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SUMMARY In the present study we examined the effects of high extracellular glucose concentrations on vasopressin (AVP) V(1A) receptor kinetics and signal transduction in cultured rat mesangial cells. Scatchard analysis of [(3) H]-AVP binding to mesangial cell plasma membranes showed that although high glucose (30 mmol/L) decreased V(1A) receptor numbers relative to cells cultured in normal glucose (10 mmol/L), receptor affinity was not affected. This V(1A) receptor downregulation was associated with an attenuated increase in AVP-stimulated cytosolic free calcium concentrations ([Ca(2+) ](i) ). In addition, high glucose increased both the basal and AVP-stimulated activity of the classic mitogen-activated protein kinase, namely extracellular signal-regulated kinase (ERK). Furthermore, high glucose induced activation of protein kinase C (PKC) in mesangial cells that could be inhibited by coincubation with the PKC inhibitor staurosporine (10 nmol/L). Staurosporine also markedly attenuated the high glucose-induced downregulation of V(1A) receptors on mesangial cells and blocked the depressed [Ca(2+) ](i) response and increased ERK activity induced by AVP. The results indicate that high extracellular glucose downregulates V(1A) receptors on rat mesangial cells and modulates their signal transduction properties via PKC activation.

A series of (4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepine-5-ylidene)acetamide derivatives were optimized to achieve potent agonistic activity, both in vitro and in vivo, for the arginine vasopressin V(2) receptor, resulting in the eventual discovery of compound 1g. Molecular modeling of compound 1g with V(2) receptor was also examined to evaluate the binding mode of this series of compounds.

A series of (4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepin-5-ylidene)acetamide derivatives was synthesized, and their structure-activity relationships were examined in order to identify potent and selective arginine vasopressin V(2) receptor agonists. Attempts to substitute other chemical groups in place of the 2-pyridilmethyl moiety of 1a led to the discovery that potent V(2) binding affinity could be obtained with a wide range of functional groups. This structural tolerance allowed for the manipulation of other attributes, such as selectivity against V(1a) receptor affinity or avoidance of the undesirable inhibition of cytochrome P450 (CYP), without losing potent affinity for the V(2) receptor. Some representative compounds obtained in this study were also found to decrease urine volume in awake rats.

SUMMARY 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.

Production of extracellular matrix proteins, such as type IV collagen and fibronectin, by mesangial cells contributes to progressive glomerulosclerosis. In this study, the ability of vasopressin (AVP), which causes mesangial cell proliferation and hypertrophy, to stimulate type IV collagen production by cultured human mesangial cells was examined using an enzyme-linked immunosorbent assay. AVP induced a concentration-dependent increase in the production of type IV collagen and this effect was potently and concentration-dependently inhibited by AVP V1A receptor antagonists, including YM218. AVP also induced a concentration-dependent increase in transforming growth factor (TGF)-beta secretion by human mesangial cells and this effect was inhibited by V1A receptor antagonists. 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 findings indicate that AVP stimulates synthesis of type IV collagen by cultured human mesangial cells through the induction of TGF-beta synthesis mediated by V1A receptors; consequently, AVP contributes to glomerular remodeling and extracellular matrix accumulation observed in glomerular diseases.

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.

Mesangial cell growth constitutes a key feature of progressive glomerular injury. Vasopressin (AVP), a potent peptide vasoconstrictor, acts on mesangial cells through the V 1A receptors, inducing contraction and cell proliferation. This study examined the effects of YM218, a nonpeptide AVP V 1A receptor-selective antagonist, on the mitogenic and hypertrophic effects of AVP in rat mesangial cells. When added to mesangial cells whose growth was arrested, AVP concentration-dependently induced hyperplasia and hypertrophy. YM218 potently prevented AVP-induced hyperplasia and hypertrophy of these cells. Furthermore, AVP stimulated endothelin (ET)-1 secretion from mesangial cells in a concentration-dependent manner and this effect was potently inhibited by YM218. ET-1 also induced hyperplasia and hypertrophy in mesangial cells and this effect was completely abolished by ET A receptor-selective antagonist YM598. In addition, AVP-induced hyperplasia and hypertrophy were partly inhibited by YM598. These results suggest that AVP may modulate mesangial cell growth not only by its direct action but also through the stimulation of ET-1 secretion. YM218 displays high potency in inhibiting the AVP-induced physiologic responses of mesangial cells via the V 1A receptors and is a potent pharmacologic probe for investigating the physiologic and pathophysiologic roles of AVP in several renal diseases.

Mesangial cells are centrally-located glomerular pericytes with contractile, endocrine, and immunity-regulating functions. These cells are thought to maintain normal glomerular function, since mesangial cell proliferation and extracellular matrix formation are hallmarks of chronic glomerular disease. Vasopressin causes mesangial cell contraction, proliferation and hypertrophy. Consequently, the effects of YM218, a potent, nonpeptide vasopressin V(1A) receptor-selective antagonist, on the growth responses of human mesangial cells to vasopressin were investigated. YM218 showed high affinity for vasopressin V(1A) receptors, exhibiting a K(i) value of 0.18 nM. Vasopressin concentration-dependently increased intracellular Ca(2+) levels and induced hyperplasia and hypertrophy in cultured mesangial cells, YM218 potently inhibited these vasopressin-induced responses. These results clearly show that YM218 has both strong affinity for human mesangial cell vasopressin V(1A) receptors and great potency in inhibiting the vasopressin-induced growth responses of mesangial cells controlled by the vasopressin V(1A) receptors. The hyperplasia and hypertrophy of mesangial cells in vitro caused by vasopressin indicate its possible in vivo role in glomerular disease pathogenesis. Therefore, YM218 is a potent pharmacologic probe to investigate the physiologic and pathophysiologic roles of vasopressin in the development of renal disease.

