Effects of veratridine on single neuronal sodium channels expressed inXenopus oocytes

(1) Chick neuronal Na⁺ channels were expressed inXenopus laevis oocytes after injection with total messenger ribonucleic acid (mRNA) isolated from chick brain. The currents were investigated with the whole cell voltage clamp and with the patch clamp technique. Activation and inactivation of the induced current, and its sensitivity towards tetrodotoxin (TTX) and veratridine were reminiscent of vertebrate neuronal Na⁺ channels. (2) In the presence of veratridine normal single channel openings often converted into small amplitude openings of long duration. These small amplitude openings persisted for hundreds of milliseconds after return to the holding potential. (3) The slope conductance of the veratridine modified open channel state was 5–6 pS as compared to the normal state with 21–25 pS in the voltage range between –35 and +5 mV. (4) The modified channel showed saturation behaviour towards Na⁺ ions. Half saturation of the single channel amplitude was observed at 330 mM Na⁺ at a membrane potential of –100 mV. (5) Final closure of the modified channel after return to the holding potential followed an exponential time course. Its potential dependence was similar to that of the time course of the veratridine induced tail currents in the whole cell configuration. (6) The properties of the Na⁺ channel derived from chick forebrain are compared with the properties of the same channel derived from chick skeletal muscle. Both were expressed in the same membrane environment, theXenopus oocyte plasma membrane. While earlier results with Na⁺ channels of muscle origin showed two channel populations, one with short and another with long mean open times, Na⁺ channels of neuronal origin were homogeneous and characterized by short open times.     

The relaxing effect of BDF 9148 on the KCl-contracted aorta isolated from normo- and hyper-tensive rats

BDF 9148, a positive cardiac inotrope, relaxes the rat isolated portal vein and the KCl-contracted rat aorta. The aims of our study were to determine the mechanism of action of BDF 9148, and to ascertain whether the relaxing effect of BDF 9148 was maintained in the presence of the hypertrophy associated with hypertension, by investigating the effects of BDF 9148 on the contractility and electrophysiology of aortae of Wistar Kyoto normotensive rats (WKY) and Spontaneously Hypertensive Rats (SHRs). High concentrations of veratridine contracted the quiescent rat aorta. BDF 9148 had no effect on the quiescent, but relaxed the KCl-contracted WKY and SHR aorta by a tetrodotoxin insensitive mechanism, and these relaxations decreased with age but were not greatly altered by hypertrophy. The verapamil relaxations of the KCl-contracted aorta were not altered by age or hypertrophy. The ability of KCl to depolarise the aorta was reversed by verapamil, but not by BDF 9148. On the contracted rat aorta, the relaxant responses to acetylcholine were abolished by removal of the endothelium but potentiated by IBMX (10^–6 M), and the responses to isoprenaline were inhibited by propranolol (10^–6 M) but potentiated by forskolin (10^–7 M). The relaxation responses of the KCl-contracted aorta to BDF 9148 were not altered by removal of the endothelium, or by propranolol, forskolin and IBMX. In summary, the effects of verapamil and BDF 9148 on the aorta are different, and thus it is unlikely that the relaxant responses to BDF 9148 on the aorta are due to calcium channel blocking activity. The mechanism of the relaxant effect of BDF 9148 on the aorta remains unknown, but we have shown the response is endothelium-independent, and not mediated by sodium channel opening, hyperpolarization, β-adrenoceptors, or by stimulating adenylate cyclase or guanylate cyclase. Received: 12 March / Accepted: 8 October 1997     

Veratridine and other depolarizing agents counteract the inhibitory effect of Mg2+ ions on N-methyl-D-aspartate (NMDA)-induced noradrenaline release in vitro

Summary Rat brain cortex slices preincubated with ³H-noradrenaline were superfused with Krebs-Henseleit solution with or without Mg²⁺. In the absence of Mg²⁺ ions, NMDA evoked ³H-noradrenaline overflow above basal efflux; this effect was concentration-dependently inhibited by Mg²⁺ (IC₅₀: 19 mol/l). Despite the presence of 1.2 mmol/l Mg²⁺, which is known to block cation influx through the ion channel coupled to the NMDA receptor, NMDA evoked ³H-noradrenaline release if the membrane was permanently kept depolarized by 20 or 25 mmol/l K⁺, 1 mol/l veratridine or 200 mol/l 3,4-diaminopyridine; the stimulant effect of NMDA was counteracted by 2-amino-5-phosphonovaleric acid (2-APV), a competitive antagonist at the NMDA receptor and by (+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyc-lohept-5,10-imine hydrogen maleate (MK 801), an antagonist acting at the cation channel associated with the NMDA receptor. In contrast, no stimulatory effect of NMDA in the presence of 1.2 mmol/l Mg²⁺ was observed when the membrane of the nerve terminals was intermittently depolarized by electrical impulses of 2 ms duration at a frequency of 1–3 Hz.It is concluded that continuous depolarization of the nerve membrane counteracts the blocking effect of Mg²⁺ on cation influx through the NMDA receptor-associated ion channel. Under this condition, noradrenaline release can be stimulated by NMDA receptor activation even in the presence of physiological Mg²⁺ concentrations. Send offprint requests to M. Göthert at the above address     

