Toshitaka Tamai

Effect of cilostazol, a cyclic AMP phosphodiesterase inhibitor, on the proliferation of rat aortic smooth muscle cells in culture.

Summary: Cilostazol, a cyclic AMP phosphodiesterase inhibitor, has been used as an antiplatelet agent. In the present study, we investigated the in vitro effect of cilostazol on DNA synthesis in rat aortic arterial smooth muscle cells (SMCs) in culture stimulated with fetal calf serum (FCS), platelet-derived growth factor (PDGF), insulin, or insulin-like growth factor-I (IGF-I). Micromolar concentrations of cilostazol inhibited [3H]thymidine incorporation into DNA and cell growth as determined by cell number and protein concentration. Treatment with cilostazol increased the intracellular concentration of cyclic AMP, suggesting that the inhibition of SMC proliferation by cilostazol may be mediated through increased levels of cyclic AMP. The results suggested that cilostazol, by interfering with the proliferation of arterial SMCs, may have potential to prevent initiation and progression of atherosclerosis.

Plasma thrombomodulin concentration in diabetes mellitus

Thrombomodulin is an endothelial cell membrane protein acting as a cofactor for the activation of plasma protein C. Recently, it was found that soluble forms of thrombomodulin exist in plasma. Although the physiological significance of circulating thrombomodulin is presently obscure, it may reflect injury of the endothelial cell. In the present study, we examined plasma thrombomodulin concentrations in 106 Type 2 (non-insulin-dependent) diabetic patients. Plasma thrombomodulin was determined by a sandwich ELISA employing monoclonal anti-thrombomodulin antibodies. The patients with proteinuria had higher plasma thrombomodulin concentrations (61.0 +/- 36.0 ng/ml) compared to the patients without proteinuria (33.6 +/- 9.5 ng/ml, P less than 0.001) and control subjects (32.8 +/- 6.5 ng/ml, P less than 0.001). Plasma thrombomodulin concentrations were positively correlated with the level of serum creatine, blood urea nitrogen, urinary albumin and urinary beta 2-microglobulin (P less than 0.001 for each), but not with fasting plasma glucose, hemoglobin A1c or fructosamine. Elevated plasma thrombomodulin was also observed in the patients with pre-proliferative (63.4 +/- 28.9 ng/ml) or proliferative retinopathy (57.4 +/- 34.7 ng/ml), but not in the patients with non-proliferative retinopathy (33.5 +/- 12.9 ng/ml) or those without retinopathy (32.4 +/- 8.9 ng/ml). Even in the 81 diabetic subjects without proteinuria as determined by a dip and read method, and whose serum creatinine was lower than 1.0 mg/dl, the plasma thrombomodulin concentration was significantly higher in the patients with pre-proliferative (41.5 +/- 4.4 ng/ml) and proliferative retinopathy (41.0 +/- 12.8 ng/ml) compared to the patients without retinopathy (32.2 +/- 8.8 ng/ml) and those with non-proliferative retinopathy (31.9 +/- 7.8 ng/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

Increase in neutral cholesteryl ester hydrolase activity produced by extralysosomal hydrolysis of high-density lipoprotein cholesteryl esters in rat hepatoma cells (H-35)

The metabolism of high-density lipoprotein-associated cholesteryl esters (HDL-CE) in liver cells is not well understood. We studied the possible role of lysosomal and extralysosomal pathways on such metabolism by measuring the uptake and hydrolysis of HDL-CE in H-35 rat hepatoma cells. Incubation of cells with [3H]cholesteryl ester-labeled HDL led to the intracellular accumulation of both 3H-free cholesterol and [3H]cholesteryl ester. The ratio of 3H-free cholesterol/[3H]cholesteryl ester increased with an increase in incubation time even in the presence of chloroquine. Because chloroquine did not inhibit the conversion of cholesteryl ester to free cholesterol, the hydrolysis of HDL-CE may have been catalyzed by an extralysosomal enzyme, perhaps by neutral cholesteryl ester hydrolase (NCEH). When we incubated cells with increasing concentrations of HDL, NCEH activity increased. This increase in enzyme activity was not inhibited by the addition of chloroquine. A complex of dimyristoylphosphatidylcholine (DMPC)/apo HDL/cholesteryl ester enhanced the activity as well as native HDL. Neither the DMPC/apo HDL nor the DMPC/cholesteryl ester complex affected the activity, suggesting that apo HDL may be required for the uptake of HDL-CE. The present study demonstrated that the extralysosomal hydrolysis by NCEH is operating in the metabolism of HDL-CE in hepatoma cells.

