Aizan Hirai

Effect of oral administration of highly purified eicosapentaenoic acid on platelet function, blood viscosity and red cell deformability in healthy human subjects

Eicosapentaenoic acid (EPA), which is abundant in seafood, has been reported to be a potent antagonist of platelet aggregation and also to reduce the incidene of cardiovascular disorders. We recently reported that EPA also reduces whole blood viscosity. A highly purified EPA, in a soft capsule (75% ethylester form of EPA; EPA-E), manufactured from sardine oil was administered to 8 healthy male subjects for 4 weeks. No side effects were observed. Platelet aggregation and platelet retention significantly decreased. The EPA content in platelet phospholipids markedly increased but docosahexaenoic acid (DHA) and arachidonic acid (AA) contents did not change. A reduction in whole blood viscosity and an increase in erythrocyte deformability were also observed after 4 weeks ingestion of EPA-E. The EPA content in erythrocyte membrane phospholipids markedly increased after 4 weeks, and was positively correlated with erythrocyte deformability. Reduction of platelet aggregation and improvement of the rheological properties of the erythrocyte might be explained by an increase in the EPA content in platelet and erythrocyte phospholipids.

The effects of the oral administration of fish oil concentrate on the release and the metabolism of [14C]arachidonic acid and [14C]eicosapentaenoic acid by human platelets

It has been suggested by several investigators that eicosapentaenoic acid (C20:5 omega 3, EPA) might have anti-thrombotic effects. In this experiment, the effect of the oral administration of EPA rich fish oil concentrate on platelet aggregation and the release and the metabolism of [1-14C]arachidonic acid and [(U)-14C]eicosapentaenoic acid by human platelets was studied. Eight healthy male subjects ingested 18 capsules of fish oil concentrate (EPA 1.4 g) per day for 4 weeks. Plasma and platelet concentrations of EPA markedly increased, while those of arachidonic acid (C20:4 omega 6, AA) and docosahexaenoic acid (C22:6 omega 3, DHA) did not change. Platelet aggregation induced by collagen and ADP was reduced. Collagen induced [14C]thromboxane B2 (TXB2) formation from [14C]AA prelabeled platelets decreased. There was no detectable formation of [14C]TXB3 from [14C]EPA prelabeled platelets, and the conversion of exogenous [14C]EPA to [14C]TXB3 was lower than that of [14C]AA to [14C]TXB2. The release of [14C]AA from [14C]AA prelabeled platelets by collagen was significantly decreased. These observations raise the possibility that the release of arachidonic acid from platelet lipids might be affected by the alteration of EPA content in platelets.

Effects of orally administered ethyl ester of eicosapentaenoic acid (EPA; C20:5, ω-3) on PGI2-like substance production by rat aorta☆

A highly purified ethyl ester of EPA (EPAEE) (74%) was manufactured from sardine oil. Sixty mg/kg/day of EPAEE was given orally to male Wistar rats for 8 weeks. No side effect or toxicity from the administration of EPAEE was observed. Plasma EPA concentration and the ratio of EPA to arachidonic acid were significantly increased, compared with control Wistar rats. An enhancement of PGI2-like substance production by aortas obtained from rats fed EPAEE was noted. Conversion of EPA to delta 17-6-keto-PGF1 alpha, a stable metabolite of PGI3, could not be detected by an incubation study of 14C-EPA and aortas either from rats fed EPAEE or from control rats. Therefore, PGI2-like substance produced by rat aorta is most likely to be PGI2 itself and not PGI3.

Pituitary adenylate cyclase-activating polypeptide protects rat-cultured cortical neurons from glutamate-induced cytotoxicity.

We have investigated the effects of pituitary adenylate cyclase-activating polypeptide with 38 residues (PACAP38) on glutamate-induced neuronal cell death in rat-cultured cortical neurons. The rat-cultured neurons were obtained from E17 day-old embryos and cultured in a chemically defined medium without serum for 10 days, after which more than 95% of the cells were stained by a specific antibody against MAP-2, a specific marker for neurons. The number of viable neurons was identified by the mitochondrial conversion of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) to formazan, which was detected by the associated change in optical density at 570 nm. Glutamate-induced neuronal cell death was suppressed by PACAP38 at concentrations as low as 10(-13) M, and at 10(-11) M maximally suppressed half of the amount of glutamate-induced cell death seen in a control situation (no PACAP38). The dose-response curve was bell-shaped. Dibutyryl cAMP (dbcAMP) also increased the number of neurons that were protected from damage with a bell-shaped dose-response curve suggesting that PACAP exerts its neuroprotective effect through the activation of a cAMP signal transduction system. However, cAMP accumulation in the media of neurons was stimulated by PACAP38 at concentrations as low as 10(-11) M, a much higher concentration than the minimal effective dose of PACAP38 required for protection against glutamate-induced neuronal cell death. Among the three neuropeptides of PACAP38, arginine vasopressin (AVP) and C-type natriuretic peptide (CNP), only PACAP38 exhibited a neurotrophic effect in the glutamate-induced neuronal cell death at the indicated concentrations. These data indicate that PACAP38 is one of the more important neuroprotective factors. The kind of intracellular signal transduction system involved in the neuroprotective effect of PACAP38 still remains to be established.

