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Nitric oxide augments voltage-activated calcium currents of crustacea (Idotea baltica) skeletal muscle
Neuroscience Letters 300 (2001) 133±136
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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 Department of Molecular Neurobiology & Cellular Physiology, Institute of Zoology, University of Salzburg, Hellbrunnerstrasse 34, A-5020 Salzburg, Austria
b
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 sarcolemmal 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 invertebrates [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 important 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 messenger on receptors, ion channels and signalling pathways [2,28]. In invertebrates synaptic integration, i.e. excitation or inhibition is processed at the muscle membrane. Invertebrate muscle, therefore, responds to a great variety of transmitters, 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 * Corresponding author. Tel.: 143-662-8044-5610; fax: 143662-8044-5698. E-mail address: [email protected] (A. Hermann). 1 Present address: NIEHS, 101/F210, Research Triangle Park, NC 27709, USA.
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 twoelectrode 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 CaCl2, 12 MgCl2, 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
0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 01 57 1- 3
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C. Erxleben, A. Hermann / Neuroscience Letters 300 (2001) 133±136
mixture of l-cysteine (100 mM) and NaNO2 (100 mM by acidi®cation with 5% (v/v) 10 N HCl, added to ASW and the pH adjusted to 7.4 with 1 N NaOH[4]. The NO donor S-nitroso-n-acetylpenicillamine (SNAP) (RBI, Natick, MA, USA) was dissolved in dimethylsulfoxide (DMSO). DMSO in the bath solution up to 1 mM concentration had no effect on membrane currents. Hydroxylamine (HA) and other drugs were dissolved in ASW as 100 mM stock solutions. Further drugs used were: methylene blue (MB), an inhibitor of soluble guanylyl cyclase, and tetracaine, a muscle relaxant. Data were analysed using pClamp software (Axon Instruments). Leak and capacitive currents were subtracted from current traces using the P/4 protocol. For preparation of ®gures data were low pass ®ltered at 3 kHz using a Gaussian software ®lter (pClamp, Axon Instruments). NO was measured using a NO-selective electrode (ISONOP probe, WPI, Berlin, FRG). The instrument (ISO-NO Mark II, WPI) was calibrated using the KNO2, KJ, H2SO4 reaction which leads NO, J, H2O and K2SO4, or by decomposition of SNAP using copper sulfate as a catalyst which yields NO and a disul®de byproduct. The results were used to convert electrode currents to NO concentrations. We also tested the decomposition of SNAP in ASW without copper sulfate. The NO concentration derived from SNAP, 1 mM in ASW (no copper sulfate added), was also assayed in the experimental bath. The actual NO concentration from 1 mM SNAP close to the site of the preparation was 0.8 ^ 0.1 mM. DMSO in ASW (1%) had no effect on the NO-sensing electrode. In experiments with muscle ®bres copper sulfate was excluded from SNAP solutions to avoid effects on membrane currents. Voltage-activated membrane currents of Idotea muscle consist of a l-type calcium inward current [7] and various types of potassium outward currents. Inward currents were pharmacologically isolated by suppressing outward currents using the potassium channel blockers tetraethylammoniumchloride (50 mM), 4-aminopyridine (5 mM), cesium-®lled microelectrodes and in some cases barium (10 mM) in place of calcium ions as charge carriers. Tetracaine (0.5 mM) was used to suppress muscle contractions [7]. Fig. 1 shows
calcium currents recorded under these conditions using voltage steps and voltage-ramp. The current activates at about 250 mV and reverses between 130 and 150 mV. NO donors, were applied after stable recordings of inward currents for at least 5 min. Fig. 2A illustrates the effect of the NO donor SNAP which substantially increased the calcium inward current. The maximum increase of inward current obtained by SNAP (1 mM) was 406 ^ 140%, n 5, (P , 0:05) with a time constant of 230 ^ 59 s. The SNAP effect was reversible after washout of the drug at a somewhat slower time scale of about 10±15 min. The plot in Fig. 2B shows the time course of the SNAP effect in a peak inward currents versus time plot. Similarly, the NO precursor, hydroxylamine (HA), 1 mM, unmasked calcium currents in ®bres which lack calcium current under control (Fig. 3). The increase of calcium current in two more experiments was about 200%. Hydroxylamine, as a cell permeable, catalase-dependent compound more closely mimics the intracellular NO increase. Fig. 4 shows current-voltage plots before and during application of the NO donor SNOC. Peak calcium currents increased from a basal level of ~120 nA within 15 min to a maximum of ~370 nA in the presence of SNOC (Fig. 4B). The increase of calcium current by SNOC (1 mM) was on average 113 ^ 41%, n 5, (P , 0:05) with a time constant of 302 ^ 55 sec. The NO effect was reversible after washing with donor free solution within 20±30 min. Reapplication of SNOC to the same
Fig. 1. Inward currents through Idotea muscle calcium channels elicited by voltage steps and voltage-ramp. (A) Barium currents elicited by 10 mV voltage-steps to 130 mV (lower traces) from a holding potential of 270 mV. (B) Current-voltage relationship of the peak inward current from (A) as a function of the step potential and current elicited by a voltage-ramp (2 mV/ms) from 270 mV (solid line).
