Biomedical subjects

M Shichiri

Publications and source records attributed to M Shichiri.

At least 55 records · Page 3Linked to original sources

PubMed1999

Glucocorticoid inhibits oxidized LDL-induced macrophage growth by suppressing the expression of granulocyte/macrophage colony-stimulating factor.

Glucocorticoid, an anti-inflammatory agent, inhibits the development of atherosclerosis in various experimental animal models. This is partially explained by its ability to inhibit smooth muscle cell migration and proliferation in the intima and to reduce chemotaxis of circulating monocytes and leukocytes into the subendothelial spaces. We have recently demonstrated that oxidized LDL (Ox-LDL) has a mitogenic activity for macrophages in vitro in which Ox-LDL-induced granulocyte/macrophage colony-stimulating factor (GM-CSF) production plays an important role. Proliferation of cellular components is one of the characteristic events in the development and progression of atherosclerotic lesions. In the present study, we investigated the effects of glucocorticoids on Ox-LDL-induced macrophage growth. Dexamethasone, prednisolone, and cortisol inhibited Ox-LDL-induced thymidine incorporation into macrophages by 85%, 70%, and 50%, respectively. Ox-LDL induced a significant production of GM-CSF by macrophages, which was effectively inhibited by dexamethasone, prednisolone, and cortisol by 80%, 65%, and 50%, respectively. Dexamethasone-mediated inhibition of Ox-LDL-induced GM-CSF mRNA expression and macrophage growth was significantly abrogated by RU-486, a glucocorticoid receptor antagonist. Our results suggest that the inhibitory effects of glucocorticoids on macrophage growth may be due to the inhibition of Ox-LDL-induced GM-CSF production through transactivation of the glucocorticoid receptor.

Animals↗

PubMed1999

Differential inhibitory actions by glucocorticoid and aspirin on cytokine-induced nitric oxide production in vascular smooth muscle cells.

Glucocorticoids and nonsteroidal antiinflammatory drugs (NSAIDs) are widely used for the treatment of inflammatory and immune diseases. Nitric oxide (NO) has a diversity of physiological functions, but its excess production has been implicated in the inflammatory process. The present study was designed to elucidate the mechanisms by which glucocorticoids and NSAIDs affect inducible nitric oxide synthase (iNOS) expression in cultured rat vascular smooth muscle cells (VSMCs). Both interleukin (IL)-1beta and tumor necrosis factor (TNF)-alpha potently stimulated nitrite/nitrate (NOx) production with a concomitant expression of iNOS mRNA and protein as demonstrated by Northern and Western blot analysis, respectively. Both IL-1beta and TNF-alpha activated nuclear factor (NF)-kappaB as demonstrated by electrophoretic mobility shift assay. Dexamethasone, salicylate and aspirin, but not indomethacin, dose dependently inhibited cytokine-stimulated NOx production and iNOS protein expression. Dexamethasone decreased cytokine-induced NF-kappaB activation and iNOS mRNA expression, but neither salicylate nor aspirin affected NF-kappaB activation or iNOS mRNA expression. IL-1beta caused a rapid increase in phosphorylated IkappaB-alpha levels and subsequent transient decrease in IkappaB-alpha levels, an inhibitor of NF-kappaB, as revealed by Western blot analysis using specific antibodies for phosphorylated and nonphosphorylated IkappaB-alpha. These effects were blocked by pretreatment with dexamethasone. Aspirin dose dependently inhibited iNOS enzymatic activity, whereas salicylate and dexamethasone had limited effect. The present study demonstrates that 1) inhibitory effect of dexamethasone on cytokine-induced iNOS expression and NO production in rat VSMCs, although potentially acting at multiple levels, is partly mediated by inhibition of NF-kappaB activation resulting from decreased phosphorylation and degradation of IkappaB-alpha, 2) both salicylate and aspirin inhibit cytokine-stimulated NO production at translational and/or posttranslational levels without affecting NF-kappaB- mediated iNOS gene expression, and 3) aspirin directly inhibits iNOS enzyme activity. These data suggest the differential inhibitory mechanisms of iNOS-mediated NO synthesis by glucocorticoids and NSAIDs in the vasculature.

