Tetrodotoxin-resistant release of3H-noradrenaline from the mouse vas deferens by high intensity electrical stimulation

Vasa deferentia of mice were preincubated with ³H-noradrenaline and then superfused with a medium containing cocaine 10 μM and phentolamine 30 μM. The tetrodotoxin-resistant outflow of tritium evoked by high intensity electrical field stimulation (0.5 Hz, 200 mA current strength, 2 ms pulse width) was studied and, in some experiments, compared with the tetrodotoxin-sensitive outflow evoked by low intensity electrical field stimulation (0.5 Hz, 50 mA, 1 ms). In contrast to the outflow evoked by low intensity stimulation, the outflow evoked by high intensity stimulation was increased in Na⁺-free medium, and was only partly dependent on the external Ca²⁺ concentration. The Ca²⁺-dependent fraction consisted mainly of ³H-noradrenaline. Again, in contrast to the outflow caused by stimulation at low intensity, that caused by stimulation at high intensity was not reduced by Mg²⁺ 20 mM, Co²⁺ 5 mM or normorphine 40 or 100 μM, and was not enhanced by tetraethylammonium 5 mM or 4-aminopyridine 1 mM. It is concluded that high intensity electrical stimulation elicits a tetrodotoxin-resistant, calciumdependent release of noradrenaline which differs in mechanism from the release elicited by action potentials.     

Ionic currents and firing patterns of mammalian vagal motoneurons in vitro

The electrophysiological properties of guinea-pig dorsal vagal motoneurons were studied in an in vitro slice preparation. Antidromic, orthodromic and direct stimulation of the neurons demonstrated that the action potential is comprised of several distinct components: a fast initial spike followed by afterdepolarization and an early and a late afterhyperpolarizations. The fast initial spike and the early afterhyperpolarization were blocked by tetrodotoxin and tetraethylammonium ions, respectively. The afterdepolarization (present on the falling phase of the spike) and the late afterhyperpolarization were blocked by the addition of ions known to block calcium conductance (CdCl2, CoCl2 or MnCl2), indicating close association between these two potentials. Prolonged outward current injection through the recording electrode produced two different firing patterns, depending on the initial level of the membrane potential. From resting potential (usually -60 mV) the firing pattern was characterized by a short train of action potentials appearing shortly after the onset of the depolarization step. By contrast, when the depolarization was delivered from a hyperpolarized membrane potential level, a short train of repetitive firing appeared after an initial delay of 300-400 ms. The membrane current responsible for this initial reduction in excitability was studied by means of a single-electrode voltage-clamp technique. The magnitude, direction and kinetics of such current flow are consistent with the presence of early potassium current (IA), partly inactive at the resting potential. Synaptic activation of vagal motoneurons could be obtained by electrical stimulation of the tissue surrounding the vagal nucleus or by direct activation of the vagal nerve. Perivagal stimulation generated excitatory and inhibitory synaptic potentials which could be reversed by shifting the membrane potential. Vagal nerve stimulation, in addition to the antidromic activation of the cells, generated depolarizing responses which were unitary in nature and did not show much sensitivity to shifts in membrane potential. Perivagal and vagal nerve-evoked depolarizations could generate action potentials as well as partial dendritic spikes. We conclude that spike electroresponsiveness in vagal motoneurons is generated by voltage-dependent Na+ and Ca2+ conductances. In addition, the Ca2+-dependent current triggers a K+ conductance which is responsible for modulating the firing frequency obtained from the normal resting level.(ABSTRACT TRUNCATED AT 400 WORDS)     

Coronary artery bypass grafting in the awake patient combining high thoracic epidural and femoral nerve block: first series of 15 patients

Background: We present a 15-patient series of awake ‘off-pump’[without cardiopulmonary bypass (CPB)] coronary artery bypassgraft surgery, facilitated by thoracic epidural analgesia (TEA)and femoral nerve block. Methods: Surgery was performed with a conventional median sternotomy.Analgesia was provided with TEA at T1–2 or 2–3 interspace,using bupivacaine 0.5% and sufentanil 1.66 µg ml^–1,initially at 20 ml litre^–1 until T1–10 dermatomalblock was achieved, then maintained at 2–14 ml litre^–1throughout surgery. Femoral nerve block was performed beforeoperation with neuro-stimulation at the saphenous vein harvestsite with 10 ml each of bupivacaine 0.25% and lidocaine 2%.Successful awake surgery, avoiding general anaesthesia (GA)with adequate surgical conditions, without CPB was the primaryend point. Results: Fifteen men, mean (SD) age of 63 (9) yr (range 49–81 yr),weight 78 (10) kg, underwent surgery. Three patients (20%) neededconversion to GA: one patient due to insufficient thoracic analgesia,another required initiation of CPB, and the third needed stabilizationof the heart for graft suturing due to profound respiratorymovements. All three were successfully extubated immediatelyafter surgery. Awake surgery was successful and uneventful in80% of cases. Conclusions: Combined TEA and femoral block is a novel anaesthetic technique,and is feasible, for cardiac surgery. However, certain technicallimitations need to be overcome to evaluate the full potentialof ‘awake’ cardiac surgery.     

