Dysregulation of dimethylargininedimethylaminohydrolase/asymmetric dimethylarginine pathway in rat type II diabetic nephropathy.

An impaired generation of nitric oxide has been associated with diabetic renal disease. In order to elucidate the underlying molecular mechanisms into how nitric oxide synthesis is impaired in diabetic renal disease, we examined changes in activities and expressions of some renal enzymes involved in nitric oxide production during the development of diabetic nephropathy in type II diabetic Otsuka Long-Evans Tokushima Fatty rats. Ten-week old Otsuka Long-Evans Tokushima Fatty (n = 40) and control Long-Evans Tokushima Otsuka rats (n = 20) were given drinking water containing 20% sucrose to accelerate the development of diabetic nephropathy. Otsuka Long-Evans Tokushima Fatty rats developed diabetic nephropathy in an age-dependent manner. Renal nitric oxide synthase activities in Otsuka Long-Evans Tokushima Fatty rats gradually declined with the progression of diabetic mellitus and were significantly lower than those of age-matched Long-Evans Tokushima Otsuka rats after 22 weeks of age. The lower activities of renal nitric oxide synthase in Otsuka Long-Evans Tokushima Fatty rats were correlated with relatively higher levels of renal free asymmetric dimethylarginine, an endogenous nitric oxide synthase inhibitor, and were also correlated with decreased activities of dimethylargininedimethylaminohydrolase which metabolizes asymmetric dimethylarginine to citrulline. These results imply that dimethylargininedimethylaminohydrolase dysregulation may play an important role in the development of diabetic nephropathy by increasing asymmetric dimethylarginine levels, which leads to inhibition of renal nitric oxide synthesis.

Inhibition of endothelial- and neuronal-type, but not inducible-type, nitric oxide synthase by the oxidized cholesterol metabolite secosterol aldehyde: Implications for vascular and neurodegenerative diseases.

The cholesterol ozonolysis products secosterol-A and its aldolization product secosterol-B were recently detected in human atherosclerotic tissues and brain specimens, and have been postulated to play pivotal roles in the pathogenesis of atherosclerosis and neurodegenerative diseases. We examined several oxidized cholesterol metabolites including secosterol-A, secosterol-B, 25-hydroxycholesterol, 5ß,6ß-epoxycholesterol and 7-ketocholesterol for their effects on the activities of three nitric oxide synthases. In contrast to other oxidized metabolites, secosterol-A was found to be a potent inhibitor against the neuronal- and endothelial-type, but not the inducible-type nitric oxide synthase, with IC(50) values of 22 ± 1 and 50 ± 5 µM, respectively. The calmodulin-binding regions of the neuronal- and endothelial-nitric oxide synthases contain lysine residues which are not present in the inducible-type nitric oxide synthase. Secosterol-A modifies proteins through the formation of a Schiff base with the lysine epsilon-amino group. It is possible that secosterol-A modifies lysine residues of constitutive nitric oxide synthases, leading to the inhibition of enzymatic activities. As nitric oxide is a critical signaling molecule in vascular function and in long-term potentiation, its reduced production through inhibition of constitutive nitric oxide synthases by secosterol-A may contribute to the development of atherosclerosis and memory impairment in particular neurodegenerative diseases.

Inhibition of L-arginine metabolizing enzymes by L-arginine-derived advanced glycation end products.

