Enhancement of a slow potassium current component by uranyl nitrate and its relation to the antagonism on β-bungarotoxin in the mouse motor nerve terminal

Neuropharmacology Vol. 34, No. 2, pp. 165-173, 1995 Copyright 0 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0028-3908195 $9.50 + 0.00

002&3908(94)00125-1

Enhancement of a Slow Potassium Current Component by Uranyl Nitrate and its Relation to the Antagonism on /I-Bungarotoxin in the Mouse Motor Nerve Terminal K. F. CHAO’ Institutes of’ Pharmacology

and zToxicology,

and

S. Y. LIN-SHIAU’,‘*

College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China

(Accepted 30 August 1994)

Summary-Uranyl nitrate (UO,(NO,),) has been shown to be capable of increasing transmitter release from the motor nerve accompanied by the potentiation of nerve evoked muscle contraction. In this paper, we have demonstrated that UOi+ induced an initial twitch depression followed by a later twitch potentiation in low (0.35 mM) Ca’+ medium. Although UO:+ has been identified as a K+-channel blocker, we have found it only partially blocked the fast KC-current (IK(,,) as recorded in the perineurial sheath of the mouse triangularis sterni preparation. Increasing the concentration of UOZ2+ to a high concentration of 0.4 mM did not further inhibit IK,, but markedly prolonged the duration of the outward current of the nerve terminals. From the time course of its appearance together with the specific inhibition by 4-aminopyridine, dendrotoxin and /I-bungarotoxin, which has been shown to be capable of blocking the K+-current of the motor nerve terminal, it was proposed that II@+ prolonged the duration of the nerve terminal spikes by an enhancement of an IK,,-like current, which was further characterized by its susceptibility to be enhanced by low K+, low Ca2+ and Cd2+ but attenuated by high K+ and high Ca2+. These cation effects not only supported UOi+-induced IK,, current but also excluded the possibility of an enhancement of Ca2+ -activated KC-current induced by UO:+ plus TEA. ‘The significance of this enhancement of IK,,, induced by UOi+ has been elucidated by the finding that dendrotoxin inhibited but tetraethylammonium potentiated not only UOi+-induced IK,,, but also UO$+-induced twitch depression. Therefore, it is concluded that in addition to the blockade of IK,,, UO:+ can also enhance IK,,-like current which accounts for presynaptic inhibition of the twitch contractions. Moreover, U02(N03)2 has been found to be a selective potent antagonist of /I-bungarotoxin (/I-BuTX) in blocking the neuromuscular transmission of mouse phrenic nerve diaphragm. In accordance with their effects on UOi+ -induced IK,,, , TEA markedly potentiated UOi+ in antagonizing the neuromuscular blocking action of /I-BuTX, while 4-AP, DTX, high K+ and Ca2+ all reversed the antagonistic action of UOz+ plus TEA against j?-BuTX. ‘These findings provide more evidence that IK,,, plays a role in the regulation of transmitter release and UOi+ antagonizes the neuromuscular blocking action of fl-BuTX through their interaction at least in part on the slow K+-channels of the motor nerve terminals. KeywordeUranyl

Uranyl

nitrate

nitrate, motor nerve terminal, B-bungarotoxin,

has been shown to be capable

of increas-

content

dendrotoxin,

of endplate

potassium current.

potentials

accompanied

by repetitive

ing the release of transmitter, not only from motor nerve terminals (Benoit and Mambrini, 1970; Lin-Shiau et al., 1979; Datyner and Gage, 1980) but also from autonomic synapses (Fu and LinShiau, 1985). In our previous papers, it was found that uranyl nitrate and 4-aminopyridine (4-AP) increased the amplitude and the quanta1

endplate potentials following a single nerve stimulation (Lin et al., 1988). In a direct observation on the nerve terminal currents, it has been reported that UO:+ produced a partial block of a fast K+ current (IK,,) which contributed to its facilitatory effect upon transmitter release (Tabti et al., 1989). By contrast, tetraethylammonium (TEA) and 4-AP completely blocked the

*To whom correspondence should be addressed, at: Department of Pharmacology, College of Medicine, National Taiwan University, No. 1, Jen Ai Road, 1st section, Taipei, Taiwan.

