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This study demonstrated that PDE1 (phosphodiesterase 1) existing in the ciliary beat frequency (CBF)-regulating metabolon regulates CBF in procaterol-stimulated lung airway ciliary cells of mouse. Procaterol (an β2-agonist) increased the ciliary bend angle (CBA) and CBF via cAMP accumulation in the ciliary cells of mice: interestingly, the time course of CBF increase was slower than that of CBA increase. However, IBMX (3-isobutyl-1-methylxanthine, an inhibitor of PDE) increased CBA and CBF in an identical time course. Lowering an intracellular Ca2+ concentration ([Ca2+]i) caused by switching to an EGTA-containing Ca2+-free solution from normal one elevated the procaterol-induced increasing rate of CBF. These observations suggest that Ca2+-dependent PDE1 controls cAMP-stimulated CBF increase. Either application of 8MmIBMX (8-methoxymethyl-IBMX, a selective PDE1 inhibitor), BAPTA-AM (an intracellular Ca2+ chelator), or calmidazolium (an inhibitior of calmodulin) alone increased CBA and CBF in the lung airway ciliary cells and increased cAMP contents in the isolated lung cells, and like IBMX, each application of the compound made the time courses of CBA and CBF increase stimulated by procaterol identical. The immunoelectron microscopic examinations revealed that PDE1A exists in the space between the nine doublet tubules ring and plasma membrane in the lung airway cilium, where the outer dynein arm (a molecular motor regulating CBF) functions. In conclusion, PDE1A is a key factor slowing the time course of the procaterol-induced increase in CBF via degradation of cAMP in the CBF-regulating metabolon of the mouse lung airway cilia.

A PPARα (peroxisome proliferation activation receptor α) agonist (GW7647) activates nitric oxide synthase 1 (NOS1) to produce NO leading to cGMP accumulation in antral mucous cells. In this study, we examined how PPARα activates NOS1. The NO production stimulated by GW7647 was suppressed by inhibitors of PI3K (wortmannin) and Akt (AKT 1/2 Kinase Inhibitor, AKT-inh), although it was also suppressed by the inhibitors of PPARα (GW6471) and NOS1 (N-PLA). GW7647 enhanced the ACh (acetylcholine)-stimulated exocytosis (Ca(2+)-regulated exocytosis) mediated via NO, which was abolished by GW6471, N-PLA, wortmannin, and AKT-inh. The Western blotting revealed that GW7647 phosphorylates NOS1 via phosphorylation of PI3K/Akt in antral mucous cells. The immunofluorescence examinations demonstrated that PPARα existing with NOS1 co-localizes with PI3K and Akt in the cytoplasm of antral mucous cells. ACh alone and AACOCF3, an analogue of arachidonic acid (AA), induced the NOS1 phosphorylation via PI3K/Akt to produce NO, which was inhibited by GW6471. Since AA is a natural ligand for PPARα, ACh stimulates PPARα probably via AA. In conclusion, PPARα activates NOS1 via PI3K/Akt phosphorylation to produce NO in antral mucous cells during ACh stimulation.

In antral mucous cells, acetylcholine (ACh, 1 μM) activates Ca2+-regulated exocytosis, consisting of a peak in exocytotic events that declines rapidly (initial phase) followed by a second slower decline (late phase) lasting during ACh stimulation. GW7647 [a peroxisome proliferation activation receptor α (PPARα) agonist] enhanced the ACh-stimulated initial phase, and GW6471 (a PPARα antagonist) abolished the GW7647-induced enhancement. However, GW6471 produced the delayed, but transient, increase in the ACh-stimulated late phase, and it also decreased the initial phase and produced the delayed increase in the late phase during stimulation with ACh alone. A similar delayed increase in the ACh-stimulated late phase is induced by an inhibitor of the PKG, Rp8BrPETcGMPS, suggesting that GW6471 inhibits cGMP accumulation. An inhibitor of nitric oxide synthase 1 (NOS1), N5-[imino(propylamino)methyl]-l-ornithine hydrochloride ( N-PLA), also abolished the GW7647-induced-enhancement of ACh-stimulated initial phase but produced the delayed increase in the late phase. However, in the presence of N-PLA, an NO donor or 8BrcGMP enhanced the ACh-stimulated initial phase and abolished the delayed increase in the late phase. Moreover, GW7647 and ACh stimulated NO production and cGMP accumulation in antral mucosae, which was inhibited by GW6471 or N-PLA. Western blotting and immunohistochemistry revealed that NOS1 and PPARα colocalize in antral mucous cells. In conclusion, during ACh stimulation, a PPARα autocrine mechanism, which accumulates NO via NOS1 leading to cGMP accumulation, modulates the Ca2+-regulated exocytosis in antral mucous cells.

