Galanthamine and other Amaryllidaceae related alkaloids are inhibitors of α7, α4β2 and α3β4 nicotinic acetylcholine receptors

Abstract Galanthamine is an Amaryllidaceae-derived acetylcholinesterase inhibitor used to treat memory impairment in Alzheimer's disease and vascular dementia. There is evidence that galanthamine, in addition to its effects on acetylcholinesterase, may enhance or inhibit brain nicotinic acetylcholine receptors, which could increase or decrease the therapeutic efficacy of galanthamine, respectively. Here, we evaluated the effects of galanthamine and two others Amaryllidaceae acetylcholinesterase inhibitors (haemanthamine and tazettine) analyzed by gas chromatography-mass spectrometry and identified by comparing their mass fragmentation patterns with literature and database NIST vs.2.0 on the agonist responses of brain nicotinic acetylcholine receptors α7, α3β4, (α4)2(β2)3 and (α4)3(β2)2. Using nicotinic acetylcholine receptors expressed heterologously in Xenopus oocytes, in conjunction with two-electrode voltage clamping, we found that galanthamine inhibits the function of nicotinic acetylcholine receptors assayed through a mix competitive and non-competitevely. Nicotinic acetylcholine receptor α7 were significantly more sensitive to inhibition (17 ± 0.6 µM) than the heteromeric receptor, α3β4 (90 ± 3.4 µM). Neither haemanthamine nor tazettine were more potent than galanthamine.

Microvascular anatomy of olfactory and accessory olfactory (vomeronasal) organs in adult Xenopus laevis: scanning electron microscopy of vascular corrosion casts / Anatomía microvascular de órganos olfatorios y accesorios (vomeronasal) en adultos Xenopus laevis: microscopía electrónica de barrido de moldes de corrosión vascular

Microvascular anatomy and histomorphology of olfactory and vomeronasal organs in adult Xenopus laevis Daudin were studied by scanning electron microscopy of vascular corrosion casts and paraplast embedded stained serial tissue sections. Results show that the arterial supply is bilaterally by terminal arterioles of the medial branch of the nasal artery and by the palatal artery. Arterioles give rise to a capillary meshwork characteristic for respiratory surfaces in principal chambers and in dorsal and caudal areas of middle chambers. Anterior and inferior areas of the middle chambers own a distinctly different capillary network with conspicuous short capillary loops. Loops have a dilated tip and extend in acute angles towards the chamber lumen. The vomeronasal organ (VNO) locates beneath the olfactory organ. It has a medial to lateral extension and attaches with its caudal circumference to the medial nasal glands. Its capillary bed displays rectangular meshes which preferentially orientate along the long axis of the VNO. Locally, capillaries form short hairpin-like or strongly twisted loops with dilated tips which point towards the lumen of the VNO. These capillaries slow-down blood velocity and may lead to an increased exchange of oxygen, nutrients and water-borne odorants in the middle chambers and of pheromones in the VNO. In the latter vascular structures are present which might serve as a vascular pump.

Se estudiaron la anatomía microvascular e histomorfología de los órganos olfatorios y vomeronasales de Xenopus laevis Daudin adultos, mediante microscopía electrónica de barrido de moldes de corrosión vascular y secciones de tejido seriadas, teñidas e incluídas en paraplast. Los resultados muestran que el suministro arterial es bilateral por arteriolas terminales de la rama medial de la arteria nasal y por la arteria palatina. Las arteriolas dan lugar a un lecho capilar característico de las superficies respiratorias en las cámaras principales y en las áreas dorsal y caudal de las cámaras intermedias. Las áreas anterior e inferior de las cámaras centrales poseen una red capilar significativamente diferente con llamativos bucles capilares cortos. Los bucles tienen una punta dilatada y se extienden en ángulos agudos hacia la luz de la cámara. El órgano vomeronasal (VNO) se ubica debajo del órgano olfatorio. Se extiende de medial a lateral y se une con su circunferencia caudal a las glándulas nasales mediales. El lecho capilar muestra mallas rectangulares que se orientan preferentemente a lo largo del eje longitudinal del VNO. Localmente, los capilares forman bucles cortos en forma de horquilla o fuertemente retorcidos con puntas dilatadas que apuntan hacia la luz del VNO. Estos capilares ralentizan la velocidad de la sangre y pueden conducir a un mayor intercambio de oxígeno, nutrientes y odorizantes, a base de agua en las cámaras intermedias y de feromonas, en el VNO. En este último, están presentes estructuras vasculares que podrían servir como una bomba vascular.

