Toshio Nakaki

Up-regulation of nitric oxide synthase by estradiol in human aortic endothelial cells

We have examined the effects of sex hormones on calcium‐dependent NO production and protein levels of NO synthase in cultured human aortic endothelial cells, which were treated with various doses of 17β‐estradiol and testosterone for 8–48 h. Treatment with 17β‐estradiol enhanced calcium‐dependent NO production, but testosterone had exerted no effect. Western blot using monoclonal anti‐human endothelial NO synthase antibody clarified that increased NO production by 17β‐estradiol treatment was accompanied by increased NO synthase protein. Our results provide evidence that human endothelial NO synthase can be regulated by estrogens.

Inhibition by nitric oxide and nitric oxide-producing vasodilators of DNA synthesis in vascular smooth muscle cells

Effects of nitric oxide (NO) and NO-producing vasodilators such as glyceryl trinitrate and sodium nitroprusside were tested on DNA synthesis in the clonal rat aortic smooth muscle cells, RACS-1. DNA synthesis was estimated by [3H]thymidine incorporation to DNA. NO and NO-producing vasodilators inhibited the DNA synthesis that was induced by 10% fetal calf serum. NO and NO-producing vasodilators also inhibited the basal level of DNA synthesis that occurred possibly as a result of autocrine mechanisms. NO-producing vasodilators also inhibited the fetal calf serum-induced proliferation of cells. Sodium nitroprusside inhibited the endothelin-mediated DNA synthesis. In another mesenchymal cell line, Chinese hamster fibroblast V79 cells, NO and NO-producing vasodilators failed to inhibit DNA synthesis, excluding the possibility of general cell toxicity. An exposure to NO and NO-producing vasodilators resulted in an increase of cyclic GMP (cGMP) content in the RACS-1 cells. A cGMP analog, 8-bromo-cGMP, inhibited DNA synthesis in the RACS-1 cells. These results suggest that EDRF/nitric oxide released from endothelium possibly contributes to inhibition of the DNA synthesis in vascular smooth muscle cells.

Elevated plasma nitrate levels in depressive states

BACKGROUND Previous studies have shown that nitric oxide (NO) synthase inhibitors show preclinical antidepressant-like properties, suggesting that NO is involved in the pathogenesis of depression. The purpose of this study is to examine whether or not NO production increases in depressed patients. METHODS Plasma nitrate concentrations, an index of NO production, were measured by high-performance liquid chromatography in depressed patients (n=17) and compared with patients suffering anxiety (n=6) and with healthy controls (n=12). RESULTS Plasma nitrate concentrations were significantly higher in depressed patients than in patients with an anxiety disorder (P<0.05) or in controls (P<0.01). LIMITATIONS The study group was small. The source of the surplus production of NO in patients with major depressive episode remains unclear. CONCLUSIONS These results suggest that NO production is increased in depression.

Endothelin-mediated stimulation of DNA synthesis in vascular smooth muscle cells

Effects of endothelin on DNA synthesis were investigated in two clones of vascular smooth muscle cells, 1YB4 and A7r5. The peptide stimulated DNA synthesis in both clones with apparent EC50 of less than 1 ng/ml. More than 17 h was required before initiating endothelin-stimulated DNA synthesis. The platelet-derived growth factor at a concentration which had no effects by itself on DNA synthesis enhanced the effect of low concentrations of endothelin. A calcium antagonist, nifedipine, inhibited endothelin-induced DNA synthesis. These data suggest that endothelin stimulates DNA synthesis in vascular smooth muscle cells through nifedipine-sensitive mechanisms that can be modulated by platelet-derived growth factor.

Musculin/MyoR is expressed in kidney side population cells and can regulate their function.

Musculin/MyoR is a new member of basic helix-loop-helix transcription factors, and its expression is limited to skeletal muscle precursors. Here, we report that musculin/MyoR is expressed in adult kidney side population (SP) cells and can regulate their function. SP phenotype can be used to purify stem cell–rich fractions. Microarray analysis clarified that musculin/MyoR was exclusively expressed in kidney SP cells, and the cells resided in the renal interstitial space. Musculin/MyoR-positive cells were decreased in acute renal failure, but infusion of kidney SP cells increased musculin/MyoR-positive cells and improved renal function. Kidney SP cells in reversible acute renal failure expressed a high level of renoprotective factors and leukemia inhibitory factor (LIF), but not in irreversible chronic renal failure. In cultured kidney SP cells, LIF stimulated gene expression of renoprotective factors, and down-regulation of musculin/MyoR augmented LIF-induced gene expression. Our results suggest that musculin/MyoR may play important roles not only in developmental processes but also in regenerative processes in adult tissue.

