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Biomedical subjects

K Waku

Publications and source records attributed to K Waku.

At least 55 records · Page 3Linked to original sources
PubMed1996

N-arachidonoylethanolamine (anandamide), an endogenous cannabinoid receptor ligand, and related lipid molecules in the nervous tissues.

The effects of N-arachidonoylethanolamine (anandamide) and related compounds on the binding of [3H]CP55940 to rat brain synaptosomes were examined. Anandamide was shown to inhibit competitively the specific binding of [3H]CP55940 to synaptosomal membranes. The Ki value was 89 nM. In contrast, N-acylethanolamines containing saturated or monoenoic fatty acids did not exhibit high binding affinity. Several structural analogues of anandamide showed some binding activity. Among them, 2-arachidonoylglycerol is noteworthy because of its occurrence in mammalian tissues. A biosynthetic study indicated that anandamide can be synthesized via two separate synthetic pathways. The first is synthesis from free arachidonic acid and ethanolamine, and the second is the formation of N-arachidonoyl phosphatidylethanolamine (PE) from diarachidonoyl phospholipids and PE and the subsequent enzymatic release of N-arachidonoylethanolamine. The latter pathway appears to explain very well the fatty acid composition of N-acylethanolamines present in mammalian tissues.

Animals↗
PubMed1996

Enzymatic synthesis of oleamide (cis-9, 10-octadecenoamide), an endogenous sleep-inducing lipid, by rat brain microsomes.

The enzymatic synthesis of a novel sleep-inducing lipid, oleamide (cis-9,10-octadecenoamide), was studied using rat brain subcellular fractions as enzyme sources. We found that oleamide was formed from oleic acid and ammonia on incubation with a brain homogenate. The enzyme activity catalyzing the formation of oleamide from oleic acid and ammonia was highest in the microsomal fraction among the subcellular fractions. Boiled microsomes did not exhibit appreciable enzyme activity. These results strongly suggest that oleamide can be synthesized enzymatically in the brains of stimulated animals.

Adenosine Triphosphate↗
PubMed1996

Enhanced expression of platelet-activating factor receptor on human eosinophils by interleukin-3, interleukin-5 and granulocyte-macrophage colony-stimulating factor.

The effects of interleukin-3 (IL-3), IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF) on the surface expression of platelet-activating factor (PAF) receptor on eosinophils was examined using a PAF receptor binding assay. Specific binding of [3H]WEB 2086, a specific ligand for PAF receptor, to peripheral blood eosinophils obtained from healthy individuals was significantly increased when the cells were cultured with IL-3, IL-5, or GM-CSF for 12 h, compared with untreated cells. The PAF-induced increase in intracellular calcium ion concentration in eosinophils was markedly augmented by exposure to IL-5 for 12 h. These results suggest that these cytokines modulate the responsiveness of eosinophils to PAF through enhancement of PAF receptor expression on the cell surface.

Calcium↗
PubMed1995

Acyl-CoA binding and acylation of UDP-glucuronosyltransferase isoforms of rat liver: their effect on enzyme activity.

When [14C]arachidonoyl-CoA was incubated with crude extracts of rat liver microsomes, [14C]arachidonic acid was incorporated into many proteins, suggesting that modification of these proteins with fatty acid, i.e. acylation, occurred. Using a [14C]arachidonyl-CoA labelling assay, 50 and 53 kDa proteins were purified from rat liver microsomes to near homogeneity by sequential chromatography on Red-Toyopearl, hydroxyapatite, heparin-Toyopearl, Blue-Toyopearl and UDP-hexanolamine-agarose. Acylation of the 50 and 53 kDa proteins occurred in the absence of any other protein, suggesting that these molecules catalyse autoacylation. The acylation was dependent on the length of the incubation period and the concentration of [14C]arachidonoyl-CoA. The 50 and 53 kDa proteins also had acyl-CoA-binding activity; initial rates of acyl-CoA binding and acylation were 0.25 and 0.004 min-1 respectively. The proteins also had weak but distinct acyl-CoA-hydrolysing activity (0.006 min-1). These results suggest that the proteins catalysed the sequential reactions of binding to acyl-CoA, autoacylation, and hydrolysis of fatty acid. N-terminal amino acid sequencing analysis showed these proteins to be UDP-glucuronosyltransferase (UDPGT) isoforms. UDPGT activity was inhibited by arachidonoyl-CoA. These results suggest that binding of acyl-CoA and acylation of UDPGT isoforms regulate the enzyme activities, implying a possible novel function for fatty acyl-CoA in glucuronidation, which is involved in the metabolism of drugs, steroids and bilirubin.

Acyl Coenzyme A↗
PubMed1995

2-Arachidonoylglycerol: a possible endogenous cannabinoid receptor ligand in brain.