In antral mucous cells, acetylcholine (ACh, 1 μM) activates Ca2+-regulated exocytosis, consisting of an initial peak that declines rapidly (initial transient phase) followed by a second slower decline (late phase) lasting during ACh stimulation. The addition of 8-bromo-cGMP (8-BrcGMP) enhanced the initial phase, which was inhibited by the protein kinase G (PKG) inhibitor guanosine 3′,5′-cyclic monophosphorothoiate, β-phenyl-1, N2-etheno-8-bromo, Rp-isomer, sodium salt (Rp-8-BrPETcGMPS, 100 nM). However, Rp-8-BrPETcGMPS produced a delayed, but transient, increase in the exocytotic frequency during the late phase that was abolished by a protein kinase A (PKA) inhibitor (PKI-amide), suggesting that Rp-8-BrPETcGMPS accumulates cAMP. The cGMP-dependent phosphodiesterase 2 (PDE2), which degrades cAMP, may exist in antral mucous cells. The PDE2 inhibitor BAY-60-7550 (250 nM) mimicked the effect of Rp-8-BrPETcGMPS on ACh-stimulated exocytosis. Measurement of the cGMP and cAMP contents in antral mucosae revealed that ACh stimulates the accumulation of cGMP and that BAY-60-7550 accumulates cAMP similarly to Rp-8-BrPETcGMPS during ACh stimulation. Analyses of Western blot and immunohistochemistry demonstrated that PDE2A exists in antral mucous cells. In conclusion, Rp-8-BrPETcGMPS accumulates cAMP by inhibiting PDE2 in ACh-stimulated antral mucous cells, leading to the delayed, but transient, increase in the frequency of Ca2+-regulated exocytosis. PDE2 may prevent antral mucous cells from excessive mucin secretion caused by the cAMP accumulation.

The purpose of this study was to investigate the minimum effective dose of recombinant canine interferon-gamma (rCaIFN-gamma) for the treatment of dogs with atopic dermatitis (AD). Thirty-four dogs with AD from 17 animal hospitals in Japan were administered half or one-fifth of the approved rCaIFN-gamma dose of 10 000 units/kg, three times a week for 4 weeks, followed by once weekly for an additional 4 weeks. Pruritus, excoriation, erythema and alopecia were evaluated and scored by the investigators on weeks 2, 4, 6, 8 and 12. The efficacy rate (number of excellent cases + number of good cases/total number of cases) at week 8 in the 2000 units/kg group was 36.4% for pruritus, 36.4% for excoriation, 45.5% for erythema and 36.4% for alopecia. In contrast, in the 5000 units/kg group, the efficacy rate was 64.3% for pruritus, 57.1% for excoriation, 78.6% for erythema and 78.6% for alopecia. The efficacy rate of the 5000 units/kg group was high for all signs evaluated and comparable to that of the 10 000 units/kg group reported in a previous study. The results of this study showed that 2000 units/kg of rCaIFN-gamma is less effective than 5000 units/kg to treat dogs with AD, and the efficacy of the 5000 units/kg dose is comparable to that of 10 000 units/kg at week 8.

The present study examined the expression pattern of FOS in the hypothalamic peptide neurons during the sleep-dominant state induced by an adenosine A2A receptor agonist. The control rats, those that received the microdialysis-perfusion of their ventral striatum with artificial cerebrospinal fluid in the dark-active phase, spent 24% of the 90-min period prior to sacrifice in non-rapid eye movement (non-REM) sleep and 2.3% of that in REM sleep. These rats exhibited FOS, a transcription factor, in 21% of their orexin neurons and in 1.0% of their melanin-concentrating hormone (MCH) neurons in the perifornical/lateral hypothalamic areas. However, the rats perfused with 50 microM CGS21680, an adenosine A2A receptor agonist, spent 60% of the 90-min period prior to sacrifice in non-REM sleep and 11% of that in REM sleep. These rats exhibited FOS in 1.7% of their orexin neurons and FOS in 0.5% of their MCH neurons. When the sleep-dominant state was disturbed by mild stimulation and the rats were kept in the sleepy state by treatment with a sleep-inducing dose of CGS21680, the rats exhibited FOS in 13.3% of their orexin neurons, which percentage was about half of that for the control rats. These results suggest that the sleep-promoting process induced by this adenosine A2A receptor agonist was associated with a decline in the activity of orexin neurons. MCH neurons are not likely to change their activities during this sleep-promoting process.

Unilateral microdialysis-perfusion of the preoptic area with 50 microM muscimol decreased the sleep period of rats to less than 3% of the baseline value over a 90 min period before death (p = 0.018 by Wilcoxon signed-rank test). These rats showed the expression of FOS in 36% of the orexin neurons located in the perifornical/lateral hypothalamic areas on the side ipsilateral to the perfusion site, but in only 3% of the orexin neurons on the side contralateral to it (p = 0.018 by Wilcoxon signed-rank test). These results suggest that subpopulations of the preoptic neurons give an inhibitory tone to the activities of the orexin neurons in the perifornical/lateral hypothalamic areas.

The present study examined whether the activities of the rostral basal forebrain neurons alter the activities of the orexin (also known as hypocretin) neurons in the tuberal part of the hypothalamus in rats. We performed microdialysis perfusion of the ventromedial portion of the rostral basal forebrain with the GABAA receptor agonist muscimol to inhibit focally the neuronal activities in the rostral basal forebrain. Then, we monitored sleep/wake behaviour and investigated the pattern of activities of orexin neurons by examining the expression of FOS as an indicator of cellular activation. Bilateral perfusion with muscimol (5, 15, and 50 micro m) produced a dose-dependent decrease in the amount of sleep. This perfusion with muscimol at 50 micro m produced FOS-like immunoreactivity in 37% of the orexin neurons located in the tuberal part of the hypothalamus, whereas the FOS-like immunoreactivity was sparse in orexin neurons of the sleeping control rats (P = 0.001 by Mann-Whitney U-test). Unilateral perfusion with muscimol (50 micro m) also suppressed sleep. In this case, FOS-like immunoreactivity was seen in 40% of the orexin neurons on the side ipsilateral to the perfusion site but only in 10% of orexin neurons on the contralateral side (P = 0.018 by Wilcoxon signed rank test). These functional data suggested that a sleep-generating element in the ventromedial part of the rostral basal forebrain provides an inhibitory influence on the activities of the orexin neurons in the tuberal part of the hypothalamus.

