Evaluation of mag-fura-5, the new fluorescent indicator for free magnesium measurements

The new fluorescent indicator, mag-fura-5, was evaluated for its ability to measure accurately physiological changes in cytosolic free magnesium. The apparent dissociation constants (K _d) of the fluorochrome for Mg²⁺, Mg²⁺/EGTA and Ca²⁺/EGTA solutions were 14.7 mM, 15.4 mM, and 1.8 mM respectively. The calculated difference in the fluorescence ratios and in the resulting pMg between the standards with low-Ca²⁺ or low H⁺ backgrounds and the corresponding samples with approximately physiological levels were not significant. In contrast, the changes due to an increased Ca²⁺ or H⁺ content were statistically significant, with mean pMg differences of 0.10±0.09 (P<0.02) and 0.33±0.26 (P<0.01) respectively. Repetitive measurements on 3 consecutive days yielded comparable data with differences not exceeding 4%. Because of the good reproducibility, it is suggested that the new fluoresecent probe may be suitable for free cytosolic magnesium determinations in isolated cells.     

穿琥宁联合病毒唑治疗小儿喘息性肺炎

目的 :观察穿琥宁联合病毒唑治疗小儿喘息性肺炎的疗效。方法 :65例喘息性肺炎患者随机分治疗组和对照组 ,治疗组 3 0例用穿琥宁、病毒唑静脉点滴 7天 ,对照组 3 5例用病毒唑、青霉素静脉点滴 7天。结果 :穿琥宁组的临床治愈率为 83 .3 % ,总有效率 96.7%。对照组临床治愈率为 5 7.1% ,总有效率 71.4%。经统计分析 ,两组疗效有显著性差异 ,χ2 =7.90 ,P <0 .0 1。结论 :穿琥宁联合病毒唑治疗喘息性肺炎治愈率高 ,疗效满意。     

Mechanism of action of the epileptogenic drug pentylenetetrazol on a cloned neuronal potassium channel

The action of the epileptogenic agent pentylenetetrazol (PTZ) on a cloned potassium channel of the rat brain was studied. The Kv1.1 channel was expressed in oocytes ofXenopus laevis and potassium currents were investigated in outside-out and inside-out membrane patches. The results show that PTZ increased the multi-channel potassium currents at strongly negative potentials and decreased them at potentials positive to −35 mV both in outside-out and inside-out membrane patches. The extent and manner of PTZ action, the concentration dependence as well as the onset and time course of the PTZ effect were the same both in outside-out and inside-out membrane patches. The single-channel potassium currents showed an increase in open probability and frequency of opening and a decrease in close time at −50 mV and vice versa at 0 mV with application of PTZ. The amplitude of single-channel current, the open time and the latency to the first channel opening remained almost unchanged under PTZ. The results indicate that PTZ acts via the cell membrane and influences the membrane-associated part of the potassium channel. Thereby, PTZ accelerates the transition from the inactivated to the open state of the channel at strongly negative potentials and reduces it at slightly negative and positive potentials. This mechanism may be the basis for a gate function which is in favour of the development of epileptic discharges.     

Neurotransmitter release evoked by α-latrotoxin in the smooth muscle of the female pig urethra