The Effects of 4-Hydroxybenzoic Acid Identified from Bamboo (Dendrocalamus asper) Shoots on Kv1.4 Channel

Background: Bamboo shoot has been used as a treatment for epilepsy in traditional Chinese medicine for generations to treat neuronal disorders such as convulsive, dizziness and headaches. 4-hydroxybenzoic acid (4-hba) is a non-flavonoid phenol found abundantly in Dendrocalamus asper shoots (bamboo), fruits (strawberries and apples) and flowers. Kv1.4 is a rapidly inactivating Shaker-related member of the voltage-gated potassium channels with two inactivation mechanisms; the fast N-type and slow C-type. It plays vital roles in repolarisation, hyperpolarisation and signaling the restoration of resting membrane potential through the regulation of the movement of K+ across the cellular membrane. Methods: Chemical compounds from Dendrocalamus asper bamboo shoots were purified and identified as major palmitic acids mixed with other minor fatty acids, palmitic acid, 4-hydroxybenzaldehyde, lauric acid, 4-hydroxybenzoic acid and cholest-4-ene-3-one. The response of synthetic 4-hydroxybenzoic acid was tested on Kv1.4 potassium channel which was injected into viable oocytes that was extracted from Xenopus laevis. The current were detected by the two-microelectrode voltage clamp, holding potential starting from −80 mV with 20 mV stepup until +80 mV. Readings of treatments with 0.1% DMSO, 4-hba concentrations and K channel blockers were taken at +60 mV. The ratio of tail/peak amplitude is the index of the activity of the Kv1.4 channels with n ≥ 6 (number of oocytes tested). The decreases of the ratios of five different concentrations (1 μM, 10 μM, 100 μM, 1 mM and 2.5 mM) were compared with 0.1% DMSO as the control. Results: All concentration showed statistically significant results with P < 0.05 except for 100 μM. The normalised current of the 4-hba concentrations were compared with potassium channel blockers (TEA and 4-AP) and all groups showed statistically significant results. This study also showed that time taken for each concentration to affect Kv1.4 does not play any significant roles. Conclusion: 4-hydroxybenzoic acid was found to be able to enhance the inactivation of Kv1.4 by lowering the membrane potential so that the abnormal neuronal firing can be inhibited. With IC50 slightly higher than 10 μM, increasing concentrations (100 μM, 1 mM and 2.5 mM) had shown to exhibit toxicity effects. The best concentration from this study is 10 μM with Hill slope of 0.1799.

The Effect of 4-hydroxybenzaldehyde on the γ-aminobutyric Acid Type A Receptor

in the mammalian cortex and hippocampus. It is expressed heterologously in the Xenopus laevis oocyte as a α1β2γ2S/L subtype for application as an in vitro model for the screening of compounds that modulate receptor activities. In fact, 4-hydroxybenzaldehyde (4-HB) has been identified as one of the major components in Dendrocalamus asper bamboo shoots in our previous study, and the current study showed that at 101.7 μM, 4-HB significantly reduced the GABA-induced chloride current of GABAA receptors expressed on Xenopus oocytes, indicating a possible GABAergic antagonistic effect at high concentrations.

