Nobuaki Taniguchi

A Role of Androgen Receptor Protein in Cell Growth of an Androgen-Independent Prostate Cancer Cell Line

Prostate cancer, which develops due to androgen and is initially responsive to androgen deprivation therapy, often comes to acquire androgen deprivation therapy resistance in short order. We investigated the role of androgen receptor (AR) protein in an androgen-independent prostate cancer cell line using AR ligands and AR siRNA. Although the androgen-independent cell line scarcely responded to AR ligands, their growth was attenuated by ablation of AR protein by siRNA.

Alterations of Renal Vasopressin V1A and V2 Receptors in Spontaneously Hypertensive Rats

To elucidate the role of arginine vasopressin (AVP) in a hypertensive state, the characteristics of renal cortex V_1A and medulla V₂ receptors in young spontaneously hypertensive rats (SHR) during the developmental phase of hypertension were compared with those of age-matched Wistar-Kyoto (WKY) rats using the radioligand receptor assay technique. The systolic blood pressure of 8-week-old SHR was statistically significantly higher than that of WKY rats (142 ± 1 vs. 125 ± 2 mm Hg). The plasma AVP levels were also significantly higher in SHR than in WKY rats (3.20 ± 0.41 vs. 1.96 ± 0.34 pg/ml). In SHR, the maximum capacity of ³H-d(CH₂)₅Tyr(Me)AVP binding to cortical V_1A receptors (B_max) was statistically significantly higher than that of WKY rats (39.7 ± 2.7 vs. 22.4 ± 0.9 fmol/mg protein). Furthermore, the B_max values of ³H-AVP binding to medullary V₂ receptors in SHR were also significantly higher than in WKY rats (40.2 ± 1.9 vs. 28.3 ± 1.3 fmol/mg protein). However, the apparent dissociation constant (K_d) values of renal cortex V_1A and medulla V₂ receptors in SHR and WKY rats were not significantly different. These results indicate that increased amounts of renal cortex V_1A and medulla V₂ receptors in SHR play an important role in the pathophysiology of hypertension.

Effects of YM471, a nonpeptide AVP V1A and V2 receptor antagonist, on human AVP receptor subtypes expressed in CHO cells and oxytocin receptors in human uterine smooth muscle cells

YM471, (Z)‐4′‐{4,4‐difluoro‐5‐[2‐(4‐dimethylaminopiperidino)‐2‐oxoethylidene]‐2,3,4,5‐tetrahydro‐1H‐1‐benzoazepine‐1‐carbonyl}‐2‐phenylbenzanilide monohydrochloride, is a newly synthesized potent vasopressin (AVP) receptor antagonist. Its effects on binding to and signal transduction by cloned human AVP receptors (V1A, V1B and V2) stably expressed in Chinese hamster ovary (CHO) cells, and oxytocin receptors in human uterine smooth muscle cells (USMC) were studied. YM471 potently inhibited specific [3H]‐AVP binding to V1A and V2 receptors with Ki values of 0.62 nM and 1.19 nM, respectively. In contrast, YM471 exhibited much lower affinity for V1B and oxytocin receptors with Ki values of 16.4 μM and 31.6 nM, respectively. In CHO cells expressing V1A receptors, YM471 potently inhibited AVP‐induced intracellular Ca2+ concentration ([Ca2+]i) increase, exhibiting an IC50 value of 0.56 nM. However, in human USMC expressing oxytocin receptors, YM471 exhibited much lower potency in inhibiting oxytocin‐induced [Ca2+]i increase (IC50=193 nM), and did not affect AVP‐induced [Ca2+]i increase in CHO cells expressing V1B receptors. Furthermore, in CHO cells expressing V2 receptors, YM471 potently inhibited the production of cyclic AMP stimulated by AVP with an IC50 value of 1.88 nM. In all assays, YM471 showed no agonistic activity. These results demonstrate that YM471 is a potent, nonpeptide human V1A and V2 receptor antagonist which will be a valuable tool in defining the physiologic and pharmacologic actions of AVP.