Neuronal regulation of smooth muscle tone in the female pig urethra has mainly been studied in vitro using electrical field stimulation (EFS) of nerves. Excitatory control is considered to be exerted by released noradrenaline, whereas inhibitory control is non-adrenergic non-cholinergic (NANC), and mediated by nitric oxide (NO), and an as yet unidentified agent. We investigated the functional and morphological effects of α-latrotoxin (αLTX), a spider neurotoxin believed to cause massive release of vesicle-stored neurotransmitters, on spontaneously developed urethral smooth muscle tone. The effects were compared to those of EFS and high potassium. In the presence of the NO-synthesis inhibitor N^ω-nitro-L-arginine (L-NOARG: 0.3 mM) both αLTX and EFS evoked contractions. After treatment with scopolamine and phentolamine, no contraction was observed, and under these conditions αLTX and EFS induced relaxation. At low frequencies (<12 Hz), the EFS-induced relaxations were rapid, whereas at higher frequencies (>12 Hz), they were biphasic, consisting of a rapid first phase followed by a more long-lasting second phase. L-NOARG abolished the relaxations at low frequencies, as well as the first phase of the biphasic relaxation. The second phase was not affected by treatment with L-NOARG, but 0.1 μM ω-conotoxin GVIA, blocker of N-type voltage-operated calcium- channels (VOCCs), markedly reduced or abolished the response. In the presence of L-NOARG or ω-conotoxin GVIA, the αLTX-induced relaxation was significantly decreased, and the combination of L-NOARG and ω-conotoxin GVIA further reduced or abolished the relaxation. In preparationstreated with tetrodotoxin or scorpion venom, believed to inactivate nerves by acting on sodium channels, αLTX and EFS had no effects. αLTX-induced relaxation was not associated with changes in cyclic GMP or cyclic AMP content. High (80 mM) potassium solution induced a triphasic response of the preparation. A transient relaxation was followed by a restoration of tone, and then by a persistent relaxation. The persistent relaxation was slightly reduced by scorpion venom or L-NOARG, but reduced by 50% by a combination of L-NOARG and ω-conotoxin GVIA. Ultrastructural analysis of the urethral circular smooth muscle layer revealed a moderate amount of nerve profiles supplying the smooth muscle. In control preparations, the nerve profiles contained both small synaptic vesicles and large dense core vesicles. αLTX caused a major loss of both types of vesicle. The present data suggest that αLTX has the ability to release not only adrenergic and cholinergic transmitters, but also NANC mediators of relaxation, including NO, from nerve terminals in the urethra. Received: 13 January 1997 / Accepted: 17 April 1997     

Protective Effect of Tetrandrine against Excitatory Amino Acids induced Neuronal Injury in Cortical Culture

兴奋性氨基酸类神经毒剂与粉防己碱（ｔｅｔｒａｎｄｒｉｎｅ，Ｔｅｔ）共同作用于原代培养胎鼠大脑皮层神经元２４小时，发现１０７，１０６ｍｏｌ·Ｌ１Ｔｅｔ明显降低５０μｍｏｌ·Ｌ１谷氨酸（ｇｌｕｔａｍａｔｅ，Ｇｌｕ），３００μｍｏｌ·Ｌ１βＮｍｅｔｈｙｌａｍｉｎｏＬａｌａｎｉｎｅ（ＢＭＡＡ，ＮＭＤＡ受体激动剂）和２０μｍｏｌ·Ｌ１βＮｏｘａｌｙｌａｍｉｎｏＬａｌａｎｉｎｅ（ＢＯＡＡ，ｎｏｎＮＭＤＡ受体激动剂）导致的培养液乳酸脱氢酶（ｌａｃｔａｔｅｄｅｈｙｄｒｏｇｅｎａｓｅ，ＬＤＨ）活性的增高；细胞形态损害减轻，细胞数量增加。对２０μｍｏｌ·Ｌ１ＮＭＤＡ介导的神经元损伤改变无影响。提示Ｔｅｔ对某些Ｇｌｕ类神经毒剂引起的胎鼠大脑皮层神经元损伤有一定保护作用，其机制可能是抑制细胞膜上的Ｎａ＋通道开放，阻止膜去极化而影响电压依赖性Ｃａ２＋通道启动。对ＮＭＤＡ受体可能亦有一定作用。     

Altered calcium metabolism in red blood cells of hypertensives: Persistent marker or sequel of essential hypertension?

