Tatsuo Watanabe

Angiotensin type 1 receptor antagonist inhibits lipopolysaccharide-induced stimulation of rat microglial cells by suppressing nuclear factor κB and activator protein-1 activation

We investigated whether angiotensin (ANG)u2003II and its receptors contribute to lipopolysaccharide (LPS)‐induced microglial activation through activation of the proinflammatory transcription factors nuclear factoru2003κB (NF‐κB) and activator protein‐1 (AP‐1). Using primary microglial cell cultures, we examined whether losartan [ANG typeu20031 receptor (AT1) antagonist] alters the effects of LPS on: the production of interleukin‐1 (IL‐1) and nitric oxide, cell morphology, and NF‐κB and AP‐1 activities. Reverse transcription‐polymerase chain reaction revealed that LPS‐stimulated microglial cells exhibited marked mRNA expression for AT1, ANG typeu20032 receptor (AT2) and the ANGu2003II precursor angiotensinogen, whereas non‐stimulated microglial cells expressed only those for AT2 and angiotensinogen. We further demonstrated marked peptide/protein expression for AT1 and ANGu2003II in LPS‐activated microglial cells. LPS (100u2003ng/mL)‐stimulated microglial cells showed increased concentrations of IL‐1 and nitrite (a relatively stable metabolite of nitric oxide), and increased expression of IL‐1 mRNA as well as a morphological change from an amoeboid shape to a multipolar (mostly bipolar but sometimes tripolar) rod shape. These effects were all significantly inhibited by losartan treatment (10−5u2003m or less). NF‐κB and AP‐1 activities were enhanced in LPS‐stimulated microglial cells, effects that were significantly suppressed by losartan (10−5u2003m). ANGu2003II application enhanced the LPS‐induced increases in IL‐1 and nitrite concentrations, as well as the LPS‐induced morphological changes and AP‐1 activation, and these enhancements were inhibited by losartan (10−5u2003m). These results suggest that endogenous ANGu2003II enhances LPS‐induced microglial activities through stimulation of the microglial AT1, which itself evokes activation of the transcription factors NF‐κB and AP‐1.

Modulating effects of prenatal stress on hyperthermia induced in adult rat offspring by restraint or LPS-induced stress.

The present study was carried out to investigate the effects of prenatal stress on stress-induced hyperthermia in adult rats. Prenatal stress was administered daily for 3 days (embryonic days 15-17) by restraining pregnant rats in a small cage either for 30 or 240 min. After birth, foster mothers raised the pups. Offspring were tested at 9-10-weeks-old. Changes in body temperature and in the plasma concentrations of corticosterone, norepinephrine (NE), and epinephrine (Epi) induced by restraint or lipopolysaccharide (LPS)-induced stress were examined. By comparison with the prenatally nonstressed control group, the 240-min stress group showed a significantly lower hyperthermia in response to restraint stress but a higher fever after injection of LPS. The 30-min stress group showed similar alterations in these hyperthermic responses but did not reach significance. Both the restraint stress and the injection of LPS evoked greater increases in the plasma level of corticosterone in the 240-min stress group than in the control group. Although restraint stress induced significant increases in NE and Epi in the control and 30-min stress groups, the plasma levels of these catecholamines did not increase in the 240-min stress group. These results demonstrate for the first time that prenatal stress has opposite effects on the hyperthermic responses to restraint and LPS injection, suggesting that different mechanisms underlie the modulating effects of prenatal stress on the responses to the two types of stressors.

