increased. Further pharmacological experiments using alpha(1)AR antagonists revealed that GRK2 inhibition of expression (GRK2KO) or activity (GRK2ct) enhanced alpha(1D)AR vasoconstriction. This is the first study to suggest that VSM alpha(1D)ARs are a GRK2 substrate in vivo.

Examination of alpha 1 adrenoceptor subtypes in the human ureter

Urinary stone disease is a common condition affecting up to 12% of the population. Speedy elimination of ureterolithiasis in the lower part of ureters is reported with the alpha 1-blocker. This study was carried out to characterize the alpha 1 adrenoceptors (AR) subtype gene and protein expression in the proximal, medial, and distal regions of the human ureter with the aim of facilitating stone expulsion. The study was carried out on 20 volunteers. Proximal, medial and distal ureter specimens were obtained as discarded tissues after nephroureterectomy for renal pelvic or ureteral carcinoma. Proximal ureter specimens were obtained as discarded tissues after nephrectomy for renal carcinoma. Using ureter samples, real-time reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical staining were performed. The alpha 1d subtype mRNA was highly expressed in each ureteral region, accounting for about 54% of the total AR Messenger Ribonucleic Acid. In the proximal and medial ureter, the distribution of ARs was alpha 1d > or = alpha 1a > alpha 1b as well as that of the total ureter. The distal ureter expressed the highest amount of alpha 1d ARs. The alpha 1d subtype expression tended to be high compared with the alpha 1a subtype expression. In the distal ureter, the distribution of ARs was alpha 1d > alpha 1a > alpha 1b. Immunohistochemical staining revealed that expressions of alpha 1a-AR and alpha 1d-AR were higher than that of alpha 1b-AR. According to our results, we should try the alpha 1d AR antagonist for the conservative expulsive therapy of distal ureteral stones in the highest region of alpha 1d subtype distribution.

Receptor subtype involved in ?1-adrenergic receptor-mediated Ca2+signaling in cardiomyocytes

The enhancement of intracellular Ca2+ signaling in response to alpha 1-adrenergic receptor (alpha 1-AR) stimulation is an essential signal transduction event in the regulation of cardiac functions, such as cardiac growth, cardiac contraction, and cardiac adaptation to various situations. The present study was intended to determine the role(s) of the alpha 1-AR subtype(s) in mediating this response.We evaluated the effects of subtype-specific agonists and antagonists of the alpha 1- AR on the intracellular Ca2+ signaling of neonatal rat ventricular myocytes using a confocal microscope.After being cultured for 48 h, the myocytes exhibited spontaneous local Ca2+ release, sparks, and global Ca2+ transients. The activation of the alpha 1-AR with phenylephrine, a selective agonist of the alpha 1-AR, dose-dependently increased the frequency of Ca2+ transients with an EC50 value of 2.3 micromol/L. Blocking the alpha 1A-AR subtype with 5-methylurapidil (5-Mu) inhibited the stimulatory effect of phenylephrine with an IC(50) value of 6.7 nmol/L. In contrast, blockade of the alpha 1B-AR and alpha 1D-AR subtypes with chloroethylclonidine and BMY 7378, respectively, did not affect the phenylephrine effect. Similarly, the local Ca2+ spark numbers were also increased by the activation of the alpha 1-AR, and this effect could be abolished selectively by 5-Mu. More importantly, A61603, a novel selective alpha 1A-AR agonist, mimicked the effects of phenylephrine, but with more potency (EC(50) value =6.9 nmol/L) in the potentiation of Ca2+ transients, and blockade of the alpha 1A-AR by 5-Mu caused abolishment of its effects.These results indicate that alpha 1-adrenergic stimulation of intracellular Ca2+ activity is mediated selectively by the alpha 1A-AR.

