l-Arginine fails to protect against myocardial remodelling in l-NAME-induced hypertension

A critical study of the different steps involved in previous procedure for hydroxyproline assay allows the direct measurement of collagen content in tissue' homogenates without losing the advantages of the method. The procedure is based on alkaline hydrolysis of the tissue homogenate and subsequent determination of the free hydroxyproline in hydrolyzates. Chloramine-T was used to oxidize the free hydroxyproline for the production of a pyrrole. The addition of Ehrlich's reagent resulted in the formation of a chromophore that can be measured at 550 nm. Optimal assay conditions were determined using tissue homogenate and purified acid soluble collagen along with standard hydroxyproline. Critical parameters such as the amount of chloramine-T, sodium hydroxide, p-dimethylaminobenzaldehyde, pH of the reaction buffer, and length of oxidation time were examined to obtain satisfactory results. The method has been applied to samples of tissue homogenate and purified acid soluble collagen, with recovery of added hydroxyproline of 101 +/- 6.5 and 104 +/- 6.0 (SD) percent, respectively. The method is highly sensitive and reproducible when used to measure the imino acid in tissue homogenates. The modified hydroxyproline assay presented in this communication will be useful for routine measurement of collagen content in extracts of various tissue specimens. In addition, the modified method can be used for batch processing of column fractions to monitor the collagen concentrations during purification.

A fast single-step lipid extraction procedure and high-performance liquid chromatography with in-line uv and electrochemical detection are used for the simultaneous quantitative determination of tocopherols, ubiquinols, and ubiquinones in blood, plasma, tissue homogenates, and subcellular fractions. The compounds of interest can be quantitatively extracted into hexane from a sodium dodecyl sulfate-treated aqueous homogenate after precipitation of protein by addition of an equal volume of ethanol. alpha-, gamma-, and delta-Tocopherol, ubiquinol 9, ubiquinol 10, and ubiquinones 9 and 10 can be well separated on a reversed phase column. Ubiquinones are detected at 275 nm by the uv detector, and ubiquinols and tocopherols by the electrochemical detector in the oxidative mode. Quantitation is done by comparing chromatographic peak heights to those of a standard solution containing known amounts of tocopherols, ubiquinols 9 and 10, and ubiquinones 9 and 10, analyzed under identical conditions. The high sensitivity of the electrochemical detection allows operation at low potentials (+0.5 V) with low detector response, but high selectivity for the easily oxidizable tocopherols and ubiquinols and decreased baseline noise. The uv detection limits the overall sensitivity of the procedure to 2 pmol ubiquinone, corresponding to 0.1 microM ubiquinone in the lipid extract. The ranges of values obtained for rat and guinea pig tissues, for rat liver mitochondria, and for blood and plasma from rats and humans are given.

Nitric oxide (NO) participates in the regulation of vascular tone and smooth muscle cell proliferation, but little is known of its effect on total protein and matrix synthesis in smooth muscle. We studied the effects of the NO-generating compounds S-nitroso-N-acetylpenicillamine (SNAP, 0.4 to 1.2 mmol/L) and sodium nitroprusside (SNP, 0.1 to 0.5 mmol/L) on total protein (using [3H]leucine) and collagen (using [3H]proline) synthesis in cultured rabbit aortic smooth muscle cells. Both agents caused dose-dependent inhibition of the relative rate of protein (maximum reduction of 87% [SNAP] and 80% [SNP]) and collagen synthesis, as measured by trichloroacetic acid-precipitated label. The magnitudes of percent inhibition of total protein and collagen synthesis were approximately equal. Inhibition of protein synthesis by SNAP and SNP was prevented by hemoglobin (10 mumol/L), suggesting that the protein synthesis inhibition was due to NO release. Inhibition of protein synthesis was reversible after removal of SNAP and SNP and was not caused by damage to the cells. These results suggest that NO may function as a modulator of vascular smooth muscle cell protein synthesis and production of extracellular matrix components.