Journal of Neuroscience Research 44:21-26 (1996)

Nitric Oxide Donor Sodium Nitroprusside Inhibits the Acetylcholine-Induced K +

Current in Identified Aplysia Neurons M. Sawada and M. Ichinose Department of Physiology, Shimane Medical University, Izumo, Japan

The effects of bath-applied sodium nitroprusside (SNP), a nitric oxide (NO) donor, on an acetylcholine (ACh)-induced K+ current recorded from identified neurons (R9 and R10) of Aplysia kurodui were inves- tigated with conventional voltage-clamp and pressure ejection techniques. Bath-applied SNP (25-50 pM) reduced the ACh-induced K+ current in the neurons without affecting the resting membrane conductance and holding current. The suppressing effects of SNP on the current were completely reversible. In- tracellular injection of 1 mM guanosine 3’,5’-cyclic monophosphate (cGMP) or bath-applied 50 pM 3-isobutyl-1-methylxanthine (IBMX), a nonspecific phosphodiesterase (PDE) inhibitor, also inhibited the ACh-induced current, thus mimicking the effect of the NO donor on the ACh-induced current. In con- trast, pretreatment with methylene blue (10 pM), an inhibitor of guanylate cyclase, and hemoglobin (50 pM), a nitric oxide scavenger, decreased the SNP- induced inhibition of the ACh-induced current. These results suggest that SNP, a NO donor, inhibits the ACh-induced K+ current, and that the mechanism of NO inhibition of the ACh-induced current recorded from identified ApZysiu neurons involves cGMP-de- pendent protein kinase.

Key words: nitric oxide, ACh-induced current, cGMP, Aplysiu neurons

o 1996 Wiley-Liss, Inc.

INTRODUCTION

Nitric oxide (NO) is known to be important mod- ulator of many cellular processes in a variety of tissues (Ignarro, 1991; Moncada et al., 1991). It has been pro- posed that NO is a central and peripheral neuronal mes- senger and represents a new class of neurotransmitter (Snyder and Bredt, 1991; Garthwaite, 1991; Snyder, 1992; Sawada et al., 1995). NO is synthesized in mam- malian neurons by Ca2 + kalmodulin activated NO syn- thase (NOS) and functions as a signaling molecule by activating soluble guanylate cyclases in target cells

(Bredt and Snyder, 1990, 1992). There is also biochem- ical evidence for the presence of the NO-synthesizing enzyme, NOS, in the central nervous system of molluscs (Elofsson et al. , 1993; Cooke et al., 1994; Moroz et al., 1994; Sanchez-Alvarez et al., 1994) and in the brain of the locust (Elphick et al. , 1993). Previous electrophysi- ological studies have shown that NO enhances the open probability of Ca2 + -activated K + channels in isolated muscle of canine colon (Thornbury et al., 1991), acti- vates a K+ channel in vascular smooth muscle of the rat by a cGMP-dependent protein kinase (Archer et al., 1994), induces a slow inward current associated with an increase in Naf conductance and mediated by an in- crease in intracellular cGMP in identified neurons of Ap- lysiu (Sawada et al., 1995), and activates feeding move- ments of the buccal mass and modulates the activity of buccal motoneurons in the molluscan nervous system (Moroz et al., 1993).

Since the half-life of NO is only about 5 sec, sev- eral molecules able to generate NO over extended peri- ods of the time have been employed. One of these is sodium nitroprusside (SNP). It has been suggested re- cently that SNP is able to amplify and modulate poten- tials recorded from mouse hippocampal slices (Bohme et al., 1991; Li and Wieraszko, 1994). However, the mech- anism(s) by which NO modulates potentials recorded from neurons has not been established. NO may modu- late the membrane potential and the firing rate of the neurons in the central nervous system (CNS) by influ- encing membrane receptors. To explore this possibility, we determined the effects of SNP on ACh-induced re- sponses in identified neurons of Aplysia. The results pre- sented here clearly demonstrate that extracellularly ap- plied SNP can reduce an ACh-induced K+ current and that this effect is mediated by an increase in intracellular cGMP.

Received June 23, 1995; revised October 2, 1995; accepted October 3, 1995.

Address reprint requests to Dr. Masashi Sawada, Department of Phys- iology, Shimane Medical University, Izumo, 693, Japan.

