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Objectives To provide patient-centred care, psychological knowledge, and skills are necessary for pharmaceutical communication. Acquisition of these communication skills is closely related to patient comprehension. Therefore, to improve pharmacist’s communication skills, pharmacist need to learn the characteristics of their medication instructions, such as posture, facial expressions, eye contact, nodding, and more. For the analysis of medical communication, there is a rating scale, functional analysis, and others. However, these methods may not match the actual emotions due to their analysis skills and the psychological stress of the patients. In this study, we examined the methods to evaluate patient-pharmacist communication using emotion recognition AI software, which recognises emotions from facial expressions. Methods With the cooperation of six simulated patients (SP) and eight pharmacists, we recorded the SP’s facial expressions during medication instruction. The facial expression video was analysed using emotion recognition AI, which can obtain emotion values (anger, contempt, disgust, fear, joy, sadness, surprise, and engagement). We compared the emotion of the extracted peaks with the feedback and calculated the emotion match rate. Key findings As a result, 33% of the emotions matched in the peak and feedback. This result indicates that emotion recognition AI cannot capture every feedback emotion. However, in joy, the result was not affected by engagement, and the match rate between peak and feedback was high. Conclusions In the future, emotion recognition AI will allow us to assess the effects of communication skills of the pharmacists on the psychological state of the patients more objectively and noninvasively.

Chemerin is an adipocytokine involved in inflammation and lipid metabolism via G protein-coupled receptor, chemokine-like receptor (CMKLR)1. Since the important nuclei regulating pressure (BP) exist in the brain, we examined the effects of acute intracerebroventricular (i.c.v.) injection of chemerin-9 on systemic BP and explored underlying mechanisms. We examined the effects of acute i.c.v. injection of chemerin-9 (10 nmol/head) on systemic BP by a carotid cannulation method in the control or CMKLR1 small interfering (si) RNA-treated Wistar rats (0.04 nmol, 3 days, i.c.v.). We examined protein expression of CMKLR1 around brain ventricles by Western blotting. We examined the effects of acute i.c.v. injection of chemerin-9 on serum adrenaline by a high performance liquid chromatography. In the control siRNA-treated rats, chemerin-9 significantly increased mean BP, which reached a peak at 2 to 4 min after injection. On the other hand, in the CMKLR1 siRNA-treated rats, chemerin-9 did not affect the mean BP. Protein expression of CMKLR1 specifically in subfornical organ (SFO) and paraventricular nucleus (PVN) from the CMKLR1 siRNA-treated rats decreased compared with the control siRNA-treated rats. In the control siRNA-treated rats, chemerin-9 increased serum adrenaline level. On the other hand, in the CMKLR1 siRNA-treated rats, chemerin-9 did not affect the serum adrenaline level. Further, pretreatment with prazosin, an α-adrenaline receptor blocker, significantly prevented the pressor responses induced by chemerin-9. In summary, we for the first time demonstrated that chemerin-9 stimulates the sympathetic nerves via CMKLR1 perhaps expressed in SFO and PVN, which leads to an increase in systemic BP.

Acute restraint stress (RS) induces sympathetic activation such as elevating plasma catecholamines, resulting in increase in blood glucose. We aimed to investigate whether glucose infusion affects the RS-induced sympathetic responses.Plasma catecholamines were measured by high-performance liquid chromatography with electrochemical detection. Blood glucose levels were measured with a glucometer and a glucose assay kit. Cardiac parameters were measured by echocardiographic and hemodynamic analysis. Prostanoid levels in the paraventricular nucleus of hypothalamus (PVN) microdialysates were measured by liquid chromatography-ion trap tandem mass spectrometry analysis.RS significantly increased plasma noradrenaline and adrenaline. Intravenous infusion of a 5% glucose solution significantly attenuated the RS-induced elevation of plasma adrenaline but did not alter the plasma noradrenaline. Glucose administration during RS suppressed the progression of cardiac impairment by attenuating the decline rates in left ventricular diastolic, end-diastolic volume, stroke volume, fractional shortening, and ejection fraction. Both Intravenous and intracerebroventricular infusion of glucose solution significantly attenuated the RS-induced elevation of thromboxane BOur results demonstrate that glucose infusion suppresses RS-induced elevation of plasma adrenaline and left ventricular dysfunction. In the brain, glucose infusion suppresses RS-induced production of TxA

