5-HT2受体介导大鼠DRG神经元膜GABA-激活电流的增强作用

目的探讨5-HT对大鼠DRG神经元膜GABA-激活电流的调节作用及其机制。方法在新鲜分离的大鼠DRG神经元标本上，以全细胞膜片钳技术记录膜电流，用排管快速换液装置行胞外给药，以胞内透析技术分析信号转导途径。结果给予GABA可使多数受检细胞产生浓度依赖性内向电流(I_GABA)。预加5-HT，可使I_GABA增加。此效应可被5-HT₂受体特异性激动剂α-methyl-5-HT(1×10^-6 mol·L^-1)所模拟，被5-HT₂受体选择性拮抗剂cyproheptadine所阻断。在部分细胞，5-HT本身可引起由5-HT₃受体介导的快速内向电流，但并未发现该电流与5-HT对I_GABA的增强作用有必然的联系。从GABA激活电流的量效曲线可见，预加5-HT后和对照曲线相比，阈浓度不变、EC₅₀值相近，I_GABA最大值增加33.6%。胞内透析GDP-β-S或H-7可取消5-HT增强I_GABA的效应，而透析H-9无效。结论5-HT可增强GABA-激活电流，其机制为5-HT₂受体激活后通过PKC引起GABA_A受体胞内磷酸化所致。     

铝对大鼠背根神经节分离神经元GABA激活电流的调制作用

目的　研究三氯化铝 (AlCl3)对大鼠背根神经节分离神经元γ 氨基丁酸 (GABA)激活电流的作用。方法　应用全细胞膜片钳技术在新鲜分离的大鼠背根神经节 (DRG)神经元上观察AlCl3 对GABA激活电流的影响。结果　大部分受检细胞 (4 6 /5 8)对GABA(1～ 10 0 0 μmol·L-1)敏感。在这 46个细胞中 ,单独加AlCl3 可引起三类膜反应 :(1)外向电流 (3/4 6 ) ;(2 )内向电流 (5 /4 6 ) ;(3)无反应 (38/4 6 )。与GABA激活的内向电流相比 ,AlCl3 激活电流幅值要小得多。预加较低浓度 (≤ 10 0 μmol·L-1)AlCl3 对GABA激活电流影响如下 :产生增强效应的细胞为 32 /38,并具有浓度依赖性 ;产生抑制作用的细胞为 4/38;无明显作用的细胞为 2 /38。较高浓度 (10 0 0 μmol·L-1)AlCl3 抑制GABA激活电流 (n2 0 0 0 0 3 16收稿 ,2 0 0 0 0 7 12修回1 江西医学院生理教研室 ,南昌　 330 0 0 6作者简介 :梁尚栋 ,男 ,41岁 ,医学硕士 ,副教授 ,主要研究方向 :神经生理学 ;李之望 ,男 ,教授 ,主要研究方向 :神经生理和药理学=8)。AlCl3 对GABA激活电流的增强作用既可增强峰电流也可增强稳态部分。结论　AlCl3 改变GABAA 受体的机能。     

川芎嗪对大鼠背根神经节细胞P2X嘌呤受体介导反应的作用

目的探讨川芎嗪(tetram ethy1pyrazine,TMP)对嘌呤2X(P2X)受体介导反应的作用。方法在大鼠新鲜分离的背根神经节(dorsal root ganglion,DRG)神经元标本上应用全细胞膜片钳技术记录川芎嗪对P2X受体激动剂激活电流的影响。结果外加ATP(1~1 000μmol.L-1)可引起DRG神经元产生激活电流(n=102),ATP-激活电流(IATP)显示快失敏和慢失敏两种形式的内向电流。预加川芎嗪(0.1~10mmol.L-1)后,大部分(89.2%,91/102)受检细胞可观察到ATP(100μmol.L-1)-激活电流出现明显的抑制作用。川芎嗪(1 mmol.L-1)使α,-βm eATP(10μmol.L-1)-激活电流减小。预加川芎嗪(1 mmol.L-1)后ATP(1~1 000μmol.L-1)激活电流的剂量-效应曲线明显下移。预加川芎嗪(1 mmol.L-1)前后ATP(100μmol.L-1)的I-V曲线反转电位值不变,均接近0 mV。川芎嗪(1 mmol.L-1)可明显抑制被前列腺素E2(100μmol.L-1)或P物质(0.1μmol.L-1)增大的ATP激活电流。通过微电极胞内透析注入PKA抑制剂H89(10μmol.L-1)至胞内,使川芎嗪(1 mmol.L-1)抑制ATP(100μmol.L-1)激活电流的作用减小。结论川芎嗪可能是通过PKA系统以及P2X受体离子通道复合体细胞外环的变构调制点影响P2X受体激动剂在大鼠DRG神经元的激活电流。     