IK,,, but the former suppressed and the latter induced a Ca*+-activated K+ current (IK,,,) respectively (Mallart, 198513). Although these three K+ channel blockers TEA and 4-AP) exhibited some differences in (Ho:+ 9 165

K. F. Chao and S. Y. Lin-Shiau

166

the alterations of these pharmacologically dissectable K+-channels in the motor nerve terminals, their effect upon the neuromuscular blocking action of /?-bungarotoxin (j?-BuTX), a specific presynaptic neurotoxin, were quite different, e.g. UOi+ antagonized but TEA and 4-AP enhanced the action of /I-BuTX. During our investigation on the effects of low Ca’+ medium on their potentiation of the nerve-evoked muscle contractions, we have found that UO:+ but not TEA nor 4-AP initially suppressed the muscle contractions. In this paper, we attempted to explore the possible mechanism of action of UO:+ in the initial twitch suppression and its antagonism on the neuromuscular blocking effect of /I-BuTX. The results obtained from this study indicate that uo;+ ) especially in the presence of TEA, markedly enhance a slow K+-current on the motor nerve terminals which accounts at least in part for the initial twitch suppression and the antagonism on /I-BuTX in neuromuscular blockade. METHODS

Nerve terminal spikes in mouse triangularis sterni nervemuscle preparation

Experiments were performed on the isolated intercostal nerve triangularis sterni muscle of adult ICR strain mice (20-25 g). The preparations were made as described by McArdle et al. (1981), and continuously perfused at a rate of 10 ml/min with an oxygenated and modified Krebs’ solution containing (mM): (NaCl 130.6, KC1 4.8, CaCl, 2.5, MgSO, 1.2, NaHCO, 12.5 and glucose 11.1). The temperature was maintained at 25527°C. In all experiments, d-tubocurarine (20 PM) was used to abolish the neuromuscular transmission. The intercostal nerve was stimulated by a glass suction electrode with supramaximal current pulses of 2Opsec duration at 0.2 Hz. Extracellular signals were picked up by a 0.5 mM NaCl-filled microelectrode inserted into the perineurial sheath of superficial nerve bundles near the endplate under visual control at 300 x magnification. The reference electrode was a piece of chlorided silver wire placed in the recording chamber. After conventional amplification, the signals were fed into an oscilloscope (Tektronix 5113A) for visual inspection as well as to a video recorder and a waveform analyzer (D6000, Data Precision, U.S.A.) for waveform storage and analysis. The waveform was drawn by X-Y plotter (Roland, model Dxy-1100). Nerve-evoked muscle contraction of mouse phrenic neruediaphragm preparation

The mouse phrenic nerve-diaphragm preparation was isolated according to the method of Bulbring (1946). Modified Krebs’ solution, gassed with 95% 0, + 5% CO, at 37 &-0.5”C, was used throughout the experiment;

the composition of the solution was as follows (mM): NaCl 130.6, KC1 4.8, CaCl, 2.5, MgSO, 1.2, NaHCO, 12.5 and glucose 11.1. The phrenic nerve was stimulated with supramaximal rectangular pulses of 0.05 msec at a rate of 12/min. The contraction was recorded isometrically with a force-displacement transducer (Grass FT. 03) on a Grass Model 7 polygraph. Drugs

/I-Bungarotoxin (b-BuTX) was isolated from the venom of Bungarus multicinctus by the method described by Lee et al. (1972). The homogeneity of the purified toxins was verified by disc gel electrophoresis (David, 1964). Dendrotoxin (DTX), 4-aminopyridine (4-AP), tetraethylammonium (TEA) and uranyl nitrate (UO,(NO,),) were purchased from Sigma (St Louis, MO, U.S.A.) and Merck (Germany) respectively. Statistics

The number of the experiments for each group was more than four and significance of difference was assessed by using one-way analysis of variance (ANOVA) as indicated in the results. Furthermore, the difference between the control and each test group was also assessed by Student’s t-test. RESULTS Eflects of UO;+ , TEA and 4-AP on the nerve terminal spike of mouse triangufaris sterni nerve-muscle preparation

The representative nerve terminal spikes recorded in the perineural sheath close to the motor nerve terminals are shown in Fig. 1. The nerve terminal spike consists of two negative peaks. The first negative peak can be attributed to Na+ influx in nodes of Ranvier (the propagating nerve action potential) and the second negative peak corresponds to a K+ outward current generated in the nerve terminals, which can be pharmacologically dissected into fast K+ current (IK&, slow K+ current (IK,,,), and Ca’+-dependent K+ current (IK,,,). In this paper, the second wave is mainly fast potassium current as described by Mallart (1985b), Penner and Dreyer (1987) and Tabti et al. (1989). Treatment with UO:+ (0.4 mM) for 30 min, partially depressed the IK,,, current but prolonged the duration of the nerve terminal spike (165 f 5% of the control (n = 9)). Further increase in the concentration of UO:+ up to 0.8 mM did not completely suppress IK,, but profoundly prolonged the duration of nerve terminal spike (Fig. 1). Addition of TEA (2 mM) for 20 min inhibited the IK,,, and IK,,,, currents of the nerve terminal spike, while an ICa current was revealed (Fig. 1). After the IK,,, and IK,,, currents were inhibited by adding 4-AP (1 mM) for 20 min, a small ICa’+ current and a Ca’+-dependent K+ curent (IKtc,J appeared (Fig. 1). UO:+ (0.4mM) induced a prolonged nerve