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.

The present experiment was designed to investigate the role of peripheral dopamine D1-like receptors and to evaluate the prophylactic effect of zelandopam, a dopamine D1-like receptor agonist, on puromycin aminonucleoside (PA)-induced nephrosis in rats. Rats were divided into six groups (n=10 per group): 0.9% saline-injected rats (control); PA-injected rats (PAN); PA-injected rats treated with the selective dopamine D1-like receptor agonist zelandopam (30, 100, 300 mg/kg p.o. twice a day); PA-injected rats treated with prednisolone (1 mg/kg p.o. once a day). Nephrosis was induced in rats with a single intravenous injection of PA at a dose of 50 mg/kg. The effects of zelandopam and prednisolone in PA nephrosis rats were evaluated before injection of PA and at 7 and 14 days after injection. PA-induced nephrosis was characterized by an increase in urinary protein excretion (proteinuria) and plasma total cholesterol. Zelandopam dose-dependently attenuated the increase in proteinuria and total cholesterol. Prednisolone significantly attenuated the increase in proteinuria and total cholesterol and resulted in a significant decrease in body weight. The present study demonstrates for the first time that zelandopam, a selective dopamine D1-like receptor agonist, is effective in blunting the development of PA-induced nephrosis, and that the effects of zelandopam are dose dependent.

To find a new series of arginine vasopressin (AVP) V1A receptor antagonists, the influence of the 2-phenyl group of 2-phenyl-4'-[(2,3,4,5-tetrahydro-1H-1-benzazepin-1-yl)carbonyl]benzanilide (7) was investigated. Replacement of the 2-phenyl group by a 2-ethyl-1H-imidazol-1-yl group was effective in yielding a V1A-selective compound. Moreover, this imidazolyl group was introduced in the same position in YM-35471 (6), and further studies of these compounds were performed. Consequently, we found that the (Z)-4'-({4,4-difluoro-5-[(N-cyclopropylcarbamoyl)methylene]-2,3,4,5-tetrahydro-1H-1-benzazepin-1-yl}carbonyl)-2-(2-ethyl-1H-1-imidazol-1-yl)benzanilide (9f) exhibited highly potent affinity and selectivity, and was the most potent antagonist for the V1A receptor among our compounds. The synthesis and pharmacological evaluation of these compounds are described in this paper.

Several factors such as proteinuria and renal fibrosis may be important in the progression of many forms of chronic renal diseases. The purposes of the current study were to investigate the progressive renal failure of the rats with surgical renal mass reduction (RMR) and the effect of the angiotensin-converting enzyme (ACE) inhibitor, lisinopril, and to document correlation of several factors associated with progressive renal failure. Rats were subtotal (5/6) nephrectomized by resection of the renal poles and sham-operated. The functional, histological and haematological changes of the rats were studied for up to 10 weeks. After 2 weeks of RMR, oral administration of lisinopril (10 mg kg −1 per day) was performed for 8 weeks. RMR resulted in progressive renal failure with proteinuria, monocyte/macrophage (ED1 + ) infiltration, anaemia as assessed by haemoglobin and haematocrit (Htc), renal hypertrophy as assessed by left kidney to body weight ratio (BKW/BW), and renal fibrosis as assessed by glomerular lesions and tubulointerstitial changes. Lisinopril exhibited renoprotection with antiproteinuric effect and inhibition of monocyte/macrophage (ED1 + ) infiltration. However, beneficial effect of lisinopril on anaemia was not observed. At 10 weeks after surgery, severity of proteinuria positively correlated with plasma creatinine (Pcr), BKW/BW, histological damage, and systolic blood pressure, and negatively correlated with haemoglobin. Severity of tubulointerstitial changes positively correlated with Pcr and blood urea nitrogen, and negatively correlated with haemoglobin and Htc. Moreover, monocyte/macrophage (ED1 + ) infiltration positively correlated with severity of proteinuria and tubulointerstitial changes. These findings strongly support that proteinuria, monocyte/macrophage infiltration and renal fibrosis appear to play principal roles in the progressive renal failure with anaemia and renoprotection of ACE inhibition may be mediated by multiple actions of ACE inhibitor. The present study confirms that rats with RMR is useful to explore target molecules for renoprotective drugs and evaluate renoprotective effect of new molecular entities.

To elucidate the role of peripheral dopamine D1 receptors in cisplatin-induced acute renal injury, effect of zelandopam (YM435, (−)-(S)-4-(3,4-dihydroxyphenyl)-7,8-dihydroxy-1,2,3,4-tetrahydroisoquinoline hydrochloride hydrate), a selective renal dopamine D1 receptor agonist, on cisplatin-induced acute renal failure in rats was studied. Rats were divided into six groups: control, cisplatin and cisplatin plus zelandopam (30, 100, 300 mg/kg p.o. twice, 75 and 15 min before cisplatin injection) or the free radical scavenger CV-3611 (2-O-octadecylascorbic acid, 10 mg/kg p.o., 75 min before cisplatin injection) treated groups. Rats received intraperitoneal injection of cisplatin at a dose of 5 mg/kg. Four days after cisplatin injection, plasma creatinine, blood urea nitrogen and body weight were measured and the kidneys were removed for histological examination. Cisplatin induced acute renal failure characterized by the increases in plasma creatinine and blood urea nitrogen with tubular damage, and decreased body weight. Zelandopam dose-dependently prevented all these changes. The free radical scavenger CV-3611 significantly attenuated a decrease in body weight and renal dysfunction without reducing tubular damage. The present study is the first demonstration for that a selective dopamine D1 receptor agonist is effective in preventing acute renal failure induced by cisplatin.

