Toshiyuki Kusayama

Effect of YM087, a potent nonpeptide vasopressin antagonist, on vasopressin-induced protein synthesis in neonatal rat cardiomyocyte

OBJECTIVE Hypertrophy of cardiomyocytes may play an important role in the pathogenesis of cardiac hypertrophy associated with various cardiovascular diseases such as congestive heart failure. The aim of this study was to investigate whether vasopressin (AVP) induces protein synthesis in cultured neonatal rat cardiomyocytes through its specific receptor and whether YM087, a newly synthesized nonpeptide AVP receptor antagonist, inhibits AVP-induced protein synthesis in vitro. METHODS AVP receptors on cardiomyocytes were characterized using the radioligand [3H] AVP. The effects of AVP and YM087 on intracellular free calcium concentration ([Ca2+]i), mitogen-activated protein (MAP) kinase and [3H]-leucine incorporation were investigated in cultured neonatal rat cardiomyocytes. RESULTS In cardiomyocytes, Scatchard analysis showed a single population of high-affinity binding sites with the expected AVP V1A receptor subtype profile. YM087 showed high affinity for cardiomyocyte V1A receptors with a Ki value of 0.63 nM. In these same cells, YM087 potently inhibited AVP-induced increases in [CA2+]I and activation of MAP kinase in a concentration-dependent manner. In addition, AVP concentration-dependently stimulated the synthesis of protein without changing the rate of DNA synthesis, and YM087 prevented AVP-induced protein synthesis in a concentration-dependent manner. CONCLUSIONS These results suggest that AVP directly causes protein synthesis and YM087 is a potent inhibitor of AVP-induced protein synthesis of cardiomyocytes and thus may have beneficial effects in the development and regression of cardiomyocytic hypertrophy.

Pharmacological characterization of the human vasopressin receptor subtypes stably expressed in Chinese hamster ovary cells

1 Three subtypes of human (h) arginine vasopressin (AVP) receptors, hV1A, hV1B and hV2, were stably expressed in Chinese hamster ovary (CHO) cells and characterized by [3H]‐AVP binding studies. In addition, the coupling of the expressed receptor protein to a variety of signal transduction pathways was investigated. 2 Scatchard analysis of saturation isotherms for the specific binding of [3H]‐AVP to membranes, prepared from CHO cells transfected with hV1A, hV1B and hV2 receptors, yielded an apparent equilibrium dissociation constant (Kd) of 0.39, 0.25 and 1.21 nm and a maximum receptor density (Bmax) of 1580 fmol mg−1 protein, 5230 fmol mg−1 protein and 7020 fmol mg−1 protein, respectively. Hill coefficients did not differ significantly from unity, suggesting binding to homogenous, non‐interacting receptor populations. 3 Pharmacological characterization of the transfected human AVP receptors was undertaken by measuring the relative ability of nonpeptide AVP receptor antagonists, YM087, OPC‐21268, OPC‐31260, SR 49059 and SR 121463A, to inhibit binding of [3H]‐AVP. At hV1A receptors, the relative order of potency was SR49059>YM087>OPC‐31260>SR 121463A>>OPC‐21268 and at hV2 receptors, YM087=SR 121463A>OPC‐31260>SR 49059>>OPC‐21268. In contrast, the relative order of potency, at hV1B receptors, was SR 49059>>SR 121463A=YM087=OPC‐31260=OPC‐21268. 4 In CHO cells expressing either hV1A or hV1B receptors, AVP caused a concentration‐dependent increase in intracellular Ca2+ concentration ([Ca2+]i) with an EC50 value of 1.13 nm and 0.90 nm, respectively. In contrast, stimulation of CHO cells expressing hV2 receptors resulted in an accumulation of cyclic AMP with an EC50 value of 2.22 nm. The potency order of antagonists in inhibiting AVP‐induced [Ca2+]i or cyclic AMP response was similar to that observed in radioligand binding assays. 5 In conclusion, we have characterized the pharmacology of human cloned V1A, V1B and V2 receptors and used these to determine the affinity, selectivity and potency of nonpeptide AVP receptor antagonists. Thus they may prove to be a valuable tool in further examination of the physiological and pathophysiological roles of AVP.

Pharmacologic characterization of the oxytocin receptor in human uterine smooth muscle cells.

[3H]‐oxytocin was used to characterize the oxytocin receptor found in human uterine smooth muscle cells (USMC). Specific binding of [3H]‐oxytocin to USMC 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 an apparent equilibrium dissociation constant (Kd) of 0.76 nM and a maximum receptor density (Bmax) of 153 fmol mg−1 protein. The Hill coefficient (nH) did not differ significantly from unity, suggesting binding to homogenous, non‐interacting receptor populations. Competitive inhibition of [3H]‐oxytocin binding showed that oxytocin and vasopressin (AVP) receptor agonists and antagonists displaced [3H]‐oxytocin in a concentration‐dependent manner. The order of potencies for peptide agonists and antagonists was: oxytocin>[Asu1,6]‐oxytocin>AVP= atosiban>d(CH2)5Tyr(Me)AVP>[Thr4,Gly7]‐oxytocin>dDAVP, and for nonpeptide antagonists was: L‐371257>YM087>SR 49059>OPC‐21268>SR 121463A>OPC‐31260. Oxytocin significantly induced concentration‐dependent increase in intracellular Ca2+ concentration ([Ca2+]i) and hyperplasia in USMC. The oxytocin receptor antagonists, atosiban and L‐371257, potently and concentration‐dependently inhibited oxytocin‐induced [Ca2+]i increase and hyperplasia. In contrast, the V1A receptor selective antagonist, SR 49059, and the V2 receptor selective antagonist, SR 121463A, did not potently inhibit oxytocin‐induced [Ca2+]i increase and hyperplasia. The potency order of antagonists in inhibiting oxytocin‐induced [Ca2+]i increase and hyperplasia was similar to that observed in radioligand binding assays. In conclusion, these data provide evidence that the high‐affinity [3H]‐oxytocin binding site found in human USMC is a functional oxytocin receptor coupled to [Ca2+]i increase and cell growth. Thus human USMC may prove to be a valuable tool in further investigation of the physiologic and pathophysiologic roles of oxytocin in the uterus.