Summary The characteristics of the increased calcium (Ca) influx observed in metabolically depleted red blood cells (RBCs) of hypertensive patients were investigated. Twenty-four normotensives, 16 untreated essential hypertensives, and 10 essential hypertensives under sufficient blood pressure control by 50–100 mg/day atenolol were studied. Free intracellular concentrations of Ca, sodium (Na), and potassium (K) were assessed using ion-selective electrodes in freeze-thawed RBCs, which were metabolically depleted by 30 mM desoxy-glucose at 37°C for 48 h. In the treated hypertensives values for Ca and K at 24 and 48 h were not different from values for the normotensives, whereas elevated Ca was found in RBCs of untreated hypertensives. Na in treated hypertensives was significantly increased at 0 and 48 h, thus, being similar to values for untreated hypertensives. Additionally, RBCs of six normals were stressed in a glass/teflon potter. Before metabolic depletion electrolytes were not affected by this procedure, while Ca at 24 and 48 h of metabolic depletion increased to significantly higher values for the hypertensive patients as compared to the controls. These results suggest that the altered Ca metabolism in the RBCs of hypertensives may reflect a secondary phenomenon due to the mechanical damage to RBCs by the elevated blood pressure.     

Niflumic acid-induced increase in potassium currents in frog motor nerve terminals: effects on transmitter release

The actions of the nonsteroidal antiinflammatory drug niflumic acid were studied on frog neuromuscular preparations by conventional electrophysiological techniques. Niflumic acid reduced the amplitude and increased the latency of endplate potentials in a concentration-dependent manner. Neuromuscular junctions pretreated with niflumic acid (0.05–0.5 mM) showed much less depression than control when they were stimulated with trains of impulses. Inhibition of acetylcholine release was reverted by raising the extracellular Ca²⁺ concentration but not by simply washing out the preparations with niflumic acid-free solutions. Pretreatment with indomethacin (0.1 mM), another nonsteroidal antiinflamatory drug, did not affect the niflumic acid-induced inhibition of evoked responses. Niflumic acid (0.1 mM) did not change the amplitude of miniature endplate potentials and had a dual action on the frequency of miniatures: it decreased their frequency at 0.1 mM whereas it produced an enormous increase in the rate of spontaneous discharge at 0.5 mM. Niflumic acid (0.1–1 mM) reversibly increased the amplitude and affected the kinetics of presynaptic voltage-activated K⁺ current and Ca²⁺-activated K⁺ current in a concentration-dependent manner. Niflumic acid (0.1–1 mM) irreversibly decreased the amplitude and reversibly affected the kinetics of the nodal Na⁺ current. Indomethacin (0.1 mM) had no effect on presynaptic currents. In conclusion, niflumic acid reduces acetylcholine release by increasing presynaptic K⁺ currents. This may shorten the depolarizing phase of the presynaptic action potential and may reduce the entry of Ca²⁺ with each impulse.     

Activation of a Ca2+-dependent K+ current by the oncogenic receptor protein tyrosine kinase v-Fms in mouse fibroblasts

We investigated the effects of the receptor-coupled protein tyrosine kinase (RTK) v-Fms on the membrane current properties of NIH3T3 mouse fibroblasts. We found that v-Fms, the oncogenic variant of the macrophage colony-stimulating factor receptor c-Fms, activates a K⁺ current that is absent in control cells. The activation of the K⁺ current was Ca²⁺-dependent, voltage-independent, and was completely blocked by the K⁺ channel blockers charybdotoxin, margatoxin and iberiotoxin with IC₅₀ values of 3nM, 18 nM and 76nM, respectively. To identify signalling components that mediate the activation of this K⁺ current, NIH3T3 cells that express different mutants of the wildtype v-Fms receptor were examined. Mutation of the binding site for the Ras-GTPase-activating protein led to a complete abolishment of the K⁺ current. A reduction of 76% and 63%, respectively, was observed upon mutation of either of the two binding sites for the growth factor receptor binding protein 2. Mutation of the ATP binding lobe, which disrupts the protein tyrosine kinase activity of v-Fms, led to a 55% reduction of the K⁺ current. Treatment of wild-type v-Fms cells with Clostridium sordellii lethal toxin or a farnesyl protein transferase inhibitor, both known to inhibit the biological function of Ras, reduced the K⁺ current amplitude to 17% and 6% of the control value, respectively. This is the first report showing that an oncogenic RTK can modulate K⁺ channel activity. Our results indicate that this effect is dependent on the binding of certain Ras-regulating proteins to the v-Fms receptor and is not abolished by disruption of its intrinsic protein tyrosine kinase activity. Furthermore, our data suggest that Ras plays a key role for K⁺ channel activation by the oncogenic RTK v-Fms. Received: 19 November 1997 / Accepted: 21 January 1998     