Antagonistic Activity of α-Conotoxin TxIB Isomers on Rat and Human α6 /α3β2β3 Nicotinic Acetylcholine Receptors / 中国药学杂志

OBJECTIVE: To investigate antagonistic activities of three isomers of α-conotoxin TxIB on rat and human α6 /α3β2β3 nicotinic acetylcholine receptors (nAChRs). METHODS: Three disulfide bond isomers were synthesized using Fmoc chemistry, which were identified by ultra performance liquid chromatography (UPLC)and confirmed by MALDI-TOF mass spectrometry. Rat and human α6/α3β2β3 nAChRs were expressed in oocytes of Xenopus laevis, which were used to test the antagonistic abilities of the 3 isomers. RESULTS: The three isomers of α-conotoxin TxIB were synthesized successfully. The retention time of each isomer of α-conotoxin TxIB was different each other significantly. The observed molecular masses of three isomers were the same, which were consistent with their theoretical molecular mass. Their hydrophilicity orders were globular > ribbon > bead. Both rat and human α6/α3β2β3 nAChRs were expressed in oocytes well. Inhibition of three isomers of α-conotoxin TxIB on rat and human α6 /α3β2β3 nAChRs were evaluated respectively. Among the three isomers of TxIB, the activity of the globular isomer was the most potent one, which had almost same activity at rat and human α6/α3β2β3 nAChRs with corresponding IC50 of 28.2 and 32.0 nmol·L-1 respectively. However, the other two isomers, ribbon and bead isomers displayed little antagonistic effect on both rat and human α6/α3β2β3 nAChRs only with an IC50 of > 10 μmol·L-1. CONCLUSION: The synthesized globular isomer of α-conotoxin TxIB in this work has a high selectivity and potent antagonistic activity on rat and human α6/α3β2β3 nAChRs, which would be helpful for its new drug development.

Effect of root saponins of Panax notoginseng on different voltage-dependent calcium and potassium ion channels / 中国药理学与毒理学杂志

OBJECTIVE To investigate the effect of root saponins of Panax notoginseng(RPNS) on different voltage-dependent calcium and potassium ion channels. METHODS By using the two-elec?trode voltage clamp (TEVC), the effect of RPNS 0.01, 0.06, 0.1, 0.6, 1 and 4 g · L- 1 was investigated on Cav1.2,and the effect of RPNS 1 g · L-1 was evaluated on Cav2.1,Cav2.2,Cav3.1, KCNH2,KCNQ1,KCNQ1/KCNE1 and BK channel. All the ion channels examined were expressed in Xenopus laevis oocytes. RESULTS TEVC suggested that the effect of RPNS on Cav1.2 exhibited the concentration-response relationship and its EC50 was 0.048 g · L-1. Compared with cell control,TEVC also showed that RPNS 1 g·L-1 had obviously inhibitory effect on Cav1.2,Cav2.2 and Cav3.1,and the inhibitory rate of RPNS 1 g · L-1 on the peak current of Cav1.2,Cav2.2 and Cav3.1 was(57.1 ± 8.6)%, (17.2 ± 0.7)% and(50.2 ± 7.7)%(P<0.01),respectively. RPNS 1 g · L-1 had obviously activated effect on BK channel,and the activated rate of RPNS 1 g·L-1 on the peak current of BK channel was(37.9± 2.7)%(P<0.01). RPNS 1 g·L-1 showed no significant effect on Cav2.1,KCNH2,KCNQ1 and KCNQ1/KCNE1. CONCLUSION RPNS may effectively inhibit Cav1.2 and Cav3.1,activate BK channel,but have little effect on Cav2.1,Cav2.2,KCNH2,KCNQ1 and KCNQ1/KCNE1.

Expression analysis of ciliary rootlet coiled coil protein mRNA during Xenopus development / 대한수의학회지

Ciliary rootlet coiled coil protein (CROCC), the structural component that originates from the basal body at the proximal end of the ciliary rootlet, plays a crucial role in maintaining the cellular integrity of ciliated cells. In the current study, we cloned Xenopus CROCC and performed the expression analysis. The amino acid sequence of Xenopus laevis was related to those of Drosophila, cow, goat, horse, chicken, mouse and human. Reverse transcription polymerase chain reaction analysis revealed that CROCC mRNA encoding a coiled coil protein was present maternally, as well as throughout early development. In situ hybridization indicated that CROCC mRNA occurred in the animal pole of embryo during gastrulation and subsequently in the presumptive neuroectoderm at the end of gastrulation. At tailbud stages, CROCC mRNA expression was localized in the anterior roof plate of the developing brain, pharyngeal epithelium connected to gills, esophagus, olfactory placode, intestine and nephrostomes of the pronephric kidney. Our study suggests that CROCC may be responsible for control of the development of various ciliated organs.

