[Practices and challenges of joint education at two medical schools utilizing online pharmacology role-playing].

The pharmacology role-play, in which students impersonate medical personnel and patients to explain illness and drug treatment, is one of the active learning of pharmacology. However, until now, it has been carried out only within one facility, and has not been carried out between different multi-facility facilities with a larger scale. However, the spread of COVID-19 infection in 2020 was a turning point that drastically changed the way of medical school education centered on traditional face-to-face lectures. Above all, remote real-time lessons using Zoom etc. have the advantage that about 300 students can be conducted at multiple facilities without having to gather them in one place at the same time. With the Korona-ka as a strange currency, the infrastructure has been set up to carry out joint education in pharmacological role-playing between different multi-institutions. We are the first in Japan to conduct a pharmacology role-play jointly by Fujita Medical University and Aichi Medical University, so we would like to introduce the contents.

Isolation of the Anti-Inflammatory Agent Myceliostatin from a Methionine-Enriched Culture of Myceliophthora thermophila ATCC 42464.

Safe and effective nonsteroidal anti-inflammatory drugs are needed. Meanwhile, addition of amino acids to cultures of microorganisms is likely to increase the possibility of novel secondary metabolite isolation. In the course of screening for anti-inflammatory agents using cellular lipopolysaccharide (LPS)-induced nitric oxide (NO) production, two new related compounds with the myceliothermophin structure from a methionine-enriched culture of Myceliophthora thermophila ATCC 42464 were isolated. The new compounds have an additional methylthio group on the myceliothermophin structure and were named myceliostatins A and B. Both compounds inhibited LPS-induced NO production at nontoxic concentrations in macrophage-like mouse monocytic leukemia RAW264.7 cells. Myceliostatin B inhibited the expression of LPS-induced iNOS, IL-6, and IL-1ß and the upstream NF-κB activity in situ and in vitro. Finally, it was found to inhibit NF-κB binding to DNA in the reconstruction system with purified p65. Myceliostatin B also inhibited LPS-induced reactive oxygen species (ROS) production. Thus, myceliostatin B, a novel compound derived from M. thermophila, was found to be a new anti-inflammatory and antioxidant compound directly inhibiting NF-κB.

Glucose infusion suppresses acute restraint stress-induced peripheral and central sympathetic responses in rats.

BACKGROUND: 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. METHODS: 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. RESULTS: 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 B2 (TxB2) (a metabolite of TxA2) levels in the PVN but did not alter prostaglandin E2 levels in the PVN. CONCLUSION: Our 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 TxA2 in the PVN.

Acute intracerebroventricular injection of chemerin-9 increases systemic blood pressure through activating sympathetic nerves via CMKLR1 in brain.

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.

Thromboxane A2 in the paraventricular hypothalamic nucleus mediates glucoprivation-induced adrenomedullary outflow.

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 ß-adrenergic receptor activation and prostanoids production in the PVN to elevate plasma catecholamine levels. We examined the effects of α- and ß-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 ß-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 ß-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.

Clostridium butyricum Modulates the Microbiome to Protect Intestinal Barrier Function in Mice with Antibiotic-Induced Dysbiosis.

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.

Prostaglandin E2 receptor EP3 subtype in the paraventricular hypothalamic nucleus mediates corticotropin-releasing factor-induced elevation of plasma noradrenaline levels in rats.

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.

GABAB receptors in the hypothalamic paraventricular nucleus mediate ß-adrenoceptor-induced elevations of plasma noradrenaline in rats.

In the brain, various neurotransmitters such as noradrenaline and GABA regulate peripheral sympathetic functions. Previously, it has been reported that both ß-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 ß-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 ß-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 ß-adrenoceptor antagonist propranolol did not alter the GABAB receptor agonist baclofen-induced elevation of plasma noradrenaline levels. We next examined the central effects of ß-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 ß-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.

Isolation of ketomycin from Actinomycetes as an inhibitor of 2D and 3D cancer cell invasion.

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-ß. 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.

Inhibition of MMP-2-mediated cellular invasion by NF-κB inhibitor DHMEQ in 3D culture of breast carcinoma MDA-MB-231 cells: A model for early phase of metastasis.

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.

Possible role of adrenoceptors in the hypothalamic paraventricular nucleus in corticotropin-releasing factor-induced sympatho-adrenomedullary outflow in rats.

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 ß-adrenoceptors, respectively, in the rat PVN.

Rational design, synthesis and in vitro evaluation of novel exo-methylene butyrolactone salicyloylamide as NF-κB inhibitor.

(-)-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)-ß-salicyloylamino-α-exo-methylene-Æ´-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.

Effects of aging on stress-related responses of serotonergic neurons in the dorsal raphe nucleus of male rats.

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 ß 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 ß on TPH expression in stressed old males differ from those in young males.

Propofol reduces liver dysfunction caused by tumor necrosis factor-α production in Kupffer cells.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Inhibition of IGF-1-Mediated Cellular Migration and Invasion by Migracin A in Ovarian Clear Cell Carcinoma Cells.