Hypocretins/orexins are neuropeptides implicated in sleep regulation and the sleep disorder narcolepsy. In order to examine how hypocretin activity fluctuates across 24 h with respect to the sleep-wake cycle, we measured changes in extracellular hypocretin-1 levels in the lateral hypothalamus and medial thalamus of freely moving rats with simultaneous sleep recordings. Hypocretin levels exhibited a robust diurnal fluctuation; levels slowly increased during the dark period (active phase), and decreased during the light period (rest phase). Levels were not correlated with the amount of wake or sleep in each period. Although an acute 4-h light-shift did not alter hypocretin levels, 6-h sleep deprivation significantly increased hypocretin release during the forced-wake period. Hypocretin activity is, thus, likely to build up during wakefulness and decline with the occurrence of sleep. These findings, together with the fact that a difficulty in maintaining wakefulness during the daytime is one of the primary symptoms of hypocretin-deficient narcolepsy, suggest that hypocretin activity may be critical in opposing sleep propensity during periods of prolonged wakefulness.

The present study has demonstrated that the sleep-promoting potency of 2-[p-(2-carboxyethyl)phenethylamino]-5′-N-ethylcarboxamido adenosine (CGS21680), a selective agonist for the adenosine A2A receptor, varies depending on the location of the administration. CGS21680 was continuously administered to rats through a chronically implanted cannula for 6 h during their active phase. The tip of the cannula was located in the subarachnoid space or the brain ventricle neighbouring the established brain areas implicated in the regulation of sleep–wake phenomena, i.e. rostral basal forebrain, medial preoptic area, lateral preoptic area, posterior hypothalamus, and dorsal tegmentum of the pons and medulla. At an infusion rate of 2.0 pmol/min, the magnitude of increase in non-rapid eye movement sleep varied from 14 min (a 15% increase) to 96 min (a 103% increase), and those of rapid eye movement sleep varied from 6 min (a 40% increase) to 28 min (a 264% increase) from the respective baseline values. The largest increases in both types of sleep occurred when CGS21680 was administered to the subarachnoid space underlying the rostral basal forebrain. These findings were interpreted to mean that the major, if not the only, site responsible for the CGS21680-inducing sleep was located in or near the rostral basal forebrain. This interpretation was supported by the findings that the administration of CGS21680 to the rostral basal forebrain produced predominant expression of Fos within the shell of the nucleus accumbens and the medial portion of the olfactory tubercle, and that the microdialysis perfusion of CGS21680 into the shell of the nucleus accumbens also exhibited a sleep-promoting effect.

We examined the sleep-modulatory effects of four adenosine agonists, namely, (1) 2-(4-(2-carboxyethyl)phenylethylamino)adenosine-5'-N-ethylcarbo xamideadenosine (CGS21680), a highly selective adenosine A2A receptor agonist; (2) 2-(4-(2-(2-aminoethylaminocarbonyl)ethyl)phenylethylamino)-5 '-N-ethylcarboxamidoadenosine (APEC), a selective adenosine A2A receptor agonist; (3) 5'-N-ethylcarboxamidoadenosine (NECA), a nonselective adenosine A1/A2 receptor agonist, and (4) N6-cyclopentyladenosine (CPA), a selective adenosine A1 receptor agonist. Each agonist was administered in the subarachnoid space underlying the rostral basal forebrain of rats through chronically implanted cannulae at the rate of 0.02, 0.2, 2.0, 12.0, or 20.0 pmol/min over a 6-h period starting from 2300 h, which period is the active phase of the animals. CGS21680, APEC, and NECA produced significant increases in the total amounts of non-rapid-eye-movement (NREM) sleep and rapid-eye-movement (REM) sleep after at least one dose within the range of administration rates. CPA did not produce any significant increase in the total amount of either type of sleep at any of the above administration rates, but instead suppressed REM sleep at the administration rates of 12.0 and 20.0 pmol/min. These results indicate that the activities of adenosine A2A receptors are crucially involved in the promotion of sleep.

Objective Antegrade central perfusion for acute Stanford type A aortic dissection prevents malperfusion and retrograde cerebral embolism during cardiopulmonary bypass. Prompt establishment of antegrade perfusion via the ascending aorta may improve surgical results of type A dissections, especially in situations of hemodynamic instability. Thus, we evaluated the safety and efficacy of cannulation of the dissected ascending aorta in acute type A dissection. Methods We reviewed the medical charts of patients undergoing repair of acute ascending aortic dissection (n = 52) from April 2010 to April 2013. Cannulation was accomplished in 29 patients via the ascending aorta (central) and in 23 patients via the femoral or axillary artery (peripheral). The ascending aorta was routinely cannulated using Seldinger technique under epiaortic ultrasound guidance. Comorbidities, mortality, complications, and durations of hospital stays were compared for the groups. Results In all cases, routine cannulation of the ascending aorta was safely performed with no resultant malperfusion or thromboembolism. Mean operative duration, cardiopulmonary bypass time, intubation time, and intensive care unit stay were significantly shorter in the central group. Two patients (6.8 %) in the central group died compared with four patients (17.3 %) in the peripheral group (P = 0.005). Conclusions Antegrade central perfusion via the ascending aorta, a simple and safe technique that enables rapid establishment of antegrade systemic perfusion, was as safe as peripheral cannulation in patients with type A acute aortic dissection.

