Demonstration of pyruvate recycling in primary cultures of neocortical astrocytes but not in neurons

Pyruvate recycling was studied in primary cultures of mouse cerebrocortical astrocytes, GABAergic cerebrocortical interneurons, and co-cultures consisting of both cell types by measuring production of [4-¹³C]glutamate from [3-¹³C]glutamate by aid of nuclear magnetic resonance spectroscopy. This change in the position of the label can only occur by entry of [3-¹³C]glutamate into the tricarboxylic acid (TCA) cycle, conversion of labeled -ketoglutarate to malate or oxaloacetate, malic enzyme-mediated decarboxylation of malate to pyruvate or phosphoenolpyruvate carboxykinase-mediated conversion of oxaloacetate to phosphoenolpyruvate and subsequent hydrolysis of the latter to pyruvate, and introduction of the labeled pyruvate into the TCA cycle, i.e., after exit of the carbon skeleton of pyruvate from the TCA cycle followed by re-entry of the same pyruvate molecules via acetyl CoA. In agreement with earlier observations, pyruvate recycling was demonstrated in astrocytes, indicating the ability of these cells to undertake complete oxidative degradation of glutamate. The recycled [4-¹³C]glutamate was not further converted to glutamine, showing compartmentation of astrocytic metabolism. Thus, absence of recycling into glutamine in the brain in vivo cannot be taken as indication that pyruvate recycling is absent in astrocytes. No recycling could be demonstrated in the cerebrocortical neurons. This is consistent with a previously demonstrated lack of incorporation of label from glutamate into lactate, and it also indicates that mitochondrial malic enzyme is not operational. Nor was there any indication of pyruvate recycling in the co-cultures. Although this may partly be due to more rapid depletion of glutamate in the co-cultures, this observation at the very least indicates that pyruvate recycling is not up-regulated in the neuronal-astrocytic co-cultures.     

Uptakes of iodide and chloride by primary cultures of mouse astrocytes and neurons

Primary cultures of both mouse astrocytes and neurons accumulate more¹²⁵I^– than³⁶Cl^– from the medium. The average cell/medium ratio of¹²⁵I^– of astrocytes (1.01) is greater than that of neurons (0.74), whereas the ratio of³⁶Cl^– of neurons (0.47) is greater than that of astrocytes (0.25). The equilibrium potentials of both¹²⁵I^– and³⁶Cl^– calculated from the cell/medium ratios in astrocytes and neurons are significantly lower than their corresponding resting transmembrane potentials which suggest that both iodide and chloride are actively transported into both cell types. With respect to different transport inhibitors, thiocyanate is more effective in inhibiting¹²⁵I^– uptake whereas furosemide is more effective in inhibiting³⁶Cl^– uptake. Radioiodide uptake by mouse astrocytes was directly proportional to the [Na⁺]_o but was not significantly affected by changes of [Cl^–]_o or [HCO ₃ ^– ]_o, except that it is low in bicarbonate-free medium. Radiochloride uptake by astrocytes was inversely related to [Cl^–]_o and [HCO ₃ ^– ]_o and was not affected [Na⁺]_o, except that it was low in sodium-free medium. Radioiodide uptake by neurons was directly related to [Na⁺]_o between 60 and 140 mM and inversely related to [HCO ₃ ^– ]_o between 10 and 40 mM, but it was not affected by [Cl^–]_o. Radiochloride uptake by neurons was directly related to [Cl^–]_o and to [Na⁺]_o between 60 and 140 mM and was not affected by [HCO ₃ ^– ]_o. However, in sodium-free medium both¹²⁵I^– and³⁶Cl^– uptakes into neurons were higher than those in [Na⁺]_o between 5 and 60 mM. These results indicate that uptake of¹²⁵I^– and³⁶Cl^– into astrocytes and neurons are different in their ion dependence and that they are under separate regulation.Special issue dedicated to Dr. Paola S. Timiras     

Functional expression of metabotropic GABAB receptors in primary cultures of astrocytes from rat cerebral cortex