0 1996 Wiley-Liss, Inc.

22 Sawada and Ichinose

MATERIALS AND METHODS Identified R9 and RlO neurons (nomenclature of

Frazier et al., 1967) in the abdominal ganglion of Aplysia kurodai (100-300 g) were used. The neurons (R9 and R10) used here are homologous to R9 and RIO of Aplysia californica (Frazier et al., 1967) in appearance (white), location, and physiological properties (regular firing and hyperpolarizing response to ACh; Koester and Kandel, 1977). These neurons innervate several major tissues controlling hemolymph pressure and flow (Rittenhouse and Price, 1986). Isolated, desheathed abdominal gan- glia were pinned, ventral surface upward, to a sylgard floor in a 0.3-ml perfusion chamber. The rate of perfu- sion was 3 ml/min. The ganglia were superfused with buffered saline [normal seawater (NSW), 587 mM Na+ , 12 mM K + , 671 mM C1-, 14 mM Ca2', and 52 mM Mg2+] at room temperature (19-20°C). The pH was adjusted to 7.6 with Tris and HCl. An identified neuron (R9 or R 10) was impaled with two microelectrodes (filled with 4 M potassium acetate) for conventional two- electrode voltage-clamp. A third blunt-tipped, double- barreled microelectrode was positioned near the soma of the neuron. One barrel was filled with acetylcholine chloride (ACh, 1 mM in NSW) or y-aminobutyric acid (GABA, 10 mM in NSW), and the other with sodium nitroprusside (SNP, 25 pM in NSW). Drugs were ap- plied by micropressure ejection using a PPM-2 pneu- matic pump (Medical System Corp., New York). SNP was also applied in the bath at concentrations of 25-50 pM. cGMP (100 mM in distilled water or 100 mM in a 200 mM KC1 solution) was injected into the neuron by pressure (100-1,000 msec, 2 kg/cm2). The intracellular concentration of cGMP was estimated according to the method described by Hara et al. (1 985) and calculated to be in the range of 10-20 p,M. IBMX was dissolved in dimethylsulfoxide (DMSO) and dispersed in NSW; the final DMSO concentration was less than 0.1%. Control perfusion with the NSW containing 0.1% DMSO was without effect. Reduced hemoglobin was prepared ac- cording to the method described by Martin et al. (1 985). Drugs applied extracellularly were added to NSW. The following drugs were used: sodium nitroprusside (SNP, Sigma, St. Louis, MO); 3-isobutyl-I-methylxanthine (IBMX, Aldrich, Milwaukee, WI); cyclic guanosine 3' ,5'-monophosphate (cGMP, Sigma); hemoglobin (bo- vine) (Hb, Sigma); methylene blue (MB, Sigma).

RESULTS Micropressure ejection of ACh onto the soma of

neuron R10 induced a slow outward current [I,(ACh)] associated with a conductance increase during voltage- clamp at -60 mV (Fig. 1A). IJACh) reached a maxi-

mum amplitude (8-10 nA) within I sec and lasted 15-20 sec. This current is due to an increased K+ conductance and hence disappears or reverses to an inward current when the neuron is hyperpolarized beyond E, (about -80 mV; Sawada et al., 1991). Previous studies have established that this current is a 4-aminopyridine-resis- tant, tetraethylammonium-sensitive K + current (Sawada et a]., 1991).

Effect of SNP on I,(ACh) SNP, an NO donor, is used frequently in pharma-

cological analyses of NO actions instead of NO itself because this compound is not easily oxidized and re- leases NO as a consequence of degradation (Feelish, 1991).

Bath-applying SNP (25 pM) for 9 min reduced I,(ACh) recorded from the R10 neuron to about 40% of control (34.8 5 4.8%, mean 2 SD, n = 5 , Fig. lA, middle trace). Furthermore, I,(ACh) recorded from the same neuron at a holding potential of -60 mV was in- hibited (to about 46% of control, n = 2) by a focal application of 25 pM SNP to the somata of the neuron from a blunt-tipped microelectrode (Fig. ID). In con- trast, bath application of 25 pM SNP for the same period did not inhibit the GABA-induced C1- outward current (Fig. 1B) recorded from neuron R9 (Sawada et al., 1992) or the ACh-induced Naf inward current (Fig. 1C) re- corded from neurons in the RB group (Sasaki, 1985). Figure 2 shows representative results from several ex- periments in which the effects of extracellularly applied SNP on I,(ACh) were measured. After two control re- sponses to ACh were recorded, 25 pM SNP was applied to the bath. Bath application of SNP for 9 min inhibited I,(ACh) about 60%. The inhibitory effect of SNP on I,(ACh) was evident within 3 min after application. This effect was concentration dependent, with a threshold of 10 pM SNP, and completely reversible (Fig. 3).