The effects of anadamide, 2-arachidonoylglycerol and related compounds on the specific binding of a radiolabeled cannabinoid receptor ligand,[3H]CP55940, to synaptosomal membranes were examined. Anandamide, an endogenous cannabinoid receptor ligand, reduced the specific binding of [3H]CP55940 to synaptosomal membranes in a dose-dependent manner: the Ki value was 89 nM. 2-Arachidonoylglycerol was also shown to bind appreciably to the cannabinoid receptor in competitive inhibition experiments. The apparent binding affinity was markedly increased when the binding assay was carried out in the presence of the esterase inhibitor DFP or at 0 degrees C. Free arachidonic acid and N-palmitoylethanolamine were almost inactive in terms of binding to the cannabinoid receptor in synaptosomal membranes. 2-Arachidonoylglycerol may be an endogenous cannabinoid receptor ligand in the brain.

Animals↗
PubMed1995

Platelet-activating factor and its structural analogues in the earthworm Eisenia foetida.

The earthworm Eisenia foetida was shown to contain large amounts of ether-containing phospholipids such as alkylacylglycerophosphocholine (61.3% of choline glycerophospholipids) and alkenylacylglycerophosphoethanolamine (66.0% of ethanolamine glycerophospholipids). We also found a substantial amount of ether-containing PAF-like lipid in this animal, its level being increased after the animal is injured. We showed evidence that this PAF-like lipid consists of PAF and PAF analogues containing short chain fatty acids other than acetic acid. Notably, a propionic acid-containing species but not PAF itself, is the most predominant species in this animal. We also confirmed that the earthworms contain enzyme activities involved in the synthesis of PAF and short chain fatty acid-containing PAF analogues. Interestingly, the acetyltransferase activity in earthworms is resistant to high concentrations of the substrate lysophospholipid. Thus, both the structure of the PAF-like lipid and the properties of the enzymes involved in the PAF-like lipid metabolism in the earthworms are somewhat different from those in mammalian tissues.

Acetyltransferases↗
PubMed1995

Coenzyme A-dependent cleavage of membrane phospholipids in several rat tissues: ATP-independent acyl-CoA synthesis and the generation of lysophospholipids.

Substantial amounts of acyl-CoA were formed when microsomes from several rat tissues were incubated with varying concentrations of free CoA and bovine serum albumin even in the absence of ATP and Mg2+. For instance, 86 nmol of acyl-CoA was produced when microsomes (5 mg protein) were incubated with 300 microM CoA for 30 min. It was calculated that 1.8% of total fatty acyl residues were converted to acyl-CoA during the incubation. No appreciable amount of acyl-CoA was formed from free fatty acid or from boiled microsomes under the same experimental conditions. These observations indicate that acyl-CoA is formed from microsomal lipids by an enzyme activity distinct from previously known long-chain fatty acyl-CoA synthetase. The apparent Km value for CoA and Vmax were 180 microM and 20 nmol/30 min per mg protein, respectively. We found that several species of acyl-CoA such as arachidonoyl-CoA were preferentially synthesized through the reaction and that several types of phospholipids actually act as acyl donors in the formation of acyl-CoA. Phosphatidylinositol and phosphatidylcholine appear to be preferred substrates. We confirmed that lysophosphatidylinositol and lysophosphatidylcholine were generated along with the formation of acyl-CoA. It seems very likely that CoA-mediated cleavage of phospholipids/ATP-independent acyl-CoA synthesis is implicated in the metabolism of certain types of fatty acyl residues of membranous phospholipids in mammalian cells.

Acyl Coenzyme A↗
PubMed1995

Coenzyme A-dependent modification of fatty acyl chains of rat liver membrane phospholipids: possible involvement of ATP-independent acyl-CoA synthesis.

Certain species of fatty acyl chains of microsomal lipids from rat hepatocytes underwent desaturation when the microsomal fraction was incubated with CoA and NADH. For instance, 18:0, 18:2(n-6), and 20:3(n-6) incorporated into membrane lipids were gradually converted to 18:1(n-9), 18:3(n-6), and 20:4(n-6), respectively. Further, 20:5(n-3) and 18:3(n-6) were metabolized to 22:5(n-3) and 20:4(n-6) respectively, through chain elongation or chain elongation plus subsequent desaturation when malonyl-CoA was present. In contrast to esterified fatty acids, negligible change was observed for fre fatty acids under the same experimental conditions. It is apparent that ATP-independent acyl-CoA synthesis is implicated in such enzymatic modification of fatty acyl chains. The presence of either gel-filtered cytosol or bovine serum albumin markedly potentiated the reaction. However, the addition of ATP and Mg2+ did not accelerate the overall reaction induced in the presence of CoA alone. These results suggest that the pathway of CoA-dependent/ATP-independent modification of fatty acyl chains is sufficiently active to account for the desaturation and chain elongation of fatty acids esterified in membrane lipids, especially phospholipids, of living cells.

