Ginsenoside Rgl promotes endothelial progenitor cell migration and proliferation

Aim： To investigate the effect of ginsenoside Rgl on the migration, adhesion, proliferation, and VEGF expression of endothelial progenitor cells （EPCs）.
Methods： EPCs were isolated from human peripheral blood and incubated with different concentrations of ginsenoside Rgl （0.1, 0.5, 1.0, and 5.0 μmol/L） and vehicle controls. EPC migration was detected with a modified Boyden chamber assay. EPC adhesion was determined by counting adherent cells on fibronectin-coated culture dishes. EPC proliferation was analyzed with the 3.（4,5.dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay. In vitro vasculogenesis was assayed using an in vitro vasculogenesis detection kit. A VEGF-ELISA kit was used to measure the amount of VEGF protein in the cell culture medium.
Results： Ginsenoside Rgl promoted EPC adhesion, proliferation, migration and in vitro vasculogenesis in a dose- and timedependent manner. Cell cycle analysis showed that 5.0 μmol/L ofginsenoside Rgl significantly increased the EPC proliferative phase （S phase） and decreased the resting phase （G0/G1 phase）. Ginsenoside Rgl increased vascular endothelial growth factor production. Conclusion： The results indicate that ginsenoside Rgl promotes proliferation, migration, adhesion and in vitro vasculogenesis.     

Hepatocyte growth factor protects endothelial cells against gamma ray irradiation-induced damage

Aim： To investigate the effect of HGF on proliferation, apoptosis and migratory ability of human vascular endothelial cells against gamma ray irradiation. Methods： ECV304 cells derived from adult human umbilical vein endothelial cells （HUVEC） were irradiated with a single gamma ray dose of 20 Gy. Immunocytochemistry and Western blot analysis were used to detect c-Met protein expression and HGF/c-Met signal pathway. In the HGF-treated groups, ECV304 cells were incubated with HGF （20 or 40 ng/mL） 3 h prior to irradiation. At 48 h post- irradiation, the proliferation of ECV304 cells was measured by MTT assay, the apoptosis was assessed by flow cytometry, and the migratory ability of ECV304 cells was measured by transwell chamber assay, Results： c-Met protein is expressed in ECV304 cells and can be activated by HGF. Gamma ray irradiation inhibits proliferation and migration of ECV304 cells in a dose-dependent manner. HGF significantly promoted the proliferation of ECV304 cells, and flow cytom- etry revealed that HGF can inhibit apoptosis of ECV304 cells. Transwell chamber assay also showed that HGF increases migration activity of endothelial cells. Conclusion： HGF may afford protection to vascular endothelial cells against gamma ray irradiation-induced damage.     

Response of mesenchymal stem cells to shear stress in tissueengineered vascular grafts

Aim： Recent studies have demonstrated that mesenchymal stem cells （MSCs） can differentiate into endothelial cells. The effect of shear stress on MSC differentiation is incompletely understood, and most studies have been based on two-dimensional systems. We used a model of tissue-engineered vascular MSC differentiation. grafts （TEVGs） to investigate the effects of shear stress on Methods： MSCs were isolated from canine bone marrow. The TEVG was constructed by seeding MSCs onto poly-ε- caprolactone and lactic acid （PCLA） scaffolds and subjecting them to shear stress provided by a pulsatile bioreactor for four days （two days at 1 dyne/cm^2 to 15 dyne/cm^2 and two days at 15 dyne/cm^2）. Results： Shear stress significantly increased the expression of endothelial cell markers, such as platelet-endothelial cell adhesion molecule-1 （PECAM-1）, VE-cadherin, and CD34, at both the mRNA and protein levels as compared with static control cells. Protein levels of alpha-smooth muscle actin （α-SMA） and calponin were substantially reduced in shear stresscultured cells. There was no significant change in the expression of α-SMA, smooth muscle myosin heavy chain （SMMHC） or calponin at the mRNA level. Conclusion： Shear stress upregulated the expression of endothelial cell-related markers and downregulated smooth muscle-related markers in canine MSCs. This study may serve as a basis for further investigation of the effects of shear stress on MSC differentiation in TEVGs.     