Systemic administration of [6]-gingerol, a pungent constituent of ginger, induces hypothermia in rats via an inhibitory effect on metabolic rate

We investigated the effects of systemic administrations of ginger (Zingiber officinale Roscoe, Zingiberaceae) or its pungent constituent, [6]-gingerol, on resting body temperature in rats. Rats given ginger-containing rat chow for 5 days showed no changes in their day-night cycle of body temperature or physical activity. However, a single intraperitoneal (i.p.) injection of [6]-gingerol (2.5 or 25 mg/kg) induced a rapid, marked drop in body temperature in a dose-related manner, with no change in physical activity. A significant decrease in metabolic rate was observed immediately after an i.p. injection of [6]-gingerol (25 mg/kg), although heat-loss responses underwent no alteration (versus vehicle). These results suggest that in rats: (a) a decrease in metabolic rate is responsible for the [6]-gingerol-induced hypothermia, and (b) [6]-gingerol modulates or interferes with the mechanisms underlying body temperature regulation, while other bioactive constituents of ginger may counteract the hypothermic effect of [6]-gingerol.

Green odor inhalation by rats down-regulates stress-induced increases in Fos expression in stress-related forebrain regions

In the present study, on rats, a quantitative analysis of Fos protein immunohistochemistry was performed as a way of investigating the effects of inhalation of green odor (a mixture of equal amounts of trans-2-hexenal and cis-3-hexenol) on the neuronal activations in stress-related forebrain regions induced by acute and repeated stress. Rats were exposed to restraint stress for 90 min each day for 1, 2, 4, 7, or 11 consecutive days. The hypothalamic paraventricular nucleus (PVN), amygdala, hippocampus and paraventricular thalamic nucleus (PVT) were examined. Both acute and repeated restraint stress increased Fos-positive cells in the entire hypothalamic PVN, in the central and medial amygdala, and in PVT, although these responses declined upon repeated exposure to such stress. The stress-induced Fos responses were much weaker in rats that inhaled green odor during each days restraint. No increases in Fos-positive cells were observed in the hippocampus in acutely stressed rats. The Fos-immunoreactive response to acute stress shown by the piriform cortex did not differ significantly between the vehicle+stress and green+stress groups. Green odor had inhibitory effects on the stress-induced corticosterone response, body-weight loss, and adrenal hypertrophy. These results suggest that in rats, green odor inhalation may, in an as yet unknown way, act on the brain to suppress activity in the neuronal networks involved in stress-related responses (such as activation of the hypothalamo-pituitary-adrenocortical axis and activation of the sympathetic nervous system, as well as stress-induced fear responses).

Novel loss-of-function putative aminotransferase alleles cause biosynthesis of capsinoids, nonpungent capsaicinoid analogues, in mildly pungent chili peppers (Capsicum chinense).

Capsinoids are a group of nonpungent capsaicinoid analogues produced in Capsicum fruits. They have similar bioactivities to capsaicinoids such as suppression of fat accumulation and antioxidant activity. They are more palatable ingredients in dietary supplements than capsaicinoids because of their low pungency. Previous studies on nonpungent Capsicum annuum cultivars showed that capsinoid biosynthesis is caused by loss-of-function putative aminotransferase (p-amt) alleles. This study showed that three mildly pungent cultivars of Capsicum chinense (Zavory Hot, Aji Dulce strain 2, and Belize Sweet) contain high levels of capsinoid. It was shown that these cultivars have novel p-amt alleles, which contain mutations that differ from those of C. annuum. Sequence analysis of p-amt in Belize Sweet revealed that a 5 bp insertion (TGGGC) results in a frameshift mutation. A transposable element (Tcc) was found in the p-amt of Zavory Hot and Aji Dulce strain 2. Tcc has features similar to those of the hAT transposon family. This was inserted in the fifth intron of Zavory Hot and in third intron of Aji Dulce strain 2. The p-amt alleles harboring Tcc cannot produce an active p-AMT. These mildly pungent cultivars will provide a new natural source of capsinoids.