Animals↗

PubMed1999

Induction of max by adrenomedullin and calcitonin gene-related peptide antagonizes endothelial apoptosis.

Adrenomedullin is a novel vasodilatory peptide originally isolated from pheochromocytoma. Recently, we found that adrenomedullin acts as an autocrine/paracrine apoptosis survival factor for rat endothelial cells. In the present study, we show that adrenomedullin induces the expression of Max, a heterodimeric partner of c-Myc, which may contribute to its ability to rescue endothelial cells from apoptosis. Max is a basic-helix-loop-helix-leucine zipper protein that forms heterodimers with its alternative partners, Mad and Mxi-1, to behave as an antagonist for Myc-Max heterodimer through competition for common DNA targets. The expression of Max is reported to be constitutive and more stable than c-Myc, and serum induces immediate c-Myc stimulation followed by modest Max up-regulation. In quiescent rat endothelial cells, adrenomedullin stimulated the expression of Max without affecting c-Myc. Quantitation with real-time quantitative PCR detected on the ABI Prism 7700 Sequence Detection System revealed that adrenomedullin and calcitonin gene-related peptide (CGRP), as well as serum, up-regulated Max mRNA levels and that down-regulation of Max mRNA after serum deprivation was prevented by adrenomedullin. Neither adrenomedullin nor CGRP affected c-Myc expression. Transfection of a Max-expressing plasmid into endothelial cells rescued the apoptosis induced by serum deprivation. Neutralization with anti-adrenomedullin antiserum or blockade with a CGRP receptor antagonist, CGRP(8-37), reduced Max mRNA levels in growing endothelial cells and enhanced apoptosis after serum starvation. Introduction of an antisense oligodeoxynucleotide against Max mRNA using transferrin receptor-operated transfer led to inhibition of both adrenomedullin-induced up-regulation of Max transcripts and its cell survival effect, whereas random, sense, or missense oligonucleotides were without effect. The negative regulation of E-box-driven transcription by adrenomedullin was demonstrated by using preproendothelin-1 promoter containing c-Myc-Max binding consensus sequence; the promoter activity of preproendothelin-1 was reduced by cotransfecting Max- and Mad-expressing plasmids as well as addition of adrenomedullin and CGRP. The present results demonstrate that adrenomedullin antagonizes serum deprivation-induced endothelial apoptosis by up-regulation of the max gene in an autocrine/ paracrine manner.

Adrenomedullin↗

PubMed1999

Possible involvement of atypical protein kinase C (PKC) in glucose-sensitive expression of the human insulin gene: DNA-binding activity and transcriptional activity of pancreatic and duodenal homeobox gene-1 (PDX-1) are enhanced via calphostin C-sensitive but phorbol 12-myristate 13-acetate (PMA) and Gö 6976-insensitive pathway.

Pancreatic and duodenal homeobox gene-1 (PDX-1) is a transcription factor which regulates the insulin gene expression. In this study, we tried to elucidate the role of PDX-1 in the glucose-induced transcriptional activation of the human insulin gene promoter in MIN6 cells. Electrophoretic mobility shift assay (EMSA) and chloramphenicol acetyltransferase (CAT) assay demonstrated that both DNA-binding activity and transcriptional activity of PDX-1 were increased with 20 mmol/l glucose more than with 2 mmol/l glucose. The DNA-binding activity of PDX-1 induced by high glucose was blocked by phosphatase treatment, suggesting the involvement of PDX-1 phosphorylation in this event. In an in vitro phosphorylation study, PDX-1 was phosphorylated by protein kinase C (PKC), but not by cAMP dependent protein kinase (PKA) or mitogen-activated protein kinase (MAPK). Furthermore, increased PDX-1 function induced by high glucose was blocked by calphostin C, an inhibitor of all PKC isoforms, but unaffected by phorbol 12-myristate 13-acetate (PMA), an activator of classical and novel PKC, or Gö 6976, an inhibitor of classical and novel PKC, which suggested that the PKC family which activated PDX-1 in MIN6 cells was atypical PKC. Western blot and immunocytochemical studies with anti-PKC zeta antibody confirmed the presence of PKC zeta, one of the isoforms of atypical PKC, in MIN6 cells. Furthermore, PKC zeta activity was significantly increased by glucose stimulation. These results suggest that high glucose increased DNA-binding activity of PDX-1 by activating atypical PKC including PKC zeta, resulting in transcriptional activation of the human insulin gene promoter.

