胰岛素对蟾蜍卵母细胞膜电位及电解质的影响

应用普通玻璃微电极和离子选择性微电极,对正常及经过胰岛素处理的中华大蟾蜍卵母细胞膜电位、细胞内Na~+、K~+、Cl~-、H~+等活度及膜对Na~+、K~+的转运系数进行了测定。结果表明,胰岛素在促进蟾蜍卵母细胞发育成熟同时,具有使膜电位降低、细胞内Na~+、Cl~-活度增加、K~+、H~+活度减少及K~+转运系数降低等作用。胰岛素的上述作用可能与膜的通透性改变及膜上钠泵活性和Na~+/H~+交换的改变有关。     

钠钾氯共转运体在γ-氨基丁酸引起的肠神经元兴奋中的作用（英文）

γ-氨基丁酸(γ-aminobutyric acid, GABA)是成年个体中枢神经系统(central nervous system, CNS)的一种抑制性神经递质,但对未成熟CNS神经元有兴奋作用。出生后GABA对CNS神经元的作用由去极化逐渐转为超极化,这种转变主要是由于大脑Na~+-K~+-2Cl~-共运体1 (Na~+-K~+-2Cl~-symporter 1, NKCC1)表达逐渐减少和K~+-Cl~-共运体2 (K~+-Cl~-cotransporter 2, KCC2)表达增加。与CNS神经元不同,肠神经系统(enteric nervous system, ENS)中未成熟和成体神经元都可被GABA去极化。GABA激发ENS神经元兴奋的分子机制尚不清楚,但与ENS神经元内的高Cl-浓度有关。本研究目的是检验一个假设,即ENS神经元的细胞内高Cl-浓度是由NKCCs活动维持的。结果显示,NKCC2免疫反应性(immunoreactivity, IR)在出生后第1天(P1)大鼠结肠ENS中出现,随后持续升高,在P14达到稳定的高水平,并在成年后保持在这一水平。NKCC1的IR出现在P14大鼠的ENS中,并维持到成年。在任何发育阶段的大鼠ENS中都检测不到KCC2的IR。在ENS神经元中,NKCC1和NKCC2均与GABAA受体共同表达。外源性GABA (1 mmol/L)可引起ENS神经元膜去极化。GABA诱导的去极化逆转电位约为-16 m V。NKCC抑制剂布美他尼(50μmol/L)或速尿(300μmol/L)可显著抑制GABA诱导的去极化。布美他尼(50μmol/L)使GABA诱导的去极化逆转电位向超极化方向移动。无论是KCC抑制剂DIOA (20μmol/L)还是Cl~-/HCO_3~-交换抑制剂DIDS (200μmol/L)对GABA诱发的去极化都没有明显影响。以上结果提示,ENS表达NKCC1和NKCC2,但是不表达KCC2。NKCC1和NKCC2在GABA引起的肠神经元兴奋中起重要作用。     

糖皮质激素引起哺乳类神经元超极化反应的离子机制

在豚鼠腹腔神经节上对383个神经元作细胞内记录,给予1μmol／L半琥珀酸皮质醇灌流,38个神经元膜电位发生超极化反应,幅度变化为2～12mV（6．3±0.1mV）,伴有膜电阻的降低,反应呈剂量效应关系。9个神经元呈去极化反应,其余336个神经元不反应。用单电极间断电压箝方法记录43个神经元在糖皮质激素作用下膜电流的变化,其中5个神经元出现外向电流,膜电导增加;1个神经元为内向电流。用低钙高镁液阻断突触传递和蛋白质合成抑制剂放线菌素D后,超极化反应仍然存在。皮质醇超极化反应的翻转电位为-79.0±4.3mV（n=5）。皮质醇超极化反应和GABA去极化反应可在同一神经元上出现,印防己毒素可拮抗GABA的去极化反应,但不能拮抗皮质醇的超极化反应。钾离子通道阻断剂四乙基铵（TEA）和4-氨基吡啶（4-AP）能拮抗皮质醇的超极化反应。我们推断皮质醇的超极化反应是细胞膜钾离子通道介导的。     