A slow terminal 0.41mM UD22+

J (A)

I

J

0.5&l

TEA

potassium

current

r_

induced

by uranyl nitrate

167

0.8 mM UC&~+

J

ImVL

(A)

L

ImV

2mM TEA

1mS

0.4mM uo22+

571&i DTX

@)

r

(C) Fig. 1. Effects of K+ channel blockers on fast K+ current of the mouse triangularis sterni. The representative subendothelial signals are shown before and 30 min after addition of UOi+ (A, 0.4 mM, n = 28 and 0.8 mM, n = 3), TEA (B, 0.5 mM and 2 mM, n = 3), and 4-AP (C, 0.3 mM and 1 mM, n = 3). Note that UO$+ only slightly reduced the amplitude but

markedly prolonged the duration of the K+ component of the spike. The Na+-spike remained unaffected.

terminal spike in normal Krebs’ solution. However, elevating the concentration of extracellular Ca2+ from 2.5 to 6mM attenuated while low Ca2+ (0.75 mM) augmented the development of the prolonged nerve terminal spike induced ibyUO$+ [Fig. 2(A) and (C)l. Moreover, pretreatment ,with DTX (57 nM) prior to the addition of UO:+ (0.4 m:M) also prevented the development of the prolonged nerve terminal spike [Fig. 2(B)], while UO:+ (0.4 mM) followed by TEA (2 mM) slightly decreased the amplitude but markedly prolonged the duration of the outward K+ current (325 f 23% of control group) (Fig. 3). Pretreatment with both 4-AP (1 mM) and UOz+ (0.4 mM) completely suppressed the IK,,, current and induced an ICa current together with an IK,,,, current. Both ICa and IK,,,, currents could be blocked by Co’+ (10mM) (Fig. 3). Whether this prolonged duration of nerve terminal of spikes induced by UO:‘- is mediated by inhibition Ca2+ influx or induction of an IK current was differentiated by applying Cd2+ (1 mM) instead of UO:+ (0.4 mM) followed by cumulative application of TEA from 2 to 10mM. Alt!hough the amplitude of IK,,, current was partially blocked by TEA, the duration of IK current was not affected as seen with UO:+ and TEA (Fig. 4).

VL Ima

I-v

h

J

Im

0.4mM uD22+

0.75mM cd+

ImV

L lms

Fig. 2. Effects of Ca*+ and dendrotoxin (DTX) on nerve spikes of mouse triangularis sterni. Nerve spikes were recorded before and 30 min after addition of UOi+ (0.4 mM) either in 6 mM Ca*+ Krebs solution (A, it = 4), dendrotoxin (B, 57 nM, n = 3) or with 0.75 mM Ca*+ Krebs solution (C, n = 9).

Efects of K+ and Ca2+ on the prolonged duration of nerve terminal spikes induced by UO$’ plus TEA As shown in Fig. 5, the prolonged outward current induced by UO:+ plus TEA could be inhibited by high K+ (10 mM), but was potentiated by removing K+ from

0.4mM uo22++2mM TFA

(4

J v 0.4dvf uo22++ld

@>

0.5mV~ 4-AP

1Od4Cd+

ykc2f ImV

&

Fig. 3. Effects of potassium channel blockers on nerve spikes of mouse triangularis sterni. Nerve spikes were recorded 20 min after addition of UO$+ (0.4 mM) and TEA (A, 2 mM, n = 24) or 4-AP (B, 1 mM, n = 4). The IK,,,, induced by UOit plus 4-AP was inhibited by Co*+ (B, 10 mM, n = 3).

K. F. Chao and S. Y. Lin-Shiau

168

ImM Cd2+

prolonged nerve terminal spikes induced by UOit, low Ca2+ [Fig. S(A)] enhanced but high Ca2+ [Fig. 8(D)] and high Kf [Fig. 8(E)] prevented the declining phase of the twitch amplitude following the addition of TEA.

2mM TEA

Eflects of UO: with or without blocking action of /I-bungarotoxin diaphragm preparation

4mM TEA

6mMTEA

B-BuTX (0.22 PM) alone progressively inhibited the twitch amplitude evoked by nerve stimulation and caused an irreversible blockade of neuromuscular transmission after treatment for 122 f 8 min (n = 12). Pretreatment with TEA (2 mM) or UO:+ (0.4 mM) for 20min prior to the addition of /?-BuTX (0.22pM) altered the neuromuscular blocking time from

lomh4TEA

Fig. 4. Effects of cumulative TEA on nerve spikes of mouse triangularis sterni in the presence of Cd’+. The concentration of Cd2+ is 1 mM. The experimental number more than three.