Animals↗

PubMed1999

[Indication of intensive insulin therapy and its practice].

DCCT and Kumamoto Study demonstrated that optimal glycemic control with intensive insulin therapy could delay the onset and progression of the early stages of diabetic microvascular complications in Japanese patients with noninsulin dependent diabetes as well as in patients with insulin dependent diabetes mellitus. To obtain optimal glycemic control, the prandial insulin supplements before each meal are recommended for insulin requiring patients with noninsulin dependent diabetes mellitus whose residual B cell functions are retained to some extent, whereas the combined basal at bedtime plus prandial insulin supplements are essential for the patients with noninsulin dependent diabetes mellitus whose residual B cell functions are severely exhausted as well as in the patients with insulin dependent diabetes mellitus. The glycemic threshold to prevent the onset and progression of diabetic microvascular complications in the Kumamoto Study was indicated as follows; HbA1c < 6.5%, fasting blood glucose concentration < 110 mg/dl, and 2-hour postprandial blood glucose concentration < 180 mg/dl.

Blood Glucose↗

PubMed1999

[Design and development strategy for wearable and implantable artificial endocrine pancreas].

The ultimate goal of development of an artificial endocrine pancreas is for long-term strict glycemic control, and therefore, the trend in development is now from bedside-type to wearable- or implantable-type. With either a miniaturized extracorporeal glucose monitoring system based on microdialysis sampling method or a ferrocene-mediated needle-type glucose sensor covered with highly biocompatible membrane, and with subcutaneous insulin infusion algorithm using short-acting insulin analogue, long-term physiological glycemic control could be obtained by wearable artificial endocrine pancreas. The next step will be directed to the implantable one. Non-invasive infrared absorbance spectroscopy to fit into an artificial tooth prosthesis, an implantable artificial endocrine pancreas, in which measured glucose concentrations are transmitted telemetrically to implanted computer and pump system, might be developed.

Equipment Design↗

PubMed1999

[Serum uric acid level and fractional excretion of urate in fluid and electrolyte disturbances].

Changes in renal urate clearance may reflect altered glomerular dynamics more precisely than other commonly used indices. Extracellular fluid volume expansion is known to be correlated with uricosuria, while volume depletion is associated with decreased urate excretion. The diagnostic usefulness of measuring serum urate levels and fractional excretions of urate in SIADH, incipient diabetic nephropathy, and prerenal azotemia have been reviewed. In SIADH, profound hypouricemia and markedly increased fractional excretion of urate (FEUA) accompany the extracellular fluid volume expansion. In prerenal azotemia, decreased FEUA may represent a reliable indicator of prerenal azotemia in the differential diagnosis of acute renal failure. In incipient diabetic nephropathy, glomerular hyperfiltration may increase renal urate clearance and lower the serum uric acid level. Hypouricemia may also predict the future progression of incipient nephropathy in type II diabetes.

Biomarkers↗

PubMed1999

Non-invasive blood glucose measurement by Fourier transform infrared spectroscopic analysis through the mucous membrane of the lip: application of a chalcogenide optical fiber system.

Non-invasive blood glucose measurement through the mucous membrane of the lip was investigated using Fourier transform infrared (FT-IR) spectroscopy with an attenuated total reflection (ATR) prism. To achieve easy attachment and easy control of attachment pressure of the ATR prism to the mucous membrane of the lip, a chalcogenide optical fiber with an ATR prism built in the tip was used. The same glucose-specific peaks at wave numbers of 1080 and 1033 cm-1 as glucose solutions were found in a spectrum through the mucous membrane of the lip. With a constant pressure of the ATR prism to the mucous membrane of the lip of 6.7 x 10(3) dyn/cm2, coefficients of variation of measurements within the day and of day-to-day measurements were 3.8 and 5.4% respectively. To eliminate baseline drifts and interference of body constituents other than glucose, the difference absorbances at 1080 cm-1 between spectra measured at the postprandial state and background spectrum obtained at the fasting state as an individual characteristic were evaluated. Following i.v. pulsatile injection of glucose, the difference absorbances at 1080 cm-1 nicely followed the changes in blood glucose concentrations with a time delay of 4 min. In daily blood glucose monitoring, a highly significant correlation between the difference absorbances and increases in blood glucose concentrations above the fasting level was obtained (r = 0.920, P < 0.01). From these experiments, it was suggested that FT-IR spectroscopy with a chalcogenide optical fiber could be useful clinically for non-invasive monitoring of glucose through the mucous membrane of the lip.