EFFECTS OF α 1-ADRENERGIC RECEPTOR SUBTYPE SELECTIVE ANTAGONISTS ON LOWER URINARY TRACT FUNCTION IN RATS WITH BLADDER OUTLET OBSTRUCTION

Antagonists of alpha 1-adrenergic receptors (alpha 1ARs) relieve obstructive and irritative symptoms in patients with bladder outlet obstruction. However, to our knowledge mechanisms underlying the relief of irritative symptoms remain unknown. Because bladder alpha 1dARs are up-regulated in some rats with bladder outlet obstruction, we investigated the effect of the alpha 1aAR antagonist 5-methyl urapidil (5MU) vs the alpha 1a/alpha 1dAR antagonist tamsulosin on urinary frequency in obstructed rats. Baseline frequency was measured using a chronic micturition recording system and then obstruction (40 rats) or sham obstruction surgery (11 rats) was performed. After 6 weeks frequency was reassessed, followed by subcutaneous implantation of osmotic pumps to deliver 5MU, tamsulosin or vehicle for 1 week. Upon the completion of drug treatment urinary frequency was again measured and the pressor response to the alpha 1AR agonist phenylephrine was documented. Obstructed bladder mass was an average of 4.9 times greater than bladder mass in sham operated rats (p <0.001). Urinary frequency was elevated in obstructed rats with a bladder mass of greater than 500 mg vs all rats with a bladder mass of under 255 mg (p = 0.01). Of rats with a bladder mass of greater than 500 mg frequency was decreased in those treated with tamsulosin (p = 0.03) but not in those treated with 5MU. Tamsulosin and 5MU inhibited the pressor response to phenylephrine. Urinary frequency is increased in rats with a bladder mass of greater than 500 mg. The combined alpha 1a/alpha 1dAR antagonist tamsulosin decreases urinary frequency more than the alpha 1aAR selective antagonist 5MU. This finding supports the hypothesis that the alpha 1dAR is important for mediating irritative symptoms.

Differential cardiovascular regulatory activities of the alpha 1B- and alpha 1D-adrenoceptor subtypes.

The regulation of cardiac and vascular function by the alpha 1B- and alpha 1D-adrenoceptors (ARs) has been assessed in two lines of transgenic mice, one over-expressing a constitutively active alpha 1B-AR mutation (alpha 1B-ARC128F) and the other an alpha 1D-AR knockout line. The advantage of using mice expressing a constitutively active alpha 1B-AR is that the receptor is tonically active, thus avoiding the use of nonselective agonists that can activate all subtypes. In hearts from animals expressing alpha 1B-ARC128F, the activities of the mitogen-activated protein kinases, extracellular signal-regulated kinase, and c-Jun N-terminal kinase were significantly elevated compared with nontransgenic control animals. Mice over-expressing the alpha 1B-ARC128F had echocardiographic evidence of contractile dysfunction and increases in chamber dimensions. In isolated-perfused hearts or left ventricular slices from alpha 1B-ARC128F-expressing animals, the ability of isoproterenol to increase contractile force or increase cAMP levels was significantly decreased. In contrast to the prominent effects on the heart, constitutive activation of the alpha 1B-AR had little effect on the ability of phenylephrine to induce vascular smooth muscle contraction in the isolated aorta. The ability of phenylephrine to stimulate coronary vasoconstriction was diminished in alpha 1D-AR knockout mice. In alpha 1D-AR knockout animals, no negative effects on cardiac contractile function were noted. These results show that the alpha1-ARs regulate distinctly different physiologic processes. The alpha 1B-AR appears to be involved in the regulation of cardiac growth and contractile function, whereas the alpha 1D-AR is coupled to smooth muscle contraction and the regulation of systemic arterial blood pressure.