大鼠耳蜗螺旋神经节神经元GABA_A受体激活电流的特性

目的研究大鼠耳蜗螺旋神经节神经元GABAA受体激活电流的特性。方法采用全细胞膜片钳记录技术,观察离体培养大鼠螺旋神经节神经元上GABAA受体激活电流的特点。结果不同浓度的GABA可诱出螺旋神经节神经元的内向电流,该电流可被10μM荷包牡丹碱可逆性阻断。GABA诱导出的内向电流幅度随着浓度（10、50、100、500μM）的增大而增大。结论耳蜗螺旋神经节神经元可诱出GABAA受体电流,GABA诱导的GABAA受体电流呈浓度依赖关系。     

莫达非尼对大鼠海马神经元I_(GABA)的抑制作用机制

目的研究新型促醒药莫达非尼对培养的大鼠海马神经元GABAA受体介导的电流(IGABA)的影响。方法在培养的海马锥体神经元上,进行全细胞膜片钳记录。结果GABA引起电流能被GABAA受体的竞争性阻断剂荷包牡丹碱阻断。单独给予莫达非尼时,明显地抑制了内向电流IGABA。而格列本脲预处理海马神经元后,再单独给予莫达非尼,发现莫达非尼对于IGABA的抑制作用被逆转。结论新型促醒药莫达非尼不同于传统的兴奋剂,莫达非尼对于神经元的保护作用可能与ATP敏感性钾离子通道的开放有关。     

戈那瑞林对大鼠脊神经节细胞GABA引起的去极化及GABA激活电流的调制作用(英文)

目的:探索戈那瑞林对大鼠初级感觉神经元膜GABA引起的去极化和GABA激活电流的调制作用。方法:应用细胞内记录和全细胞膜片钳技术分别在大鼠脊神经节(SG)标本和新鲜分离神经元进行实验。结果:GABA(10 μmol·L~(-1)—1mmol·L~(-1))在大多数神经元引起可为荷包牡丹碱(100μmol·L~(-1))所阻断的膜去极化。预加戈那瑞林(50μmol·L~(-1))可减少GABA引起的去极化,抑制率为79±22％(n=29),而戈那瑞林本身不产生膜反应或只引起轻微去极化。在11个细胞中有6个细胞GABA激活电流也为戈那瑞林的预处理所抑制,另5个细胞无改变或反应稍有增加。结论:戈那瑞林对初级感觉神经元GABA介导的去极化和GABA激活电流具有抑制作用。     

焦亚硫酸钠对大鼠背根节神经元胞内钙离子浓度的影响

目的:探讨一种食品添加剂焦亚硫酸钠(SMB)对大鼠背根节神经元(DRG)的生理作用和毒性效应。方法:急性分离大鼠背根节神经元细胞后,应用Ca2+荧光指示剂Fura-2/AM标记细胞检测大鼠DRG神经元细胞内钙离子浓度的变化。结果:SMB可增大大鼠背根神经元胞内钙离子浓度。结论:SMB可以引起大鼠背根神经元胞内钙超载,进而可能导致一系列与钙离子浓度有关的中枢神经系统损伤。     

大鼠下颌下腺神经节分离神经元P2X受体的药理学研究

目的研究大鼠下颌下腺副交感神经节分离神经元不同P2X受体的表达。方法用全细胞膜片钳的方法对大鼠下颌下腺神经节急性分离的神经元进行了药理学鉴定。结果在所有的神经元上,ATP都可以引起一个快速反应的缓慢失活的内向电流。但只在59%的神经元上,αβmeATP也可以引起一个类似的内向电流。降低pH值及同时给予Zn2+可以增强ATP及αβmeATP诱发的电流。而Ivermectin没有作用。结论在大鼠下颌下腺神经节急性分离的神经元上,主要表达同源二聚体P2X2受体和异源二聚体P2X2/3受体。     

