Katsushi Shibata

Use of recombinant α1-adrenoceptors to characterize subtype selectivity of drugs for the treatment of prostatic hypertrophy

Several alpha 1-adrenoceptor antagonists have recently been developed for the treatment of benign prostatic hypertrophy because of their less frequent systemic side-effects compared to conventional alpha 1-adrenoceptor blockers. One potential explanation for their good tolerability would be the selectivity for a certain subtype of alpha 1-adrenoceptor. Utilizing COS-7 cells expressing the rat alpha 1A, the hamster alpha 1B and the human alpha 1C-adrenoceptors, we investigated affinities of alfuzosin, doxazosin, terazosin, indoramin and (+)- and (-)-5-[2-[[2-(o-ethoxyphenoxy)ethyl] amino]propyl]-2-methoxybenzesulfonamide HCl (YM 617) compared to prazosin. Radioligand binding studies showed that the affinities of alpha 1-adrenoceptor subtypes for alfuzosin (Ki value; alpha 1A: 2.4 nM, alpha 1B:1.4 nM, alpha 1C:4.2 nM), doxazosin (Ki value; alpha 1A:2.7 nM, alpha 1B:3.2 nM, alpha 1C:7.5 nM), terazosin (Ki value; alpha 1A:2.5 nM, alpha 1B:2.7 nM, alpha 1C:7.1 nM), indoramin (Ki value; alpha 1A:69 nM, alpha 1B:21 nM, alpha 1C:13 nM) and prazosin (Ki value; alpha 1A:0.16 nM, alpha 1B:0.19 nM, alpha 1C:0.2 nM) were equipotent to the three receptor subtypes. Unlike these antagonists, both (+)- and (-)-YM617 had relatively lower affinity for alpha 1B receptors compared to the other subtypes (Ki value; for (+)-YM617, alpha 1A:22 nM, alpha 1B:96 nM, alpha 1C:4.3 nM; for (-)-YM617, alpha 1A:0.11 nM, alpha 1B:0.7 nM, alpha 1C:0.035 nM). The data suggest that alpha 1-adrenoceptor antagonists currently used for the treatment of the benign prostatic hyperplasia do not show substantial subtype selectivity.

α1a‐Adrenoceptor polymorphism: pharmacological characterization and association with benign prostatic hypertrophy

1 Two restriction fragment length polymorphisms of the human α1a‐adrenoceptor gene digested with PstI restriction enzyme exist; the nucleotide change causes the substitution of C residue for T at nucleotide 1441, thereby Arg492 to Cys492 transition, which might confer an additional putative palmitoylation site in the carboxy‐terminal segment of the α1a‐adrenoceptor. In the present study, we compared their pharmacological properties and examined whether this α1a‐adrenoceptor polymorphism is associated with benign prostatic hypertrophy (BPH). 2 The frequency of α1a‐adrenoceptor polymorphism was not differently distributed between patients with benign prostatic hypertrophy (BPH) and normal subjects in Japan; thus, the relative frequencies of the C and T alleles were 0.90: 0.10 in normal male subjects (n = 45) and 0.87: 0.13 in BPH patients (n = 222), respectively. However, the frequency distribution of this polymorphism was significantly different between the Japanese and U.S. populations; thus, C and T alleles were 0.34 and 0.66 in U.S. populations. 3 Utilizing Chinese hamster ovary (CHO) cells stably expressing the two polymorphic α1a‐adrenoceptors (Arg492 and Cys492), we compared their binding affinity and signal transduction. Radioligand binding studies with 2‐[β‐(4‐hydroxy‐3[125I]‐iodophenyl) ethylamino‐methyl]tetralone ([125I]‐HEAT) showed no marked difference in the antagonist or agonist binding affinities between the two receptors. Also, both receptors were found to be coupled to the calcium signaling, and the concentration‐cytosolic Ca2+ concentrations ([Ca2+]i response relationships for noradrenaline were similar for the two polymorphic receptors. Furthermore, the receptor‐mediated [Ca2+]i response was markedly desensitized after a 2 h exposure of phenylephrine (10 μm), and the extent of the desensitization was not significantly different between the two receptors. 4 In summary, the results showed that the two α1a‐adrenoceptors generated by genetic polymorphism have similar pharmacological characteristics, and the receptor‐mediated [Ca2+]i response can be desensitized in a similar manner. The study did not provide any evidence to support the hypothesis that α1a‐adrenoceptor gene polymorphism is associated with BPH.