In the brain, various neurotransmitters such as noradrenaline and GABA regulate peripheral sympathetic functions. Previously, it has been reported that both β-adrenoceptor activation and GABAB receptor activation in the brain are involved in the elevation of plasma noradrenaline levels. However, it is unknown whether these pathways interact with each other. In the present study, we examined the relationship between the central actions of β-adrenoceptor activation and GABAB receptor activation with regard to plasma noradrenaline responses using urethane-anesthetized rats. Intracerebroventricular pretreatment with the GABAA receptor antagonist bicuculline did not affect the β-adrenoceptor agonist isoproterenol-induced elevation of plasma noradrenaline levels. In contrast, pretreatment with the GABAB receptor antagonist CGP 35348 suppressed the isoproterenol-induced elevation of noradrenaline levels. Intracerebroventricular pretreatment with the β-adrenoceptor antagonist propranolol did not alter the GABAB receptor agonist baclofen-induced elevation of plasma noradrenaline levels. We next examined the central effects of β-adrenoceptor activation on GABA release in the paraventricular hypothalamic nucleus (PVN), the major integrative center for sympathetic regulation in the brain. Intracerebroventricular administration of isoproterenol increased GABA content in PVN dialysates. In addition, baclofen microinjected unilaterally into the PVN resulted in elevated plasma levels of noradrenaline, but not adrenaline. Finally, unilateral blockade of GABAB receptors in the PVN suppressed the isoproterenol-induced elevation of plasma noradrenaline level. Our results suggest that activation of β-adrenoceptors in the brain, likely in the PVN, induces GABA release in the PVN, which in turn activates GABAB receptors in the PVN, leading to elevated plasma noradrenaline.

Inhibitors of cancer cell migration and invasion should be useful to inhibit metastasis. Then, we have screened microbial culture filtrates for the inhibitors of cancer cell migration. As a result, we isolated an antibiotic ketomycin from a culture filtrate of Actinomycetes SF2912 as an inhibitor of cancer cell migration. It is a known antibiotic, but its biological activity on mammalian cells has not been reported. Ketomycin inhibited cellular migration and invasion in human breast carcinoma MDA-MB-231 and MCF-7 cells at the non-toxic concentrations. Ketomycin decreased the expressions of MMP-9 and MMP-11 in MDA-MB-231 cells. Knockdown of each gene by siRNA inhibited the cellular migration and invasion. Ketomycin was then found to inhibit the cellular NF-κB activity that may be involved in the upstream signaling. For the mechanism of NF-κB inhibition, ketomycin inhibited autophosphorylation of IKK-α/IKK-β. Ketomycin also inhibited the 3D-invasion of MDA-MB-231 cells at the non-toxic concentrations. Thus, ketomycin having a comparatively simple structure may become a seed of anti-metastasis agent.

Summary Clostridium butyricum MIYAIRI 588 (CBM 588) is a probiotic bacterium that has previously been used to prevent antibiotic-associated diarrhea. However, the underlying mechanism by which CBM 588 protects the gut epithelial barrier remains unclear. Here, we show that CBM 588 increased the abundance of Bifidobacterium, Lactobacillus, and Lactococcus species in the gut microbiome and also enhanced the intestinal barrier function of mice with antibiotic-induced dysbiosis. Additionally, CBM 588 significantly promoted the expansion of IL-17A-producing γδT cells and IL-17A-producing CD4 cells in the colonic lamina propria (cLP), which was closely associated with changes in the intestinal microbial composition. Additionally, CBM 588 plays an important role in controlling antibiotic-induced gut inflammation through upregulation of anti-inflammatory lipid metabolites such as palmitoleic acid, 15d-prostaglandin J2, and protectin D1. This study reveals a previously unrecognized mechanism of CBM 588 and provides new insights into gut epithelial barrier protection with probiotics under conditions of antibiotic-induced dysbiosis., Graphical Abstract, Highlights • CBM 588 increases the abundance of Bifidobacterium, Lactobacillus, and Lactococcus • Microbiota-driven TGF-β1 controls the differentiation of lymphocytes to γδT cells • CBM 588 promotes the expansion of IL-17A-producing γδT cells and CD4 cells • CBM 588 upregulates anti-inflammatory lipid metabolites, Microbiome; Clinical Microbiology

