Type 2 diabetes mellitus(T2DM)is a lifelong condition and a threat to human health.Thorough understanding of its pathogenesis is acutely needed in order to devise innovative,preventative,and potentially curative pharm...Type 2 diabetes mellitus(T2DM)is a lifelong condition and a threat to human health.Thorough understanding of its pathogenesis is acutely needed in order to devise innovative,preventative,and potentially curative pharmacological interventions.MicroRNAs(miRNA),are small,non-coding,one-stranded RNA molecules,that can target and silence around 60%of all human genes through translational repression.MiR-155 is an ancient,evolutionarily well-conserved miRNA,with distinct expression profiles and multifunctionality,and a target repertoire of over 241 genes involved in numerous physiological and pathological processes including hematopoietic lineage differentiation,immunity,inflammation,viral infections,cancer,cardiovascular conditions,and particularly diabetes mellitus.MiR-155 Levels are progressively reduced in aging,obesity,sarcopenia,and T2DM.Thus,the loss of coordinated repression of multiple miR-155 targets acting as negative regulators,such as C/EBPβ,HDAC4,and SOCS1 impacts insulin signaling,deteriorating glucose homeostasis,and causing insulin resistance(IR).Moreover,deranged regulation of the renin angiotensin aldosterone system(RAAS)through loss of Angiotensin II Type 1 receptor downregulation,and negated repression of ETS-1,results in unopposed detrimental Angiotensin II effects,further promoting IR.Finally,loss of BACH1 and SOCS1 repression abolishes cytoprotective,anti-oxidant,anti-apoptotic,and anti-inflam matory cellular pathways,and promotesβ-cell loss.In contrast to RAAS inhibitor treatments that further decrease already reduced miR-155 Levels,strategies to increase an ailing miR-155 production in T2DM,e.g.,the use of metformin,mineralocorticoid receptor blockers(spironolactone,eplerenone,finerenone),and verapamil,alone or in various combinations,represent current treatment options.In the future,direct tissue delivery of miRNA analogs is likely.展开更多
In the perifused fura-2 loaded exocrine pancreatic acinar cell line AR4-2J pulses of high potassium induced repetitive increases in intracellular calcium. Attached cells when stimulated with high potassium secreted la...In the perifused fura-2 loaded exocrine pancreatic acinar cell line AR4-2J pulses of high potassium induced repetitive increases in intracellular calcium. Attached cells when stimulated with high potassium secreted large amount of amylase. High potassium-induced secretion was dependent both on the concentration of potassium and duration of stimulation. High potassium induced increases in intracellular calcium were inhibited by voltage-dependent calcium channel antagonists with an order of potency as follows: nifedipine > ω-agatoxin IVA > ω-conotoxin GVIA. In contrast, the L-type calcium channel antagonist nifedipine almost completely inhibited potassium-induced amylase secretion, whereas the N-type channel antagonist ω-conotoxin GVIA was without effect. The P-type channel antagonist ω-agatoxin IVA had a small inhibitory effect, but this inhibition was not significant at the level of amylase secretion. In conclusion, the AR4-2J cell line possesses different voltage-dependent calcium channels (L, P,N) with the L-type predominantly involved in depolarization induced amylase secretion.展开更多
On September 10, 2015, unprecedented flood was occurred in Kinugawa River basin located on eastern Japan. It inundated 40 km2 of flood plain in Joso city, Ibaraki Prefecture, and more than 4000 people there called for...On September 10, 2015, unprecedented flood was occurred in Kinugawa River basin located on eastern Japan. It inundated 40 km2 of flood plain in Joso city, Ibaraki Prefecture, and more than 4000 people there called for help despite supposedly having sufficient time to evacuate. Some said that small initial flood before main severe flood arrived made them make a mistake in deciding whether to evacuate or stay there, despite having to actually evacuate in reality. This study focused on flood behaviour in this area, in particular, the effect of a small drainage channel lying on the flood plain which caused fast flood diffusion in case of occurring huge overflowing. Field