Electrophysiological Properties of Ion Channels in Ascaris suum Tissue Incorporated into Planar Lipid Bilayers

Ion channels are important targets of anthelmintic agents. In this study, we identified 3 types of ion channels in Ascaris suum tissue incorporated into planar lipid bilayers using an electrophysiological technique. The most frequent channel was a large-conductance cation channel (209 pS), which accounted for 64.5% of channels incorporated (n=60). Its open-state probability (Po) was ~0.3 in the voltage range of –60~+60 mV. A substate was observed at 55% of the main-state. The permeability ratio of Cl- to K+ (PCl/PK) was ~0.5 and PNa/PK was 0.81 in both states. Another type of cation channel was recorded in 7.5% of channels incorporated (n=7) and discriminated from the large-conductance cation channel by its smaller conductance (55.3 pS). Its Po was low at all voltages tested (~0.1). The third type was an anion channel recorded in 27.9% of channels incorporated (n=26). Its conductance was 39.0 pS and PCl/PK was 8.6±0.8. Po was ~1.0 at all tested potentials. In summary, we identified 2 types of cation and 1 type of anion channels in Ascaris suum. Gating of these channels did not much vary with voltage and their ionic selectivity is rather low. Their molecular nature, functions, and potentials as anthelmintic drug targets remain to be studied further.

Electrophysiological Properties of Ion Channels in Ascaris suum Tissue Incorporated into Planar Lipid Bilayers

Ion channels are important targets of anthelmintic agents. In this study, we identified 3 types of ion channels in Ascaris suum tissue incorporated into planar lipid bilayers using an electrophysiological technique. The most frequent channel was a large-conductance cation channel (209 pS), which accounted for 64.5% of channels incorporated (n=60). Its open-state probability (Po) was ~0.3 in the voltage range of –60~+60 mV. A substate was observed at 55% of the main-state. The permeability ratio of Cl- to K+ (PCl/PK) was ~0.5 and PNa/PK was 0.81 in both states. Another type of cation channel was recorded in 7.5% of channels incorporated (n=7) and discriminated from the large-conductance cation channel by its smaller conductance (55.3 pS). Its Po was low at all voltages tested (~0.1). The third type was an anion channel recorded in 27.9% of channels incorporated (n=26). Its conductance was 39.0 pS and PCl/PK was 8.6±0.8. Po was ~1.0 at all tested potentials. In summary, we identified 2 types of cation and 1 type of anion channels in Ascaris suum. Gating of these channels did not much vary with voltage and their ionic selectivity is rather low. Their molecular nature, functions, and potentials as anthelmintic drug targets remain to be studied further.

Bee venom reduces burn-induced pain via the suppression of peripheral and central substance P expression in mice

Background@#Scalding burn injuries can occur in everyday life but occur more frequently in young children. Therefore, it is important to develop more effective burn treatments. @*Objectives@#This study examined the effects of bee venom (BV) stimulation on scalding burn injury-induced nociception in mice as a new treatment for burn pain. @*Methods@#To develop a burn injury model, the right hind paw was immersed temporarily in hot water (65°C, 3 seconds). Immediately after the burn, BV (0.01, 0.02, or 0.1 mg/kg) was injected subcutaneously into the ipsilateral knee area once daily for 14 days. A von Frey test was performed to assess the nociceptive response, and the altered walking parameters were evaluated using an automated gait analysis system. In addition, the peripheral and central expression changes in substance P (Sub P) were measured in the dorsal root ganglion and spinal cord by immunofluorescence. @*Results@#Repeated BV treatment at the 2 higher doses used in this study (0.02 and 0.1 mg/kg) alleviated the pain responses remarkably and recovered the gait performances to the level of acetaminophen (200 mg/kg, intraperitoneal, once daily), which used as the positive control group. Moreover, BV stimulation had an inhibitory effect on the increased expression of Sub P in the peripheral and central nervous systems by a burn injury. @*Conclusions@#These results suggest that a peripheral BV treatment may have positive potency in treating burn-induced pain.

