Kenzo Takahashi

Protective effects of SEA0400, a novel and selective inhibitor of the Na+/Ca2+ exchanger, on myocardial ischemia-reperfusion injuries

The Na(+)/Ca(2+) exchanger (NCX) is involved in myocardial ischemia-reperfusion injuries. We examined the effects of 2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline (SEA0400), a potent and selective inhibitor of NCX, on myocardial ischemia-reperfusion injury models. In canine cardiac sarcolemmal vesicles and rat cardiomyocytes, SEA0400 potently inhibited the Na(+)-dependent 45Ca(2+) uptake with an IC(50) value of 90 and 92 nM, compared with 2-[2-[4-(4-nitrobenzyloxy)phenyl]isothiourea (KB-R7943, 7.0 and 9.5 microM), respectively. In rat cardiomyocytes, SEA0400 (1 and 3 microM) attenuated the Ca(2+) paradox-induced cell death. In isolated rat Langendorff hearts, SEA0400 (0.3 and 1 microM) improved the cardiac dysfunction induced by low-pressure perfusion followed by normal perfusion. In anesthetized rats, SEA0400 (0.3 and 1 mg/kg, i.v.) reduced the incidence of ventricular fibrillation and mortality induced by occlusion of the left anterior descending coronary artery followed by reperfusion. These results suggest that SEA0400 is a most potent NCX inhibitor in the heart and that it has protective effects against myocardial ischemia-reperfusion injuries.

Characterization of rapid and high-affinity uptake of L-serine in neurons and astrocytes in primary culture

The non‐essential amino acid L‐serine was shown to be required to support the survival of rat cerebellar Purkinje neurons because of lack of the expression of the L‐serine biosynthesis enzyme 3‐phosphoglycerate dehydrogenase in them. In the present study, we investigated L‐[3H]serine uptake in primary cultures of neurons and astrocytes from the rat telencephalon. In both neurons and astrocytes, L‐[3H]serine uptake was dependent on temperature and Na+ ions, and exhibited a single component of high‐affinity uptake sites (K m=15.0 and 17.2 μM for neurons and astrocytes, respectively). Kinetic analysis of L‐[3H]serine uptake also revealed that the uptake into neurons was faster than that into astrocytes. The selectivity of inhibition by amino acids of the L‐[3H]serine uptake resembled that of the system ASC transporters ASCT1 and ASCT2. Neutral amino acids L‐alanine, L‐serine, L‐cysteine, and L‐threonine strongly inhibited the uptake by both cell types. Furthermore, in astrocytes, but not in neurons, L‐valine and L‐proline also inhibited L‐[3H]serine uptake. Neither α‐methyl aminoisobutyric acid (a system A‐specific substrate) nor 2‐aminobicyclo(2,2,1)heptane‐2‐carboxylic acid (a system L‐specific substrate) inhibited the uptake of L‐[3H]serine in both neurons and astrocytes. Expression of ASCT transporters in both neurons and astrocytes was examined by use of reverse transcriptase polymerase chain reaction and immunoblot analysis. Whereas transcripts (mRNAs) of both ASCT1 and ASCT2 transporters were detected in astrocytes, only the mRNA of the former subtype was detected in neurons. Immunoblot analysis confirmed the presence of ASCT1 in both neurons and astrocytes. These findings indicate that neurons accumulate a high level of L‐serine by using a Na+‐dependent, high‐affinity transport system, operating predominantly through the ASCT1 transporter subtype.

Sphingomyelin analogues as inhibitors of sphingomyelinase.

To search for neutral sphingomyelinase inhibitors we designed and synthesized hydrolytically stable analogues of sphingomyelin. The novel compounds 8 and 9 which were replaced the phosphodiester moiety of sphingomyelin with the carbamate moiety showed inhibitory activity with an IC(50) value of micro M on neutral sphingomyelinase in rat brain microsomes. Compound 8i showed a selective neutral sphingomyelinase inhibitory activity.

