Effect of high concentration of glucose on dopamine release from pheochromocytoma-12 cells.

To investigate the mechanism of the action of high concentration of glucose on transmitter release from neuronal cells, we examined the effect of high concentration of glucose on dopamine release from pheochromocytoma-12 (PC12) cells. When the cells were incubated with 9.0 or 13.5 mg/mL glucose (2- or 3-fold of the optimum glucose concentration for PC12 cells), dopamine release was increased in a dose-related manner. Glucose-induced increase in dopamine release was blunted by nicardipine, a Ca2+ channel blocker. Following addition of 13.5 mg/mL glucose, intracellular Ca2+ concentration was increased, which was eliminated by nicardipine. Administration of 9.0 or 13.5 mg/mL glucose induced membrane depolarization in a dose-related manner. Glucose-induced dopamine release was inhibited by pinacidil or diazoxide, adenosine triphosphate (ATP)-sensitive K+ channel (KATP channel) openers. These results suggest that a high concentration of glucose induced ATP production, which blocked the KATP channel to induce membrane depolarization, and increased intracellular Ca2+ concentration and dopamine release. When the cells were cultured with 9.0 or 13.5 mg/mL glucose for 7 days, high potassium chloride (KCl)-induced dopamine release and 45Ca2+ uptake were increased. These results suggest that long-term incubation with a high concentration of glucose increased the capacity of Ca2+ uptake to enhance depolarization-induced dopamine release from PC12 cells. These data taken together suggest that a high concentration of glucose induced activation of the Ca2+ channel to stimulate dopamine release from PC12 cells.

Involvement of nitric oxide in glucose toxicity on differentiated PC12 cells: prevention of glucose toxicity by tetrahydrobiopterin, a cofactor for nitric oxide synthase.

Effects of high concentrations of glucose on cell survival of differentiated PC12 cells were examined. Seven day-culture with D-glucose (9.0-27.0 mg/ml as 2-6-fold of the optimal level) induced cell death in a dose-related manner but 3-day culture with high concentrations of glucose had no effect on cell viability. L-glucose had no effect on viability of PC12 cells, suggesting that D-glucose toxicity was independent of its osmolarity effect. Seven-day culture with D-glucose (13.5 mg/ml as 3-fold of the optimal level) increased nitric oxide metabolites (NOx) in the culture medium. Glucose-induced increase in NOx was eliminated by 0.1 mM L-nitro-arginine methylester (L-NAME), a nitric oxide synthase (NOS) inhibitor. Intracellular Ca(2+) concentration was increased by D-glucose in a dose-related manner, suggesting that D-glucose activated NOS by increasing intracellular Ca(2+) concentration in PC12 cells. Glucose-induced cell death was blunted by 0.1 mM L-NAME, showing that nitric oxide (NO) was involved in the glucose toxicity to PC12 cells. Tetrahydrobiopterin (BH(4)), a cofactor for NOS, attenuated both glucose-induced cell death and NOx production at 1 microM but not at 10 microM. The effects of BH(4) on glucose-induced cell death and NOx production were not mimicked by reducing agents such as ascorbate and cysteine. These results taken together suggest that high concentrations of glucose induced cell death via NO production and that low concentration of BH(4) had a protective effect against glucose neurotoxicity in differentiated PC12 cells.

Effect of tetrahydrobiopterin on nitric oxide synthase-containing cells in the rat hippocampus.

We have observed that tetrahydrobiopterin (BH4), a cofactor of nitric oxide synthase (NOS), acts as a self-protection factor against nitric oxide (NO) toxicity in PC12 cells. To further investigate the self-protection action of BH4 in vivo, the effect of deletion of endogenous BH4 on NO-producing cells was examined in the rat hippocampus. Following the peripheral infusion of 50 mM 2,4-diamino-6-hydroxypyrimidine (DAHP), an inhibitor of GTP cyclohydrolase I, using a miniosmotic pump for 14 days, BH4 content in the hippocampus decreased as compared with the control group administered with vehicle solution, which had no effect on brain BH4 content. When the rats were administered with 50 mM DAHP and 10 mM BH4, the DAHP-induced decrease in BH4 content was prevented. The extracellular concentration of NO metabolites remained unchanged following DAHP administration, suggesting that DAHP-induced decrease in BH4 content had no effect on NO production. The number of NOS-positive cells decreased following DAHP administration in the hippocampal regions, while the number of NOS-negative cells remained unchanged. The DAHP-induced decrease in the NOS-positive cell number was prevented by the administration of 10 mM BH4 and DAHP. These results suggest that endogenous BH4 may affect NOS-positive cell number in the rat hippocampus.

Effect of continuous subcutaneous administration of a low dose of G-CSF on stem cell mobilization in healthy donors: a feasibility study.

We investigated the effects of low-dose granulocyte colony-stimulating factor (G-CSF) on the mobilization of stem cells in 6 healthy subjects. When G-CSF was administered by continuous subcutaneous infusion at a rate of 72 microg/day for 5 days, the numbers of white blood cells and granulocytes rapidly increased to maximal levels. CD34+ cells were mobilized to the peripheral blood in 3 days, and the maximal level was reached 4 or 5 days after the start of treatment. We attempted to determine whether the levels of mobilized stem cells that we could obtain using this method would be sufficient for peripheral blood stem cell transplantation. Two of the 6 subjects complained of mild bone pain 4 or 5 days after the start of treatment, but the pain did not affect their daily activities. Only 1 abnormal result (for serum alkaline phosphatase) was found in the laboratory data. The present preliminary results have provided us with a framework for a prospective study comparing low-dose continuous infusion with conventional mobilization procedures.

