J. Physio. (1986), 372, pp. 303-313 303With 9 text-figuresPrinted in Great Britain

FACTORS INFLUENCING AN INCREASE IN SPONTANEOUSTRANSMITTER RELEASE BY HYPOXIA AT THE

MOUSE NEUROMUSCULAR JUNCTION

BY MASAKAZU NISHIMURAFrom the Department of Veterinary Pharmacology, College of Agriculture,

University of Osaka Prefecture, Sakai, Osaka 591, Japan

(Received 19 June 1985)

SUMMARY

1. To test a possibility that the functional buffering of intracellular Caa2+ plays aprimary role in the enhancement of spontaneous transmitter release during hypoxia,the frequency of miniature end-plate potentials (m.e.p.p.s) was examined underseveral conditions.

2. At 36 'C, hypoxia (bubbling with 95% N2 and 5% C02) increased the averagefrequency ofm.e.p.p.s from about 3 sol to 100 s-1 or more, in a standard Krebs-Ringersolution.

3. This effect declined with a decrease in the temperature and was much reducedat 24 0C.

4. Removal of external Ca2+ (addition of 2 mM-EGTA), increase of Mg2+ levels to5 mm, and treatment with 20 ,LM-ouabain, which gave a slight increase, did not reducethe rise in m.e.p.p. frequency during hypoxia.

5. Pre-incubation of the tissue in a solution containing 10 mM-KCl at 24-32 0C andits subsequent exposure to hypoxia caused a very marked increase in m.e.p.p.frequency, while incubation in 10 mM-KCl alone caused a small rise in the frequency.These data indicate that this combination potentiates the individual effects of eachtreatment.

6. These experiments suggest that the hypoxia-induced increase in spontaneoustransmitter release is primarily due to an increase in intracellular Ca2+ levels,probably because of inhibition of mechanisms which control buffering and extrusionof intracellular Ca2+. The release and influx mechanisms which elevate intraterminalCa2+ may also be involved passively in the effect of hypoxia.

INTRODUCTION

In the cat spinal cord, hypoxia transiently increases synaptic potential amplitude,this increase being followed by a block of synaptic transmission (Eccles, L0yning &Oshima, 1966). Hypoxia was found to stimulate spontaneous transmitter release atthe motor nerve terminal (Boyd & Martin, 1956; Liley, 1956; Hubbard & L0yning,1966). Since the effect of hypoxia was largely suppressed by a rise in external Mg2+concentration, it was partially explained by a reduction of active transport of Na+

and K+ and consequent depolarization of the nerve (Hubbard & L0yning, 1966).However, such a stimulatory effect of hypoxia was observed in Ca2+-free solution(Nishimura, Tsutsui, Yagasaki & Yanagiya, 1984); thus the dependence of the effectsupon extracellular Ca2+ remains to be elucidated.The frequency ofthe spontaneous release oftransmitter quanta is largely determined

by intracellular Ca2+ (Baker, 1972). A variety offactors are able to affect spontaneousrelease and many may act via a modification of intracellular Ca2+. These include:external Ca2+ and Mg2+ concentrations, depolarizing solution, temperature, andpharmacological inhibition of enzymes regulating ion balance across the cell mem-brane. Removal of Ca2+ is known to depress markedly the rate of spontaneoustransmitter release at the mammalian neuromuscular junction (Elmqvist & Feldman,1965). Divalent cations such as Mg2+, Mn2+, Co2+ and Pb2+ act as inhibitors bycompeting with Ca2+ for the binding site on the surface of the nerve terminal, therebydecreasing Ca2+ influx during depolarization of the membrane (Jenkinson, 1957).Raising external K+ can uniformly depolarize the membrane, thereby increasingspontaneous quantal release via a possible increase in Ca2+ influx by way ofvoltage-gated channels (Van der Kloot, 1978). Temperature is known to have amarked effect on spontaneous transmitter release at the mammalian neuromuscularjunction; a positive Q10 or negative Q10 can be obtained depending on the range ofits changes (Hubbard, Jones & Landau, 1971; Ward, Crowley & Johns, 1972).Ouabain, an inhibitor of the Na+-K+ exchange pump, slightly increases bothspontaneous and evoked release of transmitter from the motor nerve terminals (Birks& Cohen, 1968). Thus these factors may modify the effect of hypoxia on spontaneoustransmitter release at the neuromuscular junction through changing the intraterminalCa2+ level.The present experiments were undertaken to examine the effects of several factors

on changes in the frequency of m.e.p.p.s during hypoxia in order to determinewhether a buffering system for intracellular Ca2+ plays an important role in theincrease in spontaneous quantal release of transmitter under hypoxic conditions.