CD-832, a new dihydropyridine derivative with both nitrate-like and Ca2+ channel antagonist vasodilator activities

We investigated the effects of CD-832 ((4R)-(-)-2-(nicotinoylamino)ethyl 3-nitroxypropyl 1,4-dihydro-2,6-dimethyl-4,3-nitrophenyl, 3,5-pyridine dicarboxylate), a new dihydropyridine derivative with nitrate ester, on contraction and relaxation responses induced by various vasoactive agents in rabbit aorta. CD-832 potently inhibited the specific binding of [3H](+)-PN200-110 to rat brain membranes. The IC50 values for [3](+)-PN200-110 binding were 2.8 nM and 4.9 nM in CD-832 and nifedipine, respectively. CD-832 (10(-8) to 10(-5) M), diltiazem (10(-8) to 10(-5) M) and benidipine (10(-8) to 10(-5) M) inhibited the 64 mM KCl-induced contraction of the aortic strips in a concentration-dependent manner. Neither nitroglycerin (10(-8) to 10(-5) M) nor nicorandil (10(-8) to 10(-5) M) affected the 64 mM KCl-induced contraction in rabbit aorta. CD-832 (10(-8) to 10(-5) M), nitroglycerin (10(-8) to 10(-5) M) and nicorandil (10(-5) M) had no effect on norepinephrine-induced contraction in rabbit aorta. Nitroglycerin (10(-5) M), atrial natriuretic peptide (10(-8) M), nicorandil (10(-5) M) and CD-832 (10(-7) to 10(-5) M) augmented the isoproterenol-induced relaxation responses of rabbit aorta precontracted with endothelin-1 (1 x 10(-7) to 2 x 10(-7) M). The effects of nitroglycerin (10(-5) M), nicorandil (10(-5) M) and CD-832 (10(-5) M) on isoproterenol-induced relaxation responses were antagonized by treatment with methylene blue (10(-5) M) and oxyhemoglobin (10(-5) M).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of CD-832, a dihydropyridine derivative with a nitrate ester moiety, on rabbit femoral artery and vein

We investigated the effects of CD-832 ((4R)-(-)-2-(nicotinoyl-amino)ethyl 3-nitroxypropyl 1,4-dihydro-2,6-dimethyl-4,3-nitrophenyl, 3,5-pyridine dicarboxylate), a dihydropyridine derivative with a nitrate ester moiety, on contractile responses in rabbit femoral arteries and veins. CD-832 (10(-8) to 10(-6) M and nifedipine inhibited the 64 mM KCl-induced and 10(-6) M norepinephrine-induced contractions of rabbit femoral arteries, while nitro compounds had no effect on the contractions. CD-832 (10(-8) to 10(-6) M) and nitro compounds inhibited the 10(-6) M norepinephrine-induced contractions in rabbit femoral veins, while other Ca2+ channel antagonists had little effect. The inhibitory effects of CD-832 (10(-7) M) on norepinephrine-induced contractions were antagonized by treatment with methylene blue (10(-5) M). These results indicate that CD-832 potently relaxes venous smooth muscle, and that it may be a useful agent for the treatment of angina pectoris.

Protective effects of CD-832 on organ damage in stroke-prone spontaneously hypertensive rats

Effects of a newly developed Ca2+ channel antagonist, (4R)-(-)-2-(nicotinoylamino)ethyl 3 nitrooxypropyl 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl) 3,5-pyridine-dicarboxylate (CD-832), on hypertensive complications in stroke-prone spontaneously hypertensive rats (SHRSPs) were compared with effects of diltiazem. We examined changes in histological and hematological parameters in SHRSPs given the following treatments at 8 to 20 weeks of age: (a) CD-832; (b) diltiazem; (c) no treatment. CD-832 and diltiazem were added to the diet, in doses of 0.05 and 0.15% (approximately 30 and 100 mg/kg per day), respectively, throughout the experimental period. In untreated control SHRSPs, systolic blood pressure increased and severe renal lesions such as fibrinoid necrosis, smooth muscle proliferation, glomerular and tubular lesions and some cardiac fibrosis were observed at age 20 weeks. 12-week repeated-administration of CD-832 and diltiazem led to a comparable hypotension and decreased heart rate. CD-832 and diltiazem decreased the ratios of weights of kidney and heart to body weight and the concentration of blood urea nitrogen and creatinine in serum, compared to values in controls. In SHRSPs treated with CD-832 and diltiazem, the incidence of renal lesions and myocardial fibrosis was significantly reduced when compared with control SHRSPs. These results suggest that 12-week repeated-administration of CD-832 prevents the development of hypertension and the incidence of organ damage in SHRSPs. CD-832 and diltiazem were equally efficacious in preventing organ damage but this organ-protective effect was obtained at a lower dose for CD-832 (30 mg/kg per day) than that of diltiazem (100 mg/kg per day).

