Hiroshi Narita

Effects of imidapril and captopril on streptozotocin-induced diabetic nephropathy in mice

We investigated whether the prevention of the development of diabetic nephropathy by angiotensin-converting enzyme inhibitors is associated with decreases in renal angiotensin-converting enzyme activity and/or blood pressure in diabetic mice. C57Bl/6 mice were injected with streptozotocin (200 mg/kg, i.v.) and randomized to receive either imidapril (1 and 5 mg/kg) or captopril (10 and 50 mg/kg) or vehicle by gavage for 28 days. Each assay was performed on 8-10 mice from each treatment. At 28 days after the start of drug treatment, imidapril and captopril significantly reduced blood pressure of the diabetic mice, and this effect of captopril was stronger than that of imidapril. On the other hand, inhibition of renal angiotensin-converting enzyme activity by imidapril was stronger than that by captopril. Imidapril and captopril dose-dependently inhibited urinary albumin excretion to similar extents, but they failed to inhibit the renal hypertrophy and elevation of creatinine clearance. Total renal angiotensin-converting enzyme activity was significantly reduced in diabetic mice, but immunohistochemical localization of angiotensin-converting enzyme was intensive in the vasculature and glomeruli of the diabetic kidney. In conclusion, both effects on blood pressure and angiotensin-converting enzyme activity may be involved in the prevention of development of diabetic nephropathy by imidapril and captopril in streptozotocin-induced diabetic mice. The data suggest that the degrees of contribution of their effects on blood pressure and renal angiotensin-converting enzyme activity to the inhibition of urinary albumin excretion may be different between the two angiotensin-converting enzyme inhibitors.

Tissue Angiotensin-converting enzyme activity plays an important role in pressure overload-induced cardiac fibrosis in rats.

It has been widely assumed that the cardiac angiotensin-generating system plays an important role in the development and maintenance of cardiac remodeling caused by pressure overload. The roles of angiotensin-converting enzyme (ACE) in pressure overload–induced cardiac hypertrophy and fibrosis in rats were investigated. Pressure overload was achieved by constricting the abdominal aorta above the renal arteries. After they underwent surgery, the rats were treated with a low or high dose of the ACE inhibitor imidapril (0.07 and 0.7 mg/kg/d s.c.) with an osmotic pump for 4 weeks. High-dose imidapril prevented the increase in blood pressure, cardiac hypertrophy, and fibrosis. Low-dose imidapril inhibited only cardiac fibrosis. ACE activity in the myocardium, but not in serum, was significantly increased in the rats with the banded aorta, and ACE immunoreactivity was increased in the areas of fibrosis. These changes were markedly reduced by both doses of imidapril. These results suggest that the increased local ACE expression contributes to the development of pressure overload–induced cardiac fibrosis but is not responsible for hypertrophy in rats.

Differential effects of imidapril and candesartan cilexetil on plasminogen activator inhibitor-1 expression induced by prolonged inhibition of nitric oxide synthesis in rat hearts.

We investigated effects of the angiotensin-converting enzyme (ACE) inhibitor imidapril and the angiotensin II type 1 (AT1) antagonist candesartan cilexetil on cardiac plasminogen activator inhibitor-1 (PAI-1) expression in rats. Cardiac PAI-1 mRNA levels were increased after a 7-day treatment with the nitric oxide (NO) synthesis inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME). PAI-1 immunoreactivity was increased in the coronary arteries. Treatment with imidapril significantly prevented the L-NAME-induced increase in the gene expression and immunoreactivity of PAI-1, but candesartan cilexetil showed no such effect. This study provides the first evidence of differential effects of ACE inhibition and AT1 antagonism on cardiac PAI-1 expression in vivo.

T-477, a novel Ca2+- and Na+ channel blocker, prevents veratridine-induced neuronal injury

To evaluate the effect of (R)-(+)-2-(4-chlorophenyl)-2, 3-dihydro-4-diethyl aminoacetyl-4H-1,4-benzothiazine hydrochloride (T-477), a novel Na(+)- and Ca(2+) channel blocker, on neuronal injury in vitro, we studied veratridine-induced injury in cultured rat hippocampal neurons. Neurons swelled extensively 10 min after the addition of veratridine, and returned to their initial size within 2 h. Intracellular Na(+) and Ca(2+) concentrations and amino acid release from the cells, in particular, that of glutamate, increased after the treatment with veratridine. Approximately 70% of neurons died within 24 h. T-477 inhibited both veratridine-induced swelling and death in a concentration-dependent manner. Moreover, T-477 concentration dependently reduced the increases in Na(+) and Ca(2+) influx and amino acid release. These results suggest that T-477 prevented the veratridine-induced influx of Na(+) and, thereby, reduced neuronal swelling. This, combined with the effects of T-477 on the inhibition of Ca(2+) influx and glutamate release, possibly by the blockade of Na(+) channels, may be the mechanism by which T-477 protects neurons from death induced by veratridine.

