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Caffeine blocks the delayed K+ outward current of molluscan neurons
480
Brain Research, 211 (1981) 480-484 © Elsevier/North-Holland Biomedical Press
Caffeine blocks the delayed K + outward current of molluscan neurons
A. HERMANN* Boston University, School of Medicine, Department of Physiology, 80 East Concord Street, Boston, Mass. 02118 (U.S.A.) (Accepted December 4th, 1980) Key words: caffeine - - delayed K + current - - molluscan neuron
Extracellular application of caffeine inhibits the delayed K + outward current of Aplysia neurons in a dose dependent manner without changing the kinetics. Half-maximum blockade is produced with a concentration of 16.0 ± 0.7 mM (S.E.M.) caffeine after 1-2 rain. Intracellular injection of caffeine has an almost immediate blocking effect. The evidence suggests that the blocking site is at or close to the inner surface of the cellular membrane. Caffeine is a well k n o w n stimulant which affects nerve a n d muscle cells a n d which is used as a therapeutic d r u g a n d as an ingredient o f widely c o n s u m e d beverages. I n muscle, caffeine facilitates the release o f calcium f r o m the sarcoplasmic reticulum a n d subsequently induces contractiong,16, 23. Caffeine a n d other methylxanthines are further k n o w n to inhibit p h o s p h o d i e s t e r a s e 5, the enzyme which is responsible for the b r e a k d o w n o f cyclic A M P ( c A M P ) . I n t r a c e l l u l a r a c c u m u l a t i o n o f c A M P is t h o u g h t to act on a variety o f bioelectrical processes (for review see ref. 2). The effect o f caffeine on nerve cells is, however, less well u n d e r s t o o d . In vertebrates, caffeine a n d its derivatives have the ability to p r o d u c e stimulation o f the central nervous system (for review see refs. 18 a n d 22), a n d cause rhythmic electrical activity in isolated s y m p a t h e t i c ganglion cells14,19. In molluscan neurons, it has been r e p o r t e d t h a t caffeine a n d other methylxanthines alter the r h y t h m i c discharge o f p a c e m a k e r cells 21, but it is n o t k n o w n whether this is a direct o r indirect effect on m e m b r a n e c o n d u c t a n c e . This r e p o r t shows t h a t caffeine blocks the delayed o u t w a r d p o t a s s i u m current o f molluscan neurons. The experiments were d o n e on identified n e u r o n s (R15, L 2 - L 6 , L11, LT) which are f o u n d in the a b d o m i n a l ganglion o f the mollusc, Aplysia californica. The cells were e x p o s e d to the external b a t h solution by r e m o v a l o f the connective tissue over the cells. In some experiments the axon o f the R15 cell was cut at a distance o f a b o u t 300-500 # m f r o m the s o m a to minimize the c o n t r i b u t i o n o f a x o n a l currents. There was no difference in the caffeine results with or without the a x o n attached. The e x p e r i m e n t a l * Present address for correspondence: University of Konstanz, Fakultat ffir Biologie, D-7750 Konstanz, G.F.R.
481 voltage-clamp and intracellular injection procedures have been described elsewhere 11. Caffeine (Sigma, St. Louis, U.S.A.) was exchanged for NaCI or its replacement in the artificial sea water (ASW) bathing media which contained (in mmol): 478 NaCI; 10 KCI; 10 CaC12; 55 MgC12; and 15 Tris.HC1 at pH 7.8. The temperature of the experimental bath was kept at 16 °C. Caffeine was injected intraceUularly by pressure or by electroosmosis 6 from microelectrodes filled with 0.05 M caffeine (pH = 7.3) or with 0.1 M caffeine and 0.05 M KCI. The molluscan nerve cell membrane exhibits a number of current components when studied under voltage-clamp conditions which have been related to the movement of Na +, K + and Ca 2+ ions 1. The delayed outward K + current can be studied separately in cells only where other currents are blocked. Na + currents are eliminated
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Fig. 1. Effect of external caffeine on the delayed outward K + current of the Aplysia pacemaker neuron RI5. A: outward K ÷ currents before and after addition of caffeine 0 0 mM) measured with brief depolarizing clamp pulses to the indicated voltages from a holding potential of --45 mV in a cell injected with EGTA (500 nA for 5 rain) and in Ca 2÷ and Na ÷ free ASW. The test pulses were given in one minute intervals 10 min after caffeine application. B: current-voltage relation of peak outward currents from the same cell as in A before (closed circles) and after addition of various amounts of caffeine (1 mM, open circles; 5 raM, closed triangles; 10 mM, open triangles; 25 mM, closed squares). The leakage current was subtracted from the data. The insert plot in B is a dose-response plot of the ratio of the peak outward K + current in caffeine (IK,eaffeine) to the control outward K + current (IK) versus the logarithm of the external caffeine concentration at V = ÷ 2 0 mV. The theoretical curve drawn through the experimental points was calculated using the relation Ir~, c~neine/ I K = Keaffeine / Keaffeine "+" [caffeine] where Keaffeine is the concentration of caffeine required to produce a 50 % block of the delayed outward K ÷ current and [caffeine] is the external caffeine concentration.
