European Journal of Pharmacology, 54 (1979) 119--127 © Elsevier/North-Holland Biomedical Press
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T H E A C T I O N O F V E R A P A M I L ON T H E R A T E O F S P O N T A N E O U S R E L E A S E O F TRANSMITTER AT THE FROG NEUROMUSCULAR JUNCTION STEPHEN J. PUBLICOVER and CHRISTOPHER J. DUNCAN * Department of Zoology, University of Liverpool, P.O. Box 147, Liverpool L 69 3BX, U.K.
Received 20 June 1978, revised MS received 9 October 1978, accepted 14 November 1978
S.J. PUBLICOVER and C.J. DUNCAN, The action of verapamil on the rate of spontaneous release of transmitter at the frog neuromuscular junction, European J. Pharmacol. 54 (1979) 119--127. Verapamil is known to reduce Ca2÷ entry in a variety of cells. At 10 -s M it produces a small reduction in MEPP frequency at the frog neuromuscular junction, whereas the rate of spontaneous release rises following treatment at a concentration of 10 -4 M. This latter effect is augmented by raising/Ca2+/0 to 9 mM or, more especially, by raising the temperature from 17 to 23°C. It is argued that both these opposing effects are related to the action of verapamil in modifying/Ca 2+]i at the presynaptic terminals and it is suggested that the drug can affect both inward Ca 2+ flux (so reducing the steady-state position of [Ca2+]i) and also, at higher concentration, either inhibit the membrane Ca 2+ pump or cause the release of Ca2+ from intracellular Ca2÷ stores (so raising /Ca 2+]i). Verapamil
Neuromuscular junction
Calcium
1. I n t r o d u c t i o n It is clear t h a t t h e m a g n i t u d e o f t h e e v o k e d release at t h e frog n e u r o m u s c u l a r j u n c t i o n is primarily determined by the concentration of extracellular calcium (/Ca2÷/0) w h i c h determines t h e a m o u n t o f Ca 2÷ available f o r e n t r y at e x c i t a t i o n ( C r a w f o r d , 1 9 7 4 ) . On t h e o t h e r h a n d , evidence is n o w a c c u m u l a t i n g t h a t the f r e q u e n c y o f t h e s p o n t a n e o u s release o f q u a n t a o f t r a n s m i t t e r , r e c o r d e d as t h e m i n i a t u r e endplate p o t e n t i a l s (MEPPs), is largely g o v e r n e d b y [Ca2÷]i at t h e p r e s y n a p t i c terminals (A1naes a n d R a h a m i m o f f , 1 9 7 5 ; D u n c a n and Statham, 1977a). T h u s , MEPP f r e q u e n c y at t h e frog n e u r o m u s c u l a r j u n c t i o n can b e m o d i f i e d experim e n t a l l y b y (i) altering Ca 2÷ fluxes across t h e plasma m e m b r a n e b y changing /Ca2÷/0 ( D u n c a n and S t a t h a m , 1 9 7 7 a ) , b y t r e a t m e n t w i t h t h e divalent c a t i o n i o n o p h o r e A 2 3 1 8 7 ( S t a t h a m and D u n c a n , 1 9 7 5 ) o r b y c o m -
* To whom requests for reprints should be made.
Miniature endplate potentials
p e t i t i o n b e t w e e n extracellular Ca 2÷ and Na ÷ ( S t a t h a m and D u n c a n , 1 9 7 7 ; D u n c a n , 1 9 7 7 ) (ii) releasing Ca 2÷ f r o m t h e m i t o c h o n d r i a with u n c o u p l e r s (Alnaes and R a h a m i m o f f , 1 9 7 5 ; S t a t h a m , D u n c a n and Publicover, 1 9 7 8 ) o r b y raising e i t h e r [Na*]i or [Li*]i ( S t a t h a m and D u n c a n , 1 9 7 7 ; D u n c a n , 1 9 7 7 ) (iii) m o d i f i c a t i o n o f Ca 2÷ u p t a k e and release at intracellular Ca 2÷ stores o t h e r t h a n t h e m i t o c h o n d r i a b y such agents as t h e o p h y l l i n e or D a n t r o l e n e sodium, DaNa ( S t a t h a m and D u n c a n , 1 9 7 6 ) (iv) r e d u c i n g t h e activity o f t h e Ca 2÷ p u m p o f t h e plasma m e m b r a n e which is responsible for t h e active e f f l u x o f Ca 2÷ b y lowering t h e t e m p e r a t u r e ( S t a t h a m and D u n c a n , 1 9 7 5 ; D u n c a n , 1 9 7 6 ; D u n c a n and S t a t h a m , 1 9 7 7 b ) . One class o f drugs t h a t are k n o w n t o alter Ca 2÷ fluxes across t h e plasma m e m b r a n e are verapamil and D 6 0 0 ( m e t h o x y v e r a p a m i l ) which have b e e n described as " c a l c i u m antagonists". Verapamil was originally d e v e l o p e d as an a n t i a r r h y t h m i c drug and acts b y b l o c k i n g the u p t a k e o f Ca 2÷ b y t h e m y o cardial m e m b r a n e . Its effects are n o t sup-
120 pressed b y the prior administration of fi-blocking agents (Vohra, 1977). Similarly, either verapamil or D600 reduces Ca 2÷ uptake in fi-cells of the islets (Malaisse et al., 1977), smooth muscle cells (Mayer et al., 1972) and isolated fat cells (Martin et al., 1975); Ca 2÷ entry is also reduced during late outward current in snail neurones (Eckert and Lux, 1977) and in the "late Ca 2÷ channel" of squid axon (Baker et al., 1973). These drugs also depress the secretion of adrenal catecholamines induced b y acetylcholine (Pinto and TrifarS, 1976) and the release of oxytocin and vasopressin from the neurohypophysis (Dreifuss et al., 1973). The action of verapamil on the rate of spontaneous release of quanta of transmitter at the frog neuromuscular junction was therefore tested in the present experiments. One point that emerged from previous studies (Duncan and Statham, 1977a) was that treatments serving to m o d i f y [Ca2÷]i interacted with one another. The effect of temperature was of particular importance in this respect. For example, A23187 had no effect at 17°C b u t caused a marked increase in MEPP frequency at 25°C (Statham and Duncan, 1975); raising [Ca2+]0 had a much more pronounced effect on MEPP frequency at 23 than at 17°C (Duncan and Statham, 1977a). The results suggest that elevated temperature augments the effect of other treatments that increase Ca 2÷ influx at the presynaptic terminals. For this reason, the effect of verapamil was studied at temperatures above and below 20°C (17 and 23°C) and, again, elevated temperature had a marked effect on the action of this drug also.
2. Materials and methods Electrophysiological recordings of MEPP frequency were made from the isolated cutaneous pectoris nerve muscle preparation of the frog Rana pipiens. Frogs were maintained in the laboratory at 10°C.