Effect of XinBao pill on electrophysiological properties of human HCN4 channel / 中国生化药物杂志

Objective To explore the basic medical mechanism of XinBao pill on electrophysiological characteristics of hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4), and illustrate the mechanism of its therapeutical effect on bradycardia.Methods Human HCN4 mRNA was injected into the Xenopus laevis oocytes, after incubated for 2 ~3 days, channel current properties of HCN4 perfused with 40 mg/L XinBao Pill were observed by double electrode voltage clamp technique.Results At-90mV test potential, compared with control group (no XinBao pill), HCN4 channel peak current and tail current in 40 mg/L XinBao pill group had obvious changes, and V1/2 from ( -103.61 ±3.57)mV to ( -106.42 ±5.33)mV in XinBao pill group, from( -81.11 ±4.26)mV to( -86.36 ±7.44)mV in control group.The values of k from (15.15 ±2.23)mV to (17.33 ±3.58) mV in XinBao pill group, from(11.78 ±0.85)mV to(12.39 ±1.51)mV in control group(n=10).At test potential -90 mV, 40 mg/L XinBao pill perfusion fluid decreased the instantaneous current of(0.15 ±0.24)%, the EC50 was (30.8 ±4.8)mg/L (n=8).At test potential-140 mV~-100 mV level, 40 mg/L XinBao pill group increased the channel activation time constant compared with control group[(226.73 ±31.36)ms vs(143.67 ± 21.44)ms;-140 mV,n=10,P<0.05].40 mg/L XinBao pill group increased the channel deactivation time constant compared with control group [(1293.53 ±95.02)ms vs (647.12 ±61.35)ms;-140 mV,n=10,P<0.05].Conclusion The XinBao pill enhances the instantaneous current of HCN4 in a concentration-dependent manner, and extents channel activation and deactivation processes.

Osteocalcin Expression and Mineralization in Developing Tooth of Xenopus laevis

Osteocalcin (OC) is the most abundant noncollagenous protein of extracellular matrix in the bone. In an OC deficient mouse, bone formation rates are increased in cancellous and cortical bones. OC is known as a negative regulator of mineral apposition. OC is also expressed in the tooth of the rat, bovine, and human. However, little is known about OC during tooth development in Xenopus. The purpose of this study is to compare the expression of OC with mineralization in the developing tooth of Xenopus, by using von Kossa staining and in situ hybridization. At stage 56, the developmental stage of tooth germ corresponds to the cap stage, and an acellular zone was apparent between the dental papilla and the enamel organ. From stage 57, calcium deposition was revealed by von Kossa staining prior to OC expression, and the differentiated odontoblasts forming predentin were located at adjoining predentin. At stage 58, OC transcripts were detected in the differentiated odontoblasts. At stage 66, OC mRNA was expressed in the odontoblasts, which was aligned in a single layer at the periphery of the pulp. These findings suggest that OC may play a role in mineralization and odontogenesis of tooth development in Xenopus.

Sox9 regulates development of neural crest and otic placode in a time- and dose-dependent fashion / 동물의과학연구지

Neural crest and placodes share a number of important features, pointing to a possible common evolutionary origin. They both arise from the neural plate border, which is the boundary between the non-neural ectoderm and neural plate. The transcription factor Sox9 has been implicated in neural crest and otic placode induction in several species. To investigate the differential regulation of neural crest and otic placode induction by Sox9, a gain of function assay was performed using a hormone-inducible version of the Sox9 construct at different doses and time periods. Sox9 was expressed in both neural crest and otic placode cell populations in the same stage embryos by in situ hybridization. Using a gain of function approach, increased expression of neural crest marker (Snail2) and otic placode marker (Pax8) in Sox9-overexpressed embryos was observed. Higher dose of Sox9 reduced or eliminated both neural crest and placode cells in the embryos. Interestingly, otic placodes cells were more strongly affected as compared to neural crest cells. So, optimal dosage and timing of Sox9 expression are important for the development of the neural crest and otic placode. The development of the neural crest and otic placode are affected by Sox9 in a time- and dose-dependent manner.