482 by the addition of external tetrodotoxin (TTX) or the replacement of external Na + ions by Tris or by sucrose. Ca 2+ currents and the CaZ+-activated K ÷ current are eliminated by the replacement of external Ca 2+ by Mg 2+ ions and/or by the internal injection of E G T A (ethylenglycol-bis (fl-amino-ethylether)-N,N'-tetraacetic acid). To inactivate the fast K ÷ outward current the cells were held at - - 4 5 mV 17. Under these conditions, the remaining current measured with brief, depolarizing voltage-clamp pulses is composed of the delayed outward K ÷ current and the leakage current. Fig. 1A illustrates the effect of 10 m M external caffeine solution on the delayed outward K + current measured at 3 voltages. The peak outward K + currents in caffeine are smaller than the corresponding control currents, but the time course of activation and of inactivation is not changed. The current-voltage relation for the same cell measured in different caffeine concentrations between 1 m M and 25 m M is shown in Fig. lB. There is a dose-dependent reduction of the delayed outward K ÷ current as the external caffeine concentration is increased. The experimental values at + 20 mV are replotted versus the external caffeine concentration in the inset plot shown in Fig. 1B. The curve fitted to the experimental values is drawn assuming that one caffeine molecule interacts with one receptor site. In 5 cells, the apparent dissociation constant for the reaction of caffeine with the receptor site occurred at a concentration of 16.0 -k 0.7 mM (S.E. of the mean). The block of the delayed outward K ÷ current, however, depends on membrane voltage. The apparent dissociation constant decreases at voltages between + 2 0 mV and about + 5 0 mV, but increases at more positive potentials. The time course of the inhibitory action of caffeine and the recovery from inhibition was studied with test pulses to + 2 0 mV applied once per minute. The time necessary for a 50 ~ block decreased with increasing caffeine concentrations and was 2.5 min in 1 m M and 1 min in 25 mM caffeine. Maximum effects were obtained after 5-15 min in caffeine solution. Recovery was slightly faster at low external concentrations, but was slower and often incomplete at higher concentrations. Caffeine is a neutral molecule which is known to diffuse through cell membranes 3 and it is possible that the site of action of caffeine is on the inner rather than the outer membrane surface. To test this possibility, caffeine was injected into cells and the delayed outward K ÷ current was measured with voltage-clamp pulses before, immediately after and at various times after caffeine injection (Fig. 2). Maximal reduction of the K ÷ current occurs immediately after injection. The amount of block and the time course of recovery depends on the quantity of caffeine injected but recovery was complete within at least 5 min. Methylxanthines are inhibitors of phosphodiesterase 5. It is possible therefore that the blocking action of caffeine on K ÷ currents is a result ofintracellular accumulation of cAMP. I B M X (isobuthylmethylxanthine) is a potent inhibitor of phosphodiesterase and increases cAMP in the Aplysia abdominal ganglion at concentrations between 10 -5 M and 3 × 10 -4 M 21. When I B M X was used up to 10 -3 M concentration it had, however, no significant effect on delayed K ÷ currents after test periods of 60 min. The results provide the first clear evidence that caffeine blocks the delayed K +
483
control
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5
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100 ms Fig. 2. Effect of internal caffeine on the delayed outward K + current. Outward K + current before (control), immediately after, 2 min and 5 min after electroosmotic injection of caffeine using an injection intensity of 1000 nA for 1 min. The cell was previously injected with EGTA (500 nA/5 min) and bathed in Caz÷ free ASW containing 5 × 10 ~ M TTX. The traces show the membrane currents measured with voltage pulses to +20 mV from a holding potential of 4 5 mV. RI5 cell.