s.J. PUBLICOVER, C.J. DUNCAN All salines in which the preparations were bathed contained (mM) NaC1 110, KC1 1.87, NaH2PO4 0.032, NaHCO3 4.76, glucose 2g/1 at pH 7.1. Calcium concentration was varied; normal saline contained 1.8 mM CaC12, whilst at 5 × 10 -7 M [Ca2÷]0 a Ca2÷-EGTA buffer was used. 0.5 mM EGTA was added, together with the appropriate volume of AnalaR standard volumetric solution of CaC12, and free Ca 2÷ concentrations were calculated from the method of Portzehl et al. (1964). Stock solutions containing verapamil (1 raM), theophylline (1 mM) and Dantrolene (10 pg ml -~) were made up in appropriate saline. Solutions of Dantrolene were vigorously stirred for 1 h and the final solution was then filtered (Ellis and Carpenter, 1972; Statham and Duncan, 1976). The muscle was excised and equilibrated in the appropriate control saline for 30 min at 10°C. It was then pinned out in the experimental bath, the microelectrode inserted in the endplate region and the temperature adjusted to the experimental value. Records of MEPPs began after a further 10 rain. Electrophysiological recordings were made by the use of conventional glass microelectrodes filled with 3 M KC1; the temperature of the bath was controlled (+0.5°C) by a Peltier device. Potentials were fed through a cathode follower to a Tektronix 502A oscilloscope. MEPPs were recorded on moving film and counted. In any one experiment, the MEPPs were monitored in a single fibre at the intervals shown in the figures and at least 100 MEPPs were measured, except at very low frequencies. At least two control readings were taken before application of drugs to give a mean control rate. Studies on evoked release were made on the sartorius nerve-muscle preparation equilibrated for 30 min in saline containing 0.66-1.1 pM d-tubocurarine. EPPs were recorded on a Tektronix 5103N storage oscilloscope. All inorganic salts were AnalaR grade. Theophylline, d-tubocurarine and EGTA were obtained from Sigma Chemical Co., St. Louis, U.S.A. Dantrolene sodium was obtained from
VERAPAMIL AND MEPP FREQUENCY
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Eaton Laboratories, London. Verapamil was a gift of A b b o t t Laboratories, Queenborough, Kent. "
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3. Results The control MEPP frequency varied between different preparations, although only small oscillations (less than 10% of the initial rate) were recorded in any one preparation. In most figures, therefore, comparisons of the effects of treatments are shown by expressing MEPP frequency as a ratio of the control frequency (expressed as F~/F0, where F0 is the control frequency and F1 the frequency after treatment). As will be shown, verapamil is able to have a dual effect on MEPP frequency, causing both a fall and a rise in the rate of spontaneous release. The relative magnitude of these two opposing actions depends on the experimental conditions and also differs in different preparations. For this reason the time course of the response is shown for individual experiments, since pooling the data tended to obscure these opposing effects of the drug. The verapamil preparation used was the standard provided by A b b o t t Laboratories, containing the mixture of both isomers (i.e. both D.365 and CP.16533-1). Statistical analysis was carried out by calculating a regression coefficient and then determining the variance using the difference between successive residuals of y, using the equation
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Fig. 1. Stimulatory effect of 10 -4 M verapamil at 17°C. [ C a 2 + ] 0 = 1.8 raM, except in the experiment shown with a dashed line where [Ca2+]o=5× 10 -7 M. Mean F0 before the application of verapamil in experiments where [Ca2+]0 = 1.8 mM was 30.0 MEPPs rain -1. In all figures ordinate: MEPP frequency as a ratio of the control frequency (F]/ F0); abscissa: time (rain) after application of verapamil. Time course of individual experiments shown. MEPP frequency (P < 0.001) in normal saline ([Ca2+]0 = 1.8 × 10 -3 M) although this effect was clearly different (P < 0.001) at 17°C (fig. 1) and 23°C (fig. 2). F~/F0 rarely rose above 1.75 at 17°C whereas, at 23°C, although the
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(Bliss, 1967). 3.1. Effect of verapamil at a concentration o f 1 0 -4 M At a concentration o f 1 0 -4 M, verapamil caused a significant and consistent rise in
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Fig. 2. Effect o f 10 -4 M verapamil at 23°C. Note much greater stimulatory effect at the higher temperature. [Ca 2+]o "= 1.8 raM. Mean control F 0= 103.5 MEPPs rain -1.
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S.J. PUBLICOVER, C.J. DUNCAN
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Fig. 3. Effect of 10 -4 M verapamil at 23°C when [Ca2+]0 = 5 x 10 -7 M. Mean control F0 = 14.3 MEPPs min-1. rate o f rise was variable, m u c h higher MEPP frequencies were recorded, with F1/Fo r e a c h i n g 7 (fig. 2). At b o t h 17 and 23°C, verapamil caused an initial small fall in MEPP f r e q u e n c y in s o m e p r e p a r a t i o n s . H o w e v e r , this overall rise in t h e rate was c o n t r a r y t o e x p e c t a t i o n since, if verapamil r e d u c e d passive Ca :+ e n t r y , a fall in t h e s t e a d y - s t a t e p o s i t i o n o f [Ca2+]i, and h e n c e in MEPP rate, might have b e e n e x p e c t e d . T h e e x p e r i m e n t s were t h e r e f o r e r e p e a t e d w h e n Ca :+ in t h e b a t h i n g saline was r e d u c e d t o 5 X 10 -7 M ( w h e n [Ca2+]0 ~- [Ca2+]i), so largely eliminating t h e e f f e c t o f Ca :÷ fluxes across t h e plasma membrane. R e d u c i n g [Ca2÷]0 t o 5 X 10 -7 M causes a significant fall in MEPP f r e q u e n c y (at b o t h 17 a n d 23°C m e a n F0 fell b y a p p r o x i m a t e l y 50%), as p r e v i o u s l y r e p o r t e d ( D u n c a n and S t a t h a m , 1 9 7 7 a ) . A f t e r equilibration, however, t h e p a t t e r n o f r e s p o n s e t o 1 0 - 4 M verapamil was similar t o t h a t f o u n d at 1.8 X 1 0 - 3 M [Ca:÷]0, w i t h a significant rise in MEPP f r e q u e n c y ( P < 0.01). A t 17°C t h e slow rise in MEPP f r e q u e n c y was indistinguishable f r o m t h a t f o u n d at t h e higher Ca 2÷ conc e n t r a t i o n (fig. 1); at 2 3 ° C t h e increase in
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Fig. 4. Effect of 10 -4 M verapamil at 23°C when [Ca2+]0 = 9 mM. Mean control F 0 -- 56.7 MEPPs rain-1.
rate was again m u c h greater and m o r e variable (fig. 3); the results s h o w n in figs. 2 and 3 are n o t significantly d i f f e r e n t at 0.05. However, n o small initial fall in MEPP f r e q u e n c y was seen at e i t h e r t e m p e r a t u r e at this low value o f [Ca2+]o. Raising [Ca2+]0 t o 9 m M (5 times t h e conc e n t r a t i o n in n o r m a l saline) did n o t m a r k e d l y m o d i f y t h e a c t i o n o f verapamil w h i c h again caused a significant rise in MEPP f r e q u e n c y (P < 0.001). A t 23°C (fig. 4) t h e rate o f rise in MEPP f r e q u e n c y was variable and in some cases rapid; in o n e p r e p a r a t i o n F1/F0 r e a c h e d 5.8 a f t e r o n l y 12 min. At 17°C {fig. 5), how-
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Fig. 5. Effect of 10 -4 M verapamil at 17°C when [Ca2+]0 = 9 raM. Mean control Fo = 56.4 MEPPs rain-1.
VERAPAMIL AND MEPP FREQUENCY
ever, t h e overall rise in MEPP f r e q u e n c y was slightly greater (P < 0 . 0 0 1 ) t h a n w h e n [Ca2÷]0 = either 1.8 × 10 -3 M or 5 × 10 -7 M. Figs. 4 and 5 s h o w t h a t , as at 1 . 8 × 1 0 - 3 M , an initial fall in MEPP f r e q u e n c y was r e c o r d e d in several p r e p a r a t i o n s w h e n [Ca2÷]0 = 9 mM. T h e s e results at d i f f e r e n t t e m p e r a t u r e s and values o f [ Ca 2÷ ] 0 s h o w t h a t verapamil (10 -4 M) clearly p r o m o t e s a rise in the rate o f spont a n e o u s release o f t r a n s m i t t e r , particularly at higher t e m p e r a t u r e s (figs. 2--4), and s o m e p r e p a r a t i o n s indicate t h a t it m a y have a dual action, causing a small initial fall in F]/F0. This suggestion is s u p p o r t e d b y i n s p e c t i o n o f t h e c a l c u l a t e d regression lines; in all e x p e r i m e n t s at 1.8 mM and 9.0 mM [Ca2÷]0 t h e calculated i n t e r c e p t o n t h e o r d i n a t e lay b e l o w t h e origin (F1/Fo < 1.0) whereas, with 5 × 10 -7 M [Ca2÷]0, the i n t e r c e p t was above, suggesting t h a t t h e r e was no initial fall w h e n [Ca2÷]0 was m a i n t a i n e d very low. This initial fall in MEPP f r e q u e n c y in t h e presence o f extracellular Ca 2÷ w o u l d be m o r e in a c c o r d with t h e generally a c c e p t e d role o f verapamil, and the validity o f this initial t r a n s i e n t fall was t e s t e d b y a p p l y i n g t h e drug at a concent r a t i o n o f 10 -s M.
3.2. Effect of verapamil at a concentration o f lO-S M
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w i t h i n 5 min b u t , s u b s e q u e n t l y , a gradual, small rise in MEPP f r e q u e n c y o c c u r r e d . A t 23°C all p r e p a r a t i o n s also s h o w e d an initial fall in F]/F0 to a r o u n d 0.75, and t h e n a subs e q u e n t , b e l a t e d rise. T h e d e l a y b e f o r e this rise o c c u r r e d varied greatly, o n e p r e p a r a t i o n reaching 1.5 a f t e r o n l y 15 min whilst in o t h e r s MEPP f r e q u e n c y was depressed f o r 40 min. In no p r e p a r a t i o n s with verapamil at 10 -s M was a m a r k e d increase in MEPP f r e q u e n c y r e c o r d e d ( c o m p a r e fig. 6 with figs. 2 - 4 ) . T h e d e p e n d e n c e o f t h e rise in F~/F0 o n verapamil c o n c e n t r a t i o n was shown in an e x p e r i m e n t at 23°C. T h e p r e p a r a t i o n was e x p o s e d t o 10 -s M verapamil for 40 min and, a f t e r an initial fall, F1/F0 had risen to o n l y 1.5; a d d i t i o n o f 1 0 - 4 M verapamil n o w caused F]/F0 to rise rapidly t o 3.5.
