Aya Fujinami

Effects of dopamine agonists bromocriptine, pergolide, cabergoline, and SKF-38393 on GDNF, NGF, and BDNF synthesis in cultured mouse astrocytes

We examined the stimulatory effects of the dopamine agonists bromocriptine, pergolide, cabergoline, and SKF-38393 on the synthesis and secretion of neurotrophic factors (nerve growth factor, NGF; brain-derived neurotrophic factor, BDNF; and glial cell line-derived neurotrophic factor, GDNF) in cultured mouse astrocytes, and clarified the role of dopamine D1 and D2 receptors in these effects. Bromocriptine, a D2 agonist, elevated NGF levels in the culture medium 6.8-fold vs. control, and significantly decreased GDNF and BDNF levels, at 24 h. Both pergolide, a D1/D2 agonist, and cabergoline, a D2/weak D1 agonist, rapidly elevated NGF and GDNF levels at 4-6 h, respectively to 21- and 1.5-fold, respectively, and 84- and 9-fold, respectively, of control levels at 24 h. SKF-38393, a D1 agonist, elevated NGF and GDNF levels to 20- and 2.8-fold of controls, respectively, at 24 h. Relative levels of NGF and GDNF mRNA detected by Northern blot analysis or semiquantitative reverse transcriptase-polymerase chain reaction confirmed that increases in levels of the 2 proteins in culture medium were due to overexpression as opposed to leakage from cells. Cabergoline rapidly increased GDNF mRNA expression at 4 h, producing a potent and long-lasting increase in GDNF levels. Bromocriptine significantly suppressed GDNF synthesis. These findings suggest that stimulation of dopamine D1 receptors may be required for GDNF synthesis and secretion, and that concurrent stimulation of dopamine D1 and D2 receptors may augment synthesis and secretion of NGF and GDNF. These dopamine agonists may play a role in neuronal survival by stimulating NGF and GDNF synthesis in the brain, and as drugs are good candidates as NGF and GDNF inducers.

Increased concentration of pentosidine, an advanced glycation end product, and interleukin-6 in the vitreous of patients with proliferative diabetic retinopathy

To investigate the relationship between advanced glycation end products (AGEs) and interleukin-6 (IL-6) in the development of diabetic retinopathy, we determined the concentrations of pentosidine, a well-characterized AGE, and IL-6 in the vitreous of 62 patients with proliferative diabetic retinopathy (PDR) and 50 non-diabetic control subjects. We also investigated the effect of AGEs on the production of IL-6 by human retinal Müller cells. The levels of pentosidine and IL-6 in the vitreous of patients with PDR were significantly higher compared with controls. In patients with PDR with vitreous hemorrhage (VH), the mean vitreous concentration of IL-6 was significantly higher than that in PDR patients without VH. There was a strong positive correlation between the vitreous levels of pentosidine and IL-6. Levels of IL-6 were strikingly higher in the vitreous compared with the serum and there was no correlation between IL-6 concentrations in the two fluids. Treatment of Müller cells with AGEs for 48 h resulted in a dose-dependent increase of IL-6 in the culture medium. These results suggest that increased formation of AGEs in the vitreous may be involved in the development of diabetic retinopathy by inducing the production of IL-6 from retinal Müller cells.

The effect of dopamine agonists: The expression of GDNF, NGF, and BDNF in cultured mouse astrocytes

In Parkinsons disease, cell death is selectively induced in mesencephalic nigral dopaminergic neurons. At present, no disease modifying therapy or radical treatment has been found for this disease. Some dopamine agonists may have a neuroprotective action in cultured cells and animal models. In the present study, we examined stimulating effects of a non-ergoline D(2) dopamine agonist, ropinirole, on synthesis/secretion of neurotrophic factors, including NGF, BDNF, and GDNF, in cultured mouse astrocytes. These effects were compared with those of ergoline dopamine agonists, SKF-38393, a D(1) agonist, bromocriptine, D(2) agonist, and apomorphine, D(1)/D(2) agonist. Ropinirole elevated GDNF levels to 4-fold, and NGF levels to 6.3-fold, compared with the control group. Of the dopamine agonists examined, ropinirole produced and secreted more GDNF than a 1.8-fold greater amount of apomorphine, a lesser amount of bromocriptine, or a 2.8-fold greater amount of SKF-38393, which served as the control group.

