Cross-desensitization Reveals Pharmacological Specificity of Excitatory Amino Acid Receptors in Isolated Hippocampal Neurons

Ionic currents elicited by excitatory amino acids were studied, using the concentration clamp method, in enzymatically isolated rat hippocampal neurons. Cross-desensitization between the responses to various agonists was applied to separate the activity of two types of receptors, N-methyl-d-aspartate (NMDA) and non-NMDA. NMDA receptors were selectively activated by NMDA, l- and d-aspartate, d-glutamate and quinolinate. Kainate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate appeared to be selective, and quisqualate relatively less selective non-NMDA agonists, acting on the same receptor type. l-Glutamate, l- and d-homocysteate activated both receptor types. It is supposed that two receptor sites, activation site and desensitization site, control the action of agonists at the non-NMDA receptor. When examined in the cross-desensitization experiments, NMDA and non-NMDA receptors appear to be represented by the two homogeneous and independent receptor populations operating different ionic channels.     

Ca2+ channel actions of the non-dihydropyridine Ca2+ channel antagonist Ro 40-5967 in vascular muscle cells cultured from dog coronary and saphenous arteries

Summary We studied the membrane effects of (1S,2S)-2-(2-[[3-2(benzimidazolyl) propyllmethylamino]ethyl)-6-fluoro-1,2,3,4-tetrahydro-l-isopropyl-2-naphthyl-methoxy-acetate dihydrochloride, Ro 40-5967, a new non-dihydropyridine (DHP) Ca²⁺ channel antagonist, on dog coronary and saphenous arterial vascular muscle cells using the whole-cell patch-clamp method. Long-lasting (L-type) inward currents in 20 mM Ba²⁺ were measured over a range of test potentials (300 ms) from –50 mV to + 90 mV from a holding potential of –80 mV in the presence of 1 M Bay k8644 (a DHP Ca²⁺ agonist). Ro 40-5967 caused a concentration-dependent suppression of Ca²⁺ channel currents in muscle cells from both arteries, with greater potency on coronary than saphenous arterial cells. The concentration of Ro 40-5967 which inhibited the magnitude of peak inward currents by 50% (IC₅₀) was estimated to be 1 M (n = 5) in muscle cells from coronary artery and 10 M (n = 4) in saphenous artery. Ro 40-5967 (1 M) decreased the amplitude of the activation current-voltage relationship for coronary L-type Ca²⁺ channel currents over a wider range of membrane potentials than verapamil, diltiazem, or nifedipine. In contrast, block of Ca²⁺ channel currents in saphenous artery cells by 1 M Ro 40-5967 was only observed at command potentials positive to 0 mV. Ro 40-5967 (1 M) significantly shifted the voltage-inactivation curve downward by 40% in coronary (n = 4), but only by 18% in saphenous arterial muscle cells (n = 3). The non-parallel shift of the coronary artery inactivation curve suggests that pronounced resting channel block is a notable feature of Ro 40-5967. The marked inhibition of Ba²⁺ current by 1 M Ro 40-5967 in the inactivation protocol in coronary arterial muscle cells was found over the entire range of membrane holding potentials tested, while inhibition in the saphenous artery inactivation curve occurred only from holding potentials more positive than –40 mV. Therefore, Ro 40-5967 is unique: 1) in acting over a wider range of voltages, on both instantaneous and resting Ca²⁺ currents, than other Ca²⁺ antagonists; 2) in producing more significant resting state block; and 3) in acting with selectivity for coronary over saphenous arteries.This research was supported by National Institutes of Health grants HL38537, HL38645, and by F. Hoffmann-La Roche, Basel, Switzerland     

牛蛙球囊毛细胞电压依赖性钾通道电流的研究

目的 :研究牛蛙球囊毛细胞膜上钾通道的类型。方法 :全细胞膜片钳技术。结果 :1当钳制电位为 - 1 0 0 m V,以 1 0 m V的步距阶跃 ,阶跃从 - 70 m V至 + 2 0 m V,随着膜电位的去极化 ,可记录到一系列快速、瞬时的外向电流 ,4- Ap对其有特异性阻断作用。激活电压为 - 53.85± 7.8m V。2 A型钾通道的数量在牛蛙毛细胞间存在差异。 3当钳制电位为 - 70 m V,以 1 0 m V的步距阶跃去极化 ( - 50 m V～ + 4 0 m V) ,可产生一延迟整流性钾离子流 ,四乙基氯化氨 ( TEA)能使该电流幅度下降 59%± 1 1 %。结论 :牛蛙毛细胞膜上含有 A型钾通道和延迟整流性钾通道。     