METHODS

Media and ti88ue preparation. All experiments were performed on the isolated left hemidiaphragmpreparation of male mice of ddy strain with a body weight of 25-28 g (6-10 weeks old). Thepreparation was pinned to a silicone resin lining the bottom of a plastic chamber of about 30 mlcapacity and was soaked in Krebs-Ringer (K-R) solution, which was constantly recirculated bymeans of an 'oxygen lift' system. The K-R solution had the following composition (mM): NaCl,136; KCl, 5; CaCl2, 2; MgCl2, 1; NaHCO3, 15; glucose, 11. Ca2+-deficient (Ca2+-free) solution wasprepared with or without adding 2 mM-EGTA (ethylenediaminetetraacetic acid). The solution wasbubbled with a mixture of95% 02 and 5% C02 and kept atpH 7-3 and 36 °C except where otherwisestated. For making a hypoxic condition, the K-R solution was aerated with a mixture of 95% N2and 5% C02 instead of the normal gas mixture. The temperature of the fluid in the bath wasmonitored by a thermistor (Shibaura Electric Co., Model MGA-II) and held constant by means ofan external water jacket and a thermoregulatory device (Taiyo, Thermominder Mini 80) duringeach experiment at temperatures of 24-36 'C. Changes in the external Ca2+ or Mg2+ concentrationwere made by changing the amount of CaCl2 or MgCl2 present in the reservoir of K-R solutionsupplying the organ bath. To depolarize the presynaptic endings, the preparation was equilibratedin a medium containing 10 mm-KCl (10 mm-K+). The preparation was equilibrated in the K-Rsolution for at least 30 min before exposure to hypoxic condition.

304 M. NISHIMURA

TRANSMITTER RELEASE BY HYPOXIA 305

Drugs used: EGTA (Wako Pure Chemicals), ouabain (Merck). All other chemicals were ofanalytical grade.

Electrophy8iology. Intracellular recordings were made with glass microcapillary electrodes filledwith 3 M-KCI of 6-10 MC resistance. The electrode was inserted into fibres near end-plate regions.The signals were led through a high-impedance unity-gain preamplifier (Nihon Kohden, MEZ-8201),displayed on an oscilloscope (Nihon Kohden, VC-10) and stored on magnetic tape (Nihon Kohden,RMG-5204).

Miniature end-plate potentials (m.e.p.p.s) of 0-2 mV or larger amplitude were counted by acomputer (Nihon Kohden, DAB-1100). M.e.p.p.s were recorded for successive 1 min periods afterexposure to a given solution; from this the mean m.e.p.p. frequency (s') was calculated. Only datafrom junctions whose m.e.p.p. frequency in control solution was relatively stable for some time,were included in the experimental results. Student's t tests were used for statistical analysis anda probability of less than 0 05 was deemed statistically significant.

RESULTS

Effects of hypoxia on m.e.p.p.sThe effects of hypoxia on spontaneous transmitter release, measured as m.e.p.p.

frequency, were examined in the mouse diaphragm preparations in vitro. Hypoxiccondition was made by gassing with a mixture of 95 % N2 and 5 % CO2. Duringhypoxic condition at 36 TC the frequency of m.e.p.p.s increased (Fig. 1). The increasewas sometimes gradual, but usually, as shown in Fig. 2, it occurred in transitory burstsof high frequency, sometimes more than 200 s51. During hypoxia the m.e.p.p.frequency rose from about 3 to 100 s-1 or more within 10-20 min at most junctions.The bursts of m.e.p.p.s did not occur simultaneously at all junctions. At some stageduring hypoxia, therefore, some junctions showed frequencies in the control range,while at others it was much higher. In some preparations an increase in the amplitudeof the m.e.p.p.s was observed together with the rise in frequency (Fig. 1 D).