conductance of the neuronal membrane. The relatively slow time course of external caffeine to achieve a maximum effect at a given concentration when contrasted to the immediate maximum effect obtained after injection of caffeine suggests that the drug may have to penetrate into or through the membrane to reach a blocking site which is at or close to the inner membrane surface, although it is not possible, using the present data, to exclude the possibility of separate receptor sites. The dependence of the block on membrane potential suggests further that this site may be within the membrane electrical field. IBMX alters bursting discharge of Aplysia R15 neurons and it has been suggested that an increase of cyclic nucleotide is involved in this action 21. There is, however, no effect of IBMX on K + outward current as reported here. This suggests that cAMP is not responsible for alternation of K ÷ conductance and that caffeine which is also known to increase intracellular cAMP but blocks K + current does not involve a cAMP mechanism of action. The results explain the prolongation of the molluscan pacemaker neuron action potential 11 which may have important consequences since an increase in action potential duration in these cells prolongs the influx of Ca 2+ ions and, thus causes a greater change in the intracellular Ca ~+ concentration 10. The results may also help to explain some of the effects of caffeine and other methylxanthines on excitable cells in other tissues. For example, in Limulus photoreceptors 7 the methylxanthines reduce voltage dependent outward currents, and in heart muscle8,13 caffeine increases action potential duration. Both effects could be caused by the inhibition of the delayed K + outward current. Caffeine and other methylxanthines appear to have multiple actions on K + conductance. In sympathetic ganglion cells there is evidence that caffeine 15 and the related methylxanthine, theophylline4,20, increase the Ca 2+ activated K + current. Caffeine has a similar effect on the Ca 2÷ activated K + current of molluscan cells, but the effect takes some time to developlL The voltage dependent and Ca 2+ activated potassium conductances are involved in the regulation of the excitability and the
484 r e p e t i t i v e d i s c h a r g e o f n e u r o n s . T h e effects o f caffeine o n t h e s e t w o systems is likely to p r o v i d e a basis f o r its c o m p l e x a c t i o n o n n e u r o n a l excitability in v e r t e b r a t e a n d i n v e r t e b r a t e n e r v o u s systems. T h i s w o r k was s u p p o r t e d by U S P H S G r a n t N S 11429 a n d D F G G r a n t S F B 138. ! a m g r a t e f u l to D r . A. L. F. G o r m a n f o r v a l u a b l e c o m m e n t s o n the m a n u s c r i p t .
1 Adams, D. J., Smith, S. J. and Thompson, S. H., Ionic currents in molluscan soma, Ann. Rev. Neurosci., 3 (1980) 141-167. 2 Berridge, M. J., Modulation of nervous activity by cyclic nucleotides and calcium. In F. O. Schmitt (Ed.), The Neurosciences, 4th Study Program, 1979, pp. 873-879. 3 Bianchi, C. P., Kinetics of radiocaffeine uptake and release in frog Sartorius, J. Pharmacol. exp. Ther., 138 (1962) 41-47. 4 Busis, N. A. and Weight, F. F., Spike after-hyperpolarization of a sympathetic neurone is calcium sensitive and is potentiated by theophylline, Nature (Lond.), 263 (1976) 434-436. 5 Butcher, R. W. and Sutherland, E. W., Adenosine 3 ;5'-phosphate in biological materials - - I. Purification and properties of cyclic 3 ;5'-nucleotide phosphodiesterase and the use of this enzyme to characterize adenosine 3 ;5'-phosphate in human urine, J. bioL Chem., 237 (1962) 1244-1250. 6 Chiarandini, D. J., Reuben, J. P., Brandt, P. W. and Grundfest, H., Effects of caffeine on crayfish muscle fibres, J. gen. Physiol., 55 (1970) 640-664. 7 Corson, D. W., Fein, A. and Schmidt, J., Two effects of phosphodiesterase inhibitors on Limulus ventral photoreceptors, Brain Research, 176 (1979) 365-368. 8 De Gubareff, T. and Sleator, W. Jr., Effects of caffeine on mammalian atrial muscle, and its interaction with adenosin and calcium, J. Pharmacol. exp. Ther., 148 (1965) 202-214. 9 Endo, M., Tanaka, M. and Ogawa, J., Calcium induced release of calcium from the sarcoplasmatic reticulum of skinned skeletal muscle fibres, Nature (Lond.), 228 (1970) 34-36. 10 Gorman, A. L. F. and Thomas, M. V., Changes in the intracellular concentration of free calcium ions in a pacemaker neurone, measured with the metallochromic indicator dye arsenazo 1II, J. PhysioL (Lond.), 275 (1978) 357-376. 11 Hermann, A. and Gorman, A. L. F., Blockade of voltage-dependent and Ca~+-dependent K + current components by internal Ba 2+ in molluscan pacemaker neurons, Experientia (Basel), 35 (1979) 229-231. 