Telmisartan, an angiotensin II type 1 receptor blocker, prevents the development of diabetes in male Spontaneously Diabetic Torii rats

To assess the beneficial effects of the angiotensin II type 1 receptor blocker telmisartan on a non-obese animal model of reduced function and mass of islet beta-cells prior to the development of diabetes, Spontaneously Diabetic Torii (SDT) rats were treated with telmisartan at 8 weeks of age. At 24 weeks of age, the treatment with telmisartan dose-dependently ameliorated hyperglycemia and hypoinsulinemia, and high-dose (5 mg/kg/day) treated SDT rats did not developed diabetes. Real-time RT-PCR analysis revealed that treatment with high-dose telmisartan reduced mRNA expression of local renin-angiotensin system (RAS) components, components of NAD(P)H oxidase, transforming growth factor-beta1 and vascular endothelial growth factor in the pancreas of male SDT rats. Immunohistochemical and Western blot analyses revealed that treatment with telmisartan also reduced expression of p47(phox). These results suggest that treatment with telmisartan reduces oxidative stress by local RAS activation and protects against islet beta-cell damage and dysfunction. These findings provide at least a partial explanation for the reduced incidence of new-onset diabetes that has been observed in several clinical trials involving angiotensin II type 1 receptor blockers and ACE inhibitors.

Effect of Atorvastatin on in vitro Expression of Resistin in Adipocytes and Monocytes/Macrophages and Effect of Atorvastatin Treatment on Serum Resistin Levels in Patients with Type 2 Diabetes

Resistin is a novel cysteine-rich protein that plays a role in the development of insulin resistance and atherosclerosis. HMG-CoA reductase inhibitors (statins) possess anti-inflammatory properties that are independent of their lipid-lowering action. The aims of this study were to investigate the effect of atorvastatin on expression of resistin in vitro and to determine the effect of 6 months of treatment with atorvastatin on serum levels of resistin in patients with type 2 diabetes. 3T3-L1 adipocytes and human monocytes/macrophages and preadipocytes were incubated with 1 and 10 µmol/l atorvastatin for 24 and 48 h, followed by measurement of resistin mRNA by the quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). Serum resistin concentration in the patients with type 2 diabetes was measured at baseline and after 6 months of atorvastatin treatment (10 mg/day). qRT-PCR analysis revealed that atorvastatin decreased resistin mRNA expression in a dose- and time-dependent manner. Serum resistin concentration tended to decrease after 6 months of atorvastatin treatment, although this decrease did not reach statistical significance. In conclusion, the findings of our in vitro study contribute to the growing volume of evidence on the anti-inflammatory and anti-atherosclerotic effects of statins, and led us to suggest that statins may control inflammatory responses by inhibiting expression of resistin mRNA. It is necessary to confirm the findings of our in vitro study by an appropriately designed large-scale clinical study.

The novel catecholaminergic and serotoninergic activity enhancer R-(−)-1-(benzofuran-2-yl)-2-propylaminopentane up-regulates neurotrophic factor synthesis in mouse astrocytes

We investigated the effects of the novel catecholaminergic and serotoninergic activity enhancer R-(-)-1-(benzofuran-2-yl)-2-propylaminopentane ((-)-BPAP) on the synthesis and secretion of neurotrophins, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and glial cell line-derived neurotrophic factor (GDNF), in cultured mouse astrocytes. The protein and mRNA levels of the neurotrophic factors were measured using the enzyme-linked immunosorbent assay and reverse transcription-polymerase chain reaction methods, respectively. The amounts of NGF, BDNF, and GDNF secreted from astrocytes into the culture medium increased by up to 120, two, and seven times higher than those of the control, respectively, by treatment with 0.35 mM (-)-BPAP for 24 h. The increases in NGF and GDNF induced by the treatment with (-)-BPAP was inhibited by concomitant treatment with actinomycin D for transcriptional blockade. Furthermore, the treatment with (-)-BPAP for 6 h increased the mRNA expression of NGF, BDNF, and GDNF. These results suggest that (-)-BPAP up-regulated neurotrophic factor synthesis in cultured astrocytes.

The α-glucosidase inhibitor acarbose reduces the net electronegative charge of low-density lipoprotein in patients with newly diagnosed type 2 diabetes

BACKGROUND Several epidemiological studies have shown that postprandial hyperglycemia is associated with an increased risk of cardiovascular disease (CVD). The present study was conducted in order to compare the effects of acarbose and glimepiride treatment on serum lipoprotein profiles in patients with type 2 diabetes. METHODS A total of 37 patients with newly diagnosed type 2 diabetes were studied. The patients were assigned randomly to treatment for 12 weeks with either acarbose (n=13, 100 mg x 3/day, group A), glimepiride (n=13, 2 mg/day, group G) or diet only (n=11, group D). Lipid and lipoprotein profiles before and after each treatment were evaluated. RESULTS A significant reduction in the net electronegative charge of low-density lipoprotein (emLDL) was observed in group A (-1.8, P<0.01), whereas no significant change in emLDL was observed in groups G and D. In group A, small VLDL and very small LDL levels were also decreased significantly (P<0.05). The change in emLDL levels correlated significantly with changes in very small LDL (r=0.751, P<0.01) and oxidized LDL levels (r=0.623, P<0.05). CONCLUSION These results suggest that measurement of serum emLDL may be a sensitive and clinically useful marker for determining qualitative lipoprotein abnormalities in diabetes, and that acarbose treatment lowers CVD risk by decreasing production of emLDL.