Effect of Ca2+ removalThe increase in m.e.p.p. frequency by hypoxia was not noticeably diminished in

Ca2+-free solution containing 2 mM-EGTA (Fig. 3). At most junctions the effect wasobserved within 25 min. Thus, to a large extent the action ofhypoxia does not dependupon external Ca2+.

Effects of temperatureThe influence oflowering temperature on m.e.p.p. frequency in normal and hypoxic

conditions was studied. The mean frequency of m.e.p.p.s recorded in the mousediaphragm at 36 0C in standard solution was 2-91 + 015 s51 (n = 66). Both instandard solution and in Ca2+-free 2 mM-EGTA solution variations in temperaturecaused changes in the spontaneous discharge rate. For example, an increase oftemperature from 24 to 36 C raised the m.e.p.p. frequency from 0-69+ 0-06 s-(n = 41) to 2-91 + 015 s-1 (n =66) in standard solution, and from 0-35+0-02 s-1(n = 67) to 2-33 + 0-22 s-1 (n = 28) in Ca2+-free 2 mM-EGTA solution. The increasein frequency with temperature occurred similarly in both solutions (Fig. 4, bottom).The absolute frequencies at any given temperature were, however, appreciably lowerin the absence of Ca2+. The effect of temperature on m.e.p.p. frequency can bedescribed by a positive Q1o of, usually, about 3-8 at 24-28 0C, 3-7 at 28-32 TC and4-6 at 32-36 0C in the standard solution, and about 5-4 at 24-28 0C, 3-6 at 28-32 0C

M. NISHIMURA

A B

IAVuAuj

D 1C

_ _ - OWs- -

I I I I I

0 1 2 3 4

512

384

256

128

I I JOI I I I0 1 2 3 4 mV

Fig. 1. Effect of hypoxia on m.e.p.p.s at the mouse neuromuscular junction: A, control(normal Krebs-Ringer solution); B, 15 min after starting hypoxic condition (bubblingwith 95% N2 and 5% C02); C, amplitude histogram of control m.e.p.p.s recorded during1 min with 142 counts at a resting potential of -70 mV; D, amplitude histogram ofm.e.p.p.s from the same end-plate, 15 min afterexposure to hypoxia, showing a distributionof 5424 during 1 min at -67 mV resting potential.

and 5-3 at 32-36 0C in Ca2+-free 2 mM-EGTA solution. Activation energies were

calculated by means of the usual Arrhenius equation,

log k = Ea(1/T11/T2)k1 2-303 R

in which k1 and k2 are observed frequencies, T1 and T2 corresponding absolutetemperatures, and R the gas constant, 1-987 cal deg-' mol-h. The activation energieswere 21-6 and 27-8 kcal deg-' mol-1 in the presence and absence of external Ca2+,respectively.

In the presence of 10 mM-K+ (Fig. 4, top) an increase of the temperature from 24to 36 °C elevated the m.e.p.p. frequency from 10-6 + 0-70 s-1 (n = 40) to 28-8 + 1-93 s-(n = 40). The Q10 was about 3-7 at 24-28 °C, 4-1 at 28-32 °C and 2-8 at 32-36 'C.The effect of hypoxia in raising the frequency was also dependent on temperature,

regardless of the presence or absence of external Ca2+ (Fig. 5). The average frequencyof m.e.p.p.s was plotted, calculated from the maximum effect detected within the first

32 r

24 1-

16 F

-

c

um0

U?00.0.

8

0

306

TRANSMITTER RELEASE BY HYPOXIA

Normal soln., 36 'C200 r

150 1 a a .U a -

a

100 .

0

0

0

50 1 N2

*PE Ao

0 0

0 0

A% AU" AO o

0 5 10 15 20 25Time (min)

Fig. 2. Time course of effect of hypoxia on m.e.p.p. frequency (F, sol) at the mouseneuromuscular junction at 36 0C: three individual determinations are illustrated withdifferent symbols; ordinate, m.e.p.p. frequency; abscissa, time in minutes after startingexposure to hypoxia (N2 and arrow); frequency-elevating effect is transient and the peaktime varied from fibre to fibre.