Pressure promotes DNA synthesis in rat cultured vascular smooth muscle cells.

High blood pressure is one of the major risk factors for atherosclerosis. In this study, we examined the effects of pressure on cell proliferation and DNA synthesis in cultured rat vascular smooth muscle cells. Pressure without shear stress and stretch promotes cell proliferation and DNA synthesis in a pressure-dependent manner. Pressure-induced DNA synthesis was inhibited significantly by the phospholipase C (PLC) inhibitor 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate, the protein kinase C inhibitor H-7, 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine, staurosporine, and the tyrosine kinase inhibitor ([3,4,5-trihydroxyphenyl]methylene)propanedinitrile. To clarify whether activation of PLC and calcium mobilization are involved in pressure-induced DNA synthesis, production of 1,4,5-inositol trisphosphate (IP3) and intracellular Ca2+ was measured. Pure pressure increased IP3 and intracellular Ca2+ in a pressure-dependent manner. The increases in both IP3 and intracellular Ca2+ were inhibited significantly by 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate. This study demonstrates a novel cellular mechanism whereby pressure regulates DNA synthesis in vascular smooth muscle cells, possibly via activation of PLC and protein kinase C.

Pressure Enhances Endothelin-1 Release From Cultured Human Endothelial Cells

The effect of pure pressure without shear stress or stretch on the release of endothelin-1 was investigated. Elevation of pressure significantly enhanced endothelin-1 release from cultured human umbilical vein endothelial cells. A calcium channel blocker, nifedipine, and a putative stretch-activated channel blocker, gadolinium, did not affect the pressure-induced endothelin-1 increase. On the other hand, a phospholipase C inhibitor, 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate, and protein kinase C inhibitors, 1-5-(isoquinolinylsulfonyl)-2-methylpiperazine and chelerythrine, significantly inhibited the pressure-induced endothelin-1 increase. Moreover, pure pressure reduced basal nitric oxide release, while pretreatment with a nitric oxide synthase inhibitor, NG-monomethyl-L-arginine, had no effect on the pressure-induced endothelin-1 increase. In conclusion, our results show for the first time that pressure enhances endothelin-1 release partially through activation of phospholipase C and protein kinase.

Aortic recognition sites for serotonin (5HT) are coupled to phospholipase C and modulate phosphatidylinositol turnover

The 5HT-mediated contraction of rat thoracic aorta is competitively blocked by the specific receptor antagonist 5HT2 ketanserin. In this tissue the addition of 5HT activated the turnover of 3H-phosphatidylinositol in a ketanserin-reversible fashion. These 5HT2 recognition sites appear to be coupled to a phospholipase C mediated cleavage of phosphatidylinositol.

α2-Adrenoceptors modulating insulin release from isolated pancreatic islets

SummaryUsing rat isolated pancreatic islets, we investigated the effects of various α-adrenoceptor blocking agents on adrenaline-induced inhibition of glucose-stimulated insulin release. Yohimbine was about 100 times more potent than prazosin in antagonizing the inhibitory effect of adrenaline. At concentrations of 10 μM, phentolamine was about as effective as an antagonist as yohimbine, whereas dihydroergotamine, WB-4101 and phenoxybenzamine were less effective and prazosin produced very little antagonism. These results strongly suggest that postsynaptic α2-adrenoceptors modulate insulin release from pancreatic islets.

Serum interleukin-6 in schizophrenic patients

We examined serum interleukin-6 (IL-6) in 90 schizophrenic patients in remission and 90 normal controls using enzyme-linked immuno-sorbent assay (ELISA). We found a significant difference in variation between the schizophrenic and the control groups (F = 10.9, P less than .002). The difference in distribution was also statistically significant by Kolmogorov-Smirnov (chi-square = 45.0, P less than .001). Eight patients had aberrantly high serum levels of interleukin-6. Since the higher levels of IL-6 are characteristically found in several autoimmune disorders, our finding suggests a link between schizophrenia and immune response, which could be either autoimmune or a process induced by reactivation of viruses.