Calpain-2 as a Treatment Target in Prenatal Stress-induced Epileptic Spasms in Infant Rats

Stress can induce a serious epileptic encephalopathy that occurs during early infancy. Recent studies have revealed that prenatal stress exposure is a risk factor for the development of infantile spasms. Our previous work demonstrates that prenatal stress with betamethasone-induced alterations to the expression of the K⁺/Cl⁻ co-transporter (KCC2) in gamma-aminobutyric acid (GABA) interneurons lowers the seizure threshold in exposed animals. Here, we further investigated the mechanisms involved in this KCC2 dysfunction and explored possible treatment options. We stressed Sprague-Dawley rats prenatally and further treated dams with betamethasone on gestational day 15, which increases seizure susceptibility and NMDA (N-Methyl-D-aspartate)-triggered spasms on postnatal day 15. In this animal model, first, we evaluated baseline calpain activity. Second, we examined the cleavage and dephosphorylation of KCC2. Finally, we checked the effect of a calpain inhibitor on seizure occurrence. The phosphorylated-N-methyl-D-aspartate Receptor 2B (NR2B):non-phosphorylated NR2B ratio was found to be higher in the cortex of the prenatally stressed beta-methasone model. We further found that the betamethasone model exhibited increased phosphorylation of calpain-2 and decreased phosphorylation of KCC2 and Glutamic acid decarboxylase 67 (GAD67). After using a calpain inhibitor in prenatal-stress rats, the seizure frequency decreased, while latency increased. GABAergic depolarization was further normalized in prenatal-stress rats treated with the calpain inhibitor. Our study suggests that calpain-dependent cleavage and dephosphorylation of KCC2 decreased the seizure threshold of rats under prenatal stress. Calpain-2 functions might, thus, be targeted in the future for the development of treatments for epileptic spasms.

Developmental changes in GABAA tonic inhibition are compromised by multiple mechanisms in preadolescent dentate gyrus granule cells

The sustained tonic currents (I(tonic)) generated by γ-aminobutyric acid A receptors (GABA(A)Rs) are implicated in diverse age-dependent brain functions. While various mechanisms regulating I(tonic) in the hippocampus are known, their combined role in I(tonic) regulation is not well understood in different age groups. In this study, we demonstrated that a developmental increase in GABA transporter (GAT) expression, combined with gradual decrease in GABA(A)R α₅ subunit, resulted in various I(tonic) in the dentate gyrus granule cells (DGGCs) of preadolescent rats. Both GAT-1 and GAT-3 expression gradually increased at infantile (P₆₋₈ and P₁₃₋₁₅) and juvenile (P₂₀₋₂₂ and P₂₇₋₂₉) stages, with stabilization observed thereafter in adolescents (P₃₄₋₃₆) and young adults (P₄₁₋₄₃). I(tonic) facilitation of a selective GAT-1 blocker (NO-711) was significantly less at P₆₋₈ than after P₁₃₋₁₅. The facilitation of I(tonic) by SNAP-5114, a GAT-3 inhibitor, was negligible in the absence of exogenous GABA at all tested ages. In contrast, I(tonic) in the presence of a nonselective GAT blocker (nipecotic acid, NPA) gradually decreased with age during the preadolescent period, which was mimicked by I(tonic) changes in the presence of exogenous GABA. I(tonic) sensitivity to L-655,708, a GABA(A)R α₅ subunit inverse agonist, gradually decreased during the preadolescent period in the presence of NPA or exogenous GABA. Finally, Western blot analysis showed that the expression of the GABA(A)R α₅ subunit in the dentate gyrus gradually decreased with age. Collectively, our results suggested that the I(tonic) regulation of altered GATs is under the final tune of GABA(A)R α₅ subunit activation in DGGCs at different ages.

