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Possible mechanism of the stimulatory effect of 4-aminopyridine on toad skin
Gen. Pharmac. Vol. 20, No. 6, pp. 767-770, 1989 Printed in Great Britain.All rights reserved
0306-3623/89$3.00+ 0.00 Copyright © 1989PergamonPress pie
POSSIBLE MECHANISM OF THE STIMULATORY EFFECT OF 4-AMINOPYRIDINE ON TOAD SKIN C. S. GONZALEZ,B, C. NORRIS, J. B. CONCHAand G. M. CONTRERAS Department of Physiological Sciences, Faculty of Biological and Natural Sciences, University of Concepcion, Casilla 2407, Apartado I0, Concepci6n, Chile (Received 8 February 1989) Abstract--1. This study was undertaken to demonstrate the facilitatory effects of 4-aminopyridine on
transmitter release in the toad skin preparation. 2. A dose-dependent increase in the bioelectric parameters was found. 3. This effect was blocked by propranolol and by verapamil but not by dibenamine, and was mimicked by the calcium ionophore A-23187 but not by potassium channel blockers. 4. These results support the hypothesis that 4-aminopyridine increases presynaptic nerve terminal permeability of calcium and thus the release of noradrenaline.
INTRODUCTION
It is well known that 4-aminopyridine (4-AP) decreases K+-conductance of the cell membrane in many excitable tissues Ofeh et aL, 1976; Kenyon and Gibbons, 1979; Thesleff, 1980; Kumamoto and Kuba, 1985). It is also known that an increase in the duration of a depolarizing pulse applied to a nerve terminal augments the output of transmitter (Katz and Miledi, 1967; Glavinovic, 1987); therefore 4-AP may be expected to increase the amount of transmitter released on nerve stimulation by lengthening the action potential in the presynaptic terminals. This phenomenon has been attributed to increased Ca z+ entry into the presynaptic nerve terminal during action potentials that are prolonged due to the blockade of voltage-sensitive K + channels (Molgo et al., 1977; Lundh, 1978). However, the facilitatory effects of 4-AP on transmitter release occur at concentrations lower than those required to block K + channels (Herman and Gorman, 1981; Rogawski and Barker, 1983). The alternative hypothesis has therefore been proposed that 4-AP directly promotes Ca 2+ conductance mechanisms in presynaptic nerve terminals (Illes and Thesleff, 1978; Molgo et al., 1980; Ohkawa, 1984). The possibility that 4-AP may trigger Ca 2+ release from appropriate stores in the nerve terminals during the presynaptic action potential could be ruled out by the finding that extra-cellular Ca z+ must be present to allow transmitter release in the presence of 4-AP (Lundh and Thesleff, 1977). Rogawski and Barker (1983) in spinal neurons, demonstrated that the apparent enhancement of inward Ca 2+ current by 4-AP cannot be attributed to the suppression of any outward K + current species, indicating a direct effect on Ca z+ entry. It has been previously demonstrated (Gonzfilez et al., 1966; Rudolph et al., 1978) that the electrical stimulation of an isolated nerve-skin preparation of the toad produces variations in the potential difference (I'D) and similar changes in short-circuit current (SCC), due principally to glandular activity
(Skoglund and Sj6berg, 1977). Addition of noradrenaline to the vascular surface of the skin elicits effects which closely resemble those induced by nerve stimulation. Evidence for the sympathetic participation in the effect produced by nerve stimulation was obtained in sympathectomized and guanethidine treated toads (Gon~lez et al., 1967). The present study was undertaken to demonstrate the facilitatory effects of 4-AP on transmitter release in the neuroglandular synapsis of the toad. Part of this work has been reported in a short communication (Norris et al., 1988). MATERIALS AND METHODS
Animals and experimental procedures The abdominal skin dissected from male and female pithed Pleurodema thaul (5-10 g) toads, kept in tap water 24 hr prior to use, was carefully washed in toad Ringer's solution, stretched and mounted between modified perspex Ussing chambers. The composition of the solution was (mM): NaC1 113; KC1 1.0; CaC122.0; NaHCO 3 2.3; glucose 11.0; and phosphate buffered to pH 7.4. The reservoirs on both sides of the skin (exposed surface 0.70 cm2) were filled with 3 ml toad Ringer's solution, and constant aeration was provided. Electrical measurements The potential difference (PD) was recorded on a 2-channel Cole-Parmer recorder with calomel electrodes and agar-Ringer bridges. The short-circuit current (SCC) was measured according to Ussing and Zerahn (1951) through Ag-AgCI w i r e electrodes using a voltage-clamp ((3. M6traux). Experiments were started when the PD and the SCC had been stabilized for 30 rain. Drugs The following drugs were used: 4-AP, dibenamine, propranolol, the calcium ionophore A-23187 (all from Sigma ), verapamil (Knoll Laboratories, Luwigshaben), cesium chloride, barium chloride (Merck), amiloride (Merck, Sharp and Dohme). The organic solvent used in the solubilisation of A-23187 was dimethylsulfoxidein a final concentration of 2.3 x 10-4 M. Aliquots of this solvent were always tested before the drug was used.