The three-dimensional (3D) culture of cancer cells provides an environmental condition closely related to the condition in vivo. It would especially be an ideal model for the early phase of metastasis, including the detachment and invasion of cancer cells from the primary tumor. In one hand, dehydroxymethylepoxyquinomicin (DHMEQ), an NF-κB inhibitor, is known to inhibit cancer progression and late phase metastasis in animal experiments. In the present research, we studied the inhibitory activity on the 3D invasion of breast carcinoma cells. Breast carcinoma MDA-MB-231 cells showed the most active invasion from spheroid among the cell lines tested. DHMEQ inhibited the 3D invasion of cells at the 3D-nontoxic concentrations. The PCR array analysis using RNA isolated from the 3D on-top cultured cells indicated that matrix metalloproteinase (MMP)-2 expression is lowered by DHMEQ. Knockdown of MMP-2 and an MMP inhibitor, GM6001, both inhibited the invasion. DHMEQ was shown to inhibit the promoter activity of MMP-2 in the reporter assay. Thus, DHMEQ was shown to inhibit NF-κB/MMP-2-dependent cellular invasion in 3D-cultured MDA-MB-231 cells, suggesting that DHMEQ would inhibit the early phase of metastasis.

Glucoprivation stimulates a rapid sympathetic response to release and/or secrete catecholamines into the bloodstream. However, the central regulatory mechanisms involving adrenoceptors and prostanoids production in the paraventricular hypothalamic nucleus (PVN) that are responsible for the glucoprivation-induced elevation of plasma catecholamines are still unresolved. In this study, we aimed to clarify whether glucoprivation-induced activation of noradrenergic neurons projecting to the PVN can induce α- and/or β-adrenergic receptor activation and prostanoids production in the PVN to elevate plasma catecholamine levels. We examined the effects of α- and β-adrenergic receptor antagonists, a cyclooxygenase inhibitor, a thromboxane A synthase inhibitor, and a PGE

Corticotropin-releasing factor (CRF) plays an important role in sympathetic regulation. Centrally administered CRF elevates plasma catecholamine levels, resulting in CRF-dependent hypertension and tachycardia. We previously reported that brain thromboxane A

Responses to various stressors in the brain change with age. However, little is known about the neural mechanisms underlying age-dependent changes in stress responses. It is known that serotonin, a stress-related transmitter, is closely related with the regulation of stress responses in the brain and that serotonergic function is modulated by various factors, including estrogen, in both sexes. In the present study, to elucidate the effects of aging on stress responses in serotonergic neurons, we examined the expression levels of tryptophan hydroxylase (TPH; a marker of serotonergic neurons) in the dorsal, ventral and lateral parts of the dorsal raphe nucleus (DRN) in young and old intact male rats. In young males, repeated restraint stress significantly increased the number of TPH-positive cells in all subdivisions of the DRN. In contrast, the stress-induced increase in TPH expression was only observed in the ventral part of the DRN in old males. Pretreatment with an estrogen receptor β antagonist had no effect on the number of TPH-positive cells in the dorsal and lateral DRN in young stressed males, whereas the antagonist decreased the number of TPH-positive cells in all DRN subdivisions in old stressed males. Our results suggest that the effects of repeated stress exposure on the expression of TPH in serotonergic neurons in the DRN change with age and that estrogenic effects via estrogen receptor β on TPH expression in stressed old males differ from those in young males., Highlights • We examined the effect of aging on stress-induced TPH expression in male rats. • The effect of stress exposure on TPH expression in the DRN changed with age. • The effect of ER-β blockade on stress-induced TPH expression changed with age. • Stress experience in young adulthood changed serotonergic response in old age.

The present study, conducted in rats, investigated whether propofol attenuates lipopolysaccharide (LPS)-triggered liver dysfunction via regulation of tumor necrosis factor (TNF)-α production in activated Kupffer cells. Rats received LPS (500 μg/kg) under Urethane™ sedation (1 g/kg) in combination with propofol (5 mg/kg/h) or Intralipid™ from 1 h before to 6 h after LPS administration. Some rats were treated with 10 mg/kg gadolinium chloride (GdCl3) to induce Kupffer cell depletion. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), TNF-α mRNA and protein expression, caspase-3 activation and apoptosis were evaluated in hepatocytes. Immunofluorescence staining revealed expression of the pan-macrophage marker CD68 as well as TNF-α in Kupffer cells. ALT and AST serum levels increased approximately four-fold in LPS-exposed rats compared with Intralipid™-treated rats at 6 h after LPS administration, whereas propofol and GdCl3 reduced the LPS-induced increases. LPS simultaneously augmented TNF-α expression in Kupffer cells, followed by increased caspase-3 activity and apoptosis in hepatocytes. Immunofluorescence staining and immunoblotting assay showed that TNF-α expression in Kupffer cells was inhibited by propofol and GdCl3, resulting in a reduction of caspase-3 activity and apoptosis in LPS-treated rat hepatocytes. Propofol (5 mg/kg/h) attenuated LPS-triggered liver dysfunction via inhibition of TNF-α production in activated Kupffer cells. These results suggest that propofol is capable of inhibiting inflammation-induced liver dysfunction in vivo.