Inoculation of Lewis lung carcinoma cells enhances formalin-induced pain behavior and spinal Fos expression in mice

The incidence of lung cancer has rapidly increased and cancer patients at a later cancer stage frequently suffer from unbearable cancer-associated pain. However, the pathophysiology of lung cancer pain has not been fully described due to a lack of appropriate animal models. This study was designed to determine the effect of Lewis lung carcinoma (LLC) cell inoculation on formalin-induced pain behavior and spinal Fos expression in C57BL/6 mice. LLC cells (1.5 × 10⁵, 2.5 × 10⁵, 3.0 × 10⁵ or 5.0 × 10⁵) were inoculated into back or peri-sciatic nerve areas. Back area inoculation was adopted to determine the effect of cancer cell circulating factors and the peri-sciatic nerve area was used to evaluate the possible effects of cancer cell contacting and circulating factors on formalin-induced pain. At postinoculation day 7, LLC cell (5.0 × 10⁵) inoculations in both back and peri-sciatic nerve area significantly increased formalin-induced paw-licking time and spinal Fos expression over those in cell-media-inoculated (control) mice. Enhanced pain behavior and spinal Fos expression were significantly suppressed by ibuprofen pretreatment (250 mg/kg). The results of this study suggest that LLC cell circulating factors and inflammatory responses may be critical in enhancing pain sensation in the early stage of lung cancer cell inoculation.

Facilitation of AMPA receptor-mediated steady-state current by extrasynaptic NMDA receptors in supraoptic magnocellular neurosecretory cells

In addition to classical synaptic transmission, information is transmitted between cells via the activation of extrasynaptic receptors that generate persistent tonic current in the brain. While growing evidence supports the presence of tonic NMDA current (INMDA) generated by extrasynaptic NMDA receptors (eNMDARs), the functional significance of tonic I(NMDA) in various brain regions remains poorly understood. Here, we demonstrate that activation of eNMDARs that generate I(NMDA) facilitates the α-amino-3-hydroxy-5-methylisoxazole-4-proprionate receptor (AMPAR)-mediated steady-state current in supraoptic nucleus (SON) magnocellular neurosecretory cells (MNCs). In low-Mg2+ artificial cerebrospinal fluid (aCSF), glutamate induced an inward shift in I(holding) (I(GLU)) at a holding potential (V(holding)) of -70 mV which was partly blocked by an AMPAR antagonist, NBQX. NBQX-sensitive I(GLU) was observed even in normal aCSF at V(holding) of -40 mV or -20 mV. I(GLU) was completely abolished by pretreatment with an NMDAR blocker, AP5, under all tested conditions. AMPA induced a reproducible inward shift in I(holding) (I(AMPA)) in SON MNCs. Pretreatment with AP5 attenuated I(AMPA) amplitudes to ~60% of the control levels in low-Mg2+ aCSF, but not in normal aCSF at V(holding) of -70 mV. I(AMPA) attenuation by AP5 was also prominent in normal aCSF at depolarized holding potentials. Memantine, an eNMDAR blocker, mimicked the AP5-induced I(AMPA) attenuation in SON MNCs. Finally, chronic dehydration did not affect I(AMPA) attenuation by AP5 in the neurons. These results suggest that tonic I(NMDA), mediated by eNMDAR, facilitates AMPAR function, changing the postsynaptic response to its agonists in normal and osmotically challenged SON MNCs.

TonEBP suppresses adipocyte differentiation via modulation of early signaling in 3T3-L1 cells

TonEBP belongs to the Rel family of transcription factors and plays important roles in inflammation as well as kidney homeostasis. Recent studies suggest that TonEBP expression is also involved in differentiation of several cell types such as myocytes, chondrocytes, and osteocytes. In this study, we investigated the roles of TonEBP during adipocyte differentiation in 3T3-L1 cells. TonEBP mRNA and protein expression was dramatically reduced during adipocyte differentiation. Sustained expression of TonEBP using an adenovirus suppressed the formation of lipid droplets as well as the expression of FABP4, a marker of differentiated adipocytes. TonEBP also inhibited the expression of PPARγ, a known master regulator of adipocytes. RNAi-mediated knock down of TonEBP promoted adipocyte differentiation. However, overexpression of TonEBP did not affect adipogenesis after the initiation of differentiation. Furthermore, TonEBP expression suppressed mitotic clonal expansion and insulin signaling, which are required early for adipocyte differentiation of 3T3-L1 cells. These results suggest that TonEBP may be an important regulatory factor in the early phase of adipocyte differentiation.