To elucidate the role of arginine vasopressin (AVP) in a hypertensive state, the characteristics of renal cortex V1A 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 V1A receptors (Bmax) was statistically significantly higher than that of WKY rats (39.7 ± 2.7 vs. 22.4 ± 0.9 fmol/mg protein). Furthermore, the Bmax 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 (Kd) values of renal cortex V1A and medulla V2 receptors in SHR and WKY rats were not significantly different. These results indicate that increased amounts of renal cortex V1A and medulla V2 receptors in SHR play an important role in the pathophysiology of hypertension.

Objective To investigate the effects of YM471, a non-peptide arginine vasopressin (AVP) VIA and V 2 receptor antagonist, on the AVP-induced growth responses in human vascular smooth muscle cells (VSMCs). Methods Binding of YM471 to VIA receptors on VSMCs was measured using [ 3 H]AVP. Intracellular free Ca 2+ 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- 32 P]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 [ 3 H]AVP to VIA receptors on VSMCs, exhibiting an inhibition constant (K i ) of 0.35 nmol/l. YM471 inhibited the AVP-induced increase in intracellular free Ca 2+ concentration with an 50% inhibition concentration (IC 50 ) of 2.01 nmol/l and inhibited the activation of MAP kinase with an IC 50 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 IC 50 values of 2.31 and 0.23 nmol/l, respectively. Conclusions These results indicate that YM471 has high affinity for VIA receptors on, and potently inhibits AVP-induced physiologic responses of, human VSMCs.

Myocardial infarction often induces congestive heart failure accompanied by a significant increase in plasma vasopressin concentration. To delineate the role of vasopressin in the pathogenesis of congestive heart failure, the acute hemodynamic and aquaretic effects of conivaptan (YM087, 4'-(2-methyl-1,4,5,6-tetrahydroimidazo[4,5-d][1]benzoazepine-6-carbonyl)-2-phenylbenzanilide monohydrochloride), a combined vasopressin V(1A) and V(2) receptor antagonist, were assessed in rats with heart failure induced by myocardial infarction. Left coronary artery ligation resulted in decreased left ventricular systolic pressure and first derivatives of left ventricular developed pressure, as well as increased left ventricular end-diastolic pressure, lung and right ventricular weight. Single oral administration of conivaptan (0.3 to 3.0 mg/kg) dose-dependently increased urine volume and decreased urine osmolality in heart failure rats. Furthermore, conivaptan (3.0 mg/kg) attenuated the changes in left ventricular end-diastolic pressure, lung and right ventricular weight induced by heart failure while reducing blood pressure. These results show that vasopressin plays a significant role in elevating vascular tone through vasopressin V(1A) receptors and plays a major role in retaining free water through vasopressin V(2) receptors in this model of congestive heart failure. Additionally, conivaptan, with its dual vasopressin V(1A) and V(2) receptor-inhibiting properties, could exert a beneficial effect on cardiac function in the congestive heart failure rat model.

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. British Journal of Pharmacology (2001) 133, 746–754; doi:10.1038/sj.bjp.0704117

[(3)H]-oxytocin was used to characterize the oxytocin receptor found in human uterine smooth muscle cells (USMC). Specific binding of [(3)H]-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 (K(d)) of 0.76 nM and a maximum receptor density (B(max)) of 153 fmol mg(-1) protein. The Hill coefficient (n(H)) did not differ significantly from unity, suggesting binding to homogenous, non-interacting receptor populations. Competitive inhibition of [(3)H]-oxytocin binding showed that oxytocin and vasopressin (AVP) receptor agonists and antagonists displaced [(3)H]-oxytocin in a concentration-dependent manner. The order of potencies for peptide agonists and antagonists was: oxytocin>[Asu(1,6)]-oxytocin>AVP= atosiban>d(CH(2))(5)Tyr(Me)AVP>[Thr(4),Gly(7)]-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 Ca(2+) concentration ([Ca(2+)](i)) and hyperplasia in USMC. The oxytocin receptor antagonists, atosiban and L-371257, potently and concentration-dependently inhibited oxytocin-induced [Ca(2+)](i) increase and hyperplasia. In contrast, the V(1A) receptor selective antagonist, SR 49059, and the V(2) receptor selective antagonist, SR 121463A, did not potently inhibit oxytocin-induced [Ca(2+)](i) increase and hyperplasia. The potency order of antagonists in inhibiting oxytocin-induced [Ca(2+)](i) increase and hyperplasia was similar to that observed in radioligand binding assays. In conclusion, these data provide evidence that the high-affinity [(3)H]-oxytocin binding site found in human USMC is a functional oxytocin receptor coupled to [Ca(2+)](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. British Journal of Pharmacology (2000) 129, 131 - 139