Acetylcholine (ACh) exerts various anti-inflammatory effects through α7 nicotinic ACh receptors (nAChRs). We have previously shown that secreted lymphocyte antigen-6/urokinase-type plasminogen activator receptor-related peptide-1 (SLURP-1), a positive allosteric modulator of α7 nAChR signaling, is down-regulated both in an animal model of asthma and in human epithelial cells treated with an inflammatory cytokine related to asthma. Our aim of this study was to explore the effect of SLURP-1, signal through α7 nAChR, in the pathophysiology of airway inflammation. Cytokine production was examined using human epithelial cells. Ciliary beat frequency of murine trachea was measured using a high speed camera. The IL-6 and TNF-α production by human epithelial cells was augmented by siRNA of SLURP-1 and α7 nicotinic ACh receptor. The cytokine production was also dose-dependently suppressed by human recombinant SLURP-1 (rSLURP-1). The ciliary beat frequency and amplitude of murine epithelial cells were augmented by PNU282987, a selective α7 nAChR agonist. Those findings suggested that SLURP-1 and stimulus through α7 nicotinic ACh receptors actively controlled asthmatic condition by stimulating ciliary beating and also by suppressing airway inflammation.

Levtiracetam (Lev), an inhibitor of SV2A (synaptic vesicle protein A2), affected the ATP-dependent priming of Ca 2+ -regulated exocytosis in antral mucous cells of guinea pig. In antral mucous cells, the Ca 2+ -regulated exocytosis, which is activated by acetylcholine (ACh), consists of an initial peak that declines rapidly (initial phase) followed by a second slower decline (late phase). Dinitrophenol (DNP), which depletes ATP, inhibits the ATP-dependent priming. DNP abolished the initial phase by reducing the number of primed granules, Lev decreased the frequency of initial phase, but not in the presence of DNP. Moreover, 8-bromoguanosine 3′5′-cyclic monophosphate (8BrcGMP) accelerates the ATP-dependent priming. 8BrcGMP enhances the frequency of initial phase by increasing the number of primed granule. Lev added prior to 8BrcGMP addition decreased the frequency of initial phase, but Lev added after 8BrcGMP addition did not. Thus, Lev affected the granules in the process of priming, but it did not affect the granules already primed. Lev did not affect [Ca 2+ ] i in unstimulated or ACh-stimulated antral mucous cells. Immunohistochemistry and western blotting demonstrated that SV2A exists in antral mucous cells. The results suggest that SV2A plays an essential role in maintaining the process of ATP-dependent priming in antral mucous cells. In conclusion, Lev decreases the frequency of Ca 2+ -regulated exocytosis the number of primed granules by inhibiting SV2A functions, leading to a decrease in antral mucous cells.

Once diagnosed with diabetes mellitus, the risk of diseases, such as nephropathy, neuropathy, retinopathy and heart disease also increases. The complication of diabetes accompanying myocardial disorder is known as diabetic cardiomyopathy, which is characterized by ventricular dilation that is usually asymptomatic as diabetes progresses. Myocardial fibrosis is closely related to diastolic dysfunction. Thiamine (vitamin B1), an essential micronutrient, has been reported to attenuate diabetic complications and all diabetics may be lacking in thiamine. Thiamine is a coenzyme utilized at multiple steps of glucose metabolism. We believe that thiamine repletion under hyperglycemia might activate glucose oxidation and reduce the overflow of glucose to the hexosamine biosynthesis pathway of glucose metabolism with concomitant reduction of diabetic lesions. The aim of this article is to highlight the role of thiamine, an important factor that combats diabetic complications, especially diabetic cardiomyopathy and also elucidate its impact on O-glycosylated protein in diabetes. Finally, we discuss the ability of thiamine repletion to prevent metabolic syndrome and obesity, which are considered prediabetic states, as well as prediabetic cardiomyopathy.

A 6-hr continuous infusion of 2-[p-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenos ine (CGS21680), a selective A2a-adenosine agonist, into the subarachnoid space underlying the ventral surface region of the rostral basal forebrain, which has been defined as the prostaglandin (PG) D2-sensitive sleep-promoting zone, at rates of 0.02, 0.2, 2.0, and 12 pmol/min increased slow-wave sleep (SWS) and paradoxical sleep (PS) in a dose-dependent manner up to 183% and 202% of their respective baseline levels. The increments produced by the infusion of CGS21680 at 0.2 and 2.0 pmol/min were totally diminished when the rats had been pretreated with an i.p. injection of (E)-1,3-dipropyl-7-methyl-8-(3,4-dimethoxystyryl)xanthine (KF17837; 30 mg/kg of body weight), a selective A2-adenosine antagonist. In contrast, the infusion of N6-cyclohexyladenosine (CHA), a selective A1-adenosine agonist, at 2 pmol/min significantly suppressed SWS before causing an increase in SWS, and a decrease in PS was also markedly visible. Essentially the same effects of CGS21680 and CHA were observed when these compounds were administered to the parenchymal region of the rostral basal forebrain through chronically implanted microdialysis probes. Thus, we clearly showed that stimulation of A2a-adenosine receptors in the rostral basal forebrain promotes SWS and PS. Furthermore, i.p. injections of KF17837 at 30 and 100 mg/kg of body weight dose-dependently attenuated the magnitude of the SWS increase produced by the infusion of PGD2 into the subarachnoid space of the sleep-promoting zone, thus indicating that the A2a-adenosine receptors are crucial in the sleep-promoting process triggered by PGD2.