GABA(B) receptor subunits are widely expressed on neurons throughout the central nervous system (CNS), at both pre- and postsynaptic sites, where they mediate the late and slow component of the inhibitory response to the major inhibitory neurotransmitter GABA. Recently, GABA(B) receptors have been reported to be expressed in astrocytes and microglia in the rat CNS by immunocytochemistry. However, there are few reports available for the functional characterization of GABA(B) receptors on astrocytes. In the present study, we therefore investigated the functional expression and characteristics of GABA(B) receptors in primary cultures of astrocytes from rat cerebral cortex. In the presence of 10 microM GTP, forskolin concentration-dependently increased adenylylcyclase (AC) activity in membranes prepared from rat astrocytes. The selective GABA(B) agonist (R)-baclofen concentration-dependently reduced forskolin-stimulated AC activity in the presence of 10 microM GTP. This effect was reversed by the selective GABA(B) antagonists, CGP-55845 and CGP-54626, and was completely abolished by treatment of astrocytic membranes with pertussis toxin. In addition, RT-PCR, Western blotting, and immunocytochemistry clearly showed that metabotropic GABA(B) receptor isoforms (GABA(B)R1 and GABA(B)R2) are expressed in rat cerebrocortical astrocytes. Taken collectively, these results demonstrate that functionally active metabotropic GABA(B) receptors are expressed in rat cerebrocortical astrocytes.     

Pyruvate decarboxylation in astrocytes and in neurons in primary cultures in the presence and the absence of ammonia

Oxidative decarboxylation of [1-¹⁴C]pyruvate was studied in primary cultures of neurons and of astrocytes. The rate of this process, which is a measure of carbon flow into the tricarboxylic acid (TCA) cycle and which is inhibited by its end product, acetyl CoA, was determined under conditions which would either elevate or reduce the components of the malate-aspartate shuttle (MAS). Addition of aspartate (1 mM) was found to stimulate pyruvate decarboxylation in astrocytes whereas addition of glutamate (or glutamine) had no effect. Since aspartate is a precursor for extramitochondrial malate, and thus intramitochondrial oxaloacetate, whereas glutamate and glutamine are not, this suggests that an increase in oxaloacetate level stimulates TCA cycle activity. Conversely, a reduction of the glutamate content by 3 mM ammonia, which might reduce exchange between glutamate and aspartate across the mitochondrial membrane, suppressed pyruvate decarboxylation. This effect was abolished by addition of glutamate or glutamine or exposure to methionine sulfoximine (MSO). These findings suggest that impairment of MAS activity by removal of MAS constituents decreases TCA cycle activity whereas replenishment of these compounds restores the activity of the TCA cycle. No corresponding effects were observed in neurons.     

皮质酮对体外培养的海马神经元延迟整流钾电流的影响

目的：探讨应激激素皮质酮对海马神经元延迟整流钾电流的影响。方法：膜片钳全细胞记录测量原代培养大鼠海马神经元膜的钾离子电流。结果：在皮质酮的作用下,海马神经元膜的钾离子电流幅度明显下调,激活阈电位升高。结论：过量皮质酮激素可能通过影响延迟整流钾通道损伤海马神经元。     

Role of TASK2 potassium channels regarding volume regulation in primary cultures of mouse proximal tubules

Several papers reported the role of TASK2 channels in cell volume regulation and regulatory volume decrease (RVD). To check the possibility that the TASK2 channel modulates the RVD process in kidney, we performed primary cultures of proximal convoluted tubules (PCT) and distal convoluted tubules (DCT) from wild-type and TASK2 knockout (KO) mice. In KO mice, the TASK2 coding sequence was in part replaced by the lac-Z gene. This allows for the precise localization of TASK2 in kidney sections using beta-galactosidase staining. TASK2 was only localized in PCT cells. K+ currents were analyzed by the whole-cell clamp technique with 125 mM K-gluconate in the pipette and 140 mM Na-gluconate in the bath. In PCT cells from wild-type mice, hypotonicity induced swelling-activated K+ currents insensitive to 1 mM tetraethylammonium, 10 nM charybdotoxin, and 10 microM 293B, but blocked by 500 microM quinidine and 10 microM clofilium. These currents were increased in alkaline pH and decreased in acidic pH. In PCT cells from TASK2 KO, swelling-activated K+ currents were completely impaired. In conclusion, the TASK2 channel is expressed in kidney proximal cells and could be the swelling-activated K+ channel responsible for the cell volume regulation process during osmolyte absorptions in the proximal tubules.     

