Junko Tsukada

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.

Vasopressin increases type IV collagen production through the induction of transforming growth factor-beta secretion in rat mesangial cells

Production of extracellular matrix proteins, such as type IV collagen, by mesangial cells contributes to progressive glomerulosclerosis. Transforming growth factor-beta (TGF-beta) modulates mesangial cell growth and stimulates extracellular matrix synthesis by mesangial cells. In this study, the ability of vasopressin (AVP), which causes mesangial cell proliferation and hypertrophy, to stimulate type IV collagen production and correlation with TGF-beta secretion by cultured rat mesangial cells was examined. AVP induced a time- and concentration-dependent increase in TGF-beta secretion and mitogenic effect in rat mesangial cells. This AVP-induced increase in TGF-beta secretion was potently inhibited by AVP V(1A) receptor-selective antagonist. AVP also induced a concentration-dependent increase in the production of type IV collagen and this effect was inhibited by V(1A) receptor-selective antagonist. Furthermore, TGF-beta also induced an increase in the production of type IV collagen; the AVP-enhanced production of type IV collagen was inhibited by an anti-TGF-beta antibody. These results demonstrate that AVP stimulates synthesis of type IV collagen by cultured rat mesangial cells through the induction of TGF-beta synthesis mediated by V(1A) receptors. Therefore, AVP-induced TGF-beta secretion by proliferating mesangial cells might act as an autocrine factor to regulate synthesis of extracellular matrix; this mechanism may contribute to glomerulosclerosis in renal diseases including diabetic nephropathy.

PHARMACOLOGICAL PROFILE OF ORALLY ADMINISTERED YM087, A VASOPRESSIN ANTAGONIST, IN CONSCIOUS RATS

1. YM087 is a newly synthesized non‐peptide arginine vasopressin (AVP) antagonist that shows high affinity for both V1A and V2 receptors. In the present study, the V1A and V2 receptor antagonist effects of orally administered YM087 were assessed in conscious rats.

VASOPRESSIN STIMULATES THE PRODUCTION OF EXTRACELLULAR MATRIX BY CULTURED RAT MESANGIAL CELLS

1 Mesangial expansion, an indicator of chronic glomerular diseases, occurs as a result of the excessive accumulation of extracellular matrix (ECM) proteins, such as type IV collagen. In order to investigate the ability of vasopressin (AVP), which causes mesangial cell proliferation and hypertrophy, to induce ECM production, an enzyme‐linked immunosorbent assay was used to measure type I and IV collagen and fibronectin produced from cultured rat mesangial cells. 2 Addition of AVP (0.01–1000 nmol/L) caused a significant and concentration‐dependent production of secreted and cell‐associated ECM, type I collagen, type IV collagen and fibronectin by cultured rat mesangial cells. The AVP V1A receptor‐selective antagonist YM218 (0.01–1000 nmol/L) potently and concentration‐dependently inhibited the induced increase in ECM production caused by AVP, but the V2 receptor‐selective antagonist SR 121463A (0.1–1000 nmol/L) did not potently inhibit. 3 Vasopressin inhibited the synthesis of matrix metalloproteinase (MMP)‐2, which degrades matrix proteins, including type IV collagen, and stimulated endothelin (ET)‐1 secretion from mesangial cells. These effects were potently inhibited by YM218, but not by SR 121463A. 4 In addition, 10 nmol/L ET‐1 inhibited the synthesis of MMP‐2 and stimulated ECM production in mesangial cells. These effects were completely abolished by the ETA receptor‐selective antagonist YM598 (1 mmol/L); however, the ETB receptor‐selective antagonist BQ‐788 (1 mmol/L) and the AVP receptor antagonists YM218 and SR 121463A did not inhibit ET‐1‐induced inhibition of MMP‐2 synthesis and ECM production. In addition, AVP‐induced inhibition of MMP‐2 synthesis and ECM production were partly inhibited by YM598. 5 These findings indicate that AVP may modulate ECM production not only via a direct action on V1A receptors, but also through stimulation of ET‐1 secretion. Vasopressin may contribute to the glomerular remodelling and ECM accumulation observed in glomerular diseases.

Vasopressin induces human mesangial cell growth via induction of vascular endothelial growth factor secretion

Vasoactive hormones, growth factors, and cytokines are important in promoting mesangial cell growth, a characteristic feature of many glomerular diseases. Vascular endothelial growth factor (VEGF) is an endothelial mitogen and promoter of vascular permeability that is constitutively expressed in human glomeruli, but its role in the kidney is still unclear. In the present study, we investigated the ability of vasopressin (AVP) to stimulate VEGF secretion by and correlation with AVP-induced cell growth in human mesangial cells. AVP caused time- and concentration-dependent increases in VEGF secretion from human mesangial cells, which was in turn potently inhibited by a V(1A) receptor-selective antagonist, confirming that this secretion is a V(1A) receptor-mediated event. VEGF also induced mesangial cell growth which was completely inhibited on administration of an anti-VEGF neutralizing antibody. Further, AVP-induced mesangial cell growth was completely abolished by the V(1A) receptor-selective antagonist and partially inhibited by an anti-VEGF neutralizing antibody. These results suggest that AVP stimulates VEGF secretion by human mesangial cells via V(1A) receptors. This secreted VEGF may function as an autocrine hormone to regulate mesangial cell growth, a mechanism by which AVP might contribute to progressive glomerular diseases such as diabetic nephropathy.

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.