NT-702 (parogrelil hydrochloride, NM-702), a novel and potent phosphodiesterase 3 inhibitor, suppress the asthmatic response in guinea pigs, with both bronchodilating and anti-inflammatory effects

We evaluated the effects of NT-702 (parogrelil hydrochloride, NM-702, 4-bromo-6-[3-(4-chlorophenyl) propoxy]-5-[(pyridine-3-ylmethyl) amino] pyridazin-3(2H)-one hydrochloride), a selective phosphodiesterase 3 inhibitor, on the asthmatic response in guinea pigs. NT-702 at a concentration of 1 x 10(-7)M elevated the cyclic adenosine monophosphate content in prostaglandin E(2)-treated guinea pig tracheal smooth muscle cells. Leukotriene (LT) D(4)- and histamine-induced contraction of isolated guinea pig tracheal strips was inhibited by NT-702, with EC(50) values of 3.2 x 10(-7) and 2.5 x 10(-7)M, respectively. In an in vivo study, NT-702 suppressed LTD(4)-induced bronchoconstriction and the ovalbumin-induced immediate asthmatic response in guinea pigs through its bronchodilating effect. Furthermore, NT-702 also suppressed the ovalbumin-induced late asthmatic response, airway hyperresponsiveness, and the accumulation of inflammatory cells in the bronchoalveolar lavage fluid. These results suggest that NT-702 has an anti-inflammatory effect as well as a bronchodilating effect and might be useful as a novel potent therapeutic agent for the treatment of bronchial asthma, a new type of agent with both a bronchodilating and an anti-inflammatory effect.

In-vitro Negative Chronotropic and Inotropic Effects of a Novel Dihydropyridine Derivative, CD-832, in the Guinea-pig: Comparison with Calcium-channel Antagonists

The effects of CD‐832 (4R‐(‐)‐2‐(nicotinoylamino)ethyl‐3‐nitroxypropyl‐1,4‐dihydro‐2,6‐dimethyl‐4,3‐nitrophenyl, 3,5‐pyridine dicarboxylate), a novel dihydropyridine derivative, on guinea‐pig isolated myocardial preparations have been compared with those of Ca2+‐channel antagonists.

Vasodilation profile of CD-832, a novel dihydropyridine derivative in rabbit aorta

1. CD-832, nifedipine and nitrendipine, but not nitroglycerin and nicorandil, inhibited the KCl-induced contraction of rabbit aortas. 2. CD-832, nitroglycerin and nicorandil, but not nifedipine and nitrendipine, inhibited the norepinephrine-induced contraction of aortas. The inhibitory effects of these agents were either potentiated or inhibited by zaprinast or methylene blue, respectively. 3. On the KCl-induced contraction, the duration of CD-832-induced vasodilation was longer than that of nifedipine. Concerning the norepinephrine-induced contraction, the duration of CD-832-induced vasodilation was longer than that of nicorandil. 4. These results suggest that the mechanism of CD-832-induced vasodilation concerns both Ca(2+)-antagonistic action and a nitratelike action. Furthermore, the vasodilation is long lasting.

Overcoming of multidrug resistance by VA-033, a novel derivative of apovincaminic acid ester.

We have studied the effects of a novel derivative of apovincaminic acid ester, VA-033, on the resistance of tumors to chemotherapeutic agents. VA-033 increased the sensitivity of drug-resistant cell lines (P388/VCR, P388/ADM, AD10, and K562/ADM) to adriamycin or vincristine. The potency of VA-033 was stronger than verapamil. The drug lengthened the survival time of the P388/VCR-implanted mice treated with vincristine. VA-033 increased the intracellular accumulation of vincristine in the tumor cells, and the photolabeling of P-glycoprotein by [3H]azidopine was inhibited by VA-033. VA-033 showed a slight inhibitory effect on the L-type Ca2+ current in the ventricular myocytes, and had less effect on the cardiovascular parameters such as blood pressure, contractile force and atrio-ventricular conduction time than verapamil when administered systemically in the dog. These results suggest that VA-033 may become a beneficial compound as a modifier to the neoplastic cell resistant to multidrugs.