Hiroyuki Koike

Molecular Mechanism Underlying Inverse Agonist of Angiotensin II Type 1 Receptor

To delineate the molecular mechanism underlying the inverse agonist activity of olmesartan, a potent angiotensin II type 1 (AT1) receptor antagonist, we performed binding affinity studies and an inositol phosphate production assay. Binding affinity of olmesartan and its related compounds to wild-type and mutant AT1 receptors demonstrated that interactions between olmesartan and Tyr113, Lys199, His256, and Gln257 in the AT1 receptor were important. The inositol phosphate production assay of olmesartan and related compounds using mutant receptors indicated that the inverse agonist activity required two interactions, that between the hydroxyl group of olmesartan and Tyr113 in the receptor and that between the carboxyl group of olmesartan and Lys199 and His256 in the receptor. Gln257 was found to be important for the interaction with olmesartan but not for the inverse agonist activity. Based on these results, we constructed a model for the interaction between olmesartan and the AT1 receptor. Although the activation of G protein-coupled receptors is initiated by anti-clockwise rotation of transmembrane (TM) III and TM VI followed by changes in the conformation of the receptor, in this model, cooperative interactions between the hydroxyl group and Tyr113 in TM III and between the carboxyl group and His256 in TM VI were essential for the potent inverse agonist activity of olmesartan. We speculate that the specific interaction of olmesartan with these two TMs is essential for stabilizing the AT1 receptor in an inactive conformation. A better understanding of the molecular mechanisms of the inverse agonism could be useful for the development of new G protein-coupled receptor antagonists with inverse agonist activity.

Antihypertensive Action of Melatonin in the Spontaneously Hypertensive Rat

The effects of melatonin on blood pressure and heart rate were studied in 23-week-old male spontaneously hypertensive rats. Melatonin infused i.p. at a dose of 6 mg/rat per day for 5 days using an osmotic minipump produced a significant reduction of blood pressure and a slight but significant decrease of heart rate in the conscious and unrestrained state. These cardiovascular effects of melatonin developed gradually. Plasma renin concentration tended to decrease after melatonin treatment. These results demonstrate that melatonin has an antihypertensive action. The mechanism of the antihypertensive action of melatonin requires further study.

New pharmacologic aspects of CS-866, the newest angiotensin II receptor antagonist

CS-866 is a new angiotensin II receptor blocker that has demonstrated effectiveness for lowering blood pressure in animal models of hypertension. Given the proposed involvement of the renin-angiotensin system in diabetic nephropathy and atherosclerosis, we have tested CS-866 in animal models of these conditions. The renal protective properties of CS-866 were examined in the Zucker diabetic fatty (ZDF) rat, a model of type 2 diabetes that develops progressive hyperglycemia, glomerulosclerosis, and proteinuria. Treatment of ZDF rats with CS-866 in the diet for 19 weeks resulted in a dose-dependent reduction in urinary protein excretion compared with vehicle-treated control rats, which was independent of changes in blood pressure and glycemic state. The antiatherosclerotic properties of CS-866 were tested in 2 animal models. In the first study, cynomolgus monkeys were fed a high-cholesterol diet for 6 months while receiving CS-866 or vehicle. At the end of this period, CS-866-treated animals had 64% less plaque area in the aorta than controls. CS-866 was also tested in the Watanabe heritable hyperlipidemic (WHHL) rabbit model of atherosclerosis. WHHL rabbits were treated for 32 weeks with CS-866 (1 mg/kg), pravastatin (50 mg/kg), a combination of the 2 drugs, or vehicle. CS-866 had no effect on plasma cholesterol levels and reduced blood pressures minimally. Pravastatin alone reduced serum cholesterol but had no effect on blood pressure or lesion area. In contrast, treatment with CS-866 resulted in a 40% reduction in lesion area compared with vehicle-treated control when given alone and a 50% reduction in combination with pravastatin. On the basis of results from animal models, CS-866 may be a useful treatment for diabetic nephropathy and atherosclerosis.

Effects of captopril on urinary excretion of prostaglandins and electrolytes in spontaneously hypertensive rats

Captopril was administered to spontaneously hypertensive rats (SHR) and the acute and long-term effects on the urinary excretion of Na, K, prostaglandin (PG)E2 and PGF2 alpha were studied. Captopril (30 mg and 100 mg/kg p.o.) increased urine volume, urinary Na and K excretion on the 1st and 2nd days of its administration. The urinary K/Na ratio was decreased in the captopril-treated groups on the 1st to 3rd day. Urinary PGE2 excretion in the captopril 100 mg/kg group was significantly increased compared to that in the control group (210.6 +/- 31.4 vs. control 123.2 +/- 8.4 ng/day) on the 1st day of captopril administration. A slight but significant increase in urinary PGF2 alpha excretion was also seen in the captopril 100 mg/kg group (105.2 +/- 9.8 vs. control 75.8 +/- 2.1 ng/day). But PG excretion declined during prolonged treatment and these were no significant difference between the control and captopril-treated groups on the 3rd and 6th week. These data suggest that captopril increases renal PG only as an acute phase and that the increase of PG at least partly accounts for natriuretic affects of the agent.