Thiamine (vitamin B1), which plays an important role in glucose metabolism, can prevent diabetic complications, including those in obesity. We previously found that thiamine intervention can impact metabolic abnormalities, such as progressive obesity and metabolic disorders similar to human metabolic syndrome, in polyphagia-induced Otsuka Long-Evans Tokushima Fatty (OLETF) rats. Thiamine intervention averted obesity, mainly resulting from reduction in visceral adiposity, and prevented metabolic disorders in OLETF rats. Seventy-six genes showed at least a two-fold difference in hepatic expression with thiamine treatment. Several of these genes participated in carbohydrate metabolism, lipid metabolism, vascular physiology and carcinogenesis. Thiamine has a potential to prevent obesity and metabolic disorders in OLETF rats. Although corroboration is necessary, the present findings indicate that thiamine may be beneficial in targeting composite physiological abnormalities, rather than individual component criteria, and can be used for preventive intervention. Given the detrimental effects of obesity and the safety and cost-effectiveness of thiamine, we believe that its use offers considerably more benefits than disadvantages.

The correlation between the somnogenic effect of prostaglandin (PG) D2 and the serotoninergic system was examined in freely-moving rats (n = 64) by use of a continuous infusion method. Rats pretreated with para-chlorophenylalanine (PCPA: 450 mg/kg body weight, i.p.) or non-PCPA-pretreated rats received infusion of PGD2, serotonin, or its direct precursor, 5-hydroxytryptophan (5HTP), into their third cerebral ventricle at a rate of 100 pmol/0.2 microliter/min between 11:00 and 17:00 h. In the PCPA-pretreated insomniac rats, PGD2 infusion resulted in an immediate increase in slow-wave sleep (SWS) and an increase with a 2-h latency in paradoxical sleep (PS). The total amounts of SWS and PS during the PGD2-infusion period were 151% and 154% of the respective control values. These results indicate that inhibition of the biosynthesis of serotonin and 5HTP by PCPA marginally affects the sleep-promoting effect of PGD2. The transient sleep restoration produced by 5HTP infusion into PCPA-pretreated rats was hardly affected by the simultaneous infusion (200 pmol/0.2 microliter/min; 07:00-17:00 h) of diclofenac sodium, an inhibitor of cyclo-oxygenase, suggesting that PGD2 production is not critically involved in the sleep restoration by 5HTP. The sleep-promoting property of PGD2 is thus probably independent of the serotoninergic modulation of sleep-wake activity.

Prostaglandin (PG) D2 and four lipoxygenase-derived eicosanoids [lipoxins (LX) A4 and B4, and leukotrienes (LT) C4 and D4] were examined for their effects on sleep and brain temperature in freely-behaving rats. In the first series of experiments, PGD2 was infused into the third ventricle at four different locations between 23:00 and 05:00. In a location apposed to the medial preoptic area (MPO), PGD2 at doses 1, 10 and 100 pmol/min, increased the slow wave sleep (SWS) by 23% (p < or = 0.01), 35% (p < or = 0.05) and 44% (p < or = 0.01), respectively, during the infusion period. In the second series of experiments, LXs and LTs were infused at the location apposed to MPO. Significant increases in SWS were detected with LXA4 at 100 pmol/min (14%, p < or = 0.05), LXB4 at 100 pmol/min (20%, p < or = 0.05), and LTD at 10 pmol/min (17%, p < or = 0.05). An increase in paradoxical sleep (PS) was produced by PGD2 at 1 and 10 pmol/min infusion (p < or = 0.05), but not by any of the lipoxygenase-derived eicosanoids examined. PGD2 also elevated the mean brain temperature during infusion by 0.2 degrees C and 0.9 degrees C at infusion doses 10 and 100 pmol/min, respectively. But PGD2 infusion at 1 pmol/min did not elevate the brain temperature. LXs (excluding LXB4 at 100 pmol/min) and LTs did not alter the brain temperature significantly at the tested doses. We conclude that PGD2 is the most effective sleep promoter among the eicosanoids examined so far.

There have been many reports that P-glycoprotein expression and activity are altered during sepsis, but few of them have examined such changes over 72 h. In this study, we examined the effect of lipopolysaccharide (LPS, 5mg/kg, ip) on P-glycoprotein expression (Western blotting) and activity (rhodamine-123 (Rho123) pharmacokinetics) in liver and kidneys for 7 days. On day 1 after LPS administration, hepatic P-glycoprotein expression and activity significantly decreased. On day 3, hepatic P-glycoprotein expression significantly increased compared with the control group, while activity had returned to the control level. On day 7, hepatic P-glycoprotein expression returned to the control level. There were no significant changes in P-glycoprotein expression or activity in the kidneys after LPS administration. The amount of Rho123 excretion in urine remained unchanged with (4.2%) or without (4.0%) LPS administration, but the amount of Rho123 excretion in bile decreased from 2.0 to 0.7% with LPS administration. Our findings suggested that hepatic P-glycoprotein expression and activity decreased on day 1 but recovered within 3 days, but there were no significant differences in the kidneys after LPS administration. These results suggested that the change in P-glycoprotein activity might be due to change in P-glycoprotein expression in the liver rather than the kidneys.

Prostaglandin (PG) E 2 reportedly augmented wakefulness when continuously infused into the third ventricle of the rat brain, whereas it promoted sleep when continuously infused into the subarachnoid space of the ventral surface zone of the rostral basal forebrain, which was designated previously as a PGD 2 -sensitive sleep-promoting zone (PGD 2 -SZ). In the present study, we investigated the effects of PGE (EP)-receptor agonists on sleep-wakefulness activities by infusing agonists into the third ventricle or into the subarachnoid space of the PGD 2 -SZ. Our results indicated that the waking effect is mediated by EP 1 and EP 2 receptors situating around the third ventricle, whereas the sleep-promoting effect is brought about mainly through activation of EP4 receptors located at or near the subarachnoid space of the PGD 2 -SZ.