Previously we isolated migracin A from a Streptomyces culture filtrate as an inhibitor of cancer cell migration. In the present research, we found that migracin A inhibited migration and invasion of ovarian clear cell carcinoma ES-2 cells. In the course of our mechanistic study, migracin A was shown to enhance vasohibin-1 expression in an angiogenesis array. We also confirmed that it increased the mRNA expression of this protein. Moreover, overexpression of vasohibin-1 lowered the migration but not the invasion of ES-2 cells. Then, we looked for another target protein employing a motility array, and found that migracin A lowered the IGF-1 expression. Knockdown of IGF-1 by siRNA decreased the migration and invasion of ES-2 cells. Migracin A also decreased Akt phosphorylation involved in the downstream signaling. Crosstalk analysis indicated that overexpression of vasohibin-1 decreased the IGF-1 expression. On the other hand, it showed no direct anticancer activity in terms of the ES-2 growth in agar. Migracin A inhibited the migration and IGF-1 expression in not only ES-2 but also another ovarian clear cell carcinoma JHOC-5 cells. In addition, it also inhibited capillary tube formation of human umbilical vein endothelial cells. Since its cytotoxicity is very low, migracin A may be a candidate for an anti-metastasis agent not exhibiting prominent toxicity.

Centrally administered isoproterenol induces sympathetic outflow via brain prostaglandin E2-mediated mechanisms in rats.

Brain ß-adrenoceptor stimulation can induce elevations of plasma levels of noradrenaline. However, there have been no detailed studies related to signaling pathways downstream of ß-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 ß-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.

Changes in hypothalamic neurotransmitter and prostanoid levels in response to NMDA, CRF, and GLP-1 stimulation.

Determination of neuroactive substances, such as neurotransmitters and prostanoids, in the extracellular space of the living brain is a very important technique in neuroscience. The hypothalamic paraventricular nucleus (PVN) is one of the most important autonomic control centers in the brain. Recently, we demonstrated that thromboxane (Tx) A2 in the PVN may play an important role in adrenomedullary outflow evoked by N-methyl-D-aspartate (NMDA), corticotrophin-releasing factor (CRF), and glucagon-like peptide-1 (GLP-1) stimulation using microdialysis sampling and liquid chromatography-ion trap tandem mass spectrometry (LC-ITMS(n)). In the present study, we investigated whether centrally administered NMDA, CRF, and GLP-1 can release five neurotransmitters, acetylcholine (ACh), histamine, glutamate (Glu), γ-aminobutyric acid (GABA), and serotonin (5-HT), along with six prostanoids, TxB2, prostaglandin (PG) E2, PGD2, 15-deoxy-∆(12,14) (15d)-PGJ2, 6-keto-PGF1α, and PGF2α in rat PVN microdialysates after optimization of LC-ITMS(n) conditions . All stimulations increased the levels of 5-HT, TxB2, PGE2, and PGF2α (except for 5-HT stimulated with GLP-1). Only NMDA increased the levels of ACh, Glu, and GABA. Treatment with dizocilpine maleate (MK-801), a noncompetitive NMDA receptor antagonist, attenuated the NMDA-induced increase in the levels of neuroactive substances except for Glu. This is the first study to use LC-ITMS(n) to analyze neurotransmitters and prostanoids in the same microdialysates from rat PVN.

UGT1A1*28 is associated with greater decrease in serum K⁺ levels following oral intake of procaterol.

BACKGROUND AND OBJECTIVE: Procaterol is a potent ß2-agonist frequently used for the management of asthma and chronic obstructive pulmonary disease. The efficacy and adverse effects of ß2-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. METHODS: 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 ß2-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. RESULTS: 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. CONCLUSION: The present study indicates that significant hypokalemia may occur in carriers of UGT1A1*28 by systemic administration of procaterol and potentially by other ß2-agonists metabolized in the liver.

Enhanced expression of WD repeat-containing protein 35 via CaMKK/AMPK activation in bupivacaine-treated Neuro2a cells.

We previously reported that bupivacaine induces reactive oxygen species (ROS) generation, p38 mitogen-activated protein kinase (MAPK) activation and nuclear factor-kappa B activation, resulting in an increase in expression of WD repeat-containing protein 35 (WDR35) in mouse neuroblastoma Neuro2a cells. However, the identity of signaling upstream of p38 MAPK pathways to WDR35 expression remains unclear. It has been shown that AMP-activated protein kinase (AMPK) can activate p38 MAPK through diverse mechanisms. In addition, several kinases acting upstream of AMPK have been identified including Ca2+/calmodulin-dependent protein kinase kinase (CaMKK). Recent studies reported that AMPK may be involved in bupivacaine-induced cytotoxicity in Schwann cells and in human neuroblastoma SH-SY5Y cells. The present study was undertaken to test whether CaMKK and AMPK are involved in bupivacaine-induced WDR35 expression in Neuro2a cells. Our results showed that bupivacaine induced activation of AMPK and p38 MAPK in Neuro2a cells. The AMPK inhibitors, compound C and iodotubercidin, attenuated the bupivacaine-induced activation of AMPK and p38 MAPK, resulting in an inhibition of the bupivacaine-induced increase in WDR35 expression. Treatment with the CaMKK inhibitor STO-609 also attenuated the bupivacaine-induced activation of AMPK and p38 MAPK, resulting in an inhibition of the bupivacaine-induced increase in WDR35 expression. These results suggest that bupivacaine activates AMPK and p38 MAPK via CaMKK in Neuro2a cells, and that the CaMKK/AMPK/p38 MAPK pathway is involved in regulating WDR35 expression.