Kunihiko Saito

Platelet-activating factor in normal rat uterus

Platelet-activating factor (PAF) was found in normal rat uterus and identified as 1-0-hexadecyl/octadecenyl-2-acetyl-sn-glycero-3-phosphocholine. PAF was purified by several successive chromatographic procedures. It showed platelet aggregating activity, which was inhibited by CV 3988, and had no effect on platelets desensitized with 1-0-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine. The tert-butyldimethyl-silylderivative of 1-0-alkyl-2-acetyl-sn-glycerol, which was obtained by hydrolysis of uterine PAF with phospholipase C, was analyzed by gas chromatography-mass spectrometry. One rat uterus contained approximately 21.3 ng of 1-0-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine. This is the first report of the occurrence of a significant amount of PAF in a normal animal tissue.

Evidence for production of 1-acyl-2-acetyl-sn-glyceryl-3-phosphorylcholine concomitantly with platelet-activating factor

The presence of 1-acyl-2-acetyl-sn-glyceryl-3-phosphorylcholine in a sample of platelet-activating factor from stimulated rabbit neutrophils was demonstrated by a gas-liquid chromatography/mass spectrometry technique coupled with selected ion monitoring. The ions chosen for identification were those of acetyl and long-chain acyl moieties and molecular weight. Species containing palmitic, oleic and stearic acids were detected. A good correlation was observed between the productions of 1-acyl-2-acetyl-sn-glyceryl-3-phosphorylcholine and 1-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine by neutrophils stimulated with ionophore A23187.

Effect of human recombinant interleukin-6 on the proliferation of mouse hepatocytes in the primary culture

Effects of various cytokines on the proliferation of mouse hepatocytes were investigated. Human recombinant IL-6 not only enhanced the proliferation of mouse hepatocytes in the presence of epidermal growth factor, but also without epidermal growth factor. However, other human or mouse cytokines such as recombinant IL-1, IL-2, IL-3, IL-4, IFN-beta and IFN-gamma, which are known to regulate immune responses and/or hematopoiesis, had no effect on the proliferation of hepatocytes. These results suggest that IL-6 plays a crucial role in regulating the regeneration of hepatocytes after hepatitis or partial hepatectomy.

Phospholipase B from Penicillium notatum.

Publisher Summary Phospholipase B isolated from aqueous extracts of Penicillium notatum is a glycoprotein with a molecular weight of about 95,000. The active glycoprotein for phospholipase B has two phospholipase activities. When phospholipase B undergoes limited proteolysis by endogenous protease(s) in P. notatum —which occurs at the initial stage of purification—the B activity decreases greatly, but all of the lyso activity remains. Penicillium notatum phospholipase B activity is stimulated by diethyl ether, Triton X-100, and chlorpromazine (local anesthetic). Lyso activity is inhibited by detergents however not affected by the other reagents tested. Diisopropyl fluorophosphate (DFP) inhibits the B and lyso activities at a relatively high concentration (50 mM). Both activities are rather heat-labile. Phospholipase B in Penicillium notatum , catalyzes the complete deacylation of all kinds of natural glycerophospholipids including lysoglycerophospholipids. Therefore, the phospholipase B has both intrinsic B and lyso activities. Following proteolytic modification, the B activity is almost completely lost, however the lyso activity remains intact. The modified enzyme gives two large peptides and two small peptides by reductive cleavage with 2-mercaptoethanol. The large peptides are located at the C-terminal part, and the small peptides are at the N-terminal part of the native enzyme.

Metabolism of 1-O-alkyl-2-acetyl-sn-glycerol by washed rabbit platelets: formation of platelet activating factor.

A new type of neutral lipid, 1-O-alkyl-2-acetyl-sn-glycerol (AAG), induced a delayed aggregation pattern on interaction with washed rabbit platelets. Although far less potent on a molar basis than platelet activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, AGEPC, nevertheless this compound caused an aggregation, albeit delayed in time, remarkably similar to that exhibited by AGEPC. In view of the possible formation of AGEPC in this reaction, AAG was incubated with washed rabbit platelets, and a lipid corresponding in chromatographic behavior to AGEPC was isolated and identified as such by a combined gas-liquid chromatography/mass spectrometry technique coupled with selected ion monitoring.

Occurrence of platelet-activating factor (PAF) in normal rat stomach and alteration of PAF level by water immersion stress.