767
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768
~., 1 0 0
~,
when the preparation was not washed. Figure 1 shows the dose-response curve o f the toad skin to 4-AP expressed as percentage increase in P D and in the SCC.
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-Log 4-ominopyridine concentrotion(M) Fig. 1. The dose--responserelationship for 4-aminopyridinetreated skins. The maximum % increase in short-circuit current and in potential difference normalized to control values is plotted against the molar concentration of 4-AP: means __+SEM, N = 8.
Statistical analysis Values throughout the text refer to means _+ SEM. Statistical treatment was performed by means of Student's t-test for paired data. RESULTS
Effect of 4-aminopyridine (4-AP) on the electric properties o f the isolated toad skin Thirty min after stabilization o f the electric p r o p erties o f the skin, 4-AP was applied in the inner solution. The c o n c e n t r a t i o n range used in the experiments (1.7-7.0 x 10 -4 M) induced a reversible increase in P D and SCC across the skin. Larger doses decreased the effect o f the drug and smaller doses did n o t elicit response. A c o n c e n t r a t i o n o f 7.0 × 10 -4 M was used in the work since it was the m o s t effective in that it was the m i n i m u m dose which elicited a maximal response. The time course o f the response was a rise to peak in 4.20 _ 1.30 min (n = 8) followed by a decline to control level in 27.0 + 0.6 min (n = 8)
Effect of several agents on the toad skin response to 4-AP Adrenoceptor blockers. The effect o f 4-AP was not modified by p r e t r e a t m e n t (15-20 min) with the alpha a d r e n o c e p t o r blocker dibenamine (1 × 10 -6 M, inner surface) in eight experiments. A d d i t i o n o f the beta adrenergic blocker p r o p r a n o l o l ( 1 5 - 2 0 m i n previously, 1 x 10 -6 M, inner surface) inhibited the stimulatory effect o f 4-AP on the P D and on the SCC (Table 1). Calcium channel blocker. When verapamil (1 × 10 -5 M) was a d d e d to the inner surface o f the skin, the effect o f 4-AP on the P D and on the SCC was blocked (Table 1).