去甲肾上腺素对蟾蜍背根神经节细胞α-肾上腺素能受体的作用

在蟾蜍离体灌流背根神经节(DRG)标本上,用微电极进行细胞内记录。在51个细胞中A型神经元为46个,C型5个。此两类细胞的静息膜电位为60.06±1.34mV(x±SE)。当灌流液中滴加10~(-4)-10~(-3)mol/L去甲肾上腺素(NA)引起如下的膜电位改变:(1)超极化:幅值8.38±1.12mV(x±SE)(20/48);(2)去极化:幅值9.39±1.24mV(x±SE)(23/48);(3)无反应(5/48)。上述膜电位改变既不能由灌流液中滴加异丙基肾上腺素所拟似,也不能为心得安所阻断,因而排除了β-肾上腺能受体介导的可能性。加苯肾上腺素及可乐宁于灌流液,分别产生膜的去极化和超极化,而应用哌唑唪及育亨宾灌流,则分别阻断NA引起的膜去极化和超极化。因此认为:NA引起的DRG神经元的去极化和超极化反应分别是由胞体膜上之α_1-及α_2-肾上腺素能受体所介导的。     

乙酰胆碱不依赖NO的释放引起耳蜗螺旋动脉平滑肌细胞的超极化

目的：乙酰胆碱（ACh）不仅是神经递质,也是一种有效的血管舒张物质参与许多血管床的调节活动。本实验观察ACh引起耳蜗螺旋动脉平滑肌细胞超极化的离子机制以及NO在超极化反应中的可能作用。方法：在豚鼠离体耳蜗螺旋动脉标本上,运用细胞内微电极技术记录外源性的ACh引起的反应。结果：在保持灌流液中含有5mmol／L K^＋以及最小纵向张力的情况下,ACh（0．1—10μmol／L）引起低静息膜电位细胞明显的超极化反应,而引起高静息膜电位细胞明显的去极化反应。ACh引起的平滑肌细胞超极化反应是浓度依赖性的（ACh的浓度是1μmol／L和10／μmol／L时,分别引起超极化的幅度是22和30mV,n=7）。ACh引起的超极化反应能被阿托品（atropine,0．1～1μmol／L,n=6）或DAMP（50～100nmol／L,n=6,一种选择性的地受体的拮抗剂）所阻断,同时也可被BAPTA—AM（10μmol／L,n=7,一种可通过细胞膜的Ca^2＋螯合剂）或eharybdotoxin＋apamin（50-100nmol／L,n=4,两种Ca^2＋激活K^＋通道的阻断剂）所阻断,但是Nω-nitro-L-arginine methyl ester（L-NAME,300μmol／L,n=8,一种NO合成酶的完全抑制剂,n≥5）或glipizide（10μmol／L,ATP敏感性的K^＋通道阻断剂,n=4）或indomethacin（10μmol／L,环氧合酶的抑制剂,n=4）不能阻断ACh引起的超极化反应。结论：ACh通过激活内皮细胞的M3受体,开放钙依赖的钾通道．进而引起耳蜗螺旋动脉平滑肌细胞产生超极化反应,并且这一超极化反应与内皮细胞NO的产生和释放无关。     

血管升压素对大鼠背根神经节神经元膜的作用

为研究血管升压素(arginine vasopressin,AVP)对大鼠背根神经节(dorsal root ganglion,DRG)神经元的作用及其机制,用细胞内微电极记录技术记录离体灌流DRG神经元的膜电位。结果如下:(1)在受检的120个细胞中,大多数(81.67％)在滴加AVP后产生明显的超极化反应。(2)滴加AVP(10μmol/L)后膜电导增加约19.34％(P<0.05)。(3)灌流平衡液巾的NaCl以氯化胆碱(CH-Cl)置代和用Cd2+阻断Ca2+通道后,AVP引起超极化反应的幅值均无明显变化(P>0.05),而加入K+通道阻断剂四乙铵(TEA)后,AVP引起的超极化反应幅值明显减小(P<0.05)。(4)AVP引起的超极化反应可被AVP V.受体拈抗剂阻断。结果捉示,AVP可使DRG大多数神经元膜产生超极化,DRG神经元膜上存在AVP V,受体,且AVP引起的超极化反应是通过神经元膜上AVP V.受体介导的K+外流所致.AVP可能参与了初级感觉信息传入的调制。     