0.4mM UO22++ 2mM TEA

blockers

on the prolonged

4-AP (1 mM) not only completely suppressed the prolonged outward current induced by UO:+ plus TEA but also promoted the development of an ICa current; the duration of the ICa current was about 25msec (Fig. 6). Furthermore, the toxins, dendrotoxin (57 nM) and /?-BuTX (0.22 PM) which have been proved to be capable of suppressing the IK,, current in mouse motor nerve terminals, could suppress the prolonged current induced by combination of UO:-+ and TEA. It was noted that /?-BuTX was weaker than DTX in suppressing this current (Fig. 7). Eflects of cations, DTX, suppressing action of UO;

and

TEA

on

the

IOmM

K+

I--

ImVL 2ms

the perfusion fluid. Similarly, while high Ca*+ reduced but low Ca*+ (0.75 mM) enhanced this outward current (Fig. 5), Cd’+ (1 mM) also mimicked the low Ca2+ enhancing effect (Fig. 5). Effects of other K+-channel outward currents

TEA on neuromuscular in mouse phrenic nerve-

(W

(Cl

VT--r hVL

twitch-

In normal Krebs solution, UO:+ (0.4 mM) may facilitate twitch amplitude evoked by the nerve stimulation of mouse phrenic nerve-diaphragm preparations, but in 0.35 mM Ca*+ Krebs solution, the facilitatory phase induced by UO:+ is preceded by a suppression of the twitch amplitude. This suppression phase of twitch amplitude was prevented by pretreatment with DTX (57 nM, Fig. 8). On the other hand, addition of 2 mM TEA transiently potentiated the twitch amplitude and subsequently decreased the twitch by more than 70% of the control prior to the application of TEA [Fig. 8(C)]. This phase of reduced twitches induced by UO:+ plus TEA was supposed to be due to the augmentation by TEA of the twitch depression induced by UOzt. In agreement with the potentiation by TEA of the

2ms

Fig. 5. Effects of cations on nerve spikes of mouse triangularis sterni treated with UOz+ (0.4 mM) plus TEA (2 mM). Nerve spikes were recorded before and 15 min after treatment with 10 mM K+ (A, n = 4), removing K+ (B, n = 3), 6 mM Ca2+ (C, n = 3), 0.75 mM Ca2+ (D, n = 6), and 1 mM Cd2+ (E, n = 4). Note the augmentation by Cd2+, K+ free, and low Ca2+ but antagonism by high Ca2+ and high K+ of the prolonged K+ spike current of mouse triangularis sterni treated with UOi+

(0.4 mM) plus TEA (2 mM).

A slow terminal potassium current induced by uranyl nitrate

169

Fig. 6. Antagonism by 4-AP of slow K+ spike component of mouse triangularis stemi treated with UO:+ (0.4 mM) plus TEA (2 mM). Nerve spikes were recorded before and 15 min after cumulative addition of each concentration of 4-aminopyridine (CAP, 0.1, 0.3, and 1 mM, n = 3).

122+8min to 91+8min (n=5) and 326f19min (n = 10) respectively. Moreover, pretreatment with UO:+ (0.4 mM) plus TEA (2 mM) each for 20 min prior to the addition of /?-BuTX (0.22 PM) markedly prolonged the blocking time of /3-BuTX (655 + 40 min, n = 5) (Fig. 9).

Reversal by 4-AP, DTX, high Kf and high CaZf from the antagonistic action of UO<+ against fi-BuTX Pretreatment with 1 mM 4-AP, which markedly increased the twitch amlplitude, not only enhanced the neuromuscular blocking action of j?-BuTX (88 + 5 min, n = 6) but also attenuated the antagonistic action of UO$+ against b-BuTX (172 + 54 min, n = 4). Moreover, the combined treatment with all of UO:+ (0.4mM), TEA (2 mM), and 4-AP (1 mM), profoundly shortened the neuromuscular blocking time of p-BuTX to 143 f 38 min (n = 4). That result showed that 4-AP reversed the profound antagonistic action of UOit plus

0.4mM uo22++2l&l

TEA

0.22@4 p-BuTX

57&l D’IX

Fig. 7. Antagonism by P-BuTX and dendrotoxin of K+ spike currents of mouse triangularis stemi treated with UOi+ (0.4 mM) plus TEA (2 mM). Nerve spikes were recorded before and 4Omin after addition of b-BuTX (0.22pM), II = 3 and 10 min after dendrolloxin (DTX, 57 nM, n = 3).