二甲基-氨氯吡咪和牛黄酸对大鼠心室肌细胞Na+/Ca2+交换电流的影响

利用全细胞膜片钳技术 ,采用胶原酶B急性分离的大鼠心室肌细胞 ,研究了牛黄酸和二甲基 -氨氯吡咪对心肌细胞膜Na /Ca2 交换电流的影响。结果表明 ,10 μmol,2 0 μmol和 4 0 μmol的二甲基 -氨氯吡咪可使Ni2 敏感电流浓度依赖性地增加 ;膜电位为 5 0mV时分别增加 ( 2 7± 7) % ,( 12 1± 4 3 ) % ,和 ( 173± 68) % ;膜电位为 - 10 0mV时分别增加 ( 110±5 2 ) % ,( 2 2 1± 88) %和 ( 2 81± 80 ) %。牛黄酸对Na /Ca2 交换的内向和外向电流均无影响     

Insulin concentrations and insulin sensitivity after short-term amiloride in healthy subjects

We have evaluated the short-term effects of amiloride on insulin action in vivo, since amiloride is known to impair insulin action in vitro.Seven healthy subjects were treated according to a randomized, double-blind, cross-over protocol. The treatment periods were 3 days each with amiloride 15 mg daily and placebo. Insulin action on glucose turnover was assessed directly after each treatment period with the hyper-insulinaemic euglycaemic glucose clamp technique.At the two insulin concentrations studied ( 30 mU·l^–1 and 200 mU·l^–1), the glucose infusion rate required to maintain constant euglycaemia did not differ after either amiloride or placebo. The rates of glucose production and utilization were also similar, whereas the so-called insulin sensitivity index at the lower insulin concentration was significantly reduced (by about 15 %) after amiloride. Moreover, amiloride produced significantly higher fasting insulin and C-peptide concentrations, whereas fasting glucose and NEFA concentrations were unaltered.In conclusion, these data suggest that short-term amiloride slightly impairs insulin sensitivity with respect to glucose uptake. However, overall glucose homoeostasis does not appear to be affected, probably due to a compensatory rise in plasma insulin.     

Kainate activates Ca2+-permeable glutamate receptors and blocks voltage-gated K+ currents in glial cells of mouse hippocampal slices

Glial cells in the CA1 stratum radiatum of the hippocampus of 9- to 12-day-old mice show intrinsic responses to glutamate due to the activation of -amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/ kainate receptors. In the present study we have focused on a subpopulation of the hippocampal glial cells, the complex cells, characterized by voltage-gated Na⁺ and K⁺ channels. Activation of glutamate receptors in these cells led to two types of responses, the activation of a cationic conductance, and a longer-lasting blockade of voltage-gated K⁺ channels. In particular, the transient (inactivating) component of the outwardly rectifying K⁺ current was diminished by kainate. Concomitantly, as described in Bergmann glial cells, kainate also elevated cytosolic Ca²⁺. This increase was due to an influx via the glutamate receptor itself. In contrast to Bergmann glial cells, the cytosolic Ca²⁺ increase was not a link to the K⁺ channel blockade, since the blockade occurred in the absence of the Ca²⁺ signal and, vice versa, an increase in cytosolic Ca²⁺ induced by ionomycin did not block the transient K⁺ current. We conclude that glutamate receptor activation leads to complex and variable changes in different types of glial cells; the functional importance of these changes is as yet unresolved.     