Glucoprivation stimulates a rapid sympathetic response to release and/or secrete catecholamines into the bloodstream. However, the central regulatory mechanisms involving adrenoceptors and prostanoids production in the paraventricular hypothalamic nucleus (PVN) that are responsible for the glucoprivation-induced elevation of plasma catecholamines are still unresolved. In this study, we aimed to clarify whether glucoprivation-induced activation of noradrenergic neurons projecting to the PVN can induce α- and/or β-adrenergic receptor activation and prostanoids production in the PVN to elevate plasma catecholamine levels. We examined the effects of α- and β-adrenergic receptor antagonists, a cyclooxygenase inhibitor, a thromboxane A synthase inhibitor, and a PGE2 subtype EP3 receptor antagonist on intravenously administered 2-deoxy-D-glucose (2-DG)-induced elevation of noradrenaline in the PVN and plasma levels of catecholamine in freely moving rats. In addition, we examined whether intravenously administered 2-DG can increase prostanoids levels in the PVN microdialysates. Intracerebroventricular (i.c.v.) pretreatment with phentolamine (a non-selective α-adrenergic receptor antagonist) suppressed the 2-DG-induced increase in the plasma level of adrenaline, whereas i.c.v. pretreatment with propranolol (a non-selective β-adrenergic receptor antagonist) suppressed the 2-DG-induced elevation of the plasma level of noradrenaline. I.c.v. pretreatment with indomethacin (a cyclooxygenase inhibitor) and furegrelate (a thromboxane synthase inhibitor) attenuated the 2-DG-induced elevations of both noradrenaline and adrenaline levels. Furthermore, 2-DG administration elevated the thromboxane B2 level, a metabolite of thromboxane A2 in PVN microdialysates. Our results suggest that glucoprivation-induced activation of α- and β-adrenergic receptor in the brain including the PVN and then thromboxane A2 production in the PVN, which are essential for the 2-DG-induced elevations of both plasma adrenaline and noradrenaline levels.

In the brain, various neurotransmitters such as noradrenaline and GABA regulate peripheral sympathetic functions. Previously, it has been reported that both β-adrenoceptor activation and GABA

Brain β-adrenoceptor stimulation can induce elevations of plasma levels of noradrenaline. However, there have been no detailed studies related to signaling pathways downstream of β-adrenoceptors responsible for central sympathetic outflow. In the present study, we pharmacologically examined the possibility that centrally administered isoproterenol can induce elevations of plasma noradrenaline levels in a brain prostaglandin-dependent manner. In addition, we also examined whether or not intracerebroventricular administration of isoproterenol could release endogenously synthesized prostaglandin (PG) E2 in the hypothalamic paraventricular nucleus (PVN) by using the brain microdialysis technique combined with liquid chromatography-ion trap tandem mass spectrometry (LC-ITMS(n)). Under urethane anesthesia, a femoral venous line was inserted for infusion of saline and a femoral arterial line was inserted for collecting blood samples. Next, animals were placed in a stereotaxic apparatus for application of test agents. Catecholamines in the plasma were extracted by alumina absorption and were assayed by high-performance liquid chromatography with electrochemical detection. Quantification of PGE2 in rat PVN microdialysates was performed by the LC-ITMS(n) method. We demonstrated that centrally administered isoproterenol-induced elevations of plasma noradrenaline could be mediated via activation of β-adrenoceptors and the downstream phospholipase A2-cyclooxygenase pathway. Furthermore, PGE2 in the PVN and the PGE2 receptor EP3 subtype appear to play an important role in the process. Our results suggest that central isoproterenol-induced sympathetic outflow is mediated via brain PGE2 in a PGE2 receptor EP3 subtype-dependent manner.