Prolonging action of imidapril on the lifespan expectancy of cardiomyopathic hamsters.

We studied the effect of imidapril, a novel angiotensin-converting enzyme (ACE) inhibitor, on lifespan expectancy of cardiomyopathic (CM) hamsters of BIO 14.6 strain, one of the representative models of congestive heart failure (CHF). Imidapril was consecutively administered to hamsters by mixing it in their diet at a concentration of 480 ppm (approximately 30 mg/kg/day) or 1,600 ppm (approximately 120 mg/kg/day) from age 26 weeks. Only several control hamsters died before age 54 weeks, but their survival rate decreased to 23.7% at age 73 weeks. The survival rates of 480-ppm and 1,600-ppm imidapril groups at age 73 weeks were as high as 75.7 and 68.4%, respectively (p < 0.01 vs. control hamsters). Macroscopic and microscopic pathology in imidapril-treated groups was milder than that in control animals in general, but differences were not statistically significant when animals were divided into survivors and fatalities except for the presence of mural thrombus in the heart. We further studied the effects of imidapril on blood pressure (BP), in vivo cardiac function, cardiac beta-adrenoceptor distribution, and plasma catecholamine levels after dietary treatment with 480 ppm imidapril for 8-10 weeks from age 37 weeks. Imidapril-treated animals showed improved cardiac function under urethane anesthesia. These results indicate that imidapril prolongs lifespan expectancy of CM hamsters and suggest that a hemodynamic effect of imidapril is involved in its beneficial effect.

Fluvastatin depresses the enhanced lipid peroxidation in vitamin E-deficient hamsters

Fluvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, has recently been reported to have the antioxidative activity in vitro. However, it is still unclear whether chronic treatment with this drug actually leads to amelioration of the redox status in the body. In this study, we investigated the antioxidative effect of fluvastatin in vivo, using a vitamin E-deficient hamster model, an in vivo model of enhanced oxidative stress. After pre-treatment with a vitamin E-deficient diet for 2 months, fluvastatin, pravastatin or probucol was added to the diet for 1 month. Vitamin E deficiency caused a significant increase in the levels of plasma oxidative stress markers such as 8-iso-prostaglandin F2α (8-iso-PGF2α) and hydroperoxides. Furthermore, there was a significant increase in the oxidizability of plasma lipids in the vitamin E-deficient animals, indicating that the oxidative stress was increased in the circulation. Fluvastatin markedly depressed the above oxidative stress markers in plasma, and significantly decreased the oxidizability of plasma lipids without affecting their levels. Probucol, a reference antioxidant, also showed a similar effect while pravastatin, another HMG-CoA reductase inhibitor, showed only a weak improvement. We suggest that the treatment with fluvastatin leads to a reduction of oxidative stress in vivo, which is mainly derived from its antioxidative property rather than its lipid-lowering activity.

Subcellular fractions of cyclic AMP and cyclic AMP-dependent protein kinase and the positive inotropic effects of selective β1- and β2-adrenoceptor agonists in guinea pig hearts

We studied the effects of a full beta 1-adrenoceptor agonist (T-0509), a beta 2-adrenoceptor agonist (procaterol) and a nonselective beta-adrenoceptor agonist (isoproterenol, ISO) on subcellular cyclic AMP levels and cyclic AMP-dependent protein kinase (cyclic AMP-PK) activity in guinea pig hearts and compared them with the effect of each drug on cardiac contractility. T-0509 (10(-8)M) and ISO (3 x 10(-8)M) caused an increase of approximately 170% in dF/dtmax, whereas 10(-7) procaterol produced only a 25% increase. All these agonists significantly increased the cyclic AMP level in ventricular homogenate. Subcellular fractions were obtained by centrifugation at 100,000 g for 10 min and by Li2SO4 precipitation of the 100,000-g supernatant. In the control heart, probably salcolemmal protein, phospholamban, and 60-kDa protein in the particulate fraction and probably troponin I and troponin C in the supernatant fraction were mainly phosphorylated by the catalytic subunit of cyclic AMP-PK. In the precipitate obtained from the supernatant fraction with Li2SO4, probably all proteins described were contained. However, none of the proteins were detected in the supernatant obtained with Li2SO4. T-0509 and ISO caused significant changes in cyclic AMP levels and cyclic-PK activities in all fractions. However, procaterol increased the cyclic AMP concentrations and cyclic AMP-PK activities only in the supernatant fraction and the supernatant obtained with Li2SO4. T-0509 and ISO increased cyclic AMP level (9-16 pmol/mg protein) and cyclic AMP-PK activity ratio (0.27-0.33) significantly to the same degree in the precipitate obtained with Li2SO4, whereas the effects of T-0509 in other fractions were about twofold less than those of ISO. These results suggest that beta 1- and beta 2-adrenoceptor agonists cause differential compartmentalization of cyclic AMP and cyclic AMP-PK in the cardiac muscle.