Several studies indicate that oxytocin and vasopressin receptors in the human uterus are heterogeneous. We have investigated whether oxytocin and vasopressin bind to separate receptors or one class of receptors in human uterine smooth muscle cells. [ 3 H ]d(CH2)5Tyr(Me)AVP, the vasopressin V1A receptor selective radioligand, was used for comparison of vasopressin binding sites in human uterine and vascular smooth muscle cell membranes. Both membrane preparations exhibited one class of high-affinity binding sites with Kd values of 6.44 and 0.47 nM, Bmax values of 166 and 34.8 fmol/mg protein for uterine and vascular smooth muscle cells, respectively. In vascular preparations, the selective vasopressin V1A receptor antagonist, SR 49059 ((2S) 1-[(2R 3S)-(5-chloro-3-(2-chlorophenyl)-1-(3,4-dimethoxybenzenesulfonyl)-3-hydroxy-2,3-dihydro-1H-indole-2-carbonyl]-pyrrolidine-2-carboxamide), showed high affinity with Ki value of 0.98 nM, confirming that these receptors belong to the vasopressin V1A receptor subtype. On the contrary, in uterine preparations, binding of [ 3 H ]d(CH2)5Tyr(Me)AVP was more effectively displaced by oxytocin and the oxytocin receptor selective antagonist, L-371257, (1-{1-[4-[N-Acetyl-4-piperidinyl)oxy]2-methoxybenzoyl]piperidin-4-yl}-4H-3,1-benzoxazin-2(1H)-one), than vasopressin and SR 49059, suggesting that binding may be due to cross-reaction with the oxytocin receptors. These results suggest that human uterine smooth muscle cells express only a high density of oxytocin receptors.

Pharmacology of conivaptan hydrochloride (YM087) was investigated in in vitro and in vivo studies. In radioligand binding study, YM087 showed high affinity for both V1A and V2 receptors in animal and human species. Affinity of YM087 for V1A and V2 receptors was comparable to that of vasopressin (AVP). In functional antagonistic activity study, YM087 concentration-dependently inhibited AVP-induced intracellular Ca2+ elevation via human V1A receptors and AVP-stimulated cAMP accumulation via human V2 receptors. Intravenous administration of YM087 dose-dependently inhibited AVP-induced pressor responses and produced a dose-dependent aquaresis in rats and dogs. Oral administration of YM087 showed a potent and long-lasting antagonistic activity on V1A and V2 receptors. YM087 was effective in dogs with heart failure and in heart failure rats with hyponatremia and edema. These results reveal that YM087 is the first orally active V1A/V2 receptor antagonist and suggest that YM087 may be useful in the treatment of congestive heart failure and hyponatremia.

Arginine vasopressin (AVP) has a dual action, i.e. vasoconstriction and water reabsorption via V1A and V2 receptors, and may play a role in a number of diseases, including congestive heart failure (CHF), hypertension, renal disease, edema, and hyponatremia. We have attempted to develop a new series of AVP antagonists for both V1A and V2 receptors based on the hypothesis that the blockade of both V1A and V2 receptors might be beneficial to CHF patients. In this report, a series of compounds structurally related to 4'-[5-(substituted methylidene)-2,3,4,5-tetrahydro-1H-1-benzoazepine-1-carbonyl ]benzanilide (exo-olefin isomer) and 4'-[5-(substituted methyl)-2,3-dihydro-1H-1-benzoazepine-1-carbonyl]benzanilide (endo-olefin isomer) were synthesized and examined to have AVP antagonist activity for both V1A and V2 receptors. As a result, it was found that the (E)-exo-olefin isomers showed more potent binding affinity compared with endo-olefin isomers. Among these (E)-exo-olefin isomers, (E)-N-methyl-{1-[4-(2-methylbenzoylamino)benzoyl]-2,3,4,5-tetrahyd ro-1H-1 -benzoazepin-5-ylidene}acetamide (14) exhibited the most potent binding affinity and (E)-N-methyl-(1-{4-[2-(4-methylphenyl)benzoylamino]benzoyl}-2,3,4,5- tetrahydro-1H-1-benzoazepin-5-ylidene)acetamide (20) exhibited a high AVP antagonist activity for both V1A and V2 receptors after intravenous administration. Details of the synthesis and pharmacological properties of this series are presented.

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. 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. 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. 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. 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.

The binding characteristics of YM087, a nonpeptide vasopressin (AVP) V 1A and V 2 receptor antagonist, were studied using 3 H-AVP binding to rhesus monkey liver and kidney membrane preparations. Both membrane preparations exhibited one class of high-affinity binding sites. However each membrane’s receptors were different, with K d values of 0.57 and 1.11 nM, B max values of 59.6 and 147 fmol/mg protein for liver and kidney, respectively. AVP receptor agonist or antagonist binding inhibition studies confirmed that these receptors belong to the V 1A (liver) and V 2 (kidney) subtypes. YM087 showed high affinity for both liver V 1A and kidney V 2 receptors with K i values of 26.3 and 9.89 nM, respectively. These results show that YM087 is a potent, nonpeptide dual AVP V 1A and V 2 receptor antagonist, and would be a powerful tool for understanding the physiologic roles of AVP.

