Modulation of vertebrate neuronal calcium channels by transmitters

A large number of neurotransmitters have now been shown to reduce the amplitude and slow the activation kinetics of whole cell HVA I_Ca in a great diversity of neurons. These transmitters include l-glutamate (AMPA/kainate, metabotropic and NMDA receptors), G AB A (via GABA_B receptors, NA (via α₂ receptors), 5-HT, N A (via α₂ receptors), DA and several peptides. Both whole-cell and single-channel studies have demonstrated that the N-channel is the most common channel type to be blocked by transmitters, although an inhibition of the L-type channel has also occasionally been reported. The suppression of the N-type Ca current was commonly shown to be voltage-dependent, with a relief at large positive voltages. Strong evidence has been put forward showing that the transmitter action is mediated by a G-protein, with GDP-β-S blocking transmitter action, and GTP-γ-S directly inhibiting the Ca channel. Moreover, pertussis toxin blocked the transmitter action in most neurons, and following such block, injection of the G-protein G₀ restored transmitter action. A direct link between the G-protein and the Ca channel has been widely theorized to mediate the action of transmitters on certain neurons. There is also some evidence that certain transmitters in specific neurons mediate calcium channel inhibition through a 2nd messenger, perhaps protein kinase C.Transmitters have also been found, although uncommonly, to inhibit HVA L-type and LVA T-type channels. In addition, an enhancement of both HVA and LVA, Ca currents by transmitters has been demonstrated, and substantial evidence exists for mediation of this action by cAMP.     

Cobalt-dependent potentiation of net inward current density in Helix aspersa neurons.

A low concentration of transition metal ions Co2+ and Ni2+ increases the inward current density in neurons from the land snail Helix aspersa. The currents were measured using a single electrode voltage-clamp/internal perfusion method under conditions in which the external Na+ was replaced by Tris+, the predominant external current carrying cation was Ca2+, and the internal perfusate contained 120 mM Cs+/0 K+; 30 mM tetraethylammonium (TEA) was added externally to block K+ current. In the presence of Co2+ (3 mM) or Ni2+ (0.5 mM) inward Ca2+ currents were stimulated normally by voltage-dependent activation of Ca2+ channels. There was a 5-10% decrease in the rate of rise of the inward current. The principal effect of Co2+ and Ni2+ in increasing the current density seems to be a decrease in the rate at which the inward currents decline during a depolarizing voltage pulse. The results may be due to a decrease in a voltage-dependent or Ca(2+)-dependent outward current and/or an inhibition of Ca2+ channel inactivation. Outward current under these conditions (zero internal K+) was significant and most likely due to Cs+ efflux through the voltage-activated or Ca(2+)-activated nonspecific cation channels. Co2+ is an extremely effective blocker of this outward current. These results are not an artifact of internal perfusion or the special ionic conditions. Intracellular recording of unperfused neurons in normal Helix Ringer's solution showed that the Ca(2+)-dependent action potential duration was increased significantly by low concentrations of Co2+. This result is consistant with the Co(2+)-dependent increase in inward (depolarizing) current seen in voltage-clamp experiments.     

Analysis of a decreased Na+ conductance by tumor necrosis factor in identified neurons of Aplysia kurodai.

The ionic mechanisms of the effect of extracellularly ejected recombinant human tumor necrosis factor-alpha (rhTNF-alpha) on the membrane of identified neurons R9 and R10 of Aplysia kurodai was investigated with conventional voltage-clamp, micropressure ejection, and ion substitution techniques. Micropressure-ejected rhTNF caused a marked hyperpolarization in the unclamped neuron. Clamping the same neuron at it resting potential level (-60 mV) and reejecting rhTNF-alpha with the same dose produced a slow outward current [Io (TNF)] associated with a decrease in input membrane conductance. Io (TNF) was decreased by depolarization and increased by hyperpolarization. The extrapolated reversal potential of Io (TNF) was approximately +10 mV. Ion substitution and pharmacological experiments suggest that Io (TNF) in identified neurons R9 and R10 of A. kurodai is due to a decreased Na+ conductance but not due to an activation of the Na(+)-K+ pump. Our results demonstrate that the immunomodulator TNF can act directly on the nervous system as well as on the immune system.     

Morphological analysis of renal cell culture models of calcium phosphate stone formation

Cell culture models of calcium phosphate renal stone formation were established using the MDCK cell line. Renal microliths were detected within pseudocysts in three-dimensional soft agar cultures, and were also observed in the basal region of cells lining the cell sheet, and immediately beneath domes or blisters in monolayers and collagen gel cultures. Light and scanning electron microscopy indicated that these microliths had a similar lamellated and spherical appearance to those in humans. These microliths were first detected microscopically after 21 days of culture, and were found to be composed of calcium phosphate by X-ray and microinfrared spectroscopic analyses. These culture models may provide a powerful new tool to study the pathogenesis of renal stone diseases and/or calcium phosphate stone formation in humans and animals.     