Genipin inhibits endothelial exocytosis via nitric oxide in cultured human umbilical vein endothelial cells

Aim： Exocytosis of endothelial Weibel-Palade bodies, which contain von Willebrand factor （VWF）, P-selectin and other modulators, plays an important role in both inflammation and thrombosis. The present study investigates whether genipin, an aglycon of geniposide, inhibits endothelial exocytosis. Methods： Human umbilical vein endothelial cells （HUVECs） were isolated from umbilical cords and cultured. The concentration of VWF in cell supernatants was measured using an ELISA Kit. P-selectin translocation on the cell surface was analyzed by cell surface ELISA. Cell viability was measured using a Cell Counting Kit-8. Mouse bleeding times were measured by amputating the tail tip. Western blot analysis was used to determine the amount of endothelial nitric oxide synthase （eNOS） and phospho-eNOS present. Nitric oxide （NO） was measured in the cell supernatants as nitrite using an NO Colorimetric Assay. Results： Genipin inhibited thrombin-induced VWF release and P-selectin translocation in HUVECs in a dose- and time- dependent manner. The drug had no cytotoxic effect on the cells at the same doses that were able to inhibit exocytosis. The functional study that demonstrated that genipin inhibited exocytosis in vivo also showed that genipin prolonged the mouse bleeding time. Furthermore, genipin activated eNOS phosphorylation, promoted enzyme activation and increased NO production. L-NAME, an inhibitor of NOS, reversed the inhibitory effects of genipin on endothelial exocytosis. Conclusion： Genipin inhibits endothelial exocytosis in HUVECs. The mechanism by which this compound inhibits exocytosis may be related to its ability to stimulate eNOS activation and NO production. Our findings suggest a novel antiinflammatory mechanism for genipin. This compound may represent a new treatment for inflammation and/or thrombosis in which excess endothelial exocytosis plays a pathophysiological role.     

Dauricine inhibits insulin-like growth factor-Ⅰ-induced hypoxia inducible factor la protein accumulation and vascular endothelial growth factor expression in human breast cancer cells

Aim： To investigate the effects of dauricine （Dau） on insulin-like growth factor-Ⅰ （IGF-Ⅰ）-induced hypoxia inducible factor 1α （HIF-1α） and vascular endothelial growth factor （VEGF） expression in human breast cancer cells （MCF-7）. Methods： Serum-starved MCF-7 cells were pretreated for 1 h with different concentrations of Dau, followed by incubation with IGF-Ⅰ for 6 h. HIF-1α and VEGF protein expression levels were analyzed by Western blotting and ELISA, respectively. HIF-1α and VEGF mRNA levels were determined by real-time PCR. In vitro angiogenesis was observed via the human umbilical vein endothelial cell （HUVEC） tube formation assay. An in vitro invasion assay on HUVECs was performed. Results： Dau significantly inhibited IGF-Ⅰ-induced HIF-1α protein expression but had no effect on HIF-1α mRNA expression. However, Dau remarkably suppressed VEGF expression at both protein and mRNA levels in response to IGF-Ⅰ. Mechanistically, Dau suppressed IGF-Ⅰ-induced HIF-1α and VEGF protein expression mainly by blocking the activation of PI-3K/AKT/mTOR signaling pathway. In addition, Dau reduced IGF-Ⅰ-induced HIF-1α protein accumulation by inhibiting its synthesis as well as by promoting its degradation. Functionally, Dau inhibited angiogenesis in vitro. Moreover, Dau had a direct effect on IGF-Ⅰ-induced invasion of HUVECs. Conclusion： Dau inhibits human breast cancer angiogenesis expression, which may provide a novel potential mechanism for by suppressing HIF-1α protein accumulation and VEGF the anticancer activities of Dau in human breast cancer.     