Analysis of temporal and spatial expression patterns of Miox gene during development of Xenopus laevis embryos / 第二军医大学学报

Objective To explore the molecular evolution of myo-inositol oxygenase (Miox) gene and its temporal and spatial expression patterns during the development of Xenopus laevis embryos. Methods The temporal and spatial expression patterns of Miox gene were analyzed by semi, quantitative RT, PCR and whole, mount in situ hybridization technique, respectively. Results RT, PCR results showed that Miox gene was hardly found before stage 26; slight expression was found at stage 28, which gradually increased thereafter, reaching a high level at stage 40 and peaked at stage 41; and then it had a decrease at stage 45. Compared with stages 28, 34, stages 40, 41, and 45 had a significantly higher Miox gene expression (P<0.05). Compared with stage 40, stage 41 had a significantly higher Miox gene expression(P<0.05). But stage 45 had a significantly lower expression compared with stage 41(P<0.05). The results of whole, mount in situ hybridization showed no Miox expression before stage 30; at stage 33 weak expression was found in the pronephros, and the expression gradually increased as time went by. The results of whole, mount in situ hybridization were consistent with that of RT, PCR, with Miox expression notably increased at stage 39, 40, and then remained at that level. We also found that Miox was only expressed in the pronephros tubules during the whole embryo development period. Conclusion Miox is a kidney, specific gene during Xenopus laevis pronephros development, and it may serve as a marker for later pronephros development in organogenesis.

Na, K-ATPase beta2 isoform (atp1b2) expressed in the retina of Xenopus / 동물의과학연구지

The ubiquitous Na, K-ATPase is a membrane-bound ion pump located in the plasma membrane in all animal cells and plays an essential role in a variety of cellular functions. Studies in several organisms have shown that this protein regulates different aspects of embryonic development and is responsible for the pathogenesis of several human diseases. Na, K-ATPase is an important factor for retinal development, and combinations of the isoforms of each of its subunits are expressed in different cell types and determine its functional properties. In this study, we performed RT-PCR assay to determine temporal expression and in situ hybridization to determine spatial expression of Na, K-ATPase beta2 isoform (atp1b2) in Xenopus laevis. Focusing on retinal expression to distinguish the specific expression domain, we used retinal marker genes sox4, sox11, vsx1, and . Xenopus atp1b2 was expressed from late gastrulation to the tadpole stage. Using whole mount in situ hybridization, we showed that Xenopus atp1b2 was expressed broadly in the eye, the whole surface ectoderm, and gills. In situ hybridization on sections revealed detailed and specific expression in the outer nuclear layer of the retina, which consists of two major classes of photoreceptors, rods and cones, surface ectoderm, pharyngeal epithelium, and gills. These findings indicate that atp1b2 may play an important role for the development of Xenopus retina.

Aparición de melanina como pigmento protector en el encéfalo de xenopus laevis para protegerlo de los efectos de la radiación ultravioleta / Emerging melanin protective pigment in the brain of xenopus laevis to protect from the effects of uv radiation

Con el fin de proteger al organismo de condiciones estresantes, tales como cambios de osmolaridad y de temperatura, además de actuar como pantalla protectora en contra de los rayos ultravioleta (UV). Se ha observado que ciertos anfibios han desarrollado pigmentación en su encéfalo como una posible protección ante el aumento de la radiación UV, causada por el daño en la capa de ozono, la cual estaría alterando al ecosistema. En este trabajo se describe la presencia de pigmentación en el encèfalo de X. laevis durante el desarrollo larvario y su posible función protectora frente a la radiación UV. Para ello, se recolectaron individuos de diferentes estados larvarios, los que fueron obtenidos de distintas localidades de la región de Valparaíso (V región, Chile), para ser procesados con el método corriente H-E y el método de Lillie. En los análisis se pudo evidenciar que la pigmentación correspondía a melanina, que se encontraría en la membrana denominada leptomeninge, la cual recubre al encéfalo y estaría actuando como un filtro protector para evitar daños a nivel del desarrollo en el sistema nervioso de estos anuros. En suma, los rayos UV como agentes deletéreos estarían estimulando la producción de eumelanina en la leptomeninge de estos anfibios, para proteger parte del SNC (encéfalo), como al individuo en sí de posibles alteraciones teratogénicas y/o mutagénicas.