investigations starting on time of the disaster with high-resolution positioning system were conducted to obtain spatial maps of flood depth and height. For appropriate modelling of the effect of small channel, we applied simulation model coupling 1-dimensional (1D) and 2-dimensional (2D) hydraulic scheme on the field and compared results from the 1D/2D coupled model and model without 1D scheme. The models provided information that the flood could reach 4 hours earlier to the city central of Joso city comparing in case of model without 1D scheme. The water depth rose irregularly and it was more confusing and difficult for the victims to make appropriate evacuation act.展开更多
Objective To investigate the effect of(-)-Stepholidine(SPD)on enhancing D1 receptor mediated contraction of cardiac muscle in isolated rat heart and to examine whether SPD has a direct effect on the heart dopamine D1 ...Objective To investigate the effect of(-)-Stepholidine(SPD)on enhancing D1 receptor mediated contraction of cardiac muscle in isolated rat heart and to examine whether SPD has a direct effect on the heart dopamine D1 receptors.SPD an active ingredient of the Chinese herb Stephania intermedia,binds to dopamine D1 and D2 like receptors.Biochemical,electrophysiological and behavioural experiments have provided strong evidence that SPD is both a D(1/5)agonist and a D(2/4)antagonist,which could indicate unique antipsychotic properties.Methods Normal adult rat working hearts were isolated by Langendorff technique.Results SPD significantly increased the cardiac muscle contraction in a dose-dependent manner.The selective D1 dopamine receptor antagonist SCH23390(1 μM)blocked the SPD induced heart contraction,however,neither the β-receptor antagonist propranolol(1 μM)nor the α1-receptor antagonist prazosin(1 μM)had any effect on blocking SPD induced heart contractions.Moreover,the L-type Ca2+ channel inhibitor nimodipine(1 μM)completely blocked the effect of SPD on cardiac muscle contraction.Conclusions SPD show the effect on enhancing contraction of isolated rat heart through activating L-type Ca2+ channel mediated by heart D1 receptors.展开更多
Anoctamin 1(ANO1) or TMEM16 A gene encodes a member of Ca^(2+) activated Cl^(-) channels(CaCCs) that are critical for physiological functions,such as epithelial secretion,smooth muscle contraction and sensory signal t...Anoctamin 1(ANO1) or TMEM16 A gene encodes a member of Ca^(2+) activated Cl^(-) channels(CaCCs) that are critical for physiological functions,such as epithelial secretion,smooth muscle contraction and sensory signal transduction.The attraction and interest in ANO1/TMEM16 A arise from a decade long investigations that abnormal expression or dysfunction of ANO1 is involved in many pathological phenotypes and diseases,including asthma,neuropathic pain,hypertension and cancer.However,the lack of specific modulators of ANO1 has impeded the efforts to validate ANO1 as a therapeutic target.This review focuses on the recent progress made in understanding of the pathophysiological functions of CaCC ANO1 and the current modulators used as pharmacological tools,hopefully illustrating a broad spectrum of ANO1 channelopathy and a path forward for this target validation.展开更多
Using fura-2-acetoxymethyl ester (AM) fluorescence imaging and patch clamp techniques, we found that endothelin-1 (ET-1) significantly elevated the intracellular calcium level ([Ca2+]i) in a dose-dependent manner and ...Using fura-2-acetoxymethyl ester (AM) fluorescence imaging and patch clamp techniques, we found that endothelin-1 (ET-1) significantly elevated the intracellular calcium level ([Ca2+]i) in a dose-dependent manner and activated the L-type Ca2+ channel in cardiomyocytes isolated from rats. The effect of ET-1 on [Ca2+]i elevation was abolished in the presence of the ETA receptor blocker BQ123, but was not affected by the ETB receptor blocker BQ788. ET-1-induced an increase in [Ca2+]i, which was inhibited 46.7% by pretreatment with a high concentration of ryanodine (10 μmol/L), a blocker of the ryanodine receptor. The ET-1-induced [Ca2+]i increase was also inhibited by the inhibitors of protein kinase A (PKA), protein kinase C (PKC) and angiotensin type 1 receptor (AT1 receptor). We found that ET-1 induced an enhancement of the amplitude of the whole cell L-type Ca2+ channel current and an increase of open-state probability (NPo) of an L-type single Ca2+ channel. BQ123 completely blocked the ET-1-induced increase in calcium channel open-state probability. In this study we demonstrated that ET-1 regulates calcium overload through a series of mechanisms that include L-type Ca2+ channel activation and Ca2+-induced Ca2+ release (CICR). ETA receptors, PKC, PKA and AT1 receptors may also contribute to this pathway.展开更多