葛根素抑制大鼠背根神经节细胞河豚毒素不敏感性钠电流(英文)

目的:测定葛根素(Pue)对大鼠背根神经节(DRG)细胞河豚毒素不敏感性(TTXr)钠电流的作用。方法:采用全细胞钳制技术,记录成年Wistar大鼠DRG神经元中TTXr钠电流。结果:Pue在0.01-2mmol·L~(-1)浓度范围内,对TTXr钠电流的抑制率为9.5％-83.2％。该抑制作用为浓度依赖性,可部分洗脱,但非频率依赖性或电压依赖性。Pue不影响失活曲线,但使1/2最大激活电压由-26mV升至-16mV,说明抑制了激活过程。结论:Pue抑制大鼠DRG细胞中TTXr钠电流。     

大鼠酸感受离子通道亚基2a表达质粒的构建及生物学特性考察

目的构建大鼠酸感受离子通道亚基2a(acid-sensingion channel subunit 2a,ASIC2a)的表达质粒并研究其组成的同聚体离子通道的生物学特性。方法使用分子生物学技术构建大鼠ASIC2a亚基表达质粒;通过体外转录技术,使编码ASIC2a亚基的cRNA在爪蟾卵母细胞内表达并在膜表面形成同聚体离子通道;使用双电极电压钳技术研究ASIC2a的生物学特性。结果在注射ASIC2a亚基cRNA的爪蟾卵母细胞上,降低胞外液pH值可诱导出内向电流。H+诱发的ASIC2a内向电流具有稳态失活成分可被氨氯吡咪可逆性阻断,其pH50为5.12。提高胞外Ca2+浓度可降低H+诱发的电流幅度,其IC50为11.98 mmol.L-1。当细胞外液中无Na+时,H+基本上不能诱发出内向电流;当同时去除细胞外液中Na+和K+时,H+可诱发出外向电流。结论成功构建ASIC2a表达质粒;ASIC2a除了对Na+通透外,对K+也有一定的通透性,胞外Ca2+抑制ASIC2a孔道的开放。     

Bradykinin potentiates 5-HT3 receptor-mediated current in rat trigeminal ganglion neurons

AIM: To explore the modulatory effect of bradykinin (BK) on 5-HT(3 )receptor-mediated current in trigeminal ganglion (TG) neurons in rats. METHODS: The whole-cell patch-clamp technique was used to record 5-HT-activated currents (I(5-HT)) in neurons freshly dissociated from rat TG. Drugs were applied by rapid solution exchange. RESULTS: The majority of the neurons examined responded to 5-HT applied externally with an inward current (76.3%, 74/97) that could be blocked by the 5-HT(3 )receptor antagonist, ICS-205,930 (10(-6) mol/L). In 66 of the 74 cells sensitive to 5-HT (89.2%), pretreatment for 30 s with BK (10(-6)-10(-10) mol/L) could potentiate I(5-HT) with the maximal modulatory effect occurring at 10(-7) mol/L BK (71.6%+/-4.9%). BK shifted the 5-HT concentration-response curve upwards with an increase of 68.9%+/-7.2% in the maximal current response, but with no significant change in the EC(50) value (19.1+/-3.2 mumol/L vs 20.9+/-3.5 micromol/L; t-test, P>0.05; n=8). BK potentiated I(5-HT) in a holding potential-independent manner and did not alter the reverse potential of I(5-HT). This BK-induced potentiation of I(5-HT) was almost completely blocked by Hoe 140 (5*10(-7) mol/L), a selective B2 BK receptor antagonist, and was removed after intracellular dialysis of GF-109203X (2 micromol/L), a selective protein kinase C (PKC) inhibitor, with the re-patch clamp. CONCLUSION: Pre-application of BK exerts an enhancing effect on I(5-HT) via a PKC-dependent pathway in rat TG neurons, which may explain the peripheral mechanism of pain and hyperalgesia caused by, for example, tissue damage and inflammation.     