3.3. Effect of Dantrolene Since verapamil is believed t o r e d u c e passive Ca 2÷ influx, and y e t t h e drug at a c o n c e n t r a t i o n o f 1 0 - 4 M is able t o p r o m o t e a m a r k e d rise in MEPP f r e q u e n c y , it s e e m e d likely t h a t its a c t i o n at higher c o n c e n t r a t i o n s m i g h t be t o cause a release o f Ca 2÷ f r o m intracellular Ca 2÷ storage sites in this p r e p a r a t i o n . Such a c o n c l u s i o n is s u p p o r t e d b y t h e rise in F1/F0 even w h e n Ca 2÷ fluxes across t h e plasma m e m b r a n e are m a r k e d l y r e d u c e d b y main-
[Ca2÷]0 was raised t o 9 mM, so as t o prom o t e Ca 2÷ i n f l u x at t h e p r e s y n a p t i c terminals and, a f t e r equilibration, verapamil at 10 -s M was a d d e d t o t h e saline. At 17°C (fig. 6) F~/F0 fell in all p r e p a r a t i o n s to a r o u n d 0.7
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Fig. 6. Effect of 10 -s M verapami] at 17°C when [Ca2+]o = 9 mM. Note inhibitory action of the drug at this lower concentration. Mean control F 0 = 88.4 MEPPs min -1 .
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Fig. 7. Effect of pretreatment with Dantrolene on the action of 10 -~ M verapamil. [Ca2+]o = 1.8 mM; 23°C. Pretreatment with Dantrolene (10pg ml-1) for 45 rain. Mean F 0 after Dantrolene treatment = 49.3 MEPPs rain -1.
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taining [Ca2+]0 at 5 × 10 -7 M (fig. 3). DaNa is a skeletal muscle relaxant that acts b y reducing the release of Ca 2÷ from the sarcoplasmic reticulum at excitation (Ellis and Carpenter, 1972). It also reduces the MEPP frequency at the frog neuromuscular junction by 50--80% after 4 5 m i n treatment and it has been suggested that it acts by suppressing the release of Ca 2÷ from intracellular storage sites at the presynaptic terminals (Statham and Duncan, 1976). The experiments were carried o u t at 23°C (so causing a rise in MEPP frequency) and the preparation was pretreated with DaNa (10 pg m1-1) for 45 min which then reduced MEPP frequency. Addition of 1 0 - 4 M verapamil again caused a significant rise (P < 0.001) in the rate of spontaneous release of transmitter (fig. 7), the effect on the ratio F1/F0 being approximately intermediate between that observed at 17°C (fig. 1) and 23°C (fig. 2) in the absence of DaNa. The calculated regression lines for figs. 2 and 7 were significantly different (P < 0.001).
3.4. Effect of theophylline Theophylline has been shown to cause a rise in MEPP frequency at the frog neuro-
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muscular junction (Onodera, 1973), an approximate doubling at 25°C (Statham and Duncan, 1976). This action is independent of external Ca 2÷ and it is suggested that theophylline acts b y releasing Ca :÷ from intracellular storage sites other th~'n the mitochondria (Statham and Duncan, 1976). In the present experiments, the terminals were pretreated with theophylline (1 mM; 17°C; [Ca2+]0 = 1.8 mM) for 45 min, causing the typical rise in MEPP frequency (F0 = 67.5 MEPPs min-1). Subsequent treatment with 1 0 - 4 M verapamil (fig. 8) again produced a small, significant rise in MEPP frequency (P < 0.02) comparable with that found under these conditions, b u t in the absence of theophylline (compare figs. 1 and 8).
3.5. Evoked release Following presynaptic stimulation, muscle action potentials were recorded in the muscle after treatment with 1 0 - 4 M verapamil and this concentration of the drug did not markedly affect the magnitude of the EPP in preparations partially blocked with d-tubocurarine. Verapamil had no significant effect on muscle resting potential. However, when the action of 1 0 - 4 M verapamil on muscle twitch was studied in an indirectly stimulated sartorius nerve muscle preparation, twitch amplitude began to decline after 10 min exposure and was absent after some 30 min. Subsequent washing resulted in the reappearance of the twitch, b u t the action of verapamil was n o t completely reversible even after 40 min washing.
4. Discussion
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The marked rise in MEPP frequency is not seen when verapamil is applied at the lower concentration of 10 -s M (fig. 6) at both 17 and 23 ° C. These trials were carried o u t with an extracellular concentration of Ca 2÷ of 9 mM, so that this elevated level of [Ca2+]0 ensured that Ca 2+ influx was an important
VERAPAMIL AND MEPP FREQUENCY factor in determining the steady-state position of [Ca2+]i . Under these conditions a small initial fall in MEPP frequency was seen in most preparations (F1/F0 ~ 0.7), especially during the first 15 min of treatment. There are also suggestions of an initial depression in the records obtained at 10 -4 M verapamil, particularly under conditions where Ca 2÷ influx are important ([Ca2÷]0 = 9 mM; fig 5) or where Ca 2÷ has not been mobilized from intracellular stores (17°C; fig. 1). Such an inhibitory effect would be consistent with the established role of verapamil in reducing Ca :+ entry and hence in lowering the steadystate position of [Ca2+]i . However, it is clear that such an action is not very marked at the presynaptic terminals of the frog neuromuscular junction. Such an observation agrees with previous findings; MEPP frequency is more sensitive to alterations in [Ca2÷]i produced by a modification of the intracellular Ca 2÷ storage sites than to the changes in Ca 2÷ influx across the plasma membrane. Thus, changes in [Ca2÷]0 have only modest effects (Duncan and Statham, 1977a); the divalent cation ionophore A 2 3 1 8 7 is ineffective at 17°C and it is necessary first to raise the temperature so as to demonstrate that the ionophore is indeed promoting Ca :÷ entry (Statham and Duncan, 1975). At the higher concentration of 10 -4 M, any inhibitory effect that verapamil may have on inward flux of Ca :÷ is largely masked by the other more pronounced action of the drug, namely promoting a rise in MEPP frequency. Under normal conditions (17 ° C; [Ca 2÷] 0 = 1.8 mM), this stimulation is not impressive and usually develops slowly since MEPP frequency is less than doubled after 40 min (fig. 1). Equilibrating the terminals at a raised [Ca2+]0 of 9 m M (and hence promoting inward Ca 2÷ flux) reveals this stimulatory action at 17°C at higher verapamil concentrations more clearly; F1/F0 is greater than 2.5 after 50 min exposure (fig. 5). However, the excitatory action of 1 0 - 4 M verapamil is shown much more dramatically if the preparation is equilibrated at 23°C,
125 when the spontaneous rate of release rises progressively with time after application of the drug, and F , / F 0 may exceed 7.0 (figs. 2 - 4 ) . The effect is b u t little affected by changes in [Ca2+]0 and the same pattern of response is seen when the Ca 2+ concentration of the saline is normal (fig. 2), reduced to very low levels (5 × 10 -7 M, fig. 3) or raised to 9 mM (fig. 4). We have shown previously (Duncan and Statham, 1977a) that the effect on MEPP frequency of treatments that are believed to m o d i f y [Ca2+]i is particularly sensitive to the existing steady-state level of [Ca2+]i in the presynaptic terminals. The terminals are better able to resist experimental perturbations if [Ca2+]i is maintained low. Elevated temperature (e.g. 23--25°C) might p r o m o t e the release of Ca 2÷ from intracellular storage sites and, under these conditions, the effects of raising [Ca2÷]0 (Duncan and Statham, 1977a), changing [Na÷]0 (Statham and Duncan, 1977) or treatment with A23187 (Statham and Duncan, 1975) are all greatly augmented. This same dependency on temperature, and hence probably on the preexisting level of [Ca2÷]i, is therefore also shown in the presence of 1 0 - 4 M verapamil. This effect of temperatures above 20°C has been discussed in detail elsewhere (Duncan and Statham, 1977a); its mechanism of action is u n k n o w n but the experiments cited above are consistent with the hypothesis that it serves to raise [Ca2÷]i or that it facilitates the release mechanism, perhaps via a change in membrane fluidity. We therefore conclude that both effects of verapamil on MEPP frequency are probably explicable in terms of alterations of [Ca2+]i at the presynaptic terminals. The question remains as to how verapamil, a known Ca 2÷ antagonist, could act to raise MEPP frequency via an alteration in [ C a 2 + ] i . The steady-state position of [Ca:÷]i at the presynaptic terminals is the resultant of the following: (i) Ca 2÷ influx at the plasma membrane. (ii) Active Ca 2÷ efflux across the plasma membrane. (iii) Ca 2÷ uptake and release b y