Procaterol is a potent β2-agonist frequently used for the management of asthma and chronic obstructive pulmonary disease. The efficacy and adverse effects of β2-agonists are heterogeneous in individual patients, which may be partly caused by genetic variations in metabolizing enzymes and receptor molecules. The present study was designed to analyze the relationship between gene polymorphisms and physiological effects of procaterol in healthy subjects.Ninety-two non-smoking healthy volunteers were given 1 µg/kg body weight (max 50 µg) of procaterol as a dry syrup preparation, and the serum concentrations of procaterol, serum K(+), and the physical responses were monitored for 240 min. We genotyped β2-adrenergic receptor (ADRB2) (Arg16Gly and Gln27Glu), cytochrome P450 3A4 (rs2246709, rs4646437), and uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1) (rs4148323 [allele A, *6], rs12479045, rs4148328, rs4663971, rs12052787, rs4148329, A (TA)6/7 TAA [seven-repeat allele, *28]). Procaterol concentrations in serum were measured by liquid chromatography-tandem mass spectrometry.No gene polymorphisms affected serum procaterol concentrations. Meanwhile, overall serum K(+) level changes were significantly lower in carriers of UGT1A1*28 than in non-carriers after correcting for strong effects of serum procaterol concentrations and baseline K(+) levels. No other polymorphisms were associated with serum K(+) levels. None of polymorphisms of ADRB2 were associated with any physical responses.The present study indicates that significant hypokalemia may occur in carriers of UGT1A1*28 by systemic administration of procaterol and potentially by other β2-agonists metabolized in the liver.

The hypothalamic paraventricular nucleus (PVN) is one of the most important autonomic control centers in the brain. Several kinds of prostanoids, such as prostaglandin (PG) E2, are considered to act in the PVN as mediators of autonomic responses. In the present study, we used liquid chromatography ion trap tandem mass spectrometry (LC-ITMS(n)) to simultaneously quantify four prostanoids, thromboxane (Tx) B2, PGE2, PGD2 and 15-deoxy-∆(12,14) (15d)-PGJ2 in PVN microdialysates from urethane-anesthetized rats. The quantification limits were estimated to be 0.05ng/mL for TxB2, 0.025ng/mL for PGE2, 0.1ng/mL for PGD2, and 0.5ng/mL for 15d-PGJ2. The RSD% obtained from all prostanoids was15%, indicating an acceptable level of reproducibility. LC-ITMS(n) analysis of rat PVN microdialysates revealed that TxA2 may play an important role in adrenomedullary outflow evoked by centrally administered N-methyl-d-aspartate, corticotrophin-releasing factor and glucagon-like peptide-1. This is the first study to use LC-ITMS(n) to analyze prostanoid levels in rat PVN microdialysates. This LC-ITMS(n) method will be useful for investigating the potential involvement of prostanoids in brain function.

Corticotropin-releasing factor (CRF) plays an important role in sympathetic regulation. Centrally administered CRF elevates plasma catecholamine levels, resulting in CRF-dependent hypertension and tachycardia. We previously reported that brain thromboxane A2 mediates CRF-induced elevation of plasma adrenaline levels, whereas prostanoids other than thromboxane A2 mediate elevations in plasma noradrenaline levels. However, the mechanism by which CRF induces elevations in plasma noradrenaline levels remains unknown. Previous studies have revealed that brain prostaglandin (PG) E2, but not other PGs, causes sympathetic activation. In this study, we examined the roles of brain PGE2 and its receptors in CRF-induced elevation of plasma noradrenaline levels in rats. Our results showed that intracerebroventricular pretreatment with an antagonist of the PGE2 receptor EP3 subtype, but not other subtypes, suppressed CRF-induced elevations in plasma noradrenaline levels. We also examined the role of PGE2 and EP3 receptors in the paraventricular hypothalamic nucleus (PVN), the major integrative center for sympathetic regulation, in CRF-induced elevation of plasma noradrenaline levels. Centrally administered CRF increased PGE2 levels in PVN microdialysates, and microinjection of an EP3 receptor agonist into the PVN elevated plasma noradrenaline levels. Bilateral blockade of EP3 receptors in the PVN suppressed the elevation of plasma noradrenaline levels evoked by intracerebroventricular administration and PVN-microinjection of CRF. Our results suggest that CRF stimulates PGE2 release into the PVN that activates EP3 receptors in the PVN, resulting in the elevation of plasma noradrenaline levels.

