The essential histidine residues of bovine plasma amine oxidase.

Ethoxyformic acid anhydride and photooxidation have been used to study the function of histidine residues in bovine plasma amine oxidase. Ethoxyformic acid anhydride at pH 6.1 reacted with nearly all of the histidine residues in the enzyme in 15 min but complete enzyme inactivation occurred in several minutes. The concentration of the reagent which caused 50% inhibition was 2.2-10(-5) M under the conditions of the experiment. The diamine oxidases, Aspergillus niger and pea seedling amine oxidases were also inhibited by ethoxyformic acid anhydride. The concentrations of reagent required for 50% inhibition were 6.6-10(-5) and 3.3-10(-4) M, respectively, for the two enzymes. NH2OH could not be used to regenerate the reacted histidine residues since NH2OH itself inhibited the enzyme. Photooxidation in the presence of 0.001% Rose Bengal at pH 7.0 also inactivated bovine plasma amine oxidase. Histidine was the only amino acid destroyed by photooxidation. About six histidine residues were destroyed but in the presence of the substrate kynuramine, two less histidine residues were destroyed. Since lysine which is neither a substrate nor inhibitor of the enzyme did not protect the enzyme from photooxidation, it was concluded that two histidine residues, one in each sub-unit of the enzyme are essential for activity.

Actions of novel antidiabetic thiazolidinedione, T-174, in animal models of non-insulin-dependent diabetes mellitus (NIDDM) and in cultured muscle cells.

1. The antihyperglycaemic effect and the possible mechanism of action of T-174, a novel thiazolidinedione derivative, was determined in vivo and in vitro. 2. Oral administration of T-174 markedly improved hyperglycaemia, hyperinsulinaemia, hyperlipidaemia, and glucose intolerance in genetically obese and diabetic yellow KK (KK-Ay) mice (0.2-15.5 mg kg(-1) day(-1), for 7 days) and Zucker fatty rats (1.4-11.4 mg kg(-1) day(-1), for 6 days). The ED50 values for the glucose lowering action of T-174 and pioglitazone, another thiazolidinedione antidiabetic, were 1.8 and 29 mg kg(-1) day(-1), respectively in KK-Ay mice; T-174 was about 16 times more potent than pioglitazone. 3. The hypoglycaemic effect of exogenous insulin in KK-Ay mice was enhanced after the administration of T-174. A hyperinsulinaemic euglycaemic clamp study in Zucker fatty rats showed an amelioration of whole-body insulin resistance by the T-174 treatment. 4. Insulin-stimulated glucose metabolism was enhanced in adipocytes from KK-Ay mice treated with T-174. The insulin receptor number of the adipocytes was increased without a change in the affinity of the receptor. 5. The hypomagnesaemia in KK-Ay mice was completely restored by T-174. 6. In cultured L6 myotubes, glucose consumption and [3H]-2-deoxy-glucose transport were enhanced by T-174 (EC50; 6 and 4 microM, respectively). Combination of insulin with T-174 was additive to stimulate glucose disposal. 7. These results suggest that the antihyperglycaemic effect of T-174 was mediated by enhanced insulin action. This was associated with amelioration of the hypomagnesaemia and T-174 directly increased basal and insulin-stimulated glucose utilization by cultured muscle cells.

Effects of (-)-(R)-1-(p-hydroxyphenyl)-2-[3,4-dimethoxyphenethyl)amino]ethanol (TA-064), a new cardiotonic agent, on circulating parameters of carbohydrate and lipid metabolism in the rat.

Effects of the new cardiotonic and selective beta 1-adrenergic agonist TA-064, (-)-(R)-1-(p-hydroxyphenyl)-2-[(3,4-dimethoxyphenethyl)amino] ethanol, on circulating concentrations of glucose, lactate, free fatty acids (FFA), glycerol, cyclic AMP and the pancreatic hormones insulin (IRI) and glucagon (IRG) were examined in rats. TA-064, administered orally or intraperitoneally at the dose of 10 mg/kg (ca. 50 times the therapeutic dose) or higher, caused a slight transient rise followed by a persistent lowering of blood glucose concentrations, but it did not affect blood lactate levels at all. The same treatment with TA-064 elevated the concentrations of blood FFA, glycerol and plasma IRI and IRG. These changes induced by TA-064 were inhibited by pretreatment with propranolol (a non-selective beta-adrenergic antagonist) and practolol (a selective beta 1-adrenergic antagonist). The non-selective beta-adrenergic agonist isoproterenol and the selective beta 2-adrenergic agonist terbutaline elevated both blood glucose and lactate when administered intraperitoneally. They also brought about sustained rises in blood glycerol and plasma IRI, but only transiently increased the plasma IRG level. The cardiotonic agent prenalterol, claimed to be a selective beta 1-agonist, elevated blood glucose, lactate, and glycerol only slightly, and plasma IRI significantly, but it had no effect on plasma IRG. The cardiotonic agents dobutamine and amrinone also elevated blood glucose. Thus, TA-064 is unique among the beta-adrenergic and other cardiotonic agents in that it produces sustained hypoglycemia while it has no lactacidemic effect. Since this hypoglycemic action of TA-064 was always preceded by a rise in plasma IRI and abolished in streptozotocin-diabetic rats, we conclude that increased secretion of pancreatic insulin and the lack of hyperglycemic action are responsible for the hypoglycemia by high doses of TA-064.