Vasopressin increases vascular endothelial growth factor secretion from human vascular smooth muscle cells

Vascular endothelial growth factor (VEGF) is a potent and specific mitogen of vascular endothelial cells which promotes neovascularization in vitro. To determine whether vasopressin induces VEGF secretion in human vascular smooth muscle cells, we performed enzyme-linked immunosorbent assays. Vasopressin potently induced a time-dependent and concentration-dependent (maximal, 10(-7) M) increase in VEGF secretion by human vascular smooth muscle cells that was maximal after 24 h. Furthermore, vasopressin also concentration-dependently caused mitogenic effect, as reflected by total protein content of cells per culture well. These vasopressin-induced VEGF secretion increase and mitogenic effect of these cells were potently inhibited by vasopressin V1A receptor antagonists, confirming this is a vasopressin V1A receptor-mediated event. These results indicate that vasopressin increases VEGF secretion in human vascular smooth muscle cells, the magnitude of VEGF secretion being temporally related to the mitogenic effect of vascular smooth muscle cells and the potency of the growth-promoting stimulus. Vasopressin-induced VEGF secretion by proliferating vascular smooth muscle cells could act as a paracrine hormone to powerfully influence the permeability and growth of the overlying vascular endothelium, vasopressin play a more fundamental role in the regulation of vascular function than has previously been recognized.

Effect of YM087, a potent nonpeptide vasopressin antagonist, on vasopressin-induced hyperplasia and hypertrophy of cultured vascular smooth-muscle cells

We investigated the effects of YM087, a potent nonpeptide V1A and V2 vasopressin (AVP)-receptor antagonist, in binding and functional studies on rat vascular smooth-muscle cells (VSMCs). V1A AVP receptors on VSMCs were characterized by using the radioligand [3H]AVP. Specific binding of [3H]AVP was time dependent, reversible, and saturable. A single class of high-affinity binding sites with the expected V1A profile was identified. YM087 showed high affinity for V1A receptors with an inhibitory dissociation constant (Ki) value of 0.24 nM. In addition, YM087 potently and concentration-dependently inhibited AVP-induced increase in intracellular free calcium concentration and activation of mitogen-activated protein kinase. When added to growth-arrested VSMCs, AVP concentration-dependently induced hyperplasia and hypertrophy. YM087 prevented AVP-induced hyperplasia and hypertrophy of these cells in a concentration-dependent manner. YM087 had no agonistic activity in any biological assays used. These results suggest that YM087 displays high affinity for V1A receptors on VSMCs and high potency in inhibiting the AVP-induced physiological response. YM087 is a potent pharmacologic probe for investigating the physiologic and pathophysiologic roles of AVP in several diseases.

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.

N-Methylbenzanilide derivatives as a novel class of selective V1A receptor antagonists

During our efforts to develop a novel class of selective V(1A) receptor antagonists, the N-methylbenzanilide structure was applied to a 4,4-difluoro-1-benzazepine derivative, 4, which is a selective V(1A) receptor antagonist. Further structural modifications gave 16a with high V(1A) affinity and V(2)/V(1A) selectivity (K(i)=5.71 nM, V(2)/V(1A)=140) and potent V(1A) receptor antagonist activity (ID(50)=0.0080 mg/kg iv).

Characterization of rodent liver and kidney AVP receptors: pharmacologic evidence for species differences

Radioligand binding studies with [3H]vasopressin (AVP) were used to determine the affinities of AVP receptor agonists and antagonists for mouse liver and kidney plasma membrane preparations. Both membrane preparations exhibited one class of high-affinity binding site. AVP ligand binding inhibition studies confirmed that mouse liver binding sites belong to the V1A subtype while kidney binding sites belong to the V2 receptor subtype. The affinity of each ligand for mouse V1A receptors was very similar to that for rat V1A receptors, showing differences in Ki values of less than 3-fold. In contrast, several peptide (d(CH2)5Tyr(Me)AVP) and nonpeptide (OPC-21268 and SR 49059) ligands had different affinities for mouse and rat kidney V2 receptors, with differences in Ki values ranging from 14- to 17-fold. These results indicate that mouse and rat kidney V2 receptors show significant pharmacologic differences.

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.

AVP-induced mitogenic responses of Chinese hamster ovary cells expressing human V1A or V1B receptors.

Abstract Arginine vasopressin (AVP) induces cell proliferation and hypertrophy; however, the human receptor subtype and the intracellular signaling pathways responsible for this mitogenic activity remain unclear. Experiments were conducted to determine which AVP receptor is linked to mitogen-activated protein (MAP) kinase activation and the mitogenic effect seen in Chinese hamster ovary (CHO) cells expressing human V1A or V1B receptors. Adding AVP to CHO cells transfected with human V1A or V1B cDNA significantly and concentration-dependently induced activation of MAP kinase and increased DNA synthesis, as measured by [3H]thymidine incorporation. These effects were inhibited by AVP receptor antagonists and the potency order of antagonists in vitro was similar to that observed in radioligand binding assays. These results suggest that AVP induces the MAP kinase cascade leading to cell proliferation through either human V1A or V1B receptors, and that these cloned, expressed AVP receptors may prove an invaluable tool for probing the physiologic and pathophysiologic effects of AVP.