Modulatory effect of substance P on GABA-activated currents from rat dorsal root ganglion

INTRODUCTIONSP has been reported to serve as a pain transmit-ter or a modulator of noxious transmission in the dorsalhorn of the spinal cord[1,2]. Application of SP to neu-rons of the DRG produced a depolarizing response[3-5]and activated an inward current[6,7] associated with anincrease in neuronal excitability. g-Aminobutyric acid(GABA) is the primary inhibitory transmitter in the spi-nal cord, which causes a reduction of the release ofexcitatory transmitter from primary afferent n…     

Modulation by oxytocin of ATP-activated currents in rat dorsal root ganglion neurons

The modulatory effect of oxytocin (OT) on ATP-activated currents (I(ATP)) was studied in freshly isolated dorsal root ganglion (DRG) neurons of rats using whole cell clamp technique. In most of the neurons examined (50/70, 71.4%) extracellular application of OT (10(-9)-10(-5) mol/L) suppressed I(ATP) while in the rest (20/70, 28.6%) no modulatory effect was observed. OT shifted the ATP concentration-response curve downwards with a decrease of 39.8+/-4.2% in the maximal current response and with no significant change of Kd value. This OT-induced inhibition of I(ATP) showed no voltage dependence, and could be blocked by [d(CH(2))(5),Tyr(Me)(2),Thr(4),Tyr-NH(2)(9)]-OVT (d(CH(2))(5)-OVT) (10(-8) mol/L), a specific OT receptor antagonist. Intracellular application of H-9 (4 x 10(-5) mol/L, an inhibitor of protein kinase A) (n=12), BAPTA (10(-2) mol/L, a chelator of calcium ions) (n=4) could reverse the inhibitory effect of extracellular OT (10(-7) mol), while inclusion of H-7 (2 x 10(-5) mol/L, a protein kinase C inhibitior) (n=8) and KN-93 (10(-5) mol/L, an inhibitor of CaMKII) (n=9) in the recording pipette did not affect this effect. The results suggested that OT inhibition on ATP-activated currents was mediated by OT receptors in the membrane of DRG neurons; and this inhibitory effect involved the transduction of intracellular cAMP-PKA and Ca(2+).     

5-HT1-like receptors mediate potentiation of cholinergic nerve-mediated contraction of isolated mouse trachea.

While it had no effect on the resting tension of mouse tracheal segments, 5-HT (10(-8)-10(-4) M) potentiated concentration dependently the contractions induced by electrical field stimulation (EFS). The maximal potentiation was 105 +/- 38% and the EC50 value was 1.4 +/- 0.6 x 10(-6) M (n = 6). The responsiveness of mouse trachea to acetylcholine was not altered by 5-HT (10(-5) M). The 5-HT1A,B antagonist pindolol (10(-6) M), the combined 5-HT2 and 5-HT1C receptor antagonist, ketanserin (10(-6) M), or the combined 5-HT1 and 5-HT2 receptor antagonist, methysergide (10(-6) M), all partially inhibited the effect of 5-HT on the twitch responses. Blockade of 5-HT3 receptors by GR 38032F (10(-6) M) did not affect the potentiation by 5-HT. Antagonism of 5-HT3 and 5-HT4 receptors by ICS 205,930 (3 x 10(-6) M) increased the potentiation of the twitch responses by 5-HT, this was probably due to a decrease of the baseline EFS-induced twitch response by ICS 205,930. Alkylation of the 5-HT2 receptor by phenoxybenzamine (3 x 10(-7) M) treatment did not significantly affect the potentiation of the twitch responses by 5-HT. The beta-adrenoceptor antagonist, timolol (10(-6) M), and the alpha-adrenoceptor antagonist, phentolamine (10(-6) M), did not influence the potentiation of the twitch responses by 5-HT, excluding the involvement of the adrenergic system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Potentiation of 5-HT3 receptor function by the activation of coexistent 5-HT2 receptors in trigeminal ganglion neurons of rats