Ca2l-free, 2 mM-EGTA, 36 0C

0

Y0

0

0 a

* I

a i ia

T m~~~idiAjiw

0

0 5 10 15 20 25Time (min)

Fig. 3. Effect of hypoxia on m.e.p.p. frequency at the mouse neuromuscular junction inCa2+-free, 2 mM-EGTA solution at 36 0C: three results are shown with different symbols;ordinate, m.e.p.p. frequency (F, sol); abscissa, time in minutes after starting hypoxia (N2and arrow).

307

F(s ')

* a .U

a -a a

A

A A

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150

F(s ')

100 . 0 UU

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a

a

50

0

a a

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A

308 M. NISHIMURA

30

20 10 mM-K+F

10*

Control

0 0 0 0

3l

Normal soan.2

F

Ca2+-free, 2 mM-EGTA

0

24 28 32 36

Temperature (0C)

Fig. 4. Effect of temperature on m.e.p.p. frequency (F, sol) at the mouse neuromuscularjunction in normal, Ca2+-free (2 mM-EGTA) and depolarizing solutions: Ca2+-free,2 mM-EGTA, Ca2+-free solution containing 2 mM-EGTA; 10 mm-K+, depolarizing solutionwhich was made by adding 10 mM-KCl without any substitution; ordinate, m.e.p.p.frequency; abscissa, temperature (CC) of incubation medium; vertical bar shows s.E. ofthe mean of 12-124 determinations.

180 HypoxiaCa2+-free, 2 mM-EGTA

120

F Normal soln.

60

0 Ad - Control

24 28 32 36

Temperature (0C)Fig. 5. Effect of temperature on m.e.p.p. frequency at the mouse neuromuscular junctionin normal (open symbols) and hypoxic (filled symbols) solutions: circles represent normalsolution; squares in Ca2+-free, 2 mM-EGTA solution; vertical bar shows s.E. of the meanof 9-124 determinations, where it is possible.

TRANSMITTER RELEASE BY HYPOXIA

801-

F

(S-i)

4010 mM-K++5 mM

U4 4

-Mg2+2

0

24 28 32 36

Hypoxia

Hypoxia+5 mM-Mg2"

24 28 32 36

Temperature (0C)

Fig. 6. Effect ofMgCl2 (5 mM) on m.e.p.p. frequency at the mouse neuromuscular junctionin the presence of 10 mM-KCl or under hypoxia: ordinate, m.e.p.p. frequency (F, s-1);abscissa, temperature (TC) of incubation medium; all the determinations were obtainedin normal solutions; vertical bar indicates S.E. of the mean of 33-124 observations.

Control

Hypoxia

Ouabain

Ouabain+ hypoxia

0 40 80 120F (s -1)

Fig. 7. Effect of20 /tM-ouabain on m.e.p.p. frequency at the mouse neuromuscular junctionunder normoxia and hypoxia: abscissa, m.e.p.p. frequency (F, s1); horizontal bar showsS.E. of the mean of thirty-nine to sixty-six determinations.

25 min of hypoxia at individual end-plates. At 36 'C, mean peak frequency ofm.e.p.p.s during hypoxia was 112-2 and 148-8 s-' in the presence and absence ofexternal Ca2+, respectively. Increasing the temperature from 24 to 36 TC raised thefrequency from 5-80+0-86 s-1 (n = 124) to 112-2+9-51 s-1 (n = 53) in the presence

of Ca2+ and from 5-77 +0-67 s-1 (n = 46) to 148-8 + 17-0 s-1 (n = 9) in the absence ofCa2+. Thus the effect of hypoxia was much reduced at 24 TC and greatly dependenton temperature, but barely on external CaS+.

Effects of Mg2+ and ouabainThe frequency of m.e.p.p.s during hypoxia was studied in solutions containing high

Mg2+ or ouabain. An elevation of external Mg2+ to 5 mm had little effect on m.e.p.p.s

309

30 r

20 1

F(s-')

10

0

a & I a

M. NISHIMURA

100.