Lamotrigine inhibits basal and Na+-stimulated, but not Ca2+-stimulated, release of corticotropin-releasing hormone from the rat hypothalamus

Rationale Corticotropin-releasing hormone (CRH) is a peptide neurotransmitter involved in the pathogenesis of anxiety and depressive disorders; CRH receptor antagonists are currently developed as anxiolytic and antidepressive agents, and several antidepressants negatively modulate CRH in the central nervous system.Objectives and methods Originally marketed as an antiepileptic drug, lamotrigine (LTG) was subsequently shown to be effective as a mood stabilizer and an antidepressant. In this study, we used acute rat hypothalamic explants to investigate the effects of LTG on the gene expression and secretion of CRH from the hypothalamus in short-term incubation experiments.Results We found that LTG reduces basal CRH release in a time- and concentration-dependent manner. LTG also inhibits veratridine-stimulated, but not K⁺-stimulated, CRH release in 1-h experiments. Moreover, LTG tended to reduce CRH mRNA expression in 1-h experiments, regardless of whether the drug was given alone or in combination with veratridine or high K+ concentrations; such reduction achieved statistical significance after 3 h of incubation.Conclusion In 1-h experiments, LTG reduces CRH release from CRH-containing neurons in the rat hypothalamus by interfering with Na⁺-driven secretion mechanisms. After 3-h incubations, the reduction in CRH release is also accounted for by LTG-induced decrease in CRH gene expression.     

Type B brevetoxins show tissue selectivity for voltage-gated sodium channels: comparison of brain, skeletal muscle and cardiac sodium channels.

Brevetoxins and ciguatoxins are two classes of phycotoxins which exert their toxic effect by binding to site-5 of voltage-gated sodium channels. Sodium channels, a family of at least 10 structurally different proteins, are responsible for the rising phase of the action potential in membranes of neuronal, cardiac and muscular excitable cells. This work is a comparative study of the binding properties and the cytotoxic effects of ciguatoxins and brevetoxins on human embryonic cells (HEK) stably expressing either the skeletal muscle (Na(v)1.4), or the cardiac (Na(v)1.5) sodium channel alpha-subunit isoforms. We report that type A (PbTx-1) and type B (PbTx-3 and PbTx-2) brevetoxins as well as ciguatoxins target both cardiac and muscle channels; type B brevetoxins show isoform selectivity, presenting a lower affinity for the heart than the skeletal muscle channel. The lower selectivity of type B brevetoxins for heart sodium channels may result from a more rigid backbone structure than is found in type A brevetoxins and ciguatoxins.     

Calcium-dependent, sustained enhancement of excitability during washout of aconitine in rat hippocampal slices

 Extracellular recording of the stimulus-evoked population spike was performed in the CA1 area of rat hippocampal slices in order to investigate delayed effects of the plant alkaloids aconitine and veratridine. Veratridine (1 μM and 10 μM) suppressed the orthodromic and antidromic population spike. After washout of the drug, only a partial recovery was obtained. Aconitine (1 μM) exerted the same inhibitory action as veratridine. However, after washout, the spike amplitude was enhanced compared with the control. This enhancement of the spike amplitude was dependent on the concentration of aconitine and was maintained during the observation period of at least 2 h. Lowering the Ca²⁺ concentration of the bathing medium from 2.5 mM to 1.25 mM during application of aconitine attenuated recovery and prevented the enhancement observed during washout of the drug. Application of aconitine in the presence of CdCl₂ as well as in the presence of inhibitors of protein kinase C and Ca²⁺/calmodulin-dependent protein kinase II prevented the increase in spike amplitude during washout with standard artificial cerebrospinal fluid. In contrast, the N-methyl-d-aspartate (NMDA) receptor antagonists D-AP5 and MK-801 as well as the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione were ineffective in abolishing the aconitine-induced enhancement. These data support the conclusion that different modes of action are involved in the effects of aconitine but not veratridine. It is concluded that the aconitine-induced increase in neuronal activity is mediated by intracellular Ca²⁺-dependent mechanisms leading to an activation of Ca²⁺-dependent protein kinases. This effect is independent of Ca²⁺ entrance through NMDA and non-NMDA receptors. Received: 3 July 1996 / Accepted: 14 November 1996