Effect of “Rose Essential Oil” Inhalation on Stress-Induced Skin-Barrier Disruption in Rats and Humans

In stressed animals, several brain regions (e.g., hypothalamic paraventricular nucleus [PVN]) exhibit neuronal activation, which increases plasma adrenocorticotropic hormone (ACTH) and glucocorticoids. We previously reported that so-called green odor inhibits stress-induced activation of the hypothalamo-pituitary-adrenocortical axis (HPA axis) and thereby prevents the chronic stress-induced disruption of the skin barrier. Here, we investigated whether rose essential oil, another sedative odorant, inhibits the stress-induced 1) increases in PVN neuronal activity in rats and plasma glucocorticoids (corticosterone [CORT] in rats and cortisol in humans) and 2) skin-barrier disruption in rats and humans. The results showed that in rats subjected to acute restraint stress, rose essential oil inhalation significantly inhibited the increase in plasma CORT and reduced the increases in the number of c-Fos-positive cells in PVN. Inhalation of rose essential oil significantly inhibited the following effects of chronic stress: 1) the elevation of transepidermal water loss (TEWL), an index of the disruption of skin-barrier function, in both rats and humans and 2) the increase in the salivary concentration of cortisol in humans. These results suggest that in rats and humans, chronic stress-induced disruption of the skin barrier can be limited or prevented by rose essential oil inhalation, possibly through its inhibitory effect on the HPA axis.

Angiotensin II: its effects on fever and hypothermia in systemic inflammation.

Angiotensin II (ANG II), a bioactive peptide that plays important roles in blood-pressure and body-fluid regulation, has recently been reported to be involved in normal thermoregulation and fever. In the case of thermoregulation, ANG II lowers body temperature when administered centrally or systemically (i.e. exogenous ANG II acts as a hypothermia-inducing agent). In contrast, endogenous ANG II is involved both in heat-loss responses in a hot environment and in thermogenesis in the cold. It therefore seems likely that endogenous ANG II is involved in maintaining body temperature at the set-point. In the case of fever, it has been reported that endogenous brain ANG II and its type 1 receptor mediate or modulate the fever induced by restraint stress. At the final step in pyrogen-induced fever, brain ANG II facilitates the fever induced by prostaglandin E2 (PGE2) through its action on the type 2 receptor, whereas at its first step the lipopolysaccharide (LPS, 2 microg/kg, i.v.)-induced production of pyrogenic cytokines [such as interleukin-1 (IL-1)] involves an action of endogenous ANG II through its type 1 receptor. On the other hand, it is well known that a very high dose of LPS (50-5000 microg/kg) injected systemically induces hypothermia in rodents. This hypothermia is presumably initiated by tumor necrosis factor (TNF). Since ANG II contributes to the LPS-induced production of cytokines such as IL-1beta, as described above, it is possible that the generation of TNF by LPS involves an action of ANG II, too, and that this TNF production leads to the LPS-induced hypothermia. Together, these findings suggest that ANG II and its receptors make a number of contributions to normal thermoregulation, to fever, and to the hypothermia in systemic inflammation.

Oxidative stress exaggerates skeletal muscle contraction-evoked reflex sympathoexcitation in rats with hypertension induced by angiotensin II

Muscle contraction stimulates thin fiber muscle afferents and evokes reflex sympathoexcitation. In hypertension, this reflex is exaggerated. ANG II, which is elevated in hypertension, has been reported to trigger the production of superoxide and other reactive oxygen species. In the present study, we tested the hypothesis that increased ANG II in hypertension exaggerates skeletal muscle contraction-evoked reflex sympathoexcitation by inducing oxidative stress in the muscle. In rats, subcutaneous infusion of ANG II at 450 ng·kg(-1)·min(-1) for 14 days significantly (P < 0.05) elevated blood pressure compared with sham-operated (sham) rats. Electrically induced 30-s hindlimb muscle contraction in decerebrate rats with hypertension evoked larger renal sympathoexcitatory and pressor responses [+1,173 ± 212 arbitrary units (AU) and +35 ± 5 mmHg, n = 10] compared with sham normotensive rats (+419 ± 103 AU and +13 ± 2 mmHg, n = 11). Tempol, a SOD mimetic, injected intra-arterially into the hindlimb circulation significantly reduced responses in hypertensive rats, whereas this compound had no effect on responses in sham rats. Tiron, another SOD mimetic, also significantly reduced reflex renal sympathetic and pressor responses in a subset of hypertensive rats (n = 10). Generation of muscle superoxide, as evaluated by dihydroethidium staining, was increased in hypertensive rats. RT-PCR and immunoblot experiments showed that mRNA and protein for gp91(phox), a NADPH oxidase subunit, in skeletal muscle tissue were upregulated in hypertensive rats. Taken together, hese results suggest that increased ANG II in hypertension induces oxidative stress in skeletal muscle, thereby exaggerating the muscle reflex.