Cloning, functional expression and tissue distribution of human α1C-adrenoceptor splice variants

We report the cloning and characterization of two isoforms of human α 1C‐adrenoceptor cDNA (α 1C‐2, α C‐3). These isoforms are generated by alternative splicing and differ from the clone we previously isolated (α 1C‐1) in their length and sequences of the C‐terminal domain. Tissue distribution of mRNAs showed that these variants co‐express with α 1C‐1 in the human heart, liver, cerebellum and cerebrum. Despite the structural differences, functional experiments in transfected CHO cells showed that the three isoforms have similar ligand binding properties, and all couple with phospholipase C/Ca2+ signaling pathway.

Insights into α1 adrenoceptor function in health and disease from transgenic animal studies

Alpha(1)-adrenoceptors (ARs) mediate some of the main actions of the natural catecholamines, epinephrine and norepinephrine, and have a crucial role in the regulation of arterial blood pressure. Since alpha(1)-AR was subdivided into three subtypes (alpha(1A)-AR, alpha(1B)-AR and alpha(1D)-AR), the search has been on to discover subtype-specific physiological roles and to develop subtype-selective agonists and antagonists. Recently, several strains of genetically engineered mice have become available. Studies with these mice have provided several clues to help elucidate subtype-specific physiological functions; for instance, alpha(1A)-AR and alpha(1D)-AR subtypes play an important role in the regulation of blood pressure, suggesting that subtype-selective antagonists might be desirable antihypertensive agents. The ability to study subtype-specific functions in different mouse strains by altering the same alpha(1)-AR in different ways strengthens the conclusions drawn from pharmacological studies. Although these genetic approaches have limitations, they have significantly increased our understanding of the functions of alpha(1)-AR subtypes.

Negative Chronotropic Effect of Endothelin 1 Mediated Through ETA Receptors in Guinea Pig Atria

Endothelins exert potent excitatory cardiac effects by acting on specific receptors on myocytes. In this study, we have examined the signal transduction mechanism for the chronotropic effect of endothelins in guinea pig atria. A competition binding of [125I]endothelin 1 ([125I]ET-1) using the recently developed ETA receptor-selective antagonist BQ123 showed the presence of almost equal populations of ETA (44%) and ETB (56%) receptors in the guinea pig right atria. In a concentration-response study, endothelin 3 (ET-3), an agonist with higher affinity to ETB receptors than to ETA receptors, and sarafotoxin S6c (STXS6c), an ETB receptor-selective agonist, increased the rate of spontaneous beating at all concentrations tested (10 pmol/L to 100 nmol/L). In contrast, ET-1, a nonselective agonist, increased the heart rate at lower concentrations (10 pmol/L to 10 nmol/L) but decreased it at higher concentrations (30 to 100 nmol/L). When ET-1 (100 nmol/L) was applied in a single amount, heart rate was strongly increased; however, this increase was followed by a rapid decline in the response. ET-1 (100 nmol/L) but not ET-3 or STXS6c significantly reduced the heart rate when it was raised by isoproterenol (ISO, 300 nmol/L) either in the absence or presence of a phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX). Correspondingly, ET-1 significantly reduced the ISO-induced elevation of cAMP accumulation (19.1 +/- 1.7 pmol/mg protein [n = 8] and 12.6 +/- 1.2 pmol/mg protein [n = 7] in the absence and presence of ET-1, respectively; P < .01), which was also observed even in the presence of IBMX.(ABSTRACT TRUNCATED AT 250 WORDS)

NS-49, a novel α1a-adrenoceptor-selective agonist characterization using recombinant human α1-adrenoceptors

