Novel prostaglandin derivative formed from arachidonic acid by rat stomach homogenates

Abstract

pseudouridine was prepared using the ribonuclease-catalyzed reaction described by Heppel et al. (1955). Cytidine 2',3'cyclic phosphate (0.1 mmole) and pseudouridine (100 mg) were dissolved in 0.015 M Tris-chloride buffer (pH 7; 5 ml) and treated with pancreatic ribonuclease (0.1 mg) at 37" for 75 min. The mixture was then shaken with isoamyl alcohol (0.6 ml) and chloroform (0.15 ml) and streaked on Whatman No. 3MM chromatographic paper (66 cm). The components of the mixture were separated with solvent system B and the dinucleoside phosphate band was cut out and eluted (yield, 30 ODU). This product was found to be completely degradable by ribonuclease to cytidine 3'-phosphate and pseudouridine. The dinucleoside phosphate (20 ODU) was dissolved in 0.05 M sodium borate buffer (pH 8.5 ; 0.3 ml) and treated with Cmcp-toluenesulfonate (40 mg). The mixture was allowed to stand for 20 hr. At this time paper chromatography in solvent systems A and B showed that about 70% of the material had been converted to Cmc derivatives. The dinucleoside phosphate and its mono-Cmc derivative had RF values 0.41 and 0.61 (solvent system A); 0.18 and 0.26 (solvent system B), respectively. The mixture was treated with concentrated ammonia for 2 hr and then applied to Whatman No. 3MM chromatographic paper and then separated in solvent system B. The Cmc-dinucleoside phosphate (6 ODU) was dissolved in 0.02 M sodium phosphate buffer (pH 7.0; 0.2 ml) and treated with pancreatic ribonuclease (2.5 pg) at 37" for 4 hr. Paper chromatography in solvent system A showed that about 70% of the Cmc derivative had been converted into a mixture of cytidine 3'-phosphate and Cmc-pseudouridine. With twice the amount of enzyme under the same conditions it was possible to hydrolyze all the dinucleoside phosphate to its components.

A novel prostaglandin is the major product of arachidonic acid metabolism in rabbit heart.

“…However, when similarly obtained samples were chromatographed in system BDA, two peaks were observed ( Fig. 3B and D (14,16) have conclusively demonstrated, with gas chromatography-mass spectrometric methods, that the primary arachidonate products produced by homogenates of rat gastric fundus are 6(9)oxy-PGF and 6-keto-PGFIa. We used the rat stomach preparation to generate this material for comparison to the cardiac compound.…”

“…Although the data do not allow complete structural identification of the compound, they do suggest the lack of a #-OH-ketone structure (as in PGE2 and PGD2, which are sensitive to alkali treatment). Recently, Pace-Asciak and Wolfe (14,16) have reported the structures (determined by gas chromatography-mass spectrometry) of two novel PGs formed by rat fundus homogenates: (f) 6(9)oxy-PGF, and (ii) 6-keto-PGFI.. Both of these PGs have certain properties in common with the unknown compound reported here i.e., chromatographic mobility similar to that of PGE2 in most solvent systems and resistance to alkali conversion to PGB2.…”

“…Solvent (14), with 2.4 mM norepinephrine included in each incubation. RESULTS Products from Exogenous Arachidonic Acid.…”

Cardiac and Renal Prostaglandin I2

“…Bovine coronary artery was excised from freshly removed hearts and spiral strips were prepared (5). The bovine coronary artery strip is contracted by PGE2 (5-7) and PGH, (12) and relaxed uponl exposuire to PGH2, PGI2, or PGE, (12,14). 6-Keto-PGF,1 (0.1-5 ,ug) caused no detectable response on the bovine coronary strips.…”

“…In additioni, inhibition of PG eyclooxygeniase in isolated coronary artery strips led to a rapid and sustained inerease in coronary tonie (5,7,8). When the enidogeniouis phos-pholipids of the isolated rabbit heart or of isolated bovine coronary artery strips were labeled with [14C]AA (9,10), the primary metabolite of AA released had chemical and chromatographic properties identical with the biologically inactive 6-keto-PGF1a (10,12). Exogenious PGH2 also relaxed bovine coronary artery strips, but the endoperoxide itself was rapidly degraded (13)(14)(15).…”