Background Oestrogen is known to play a cardioprotective role in cardiovascular diseases, as demonstrated in a number of animal studies. However, few human studies have investigated sex-based differences with regard to cardiac glucose uptake using 18 F-fluorodeoxyglucose-positron emission tomography/computed tomography (FDG–PET/CT). Methods Therefore, we evaluated healthy male and female subjects who underwent FDG–PET/CT examination to determine whether there was a sex-related difference in cardiac glucose uptake with age. Results In females, the prevalence of maximal FDG uptake (PET score 2) demonstrated a convex pattern with ageing, and it peaked at age 51–60years in the females, gradually decreasing to a minimum at age >70years. In contrast, the prevalence of maximal FDG uptake by age in males was a mirror image of that in females, i.e. it formed a concave pattern with a nadir at 61–70years, followed by an increase in the prevalence. These findings suggest that female hearts depend more on glucose as an energy substrate as they age, however, efficient glucose uptake is attenuated with increasing age. In contrast, the male heart sustains its glucose uptake capacity at age >70years. Conclusion This characteristic sex-based difference in cardiac glucose uptake might be related to the female predominance of Takotsubo cardiomyopathy.

(-)-Dehydroxymethylepoxyquinomicin ((-)-DHMEQ, 1) is a specific inhibitor of NF-κB. It binds to SH group in the specific cysteine residue of NF-κB components with its epoxide moiety to inhibit DNA binding. In the present research, we have designed and synthesized an epoxide-free analog called (S)-β-salicyloylamino-α-exo-methylene-ƴ-butyrolactone (SEMBL, 3). SEMBL inhibited DNA binding of NF-κB component p65 in vitro. It inhibited LPS-induced NF-κB activation, iNOS expression, and inflammatory cytokine secretions. It also inhibited NF-κB and cellular invasion in ovarian carcinoma ES-2 cells. Moreover, its stability in aqueous solution was greatly enhanced compared with (-)-DHMEQ. Thus, SEMBL has a potential to be a candidate for a new anti-inflammatory and anticancer agent.

Aims A functional interaction between the corticotropin-releasing factor (CRF) system and noradrenergic neurons in the brain has been suggested. In the present study, we investigated the interrelationship between the central CRF-induced elevation of plasma catecholamines and adrenoceptor activation in the paraventricular nucleus of the hypothalamus (PVN) using urethane-anesthetized rats. Main methods In rats under urethane anesthesia, a femoral venous line was inserted for infusion of saline, and a femoral arterial line was inserted for collecting blood samples. Next, animals were placed in a stereotaxic apparatus for the application of test agents. Catecholamines in the plasma were extracted by alumina absorption and were assayed with high-performance liquid chromatography with electrochemical detection. Quantification of noradrenaline in rat PVN microdialysates was performed with high-performance liquid chromatography with electrochemical detection. Key findings We showed that centrally administered CRF elevated noradrenaline release in the PVN. Furthermore, we demonstrated that microinjection of phenylephrine into the PVN induced elevation of plasma levels of adrenaline, but not of noradrenaline, whereas microinjection of isoproterenol into the PVN induced elevation of plasma levels of noradrenaline, but not of adrenaline. Bilateral blockade of adrenoceptors in the PVN revealed that phentolamine significantly suppressed the CRF-induced elevation of plasma adrenaline level, while propranolol significantly CRF-induced elevation of plasma noradrenaline level. Significance Our results suggest that centrally administered CRF-induced elevation of plasma levels of adrenaline and noradrenaline can be mediated via activation of α-adrenoceptors and β-adrenoceptors, respectively, in the rat PVN.

