Toyoki Mori

Characterization of a novel aquaretic agent, OPC‐31260, as an orally effective, nonpeptide vasopressin V2 receptor antagonist

1 OPC‐31260, a benzazepine derivative, has been studied for its ability to antagonize the binding of arginine vasopressin (AVP) to receptors in rat liver (V1) and kidney (V2) plasma membranes, for antagonism of the antidiuretic action of AVP in alcohol‐anaesthetized rats and for diuretic action in conscious normal rats. 2 OPC‐31260 caused a competitive displacement of [3H]‐AVP binding to both V1 and V2 receptors with IC50 values of 1.2 ± 0.2 × 10−6 m and 1.4 ± 0.2 × 10−8 m, respectively. 3 OPC‐31260 at doses of 10 to 100 μg kg−1, i.v., inhibited the antidiuretic action of exogenously administered AVP in water‐loaded, alcohol‐anaesthetized rats in a dose‐dependent manner. OPC‐31260 did not exert an antidiuretic activity suggesting that it is not a partial V2 receptor agonist. 4 After oral administration at doses of 1 to 30 mg kg−1 in normal conscious rats, OPC‐31260 dose‐dependently increased urine flow and decreased urine osmolality. The diuretic action of OPC‐31260 was characterized as aquaresis, the mode of diuretic action being different from previously known diuretic agents such as furosemide, hydrochlorothiazide and spironolactone. 5 The results indicate that OPC‐31260 is a selective V2 receptor antagonist and behaves as an aquaretic agent. OPC‐31260 will be a useful tool in studying the physiological role of AVP and in the treatment of various conditions characterized by water retention.

The vasopressin V1b receptor critically regulates hypothalamic-pituitary-adrenal axis activity under both stress and resting conditions

The neurohypophyseal peptide [Arg(8)]-vasopressin (AVP) exerts major physiological actions through three distinct receptor isoforms designated V1a, V1b, and V2. Among these three subtypes, the vasopressin V1b receptor is specifically expressed in pituitary corticotrophs and mediates the stimulatory effect of vasopressin on ACTH release. To investigate the functional roles of V1b receptor subtypes in vivo, gene targeting was used to create a mouse model lacking the V1b receptor gene (V1bR-/-). Under resting conditions, circulating concentrations of ACTH and corticosterone were lower in V1bR-/- mice compared with WT mice (V1bR+/+). The normal increase in circulating ACTH levels in response to exogenous administration of AVP was impaired in V1bR-/- mice, while corticotropin-releasing hormone-stimulated ACTH release in the V1bR-/- mice was not significantly different from that in the V1bR+/+ mice. AVP-induced ACTH release from primary cultured pituitary cells in V1bR-/- mice was also blunted. Furthermore, the increase in ACTH after a forced swim stress was significantly suppressed in V1bR-/- mice. Our results clearly demonstrate that the V1b receptor plays a crucial role in regulating hypothalamic-pituitary-adrenal axis activity. It does this by maintaining ACTH and corticosterone levels, not only under stress but also under basal conditions.

V1a vasopressin receptors maintain normal blood pressure by regulating circulating blood volume and baroreflex sensitivity

Arginine-vasopressin (AVP) is a hormone that is essential for both osmotic and cardiovascular homeostasis, and exerts important physiological regulation through three distinct receptors, V1a, V1b, and V2. Although AVP is used clinically as a potent vasoconstrictor (V1a receptor-mediated) in patients with circulatory shock, the physiological role of vasopressin V1a receptors in blood pressure (BP) homeostasis is ill-defined. In this study, we investigated the functional roles of the V1a receptor in cardiovascular homeostasis using gene targeting. The basal BP of conscious mutant mice lacking the V1a receptor gene (V1a−/−) was significantly (P < 0.001) lower compared to the wild-type mice (V1a+/+) without a notable change in heart rate. There was no significant alteration in cardiac functions as assessed by echocardiogram in the mutant mice. AVP-induced vasopressor responses were abolished in the mutant mice; rather, AVP caused a decrease in BP, which occurred in part through V2 receptor-mediated release of nitric oxide from the vascular endothelium. Arterial baroreceptor reflexes were markedly impaired in mutant mice, consistent with a loss of V1a receptors in the central area of baroreflex control. Notably, mutant mice showed a significant 9% reduction in circulating blood volume. Furthermore, mutant mice had normal plasma AVP levels and a normal AVP secretory response, but had significantly lower adrenocortical responsiveness to adrenocorticotropic hormone. Taken together, these results indicate that the V1a receptor plays an important role in normal resting arterial BP regulation mainly by its regulation of circulating blood volume and baroreflex sensitivity.