The peroxisome proliferator-activated receptor-α （PPAR-α） agonist,AVE8134, attenuates the progression of heart failure and increases survival in rats

Aim： To investigate the efficacy of the peroxisome proliferator-activated receptor-α （PPARa） agonist, AVE8134, in cellular and experimental models of cardiac dysfunction and heart failure.
Methods： In Sprague Dawley rats with permanent ligation of the left coronary artery （post-MI）, AVE8134 was compared to the PPARy agonist rosiglitazone and in a second study to the ACE inhibitor ramipril. In DOCA-salt sensitive rats, efficacy of AVE8134 on cardiac hypertrophy and fibrosis was investigated. Finally, AVE8134 was administered to old spontaneously hypertensive rats （SHR） at a nonblood pressure lowering dose with survival as endpoint. In cellular models, we studied AVE8134 on hypertrophy in rat cardiomyocytes nitric oxide signaling in human endothelial cells （HUVEC） and LDL-uptake in human MonoMac-6 cells.
Results： In post-MI rats, AVE8134 dose-dependently improved cardiac output, myocardial contractility and relaxation and reduced lung and left ventricular weight and fibrosis. In contrast, rosiglitazone exacerbated cardiac dysfunction. Treatment at AVE8134 decreased plasma proBNP and arginine and increased plasma citrulline and urinary NOx/creatinine ratio. In DOCA rats, AVE8134 prevented development of high blood pressure, myocardial hypertrophy and cardiac fibrosis, and ameliorated endothelial dysfunction. Compound treatment increased cardiac protein expression and phosphorylation of eNOS. In old SHR, treatment with a low dose of AVE8134 improved cardiac and vascular function and increased life expectancy without lowering blood pressure. AVE8134 reduced phenylephrine-induced hypertrophy in adult rat cardiomyocytes. In HUVEC, Ser-1177-eNOS phosphorylation but not eNOS expression was increased. In monocytes, AVE8134 increased the expression of CD36 and the macrophage scavenger receptor 1, resulting in enhanced uptake of oxidized LDL.
Conclusion： The PPARa agonist AVE8134 prevents post-MI myocardial hypertrophy, fibrosis and cardiac dysfunction. AVE8134 has beneficial effects against hy     

Macrophage receptors of polysaccharide isolated from a marine filamentous fungus Phoma herbarum YS4108

YCP, a novel （1,4）-α-D-glucan, was isolated from the mycelium of the marine filamentous fungus Phoma herbarum YS4108. In this work, we investigated a YCP-binding cellular receptor expressed by macrophages and the intracellular signal transduction pathways involved in YCP-induced macrophage activation.
Methods： Fluorescence-labeled YCP （fl-YCP） was prepared using the CDAP-activation method. Fluorescence confocal laser microscopy and fluorescence-activated cell sorting （FACS） were used to analyze the effect of fl-YCP on macrophages. To characterize the properties of the YCP receptor, carbohydrates and antibodies were used to inhibit the binding of fl-YCP to macrophages. Moreover, we investigated the role of membrane receptors Toll-like receptor 2 （TLR2）, Toll-like receptor 4 （TLR4）, Toll-like receptor 6 （TLR6） and complement receptor 3 （CR3）. We also examined the role of the p38 kinase pathway in mediating nitric oxide （NO） production.
Results： YCP had an in vitro stimulatory effect on the release of NO in macrophage, and fI-YCP can bind directly to receptors on the surface of macrophages in a time- and dose-dependent manner. Competition studies show that LPS, laminarin, anti-TLR4 antibody and anti-CD11b （CR3） antibody could inhibit fl-YCP binding to macrophages. Conversely, mannose, anti-TLR2 and anti-TLR6 antibody could not. Treatment of RAW264.7 cells with YCP resulted in significant activation of p38 in a time-dependent manner. The specific p38 inhibitor SB203580 abrogated YCP-induced NO generation. Treatment of RAW264.7 cells with anti-TLR4 antibody and anti-CR3 antibody significantly reduced YCP-induced NO production and p38 activation.
Conclusion： We have demonstrated that YCP-induced NO production occurs through the TLR4 and CR3 membrane receptors in a p38 kinase-dependent manner in macrophages.     