12 Hermann, A. and Gorman, A. L. F., Dual action of caffeine on K+-conductance, Pfliiger's Arch., 483 (1980) R-12. 13 Kimoto, Y., Saito, M. and Goto, M., Effects of caffeine on the membrane potentials membrane currents and contractility of bull-frog atrium, Jap. J. Physiol., 24 (1974) 531-542. 14 Kuba, K. and Nishi, S., The rhythmic hyperpolarizations and the depolarization of sympathetic ganglion cells induced by caffeine, J. Neurophysiol., 39 (1976) 547-563. 15 Kuba, K., Release of calcium ions linked to the activation of potassium conductance in a caffeinetreated sympathetic neurone, J. Physiol. (Lond.), 298 (1980) 251-269. 16 L0ttgau, H. C. and Oetliker, H., The action of caffeine on the activation of the contractile mechanism in striated muscle fibres, J. Physiol. (Lond.), 194 (1968) 51-74. 17 Neher, E., Two fast transient current components during voltage clamp on snail neurons, J. gem Physiol., 58 (1971) 36-53. 18 Ritchie, J. M., The xanthines. In L. S. Goodman and A. Gilman (Eds.), The Pharmacological Basis of Therapeutics, 5th edn., Macmillan, N.Y., 1975, rip. 358-370. 19 Skok, V. J., Storch, N. N. and Nishi, S., The effect of caffeine on the neurons of a mammalian sympathetic ganglion, Neuroscience, 3 (1978)697-708. 20 Smith, P. A., Weight, F. F. and Lehne, R. A., Potentiation of Ca2+-dependent K+-activation by theophylline is independent of cyclic nucleotide elevation, Nature (Lond.), 280 (1979) 400-402. 21 Treistman, S. N. and Levitan, J. B., Alteration of electrical activity in molluscan neurons by cyclic nucleotides and peptide factors, Nature (Lond.), 261 (1976) 62-64. 22 Truitt, E. B., Jr., The xanthines. In J. R. Di Palma (Ed.), Drill's Pharmacology in Medicine, McGraw-Hill, N. Y., 1970, pp. 533-556. 23 Weber, A. and Herz, R., The relationship between caffeine contracture of intact muscle and the effect of caffeine on reticulum, J. gen. Physiol., 52 (1968) 750-759.
Brain Research, 211 (1981) 480-484 © Elsevier/North-Holland Biomedical Press
Caffeine blocks the delayed K + outward current of molluscan neurons
A. HERMANN* Boston University, School of Medicine, Department of Physiology, 80 East Concord Street, Boston, Mass. 02118 (U.S.A.) (Accepted December 4th, 1980) Key words: caffeine - - delayed K + current - - molluscan neuron
Extracellular application of caffeine inhibits the delayed K + outward current of Aplysia neurons in a dose dependent manner without changing the kinetics. Half-maximum blockade is produced with a concentration of 16.0 ± 0.7 mM (S.E.M.) caffeine after 1-2 rain. Intracellular injection of caffeine has an almost immediate blocking effect. The evidence suggests that the blocking site is at or close to the inner surface of the cellular membrane. Caffeine is a well k n o w n stimulant which affects nerve a n d muscle cells a n d which is used as a therapeutic d r u g a n d as an ingredient o f widely c o n s u m e d beverages. I n muscle, caffeine facilitates the release o f calcium f r o m the sarcoplasmic reticulum a n d subsequently induces contractiong,16, 23. Caffeine a n d other methylxanthines are further k n o w n to inhibit p h o s p h o d i e s t e r a s e 5, the enzyme which is responsible for the b r e a k d o w n o f cyclic A M P ( c A M P ) . I n t r a c e l l u l a r a c c u m u l a t i o n o f c A M P is t h o u g h t to act on a variety o f bioelectrical processes (for review see ref. 2). The effect o f caffeine on nerve cells is, however, less well u n d e r s t o o d . In vertebrates, caffeine a n d its derivatives have the ability to p r o d u c e stimulation o f the central nervous system (for review see refs. 18 a n d 22), a n d cause rhythmic electrical activity in isolated s y m p a t h e t i c ganglion cells14,19. In molluscan neurons, it has been r e p o r t e d t h a t caffeine a n d other methylxanthines alter the r h y t h m i c discharge o f p a c e m a k e r cells 21, but it is n o t k n o w n whether this is a direct o r indirect effect on m e m b r a n e c o n d u c t a n c e . This r e p o r t shows t h a t caffeine blocks the delayed o u t w a r d p o t a s s i u m current o f molluscan neurons. The experiments were d o n e on identified n e u r o n s (R15, L 2 - L 6 , L11, LT) which are f o u n d in the a b d o m i n a l ganglion o f the mollusc, Aplysia californica. The cells were e x p o s e d to the external b a t h solution by r e m o v a l o f the connective tissue over the cells. In some experiments the axon o f the R15 cell was cut at a distance o f a b o u t 300-500 # m f r o m the s o m a to minimize the c o n t r i b u t i o n o f a x o n a l currents. There was no difference in the caffeine results with or without the a x o n attached. The e x p e r i m e n t a l * Present address for correspondence: University of Konstanz, Fakultat ffir Biologie, D-7750 Konstanz, G.F.R.