Adult↗

PubMed1999

[Endothelin].

Explore the source record for details and available documents.

Endothelin-1↗

PubMed1998

Induction of murine macrophage growth by oxidized low density lipoprotein is mediated by granulocyte macrophage colony-stimulating factor.

We have examined whether certain secreted factor(s) is involved in oxidized low density lipoprotein (Ox-LDL)-induced murine macrophage growth. An antibody against granulocyte-macrophage colony-stimulating factor (GM-CSF) effectively inhibited Ox-LDL-induced macrophage growth by >80%. Ox-LDL as well as phospholipase A2-treated acetylated LDL enhanced mRNA levels and protein release of GM-CSF from macrophages, while neither acetylated LDL nor lysophosphatidylcholine (lyso-PC) showed such effects. The maximal induction of GM-CSF by Ox-LDL was noted at 4 h, followed by a time-dependent decrease to a basal level within 24 h. Ox-LDL-induced macrophage growth was inhibited by 75% by replacement of the culture medium at 24 h by a fresh medium containing the same concentration of Ox-LDL, when GM-CSF had already returned to the basal level. Thus, a cytokine(s) other than GM-CSF is also expected to participate in Ox-LDL-induced macrophage growth in a later phase. The Ox-LDL-induced GM-CSF release was inhibited by calphostin C, a protein kinase C inhibitor, and was significantly reduced in macrophages from the knockout mice lacking class A, type I and type II macrophage scavenger receptors (MSR-AI/AII). These results taken together indicate that effective endocytosis of lyso-PC of Ox-LDL by macrophages through MSR-AI/AII and subsequent protein kinase C activation have led to GM-CSF release into the medium which may play a priming role in conjunction with other cytokines in Ox-LDL-induced macrophage growth.

Animals↗

PubMed1998

Transfection of inducible nitric oxide synthase gene causes apoptosis in vascular smooth muscle cells.

BACKGROUND: Excess production of nitric oxide (NO) by inducible NO synthase (iNOS) has been implicated in a variety of physiological processes including vascular remodeling. To elucidate whether endogenous NO generated by iNOS is involved in the programmed cell death (apoptosis) of the vasculature, iNOS cDNA- expressing construct was transfected into rat and human vascular smooth muscle cells (VSMCs) by lipofection. METHODS AND RESULTS: VSMCs transiently transfected with iNOS cDNA functionally expressed 130 kd iNOS protein with full catalytic activity to generate massive NO in proportion to the doses of cDNA used; its enzymatic activity as well as NO production was completely blocked by an NOS inhibitor, NG-monomethyl-L-arginine (LNMMA). Overexpression of iNOS led to a marked inhibition of DNA synthesis as well as induction of apoptosis in VSMCs. Evidence for apoptotic cell death was provided by internucleosomal DNA fragmentation by agarose gel electrophoresis, positive staining for TdT-mediated dUTP biotin nick end-labeling, and appearance of hypodiploid cells by flow cytometry analysis. Apoptosis after transfection with iNOS cDNA was abrogated by LNMMA. Transfection of iNOS cDNA caused accumulation of the tumor suppressor gene p53 but not of bcl-2, which was also blocked by LNMMA. CONCLUSIONS: These results demonstrate that massive generation of endogenous NO derived from iNOS overexpression leads to a marked apoptosis in VSMCs, thus suggesting an important role of NO as a proapoptotic factor for VSMCs in the process of vascular remodeling.

Animals↗

PubMed1998

Down-regulation of adenylate cyclase coupled to adrenomedullin receptor in vascular smooth muscle cells.