TEA on j?-BuTX (Fig. 9). Similarly, DTX mimicked 4-AP in augmenting the neuromuscular blocking action of /3-BuTX (Fig. 9). On the other hand, high K+ (10 mM) alone did not significantly change the neuromuscular blocking action of /3-BuTX (133 f 11 min, n = 4), but markedly attenuated the antagonistic action of uo:+ against j-BuTX whether in the absence (155 + 4 min, n = 5) or the presence (185 + 11 min, n = 4) of 2 mM TEA. Furthermore, high Ca*+ (6 mM) has a similar effect in depressing the antagonistic action of UO:+ against /I-BuTX (Ca2+ + j?-BuTX: 118 + 10 min, n = 4; Ca2+ + UOz+ + fi-BuTX: 164 f 36 min, n = 4; Ca*+ + UOit + TEA + /?-BuTX: 145 + 22min, n =4) (Fig. 9).

DISCUSSION By means of extracellular recording in the perineurial sheath of the motor nerve terminal, Mallart (1985a, b) first demonstrated the motor nerve terminal spikes, which could pharmacologically be characterized as Na+ , Kf and Ca2+ currents. The initial negative spike could be blocked by tetrodotoxin and was recognized as Na+-current (INa), whereas the second negative spike could be blocked by TEA and 4-AP and was designed as fast K+-current (I#,,). Both Ca2+ current (ICa) and slow K+-current (IK,,,) were revealed after complete blockade of IK,,, by TEA (Tabti et al., 1989). Treatment with 4-AP abolished IK,,, but instead induced IK,,,, due to the increase of the intracellular Ca2+ concentration (Mallart, 1985b). In this paper, we have demonstrated that UOzt alone, unlike TEA or 4-AP, only partially blocked IK,,, even at a high concentration of 0.8 mM, but significantly prolonged the duration of the nerve terminal spikes. This peculiar prolongation of the nerve terminal spikes induced by UO:+ could be further enhanced by TEA but abolished by 4-AP. According to these results, together with the time course of its appearance, it is suggested that this prolonged duration of nerve terminal spike is due to the enhancement of IK,,,

170

K. F. Chao and S. Y. Lin-Shiau

6% .

0 35m-M Ca2+

0

0.41nM-UO2~’

;

2n1M’TEA kmL

1 0.3Sm:

ca2+

. 10

;

57aM DTX

. 20

0.4m~‘“.O2~+

o.‘gmL . . . . .

o.4n,M’“O~2+ f

I5

30

w

160

220

03 loll1

(E)

IOmMKC 0.4mMA”022+

; I

io

;5

2mM TEA

Fig. 8. Effects of dendrotoxin, cations and tetraethylammonium on the nerve-evoked twitches of the mouse phrenic nerve-diaphragm. The muscle contractions of the mouse diaphragm were evoked by electrical stimulation of the phrenic nerve and recorded isometrically. The diaphragm was pretreated with the cations as indicated for 20min prior to the application of 0.4 mM UO,2f. Note that UOi+-induced twitch depression (A) can be inhibited by dendrotoxin (DTX) and the twitch decline phase induced by UO$+ plus tetraethylammonium (TEA) can be enhanced by low Ca2+ (A) but prevented by 1OmM Ca2+ (D) or 1OmM K+ (E).

induced by UOi+ and designated as IKo,-like current in this paper. In order to further characterize this IK,,-like current, we first tried to exclude the possibility of the involvement The first evidence for this concentration was of IKw. provided by the fact that both high K+ (10 mM) and high Ca2+ (6 mM) inhibited, but low K+, low Ca2+ and Cd’+ enhanced this IK,,-like current. On the contrary, Mallart (1985b) has reported that IK,,,, current could be abolished by the addition of inorganic Ca2+ channel blockers (Cd2+) but increased by elevation of the extracellular Ca2+ concentration. This opposite effect of Ca2+ on IK,,, and IK,,,, supports the notion that UO:+ induced an IK,,,-like current, but not IKcc,). Further-