原癌基因c-myb在体外孕酮诱导小鼠卵母细胞成熟中的作用

目的：本文采用体外培养技术观察原癌基因c-myb对孕酮诱导的生发泡（GV）期小鼠裸卵体外成熟的影响。方法：建立小鼠裸卵体外培养模型,用不同浓度反义C-myb寡脱氧核苷酸（c-myb ASODNs）与GV期小鼠裸卵共孵育观察其对孕酮诱导的小鼠裸卵体外成熟的影响并探讨其机制。结果：在M199培养液中体外培养GV期小鼠裸卵24h,10μmol／L孕酮组与5μmol／L孕酮组比较有显著性差别（2 h GVBD％ P〈0.05,8 h PBI％ P〈0.05）．与20μmol／L孕酮组比较无显著性差别。10μmol／L c-myb AFODNs能抑制孕酮（10μmol／L）诱导的小鼠裸卵体外成熟（2 h GVBD％ P〈0.05,8 h PBI％ P〈0.01）。1×10^-4μmol／L dbcAMP、10μg／ml肝素钠可分别单独抑制孕酮诱导的GV期小鼠卵母细胞体外成熟（2 h GVBD％均P〈0.01,8 h PBI％均P〈0.01）．也可和反义c-myb ODN协同抑制孕酮诱导的卵母细胞体外成熟（2 h GVBD％均P〈0.01,8 h PBI％均P〈0.01）。结论：孕酮、原癌基因c-myb和cAMP、Ca^2＋参与了GV期小鼠卵母细胞的体外成熟,孕酮、cAMP和Ca^2＋调控卵母细胞成熟的机理可能与原癌基因c-myb表达有关。     

一氧化氮和K+通道参与乙酰胆碱引起的大鼠离体输精管平滑肌细胞超极化

本文旨在探讨大鼠新鲜离体输精管平滑肌细胞中乙酰胆碱（acetylcholine,ACh）引起超极化反应的机制,采用细胞内微电极记录技术和细胞内荧光标记技术研究ACh对大鼠输精管不同走行方向平滑肌细胞的作用。用尖端含0．1％碘化吡啶（propidium iodide,PI）的记录电极标记电生理记录后的平滑肌细胞,其中37个为外层纵行细胞,17个为内层环行细胞。它们的平均静息膜电位分别为（-53．56±3．88）mV和（-51．62±4．27）mV,膜输入阻抗分别为（2245．60±372．50）MQ和（2101．50±513．50）MQ。ACh引起的膜超极化反应是浓度依赖性的,EC50为36 μmol／L。ACh引起的超极化反应可被非选择性的毒草碱（muscarinic receptor,M）受体阻断剂阿托品（atropine,1 μmol／L）和选择性的M3受体阻断剂diphenylacetoxy-N-methylpiperidine-methiodide（DAMP,100nmol/L）阻断。ACh引起的超极化还能被一氧化氮合酶抑制剂L-硝基-精氨酸甲酯（N-nitro-L-arginine methylester,L．NAME,300μmol／L）阻断,并可被ATP敏感的钾通道阻断剂glipizide（5μmol／L）或内向整流钾通道阻断剂钡离子（50μmol／L）部分阻断。Glipizide和钡离子联合使用可完全阻断ACh引起的超极化反应。上述结果表明：ACh通过作用于大鼠输精管平滑肌细胞膜上的M3受体引起超极化反应,一氧化氮、ATP敏感性钾通道和内向整流钾通道参与了ACh引起的超极化反应。     

GABA_A和GABA_B受体介导的蟾蜍背根神经节神经元胞体膜反应

实验在蟾蜍离体背根神经节(DRG)标本进行细胞内记录。浴槽滴加10~(-4)-10~(-3)mol/LGABA引起膜电位改变如下:(1)去极化(79/100);(2)双相反应;先为去极化,继后为超极化(10/100);(3)无反应(11/100)。以上去极化反应均可为荷包牡丹碱所阻断。GABA-去极化时膜电导增加,逆转电位值为-15——25mV。低Cl~-和高Cl~-任氏液分别使GABA-去极化反应增大和减小。10~(-4)mol/Lbaclofen不引起膜电位改变。在GABA-去极化期间,观察到大部分细胞的动作电位时程(ApD)缩短。ApD的此种变化可为baclofen所模拟,但不为荷包牡丹碱所阻断。结果提示:蟾蜍DRG神经元胞体膜有GABA_A和GABA_B受体共存,前者介导膜电位的改变,后者介导ApD的缩短。本文并联系到初级传入终末的突触前抑制的产生机制进行了讨论。     