Abstract α1-Adrenoceptors comprise a heterogeneous family and subtype-selective ligands are valuable in studying the functional role of each receptor subtype. Using the Chinese hamster ovary (CHO) cells stably expressing the cloned human α1-adrenoceptor subtypes (α1a, α1b, and α1d)1, we have compared a newly synthesized phenethylamine class agonist (R)-(−)-3′-(2-amino-1-hydroxythyl)-4′- fluoromethanesulfonanilide hydrochloride (NS-49) with imidazoline class agonist oxymetazoline in their binding affinities and intrinsic activities in causing transient increases of cytosolic Ca2+ concentrations ([Ca2+]1 response). Radioligand binding sites with 2-[β-(4-hydroxy-3-[125I]iodophenyl)ethylamino-methyl]tetralone ([125I]HEAT) showed NS-49 and oxymetazoline had higher affinities at α1a- than at α1b- and α1d-subtypes (−log Ki values at α1a−, α1b− and α1d-subtype: 6.18, 5.13, and 5.38 for NS-49; 8.19, 6.50, and 6.44 for oxymetazoline, respectively). In functional studies, both oxymetazoline and NS-49 worked as a selective and partial agonist at α1a-subtype; however, NS-49 is more efficacious than oxymetazoline. NS-49 is the phenethylamine class of α1-adrenoceptor partial agonist relatively selective and efficacious for the human α1a-adrenoceptor subtype. NS-49 would be potentially useful for studying the physiological role of α1-adrenoceptor subtype.

Restoration of E-cadherin expression by selective Cox-2 inhibition and the clinical relevance of the epithelial-to-mesenchymal transition in head and neck squamous cell carcinoma

BackgroundThe epithelial-to-mesenchymal transition (EMT) accompanied by the downregulation of E-cadherin has been thought to promote metastasis. Cyclooxygenase-2 (Cox-2) is presumed to contribute to cancer progression through its multifaceted function, and recently its inverse relationship with E-cadherin was suggested. The aim of the present study was to investigate whether selective Cox-2 inhibitors restore the expression of E-cadherin in head and neck squamous cell carcinoma (HNSCC) cells, and to examine the possible correlations of the expression levels of EMT-related molecules with clinicopathological factors in HNSCC.MethodsWe used quantitative real-time PCR to examine the effects of three selective Cox-2 inhibitors, i.e., celecoxib, NS-398, and SC-791 on the gene expressions of E-cadherin (CDH-1) and its transcriptional repressors (SIP1, Snail, Twist) in the human HNSCC cell lines HSC-2 and HSC-4. To evaluate the changes in E-cadherin expression on the cell surface, we used a flowcytometer and immunofluorescent staining in addition to Western blotting. We evaluated and statistically analyzed the clinicopathological factors and mRNA expressions of Cox-2, CDH-1 and its repressors in surgical specimens of 40 patients with tongue squamous cell carcinoma (TSCC).ResultsThe selective Cox-2 inhibitors upregulated the E-cadherin expression on the cell surface of the HNSCC cells through the downregulation of its transcriptional repressors. The extent of this effect depended on the baseline expression levels of both E-cadherin and Cox-2 in each cell line. A univariate analysis showed that higher Cox-2 mRNA expression (p = 0.037), lower CDH-1 mRNA expression (p = 0.020), and advanced T-classification (p = 0.036) were significantly correlated with lymph node metastasis in TSCC. A multivariate logistic regression revealed that lower CDH-1 mRNA expression was the independent risk factor affecting lymph node metastasis (p = 0.041).ConclusionsThese findings suggest that the appropriately selective administration of certain Cox-2 inhibitors may have an anti-metastatic effect through suppression of the EMT by restoring E-cadherin expression. In addition, the downregulation of CDH-1 resulting from the EMT may be closely involved in lymph node metastasis in TSCC.