Tumor necrosis factor-alpha (TNFalpha) was infused into the subarachnoid space of the rat rostral basal forebrain, which was previously defined as a prostaglandin (PG) D2-sensitive, sleep-promoting zone. TNFalpha increased the amount of slow-wave sleep (SWS), decreased that of paradoxical sleep (PS), and caused fever and anorexia. The TNFalpha-induced SWS enhancement, fever and anorexia were all blocked by co-infusion of diclofenac, a non-selective cyclooxygenase (COX) inhibitor, and by pretreatment with NS-398, a COX-2-specific inhibitor. In striking contrast, the TNFalpha-induced suppression of PS was not affected by the inhibitors. These results indicate that COX-2-mediated hyperproduction of PGs is critically involved in the enhancement of SWS, fever, and anorexia but not in the suppression of PS, caused by TNFalpha infused into the PGD2-sensitive zone.

To determine the site of action of the sleep-promoting effect of interleukin-1 (IL-1), we continuously infused (between 11 P.M. and 5 A.M.) murine recombinant IL-1β into seven different locations in the ventricular and subarachnoid systems of the brain in freely moving rats. When IL-1 was infused at 10 ng/6 hr into the subarachnoid space underlying the ventral surface of the rostral basal forebrain, which previously was defined as the “prostaglandin (PG) D(2)-sensitive sleep-promoting zone” (PGD(2)-SZ), the total amount of slow-wave sleep (SWS) increased by 110.7 min (IL-1 was 208.1 ± 14.3 min vs control at 97.4 ± 9.3 min;n = 8; p < 0.01 by paired Student’s t test) from the baseline control level obtained under continuous infusion of saline vehicle. The hourly SWS during the infusion period reached the level of daytime SWS, the physiological maximum, whereas paradoxical sleep (PS) was decreased transiently. This site of action for the SWS promotion was dissociated from the site in the third ventricle sensitive to the IL-1-mediated PS suppression, fever, and anorexia. The SWS increase caused by IL-1 infusion into the PGD(2)-SZ was blocked completely by coadministered diclofenac, a nonselective cyclooxygenase (COX) inhibitor. Pretreatment of rats with NS-398 or piroxicam (3 mg/kg of body weight, i.p.), which are said, respectively, to possess high and relative specificity for the COX-2 enzyme, also blocked the SWS-promoting effect of IL-1. We present a hypothesis that IL-1 induces SWS, at least in part, via COX-2-mediated PG production in the PGD(2)-SZ.

The concentration of PGD2, PGE2, and of PGF2 alpha was measured in the cerebrospinal fluid (CSF) collected from the cisterna magna of conscious rats (n = 29), which, chronically implanted with a catheter for the CSF sampling, underwent deprivation of daytime sleep. Significant elevation of the CSF level of PGD2 was observed following 2.5-h sleep deprivation (SD), and the elevation became more marked following 5- and 10-h SD, apparently reaching the maximum at 5-h SD (703 +/- 140 pg/ml (mean +/- S.E.M.) for baseline vs. 1734 +/- 363 pg/ml for SD, n = 10). The levels of PGE2, and PGF2 alpha also significantly increased following 5- and 10-h SD, but not following 2.5-h SD. It is unlikely that these changes were simply caused by some responses of the animals to stress stimuli, because stress stimuli derived from restraint of the animal at the supine position to a board for 1 h did not produce any acute responses in the CSF levels of prostaglandins (n = 13). In a different group of animals (n = 11) implanted with electrodes for recording electroencephalogram (EEG) and electromyogram (EMG) in addition to the catheter, the levels of the prostaglandins in CSF were determined for slow-wave sleep (SWS) and wakefulness in the day and for SWS and wakefulness in the night. The highest PGD2 value was obtained at daytime SWS, whereas the lowest was at night wakefulness; furthermore, a significant difference was observed between SWS and wakefulness rather than between day and night. The CSF level of PGE2 also showed a similar tendency. In an additional group of animals (n = 6), not only PGD2 but also PGE2 and PGF2 alpha significantly increased the sleeping time of the animal when applied into the subarachnoid space underlying the ventral surface area of the rostral basal forebrain, the previously defined site of action for the sleep-promoting effect of PGD2. The promotion of sleep by PGE2 applied to the subarachnoid space was an effect completely opposite to the well-established awaking effect of the same prostaglandin demonstrated in the hypothalamic region in a series of previous studies. Based on these results, we conclude that increases in CSF levels of prostaglandins, especially that of PGD2, are correlated in rats with heightened propensity towards sleep and further with the depth of sleep under normal as well as SD conditions.

Recent biochemical, molecular biological, and pharmacological experiments revealed that prostaglandin D synthase as well as prostaglandin D2 circulated in the ventricular system, subarachnoidal space, and extracellular space in the brain. Prostaglandin D2 then interacts with chemosensors or receptors on the ventro-medial surface of the rostral basal forebrain to initiate the signal to promote sleep. Prostaglandin D2 is, therefore, not a typical neurotransmitter but rather a ‘neurohormone’ or an ‘informational substance’ that circulates through the cerebrospinal fluid and transmits certain chemical messages to promote sleep. The mode of communication through the cerebrospinal fluid in the ventricular system and the extracellular space has advantages for global regulation of the brain to induce sleep.