Possible involvement of tetrahydrobiopterin in the trophic effect of insulin-like growth factor-1 on rat pheochromocytoma-12 (PC12) cells.

Tetrahydrobiopterin (BH(4)) has a trophic effect on pheochromocytoma-12 (PC12) cells such as insulin-like growth factor-1 (IGF-1). We investigated involvement of BH(4) in the trophic effect of IGF-1 on PC12 cells. IGF-1 (10-300 ng/ml) increased cellular BH(4) content in a dose-related manner. Cellular BH(4) content increased after 6-36 h incubation with IGF-1. IGF-1-induced increase in the cellular BH(4) content was blunted by 0.3 mM 2,4-diamino-6-hydroxypyrimidine (DAHP), an inhibitor for BH(4) synthesis. IGF-1 protected PC12 cells from the cell death induced by depletion of serum and nerve growth factor, which was attenuated by DAHP. The effects of IGF-1 on the cellular BH(4) content and cell viability were eliminated by 0.2 microM wortmannin. These results suggest that BH(4) is involved in the trophic effect of IGF-1 on PC12 cells and that the effect of IGF-1 on BH(4) synthesis is mediated by phosphatidylinositol 3-kinase.

Monitoring of urine nitric oxide (NO) related substrates and immunological competence in hematological malignancy.

It has been reported that concentrations of neopterin in the urine are changed according to the host immunological conditions. In the present study, we measured urinary concentration of neopterin in patients with malignant hematological disorders and investigated the relationship between urinary neopterin levels and laboratory indices for cellular immunity. Urine neopterin levels were correlated with serum sIL-2R levels in the patients with malignant lymphoma, and inversely correlated with lymphocyte reactivity with ConA in the patients with acute myelocytic leukemia. However, no significant correlation was observed between urine neopterin levels and lymphocyte reactivity with phytohemagglutinin and pokeweed mitogen, CD4/8 ratio, CD56+ 16+ subset or serum IFN-gamma levels. In the patients with malignant lymphoma, parallel changes in serum sIL-2R and urine neopterin were observed. The presented results suggest that urine neopterin levels are related to the activation of T cells in malignant lymphoma.

Cytotoxic effect of nitric oxide on human hematological malignant cells.

We investigated the cytotoxic effect of nitric oxide (NO) on primary culture of human hematological malignant cells. Sodium nitroprusside (SNP), an NO donor, had cytotoxic effects on the cells of some patients with malignant lymphoma (ML), acute myelocytic leukemia (AML) or chronic myelomonocytic leukemia (CMMoL), but not with multiple myeloma. Cultured cells from the ML patient remained sensitive to SNP after the cells became resistant to anti-cancer drugs. In contrast, the cells from the patients with AML and CMMoL became resistant to SNP while anti-cancer drugs remained effective. In samples of the cells of the patients with ML and AML, the number of CD3 positive lymphoma cell was decreased by SNP and the number of CD33 negative cells and normal B lymphocytes (CD19 positive cells) were increased. In the cells of the patient with ML, apoptosis was induced by SNP. SNP had no effect on lymphocytes of healthy volunteers. These results suggest that SNP had an anti-tumor effect on human hematological malignant cells.

Growth hormone-releasing hormone and gonadotropin-releasing hormone stimulate nitric oxide production in 17beta-estradiol-primed rat anterior pituitary cells.

It was reported that neuronal nitric oxide synthase (nNOS) was expressed only in gonadotrophs and folliculo-stellate cells in the anterior lobe of the pituitary gland. However, recent studies have demonstrated the occurrence of nNOS in the somatotrophs and lactotrophs. In the present study, we investigated effects of growth hormone-releasing hormone (GHRH), gonadotropin-releasing hormone (GnRH), and 17beta-estradiol on nitric oxide (NO) release in cultured rat anterior pituitary cells in vitro. The NO2- level in the incubation medium of the rat anterior pituitary cells was dependent on the cell density. Pretreatment with 10 microM 17beta-estradiol resulted in an increase in medium NO2- level. GHRH and GnRH failed to change medium NO2- levels, but they elicited increases in medium NO2- levels in estrogen-treated cells. The GHRH-induced increase in NO2- level was inhibited by Nomega-nitro-L-arginine methyl ester, a NOS inhibitor. These findings suggest that GnRH and GHRH could activate nNOS in the gonadotrophs and the somatotrophs, respectively.

Relationship between visceral fat accumulation and anti-lipolytic action of insulin in patients with type 2 diabetes mellitus.

Insulin resistance is closely related to developing type 2 diabetes mellitus. Visceral fat accumulation is associated with insulin resistance, which affects the free fatty acid (FFA) metabolism. We investigated the interactions among visceral fat accumulation, FFA metabolism and insulin resistance in 20 patients with type 2 diabetes mellitus, including 11 obese and 9 non-obese subjects. Body fat distribution was estimated by measuring the areas of both subcutaneous and visceral fat mass on abdominal computed tomography at the umbilical level. Glucose infusion rate (GIR) and plasma FFA responses to insulin were determined as an index of insulin resistance and anti-lipolytic action, respectively, in a euglycemic hyperinsulinemic clamp study. There was an inverse correlation between GIR and insulin-induced decrease in plasma FFA in all diabetic patients (r = -0.652, P < 0.01). Visceral fat mass area was well correlated with GIR (r = -0.583, P < 0.01) and insulin-induced decrease in plasma FFA (r = 0.724, P < 0.001), whereas subcutaneous fat mass area was not correlated either with GIR or plasma FFA decrease. These findings suggest that visceral fat accumulation results in increasing the resistance against the anti-lipolytic action of insulin, and that FFA metabolism is closely related with glucose utilization in patients with type 2 diabetes mellitus.