Effects of the anion transport inhibitor,SITS, on the proximal straight tubule of the rabbit perfusedin vitro

Summary Conventional microelectrodes were used to study the effects of SITS (4-acetamido-4-isothiocyanostilbene-2,2-disulfonate) on the basolateral membrane potentialVbl of the superficial proximal straight tubule (PST) of the rabbit kidney perfusedin vitro. Addition of 0.1mm SITS to the bathing solution resulted in a slow and irreversible hyperpolarization ofVbl from –42.5±1.17 (37) mV to –77.3±0.83 (52) mV. The new steady-state potential was reached in 10 to 15 min and was accompanied by visible cell swelling. Associated with thisVbl hyperpolarization was: 1) an increased steady-state depolarization (from 6.2±0.77 (17) mV to 25.7±0.83 (29) mV) in response to increasing bath potassium concentration from 5 to 16.7mm (HK); 2) a decreased transient depolarization (from 19.8±1.88 (8) mV to 0.43±0.37 (8) mV) in response to decreasing bath bicarbonate concentration from 22 to 6.6mm at constant bath pH (L-HCO₃); and 3) inhibition of a depolarizing overshoot and a decreased steady-state depolarization (from 35.9±1.84 (12) mV to 4.7±1.37 (13) mV) in response to reducing bath sodium concentration from 144 to zero (0-Na). Sodium, chloride and NMDG (N-methyl-d-glucamine) were used as the substituting ions, respectively. These results are consistent with the presence of a coupled sodium-bicarbonate carrier in the basolateral membrane which is electrogenic and SITS inhibitable. Comparison of the time course of SITS effects on these ion-substitution responses suggests that the inhibition of the bicarbonate exit pathway(s) is the primary event and that the changes inVbl and in the steady-stateVbl responses to HK and 0-Na are secondary events which may be related to changes in intracellular composition and/or basolateral membrane properties.     

Effects of Vinpocetine on mitochondrial function and neuroprotection in primary cortical neurons

Vinpocetine (ethyl apovincaminate), a synthetic derivative of the Vinca minor alkaloid vincamine, is widely used for the treatment of cerebrovascular-related diseases. One of the proposed mechanisms underlying its action is to protect against the cytotoxic effects of glutamate overexposure. Glutamate excitotoxicity leads to the disregulation of mitochondrial function and neuronal metabolism. As Vinpocetine has a binding affinity to the peripheral-type benzodiazepine receptor (PBR) involved in the mitochondrial transition pore complex, we investigated whether neuroprotection can be at least partially due to Vinpocetine’s effects on PBRs.Neuroprotective effects of PK11195 and Ro5-4864, two drugs with selective and high affinity to PBR, were compared to Vinpocetine in glutamate excitotoxicity assays on primary cortical neuronal cultures. Vinpocetine exerted a neuroprotective action in a 1–50 μM concentration range while PK11195 and Ro5-4864 were only slightly neuroprotective, especially in high (>25 μM) concentrations. Combined pretreatment of neuronal cultures with Vinpocetine and PK11195 or Ro5-4864 showed increased neuroprotection in a dose-dependent manner, indicating that the different drugs may have different targets. To test this hypothesis, mitochondrial membrane potential (MMP) of cultured neurons was measured by flow cytometry. 25 μM Vinpocetine reduced the decrease of mitochondrial inner membrane potential induced by glutamate exposure, but Ro5-4864 in itself was found to be more potent to block glutamate-evoked changes in MMP. Combination of Ro5-4864 and Vinpocetine treatment was found to be even more effective.In summary, the present results indicate that the neuroprotective action of vinpocetine in culture can not be explained by its effect on neuronal PBRs alone and that additional drug targets are involved.     