Fig. 3. NO donors can unmask large calcium currents in ®bers which lack calcium current under control conditions. (A) Inward currents elicited by voltage-ramps (2 mV/ms) from 270 mV before and during exposure to the NO donor hydroxylamine (four lower traces). (B) Recording of stable currents measured under control condition and time course of peak inward current during application of hydroxylamine.
Fig. 2. The NO donor SNAP potentiates calcium currents in Idotea muscle ®bers. (A) Currents elicited by voltage-steps from 270 mV holding potential to 0 mV before and after bath application of 5 mM SNAP. (B) Time course of the SNAP effect on the peak inward calcium currents. The asterisks mark the time for the traces shown in (A).
C. Erxleben, A. Hermann / Neuroscience Letters 300 (2001) 133±136
Fig. 4. Potentiation of calcium currents in Idotea muscle ®bers by the NO donor SNOC is reversible and can be blocked by an inhibitor of guanylyl cyclase, methylene blue (MB). (A) time course of the SNOC effect on the peak inward calcium current and inhibition by methylene blue (MB). (B,C) Calcium currents elicited by voltage-ramps from 270 mV with current traces at times as indicated by the numbers in (A).
preparation again increased the inward current (Fig. 4A,C), although to a lesser extent. Application of methylenblue (50 mM), an inhibitor of soluble guanylyl cyclase, blocked the increase of inward current activated by SNOC (Fig. 4C) (n 4). NO has been reported in a number of preparations to either activate or to inhibit calcium channels in a cGMPdependent or independent manner. For example, in human atrial myocytes NO increases calcium in¯ux [18] and NO signaling activates calcium channels in cat atrial myocytes [29]. NO also induces calcium in¯ux into mouse cerebral cortical neurons via opening of voltage-dependent, L- and P-type calcium channels but inhibits the function of N-type calcium channels [23]. A NO-cGMP-dependent signaling pathway mediating calcium in¯ux and/or intracellular calcium mobilisation is indicated in rat sympathetic neurons [6], in glial cells [30], and in retinal ganglion cells, where NO causes activation of N-type calcium channels via a mechanism involving guanylate cyclase/protein kinase Gdependent phosphorylation [12]. On the other hand, NO has been reported to inhibit the activity of calcium current from ferret ventricular myocytes [5], of macroscopic and single channel calcium currents of smooth muscle cells from guinea pig basilar artery [27], and to modulate basal ltype calcium current of guinea-pig ventricular cells [9]. Furthermore, NO through a cGMP-dependent pathway suppresses L-type calcium channels in alveolar epithelial cells [25], of both L-type and P/Q-type calcium channels in rat insulinoma cells [11], and of voltage-dependent calcium current in rat dorsal root ganglion cells [17]. It appears interesting to note that cGMP plays a crucial role in maintaining basal calcium in¯ux in new-born rabbit ventricular cells but not in adult cells [20]. NO inhibits Ltype calcium channels from adult rabbit glomus [26] and
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blocks current of expressed cardiac L-type channels [13] via a cGMP-independent mechanism. The present study using different NO-donors indicates that NO causes an augmentation of calcium inward current. The target of NO action remains to be identi®ed. Methylene blue, which has been widely used as guanylyl cyclase blocker [14,16], inhibits the NO-activated calcium inward current in our experiments which may provide some indication for a NO-cGMP pathway. However, since methylene blue is a rather non-speci®c compound, which also inhibits NO-synthase and can inactivate NO by generating superoxide anions, more detailed investigation