126
the mitochondria. (iv) Ca 2÷ exchange at other intracellular stores. Since the major stimulatory effect of verapamil is still seen when [Ca2÷]0 is reduced to 5 X 10 -7 M (and [Ca2+]0 ~ [Ca2+]i) it seems unlikely that this drug at 10 -4 M is acting to promote Ca :+ influx as in (i) above (note the similarity of figs. 2 and 3 which are not significantly different at 0.05). Such considerations also show that it is unlikely that verapamil has a depolarizing effect which, in turn, promotes Ca 2~ influx. Verapamil has been shown to have an action on isolated mitochondria, inhibiting net Ca 2÷ uptake (Frey and Janke, 1975) and oxidative phosphorylation (Silveira and Campello, 1975). However, concentrations of verapamil of at least 10-3M are necessary to demonstrate this effect and we believe that it is unlikely that the mitochondria (paragraph (iii) above) are the site of action of externally applied 10-4 M verapamil. Verapamil at 10 -4 M still exhibits a stimulatory effect after pretreatment of the terminals with either theophylline or DaNa. These agents are believed to act at the intracellular storage sites, DaNa serving to suppress Ca 2+ leakage (and so reduce MEPP frequency) and theophyUine promoting Ca 2÷ release (and so raising MEPP frequency) (Statham and Duncan, 1976). Pretreatment with either of these agents therefore alters [Ca2+]i, and hence F0, so that direct comparison with untreated terminals of the subsequent effect of verapamil is not possible. However, it is clear that, at 10 -4 M, the drug is still effective in raising MEPP frequency, even when the Ca 2+ stores have been partially emptied with theophylline (compare figs. 8 and 1) or when leakage has been reduced with DaNa (compare figs. 7 and
2). We therefore tentatively conclude that verapamil probably does n o t act at intracellular Ca 2÷ storage sites and it may prove ultimately t h a t its target is the Ca 2+ pump at the plasma membrane (paragraph (ii) above). It is known to inhibit active Ca 2÷ transport by everted rat d u o d e n u m sacs (Wrdbel and Michalska, 1977). Thus, verapamil reduces Ca 2÷ influx in m a n y
S.J. P U B L I C O V E R , C.J. D U N C A N
different cells at low concentration; at higher concentrations it may inhibit active Ca 2÷ efflux. Such speculation raises the possibility of a similarity between the passive Ca 2+ channel and the membrane Ca 2÷ pump, since both would be binding verapamil, a suggestion that is in accord with the hypothesis developed in detail by Shamoo and Goldstein (1977). 10 -4 M verapamil had no marked effect on the magnitude of the EPP when the neuromuscular junction was partially blocked with d-tubocurarine. Muscle resting potential was not significantly affected. Action potentials were also recorded post-synaptically in the absence of d-tubocurarine but in the presence of verapamil, and we conclude that the drug has little effect on the excited Ca z÷ channels of muscle during evoked release. However, Bondi (1978) has also found that verapamil, at concentrations of 10 -4 M and above, causes a reduction and, eventually, a total inhibition of twitch in a directly stimulated frog muscle. The time course of the response was similar to that in our experiments and reversal of the effect was again difficult to achieve. The findi~gs suggest that verapamil may have more than one site of action in muscle also and that it may, at high concentration, serve to block the Ca 2+ release mechanism of the S.R. MEPP frequency is of little importance in vivo, but if verapamil is able to have similar subliminal actions at other synapses (e.g. in the CNS where it could modify excitability) and other secretory systems, these findings might be of clinical significance. Acknowledgements We are grateful t o Miss S. S c o t t for assistance in t h e p r e p a r a t i o n o f t h e m a n u s c r i p t a n d t o Dr. M.E. B e g o n for advice w i t h statistics. This w o r k was s u p p o r t e d , in p a r t , b y t h e M u s c u l a r D y s t r o p h y G r o u p of G r e a t Britain.
References Alnaes, E. a n d R. R a h a m i m o f f , 1 9 7 5 , On t h e role o f m i t o c h o n d r i a in t r a n s m i t t e r release f r o m m o t o r
VERAPAMIL AND MEPP FREQUENCY nerve terminals, J. Physiol. 248, 285. Baker, P.F., H. Meves and E.B. Ridgway, 1973, Calcium entry in response to maintained depolarization of squid axons, J. Physiol. 231, 527. Bliss, C.I., 1967, Statistics in Biology, Vol. 1 (McGraw-Hill, New York). Bondi, A.Y., 1978, Effects of verapamil on excitation-contraction coupling in frog sartorius muscle, J. Pharmacol. Exptl. Therap. 205, 49. Crawford, A.C., 1974, The dependence of evoked transmitter release on external calcium ions at very low mean quantal contents, J. Physiol. 240, 255. Dreifuss, J.J., J.D. Grau and J.D. Nordmann, 1973, Effects on the isolated neurohypophysis of agents which affect the membrane permeability to calcium, J. Physiol. 231, 96P. Duncan, C.J., 1976, Properties of the Ca2+-ATPase activity of mammalian synaptic membrane preparations, J. Neurochem. 27, 1277. Duncan, C.J., 1977, The action of ouabain in promoting the release of catecholamines, Experientia 33, 923. Duncan, C.J. and H.E. Statham, 1977a, Interacting effects of temperature and extracellular calcium on the spontaneous release of transmitter at the frog neuromuscular junction, J. Physiol. 268, 319. Duncan, C.J. and H.E. Statham, 1977b, The effect of temperature on spontaneous release of transmitter at the mammalian neuromuscular junction, J. Therm. Biol. 2, 23. Eckert, R. and H.D. Lux, 1977, Calcium-dependent depression of a late outward current in snail neurons, Science 197,472. Ellis, K.O. and J.F. Carpenter, 1972, Studies on the mechanism of action of dantrolene sodium, Naunyn-Schmiedeb. Arch. Pharmacol. 275, 83. Frey, M. and J. Janke, 1975, The effect of organic Ca-antagonists (verapamil, prenylamine) on the calcium transport system in isolated mitochondria of rat cardiac muscle, Pflugers Arch. 359, R26. Malaisse, W.J., A. Herchuelz, J. Levy and A. Sener, 1977, Calcium antagonists and islet function-III The possible site of action of verapamil, Biochem. Pharmacol. 26, 735. Martin, B.R., T. Clausen and J. Gliemann, 1975, Relationships between the exchange of calcium and
127 phosphate in isolated fat-cells, Biochem. J. 152, 121. Mayer, C.J., C. Van Breemen and R. Casteels, 1972, The action of lanthanum and D-600 on the calcium exchange in the smooth muscle cells of the guinea-pig taenia coli, Pflfgers Arch. ges. Physiol. 337,333. Onodera, K., 1973, Effect of caffeine on the neuromuscular junction of the frog and its relation to external calcium concentration, Jap. J. Physiol. 23,587. Pinto, J.E.B. and J.M. Trifar6, 1976, The different effects of D-600 (methoxyverapamil) on the release of adrenal catecholamines induced by acetylcholine, high potassium or sodium deprivation, Brit. J. Pharmacol. 57,127. Portzehl, H., P.C. Caldwell and J.C. Riiegg, 1964, The dependence of contraction and relaxation of muscle fibres from the crab Maia squinado on the internal concentration of free calcium ions, Biochim. Biophys. Acta 79, 581. Shamoo, A.E. and D.A. Goldstein, 1977, Isolation of ionophores from ion transport systems and their role in energy transduction, Biochim. Biophys. Acta 472, 13. Silveira, O. and A.P. Campello, 1975, Effects of iproveratril on isolated heart mitochondria, Res. Commun. Chem. Pathol. Pharmacol. 10, 149. Statham, H.E. and C.J. Duncan, 1975, The action of ionophores at the frog neuromuscular junction, Life Sci. 17, 1401. Statham, H.E. and C.J. Duncan, 1976, Dantrolene and the neuromuscular junction: evidence for intracellular calcium stores, European J. Pharmacol. 39, 143. Statham, H.E. and C.J. Duncan, 1977, The effect of sodium ions on MEPP frequency at the frog neuromuscular junction, Life Sci. 20, 1839. Statham, H.E., C.J. Duncan and S.J. Publicover, 1978, Dual effect of 2,4-Dinitrophenol on the spontaneous release of transmitter at the frog neuromuscular junction, Biochem. Pharmacol. 27, 2199. Vohra, J.K., 1977, Clinical use of verapamil, Drugs 13,219. Wr6bel, J. and L. Michalska, 1977, The effect of verapamil on intestinal calcium transport, European J. Pharmacol. 4 5 , 3 8 5 .