Effect of Ca 2+ ionophore and of K ÷ channel blockers on the PD and SCC o f the isolated toad skin Calcium ionophore (A-23187). Exposure o f the inner surface o f the skin to the calcium i o n o p h o r e A-23187 (1 × 10 -6 M ) induced an effect similar to that o f 4-AP in 8 skins (Table 2). Potasium channel blockers. Since it has been s h o w n (Rogawski and Barker, 1983) that the apparent e n h a n c e m e n t o f inward Ca 2÷ current by 4-AP c a n n o t be attributed to the suppression o f o u t w a r d K ÷ current, eight skins were exposed to the effects o f CsCI, a K ÷ channel blocker and o f BaCI 2, a Ca 2÷d e p e n d e n t K ÷ channel blocker. Table 2 shows that b o t h CsCI (2 × 1 0 - 3 M ) and BaCI: (2 × 1 0 - 3 M ) applied in the inner surface induce a significant decrease in P D and SCC, an effect opposite to that o f 4-AP. DISCUSSION The present experiments show that 4-AP significantly increases the P D and the SCC in the isolated skin o f Pleurodema thaul. If this increase in bioelectric parameters is due to the release o f noradrenaline, which acts on beta adrenergic receptors ( R u d o l p h et al., 1978), it follows that propranolol, a beta adrenergic blocker, should inhibit the skin response
Table 1. Effectof adrenergic blockers and of a calcium channel blocker on the isolated toad skin response to 7.0 x 10 4M 4-aminopyridine (4-AP, inner surface) Control Agent Dibenamine (10-6 M) Propranolol (10-6 M) Verapamil (10-5 M)
PD (mY) 25.0 + 5.1 22.9+:2.2 26.2 +: 2.6
SCC (~ A/cm2) 39.2+:2.1 26.2+ 1.8 27.3 + 1.4
Effect of 4-AP PD SCC (mV) (~A/cm2) 50.1 + 4.1:[ 50.2 +_3.3:[ 27.9_+3.1" 35.6 + 3.1~: 36.7 + 3.3t 38.8 + 2.0~:
Effect of 4-AP after agent PD SCC (mV) (/~A/cm2) 45.8_+3.4:[: 46.4_+ 3.5t 25.0+: 1 . 1 § 27.3_+2.1§ 29.7 + 2.3§ 28.6 + 3.1§
Values are means _+SEM of 8 experiments; *P < 0.05, *P < 0.02, ~tP < 0.01 in contrast to control values §not significant. Table 2. Effectof Ca2÷ ionophore (A-23187)and ofK + channel blockers(inner surface)on the bioelectric parameters of the isolated toad skin Control Effect of agent PD SCC PD SCC Agent (mV) (# A/cm2) (mV) (/~A/cm:) A-23187(1 x 10-6M) 35.2+ 3.1 32.4+3.3 46.9+2.8* 45.1 + 1.9"* BaCI (2 x 10-s M) 25.2+3.0 27.2+:2.8 1 8 . 6 + : 3 . 2 " 19.1+:3.0" CsCI(2 x 10-~M) 23.2+: 1.6 28.4+2.1 1 5 . 6 + : 2 . 6 " 17.2_+3.1"* Values are means + SEM of 8 experiments; *P < 0.02, **P < 0.01 in contrast to control values.
4-Aminopyridine on toad skin lOmin I
-
I
4-AP
o u'~-r~ . . . . . . . . . . . . . . . . .
~o olll
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769
tance mechanism in the toad skin presynaptic nerve terminals. 2.4-aminopyridine produced a dose-dependent increase in the potential difference and in the shortcircuit current of the skin. This effect was also present in amiloride-treated skins. 3. The increase in bioelectric parameters was blocked by propranolol and by verapamil but not by dibenamine. 4. Exposure of the skin to the calcium ionophore A-23187 induced an effect similar to that of 4-aminopyridine. The potassium channel blockers cesium and barium chloride induced a significant decrease in the bioelectric parameters, an effect opposite to that of 4-aminopyridine. 5. The preceding evidence supports the hypothesis that 4-aminopyridine increases presynaptic nerve terminal permeability to calcium, thus releasing noradrenaline which then promotes the rise in potential difference and in short-circuit current across the skin.