into the question of NO targets, i.e. guanylyl cyclase activation and/or nitrosylation is required. From a different study we have indication that NO donors increase potassium outward currents of Idotea muscle in a cGMP-dependent manner (Hermann and Erxleben, unpublished). At ®rst sight this appears puzzling and raises the question about the biological meaning of NO increasing both, inward as well as outward currents. At closer view, however, this may constitute an interesting mechanism of modulation since by activation of both, inward and outward currents it is expected that the principal characteristics of the muscle response remain unchanged. The NO-activated increase of calcium current, i.e. during the depolarising phase of an action potential, is expected to cause a more rapid increase of the intracellular calcium concentration, which in invertebrate muscle muscle (similar to vertebrate heart muscle) is directly involved in the activation of muscle contraction. The following NO-induced increase of outward current drives the membrane potential in hyperpolarizing direction, and hence, will cause a rapid termination of calcium in¯ux. Taken together the NO effects on ion currents predict a positive iontropic effect which will accentuate muscle contraction and hence allow for adjustment of muscle force. Supported by the Medical Research Coordination Center, Salzburg. [1] Andrade, F.H., Reid, M.B., Allen, D.G. and Westerblad, H., Effect of nitric oxide on single skeletal muscle ®bres from the mouse, J. Physiol., 509 (1998) 577±586. [2] Bredt, D.S. and Snyder, S.H., Nitric oxide: a physiologic messenger molecule, Annu. Rev. Biochem., 63 (1994) 175±195. [3] Bredt, D.S., Endogenous nitric oxide synthesis: biological functions and pathophysiology, Free Radic. Res., 31 (1999) 577±596. [4] Brorson, J.R., Schuhmacker, P.T. and Zhang, H., Nitric oxide acutely inhibits neuronal energy production, J. Neurosci., 19 (1999) 147±158. [5] Campbell, D.L., Stamler, J.S. and Strauss, H.C., Redox modulation of l-type calcium channels in ferret ventricular myocytes. Dual mechanism regulation by nitric oxide and S-nitrosothiols, J. Gen. Physiol., 108 (1996) 277±293. [6] Chen, C. and Scho®eld, G.G., Nitric oxide donors enhanced Ca 21 currents and blocked noradrenaline-induced Ca 21
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C. Erxleben, A. Hermann / Neuroscience Letters 300 (2001) 133±136 current inhibition in rat sympathetic neurons, J. Physiol., 482(3) (1995) 521±531. Erxleben, C. and Rathmayer, W., A dihydropyridine-sensitive voltage-dependent calcium channel in the sarcolemmal membrane of crustacean muscle, J. Gen. Physiol., 109 (1997) 313±326. Erxleben, C.F.J., deSantis, A. and Rathmayer, W., Effects of proctolin on contractions, membrane resistance, and nonvoltage-dependent sarcolemmal ion channels in crustacean muscle ®bres, J. Neurosci., 15 (1995) 4356±4369. Gallo, M.P., Ghigo, D., Bosia, A., Alloatti, G., Costamagna, C., Penna, C. and Levi, R.C., Modulation of guinea-pig cardiac L-type calcium current by nitric oxide synthase inhibitors, J. Physiol. (Lond.), 506 (1998) 639±651. Garthwaite, J. and Boulton, C.L., Nitric oxide signaling in the central nervous system, Annu. Rev. Physiol., 57 (1995) 683±706. Grassi, C., D'Ascenzo, M., Valente, A. and Battista Azzena, G., Ca 21 channel inhibition induced by nitric oxide in rat insulinoma RINm5F cells, P¯uÈg. Arch. Eur. J. Physiol., 437 (1999) 241±247. Hirooka, K., Kourennyi, D.E. and Barnes, S., Calcium channel activation facilitated by nitric oxide in retinal ganglion cells, J. Neurophysiol., 83 (2000) 198±206. Hu, H., Chiamvimonvat, N., Yamagishi, T. and Marban, E., Direct inhibition of expressed cardiac L-type Ca 21 channels by S-nitrosothiol nitric oxide donors, Circ. Res., 