Adenovirus-mediated gene transfer of glial cell line-derived neurotrophic factor prevents ischemic brain injury after transient middle cerebral artery occlusion in rats

To examine a possible protective effect of exogenous glial cell line-derived neurotrophic factor (GDNF) gene expression against ischemic brain injury, a replication-defective adenoviral vector containing GDNF gene (Ad-GDNF) was directly injected into the cerebral cortex at 1 day before 90 minutes of transient middle cerebral artery occlusion (MCAO) in rats. 2,3,5-Triphenyltetrazolium chloride staining showed that infarct volume of the Ad-GDNF-injected group at 24 hours after the transient MCAO was significantly smaller than that of vehicle- or Ad-LacZ-treated group. Enzyme-linked immunosorbent assay (ELISA) for immunoreactive GDNF demonstrated that GDNF gene products in the Ad-GDNF-injected group were higher than those of vehicle-treated group at 24 hours after transient MCAO. Immunoreactive GDNF staining was obviously detected in the cortex around the needle track just before or 24 hours after MCAO in the Ad-GDNF group, whereas no or slight GDNF staining was detected in the vehicle group. The numbers of TUNEL, immunoreactive caspase-3, and cytochrome c-positive neurons induced in the ipsilateral cerebral cortex at 24 hours after transient MCAO were markedly reduced by the Ad-GDNF group. These results suggest that the successful exogenous GDNF gene transfer ameliorates ischemic brain injury after transient MCAO in association with the reduction of apoptotic signals.

Immunoreactive Akt, PI3-K and ERK protein kinase expression in ischemic rat brain.

In order to clarify the role of protein kinases in ischemic brain injury, the spatiotemporal expression of immunoreactive serine-threonine kinase Akt, phosphatidylinositol 3-kinase (PI3-K) and extracellular signal-regulated kinase (ERK) were examined at 3, 8, or 24 h after permanent middle cerebral artery occlusion (MCAO) in rats. Weak staining for these protein kinases was found in both cortical and caudate neurons in sham controls. The staining for Akt-1 and PI3-K was increased at 3-8 h in the ischemic penumbral region and declined at 24 h. A slight induction of these kinases was observed in the ischemic core region. Robust expression of ERK was noted at 3-8 h in most neurons in the area of ischemia. At 24 h, ERK continued to be expressed in the ischemic penumbra, but decreased in the ischemic core. These findings suggest that the signaling for Akt and PI3-K are different from the ERK dependent signal transduction during ischemic brain injury.

7-Chloro-5-hydroxy-1-[2-methyl-4-(2-methylbenzoyl-amino)benzoyl ]-2,3,4,5-tetrahydro-1H-1-benzazepine (OPC-41061): a potent, orally active nonpeptide arginine vasopressin V2 receptor antagonist.

We previously reported a series of benzazepine derivatives as orally active nonpeptide arginine vasopressin (AVP) V2 receptor antagonists. After the lead structure OPC-31260 was structurally evaluated and optimized, the introduction of the 7-Cl moiety on the benzazepine and 2-CH3 on the aminobenzoyl moiety enhanced its oral activity. The new AVP-V2 selective antagonist OPC-41061 was determined to be a potent and orally active agent.

Cilostazol reduces brain lesion induced by focal cerebral ischemia in rats--an MRI study.

To investigate the effects of cilostazol on the hemispheric ischemic lesion, we monitored the apparent diffusion coefficient (ADC) and T2 images by MRI techniques in comparison with histology at the terminal of and after 24-h reperfusion following 2-h occlusion of middle cerebral artery (MCA). The ADC values of tissue water and T2-weighted images were quantified by high field magnetic resonance. No significant difference was observed by ADC image among vehicle and cilostazol treatment groups when measured during MCA occlusion. Oral treatment with cilostazol 30 mg/kg two times at 5 min and 4 h significantly suppressed the hemispheric lesion area and volumes when detected by ADC, T2 images and histology, but 3 and 10 mg/kg cilostazol were without effect. Cilostazol (30 mg/kg) significantly reduced the increased cerebral water content at the ischemic hemisphere compared with vehicle group. In line with these results, the neurological deteriorations were much improved in the cilostazol-treated group. Taken together, it is concluded that post-treatment with cilostazol exerts a potent protective effect against cerebral infarct size by reducing the cytotoxic edema.