Allosteric activation mechanism of the cys-loop receptors

Binding of a neurotransmitter to its ionotropic receptor opens a distantly located ion channel, a process termed allosteric activation. Here we review recent advances in the molecular mechanism by which the cys-loop receptors are activated with emphasis on the best studied nicotinic acetylcholine receptors （nAChRs）. With a combination of affinity labeling, mutagenesis, electrophysiology, kinetic modeling, electron microscopy （EM）, and crystal structure analysis, the allosteric activation mechanism is emerging. Specifically, the binding domain and gating domain are interconnected by an allosteric activation network. Agonist binding induces conformational changes, resulting in the rotation of a β sheet of aminoterminal domain and outward movement of loop 2, loop F, and cys-loop, which are coupled to the M2-M3 linker to pull the channel to open. However, there are still some controversies about the movement of the channel-lining domain M2. Nine angstrom resolution EM structure of a nAChR imaged in the open state suggests that channel opening is the result of rotation of the M2 domain. In contrast, recent crystal structures of bacterial homologues of the cys-loop receptor family in apparently open state have implied an M2 tilting model with pore dilation and quaternary twist of the whole pentameric receptor. An elegant study of the nAChR using protonation scanning of M2 domain supports a similar pore dilation activation mechanism with minimal rotation of M2. This remains to be validated with other approaches including high resolution structure determination of the mammalian cys-loop receptors in the open state.     

Addictive evaluation of cholic acid-verticinone ester,a potential cough therapeutic agent with agonist action of opioid receptor

Aim： The purpose of this work was to search for potential drugs with potent antitussive and expectorant activities as well as a low toxicity, but without addictive properties. Cholic acid-verticinone ester （CA-Ver） was synthesized based on the clearly elucidated antitussive and expectorant activities of verticinone in bulbs of Fritillaria and different bile acids in Snake Bile. In our previous study, CA-Ver showed a much more potent activity than codeine phosphate. This study was carried out to investigate the central antitussive mechanism and the addictive evaluation of CA-Ver. Methods： Testing on a capsaicin-induced cough model of mice pretreated with naloxone, a non-selective opioid receptor antagonist, was performed for the observation of CA-Ver＇s central antitussive mechanism. We then took naloxone-induced withdrawal tests of mice for the judgment of CA-Ver＇s addiction. Lastly, we determined the opioid dependence of CA-Ver in the guinea pig ileum. Results： The test on the capsaicin-induced cough model showed that naloxone could block the antitussive effect of CA-Ver, suggesting the antitussive mechanism of CA-Ver was related to the central opioid receptors. The naloxone-urged withdrawal tests of the mice showed that CA-Ver was not addictive, and the test of the opioid dependence in the guinea pig ileum showed that CA-Ver had no withdrawal response. Conclusion： These findings suggested that CA-Ver deserved attention for its potent antitussive effects related to the central opioid receptors, but without addiction, and had a good development perspective.     

Nicotinic acetylcholine receptor α7 subunits with a C2 cytoplasmic loop yellow fluorescent protein insertion form functional receptors

Aim： Several nicotinic acetylcholine receptor （nAChR） subunits have been engineered as fluorescent protein （FP） fusions and exploited to illuminate features of nAChRs. The aim of this work was to create a FP fusion in the nAChR α7 subunit without compromising formation of functional receptors, Methods： A gene construct was generated to introduce yellow fluorescent protein （YFP）, in frame, into the otherwise unaltered, large, second cytoplamsic loop between the third and fourth transmembrane domains of the mouse nAChR α7 subunit （α7Y）. SH-EP1 cells were transfected with mouse nAChR wild type α7 subunits （α7） or with α7Y subunits, alone or with the chaperone protein, hRIC-3. Receptor function was assessed using whole-cell current recording. Receptor expression was measured with 125I-labeled α-bungarotoxin （I-Bgt） binding, laser scanning confocal microscopy, and total internal reflectance fluorescence （TIRF） microscopy. Results： Whole-cell currents revealed that α7Y nAChRs and α7 nAChRs were functional with comparable EC50 values for the α7 nAChR-selective agonist, choline, and IC50 values for the α7 nAChR-selective antagonist, methyllycaconitine. I-Bgt binding was detected only after co-expression with hRIC-3. Confocal microscopy revealed that α7Y had primarily intracellular rather than surface expression. TIRF microscopy confirmed that little α7Y localized to the plasma membrane, typical of α7 nAChRs. Conclusion： nAChRs composed as homooligomers of α7Y subunits containing cytoplasmic loop YFP have functional, ligand binding, and trafficking characteristics similar to those of α7 nAChRs, α7Y nAChRs may be used to elucidate properties of α7 nAChRs and to identify and develop novel probes for these receptors, perhaps in high-throughput fashion.     