cGMP and IBMX Mimic the Effect of SNP on I,(ACh)

It is well recognized that NO-induced vascular smooth muscle relaxation is mediated via elevation of intracellular cGMP by activation of soluble guanylate cyclase (Ignarro, 1991). Recently, the NO donor SNP has been shown to enhance a Ca2+ current in rat sym- pathetic neurons via stimulation of cGMP formation (Chen and Schofield, 1995). To investigate the possible involvement of the guanylate cyclase-cGMP cascade in the SNP-induced inhibition of I,(ACh), we tested the effect of intracellular injection of cGMP and bath appli- cation of IBMX, an inhibitor of phosphodiesterase (PDE). Intracellular injection of cGMP specifically in- hibited the ACh-induced K+ current amplitude by 40% (41.8 -+ 4.3%, mean i- SD, n = 4), except for a slight

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Fig. 1. Effects of 25 pM SNP on the ACh-induced K f current ([IJACh)]) recorded from neuron R10 (A), the GABA-in- duced C1- current (B) recorded from neuron R9, and the ACh- induced Na+ current (C) recorded from the neuron in the RB group at holding potentials of -60 mV (A), -45 mV (B), and -60 mV (C), respectively. SNP was applied to the bath for 9 min. ACh and GABA were ejected by a constant pressure pulse (200 msec duration, 2 kg/cm2 intensity in A, C, and D; 150 msec, 2 kg/cm2 in B). D: Effect of focal application of 25 pM SNP to neuron R9. SNP was extracellularly ejected by pressure pulses (200 msec, 2 kglcm’, indicated by small bars). A-D: Left, control; center, exposure to test solutions; right, 15-18 min after washout. NSW, normal seawater. Numbers on the lowest traces represent the duration of the pressure pulse (msec) in this and subsequent figures.

inward shift (0.2 nA) of the baseline holding current (Fig. 4B). Moreover, I,(ACh) was reduced to about 60% (56.3 2 7.5%, mean * SD, n = 3) of control by bath application of 50 pM IBMX (Fig. 4C). In the presence of 50 pM IBMX, the magnitude of the SNP-induced inhibition of I,(ACh) was 55-68% greater than the amount of inhibition produced by SNP alone (Fig. 4A,C). The synergistic inhibition in I,(ACh) caused by addition of SNP to IBMX-treated neurons mirrored the higher cGMP level in the neuron treated with SNP and IBMX compared to the neurons treated with IBMX alone. The fact that intracellular injection of cGMP or bath application of IBMX mimicked the inhibitory effect of an NO donor on I,(ACh) suggests that NO modulates

NO Inhibits ACh-Induced Current 23

( 7 0 )

100

80

- 60 S u Q - 0 -

LO

20

0 -3 0 3 6 9 12 I5 18 21 2 4 2 7 30 3 3 36 39 4 2 45

T ime (min)

Fig. 2 . Inhibitory effects of 25 pM SNP on I,(ACh) (mean * SD, n = 5, two R9s and three RlOs). Results are normalized to I,(ACh) = 100% before exposure to 25 pM SNP.

the ACh-induced K + current of identified neurons of Aplysia via stimulation of cGMP formation.