STIM1 is an endoplasmic reticulum(ER) protein with a key role in Ca^(2+)mobilization. Due to its ability to act as an ER-intraluminal Ca^(2+) sensor, it regulates store-operated Ca^(2+) entry(SOCE), which is a Ca^(2+)...STIM1 is an endoplasmic reticulum(ER) protein with a key role in Ca^(2+)mobilization. Due to its ability to act as an ER-intraluminal Ca^(2+) sensor, it regulates store-operated Ca^(2+) entry(SOCE), which is a Ca^(2+) influx pathway involved in a wide variety of signalling pathways in eukaryotic cells. Despite its important role in Ca^(2+) transport, current knowledge about the role of STIM1 in neurons is much more limited. Growing evidence supports a role for STIM1 and SOCE in the preservation of dendritic spines required for long-term potentiation and the formation of memory. In this regard, recent studies have demonstrated that the loss of STIM1, which impairs Ca^(2+) mobilization in neurons, risks cell viability and could be the cause of neurodegenerative diseases. The role of STIM1 in neurodegeneration and the molecular basis of cell death triggered by low levels of STIM1 are discussed in this review.展开更多
The purpose of this study was to examine the relaxation effect of CY on the vascular smooth muscle （VSM） from rabbits. Experiments were carried out on isolated thoracic aorta of rabbits. CY （3 x 103 mM- 3 mM） coul...The purpose of this study was to examine the relaxation effect of CY on the vascular smooth muscle （VSM） from rabbits. Experiments were carried out on isolated thoracic aorta of rabbits. CY （3 x 103 mM- 3 mM） could relax the VSM preparations pre-contracted by adrenaline （AD）, noradrenaline （NE）, high-K^＋ solution or BaCl2 with respective EC50 values of （0.3 1±0.11） mM, 0.19±0.03 mM, 0.20±0.04 mM and 0.25±0.04 mM. Moreover, CY （10-2 mM, 0.1 mM and 1 mM） inhibited norepinephrine （NE）, CaCl2 and KCl-induced vasoconstriction in a concentration dependent manner. The phasic contraction produced by NE was concentration dependently attenuated with CY （10^-2 mM, 0.1 mM and 1 mM） in calcium-free medium, similar to that caused by verapamil. The present findings suggest that CY relaxed thoracic aortic rings by blocking voltage-dependent Ca^2＋ channels. The inhibition of intracellular Ca^2＋ release may be one of the main vasorelaxant mechanisms of CY.展开更多
Objective Presynaptic voltage-gated Ca^2＋ channels mediate rapid Ca^2＋ influx into the synaptic terminal which triggers synaptic vesicle exocytosis and neurotransmitter release. The FM 1-43 dye was firstly introduce...Objective Presynaptic voltage-gated Ca^2＋ channels mediate rapid Ca^2＋ influx into the synaptic terminal which triggers synaptic vesicle exocytosis and neurotransmitter release. The FM 1-43 dye was firstly introduced as a fluorescence probe by Betz and his colleagues in 1992, and has been used to monitor exocytosis, endocytosis and endosomal traffic in a variety of cell types. The present study aims to investigate the feasibility of applying the FM 1-43 dye in the functional analysis of calcium channel-mediated exocytosis in synaptic terminals. Methods The hippocampi were isolated from embryos of pregnant rats, and hippocampal neurons were then transfected with Ds-Red conjugated plasmid. The neurons were then loaded with 8 μmol/L FM 1-43 and 47 mmol/L KCl for 90 s after transfection. After that, 90 mmol/L KCI was employed to induce FM dye destaining, which was recorded by FM imaging system. Results The neuron synaptic terminals of rat hippocampus could be effectively stained by the FM 1-43 dye. Besides, the destaining of the labeled neuron terminals was in accordance with the transmitter release, which could be blocked by the application of nifedipine （inhibitor for L-type calcium channel）. Conclusion The FM imaging technique is an advanced and effective method for analyzing synaptic vesicle exocytosis and neurotransmitter release, and can be applied in various synaptic functional studies.展开更多