Small Molecules with Similar Structures Exhibit Agonist, Neutral Antagonist or Inverse Agonist Activity toward Angiotensin II Type 1 Receptor

Small differences in the chemical structures of ligands can be responsible for agonism, neutral antagonism or inverse agonism toward a G-protein-coupled receptor (GPCR). Although each ligand may stabilize the receptor conformation in a different way, little is known about the precise conformational differences. We synthesized the angiotensin II type 1 receptor blocker (ARB) olmesartan, R239470 and R794847, which induced inverse agonism, antagonism and agonism, respectively, and then investigated the ligand-specific changes in the receptor conformation with respect to stabilization around transmembrane (TM)3. The results of substituted cysteine accessibility mapping studies support the novel concept that ligand-induced changes in the conformation of TM3 play a role in stabilizing GPCR. Although the agonist-, neutral antagonist and inverse agonist-binding sites in the AT1 receptor are similar, each ligand induced specific conformational changes in TM3. In addition, all of the experimental data were obtained with functional receptors in a native membrane environment (in situ).

Effects of long-term blockade of angiotensin converting enzyme with captopril on blood pressure and aortic prolyl hydroxylase activity in spontaneously hypertensive rats

In order to determine if long-term treatment with captopril affects vascular collagen biosynthesis we administered the agent at a dose of 30 or 100 mg/kg/day p.o. for 6 weeks to spontaneously hypertensive rats. At the 6th week of captopril treatment the aorta was excised and the activity of prolyl hydroxylase, the rate-limiting enzyme of collagen biosynthesis, was measured. Vascular enzyme activity was decreased by 7.0% and 34.6% in the low- and high-dose groups, respectively. A single oral administration of captopril, on the other hand, did not decrease the enzyme activity in the aorta. Furthermore, captopril had no direct inhibitory effect on the enzyme activity in vitro. These findings suggest that the decrease in vascular prolyl hydroxylase activity resulted from a sustained decrease in blood pressure.

Relationship between inhibition of pressor response to angiotensin I and blood concentration of captopril following a single oral administration to dogs

The time courses for the inhibition of the pressor response to AI and for the blood concentration of captopril and its metabolites were determined following a single oral administration of captopril at a dose of 0.8 or 2.5 mg/kg to normal dogs. Captopril was rapidly absorbed from the gastro-intestinal tract attaining a maximum blood concentration 1-1.5h after its administration. The inhibition of the pressor response to AI was closely related with the blood concentration of unchanged captopril but not of total captopril. The blood concentrations of captopril which produced 50 and 100% inhibition were estimated to be 0.1 and 0.4 millimicron/ml respectively. At 2.5 mh/kg the maximum blood concentration attained was 4 times higher than the concentration required to completely inhibit the pressor response to AI, suggesting that a fraction of the absorbed captopril was in excess of the therapeutic requirement.

Antihypertensive effects of captopril in combination with diuretics in spontaneously hypertensive rats.

To examine the antihypertensive effects of captopril, when used in combination with diuretics, an oral dose of the agent (30 mg/kg) was given to spontaneously hypertensive rats pretreated for 1 week with trichlormethiazide (TCTZ), hydrochlorothiazide (HCTZ), furosemide, or water. One-week treatment with diuretics did not lower blood pressure, but potentiated the anti-hypertensive action of captopril in magnitude as well as duration. The degree of potentiation was in the following order: furosemide ≥ HCTZ > TCTZ. Plasma renin concentration (PRC) prior to the administration of captopril was highest in the furosemide group, followed by the HCTZ, TCTZ, and control groups, in this order. When data from four groups were taken together, the reduction of blood pressure following captopril administration was closely related to PRC before administration. These data suggest that repeated administration of diuretics renders the maintenance of blood pressure more dependent on the renin-angiotensin system without affecting blood pressure and that this situation underlies the potentiation of the antihypertensive action of captopril by combined use of diuretics. There was no qualitative difference between furosemide and thiazides in the potentiation of anti-hypertensive action of captopril.

Long-Term Effects of Captopril on Passive Sodium Permeability and Contractility in Vascular Smooth Muscles of Spontaneously Hypertensive Rats

Long-term effects of captopril on passive sodium permeability and cellular sodium concentration in the aorta of spontaneously hypertensive rats were examined in terms of contractile properties of the muscle. In cold lithium solution, cellular sodium in the aorta isolated from spontaneously hypertensive rats leaked more rapidly than did that isolated from Wistar Kyoto rats (WKY), suggesting an increased sodium permeability in the aorta of spontaneously hypertensive rats. Six weeks of treatment with captopril reduced the increased sodium permeability after hydralazine had failed. Inhibition of the sodium pump by incubating the aorta in potassium-free solution produced a slowly developing contraction which was associated with accumulation of cellular sodium. Both the magnitude of contraction and the accumulation of sodium during exposure to potassium-free solution for 1 hour were significantly less in the aorta isolated from spontaneously hypertensive rats treated with captopril compared with those from control spontaneously hypertensive rats. These data suggest that after prolonged administration, captopril alters the abnormal sodium permeability of vascular smooth muscle in spontaneously hypertensive rats and that the restoration of normal permeability reduces vascular tone.