Heparin cofactor II inhibits thrombus formation in a rat thrombosis model.

Heparin cofactor II is postulated to be an extravascular thrombin inhibitor that is physiologically stimulated by dermatan sulfate. However, the role of heparin cofactor II has not yet been clearly demonstrated in vivo. In this study, we estimated the antithrombotic effect of heparin cofactor II administered exogenously in a rat model of thrombosis. Thrombus was induced in the rat femoral artery by endothelial damage due to the photochemical reaction between systemically injected rose bengal and transillumination with green light. Pretreatment with heparin cofactor II significantly prolonged the time required to occlude the femoral artery (occlusion time) in a dose-dependent manner. At an effective dose in this thrombosis model, heparin cofactor II did not prolong the activated partial thromboplastin time and the prothrombin time in normal rats. Argatroban, a selective synthetic thrombin inhibitor, significantly prolonged the occlusion time. However, argatroban also prolonged the activated partial thromboplastin time and prothrombin time at an effective dose. These results suggest that the administration of heparin cofactor II in vivo effectively inhibited thrombus formation on the vessel walls whose endothelium is damaged without a prolongation of the coagulation time while heparin cofactor II may also inhibit the thrombin activity in the subendothelial tissue in vivo.

Calcium channel blocking and vasodilating actions of the novel dihydropyridine derivative AE0047.

1. The pharmacological characteristics of AE0047, a newly synthesized dihydropyridine (DHP) derivative, were investigated in vitro. 2. In bovine aortic membrane, AE0047 and other DHP calcium channel blockers (nitrendipine, nicardipine) displayed concentration-dependent antagonism to specific [3H]-PN200-110 binding sites with the following values for inhibition constants (Ki) obtained: 20.8 +/- 8.9, 12.3 +/- 4.5 and 3.9 +/- 1.0 nmol/L for AE0047, nitrendipine and nicardipine, respectively. 3. In guinea-pig ventricular myocytes, AE0047 blocked the L-type calcium current, with values for the dissociation constant (Kd) and Hill coefficient of 11.4 +/- 5.7 nmol/L and 0.852 +/- 0.061, respectively, indicating in the terms of Hill's hypothesis that one drug molecule blocks one calcium channel molecule. 4. In rat aorta, AE0047 inhibited 45Ca uptake induced by high K+ (100 mmol/L) by 55%. 5. AE0047 and nitrendipine concentration dependently relaxed rat aortic strips contracted with 30 mmol/L KCl. The response to nitrendipine reached a plateau within 60 min and disappeared after drug washing. Interestingly, AE0047 required 5 h or more to produce a plateau of response, with no effect of drug washing. This confirmed the slow onset and long duration of its vasodilating action. 6. With AE0047, tissue content in rat aorta increased more slowly than with nitrendipine and release of AE0047 from tissue was also slower. 7. The data suggest that AE0047 is incorporated slowly into smooth muscle membranes, approaches receptors slowly through the membrane bilayer and accumulates in the membrane because of its high lipophilicity, resulting in an anti-hypertensive action that is slow in onset and of long duration.

AE0047-mediated calcium channel blocking in vascular smooth muscles.

1. This experiment was designed to pharmacologically characterize a novel calcium channel blocker, AE0047. 2. After 1-hr treatment with each drug (10(-6) M), K(+)-induced contraction in rat aortic strip was clearly depressed by nifedipine and manidipine and slightly depressed by AE0047. After a wash out of the preparation in drug-free medium, the inhibition of K(+)-induced contraction by nifedipine or manidipine was abolished or unchanged, respectively. In contrast, AE0047-produced inhibition was reinforced with time after removal of the drug. 3. A cell membrane depolarization-induced 45Ca uptake into tissue was depressed completely by nifedipine, but, if it was washed out, merely 20% inhibition of control remained. AE0047-produced inhibition became prominent after drug removal. Manidipine did not have the same inhibitory effect after wash out. 4. A receptor-binding study indicated that affinity of AE0047 and manidipine for the dihydropyridine-sensitive Ca channel receptor was lower than that of nifedipine. AE0047, unlike nifedipine and manidipine, inhibited [3H]PN200-110 binding more strongly when a 4-hr preincubation was used than without extended incubation. 5. The drug molecule of AE0047 was highly partitioned into the lipid bilayer of the synaptosome in canine cerebral cortices. In the synaptic membrane and liposomes, both prepared from canine cerebral cortices, the respective partition coefficients of the drug were 6997 +/- 2309 and 422 +/- 28 against 1395 +/- 161 and 24 +/- 2 of nitrendipine. 6. AE0047 showed slower onset of inhibition against K(+)-induced contraction and enhanced Ca influx compared with manidipine and nifedipine. These results may suggest that AE0047 requires a long period of time to occupy the dihydropyridine-sensitive sites within the Ca channel, which was detected by decreased specific [3H]PN200-110 binding, and to inhibit K(+)-induced Ca influx into rat aorta.