A series of compounds structurally related to 2-phenyl-4'-(2,3,4,5-tetrahydro-1H-1,5-benzodiazepine-1-carbonyl) benzanilide was synthesized and demonstrated to have arginine vasopressin (AVP) antagonist activity for both V1A and V2 receptors. The introduction of a hydrophilic substituent group into the 5-position of the benzodiazepine ring resulted in an increase in oral availability. Especially, the (3-pyridyl)methyl (31b), the 2-(4-methyl-1,4-diazepan-1-yl)-2-oxoethyl (32i), and the 2-(4-methylpiperazin-1-yl)ethyl (33g) derivatives exhibited high antagonist activities and high oral availability. Details of the synthesis and pharmacological properties of this series are presented.

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.

1. 1. The renal vasodilatory effect of YM435 was used as an index of its dopamine DA1 receptor agonist activity and compared with that of dopamine in pentobarbital-anesthetized dogs. 2. 2. Intrarenal arterial administration of YM435 (0.1 to 10 μg) and dopamine (1 to 100 μg) produced a dose-dependent increase in renal blood flow. The doses of YM435 and dopamine required to cause a 30-ml/min increase in renal blood flow were 2.0 and 26.8 μg intra-arterially (IA), respectively. YM435 was therefore 13 times more potent than dopamine in this effect. 3. 3. The selective dopamine DA1 receptor antagonist, SCH 23390, but not the selective dopamine DA2 receptor antagonist, nemonapride, caused dose-dependent, parallel shifts to the right in the doseresponse curve of YM435. 4. 4. The present results demonstrate that YM435 is a potent and selective dopamine DA1 receptor agonist.

The cardiovascular effects of YM435, a dopamine DA1 receptor agonist, were compared with those of dopamine in open-chest anesthetized dogs. Intravenous infusion of YM435 (0.1-3 microg/kg/min) increased renal blood flow and cardiac output and reduced renal vascular resistance and total peripheral vascular resistance, with a decrease in mean blood pressure, in a dose-dependent manner, with little change in heart rate. At 1 microg/kg/min i.v., renal blood flow increased by 20 +/- 7%, cardiac output increased by 14 +/- 6%, renal vascular resistance decreased by 22 +/- 4%, total peripheral vascular resistance decreased by 18 +/- 4%, and mean blood pressure decreased by 7 +/- 1%. The striking difference between the cardiovascular effects of YM435 and those of dopamine was that YM435 caused no vasoconstriction or increase in heart rate, even at high doses. The cardiovascular effects of YM435 (1 microg/kg/min i.v.) were almost completely inhibited by treatment with SCH 23390, a selective dopamine DA1 receptor antagonist. Furthermore, intravenous infusion of YM435 (0.1-3 microg/kg/min) dose-dependently reversed the increase in blood pressure and renal vascular resistance induced by angiotensin II or norepinephrine in closed-chest anesthetized dogs. Our results demonstrate that intravenous infusion of YM435 produces dose-dependent renal vasodilating and hypotensive effects by stimulation of dopamine DA1 receptors and suggest that YM435 may be useful for the parenteral treatment of acute elevation of blood pressure.

Hyperglycemia is a principal characteristic of diabetes and influences many cellular functions. Diabetic nephropathy is characterized by glomerular mesangial expansion which could result from increased mesangial cell extracellular matrix synthesis induced by hyperglycemia.To investigate whether the physiological functions of mesangial cells are altered in a diabetic environment, we evaluated the effect of high extracellular glucose concentration on thymidine/leucine incorporation, hyperplasia/hypertrophy, and type IV collagen synthesis, induced by vasopressin (AVP), in cultured rat mesangial cells.The exposure of mesangial cells to a high glucose concentration (30 mM) significantly reduced AVP-induced thymidine incorporation and hyperplasia compared with normal glucose (10 mM). By contrast, treatment of mesangial cells with AVP in the presence of high extracellular glucose significantly increased leucine incorporation, hypertrophy, and type IV collagen synthesis compared with those at normal glucose levels. The administration of staurosporine, a protein kinase C inhibitor, reversed these effects of high-glucose conditions. Furthermore, the nonpeptide AVP V(1A) receptor-selective antagonists potently inhibited these AVP-induced physiological responses in mesangial cells cultured in high-glucose conditions.These results demonstrate that high glucose suppresses mesangial cell proliferation but enhances hypertrophy and type IV collagen synthesis induced by AVP. This increased mesangial cell hypertrophy and extracellular matrix synthesis may play a crucial role in the glomerular mesangial expansion common to diabetic nephropathy.

Arginine vasopressin (AVP) has a dual action mainly in the periphery, i.e., vasoconstriction and water reabsorption via V1A and V2 receptors; it may play a role in a number of diseases, including congestive heart failure (CHF), hypertension, renal disease, edema, and hyponatremia. We have attempted to develop a new series of orally active AVP antagonists for both V1A and V2 receptors based on the hypothesis that the blockade of both V1A and V2 receptors might be beneficial to CHF patients. In this report, a series of compounds structurally related to 4'-(1,4,5,6-tetrahydroimidazo[4,5-d][1]benzoazepine-6- carbonyl)benzanilide and 4'-(5,6-dihydro-4H- thiazolo[5,4-d][1]benzoazepine-6-carbonyl)benzanilide were synthesized and examined for AVP antagonist activity for both V1A and V2 receptors. As a result, it was found that the 4'-(1,4,5,6-tetrahydroimidazo[4,5-d][1]benzoazepine-6-carbon yl)-2- phenylbenzanilide derivatives showed potent binding affinity for both V1A and V2 receptors. Especially, 4'-(2-methyl-1,4,5,6- tetrahydroimidazo[4,5-d][1]benzoazepine-6-carbonyl)-2-phe nylbenzanilide monohydrochloride (18, YM087 = conivaptan hydrochloride) exhibited potent binding affinity and AVP antagonist activity, after intravenous administration, for both V1A and V2 receptors. Furthermore, YM087 exhibited the most potent oral activity for the V2 receptor. Details of the synthesis and pharmacological properties of this series are presented.