Geranylgeranyl-Pyrophosphate, an Isoprenoid of Mevalonate Cascade, Is a Critical Compound for Rat Primary Cultured Cortical Neurons to Protect the Cell Death Induced by 3-Hydroxy-3-Methylglutaryl-CoA Reductase Inhibition

We investigated the role of the intrinsic mevalonate cascade in the neuronal cell death (NCD) induced by the inhibition of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase in rat primary cortical neurons cultured from the brains of 17-d-old fetal SD rats. HMG-CoA reductase inhibitors induced NCD [HMG-CoA reductase inhibitor-induced NCD (H-NCD)] in time- and dose-dependent manners. The apoptotic characteristics were revealed by the formation of the DNA ladder and by the electron microscopical observation. During the progression of H-NCD, p53 was induced followed by the expression of Bax. Although the mevalonate completely inhibited H-NCD, the cholesterol did not. Thus, we examined two major metabolites of mevalonate, geranylgeranyl-pyrophosphate (GGPP) and farnesyl-pyrophosphate (FPP), using a novel liposome system for uptake into the cells. GGPP, not FPP, prohibited H-NCD with inhibition of the induction of p53 and Bax. The inhibition of HMG-CoA reductase decreased the amount of membrane-associated Rho small GTPase families, but not Ras small GTPase, and GGPP restored the blockage by HMG-CoA reductase inhibitor in the translocation or redistribution of Rho small GTPase families to membrane. These data indicated that (1) the inhibition of the intrinsic mevalonate cascade induces the apoptotic NCD with the induction of p53 followed by that of Bax, (2) the inhibition of HMG-CoA reductase concomitantly causes blockage of the translocation or redistribution of Rho small GTPase families, not Ras small GTPase, to membrane, and (3) GGPP, not FPP, is one of the essential metabolites in the mevalonate cascade for protecting neurons from H-NCD.

Role of p27Kip1 and Cyclin-Dependent Kinase 2 in the Proliferation of Non-Small Cell Lung Cancer

The cell cycle is governed by a family of cyclin-dependent kinases (Cdks). Cdk2 forms a functional complex with cyclin E and plays a pivotal role in the regulation of G1/S transition. Cdk2 activity is negatively regulated by interactions with inhibitors. p27Kip1, one of the most potent inhibitors of Cdk2, was recently identified as a powerful negative prognostic marker in non-small cell lung cancer as well as in colorectal and breast cancer. In the present study, the expression of p27 and Ki-67 antigen in nonneoplastic and cancerous lung tissues was determined by immunohistochemistry. After establishing that the antibody-measured p27 labeling index was a good reflection of the level of p27 expression measured by Western blotting, we show that p27 labeling index is decreased in cancerous lung tissues, compared with nonneoplastic lung tissues, and exhibits a significant inverse relation to the proliferation marker Ki-67 antigen, detected with monoclonal antibody MIB-1. Consistent with these data, all cancerous lung tissues showed enhanced degradation activity of p27 compared with nonneoplastic lung tissues and, in addition, increased levels of the phosphorylated form of Cdk2, as determined with Western blot analysis. The H1 histone kinase activity associated with Cdk2 was also increased in non-small cell lung cancers. Statistical analysis showed that proliferative activity as measured by MIB-1 labeling index was highly correlated with Cdk2 activity (r = 0.767, P < 0.0015). These results suggest that p27 and Cdk2 may play an important role in the proliferation of non-small cell cancer.

Eicosapentaenoic acid and adult diseases in Japan: epidemiological and clinical aspects

Abstract. Residents of a coastal fishing village in Japan consume larger amounts of fresh fish rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) than those in inland farming villages. A higher content of EPA and DHA in the plasma and reduced platelet aggregability was observed in the residents of the fishing village than in farmers. Incidence of thrombotic cardiovascular disorders was lower in the fishing area than in the farming area. Oral ingestion of highly purified EPA or DHA reduced platelet aggregability and improved serum lipid profile in healthy subjects and in hyperlipidaemic patients, though DHA seems to be much less potent. In clinical studies with highly purified EPA, improvement of clinical features was noted in patients with thrombotic cardiovascular disorders. It is also shown that EPA and DHA have an anti‐inflammatory effect in man although EPA is far more active than DHA.