The α2δ-1 subunit of the voltage-gated Ca2＋ channel （VGCC） is a molecular target of gabapentin （GBP）, which has been used as a first-line drug for the relief of neuropathic pain. GBP exerts its anti-nociceptive...The α2δ-1 subunit of the voltage-gated Ca2＋ channel （VGCC） is a molecular target of gabapentin （GBP）, which has been used as a first-line drug for the relief of neuropathic pain. GBP exerts its anti-nociceptive effects by disrupting trafficking of the α2δ-1 subunit to the presynaptic membrane, resulting in decreased neurotrans- mitter release. We previously showed that GBP has an anti- allodynic effect in the first two weeks; but this is followed by insensitivity in the later stage after repeated adminis- tration in a rat model of central post-stroke pain （CPSP） hypersensitivity induced by intra-thalamic hemorrhage. To explore the mechanisms underlying GBP insensitivity, the cellular localization and time-course of expression of the α2δ-1 subunit in both the thalamus and spinal dorsal horn were studied in the same model. We found that the α2δ-1 subunit was mostly localized in neurons, but not astrocytes and microglia. The level of α2δ-1 protein increased in the first two weeks after injury but then decreased in the third week, when GBP insensitivity occurred. Furthermore, the c^2g-1 down-regulation was likely caused by later neuronal loss in the injured thalamus through a mechanism other than apoptosis. In summary, the present results suggest that the GBP receptor ~2^-1 is mainly expressed in thalamic neurons in which it is up-regulated in the early stage of CPSP but this is followed by dramatic down-regulation, which is likely associated with GBP insensitivity after long-term use.展开更多
AIM:To investigate the pathway(s)mediating rat antral circular smooth muscle contractile responses to the cholinomimetic agent,bethanechol and the subtypes of muscarinic receptors mediating the cholinergic contraction...AIM:To investigate the pathway(s)mediating rat antral circular smooth muscle contractile responses to the cholinomimetic agent,bethanechol and the subtypes of muscarinic receptors mediating the cholinergic contraction. METHODS:Circular smooth muscle strips from the antrum of Sprague-Dawley rats were mounted in muscle baths in Krebs buffer.Isometric tension was recorded.Cumulative concentration-response curves were obtained for(+)-cis- dioxolane(cD),a nonspecific muscarinic agonist,at 10^(-8)- 10^(-4)mol/L,in the presence of tetrodotoxin(TTX,10^(-7)mol/L). Results were normalized to cross sectional area.A repeat concentration-response curve was obtained after incubation of the muscle for 90 min with antagonists for M1(pirenzepine), M2(methoctramine)and M3(darifenadn)muscarinic receptor subtypes.The sensitivity to PTX was tested by the ip injection of 100 mg/kg of PTX 5 d before the experiment.The antral circular smooth muscles were removed from PTX-treated and non-treated rats as strips and dispersed smooth muscle cells to identify whether PTX-linked pathway mediated the contractility to bethanechol. RESULTS:A dose-dependent contractile response observed with bethanechol,was not affected by TTx.The pretreatment of rats with pertussis toxin decreased the contraction induced by bethanechol.Lack of calcium as well as the presence of the L-type calcium channel blocker,nifedipine,also inhibited the cholinergic contraction,with a reduction in response from 2.5±0.4 g/mm^2 to 1.2±0.4 g/mm^2(P<0.05).The dose- response curves were shifted to the right by muscarinic antagonists in the following order of affinity:darifenacin (M_3)>methocramine(M_2)>pirenzepine(M_1). CONCLUSION:The muscarinic receptors-dependent contraction of rat antral circular smooth muscles was linked to the signal transduction pathway(s)involving pertussis-toxin sensitive GTP-binding proteins and to extracellular calcium via L-type voltage gated calcium channels.The presence of the residual contractile response after the treatment with nifedipine,suggests that an additional pathway could mediate the cholinergic contraction.The involvement of more than one muscarinic receptor(functionally predominant type 3 over type 2)also suggests more than one pathway mediating the cholinergic contraction in rat antrum.展开更多
