The effect of intermittent administration of sustained release isosorbide dinitrate (sr-ISDN) in rats with volume overload heart.

Recently, it has been reported that intermittent administration of nitrate, with a nitrate-free interval of 10 to 12 hr eliminated expression of tolerance, and maintained its hypotensive effect. In the present study, we evaluated whether nitrate tolerance developed or not with an intermittent administration of sr-ISDN (5 mg/kg/ once a day) in Wistar rats. The effect of this administration protocol for sr-ISDN on the volume overload heart model, aortovenous fistula, was also examined. Furthermore, blood pressure was monitored by radio telemetry during sr-ISDN (5 mg/kg/once a day) administration. Nitrate tolerance did not develop, and eccentric hypertrophy due to volume overload was moderated by sr-ISDN administration. Sr-ISDN administration maintained blood pressure lower level than the placebo group. In conclusion, prolonged intermittent administration of sr-ISDN maintained its hypotensive effect during the entire experiment period, without developing tolerance, and moderated efferent hypertrophy with attenuated volume overload.

Functional analysis of Chinese hamster phosphatidylserine synthase 1 through systematic alanine mutagenesis.

PtdSer (phosphatidylserine) synthesis in mammalian cells occurs through the exchange of L-serine with the base moieties of phosphatidylcholine and phosphatidylethanolamine, which is catalysed by PSS (PtdSer synthase) 1 and 2 respectively. PtdSer synthesis in intact cells and an isolated membrane fraction was inhibited by exogenous PtdSer, indicating that feedback control is involved in the regulation of PtdSer biosynthesis. PSS 1 and 2 are similar in amino acid sequence, with an identity of 32%; however, due to a lack of homology with other known enzymes, their amino acid sequences do not provide information on their catalytic and regulatory mechanisms. In the present study, to identify amino acid residues crucial for the activity and/or regulation of PSS 1, we systematically introduced mutations into a Chinese hamster PSS 1 cDNA clone; namely, each of the 66 polar amino acid residues common to PSS 2 was replaced with an alanine residue. On analysis of Chinese hamster ovary cells transfected with each of the alanine mutant clones, we identified eight amino acid residues (His-172, Glu-197, Glu-200, Asn-209, Glu-212, Asp-216, Asp-221 and Asn-226) as those crucial for the enzyme reaction or the maintenance of the correct structure required for serine base-exchange activity. Among these residues, Asn-209 was suggested to be involved in the recognition and/or binding of free L-serine. We also identified six amino acid residues (Arg-95, His-97, Cys-189, Arg-262, Gln-266 and Arg-336) as those important for regulation of PSS 1. In addition, we found that the alanine mutations at Tyr-111, Asp-166, Arg-184, Arg-323, and Glu-364 affected the production and/or stability of PSS 1 in Chinese hamster ovary cells.

Purification and characterization of Chinese hamster phosphatidylserine synthase 2.

Phosphatidylserine (PtdSer) in mammalian cells is synthesized through the action of PtdSer synthase (PSS) 1 and 2, which catalyze the conversion of phosphatidylcholine and phosphatidylethanolamine, respectively, to PtdSer. The PtdSer synthesis in intact cells and an isolated membrane fraction is inhibited by exogenous PtdSer, indicating that inhibition of PtdSer synthases by PtdSer is important for the regulation of PtdSer biosynthesis. In this study, to examine whether the inhibition occurs through the direct interaction of PtdSer with the synthases or is mediated by unidentified factor(s), we purified a FLAG and HA peptide-tagged form of Chinese hamster PSS 2 to near homogeneity. The purified enzyme, as well as the crude enzyme in a membrane fraction, was inhibited on the addition of PtdSer to the enzyme assay mixture. In contrast to PtdSer, phosphatidylcholine and phosphatidylethanolamine did not significantly inhibit the purified enzyme. Furthermore, PtdSer-resistant PtdSer synthesis was observed on cell-free assaying of the membrane fraction prepared from a Chinese hamster ovary cell strain whose PtdSer synthesis in vivo is not inhibited by exogenous PtdSer. These results suggested that the interaction of PtdSer with PSS 2 or a very minor protein co-purified with PSS 2 was critical for the regulation of PSS 2 activity in intact cells.