IIIlllllll
Fig. 2. Effect of 4-aminopyridine (4-AP, 7 x 10-4 M, serosal surface) on the short-circuit current (SCC) and on the potential difference (PD) of the isolated toad skin treated 15-20 rain previously with amiloride (1 x 10-4 M, mucosal surface). to 4-AP, which is in fact the case. Table 1 shows that dibenamine, an alpha adrenergic blocker, does not alter the effect of 4-AP on the skin. Thompson and Mills (1981, 1983) established that beta adrenergic agonists stimulate an amilorideinsensitive increase in current across the isolated frog skin, due to C1- transport by cells of the mucosal glands, innervated by postganglionic sympathetic fibres. Thus in our experiments, following the addition of 4-AP to the inner surface, the PD and the SCC increased significantly in skins pretreated (15-20min) with amiloride added to the mucosal surface (Fig. 2). That 4-AP exerts its action on the Ca 2+ channels of the presynaptic nerve terminals, and not by blocking K + channels of the sympathetic postganglionic fibres, is supported by the following findings: BaC12, a Ca2+-dependent K + channel blocker, and CsC1, a direct K ÷ channel blocker (Latorre and Miller, 1983; Schwarz and Passow, 1983; Kumamoto and Kuba, 1985) significantly decreased the bioelectric parameters of the toad skin, an effect opposite to that of 4-AP. Therefore, 4-AP in the concentration range used in this work does not block the K ÷ channels in the sympathetic fibres which innervate the skin. On the other hand, verapamil, a Ca 2÷ channel blocker significantly decreased the skin response to 4-AP: it is therefore possible that this drug acts presynaptically on the mechanism of noradrenaline release and not postsynaptically as is the case with propranolol which prevents the action of noradrenaline on the beta adrenergic receptors of the glands (Thompson and Mills, 1981). In addition, the Ca 2+ ionophore A-23187 produces an effect similar to that 4-AP. In preliminary experiments, we have found that the bioelectric responses to stimulation of the isolated nerve-skin preparation of the toad Caudiverbera caudiverbera, are significantly enhanced by the addition of 4-AP. These results are in agreement with our hypothesis and that of other authors (Rogawski and Barker, 1983) that 4-AP directly increases presynaptic nerve terminal permeability to Ca 2+, releasing noradrenaline which is responsible for the rise in PD and SCC in this skin preparation.
SUMMARY
1. This work is concerned with the hypothesis that 4-amino-pyridine directly promotes Ca 2+ conduc-
Acknowledgements--This work received financial support
from grants 20.33.35, 20.33.30 and 20.33.33, University of Concepci6n, Chile. The authors wish to thank Merck, Sharp and Dohme for the gift of amiloride. The authors also thank Mrs Maria Cecilia Nova and Mr Oscar Sepfilveda for excellent technical assistance. REFERENCES
Glavinovic M. I. (1987) Differences in presynaptic action of 4-aminopyridine and tetraethylammonium at frog neuromuscular junction. Can J. Physiol. PharmacoL 65, 747-752. Gonz~lez C., S/mchez J. and Concha J. (1966) Changes in potential difference and short-circuit current produced by electrical stimulation in a nerve-skin preparation of the toad. Biochem. Biophys, Acta. 120, 186-188. Gonz/dez C., S~chez J. and Concha J. (1967) Further evidence for the release of noradrenaline under nerve stimulation and its effects on the potential differencein a toad nerve-skin preparation. Biochirn. Biophys. Acta 135, 167-170. Hermann A. and Gorman A. L. P. (1981) Effects of 4-aminopyridine on potassium currents in a molluscan neuron. J. Gen. Physiol. 78, 63-86. Iles P. and Thesleff S. (1978) 4-aminopyridine and evoked transmitter release from motor nerve endings. Br. J. Pharmac. 