It has been observed that certain amphibians have developed pigmentation in brain as a possible increased protection against UV radiation, caused by damage to the ozone layer, which would alter the ecosystem. In this paper we describe the presence of pigment in the brain of X. laevis during larval development and possible protective function against UV radiation. To do this, we collected individuals at various larval stages, which were obtained from different locations in Valparaiso (V Region, Chile), to be processed with HE and the method of Lillie. In the analysis it was evident that pigmentation corresponded to melanin, which would be in the membrane called leptomeninges, which covers the brain and would be acting as a protective filter to prevent damage to the level of development in the nervous system of these frogs. In addition, UV rays would be deleterious agents stimulating production of eumelanin in the leptomeninges of these amphibians, to protect the CNS (brain), and the individual itself of potential teratogenic or mutagenic alterations.

Activation of Lysophosphatidic Acid Receptor Is Coupled to Enhancement of Ca(2+)-Activated Potassium Channel Currents

The calcium-activated K+ (BKCa) channel is one of the potassium-selective ion channels that are present in the nervous and vascular systems. Ca2+ is the main regulator of BKCa channel activation. The BKCa channel contains two high affinity Ca2+ binding sites, namely, regulators of K+ conductance, RCK1 and the Ca2+ bowl. Lysophosphatidic acid (LPA, 1-radyl-2-hydroxy-sn-glycero-3-phosphate) is one of the neurolipids. LPA affects diverse cellular functions on many cell types through G protein-coupled LPA receptor subtypes. The activation of LPA receptors induces transient elevation of intracellular Ca2+ levels through diverse G proteins such as Galphaq/11, Galphai, Galpha12/13, and Galphas and the related signal transduction pathway. In the present study, we examined LPA effects on BKCa channel activity expressed in Xenopus oocytes, which are known to endogenously express the LPA receptor. Treatment with LPA induced a large outward current in a reversible and concentration-dependent manner. However, repeated treatment with LPA induced a rapid desensitization, and the LPA receptor antagonist Ki16425 blocked LPA action. LPA-mediated BKCa channel activation was also attenuated by the PLC inhibitor U-73122, IP3 inhibitor 2-APB, Ca2+ chelator BAPTA, or PKC inhibitor calphostin. In addition, mutations in RCK1 and RCK2 also attenuated LPA-mediated BKCa channel activation. The present study indicates that LPA-mediated activation of the BKCa channel is achieved through the PLC, IP3, Ca2+, and PKC pathway and that LPA-mediated activation of the BKCa channel could be one of the biological effects of LPA in the nervous and vascular systems.

Inhibitory Effects of Ginsenoside Metabolites, Compound K and Protopanaxatriol, on GABAC Receptor-Mediated Ion Currents