Direct fusion of subunits of heterodimeric nitric oxide sensitive guanylyl cyclase leads to functional enzymes with preserved biochemical properties: Evidence for isoform specific activation by ciguates

Nitric oxide sensitive guanylyl cyclase (NOsGC) is a heterodimeric enzyme consisting of an α and a β subunit. Two heterodimeric enzymes are known to be important for NO-signalling in humans: α₁/β₁ and α₂/β₁. No difference had so far been detected with respect to their pharmacological properties, but as we show in the present paper the new drugs cinaciguat and ataciguat activate the α₁/β₁ form more effectively. Recent evidence suggests that homodimeric complexes of α and β subunits exist in vivo and that these non-heterodimerizing subunits have a separate function from cGMP signaling. To isolate the effect of the α₁/β₁ or α₂/β₁ heterodimeric enzyme in overexpression experiments from potential effects of non-heterodimerizing α₁, β₁ or α₂ subunits, we cloned constructs that guarantee a 1:1 stochiometry between α and β subunits and rule out the presence of homodimers. The carboxy-terminus of the β₁ subunit was directly fused to the amino-terminus of either the α₁ or α₂ subunit. The two different “conjoined” NOsGCs faithfully reproduced the biochemical and pharmacological properties of the α₁/β₁ and α₂/β₁ heterodimeric enzymes including the differential activation by ciguat-activators. Conjoined NOsGCs can be used for isoform specific overexpression in transgenic animals and therapeutic overexpression may be an application in the future. In both cases possible side effects of homodimeric α or β subunits are avoided. Crystallization with the goal of structure determination may also be easier for conjoined NOsGCs because enzyme preparations are more homogenous and are free of “contaminating” homodimers.     

Axonal ion channels from bench to bedside: A translational neuroscience perspective

Over recent decades, the development of specialised techniques such as patch clamping and site-directed mutagenesis have established the contribution of neuronal ion channel dysfunction to the pathophysiology of common neurological conditions including epilepsy, multiple sclerosis, spinal cord injury, peripheral neuropathy, episodic ataxia, amyotrophic lateral sclerosis and neuropathic pain. Recently, these insights from in vitro studies have been translated into the clinical realm. In keeping with this progress, novel clinical axonal excitability techniques have been developed to provide information related to the activity of a variety of ion channels, energy-dependent pumps and ion exchange processes activated during impulse conduction in peripheral axons. These non-invasive techniques have been extensively applied to the study of the biophysical properties of human peripheral nerves in vivo and have provided important insights into axonal ion channel function in health and disease. This review will provide a translational perspective, focusing on an overview of the investigational method, the clinical utility in assessing the biophysical basis of ectopic symptom generation in peripheral nerve disease and a review of the major findings of excitability studies in acquired and inherited neurological disease states.     

SNAP-25, a SNARE protein, inhibits two types of K channels in esophageal smooth muscle

BACKGROUND & AIMS: The plasma membrane-associated soluble N-ethylmaleimide-sensitive factors attachment protein receptors (SNAREs), synaptosome-associated protein of 25 kilodaltons (SNAP-25), and syntaxin 1A, have been found to physically interact with and functionally modify membrane-spanning ion channels. Studies were performed in cat esophageal body and lower esophageal sphincter (LES) smooth muscle to (1) show the presence of SNAP-25, and (2) determine whether SNAP-25 affects K+ channel activity. METHODS: Single circular muscle cells from the esophageal body and sphincter were studied. Cellular localization of SNAP-25 and K+ channel activity were assessed. RESULTS: SNAP-25 was found in the plasma membrane of all regions examined. Outward K+ currents in body circular muscle were mainly composed of large conductance Ca2+-activated channel currents (K(Ca), 40.1%) and delayed rectifier K+ channel currents (K(V), 54.2%). Microinjection of SNAP-25 into muscle cells caused a dose-dependent inhibition of both outward K+ currents, maximal 44% at 10(-8) mol/L. Cleavage of endogenous SNAP-25 by dialyzing botulinum neurotoxin A into the cell interior resulted in a 35% increase in outward currents. CONCLUSIONS: SNAP-25 protein is present in esophageal smooth muscle cells, and inhibits both K(V) and K(Ca) currents in circular muscle cells. The findings suggest a role for SNAP-25 in regulation of esophageal muscle cell excitability and contractility, and point to potential new targets for treatment of esophageal motor disorders.     