119
T H E A C T I O N O F V E R A P A M I L ON T H E R A T E O F S P O N T A N E O U S R E L E A S E O F TRANSMITTER AT THE FROG NEUROMUSCULAR JUNCTION STEPHEN J. PUBLICOVER and CHRISTOPHER J. DUNCAN * Department of Zoology, University of Liverpool, P.O. Box 147, Liverpool L 69 3BX, U.K.
Received 20 June 1978, revised MS received 9 October 1978, accepted 14 November 1978
S.J. PUBLICOVER and C.J. DUNCAN, The action of verapamil on the rate of spontaneous release of transmitter at the frog neuromuscular junction, European J. Pharmacol. 54 (1979) 119--127. Verapamil is known to reduce Ca2÷ entry in a variety of cells. At 10 -s M it produces a small reduction in MEPP frequency at the frog neuromuscular junction, whereas the rate of spontaneous release rises following treatment at a concentration of 10 -4 M. This latter effect is augmented by raising/Ca2+/0 to 9 mM or, more especially, by raising the temperature from 17 to 23°C. It is argued that both these opposing effects are related to the action of verapamil in modifying/Ca 2+]i at the presynaptic terminals and it is suggested that the drug can affect both inward Ca 2+ flux (so reducing the steady-state position of [Ca2+]i) and also, at higher concentration, either inhibit the membrane Ca 2+ pump or cause the release of Ca2+ from intracellular Ca2÷ stores (so raising /Ca 2+]i). Verapamil
Neuromuscular junction
Calcium
1. I n t r o d u c t i o n It is clear t h a t t h e m a g n i t u d e o f t h e e v o k e d release at t h e frog n e u r o m u s c u l a r j u n c t i o n is primarily determined by the concentration of extracellular calcium (/Ca2÷/0) w h i c h determines t h e a m o u n t o f Ca 2÷ available f o r e n t r y at e x c i t a t i o n ( C r a w f o r d , 1 9 7 4 ) . On t h e o t h e r h a n d , evidence is n o w a c c u m u l a t i n g t h a t the f r e q u e n c y o f t h e s p o n t a n e o u s release o f q u a n t a o f t r a n s m i t t e r , r e c o r d e d as t h e m i n i a t u r e endplate p o t e n t i a l s (MEPPs), is largely g o v e r n e d b y [Ca2÷]i at t h e p r e s y n a p t i c terminals (A1naes a n d R a h a m i m o f f , 1 9 7 5 ; D u n c a n and Statham, 1977a). T h u s , MEPP f r e q u e n c y at t h e frog n e u r o m u s c u l a r j u n c t i o n can b e m o d i f i e d experim e n t a l l y b y (i) altering Ca 2÷ fluxes across t h e plasma m e m b r a n e b y changing /Ca2÷/0 ( D u n c a n and S t a t h a m , 1 9 7 7 a ) , b y t r e a t m e n t w i t h t h e divalent c a t i o n i o n o p h o r e A 2 3 1 8 7 ( S t a t h a m and D u n c a n , 1 9 7 5 ) o r b y c o m -
* To whom requests for reprints should be made.
Miniature endplate potentials
p e t i t i o n b e t w e e n extracellular Ca 2÷ and Na ÷ ( S t a t h a m and D u n c a n , 1 9 7 7 ; D u n c a n , 1 9 7 7 ) (ii) releasing Ca 2÷ f r o m t h e m i t o c h o n d r i a with u n c o u p l e r s (Alnaes and R a h a m i m o f f , 1 9 7 5 ; S t a t h a m , D u n c a n and Publicover, 1 9 7 8 ) o r b y raising e i t h e r [Na*]i or [Li*]i ( S t a t h a m and D u n c a n , 1 9 7 7 ; D u n c a n , 1 9 7 7 ) (iii) m o d i f i c a t i o n o f Ca 2÷ u p t a k e and release at intracellular Ca 2÷ stores o t h e r t h a n t h e m i t o c h o n d r i a b y such agents as t h e o p h y l l i n e or D a n t r o l e n e sodium, DaNa ( S t a t h a m and D u n c a n , 1 9 7 6 ) (iv) r e d u c i n g t h e activity o f t h e Ca 2÷ p u m p o f t h e plasma m e m b r a n e which is responsible for t h e active e f f l u x o f Ca 2÷ b y lowering t h e t e m p e r a t u r e ( S t a t h a m and D u n c a n , 1 9 7 5 ; D u n c a n , 1 9 7 6 ; D u n c a n and S t a t h a m , 1 9 7 7 b ) . One class o f drugs t h a t are k n o w n t o alter Ca 2÷ fluxes across t h e plasma m e m b r a n e are verapamil and D 6 0 0 ( m e t h o x y v e r a p a m i l ) which have b e e n described as " c a l c i u m antagonists". Verapamil was originally d e v e l o p e d as an a n t i a r r h y t h m i c drug and acts b y b l o c k i n g the u p t a k e o f Ca 2÷ b y t h e m y o cardial m e m b r a n e . Its effects are n o t sup-
120 pressed b y the prior administration of fi-blocking agents (Vohra, 1977). Similarly, either verapamil or D600 reduces Ca 2÷ uptake in fi-cells of the islets (Malaisse et al., 1977), smooth muscle cells (Mayer et al., 1972) and isolated fat cells (Martin et al., 1975); Ca 2÷ entry is also reduced during late outward current in snail neurones (Eckert and Lux, 1977) and in the "late Ca 2÷ channel" of squid axon (Baker et al., 1973). These drugs also depress the secretion of adrenal catecholamines induced b y acetylcholine (Pinto and TrifarS, 1976) and the release of oxytocin and vasopressin from the neurohypophysis (Dreifuss et al., 1973). The action of verapamil on the rate of spontaneous release of quanta of transmitter at the frog neuromuscular junction was therefore tested in the present experiments. One point that emerged from previous studies (Duncan and Statham, 1977a) was that treatments serving to m o d i f y [Ca2÷]i interacted with one another. The effect of temperature was of particular importance in this respect. For example, A23187 had no effect at 17°C b u t caused a marked increase in MEPP frequency at 25°C (Statham and Duncan, 1975); raising [Ca2+]0 had a much more pronounced effect on MEPP frequency at 23 than at 17°C (Duncan and Statham, 1977a). The results suggest that elevated temperature augments the effect of other treatments that increase Ca 2÷ influx at the presynaptic terminals. For this reason, the effect of verapamil was studied at temperatures above and below 20°C (17 and 23°C) and, again, elevated temperature had a marked effect on the action of this drug also.
2. Materials and methods Electrophysiological recordings of MEPP frequency were made from the isolated cutaneous pectoris nerve muscle preparation of the frog Rana pipiens. Frogs were maintained in the laboratory at 10°C.