Adrenomedullin activates receptor-mediated adenylate cyclase to cause vasorelaxation. To elucidate whether desensitization of adenylate cyclase coupled to vascular adrenomedullin receptors occurs, we studied the adenylate cyclase activity after treatment with rat adrenomedullin in cultured rat aortic vascular smooth muscle cells. Cyclic AMP (cAMP) generation induced by adrenomedullin was markedly decreased by pretreatment with adrenomedullin: a maximal reduction (approximately 80%) was induced after 2 h and persisted during 24 h. Desensitization was independent of protein kinase A, protein kinase C, protein tyrosine kinase or receptor sequestration, because pretreatment with either isoproterenol, forskolin, tetradecanoylphorbol acetate, cytochalasin D, or colchicine did not affect the adrenomedullin-stimulated cAMP response. Furthermore, preincubation with inhibitors for these protein kinases prior to pretreatment with adrenomedullin failed to affect the adrenomedullin-induced decrease in cAMP response following the second stimulation with adrenomedullin. The present results provide the evidence for the existence of desensitization of adenylate cyclase coupled to vascular adrenomedullin receptors.

Adenylyl Cyclases↗

PubMed1998

Molecular cloning, functional expression and tissue distribution of rat acyl-coenzyme A:cholesterol acyltransferase.

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is an enzyme catalyzing the intracellular formation of cholesteryl esters from free cholesterol and fatty acyl-CoA. In the present study, we cloned rat ACAT cDNA and determined its tissue distribution. Rat ACAT cDNA, having a coding region of 1635 bp with its deduced protein sequence of 545 amino acids and two typical motifs such as signature sequences and leucine heptad motif, showed 83, 92 and 90% identity with human, mouse, and hamster ACAT, respectively. Expression of rat ACAT cDNA in A293 cells and CHO cells resulted in a 3.0 to 3.5-fold increase in the enzyme activity. Among twelve tissues examined, ACAT activity was highest in adrenal followed by liver and intestine while that of aorta was extremely low. The mRNA level was also the highest in adrenal among four tissues examined. However, in contrast to its high ACAT activity, the liver mRNA level was extremely low (adrenal >> intestine > aorta >> liver). Consistent with mRNA levels, immunohistochemical analyses with a specific ACAT antibody detected significant ACAT signals in adrenal and intestine but a negligible signal in liver. These results indicate that adrenal most abundantly expresses ACAT in rat. Furthermore, rat liver showed a high ACAT activity but an extremely low ACAT mRNA and negligible immunohistochemical reactivity, suggesting the presence of a structurally different ACAT protein(s) in rat liver.

Amino Acid Sequence↗

PubMed1998

Cellular characterization of pituitary adenoma cell line (AtT20 cell) transfected with insulin, glucose transporter type 2 (GLUT2) and glucokinase genes: insulin secretion in response to physiological concentrations of glucose.

We investigated the mechanisms of insulin secretion by transfecting into a pituitary adenoma cell line (AtT20) a combination of genes encoding human insulin (HI), glucose transporter type 2 (GLUT2) and glucokinase (GK), followed by studying the characteristics of these cells. In static incubation, a cell line transfected with insulin gene alone (AtT20HI) secreted mature human insulin but this was not in a glucose-dependent manner. Other cell lines transfected with insulin and GLUT2 genes (AtT20HI-GLUT2-3) or with insulin and GK genes (AtT20HI-GK-1) secreted insulin in response to glucose concentrations of only less than 1 mmol/l. In contrast, cell lines transfected with insulin, GLUT2 and GK genes (AtT20HI-GLUT2-GK-6, AtT20HI-GLUT2-GK-7 and AtT20HI-GLUT2-GK-10) showed a glucose-dependent insulin secretion up to 25 mmol/l glucose. Glucose utilization and oxidation were increased in AtT20HI-GLUT2-GK cell lines but not in AtT20HI, AtT20HI-GLUT2-3 and AtT20HI-GK-1 cells at physiological glucose concentrations, compared with AtT20 cells. Diazoxide, nifedipine and 2-deoxy glucose suppressed (p < 0.05) glucose stimulated insulin secretion in AtT20HI-GLUT2-GK-6 cells. Glibenclamide, KCl or corticotropin releasing factor (CRF) stimulated (p < 0.05) insulin secretion both in AtT20HI and AtT20HI-GLUT2-GK-6 cells. Insulin secretion stimulated by glibenclamide, KCl or CRF was further enhanced by the addition of 25 mmol/l glucose in AtT20HI-GLUT2-GK-6 cells but not in AtT20HI cells. In perifusion experiments, a stepwise increase in glucose concentration from 5 to 25 mmol/l stimulated insulin secretion in AtT20HI-GLUT2-GK cell lines but the response lacked a clear first phase of insulin secretion. Our results suggest that both GLUT2 and glucokinase are necessary for the glucose stimulated insulin secretion in at least rodent cell lines, and that other element(s) are necessary for a biphasic insulin secretion typically observed in beta cells.