Blockade of BDNF signaling turns chemically‐induced long‐term potentiation into long‐term depression

Long‐term potentiation (LTP) is accompanied by increased spine density and dimensions triggered by signaling cascades involving activation of the neurotrophin brain‐derived neurotrophic factor (BDNF) and cytoskeleton remodeling. Chemically‐induced long‐term potentiation (c‐LTP) is a widely used cellular model of plasticity, whose effects on spines have been poorly investigated. We induced c‐LTP by bath‐application of the N‐methyl‐d ‐aspartate receptor (NMDAR) coagonist glycine or by the K⁺ channel blocker tetraethylammonium (TEA) chloride in cultured hippocampal neurons and compared the changes in dendritic spines induced by the two models of c‐LTP and determined if they depend on BDNF/TrkB signaling. We found that both TEA and glycine induced a significant increase in stubby spine density in primary and secondary apical dendrites, whereas a specific increase in mushroom spine density was observed upon TEA application only in primary dendrites. Both TEA and glycine increased BDNF levels and the blockade of tropomyosin‐receptor‐kinase receptors (TrkRs) by the nonselective tyrosine kinase inhibitor K‐252a or the selective allosteric TrkB receptor (TrkBR) inhibitor ANA‐12, abolished the c‐LTP‐induced increase in spine density. Surprisingly, a blockade of TrkBRs did not change basal spontaneous glutamatergic transmission but completely changed the synaptic plasticity induced by c‐LTP, provoking a shift from a long‐term increase to a long‐term depression (LTD) in miniature excitatory postsynaptic current (mEPSC) frequency. In conclusion, these results suggest that BDNF/TrkB signaling is necessary for c‐LTP‐induced plasticity in hippocampal neurons and its blockade leads to a switch of c‐LTP into chemical‐LTD (c‐LTD). © 2013 Wiley Periodicals, Inc.     

黄芪皂甙对CVB_3病毒引起Vero细胞凋亡的研究

目的：细胞水平上建立病毒性心肌炎（Viral Myocarditis,VMC）模型,通过相关实验得出黄芪皂甙（total extract astragalus,TEA）具有抗病毒能力,进一步明确VMC发病过程中是否存在细胞凋亡以及黄芪皂甙是否能够抑制细胞凋亡。方法：体外培养Vero细胞和出生1-3 d乳鼠心肌细胞,通过细胞病变抑制实验、病毒繁殖抑制实验测定黄芪皂甙抗病毒能力,采用AnnexinV-FITC染色观察VMC发病过程中心肌细胞是否有凋亡,采用流式细胞仪检测TEA是否抑制细胞凋亡。结果：通过MTT染色分析发现TEA可有效保护细胞免受CVB3病毒侵袭,病毒繁殖抑制实验显示中、高剂量组TCID50比病毒组相差一个以上对数值,且TEA对细胞凋亡有抑制作用。结论：实验证实黄芪皂甙对心肌细胞有一定的保护作用,并且在VMC发病过程中存在细胞凋亡,且TEA能抑制心肌细胞凋亡。     

Inhibition of choline acetyltransferase by quaternary ammonium analogues of choline.

The effects of the choline analogues hemicholinium-3, triethylcholine, tetraethylammonium and acetylcholine on the synthesis of ACh by choline acetyltransferase have been studied in vitro. Hemicholinium-3 is shown to be an inhibitor of choline acetyltransferase with a K_i of 2·5 mM. HC-3, TEA and ACh are competitive with choline. Michaelis constants for HC-3 and TEC are also calculated and show that these two compounds are competitive substrates. A comparison of the in vitro with the known in vivo behaviour of these compounds is made and the probability stressed that their in vivo inhibitory effect on ACh synthesis is due to inhibition of ChAc itself. The results favour this explanation than the hypothesis that inhibition occurs because of interference with choline transport.