Ginsenosides, one of the active ingredients of Panax ginseng, show various pharmacological and physiological effects, and they are converted into compound K (CK) or protopanaxatriol (M4) by intestinal microorganisms. CK is a metabolite derived from protopanaxadiol (PD) ginsenosides, whereas M4 is a metabolite derived from protopanaxatriol (PT) ginsenosides. The gamma-aminobutyric acid receptorC (GABAC) is primarily expressed in retinal bipolar cells and several regions of the brain. However, little is known of the effects of ginsenoside metabolites on GABAC receptor channel activity. In the present study, we examined the effects of CK and M4 on the activity of human recombinant GABAC receptor (rho1) channels expressed in Xenopus oocytes by using a 2-electrode voltage clamp technique. In oocytes expressing GABAC receptor cRNA, we found that CK or M4 alone had no effect in oocytes. However, co-application of either CK or M4 with GABA inhibited the GABA-induced inward peak current (IGABA). Interestingly, pre-application of M4 inhibited IGABA more potently than CK in a dose-dependent and reversible manner. The half-inhibitory concentration (IC50) values of CK and M4 were 52.1+/-2.3 and 45.7+/-3.9 microM, respectively. Inhibition of IGABA by CK and M4 was voltage-independent and non-competitive. This study implies that ginsenoside metabolites may regulate GABAC receptor channel activity in the brain, including in the eyes.

Resveratrol Inhibits GABAC rho Receptor-Mediated Ion Currents Expressed in Xenopus Oocytes

Resveratrol is a phytoalexin found in grapes, red wine, and berries. Resveratrol has been known to have many beneficial health effects, such as anti-cancer, neuroprotective, anti-inflammatory, and life-prolonging effects. However, relatively little is known about the effects of resveratrol on the regulation of ligand-gated ion channels. We have previously reported that resveratrol regulates subsets of homomeric ligand-gated ion channels such as those of 5-HT3A receptors. The gamma-aminobutyric acidC (GABAC) receptor is mainly expressed in retinal bipolar cells and plays an important role in visual processing. In the present study, we examined the effects of resveratrol on the channel activity of homomeric GABAC receptor expressed in Xenopus oocytes injected with cRNA encoding human GABAC rho subunits. Our data show that the application of GABA elicits an inward peak current (IGABA) in oocytes that express the GABAC receptor. Resveratrol treatment had no effect on oocytes injected with H2O or with GABAC receptor cRNA. Co-treatment with resveratrol and GABA inhibited IGABA in oocytes with GABAC receptors. The inhibition of IGABA by resveratrol was in a reversible and concentration-dependent manner. The IC50 of resveratrol was 28.9+/-2.8 microM in oocytes expressing GABAC receptor. The inhibition of IGABA by resveratrol was in voltage-independent and non-competitive manner. These results indicate that resveratrol might regulate GABAC receptor expression and that this regulation might be one of the pharmacological actions of resveratrol on the nervous system.

The expression of melanopsin and clock genes in Xenopus laevis melanophores and their modulation by melatonin

Vertebrates have a central clock and also several peripheral clocks. Light responses might result from the integration of light signals by these clocks. The dermal melanophores of Xenopus laevis have a photoreceptor molecule denominated melanopsin (OPN4x). The mechanisms of the circadian clock involve positive and negative feedback. We hypothesize that these dermal melanophores also present peripheral clock characteristics. Using quantitative PCR, we analyzed the pattern of temporal expression of Opn4x and the clock genes Per1, Per2, Bmal1, and Clock in these cells, subjected to a 14-h light:10-h dark (14L:10D) regime or constant darkness (DD). Also, in view of the physiological role of melatonin in the dermal melanophores of X. laevis, we determined whether melatonin modulates the expression of these clock genes. These genes show a time-dependent expression pattern when these cells are exposed to 14L:10D, which differs from the pattern observed under DD. Cells kept in DD for 5 days exhibited overall increased mRNA expression for Opn4x and Clock, and a lower expression for Per1, Per2, and Bmal1. When the cells were kept in DD for 5 days and treated with melatonin for 1 h, 24 h before extraction, the mRNA levels tended to decrease for Opn4x and Clock, did not change for Bmal1, and increased for Per1 and Per2 at different Zeitgeber times (ZT). Although these data are limited to one-day data collection, and therefore preliminary, we suggest that the dermal melanophores of X. laevis might have some characteristics of a peripheral clock, and that melatonin modulates, to a certain extent, melanopsin and clock gene expression.