We detected platelet‐activating substance in gastrointestinal areas, which was confirmed to be platelet‐activating factor (PAF) on the basis of the following findings: 1) it comigrated with authentic PAF on thin‐layer chromatography; 2) it did not aggregate PAF‐desensitized platelets; and 3) its activity was completely antagonized by the receptor antagonists CV3988 and L‐652,731. The level of PAF was determined with a bioassay method based on the release of [3H] serotonin from washed rabbit platelets. In the normal rat stomach, the level of PAF was high in the antrum (940 ± 200 nmol PAF/mol phosphorus of original phospholipids), especially in the antral mucosa (1801 ± 426 nmol/mol phosphorus of original phospholipids). The stomach PAF level was significantly altered by water immersion stress. Stress for a period of 1 h was associated with a decrease in the antral PAF level to 39 ± 7% of that of untreated controls. This low PAF level persisted during stress. On the other hand, in the corpus, stress for periods of 1 and 3 h was associated with decreases in the PAF content, and further stress (7 h) resulted in restoration of the PAF level to normal. Furthermore, 7 h of stress was associated with distinct hemorrhagic lesions, which were prevented by CV3988 infused i.v. before the stress. This is the first report of an association between a decrease of the endogenous PAF level in animal tissues and tissue damage.—Sugatani, J.; Fujimura, K.; Miwa, M.; Mizuno, T.; Sameshima, Y.; Saito, K. Occurrence of platelet‐activating factor (PAF) in normal rat stomach and alteration of PAF level by water immersion stress. FASEB J. 3: 65‐70; 1989.

Studies on trimethylsilyl derivatives of 1,2-dialkylglycerols by gas-liquid chromatography mass spectrometry.

Trimethylsilyl derivatives of 1,2-dihexadecyl- and 1,2-dioctadecyl-glycerols were subjected to analysis by a gas chromatograph mass spectrometer system. The mass chromatographic identification of four kinds of glycerophospholipids, 1,2-dihexadecyl, 1-hexadec-1-enyl-2-hexadecanoyl, 1-hexadecyl-2-hexadecanoyl- and 1,2-dihexadecanoyl-glycerol is also described.

Effect of prostaglandins and their analogues on hormone-stimulated glycogenolysis in primary cultures of rat hepatocytes

Hepatocytes were isolated by collagenase perfusion method from adult male rats, cultured and then prelabeled with [14C]glucose. The [14C]glycogen-labeled cells were used in experiments for effect of prostaglandins on hormone-stimulated glycogenolysis. Prostaglandin E1, prostaglandin E2 and 16,16-dimethylprostaglandin E2, but not prostaglandin D2 or prostaglandin F2 alpha, inhibited glycogenolysis stimulated by glucagon, epinephrine, isoproterenol (beta-adrenergic agonist) or epinephrine in the presence of propranolol (beta-antagonist) in primary cultured hepatocytes. The inhibitory effects on day 2 of cultures were approx. twice those on day 1. Dimethylprostaglandin E2 (10(-6)M) caused 60-70% inhibitions of the stimulations by these substances. In the case of the stimulation by glucagon, the inhibition further increased by 80-100% on day 3 of culture. Prostaglandin E1 and prostaglandin E2 caused less inhibition than dimethylprostaglandin E2 of all these stimulations. Dinorprostaglandin E1 (9 alpha,13-dihydroxy-7-ketodinorprost-11-enoic acid), which is a hepatocyte-metabolite of prostaglandin E1 and prostaglandin E2, and arachidonic acid did not have any inhibitory effects. These data indicate that the E series of prostaglandins may function as the regulation of hepatic glycogenolysis stimulated by epinephrine and glucagon, and that their rapid degradation system may contribute to the modulation of the action in liver.

1-Acyl-2-acetyl-sn-glycero-3-phosphocholine from stimulated human polymorphonuclear leukocytes

Abstract1-Acyl-2-acetyl-sn-glycero-3-phosphocholine (1-acyl-2-acetyl GPC) was found in the fraction of platelet-activating factor obtained from stimulated human polymorphonuclear leukocytes (PMN). The amount of 1-acyl-2-acetyl GPC obtained from 1×107 PMN stimulated with ionophore A23187 at 37 C for 15 min ranged from 8 to 56 pmol (32±10 pmol, mean±standard error; n=4). The main species was 16∶0 palmitoyl (17±5 pmol), followed by 18∶0 stearoyl (8±3 pmol) and 18∶1 oleoyl (7±3 pmol).Although the physiological significance is unknown, 1-acyl-2-acetyl GPC was always detected when 1-alkyl-2-acetyl GPC was detected.

Effect of 17β-estradiol on PAF and prostaglandin levels in oophorectomized rat uterus

The effects of 17 beta-estradiol on the levels of platelet-activating factor (PAF) and prostaglandins and their precursor phospholipid in the uterus of oophorectomized rats were studied. Oophorectomy results in the decrease in the uterine PAF level to one-third of that in natural estrus. This level was recovered by subcutaneous administration of 17 beta-estradiol. The level of uterine phospholipids, which are rich in arachidonic acid, was significantly decreased by estradiol treatment. More arachidonate-PC was depleted than arachidonate-PE. The molecular structure was confirmed by gas chromatography-mass spectrometry. The amount of PGF2 alpha in the oophorectomized uterine tissue was 10-times that of PAF, but like the latter, increased 3-4 times on estradiol treatment. The chemical structures of PAF and PGF2 alpha formed on estradiol treatment were confirmed by mass spectrometry. The present data strongly suggest a correlation between the formations of PAF and PGF2 alpha, and indicate that estradiol may regulate the physiological formations of PAF and PGs in non-pregnant rat uterus.