Takamura Muraki

Vasculo-protective effects of insulin sensitizing agent pioglitazone in neointimal thickening and hypertensive vascular hypertrophy

A novel insulin sensitizing agent, thiazolidine, has been demonstrated to inhibit the growth of cultured vascular smooth muscle cells (VSMC) in vitro. This study was undertaken to examine the in vivo effects of the thiazolidine compound pioglitazone (PIO) on carotid neointimal thickening, after endothelial injury in Wistar rats and vascular hypertrophy in stroke-prone spontaneously hypertensive rats (SHR-SP/Izm). PIO treatment (3 mg/kg/day for 1 week prior to endothelial injury and 2 weeks postendothelial injury) remarkably decreased neointimal cross-sectional areas in treated animals (63.8 +/- 4.9 x 10(3) microm2) versus controls (196 +/- 7.6 x 10(3) microm2, P < 0.05). Bromodeoxyuridine uptake in the neointima, a marker of DNA synthesis, was also decreased after treatment compared with controls. In SHR-SP/Izm but not in Wistar rats, PIO treatment decreased blood pressure and plasma insulin levels. PIO treatment in SHR-SP/Izm (3 mg/kg/day from 4 weeks of age for 7 weeks) significantly decreased the medial wall thickness of the mesenteric artery (10.4 +/- 1.2 x 10(3) microm2 versus control, 21.2 +/- 2.4 x 10(3) microm2, P < 0.05). In addition, PIO treatment significantly decreased the expression of EIIIA fibronectin both in the carotid neointima of Wistar rats and the media of the mesenteric artery in SHR-SP/Izm compared with their respective controls (P < 0.05). These results suggest that PIO has vasculo-protective effects in both acute and chronic vascular injury in vivo through inhibition of VSMC proliferation.

Uraemic toxins induce proximal tubular injury via organic anion transporter 1-mediated uptake.

A direct effect of uraemic toxins in promoting progression of chronic renal disease has not been established. In this study, we investigated the toxic effects of organic anions which characteristically appeared in the patients with progressive renal disease on renal proximal tubular cells expressing human organic anion transporter (hOAT) 1. A renal proximal tubular cell line, opossum kidney (OK) cells, was transformed with hOAT1. Among the organic anions examined, hippuric acid, para‐hydroxyhippuric acid, ortho‐hydroxyhippuric acid, indoxyl sulphate and indoleacetic acid showed a high affinity for hOAT1 expressed in the OK cells. Indoxyl sulphate and indoleacetic acid concentration‐dependently inhibited proliferation of the hOAT1‐transformed cells. The h.p.l.c. analysis demonstrated that cellular uptake of these organic anions was significantly elevated in hOAT1‐transformed cells. These organic anions also concentration‐dependently stimulated cellular free radical production. The degrees of inhibition of cell proliferation and the stimulation of free radical production induced by the organic anions were significantly higher in the hOAT1‐transformed cells than vector‐transformed cells. The stimulatory effect of indoxyl sulphate on free radical production was abolished by anti‐oxidants and probenecid. Less free radical production was observed in the hOAT1‐transformed cells treated with p‐hydroxyhippuric acid, o‐hydroxyhippuric acid compared with indoxyl sulphate and indoleacetic acid. Hippuric acid had little effect on free radical production. Organic anions present in the serum of patients with progressive renal disease may cause proximal tubular injury via hOAT1‐mediated uptake. The mechanism of cellular toxicity by these uraemic toxins involves free radical production. Thus, some uraemic toxins may directly promote progression of chronic renal disease.

Regulation of plasma insulin level by α2-adrenergic receptors

Phentolamine, yohimbine or dihydroergotamine markedly increased plasma immunoreactive insulin (IRI) and inhibited epinephrine-induced hyperglycemia in fasted mice. On the other hand, phenoxybenzamine or prazonsin only slightly increased plasma IRI and enhanced epinephrine-induced hyperglycemia. These results indicate that there is a distinct difference in the effects of α-adrenergic blockers on the plasma IRI and glucose levels, and that α-adrenergic receptors responsible for the plasma IRI level resemble α2-adrenergic receptors more closely.