1α,25-dihydroxyvitamin D3 induces very low density lipoprotein receptor mRNA expression in HL-60 cells in association with monocytic differentiation

Expression of VLDL receptor mRNA during differentiation of HL-60 cells was investigated by Northern analysis. The expression induced in 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3)-treated cells was 3 times that in untreated cells, while LDL receptor mRNA expression was unchanged. VLDL receptor mRNA levels were not changed in macrophages caused to differentiate from HL-60 cells by treatment with phorbol 12-myristate 13-acetate (PMA). Treatment of sarcoma cells which possess the vitamin D receptor (MG-63 cell line) with 1 alpha,25(OH)2D3 did not affect VLDL receptor mRNA levels. Therefore, 1 alpha,25(OH)2D3 induces VLDL receptor mRNA in HL-60 cells through differentiation-dependent mechanisms.

Decreased ketonaemia in the monosodium glutamate-induced obese rats

Plasma concentrations of total ketone bodies, acetoacetate (AcAc) and 3-hydroxybutyrate (3-OHBA) in monosodium glutamate (MSG)-induced obese rats were measured. MSG-treated rats showed higher Lees indices, shorter naso-anal and tail length, and a more marked intraperitoneal fat deposition than control rats. Plasma concentrations of glucose, free fatty acid, triglyceride and phospholipids were significantly increased in the MSG-treated rats as compared to the control rats (24 weeks-old). Plasma levels of total ketone bodies, AcAc and 3-OHBA were all decreased in the MSG-treated rats as compared to control rats. The ratio, 3-OHBA/AcAc in the MSG-treated rats were not different from those in the control rats.

Characteristics of lipoprotein receptors of the isolated liver parenchymal cells prepared from the streptozotocin-induced diabetic rats

Characteristics of lipoprotein receptors of the isolated liver parenchymal cells prepared from the streptozotocin-induced diabetic rats were investigated. Streptozotocin-induced diabetic rats fed 1.0% cholesterol showed the exaggerated hypercholesterolemia as compared to control rats fed 1.0% cholesterol. The present study was designed to elucidate the role of lipoprotein receptor mechanisms of liver parenchymal cells in the diabetic dyslipoproteinemia. 125I-labeled lipoproteins (rat beta-VLDL, human LDL2 or rat HDL3) were incubated with liver parenchymal cells isolated by liver perfusion using collagenase. According to the Scatchard analysis, the apparent dissociation constant (kd) and maximum beta-VLDL binding (Bmax) for the higher affinity binding site in the diabetic rats (n = 6) were (11.9 +/- 5.1) X 10(2) ng/ml and 307.5 +/- 145.2 ng/10(6) cells, respectively. These binding characteristics of the diabetic rats were not significantly different from the control rats. Furthermore, there were no significant differences in the binding characteristics of human LDL2 and rat HDL3 between the diabetic rats and the control rats. The data presented suggest that significant role of alteration of lipoprotein receptor characteristics in liver parenchymal cells is not played in the diabetic dyslipoproteinemia.

Effects of nicorandil on cell proliferation and cholesteryl ester accumulation in arterial smooth muscle cells in culture