文摘Type 2 diabetes mellitus(T2DM)is a lifelong condition and a threat to human health.Thorough understanding of its pathogenesis is acutely needed in order to devise innovative,preventative,and potentially curative pharmacological interventions.MicroRNAs(miRNA),are small,non-coding,one-stranded RNA molecules,that can target and silence around 60%of all human genes through translational repression.MiR-155 is an ancient,evolutionarily well-conserved miRNA,with distinct expression profiles and multifunctionality,and a target repertoire of over 241 genes involved in numerous physiological and pathological processes including hematopoietic lineage differentiation,immunity,inflammation,viral infections,cancer,cardiovascular conditions,and particularly diabetes mellitus.MiR-155 Levels are progressively reduced in aging,obesity,sarcopenia,and T2DM.Thus,the loss of coordinated repression of multiple miR-155 targets acting as negative regulators,such as C/EBPβ,HDAC4,and SOCS1 impacts insulin signaling,deteriorating glucose homeostasis,and causing insulin resistance(IR).Moreover,deranged regulation of the renin angiotensin aldosterone system(RAAS)through loss of Angiotensin II Type 1 receptor downregulation,and negated repression of ETS-1,results in unopposed detrimental Angiotensin II effects,further promoting IR.Finally,loss of BACH1 and SOCS1 repression abolishes cytoprotective,anti-oxidant,anti-apoptotic,and anti-inflam matory cellular pathways,and promotesβ-cell loss.In contrast to RAAS inhibitor treatments that further decrease already reduced miR-155 Levels,strategies to increase an ailing miR-155 production in T2DM,e.g.,the use of metformin,mineralocorticoid receptor blockers(spironolactone,eplerenone,finerenone),and verapamil,alone or in various combinations,represent current treatment options.In the future,direct tissue delivery of miRNA analogs is likely.
文摘In the perifused fura-2 loaded exocrine pancreatic acinar cell line AR4-2J pulses of high potassium induced repetitive increases in intracellular calcium. Attached cells when stimulated with high potassium secreted large amount of amylase. High potassium-induced secretion was dependent both on the concentration of potassium and duration of stimulation. High potassium induced increases in intracellular calcium were inhibited by voltage-dependent calcium channel antagonists with an order of potency as follows: nifedipine > ω-agatoxin IVA > ω-conotoxin GVIA. In contrast, the L-type calcium channel antagonist nifedipine almost completely inhibited potassium-induced amylase secretion, whereas the N-type channel antagonist ω-conotoxin GVIA was without effect. The P-type channel antagonist ω-agatoxin IVA had a small inhibitory effect, but this inhibition was not significant at the level of amylase secretion. In conclusion, the AR4-2J cell line possesses different voltage-dependent calcium channels (L, P,N) with the L-type predominantly involved in depolarization induced amylase secretion.
文摘On September 10, 2015, unprecedented flood was occurred in Kinugawa River basin located on eastern Japan. It inundated 40 km2 of flood plain in Joso city, Ibaraki Prefecture, and more than 4000 people there called for help despite supposedly having sufficient time to evacuate. Some said that small initial flood before main severe flood arrived made them make a mistake in deciding whether to evacuate or stay there, despite having to actually evacuate in reality. This study focused on flood behaviour in this area, in particular, the effect of a small drainage channel lying on the flood plain which caused fast flood diffusion in case of occurring huge overflowing. Field investigations starting on time of the disaster with high-resolution positioning system were conducted to obtain spatial maps of flood depth and height. For appropriate modelling of the effect of small channel, we applied simulation model coupling 1-dimensional (1D) and 2-dimensional (2D) hydraulic scheme on the field and compared results from the 1D/2D coupled model and model without 1D scheme. The models provided information that the flood could reach 4 hours earlier to the city central of Joso city comparing in case of model without 1D scheme. The water depth rose irregularly and it was more confusing and difficult for the victims to make appropriate evacuation act.