Role of nitric oxide and prostaglandins in lipopolysaccharide-induced increase in vascular permeability in mouse skin

To examine the possible role of increased vascular permeability in the circulatory shock induced by endotoxin (lipopolysaccharide), we examined whether lipopolysaccharide elicits plasma extravasation in the skin of ddY strain mice. We also studied whether nitric oxide (NO) and prostaglandins may mediate the lipopolysaccharide-induced increase in vascular permeability. Subcutaneous injection of lipopolysaccharide (100-400 micrograms/site) induced a dose-related and delayed increase in vascular permeability at the injection site as determined by the leakage of pontamine sky blue. Concurrent administration of aminoguanidine (a putative inducible NO synthase inhibitor) (10 mg/kg, i.v.) inhibited the lipopolysaccharide (400 micrograms/site)-induced dye leakage by 71%. N(G)-Nitro-L-arginine methyl ester (an inhibitor for both constitutive and inducible NO synthase) (10 and 20 mg/kg, i.v.) inhibited the lipopolysaccharide-induced dye leakage by 36% and 54%, respectively, whereas the inactive enantiomer, N(G)-nitro-D-arginine methyl ester (10 mg/kg, i.v.), had no effect. Pretreatment with an intraperitoneal injection of dexamethasone (500 micrograms/kg) or indomethacin (a cyclooxygenase-1 and -2 inhibitor) (5 mg/kg) almost completely inhibited the response induced by lipopolysaccharide, by 96% and 84%, respectively. [N-(2-Cyclohexyloxy-4-nitrophenyl) methanesulphonamide (a cyclooxygenase-2-specific inhibitor) (0.01-1 mg/kg, i.p.) also induced a dose-related inhibition of dye leakage elicited by lipopolysaccharide: 38% and 80% suppression at the doses of 0.1 and 1 mg/kg, respectively. Cycloheximide (a protein biosynthesis inhibitor) (35 mg/kg, s.c.) suppressed the effect of lipopolysaccharide by 74%. These results suggest that the increase in vascular permeability induced by lipopolysaccharide is mediated by both NO and prostaglandins and that synthesis of inducible NO synthase and cyclooxygenase-2 may be involved in this effect of lipopolysaccharide.

Potentiation of Natriuretic Peptide Action by the β -Adrenergic Blocker Carvedilol in Hypertensive Rats: A New Antihypertensive Mechanism1

Treatment with a β-adrenergic blocker (β-blocker) in hypertension is associated with increased plasma atrial natriuretic peptide (ANP) levels despite a decrease in cardiac overload. The mechanism and pathophysiological significance of the phenomenon remain unclear. To clarify the role of the ANP system in the antihypertensive effects of the β-blocker, we investigated the effects of carvedilol (30 mg/kg·day, orally, for 4 weeks) on the ANP system in stroke-prone spontaneously hypertensive rats (SHR-SP/Izm). Plasma ANP levels showed a significant increase despite a significant decrease in blood pressure and heart rate in the carvedilol group. Although ANP messenger RNA levels in the heart did not change, messenger RNA levels of the natriuretic peptide-C (NP-C) receptor as a clearance receptor showed a significant decrease in both the aorta and lung in the carvedilol group. NP-C receptor densities were also significantly decreased in the lung in this group. The biological half-life of exogenous ANP in circul...