F 50(s ')

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0 10 20 30 40 50 60 min0z/ N2 1 N.N2

Ca2l m - 4mM

.`N5 H0

Fig. 8. Effect of 10 mM-KCl on m.e.p.p. frequency at the mouse neuromuscular junctionexposed to hypoxia in solution containing 4 mM-CaCl2 at 24 'C. Ordinate, m.e.p.p.frequency (F, s1); abscissa, time in minutes after exposure to hypoxia; N5, KCl level of5 mm in normal Krebs-Ringer solution; H10, 10 mM-KCl added to the normal Krebs-Ringer solution; representative result out of three similar observations is shown.

in a standard solution in the range of the temperature tested: 0-67 + 0-07 s-' (n = 33)at 24 C, 0-88+012 s-1 (n = 25) at 28 C, 1-38+008 s-1 (n = 26) at 32 0C, and2-50 + 014 $-1 (n = 49) at 36 0C. The high Mg2+ concentration largely depressed thestimulatory effect of 10 mM-K+ on m.e.p.p. frequency (Fig. 6, left), indicative of anexternal Ca2+-dependent origin of the effect. However, the effect of hypoxia did notchange in the presence of a high concentration of Mg2+, which indicates independenceof depolarization of the nerve terminals.The effect of 20 /M-ouabain on m.e.p.p. frequency during normal or hypoxic

condition at 36 'C is shown in Fig. 7. Measurements were made within the first 25 minof hypoxia and within 10-20 min after addition of 20 /LM-ouabain. Hypoxia started30 min after ouabain application. In the standard solution, hypoxia elevated them.e.p.p. frequency about 40-fold. Ouabain alone (20 #sM) raised the frequency onlyslightly (5f26 + 0 55 s-1, n = 30). The effect of hypoxia was not affected by this agent.

Effect of high K+ concentrationAt 24 0C, the majority of junctions showed m.e.p.p. frequencies within the control

range during 25-30 min of hypoxia, in the presence or absence of external Ca2+. Thisraised the possibility that such nerve terminals may have ceased to respond to stimuliat the low temperature. To test this, the effect of 10 mM-K+ on m.e.p.p. frequencywas examined in the presence of Ca2+ at such a 'silent junction', at 24 0C underhypoxic condition. A typical experiment is illustrated in Fig. 8. After recording them.e.p.p. frequency in a Ca2+-free solution at 24 'C for the first 30 min of hypoxia,4 mM-Ca2+ was added, and 10 min later the K+ concentration was raised to 10 mM.The m.e.p.p. frequency remained low and constant during the first 30 min exposure

310

TRANSMITTER RELEASE BY HYPOXIA

180

Hypoxia+10 mM-K+

120

(s-1) Hypoxia60

0o 8 Normal+10 mM-K+0 0

24 28 32 36"CTemperature (0C)

Fig. 9. Potentiating action of 10 mM-KCl combined with hypoxia on m.e.p.p. frequencyat the mouse neuromuscular junction at several temperatures. All determinations weremade in normal solutions; El, 10 mM-KCI at normal oxygenation; 0, hypoxia;0, 10 mM-KCl combined with hypoxia; ordinate, m.e.p.p. frequency (F, s-1); abscissa,temperature ("C) of medium.

to hypoxia in the absence of Ca2+, and it was not affected by addition of 4 mM-Ca2+alone but was greatly increased by 10 mM-K+. Without 10 mm-K+, the frequency ofm.e.p.p.s at such a junction stayed within the control range.The effect of hypoxia on m.e.p.p.s in a standard solution containing 10 mM-K+ was

studied at different temperatures. Results are shown in Fig. 9 and compared withthe effect of hypoxia or 10 mM-K+ alone. At 24-32 TC the stimulatory effect ofhypoxia or 10 mM-K+ alone was much less than that at 36 'C. However, acombination of hypoxia with 10 mM-K+ caused a very marked increase in m.e.p.p.frequency. Thus this combination potentiates the effects of each individualtreatment.

DISCUSSION

Cytosolic Ca2+ activity appears to be the trigger of the transmitter releasemechanism (Katz & Miledi, 1967). M.e.p.p. frequency is also largely determined byintraterminal Ca2+ (Alnaes & Rahamimoff, 1975). Based on this aspect m.e.p.p.frequency rise by hypoxia may include an increase in intraterminal Ca2+ level. Theintracellular Ca2+ can be elevated by stimulating influx or by inhibiting sequestrationor extrusion, resulting always in increased quantal release (Van der Kloot, 1978).