more, this fact together with the finding that high K+ inhibited but low K+ enhanced the IK,,-like current, also supports the enhancement of K+ current by UO:+ through the K+ concentration gradient in the nerve terminals. Because these extracellular recordings in perineurial nerve sheath show only net current, the current at the end of the INa corresponds to an exact balance of outward K+ and inward Ca2+ currents. And, as shown in Fig. 1, TEA (2 mM) alone is high enough to suppress IK,,, and IK,,,, and then to promote the development of ICa. UO:+ was also defined as a K+ channel blocker which might increase Ca2+ influx (Tabti et al., 1989). Interestingly, we have found that in combined application with UO:+ on the nerve terminal, TEA could not completely abolish the residue IK nor could it create an ICa. It is postulated that UO,‘+ possesses another action on the nerve terminal spike, either an enhancement of IK or a reduction of ICa. Therefore, we use Cd2+ to distinguish these two possibilities. The possibility that UO:+ prolonged the duration of the nerve terminal spikes due to the inhibition of ICa rather than the enhancement of IK,,, could be excluded by the finding that Cd2+, a well established inorganic Ca2+ channel blocker, followed by addition of TEA did not mimic the action of UO:+ to produce IK,,,. Thus, it is concluded that UO:+ directly enhances IK,,, rather than acting indirectly through the inhibition of ICa. Dendrotoxin (DTX) has been found to be a K+channel blocker specifically inactivating an outward current which shows slow activation and incomplete inactivation in rat visceral sensory neurons (Stansfield et al., 1986), in guinea pig dorsal root ganglion (Penner et al., 1986) and in mouse motor nerve terminal (Penner and Dreyer, 1987). Similarly, /I-bungarotoxin has also been reported to be capable of blocking an IK,,, in guinea pig dorsal root ganglion (Petersen et al., 1986), in frog motor nerve terminal (Rowan and Harvey, 1988) and in mouse motor nerve terminal (Penner and Dreyer, 1987). In this paper, we have used these two toxins to further characterize the UO; f -induced IKe,-like current. Pretreatment with either DTX or j?-BuTX was able to block UO:+ in inducing IK,,,-like current. In addition, DTX pretreatment allowed UO:+ to completely block IK,,, which was only partially blocked by UO:+ alone because of the immediate appearance of IK,,, (Fig. 2). This finding provides additional evidence for supporting the idea that the prolonged duration of the nerve terminal spike induced by UO:+ was due to the enhancement of IK,,,. An extensive mutational analysis of voltage-gated K+-channels has provided important clues about the binding sites of the K+-channel blockers (Pongs, 1992). The amino acid residues l(thr) and 19(glu) of the H5 region of K+-channels have been found to be located near the entrance of the channel pore which were identified as the binding sites of TEA, while the binding site of dendrotoxin and aminopyridines would be closely

A slow terminal potassium current induced by uranyl nitrate

(A)

1mM 4-AP

+

+

+

++ ++

uog+ TEA P-BuTX

(B)

+ +

+ +

+’ :

+

57nM DT;

uog+

TEA P-BuTX

+ +

: !z.

600 500

3 iz ,”

400 300

.

200

B G

100 Inl

10mM ICt uo2:!+

TEA P-BuTX

+

+

+ +

+ +

.1 1 +

++

++

+ +

++ + +

700 z .5

600 500

8

:i;

z

200

.g b

100

.

0 L_c&

6mM Ca2+

+

uo$+ TEA P-BuTX

+ +

.IL + .IL +

++ ++ @I

CC) 700

+

171

+

++ + +

++

++

+ +

++ + +

Fig. 9. Effects of cations and K+-channel blockers on the neuromuscular blocking action of /3-bungarotoxin in the mouse phrenic-nerve diaphragm pretreated with or without UOi+. The mouse phrenic nerve-diaphragm is electrically stimulated on the nerve in Krebs solution at 37°C and pretreated with either 4-aminopyridine (4-AP, 1 mM, A) or dendrol.oxin (DTX, 57 nM, B) or high K+ (10 mM, C) or high Ca2+ (6 mM, D) for 20 min prior to the addition of UOz+ (0.4 mM) or UOi+ (0.4 mM) plus Tetraethylammonium (TEA, 2 mM). Note that all of these pretreatments significantly reversed the antagonism by UOs+ of the neuromuscular blocking action of p-bungarotoxin (/?-BuTX, 0.22 PM).

related but were different from those of TEA, which were also located on the extracellular surface of K+-channels. A distinct negative charged amino acid which lies Nterminal to the H5 region appears to be important for dendrotoxin binding. This useful information coupled with the findings of this study, allow us tentatively to postulate that UO:+ might bind by its positive charge with the negatively charged amino acid of the dendrotoxin binding site in the H5 region of the K+-channel. In the report of Tabti et al. (1989), only a short treatment of UO:+ for 15 min was used and this may be considered to be inadequate to fully enhance the channel opening. TEA binding to the H5 region, close to the binding site may further facilitate the channel opening. of uo:+, Further study is needed for the elucidation of their exact binding sites. In consideration of the possible functional role of the enhanced IKe,-like current induced by UO:+, we have studied the effect of TEA, Ca2+, K+ and DTX muscle contracon UO:+ in affecting the nerve-evoked tion. DTX inhibited the action of UO:+ not only in inducing IK,,, but also in :blocking the twitch depression In general, UO:+-induced in 0.35 mM Ca2+ medium. twitch depression was always masked by the facilitatory