N(omega)-Carboxymethyl-arginine (CMA), N(omega)-carboxyethyl-arginine (CEA) and N(delta)-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1) have been identified as L-arginine-derived advanced glycation end products (AGEs) formed by non-enzymatic reactions between reducing sugars such as glucose and amino groups in proteins. These AGEs are structurally analogous to endogenous inhibitors of nitric oxide synthases (NOS) including N(G)-monomethyl-L-arginine (L-NMMA) and asymmetric N(G),N(G)-dimethyl-L-arginine (ADMA). Increased plasma levels of these NOS inhibitors, and thus impaired generation of NO in vivo has been associated with the pathogenesis of vascular complications such as kidney failure and atherosclerosis. For these reasons we examined whether L-arginine-derived AGEs inhibit the activities of three L-arginine metabolizing enzymes including three isoforms of NOS (endothelium, neuronal and inducible NOS), dimethylarginine dimethylaminohydrolase (DDAH) that catalyzes the hydrolytic degradation of L-NMMA and ADMA to L-citrulline, and arginase that modulates intracellular L-arginine bioavailability. We found that AGEs inhibited the in vitro activities of endothelium type NOS weakly (IC(50) values of CMA, CEA and MG-H1 were 830, 3870 and 1280 microM, respectively) and were also potential endogenous inhibitors for arginase (IC(50) values of CMA and CML were 1470 and 1060 microM), but were poor inhibitors for DDAH. These results suggest that the tested L-arginine- and L-lysine-derived AGEs appear not to impair NO biosynthesis directly.

Phytocomponent p-hydroxycinnamic acid stimulates mineralization in osteoblastic MC3T3-E1 cells.

Phytocomponent p-hydroxycinnamic acid (HCA) has been shown to have stimulatory effects on bone calcification and inhibitory effects on bone resorption in rat femoral tissues in vitro. Whether HCA has a stimulatory effect on mineralization in osteoblastic cells is unknown. This study was undertaken to determine the effect of HCA on mineralization in osteoblastic MC3T3-E1 cells in vitro. Cells were cultured for 72 h in a minimum essential medium (alpha-MEM) containing 10% fetal bovine serum (FBS), and the cells with subconfluency were changed to a medium containing either vehicle or HCA (10(-7)-10(-5) M) without FBS. Culture with HCA (10(-7)-10(-5) M) did not have a significant effect on cell proliferation and cell death. Deoxyribonucleic acid (DNA) content in osteoblastic cells was significantly increased after culture with HCA (10(-6) or 10(-5) M) for 48 or 72 h. Alkaline phosphatase activity in osteoblastic cells was significantly increased after culture with HCA (10(-7)-10(-5) M) for 24, 48, or 72 h. The results with Alizarin red staining for calcium showed that mineralization was significantly stimulated after culture with HCA (10(-8)-10(-5) M) for 7, 14, or 21 days. This study demonstrates that HCA has stimulatory effects on mineralization in osteoblastic MC3T3-E1 cells.

Oral administration of phytocomponent p-hydroxycinnamic acid prevents bone loss in ovariectomized rats.

The preventive effect of phytocomponent p-hydroxycinnamic acid (HCA) on ovariectomy (OVX)-induced bone loss was investigated. HCA (250 or 500 microg/100 g body weight) was orally administered once daily for 30 days to OVX rats. The analysis using a peripheral quantitative computed tomography (pQCT) showed that OVX caused bone loss in the femoral-metaphyseal tissues. This change was significantly restored after the administration of HCA (250 or 500 microg/100 g body weight) to OVX rats. Mineral content, mineral density, and polar strength strain index in the femoral-metaphyseal tissues were significantly decreased in OVX rats. These decreases were significantly restored after the administration of HCA (500 microg/100 g) to OVX rats. Moreover, OVX caused a significant decrease in calcium content or alkaline phosphatase activity in the femoral-diaphyseal and -metaphyseal tissues. These decreases were significantly restored after the administration of HCA (250 or 500 microg/100 g) to OVX rats. Deoxyribonucleic acid (DNA) content in the diaphyseal or metaphyseal tissues was significantly increased in OVX rats. These increases were significantly restored after oral administration of HCA (500 microg/100 g). This study demonstrates that HCA has preventive effects on OVX-induced bone loss of rats in vivo.

Oral administration of phytocomponent p-hydroxycinnamic acid has a preventive effect on bone loss in streptozotocin-induced diabetic rats.