Effect of MB and Hb on the SNP-Induced Inhibition of IJACh)

To further confirm that the SNP-induced inhibition of the ACh-induced K+ current was due to elevation of intracellular cGMP by activation of soluble guanylate cyclase, methylene blue (MB), an inhibitor of guanylate cyclase (Gruetter et al., 1981b; Moritoki et al., 1992), was used. In the presence of 10 pM methylene blue, I,(ACh) recorded from R9 neuron, held at -60 mV, was not inhibited by bath application of 50 pM SNP (Fig. 5C, n = 3). To test whether the SNP-induced inhibition of I,(ACh) is mediated via the generation of NO, the ganglia were superfused with hemoglobin (50 pM) for 10-15 min prior to bath application of SNP. Hemoglobin binds NO (Garthwaite et al., 1988). Bath-applied hemo- globin, a nitric oxide scavenger, almost completely blocked the SNP-induced inhibition of I,(ACh) (Fig. 5B, n = 3). These results suggest that NO inhibits I,(ACh) via stimulation of cGMP formation and that NO gener- ated from sodium nitroprusside is responsible for the SNP-induced inhibition of I,(ACh).

DISCUSSION The hypothesis that cGMP mediates the inhibitory

effect of the NO donor SNP is supported by the follow- ing observations. 1) Bath and focal application of SNP inhibit the ACh-induced K + current in a dose-dependent manner. 2) Either intracellular injection of cGMP or bath

24 Sawada and Ichinose

8

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2

10 s

-6 0 6 12 18 2L 30 3C L2 4 0 5L GO G G 72 70 01 90 Time ( m i n )

Fig. 3. Time course of the inhibitory effects of SNP on I,(ACh) in neuron R10 voltage- clamped at -60 mV. ACh was ejected by a constant pressure pulse (200 msec, 2 kg/cm2).

application of IBMX mimics the SNP-induced inhibition of I,(ACh). 3) Hemoglobin, a NO scavenger (Shibuki, 1990), blocks the SNP-induced inhibition of I,(ACh). 4) Methylene blue, an inhibitor of soluble guanylate cyclase (Mayer et al., 1993), significantly blocks the SNP-in- duced inhibition of I,,(ACh).

It has been reported that purified astrocytes from the cerebellum contain high levels of SNP-sensitive gua- nylate cyclase (Bunn et al., 1986). Immunocytochemical studies on rat cerebellum slices have shown that SNP induces a striking accumulation of cGMP in astrocyte cell bodies and in the granule cell layer (De Vente et al., 1990), and that NO synthase is present in granule cells (Bredt and Snyder, 1990). Furthermore, studies of NADPH-diaphorase histochemistry have shown the pres- ence of NOS in both the central nervous system and peripheral tissues of Aplysia (Lukowiak et al., 1993). Recently it has been shown that in the invertebrate brain the NO generator (hydroxylamine) increased the cGMP content of brain extracts (Elphick et al., 1993). These findings raise the possibility that NO may act as an in- tercellular signaling molecule or a neuromodulator in in- vertebrates. Indeed, in the present study, NO generated from SNP acts as a neuromodulator inhibiting the ACh- induced K + outward current by increasing intracellular cGMP. The NO donors (sodium nitroprusside and S-ni- troso-N-acetylpenicillamine) enhance Ca2 + currents and block noradrenaline-induced Ca2 + current inhibition in rat sympathetic neurons via stimulation of cGMP forma- tion (Chen and Schofield, 1995). Previous experiments on snail neurons showed that cGMP-induced enhance- ment of neuronal Ca2+ channel currents is via a cGMP- dependent protein kinase (Paupardin-Tritsch et al., 1986). In addition, another NO donor, SIN-1 (3-mor- pholino-sydnonimine) inhibits mammalian cardiac Ca2'

current through a cGMP-dependent protein kinase (Wahler and Dollinger, 1995). cGMP and the cGMP- dependent protein kinase are capable of modulating membrane potentials and ion channels (Butt et al., 1993). It is possible that NO-induced inhibition of the ACh-induced K + current results from stimulating the formation of cGMP, which, in turn, activates cGMP- dependent protein kinase, which then results in ion chan- nel protein phosphorylation (Butt et al., 1993).

Our data from direct intracellular injection of cGMP support the hypothesis that guanylate cyclase me- diates the SNP-induced inhibition of I,(ACh), because in contrast to its blocking effect on I,(ACh), methylene blue did not block either an ACh-induced inward current (an increase in Na+ conductance) or a GABA-induced outward current (an increase in C1- conductance, data not shown). Thus, methylene blue specifically blocks the SNP-induced inhibition of I,(ACh). Methylene blue is a well-known vital stain for neuronal tissue. The heme group of soluble guanylate cyclase has been suggested as a target site of methylene blue, resulting in a reduced responsiveness of the enzyme to activation by NO (Gru- etter et al., 1981a).