Portal hypertension is associated with increased mRNA levels of vasopressor G‐protein‐coupled receptors in human hepatic arteries

The contractile response of human splanchnic vessels to different vasoconstrictors is attenuated in cirrhosis. Functional studies indicate a cellular signalling defect upstream of the G-protein level. The aim of the present study was to analyze expression and mRNA levels of the following most relevant vasopressor receptors in the smooth musculature of human hepatic arteries: alpha1 adrenoceptor (AR) subtypes a, b and d, angiotensin II type 1 receptor (AT1), arginine vasopressin receptor type 1a (V1a), endothelin receptor type A (ETA) and B (ETB). Hepatic arteries were collected from 10 donors (noncirrhotic) and 14 recipients (cirrhotic) at liver transplantations. Real-time-PCR was performed to quantify steady-state levels of receptor mRNAs. alpha 1aAR mRNA levels showed no significant difference between the cirrhotic arteries and the controls while the mRNA levels of the other vasoactive receptors were significantly higher in the cirrhotic hepatic arteries (alpha 1bAR: 4-fold, P = 0.013; AT1: 16-fold, P = 0.024; V1a: 23-fold, P = 0.001; ETA: 4-fold, P = 0.02; ETB: 8-fold, P = 0.008). No mRNA for the alpha 1dAR was detected either in the donor or recipient hepatic arteries. We conclude that vascular hyporeactivity to the most relevant endogenous vasoconstrictors of cirrhotic hepatic arteries is not caused by a receptor down-regulation at mRNA levels. In contrast they were up-regulated.

Distribution of α&lt;sub&gt;1&lt;/sub&gt;-adrenoceptor subtype proteins in different tissues of neonatal and adult rats

Distribution of alpha(1)-adrenoceptor (alpha(1)AR) subtype (alpha(1A), alpha(1B), alpha(1D)) proteins in brain, heart, kidney, and liver of 1-week-old rats and in brain, heart, aorta, kidney, liver, vas deferens, prostate, and adrenal glands of adult rats was investigated by Western analysis, using receptor subtype specific polyclonal antibodies. High levels of immunoreactive alpha(1A)AR and alpha(1D)AR in brain and heart and of alpha(1B)AR in liver and heart of neonatal rats were detected. In adult rat tissues, the abundance of alpha(1A)AR protein was most marked in the brain, intermediate in heart, aorta, liver, vas deferens, and adrenals, and minimal in the kidney and prostate; relative to other tissues, the expression of alpha(1B)AR was higher in brain and heart and that of alpha(1D)AR in brain. All the three receptor subtypes increased with age in the brain cortex, whereas the abundance of alpha(1B)AR increased in the heart but decreased in the liver; alpha(1A)AR and alpha(1D)AR in liver, kidney, and heart were not affected by age. It is concluded that alpha(1)AR subtypes are widely expressed in different neonatal and adult rat tissues.

Cardiovascular α&lt;sub&gt;1&lt;/sub&gt;-adrenoceptor subtypes: functions and signaling

alpha-Adrenoceptors (alpha1AR) are G protein-coupled receptors and include alpha1A, alpha1B, and alpha1D subtypes corresponding to cloned alpha1a, alpha1b, and alpha1d, respectively. alpha1AR mediate several cardiovascular actions of sympathomimetic amines such as vasoconstriction and cardiac inotropy, hypertrophy, metabolism, and remodeling. alpha1AR subtypes are products of separate genes and differ in structure, G protein-coupling, tissue distribution, signaling, regulation, and functions. Both alpha(1A)AR and alpha(1B)AR mediate positive inotropic responses. On the other hand, cardiac hypertrophy is primarily mediated by alpha(1A)AR. The only demonstrated major function of alpha(1D)AR is vasoconstriction. alpha1AR are coupled to phospholipase C, phospholipase D, and phospholipase A2; they increase intracellular Ca2+ and myofibrillar sensitivity to Ca2+ and cause translocation of specific phosphokinase C isoforms to the particulate fraction. Cardiac hypertrophic responses to alpha1AR agonists might involve activation of phosphokinase C and mitogen-activated protein kinase via Gq x alpha1AR subtypes might interact with each other and with other receptors and signaling mechanisms.