中华大蟾蜍卵母细胞质膜的外向整流型钾离子通道

我们用电压箝方法研究了中华大蟾蜍卵母细胞的膜生理特性。发现卵母细胞膜去极化至-30mV及更偏正时,有一持续的外向电流出现,该电流与去极化程度约呈正比增加,当膜电位箝在20mV时其峰值达3.7±1.4μA。该电流被钾离子通道拮抗剂TEA和4-AP抑制,TEA半抑制浓度为2.6mmol/L。氯通道拮抗剂9-AC(2.5mmol/L)无抑制作用。无钙的或钙离子浓度增加三倍的胞外灌流液均对该电流无影响、该外向电流的逆转电位随胞外钾离子浓度的改变而变化。胞外钾离子浓度增加十倍,逆转电位约增加47.3mV,而胞外钠、钙或氯离子浓度的改变对逆转电位基本上无影响,因此该电流可被认为主要是电压依赖性钾离子流。取自冬眠蟾蜍的卵母细胞经孕酮诱发成熟后,电压依赖性钾离子流减小,仅为原来的1/20-1/30,而取自全年在高温饲养的蟾蜍的卵母细胞经孕酮处理后未见成熟,其电压依赖性钾离子流仅减小至原来的三分之一。     

Changes in membrane properties during in-vitro meiotic maturation of the limpet Patella vulgata

Changes in the membrane properties of the oocyte of the mollusk, Patella vulgata, were analyzed following the induction of meiosis reinitiation by paleopedial ganglia extract or by the weak base ammonia. During maturation it was possible to distinguish between an early phase characterized by an initial hyperpolarization and a late phase consisting of a depolarization which triggers an action potential with a long-term overshoot (20 minutes) of the membrane potential. Major changes in individual ionic permeabilities were studied using both current and voltage clamp conditions. The depolarizing phase appears to depend on decreases in K+ membrane permeability. Finally we observed that the overshoot did not appear to be directly related to germinal vesicle breakdown (GVBD) since it was absent in Na-deprived artificial sea water and could be elicited in the presence of TEA bromide, which did not induce maturation. This last observation suggests that it may result from a change in specific K+ ion permeability due to the possible activation of stretch channels.     

Heterotrophic Potential for Amino Acid Uptake in a Naturally Eutrophic Lake

The uptake of sixteen (14)C-labeled amino acids by the indigenous heterotrophic microflora of Upper Klamath Lake, Oregon, was measured using the kinetic approach. The year-long study showed a seasonal variation in the maximum uptake velocity, V(max), of all the amino acids which was proportional to temperature. The maximum total flux of amino acids by the heterotrophic microflora ranged from 1.2 to 11.9 mumol of C per liter per day (spring to summer). Glutamate, asparagine, aspartate, and serine had the highest V(max) values and were respired to the greatest extent. The percentages of the gross (net + respired) uptake of the amino acids which were respired to CO(2) ranged from 2% for leucine to 63% for glutamate. Serine, lysine, and glycine were the most abundant amino acids found in Upper Klamath Lake surface water; at intermediate concentrations were alanine, aspartate, and threonine; and the remaining amino acids were always below 7.5 x 10(-8) M (10 mug/liter). The amino acid concentrations determined chemically appear to be the sum of free and adsorbed amino acids, since the values obtained were usually greater than the (K(t) + S(n)) values obtained by the heterotrophic uptake experiments.     

Electrophysiology of L-lysine entry across the brush-border membrane of Necturus intestine