The impact of extracellular and intracellular Ca2＋ on ethanol-induced smooth muscle contraction

Aim： To evaluate the impact of extracellular and intracellular Ca2＋ on contractions induced by ethanol in smooth muscle. Methods： Longitudinal smooth muscle strips were prepared from the gastric fundi of mice. The contractions of smooth muscle strips were recorded with an isometric force displacement transducer. Results： Ethanol （164 mmol/L） produced reproducible contractions in isolated gastric fundal strips of mice. Although lidocaine （50 and 100 μmol/L）, a local anesthetic agent, and hexamethonium （100 and 500 μmol/L）, a ganglionic blocking agent, failed to affect these contractions, verapamil （1-50 μmol/L） and nifedipine （1-50 pmol/L）, selective blockers of L-type Ca2＋ channels, significantly inhibited the contractile responses of ethanol. Using a Ca2＋-free medium nearly eliminated these contractions in the same tissue. Ryanodine （1-50 μmol/L） and ruthenium red （10-100 pmol/L）, selective blockers of intracellular Ca2＋ channels/ryanodine receptors; cyclopiazonic acid （CPA; 1-10 μmol/L）, a selective inhibitor of sarcoplasmic reticulum （SR） Ca2＋-ATPase; and caffeine （0.5-5 mmol/L）, a depleting agent of intracellular Ca2＋ stores, significantly inhibited the contractile responses induced by ethanol. In addition, the com- bination of caffeine （5 mmol/L） plus CPA （10 μmol/L）, and ryanodine （10 μmol/L） plus CPA （10 μmol/L）, caused further inhibition of contractions in response to ethanol. This inhibition was significantly different from those associated with caffeine, ryanodine or CPA. Furthermore the combination of caffeine （5 mmol/L）, ryanodine （10 μmol/L） and CPA（IO pmol/L） eliminated the contractions induced by ethanol in isolated gastric fundal strips of mice. Conclusion： Both extracellular and intracellular Ca2＋ may have important roles in regulating contractions induced by ethanol in the mouse gastric fundus.     

Central cholinergic regulation of respiration： nicotinic receptors

Nicotinic acetylcholine receptors （nAChRs） are expressed in brainstem and spinal cord regions involved in the control of breathing. These receptors mediate central cholinergic regulation of respiration and effects of the exogenous ligand nicotine on respiratory pattern. Activation of α4＊ nAChRs in the preBotzinger Complex （preBotC）, an essential site for normal respiratory rhythm generation in mammals, modulates excitatory glutamatergic neurotransmission and depolarizes preBotC inspiratory neurons, leading to increases in respiratory frequency, nAChRs are also present in motor nuclei innervating respiratory muscles. Activation of post- and/or extra-synaptic α4＊ nAChRs on hypoglossal （XII） motoneurons depolarizes these neurons, potentiating tonic and respiratory-related rhythmic activity. As perinatal nicotine exposure may contribute to the pathogenesis of sudden infant death syndrome （SIDS）, we discuss the effects of perinatal nicotine exposure on development of the cholinergic and other neurotransmitter systems involved in control of breathing. Advances in understanding of the mechanisms underlying central cholinergic/nicotinic modulation of respiration provide a pharmacological basis for exploiting nAChRs as therapeutic targets for neurological disorders related to neural control of breathing such as sleep apnea and SIDS.     