Aim: Lipopolysaccharide (LPS) causes apoptosis of hepatocytes, which is probably mediated by inflammatory substances released from Kupffer cells (KCs). Recently, we have reported that naofen, a newly found intracellular WD40-repeat protein, has a role in inducing the apoptosis in HEK293 cells. Hence, the present study was undertaken to investigate a role of naofen in the LPS-induced apoptosis of rat hepatocytes. Methods: Rats were treated with i.v. injections of LPS, and livers were extirpated to evaluate expression of naofen and apoptosis. In in vitro experiments, hepatocytes and KCs were separately isolated from rat livers. The incubation medium for KCs treated with LPS (KC-CM) was used for hepatocyte culture. Results: Intravenous injections of LPS enhanced the expression of naofen in the livers. Livers showed terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive staining, and elevated caspase-3 activity. In isolated KCs or hepatocytes, LPS hardly affected naofen expression and caspase-3 activity, whereas incubation of hepatocytes with KC-CM enhanced both naofen expression and caspase-3 activation. Transfection of hepatocyte with naofen siRNA prevented such effects of KC-CM, and clearly eliminated KC-CM-induced reduction of Bcl-2 and Bcl-xL. In contrast, overexpression of naofen in hepatocytes downregulated Bcl-2 and Bcl-xL, released cytochrome c from mitochondria, and activated caspase-3. Conclusion: These results indicate that LPS may induce the hepatic apoptosis in association with enhanced naofen expression, and that naofen may mediate the activation of caspase-3 through downregulating the Bcl-2 and Bcl-xL expression, and releasing cytochrome c from mitochondria to cytoplasm.

The reaction of some physiologically active peptides with bacterial strain B-9 has been investigated. Bradykinin, β-endorphin, and [Leu(5)]enkephalin were quickly degraded, with half-lives of5 min. Somatostatin, substance P, and angiotensin I were degraded relatively smoothly, with half-lives of 10 min to 1 h, whereas oxytocin and insulin were slowly degraded, with half-lives of 1 and 4 days, respectively. Vasopressin was barely degraded, with a half-life of7 days. Linearized vasopressin, prepared by the reductive cleavage of the disulfide bond followed by alkylation with iodoacetamide, was degraded significantly faster than intact vasopressin, with a half-life of 2.5 h. A loop formed by disulfide bond formation was regarded as one of the degradation-resistant factors. Hydrolysis of the peptides in this study took place through cleavage of various peptide bonds, and the strain B-9 may bear similarities to the neutral endopeptidase in terms of its broad selectivity.

Stress is one of the important factors to activate the sympathetic nervous system. We recently reported that central administration of corticotropin-releasing factor (CRF), known as a stress-related neuropeptide, increases the expression of both cyclooxygenase (COX) and nitric oxide synthase (NOS) in presympathetic neurons in the paraventricular hypothalamic nucleus (PVN). In the present study, therefore, we investigated whether brain COX and NOS can also mediate restraint stress (RS)-induced sympathetic activation by assessing the plasma catecholamine levels and neuronal activation of presympathetic neurons in the PVN. In addition, we examined effects of RS on the expression of both COX and NOS isozymes in the presympathetic PVN neurons. Intraperitoneal administration of an inhibitor for COX-1, COX-2 or inducible NOS (iNOS), but not for neuronal NOS (nNOS), reduced RS-induced elevation of plasma catecholamine levels and Fos expression in the presympathetic PVN neurons. Moreover, RS increased the expression of COX-1, COX-2 and iNOS in the presympathetic PVN neurons, whereas nNOS expression did not change. These results suggest that COX-1, COX-2 and iNOS in the presympathetic PVN neurons mediate acute RS-induced sympathetic activation.

N-methyl-D-aspartate (NMDA) has been implicated in the regulation of several autonomic responses in the brain. The present study determined whether activation of alpha1-adrenoceptors is involved in the centrally administered NMDA-induced adrenomedullary catecholamine outflow, using urethane-anesthetized rats. The NMDA (5.0 nmol/animal, i.c.v.)-induced elevation of plasma levels of noradrenaline and adrenaline was reduced by phentolamine (0.33 micromol/animal, i.c.v.), a non-selective alpha-adrenoceptor antagonist, and by 2-{[b-(4-hydroxyphenyl)ethyl]aminomethyl}-1 tetralone (HEAT) (90.0 nmol/animal, i.c.v.), a selective alpha1-adrenoceptor antagonist. In contrast, sotalol (0.8 micromol/animal, i.c.v.), a non-selective beta-adrenoceptor antagonist, did not alter the responses. In addition, U-73122, a phospholipase C inhibitor (5.0 nmol/animal, i.c.v.), RHC-80267, a diacylglycerol lipase inhibitor (1.3 micromol/animal, i.c.v.) and URB 602, a monoacylglycerol lipase inhibitor (0.85 and 1.7 micromol/animal, i.c.v.), reduced the NMDA-induced plasma elevation of both catecholamines. Furthermore, perfusion of the hypothalamic paraventricular nucleus with NMDA (0.3 and 1.0 mM) dose-dependently elevated both noradrenaline levels in the hypothalamic paraventricular nucleus and plasma catecholamine levels. These responses were abolished by co-administration of dizocilpine malate (MK-801, 0.1 mM), a selective non-competitive antagonist of the NMDA receptor and by co-administration of (+)-S-145 (2.5 mM), a selective competitive antagonist of the thromboxane A2 receptor. These results suggest that activation of central alpha1-adrenoceptors is involved in the centrally administered NMDA-induced activation of adrenomedullary catecholamine outflow in rats. Furthermore, signaling cascades downstream of the alpha1-adrenoceptor in the hypothalamic paraventricular nucleus may play an important role in the process.