Role of nitric oxide, prostaglandins and tyrosine kinase in vascular endothelial growth factor-induced increase in vascular permeability in mouse skin

Abstract We investigated role of nitric oxide (NO), prostaglandins (PG) and tyrosine kinase in vascular endothelial growth factor (VEGF)-induced increase in vascular permeability in mouse skin. Subcutaneous injection of VEGF (0.5–2.0 ng/site) induced dose- and time-dependent increase in vascular permeability at the injection site determined by a leakage of Pontamine sky blue. VEGF (1 ng/site)-induced dye leakage was partially inhibited by NG-nitro-l-arginine methyl ester (an inhibitor for both constitutive and inducible NO synthase) (5 and 10 mg/kg, i.v.) and by aminoguanidine (a selective inducible NO synthase inhibitor) (5–20 mg/kg, i.v.), but not by an inactive enantiomer, NG-nitro-d-arginine methyl ester (10 mg/kg, i.v.). Pretreatment with an intraperitoneal injection of indomethacin (a nonselective cyclooxygenase inhibitor) (5 mg/kg) or N-(2-cyclohexyloxy-4-nitrophenyl) methanesulphonamide (a cyclooxygenase-2 selective inhibitor) (1–100 μg/kg) almost completely inhibited the effect of VEGF (1 ng/site). Coadministration of PGE2 (3 and 30 nmol/site) with VEGF did not restore the inhibitory effect of indomethacin on VEGF (1 ng/site)-induced increase in vascular permeability. Lavendustin A (a selective tyrosine kinase inhibitor) (10 and 50 μg/kg, s.c.) dose-relatedly inhibited the VEGF (1 ng/site)-induced increase in dye leakage, whereas its negative control, lavendustin B (10 μg/kg, s.c.) had no effect. Another tyrosine kinase inhibitor, genistein (2.5 mg/kg, s.c.) also inhibited the response. Cycloheximide (a protein biosynthesis inhibitor) (35 mg/kg, s.c.) suppressed the response of VEGF (1 ng/site). Histologically, no cellular infiltration was observed in the area of VEGF injection. These results suggest that increased vascular permeability induced by VEGF is mediated by local production of NO and arachidonic acid metabolites other than PGE2, which are most probably produced by inducible NO synthase and cyclooxygenase-2, respectively. Protein tyrosine kinase-mediated phosphorylation and synthesis of any new proteins are likely to be required in this effect of VEGF in mouse skin.

Possible role of nitric oxide in 5-hydroxytryptamine-induced increase in vascular permeability in mouse skin

In order to test the hypothesis that a 5-hydroxytryptamine (5-HT)-induced increase in vascular permeability results from a cascade triggered by activation of the synthesis of nitric oxide (NO), the vascular permeability was investigated using the Pontamine sky blue leakage method in male mice. Subcutaneous injection of 5-HT induced a dose-related increase of vascular permeability at the injection site. The vascular permeability induced by 5-HT was inhibited by pretreatment with intraperitoneal injection of ketanserin (5-HT2A antagonist) and methysergide (5-HT1/2A antagonist), less efficiently by 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl] piperazine (NAN-190) (5-HT1A antagonist), but not by granisetron (5-HT3 antagonist). Increase in vascular permeability induced by 5-HT was inhibited by concurrent intravenous administration of NO synthase inhibitors NG-nitro-Lrarginine methyl ester (L-NAME) and methylene blue but not by the inactive enantiomer NG-nitro-D-arginine methyl ester (D-NAME). These results suggest that 5-HT increases vascular permeability by activating the 5-HT receptors and that endogenous NO is involved in this effect of 5-HT.

Nitric oxide mediates down regulation of lipoprotein lipase activity induced by tumor necrosis factor-α in brown adipocytes