Relationship of thromboxane generation to the aggregation of platelets from humans: Effects of eicosapentaenoic acid

A non-linear relationship between the percent aggregation of human platelets and the amount of TXB2 generated requires investigators to use caution when using the data to assess antiplatelet regimens. The relationship approximates a hyperbola with a roughly linear relationship from 0 to 70% aggregation and 0 to 50 ng TXB2 per ml of platelet-rich plasma. Above these values, the amount of TXB2 produced may increase up to 500 ng per ml of platelet-rich plasma with no clear relationship to the observed platelet function of aggregation. Also, appreciable inhibition of TXB2 formation can occur at high TXB2 levels with no detectable decrease in aggregation. Thus, assessment of antiplatelet regimens using TXB2 formation alone are unlikely to be interpretable without reference to this non-linear property of platelet function. We applied this concept when evaluating a study of forty subjects with dietary supplements of 1.8 g or 2.7 g of ethyl eicosapentaenoate (20:5n-3) for four weeks. There was a moderate, but statistically significant decrease in average values for the percent aggregation (60 +/- 15 to 45 +/- 30) and thromboxane production (51 +/- 30 to 33 +/- 31 ng/ml). Although the differences in mean values were slight relative to the overall standard deviations, reductions of platelet function were clearly evident in 31 of 40 subjects when paired results were examined relative to the recognized hyperbolic relationship.

Regulation of FRTL-5 Thyroid Cell Growth by Phosphatidylinositol (OH) 3 Kinase-Dependent Akt-Mediated Signaling

Thyrotropin (TSH)-initiated cell cycle progression from G1 to S phase in FRTL-5 thyroid cells requires serum, insulin, or insulin-like growth factor 1 (IGF-1) and involves activation of 3-hydroxy-3-methylglutaryl-CoA reductase, geranylgeranylation of RhoA, p27Kip1 degradation, and activation of cyclin-dependent kinase (cdk) 2. In the present report, we show that the serine-threonine kinase Akt is an important mediator of insulin/IGF-1/serum effects on cell cycle progression in FRTL-5 thyroid cells. The phosphoinositol (OH) 3 kinase inhibitors, Wortmannin (WM) and Ly294002 (LY), block the ability of insulin/IGF-1 to reduce p27 expression, to induce expression of cyclins E, D1, and A as well as cdk 2 and 4, and to phosphorylate retinoblastoma protein. They also inhibit insulin/IGF-1-increased DNA synthesis and cell cycle entrance (S+G2/M). Insulin/IGF-1 rapidly induced activation of Aktl in a PI3 kinase-dependent manner, and increased Aktl RNA levels. Most importantly, FRTL-5 cells transfected with a constitutively active form of Aktl have higher basal rates of DNA synthesis and no longer require exogenous insulin/IGF-1 or serum for TSH-induced growth. In sum, Aktl appears to have an important role in insulin/IGF-1 regulation of FRTL-5 thyroid cell growth and cell cycle progression.

Pituitary adenylate cyclase activating polypeptide (PACAP) stimulates mitogen-activated protein kinase (MAPK) in cultured rat astrocytes

Astrocytes, a subtype of glial cells, have been demonstrated to have an abundant number of receptors for pituitary adenylate cyclase activating polypeptide (PACAP), a neuropeptide of the VIP/secretin family which stimulates cAMP accumulation 1000 times more potent than VIP in astrocytes. PACAP is reported to stimulate the proliferation of astrocytes at low concentrations at which it does not yet stimulate the cAMP accumulation. In the present study, we examined the effect of PACAP on the activation of mitogen-activated protein kinase (MAPK), one of the important intracellular signals for the proliferation, and compared it with that of epidermal growth factor (EGF). To investigate the activation of MAPK, we focused on ERK2, one of MAPK, in cultured rat astrocytes. The activation of ERK2 was determined by immunoblotting and measurement of the activity in terms of the phosphorylating activity of immunoprecipitates with MAPK antibody on myelin basic protein. One pM of PACAP38 temporarily activated ERK2 at 10 min. In contrast, EGF activated ERK2 from 10 min to 60 min continuously. As for the dose-response effect, PACAP stimulated ERK2 at as low a concentration as 10-14 M and peaked at 10-12 M. Thereafter, its activating effect gradually decreased at 10-10 M and returned to the basal level at 10-8 M, forming a bell-shaped dose-dependency. Neither an inhibitor of PKA (H89) nor inhibitors of PKC (staurosporine and calphostin C) had any effect on the ERK2 activation induced by 1 pM PACAP38. Dibutyryl cAMP suppressed ERK2 activity in a dose-dependent manner. These data clearly demonstrated that PACAP stimulates MAPK in both a PKA- and a PKC-independent manner in cultured rat astrocytes.