The phytocomponent p-hydroxycinnamic acid (HCA) has been shown to have a stimulatory effect on bone formation and an inhibitory effect on bone resorption in rat femoral tissues in vitro. The preventive effect of HCA on bone loss induced in streptozotocin (STZ)-diabetic rats was investigated in vivo. Rats received a single subcutaneous administration of STZ (6.0 mg/100 g body weight), and then the animals were orally administered HCA (0.25, 0.5, or 1.0 mg/100 g body weight) once daily for 14 days. STZ administration caused a significant decrease in body weight and a significant increase in serum glucose, triglyceride, and calcium levels, indicating a diabetic state. These alterations were significantly prevented by administration of HCA (0.25, 0.5, or 1.0 mg/100 g). Calcium content in the femoral-diaphyseal and -metaphyseal tissues was significantly decreased in STZ-diabetic rats. This decrease was significantly prevented after administration of HCA (0.25, 0.5, or 1.0 mg/100 g). Alkaline phosphatase activity in the diaphyseal and metaphyseal tissues was significantly decreased in STZ-diabetic rats. The decrease in diaphyseal alkaline phosphatase activity in STZ-diabetic rats was significantly prevented after administration of HCA (0.5 and 1.0 mg/l00 g). The diaphyseal DNA content was also significantly decreased in STZ-diabetic rats. Administration of HCA (0.25, 0.5, or 1.0 mg/100 g) caused a significant increase in DNA content in the diaphyseal and metaphyseal tissues in STZ-diabetic rats. This study demonstrates that the intake of HCA has preventive effects on bone loss in STZ-diabetic rats, and that the intake has partially restorative effects on serum biochemical findings in the diabetic state.

Phytocomponent p-hydroxycinnamic acid inhibits osteoclast-like cell formation in mouse bone marrow cultures.

The phytocomponent p-hydroxycinnamic acid (HCA) has been shown to have inhibitory effects on bone-resorbing factor-stimulated bone resorption in rat femoral tissues in vitro. The effects of HCA on osteoclast-like cell formation in mouse bone marrow cultures in vitro were investigated. The bone marrow cells were cultured for 7 days in alpha-minimal essential medium containing a bone-resorbing agent [parathyroid hormone (1-34)] (PTH), prostaglandin E2 (PGE2), or tumor necrosis factor-alpha (TNF-alpha) in effective concentrations. Osteoclast-like cell formation was estimated by staining for tartrate-resistant acid phosphatase, a marker enzyme of osteoclasts. The presence of PTH (10(-7) M), PGE2 (10(-5) M), or TNF-alpha (10 ng/ml) induced a remarkable increase in osteoclast-like multinucleated cells. These increases were significantly inhibited in the presence of HCA (10(-8)-10(-5) M). HCA (10(-6) or 10(-5) M) significantly inhibited osteoclast-like cell formation induced by dibutyryl cyclic adenosine monophosphate (10(-5) M) or phorbol 12-myristate 13-acetate (10(-6) M), an activator of protein kinase C. Also, HCA (10(-8)-10(-5) M) had a significant inhibitory effect on osteoclast-like cell formation induced by the receptor activator of NF-kappaB ligand (RANKL) (10 ng/ml) in the presence of macrophage colony-stimulating factor (M-CSF) (10 ng/ml). The inhibitory effect of HCA (10(-6) or 10(-5) M) on RANKL plus M-CSF-induced osteoclast-like cell formation was not observed in the presence of cycloheximide (10(-7) M), an inhibitor of protein synthesis in the transcriptional process, or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (10(-6) M), an inhibitor of transcription. This study demonstrates that HCA has a potent inhibitory effect on osteoclast-like cell formation in mouse bone marrow cultures. The inhibitory action of HCA may partly involve a newly synthesized protein component which is related to RANKL stimulation in osteoclastogenesis.

Phytocomponent p-hydroxycinnamic acid stimulates bone formation and inhibits bone resorption in rat femoral tissues in vitro.