64, 623-629. Katz B. and Miledi T. (1967) The release of acetylcholine from nerve endings by graded electric pulses. Proc. R. Lond Ser. Biol. Sci. 167, 23-28. Kenyon J. L. and Gibbons W, R. (1979) 4-aminopyridine and the early outward current of sheep cardiac Purkinje fibres. J. Gen. Physiol. 73, 139-157. Kumamoto E, and Kuba K. (1985) Effects of K + channel blockers on transmitter release in bullfrog sympathetic ganglia. J. Pharmac. Exp. Ther. 235, 241-247. Latorre R. and Miller C. (1983) Conduction and selectivity in potassium channels. J. Membr. Biol. 71, 11-30. Lundh H. and Thesleff S. (1977) The mode of action of 4-amino-pyridine and guanidine on transmitter release from motor nerve terminals. Fur. J. Pharmac. 42, 411-412. Lundh H. (1978) Effects of 4-aminopyridine on neuromuscular transmission. Brain Res. 153, 307-318. Molgo J., Lemeignan M. and Lechat P. (1977) Effects of 4-amino-pyridine at the frog neuromuscular junction. J. Pharmac. Exp. Ther. 203, 653-663. Molgo J., Lundh H. and Thesleff S. (1980) Potency of 3,4-diamino-pyridine and 4-aminopyridine on maria-
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malian neuromuscular transmission and the effect of pH changes. Eur. J. Pharmac. 61, 24-34. Norris B. C., Gonz~lez C. S., Concha J. B. and Contreras G. M. (1988) Effect of 4-aminopyridine on the bioelectric parameters of the toad skin. Med. Sci. Res. 16, 887888. Ohkawa H. (1984) Effects of 4-aminopyridine on the nonadrenergic inhibitory neurotransmission in the guinea pig duodenum. Jpn. J. Physiol. 34, 893-905. Rogawski M. A. and Barker J. L. (1983) Effects of 4aminopyridine on calcium action potentials and calcium current under voltage clamp in spinal neurons. Brain Res. 280, 180-185. Rudolph I., Norris B., Concha J. and Gonz/dez C. (1978) Studies on the electrical responses of a toad nerve skin preparation. Cell. Molec. Biol. 24, 17-27. Schwarz W. and Passow H. (1983) Ca 2+ activated K +
channels in erithrocytes and excitable cells. Ann. Rev. Physiol. 45, 359-374. Skoglund C. R. and Sj6berg E. (1977) In vitro studies of frog mucous glands. Acta Physiol. Scand. 100, 457 470. Thesleff S. (1980) Aminopyridines and synaptic transmission. Neuroscience 5, 1413-1419. Thompson I. G. and Mills J. W. (1981) Isoproterenolinduced current in glands of frog skin. Am. J. Physiol. 241, C250~257. Thompson I. G. and Mills J. W. (1983) Chloride transport in glands of frog skin. Am. J. Physiol. 244, C221-C226. Ussing H. H. and Zerahn K. (1951) Active transport of sodium as the source of electric current in the shortcircuited frog skin. Acta Physiol. Scand. 23, 110-127. Yeh J. Z., Oxford G. S., Wu C. H. and Narahashi T. (1976) Dynamics of aminopyridine block of potassium channels in squid axon membrane. J. Gen. Physiol. 68, 519-535.
0306-3623/89$3.00+ 0.00 Copyright © 1989PergamonPress pie
POSSIBLE MECHANISM OF THE STIMULATORY EFFECT OF 4-AMINOPYRIDINE ON TOAD SKIN C. S. GONZALEZ,B, C. NORRIS, J. B. CONCHAand G. M. CONTRERAS Department of Physiological Sciences, Faculty of Biological and Natural Sciences, University of Concepcion, Casilla 2407, Apartado I0, Concepci6n, Chile (Received 8 February 1989) Abstract--1. This study was undertaken to demonstrate the facilitatory effects of 4-aminopyridine on
transmitter release in the toad skin preparation. 2. A dose-dependent increase in the bioelectric parameters was found. 3. This effect was blocked by propranolol and by verapamil but not by dibenamine, and was mimicked by the calcium ionophore A-23187 but not by potassium channel blockers. 4. These results support the hypothesis that 4-aminopyridine increases presynaptic nerve terminal permeability of calcium and thus the release of noradrenaline.