Microelectrode measurements of apical membrane potentials (Va) in absorptive cells of isolated Necturus intestine showed that, in the presence or absence of external Na+, 10 mM lysine added to the mucosal medium caused rapid depolarization followed by slower repolarization of Va. In Na+-free media the effects of 10 mM lysine on Va were abolished by 10 mM leucine which alone had no effect on Va under these conditions. This indicates that uncoupled electrodiffusion of lysine plays little or no role in lysine entry across the brush-border membrane. When external Na+ was greater than 10 mM the maximum depolarization of Va (delta Va') induced by [Lys] ranging from 5 to 30 mM was a simple saturable function of [Lys]. In Na+-free media, the relationship between delta Va' and [Lys] was biphasic. At first, delta Va' increased with increasing [Lys] reaching a maximum at 10 mM lysine. When [Lys] was further increased, delta Va' declined progressively to reach zero or near zero values. A single transport pathway model is proposed to account for rheogenic lysine entry across the brush-border membrane in the presence and absence of Na+. This postulates an amino acid transporter in the membrane with two binding sites. One is an amino acid site specific for the alpha-amino-alpha-carboxyl group. The other is a Na+ site. Neutral amino acids (e.g. leucine) compete with lysine for the amino acid site. The Na+ site has some affinity for the epsilon-amino group of lysine. When external Na+ is high the Na+ site is essentially 'saturated' with Na+ and formation of a mobile complex between an amino acid and the transporter depends in a saturable fashion on amino acid concentration. In Na+-free media or in media containing low [Na+]; at low external [Lys] the epsilon-amino group of a lysine molecule (simultaneously attached to the amino acid site) interacts with the Na+ site to form a mobile complex, as external [Lys] is increased, attachment of different lysine molecules to each site of an increasing number of transporters to form nontransported or poorly transported complexes results in substrate inhibition of the rheogenic lysine transport process.     

Intrafollicular amino acid concentration and the effect of amino acids in a defined maturation medium on porcine oocyte maturation, fertilization, and preimplantation development

The objective of this study was to determine the intrafollicular concentrations of free amino acids in pigs and to examine the effect of amino acids in a chemically defined maturation medium on oocyte maturation, in vitro fertilization (IVF), and embryo development in vitro. Pooled follicular fluid aspirated separately from small (<3mm in diameter), medium (3-8mm), and large follicles (>8mm) in three pairs of ovaries was analyzed for amino acid concentration. In addition, oocyte maturation, fertilization, and embryo development were examined after in vitro maturation (IVM) of oocytes in a defined maturation medium supplemented individually with glutamate (GLU), glutamine (GLN), glycine (GLY), aspartate (ASP), asparagine (ASN), arginine (ARG), alanine (ALA), leucine (LEU), lysine (LYS), proline (PRO), and valine (VAL). Irrespective of follicle size, GLY, GLU, ALA, GLN, and PRO were the most abundant amino acids in pig follicular fluid (pFF). Sperm penetration was not altered by amino acid treatment during IVM, but monospermic fertilization was increased (P<0.05) by GLN, ASP, and VAL. All amino acids except ASP and ASN stimulated (P<0.05) male pronuclear formation after IVF. ARG and ALA treatment during IVM improved (P<0.05) blastocyst formation. In conclusion, GLY, GLU, ALA, GLN, and PRO were the most abundant amino acids in pFF and amino acids in a defined medium improved porcine monospermic fertilization, male pronuclear formation, and preimplantation development.     

Expression of amino acid transport systems in Xenopus oocytes injected with mRNA of rat small intestine and kidney

Xenopus and Cynops oocytes were injected with exogenous mRNA prepared from rat small intestine and kidney and their electrical responses to amino acids were measured by both the current clamped and the voltage clamped methods. Oocytes injected with mRNA of rat small intestine showed a depolarization response to several neutral and basic amino acids, and almost no response to acidic amino acids. The responses to amino acids increased with incubation time after injection of mRNA, and followed Michaelis-Menten type kinetics. The responses were dependent on both Na+ concentration and membrane potential, and were inactivated by a sulfhydryl reagent, 5,5-dithiobis(2-nitrobenzoate). These results are interpreted as due to the expression of Na+/amino acid cotransporter(s) in oocytes injected with rat small intestine mRNA. On the other hand, the oocyte injected with rat kidney mRNA showed a hyperpolarization response to neutral amino acids, a depolarization response to basic ones, and almost no response to acidic ones in frog Ringer solution. These responses were independent of Na+ concentration and followed Michaelis-Menten type kinetics. These amino acid response characteristics in oocytes injected with rat kidney mRNA are interpreted as due to the expression of facilitated diffusion carrier protein(s) (uniporter) of amino acids in the oocyte.     