5-HT receptor subtypes are widely expressed in primary sensory neurons, yet so far little is known about the interaction among them. This study aimed to investigate whether the activation of 5-HT2 and 5-HT1 receptors could modulate 5-HT3 receptor mediated current in rat trigeminal ganglion (TG) neurons using whole-cell patch clamp technique. The majority of TG neurons examined responded to 5-HT (10(-7)-10(-3) M) with a fast activating and rapid desensitizing inward current (77.2%, 71/92). This 5-HT activated current (I(5-HT)) was blocked by ICS 205-930 and mimicked by 2-methyl-5-HT, indicating that it was mediated by 5-HT3 receptor. With alpha-methyl-5-HT applied prior to 5-HT application, I(5-HT) was potentiated in a concentration-dependent manner, with the maximal modulatory effect at 10(-9) M of alpha-methyl-5-HT. The concentration-response curve for I(5-HT) pretreated with alpha-methyl-5-HT shifts upwards compared with that for I(5-HT) without alpha-methyl-5-HT pretreatment, the maximal I(5-HT) value having increased by (60.3 +/- 5.7)% of its control while the EC50 values of the two curves being very close, i.e. (2.0 +/- 0.3) x 10(-5) M vs (1.7 +/- 0.2) x 10(-5) M, respectively. The alpha-methyl-5-HT potentiation of I(5-HT) was removed by intracellular dialysis of either GDP-beta-S, a non-hydrolyzable GDP analog, or GF109203X, a selective PKC inhibitor, almost completely. Preapplication of R-(+)-UH-301, a selective agonist of 5-HT(1A) receptor, had no modulatory effect on I(5-HT). These results suggest that in the membrane of TG neurons, the activation of 5-HT2 receptors can exert an enhancing effect on the function of coexistent 5-HT3 receptors while that of 5-HT(1A) receptors cannot.     

A pre-junctional action of 5-hydroxytryptamine and methysergide on noradrenergic nerves in dog isolated saphenous vein

Electrical stimulation (2 Hz for 2 min) of dog isolated saphenous vein strips pre-incubated with tritiated noradrenaline increased the overflow of tritium of which about 80% was noradrenaline. 5-Hydroxytryptamine (5-HT; 1.0 x 10-9-1.0 x 10-7 mol litre-1) and methysergide (3.0 x 10-8-3.0 x 10-6 mol litre-1) inhibited the induced overflow of total tritium by a maximum of 78 +/- 4% and 47 +/- 7% respectively (mean +/- s.e. mean, n=6 for each). Methysergide was about 30 times less potent than 5-HT and the maximum inhibition obtained was less than with 5-HT. Both compounds inhibited electrically-induced contractions and overflow of tritiated noradrenaline. Their inhibitory actions on tritium overflow were little affected by phentolamine (1.0 x 10-6 mol litre-1) or cyproheptadine (1.0 x 10-6 mol litre-1), nor was the inhibitory effect of methysergide on electrically induced contractions antagonized by atropine, mepyramine, cimetidine or propranolol. The findings suggest that the prejunctional inhibitory effect of methysergide may be mediated via stimulation of a 5-HT receptor which, unlike the D-receptor, is not blocked by cyproheptadine. The possibility that the pre-junctional 5-HT receptor in the dog saphenous vein is the same as the post-junctional receptor in this preparation is discussed.     

Presynaptic modulation of synaptic gamma-aminobutyric acid transmission by tandospirone in rat basolateral amygdala

Nystatin-perforated patch recordings were made from mechanically dissociated neurons (in which functional native presynaptic nerve terminals are preserved), isolated from the basolateral amygdala regions to investigate the effects of tandospirone on gamma-aminobutyric acidergic (GABAergic) inhibition. Two types of neurons, ovoid-shaped and pyramidal-shaped neurons, were obtained from the basolateral amygdala nuclei and the electrophysiological characteristics of these two types of neurons supported the morphological classification of these isolated neurons. From the ovoid-shaped neurons, bicuculline-sensitive GABA(A)ergic miniature inhibitory postsynaptic currents (miniature IPSC) were recorded in the presence of tetrodotoxin, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and DL-2-amino-5-phosphovaleric acid (DL-AP5). Tandospirone (10 microM) reversibly and continuously inhibited the GABAergic miniature synaptic events to 66.3+/-2.1% of control (P<0.01, n=17) without affecting the miniature IPSC amplitude (104.0+/-3.1% of control, n=17). The similar inhibition of miniature IPSC frequency was mimicked by a specific 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propylamino) tetralin (8-OH-DPAT, 1 microM), and the effects of tandospirone were prevented in the presence of a specific 5-HT1A receptor antagonist 1-(2-methoxyphenyl)-4-[4-(2-phthalimido)butyl] piperazine hydrobromide (NAN-190, 1 microM). Activation of 5-HT1A receptors by 8-OH-DPAT (1 microM) evoked no direct postsynaptic effects in enzyme-treated isolated basolateral amygdala neurons, suggesting that tandospirone acts at presynaptic 5-HT1A receptors. Furthermore, this presynaptic inhibition by tandospirone was prevented after treatment with a pertussis toxin-sensitive GTP-binding protein (G-protein) inhibitor, N-ethylmaleimide (at 3 microM for 5 min). In conclusion, in the basolateral amygdala nuclei, tandospirone activated presynaptic 5-HT1A receptors on the GABAergic nerve terminals projecting to ovoid-shaped neurons and inhibited synaptic GABA transmission via G-proteins.     