磷脂酶A2激活在鼠急性缺血性脑损伤中的作用机制

目的 探讨急性脑缺血后脑组织内磷脂酶A2（PLA2）激活及细胞内[Ca^2 ]i与脑损伤的关系，为预防和治疗急性缺血性脑损伤提供理论基础和新的思路。方法 将局灶性脑缺血模型大鼠分5组（假手术组、缺血30、60、90、120min组），测定脑组织PLA2活力、脑细胞[Ca^2 ]i、脑含水量及缺血120min组脑组织PLA2表达量的改变。结果 脑缺血120min脑组织PLA2活性、[Ca^2 ]i、脑含水量较假手术组明显升高，并与时间呈正相关，缺血120min后脑组织中出现sPLA2-ⅡAmRNA表达，且cPLA2-ⅣmRNA表达水平较假手术组明显增强。结论 磷脂酶A2激活参与了脑缺血后神经细胞内钙超载及脑损伤的整人病理过程。     

离子色谱法测定硫酸软骨素的含量

建立了用离子色谱法测定硫酸软骨素的方法。在６ｍｏｌ／Ｌ盐酸介质中将硫酸软骨素水解生成等摩尔的硫酸根离子，然后用ＡｎｉｏｎＨＣ离子色谱柱进行分离，以硼酸钠／葡萄糖酸钠淋洗液为流动相，流速１．５ｍｌ／ｍｉｎ，电导检测器检测。硫酸根离子在０．００１～０．０８０ｍｇ／ｍｌ浓度范围内线性关系良好，相关系数为０．９９９４，最低检测浓度为０．０００６ｍｇ／ｍｌ。样品中硫酸根离子的加样回收率为９８．５１％～９９．５２％，硫酸软骨素原料药和注射液及鲨鱼软骨粉胶囊中硫酸软骨素测定的相对标准偏差分别为０．２５％，１．０６％和１．９６％。该方法已应用于多种硫酸软骨素制剂中硫酸软骨素的含量测定     

胆结石对肝脏细胞钙稳态影响与调整的实验研究

用低蛋白饮食方法建立豚鼠胆色素结石模型，共设对照、致石、维生素Ｃ修复、丹参修复和对照修复等５组，规定时间内处死动物，用放射免疫、固相酶联免疫、生物化学等方法检测肝细胞内环—磷酸腺甙（ｃＡＭＰ）、环—磷酸鸟嘌呤（ｃＧＭＰ）、钙调素（ＣａＭ）、钙，三磷酸腺甙酶（Ｃａ２＋－ＡＴＰａｓｅ）、磷酸化酶ａ等水平。致石组豚鼠肝脏细胞内ｃＡＭＰ和磷酸化酶ａ升高，而ｃＧＭＰ，ＣａＭ和Ｃａ２＋－ＡＴＰａｓｅ下降，表明肝细胞钙稳态呈失调状态。维生素Ｃ和丹参可调整肝细胞的上述改变，说明维生素Ｃ和丹参具有维持肝细胞钙稳态的作用。     

Calcium incorporation in cultured chondroblasts perturbed by an electromagnetic field

We tested the hypothesis that electric perturbation influences 45Ca incorporation in extracellular matrix (ECM) of cartilage in vitro. Hypertrophic chondroblasts of tibial epiphyses (HC), sternum (SC), and skin fibroblasts (F) were cultured from chick embryos. HC, SC, and F cells were micromass seeded three times per week and maintained at 37.5 degrees C with 5% CO2 for two weeks. Cultures were randomly designated control (C) or exposed (E) to a pulsed electromagnetic field (PEMF). A time course experiment of calcium incorporation for all cultured groups showed that 24 h of exposure produced the largest biological response in chondroblasts. Calcium incorporation required supplemental phosphate. Autoradiography data indicated that the calcium incorporation into macromolecules largely occurred in the ECM. 45Ca steady-state perturbation was enhanced by Streptomyces hyaluronidase (SH) but not by testicular hyaluronidase (TH). 45Ca incorporation experiments tested the effects of phosphate, SH, TH, and PEMF alone and in various combinations on these cultures. Only PEMF or SH plus PEMF with phosphate enhanced 45Ca incorporation. Other experiments examined the effect of rotenone or freeze-thawing on cells exposed to PEMF. PEMF plus freeze-thaw enhanced calcium incorporation in HC only. PEMF appeared to cause disruption of the ECM, enhancing the probability of matrix calcification.     

Model Features of a Cardiac lontophoretic Drug Delivery Implant

Pharmaceutical Research -     

In vivo measurements of fibrin formation and degradation in nephrotic patients

Summary Intraglomerular fibrin deposition has been implicated as an important pathogenetic mechanism in patients with glomerular diseases and the nephrotic syndrome. To investigate fibrin formation and degradation in nephrosis, we measured fibrinopeptide A by radio-immunoassay and D-dimer by enzyme-linked immunosorbent assay in the plasma of 30 consecutive adult patients with the nephrotic syndrome; in 10 the serum creatinine was more than 2 mg/dl. Both fibrinopeptide A and D-dimer were abnormally elevated in the majority of nephrotics (P<0.001 vs. healthy controls), providing evidence of increased fibrin generation and lysis “in vivo.” A positive correlation was found between fibrinopeptide A and D-dimer (correlation coefficient 0.64,P<0.001), suggesting a close relationship between fibrin formation and degradation. Calcium heparin, administered to 12 nephrotics, caused a marked decrease in plasma fibrinopeptide A, due to a reduction of in vivo thrombin activity. As enhanced thrombin activity can favor fibrin deposition within the renal parenchyma, as well as vascular complications, it is reasonable to assume that an antithrombotic treatment aimed at controlling thrombin generation may ameliorate the natural history of nephrosis.