[Antihypertensive effects of a novel calcium antagonist, AE0047, in various hypertensive models].

The antihypertensive effects of oral or intravenous administration of AE0047, a novel 1,4-dihydropyridine-type calcium antagonist, were investigated in spontaneously hypertensive rats (SHR/crj), one kidney-one clip renal hypertensive rats (RHR), deoxycorticosterone acetate (DOCA)-salt hypertensive rats (DHR) and two kidney-one clip renal hypertensive dogs (RHD). AE0047 (1, 3, 10 mg/kg, p.o.) caused a dose-related reduction of systolic blood pressure (SBP) with low reflex tachycardia in SHR/crj and RHR. The effect reached its maximum at 2-4 hr after administration and was sustained for a long time. In DHR, AE0047 (0.3, 1, 3 mg/kg, p.o.) similarly showed the antihypertensive effects at 2-7 hr with no significant changes in heart rates (HR). The doses (ED30) of AE0047 required to decrease SBP by 30% were 2.6, 3.4 and 0.68 mg/kg in SHR/crj, RHR and DHR, respectively. In RHD, and AE0047 capsule (GJ-0956: 4, 8, 16, 32 mg/body, p.o.) produced dose-dependent and long lasting effects with a transient and slight increase in HR. Furthermore, the intravenous administration of AE0047 (10, 30, 100 micrograms/kg) produced the antihypertensive action slowly, reached a plateau 10 min later and then maintained for many hours. In contrast, nitrendipine (3-100 mg/kg, p.o., 3-30 micrograms/kg, i.v.) and nicardipine (1-30 mg/kg, p.o., 3-30 micrograms/kg, i.v.) exhibited a similar potency to AE0047, but these maximal effects were produced at 1-2 hr and 0.5-1 min in the case of oral and intravenous administration, respectively, with a rapid recovery in the above hypertensive rats. These results indicate that AE0047 exhibits an antihypertensive effect with a slow onset and long-lasting profile.

Aging decreases the production of PGI2 in rat aortic endothelial cells.

It has been suggested that progressive pathophysiologic modifications of endothelium are associated with aging. Aging has been shown to influence some specific functions at the cellular level. In the present study, the effects of aging on levels of prostacyclin (PGI2) production were examined in cultured rat aortic endothelial cells from young (six-week-old) and old (100-week-old) Wistar rats. The level of PGI2 production from rat aortic endothelial cells decreased significantly with increasing age, suggesting decreased function of the endothelial cells. The production of PGI2 stimulated by thrombin was decreased in old rat aortic endothelial cells compared to young rat aortic endothelial cells, whereas there was no difference in the rate of intracellular calcium mobilization caused by thrombin. These data indicate that aging nonuniformly affects both basal and agonist-induced levels of PGI2 production in rat aortic endothelial cells, and that this diminution in PGI2 production may be related to the age-related potentiation of various thrombotic events.

Antithrombin III prevents blood pressure elevation and proteinuria induced by high salt intake in pregnant stroke-prone spontaneously hypertensive rats.

In pregnant stroke-prone spontaneously hypertensive rats, salt-loading causes symptoms similar to those of human preeclampsia, such as hypertension and proteinuria. To seek evidence of the therapeutic potential in preeclampsia of antithrombin III (AT III), which is a serine protease inhibitor active on various enzymes of the coagulation cascade, we examined the effect of consecutive treatment with AT III on hypertension and proteinuria in this animal model. Salt-loading (2% NaCl diet) caused a significant elevation of systolic blood pressure on day 15-17 and of urinary protein excretion on day 17-19 of gestation, as compared with animals fed a normal diet. AT III, administered i.v. at a dose of 60 or 300 U/kg/d for 10 d from day 9-11 to 18-20, attenuated these pathological changes in a dose-dependent manner. Histological examination of the kidney revealed that AT III prevented the occurrence of arteriosclerosis and thickening of the capillary basement membrane. However, the pathological changes induced by salt-loading were not attributable to activation of the blood coagulation system. These results demonstrate that AT III has preventive action against salt-induced hypertension and proteinuria in pregnancy through a mechanism largely independent of its anticoagulant action. AT III may thus be beneficial for the treatment of clinical symptoms of preeclampsia.

Diuretic and vasodilating actions of torasemide.

The pharmacological profile of torasemide was examined in experimental animals. In normotensive Wistar rats, torasemide produced less kaliuresis at doses that were equipotent with furosemide in terms of natriuresis. Torasemide but not furosemide exerted a significant diuretic action in rats treated with deoxycorticosterone acetate. Both torasemide and furosemide increased plasma renin activity and aldosterone concentration, but only torasemide significantly inhibited aldosterone-receptor binding in rat kidney. Torasemide also inhibited vasoconstriction induced by thromboxane A2 in isolated canine coronary artery.

A novel loop diuretic, torasemide, inhibits thromboxane A2-induced contraction in the isolated canine coronary artery.

