Effects of chronic in-vivo treatments with protease-activated receptor 2 agonist on endothelium function and blood pressures in mice.

Short-term treatments with protease-activated receptor 2-activating peptides (PAR2-AP) induce endothelium-dependent vasodilation and decrease blood pressure. In this study, we tested the effect of chronic in-vivo treatment with PAR2-AP on the blood pressure and endothelium function of mice. Male PAR2 wild-type (WT) and par2-deficient (KO) mice received subcutaneous infusions of either saline, low (PAR2-LD), or high (PAR2-HD) doses of 2-furoyl-LIGRLO-amide for 1 or 2 weeks. In each treatment group, endothelium function was assessed in isolated arteries. Blood pressure, heart rate, and locomotor activity were recorded by radiotelemetry, and levels of tumour nercrosis factor α (TNF-α) and interkeukin 1ß (IL-1ß) were measured in plasma samples by ELISA. The relaxation of WT aortas and mesenteric arteries induced by PAR2-AP was decreased by PAR2-LD and PAR2-HD. In mesenteric arteries, PAR2-LD and PAR2-HD decreased the relaxation induced by acetylcholine, but not by nitroprusside; in aortas, PAR2-LD and PAR2-HD caused differential decreases in the relaxations induced by acetylcholine and nitroprusside. Only PAR2-HD lowered systolic arterial pressures in WT, when compared with all of the other groups. TNF-α and IL-1ß plasma concentrations were not different among the groups. We conclude that the systolic blood pressure of unrestrained mice can be lowered by chronic in-vivo activation of PAR2; however, this effect is countered by receptor desensitization and the concomitant development of endothelium and vascular dysfunction.

Protection of protease-activated receptor 2 mediated vasodilatation against angiotensin II-induced vascular dysfunction in mice.

BACKGROUND: Under conditions of cardiovascular dysfunction, protease-activated receptor 2 (PAR2) agonists maintain vasodilatation activity, which has been attributed to increased cyclooxygenase-2, nitric oxide synthase and calcium-activated potassium channel (SK3.1) activities. Protease-activated receptor 2 agonist mediated vasodilatation is unknown under conditions of dysfunction caused by angiotensin II. The main purpose of our study was to determine whether PAR2-induced vasodilatation of resistance arteries was attenuated by prolonged angiotensin II treatment in mice. We compared the vasodilatation of resistance-type arteries (mesenteric) from angiotensin II-treated PAR2 wild-type mice (WT) induced by PAR2 agonist 2-furoyl-LIGRLO-amide (2fly) to the responses obtained in controls (saline treatment). We also investigated arterial vasodilatation in angiotensin II-treated PAR2 deficient (PAR2-/-) mice. RESULTS: 2fly-induced relaxations of untreated arteries from angiotensin II-treated WT were not different than saline-treated WT. Treatment of arteries with nitric oxide synthase inhibitor and SK3.1 inhibitor (L-NAME + TRAM-34) blocked 2fly in angiotensin II-treated WT. Protein and mRNA expression of cyclooxygenase-1 and -2 were increased, and cyclooxygenase activity increased the sensitivity of arteries to 2fly in only angiotensin II-treated WT. These protective vasodilatation mechanisms were selective for 2fly compared with acetylcholine- and nitroprusside-induced relaxations which were attenuated by angiotensin II; PAR2-/- were protected against this attenuation of nitroprusside. CONCLUSIONS: PAR2-mediated vasodilatation of resistance type arteries is protected against the negative effects of angiotensin II-induced vascular dysfunction in mice. In conditions of endothelial dysfunction, angiotensin II induction of cyclooxygenases increases sensitivity to PAR2 agonist and the preserved vasodilatation mechanism involves activation of SK3.1.

Suckling rat pups accumulate creatine primarily via de novo synthesis rather than from dam milk.

During lactation, there may be a higher need for creatine replacement due to the provision of creatine to the milk. Our objectives were to: 1) quantify the creatine concentration in rat milk; 2) determine the origin of milk creatine; 3) determine the activities of the enzymes of creatine synthesis in lactating rats and pups; and 4) quantify the origin of the creatine that accumulates in rat pups. The origin of milk creatine was determined in 4 dams following the administration of (14)C-creatine by measuring the isotopic enrichment of creatine in the milk and plasma. The activities of the 2 enzymes involved in creatine synthesis were compared in lactating and virgin females (n = 7). For all experiments, the litter size was standardized to 8 pups. The data indicated that the mammary gland extracts creatine from the circulation rather than synthesizing it. This was confirmed by our failure to find substantial activities of the enzymes of creatine synthesis in mammary glands. The provision of milk creatine requires an additional 35-55% of creatine above the daily requirement by lactating rat dams. However, there was no increased creatine synthesis by these dams; the additional creatine was largely provided by hyperphagia, because creatine is present in commercial rat diet. There was a substantial accumulation of creatine in the growing pups, but only approximately 12% was obtained from milk. The great bulk of creatine accretion was via de novo synthesis by the pups, which imposed a substantial metabolic burden on them.

Creatine synthesis is a major metabolic process in neonatal piglets and has important implications for amino acid metabolism and methyl balance.