文摘Objective To investigate the effect of(-)-Stepholidine(SPD)on enhancing D1 receptor mediated contraction of cardiac muscle in isolated rat heart and to examine whether SPD has a direct effect on the heart dopamine D1 receptors.SPD an active ingredient of the Chinese herb Stephania intermedia,binds to dopamine D1 and D2 like receptors.Biochemical,electrophysiological and behavioural experiments have provided strong evidence that SPD is both a D(1/5)agonist and a D(2/4)antagonist,which could indicate unique antipsychotic properties.Methods Normal adult rat working hearts were isolated by Langendorff technique.Results SPD significantly increased the cardiac muscle contraction in a dose-dependent manner.The selective D1 dopamine receptor antagonist SCH23390(1 μM)blocked the SPD induced heart contraction,however,neither the β-receptor antagonist propranolol(1 μM)nor the α1-receptor antagonist prazosin(1 μM)had any effect on blocking SPD induced heart contractions.Moreover,the L-type Ca2+ channel inhibitor nimodipine(1 μM)completely blocked the effect of SPD on cardiac muscle contraction.Conclusions SPD show the effect on enhancing contraction of isolated rat heart through activating L-type Ca2+ channel mediated by heart D1 receptors.
基金supported by grants from the National Natural Science foundation of China (81573410)the Ministry of Science and Technology of China (2018ZX09711001-004-006)+2 种基金Shandong Provincial Medicine and Health Technology Development Plan (2019WS145, China)Medical Research Guidance Plan of Qingdao Municipal Health Committee (2019-WJZD091, China)Qingdao Excellent Young Medical Talent Training Project。
文摘Anoctamin 1(ANO1) or TMEM16 A gene encodes a member of Ca^(2+) activated Cl^(-) channels(CaCCs) that are critical for physiological functions,such as epithelial secretion,smooth muscle contraction and sensory signal transduction.The attraction and interest in ANO1/TMEM16 A arise from a decade long investigations that abnormal expression or dysfunction of ANO1 is involved in many pathological phenotypes and diseases,including asthma,neuropathic pain,hypertension and cancer.However,the lack of specific modulators of ANO1 has impeded the efforts to validate ANO1 as a therapeutic target.This review focuses on the recent progress made in understanding of the pathophysiological functions of CaCC ANO1 and the current modulators used as pharmacological tools,hopefully illustrating a broad spectrum of ANO1 channelopathy and a path forward for this target validation.
基金Supported by the National Natural Science Foundation of China (Grant No. 200830870910).
文摘Using fura-2-acetoxymethyl ester (AM) fluorescence imaging and patch clamp techniques, we found that endothelin-1 (ET-1) significantly elevated the intracellular calcium level ([Ca2+]i) in a dose-dependent manner and activated the L-type Ca2+ channel in cardiomyocytes isolated from rats. The effect of ET-1 on [Ca2+]i elevation was abolished in the presence of the ETA receptor blocker BQ123, but was not affected by the ETB receptor blocker BQ788. ET-1-induced an increase in [Ca2+]i, which was inhibited 46.7% by pretreatment with a high concentration of ryanodine (10 μmol/L), a blocker of the ryanodine receptor. The ET-1-induced [Ca2+]i increase was also inhibited by the inhibitors of protein kinase A (PKA), protein kinase C (PKC) and angiotensin type 1 receptor (AT1 receptor). We found that ET-1 induced an enhancement of the amplitude of the whole cell L-type Ca2+ channel current and an increase of open-state probability (NPo) of an L-type single Ca2+ channel. BQ123 completely blocked the ET-1-induced increase in calcium channel open-state probability. In this study we demonstrated that ET-1 regulates calcium overload through a series of mechanisms that include L-type Ca2+ channel activation and Ca2+-induced Ca2+ release (CICR). ETA receptors, PKC, PKA and AT1 receptors may also contribute to this pathway.