Antiplatelet mechanisms of TA-993 and its metabolite MB3 in ADP-induced platelet aggregation

We investigated the antiplatelet mechanisms of TA-993 [(-)-cis-3-acetoxy-5-(2-(dimethylamino)ethyl)-2, 3-dihydro-8-methyl-2-(4-methylphenyl)-1,5-benzothiazepin-4(5H)-one maleate] and its metabolite MB3 (deacetyl and N-monomethyl TA-993) in human platelets stimulated by ADP in vitro. TA-993 and MB3 concentration-dependently inhibited fibrinogen binding to the ADP-stimulated platelets as well as inhibiting platelet aggregation. The antiplatelet effect of MB3 was about 300 times more potent than those of TA-993 and a glycoprotein IIb/IIIa receptor antagonist, Arg-Gly-Asp-Ser (RGDS). Aggregation of ADP-treated fixed platelets caused by the addition of fibrinogen was inhibited by RGDS but not by TA-993 and MB3. TA-993 and MB3 inhibited ADP-induced polymerization of actin filaments. Neither TA-993 nor MB3 affected cyclic AMP and cyclic GMP levels in resting platelets, and nor suppressed the increase in intracellular Ca(2+) concentration induced by ADP. These results suggest that the antiplatelet mechanisms of TA-993 and MB3 may involve inactivation of glycoprotein IIb/IIIa receptors via inhibition of the polymerization of actin.

Cardiovascular effect of a new 1,5-benzothiazepine derivative TA-993 in anesthetized dogs.

TA-993 is a new 1,5-benzothiazepine derivative having l-cis configuration and shows a potent antiplatelet aggregating action. We studied its cardiovascular effect in anesthetized dogs by using diltiazem as a reference compound. TA-993 (> or = 10 microg/kg, i.v.) significantly increased blood flows of common carotid, brachial, and femoral arteries. The peak of its effect was observed approximately 60 min after the administration, and the peak level was maintained until > or = 300 min after the administration. TA-993 (100 microg/kg, i.v.) slightly increased cardiac output in the same manner. However, TA-993 did not cause any persistent effects on arterial pressure, LVdP/dtmax, or vertebral, coronary, superior mesenteric, and renal blood flows. TA-993 caused concentration-dependent vasorelaxation in the isolated canine femoral artery contracted with 40 mM K+, but its potency was approximately 1/20 that of diltiazem. The increasing action of TA-993 on femoral blood flow was completely inhibited by pretreatment with hexamethonium in anesthetized dogs. These results indicate that TA-993 has a selective increasing action on common carotid, brachial, and femoral blood flows and suggest that the action is mediated by the autonomic nervous system.

Functional and binding studies of insurmountable antagonism of 606A, a novel AT1-receptor antagonist in rabbit tissues.

Angiotensin AT1-receptor antagonists can be classified into two types, surmountable and insurmountable ones, based on the way they inhibit angiotensin II (AII)-induced vasoconstriction. To elucidate the causes of the difference, we studied how several antagonists associate with and dissociate from AT1-receptor sites by using rabbit adrenal cortical membrane. Four antagonists, 606A, EXP3174, CV11974, and E4177, showed equipotent competitive antagonism when they were added simultaneous with [125I]-AII in binding experiments. However, in AII-induced contraction studies with rabbit aorta, 606A, EXP3174, and CV11974 inhibited the contraction noncompetitively, whereas E4177 inhibited competitively. The longer the pretreatment period with EXP3174 or CV11974, the more effectively the antagonists suppressed AII-induced contraction. However, the suppression of contraction by 606A and E4177 changed little with the length of the pretreatment period. AII-induced contraction of 606A- or E4177-treated aorta recovered easily by washout, but that of CV11974-treated aorta was hard to recover by washout. These results obtained in the aorta were consistent with their characteristics observed in the AII binding study in the rabbit adrenal cortical membrane in cases of EXP3174 and CV11974. The differences between association rate with and dissociation rate from the AT1 receptor of E4177 and 606A were slight, in spite of the clear difference between their action in the contraction study. Because of the variations observed with the four compounds, mechanisms of insurmountable antagonism may not be uniform among AT1-receptor antagonists.