Seasonal changes in the in vivo levels of the prostaglandins (PGs) PGD2, PGE2, and PGF2(alpha) were measured in the brain of the male Asian chipmunk, Tamias asiaticus (n = 111), which underwent hibernation during the period between November and March. The mean level of PGD2 ranged from 36.0 to 85.2 pg/g tissue from June to October and remained essentially unchanged (80.5 pg/g tissue) in December. However, the mean PGD2 level rose significantly to 128.6 pg/g tissue in February, and returned to 75.2 pg/g tissue in the following April, suggesting a correlation between PGD2 and hibernation phenomenon. While PGE2 level did not vary significantly throughout the year, PGF2(alpha), which appeared to be the most abundant among the three prostanoids, showed a marked circannual rhythm with a trough of 51.6 pg/g tissue in July, rising to 391.6 pg/g tissue in February and reaching the peak value of 492.7 pg/g tissue in April, the reproduction period.

Effects of noradrenaline (NA) on the activity of sleep-related neurons in the preoptic area (POA) and the neighboring basal forebrain were examined in the rat. Of 36 sleep-active neurons tested, 19 were inhibited and the other 17 were unaffected by NA applied through a multibarrel pipette. The alpha 2-agonist clonidine inhibited 11 of 14 sleep-active neurons and did not affect the other 3 neurons, whereas the alpha 1-agonist methoxamine (n = 13) and the beta-agonist isoproterenol (n = 11) had no effect on any of the sleep-active neurons tested. Thus, alpha 2-receptors mediated the NA-induced inhibition. Of 22 waking-active neurons tested, NA excited 10, inhibited 1, and had no effect on the remaining 11. Methoxamine excited 4 of 13 waking-active neurons tested, whereas isoproterenol (n = 9) and clonidine (n = 4) were without effect on any of the waking-active neurons tested. Accordingly, alpha 1-receptors probably mediated the NA-induced excitation. Seventy-seven state-indifferent neurons, which lacked activity related to the sleep-waking state, and 20 paradoxical sleep-active neurons were mostly (65%-70%) insensitive to NA. These results suggest that NA promotes wakefulness by inhibiting sleep-active neurons and by exciting waking-active neurons.

The concept of humoral regulation of sleep was initially proposed by a French neuroscientist, Henri Pieron of Paris, in the first decade of this century. He and his associate Legendre were the first to show the presence of a sleep-inducing substance in the cerebrospinal fluid (CSF) of sleep-deprived dogs. Concurrently and independently, Kuniomi Ishimori of Nagoya University, Nagoya, Japan, employing a similar experimental approach, also demonstrated a sleep-inducing substance in the CSF of sleep-deprived dogs. During the next 80 years or so, more than 30 so-called endogenous sleep substances have been reported to exist in the brain by numerous investigators, CSF, and other organs and tissues of mammals. However, their physiological relevance has remained uncertain in most instances. In this review, we shall focus upon our own work concerning the molecular mechanisms of sleep-wake regulation by prostaglandins (PGs) D2 and E2, with special emphasis on the recent developments during the last several years.

The level of hypocretin-1 (orexin A) is reported to be low in the cerebrospinal fluid (CSF) of subjects with narcolepsy. We measured the CSF level of hypocretin-1 of two young hypersomniacs. The first subject was a 9-year-old boy who exhibited cataplexy, and was demonstrated to be human leukocyte antigen-DR2 (HLA-DR2) positive. The second subject was a 16-year-old boy who exhibited no cataplexy and was HLA-DR2 negative. When excessive daytime sleepiness ensued, appetite and bodyweight were increased in case 1 but not in case 2. The level of CSF hypocretin-1 was undetectably low and within normal limits in case 1 and case 2, respectively. It appears to be important to measure the CSF level of hypocretin-1 in the diagnosis and treatment of narcolepsy.

The site of action for the sleep-promoting effect of prostaglandin (PG) D2 was extensively examined in the brain of adult male rats (n = 231). PGD2 was administered at 100 pmol/0.2 microliter per min for 6 hr (2300-0500 hr) through chronically implanted microdialysis probes or infusion cannulae. Among the administrations of PDG2 by dialysis probes (n = 176), only those (n = 8) to a ventro-rostral part of the basal forebrain by the probes implanted on the midline consistently increased slow-wave sleep (SWS), by 51 +/- 6 min (mean +/- SEM) above the baseline value (111 +/- 11 min). Since this area is separated by a cleft into right and left regions, the results were interpreted to mean that, through this cleft, PGD2 diffused in the subarachnoid space over the adjacent ventral surface, where it had the effect of promoting sleep. When PGD2 was directly infused into the subarachnoid space (n = 55), extraordinary increases exceeding 90 min were consistently attained for the SWS at sites located between 0.5 and 2 mm rostral to the bregma and between 0 and 1.2 mm lateral to the midline defined according to the stereotaxic coordinates adopted from the brain atlas of Paxinos and Watson [Paxinos, G. & Watson, C. (1986) The Rat Brain in Stereotaxic Coordinates (Academic, San Diego)]. Thus, we demarcated a "PGD2-sensitive, sleep-promoting zone" within this region in the ventral surface of the rostral basal forebrain. During the bilateral infusion of PGD2 into the subarachnoid space of this zone, the hourly mean SWS level of the nocturnal animals (n = 6) in the night reached the maximum at the second hour of the infusion period; this maximum hourly SWS level, corresponding to the daytime level of the same animals, lasted until the end of PGD2 infusion.