Our objectives in this study were as follows: 1) to determine the rate of creatine accretion by the neonatal piglet; 2) identify the sources of this creatine; 3) measure the activities of the enzymes of creatine synthesis; and 4) to estimate the burden that endogenous creatine synthesis places on the metabolism of the 3 amino acids required for this synthesis: glycine, arginine, and methionine. We found that piglets acquire 12.5 mmol of total creatine (creatine plus creatine phosphate) between 4 and 11 d of age. As much as one-quarter of creatine accretion in neonatal piglets may be provided by sow milk and three-quarters by de novo synthesis by piglets. This rate of creatine synthesis makes very large demands on arginine and methionine metabolism, although the magnitude of the demand depends on the rate of remethylation of homocysteine and of reamidination of ornithine. Of the 2 enzymes of creatine synthesis, we found high activity of l-arginine:glycine amidinotransferase in piglet kidneys and pancreas and of guanidinoacetate methyltransferase in piglet livers. Piglet livers also had appreciable activities of methionine adenosyltransferase, which synthesizes S-adenosylmethionine, and of betaine:homocysteine methyltransferase, methionine synthase, and methylene tetrahydrofolate reductase, which are required for the remethylation of homocysteine to methionine. Creatine synthesis is a quantitatively major metabolic process in piglets.

Homocysteine metabolism in ZDF (type 2) diabetic rats.

Mild hyperhomocysteinemia is a risk factor for many diseases, including cardiovascular disease. We determined the effects of insulin resistance and of type 2 diabetes on homocysteine (Hcy) metabolism using Zucker diabetic fatty rats (ZDF/Gmi fa/fa and ZDF/Gmi fa/?). Plasma total Hcy was reduced in ZDF fa/fa rats by 24% in the pre-diabetic insulin-resistant stage, while in the frank diabetic stage there was a 59% reduction. Hepatic activities of several enzymes that play a role in the removal of Hcy:cystathionine beta-synthase (CBS), cystathionine gamma-lyase, and betaine:Hcy methyltransferase (BHMT) were increased as was methionine adenosyltransferase. CBS and BHMT mRNA levels and the hepatic level of S-adenosylmethionine were also increased in the ZDF fa/fa rats. Studies with primary hepatocytes showed that Hcy export and the transsulfuration flux in cells from ZDF fa/fa rats were particularly sensitive to betaine. Interestingly, liver betaine concentration was found to be significantly lower in the ZDf fa/fa rats at both 5 and 11 weeks. These results emphasize the importance of betaine metabolism in determining plasma Hcy levels in type 2 diabetes.

Effects of diabetes and insulin on betaine-homocysteine S-methyltransferase expression in rat liver.

Elevation of plasma homocysteine levels has been recognized as an independent risk factor for the development of cardiovascular disease, a major complication of diabetes. Plasma homocysteine reflects a balance between its synthesis via S-adenosyl-L-methionine-dependent methylation reactions and its removal through the transmethylation and the transsulfuration pathways. Betaine-homocysteine methyltransferase (BHMT, EC 2.1.1.5) is one of the enzymes involved in the remethylation pathway. BHMT, a major zinc metalloenzyme in the liver, catalyzes the transfer of methyl groups from betaine to homocysteine to form dimethylglycine and methionine. We have previously shown that plasma homocysteine levels and the transsulfuration pathway are affected by diabetes. In the present study, we found increased BHMT activity and mRNA levels in livers from streptozotocin-diabetic rats. In the rat hepatoma cell line (H4IIE cells), glucocorticoids (triamcinolone) increased the level and rate of BHMT mRNA synthesis. In the same cell line, insulin decreased the abundance of BHMT mRNA and the rate of de novo mRNA transcription of the gene. Thus the decreased plasma homocysteine in various models of diabetes could be due to enhanced homocysteine removal brought about by a combination of increased transsulfuration of homocysteine to cysteine and increased remethylation of homocysteine to methionine by BHMT.

Amino acid metabolism in the Zucker diabetic fatty rat: effects of insulin resistance and of type 2 diabetes.

We investigated amino acid metabolism in the Zucker diabetic fatty (ZDF Gmi fa/fa) rat during the prediabetic insulin-resistant stage and the frank type 2 diabetic stage. Amino acids were measured in plasma, liver, and skeletal muscle, and the ratios of plasma/liver and plasma/skeletal muscle were calculated. At the insulin-resistant stage, the plasma concentrations of the gluconeogenic amino acids aspartate, serine, glutamine, glycine, and histidine were decreased in the ZDF Gmi fa/fa rats, whereas taurine, alpha-aminoadipic acid, methionine, phenylalanine, tryptophan, and the 3 branched-chain amino acids were significantly increased. At the diabetic stage, a larger number of gluconeogenic amino acids had decreased plasma concentrations. The 3 branched-chain amino acids had elevated plasma concentrations. In the liver and the skeletal muscles, concentrations of many of the gluconeogenic amino acids were lower at both stages, whereas the levels of 1 or all of the branched-chain amino acids were elevated. These changes in amino acid concentrations are similar to changes seen in type 1 diabetes. It is evident that insulin resistance alone is capable of bringing about many of the changes in amino acid metabolism observed in type 2 diabetes.