phase of the twitches resulting from the inhibition of IK,,,. However, an addition of TEA to the UO:+pretreated mouse diaphragm definitely produced a twitch decline phase following a rapid twitch potentiation. This twitch decline phase was correlated with the enhancement by TEA of UO:+-induced IK,,-like current. Moreover, the augmentation by low Ca2+ but inhibition by high Ca2+ and high K+ on this twitch decline phase were also in concert with their effects on IK,,-like current induced by UO:+ plus TEA. All of these findings suggest that UO:+ induced an enhancement of presynaptic IK,,,-like current which resulted in the twitch depression. One possibility that these twitch depressions induced by either UO:+ alone in low Ca2+ medium or UO:+ followed by TEA were due to a postsynaptic inhibition has been excluded by the previous reports that muscle contractions evoked by direct muscle stimulations were persistently potentiated by either UO:+ alone or UOz+ plus TEA for at least 60 min (Fu et al., 1989; Lin-Shiau et al., 1991). Based on these results, we tentatively concluded that UO:+ inhibited IK(,, on the one hand but enhanced IK,,, on the other, which contributed to the presynaptic inhibition resulting in the twitch depression.

172

K. F. Chao and S. Y. Lin-Shiau

/I-BuTX is a selective presynaptic neurotoxin which inhibits acetylcholine release from the motor nerve terminals (Chang, 1985). The mechanism of the inhibitory action of /I-BuTX on acetylcholine release is still not known. UO$+ has been found to be a selective and potent antagonist of b-BuTX both in vitro and in vivo (Lin-Shiau, 1983; Lin-Shiau et al., 1983, 1984; Lin-Shiau and Fu, 1986; Lin et al., 1988). Evidence obtained suggested that UO:+ antagonized a-BuTX both in the binding and the post-binding effect of fl-BuTX (Lin-Shiau and Fu, 1986). Although /I-BuTX and UOz+ also possessed a blocking action on K+-channels of the motor nerve terminals (Penner and Dreyer, 1987; Petersen et al., 1986), the mechanism of the antagonistic action of UO:+ on j?-BuTX still needs to be clarified. In this paper, we have demonstrated that UO:+ can enhance the IK,,, of the motor nerve terminals in the mouse triangularis sterni. 4-AP, a high concentration of Ca2+ K+ and DTX not only can prevent the enhancement’ of the IK,,, current induced by UO:+ plus TEA (Figs 5, 6 and 7) but also reverse the antagonism by UO:+ either with or without TEA against /I-BuTX in the induction of neuromuscular blockade. These results indicated to us that the enhancement of IK,,, current by UO;+ must be involved in the antagonism on the neuromuscular blocking action of /I-BuTX. In summary, we have compared the effects of three K+-channel blockers (UOz+, TEA and 4-AP) on the nerve terminal spikes of mouse triangularis sterni. UOg+ alone was found to partially block IK,,, but profoundly prolonged the duration of the nerve terminal spike. This prolonged spike was characterized to be due to an enhancement of IK,,, . The induction of this presynaptic IK,,, by UO:+ was correlated with the twitch depression phase by UOz+, since DTX, high Ca*+ and high K+ inhibited, but TEA, low Ca2+, low K+ enhanced not only the UO:+-induced IK,,, but also the twitch depression. Moreover, both UO:+-enhanced IK,,, current of the motor nerve terminals and its antagonism against /I-BuTX in blocking the neuromuscular transmission whether in the presence or absence of TEA all could be reversed by 4-AP, DTX, high K+ and high Ca*+. Therefore, it is inferred that UO:+ antagonized the neuromuscular blocking action of /I-BuTX at least in part through an enhancement of IK,,, current of the motor nerve terminals.

Chang C. C. (1985) Neurotoxins with phospholipase A2 activity in snake venoms. Proc. Natn. Sci. Count., ROC 9: 126142. David B. J. (1964) Disc electrophoresis II method and application to human serum proteins. Ann. N. Y. Acad. Sci. U.S.A. 121: 404-427.

Datyner N. B. and Gage P. W. (1980) Phasic secretion of acetylcholine at a mammalian neuromuscular junction. J. Physiol., Lond. 303: 299-314.

