Naoki Ohara

Blood coagulation and osmolar tolerance of erythrocytes in stroke-prone spontaneously hypertensive rats given rapeseed oil or soybean oil as the only dietary fat

The life-span of stroke-prone spontaneously hypertensive rats (SHRSP) has been reported to become shorter by ingestion of some vegetable oils, including rapeseed oil, when given as the sole dietary fat. The present study was undertaken to examine if the ingestion of rapeseed (canola) oil affects blood coagulating time and erythrocyte membranes. Namely, SHRSP were orally given canola oil or soybean oil as the only dietary fat (10% of diet) for 4 weeks. After the 4-week feeding, activated partial thromboplastin time (APTT) in the canola oil group (19.9+/-0.5 s, N=8) was significantly shorter than that in the soybean oil group (21.6+/-0.6 s, N=8, P<0. 05), though there were no between-group differences in plasma Ca(2+), platelet density and platelet aggregation. Erythrocytes from the canola oil group were less tolerant to low osmotic pressure than those from soybean oil group; the EC(50) values for NaCl concentration to cause hemolysis were 0.42+/-0.004 and 0.40+/-0.005% in the canola oil and the soybean oil groups, respectively (N=10, P<0.01). The canola oil-induced shortening of blood coagulation time and increased fragility in erythrocyte membranes may have relevance to the promotion of strokes in SHRSP.

Rapeseed oil ingestion and exacerbation of hypertension-related conditions in stroke prone spontaneously hypertensive rats

Two groups of 20 stroke prone spontaneously hypertensive rats (SHRSP) at 5 weeks old were fed a diet containing 10 w/w% rapeseed (canola) oil or soybean oil as the only dietary fat, and given drinking water containing 1% NaCl. Life span of the canola oil group (62+/-2 days) was shorter than that of the soybean oil group (68+/-3 days). Stroke-related symptoms were observed in every animal, but the onset of those in the canola oil group, at 47+/-1 days after starting the administration was earlier than that in the soybean oil group, 52+/-2 days. Incidence of cerebral hemorrhage was similar in these groups, and no differences were found between lesions of organs in the groups. In another experiment, two groups of ten SHRSP at 5 weeks of age were fed the defatted diet and given canola oil or soybean oil by gavage at 10 w/w% of consumed food for 4 weeks without NaCl loading. After the 4-week administration, mean systolic blood pressure in the canola oil group and the soybean oil group were 233+/-2 and 223+/-0.3 mmHg, respectively. Phytosterol levels in both plasma and erythrocyte membranes reflected those contained in the oils ingested. Na(+), K(+)-ATPase activities in the brain, heart and kidney were enhanced in the canola oil group. These results indicate that promotion of hypertension-related deterioration in organs is likely to have relevance to the short life span in the canola oil group. Enhanced Na(+), K(+)-ATPase activity by phytosterols in the oil ingested may play a role in these changes.

Thirteen-week dietary intake of rapeseed oil or soybean oil as the only dietary fat in Wistar Kyoto rats—change in blood pressure

Wistar Kyoto (WKY) rats were fed a diet containing 10% rapeseed (canola) oil or soybean oil as the only dietary fat for 13 weeks. From week 5 of feeding, systolic blood pressure of the canola oil group became higher than that of the soybean oil group. The 13-week canola oil intake increased plasma levels of Na(+) and lipids, and decreased the level of K(+) compared to those in the soybean oil group. The canola oil group also showed a high density of neutrophils and a low density of platelets compared to the soybean oil group. Moreover, the activities of catalase and superoxide dismutase in the hepatic cytosol were depressed in the canola oil group. The mechanisms for the higher blood pressure are unclear. However, an increase in body fluid via activation of Na(+) pump or Na(+), K(+)-ATPase and/or a blunt endothelium-dependent vasodilation by increased superoxide might have relevance to the elevated blood pressure. The increased plasma lipids and the changes in the densities of platelets and neutrophils appear not to be critical in WKY rats. However, these would tend to promote peripheral vascular lesions in the strains, such as spontaneously hypertensive rats and stroke-prone spontaneously hypertensive rats, which are prone to present atheroscrelotic vascular injury.