基金Supported by the Spanish Ministerio de Ciencia,Innovación y Universidades,No.BFU2017-82716-P
文摘STIM1 is an endoplasmic reticulum(ER) protein with a key role in Ca^(2+)mobilization. Due to its ability to act as an ER-intraluminal Ca^(2+) sensor, it regulates store-operated Ca^(2+) entry(SOCE), which is a Ca^(2+) influx pathway involved in a wide variety of signalling pathways in eukaryotic cells. Despite its important role in Ca^(2+) transport, current knowledge about the role of STIM1 in neurons is much more limited. Growing evidence supports a role for STIM1 and SOCE in the preservation of dendritic spines required for long-term potentiation and the formation of memory. In this regard, recent studies have demonstrated that the loss of STIM1, which impairs Ca^(2+) mobilization in neurons, risks cell viability and could be the cause of neurodegenerative diseases. The role of STIM1 in neurodegeneration and the molecular basis of cell death triggered by low levels of STIM1 are discussed in this review.
文摘The purpose of this study was to examine the relaxation effect of CY on the vascular smooth muscle （VSM） from rabbits. Experiments were carried out on isolated thoracic aorta of rabbits. CY （3 x 103 mM- 3 mM） could relax the VSM preparations pre-contracted by adrenaline （AD）, noradrenaline （NE）, high-K^＋ solution or BaCl2 with respective EC50 values of （0.3 1±0.11） mM, 0.19±0.03 mM, 0.20±0.04 mM and 0.25±0.04 mM. Moreover, CY （10-2 mM, 0.1 mM and 1 mM） inhibited norepinephrine （NE）, CaCl2 and KCl-induced vasoconstriction in a concentration dependent manner. The phasic contraction produced by NE was concentration dependently attenuated with CY （10^-2 mM, 0.1 mM and 1 mM） in calcium-free medium, similar to that caused by verapamil. The present findings suggest that CY relaxed thoracic aortic rings by blocking voltage-dependent Ca^2＋ channels. The inhibition of intracellular Ca^2＋ release may be one of the main vasorelaxant mechanisms of CY.
基金supported by National Basic Research Development Program of China (No.2006CB705600)National Natural Science Foundation of China(No. 30700253+2 种基金No. 30800355)the Scientific Research Foundation for the Returned Overseas Chinese Scholars, Ministry of Education, China (No. 2008101)Program of Changjiang scholar and innovative research team in University of China(No. IRT 0734)
文摘Objective Presynaptic voltage-gated Ca^2＋ channels mediate rapid Ca^2＋ influx into the synaptic terminal which triggers synaptic vesicle exocytosis and neurotransmitter release. The FM 1-43 dye was firstly introduced as a fluorescence probe by Betz and his colleagues in 1992, and has been used to monitor exocytosis, endocytosis and endosomal traffic in a variety of cell types. The present study aims to investigate the feasibility of applying the FM 1-43 dye in the functional analysis of calcium channel-mediated exocytosis in synaptic terminals. Methods The hippocampi were isolated from embryos of pregnant rats, and hippocampal neurons were then transfected with Ds-Red conjugated plasmid. The neurons were then loaded with 8 μmol/L FM 1-43 and 47 mmol/L KCl for 90 s after transfection. After that, 90 mmol/L KCI was employed to induce FM dye destaining, which was recorded by FM imaging system. Results The neuron synaptic terminals of rat hippocampus could be effectively stained by the FM 1-43 dye. Besides, the destaining of the labeled neuron terminals was in accordance with the transmitter release, which could be blocked by the application of nifedipine （inhibitor for L-type calcium channel）. Conclusion The FM imaging technique is an advanced and effective method for analyzing synaptic vesicle exocytosis and neurotransmitter release, and can be applied in various synaptic functional studies.