Characterization of alpha 1-adrenoceptors expressed in a novel vascular smooth muscle cell line cloned from p53 knockout mice, P53LMAC01 (AC01) cells.

1. We pharmacologically studied the alpha 1-adrenoceptor (AR) subtype(s) involved in receptor-mediated signalling in a novel vascular smooth muscle cell line cloned from p53 knockout mice, P53LMAC01 (AC01) cells. 2. Radioligand binding studies with [125I]-HEAT showed the existence of a homogeneous population of binding site with an affinity (Kd value) of 0.4 nM and a maximum number of binding sites (Bmax) of 100 fmol mg-1 protein. Catecholamines competed for [125I]-HEAT binding stereospecifically and with the characteristic alpha 1-AR potency series. 3. Displacement curves for BMY-7378 and KMD-3213 best fitted a one-site model with a pKi value (-log10 (equilibrium inhibition constant)) of 6.06 and 7.07, respectively. 4. Reverse transcription-polymerase chain reaction (RT-PCR) assay detected alpha 1B- and alpha 1D-AR, but not alpha 1A-AR transcript. 5. Chlorethylclonidine (CEC) treatment nearly abolished (-)noradrenaline (NA) (10 microM)-induced inositol[1,4,5]trisphosphate (IP3) production, and BMY-7378 inhibited the response with a Ki value of 0.3 nM, which value was similar to that obtained in the cells expressing alpha 1D-AR. In both AC01 cells and cells expressing alpha 1D-AR, BMY-7378 protected alpha 1-ARs from CEC alkylation while it had little protective effect on CEC alkylation and NA-induced IP3 production in cells expressing alpha 1B-AR. 6. The results indicate that AC01 cells contain predominantly alpha 1B-ARs and a small population of alpha 1D-ARs; however, phosphoinositide (PI)/Ca2+ signalling is mainly mediated through the minor population of alpha 1D-ARs, rather than the alpha 1B-ARs.

Alpha 1-ADRENERGIC RECEPTOR SUBTYPES IN HUMAN DETRUSOR

Purpose To identify and quantitate alpha 1-adrenergic receptor (alpha 1 AR) subtype expression in human detrusor. Materials and Methods Initial studies to determine alpha 1 AR expression in human detrusor were performed using saturation binding with [( 125) I]HEAT. Once the presence of alpha 1 ARs was documented, subtype (alpha 1a, alpha 1b, alpha 1d) expression at the mRNA level (and comparison with rat) was determined with RNase protection assays (human detrusor) and RT-PCR (human detrusor, rat whole bladder). Competition binding analysis with the alpha 1d AR-selective ligand BMY7378 was used to measure alpha 1 AR subtype expression at a protein level in human detrusor. Results alpha 1 AR expression in human detrusor was low but reproducible (6.3 +/- 1.0 fmol./mg. total protein). RNase protection assays performed on total RNA extracted from human detrusor revealed the following alpha 1 AR subtype expression: alpha 1d (66%) > alpha 1a (34%), and no alpha 1b. RT-PCR confirmed alpha 1 AR subtype mRNA distribution in human detrusor with alpha 1d ([nearly =] 60-70%) > alpha ( 1a) ([nearly =] 30-40%), and a lack of alpha 1b mRNA. Rat whole bladder expressed different alpha 1 AR subtype mRNA than human detrusor, with alpha 1a [nearly =] alpha 1b [nearly =] alpha 1d. The presence of alpha 1d > alpha 1a expression in human detrusor was confirmed at a protein level by competition analysis utilizing BMY7378 which revealed a two-site fit, with K i and high affinity binding (66%) consistent with the alpha 1d AR subtype. Conclusions Human detrusor contained two alpha 1 AR subtypes (alpha ( 1d) > alpha 1a), a finding that is different from rat, another commonly used animal model. Since non-subtype selective alpha 1 AR antagonists ameliorate irritative bladder symptoms (in men and women with/without outlet obstruction), and Rec 15/2739 (alpha 1a selective antagonist) does not improve symptom scores in BPH, our findings suggest bladder alpha 1d ARs may provide a potentially novel mechanism underlying these therapeutic benefits.