Adenoma↗

PubMed1998

Serotonin enhances the production of type IV collagen by human mesangial cells.

BACKGROUND: The plasma concentration of 5-hydroxytryptamine (5-HT) in diabetic patients is higher than that in normal subjects. Since recent reports have demonstrated the presence of 5-HT2A receptor in glomerular mesangial cells, it is possible that 5-HT may be involved in the development of diabetic nephropathy through the 5-HT2A receptor in mesangial cells. Because expansion of the glomerular mesangial lesion is a characteristic feature of diabetic nephropathy, we examined the effect of 5-HT on the production of type IV collagen by human mesangial cells. METHODS: Human mesangial cells were incubated with 5-HT with or without 5-HT receptor antagonists, protein kinase C (PKC) inhibitor or transforming growth factor-beta (TGF-beta) antibody. Type IV collagen mRNA and protein concentration in medium were measured by Northern blot analysis and enzyme-linked immunosorbent assay (ELISA), respectively. TGF-beta mRNA and bioactivity in the medium were measured by Northern blot analysis and bioassay using mink lung epithelial cells, respectively. RESULTS: 5-HT stimulated the production of type IV collagen by human mesangial cells, which was inhibited by ketanserin and sarpogrelate hydrochloride, 5-HT2A receptor antagonists, but not by ondansetron, a 5-HT3 receptor antagonist. 5-HT increased the bioactivities of both active and total TGF-beta. However, the 5-HT-enhanced production of type IV collagen was completely inhibited by an anti-TGF-beta antibody. Furthermore, a PKC inhibitor, calphostin C, inhibited the 5-HT-induced increase in type IV collagen secretion, and the activity of membrane PKC was increased by 5-HT. Phorbol ester activated type IV collagen production as well as active and total TGF-beta. Calphostin C completely inhibited the 5-HT-enhanced activity of active TGF-beta, but did not inhibit exogenous TGF-beta-induced increase in type IV collagen secretion. CONCLUSIONS: Our results suggest that 5-HT-enhanced production of type IV collagen by human mesangial cells is mediated by activation of PKC and subsequent increase in active TGF-beta activity.

Cells, Cultured↗

PubMed1998

Enhanced, simplified glucose sensors: long-term clinical application of wearable artificial endocrine pancreas.

At present, 2 major problems should be solved before long-term application of the wearable artificial endocrine pancreas, the development of a reliable and stable glucose monitoring system and the development of a subcutaneous insulin infusion algorithm. With either a miniaturized extracorporeal glucose monitoring system based on microdialysis sampling method or a ferrocene-mediated needle-type glucose sensor covered with highly biocompatible membrane, poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (poly[MPC-co-BMA]) membrane, subcutaneous glucose concentrations could be monitored for 7 days without any in vivo calibrations, followed by 14 days with one point calibration. Considering the management and safety of the insulin delivery route, subcutaneous insulin infusion is obligatory. With the subcutaneous insulin infusion algorithm using a short acting insulin analogue (Insulin Lispro), near physiological glycemic control could be established in diabetic patients without showing any delayed hyperinsulinemia or hypoglycemia. The wearable artificial endocrine pancreas is now recognized as an excellent therapeutic tool for regulating blood glucose excursions physiologically in ambulatory diabetic patients on a long-term basis.

Animals↗

PubMed1998

Endothelin-B receptor-mediated suppression of endothelial apoptosis.