7,8-Dihydroxy-4-(3,4-dihydroxyphenyl)-1,2,3,4-tetrahydroisoquinoline (1) and 4-(3,4-dihydroxyphenyl)-7-hydroxy-8-methyl-1,2,3, 4-tetrahydroisoquinoline (2) are potent renal vasodilators which selectively stimulate DA1 (peripheral dopamine receptor-1) receptors. Especially, (S)-(-)-1 is the most potent. Its DA1 agonist activity is about 10 times stronger than dopamine for increasing renal blood flow in anesthetized dogs. The renal and cardiovascular effects of (S)-(-)-1 may be suitable for the treatment of patients with renal insufficiency, heart failure and hypertension.

The present work describes the discovery of novel series of (4,4-difluoro-1,2,3,4-tetrahydro-5H-1-benzazepine-5-ylidene)acetamide derivatives as arginine vasopressin (AVP) V(2) receptor agonists. By replacing the amide juncture in YM-35278 with a direct ring connection gave compound 10a, which acts as a V(2) receptor agonist. These studies provided the potent, orally active non-peptidic V(2) receptor agonists 10a and 10j.

Vasopressin (AVP) causes mesangial cell contraction, proliferation and hypertrophy. The present study investigated the effects of YM218, a potent, nonpeptide AVP V(1A) receptor-selective antagonist, on rat mesangial cells using binding, signal transduction and cell growth assays. Specific binding of (3)H-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 V(1A) receptor profile. YM218 showed high affinity for V(1A) receptors, exhibiting a K(i) value of 0.19 nmol/l. AVP concentration-dependently increased intracellular Ca(2+) ([Ca(2+)](i)) levels, stimulated mitogen-activated protein (MAP) kinase and induced hyperplasia. Conversely, YM218 potently suppressed [Ca(2+)](i) elevation, activation of MAP kinase and hyperplasia induced by AVP. These results indicate that YM218 displays both high affinity for rat mesangial cell V(1A) 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.

To find potent and selective antagonists of the arginine vasopressin (AVP) V1A receptor, optimization studies of compounds structurally related to (Z)-N-{4′-[(4,4-difluoro-5-carbamoylmethylidene-2,3,4,5-tetrahydro-1H-1-benzazepin-1-yl)carbonyl]phenyl}carboxamide were performed. The synthesis and pharmacological properties of these compounds are described. We first investigated the effect of the carboxamide moiety, and found that a 2-methylfuran-3-carbonyl group at this position increased V1A binding affinity and selectivity for the V1A receptor versus the V2 receptor. The amino group of the 5-carbamoylmethylidene moiety was also examined, and a 4-piperidinopiperidino group was found to be optimal at this position. The hemifumarate of compound 12l (YM218) was shown to exhibit potent binding affinity, V1A receptor selectivity, and in vivo antagonist activity.

Using a continuous superfusion system of dog renal cortical slices, we studied the role of prostacyclin in the control of renin release. Superfusate renin activity and prostacyclin as 6-keto-prostaglandin F1alpha, a stable metabolite of prostacyclin, concentrations were measured by radioimmunoassay. Exogenous prostacyclin (0.1, 1, 10 microM) produced a concentration dependent and significant increase in renin release. The calcium ionophore A23187 (10 microM) produced a significant increase in 6-keto-prostaglandin F1alpha release and a significant decrease in renin release. A23187 (10 microM) hardly produced changes of 6-keto-prostaglandin F1alpha release and renin release in the absence of Ca2+. Pretreatment with indomethacin (10 microM) completely abolished the stimulatory effect of A23187 (10 microM) on 6-keto-prostaglandin F1alpha release. On the other hand, the inhibitory effect of A23187 on renin release in the pretreatment with indomethacin was almost equal to that in the "untreatment" with indomethacin. Moreover, we found that there was no association of 6-keto-prostaglandin F1alpha liberation and renin activity. These results indicate that exogenous prostacyclin promotes renin release, and suggest that renin release is not to be modulated by A23187-induced prostacyclin synthesis in dog renal cortical slices.