The effect of cinnamic acid or its related compounds, which is present in many plants, on bone metabolism has not been clarified yet. The effect of cinnamic acid, p-hydroxycinnamic acid (HCA), ferulic acid, caffeic acid, or 3,4-dimethoxycinnamic acid (DCA) on bone calcium content in vitro was investigated. Rat femoral-diaphyseal (cortical bone) and -metaphyseal (trabecular bone) tissues were cultured for 48,h in Dulbecco's modified Eagle's medium (high glucose, 4.5%) supplemented with antibiotics and bovine serum albumin. The presence of HCA (10(-5) or 10(-4),M) caused a significant increase in calcium content in the diaphyseal or metaphyseal tissues. Such an effect was not observed in the presence of cinnamic acid or other compounds at the concentration of 10(-5) or 10(-4),M. Alkaline phosphatase activity and deoxyribonucleic acid (DNA) content in the diaphyseal or metaphyseal tissues was significantly increased in the presence of HCA (10(-5) or 10(-4),M). The effect of HCA (10(-4),M) in increasing calcium content, alkaline phosphatase activity, and DNA content in the diaphyseal or metaphyseal tissues was completely prevented in the presence of cycloheximide (10(-6),M), an inhibitor of protein synthesis. Thus HCA had anabolic effects on bone components. The presence of parathyroid hormone (PTH; 10(-7),M), a bone-resorbing factor, caused a significant decrease in calcium content and a corresponding elevation in medium glucose consumption, lactic acid production or tartrate-resistant acid phosphatase (TRACP) activity in the diaphyseal or metaphyseal tissues. These alterations were completely prevented in the presence of HCA (10(-5) or 10(-4),M). This study demonstrates that p-hydroxycinnamic acid (HCA) has stimulatory effects on bone formation and inhibitory effects on bone resorption in tissue culture in vitro.

Effects of copper on bone component in the femoral tissues of rats: anabolic effect of zinc is weakened by copper.

The effects of copper on biochemical components in the femoral-diaphyseal (cortical bone) and -metaphyseal (trabecullar bone) tissues of rats in vivo and in vitro were investigated. Rats were orally administered copper sulfate (50, 100, or 200 microg Cu/100 g body weight) once daily for 7 d. Calcium content in the diaphyseal and metaphyseal tissues was significantly decreased with the administration of copper (200 microg/100 g), while alkaline phosphatase activity in these tissues was not significantly changed by copper administration. The diaphyseal DNA content was significantly decreased with the administration of copper (50, 100, or 200 microg/100 g). Moreover, the femoral-diaphyseal and -metaphyseal tissues were cultured for 48 h in serum-free medium containing either vehicle or copper (10(-7) - 10(-4) M). Culture with copper (10(-7) - 10(-4) M) caused a significant decrease in alkaline phosphatase activity in the diaphyseal and metaphyseal tissues, while calcium and DNA contents in these tissues were not significantly changed. Culture with parathyroid hormone [PTH (1-34); 10(-7) M], a bone-resorbing factor, caused a significant decrease in calcium content in the diaphyseal and metaphyseal tissues. This decrease was completely inhibited in the presence of copper (10(-6) or 10(-5) M). Culture with zinc sulfate (10(-4) M) caused a significant increase in calcium content and alkaline phosphatase activity in the diaphyseal and metaphyseal tissues. The effects of zinc (10(-4) M) in increasing femoral calcium content and alkaline phosphatase activity were not seen in the presence of cycloheximide (10(-6) M), an inhibitor of protein synthesis, suggesting that the effects of zinc are involved in newly synthesized protein components. The effects of zinc in increasing calcium content and alkaline phosphatase activity in the diaphyseal and metaphyseal tissues were significantly weakened in the presence of copper (10(-4) M). The inhibitory effects of copper were further enhanced in the presence of cycloheximide. This study demonstrates that supplementation with copper in adequate copper nutrition does not have anabolic effects on bone components in vivo and in vitro and that copper weakens the anabolic effects of zinc in vitro.