Beta 1-adrenergic selectivity of the new cardiotonic agent denopamine in its stimulating effects on adenylate cyclase.

Effects of the new selectively beta 1-adrenergic cardiotonic drug denopamine (TA-064), (-)-(R)-1-(p-hydroxyphenyl)-2-[(3,4-dimethoxyphenethyl)amino]ethanol, on the adenylate cyclase-adenosine-3',5'-monophosphate (c-AMP) system of various tissues and cells in rats and guinea pigs were investigated in comparison with those of isoproterenol. Denopamine at concentrations above 10(-6) M stimulated lipolysis in vitro, and, above 10(-5) M, elevated the c-AMP level in isolated rat fat cells. The c-AMP level of guinea-pig heart ventricular muscle was also elevated when the heart was perfused with 3 X 10(-6) M denopamine or when slices of ventricular muscle were incubated with 10(-6) M denopamine. These changes were abolished in the presence of beta-adrenergic antagonists. Incubation with denopamine did not cause substantial elevation of c-AMP levels in rat reticulocytes and diaphragm. Denopamine activated adenylate cyclase of the rat cell membranes in a concentration-dependent manner. Although dose dependence was less apparent, denopamine also activated adenylate cyclase of the membrane fraction from guinea pig cardiac muscle, but it hardly activated the same enzyme from rat reticulocytes. Isoproterenol, on the other hand, showed marked concentration-dependent activation of adenylate cyclase in all these preparations. Denopamine did not inhibit c-AMP phosphodiesterase of both particulate and supernatant fractions of guinea-pig cardiac muscle. The stimulation of lipolysis by denopamine was observed even when elevation of the c-AMP level was not detected, while the stimulation of lipolysis by isoproterenol was always accompanied with an elevation of c-AMP. When guinea-pig hearts were perfused with 3 X 10(-6) M denopamine or 10(-7) M isoproterenol, their cardiotonic effects were of the same magnitude whereas the degree of c-AMP elevation in the ventricular tissue by denopamine was significantly less than that by isoproterenol. It was concluded that stimulation of the adenylate cyclase-c-AMP system by denopamine was restricted to the tissues whose receptors were predominantly of the beta 1-type, and that the elevation of c-AMP levels in these tissues by denopamine was less marked than by isoproterenol, suggesting that the stimulation of lipolysis and heart by denopamine may be mediated by a special pool of c-AMP or some other unknown factor(s).

Hypoglycemic activity of 1-alpha-(3,4-dimethoxyphenethylaminomethyl) -2-hydroxybenzylalcohol 1/2 fumarate (TA-078) in the mouse, rat and dog.

1-alpha-(3,4-Dimethoxyphenethylaminomethyl)-2-hydroxybenzylalcohol 1/2 fumarate (TA-078) is a new hypoglycemic agent structurally different from any existing hypoglycemic drug. It depresses the rise of blood glucose when it is orally administered to glucose-loaded mice, rats and beagle dogs at minimal doses of 1, 10 and 2.5 mg/kg, respectively. In contrast with tolbutamide, TA-078 hardly affected fasting blood glucose levels in rats and dogs and only weakly reduced fasting blood glucose levels in mice. Oral administration of TA-078 to KK mice also improved glucose tolerance, while no improvement was observed in streptozotocin-diabetic rats. TA-078 elevated plasma immunoreactive insulin (IRI) levels in mice and rats soon after its oral administration. In fasted rats, TA-078 caused only a transient increase in plasma IRI but did not affect plasma immunoreactive glucagon (IRG) levels in the early phase after its administration. On the other hand, tolbutamide induced a sustained increase in plasma IRI and a transient but marked decrease in plasma IRG. In perfused rat pancreas, TA-078 stimulated insulin secretion. The stimulation by 10 micrograms/ml TA-078 in the perfusion liquid required the presence of a normal concn (5.6 mM) of glucose, whereas the same concn of tolbutamide stimulated insulin release even at a low glucose concn (2.8 mM).