Effects of garcinol on free radical generation and NO production in embryonic rat cortical neurons and astrocytes

Garcinol (camboginol) is a polyisoprenylated benzophenone derivative isolated from fruit rind of Garcinia indica. This study was to elucidate the anti-oxidative and neuroprotective properties of garcinol in rat cortical neuron cultures. First, garcinol protects DNA from Fenton reaction-induced breakage in a dose-dependent manner, with an IC(50) value of 0.32 microM. Garcinol also inhibits xanthine oxidase activity with an IC(50) value of 52 microM and exhibits competitive inhibition. To further ascertain the neuroprotective effects of garcinol in inflammatory-mediated neurotoxicity, we utilized primary neuron/astrocyte co-cultures treated with LPS or cytokine. Our data implicate that treatment with garcinol (5 microM) for 7 days promotes neuronal attachment and neurite extension. The formation of nitric oxide (NO) by LPS in rat astrocytes has been suggested to correlate with the neurodegenerative process. In identifying the effect of neuroprotection, we found that garcinol prevented NO accumulation in LPS-treated astrocytes. Garcinol significantly reduced the expression of LPS-induced inflammatory mediators, such as iNOS and COX-2. Consequently, our results suggest that the neuroprotective effects of garcinol are associated with anti-oxidation and inhibition of iNOS induction in astrocytic cells. Garcinol may exert a similar anti-inflammatory effect and may be neuroprotective against brain injury.     

Elevated intracellular pH appears in aged oocytes and causes oocyte aneuploidy associated with the loss of cohesion in mice

Increases in the aneuploidy rate caused by the deterioration of cohesion with increasing maternal age have been well documented. However, the molecular mechanism for the loss of cohesion in aged oocytes remains unknown. In this study, we found that intracellular pH (pH_i) was elevated in aged oocytes, which might disturb the structure of the cohesin ring to induce aneuploidy. We observed for the first time that full-grown germinal vesicle (GV) oocytes displayed an increase in pH_i with advancing age in CD1 mice. Furthermore, during the in vitro oocyte maturation process, the pH_i was maintained at a high level, up to ～7.6, in 12-month-old mice. Normal pH_i is necessary to maintain protein localization and function. Thus, we put forward a hypothesis that the elevated oocyte pH_i might be related to the loss of cohesion and the increased aneuploidy in aged mice. Through the in vitro alkalinization treatment of young oocytes, we observed that the increased pH_i caused an increase in the aneuploidy rate and the sister inter-kinetochore (iKT) distance associated with the strength of cohesion and caused a decline in the cohesin subunit SMC3 protein level. Young oocytes with elevated pH_i exhibited substantially the increase in chromosome misalignment.     

硅钙钾镁肥对南方稻田土壤酸性和盐基离子动态变化的影响

南方稻田土壤大面积酸化是水稻生产的主要限制因子.尽管石灰作为酸化土壤调理剂已广泛应用,但大量或长期施用石灰不仅会引起土壤板结,而且会导致土壤钙、钾、镁等元素的平衡失调.硅钙钾镁肥由于溶解度更低、养分全面是良好的替代材料.为了明确硅钙钾镁肥阻控土壤酸化的效果和作用,本研究采用连续4年的硅钙钾镁肥田间定位试验,以农民习惯施肥为对照,分析在农民习惯施肥基础上增施750、1125、1500和1875 kg·hm^-2硅钙钾镁肥下稻田土壤pH、交换性酸、交换性盐基离子和有效硅的动态变化.结果表明: 农民习惯施肥导致土壤pH、土壤交换性盐基和盐基饱和度逐年下降,土壤交换性酸逐年增加.与之相反,硅钙钾镁肥处理显著提高了土壤pH值,提高幅度随硅钙钾镁肥施用次数或用量的增加而增大.连续多次施用硅钙钾镁肥有效促进了盐基离子在土壤中的累积和土壤交换酸的消耗,特别是土壤交换性Ca²⁺、Mg²⁺的累积和土壤交换性Al³⁺的消耗,硅钙钾镁肥用量越大,积累或消耗的量越多,但速率相对越慢.土壤交换性酸消耗量中,硅钙钾镁肥释放的交换性盐基离子和相应碱贡献了108.8%,是交换性酸减少的主要途径.硅钙钾镁肥在改良稻田土壤酸性的同时,土壤有效硅含量逐年增加,增幅随硅钙钾镁肥施用量的增加而显著增大.总之,农民习惯施肥导致土壤持续酸化,酸化率为2.86 kmol H⁺·hm^-2·a^-1,硅钙钾镁肥能有效阻控酸化过程,产生了大量碱(9.69~18.44 kmol OH^-·hm^-2·a^-1),释放的Ca²⁺、Mg²⁺盐基离子和相应碱是土壤酸化阻控的主要作用因子.     