INTRODUCTION
It is well known that 4-aminopyridine (4-AP) decreases K+-conductance of the cell membrane in many excitable tissues Ofeh et aL, 1976; Kenyon and Gibbons, 1979; Thesleff, 1980; Kumamoto and Kuba, 1985). It is also known that an increase in the duration of a depolarizing pulse applied to a nerve terminal augments the output of transmitter (Katz and Miledi, 1967; Glavinovic, 1987); therefore 4-AP may be expected to increase the amount of transmitter released on nerve stimulation by lengthening the action potential in the presynaptic terminals. This phenomenon has been attributed to increased Ca z+ entry into the presynaptic nerve terminal during action potentials that are prolonged due to the blockade of voltage-sensitive K + channels (Molgo et al., 1977; Lundh, 1978). However, the facilitatory effects of 4-AP on transmitter release occur at concentrations lower than those required to block K + channels (Herman and Gorman, 1981; Rogawski and Barker, 1983). The alternative hypothesis has therefore been proposed that 4-AP directly promotes Ca 2+ conductance mechanisms in presynaptic nerve terminals (Illes and Thesleff, 1978; Molgo et al., 1980; Ohkawa, 1984). The possibility that 4-AP may trigger Ca 2+ release from appropriate stores in the nerve terminals during the presynaptic action potential could be ruled out by the finding that extra-cellular Ca z+ must be present to allow transmitter release in the presence of 4-AP (Lundh and Thesleff, 1977). Rogawski and Barker (1983) in spinal neurons, demonstrated that the apparent enhancement of inward Ca 2+ current by 4-AP cannot be attributed to the suppression of any outward K + current species, indicating a direct effect on Ca z+ entry. It has been previously demonstrated (Gonzfilez et al., 1966; Rudolph et al., 1978) that the electrical stimulation of an isolated nerve-skin preparation of the toad produces variations in the potential difference (I'D) and similar changes in short-circuit current (SCC), due principally to glandular activity
(Skoglund and Sj6berg, 1977). Addition of noradrenaline to the vascular surface of the skin elicits effects which closely resemble those induced by nerve stimulation. Evidence for the sympathetic participation in the effect produced by nerve stimulation was obtained in sympathectomized and guanethidine treated toads (Gon~lez et al., 1967). The present study was undertaken to demonstrate the facilitatory effects of 4-AP on transmitter release in the neuroglandular synapsis of the toad. Part of this work has been reported in a short communication (Norris et al., 1988). MATERIALS AND METHODS
Animals and experimental procedures The abdominal skin dissected from male and female pithed Pleurodema thaul (5-10 g) toads, kept in tap water 24 hr prior to use, was carefully washed in toad Ringer's solution, stretched and mounted between modified perspex Ussing chambers. The composition of the solution was (mM): NaC1 113; KC1 1.0; CaC122.0; NaHCO 3 2.3; glucose 11.0; and phosphate buffered to pH 7.4. The reservoirs on both sides of the skin (exposed surface 0.70 cm2) were filled with 3 ml toad Ringer's solution, and constant aeration was provided. Electrical measurements The potential difference (PD) was recorded on a 2-channel Cole-Parmer recorder with calomel electrodes and agar-Ringer bridges. The short-circuit current (SCC) was measured according to Ussing and Zerahn (1951) through Ag-AgCI w i r e electrodes using a voltage-clamp ((3. M6traux). Experiments were started when the PD and the SCC had been stabilized for 30 rain. Drugs The following drugs were used: 4-AP, dibenamine, propranolol, the calcium ionophore A-23187 (all from Sigma ), verapamil (Knoll Laboratories, Luwigshaben), cesium chloride, barium chloride (Merck), amiloride (Merck, Sharp and Dohme). The organic solvent used in the solubilisation of A-23187 was dimethylsulfoxidein a final concentration of 2.3 x 10-4 M. Aliquots of this solvent were always tested before the drug was used.
767
C.S. GONZ,~LEZet al.
768
~., 1 0 0
~,
when the preparation was not washed. Figure 1 shows the dose-response curve o f the toad skin to 4-AP expressed as percentage increase in P D and in the SCC.
;
80
I
C "--
~
.a_
C
40-
"-
8
60 O
40 --
•
0"//
I
5
. ~ -
20
I 3
4
y
0
-Log 4-ominopyridine concentrotion(M) Fig. 1. The dose--responserelationship for 4-aminopyridinetreated skins. The maximum % increase in short-circuit current and in potential difference normalized to control values is plotted against the molar concentration of 4-AP: means __+SEM, N = 8.