SummaryCa2+ regulates a variety of cellular mechanisms in vascular cells as well as in platelets. Nicorandil interacts with the intracellular Ca2+-activated processes in vascular smooth muscle cells, while Ca2+ channel blockers such as verapamil and diltiazem block voltage-dependent Ca2+ channels. The effects of nicorandil are due to the hyperpolarization of the membrane, interference with mobilization of Ca2+ from the intracellular storage sites, and blockade of receptor-operated Ca2+ channels. In the present study, the effects of nicorandil on cell proliferation and cholesteryl ester accumulation in rat arterial smooth muscle cells in culture were compared to Ca2+ channel blockers. Smooth muscle cells were prepared from rat thoracic aorta, and the rate of proliferation was determined by measuring the cell number and by [3H]-thymidine incorporation into cellular DNA. The effect of nicorandil on cholesteryl ester content in smooth muscle cells was determined by thin-layer chromatography of the cell extracts. Nicorandil at concentrations of 10−6 to 10−4 M, as well as Ca2+ channel blockers (verapamil and diltiazem) inhibited the proliferation and DNA synthesis of cultured smooth muscle cells. The acute inhibitory effects on cell proliferation were observed significantly 16 hours after the addition of the three agents in serum-stimulated cells. These effects were dose dependent, both in acute and in chronic treatment with the three agents. Addition of 10−5 M nicorandil to medium supplemented with 10% serum resulted in a decrease of the net cholesteryl ester content by 18±1%, while cellular free cholesterol content was the same as control. Similar results were also obtained in the presence of verapamil and diltiazem. These data suggest that nicorandil may suppress atheroma formation, not only by inhibiting cell proliferation but also by decreasing cholesteryl ester accumulation in arterial smooth muscle cells.

Normoreninemic Hypoaldosteronism in a Case of Isolated ACTH Deficiency

This paper documents the rare and hitherto unreported association between isolated ACTH deficiency and normoreninemic hypoaldosteronism in a 63-year-old woman. Baseline plasma aldosterone and 18-hydroxycorticosterone were extremely low. Both steroids did not respond to exogenous angiotensin II infusion, whereas they were increased in parallel to ACTH stimulation. Thus, acquired dysfunction or congenital dysgenesis of the zona glomerulosa was suspected. The upright posture-furosemide test showed a subnormal but definite plasma aldosterone response coupled with a normal increase in plasma renin activity, indicating that there may be a yet unidentified mechanism(s) underlying the postural increase of aldosterone.

Regulation of lipoprotein receptors on a rat hepatoma cell line

The rat H-35 cultured hepatoma cell line expresses receptors for homologous lipoproteins. In previously reported experiments distinct receptors were identified for chylomicron remnants, HDL and LDL, by direct binding studies that yielded distinctive binding constants, cross competition assays, and by differential inhibitory effects of EDTA and suramin. In the present experiments, the regulation of expression of these receptors was assessed by growing cells either in the presence or absence of lipoproteins in the media and by growing cells to different densities (50-800 micrograms cell protein/dish). LDL binding to cells was increased by lipoprotein deprivation at all cell densities. LDL binding was inversely related to cell density when cells were grown in lipoprotein deficient serum (LPDS) but cell density did not affect LDL binding by cells grown in newborn calf serum (NBCS). By contrast HDL binding was not appreciably different whether cells were grown in NBCS or in LPDS. However, HDL binding was inversely related to cell density by cells grown either in LPDS or in NBCS. Binding of chylomicron remnants was increased by growth in LPDS at all densities, but altering growth density in either culture medium had little effect on the cellular binding of chylomicron remnants. The distinctive effects of these experimental perturbations on the binding of the 3 lipoprotein classes tend to confirm the presence of 3 separate receptor activities. The experiments also demonstrate that the responses at least of some of the receptors of the hepatoma cells in culture resemble those of hepatocytes in vivo and in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of Alpha-Glucosidase Inhibitors by Once a Day Administration on the Blood Glucose Level.

The protective effect of α-glucosidase inhibitor (GI) by once a day administration against the increase of postprandial blood glucose (ΔPBG) was examined. Using a control group of 5 hospitalized patients with non-insulin-dependent diabetes mellitus (NIDDM) consisting who received only dietary treatment, we demonstrated the effect of GI while considering the influence of an inproved of living environment during hospitalization. The effects on the blood glucose level were studied using the GI treatment group, which consisted of 8 hospitalized NIDDM patients who received medical treatment with GI, and compared the findings before and after GI treatment. In addition, we also compared two GI treatment groups, one which received treatment 3 times a day and the other which was treated only once a day. Blood samples were obtained at 30 min before and 2h after each meal, before sleep and early the next morning. The increase in the postprandial blood glucose levels decreased significantly by the once a day administration of GI, but no significant changes were observed in the control group. The in crease in (ΔPBG) level was also reduced by the once a day administration of GI, and the effect was also similar to that after the 3 times a day administration.Our findings in this small group of patients suggest that good glycemic control can be obtained all day long by the once a day administration of GI.