Pivotal effects of phosphodiesterase inhibitors on myocyte contractility and viability in normal and ischemic hearts

Phosphodiesterases （PDEs） are enzymes that degrade cellular cAMP and cGMP and are thus essential for regulating the cyclic nucleotides. At least 11 families of PDEs have been identified, each with a distinctive structure, activity, expression, and tissue distribution. The PDE type-3, -4, and -5 （PDE3, PDE4, PDE5） are localized to specific regions of the cardiomyocyte, such as the sarcoplasmic reticulum and Z-disc, where they are likely to influence cAMP/cGMP signaling to the end effectors of contractility. Several PDE inhibitors exhibit remarkable hemodynamic and inotropic properties that may be valuable to clinical practice. In particular, PDE3 inhibitors have potent cardiotonic effects that can be used for short-term inotropic support, especially in situations where adrenergic stimulation is insufficient. Most relevant to this review, PDE inhibitors have also been found to have cytoprotective effects in the heart. For example, PDE3 inhibitors have been shown to be cardioprotective when given before ischemic attack, whereas PDE5 inhibitors, which include three widely used erectile dysfunction drugs （sildenafil, vardenafil and tadalafil）, can induce remarkable cardioprotection when administered either prior to ischemia or upon reperfusion. This article provides an overview of the current laboratory and clinical evidence, as well as the cellular mechanisms by which the inhibitors of PDE3, PDE4 and PDE5 exert their beneficial effects on normal and ischemic hearts. It seems that PDE inhibitors hold great promise as clinically applicable agents that can improve cardiac performance and cell survival under critical situations, such as ischemic heart attack, cardiopulmonary bypass surgery, and heart failure.     

Expression pattern of neural synaptic plasticity marker-Arc in different brain regions induced by conditioned drug withdrawal from acute morphine-dependent rats

Aim： The immediate early gene Arc （activity-regulated cytoskeletal-associated protein） mRNA and protein are induced by strong synaptic activation and rapidly transported into dendrites, where they localize at active synaptic sites. Thus, the Arc mRNA and protein are proposed as a marker of neuronal reactivity to map the neural substrates that are recruited by various stimuli. In the present study, we examined the expression of Arc protein induced by conditioned naloxone-precipitated drug withdrawal in different brain regions of acute morphine-dependent rats. The objective of the present study was to address the specific neural circuits involved in conditioned place aversion （CPA） that has not yet been well characterized. Methods： Place aversion was elicited by conditioned naloxone-precipitated drug withdrawal following exposure to a single dose of morphine. An immunohistochemical method was employed to detect the expression of Arc, which was used as a plasticity marker to trace the brain areas that contribute to the formation of the place aversion. Results： Marked increases in Arc protein levels were found in the medial and lateral prefrontal cortex, the sensory cortex, the lateral striatum and the amygdala. This effect was more pronounced in the basolateral amygdala （BLA）, the central nucleus of the amygdala （CeA）, and the bed nucleus of the striatal terminals （BNST） when compared with the control group. Conclusion： Our results suggest that these brain regions may play key roles in mediating the negative motivational component of opiate withdrawal.     

Glutamate receptors in the dorsal hippocampus mediate the acquisition,but not the expression,of conditioned place aversion induced by acute morphine withdrawal in rats

Aim： To evaluate the role of glutamate receptors in the dorsal hippocampus （DH） in the motivational component of morphine withdrawal. Methods： NMDA receptor antagonist D-AP5 （5 pg/0.5 pL per side） or AMPA receptor antagonist NBQX （2 pg/0.5 pL per side） was micro- injected into DH of rats. Conditioned place aversion （CPA） induced by naloxone-precipitated morphine withdrawal were assessed. Results： Preconditioning microinjection of D-AP5 or NBQX into the DH impaired the acquisition of CPA in acute morphine-dependent rats. However, intra-DH microinjection of D-AP5 or NBQX after conditioning but before the testing session had no effect on the expres- sion of CPA. Conclusion： Our results suggest that NMDA and AMPA receptors in the dorsal hippocampus are involved in the acquisition, but not in the expression, of the negative motivational components of acute morphine withdrawal in rats.     