s.J. PUBLICOVER, C.J. DUNCAN All salines in which the preparations were bathed contained (mM) NaC1 110, KC1 1.87, NaH2PO4 0.032, NaHCO3 4.76, glucose 2g/1 at pH 7.1. Calcium concentration was varied; normal saline contained 1.8 mM CaC12, whilst at 5 × 10 -7 M [Ca2÷]0 a Ca2÷-EGTA buffer was used. 0.5 mM EGTA was added, together with the appropriate volume of AnalaR standard volumetric solution of CaC12, and free Ca 2÷ concentrations were calculated from the method of Portzehl et al. (1964). Stock solutions containing verapamil (1 raM), theophylline (1 mM) and Dantrolene (10 pg ml -~) were made up in appropriate saline. Solutions of Dantrolene were vigorously stirred for 1 h and the final solution was then filtered (Ellis and Carpenter, 1972; Statham and Duncan, 1976). The muscle was excised and equilibrated in the appropriate control saline for 30 min at 10°C. It was then pinned out in the experimental bath, the microelectrode inserted in the endplate region and the temperature adjusted to the experimental value. Records of MEPPs began after a further 10 rain. Electrophysiological recordings were made by the use of conventional glass microelectrodes filled with 3 M KC1; the temperature of the bath was controlled (+0.5°C) by a Peltier device. Potentials were fed through a cathode follower to a Tektronix 502A oscilloscope. MEPPs were recorded on moving film and counted. In any one experiment, the MEPPs were monitored in a single fibre at the intervals shown in the figures and at least 100 MEPPs were measured, except at very low frequencies. At least two control readings were taken before application of drugs to give a mean control rate. Studies on evoked release were made on the sartorius nerve-muscle preparation equilibrated for 30 min in saline containing 0.66-1.1 pM d-tubocurarine. EPPs were recorded on a Tektronix 5103N storage oscilloscope. All inorganic salts were AnalaR grade. Theophylline, d-tubocurarine and EGTA were obtained from Sigma Chemical Co., St. Louis, U.S.A. Dantrolene sodium was obtained from
VERAPAMIL AND MEPP FREQUENCY
121
Eaton Laboratories, London. Verapamil was a gift of A b b o t t Laboratories, Queenborough, Kent. "
/
3. Results The control MEPP frequency varied between different preparations, although only small oscillations (less than 10% of the initial rate) were recorded in any one preparation. In most figures, therefore, comparisons of the effects of treatments are shown by expressing MEPP frequency as a ratio of the control frequency (expressed as F~/F0, where F0 is the control frequency and F1 the frequency after treatment). As will be shown, verapamil is able to have a dual effect on MEPP frequency, causing both a fall and a rise in the rate of spontaneous release. The relative magnitude of these two opposing actions depends on the experimental conditions and also differs in different preparations. For this reason the time course of the response is shown for individual experiments, since pooling the data tended to obscure these opposing effects of the drug. The verapamil preparation used was the standard provided by A b b o t t Laboratories, containing the mixture of both isomers (i.e. both D.365 and CP.16533-1). Statistical analysis was carried out by calculating a regression coefficient and then determining the variance using the difference between successive residuals of y, using the equation
0
" ..
,
..,:,-%,/__ ..,." ,t
I
I
I
I
I
I
0
10
20
30
40
50
Fig. 1. Stimulatory effect of 10 -4 M verapamil at 17°C. [ C a 2 + ] 0 = 1.8 raM, except in the experiment shown with a dashed line where [Ca2+]o=5× 10 -7 M. Mean F0 before the application of verapamil in experiments where [Ca2+]0 = 1.8 mM was 30.0 MEPPs rain -1. In all figures ordinate: MEPP frequency as a ratio of the control frequency (F]/ F0); abscissa: time (rain) after application of verapamil. Time course of individual experiments shown. MEPP frequency (P < 0.001) in normal saline ([Ca2+]0 = 1.8 × 10 -3 M) although this effect was clearly different (P < 0.001) at 17°C (fig. 1) and 23°C (fig. 2). F~/F0 rarely rose above 1.75 at 17°C whereas, at 23°C, although the
,
j-,
s
!
4
V(b~) = Z { A 2 ( y - - Y ) / A x } / f ~ (Ax) where f sets of readings were made at each time interval and bc = the slope of the line
(Bliss, 1967). 3.1. Effect of verapamil at a concentration o f 1 0 -4 M At a concentration o f 1 0 -4 M, verapamil caused a significant and consistent rise in
2
:,./
./
I
I
] I
0
I
20
I
40
Fig. 2. Effect o f 10 -4 M verapamil at 23°C. Note much greater stimulatory effect at the higher temperature. [Ca 2+]o "= 1.8 raM. Mean control F 0= 103.5 MEPPs rain -1.
122
S.J. PUBLICOVER, C.J. DUNCAN
/\,J" 5
°
/.
4
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•
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i 20
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i 40
Fig. 3. Effect of 10 -4 M verapamil at 23°C when [Ca2+]0 = 5 x 10 -7 M. Mean control F0 = 14.3 MEPPs min-1. rate o f rise was variable, m u c h higher MEPP frequencies were recorded, with F1/Fo r e a c h i n g 7 (fig. 2). At b o t h 17 and 23°C, verapamil caused an initial small fall in MEPP f r e q u e n c y in s o m e p r e p a r a t i o n s . H o w e v e r , this overall rise in t h e rate was c o n t r a r y t o e x p e c t a t i o n since, if verapamil r e d u c e d passive Ca :+ e n t r y , a fall in t h e s t e a d y - s t a t e p o s i t i o n o f [Ca2+]i, and h e n c e in MEPP rate, might have b e e n e x p e c t e d . T h e e x p e r i m e n t s were t h e r e f o r e r e p e a t e d w h e n Ca :+ in t h e b a t h i n g saline was r e d u c e d t o 5 X 10 -7 M ( w h e n [Ca2+]0 ~- [Ca2+]i), so largely eliminating t h e e f f e c t o f Ca :÷ fluxes across t h e plasma membrane. R e d u c i n g [Ca2÷]0 t o 5 X 10 -7 M causes a significant fall in MEPP f r e q u e n c y (at b o t h 17 a n d 23°C m e a n F0 fell b y a p p r o x i m a t e l y 50%), as p r e v i o u s l y r e p o r t e d ( D u n c a n and S t a t h a m , 1 9 7 7 a ) . A f t e r equilibration, however, t h e p a t t e r n o f r e s p o n s e t o 1 0 - 4 M verapamil was similar t o t h a t f o u n d at 1.8 X 1 0 - 3 M [Ca:÷]0, w i t h a significant rise in MEPP f r e q u e n c y ( P < 0.01). A t 17°C t h e slow rise in MEPP f r e q u e n c y was indistinguishable f r o m t h a t f o u n d at t h e higher Ca 2÷ conc e n t r a t i o n (fig. 1); at 2 3 ° C t h e increase in
I
20
I
40
Fig. 4. Effect of 10 -4 M verapamil at 23°C when [Ca2+]0 = 9 mM. Mean control F 0 -- 56.7 MEPPs rain-1.
rate was again m u c h greater and m o r e variable (fig. 3); the results s h o w n in figs. 2 and 3 are n o t significantly d i f f e r e n t at 0.05. However, n o small initial fall in MEPP f r e q u e n c y was seen at e i t h e r t e m p e r a t u r e at this low value o f [Ca2+]o. Raising [Ca2+]0 t o 9 m M (5 times t h e conc e n t r a t i o n in n o r m a l saline) did n o t m a r k e d l y m o d i f y t h e a c t i o n o f verapamil w h i c h again caused a significant rise in MEPP f r e q u e n c y (P < 0.001). A t 23°C (fig. 4) t h e rate o f rise in MEPP f r e q u e n c y was variable and in some cases rapid; in o n e p r e p a r a t i o n F1/F0 r e a c h e d 5.8 a f t e r o n l y 12 min. At 17°C {fig. 5), how-
4
•
-
\I/-I
0
I
I
20
I
I
40
I
I
I
60
Fig. 5. Effect of 10 -4 M verapamil at 17°C when [Ca2+]0 = 9 raM. Mean control Fo = 56.4 MEPPs rain-1.
VERAPAMIL AND MEPP FREQUENCY
ever, t h e overall rise in MEPP f r e q u e n c y was slightly greater (P < 0 . 0 0 1 ) t h a n w h e n [Ca2÷]0 = either 1.8 × 10 -3 M or 5 × 10 -7 M. Figs. 4 and 5 s h o w t h a t , as at 1 . 8 × 1 0 - 3 M , an initial fall in MEPP f r e q u e n c y was r e c o r d e d in several p r e p a r a t i o n s w h e n [Ca2÷]0 = 9 mM. T h e s e results at d i f f e r e n t t e m p e r a t u r e s and values o f [ Ca 2÷ ] 0 s h o w t h a t verapamil (10 -4 M) clearly p r o m o t e s a rise in the rate o f spont a n e o u s release o f t r a n s m i t t e r , particularly at higher t e m p e r a t u r e s (figs. 2--4), and s o m e p r e p a r a t i o n s indicate t h a t it m a y have a dual action, causing a small initial fall in F]/F0. This suggestion is s u p p o r t e d b y i n s p e c t i o n o f t h e c a l c u l a t e d regression lines; in all e x p e r i m e n t s at 1.8 mM and 9.0 mM [Ca2÷]0 t h e calculated i n t e r c e p t o n t h e o r d i n a t e lay b e l o w t h e origin (F1/Fo < 1.0) whereas, with 5 × 10 -7 M [Ca2÷]0, the i n t e r c e p t was above, suggesting t h a t t h e r e was no initial fall w h e n [Ca2÷]0 was m a i n t a i n e d very low. This initial fall in MEPP f r e q u e n c y in t h e presence o f extracellular Ca 2÷ w o u l d be m o r e in a c c o r d with t h e generally a c c e p t e d role o f verapamil, and the validity o f this initial t r a n s i e n t fall was t e s t e d b y a p p l y i n g t h e drug at a concent r a t i o n o f 10 -s M.