Ion channel expression by white matter glia: I. Type 2 astrocytes and oligodendrocytes

White matter is a compact structure consisting primarily of neuronal axons and glial cells. As in other parts of the nervous system, the function of glial cells in white matter is poorly understood. We have explored the electrophysiological properties of two types of glial cells found predominantly in white matter: type 2 astrocytes and oligodendrocytes. Whole-cells and single-channel patch-clamp techniques were used to study these cell types in postnatal rat optic nerve cultures prepared according to the procedures of Raff et al. (Nature, 303:390-396, 1983b). Type 2 astrocytes in culture exhibit a "neuronal" channel phenotype, expressing at least six distinct ion channel types. With whole-cell recording we observed three inward currents: a voltage-sensitive sodium current qualitatively similar to that found in neurons and both transient and sustained calcium currents. In addition, type 2 astrocytes had two components of outward current: a delayed potassium current which activated at 0 mV and an inactivating calcium-dependent potassium current which activated at -30 mV. Type 2 astrocytes in culture could be induced to fire single regenerative potentials in response to injections of depolarizing current. Single-channel recording demonstrated the presence of an outwardly rectifying chloride channel in both type 2 astrocytes and oligodendrocytes, but this channel could only be observed in excised patches. Oligodendrocytes expressed only one other current: an inwardly rectifying potassium current that is mediated by 30- and 120-pS channels. Because these channels preferentially conduct potassium from outside to inside the cell, and because they are open at the resting potential of the cell, they would be appropriate for removing potassium from the extracellular space; thus it is proposed that oligodendrocytes, besides myelinating axons, play an important role in potassium regulation in white matter. The conductances present in oligodendrocytes suggest a "modulated Boyle and Conway mechanism" of potassium accumulation.     

Transient outward current (I A) in clonal anterior pituitary cells: blockade by aminopyridine analogs

Summary Whole cell voltage-clamp recordings from GH₃ cells, a clonal cell line derived from a rat anterior pituitary tumor, demonstrated a rapidly activating and inactivating (transient) voltage-dependent outward current. This current, referred to as I _A, was elicited by step depolarization from holding potentials negative to –50 mV, showed strong outward rectification at potentials positive to –30 mV, and exhibited steady state inactivation with V _1/2 near –64 mV. The current rose to a peak within < 10–20 ms following depolarization and decayed in two exponential phases, I _Af and IA _AS with time constants of 30–50 and 500–700 ms, respectively. Both I _A components exhibited similar voltage dependencies for activation and inactivation. Aminopyridines (2 mol/l – –5 mmol/l) produced a dose dependent, reversible blockade of I _A (70% inhibition at 0.5 to 2 mmol/l) with the following rank order of potencies: 4-aminopyridine > 3,4-diaminopyridine = 3-aminopyridine > 2-aminopyridine. These drugs reduced the peak conductance of I _A, and produced complex effects on its time-dependent decay. With submaximal degrees of block, there was an increase in the inactivation rate, suggesting that open channels are preferentially blocked by the drugs. It is concluded that GH₃ pituitary cells possess an aminopyridine-sensitive transient outward current comparable to the A-current in neural cells. However, this cell line is unusual in that it expresses both rapidly and slowly decaying A-current components.Abbreviations n-AP n-aminopyridine - 3,4-DAP 3,4-diaminopyridine - TEA tetraethylammonium - EGTA ethylene glycol bis(-aminoethyl ether)N,N-tetraacetic acid - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid Send offprint requests to M. A. Rogawski at the above address