Statistical analysis Values throughout the text refer to means _+ SEM. Statistical treatment was performed by means of Student's t-test for paired data. RESULTS
Effect of 4-aminopyridine (4-AP) on the electric properties o f the isolated toad skin Thirty min after stabilization o f the electric p r o p erties o f the skin, 4-AP was applied in the inner solution. The c o n c e n t r a t i o n range used in the experiments (1.7-7.0 x 10 -4 M) induced a reversible increase in P D and SCC across the skin. Larger doses decreased the effect o f the drug and smaller doses did n o t elicit response. A c o n c e n t r a t i o n o f 7.0 × 10 -4 M was used in the work since it was the m o s t effective in that it was the m i n i m u m dose which elicited a maximal response. The time course o f the response was a rise to peak in 4.20 _ 1.30 min (n = 8) followed by a decline to control level in 27.0 + 0.6 min (n = 8)
Effect of several agents on the toad skin response to 4-AP Adrenoceptor blockers. The effect o f 4-AP was not modified by p r e t r e a t m e n t (15-20 min) with the alpha a d r e n o c e p t o r blocker dibenamine (1 × 10 -6 M, inner surface) in eight experiments. A d d i t i o n o f the beta adrenergic blocker p r o p r a n o l o l ( 1 5 - 2 0 m i n previously, 1 x 10 -6 M, inner surface) inhibited the stimulatory effect o f 4-AP on the P D and on the SCC (Table 1). Calcium channel blocker. When verapamil (1 × 10 -5 M) was a d d e d to the inner surface o f the skin, the effect o f 4-AP on the P D and on the SCC was blocked (Table 1).
Effect of Ca 2+ ionophore and of K ÷ channel blockers on the PD and SCC o f the isolated toad skin Calcium ionophore (A-23187). Exposure o f the inner surface o f the skin to the calcium i o n o p h o r e A-23187 (1 × 10 -6 M ) induced an effect similar to that o f 4-AP in 8 skins (Table 2). Potasium channel blockers. Since it has been s h o w n (Rogawski and Barker, 1983) that the apparent e n h a n c e m e n t o f inward Ca 2÷ current by 4-AP c a n n o t be attributed to the suppression o f o u t w a r d K ÷ current, eight skins were exposed to the effects o f CsCI, a K ÷ channel blocker and o f BaCI 2, a Ca 2÷d e p e n d e n t K ÷ channel blocker. Table 2 shows that b o t h CsCI (2 × 1 0 - 3 M ) and BaCI: (2 × 1 0 - 3 M ) applied in the inner surface induce a significant decrease in P D and SCC, an effect opposite to that o f 4-AP. DISCUSSION The present experiments show that 4-AP significantly increases the P D and the SCC in the isolated skin o f Pleurodema thaul. If this increase in bioelectric parameters is due to the release o f noradrenaline, which acts on beta adrenergic receptors ( R u d o l p h et al., 1978), it follows that propranolol, a beta adrenergic blocker, should inhibit the skin response
Table 1. Effectof adrenergic blockers and of a calcium channel blocker on the isolated toad skin response to 7.0 x 10 4M 4-aminopyridine (4-AP, inner surface) Control Agent Dibenamine (10-6 M) Propranolol (10-6 M) Verapamil (10-5 M)
PD (mY) 25.0 + 5.1 22.9+:2.2 26.2 +: 2.6
SCC (~ A/cm2) 39.2+:2.1 26.2+ 1.8 27.3 + 1.4
Effect of 4-AP PD SCC (mV) (~A/cm2) 50.1 + 4.1:[ 50.2 +_3.3:[ 27.9_+3.1" 35.6 + 3.1~: 36.7 + 3.3t 38.8 + 2.0~:
Effect of 4-AP after agent PD SCC (mV) (/~A/cm2) 45.8_+3.4:[: 46.4_+ 3.5t 25.0+: 1 . 1 § 27.3_+2.1§ 29.7 + 2.3§ 28.6 + 3.1§
Values are means _+SEM of 8 experiments; *P < 0.05, *P < 0.02, ~tP < 0.01 in contrast to control values §not significant. Table 2. Effectof Ca2÷ ionophore (A-23187)and ofK + channel blockers(inner surface)on the bioelectric parameters of the isolated toad skin Control Effect of agent PD SCC PD SCC Agent (mV) (# A/cm2) (mV) (/~A/cm:) A-23187(1 x 10-6M) 35.2+ 3.1 32.4+3.3 46.9+2.8* 45.1 + 1.9"* BaCI (2 x 10-s M) 25.2+3.0 27.2+:2.8 1 8 . 6 + : 3 . 2 " 19.1+:3.0" CsCI(2 x 10-~M) 23.2+: 1.6 28.4+2.1 1 5 . 6 + : 2 . 6 " 17.2_+3.1"* Values are means + SEM of 8 experiments; *P < 0.02, **P < 0.01 in contrast to control values.