81 (1997) 742±752. Iadecola, C., Li, J., Ebner, T.J. and Xu, X., Nitric oxide contributes to functional hyperemia in cerebellar cortex, Am. J. Physiol., 268 (1995) R1153±R1162. Jacklet, J.W., Nitric oxide signaling in invertebrates, Invertebrate Neurosci., 3 (1997) 1±14. Kannan, M.S. and Johnson, D.E., Modulation of nitric oxide-dependent relaxation of pig tracheal smooth muscle by inhibitors of guanylyl cyclase and calcium activated potassium channels, Life Sci., 56 (1995) 2229±2238. Kim, S.J., Song, S.K. and Kim, J., Inhibitory effect of nitric oxide on voltage-dependent calcium currents in rat dorsal root ganglion cells, Biochem. Biophys. Res. Commun., 271 (2000) 509±514. Kirstein, M., Rivet-Bastide, M., Hatem, S., Benardeau, A., Mercadier, J.J. and Fischmeister, R., Nitric oxide regulates
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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 Department of Molecular Neurobiology & Cellular Physiology, Institute of Zoology, University of Salzburg, Hellbrunnerstrasse 34, A-5020 Salzburg, Austria
b
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 sarcolemmal 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 invertebrates [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 important 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 messenger on receptors, ion channels and signalling pathways [2,28]. In invertebrates synaptic integration, i.e. excitation or inhibition is processed at the muscle membrane. Invertebrate muscle, therefore, responds to a great variety of transmitters, 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 * Corresponding author. Tel.: 143-662-8044-5610; fax: 143662-8044-5698. E-mail address: [email protected] (A. Hermann). 1 Present address: NIEHS, 101/F210, Research Triangle Park, NC 27709, USA.
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 twoelectrode 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 CaCl2, 12 MgCl2, 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
0304-3940/01/$ - see front matter q 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S03 04 - 394 0( 0 1) 01 57 1- 3
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C. Erxleben, A. Hermann / Neuroscience Letters 300 (2001) 133±136
mixture of l-cysteine (100 mM) and NaNO2 (100 mM by acidi®cation with 5% (v/v) 10 N HCl, added to ASW and the pH adjusted to 7.4 with 1 N NaOH[4]. The NO donor S-nitroso-n-acetylpenicillamine (SNAP) (RBI, Natick, MA, USA) was dissolved in dimethylsulfoxide (DMSO). DMSO in the bath solution up to 1 mM concentration had no effect on membrane currents. Hydroxylamine (HA) and other drugs were dissolved in ASW as 100 mM stock solutions. Further drugs used were: methylene blue (MB), an inhibitor of soluble guanylyl cyclase, and tetracaine, a muscle relaxant. Data were analysed using pClamp software (Axon Instruments). Leak and capacitive currents were subtracted from current traces using the P/4 protocol. For preparation of ®gures data were low pass ®ltered at 3 kHz using a Gaussian software ®lter (pClamp, Axon Instruments). NO was measured using a NO-selective electrode (ISONOP probe, WPI, Berlin, FRG). The instrument (ISO-NO Mark II, WPI) was calibrated using the KNO2, KJ, H2SO4 reaction which leads NO, J, H2O and K2SO4, or by decomposition of SNAP using copper sulfate as a catalyst which yields NO and a disul®de byproduct. The results were used to convert electrode currents to NO concentrations. We also tested the decomposition of SNAP in ASW without copper sulfate. The NO concentration derived from SNAP, 1 mM in ASW (no copper sulfate added), was also assayed in the experimental bath. The actual NO concentration from 1 mM SNAP close to the site of the preparation was 0.8 ^ 0.1 mM. DMSO in ASW (1%) had