We investigated the effects of intravenously administered conivaptan hydrochloride, a dual vasopressin V1A and V2 receptor antagonist, on cardiac function in rats with congestive heart failure following myocardial infarction, and compared results with those for the selective vasopressin V2 receptor antagonist SR121463A. Rats were subjected to left coronary artery occlusion to induce myocardial infarction, which in turn led to congestive heart failure. At 4 weeks after coronary occlusion, conivaptan (0.03, 0.1 and 0.3 mg/kg i.v.) dose-dependently increased urine volume and reduced urine osmolality in both myocardial infarction and sham-operated rats. SR121463A (0.3 mg/kg i.v.) also increased urine volume and decreased urine osmolality in myocardial infarction rats, to a degree comparable to that by conivaptan (0.3 mg/kg i.v.). At 6 weeks after surgery, myocardial infarction rats showed increases in right ventricular systolic pressure, right atrial pressure, left ventricular end-diastolic pressure and relative weights of the heart and the lungs, and a decrease in first derivative of left ventricular pressure (dP/dt(max))/left ventricular pressure, showing that congestive heart failure was well established. Conivaptan (0.3 mg/kg i.v.) significantly reduced right ventricular systolic pressure, left ventricular end-diastolic pressure, lung/body weight and right atrial pressure in myocardial infarction rats. Moreover, conivaptan (0.3 mg/kg i.v.) significantly increased dP/dt(max)/left ventricular pressure. SR121463A at a dose of 0.3 mg/kg i.v. significantly decreased left ventricular end-diastolic pressure and right atrial pressure, and tended to decrease right ventricular systolic pressure and relative lung weight in myocardial infarction rats. Although the aquaretic and preload-reducing effects of SR121463A were similar to those of conivaptan, SR121463A failed to improve dP/dt(max)/left ventricular pressure. These results suggest that dual vasopressin V1A and V2 receptor antagonists provide greater benefit than selective vasopressin V2 receptor antagonists in the treatment of congestive heart failure.

A series of compounds structurally related to 4'-[(4,4-difluoro-5-methylidene-2,3,4,5-tetrahydro-1H-1-benzoazepin-1-yl)carbonyl]benzanilide were synthesized and evaluated for arginine vasopressin (AVP) antagonistic activity. Compounds with alkoxy groups (especially ethoxy group) at the 2'-position of benzanilide possessed potent affinity and selectivity for the V1A receptor versus V2 receptor. Further study has shown that the introduction of 4,4-dimethylaminopiperidino and morpholino groups at carbonylmethylene exhibited more potent affinity and selectivity for V1A receptors. Consequently, we found that the (Z)-4'-([4,4-Difluoro-5-[(4-dimethylaminopiperidino)carbonylmethylene]-2,3,4,5-tetrahydro-1H-1-benzoazepin-1-yl]carbonyl)-2-ethoxybenzanilide monohydrochloride (8d) and the (Z)-4'-[(4,4-Difluoro-5-morpholinocarbamoylethylene-2,3,4,5-tetrahydro-1H-1-benzoazepin-1-yl)carbonyl]-2-ethoxybenzanilide (8q) exhibited potent and selective V1A receptor antagonist activity. The synthesis and pharmacological properties of these compounds are detailed in this paper.

Using a continuous superfusion system of dog renal cortical slices, we studied the role of Ca(2+) in the intracellular control mechanism for renin release. The calcium ionophore A23187 (10 microM) produced a significant decrease in renin release. This effect was abolished in the absence of extracellular Ca(2+). Moreover, pretreatment with the calmodulin inhibitor W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide, 20 microM) completely prevented the inhibitory effect of A23187 (10 microM). The beta-adrenoceptor agonist isoproterenol (1, 10 and 100 microM) produced a concentration-dependent increase in renin release. Pretreatment with W-7 (20 microM) potentiated the stimulation of renin release induced by isoproterenol (1 microM). These results suggest that A23187-induced inhibition of renin release is mediated by the activation of calmodulin via an increase in intracellular Ca(2+) and beta-adrenoceptor-stimulated renin release is modulated by intracellular Ca(2+) mobilization.

The neurohormonal factor arginine vasopressin (AVP) produces potent systemic vasoconstriction as well as water retention in the kidneys via the V(1a) and V(2) receptors, respectively. Therefore, AVP may be considered as an aggravating factor of cardiac failure. In the present study, the effects of intravenous (i.v.) infusion of AVP on cardiovascular parameters and the effect of conivaptan (YM087, 4'-(2-methyl-1,4,5,6-tetrahydroimidazo[4,5-d][1]benzoazepine-6-carbonyl)-2-phenylbenzanilide monohydrochloride), a vasopressin V(1a)/V(2) receptor antagonist, on AVP-induced cardiac and haemodynamic changes were investigated in pentobarbitone-anaesthetised dogs. The i.v. infusion of AVP (0.12-4mUkg(-1)min(-1)) dose-dependently produced decreases in the cardiac contractility indicator LV dP/dt(max) and cardiac output (CO) and increases in left ventricular end-diastolic pressure (LVEDP) and total peripheral resistance (TPR). These changes accurately mimic the cardiovascular symptoms of congestive heart failure. The i.v. bolus injection of conivaptan (0.1mgkg(-1)) rapidly attenuated the AVP (4mUkg(-1)min(-1))-induced decrease in CO and reversed the AVP-induced elevation in both LVEDP and TPR. In conclusion, i.v. infusion of AVP produced cardiac dysfunction and vasoconstriction in pentobarbitone-anaesthetised dogs. Conivaptan demonstrated the ability to dramatically improve the impaired cardiovascular parameters induced by AVP. The results suggest the potential usefulness of conivaptan in treating congestive heart failure.

The antagonistic properties of YM471, a potent nonpeptide vasopressin (AVP) V1A and V2 receptor antagonist, were characterized using human coronary artery smooth muscle cells (CASMC). YM471 potently inhibited specific binding of 3 H -AVP to V1A receptors on human CASMC, exhibiting a Ki value of 0.49 nM. Furthermore, YM471 inhibited the AVP-induced increase in intracellular free Ca2+ concentration with an IC50 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 IC50 values of 0.93 and 2.64 nM, respectively. These results indicate that YM471 has high affinity for V1A receptors on, and high potency in inhibiting AVP-induced physiologic responses of, human CASMC.

