Shuichi Hamano

Adiponectin inhibits insulin-like growth factor-1-induced cell migration by the suppression of extracellular signal-regulated kinase 1/2 activation, but not Akt in vascular smooth muscle cells.

Adiponectin, an adipocyte-derived hormone, has been proposed to show antiatherogenic properties through the inhibitory effects against various growth factors. Insulin-like growth factor-1 (IGF-1) is one of the potent mitogens, which has been considered to play important roles in both atherogenesis and plaque stabilization in accordance to the phase of atherosclerosis. The aim of this study is to elucidate the adiponectin effects on IGF-1-induced cell migration and its intracellular signaling pathways in vascular smooth muscle cells (VSMCs). In this study, we assessed cell migration and several kinase activities in cultured rat aortic smooth muscle cells (RASMCs). Adiponectin pretreatment suppressed IGF-1-induced cell migration and extracellular signal-regulated kinase (ERK)1/2 activation, which is one of the major mediators for IGF-1-induced cell migration. In RASMCs, adiponectin and 5-aminoimidazole-4-carboxamide riboside (AICAR), a 5′-AMP-activated protein kinase (AMPK) activator, stimulated AMPK activation. AMPK activation by AICAR inhibited IGF-1-induced ERK1/2 activation and cell migration in RASMCs. On the other hand, phosphorylation of Akt and Bad, proapoptotic molecules of the Bcl-2 family, which were increased by IGF-1 stimulation, was not diminished by the pretreatment with adiponectin. It was shown that adiponectin inhibited IGF-1-induced VSMC migration through suppression of ERK1/2 activation, which might be implicated in AMPK activation. Furthermore, adiponectin selectively inhibited ERK1/2 pathway, not Akt–Bad pathway, stimulated by IGF-1. From these findings, it was implied that adiponectin suppressed IGF-1-induced VSMC migration and its signaling selectivity.

Myosin light-chain kinase inhibitor, 1-(5-chlor-naphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9), inhibits catecholamine secretion from adrenal chromaffin cells by inhibiting Ca2+ uptake into the cells

For determination of whether myosin light-chain kinase (MLCK) is involved in the secretory mechanism of adrenal chromaffin cells, the effect of a preferential inhibitor of the enzyme, 1-(5-chlornaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9), on catecholamine secretion from cultured bovine adrenal chromaffin cells was studied. ML-9 did not affect basal catecholamine secretion, but inhibited catecholamine secretion stimulated by acetylcholine, high K+, veratridine or palytoxin. At similar concentrations to those inhibiting the secretion of catecholamine, ML-9 also inhibited increased [45Ca]2+ uptake by the cells induced by these stimulants. However, it did not inhibit catecholamine secretion induced by the Ca2+ ionophore A23187. Moreover, it did not affect catecholamine secretion from digitonin-permeabilized cells induced by a micromolar Ca2+ concentration in the presence of Mg ATP. These results indicate that ML-9 inhibits catecholamine secretion from adrenal chromaffin cells by inhibiting the transmembrane Ca2+ uptake mechanism, but not by inhibiting the intracellular Ca2+-dependent mechanism. The possible role of MLCK in stimulus-secretion coupling in adrenal chromaffin cells is discussed.

Detection of histo-blood group ABO mRNA in human chronic myeloid leukemia cell lines using reverse transcription-polymerase chain reaction (RT-PCR)

ABH carbohydrate antigens are cell surface carbohydrates which occur in three allelic forms, namely A, B and O blood groups. It is unknown how the ABO blood group is expressed in hemopoietic stem cells. In an attempt to verify the ABO mRNA expression in hemopoietic precursor cells, mRNAs were isolated from human chronic myeloid leukemia (CML) cell lines which are believed to be at the most immature level of hemopoietic differentiation among hemopoietic malignancies. In particular, K-562 and KOPM-28 cells were used with the reverse transcription-polymerase chain reaction (RT-PCR) technique for amplifying ABO gene transcripts. The amplified ABO cDNAs from two cell lines were characterized by the digestion of Kpn-I restriction enzyme. The blood types were determined by polymerase chain reaction of the specific allele (PASA) method. Both of the human chronic myeloid leukemia cell lines expressed ABO mRNA. The quantity of ABO mRNA in the K-562 cell line is significantly higher than that of the KOPM-28 cell line. The ABO blood type of these two cell lines was type O. Because the CML cell lines are presumed to be at the immature stem cell level of hematopoietic cell differentiation and because it is believed that the cultured cell lines from hematologic malignancy reflect the characteristics of normal corresponding hemopoietic cells, the hemopoietic stem cells should express mRNA of the ABO blood group.