Analysing ion channels with hidden Markov models

Ion channel current amplitudes () and open probabilities (P _o) have been analysed so far by defining a 50% threshold to distinguish between open and closed states of the channels. With this standard method (SM) it is very difficult or even impossible to analyse channels of different size in one membrane patch correctly. A stochastical model, named the hidden Markov model (HMM), separates between observation noise and the stochastic process of opening and closing of ion channels. The HMM allows the independent analysis of , P _o, and mean dwell times () of different channels in one membrane patch, without defining threshold levels. Using this method errors in the analysis are not summarized like in the SM because all different analysing procedures (e. g. filtering, setting of threshold, fitting processes) are done in one step. Two different K⁺ channels in excised basolateral membranes of the cortical collecting duct of rat (CCD) were analysed by the SM and the HMM. The value of the intermediate-conductance K⁺ channel (i-K⁺) was 3.9±0.1 pA (SM) and 3.8±0.2 pA (HMM) for 11 observations. The P _o value of this channel was 10.2±4.2% (SM) and 10.1±4.0% (HMM). The mean values were 5.4±0.6 ms for the open state and 9.6±2.2 ms and 145±21 ms for the closed states (SM) and 7.8±1.1 ms, 7.7±0.9 ms and 148±24 ms (HMM), respectively. For seven small-conductance K⁺ (s-K⁺) channels, which were found in the same membrane patches as the i-K⁺, an accurate analysis of P _o and was not possible with the SM. The value was 1.0±0.1 (SM), 0.9±0.1 (HMM) pA. P _o was 16.6±4.6%, the open value was 11.1±2.8 ms, and the closed value was 34.9±8.5 ms. The HMM allows the analysis of single-channel currents, P _o, and mean values when different or more than one ion channel(s) are colocalized in one membrane patch. Where analysis with the SM was possible results did not significantly differ from those obtained with the HMM. Thus for this kind of analysis the method of setting a 50% threshold appears justified.     

Tonic inhibition of neuronal calcium channels by G proteins removed during whole-cell patch-clamp experiments

The barium current through voltage-dependent calcium channels was recorded from cultured rat cortical neurons with the whole-cell configuration of the patch-clamp technique. The maximal current evoked by depolarising pulses from –80 mV to 0 mV was divided into inactivating and non-inactivating fractions. During the first minutes of whole-cell recording, the amplitude of the inactivating fraction increased from less than 0.1 nA to an average value of 1 nA, whereas the amplitude of the non-inactivating component remained essentially the same. This increase in amplitude was prevented when the perforated-patch technique was used, suggesting that some intracellular factor that inhibited the barium current was lost or destroyed during conventional whole-cell experiments. When GTP[-S] or GTP was added to the pipette solution, no increase or only a weak rise of the inactivating current was seen, whereas GDP[-S] accelerated its increase. The results suggest that some of the calcium channels expressed in cultured cortical neurons are inhibited by a G protein even in the absence of added neurotransmitter. The current increase observed during whole-cell recordings may be due to a loss of intracellular GTP and the subsequent inactivation of an inhibitory G protein.     

跨上皮电位差对蛙直肠上皮顶膜氯通道的作用

氯离子跨紧密上皮转运主要采取细胞通路,其转运调控的关键部位是上皮细胞顶膜氯通道。应用上皮电压钳技术、高钾除极化技术和Ｕｓｓｉｎｇ上皮组织灌流系统结合测定氯离子通量,观察跨上皮电位差对氯离子跨蛙直肠上皮转运的影响。实验结果表明跨上皮电位差对氯离子跨蛙直肠上皮吸收是必不可少的。跨上皮电位差作为转运驱动力外,还能激活蛙直肠上皮细胞顶膜的氯通道。     

Proton-gated ion channels in cultured mouse cortical neurons

Proton-gated ion channels in cultured mouse cortical neurons were characterized using the patch clamp technique. In voltage clamp, rapid shifts of the extracellular saline from pH 7.4 to <7.0 invariably triggered inward currents carried by sodium. The currents were inhibited by Amiloride (IC₅₀: 6.2 μM). In current clamp, acidic saline depolarized the neurons and triggered trains of action potentials. Concentration–response experiments revealed an extreme intercell variance as the EC₅₀-value for protons varied from pH 6.8 to 5.6, indicating heterogeneity in channel type expression from cell to cell. The possible involvement of acid sensing ion channels in ischemic neurodegeneration is discussed.