Contributions in astrocytes of SMIT1/2 and HMIT to myo-inositol uptake at different concentrations and pH

myo-Inositol is important for cell signaling both in cytoplasm and in intracellular organelles. It is required in the plasma membrane and cytoplasm for maintained synthesis of the second messengers, inositoltrisphosphate (IP(3)) and diacylglycerol (DAG) from phosphatidylinositol bisphosphate (PIP(2)), and in organelles as precursor for synthesis of complex signaling phospholipids and inositolphosphates from IP(3) and PIP(2). myo-Inositol must be taken up into the cell where its is used, because neither neurons nor astrocytes synthesize it. It is also an osmolyte, taken up in response to surrounding hyperosmolarity and released during hypo-osmolarity. There are three myo-inositol transporters, the Na(+)-dependent SMIT1 and SMIT2, and HMIT, which co-transports myo-inositol with H(+). Their relative expressions in astrocytes and neurons are unknown. Uptake kinetics for myo-inositol in astrocytes has repeatedly been determined, but always on the assumption of only one component, leaving kinetics for the individual transporters unknown. This paper demonstrates that astrocytes obtained directly from the brain express SMIT1 and HMIT, but little SMIT2, and that all three transporters are expressed in neurons. Cultured mouse astrocytes show a high-affinity/low-capacity myo-inositol uptake (V(max): 60.0 ± 3.0 pmol/min per mg protein; K(m): 16.7 ± 2.6 μM), mediated by SMIT1 and perhaps partly by SMIT2. It was determined in cells pre-treated with HMIT-siRNA and confirmed by specific inhibition of SMIT. However at physiologically relevant myo-inositol concentrations most uptake is by a lower-affinity/higher-capacity uptake, mediated by HMIT (V(max): 358 ± 60 pmol/min per mg protein; K(m): 143 ± 36 μM) and determined by subtraction of SMIT-mediated from total uptake. At high myo-inositol concentrations, its uptake is inhibited by incubation in medium with increased pH, and increased during intracellular acidification with NH(4)Cl. This is in agreement with literature data for HMIT alone. At low concentration, where SMIT1/2 activity gains importance, myo-inositol uptake is reduced by ammonia-induced intracellular acidification, consistent with the transporter's pH sensitivity reported in the literature.     

三氟氯氰菊酯对棉铃虫神经细胞延迟整流钾电流的抑制作用

用膜片钳技术首次研究了三氟氯氰菊酯对离体培养的棉铃虫中枢神经细胞延迟整流钾通道电流的影响。结果表明,药物作用前有81%和39%的细胞的通道分别在-30 mV 和 -40 mV 激活（n=21）。三氟氯氰菊酯（10^-5 mmol/L）作用15 min后,有63%和38%细胞的通道分别在-40 mV 和 -50 mV 激活（n=8）;作用1 min后电流幅值明显降低,抑制率达到了37.7%（n=19）;加药后激活曲线明显左移且Vh 值变化显著,但k值没有明显变化。实验结果说明,三氟氯氰菊酯作用后,通道更容易激活,但显著抑制电流峰值,导致神经敏感性降低,棉铃虫中枢神经细胞钾通道也是拟除虫菊酯类药物的作用靶标之一。     

Metabolic differences between primary cultures of astrocytes and neurons from cerebellum and cerebral cortex. Effects of fluorocitrate

Astrocytes and neurons cultured from mouse cerebellum and cerebral cortex were analyzed with respect to content and synthesis of amino acids as well as export of metabolites to the culture medium and the response to fluorocitrate, an, inhibitor of aconitase. The intracellular levels of amino acids were similar in the two astrocytic populations. The release of citrate, lactate and glutamine, however, was markedly higher from cerebellar than from cortical astrocytes. Neurons contained higher levels of glutamate, aspartate and GABA than astrocytic cultures. Cortical neurons were especially high in GABA and aspartate, and the level of aspartate increased specifically when the extracellular level of glutamine was elevated. Fluorocitrate inhibited the TCA cycle in the astrocytes, but was less effective in cerebellar neurons. Whereas neurons responded to fluorocitrate with an increase in the formation of lactate, reflecting, glycolysis, astrocytes decreased the formation of lactate in the presence of fluorocitrate, indicating that astrocytes to a high degree synthesize pyruvate and hence lactate from TCA cycle intermediates.     