Proximal nephron Na+/H+ exchange is regulated by alpha 1A- and alpha 1B-adrenergic receptor subtypes.

Activation of alpha 1-adrenergic receptors (alpha 1-AR) increases Na+/H+ exchange (NHE) in proximal tubule. NHE mediates the majority of active Na+ absorption in the proximal tubule. Three alpha 1-AR subtypes have been detected in kidney by molecular and binding techniques. We detected message for all three alpha 1-AR subtypes in mouse proximal tubule cells through reverse transcription-polymerase chain reaction and Northern analysis. To determine the alpha 1-AR subtypes that regulate NHE in mouse proximal tubule cells, two strategies were used: (i) antisense oligodeoxynucleotides (ODNs) to selectively inhibit expression of alpha 1A-, alpha 1B-, and alpha 1D-AR subtypes and (ii) subtype-selective alpha 1-AR antagonists. Streptolysin-O permeabilization was used to introduce antisense and sense ODNs into cells three times over 72 hr. Western blot analysis of membranes prepared from cells treated with alpha 1B-AR antisense ODN demonstrated that alpha 1B-AR protein expression was reduced by 90% at 72 hr compared with control or sense ODN treatments. Functional regulation of NHE by alpha 1-ARs was determined by alpha 1-AR agonist changes in intracellular pH (pHi) in cells grown on coverslips and loaded with 2',7'-bis(2-carboxyethyl)-5(6)carboxyfluorescein-acetoxymethyl ester. Antisense ODNs for alpha 1B-AR significantly reduced phenylephrine (PHE)-induced maximal changes in pHi by 49%. The PHE-induced changes in pHi observed in cells treated with alpha 1A-AR antisense ODNs was reduced by 42%. The selective alpha 1A-AR antagonist WB-4101 and the alpha 1B-AR antagonist spiperone reduce PHE-induced pHi increases to a comparable extent. No significant changes in pHi were observed with cells treated with alpha 1D-AR antisense ODNs or the alpha 1D-AR antagonist BMY 7378 compared with untreated cells. Combined treatment with alpha 1A- and alpha 1B-AR antisense ODNs and antagonists additively inhibits PHE-induced delta pHi by 90%. We conclude that alpha 1A and alpha 1B-AR but not alpha 1D-ARs regulate NHE in proximal tubule cells.

N-arylpiperazinyl-N'-propylamino derivatives of heteroaryl amides as functional uroselective alpha 1-adrenoceptor antagonists.

Novel arylpiperazines were identified as alpha 1-adrenoceptor (AR) subtype-selective antagonists by functional in vitro screening. 3-[4-(ortho-Substituted phenyl)piperazin-1-yl]propylamines were derivatized with N,N-dimethyl anthranilamides, nicotinamides, as well as carboxamides of quinoline, 1,8-naphthyridine, pyrazolo[3,4-b]pyridine, isoxazolo[3,4-b]pyridine, imidazo[4,5-b]pyridine, and pyrazolo[1,5-a]pyrimidines. Strips of rabbit bladder neck were employed as a predictive assay for antagonism in the human lower tract. Rings of rat aorta were used as a "negative screen" for the test antagonists. Binding to alpha 1-ARs was relatively sensitive to size and electronic features of the arylpiperazine portion of the antagonists and permissive to these features on the heteroaryl carboxamide side. These structure-affinity findings were exploited to produce nicotinamides (e.g. 13ii and 25x) and pyrazolo[3,4-b]pyridines (e.g. 37f and 37y) ligands with nanomolar affinity at the alpha 1-AR subtype prevalent in the human lower urinary tract(pA2 values: 8.8, 10.7, 9.3, and 9.9, respectively) and displaying 2-3 orders of magnitude selectivity over the alpha 1D-AR.