Angiotensin II Receptor Blocker Improves Tumor Necrosis Factor-α-Induced Cytotoxicity via Antioxidative Effect in Human Glomerular Endothelial Cells

Background/Aims: Tumor necrosis factor-α (TNF-α) is known to involve the progression of renal dysfunction through its cytotoxicity and proinflammatory effects such as the induction of intercellular adhesion molecule (ICAM)-1 expression in vascular endothelial cells (ECs). Olmesartan, one of the angiotensin II type 1 receptor blockers (ARBs), has been reported to show protective effects on injured ECs by some causal factors of renal disorder other than angiotensin II. However, the effects of olmesartan on TNF-α-induced glomerular EC damage have not been investigated. In the present study, we investigated the effects of RNH-6270, an active metabolite of olmesartan, on TNF-α-induced human glomerular EC (HGEC) damage to clarify the renoprotective mechanisms of ARBs. Methods: Cultured HGECs were stimulated by TNF-α, and then cell viability and cytotoxicity were measured by MTT assay and lactate dehydrogenase release assay, respectively. TNF-α-induced oxidative stress was estimated by dihydroethidium assay and lucigenin chemiluminescence assay. ICAM-1 expression and the phosphorylations of mitogen-activated protein kinases were measured using Western blotting assay. Results: RNH-6270 suppressed cell death and the increase in ICAM-1 expression induced by TNF-α via the inhibition of reactive oxygen species in HGECs. Conclusion: Our findings suggested that olmesartan might have protective effects against TNF-α-induced glomerular EC dysfunction.

Angiotensin II receptor blocker attenuates PDGF-induced mesangial cell migration in a receptor-independent manner

BACKGROUND Clinical studies have shown that angiotensin II (Ang II) type 1 (AT1) receptor blockers (ARBs) are able to provide renoprotection independent of their blood pressure lowering effects. ARBs also are reported to suppress oxidative stress, inflammation and certain other cellular responses in a receptor-independent manner. We investigated the effects of an ARB, olmesartan, on the cell migration induced by platelet-derived growth factor (PDGF), a major mitogen involved in the pathogenesis of glomerulonephritis in rat mesangial cells (RMCs). METHODS Cell migration was determined by a modified Boyden chamber assay. The intracellular signalling pathway was examined by western blotting. AT1 receptor expression was knocked down by small interfering RNAs. The intracellular reactive oxygen species (ROS) was measured by using a fluorescent probe. The O(2)(.-) scavenging activities were studied by the electron paramagnetic resonance-spin trapping method. RESULTS PDGF-induced cell migration was inhibited by olmesartan in AT1 receptor knockdown RMCs. Olmesartan attenuated big mitogen-activated protein (MAP) kinase 1 (BMK1) and Src activation by PDGF in AT1 receptor knockdown RMCs. PDGF-induced BMK1 activation was suppressed by the Src family tyrosine kinase inhibitors, indicating that Src exists upstream of BMK1. The NADPH oxidase inhibitors inhibited not only PDGF-induced BMK1 and Src activation but also RMC migration. The elevation in ROS generation induced by PDGF was decreased by olmesartan. Olmesartan displayed neither directly ROS scavenging activity nor the inhibition of ROS-mediated intracellular signalling in RMCs. CONCLUSIONS Olmesartan attenuates ROS generation by PDGF, leading to the subsequent inhibition of Src/ BMK1/migration in an AT1 receptor-independent manner in RMCs.

Cytocidal activity of PBL, LAK, and IDEC-C2B8 and expression of HLA class 1, ICAM-1, and CD20 in vincristine-resistant hematologic cell lines.

This study was designed to determine whether the cytocidal activity of immunotherapy such as cytotoxic peripheral blood lymphocytes (PBL), lymphokine-activated killer (LAK) cells, and chimeric anti-CD20 mouse/human monoclonal antibody, IDEC-C2B8, overcome vincristine (VCR) resistance in cultured cell lines derived from human leukemia/lymphoma. In addition, the relation between the susceptibility to these immunotherapies and the expression levels of HLA class 1 and ICAM-1 as well as CD20 on the cell surface was analyzed. Three of six VCR-resistant cell lines were less susceptible to PBL cytotoxicity compared with wild-type cells, whereas the susceptibility was kept in the other three VCR-resistant cell lines. Four of six VCR-resistant cell lines were less susceptible to LAK activity and the other two cell lines were as sensitive to LAK cells as their wild-type counterparts. There was no correlation between the susceptibility for PBL cytotoxicity and the expression of HLA class 1 in both wild and VCR-resistant cells. In contrast, ICAM-1 in the two cell lines that showed decreased susceptibility for LAK cytotoxicity disappeared, although that in one cell line increased. IDEC-C2B8 was effective only against B-cell lines expressing CD20. One cell line in which the expression of CD20 increased was nearly six times more sensitive to IDEC-C2B8 than wild type. Thus, we concluded that the resistance to VCR in some tumor cell lines is associated with modified susceptibility for immunotherapies by the different expression of target molecules from those of wild-type counterparts.