We examined the effect of a novel antihypertensive diuretic, torasemide, on the vasoconstriction induced by TXA2 in the isolated canine coronary artery. Carbocyclic thromboxane A2 (CTA2), a stable analogue of the potent coronary vasoconstrictor thromboxane A2, exhibited a slow onset and progressive contraction of isolated canine coronary arteries at 2 x 10(-8) M. Torasemide (10(-7) approximately 10(-4) M) elicited a dose-dependent vasodilating action in the isolated canine coronary arteries contracted by CTA2, whereas indapamide or furosemide had little effect on this preparation. The maximum vasodilating response to torasemide was 45 +/- 12% of vasodilating effect induced by 10(-4) M papaverine. These results suggest that torasemide is a promising antihypertensive agent with a coronary protective effect.

Torasemide, but not frusemide, increases intracellular cAMP and cGMP content in the aorta of the renal hypertensive rat.

Repeated oral administration of the novel loop diuretic torasemide (3 mg kg-1) and frusemide (30 mg kg-1) for 7 days, elicited a significant fall in the systolic blood pressure in the one-kidney, one-clip Goldblatt renal hypertensive rat (RHR). The hypotensive action was greater in the torasemide group than in the frusemide group. Furthermore torasemide increased intracellular cAMP and cGMP content in aorta of RHR. Frusemide caused no effect. It is hypothesized that the increase in adenosine- or guanosine-nucleotides is involved in the antihypertensive action of torasemide, but not in that of frusemide.

Anti-aldosteronergic effect of torasemide.

The diuretic actions of torasemide and furosemide were studied in normotensive rats and in deoxycorticosterone acetate (DOCA)-saline-loaded hypertensive rats. Torasemide (0.3-3 mg/kg) and furosemide (3-30 mg/kg) had a dose-dependent and significant diuretic action in normotensive rats. Potassium retention was only observed in the case of torasemide. Torasemide also had a dose-dependent and significant diuretic action in DOCA-saline-loaded hypertensive rats, whereas furosemide did not. Higher doses of torasemide (10 mg/kg) and furosemide (100 mg/kg) increased both plasma renin activity and aldosterone concentration in normotensive rats in a similar manner. In vivo aldosterone receptor binding was determined to test the possible anti-aldosteronergic effect of torasemide. Torasemide inhibited the binding of aldosterone to its receptor in the cytoplasmic fraction of rat kidney in a dose-dependent manner, while furosemide produced no effect. These results suggest strongly that an anti-aldosteronergic action of torasemide contributes to producing less kaliuresis.

Metabolism of 7 beta-alkyl chenodeoxycholic acid analogs and their effect on cholesterol metabolism in hamsters.

The metabolism of 7-ethyl- and 7-propyl-chenodeoxycholic acids was studied in hamsters. Both bile acid analogs were absorbed efficiently by the intestine and secreted into the bile at rates similar to those of chenodeoxycholic acid. After intraduodenal administration into bile fistula hamsters, the 7-alkyl analogs were present in bile as the glycine and taurine conjugates. The glycine/taurine ratios were: chenodeoxycholic acid, 1.9; 7-ethyl analog, 0.3; and 7-propyl analog, 0.2. After oral administration, during a 21-day feeding experiment, the 14C-labeled analogs were recovered quantitatively in the feces. Chenodeoxycholic acid was largely 7-dehydroxylated to lithocholic acid in the intestinal tract. In contrast, the 7 alpha-hydroxy group of the 7-alkyl bile acids was completely resistant to bacterial action. 7-Ethyl-chenodeoxycholic acid was transformed in part to a compound tentatively identified as 7 alpha-hydroxy-3-oxo- 7 beta-ethyl-5 beta-cholanoic acid while 7-propyl-chenodeoxycholic acid was excreted unchanged. In the hamsters used, the 7-alkyl bile acid analogs did not inhibit the bacterial dehydroxylation of chenodeoxycholic acid. At the end of the 21-day feeding period, analysis of the gallbladder bile showed that 7-methyl-, 7-ethyl-, and 7-propyl-chenodeoxycholic acids accounted for 38, 31, and 12% of total bile acids, respectively. The 7-alkyl bile acids decreased cholesterol absorption; the 7-propyl analog caused significant decrease in serum and liver cholesterol concentration. These experiments demonstrate that the 7-ethyl- and 7-propyl chenodeoxycholic acids, just like the 7-methyl-analog, are absorbed by the intestine and participate in the enterohepatic circulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis of bile acid analogs: 7-alkylated chenodeoxycholic acids.

This paper describes a method for the preparation of 7-alkylated chenodeoxycholic acids from 3 alpha-hydroxy-7-oxo-5 beta-cholanoic acid. The synthetic procedure is based upon a Grignard reaction between the keto bile acid and an alkyl magnesium halide. Under the conditions employed, the introduction of alkyl groups is highly stereoselective. Only 7 beta-alkylated epimers are obtained. The overall yield is several-fold higher than that obtained by the previous method, which involved the preparation of an oxazoline intermediate.