Effect of YM471, an orally active non-peptide arginine vasopressin receptor antagonist, on human vascular smooth muscle cells.

Objective To investigate the effects of YM471, a non-peptide arginine vasopressin (AVP) V1A and V2 receptor antagonist, on the AVP-induced growth responses in human vascular smooth muscle cells (VSMCs). Methods Binding of YM471 to V1A receptors on VSMCs was measured using [3H]AVP. Intracellular free Ca2+ concentration was measured by fura 2 fluorescence. Mitogen-activated protein (MAP) kinase activity was determined using the p42/p44 MAP kinase specific peptide and [γ-32P]ATP as substrates. The effect of AVP on hyperplasia and hypertrophy of VSMCs was determined by cell number and protein content measurements. Results YM471 potently and concentration-dependently inhibited the specific binding of [3H]AVP to V1A receptors on VSMCs, exhibiting an inhibition constant (Ki) of 0.35 nmol/l. YM471 inhibited the AVP-induced increase in intracellular free Ca2+ concentration with an 50% inhibition concentration (IC50) of 2.01 nmol/l and inhibited the activation of MAP kinase with an IC50 of 6.11 nmol/l. In addition, AVP concentration-dependently induced hyperplasia and hypertrophy in VSMCs, but YM471 prevented these AVP-induced growth effects, exhibiting IC50 values of 2.31 and 0.23 nmol/l, respectively. Conclusions These results indicate that YM471 has high affinity for V1A receptors on, and potently inhibits AVP-induced physiologic responses of, human VSMCs.

Binding and signal transduction characteristics of the nonpeptide vasopressin V1A receptor-selective antagonist YM218 in cultured rat mesangial cells.

Vasopressin (AVP) causes mesangial cell contraction, proliferation and hypertrophy. The present study investigated the effects of YM218, a potent, nonpeptide AVP V1A receptor-selective antagonist, on rat mesangial cells using binding, signal transduction and cell growth assays. Specific binding of 3H-AVP to rat mesangial cell plasma membranes was dependent upon time, temperature and membrane protein concentration. Scatchard plot analysis of equilibrium binding data revealed the existence of a single class of high-affinity binding sites with the expected V1A receptor profile. YM218 showed high affinity for V1A receptors, exhibiting a Ki value of 0.19 nmol/l. AVP concentration-dependently increased intracellular Ca2+ ([Ca2+]i) levels, stimulated mitogen-activated protein (MAP) kinase and induced hyperplasia. Conversely, YM218 potently suppressed [Ca2+]i elevation, activation of MAP kinase and hyperplasia induced by AVP. These results indicate that YM218 displays both high affinity for rat mesangial cell V1A receptors and high potency in inhibiting AVP-induced signal transduction and growth response. Therefore, YM218 is a useful pharmacologic tool for investigating the physiologic and pathophysiologic roles of AVP in kidney, and may have clinical application in the prevention or regression of mesangial cell growth.

Effect of YM471, a nonpeptide AVP receptor antagonist, on human coronary artery smooth muscle cells.

The antagonistic properties of YM471, a potent nonpeptide vasopressin (AVP) V(1A) and V(2) receptor antagonist, were characterized using human coronary artery smooth muscle cells (CASMC). YM471 potently inhibited specific binding of 3H-AVP to V(1A) receptors on human CASMC, exhibiting a K(i) value of 0.49 nM. Furthermore, YM471 inhibited the AVP-induced increase in intracellular free Ca(2+) concentration with an IC(50) value of 1.42 nM, but exerted no agonistic activity on CASMC. Additionally, while AVP concentration-dependently induced hyperplasia and hypertrophy in CASMC, YM471 prevented these AVP-induced growth effects, exhibiting IC(50) values of 0.93 and 2.64 nM, respectively. These results indicate that YM471 has high affinity for V(1A) receptors on, and high potency in inhibiting AVP-induced physiologic responses of, human CASMC.