Ionic factors affecting the association of tyrosine hydroxylase with chromaffin granules in the adrenal medullary cell

Chromaffin cells were treated with digitonin in medium containing various ions and the efflux of tyrosine hydroxylase from these permeabilized cells was then determined to elucidate a possible influence of cytoplasmic ionic environment on the association of this enzyme with the chromaffin granule. The enzyme efflux was observed with a distinct lag during exposure to low concentrations of digitonin in the medium containing isotonic sucrose. In contrast, a larger extent of the enzyme efflux was observed without any notable delay in the presence of isotonic NaCl. The results were thought to indicate the possibility that the dissociation of soluble enzyme from the granule surface within the permeabilized cells might occur in the presence of NaCl. Furthermore, the interaction between tyrosine hydroxylase and isolated chromaffin granule membranes was directly examined, and this interaction was shown to be inhibited by NaCl. However, the enzyme-granule membrane interaction was also inhibited by KCl and choline chloride. It therefore seems possible to consider that the inhibitory action of NaCl on the association of soluble enzyme with the granule may not be due to the specific action of Na+, but presumably due to the non-specific chaotropic effect of Cl-. On the other hand, the enzyme efflux was markedly reduced by the presence of Ca2+ in the permeabilizing medium, but the enzyme-granule membrane interaction was not affected by Ca2+ at the same concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Further studies on the relationship between tyrosine supply and catecholamine production in cultured adrenal chromaffin cells

To elucidate a possible role of tyrosine supply as a factor modulating catecholamine biosynthesis in the adrenergic cell, the transport of [14C]tyrosine into cultured bovine adrenal chromaffin cells was first examined, and the relationship between [14C]tyrosine transport and [14C]catecholamine formation was then investigated. Under the conditions which were routinely employed to determine the rate of catecholamine biosynthesis, tyrosine was taken up into the cells in a manner independent of extracellular Na+ and Ca2+, and this uptake was also insensitive to ouabain and various metabolic inhibitors. The stimulation of these cells with high K+ and other secretagogues caused no significant alteration in the uptake. While, tyrosine transport was markedly inhibited by tyrosine analogues and other L-aromatic amino acids, and this inhibition was accompanied by the reduction of [14C]catecholamine formation. In contrast, tyrosine transport was markedly enhanced by flavone, and this enhancement was also accompanied by the augmentation of catecholamine production under the same experimental conditions. These results seem to indicate that the transport of tyrosine into the cells may be closely related to catecholamine formation within the cells, thus providing an evidence for a possible role of tyrosine supply as one of the factors affecting catecholamine production in the adrenal chromaffin cell.

Expression levels of H-type alpha(1,2)-fucosyltransferase gene and histo-blood group ABO gene corresponding to hematopoietic cell differentiation

BACKGROUND : The expression of ABO antigens on the surface of RBCs is regulated by ABO gene‐encoded ABO transferase activity after the formation of the H antigen. The molecular mechanisms that control the expression of the ABO blood group antigens along with erythroid differentiation are one of the most important subjects of study in transfusion science.

Effects of bioflavonoids on exocytotic release of catecholamines from digitonin-permeabilized chromaffin cells: A comparison with the effects of other protein kinase C inhibitors.

The effects of bioflavonoids on catecholamine release from permeabilized adrenal chromaffin cells were examined to show their intracellular actions on exocytosis. The release from these permeabilized cells in response to a direct calcium challenge was shown to be markedly inhibited by quercetin in a manner dependent on its concentration. Apigenin was also shown to cause a moderate inhibitory action, but flavone caused no significant effect on the release under the experimental conditions used here. Furthermore, the inhibitory actions of these flavonoids on the phorbol ester-dependent fraction of catecholamine release were shown to be more pronounced than those on the calcium-dependent fraction. The effects of bioflavonoids on the calcium-dependent and the phorbol ester-dependent releases were then compared with those of other protein kinase C inhibitors, and quercetin was shown to cause a potent inhibitory action on the exocytotic secretory process, which was almost equivalent to those caused by polymyxin B and neomycin. Both quercetin and apigenin were clearly shown to inhibit the phorbol ester-dependent as well as the calcium-dependent release of catecholamines from digitonin-permeabilized chromaffin cells. The inhibitory actions of these compounds were therefore thought to be attributed to their inhibitory actions on protein kinase C in the cytoplasmic space of the permeabilized cells. Thus, these results seem to provide further evidence for a possible involvement of protein kinase C as one of the sites for calcium action in the intracellular mechanism of exocytotic secretion.