Acute Functional Enhancement of Circulatory Neutrophils After Intratracheal Instillation with Diesel Exhaust Particles in Rats

Abstract We have previously demonstrated that intratracheal instillation (IT) with diesel exhaust particles (DEP) exacerbates myocardial ischemia/reperfusion-induced arrhythmia in rats. Since activated neutrophils play a pivotal role in ischemia/reperfusion arrhythmia, in the present study we investigated the effects of DEP on peripheral neutrophil count and on the oxyradical production (ORP) of neutrophils in rats. We also determined the production of cytokines for better understanding of the relationship between pulmonary inflammation and neutrophil function. Instillation with 5 mg DEP elevated circulatory neutrophil counts (CNC) at 12 and 24 h post-instillation to levels approximately 2.1- and 2.3-fold those in the vehicle-treated animals, respectively. On the other hand, 1-mg DEP caused an approximately 0.4-fold increase in CNC at 6 h. 12-O-Tetradecanoylphorbol 13-acetate-induced ORP in the isolated neutrophil was enhanced at 12 and 24 h after instillation with 5 mg DEP. Cytokine-induced neutrophil chemoattractant-1 (CINC-1), tumor necrosis factor-α (TNFα) and macrophage inflammatory protein-2 (MIP-2) levels were increased in the bronchoalveolar lavage fluid (BALF) collected from animals that received 5 mg DEP. In serum, a marked elevation of CINC-1 and a slight elevation of MIP-2 were also observed, while TNFα was not detected. Granulocyte–macrophage colony-stimulating factor (GM-CSF) was detected in neither BALF nor serum for 24 h after the instillation. These results suggest that IT instillation of DEP enhances systemic oxidative stress by increasing neutrophil count and ORP in the acute period.

Dietary intake of rapeseed oil or soybean oil as the only fat nutrient in spontaneously hypertensive rats and Wistar Kyoto rats — blood pressure and pathophysiology

Spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) rats were fed a diet containing 10% rapeseed (canola) oil or soybean oil as dietary fat, and given drinking water containing 1% NaCl for 26 weeks. From the 10th week and later, systolic blood pressure in the canola oil group became higher than that in the soybean oil group in each strain. The 26-week feeding of canola oil increased plasma lipids and the neutrophil counts, and decreased the platelet counts. In the canola oil group the heart and kidney tended to become heavier with sporadically found histologic lesions. Acetylcholine- and nitroprusside-induced dilating responses of isolated aortic rings and norepinephrine- and veratridine-induced increases in vascular tone of isolated perfused mesenteric arteries were not different between the two groups in each strain. These results demonstrate that canola oil intake as the only dietary fat elevates blood pressure of the rat provided with drinking water containing 1% NaCl through mechanisms other than blunt dilating response of the blood vessel due to dysfunction of the endothelium or vascular smooth muscle, the augmented response to norepinephrine in the arteries and the increased amount of norepinephrine in the sympathetic nerve endings. The lesions in the heart and kidney in SHR may be related to a strain-specific peripheral vascular deterioration which was disclosed by the extremely high blood pressure in the canola oil group.

Age-related differences and roles of endothelial nitric oxide and prostanoids in angiotensin II responses of isolated, perfused mesenteric arteries and veins of rats

