Masanori Nakata

Inhibition by simvastatin, but not pravastatin, of glucose‐induced cytosolic Ca2+ signalling and insulin secretion due to blockade of L‐type Ca2+ channels in rat islet β‐cells

Hypercholesterolaemia often occurs in patients with type 2 diabetes, who therefore encounter administration of HMG‐CoA reductase inhibitors. Alteration of pancreatic β‐cell function leading to an impaired insulin secretory response to glucose plays a crucial role in the pathogenesis of type 2 diabetes. Therefore, it is important to examine the effects of HMG‐CoA reductase inhibitors on β‐cell function. Cytosolic Ca2+ concentration ([Ca2+]i) plays a central role in the regulation of β‐cell function. The present study examined the effects of HMG‐CoA reductase inhibitors on the glucose‐induced [Ca2+]i signalling and insulin secretion in rat islet β‐cells. Simvastatin, a lipophilic HMG‐CoA reductase inhibitor, at 0.1–3u2003μgu2003ml−1 concentration‐dependently inhibited the first phase increase and oscillation of [Ca2+]i induced by 8.3u2003mM glucose in single β‐cells. The less lipophilic inhibitor, simvastatin‐acid, inhibited the first phase [Ca2+]i increase but was two orders of magnitude less potent. The hydrophilic inhibitor, pravastatin (100u2003μgu2003ml−1), was without effect on [Ca2+]i. Simvastatin (0.3u2003μgu2003ml−1), more potently than simvastatin‐acid (30u2003μgu2003ml−1), inhibited glucose‐induced insulin secretion from islets, whereas pravastatin (100u2003μgu2003ml−1) had no effect. Whole‐cell patch clamp recordings demonstrated a reversible inhibition of the β‐cell L‐type Ca2+ channels by simvastatin, but not by pravastatin. Simvastatin also inhibited the [Ca2+]i increases by L‐arginine and KCl, agents that act via opening of L‐type Ca2+ channels. In conclusion, lipophilic HMG‐CoA reductase inhibitors can inhibit glucose‐induced [Ca2+]i signalling and insulin secretion by blocking L‐type Ca2+ channels in β‐cells, and their inhibitory potencies parallel their lipophilicities. Precaution should be paid to these findings when HMG‐CoA reductase inhibitors are used clinically, particularly in patients with type 2 diabetes.

Anandamide induces apoptosis of PC‐12 cells: involvement of superoxide and caspase‐3

Anandamide (arachidonoylethanolamide), an endogenous cannabinoid receptor ligand has been suggested to have physiological role in mammalian nervous system. However, little is known about the role of anandamide on neuronal cells. Here, we demonstrate that anandamide causes death of PC‐12 cells, showing marked DNA condensation and fragmentation, appearance of cells at sub‐G0/G1 and redistribution of phosphatidyl serine, the hallmark features of apoptosis. Anandamide raised intracellular superoxide level and CPP32‐like protease activity in PC‐12 cells markedly. Furthermore, antioxidant N‐acetyl cysteine prevented anandamide‐induced superoxide anion formation and cell death, implying that intracellular superoxide is a novel mediator of anandamide‐induced apoptosis of PC‐12 cells.

Polymyxin B binds to anandamide and inhibits its cytotoxic effect

Anandamide (ANA), an endogenous cannabinoid, can be generated by activated macrophages during endotoxin shock and is thought to be a paracrine contributor to hypotension. We discovered that ANA in saline/ethanol solution and in serum was efficiently adsorbed in a polymyxin B (PMB)‐immobilized beads column and eluted with ethanol. We confirmed the direct binding of PMB to ANA by using surface plasmon resonance. The adsorption of ANA by PMB may abolish the diverse effects of ANA such as hypotension, immunosuppression, and cytotoxicity, and may suggest a new therapeutic strategy for endotoxin shock.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is an islet substance serving as an intra-islet amplifier of glucose-induced insulin secretion in rats.

1 We examined whether pituitary adenylate cyclase‐activating polypeptide with 38 or 27 residues (PACAP‐38 or PACAP‐27) serves as an intra‐islet regulator of glucose‐induced insulin secretion in rats. PACAP antiserum specific for PACAP‐38 and PACAP‐27 was used to neutralize the effect of endogenous PACAP in islets. PACAP release from islets was bioassayed using the response of cytosolic Ca2+ concentration ([Ca2+]i) in single β‐cells, monitored by dual‐wavelength fura‐2 microfluorometry. Expression of PACAP mRNA was studied by reverse transcription‐polymerase chain reaction (RT‐PCR), while expression of PACAP was studied by metabolic labelling and immunoblotting. Localization of PACAP receptors was studied immunohistochemically. 2 High glucose‐stimulated insulin release from isolated islets was attenuated by PACAP anti‐serum but not by non‐immune sera. 3 The islet incubation medium with high glucose (Med) possessed a capacity, which was neutralized by PACAP antiserum, to increase [Ca2+]i in β‐cells. PACAP antiserum also neutralized the [Ca2+]iincreasing action of synthetic PACAP‐38 and PACAP‐27, but not that of vasoactive intestinal polypeptide (VIP) and glucagon. 4 Both Med and synthetic PACAP increased [Ca2+]i in β‐cells only in the presence of stimulatory, but not basal, glucose concentrations. In contrast, ATP, a substance that is known to be released from β‐cells, increased [Ca2+]i in β‐cells at both basal and stimulatory glucose concentrations. 5 Expression of PACAP mRNA and biosynthesis of PACAP‐38 were detected in islets and a β‐cell line, MIN6. 6 Immunoreactivity for PACAP‐selective type‐I receptor was observed in islets. 7 [Ca2+]i measurements combined with immunocytochemistry with insulin antiserum revealed a substantial population of glucose‐unresponsive β‐cells, many of which were recruited by PACAP‐38 into [Ca2+]i responses. 8 These results indicate that PACAP‐38 is a novel islet substance that is synthesized and released by islet cells and then, in an autocrine and/or paracrine manner, potentiates and arouses β‐cells responses to glucose, thereby amplifying glucose‐induced insulin secretion in islets.