The small molecular size, lipophilic nature, and chemical instability of NO make it well suited for its probable role in local transcellular communication. Like oxygen and carbon dioxide, NO can cross cell membranes easily by diffusion. In contrast to other known neuro- transmitters or neuromodulators, NO may act as a neu- romodulator by directly activating soluble guanylate cy- clase instead of binding to a specific receptor in parallel to its site of action. Physiological evidence supporting a role for NO as a neurotransmitter or neuromodulator in the nervous system of molluscs includes the findings of Moroz et al. (1993) that S-nitrocysteine, a NO generator,

NO Inhibits ACh-Induced Current 25

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, 200

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ACh

SNP

A I

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A

IBMX

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SNP + IBMX IBMX

Fig. 4. Effects of bath-applied SNP (25 pM, for 9 min, A), intracellular injection of cGMP (B), and pretreatment with 50 pM IBMX (C) on I,(ACh) recorded from neuron R10 at a holding potential of -60 mV. ACh was ejected by a constant pressure pulse (200 msec, 2 kg/cm2). cGMP was intracellularly injected into the neuron by ten pressure pulses (50 msec, 2 kg/cm2). Intracellularly injected cGMP inhibited I,(ACh) ex- cept for a slight inward shift (0.2 nA) of the baseline holding current (B). Note that the magnitude of the SNP-induced in- hibition of I,(ACh) was enhanced in the neuron treated with SNP + IBMX compared to the neuron treated with IBMX alone (C).

NSW SNP NSW

, 200 I L A A A

ACh

NSW SNP+ Hb NSW

B k , 200 I L

A ACh

NSW SNPfMB NSW

, 200 b II 2 kglcrnz A A A -

ACh 10 s

Fig. 5. Effects of bath-applied SNP (50 pM, for 9 min, A), SNP in the presence of 50 pM hemoglobin (Hb, B), and SNP in the presence of 10 pM methylene blue (MB, C) on I,(ACh) recorded from neuron R9, held at -60 mV. The ganglia were superfused with Hb or MB for 10-15 min prior to bath appli- cation of SNP. ACh was ejected by a constant pressure pulse (200 msec, 2 kg/cm2). A-C: Left: control; center, exposure to test solutions; right: 18-20 min after washout. Note that SNP in the presence of Hb + MB did not inhibit the ACh-induced KC current.

can modulate or activate patterned motor activity in the buccal ganglia of the nervous system of Lymmaea stag- nalis. In addition, Sawada et al. (1995) have shown that

extracellular hydroxylamine or SNP (NO generator) can induce a slow inward current associated with an increase in Nat conductance, mediated by an increase in intra- cellular cGMP. Furthermore, it has been demonstrated that NO enhances synthesis of cGMP, and 8-bromo cGMP mimics the membrane hyperpolarization or inhi- bition of membrane excitability in the dog colon (Ward et al., 1992). Therefore, a major molecular event in signal transduction by NO seems to be activation of guanylate cyclase. This enhances production of cGMP, which is linked with a series of physiological phenomena.

Previously, the NO-cGMP signaling pathway has only been described in mammalian nervous systems, so our findings indicate, for the first time, that NO is an evolutionarily ancient neuromodulator with a widespread phylogenetic distribution. The physiological role of the SNP-induced inhibition of the ACh-induced K + current in the central nervous system of Aplysia is unknown at the present time. It is conceivable that NO may act as a neuromodulator that regulates the firing pattern of iden- tified neurons (R9 and R10). It has been shown that the neurons (R3 -R14) of Aplysia californicu innervate sev- eral major tissues controlling hemolymph pressure and flow, namely the heart, gill vein, and major aortae, sug- gesting their significant involvement in hemodynamics (Rittenhouse and Price, 1986).

ACKNOWLEDGMENTS This work was entrusted to Shimane Medical Uni-

versity by the Science and Technology Agency, using the Special Coordination Funds for Promoting Science and Technology. We thank Dr. D.J. McAdoo for reading the manuscript, Mrs. Y. Takeda for typing, and Mr. S. Saito for supplying Aplysia kurodai.

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