Acyl Coenzyme A↗
PubMed1994

Induction of coenzyme A-dependent transacylation activity in rat liver microsomes by administration of clofibrate.

The effect of administration of clofibrate on the activity of coenzyme A-dependent (CoA-dependent) transacylation of 1-acyl-glycerophosphocholine (1-acyl-GPC) was examined in rat liver microsomes. Administration of clofibrate to rats increased the activity of Co-A-dependent transacylation of 1-[14C]acyl-GPC and the activity reached a value (8.37 nmol/min per mg protein) twice that in control rats (3.95 nmol/min per mg protein) without any changes in apparent Km values for CoA (1.2 microM in control and 1.0 microM in clofibrate-treated) and 1-acyl-GPC (33.4 microM in control and 27.8 microM in clofibrate-treated). The rate of CoA-dependent transfer of [14C]arachidonic acid (20:4) from 1-acyl-2-[14C]20:4-glycerophosphoethanolamine (GPE) or 1-acyl-2-[14C]20:4-glycerophosphoinositol (GPI) to 1-acyl-GPC (synthesis of 1-acyl-2-[14C]20:4-GPC) was also increased by treatment with clofibrate (1.9-fold and 1.5-fold increases, respectively). These results suggest that a CoA-dependent transacylation system of 1-acyl-GPC was induced by treatment with clofibrate.

Acyltransferases↗
PubMed1994

Mercuric chloride induces the production of leukotriene B4 by rabbit alveolar macrophages.

The effects of HgCl2 on the production of leukotriene B4 (LTB4) by rabbit alveolar macrophages were investigated. Alveolar macrophages synthesized only a small amount of LTB4 when incubated in Hanks' balanced salt solution (HBSS) in the absence of stimuli or chemical reagents (0.2 +/- 0.1 ng/10(6) cells). The amount of LTB4 synthesized was greatly enhanced when macrophages were incubated with 5 x 10(-6) M HgCl2 (5.3 +/- 0.2 ng/10(6) cells). Increases in levels of free arachidonic acid (20:4), a precursor of eicosanoids, were also observed on treatment of [3H]20:4-labeled cells with HgCl2 in a concentration-dependent manner. We confirmed that [3H]20:4 was mainly liberated from the alkenyl subclass of ethanolamine glycerophospholipids (EGP). Degradation of alkenyl-EGP was also observed when HgCl2 and [3H]20:4-labeled lipids were incubated in HBSS, resulting in an increase in the level of [3H]20:4-labeled lysoEGP. When a microsomal fraction prepared from macrophages was added to the same mixture, [3H]20:4 was liberated from [3H]20:4-containing lysoEGP. These results suggest that the alkenyl linkage of EGP was degraded in the presence of HgCl2 with resultant formation of lysoEGP which was subsequently hydrolyzed by lysophospholipase. As a consequence, the level of free 20:4 in and the rate of production of LTB4 by alveolar macrophages were elevated.

Acylation↗
PubMed1993

Aspirin induces non-enzymatic formation of platelet-activating factor from lyso platelet-activating factor.

Substantial amounts of platelet-activating factor (PAF) were formed when lysoPAF was mixed with aspirin (e.g. 0.04% of added lysoPAF (200 nmol) was converted to PAF when mixed with aspirin (2 mumol) for 1 h). Non-enzymatic formation of PAF from aspirin and lysoPAF also occurs in the aqueous solution or in organic solvents in time-dependent and dose-dependent manners. Possible meanings of the non-enzymatic formation of PAF are discussed.

Animals↗
PubMed1993

Inositol 1,4,5-trisphosphate accumulation in brain of lithium-treated rats.

The mechanism of action of lithium as a drug for patients with affective disorders was investigated. Three-week-old male rats were orally administered 2.7 mEq Li2CO3/kg/d for 1 or 3 wk, and phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP), phosphatidylinositol 4,5-bisphosphate (PIP2), inositol phosphate (IP), inositol diphosphate (IP2) and inositol triphosphate (IP3) levels in brain were measured. The levels of IP were increased 1.7 and 2.4 times after 1 wk and 3 wk of lithium administration, respectively, while PI, PIP, PIP2, IP2 and IP3 levels were not altered. IP3 was further fractionated by high-performance liquid chromatography into I-1,3,4-P3 and I-1,4,5-P3. In the control rat brain, the relative percentages of I-1,3,4-P3 and I-1,4,5-P3 were 95.8 and 4.2, respectively. However, after 3 wk of lithium administration, the values were changed to 69.6 and 30.3%, respectively. This increase in the neurotransducer I-1,4,5-P3 in the brain may be relevant to the mechanism of action in the lithium treatment of patients with manic-depressive disorders.