APE1/Ref-1 as a Serological Biomarker for the Detection of Bladder Cancer / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1) is a multifunctional protein that shows elevated expression in a number of cancers. We attempted to determine whether serum APE1/Ref-1 is elevated in patients with bladder cancer. MATERIALS AND METHODS: Serum APE1/Ref-1 levels were determined using enzyme-linked immunosorbent assay in serum from patients with bladder cancer who had not received chemotherapy or radiotherapy (n=51) and non-tumor controls (n=55). The area under the receiver operating characteristic area under the curve was applied to determine the correlation between clinical factors and the serum levels of APE1/Ref-1. RESULTS: Serum levels of APE1/Ref-1 in bladder cancer patients were significantly elevated compared to those of the control group (3.548+/-0.333 ng/100 muL [n=51] for bladder cancer vs. 1.547+/-0.319 ng/100 muL [n=55] for the control group), with a sensitivity and specificity of 93% and 59%, respectively. Serum APE1/Ref-1 levels are associated with tumor stage, grade, muscle invasion, and recurrence. CONCLUSION: Serum APE1/Ref-1 might be useful as a potential serologic biomarker for bladder cancer.

Antinociceptive Effect of Cyperi rhizoma and Corydalis tuber Extracts on Neuropathic Pain in Rats

In this study, we examined the antinociceptive effect of Cyperi rhizoma (CR) and Corydalis tuber (CT) extracts using a chronic constriction injury-induced neuropathic pain rat model. After the ligation of sciatic nerve, neuropathic pain behavior such as mechanical allodynia and thermal hyperalgesia were rapidly induced and maintained for 1 month. Repeated treatment of CR or CT (per oral, 10 or 30 mg/kg, twice a day) was performed either in induction (day 0~5) or maintenance (day 14~19) period of neuropathic pain state. Treatment of CR or CT at doses of 30 mg/kg in the induction and maintenance periods significantly decreased the nerve injury-induced mechanical allodynia. In addition, CR and CT at doses of 10 or 30 mg/kg alleviated thermal heat hyperalgesia when they were treated in the maintenance period. Finally, CR or CT (30 mg/kg) treated during the induction period remarkably reduced the nerve injury-induced phosphorylation of NMDA receptor NR1 subunit (pNR1) in the spinal dorsal horn. Results of this study suggest that extracts from CR and CT may be useful to alleviate neuropathic pain.

Tat-Mediated p66shc Transduction Decreased Phosphorylation of Endothelial Nitric Oxide Synthase in Endothelial Cells

We evaluated the role of Tat-mediated p66shc transduction on the activation of endothelial nitric oxide synthase in cultured mouse endothelial cells. To construct the Tat-p66shc fusion protein, human full length p66shc cDNA was fused with the Tat-protein transduction domain. Transduction of TAT-p66shc showed a concentration- and time-dependent manner in endothelial cells. Tat-mediated p66shc transduction showed increased hydrogen peroxide and superoxide production, compared with Tat-p66shc (S/A), serine 36 residue mutant of p66shc. Tat-mediated p66shc transduction decreased endothelial nitric oxide synthase phosphorylation in endothelial cells. Furthermore, Tat-mediated p66shc transduction augmented TNF-alpha-induced p38 MAPK phosphorylation in endothelial cells. These results suggest that Tat-mediated p66shc transduction efficiently inhibited endothelial nitric oxide synthase phosphorylation in endothelial cells.