Na(+)-glucose cotransporter inhibitors as antidiabetics. I. Synthesis and pharmacological properties of 4'-dehydroxyphlorizin derivatives based on a new concept.

Based on our new concept that inhibitors of the Na(+)-glucose cotransporter (SGLT) would be useful as antidiabetics, 4'-dehydroxyphlorizin derivatives 1a--f were designed, synthesized, and examined for various pharmacological properties related to antidiabetic activity. In normal rats, 1a, e and phlorizin showed a strong SGLT-inhibitory effect and significantly increased urinary glucose on intraperitoneal administration at 10 mg/kg, though only 1a resulted in excretion of large quantities of urinary glucose on oral administration at 100 mg/kg. Compounds 1a, e, and phlorizin markedly inhibited glucose uptake in the small intestine during enteric perfusion in normal rats. Compound 1a had a significant reducing effect on blood glucose in the glucose tolerance test in mice when administered orally and also lowered blood glucose in streptozotocin-induced diabetic rats. The aglycons 2a, e of 1a, e, and 1a showed weak inhibitory effects on the facilitated glucose transporter-1 (GLUT-1) in human erythrocytes, while phloretin had a strong inhibitory effect on GLUT-1. Compound 1a caused no apparent renal damage in rats when administered orally at 1 g/kg for 4 successive weeks. Thus, 1a was considered to be a promising candidate as a lead compound for antidiabetics of a new type, and was selected for further pharmacological evaluation.

[Effects of diltiazem on developing blood pressure and accompanying cardiac and vascular hypertrophy in SHR].

To study the effects of Ca antagonists on hypertension induced cardiac and vascular hypertrophy, diltiazem, at the doses of 30 and 60 mg/kg, p.o., twice a day was administered to SHR from 4 to 16 weeks of age. In addition to suppression of developing blood pressure, left ventricular wet weight was significantly decreased in diltiazem treated SHR. Calcium contents of the abdominal aorta was significantly decreased in this group. On the other hand, in SHR, diltiazem at the dose of 60 mg/kg, p.o., decreased mean blood pressure about 45 mmHg and decreased heart rate about 70 beats/min. Plasma renin activity was significantly elevated, but plasma aldosterone concentration was not changed under this condition. In conclusion, diltiazem can suppress hypertension induced cardiac hypertrophy. In addition to the decrease in the afterload, moderate baroreflex induced augmentation of sympathetic drive and lack of volume overload through the renin-angiotensin-aldosterone system was shown to contribute to this effect.

Human T-cell lymphotropic virus type I (HTLV-I) antibodies in pre-diagnostic serum of patients with familial adult T-cell leukemia/lymphoma (ATL).

In a study of 7498 American men of Japanese ancestry in Hawaii, 26 incident cases of leukemia or non-Hodgkin's lymphoma were identified after a follow-up period of 19 years. Two of the cases, who were brothers, were diagnosed with adult T-cell leukemia/lymphoma (ATL). Both of these brothers had human T-cell lymphotropic virus type I (HTLV-I) antibodies in their stored serum which were obtained 4 and 18 years before diagnosis. None of the 24 patients with other hematologic malignancies or the 26 matched controls were HTLV-I antibody positive. This finding lends further support for a role of HTLV-I in the etiology of adult T-cell leukemia/lymphoma.

Novel benzoxazole 2,4-thiazolidinediones as potent hypoglycemic agents. Synthesis and structure-activity relationships.

A new series of benzoxazole 2,4-thiazolidinediones was synthesized and evaluated for hypoglycemic activity in genetically obese and diabetic yellow KK mice. 2-Arylmethyl- and 2-(heteroarylmethyl)benzoxazole derivatives showed far more potent activity than known 2,4-thiazolidinedione derivatives such as ciglitazone, troglitazone and pioglitazone. A facile synthesis of benzoxazole 2,4-thiazolidinediones was also established using aminophenol 2,4-thiazolidinedione (11) as a key intermediate. Details of synthesis and structure-activity relationships for this series are described.