We examined whether or not cyclo-oxygenase products of arachidonic acid and endothelium-derived relaxing factor (nitric oxide, NO) regulate the vascular response to angiotensin II differently with aging or development. For this purpose angiotensin II responses of isolated, perfused rat mesenteric vascular beds were compared between rats aged 4 weeks and 32 weeks. Angiotensin II increased perfusion pressure in arteries and veins of both rats aged 4 weeks and 32 weeks. In the arteries of rats aged 32 weeks the increase was slight, and less than that in rats aged 4 weeks. In contrast, the veins showed similar increases in perfusion pressure in rats aged 4 weeks and 32 weeks. Indomethacin, an inhibitor of cyclo-oxygenase, at 5 x 10(-6) M depressed the increase in perfusion pressure only in the arteries of rats aged 32 weeks. NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthase, applied at 5 x 10(-6) M in the presence of indomethacin enlarged the perfusion pressure increase in the arteries of both rats aged 4 weeks and 32 weeks, while it failed to modify that in the veins. After removal of the endothelium from the blood vessels, the perfusion pressure responses in arteries were increased in both rats aged 4 weeks and 32 weeks, whereas those in veins were not affected. Regardless of the endothelium being intact or removed, the increase in arterial perfusion pressure of rats aged 32 weeks all but disappeared with 5 x 10(-6) M furegrelate, an inhibitor of thromboxane A2 synthase, and with a combined application of furegrelate and 10(-6) M SQ29,548, a blocker of thromboxane A2/prostaglandin H2 receptors. These results indicate the following: in rat mesenteric vascular beds the angiotensin II response in the arteries appears to diminish with aging or development, whereas that in the veins does not change. The NO released from the endothelium regulates the arterial response but vasodilating prostanoids have no role in the response. Moreover, in the arteries of rats aged 32 weeks, vasoconstricting prostanoids, such as prostaglandin H2 and thromboxane A2, seem to play a role in angiotensin II-induced vasoconstriction. With aging or development, and depending on the type of blood vessel, NO and prostanoids appear to modify the angiotensin II response differently.

Dietary Lipids Impacts on Healthy Ageing

Healthy ageing is gaining attention in the lipid nutrition field. As in vivo biomarkers of healthy ageing, we have evaluated the survival, learning/memory performance, and physical potencies in rodents fed a diet supplemented with high-linoleic acid (LNA, ω6) safflower oil or high-α-linolenic acid (ALA, ω3) perilla oil for long periods. The results suggested that perilla oil with a low ω6/ω3 ratio is beneficial for healthy ageing. In order to address this issue further, we determined the survival of stroke-prone SHR (SHRSP) rats fed a conventional rodent diet supplemented with 10% fat or oil. Survival was longer with ω3-rich oils compared with ω6-rich oils. However, some kinds of vegetable oils and hydrogenated oils shortened the survival of SHRSP rats to an unusual degree (ca. 40% compared with that of ω6-rich oil) that could not be accounted for by the fatty acid and phytosterol composition of the oils. The observed decrease in platelet counts was associated with pathological changes in the kidney and other organs. Dihydro-vitamin K1 is proposed as a likely candidate as a stroke-stimulating factor in hydrogenated oils. Thus, factors other than fatty acids (ω6/ω3 balance) and phytosterols must be taken into account when fats and oils are evaluated in relation to healthy ageing.

Regional differences of the mouse brain in response to an α-linolenic acid-restricted diet: neurotrophin content and protein kinase activity.

AIMS The purpose of this study was to determine a relatively short-term effect of feeding an α-linolenic acid (ALA, n-3)-restricted, linoleic acid (LA, n-6)-adequate diet on neurotrophin contents and protein kinase activities in brain regions of the mouse. MAIN METHODS After feeding mice a safflower oil (SAF) diet (ALA-restricted, LA-adequate) or perilla oil (PER) diet (containing adequate amounts of ALA and LA) for 4 weeks from weaning, the fatty acid compositions of brain regions were analyzed by capillary column gas-liquid chromatography, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) contents were measured using enzyme-linked immunosorbent assay. KEY FINDINGS The striatum and hippocampus, but not the cerebral cortex, from the SAF group, contained a smaller amount of docosahexaenoic acid (DHA, 22:6n-3) than those from the PER group. The NGF contents in these brain regions were not different between the two dietary groups. However, the striatal BDNF content of the SAF group was significantly lower than that of the PER group. Protein kinase A, protein kinase C, and p44/42 mitogen-activated protein kinase (p44/42 MAPK) activities in brain regions showed no significant difference between the two dietary groups. However, the striatal p38 MAPK activity was significantly lower in the SAF group than in the PER group. No such differences were observed in the hippocampus or the cerebral cortex. SIGNIFICANCE A relatively short-term feeding of an α-linolenic acid-restricted, linoleic acid-adequate diet was found to lower the DHA content, BDNF content and p38 MAPK activity in the mouse striatum.

