Nitric oxide augments voltage-activated calcium currents of crustacea (Idotea baltica) skeletal muscle Christian Erxleben a,1 , Anton Hermann b, * a Neurobiology Lab, Stazione Zoologica Anton Dohrn, Villa Comunale, I-80121 Napoli, Italy b Department of Molecular Neurobiology & Cellular Physiology, Institute of Zoology, University of Salzburg, Hellbrunnerstrasse 34, A-5020 Salzburg, Austria Received 22 December 2000; received in revised form 19 January 2001; accepted 19 January 2001 Abstract Invertebrate skeletal muscle contraction is regulated by calcium in¯ux through voltage-dependent calcium channels in the sarcolemmal membrane. In present study we investigated the effects of nitric oxide (NO) donors on calcium currents of single skeletal muscle ®bres from the marine isopod, Idotea baltica, using two-electrode voltage clamp recording techniques. The NO donors, S-nitrosocysteine, S-nitroso-N-acetyl-penicillamine or hydroxylamine reversibly increased calcium inward currents in a time dependent manner. The increase of the current was prevented by methylene blue. Our experiments suggest that NO increases calcium inward currents. NO, by acting on calcium ion channels in the sarco- lemmal membrane, therefore, may directly be involved in the modulation of muscle contraction. q 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Calcium current; Skeletal muscle; Nitric oxide; Idotea baltica (crustacea, isopoda); Invertebrate; Voltage clamp Nitric oxide (NO), has been implicated as a modulator of cellular activity in a great number of vertebrates and inver- tebrates [3,10,15,22,28]. Although NO has originally been described as a relaxing factor in smooth muscle it was appreciated only more recently that it also plays an impor- tant role in skeletal muscle [1,19,21,24]. Cellular NO production is mediated by calcium/calmodulin activated nitric oxide synthase (NOS) and various cofactors by converting the amino acid l-arginine to l-citrulline. All major isoforms of NO-synthase (nNOS, eNOD and iNOS), the key enzyme in the generation of NO, are found in muscle tissue [21]. As a free radical gas NO readily penetrates membranes and acts as a multifunctional messen- ger on receptors, ion channels and signalling pathways [2,28]. In invertebrates synaptic integration, i.e. excitation or inhibition is processed at the muscle membrane. Inverte- brate muscle, therefore, responds to a great variety of trans- mitters, modulators and hormones and ion channel activity in the sarcolemmal membrane is directly involved in the regulation of muscle contraction. In the present study we investigated the effects of NO as a potential modulator of calcium inward currents of crustacea skeletal muscle ®bres. Specimens of marine isopods (crustacea), Idotea baltica, were collected in the gulf of Naples, kept in seawater tanks and fed with seaweed. Preparation of animals and muscle anatomy has been described in detail previously [8]. In brief: animals were decapitated, dissected and the dorsal extensor musculature of the last posterior segment exposed. These muscle ®bres are particularly well suited for two- electrode voltage clamp recording since their dimensions allow for isopotential conditions [7]. For experiments an Axoclamp 2B (Axon Instruments Inc., Foster City, CA, USA) was used. Glasmicropipettes for recording of membrane potentials were ®lled with 3 M KCl or a 50/50 vol% mixture of 3 M KCl and 1.5 M K-citrat, and for current passing electrodes with 3 M cesium. The electrodes had resistances of 1.5±3.5 MV. The membrane potential was recorded differentially with respect to an extracellular bath reference electrode. The experimental bath with a volume of 0.5 ml was continuously perfused and drugs added to the perfusate. Recordings were obtained in arti®cial seawater (ASW) containing in mM/l: 490 NaCl, 8 KCl, 10 CaCl 2 , 12 MgCl 2 , and 20 mM TRIS (pH 7.4). Drugs were from Sigma (Vienna or Milano) unless stated otherwise. The NO donor S-nitrosocysteine (SNOC) was produced from a Neuroscience Letters 300 (2001) 133±136 0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S0304-3940(01)01571-3 www.elsevier.com/locate/neulet * Corresponding author. Tel.: 143-662-8044-5610; fax: 143- 662-8044-5698. E-mail address: anton.hermann@sbg.ac.at (A. Hermann). 1 Present address: NIEHS, 101/F210, Research Triangle Park, NC 27709, USA.