3.2. Effect of verapamil at a concentration o f lO-S M
123
w i t h i n 5 min b u t , s u b s e q u e n t l y , a gradual, small rise in MEPP f r e q u e n c y o c c u r r e d . A t 23°C all p r e p a r a t i o n s also s h o w e d an initial fall in F]/F0 to a r o u n d 0.75, and t h e n a subs e q u e n t , b e l a t e d rise. T h e d e l a y b e f o r e this rise o c c u r r e d varied greatly, o n e p r e p a r a t i o n reaching 1.5 a f t e r o n l y 15 min whilst in o t h e r s MEPP f r e q u e n c y was depressed f o r 40 min. In no p r e p a r a t i o n s with verapamil at 10 -s M was a m a r k e d increase in MEPP f r e q u e n c y r e c o r d e d ( c o m p a r e fig. 6 with figs. 2 - 4 ) . T h e d e p e n d e n c e o f t h e rise in F~/F0 o n verapamil c o n c e n t r a t i o n was shown in an e x p e r i m e n t at 23°C. T h e p r e p a r a t i o n was e x p o s e d t o 10 -s M verapamil for 40 min and, a f t e r an initial fall, F1/F0 had risen to o n l y 1.5; a d d i t i o n o f 1 0 - 4 M verapamil n o w caused F]/F0 to rise rapidly t o 3.5.
3.3. Effect of Dantrolene Since verapamil is believed t o r e d u c e passive Ca 2÷ influx, and y e t t h e drug at a c o n c e n t r a t i o n o f 1 0 - 4 M is able t o p r o m o t e a m a r k e d rise in MEPP f r e q u e n c y , it s e e m e d likely t h a t its a c t i o n at higher c o n c e n t r a t i o n s m i g h t be t o cause a release o f Ca 2÷ f r o m intracellular Ca 2÷ storage sites in this p r e p a r a t i o n . Such a c o n c l u s i o n is s u p p o r t e d b y t h e rise in F1/F0 even w h e n Ca 2÷ fluxes across t h e plasma m e m b r a n e are m a r k e d l y r e d u c e d b y main-
[Ca2÷]0 was raised t o 9 mM, so as t o prom o t e Ca 2÷ i n f l u x at t h e p r e s y n a p t i c terminals and, a f t e r equilibration, verapamil at 10 -s M was a d d e d t o t h e saline. At 17°C (fig. 6) F~/F0 fell in all p r e p a r a t i o n s to a r o u n d 0.7
i --
01-: 0
0 20
40
Fig. 6. Effect of 10 -s M verapami] at 17°C when [Ca2+]o = 9 mM. Note inhibitory action of the drug at this lower concentration. Mean control F 0 = 88.4 MEPPs min -1 .
] 0
e ~ I .,.,_.. O / I ,,..,.. i ~
i/I
I
I 40
I 20
I
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I
Fig. 7. Effect of pretreatment with Dantrolene on the action of 10 -~ M verapamil. [Ca2+]o = 1.8 mM; 23°C. Pretreatment with Dantrolene (10pg ml-1) for 45 rain. Mean F 0 after Dantrolene treatment = 49.3 MEPPs rain -1.
124
S.J. P U B L I C O V E R , C.J. D U N C A N
taining [Ca2+]0 at 5 × 10 -7 M (fig. 3). DaNa is a skeletal muscle relaxant that acts b y reducing the release of Ca 2÷ from the sarcoplasmic reticulum at excitation (Ellis and Carpenter, 1972). It also reduces the MEPP frequency at the frog neuromuscular junction by 50--80% after 4 5 m i n treatment and it has been suggested that it acts by suppressing the release of Ca 2÷ from intracellular storage sites at the presynaptic terminals (Statham and Duncan, 1976). The experiments were carried o u t at 23°C (so causing a rise in MEPP frequency) and the preparation was pretreated with DaNa (10 pg m1-1) for 45 min which then reduced MEPP frequency. Addition of 1 0 - 4 M verapamil again caused a significant rise (P < 0.001) in the rate of spontaneous release of transmitter (fig. 7), the effect on the ratio F1/F0 being approximately intermediate between that observed at 17°C (fig. 1) and 23°C (fig. 2) in the absence of DaNa. The calculated regression lines for figs. 2 and 7 were significantly different (P < 0.001).
3.4. Effect of theophylline Theophylline has been shown to cause a rise in MEPP frequency at the frog neuro-
v~
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/
v
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muscular junction (Onodera, 1973), an approximate doubling at 25°C (Statham and Duncan, 1976). This action is independent of external Ca 2÷ and it is suggested that theophylline acts b y releasing Ca :÷ from intracellular storage sites other th~'n the mitochondria (Statham and Duncan, 1976). In the present experiments, the terminals were pretreated with theophylline (1 mM; 17°C; [Ca2+]0 = 1.8 mM) for 45 min, causing the typical rise in MEPP frequency (F0 = 67.5 MEPPs min-1). Subsequent treatment with 1 0 - 4 M verapamil (fig. 8) again produced a small, significant rise in MEPP frequency (P < 0.02) comparable with that found under these conditions, b u t in the absence of theophylline (compare figs. 1 and 8).
3.5. Evoked release Following presynaptic stimulation, muscle action potentials were recorded in the muscle after treatment with 1 0 - 4 M verapamil and this concentration of the drug did not markedly affect the magnitude of the EPP in preparations partially blocked with d-tubocurarine. Verapamil had no significant effect on muscle resting potential. However, when the action of 1 0 - 4 M verapamil on muscle twitch was studied in an indirectly stimulated sartorius nerve muscle preparation, twitch amplitude began to decline after 10 min exposure and was absent after some 30 min. Subsequent washing resulted in the reappearance of the twitch, b u t the action of verapamil was n o t completely reversible even after 40 min washing.
4. Discussion
0
I
I
I
I
I
I
0 20 40 Fig. 8. E f f e c t o f p r e t r e a t m e n t w i t h t h e o p h y l l i n e "on the a c t i o n o f 10 -4 M verapamil. [Ca2+]0 = 1.8 mM; 17°C. P r e t r e a t m e n t w i t h t h e o p h y l l i n e (1 raM) for 4 5 r a i n . Mean F 0 after theophylline treatment = 85.2 MEPPs rain -1.
