Takahide Nomura

Background: Tetrahydrobiopterin (BH4) is an essential cofactor of nitric oxide synthase, and GTP cyclohydrolase I (GCHI) is a rate-limiting enzyme in the biosynthesis of BH4. The expression of inducible nitric oxide synthase (NOS) was earlier demonstrated in the ventricles of patients with dilated cardiomyopathy (DCM) although that of GCHI was not clarified. The present study was designed to determine the GCHI mRNA expression as well as to confirm NOS mRNA expression in endomyocardial biopsy specimens from patients with DCM. Methods: Clinical details were assessed in 19 patients with DCM and in 9 control subjects. The real-time reverse transcription polymerase chain reaction (PCR) was performed on total RNA extracted from endomyocardial biopsy specimens. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA was quantified for use as an internal control. Results: iNOS/GAPDH for the DCM samples was 4.8-fold greater than that for the control ones (P < 0.01), whereas the GCHI/GAPDH for the DCM samples was reduced to 31.1% of the control (P < 0.05). Conclusions: The increased expression of NOS mRNA was confirmed in endomyocardial biopsy specimens from patients with DCM. The GCHI mRNA level was suppressed in these specimens. (C) 2004 Elsevier B.V. All rights reserved.

Although endothelial dysfunction deteriorates diabetic angiopathy, the mechanisms are obscure. We revealed that high glucose augmented eNOS through stimulation of eNOS mRNA in cultured BAECs. NO was decreased and O-2(-) was increased simultaneously. NOS inhibitor, inhibited O-2(-) release, so did NADPH oxidase inhibitor. The effects were synergistic. Both intracellular BH4 level and GTPCH1 activity were decreased by high glucose, in line with decrease of GTPCH1 mRNA. HMG-CoA, reductase inhibitor, atorvastatin increased GTPCH1 mRNA and activity, and BH4 level. Conclusively, high glucose leads to eNOS dysfunction by inhibiting BH4 synthesis and atorvastatin stimulate BH4 synthesis directly, and it may work as atherogenic process. (C) 2004 Elsevier Inc. All rights reserved.

Studies were conducted to clarify the effects of nitric oxide donors NOR 3 ((+/-)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamide, FK409), SIN-1 (3-morpholinosydnonimine) and SNAP (S-nitroso-N-acetylpenicillamine) on the accumulation of cGMP and cAMP and Ca2+ mobilization as well as ketogenesis from oleate in isolated rat hepatocytes. NOR 3 caused inhibition of ketogenesis from oleate along with stimulation of cGMP accumulation in rat hepatocytes, whereas SIN-1 and SNAP exerted no effect on ketogenesis despite their marked stimulation of cGMP accumulation. Although the nitric oxide frapping agent, carboxy-PTIO (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide), antagonized the stimulation by NOR 3 of cGMP accumulation, it failed to modulate the anti-ketogenic action of NOR 3. Furthermore, neither 8-bromoguanosine-3',5'-cyclic monophosphate nor N-2,2'-O-dibutyrylguanosine-3',5'-cyclic monophosphate mimicked the anti-ketogenic action of NOR 3. It is concluded in the present study that NOR 3-induced inhibition of ketogenesis in rat hepatocytes is not mediated by cGMP. The present study revealed that the remaining structure of NOR 3 from which nitric oxide had been spontaneously released had no anti-ketogenic action. We first and clearly demonstrated that nitrite production was dramatically enhanced when NOR 3 was incubated in the presence of rat hepatocytes. The mechanism whereby NOR 3 inhibits ketogenesis in rat hepatocytes will be discussed.

We examined the metabolic effects of glibenclamide, a potent second-generation sulfonylurea, in isolated rat hepatocytes incubated in the absence of extracellular Ca2+. We first demonstrated in the present study that glibenclamide caused a significant increase in basal glucose release and lactate production without any modification of intracellular Ca2+ concentration or cAMP levels in isolated rat hepatocytes. Furthermore, glibenclamide inhibited the noradrenaline-induced increase in cAMP accumulation, while activation of glycogenolysis by noradrenaline was not suppressed by this agent. Our data indicate that glibenclamide exerts its metabolic effects independent of intracellular Ca2+ mobilization and cAMP accumulation.