Flow cytometry analysis of α 1-adrenoceptor subtypes

To characterize the α 1‐adrenoceptor subtypes, we developed a flow cytometry method using the fluorescent ligand BODIPY‐FL prazosin and the anti‐peptide antibody against the α 1b‐adrenoceptor amino terminus (designated 1B‐N1‐C) as probes. Three α 1‐adrenoceptors (α 1a, β 1b and α 1d) expresed in CHO cells were detected by BODIPY‐FL prazosin; however, only α 1b‐adrenoceptor subtype was detected by the anti‐peptide antibody 1B‐N1‐C. Furthermore, the flow cytometry analysis with 1B‐N1‐C specifically identified α 1b‐adrenoceptor in native cells of hamster DDT1‐MF2 cells, rat hepatocytes and1 cardiomyocytes.

Inhibition of heat shock protein 90 attenuates adenylate cyclase sensitization after chronic morphine treatment.

Cellular adaptations to chronic opioid treatment result in enhanced responsiveness of adenylate cyclase and an increase in forskolin- or agonist-stimulated cAMP production. It is, however, not known whether chaperone molecules such as heat shock proteins contribute to this adenylate cyclase sensitization. Here, we report that treatment of cells with geldanamycin, an inhibitor of heat shock protein 90 (Hsp90), led to effective attenuation of morphine-induced adenylate cyclase sensitization. In SK-N-SH human neuroblastoma cells, morphine significantly increased RNA transcript and protein levels of type I adenylate cyclase, leading to sensitization. Whole-genome tiling array analysis revealed that cAMP response element-binding protein, an important mediator for cellular adaptation to morphine, associated with the proximal promoter of Hsp90AB1 not only in SK-N-SH cells but also in rat PC12 and human embryonic kidney cells. Hsp90AB1 transcript and protein levels increased significantly during morphine treatment, and co-application of geldanamycin (0.1-10 nM) effectively suppressed the increase in forskolin-activated adenylate cyclase activation by 56%. Type I adenylate cyclase, but not Hsp90AB1, underwent significant degradation during geldanamycin treatment. These results indicate that Hsp90 is a new pharmacological target for the suppression of adenylate cyclase sensitization induced by chronic morphine treatment.

Both α1A- and α1B-adrenergic receptor subtypes couple to the transient outward current (ITo) in rat ventricular myocytes

Regulation of transient outward current (ITo) by α1‐adrenergic (α1AR) plays a key role in cardiac repolarization. α1ARs comprise a heterogeneous family; two natively expressed subtypes (α1A and α1B) and three cloned subtypes (α1a, α1b and α1d) can be distinguished. We have examined the electrophysiological role of each α1AR subtype in regulating ITo in isolated rat ventricular myocytes. Reverse transcription‐PCR study revealed the presence of three subtype mRNAs (α1a, α1b and α1d) in rat myocytes. Radioligand binding assay using [125I]‐HEAT showed that the inhibition curves for α1AAR‐selective antagonists (WB4101, 5‐methylurapidil, (+)‐niguldipine and KMD‐3213) in rat ventricles best fit a two‐site model, with 30% high and 70% low affinity binding sites. The high affinity sites were resistant to 100 μM chloroethylclonidine (CEC), while the low affinity sites were highly inactivated by CEC. Whole cell voltage clamp study revealed that methoxamine reduced a 4‐aminopyridine(4‐AP)‐sensitive component of ITo in the isolated rat ventricle myocytes. Lower concentrations of KMD‐3213 (1 nM) or 5‐MU (10 nM) did not affect the methoxamine‐induced reduction of ITo. On the other hand, CEC treatment (100 μM) of isolated myocytes reduced the methoxamine‐induced reduction of ITo by 46%, and the remaining response was abolished by lower concentrations of KMD‐3213 or 5‐MU. The results indicate that rat ventricular myocytes express transcripts of the three α1AR subtypes (α1a, α1b and α1d); however, two pharmacologically distinct α1AR subtypes (α1A and α1B) are predominating in receptor populations, with approximately 30% α1AAR and 70% α1BAR. Although both α1A and α1BAR subtypes are coupled to the cardiac ITo, α1BARs predominantly mediate α1AR‐induced effect.