Inhibition of Excessive Cell Proliferation by Calcilytics in Idiopathic Pulmonary Arterial Hypertension

Idiopathic pulmonary arterial hypertension (IPAH) is a rare and progressive disease of unknown pathogenesis. Vascular remodeling due to excessive proliferation of pulmonary arterial smooth muscle cells (PASMCs) is a critical pathogenic event that leads to early morbidity and mortality. The excessive cell proliferation is closely linked to the augmented Ca2+ signaling in PASMCs. More recently, we have shown by an siRNA knockdown method that the Ca2+-sensing receptor (CaSR) is upregulated in PASMCs from IPAH patients, involved in the enhanced Ca2+ response and subsequent excessive cell proliferation. In this study, we examined whether pharmacological blockade of CaSR attenuated the excessive proliferation of PASMCs from IPAH patients by MTT assay. The proliferation rate of PASMCs from IPAH patients was much higher (~1.5-fold) than that of PASMCs from normal subjects and patients with chronic thromboembolic pulmonary hypertension (CTEPH). Treatment with NPS2143, an antagonist of CaSR or calcilytic, clearly suppressed the cell proliferation in a concentration-dependent manner (IC50 = 2.64 μM) in IPAH-PASMCs, but not in normal and CTEPH PASMCs. Another calcilytic, Calhex 231, which is structurally unrelated to NPS2143, also concentration-dependently inhibited the excessive proliferation of IPAH-PASMCs (IC50 = 1.89 μM). In contrast, R568, an activator of CaSR or calcimimetic, significantly facilitated the proliferation of IPAH-PASMCs (EC50 = 0.33 μM). Similar results were obtained by BrdU incorporation assay. These results reveal that the excessive PASMC proliferation was modulated by pharmacological tools of CaSR, showing us that calcilytics are useful for a novel therapeutic approach for pulmonary arterial hypertension.

Delayed Exacerbation of Acute Myocardial Ischemia/Reperfusion-Induced Arrhythmia by Tracheal Instillation of Diesel Exhaust Particles

For understanding the relationship between the increased incidence of sudden cardiac death and air pollution, we examined the effects of intratracheal instillation of diesel exhaust particles (DEP) on acute myocardial ischemia/reperfusion-induced arrhythmia in rats. The animals received 1 mg DEP 24–48 h before the ischemia/reperfusion (DEP-pretreated group, DEP-PRE), and were subjected to 3 successive brief ischemia/reperfusion (3 min ischemia followed by 5 min reperfusion) procedures. These were to make the animals tolerant to ischemia/reperfusion-related myocardial deterioration. Thereafter the animals were subjected to a 10-min ischemia followed by a 30-min reperfusion. In the experiments, an increased mortality was observed in the DEP-PRE group compared to the vehicle (0.05% Tween 80–PBS)-treated group. Forty-six percent of the animals in DEP-PRE died during the first 3-min reperfusion period. The animals of other groups were intratracheally instilled with DEP at the beginning of ischemia/reperfusion experiment, or were pretreated with polyethylene glycol-conjugated superoxide dismutase (1000 IU kg−1, iv). In these animals, incidences of both arrhythmia and mortality were similar to those in the animals treated with the vehicle. In experiments to investigate the effects of DEP on the biochemical and hematological parameters, neutrophil count was elevated by a higher dose (5 mg) of DEP at 24 h after the intratracheal instillation, and oxygen radical production, which was induced by 12-O-tetradecanoylphorbol 13-acetate, was enhanced at 72 h. These results indicate that intratracheal DEP instillation exacerbates short-period ischemia/reperfusion-induced arrhythmia. Delivery and activation of peripheral neutrophils and oxygen radicals produced in neutrophils might participate in this exacerbation. This is the first article that demonstrates the arrhythmogenicity of DEP using intratracheal instillation in rats.