The pharmacologic profile of 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), a novel potent vasopressin V(1A) and V(2) receptor antagonist, was investigated using several in vitro and in vivo techniques. YM471 showed high affinity for rat vasopressin V(1A) and V(2) receptors, exhibiting K(i) values of 0.16 and 0.77 nM, respectively. In contrast, YM471 exhibited much lower affinity for rat vasopressin V(1B) and oxytocin receptors, with K(i) values of 10.5 microM and 31.0 nM, respectively. In conscious rats, oral administration of YM471 (0.1-3.0 mg/kg) produced dose-dependent inhibition of the pressor response caused by exogenous vasopressin and increased urine excretion and decreased urine osmolality; this effect lasted more than 8 h. In all biological assays used, YM471 exhibited no agonistic activity. These results demonstrate that YM471 exerts potent and long-lasting antagonistic activity on both vasopressin V(1A) and V(2) receptors, and that this compound may be a useful tool for clarifying the physiologic and pathophysiologic roles of vasopressin and the therapeutic usefulness of the vasopressin receptor antagonist.

To elucidate the role of arginine vasopressin (AVP) in a hypertensive state, the characteristics of renal cortex V(1A) and medulla V(2) 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 (3)H-d(CH(2))(5)Tyr(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 (3)H-AVP binding to medullary V(2) 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 V(2) receptors in SHR and WKY rats were not significantly different. These results indicate that increased amounts of renal cortex V(1A) and medulla V(2) receptors in SHR play an important role in the pathophysiology of hypertension.

We examined the change in cardiac sympathetic function in the hibernating heart. To induce hibernating hearts in dogs, we placed a nylon tube via the carotid artery in the left circumflex artery (LCx) and obstructed the LCx flow. The plasma norepinephrine (NE) and epinephrine (E) concentrations in the coronary sinus and the aorta were measured before and 1 week after the tube placement to evaluate the catecholamine release from the heart. The wall motion was followed by echocardiography and. 1 week after the tube placement, regional myocardial blood flow (RBF) was measured using colored microspheres. Also. the restorability of myocardial dysfunction was examined in other dogs by extracting the LCx tube 1 week after the placement. Finally, the heart was removed for pathological observation and dogs showing myocardial infarction were excluded. One week after placing the tube, wall thickening was reduced in the LCx area, but was not in the left anterior descending (LAD) area. Compared with the LAD area, RBF in the LCx area was decreased in the endocardium (p < 0.05), but was not in the epicardium. In other dogs, the reduced wall thickening in the LCx area was restored to normal levels 1 or 2 weeks after the tube extraction. Thereby, our dogs with the tube placed were considered to be models of myocardial hibernation. The plasma NE and E concentrations were not significantly changed by placing the tube, but NE release from the heart was increased after the tube placement (p < 0.05). E uptake from the heart did not differ. Therefore, it is suggested that NE release is increased in the hibernating heart and may contribute to its mechanism.

A series of compounds structurally related to 4'-1(4,4-difluoro-5-methylidene-2,3,4,5-tetrahydro-1H-1.-benzoazepin-1-yl)carbonyl]-2-phenylbenzanilide were synthesized and evaluated for arginine vasopressin (AVP) antagonistic activity. Compounds with a (Z)-olefin geometry at the 5-position of benzoazepine possessed potent affinity for both the V 1A and V 2 receptors. Further study has shown that one of these derivatives, (Z)-4'-({4,4-difluoro-5-[(4-dimethylaminopiperidino)carbonylmethylenel-2,3,4,5-tetrahydro-1H-1-benzoazepin-1-yl}carbonyl))-2-phenylbenzanilide monohydrochloride (29, YM-35471), exhibits exceptionally potent affinity for both of VIA and V 2 receptors, even when administered orally. The synthesis and pharmacological properties of this compound are detailed in this paper.

Arginine vasopressin (AVP) has a dual action mainly in the periphery, i.e., vasoconstriction and water reabsorption via V1A and V2 receptors; it may play a role in a number of diseases, including congestive heart failure (CHF), hypertension, renal disease, edema, and hyponatremia. We have attempted to develop a new series of orally active AVP antagonists for both V1A and V2 receptors based on the hypothesis that the blockade of both V1A and V2 receptors might be beneficial to CHF patients. In this report, a series of compounds structurally related to 4'-(1,4,5,6-tetrahydroimidazo[4,5-d][1]benzoazepine-6- carbonyl)benzanilide and 4'-(5,6-dihydro-4H- thiazolo[5,4-d][1]benzoazepine-6-carbonyl)benzanilide were synthesized and examined for AVP antagonist activity for both V1A and V2 receptors. As a result, it was found that the 4'-(1,4,5,6-tetrahydroimidazo[4,5-d][1]benzoazepine-6-carbon yl)-2- phenylbenzanilide derivatives showed potent binding affinity for both V1A and V2 receptors. Especially, 4'-(2-methyl-1,4,5,6- tetrahydroimidazo[4,5-d][1]benzoazepine-6-carbonyl)-2-phe nylbenzanilide monohydrochloride (18, YM087 = conivaptan hydrochloride) exhibited potent binding affinity and AVP antagonist activity, after intravenous administration, for both V1A and V2 receptors. Furthermore, YM087 exhibited the most potent oral activity for the V2 receptor. Details of the synthesis and pharmacological properties of this series are presented.

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.