Expression of vascular endothelial growth factor in sera and lymph nodes of the plasma cell type of Castleman's disease

To evaluate the possible involvement of vascular endothelial growth factor (VEGF) in the pathogenesis of Castlemans disease, we studied VEGF levels in sera and supernatants of cultured lymph nodes from two patients with the plasma cell type of Castlemans disease, and analysed the expression of VEGF immunohistochemically in the lymph nodes. Clinically, one patient was classified as the localized type and the other as the multicentric type. Histologically, mature plasma cells and hyalinized vessels were prominent in the interfollicular region. The VEGF levels of the sera and the supernatants of cultured lymph nodes of both patients were higher than those of normal controls. VEGF was strongly expressed in plasma cells in the interfollicular region of the lymph nodes of both patients, but rarely in normal lymph nodes. Our results suggest that VEGF may be involved in the marked vascular proliferation in the interfollicular region of the lymph nodes of the plasma cell type of Castlemans disease.

Effect of Leptin in Platelet and Endothelial Cells: Obesity and Arterial Thrombosis

We demonstrated that leptin showed effects on both platelets and endothelial cells through its functional receptor. These effects are the vector to inducing thrombotic tendency. Leptin concentrations used in our experiments correspond to that of leptin in the circulation of obese individuals. Thus we suggest that increased leptin may act as a risk factor for thrombosis in obese individuals.

New quinolones, ofloxacin and levofloxacin, inhibit telomerase activity in transitional cell carcinoma cell lines

It has been demonstrated that some quinolone antibiotics inhibit cell proliferation in vitro. This study showed that ofloxacin and levofloxacin, two well-known new quinolones, had an inhibitory effect on the proliferation of transitional cell carcinoma cell lines at high concentrations (>200 microg/ml). At relatively low concentrations (10-100 microg/ml), however, there was no apparent antiproliferative effect. Despite this, decreased absorbance in the MTT assay was observed at low concentrations and telomerase activity was significantly decreased. These results suggest that the antiproliferative effect of both ofloxacin and levofloxacin may be related to impairment of telomerase activity by some unknown mechanism.

Inhibition of migration of human glioblastoma cells by cerivastatin in association with focal adhesion kinase (FAK)

To investigate the biological effect of cerivastatin on glioblastoma cells, we exposed them to various concentrations of cerivastatin. Cerivastatin exhibited dual effects on glioblastoma cells in a dose-dependent manner. Immunofluorescence microscopy showed disruption of actin stress fibers and focal adhesion plaques even at nanomolar concentrations. Matrigel assay demonstrated marked inhibition of glioblastoma cell invasion. Immunoblot analysis using a phosphospecific antibody against focal adhesion kinase (FAK) showed that inhibition of migration was associated with the down-regulation of tyrosine phosphorylation of FAK. Our data suggest that cerivastatin may be beneficial for combination therapy with conventional anti-cancer drugs by inhibiting the invasion of glioblastoma.

Inhibition of caspase-3 activation by SB 203580, p38 mitogen-activated protein kinase inhibitor in nitric oxide-induced apoptosis of PC-12 cells

Mitogen-activated protein kinase (MAPK) p38 plays pivotal role in cell proliferation, differentiation, and apoptosis when cysteine protease caspase induces apoptosis in different cell systems. SB 203580 (4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1 H-imidazole) is widely used as a specific inhibitor of p38 MAPK, and prevents apoptosis induced by various agents. The effect of SB 203580 on nitric oxide(NO)- or peroxynitrite-induced cell death is not known. Western blotting results indicate that p38 MAPK was activated significantly in NO- or peroxynitrite-induced cell death in a time-dependent manner, and subsequently this cell death was markedly inhibited by SB 203580, as determined by fluorescence-activated cell sorting (FACS)-can analyzer. Furthermore, NO/peroxynitrite-induced caspase-3 activation was notably inhibited by SB 203580, however, phosphorylation of either p38 MAPK or p44/42 was not influenced by SB 203580. Thus, it is likely that SB 203580 prevents NO/peroxynitrite-induced cell death by inhibiting caspase-3 activation in PC-12 cells.

Insulinotropin PACAP potentiates insulin-stimulated glucose uptake in 3T3 L1 cells☆ ☆

Pituitary adenylate cyclase-activating polypeptide (PACAP) is localized in pancreatic nerve fibers and islets and potently augments glucose-induced insulin secretion. The present study explored a possible extra-pancreatic action of PACAP. The specific PACAP receptor (PAC1 receptor) was expressed in the rat fat tissue and 3T3-LI adipocytes. PACAP-38 (10 nM) significantly enhanced insulin-induced 2-deoxyglucose uptake by 3T3-L1 adipocytes. Insulin-stimulated phosphatidylinositol 3-kinase activity was further increased by PACAP-38, whereas the tyrosine-phosphorylation of insulin receptor beta-subunit and insulin receptor substrate-1 was unaltered by PACAP-38. These results reveal that PACAP-38 enhances insulin-induced glucose uptake, an effect probably mediated by insulin-stimulated phosphatidyl-inositol 3-kinase, and that PACAP potentiates not only insulin secretion, but also insulin action in adipocytes.