Ca2+ can enter into the nerve terminal through voltage-gated channels, therebystimulating transmitter release. This pathway is sensitive to high Mg2+ concentrations(del Castillo & Engbaek, 1954). An increase in m.e.p.p. frequency by hypoxia at theratneuromuscular junction was reported to be much lesspronounced in Mg2+-paralysedpreparations than in unblocked preparations (Hubbard & L0yning, 1966). Thoughthe data were not quantitative, the effects were suggested to be due to depolarizationof the nerve terminals (Hubbard & L0yning, 1966), since the rate of quantal release

311

M. NISHIMURA

is increased by depolarization of the nerve terminals, an effect which is sensitive tohigh Mg2+ (del Castillo & Katz, 1954, Liley, 1956). However, the present experimentsindicated that the m.e.p.p. frequency rise during hypoxia was not significantlyreduced by removing external Ca2+ and was hardly sensitive to a high Mg2+concentration which greatly reduced the stimulatory effect of 10 mm-K+. Thus itseems most probable that the main effect of hypoxia in elevating m.e.p.p. frequencyis independent of extracellular Ca2+ in the standard solution.

It is well known that hypoxia reduces the active transport of Na+ and K+ in nerve

(Shanes & Berman, 1955; Ito & Oshima, 1964). This depression of active transportwas suggested to be the probable cause of the depolarization of the nerve and theresulting rise in the rate of quantal release because of the associated alteration ofintracellular ionic concentrations (Hubbard & L0yning, 1966). In the presentexperiments, the m.e.p.p. frequency rise during hypoxia was not significantly affectedby pre-incubation with ouabain which by itself raised the frequency much less thandid hypoxia. This shows that though an inhibition of Na+-K+-ATPase may be ableto enhance the quantal release of transmitter, the m.e.p.p. frequency rise due tohypoxia is, if anything, only very slightly dependent on its depressing effect on theactive transport of Na+ and K+ in the nerve terminals.Temperature is known to play an important role in quantal transmitter release

(Fatt & Katz, 1952; Takeuchi, 1958; Hubbard et al. 1971; Ward et al. 1972; Duncan& Statham, 1977). In the present experiments, m.e.p.p. frequency increased withtemperature. This occurred equally in Ca2+-free solution. The Ca2+ independence ofthis activation enables one to reject the possibility that the m.e.p.p. responses totemperature resulted from depolarization of the nerve terminals (del Castillo & Katz,1954; Hofman, Parsons & Feigen, 1966). Between 24 and 36°C, similar values forQ10 and the activation energies were obtained in the presence or absence of externalCa2 . It has been suggested that the main effect of temperature is to modifyintracellular Ca2+ (Duncan & Statham, 1977); it was concluded that the major effectsof temperature are (i) to raise intracellular Ca2+ by an action on intracellular Ca2+stores, promoting efflux from either the mitochondria, or from other sites, and (ii)to stimulate the Ca2+ pumps of the plasma membrane, a factor that would serve tooppose the effects of mobilization of intracellular Ca2+ and hence to lower m.e.p.p.

frequency. According to this hypothesis, the temperature dependence of the hypoxicactivation of quantal release would be due to variations in the rate of release of Ca2+stores.The second mechanism proposed by Duncan & Statham(1 977), namely temperature-

dependent stimulation of a Ca2+ pump could play an important role in the effectsof hypoxia, since inhibition of the mechanisms which lower intracellular Ca2+ wouldserve to increase quantal release of transmitter. In the low-temperature range testedhere, hypoxia and 10 mM-K+ together stimulated quantal release, but they were muchless potent when applied separately. If hypoxia reduces the capacity of the nerve

terminal to extrude and lower intracellular Ca2+, then intracellular accumulation ofCa2+ introduced by 10 mM-K+ would reach a much higher level, thereby markedlyincreasing the rate of quantal release. The Ca2+-inactivation mechanisms have beenconsidered to be composed of (1) a buffering system including high-affinity Ca2+binding sites and mitochondrial and non-mitochondrial sites and (2) an extrusionsystem through a Ca2+ pump in the presynaptic terminals (Blaustein, Ratzlaff &

312

TRANSMITTER RELEASE BY HYPOXIA

Schweitzer, 1980). Thus it is probable that hypoxia inhibits such buffering and/orextrusion mechanisms for intracellular Ca2+. The present observations support theidea that these mechanisms are important in terminating enhanced quantal release.

This work was supported by a grant-in-aid for Scientific Research from the Ministry ofEducation,Science and Culture, Japan (no. 60560333).

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