Membrane potential,anion and cation conductances in Ehrlich ascites tumor cell

Summary The fluorescence intensity of the dye 1,1-dipropyloxadicarbocyanine (DiOC₃-(5)) has been measured in suspensions of Ehrlich ascites tumor cells in an attempt to monitor their membrane potential (V _m ) under different ionic conditions, after treatment with cation ionophores and after hypotonic cell swelling. Calibration is performed with gramicidin in Na⁺-free K⁺/choline⁺ media, i.e., standard medium in which NaCl is replaced by KCl and cholineCl and where the sum of potassium and choline is kept constant at 155mm. Calibration by the valinomycin null point procedure described by Lariset al. (Laris, P.C., Pershadsingh, A., Johnstone, R.M., 1976,Biochim. Biophys. Acta 436:475–488) is shown to be valid only in the presence of the Cl^–-channel blocker indacrinone (MK196). Distribution of the lipophilic anion SCN^– as an indirect estimation of the membrane potential is found not to be applicable for the fast changes inV _m reported in this paper. Incubation with DiOC₃-(5) for 5 min is demenstrated to reduce the Cl^– permeability by 26±5% and the NO ₃ ^– permeability by 15±2%, while no significant effect of the probe could be demonstrated on the K⁺ permeability. Values forV _m , corrected for the inhibitory effect of the dye on the anion conductance, are estimated at –61±1 mV in isotonic standard NaCl medium, –78±3 mV in isotonic Na⁺-free choline medium and –46±1 mV in isotonic NaNO₃ medium. The cell membrane is depolarized by addition of the K⁺ channel inhibitor quinine and it is hyperpolarized when the cells are suspended in Na⁺-free choline medium, indicating thatV _m is generated partly by potassium and partly by sodium diffusion. Ehrlich cells have previously been shown to be more permeable to nitrate than to chloride. Substituting NO ₃ ^– for all cellular and extracellular Cl^– leads to a depolarization of the membrane, demonstrating thatV _m is also generated by the anions and that anions are above equilibrium. Taking the previously demonstrated single-file behavior of the K⁺ channels into consideration, the membrane conductances in Ehrlich cells are estimated at 10.4 S/cm² for K⁺, 3.0 S/cm² for Na⁺, 0.6 S/cm² for Cl^– and 8.7 S/cm² for NO ₃ ^– . Addition of the Ca²⁺-ionophore A23187 results in net loss of KCl and a hyperpolarization of the membrane, indicating that the K⁺ permeability exceeds the Cl^– permeability also after the addition of A23187. The K⁺ and Cl^– conductances in A23187-treated Ehrlich cells are estimated at 134 and 30 S/cm², respectively. The membrane potential is depolarized in hypotonically swollen cells, confirming that the increase in the Cl^– permeability following hypotonic exposure exceeds the concommitant increase in the K⁺ permeability. In control experiments where the membrane potentialV _m =E _K =E _Cl =E _Na , it is demonstrated that cell volume changes has no significant effect on the fluorescence signal, apparently because of a large intracellular buffering capacity. The increase in the Cl^– conductances is 68-fold when cells are transferred to a medium with half the osmolarity of the standard medium, as estimated from the net Cl^– efflux and the change inV _m . The concommitant increase in the K⁺ conductance, as estimated from the net K⁺ efflux, is only twofold.     