Quercetin Inhibits alpha3beta4 Nicotinic Acetylcholine Receptor-Mediated Ion Currents Expressed in Xenopus Oocytes

Quercetin mainly exists in the skin of colored fruits and vegetables as one of flavonoids. Recent studies show that quercetin, like other flavonoids, has diverse pharmacological actions. However, relatively little is known about quercetin effects in the regulations of ligand-gated ion channels. In the previous reports, we have shown that quercetin regulates subsets of homomeric ligand-gated ion channels such as glycine, 5-HT3A and alpha7 nicotinic acetylcholine receptors. In the present study, we examined quercetin effects on heteromeric neuronal alpha3beta4 nicotinic acetylcholine receptor channel activity expressed in Xenopus oocytes after injection of cRNA encoding bovine neuronal alpha3 and beta4 subunits. Treatment with acetylcholine elicited an inward peak current (IACh) in oocytes expressing alpha3beta4 nicotinic acetylcholine receptor. Co-treatment with quercetin and acetylcholine inhibited IACh in oocytes expressing alpha3beta4 nicotinic acetylcholine receptors. The inhibition of IACh by quercetin was reversible and concentration-dependent. The half-inhibitory concentration (IC50) of quercetin was 14.9+/-0.8 microM in oocytes expressing alpha3beta4 nicotinic acetylcholine receptor. The inhibition of IACh by quercetin was voltage-independent and non-competitive. These results indicate that quercetin might regulate alpha3beta4 nicotinic acetylcholine receptor and this regulation might be one of the pharmacological actions of quercetin in nervous systems.

Inhibitory Effects of Quercetin on Muscle-type of Nicotinic Acetylcholine Receptor-Mediated Ion Currents Expressed in Xenopus Oocytes

The flavonoid quercetin is a low molecular weight compound generally found in apple, gingko, tomato, onion and other red-colored fruits and vegetables. Like other flavonoids, quercetin has diverse pharmacological actions. However, relatively little is known about the influence of quercetin effects in the regulation of ligand-gated ion channels. Previously, we reported that quercetin regulates subsets of nicotinic acetylcholine receptors such as alpha3beta4, alpha7 and alpha9alpha10. Presently, we investigated the effects of quercetin on muscle-type of nicotinic acetylcholine receptor channel activity expressed in Xenopus oocytes after injection of cRNA encoding human fetal or adult muscle-type of nicotinic acetylcholine receptor subunits. Acetylcholine treatment elicited an inward peak current (IACh) in oocytes expressing both muscle-type of nicotinic acetylcholine receptors and co-treatment of quercetin with acetylcholine inhibited IACh. Pre-treatment of quercetin further inhibited IACh in oocytes expressing adult and fetal muscle-type nicotinic acetylcholine receptors. The inhibition of IACh by quercetin was reversible and concentration-dependent. The IC50 of quercetin was 18.9+/-1.2 microM in oocytes expressing adult muscle-type nicotinic acetylcholine receptor. The inhibition of IACh by quercetin was voltage-independent and non-competitive. These results indicate that quercetin might regulate human muscle-type nicotinic acetylcholine receptor channel activity and that quercetin-mediated regulation of muscle-type nicotinic acetylcholine receptor might be coupled to regulation of neuromuscular junction activity.

xCITED2 Induces Neural Genes in Animal Cap Explants of Xenopus Embryos

Neural tissue is arisen from presumptive ectoderm via inhibition of bone morphogenetic protein (BMP) signaling during Xenopus early development. Previous studies demonstrate that ectopic expression of dominant negative BMP4 receptor (DNBR) produces neural tissue in animal cap explants (AC) and also increases the expression level of various genes involved in neurogenesis. To investigate detail mechanism of neurogenesis in transcriptional level, we analyzed RNAs increased by DNBR using total RNA sequencing analysis and identified several candidate genes. Among them, xCITED2 (Xenopus CBP/p300-interacting transcription activator) was induced 4.6 fold by DNBR and preferentially expressed in neural tissues at tadpole stage. Ectopic expression of xCITED2 induced anterior neural genes without mesoderm induction and reduced BMP downstream genes, an eye specific marker and posterior neural marker. Taken together, these results suggest that xCITED2 may have a role in the differentiation of anterior neural tissue during Xenopus early development.