Abstract We previously reported that tumor necrosis factor- α (TNF- α )/cachectin suppresses lipoprotein lipase activity and its gene expression in brown adipocytes differentiated in culture. Recent evidence suggests that the effect of TNF- α over various cells is related to the enhanced production of nitric oxide (NO). The present study examined whether the suppressive effect of TNF- α on lipoprotein lipase activity is mediated by production of NO in the brown adipocytes. A reverse transcription-polymerase chain reaction (RT-PCR) assay revealed that TNF- α caused a concentration- and time-dependent expression of inducible NO synthase in brown adipocytes. Increasing concentrations of TNF- α (0.5–50 ng/ml) for 24 h resulted in a concentration-dependent decrease in lipoprotein lipase activity with reciprocal increase in nitrite production in the medium. The suppressive effect of TNF- α on lipoprotein lipase activity was significantly prevented by NO synthase inhibitors, N G -nitro- l -arginine methyl ester ( l -NAME) and aminoguanidine, but not by d -NAME, an inactive isomer. Furthermore, 8-bromoguanosine 3′,5′-cyclic monophosphate, cell permeant cGMP, suppressed lipoprotein lipase activity and 1 H -[1,2,4] oxadiazolo[4,3- a ]quinoxalin-1-one, a selective inhibitor for soluble guanylate cyclase, restored the TNF- α -suppressed lipoprotein lipase activity. These results suggest that TNF- α stimulates brown adipocytes to express inducible NO synthase, followed by production of NO, which in turn mediates the suppressive effect of TNF- α on lipoprotein lipase activity. The effect of NO is mediated, at least partly, through production of cGMP.

Acetyl-seryl-aspartyl-lysyl-proline is a novel natural cell cycle regulator of renal cells.

A natural tetrapeptide, acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is a physiological negative regulator of hematopoiesis. The precursor of AcSDKP, thymocin beta 4, is expressed in many tissues including kidney. The present study examined the antiproliferative effect of AcSDKP in two renal cell lines, namely, renal interstitial fibloblasts cell line (NRK 49F) and renal proximal tubular epitherial cells (LLC-PK1). An addition of AcSDKP for 48 hours in theses cells resulted in a concentration-dependent attenuation in the proliferation rate (significant difference to non-treated cells was observed at 10(-9) to 10(-5) M AcSDKP) determined by a colorimetry of alamer blue oxidation. The cell cycle analysis of NRK 49F cells treated with AcSDKP showed that AcSDKP significantly reduced the ratio of S-phase to G2/M-phases. Thus, physiological concentrations of AcSDKP is capable of altering cell cycle to inhibit the proliferation of renal cells.

Anti-NO action of carvedilol in cell-free system and in vascular endothelial cells

Carvedilol, an adrenoceptor blocker with antioxidant activity, was studied for its ability to interact with NO in a cell‐free condition and in an endothelial cell line (ECV304). In a cell‐free system, carvedilol attenuated NO‐dependent reduction of carboxy‐2‐phenyl‐4,4,5,5‐tetramethyl‐imidazoline‐1‐oxyl‐3‐oxide induced by a NO donor, 1‐hydroxy‐2‐oxo‐3‐(aminopropyl)‐3‐isopropyl‐1‐triazene (NOC5), which was determined by electron paramagnetic resonance (EPR) spectrometry. The EPR study also showed that nitrosylhaemoglobin formation in rat red blood cells by the addition of NO‐saturated solution was attenuated by prior incubation with 0.1–10 μM carvedilol. NO‐induced fluorescence in 4,5‐diaminofluorescein‐2 diacethyl (DAF‐2DA)‐loaded ECV304 cells was attenuated by carvedilol but not by labetalol. The IC50 of carvedilol for NOC5 or sodium nitroprusside‐induced fluorescence of DAF‐2DA in ECV304 cells was 1.0×10−7 M, which was similar to the reported IC50 of carvedilol for the antioxidant effect. Cell toxicity induced by a NO donor determined by the number of viable cells after 24 h treatment with 2‐2′(hydroxynitrosohydrazino)bis‐ethanamine was significantly attenuated by pretreatment with 1 μM carvedilol. Both free and cell‐associated carvedilol quenched NO. Because NO mediates both physiological and pathophysiological processes, NO quenching by the drug may have diverse clinical implications depending upon specific functions of local NO in tissues where carvedilol is distributed.