no effect on the NO-sensing electrode. In experiments with muscle ®bres copper sulfate was excluded from SNAP solutions to avoid effects on membrane currents. Voltage-activated membrane currents of Idotea muscle consist of a l-type calcium inward current [7] and various types of potassium outward currents. Inward currents were pharmacologically isolated by suppressing outward currents using the potassium channel blockers tetraethylammoniumchloride (50 mM), 4-aminopyridine (5 mM), cesium-®lled microelectrodes and in some cases barium (10 mM) in place of calcium ions as charge carriers. Tetracaine (0.5 mM) was used to suppress muscle contractions [7]. Fig. 1 shows
calcium currents recorded under these conditions using voltage steps and voltage-ramp. The current activates at about 250 mV and reverses between 130 and 150 mV. NO donors, were applied after stable recordings of inward currents for at least 5 min. Fig. 2A illustrates the effect of the NO donor SNAP which substantially increased the calcium inward current. The maximum increase of inward current obtained by SNAP (1 mM) was 406 ^ 140%, n 5, (P , 0:05) with a time constant of 230 ^ 59 s. The SNAP effect was reversible after washout of the drug at a somewhat slower time scale of about 10±15 min. The plot in Fig. 2B shows the time course of the SNAP effect in a peak inward currents versus time plot. Similarly, the NO precursor, hydroxylamine (HA), 1 mM, unmasked calcium currents in ®bres which lack calcium current under control (Fig. 3). The increase of calcium current in two more experiments was about 200%. Hydroxylamine, as a cell permeable, catalase-dependent compound more closely mimics the intracellular NO increase. Fig. 4 shows current-voltage plots before and during application of the NO donor SNOC. Peak calcium currents increased from a basal level of ~120 nA within 15 min to a maximum of ~370 nA in the presence of SNOC (Fig. 4B). The increase of calcium current by SNOC (1 mM) was on average 113 ^ 41%, n 5, (P , 0:05) with a time constant of 302 ^ 55 sec. The NO effect was reversible after washing with donor free solution within 20±30 min. Reapplication of SNOC to the same
Fig. 1. Inward currents through Idotea muscle calcium channels elicited by voltage steps and voltage-ramp. (A) Barium currents elicited by 10 mV voltage-steps to 130 mV (lower traces) from a holding potential of 270 mV. (B) Current-voltage relationship of the peak inward current from (A) as a function of the step potential and current elicited by a voltage-ramp (2 mV/ms) from 270 mV (solid line).
Fig. 3. NO donors can unmask large calcium currents in ®bers which lack calcium current under control conditions. (A) Inward currents elicited by voltage-ramps (2 mV/ms) from 270 mV before and during exposure to the NO donor hydroxylamine (four lower traces). (B) Recording of stable currents measured under control condition and time course of peak inward current during application of hydroxylamine.
Fig. 2. The NO donor SNAP potentiates calcium currents in Idotea muscle ®bers. (A) Currents elicited by voltage-steps from 270 mV holding potential to 0 mV before and after bath application of 5 mM SNAP. (B) Time course of the SNAP effect on the peak inward calcium currents. The asterisks mark the time for the traces shown in (A).
C. Erxleben, A. Hermann / Neuroscience Letters 300 (2001) 133±136
Fig. 4. Potentiation of calcium currents in Idotea muscle ®bers by the NO donor SNOC is reversible and can be blocked by an inhibitor of guanylyl cyclase, methylene blue (MB). (A) time course of the SNOC effect on the peak inward calcium current and inhibition by methylene blue (MB). (B,C) Calcium currents elicited by voltage-ramps from 270 mV with current traces at times as indicated by the numbers in (A).