基金supported by the National Natural Science Foundation of China(81171049)the National Basic Research Development Program of China(2011CB504100 and2013CB835100)+1 种基金the National Key Technology R&D Program of China(2013BAI04B04)the Twelfth Five-Year Project of China(AWS12J004)
文摘The α2δ-1 subunit of the voltage-gated Ca2＋ channel （VGCC） is a molecular target of gabapentin （GBP）, which has been used as a first-line drug for the relief of neuropathic pain. GBP exerts its anti-nociceptive effects by disrupting trafficking of the α2δ-1 subunit to the presynaptic membrane, resulting in decreased neurotrans- mitter release. We previously showed that GBP has an anti- allodynic effect in the first two weeks; but this is followed by insensitivity in the later stage after repeated adminis- tration in a rat model of central post-stroke pain （CPSP） hypersensitivity induced by intra-thalamic hemorrhage. To explore the mechanisms underlying GBP insensitivity, the cellular localization and time-course of expression of the α2δ-1 subunit in both the thalamus and spinal dorsal horn were studied in the same model. We found that the α2δ-1 subunit was mostly localized in neurons, but not astrocytes and microglia. The level of α2δ-1 protein increased in the first two weeks after injury but then decreased in the third week, when GBP insensitivity occurred. Furthermore, the c^2g-1 down-regulation was likely caused by later neuronal loss in the injured thalamus through a mechanism other than apoptosis. In summary, the present results suggest that the GBP receptor ~2^-1 is mainly expressed in thalamic neurons in which it is up-regulated in the early stage of CPSP but this is followed by dramatic down-regulation, which is likely associated with GBP insensitivity after long-term use.
文摘AIM:To investigate the pathway(s)mediating rat antral circular smooth muscle contractile responses to the cholinomimetic agent,bethanechol and the subtypes of muscarinic receptors mediating the cholinergic contraction. METHODS:Circular smooth muscle strips from the antrum of Sprague-Dawley rats were mounted in muscle baths in Krebs buffer.Isometric tension was recorded.Cumulative concentration-response curves were obtained for(+)-cis- dioxolane(cD),a nonspecific muscarinic agonist,at 10^(-8)- 10^(-4)mol/L,in the presence of tetrodotoxin(TTX,10^(-7)mol/L). Results were normalized to cross sectional area.A repeat concentration-response curve was obtained after incubation of the muscle for 90 min with antagonists for M1(pirenzepine), M2(methoctramine)and M3(darifenadn)muscarinic receptor subtypes.The sensitivity to PTX was tested by the ip injection of 100 mg/kg of PTX 5 d before the experiment.The antral circular smooth muscles were removed from PTX-treated and non-treated rats as strips and dispersed smooth muscle cells to identify whether PTX-linked pathway mediated the contractility to bethanechol. RESULTS:A dose-dependent contractile response observed with bethanechol,was not affected by TTx.The pretreatment of rats with pertussis toxin decreased the contraction induced by bethanechol.Lack of calcium as well as the presence of the L-type calcium channel blocker,nifedipine,also inhibited the cholinergic contraction,with a reduction in response from 2.5±0.4 g/mm^2 to 1.2±0.4 g/mm^2(P<0.05).The dose- response curves were shifted to the right by muscarinic antagonists in the following order of affinity:darifenacin (M_3)>methocramine(M_2)>pirenzepine(M_1). CONCLUSION:The muscarinic receptors-dependent contraction of rat antral circular smooth muscles was linked to the signal transduction pathway(s)involving pertussis-toxin sensitive GTP-binding proteins and to extracellular calcium via L-type voltage gated calcium channels.The presence of the residual contractile response after the treatment with nifedipine,suggests that an additional pathway could mediate the cholinergic contraction.The involvement of more than one muscarinic receptor(functionally predominant type 3 over type 2)also suggests more than one pathway mediating the cholinergic contraction in rat antrum.