Animals↗
PubMed1992

Distribution of alkyl and alkenyl ether-linked phospholipids and platelet-activating factor-like lipid in various species of invertebrates.

The levels of alkenylacyl, alkylacyl and diacyl subclasses of choline glycerophospholipid (CGP) and ethanolamine glycerophospholipid (EGP) fractions in 28 species of various invertebrates were studied. We found that only small amounts of either 1-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkylacyl-GPC) or 1-alkenyl-2-acyl-sn-glycero-3-phosphoethanolamine (alkenylacyl-GPE) are present in most species of insects. On the other hand, almost all species examined in various phyla other than Arthropoda were shown to contain large amounts of both alkylacyl-GPC and alkenylacyl-GPE. The highest proportion of alkylacyl subclass in CGP was noted in sponge, Halichondria japonica (81.8% of CGP) and the highest proportion of alkenylacyl subclass in EGP was found in clam worm, Marphysa sanguinea (88.7% of EGP). We next surveyed the presence of platelet-activating factor (PAF)-like lipid in 45 species of invertebrates. PAF-like lipid was widely distributed among various lower animals. The highest value was obtained for sea cucumber, Stichopus japonicus, in which PAF-like lipid was present throughout the body. We also confirmed the presence of acetyltransferase activity in several lower animals. These results suggest that alkyl and alkenyl ether-linked phospholipids including PAF are physiologically important molecules particularly for invertebrates belonging to lower phyla.

Animals↗
PubMed1992

Inhibition of the liberation of arachidonic acid by cadmium ions in rabbit alveolar macrophages.

The effects of CdCl2 on the liberation of arachidonic acid (20:4) from membrane phospholipids of A23187-stimulated rabbit alveolar macrophages and on the activity of phospholipase A2 (PLA2) in a cytosolic fraction were studied. Alveolar macrophages were prelabeled with [3H]arachidonic acid (20:4) and then treated with A23187. This treatment resulted in a remarkable increase in the liberation of [3H]20:4 from their phospholipids. Exposure of cells to Cd2+ inhibited the liberation of [3H]20:4 in a dose-dependent manner. Liberation of [3H]20:4 from cell lipids was calcium dependent and the inhibitory effect of Cd2+ competed with the stimulatory effect of Ca2+. When Ca2+ was removed from the incubation medium, Cd2+ did not influence the liberation of [3H]20:4. Entry of 45Ca2+ into cells was enhanced by treatment of A23187. However, Cd2+ did not influence the cellular uptake of 45Ca2+. Treatment with A23187 markedly enhanced entry of 109Cd2+ into cells. The effect of Cd2+ on the activity of phospholipase A2 was determined with 1-palmitoyl-2-[14C]arachidonoyl-sn-glycero-3-phosphocholine as substrate. Calcium-dependent activation of PLA2 was observed and Cd2+ inhibited activation in a dose-dependent manner. These results suggest that exposure of alveolar macrophages to Cd2+ causes a reduction in the rate of liberation of 20:4 from cell lipids, as a possible result of the inhibition of PLA2 activity by Cd2+.

Animals↗
PubMed1992

[The composition of phospholipid and biosynthesis of platelet-activating factor in earthworm (Eisenia foetida)].

Platelet-activating factor (PAF) was for the first time confirmed to exist in a lower animal, earthworm (Eisenia foetida). It amounts to 10.7 +/- 6.1 pmol/g wet body weight, and varied seasonally. Phospholipid analysis revealed that 1-O-alkyl-2-acyl-sn-glycerophosphocholine, a stored form of PAF precursor, accounted for 61.4% of the choline glycerophospholipids. Two kinds of enzyme activities operating in PAF generation in mammalian cells were also detected from this species. The PAF level increased markedly under some injurious stimuli such as cutting and pricking. The results suggest that PAF may be a primary mediator involved in pathological and physiological reactions even in lower animal like earthworm. The findings also cast a new light on the mechanisms underlying the antihypertensive and other effects of the Chinese medicinal earthworm, pheretima.

Animals↗
PubMed1992

Differential effects of platelet-activating factor on superoxide anion production in human eosinophils and neutrophils.

The effect of platelet-activating factor (PAF) on the generation of superoxide anion (O2-) in human eosinophils and neutrophils was examined. The presence of PAF potentiated O2- production in either opsonized zymosan- or formyl-methionyl-leucyl-phenylalanine (FMLP)-stimulated cells. The effect of PAF was prominent in opsonized zymosan-stimulated eosinophils and in FMLP-stimulated neutrophils. We also found that eosinophils generate substantial amounts of O2- when treated with PAF alone. The high responsiveness of unstimulated or opsonized zymosan-stimulated eosinophils to PAF to generate O2- may be relevant to the pathological changes at the loci of allergic reactions where eosinophils and PAF are crucially involved.

Eosinophils↗