Fu W. M., Day S. Y. and Lin-Shiau S. Y. (1989) Studies on cadmium-induced myotonia in the mouse diaphragm. Nauyn-Schmiedeberg’s

Archs Pharmac. 340: 191-195.

Fu W. M. and Lin-Shiau Y. S. (1985) Mechanism of rhythmic contractions induced by uranyl ion in the ileal longitudinal muscle of guinea-pig. Eur. J. Pharmac. 113: 199-204. Lee C. Y., Chang S. L., Kau S. T. and Luh S. Y. (1972) Chromatographic separation of the venom of Bungarus multicinctus and characterization of its components. J. Chromat. 72: 71-81.

Lin R. H., Fu W. M. and Lin-Shiau S. Y. (1988) Presynaptic action of uranyl nitrate on the phrenic nerve-diaphragm preparation of the mouse. Neuropharmacology 27: 857-863.

Lin-Shiau S. Y. (1983) Selective antagonism by uranyl nitrate against Formosan krait venom in chicks-Part 1. J. Formosan Med. Assoc. 82: 64-3.

Lin-Shiau S. Y., Chen C. C. and Fu W. M. (1983) Selective antagonism by uranyl nitrate against Formosan krait venom in mice-Part 2. J. Formosan Med. Assoc. 82: 1099-l 103.

Lin-Shiau S. Y., Fu W. M. and Chen-Mao C. J. (1984) Selective antagonism by uranyl nitrate against Formosan krait venom-Part 3. Mechanism of action of uranyl nitrate. J. Formosan Med. Assoc. 83: 21-26.

Lin-Shiau S. Y., Day S. Y. and Fu W. M. (1991) Use of ion channel blockers in studying the regulation of skeletal muscle contractions. Nauyn-Schmiedeberg’s Archs Pharmac. 344: 691-697.

Lin-Shiau S. Y., Fu W. M. and Lee C. Y. (1979) Effects of uranyl ions on neuromuscular transmission of chick biventer cervicis muscle. Arch Znt. Pharmacodyn. Ther. 241: 332-343. Lin-Shiau S. Y. and Fu W. M. (1986) Antagonistic action of uranyl nitrate on presynaptic neurotoxins from snake venoms. Neuropharmacology 25: 95-105. McArdle J. J., Angaut-Detit D., Mallart A., Bournaud R., Faile L. and Brigant J. L. (1981) Advantages of the triangularis sterni muscle in the mouse for investigations of synaptic phenomena. J. Neurosci. Meth. 4: 109-116. Mallart A. (1985a) Electric current flow inside perineurial sheaths of mouse motor nerves. J. Physiol., Lond. 368: 565-575.

Acknowledgement-This work was supported by a grant (NSC 79-0412-B002-28) from the National Science Council of Republic of China. REFERENCES

Benoit P. R. and Mambrini J. (1970) Modification of transmitter release by ions which prolong the presynaptic action potential. J. Physiol., Lond. 210: 681695. Bulbring E. (1946) Observation of the isolated phrenic nerve diaphragm preparation of the rat. Br. J. Pharmac. 1: 3861.

Mallart A. (1985b) A calcium-activated potassium current in motor nerve terminals of the mouse. J. Physiol., Lond. 368: 577-591.

Penner R., Petersen M., Pierau F. K. and Dreyer F. (1986) Dendrotoxin: a selective blocker of a non-inactivating potassium current in guinea pig dorsal root ganglion neurons. PjZug. Arch. 407: 365-369.

Penner R. and Dreyer F. (1987) The actions of presynaptic snake toxins on membrane currents of mouse motor nerve terminals. J. Physiol., Lond. 386: 455-463. Petersen M., Penner R., Pierau Fr. K. and Dreyer F. (1986) fl-Bungarotoxin inhibits a non-inactivating potassium

A slow terminal potassium current induced by uranyl nitrate current in guinea pig dorsal root ganglion neurons. Neurosci. Lett. 68: 141-145. Pongs 0. (1992) Structural basis of voltage-gated K+-channel pharmacology. Trends Pharmac. Sci. 13: 359-365. Rowan E. G. and Harvey A. L. (1988) Potassium channel blocking actions of /3-Bungarotoxin and related toxins on mouse and frog motor nerve terminals. Br. J. Pharmac. 94: 839-847.

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Stansfeld C. E., Marsh S. J., Halliwell J. V. and Brown D. A. (1986) 4-Aminopyridine and dendrotoxin induce repetitive firing in rat viseral sensory neurones by blocking a slowly inactivating outward current. Neurosci. Lett. 64: 299-304.

Tabti N., Bourret C. and Mallart A. (1989) Three potassium currents in mouse motor nerve terminals. P$ug. Arch. 413: 395-400.