Endothelin (ET)-1 is an endothelium-derived vasoconstrictor as well as a mitogen. We have recently described a novel role of ET-1 as a survival factor for rat endothelial cells from serum deprivation-induced apoptosis. The present study was designed to determine which receptor subtype (ETA or ETB) is responsible for and what intracellular mediators are involved in endothelial apoptosis. Apoptotic cell death was evaluated by nucleosomal ladders on agarose gel electrophoresis and immunohistochemical study using anti-single-stranded DNA antiserum. ET-1 and an ETB receptor agonist suppressed endothelial apoptosis, whose effects were abrogated by an ETB receptor antagonist but not by an ETA receptor antagonist. Addition of an ETB receptor antagonist or nonselective ETA/B receptor antagonists, but not an ETA receptor antagonist, enhanced the apoptotic events caused by serum deprivation, suggesting an autocrine/paracrine role of endogenous ET-1 in protecting against endothelial apoptosis. The effect of ET-1 in suppressing apoptosis was unaffected by any of the following reagents: a phospholipase C inhibitor (U73122), a tyrosine kinase inhibitor (ST638), an MEK inhibitor (PD98059), a phosphatidylinositol-3 kinase inhibitors (wortmannin, LY294002). Taken together, these results confirm a role for ET-1 as an autocrine/paracrine survival factor for rat endothelial cells, in which neither phospholipase C, tyrosine kinase, MAP kinase, nor phosphatidylinositol-3 kinase is involved in mediating the antiapoptotic effect of ET-1.

Animals↗

PubMed1998

NO inhibits cytokine-induced iNOS expression and NF-kappaB activation by interfering with phosphorylation and degradation of IkappaB-alpha.

Nitric oxide (NO) is known to have antiatherogenic and anti-inflammatory properties, but its effects on the cytokine-induced nuclear factor-kappa B (NF-kappaB) activation pathway in relation to the regulation of inducible nitric oxide synthase (iNOS) gene in vascular smooth muscle cells (VSMCs) remain elusive. To elucidate the roles of NO in the regulation of cytokine-induced NF-kappaB activation and consequent iNOS gene expression, we studied the effects of NO donors [(+/-)-(E)-ethyl-2-[(E)-hydroxyamino]-5-nitro-3-hexeneamide (NOR3) and sodium nitroprusside] on interleukin (IL)-1beta-induced NF-kappaB activation and IkappaB-alpha degradation and subsequent iNOS expression in rat VSMCs. Northern blot and Western blot analyses demonstrated that NO donors decreased IL-1beta-induced iNOS mRNA and protein expression. Electrophoretic mobility shift assay using synthetic oligonucleotide corresponding to the downstream NF-kappaB site of rat iNOS promoter as a probe showed that NOR3 inhibited IL-1beta-induced NF-kappaB activation and its nuclear translocation, as demonstrated with immunocytochemical study. These effects were independent of guanylate cyclase activation; an inhibitor of soluble guanylate cyclase (1H-oxadiazolo-1,2,4-[4,3-alpha]quinoxaline-1-one) had no effect on NOR3-induced inhibition of NF-kappaB activation or iNOS mRNA expression by IL-1beta, and a cGMP derivative (8-bromo-cGMP) failed to mimic the effects of NO donors. Western blot analysis using anti-IkappaB-alpha and anti-phospho-IkappaB-alpha antibodies revealed that IL-1beta induced a transient degradation of IkappaB-alpha preceded by a rapid appearance of phosphorylated IkappaB-alpha, both of which were completely blocked by NOR3. A proteasome inhibitor (MG115) blocked IL-1beta-induced transient degradation of IkappaB-alpha and stabilized the appearance of phosphorylated IkappaB-alpha stimulated by IL-1beta. NOR3 inhibited the appearance of IL-1beta-induced phosphorylated IkappaB-alpha even in the presence of MG115. Our results indicate that an inhibitory action by NO on cytokine-induced NF-kappaB activation and iNOS gene expression is due to its direct blockade on phosphorylation and subsequent degradation of IkappaB-alpha via the cGMP-independent pathway in rat VSMCs.

Animals↗