The marked rise in MEPP frequency is not seen when verapamil is applied at the lower concentration of 10 -s M (fig. 6) at both 17 and 23 ° C. These trials were carried o u t with an extracellular concentration of Ca 2÷ of 9 mM, so that this elevated level of [Ca2+]0 ensured that Ca 2+ influx was an important
VERAPAMIL AND MEPP FREQUENCY factor in determining the steady-state position of [Ca2+]i . Under these conditions a small initial fall in MEPP frequency was seen in most preparations (F1/F0 ~ 0.7), especially during the first 15 min of treatment. There are also suggestions of an initial depression in the records obtained at 10 -4 M verapamil, particularly under conditions where Ca 2÷ influx are important ([Ca2÷]0 = 9 mM; fig 5) or where Ca 2÷ has not been mobilized from intracellular stores (17°C; fig. 1). Such an inhibitory effect would be consistent with the established role of verapamil in reducing Ca :+ entry and hence in lowering the steadystate position of [Ca2+]i . However, it is clear that such an action is not very marked at the presynaptic terminals of the frog neuromuscular junction. Such an observation agrees with previous findings; MEPP frequency is more sensitive to alterations in [Ca2÷]i produced by a modification of the intracellular Ca 2÷ storage sites than to the changes in Ca 2÷ influx across the plasma membrane. Thus, changes in [Ca2÷]0 have only modest effects (Duncan and Statham, 1977a); the divalent cation ionophore A 2 3 1 8 7 is ineffective at 17°C and it is necessary first to raise the temperature so as to demonstrate that the ionophore is indeed promoting Ca :÷ entry (Statham and Duncan, 1975). At the higher concentration of 10 -4 M, any inhibitory effect that verapamil may have on inward flux of Ca :÷ is largely masked by the other more pronounced action of the drug, namely promoting a rise in MEPP frequency. Under normal conditions (17 ° C; [Ca 2÷] 0 = 1.8 mM), this stimulation is not impressive and usually develops slowly since MEPP frequency is less than doubled after 40 min (fig. 1). Equilibrating the terminals at a raised [Ca2+]0 of 9 m M (and hence promoting inward Ca 2÷ flux) reveals this stimulatory action at 17°C at higher verapamil concentrations more clearly; F1/F0 is greater than 2.5 after 50 min exposure (fig. 5). However, the excitatory action of 1 0 - 4 M verapamil is shown much more dramatically if the preparation is equilibrated at 23°C,
125 when the spontaneous rate of release rises progressively with time after application of the drug, and F , / F 0 may exceed 7.0 (figs. 2 - 4 ) . The effect is b u t little affected by changes in [Ca2+]0 and the same pattern of response is seen when the Ca 2+ concentration of the saline is normal (fig. 2), reduced to very low levels (5 × 10 -7 M, fig. 3) or raised to 9 mM (fig. 4). We have shown previously (Duncan and Statham, 1977a) that the effect on MEPP frequency of treatments that are believed to m o d i f y [Ca2+]i is particularly sensitive to the existing steady-state level of [Ca2+]i in the presynaptic terminals. The terminals are better able to resist experimental perturbations if [Ca2+]i is maintained low. Elevated temperature (e.g. 23--25°C) might p r o m o t e the release of Ca 2÷ from intracellular storage sites and, under these conditions, the effects of raising [Ca2÷]0 (Duncan and Statham, 1977a), changing [Na÷]0 (Statham and Duncan, 1977) or treatment with A23187 (Statham and Duncan, 1975) are all greatly augmented. This same dependency on temperature, and hence probably on the preexisting level of [Ca2÷]i, is therefore also shown in the presence of 1 0 - 4 M verapamil. This effect of temperatures above 20°C has been discussed in detail elsewhere (Duncan and Statham, 1977a); its mechanism of action is u n k n o w n but the experiments cited above are consistent with the hypothesis that it serves to raise [Ca2÷]i or that it facilitates the release mechanism, perhaps via a change in membrane fluidity. We therefore conclude that both effects of verapamil on MEPP frequency are probably explicable in terms of alterations of [Ca2+]i at the presynaptic terminals. The question remains as to how verapamil, a known Ca 2÷ antagonist, could act to raise MEPP frequency via an alteration in [ C a 2 + ] i . The steady-state position of [Ca:÷]i at the presynaptic terminals is the resultant of the following: (i) Ca 2÷ influx at the plasma membrane. (ii) Active Ca 2÷ efflux across the plasma membrane. (iii) Ca 2÷ uptake and release b y
126
the mitochondria. (iv) Ca 2÷ exchange at other intracellular stores. Since the major stimulatory effect of verapamil is still seen when [Ca2÷]0 is reduced to 5 X 10 -7 M (and [Ca2+]0 ~ [Ca2+]i) it seems unlikely that this drug at 10 -4 M is acting to promote Ca :+ influx as in (i) above (note the similarity of figs. 2 and 3 which are not significantly different at 0.05). Such considerations also show that it is unlikely that verapamil has a depolarizing effect which, in turn, promotes Ca 2~ influx. Verapamil has been shown to have an action on isolated mitochondria, inhibiting net Ca 2÷ uptake (Frey and Janke, 1975) and oxidative phosphorylation (Silveira and Campello, 1975). However, concentrations of verapamil of at least 10-3M are necessary to demonstrate this effect and we believe that it is unlikely that the mitochondria (paragraph (iii) above) are the site of action of externally applied 10-4 M verapamil. Verapamil at 10 -4 M still exhibits a stimulatory effect after pretreatment of the terminals with either theophylline or DaNa. These agents are believed to act at the intracellular storage sites, DaNa serving to suppress Ca 2+ leakage (and so reduce MEPP frequency) and theophyUine promoting Ca 2÷ release (and so raising MEPP frequency) (Statham and Duncan, 1976). Pretreatment with either of these agents therefore alters [Ca2+]i, and hence F0, so that direct comparison with untreated terminals of the subsequent effect of verapamil is not possible. However, it is clear that, at 10 -4 M, the drug is still effective in raising MEPP frequency, even when the Ca 2+ stores have been partially emptied with theophylline (compare figs. 8 and 1) or when leakage has been reduced with DaNa (compare figs. 7 and
2). We therefore tentatively conclude that verapamil probably does n o t act at intracellular Ca 2÷ storage sites and it may prove ultimately t h a t its target is the Ca 2+ pump at the plasma membrane (paragraph (ii) above). It is known to inhibit active Ca 2÷ transport by everted rat d u o d e n u m sacs (Wrdbel and Michalska, 1977). Thus, verapamil reduces Ca 2÷ influx in m a n y
S.J. P U B L I C O V E R , C.J. D U N C A N
different cells at low concentration; at higher concentrations it may inhibit active Ca 2÷ efflux. Such speculation raises the possibility of a similarity between the passive Ca 2+ channel and the membrane Ca 2÷ pump, since both would be binding verapamil, a suggestion that is in accord with the hypothesis developed in detail by Shamoo and Goldstein (1977). 10 -4 M verapamil had no marked effect on the magnitude of the EPP when the neuromuscular junction was partially blocked with d-tubocurarine. Muscle resting potential was not significantly affected. Action potentials were also recorded post-synaptically in the absence of d-tubocurarine but in the presence of verapamil, and we conclude that the drug has little effect on the excited Ca z÷ channels of muscle during evoked release. However, Bondi (1978) has also found that verapamil, at concentrations of 10 -4 M and above, causes a reduction and, eventually, a total inhibition of twitch in a directly stimulated frog muscle. The time course of the response was similar to that in our experiments and reversal of the effect was again difficult to achieve. The findi~gs suggest that verapamil may have more than one site of action in muscle also and that it may, at high concentration, serve to block the Ca 2+ release mechanism of the S.R. MEPP frequency is of little importance in vivo, but if verapamil is able to have similar subliminal actions at other synapses (e.g. in the CNS where it could modify excitability) and other secretory systems, these findings might be of clinical significance. Acknowledgements We are grateful t o Miss S. S c o t t for assistance in t h e p r e p a r a t i o n o f t h e m a n u s c r i p t a n d t o Dr. M.E. B e g o n for advice w i t h statistics. This w o r k was s u p p o r t e d , in p a r t , b y t h e M u s c u l a r D y s t r o p h y G r o u p of G r e a t Britain.
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127 phosphate in isolated fat-cells, Biochem. J. 152, 121. Mayer, C.J., C. Van Breemen and R. Casteels, 1972, The action of lanthanum and D-600 on the calcium exchange in the smooth muscle cells of the guinea-pig taenia coli, Pflfgers Arch. ges. Physiol. 337,333. Onodera, K., 1973, Effect of caffeine on the neuromuscular junction of the frog and its relation to external calcium concentration, Jap. J. Physiol. 23,587. Pinto, J.E.B. and J.M. Trifar6, 1976, The different effects of D-600 (methoxyverapamil) on the release of adrenal catecholamines induced by acetylcholine, high potassium or sodium deprivation, Brit. J. Pharmacol. 57,127. Portzehl, H., P.C. Caldwell and J.C. Riiegg, 1964, The dependence of contraction and relaxation of muscle fibres from the crab Maia squinado on the internal concentration of free calcium ions, Biochim. Biophys. Acta 79, 581. Shamoo, A.E. and D.A. Goldstein, 1977, Isolation of ionophores from ion transport systems and their role in energy transduction, Biochim. Biophys. Acta 472, 13. Silveira, O. and A.P. Campello, 1975, Effects of iproveratril on isolated heart mitochondria, Res. Commun. Chem. Pathol. Pharmacol. 10, 149. Statham, H.E. and C.J. Duncan, 1975, The action of ionophores at the frog neuromuscular junction, Life Sci. 17, 1401. Statham, H.E. and C.J. Duncan, 1976, Dantrolene and the neuromuscular junction: evidence for intracellular calcium stores, European J. Pharmacol. 39, 143. Statham, H.E. and C.J. Duncan, 1977, The effect of sodium ions on MEPP frequency at the frog neuromuscular junction, Life Sci. 20, 1839. Statham, H.E., C.J. Duncan and S.J. Publicover, 1978, Dual effect of 2,4-Dinitrophenol on the spontaneous release of transmitter at the frog neuromuscular junction, Biochem. Pharmacol. 27, 2199. Vohra, J.K., 1977, Clinical use of verapamil, Drugs 13,219. Wr6bel, J. and L. Michalska, 1977, The effect of verapamil on intestinal calcium transport, European J. Pharmacol. 4 5 , 3 8 5 .