Regulation of insulin release by ionic and electrical events in B cells

This review article is an attempt to schematize the major alterations in ionic fluxes and B cell membrane potential that underlie the changes in insulin release brought about by glucose and by other stimulators or inhibitors. Glucose metabolism in B cells leads to closure of K channels in the plasma membrane. The resulting decrease in K+ permeability causes depolarization with activation of voltage-dependent Ca channels. An increase in Ca2+ influx ensues, which raises the cytoplasmic concentration of free Ca2+ and ultimately triggers insulin release. Tolbutamide induces a similar sequence of events by a direct action on K channels. In contrast, diazoxide antagonizes the effects of glucose by increasing K+ permeability of the B cell membrane. Among amino acids, leucine largely mimics the effects of glucose, whereas arginine depolarizes the B cell membrane because of its transport in a positively charged form.     

Transport of neutral and cationic amino acids across the brush-border membrane of the rabbit ileum

Summary The transport of sugars and amino acids across the brush-border membrane of the distal rabbit ileum has been studied. The kinetics of the transport of glucose demonstrated that the data obtained with the present technique are less distorted by unstirred layers than those obtained with the same technique adapted to the use of magnetic stirring. The role of depolarization of the electrical potential difference across the brush-border membrane in mutual inhibition between different classes of amino acids was estimated by measurements of the effects of high concentrations of alanine and lysine on the transport of galactose. It was found that this role would be insignificant in the present study. By measurements of the transport of alanine, leucine and lysine and the inhibitory interactions between these amino acids the function of three transport systems has been delineated. The transport of lysine is resolved in a high- and a low-affinity contribution. At 140mm sodium these transport systems may also function as respectively high- and low-affinity contributors to the transport of neutral amino acids. At 0mm sodium the high-affinity system remains a high-affinity system for cationic and neutral amino acids with reduced capacity especially for the neutral amino acids. At 0mm sodium the low-affinity system's affinity for lysine is reduced and it is inaccessible to neutral amino acids. In addition to the two systems for lysine transport the existence of a lysine-resistant, sodium-dependent, high-affinity system for the transport of neutral amino acids has been confirmed. It seems unlikely that the distal ileum is equipped with a low-affinity, sodium-independent system for the transport of neutral amino acids.     

Electrophysiological effects of extracellular ATP on Necturus gallbladder epithelium

The effects of addition of ATP to the mucosal bathing solution on transepithelial, apical, and basolateral membrane voltages and resistances in Necturus gallbladder epithelium were determined. Mucosal ATP (100 microM) caused a rapid hyperpolarization of both apical (Vmc) and basolateral (Vcs) cell membrane voltages (delta Vm = 18 +/- 1 mV), a fall in transepithelial resistance (Rt) from 142 +/- 8 to 122 +/- 7 omega.cm2, and a decrease in fractional apical membrane resistance (fRa) from 0.93 +/- 0.02 to 0.83 +/- 0.03. The rapid initial hyperpolarization of Vmc and Vcs was followed by a slower depolarization of cell membrane voltages and a lumen-negative change in transepithelial voltage (Vms). This phase also included an additional decrease in fRa. Removal of the ATP caused a further depolarization of membrane voltages followed by a hyperpolarization and then a return to control values. fRa fell to a minimum after removal of ATP and then returned to control values as the cell membrane voltages repolarized. Similar responses could be elicited by ADP but not by adenosine. The results of two-point cable experiments revealed that ATP induced an initial increase in cell membrane conductance followed by a decrease. Transient elevations of mucosal solution [K+] induced a larger depolarization of Vmc and Vcs during exposure to ATP than under control conditions. Reduction of mucosal solution [Cl-] induced a slow hyperpolarization of Vmc and Vcs before exposure to ATP and a rapid depolarization during exposure to ATP. We conclude that ATP4- is the active agent and that it causes a concentration-dependent increase in apical and basolateral membrane K+ permeability. In addition, an apical membrane electrodiffusive Cl- permeability is activated by ATP4-.     

酪氨酸、hCG对蟾蜍卵母细胞膜电位的影响

作者用微电极记录了蟾蜍卵母细胞的膜电位。当用含hCG的溶液培灌时,蟾蜍卵母细胞膜电位呈去极化变化;当用含酪氨酸溶液培灌时膜电位呈超极化变化,并能抑制hCG的去极化作用。超微结构的变化与膜电位变化相一致。因此我们认为,酪氨酸可能在蟾蜍卵母细胞有对抗hCG的作用。