Alpha 1-adrenoceptor subtypes in rat aorta and mesenteric small arteries are preserved during left ventricular dysfunction post-myocardial infarction.

In heart failure, homologous downregulation of beta-adrenoceptors contributes to impaired adrenergic responsiveness of the myocardium. We evaluated alpha 1-adrenoceptors (alpha 1-AR) in a sparsely innervated and a densely innervated peripheral artery in an experimental model of left ventricular dysfunction post-myocardial infarction. [3H]Prazosin binding was determined in arterial segments of Wistar-Kyoto rats (WKY), and of Wistar rats 5 weeks after myocardial infarction (MI) or sham operation (SHAM). In the thoracic aorta (TAO) of WKY, specific prazosin binding was: (i) prevented by the irreversible alpha 1B-AR and relatively selective alpha 1D-AR antagonist, chloroethylclonidine (CEC); (ii) displaced with low affinity (pKi 6.25) by the alpha 1A-AR selective ligand, (+)-niguldipine; and (iii) displaced with both high (pKi 10.4) and low (pKi 7.37) affinity by the alpha 1D-AR antagonist, BMY 7378. In mesenteric small arteries (MSA) of WKY, prazosin binding was: (i) reduced 50% by CEC; (ii) displaced in a biphasic fashion by (+)-niguldipine (pKi 8.60 and pKi 6.22); and (iii) displaced by BMY 7378 with low affinity only (pKi 6.86). Also in TAO of SHAM. prazosin binding was prevented by CEC, but neither 30 nM (+)-niguldipine nor 1 nM BMY 7378 affected it. In MSA of SHAM, prazosin binding was virtually abolished in the presence of 30 nM (+)-niguldipine and was not reduced by 1 nM BMY 7378. In TAO and MSA of MI, compared to SHAM, the density of binding sites tended to be increased rather than decreased and neither the affinity for the ligand nor the effects of alpha 1-AR subtype selective tools were significantly modified. These findings indicate that: (i) radioligand binding can be applied in intact arterial segments to quantify and characterize alpha 1-AR; (ii) although differences seem to exist between rat strains, alpha 1B-AR and alpha 1D-AR predominate in rat thoracic aorta and alpha 1A-AR and alpha 1B-AR in mesenteric small arteries; and (iii) alpha 1-AR density is not reduced in the poorly innervated aorta and the densely innervated mesenteric small arteries of rats with heart failure due to myocardial infarction.

Phenylalanine in the second membrane-spanning domain of alpha 1A-adrenergic receptor determines subtype selectivity of dihydropyridine antagonists.

The alpha 1-adrenergic receptors (alpha 1-AR) belong to the G-protein coupled seven-transmembrane biogenic amine receptor family. Three subtypes have been successfully cloned in the alpha 1-adrenergic receptor family, and they share 50% identical amino acid sequences and 70% similarity. We have constructed seven chimeric receptors of the alpha 1A-AR. Each of the chimeras contains alpha 1D-subtype amino acid sequences within the membrane-spanning domains. Comparisons of ligand affinities with these chimeras has provided information on the importance of certain amino acid residues in determining receptor subtype specificity in the alpha 1A- and alpha 1D-ARs. With ligands in the dihydropyridine series, the niguldipine analog 1 was found to have respective pKi's of 9.32 +/- 0.17 for alpha 1A-AR; 6.84 +/- 0.24 for alpha 1D-AR; and 6.76 +/- 0.28 for alpha 1A/D(TM2), respectively. This trend was also exhibited by two other niguldipine analogs, 2 and 3, which had similar pKi's toward alpha 1D-AR and alpha 1A/D(TM2). This subtype selectivity was also maintained in the piperdine derivative, 4, and alpha 1A-AR selective ligand, which showed the same parallel trends in binding affinities with alpha 1A-AR and the six chimeras as the niguldipine analogs. Since in considering the second membrane-spanning domain, the alpha 1A- and alpha 1D-ARs only differ at positions 76, 77, 85, and 86, we were able to show through mutational studies that phenylalanine 86 is solely responsible for the selectivity found in the chimeric receptor alpha 1A/D(TM2) exhibited against the ligands 1-4 used in this study. A model based on the rhodopsin structure places the amino acid at position 86 in the final turn toward the extracellular region. This is four helical turns above aspartic acid-79, a conserved amino acid in the second membrane-spanning domain. This is the first report that suggests a significant involvement of the second membrane-spanning domain in antagonist binding in the biogenic amines class of the superfamily of seven-transmembrane receptors.