Delta9-tetrahydrocannabinol and endogenous cannabinoid anandamide directly potentiate the function of glycine receptors

Anandamide (AEA) and delta9-tetrahydrocannabinol (THC) are endogenous and exogenous ligands, respectively, for cannabinoid receptors. Whereas most of the pharmacological actions of cannabinoids are mediated by CB1 receptors, there is also evidence that these compounds can produce effects that are not mediated by the activation of identified cannabinoid receptors. Here, we report that THC and AEA, in a CB1 receptor-independent manner, cause a significant potentiation of the amplitudes of glycine-activated currents (I(Gly)) in acutely isolated neurons from rat ventral tegmental area (VTA) and in Xenopus laevis oocytes expressing human homomeric (alpha1) and heteromeric (alpha1beta1) subunits of glycine receptors (GlyRs). The potentiation of I(Gly) by THC and AEA is concentration-dependent, with respective EC50 values of 86 +/- 9 and 319 +/- 31 nM for alpha1 homomeric receptors, 73 +/- 8 and 318 +/- 24 nM for alpha1beta1 heteromeric receptors, and 115 +/- 13 and 230 +/- 29 nM for native GlyRs in VTA neurons. The effects of THC and AEA are selective for I(Gly), because GABA-activated current in VTA neurons or in X. laevis oocytes expressing alpha2beta3gamma2 GABA(A) receptor subunits were unaffected by these compounds. The maximal potentiation by THC and AEA was observed at the lowest concentration of glycine; with increasing concentrations of glycine, the potentiation significantly decreased. The site for THC and AEA seems to be distinct from that of the alcohol and volatile anesthetics. The results indicate that THC and AEA, in pharmacologically relevant concentrations, directly potentiate the function of GlyRs through an allosteric mechanism.     

Two actions of gamma-aminobutyric acid on the responses of the isolated basilar artery from the rabbit.

1 In the isolated basilar artery of the rabbit, gamma-aminobutyrate acid (GABA) (ED50 +/- s.e. mean, 2.4 +/- 1.1 x 10(-5) M) produced a relaxation, if the tone had been increased with 5-hydroxytryptamine (5-HT). 2 3-Aminoproprane sulphonic acid (3-APS) produced a similar, but smaller relaxation, while baclofen had no effect. The relaxation produced by GABA was inhibited by bicuculline. 3 Transmural electrical stimulation produced a reproducible contraction of the isolated basilar artery. In 9 out 14 preparations GABA (ED50 +/- s.e. mean, 5.6 +/- 2.1 x 10(-7) M) caused a reduction of the response, with a maximum of 49.2 +/- 4.3%. Bicuculline did not inhibit these responses to GABA. 4 Baclofen (ED50 +/- s.e. mean, 6.8 +/- 1.4 x 10(-7) M) produced a similar inhibition (47.4 +/- 3.2% maximum) but 3-APS had no effect. 5 GABA (10(-4) M) had no effect on the tone of isolated mesenteric or internal carotid arteries from the rabbit, whether or not the tone was increased with 5-HT. Similarly, GABA (10(-4) M) did not produce any change in the responses to transmural stimulation in isolated mesenteric or internal carotid arteries. 6 These findings are consistent with the presence of two types of GABA receptor on the rabbit basilar artery.