Frequency of anti-AChR epsilon subunit-specific antibodies in MG

A definite diagnosis of myasthenia gravis (MG) relies heavily on acetylcholine receptor (AChR) antibody testing. The relatively high number of antibody-negative patients therefore, causes frequent uncertainty in confirming the diagnosis. We evaluated the sensitivity and specificity of a new, commercially available AChR antibody test that uses an approximately equal mixture of AChR from TE671-ϵ (adult type) and TE671-γ (fetal type) cells. This assay was used to re-examine 365 seronegative MG sera in which AChR antibody had not been detected by the standard assay that uses fetal type AChR. The new assay detected anti-AChR antibodies in 17 (15.5%) of 110 patients with ocular type and in 33 (12.9%) of 255 patients with generalized type MG. Anti-AChR ϵ subunit-specific antibodies were present in 13.7% of the patients in whom no AChR antibody had been detected by the standard assay, showing an increase from 79 to 82% in overall diagnostic sensitivity.

Simultaneous determination of estriol and estriol 3-sulfate in serum by column-switching semi-micro high-performance liquid chromatography with ultraviolet and electrochemical detection

A column-switching HPLC with semi-microcolumn enabled us a direct and simultaneous analysis of estriol (E3) and estriol 3-sulfate (E3 S) in human serum in combination with ultraviolet (for E3 S) and electrochemical (for E3) detectors. The mobile phases (phosphate buffer pH 7.0) contained 5 mM tetra-n-butylammonium ion (TBA) as a counter ion for E3 S. Serum samples were diluted with 200 mM phosphate buffer (pH 7.0) containing 100 mM TBA, then injected to the pre-column. After serum proteins had flowed out from the pre-column, E3 and E3 S were transferred to the enrichment column. Subsequently the analytes were eluted to the analytical column. Detection limits of E3 and E3 S in human serum were 2.5 ng/ml and 295 ng/ml. Serum E3 and E3 S levels (mean +/- SD) of umbilical artery from 18 full-term healthy neonates were 33+/-23 ng/ml and 1.26+/-0.69 microg/ml, respectively.

Heterologous enzyme immunoassay for serum androstenediol

A heterologous enzyme immunoassay for serum androstenediol (Adiol: 3beta, 17beta-dihydroxy-androst-5-ene) was established. The combination of anti-Adiol antiserum raised in rabbit against Adiol 7-O-(carboxymethyl)oxime (Adiol 7-CMO) conjugated bovine serum albumin (Adiol 7-CMO-BSA) and Adiol 7-iminomethylcarboxylic acid conjugated alkaline phosphatase was used for the assay. The sensitivity of the heterologous assay system was superior to that of a homologous assay system in which an antibody raised in rabbit against Adiol 7-CMO-BSA and enzyme labeled antigen, Adiol 7-CMO conjugated alkaline phosphatase, were used. The minimal amount of Adiol detected was 0.4 ngml(-1) and the measurable range was from 0. 4 to 150 ngml(-1). Intra-assay coefficients of variation (C.V.) were 8.6% (1.52+/-0.13 ngml(-1), mean+/-S.D., n=10) and 6.7% (13.4+/-0.9 ngml(-1), n=10). Inter-assay C.V. were 12.9% (1.63+/-0.21 ngml(-1), n=8) and 11.5% (12.2+/-1.4 ngml(-1), n=8). A linear relation was observed between the serum sample dilution and the Adiol concentration. For recovery study, authentic Adiol was added to serum sample (original concentration: 1.43 ngml(-1)). The calculated final Adiol concentration was 2.99 ngml(-1). The recovery was 98.6% (n=5). The Adiol concentrations in healthy subjects measured by the proposed assay (male: 1.1+/-0.3 ngml(-1) (mean+/-S.D.), range: 0.7-1. 7 ngml(-1), age: 22-50, n=10; female: 0.6+/-0.4 ngml(-1), range: 0. 2-1.6 ngml(-1), age: 23-48, n=20) were consistent with reported values.