Effects of Anthopleurin-Q on the Intracellular Free Ca²⁺ Concentration in Cultured Rat Cortical Neurons

Abstract

The present study was designed to investigate the mechanism underlying the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) modulated by Anthopleurin-Q (AP-Q), a sea anemone toxin, using whole-cell patch clamp and fluorescence digital imaging techniques. Results indicated that the overall Ca²⁺ concentration could be augmented in presence of AP-Q. The increase of [Ca²⁺]_i induced by AP-Q was eliminated in Na⁺-free solution, Ca²⁺-free solution or in presence of TTX. However, the Ca²⁺ increase induced by AP-Q could not be influenced by cyclopiazonic acid (CPA), a specific inhibitor of the endoplasmic reticulum Ca²⁺-ATPase pump. We furthermore demonstrated that voltage-gated calcium channels (VGCCs) blocker verapamil, or inhibitor of the reverse operation Na⁺-Ca²⁺ exchanger NiCl₂ attenuated AP-Q-induced [Ca²⁺]_i elevation. Furthermore, the inactivation process of Na⁺ current (I _Na ) was significantly delayed with slightly change of its amplitude by AP-Q. These findings demonstrated that neuron voltage-gated Na⁺ channels are also targets of AP-Q. Overall, the present results suggested that AP-Q induced calcium influx via Na⁺-dependent activation of voltage-gated sodium channels (VGSCs), VGCCs and reverse operation of the Na⁺/Ca²⁺exchanger.

Key words

anthopleurin-Q - patch clamp technique - cultured cortical neuron - sodium current

• References

• 1 Norton RS. Structure and structure-function relationships of sea anemone protein that interacts with the sodium channel. Toxicon 1991; 29: 1050-1084
• 2 Gregor A, Peter M. Cytolytic peptide and protein toxin from sea anemones. Toxicon 2002; 40: 111-124
• 3 Zhou J, Fu LY, Yao WX et al. Effect of anthopleurin-Q on myocardial hypertrophy in rats and physiologic properties of isolated aria in guinea pigs. Acta Phamacol Sin 2002; 23: 924-929
• 4 Alsen C, Béress L, Fischer K et al. The action of a toxin from the sea anemone Anemonica Sulcato upon mammalian heart muscle. Naunyn-Schmiedeberg’s Arch Pharmacol 1976; 295: 55-62
• 5 Beress L, Beress R, Wunderer G. Isolation and characterization of the three polypeptides with neurotoxin activity from Anemonica Sulcato. FEBS Lett 1975; 50: 311-314
• 6 Gross GJ, Warltier DC, Hardman HF et al. Cardiotonic effects of anthopleurin-A (Ap-A), a poly peptide from a sea anemone, in dog with a coronary artery stenosis. Eur J Pharmacol 1985; 110: 271-276
• 7 Massimo M, Silvana F, Giuliano A. Anemone toxin (ATX II)-induced increase in persistent sodium current: effects on the firing properties of rat neocortical pyramidal neurons. J Phsiol 1998; 507: 105-116
• 8 Sabine B, Tomas S, Christian A. Enhancement of persistent Na⁺ current by sea anemone toxin (ATX II) exerts dual action on hippocampal excitability. Euro J Neurosci 2000; 12: 2387-2396
• 9 Santana AN, Trindade-Filho EM, Cunha RB et al. Behavial and Electroencephalographic Analysis of Seizures Induced by Intra-hippocapmal Injection of Granulitoxin, A Neurotoxic Peptide from the Sea Anemone Bunodosoma Graulifera. Brazil J Med Biol Res 2001; 34: 797-801
• 10 Berridge MJ, Lipp P, Bootman MD. The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol 2000; 1: 11-21
• 11 Berridge MJ. Neuronal calcium signaling. Neuron 1998; 21: 13-26
• 12 Ghosh A, Greenberg ME. Calcium signaling in neurons: molecular mechanisms and cellular consequences. Science 1995; 268: 239-247
• 13 Ragsdale DS, Avoli M. Sodium channels as molecular targets for antiepileptic drugs. Brain Res Brain Res Rev 1998; 26: 16-28
• 14 Badea T, Goldberg J, Mao B et al. Calcium imaging of epileptiform events with single-cell resolution. J Neurobiol 2001; 48: 215-227
• 15 Liu J, Wang F, Huang C et al. Activation of phosphatidylinositol-linked novel D1 dopamine receptor contributes to the calcium mobilization in cultured rat prefrontal cortical astrocytes. Cell Mol Neurobiol 2009; 29: 317-328
• 16 Fu LY, Li Y, Cheng L et al. Effect of sea anemone toxin anthopleurin-Q on sodium current in guinea pig ventricular myocytes. Acta Pharmacol Sin 2001; 2: 1107-1112
• 17 El-Sherif N, Fozzard HA, Hanck Da. Dose-dependent modulation of the cardiac sodium channel by sea anemone toxin ATXII. Circ Res 1992; 70: 285-301
• 18 Rodon DM, Balser JR, George AL et al. Cardiac ion channels. Annu Rev Physil 2002; 64: 431-475
• 19 HESS P. Calcium channels in vertebrate cells. Annu Rev Neurosci 1990; 13: 337-356
• 20 Castle NA, Strichartz GR. Palytoxin induces a relatively non-selective cation permeability in frog sciatic nerve which can be inhibited by cardiac glycosides. Tosicon 1988; 26: 941-951
• 21 Levi AJ, Brooksby P, Hancox JC. A role for depolarisation induced calcium entry on the Na-Ca exchange in triggering intracellular calcium release and contraction in rat ventricular myocytes. Cardiovasc Res 1993; 27: 1677-1690
• 22 Deshpande SB, Fukuda A, Nishino H. 3-Nitropropionic acid increase the intracellular Ca²⁺ in cultured astrocytes by reverse operation of the Na⁺-Ca²⁺ exchanger. Exp Neurol 1997; 145: 38-45
• 23 Kupittayanant P, Trafford AW, Diaz ME et al. A mechanism distinct from the L-type Ca current or Na⁺-Ca²⁺ exchange contributes to Ca entry in rat ventricular myocytes. Cell Calcium 2006; 39: 417-423
• 24 Frelin C, Van Renterghem C. Palytoxin. Recent electrophysiological and pharmacological evidence for several mechanism of action. Gen Pharmacol 1995; 26: 33-37
• 25 Vale C, Alfonso A, Sunol C et al. Modulation of calcium entry and glutamate release in cultured cerebellar granule cells by palytoxin. J Neurosci Res 2006; 83: 1393-1406