Vasorelaxing Activity of Ulmus davidiana Ethanol Extracts in Rats: Activation of Endothelial Nitric Oxide Synthase

Ulmus davidiana var. japonica Rehder (Urticales: Ulmaceae) (UD) is a tree widespread in northeast Asia. It is traditionally used for anticancer and anti-inflammatory therapy. The present study investigated the effect of an ethanol extract of UD on vascular tension and its underlying mechanism in rats. The dried root bark of UD was ground and extracted with 80% ethanol. The prepared UD extract was used in further analysis. The effect of UD on the cell viability, vasoreactivity and hemodynamics were investigated using propidium iodide staining in cultured cells, isometric tension recording and blood pressure analysis, respectively. Low dose of UD (10~100microg/ml) did not affect endothelial cell viability, but high dose of UD reduced cell viability. UD induced vasorelaxation in the range of 0.1~10microg/ml with an ED50 value of 2microg/ml. UD-induced vasorelaxation was completely abolished by removal of the endothelium or by pre-treatment with L-NAME, an inhibitor of nitric oxide synthase. UD inhibited calcium influx induced by phenylephrine and high K+ and also completely abolished the effect of L-NAME. Intravenous injection of UD extracts (10~100 mg/kg) decreased arterial and ventricular pressure in a dose-dependent manner. Moreover, UD extracts reduced the ventricular contractility (+dP/dt) in anesthetized rats. However, UD-induced hypotensive actions were minimized in L-NAME-treated rats. Taken together, out results showed that UD induced vasorelaxation and has antihypertensive properties, which may be due the activation of nitric oxide synthase in endothelium.

Direct Corticosteroid Modulation of GABAergic Neurons in the Anterior Hypothalamic Area of GAD65-eGFP Mice

Corticosterone is known to modulate GABAergic synaptic transmission in the hypothalamic paraventricular nucleus. However, the underlying receptor mechanisms are largely unknown. In the anterior hypothalamic area (AHA), the sympathoinhibitory center that project GABAergic neurons onto the PVN, we examined the expression of glucocorticoid receptor (GR) and mineralocorticoid receptor (MR) of GABAergic neurons using intact GAD65-eGFP transgenic mice, and the effects of corticosterone on the burst firing using adrenalectomized transgenic mice. GR or MR immunoreactivity was detected from the subpopulations of GABAergic neurons in the AHA. The AHA GABAergic neurons expressed mRNA of GR (42%), MR (38%) or both (8%). In addition, in brain slices incubated with corticosterone together with RU486 (MR-dominant group), the proportion of neurons showing a burst firing pattern was significantly higher than those in the slices incubated with vehicle, corticosterone, or corticosterone with spironolactone (GR-dominant group; 64 vs. 11~14%, p<0.01 by chi2-test). Taken together, the results show that the corticosteroid receptors are expressed on the GABAergic neurons in the AHA, and can mediate the corticosteroid-induced plasticity in the firing pattern of these neurons. This study newly provides the experimental evidence for the direct glucocorticoid modulation of GABAergic neurons in the AHA in the vicinity of the PVN.

Redox Factor-1 Inhibits Cyclooxygenase-2 Expression via Inhibiting of p38 MAPK in the A549 Cells

In this study, we evaluated the role of apurinic/apyrimidinic endonuclease1/redox factor-1 (Ref-1) on the tumor necrosis factor-alpha (TNF-alpha) induced cyclooxygenase-2 (COX-2) expression using A549 lung adenocarcinoma cells. TNF-alpha induced the expression of COX-2 in A549 cells, but did not induce BEAS-2B expression. The expression of COX-2 in A549 cells was TNF-alpha dose-dependent (5~100 ng/ml). TNF-alpha-stimulated A549 cells evidenced increased Ref-1 expression in a dose-dependent manner. The adenoviral transfection of cells with AdRef-1 inhibited TNF-alpha-induced COX-2 expression relative to that seen in the control cells (Ad beta gal). Pretreatment with 10 micrometer of SB203580 suppressed TNF-alpha-induced COX-2 expression, thereby suggesting that p38 MAPK might be involved in COX-2 expression in A549 cells. The phosphorylation of p38 MAPK was increased significantly after 5 minutes of treatment with TNF-alpha, reaching a maximum level at 10 min which persisted for up to 60 min. However, p38MAPK phosphorylation was markedly suppressed in the Ref-1-overexpressed A549 cells. Taken together, our results appear to indicate that Ref-1 negatively regulates COX-2 expression in response to cytokine stimulation via the inhibition of p38 MAPK phosphorylation. In the lung cancer cell lines, Ref-1 may be involved as an important negative regulator of inflammatory gene expression.