4-Aminopyridine on toad skin lOmin I
-
I
4-AP
o u'~-r~ . . . . . . . . . . . . . . . . .
~o olll
4-AP
769
tance mechanism in the toad skin presynaptic nerve terminals. 2.4-aminopyridine produced a dose-dependent increase in the potential difference and in the shortcircuit current of the skin. This effect was also present in amiloride-treated skins. 3. The increase in bioelectric parameters was blocked by propranolol and by verapamil but not by dibenamine. 4. Exposure of the skin to the calcium ionophore A-23187 induced an effect similar to that of 4-aminopyridine. The potassium channel blockers cesium and barium chloride induced a significant decrease in the bioelectric parameters, an effect opposite to that of 4-aminopyridine. 5. The preceding evidence supports the hypothesis that 4-aminopyridine increases presynaptic nerve terminal permeability to calcium, thus releasing noradrenaline which then promotes the rise in potential difference and in short-circuit current across the skin.
IIIlllllll
Fig. 2. Effect of 4-aminopyridine (4-AP, 7 x 10-4 M, serosal surface) on the short-circuit current (SCC) and on the potential difference (PD) of the isolated toad skin treated 15-20 rain previously with amiloride (1 x 10-4 M, mucosal surface). to 4-AP, which is in fact the case. Table 1 shows that dibenamine, an alpha adrenergic blocker, does not alter the effect of 4-AP on the skin. Thompson and Mills (1981, 1983) established that beta adrenergic agonists stimulate an amilorideinsensitive increase in current across the isolated frog skin, due to C1- transport by cells of the mucosal glands, innervated by postganglionic sympathetic fibres. Thus in our experiments, following the addition of 4-AP to the inner surface, the PD and the SCC increased significantly in skins pretreated (15-20min) with amiloride added to the mucosal surface (Fig. 2). That 4-AP exerts its action on the Ca 2+ channels of the presynaptic nerve terminals, and not by blocking K + channels of the sympathetic postganglionic fibres, is supported by the following findings: BaC12, a Ca2+-dependent K + channel blocker, and CsC1, a direct K ÷ channel blocker (Latorre and Miller, 1983; Schwarz and Passow, 1983; Kumamoto and Kuba, 1985) significantly decreased the bioelectric parameters of the toad skin, an effect opposite to that of 4-AP. Therefore, 4-AP in the concentration range used in this work does not block the K ÷ channels in the sympathetic fibres which innervate the skin. On the other hand, verapamil, a Ca 2÷ channel blocker significantly decreased the skin response to 4-AP: it is therefore possible that this drug acts presynaptically on the mechanism of noradrenaline release and not postsynaptically as is the case with propranolol which prevents the action of noradrenaline on the beta adrenergic receptors of the glands (Thompson and Mills, 1981). In addition, the Ca 2+ ionophore A-23187 produces an effect similar to that 4-AP. In preliminary experiments, we have found that the bioelectric responses to stimulation of the isolated nerve-skin preparation of the toad Caudiverbera caudiverbera, are significantly enhanced by the addition of 4-AP. These results are in agreement with our hypothesis and that of other authors (Rogawski and Barker, 1983) that 4-AP directly increases presynaptic nerve terminal permeability to Ca 2+, releasing noradrenaline which is responsible for the rise in PD and SCC in this skin preparation.
SUMMARY
1. This work is concerned with the hypothesis that 4-amino-pyridine directly promotes Ca 2+ conduc-
Acknowledgements--This work received financial support
from grants 20.33.35, 20.33.30 and 20.33.33, University of Concepci6n, Chile. The authors wish to thank Merck, Sharp and Dohme for the gift of amiloride. The authors also thank Mrs Maria Cecilia Nova and Mr Oscar Sepfilveda for excellent technical assistance. REFERENCES
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