Natural products as promising drug candidates for the treatment of hepatitis B and C

Hepatitis B virus （HBV） or hepatitis C virus （HCV） infections are a major threat worldwide. Combination therapy of interferon-α and ribavirin is currently the treatment of choice for HCV-infected patients. However, this regimen is only effective in approximately 50% of patients and provokes severe side-effects. Numerous natural alternatives for treating HCV have been suggested. Deoxynojirimycin and its derivatives are iminosugars which exert anti-HCV activity by inhibiting α-glucosidases. A non-immunosuppressive derivate of cyclosporine A, NIM811, exerts anti-HCV activity by binding to cyclophilin. Other natural products with promising anti-HCV activity are 2-arylbenzofuran derivatives, Mellein, and pseudoguaianolides. For HBV treatment, several drugs are available, specifically targeting the virus polymerase （lamivudine, entecavir, telbivudine, and adefovir dipivoxil）. The efficacy of these drugs is hampered by the development of resistance due to point mutations in the HBV polymerase. Due to drug resistance and adverse side-effects, the search for novel drugs is mandatory. Wogonin, ellagic acid, artemisinin and artesunate, chrysophanol 8-O-β-D-glucoside, saikosaponin C, and protostane triterpenes are active against HBV. Natural products need to be investigated in more detail to explore their potential as novel adjuncts to established HBV or HCV therapy.     

Increase in sphingolipid catabolic enzyme activity during aging

Aim： To understand the contribution of sphingolipid metabolism and its metabolites to development and aging. Methods： A systemic analysis on the changes in activity of sphingolipid metabolic enzymes in kidney, liver and brain tissues during development and aging was conducted. The study was conducted using tissues from 1-day-old to 720-day-old rats. Results： Catabolic enzyme activities as well as the level of sphingomyelinase （SMase） and ceramidase （CDase） were higher than that of anabolic enzyme activities, sphingomyelin synthase and ceramide synthase. This suggested an accumulation of ceramide and sphingosine during development and aging. The liver showed the highest neutral-SMase activity among the tested enzymes while the kidney and brain exhibited higher neutral-SMase and ceramidase activities, indicating a high production of ceramide in liver and ceramide/sphingosine in the kidney and brain. The activities of sphingolipid metabolic enzymes were significantly elevated in all tested tissues during development and aging, although the onset of significant increase in activity varied on the tissue and enzyme type. During aging, 18 out of 21 enzyme activities were further increased on day 720 compared to day 180. Conclusion： Differential increases in sphingolipid metabolic enzyme activities suggest that sphingolipids including ceramide and sphingosine might play important and dynamic roles in proliferation, differentiation and apoptosis during development and aging.     

Functional study of the effect of phosphatase inhibitors on KCNQ4 channels expressed in Xenopus oocytes

Aim： KCNQ4 channels play an important part in adjusting the function of cochlear outer hair cells. The aim of this study was to investigate the effects of ser/thr phosphatase inhibitors on human KCNQ4 channels expressed in Xenopus laevis oocytes. Methods： Synthetic cRNA encoding human KCNQ4 channels was injected into Xenopus oocytes. We used a two-electrode voltage clamp to measure the ion currents in the oocytes. Results： Wild-type KCNQ4 expressed in Xenopus oocytes showed the typical properties of slow activation kinetics and low threshold activation. The outward K~ current was almost completely blocked by a KCNQ4 blocker, linopirdine （0.25 mmol/L）. BIMI （a PKC inhibitor） prevented the effects of PMA （a PKC activator） on the KCNQ4 current, indicating that PKC may be involved in the regulation of KCNQ4 expressed in the Xenopus oocyte system. Treatment with the ser/thr phosphatase inhibitors, cyclosporine （2 pmol/L）, calyculin A （2 pmol/L） or okadaic acid （1 pmol/L）, caused a significant positive shift in V1/2 and a decrease in the conductance of KCNQ4 channels. The V1/2 was shifted from -14.6±0.5 to -6.4±0.4 mV by cyclosporine, -18.8±0.5 to -9.2_＋0.4 mV by calyculin A, and -14.1±0.5 to -0.7＋0.6 mV by okadaic acid. Moreover, the effects of these phosphatase inhibitors （okadaic acid or calyculin A） on the induction of a positive shift of V1/2 were augmented by further addition of PMA. Conclusion： These results indicate that ser/thr phosphatase inhibitors can induce a shift to more positive potentials of the activation curve of the KCNQ4 current. It is highly likely that the phosphatase functions to balance the phosphorylated state of substrate protein and thus has an important role in the regulation of human KCNQ4 channels expressed in Xenopus oocytes.