Induction of glial cell line-derived neurotrophic factor receptor proteins in cerebral cortex and striatum after permanent middle cerebral artery occlusion in rats.

In an attempt to elucidate whether glial cell line-derived neurotrophic factor (GDNF) receptors are induced after ischemic brain injury, possible expression of immunoreactive GDNF receptor-alpha1 (GFRalpha-1) and c-ret (RET) was examined at 3, 8, or 24 h after permanent middle cerebral artery occlusion (MCAO) in rats. Immunohistochemical study showed that both GFRalpha-1 and RET staining cells which were not detected in sham control brain, were present in the ipsilateral cortex and caudate at 3 to 8 h after permanent MCAO, and then decreased but remained to some extent at 24 h. Positive cells for both GDNF receptors were predominantly in cortical neurons of ischemic penumbral area. Western blot analysis confirmed the induction of those receptors after permanent MCAO. This rapid induction of GFRalpha-1 and RET, which correlates with the similar induction of GDNF under these conditions, may play a role in the early response to ischemic brain injury.

OPC-21268, a vasopressin V1 antagonist, produces hypotension in spontaneously hypertensive rats.

We studied the hypotensive effects of OPC-21268, an orally effective nonpeptide vasopressin V1 receptor antagonist, in spontaneously hypertensive rats (SHR) and stroke-prone SHR (SHRSP). OPC-21268 was given intravenously to conscious, freely moving SHR and SHRSP. We used young and aged animals to examine the contribution of vasopressin to the development and maintenance of hypertension in both types of rats. In SHR, hypertension was fully established at 38 weeks of age, and intravenous injection of OPC-21268 produced slight hypotensive effects at either 38 or 70 weeks of age. In SHRSP, hypertension developed at 25 weeks of age, and blood pressure was sustained at a high level (approximately 250 mm Hg systolic blood pressure) thereafter. Intravenous administration of OPC-21268 did not cause hypotensive effects in young rats at 15 weeks, but at 25 weeks a significant decrease in blood pressure was observed. Furthermore, in the malignant state of SHRSP (35 to 41 weeks), OPC-21268 significantly decreased mean blood pressure by 32.4 +/- 7.9 mm Hg (mean +/- SEM) at 3 mg/kg IV, and the decrease was dose dependent (0.3 to 3.0 mg/kg). Plasma vasopressin concentrations were increased in a more malignant phase of SHRSP at 45 weeks of age, whereas at other ages of SHRSP or in SHR, plasma vasopressin levels were not increased. These results suggest that vasopressin plays an important role through V1 receptors in the maintenance of hypertension, at least in the malignant phase of SHRSP, and OPC-21268 may be therapeutically useful in the treatment of some types of hypertension.

Cardiovascular actions of OPC-18790: A novel positive inotropic agent with little chronotropic action

SummaryOPC-18790 [(±)-6-[3-(3,4-dimethoxy-benzylamino)- 2 - hydroxypropoxy] - 2(1H) - quinolinone], a novel positive inotropic agent, was investigated in several in vitro and in vivo experiments to elucidate its cardiovascular effects and its mechanism of action. In isolated blood-perfused dog heart preparations, OPC-18790 increased contractile force at 10 to 1,000 nmol i.a.; increased coronary arterial blood flow at 30 to 1,000 nmol; and deceased sinus rate slightly at 1,000 nmol. Atrio-ventricular nodal conduction was slightly facilitated with OPC-18790 (10 to 1,000 nmol), whereas ventricular automaticity tended to decrease. OPC-18790 (10−6 to 10−4 M) increased contractile force in isolated ventricular muscles of dogs, cats, rabbits and guinea pigs but not rats. OPC-18790 increased left ventricular contractile force dosedependently in anesthetized open-chest dogs and in conscious dogs with slight or no changes in heart rate and blood pressure. The positive inotropic effect of OPC-18790 was not affected by β-blockade. OPC-18790 (10−5 to 10−4 M) prolonged the duration of action potential in guinea pig papillary muscles. Na+, K+-ATPase was not inhibited, but peak-III phosphodiesterase (low Km cyclic AMP specific fraction, inhibited by cyclic GMP) was inhibited by OPC-18790 (IC50 = 0.41 × 10−6 M) in dog myocardium. However, such an inhibitory action of phosphodiesterase can hardly be reconciled with the lack of a positive chronotropic effect shown by OPC-18790. Thus, these results suggest that OPC-18790 may have an additional mechanism. The cardiovascular effects revealed by this study suggest that OPC-18790 may exert a beneficial effect in the treatment of congestive heart failure.