Responses of sleep-related neurons in the preoptic area (POA) to stimulation of the locus coeruleus (LC) and the ventrolateral medulla (VLM), components of the reticular activating system, were recorded in the unanesthetized, head-restrained rat. Single-pulse stimulation of the LC and the VLM, respectively, inhibited 50% and 54% of 30 sleep-active neurons and excited 47% and 67% of 34 waking-active neurons. The remaining neurons were mostly unaffected. Seventy-three neurons that were not related to a sleep-wake state were mostly (i.e., 73-80%) unresponsive to stimulation. The high incidence of responses by sleep-related neurons suggests that neural inputs from the LC and VLM regulate the hypnogenic mechanisms in the POA. Stimulation of the LC antidromically activated 15% of sleep-active neurons and 11% of waking-active neurons. Thus, some of the sleep-related neurons in the POA may regulate LC neurons. In a later stage of the experiment, we used isoflurane-anesthetized rats that had been used for recording sleep-related neurons. Antagonists for adrenoceptors at a concentration of 10 microM were applied to neurons through a multibarrel micropipette to examine the involvement of noradrenaline in the responses as a neurotransmitter. Application of the alpha 2-blocker, yohimbine, attenuated the inhibitory responses in all 7 neurons tested. The beta-blocker, timolol, and the alpha 1-blocker, prazosin, did not alter any of the inhibitory responses. On the other hand, timolol attenuated the excitatory responses in 4 of 7 neurons, and prazosin attenuated the excitatory responses in 5 of 12 neurons. Yohimbine did not affect the excitatory responses. Thus, the LC and the VLM probably inhibit sleep-active neurons through alpha 2-adrenoceptors and excite waking-active neurons through either beta- or alpha 1-adrenoceptors.

Prostaglandin (PG) D2 has been postulated to be an endogenous sleep-promoting factor. Biosynthesis of PGD2 is catalyzed by PGD synthase (prostaglandin-H2 D-isomerase, EC 5.3.99.2), the activity of which is inhibited by inorganic selenium compounds such as SeCl4 and Na2SeO3. We recently examined the effect of intracerebroventricular administration of these selenium compounds on sleep in rats, and demonstrated time- and dose-dependent sleep inhibition. To establish whether this effect of selenium is also produced when the compound is administered systemically, we devised a procedure for intravenous catheterization and examined the effect of these selenocompounds on sleep-wake activity in freely moving rats (n = 35). Each test compound was administered into the inferior vena cava continuously between 11.00 and 17.00 h on the experimental day. SeCl4 time- and dose-dependently inhibited sleep at infusion rates of 5, 7.5, 10 and 20 nmol/microliters per min. During the SeCl4 infusion at 20 nmol/microliters per min, slow-wave sleep and paradoxical sleep were reduced to 63% and 50% of their respective baseline values. Na2SeO3 exhibited a similar sleep inhibition, though Na2SO3 was ineffective. Infusion of SeCl4 at 10 nmol/microliters per min or below produced no consistent changes in the mean brain temperature, or food and water intake during the infusion period. During the nocturnal period subsequent to SeCl4 infusion, sleep was increased by a rebound phenomenon, while a decrease in brain temperature and inhibition of food and water intake dose-dependently occurred. We conclude that systemic administration of these PGD synthase inhibitors has a sleep-reducing potency.

Prostaglandin (PG) D2 has been postulated to be an endogenous sleep-promoting factor in rats, and SeCl4 and Na2SeO3 recently have been shown to inhibit the PGD synthase (prostaglandin-H2 D-isomerase, EC 5.3.99.2) activity of rat brain. The effect of these selenium compounds on sleep-wake activities was examined in freely moving rats along with their effects on brain temperature, food and water intake, and behavior. Test substances were administered for 6 hr into the third ventricle of rats, using a microdialysis technique. SeCl4, time- and dose-dependently, inhibited sleep at perfusion rates of 60 pmol/0.2 microliter per min and higher, and the inhibition was almost complete at rates greater than 200 pmol/0.2 microliter per min. The effect was reversible and was followed by a rebound. Na2SeO3 exhibited similar effects, but Na2SO3 did not show any effect on sleep. Simultaneous administration of dithiothreitol eliminated the sleep-inhibiting effects of these selenium compounds. These findings indicate that the decrease in sleep is due to inhibition of the PGD synthase activity in the brain by SeCl4 as well as Na2SeO3. During the inhibition of sleep, the rats in general showed an activation of behavior with moderate elevation of brain temperature and a detectable increase in food and water intake, suggesting that the sleep-inhibited state of the rats was similar to the physiological state of wakefulness and that the inhibitory effect was not due to the general toxicity of selenium.

We reported in previous studies that prostaglandin E2 (PGE2) has central effects of augmenting wakefulness and suppressing slow-wave sleep (SWS) and paradoxical sleep (PS) in rats. In the present study, we tested the effect of AH 6809, an antagonist of PGE2 receptors, on sleep-wake activities. AH 6809 in saline was infused continuously into the third ventricle of freely moving rats at a rate of 2.1, 6.3, and 21 pmol/min from 2300 to 0500 hr. During the infusion at 21 pmol/min, wakefulness decreased to 82%, and SWS and PS increased to 122% and 161%, of the respective baseline values. These changes can be explained by AH 6809 antagonizing the endogenous PGE2 that acts to augment wakefulness in the brain. This explanation is supported by the fact that the infusion of AH 6809 at 21 pmol/min inhibited the wakefulness-promoting effect of PGE2 infused at 10 pmol/min. Moreover, the PGE2-related mechanisms for regulating sleep-wake activities may be different from those producing hyperthermia, because AH 6809 at 21 pmol/min had no primary effect on brain temperature and did not antagonize the hyperthermia produced by the PGE2 infusion. A diurnal infusion (1200 to 1800 hr) of AH 6809 at 21 pmol/min produced similar effects on sleep-wake activities compared with the nocturnal infusion (2300 to 0500 hr), although the PS increase was not significant, suggesting that the PGE2-related mechanisms are acting all day long with or without a circadian rhythm. These findings strongly suggest that endogenous PGE2 in the brain is involved in the physiological mechanisms for regulating sleep-wake activities.