Identification of Critical Determinants of α1-Adrenergic Receptor Subtype Selective Agonist Binding

alpha 1-Adrenergic receptor (AR) subtypes mediate many effects of the sympathetic nervous system. The three cloned subtypes (alpha 1a-AR, alpha 1b-AR, alpha 1d-AR), although structurally similar, bind a series of ligands with different relative potencies. This is particularly true for the alpha 1a-AR, which recognizes a number of agonists and antagonists with 5-50-fold higher affinity than the alpha 1b- or alpha 1d- subtypes. Since ligands bind to receptor-residues that are located in the transmembrane spanning domains, we hypothesize that subtype differences in ligand recognition are due to differences in the binding properties of nonconserved transmembrane residues. Using site-directed mutagenesis, selected putative ligand-binding residues in the alpha 1b-AR were converted, either individually or in combination, to the corresponding residues in the alpha 1a-AR. Mutation of two such residues (of approximately 172 amino acids in the transmembrane domains) converted the agonist binding profile entirely to that of the alpha 1a-AR. Over 80% of this conversion was due to an Ala204-->Val substitution; the remainder was due to the additional substitution of Leu314-->Met. To confirm that Ala204 and Leu314 are indeed critical for agonist subtype-selectivity, the equivalent residues in the alpha 1a-AR (Val185 and Met293) were reversed of that of the alpha 1b-AR. Correspondingly, the agonist-binding profile of this double alpha 1a-AR mutant reverted to that of the alpha 1b-AR. From these data, in conjunction with macromolecular modeling of the ligand-binding pocket, a model has been developed, which indicates that the determinants of these two residues for agonist subtype-selectivity are due not only to interactions between their side chains and specific ligand moieties but also to a critical interaction between these two amino acids.

Alpha 1a-adrenergic receptor mediated positive chronotropic effect in right atria isolated from rats.

Experiments in right atria isolated from adult male rats were designed to determine which of the alpha 1-adrenergic receptor (alpha 1-AR) subtypes are involved in the positive chronotropic effect of phenylephrine, an alpha 1-AR agonist. Chloroethylclonidine (CEC), an irreversible alpha 1b-, alpha 1c-, and alpha 1d-AR antagonist, did not alter the efficacy or potency of phenylephrine; however, CEC did elicit a concentration-dependent negative chronotropic effect and reduce the absolute maximum spontaneous rate observed in the presence of phenylephrine. WB4101, a competitive alpha 1a- and alpha 1c-AR-selective antagonist, did not alter basal spontaneous rate or the efficacy of phenylephrine, but it did produce a significant rightward shift of the phenylephrine concentration-response curve. Phenoxybenzamine, an irreversible nonselective alpha-AR antagonist, elicited a concentration-dependent negative chronotropic effect, a significant rightward shift of the phenylephrine concentration-response curve, and a reduction in the efficacy of phenylephrine. The chronotropic action of the beta-adrenergic agonist isoproterenol was not affected by CEC, WB4101, or phenoxybenzamine. These data suggest that the positive chronotropic effect of alpha 1-adrenergic agonists in rat right atria is mediated via stimulation of alpha 1a-ARs.