Overexpression of Ref-1 Inhibits Lead-induced Endothelial Cell Death via the Upregulation of Catalase

The role of apurinic/apyrimidinic endonuclease1/redox factor-1 (Ref-1) on the lead (Pb)-induced cellular response was investigated in the cultured endothelial cells. Pb caused progressive cellular death in endothelial cells, which occurred in a concentration- and time-dependent manner. However, Ref-1 overexpression with AdRef-1 significantly inhibited Pb-induced cell death in the endothelial cells. Also the overexpression of Ref-1 significantly suppressed Pb-induced superoxide and hydrogen peroxide elevation in the endothelial cells. Pb exposure induced the downregulation of catalase, it was inhibited by the Ref-1 overexpression in the endothelial cells. Taken together, our data suggests that the overexpression of Ref-1 inhibited Pb-induced cell death via the upregulation of catalase in the cultured endothelial cells.

Alternative Isoforms of TonEBP with Variable N-termini are Expressed in Mammalian Cells

Hypertonicity imposes a great deal of stress to cells since it causes rise in cellular ionic strength, which can be reduced by the accumulation of compatible osmolytes. TonEBP plays a central role in the cellular accumulation of compatible osmolytes via transcriptional stimulation of membrane transporters and aldose reductase. Alternatively spliced forms of TonEBP mRNA have previously been reported and two of them showed different transcriptional activity. In the present study, isoform-specific antibodies were produced to confirm the translation of the spliced mRNA to protein. TonEBP was immunoprecipitated by using anti-TonEBP antibody and then immunoblotted using anti-TonEBP or isoform specific antibodies to find out the expression profile of TonEBP isoforms in basal or stimulated condition. From these results, we conclude that all TonEBP isoforms are expressed in mammalian cells and their expression patterns are not same in every cells.

p66shc Adaptor Protein Suppresses the Activation of Endothelial Nitric Oxide Synthase in Mouse Embryonic Fibroblasts

Among the Shc proteins, p66shc is known to be related to oxidative stress responses and regulation of the production of reactive oxygen species (ROS). The present study was undertaken to investigate the role of p66shc on endothelial nitric oxide synthase (eNOS) activity in the mouse embryonic fibroblasts (MEFs). When wild type (WT) or p66shc (-/-) MEFs were transfected with full length of eNOS cDNA, the expression and activity of eNOS protein were higher in the p66shc (-/-) MEFs. These phenomena were reversed by reconstitution of p66shc cDNA transfection in the p66shc (-/-) MEFs. The basal superoxide production in the p66shc (-/-) MEFs was not significantly different from that of WT of MEFs. However, superoxide production induced by NADPH in the p66shc (-/-) MEF was lesser than that in WT MEFs. When compared with WT MEFs, cell lysate of p66shc (-/-) MEFs showed significantly increased H-ras activity without change of endogenous H-ras expression. Our findings suggest the pivotal role of p66shc adaptor protein played in inhibition of endothelial nitric oxide production via modulation of the expression and/or activity of eNOS protein.