Effects of chronic exposure to ammonia on glutamate and glutamine interconversion and compartmentation in homogeneous primary cultures of mouse astrocytes

Accumulation of radioactivity was studied in primary cultures of mouse astrocytes as a function of time of exposure (4–60 min) to 50 M glutamate and 200 M glutamine (initial concentrations), of whicheither glutamateor glutamine was¹⁴C-labeled. Both the glutamate pool and the glutamine pool were compartmentalized. Initially, by far the major intracellular glutamate pool (90%) was derived from extracellular glutamate and could be converted to glutamine. This allowed a rather accurate determination of metabolic flux from glutamate to glutamine, which under control conditions amounted to 2.0–2.2 nmol/min per mg protein. After chronic exposure to 3 mM ammonia for 3 days this flux was significantly increased to 3.1–3.6 nmol/min per mg protein. Acute exposure to ammonia caused a smaller, apparent increase, which was not statistically significant. The glutamine content was compartmentalized at all stages of the incubation. It consisted of at least two different pools. One of these was accessible to extracellular glutamine and could be converted to intracellular glutamate (constituting a sizeable fraction of the total glutamate pool after longer incubation), whereas the other constituted endogenously derived glutamine, formed from accumulated glutamate. The specific activity of the precursor pool for glutamate synthesis could not beaccurately determined and relatively exact fluxes therefore not be calculated. There was, however, no evidence that chronic exposure to ammonia decreases the rate of glutamine hydrolysis.     

Periaxonal surface calcium binding and distribution of charge on the faces of squid axon potassium channel molecules

Summary The calcium binding constant associated with external surface charge in a position to influence the voltage sensing charges for potassium channel gating appears to be 30 molar^–1, a value much larger than previously thought and in approximate agreement with that found for artificial membranes composed of the lipid brain phosphatidylserene. Fixed charge on the periaxonal membrane surface is distributed in such a way that much larger charges occur at a distance of at least 8 angstroms from the channel pore openings. The separation between the ion pathway and the channel gating charge appears to be greater than or equal to 8 angstroms. Periaxonal surface charge which is in a position to determine the surface potential for gating has a magnitude greater than or equal to one (negative) electronic charge per 182 square angstrom before calcium binding, which is reduced to –e/625 Å in a normal divalent ionic environment. With the normal divalent ionic composition of seawater the surface potential at a position to influence the gating voltage sensor is –15 millivolts relative to the bulk external potential. The external surface potential is –3 mV at the pore mouth. There appears to be a negligible amount of fixed charge on the axoplasmic surface in the vicinity of the ion channel opening. Further, our results confirm earlier measurements that have given a negligible amount of axoplasmic surface fixed charge whose field components would be in a position to influence the channel gating charges.     

Light and electron microscopic localization of GABAA-receptors on cultured cerebellar granule cells and astrocytes using immunohistochemical techniques

GABA_A-receptors were localized in explant cultures of rat cerebellum and in dissociated primary cultures of rat cerebellar granule cells and rat cerebellar astrocytes using the monoclonal antibody bd-17 directed against the -subunit of the GABA_A/benzodiazepine/chloride channel complex. At the light microscope level specific staining of GABA_A-receptors was localized in various types of neurones in explant cultures of rat cerebellum using the indirect peroxidase-antiperoxidase (PAP) technique, whereas no specific staining was found in astrocytes. At the electron microscope level labeling of GABA_A-receptors was observed in the plasma membrane of both the cell bodies and processes in dissociated primary cultures of cerebellar granule cells using an indirect preembedding immunogold staining technique which in contrast to the classical PAP technique allows quantitative estimations to be performed. Quantification of the labeling intensity revealed a higher concentration of GABA_A-receptors per m plasma membrane in the cell bodies than in the processes. In discrete areas an extremely high density of the GABA_A-receptors was observed. No specific labeling of GABA_A-receptors was observed in dissociated primary cultures of cerebellar astrocytes.Special issue dedicated to Dr. Eugene Roberts.     

Effects of cyhalothrin on the transient outward potassium current in central neurons of Helicoverpa armigera

The effects of cyhalothrin on the transient outward potassium current in central neurons of Helicoverpa armigera were studied by using the patch clamp techniques. The results showed that before using cyhalothrin （10.5 mmol/L）, activation potential was approximately -40 mV, after application of the drug, the activation potential shifted roughly 10 mV to the negative potential direction, so channels can be activated more easily. Before and after cyhalothrin application, the change of current amplitude was insignificant. The value of V1/2 and k of activation curves did not change significantly, however, the V1/2 of the inactivation curves changed significantly. Inactivation curves significantly shifted to a negative direction, so that inactivation of the channels was hastened. It is indicated that there may exit a primary way in which cyhalothrin provides neurotoxicity to the nervous system through the regulation of activation potentials and inactivation state of IA channels.