Orthovanadate-Induced Vasoconstriction of Rat Mesenteric Arteries Is Mediated by Rho Kinase-Dependent Inhibition of Myosin Light Chain Phosphatase.

Orthovanadate (OVA), a protein tyrosine phosphatase inhibitor, induces vasoconstriction in a Rho kinase-dependent manner. The aim of this study was to determine the mechanism underlying OVA-induced vasoconstriction of rat mesenteric arteries. OVA-induced constriction of mesenteric arterial rings treated with N(G)-nitro-L-arginine methyl ester (L-NAME, 0.1 mM), a nitric oxide synthase inhibitor, was significantly blocked by the Rho kinase inhibitor Y-27632 (R-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, 10 µM), extracellular signal-regulated kinase 1 and 2 (Erk1/2) inhibitor FR180204 (5-(2-phenyl-pyrazolo[1,5-a]pyridin-3-yl)-1H-pyrazolo[3,4-c]pyridazin-3-ylamine, 10 µM), Erk1/2 kinase (MEK) inhibitor PD98059 (2'-amino-3'-methoxyflavone, 10 µM), epidermal growth factor receptor (EGFR) inhibitor AG1478 (4-(3-chloroanilino)-6,7-dimethoxyquinazoline, 10 µM), and Src inhibitor PP2 (4-amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine, 3 µM). However, the myosin light chain kinase inhibitor ML-7 (1-(5-iodonaphthalene-1-sulfonyl)-homopiperazine, 10 µM) did not affect OVA-induced constriction. Phosphorylation of myosin phosphatase target subunit 1 (MYPT1, an index of Rho kinase activity) was abrogated by inhibitors of Src, EGFR MEK, Erk1/2, and Rho kinase. OVA-stimulated Erk1/2 phosphorylation was blocked by inhibitors of EGFR, Src, MEK, and Erk1/2 but not affected by an inhibitor of Rho kinase. OVA-induced Src phosphorylation was abrogated by an Src inhibitor but not affected by inhibitors of EGFR, MEK, Erk1/2, and Rho kinase. In addition, the metalloproteinase inhibitor TAPI-0 (N-(R)-[2-(hydroxyaminocarbonyl)methyl]-4-methylpentanoyl-L-naphthylalanyl-L-alanine amide, 10 µM) and an inhibitor of heparin/epidermal growth factor binding (CRM 197, 10 µg/mL) did not affect OVA-induced contraction of rat mesenteric arterial rings. These results suggest that OVA induces vasoconstriction in rat mesenteric arteries via Src, EGFR, MEK, and Erk1/2 activation, leading to the inactivation of myosin light chain phosphatase through phosphorylation of MYPT1.

Differential effects of pyrazinamide and clofibrate on gene expression of rat hepatic alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase, a key enzyme of the tryptophan-NAD pathway.

Hepatic alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD) [EC4.1.1.45] plays a key role in regulating NAD biosynthesis from tryptophan. The aim of this study was to evaluate the ACMSD mRNA expression after pyrazinamide or peroxisome proliferators ingestion. When rats were fed a control (pyrazinamide- and clofibrate-free) diet, 1% pyrazinamide- or 0.24% clofibrate-containing diets for 8 days, hepatic ACMSD activity and mRNA in rats consuming the clofibrate-containing diet was strongly suppressed, as compared with those fed the control and pyrazinamide diet. Pyrazinamide suppressed liver and kidney ACMSD activities, but did not affect ACMSD mRNA. Blood NAD was increased in the clofibrate and pyrazinamide groups. Shifting from the control diet to a clofibrate diet suppressed ACMSD mRNA strongly at day 1 and continued through day 4. However ACMSD activity decreased gradually. In rats fed with several kinds of peroxisome-proliferator-containing diets such as phthalate ester, bezafibrate, Wy-14,643, 2-(-4-chlorophenoxy) propionic acid, or dehydroisoandrosterone for 8 days, hepatic ACMSD mRNA was drastically decreased by all the peroxisome proliferators. These results suggest that the transcription level of hepatic ACMSD is modulated by peroxisome proliferators, and the fluctuation of the hepatic ACMSD mRNA expression was followed by that of the ACMSD activity. However, pyrazinamide does not affect the transcription level of hepatic ACMSD.

Regulation of mouse hepatic alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase, a key enzyme in the tryptophan-nicotinamide adenine dinucleotide pathway, by hepatocyte nuclear factor 4alpha and peroxisome proliferator-activated receptor alpha.

Nicotinamide adenine dinucleotide (NAD) plays a critical role in the maintenance of cellular energy homeostasis. alpha-Amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD) is the key enzyme regulating de novo synthesis of NAD from l-tryptophan (Trp), designated the Trp-NAD pathway. Acmsd gene expression was found to be under the control of both hepatocyte nuclear factor 4alpha (HNF4alpha) and peroxisome proliferator-activated receptor alpha (PPARalpha). Constitutive expression of ACMSD mRNA levels were governed by HNF4alpha and downregulated by activation of PPARalpha by the ligand Wy-14,643 ([4-chloro-6-(2,3-xylidino)-2-pyrimidinylthio]acetic acid]), as revealed by studies with hepatic HNF4alpha-null mice and PPARalpha-null mice, respectively. Transient transfection and electrophoretic mobility shift analyses showed an HNF4alpha binding site in the Acmsd gene promoter that directed transactivation of reporter gene constructs by HNF4alpha. The Acmsd promoter was not responsive to PPARalpha in transactivation assays. Wy-14,643 treatment decreased HNF4alpha protein levels in wild-type, but not PPARalpha-null, mouse livers, with no changes in HNF4alpha mRNA. These results show that Wy-14,643, through PPARalpha, post-transcriptionally down-regulates HNF4alpha protein levels, leading to reduced expression of the HNF4alpha target gene Acmsd.

NAD levels in the rat primary cultured hepatocytes affected by peroxisome-proliferators.

The effect of peroxisome-proliferators (PPs) on the NAD level in primary cultured rat hepatocytes was investigated and compared with that in the liver of rat administered with PPs. Various PPs, including fibrates, phthalate esters and steroid, increased NAD level in the cultured hepatocytes as in whole animal with a little exception. The NAD level decreased after once reaching the peak by the addition of most PPs. The gradual decrease of NAD observed in primary cultured hepatocytes, was partially inhibited by the addition of poly(ADP-ribose) polymerase and/or NAD glycohydrolase inhibitors. In the presence of these inhibitors, the increase of NAD by PPs still remained. The mechanism of increasing NAD level by PPs will be due to the stimulation of tryptophan (Trp)-NAD biosynthesis, with the possibility of the transcriptional regulation of genes related to Trp-NAD pathway by PPs. This in vitro system, therefore, can be used to clarify detailed mechanism of the stimulation of hepatic NAD biosynthesis in rats administered PPs.