Green odor inhalation by stressed rat dams reduces behavioral and neuroendocrine signs of prenatal stress in the offspring

Chronic maternal stress during pregnancy results in the prenatally stressed offspring displaying behavioral and neuroendocrine alterations that persist into adulthood. We investigated how inhalation of green odor (a mixture of equal amounts of trans-2-hexenal and cis-3-hexenol) by stressed dams might alter certain indices of prenatal stress in their offspring. These indices were depression-like behavior (increased immobility time in the forced-swim test) and acute restraint stress-induced changes in hypothalamo-pituitary-adrenocortical (HPA) axis activity [plasma corticosterone (CORT) and ACTH levels and the number of Fos-immunoreactive cells in the hypothalamic paraventricular nucleus (an index of neuronal activity)]. Pregnant rats were exposed to restraint stress for 60 min/day for 10 days (gestational days 10-19). The prenatally stressed offspring exhibited significant increases in depression-like behavior and in restraint stress-induced ACTH, CORT, and Fos responses, unless their dam had been exposed to green odor. The behavioral effect of the odor was also seen in offspring that were fostered by unstressed dams. The results obtained in the dams themselves were as follows. In vehicle-exposed stressed dams, but not in green odor-exposed ones, total body and adrenal weights were significantly decreased or increased, respectively. Depression-like behavior was not observed in the vehicle-exposed stressed dams themselves. Green odor inhalation prevented the impairment of maternal behavior induced by restraint stress. Thus, exposure of dams to stress may affect both the fetal brain and fetal HPA axis, and also maternal behavior, leading to altered behavioral and neuroendocrine responses in the offspring. Such effects may be prevented by the stressed dams inhaling green odor.

Green odor and depressive-like state in rats: Toward an evidence-based alternative medicine?

It is widely accepted that mental stress is an important factor in the development of psychological disorders such as depression. On pre-existing evidence, the so-called green odor may have a relieving and sedative effect on animals exposed to stressful situations. Using two behavioral models of depression, the forced-swim test and learned helplessness paradigm, we investigated whether inhalation of green odor (a 50:50 mixture of trans-2-hexenal and cis-3-hexenol) might alleviate and/or prevent experimentally induced depressive-like states in rats. A 3-min swim every day for 7 days resulted in significant prolongation of immobility time (vs. day 1). Inhaling green odor, but not vehicle, thereafter for 10 days (without swimming) led to the prolonged immobility time being significantly reduced and the hippocampal level of brain-derived neurotrophic factor (BDNF) being significantly increased. In the learned helplessness paradigm, the failure number and time spent in the shock compartment seen in the active avoidance test were both significantly attenuated in those rats that inhaled green odor for 11 days after the postshock screening test (vs. vehicle-exposed rats). Finally, for 10 consecutive days rats continuously exposed to green odor or vehicle swam for 3 min/day. Immobility time was significantly shorter in the green-odor group than in the vehicle-exposed group on days 6-10. These results suggest that green odor has not only a therapeutic, but also a preventive effect on depressive-like states in rats. These effects may be at least in part due to a green odor-induced upregulation of BDNF in the hippocampus.