NADPH Oxidase and Mitochondrial ROS are Involved in the TNF-alpha-induced Vascular Cell Adhesion Molecule-1 and Monocyte Adhesion in Cultured Endothelial Cells

Atherosclerosis is considered as a chronic inflammatory process. However, the nature of the oxidant signaling that regulates monocyte adhesion and its underlying mechanism is poorly understood. We investigated the role of reactive oxygen species on the vascular cell adhesion molecule-1 (VCAM-1) and monocyte adhesion in the cultured endothelial cells. TNF-alpha at a range of 1~30 ng/ml induced VCAM-1 expression dose-dependently. BCECF-AM-labeled U937 cells firmly adhered on the surface of endothelial cells when the endothelial cells were incubated with TNF-alpha (15 ng/ml). Ten micromol/L of SB203580, an inhibitor of p38 MAPK, significantly reduced TNF-alpha-induced VCAM-1 expression, compared to the JNK inhibitor (40micromol/L of SP60015) or ERK inhibitor (40micrommol/L of U0126). Also, SB203580 significantly inhibited TNF-alpha-induced monocyte adhesion in HUVEC. Superoxide production was minimal in the basal condition, however, treatment of TNF-alpha induced superoxide production in the dihydroethidine-loaded endothelial cells. Diphenyleneiodonium (DPI, 10micromol/L), an inhibitor of NADPH oxidase, and rotenone (1micromol/L), an inhibitor of mitochondrial complex I inhibited TNF-alpha-induced superoxide production, VCAM-1 expression and monocyte adhesion in the endothelial cells. Taken together, our data suggest that NADPH oxidase and mitochondrial ROS were involved in TNF-alpha-induced VCAM-1 and monocyte adhesion in the endothelial cells.

SM709, Ingredient of Antimelanogenic Bamboo Extract, Blocks Endothelin-1-induced Ca2+i Increase in Human Melanocytes

Endothelins secreted from keratinocytes are intrinsic mitogens and melanogens of human melanocytes in UVB-induced hyperpigmentation. To elucidate the cellular mechanism of antimelanogenic activity of bamboo extract, the effects of three ingredients of bamboo extract on endothelin 1 (ET-1) -induced Ca2+ mobilization were investigated in cultured human melanocytes. ET-1 receptors in human melanocytes were characterized by using specific antagonist, and ET-1 was found to increase intracellular Ca2+ concentration ([Ca2+]i) by activating ET-B receptor. SM709 (1, 2-O-diferulyl-glycerol), an ingredient of bamboo extract, inhibited ET-1-induced [Ca2+]i increase in a concentration- and time-dependent manner, although another ingredients SM707 and SM708 had no effect on ET-1-induced [Ca2+]i increase in human melanocytes. SM709 (100 microM), however, did not affect [Ca2+]i increase induced by thapsigargin and caffeine, suggesting that SM709 has no effect on the Ca2+ store in melanocytes. Furthermore, SM709 did not affect [Ca2+]i increase induced by LPA or ATP, known as G protein-mediated PLC activators like ET-1. Taken together, it is suggested that SM709 antagonizes ET-1-induced transmembrane signaling through ET-B receptor, which maybe a possible underlying mechanism of antimelanogenic activity of bamboo extract in human melanocytes.

Enhancement of ATP-induced Currents by Phospholipase D1 Overexpressed in PC12 Cells

Using phospholipase D1 (PLD1) -overexpressing PC12 (PLD1-PC12) cells, the regulatory roles of PLD1 on ATP-induced currents were investigated. In control and PLD1-PC12 cells, ATP increased PLD activity in an external Ca2+ dependent manner. PLD activity stimulated by ATP was substantially larger in PLD1-PC12 cells than in control cells. In whole-cell voltage-clamp mode, ATP induced transient inward and outward currents. The outward currents inhibited by TEA or charybdotoxin were significantly larger in PLD1-PC12 cells than in control cells. The inward currents known as Ca2+ permeable nonselective cation currents were also larger in PLD1-PC12 cells than in control cells. However, the difference between the two groups of cells disappeared in Ca2+ -free external solution, where ATP did not activate PLD. Finally, ATP-induced 45Ca uptakes were also larger in PLD1-PC12 cells than in control cells. These results suggest that PLD enhances ATP-induced Ca2+ influx via Ca2+ permeable nonselective cation channels and increases subsequent Ca2+ -activated K+ currents in PC12 cells.