The sympathetic nervous system regulates peripheral organs via the adrenal chromaffin cells containing adrenaline (A-cells) or noradrenaline (NA-cells) and the sympathetic ganglia. We examined the effect of intracerebroventricularly administered bombesin on neuronal activities of adrenal A-cells and NA-cells and several kinds of sympathetic ganglia (superior cervical, stellate and celiac ganglia) using c-Fos (a marker for neuronal activation), with regard to brain prostanoid, in anesthetized rats. Bombesin induced c-Fos in both adrenal A-cells and NA-cells, but not in any of the sympathetic ganglia. Central pretreatment with either indomethacin (a cyclooxygenase inhibitor) or furegrelate (a thromboxane A 2 synthase inhibitor) abolished all bombesin-induced responses. These results suggest that bombesin centrally activates adrenal A-cells and NA-cells by brain thromboxane A 2 -mediated mechanisms in rats.

The sympathetic efferent pathway projects to the sympathetic ganglia and the adrenal medulla. In this study, we examined centrally administered corticotropin-releasing factor (CRF)-induced neuronal activation of noradrenergic postganglionic neurons in several kinds of the sympathetic ganglia (superior cervical, stellate and celiac ganglia) in anesthetized rats. CRF significantly increased c-Fos expression in the celiac and stellate ganglia, with more pronounced effect on the celiac ganglion. On the other hand, CRF had no effect on c-Fos expression in the superior cervical ganglion even at a higher dose. These results suggest that brain CRF selectively regulates neuronal activity of each sympathetic ganglion.

The present study was undertaken to clarify the central mechanisms involved in the intracerebroventricularly administered corticotropin-releasing factor-induced elevation of plasma corticosterone in urethane- and alpha-chloralose-anesthetized rats using microdialysis and immunohistochemical techniques. When corticotropin-releasing factor was given at 0.5, 1.5, and 3.0 nmol/animal intracerebroventricularly, it dose-dependently increased noradrenaline release but not adrenaline release in the hypothalamic paraventricular nucleus. The 1.5 nmol/animal dose of corticotropin-releasing factor-induced noradrenaline release was attenuated by CP-154,526 (butyl-ethyl-{2,5-dimethyl-7-(2,4,6 trimethylphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl}amine), a selective corticotropin-releasing factor receptor 1 antagonist, at 1.3 micromol/animal, intracerebroventricularly, and was also abolished by phentolamine at 0.66 micromol/animal, intracerebroventricularly. In addition, the corticotropin-releasing factor-induced elevation of noradrenaline release in the hypothalamic paraventricular nucleus and plasma corticosterone were abolished by hexamethonium, a non-selective nicotinic acetylcholine receptor antagonist, at 1.8 micromol/animal, intracerebroventricularly, and alpha-conotoxin MII, a potent alpha(3)beta(2) nicotinic acetylcholine receptor antagonist, at 30 nmol/animal, i.c.v. Corticotropin-releasing factor at 1.5 nmol/animal, i.c.v. evoked a significant expression of Fos, an immediate-early transcription factor in neurons, on the dopamine-beta-hydroxylase-containing neurons and alpha(3) nicotinic acetylcholine receptor subunit-expressing neurons in the locus coeruleus, but not in the medullary A(1) and A(2) regions containing noradrenergic neurons. These results suggest that centrally administered corticotrophin-releasing factor elevates plasma corticosterone by the corticotropin-releasing factor 1 receptor and alpha(3) subunit-containing nicotinic acetylcholine receptor (probably alpha(3)beta(2) nicotinic acetylcholine receptor) mediated activation of the locus coeruleus noradrenergic neurons projecting to the paraventricular nucleus in rats.