preparation again increased the inward current (Fig. 4A,C), although to a lesser extent. Application of methylenblue (50 mM), an inhibitor of soluble guanylyl cyclase, blocked the increase of inward current activated by SNOC (Fig. 4C) (n 4). NO has been reported in a number of preparations to either activate or to inhibit calcium channels in a cGMPdependent or independent manner. For example, in human atrial myocytes NO increases calcium in¯ux [18] and NO signaling activates calcium channels in cat atrial myocytes [29]. NO also induces calcium in¯ux into mouse cerebral cortical neurons via opening of voltage-dependent, L- and P-type calcium channels but inhibits the function of N-type calcium channels [23]. A NO-cGMP-dependent signaling pathway mediating calcium in¯ux and/or intracellular calcium mobilisation is indicated in rat sympathetic neurons [6], in glial cells [30], and in retinal ganglion cells, where NO causes activation of N-type calcium channels via a mechanism involving guanylate cyclase/protein kinase Gdependent phosphorylation [12]. On the other hand, NO has been reported to inhibit the activity of calcium current from ferret ventricular myocytes [5], of macroscopic and single channel calcium currents of smooth muscle cells from guinea pig basilar artery [27], and to modulate basal ltype calcium current of guinea-pig ventricular cells [9]. Furthermore, NO through a cGMP-dependent pathway suppresses L-type calcium channels in alveolar epithelial cells [25], of both L-type and P/Q-type calcium channels in rat insulinoma cells [11], and of voltage-dependent calcium current in rat dorsal root ganglion cells [17]. It appears interesting to note that cGMP plays a crucial role in maintaining basal calcium in¯ux in new-born rabbit ventricular cells but not in adult cells [20]. NO inhibits Ltype calcium channels from adult rabbit glomus [26] and
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blocks current of expressed cardiac L-type channels [13] via a cGMP-independent mechanism. The present study using different NO-donors indicates that NO causes an augmentation of calcium inward current. The target of NO action remains to be identi®ed. Methylene blue, which has been widely used as guanylyl cyclase blocker [14,16], inhibits the NO-activated calcium inward current in our experiments which may provide some indication for a NO-cGMP pathway. However, since methylene blue is a rather non-speci®c compound, which also inhibits NO-synthase and can inactivate NO by generating superoxide anions, more detailed investigation into the question of NO targets, i.e. guanylyl cyclase activation and/or nitrosylation is required. From a different study we have indication that NO donors increase potassium outward currents of Idotea muscle in a cGMP-dependent manner (Hermann and Erxleben, unpublished). At ®rst sight this appears puzzling and raises the question about the biological meaning of NO increasing both, inward as well as outward currents. At closer view, however, this may constitute an interesting mechanism of modulation since by activation of both, inward and outward currents it is expected that the principal characteristics of the muscle response remain unchanged. The NO-activated increase of calcium current, i.e. during the depolarising phase of an action potential, is expected to cause a more rapid increase of the intracellular calcium concentration, which in invertebrate muscle muscle (similar to vertebrate heart muscle) is directly involved in the activation of muscle contraction. The following NO-induced increase of outward current drives the membrane potential in hyperpolarizing direction, and hence, will cause a rapid termination of calcium in¯ux. Taken together the NO effects on ion currents predict a positive iontropic effect which will accentuate muscle contraction and hence allow for adjustment of muscle force. Supported by the Medical Research Coordination Center, Salzburg. [1] Andrade, F.H., Reid, M.B., Allen, D.G. and Westerblad, H., Effect of nitric oxide on single skeletal muscle ®bres from the mouse, J. Physiol., 509 (1998) 577±586. [2] Bredt, D.S. and Snyder, S.H., Nitric oxide: a physiologic messenger molecule, Annu. Rev. Biochem., 63 (1994) 175±195. [3] Bredt, D.S., Endogenous nitric oxide synthesis: biological functions and pathophysiology, Free Radic. Res., 31 (1999) 577±596. [4] Brorson, J.R., Schuhmacker, P.T. and Zhang, H., Nitric oxide acutely inhibits neuronal energy production, J. Neurosci., 19 (1999) 147±158. [5] Campbell, D.L., Stamler, J.S. and Strauss, H.C., Redox modulation of l-type calcium channels in ferret ventricular myocytes. Dual mechanism regulation by nitric oxide and S-nitrosothiols, J. Gen. Physiol., 108 (1996) 277±293. [6] Chen, C. and Scho®eld, G.G., Nitric oxide donors enhanced Ca 21 currents and blocked noradrenaline-induced Ca 21
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