481 voltage-clamp and intracellular injection procedures have been described elsewhere 11. Caffeine (Sigma, St. Louis, U.S.A.) was exchanged for NaCI or its replacement in the artificial sea water (ASW) bathing media which contained (in mmol): 478 NaCI; 10 KCI; 10 CaC12; 55 MgC12; and 15 Tris.HC1 at pH 7.8. The temperature of the experimental bath was kept at 16 °C. Caffeine was injected intraceUularly by pressure or by electroosmosis 6 from microelectrodes filled with 0.05 M caffeine (pH = 7.3) or with 0.1 M caffeine and 0.05 M KCI. The molluscan nerve cell membrane exhibits a number of current components when studied under voltage-clamp conditions which have been related to the movement of Na +, K + and Ca 2+ ions 1. The delayed outward K + current can be studied separately in cells only where other currents are blocked. Na + currents are eliminated
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Fig. 1. Effect of external caffeine on the delayed outward K + current of the Aplysia pacemaker neuron RI5. A: outward K ÷ currents before and after addition of caffeine 0 0 mM) measured with brief depolarizing clamp pulses to the indicated voltages from a holding potential of --45 mV in a cell injected with EGTA (500 nA for 5 rain) and in Ca 2÷ and Na ÷ free ASW. The test pulses were given in one minute intervals 10 min after caffeine application. B: current-voltage relation of peak outward currents from the same cell as in A before (closed circles) and after addition of various amounts of caffeine (1 mM, open circles; 5 raM, closed triangles; 10 mM, open triangles; 25 mM, closed squares). The leakage current was subtracted from the data. The insert plot in B is a dose-response plot of the ratio of the peak outward K + current in caffeine (IK,eaffeine) to the control outward K + current (IK) versus the logarithm of the external caffeine concentration at V = ÷ 2 0 mV. The theoretical curve drawn through the experimental points was calculated using the relation Ir~, c~neine/ I K = Keaffeine / Keaffeine "+" [caffeine] where Keaffeine is the concentration of caffeine required to produce a 50 % block of the delayed outward K ÷ current and [caffeine] is the external caffeine concentration.
482 by the addition of external tetrodotoxin (TTX) or the replacement of external Na + ions by Tris or by sucrose. Ca 2+ currents and the CaZ+-activated K ÷ current are eliminated by the replacement of external Ca 2+ by Mg 2+ ions and/or by the internal injection of E G T A (ethylenglycol-bis (fl-amino-ethylether)-N,N'-tetraacetic acid). To inactivate the fast K ÷ outward current the cells were held at - - 4 5 mV 17. Under these conditions, the remaining current measured with brief, depolarizing voltage-clamp pulses is composed of the delayed outward K ÷ current and the leakage current. Fig. 1A illustrates the effect of 10 m M external caffeine solution on the delayed outward K + current measured at 3 voltages. The peak outward K + currents in caffeine are smaller than the corresponding control currents, but the time course of activation and of inactivation is not changed. The current-voltage relation for the same cell measured in different caffeine concentrations between 1 m M and 25 m M is shown in Fig. lB. There is a dose-dependent reduction of the delayed outward K ÷ current as the external caffeine concentration is increased. The experimental values at + 20 mV are replotted versus the external caffeine concentration in the inset plot shown in Fig. 1B. The curve fitted to the experimental values is drawn assuming that one caffeine molecule interacts with one receptor site. In 5 cells, the apparent dissociation constant for the reaction of caffeine with the receptor site occurred at a concentration of 16.0 -k 0.7 mM (S.E. of the mean). The block of the delayed outward K ÷ current, however, depends on membrane voltage. The apparent dissociation constant decreases at voltages between + 2 0 mV and about + 5 0 mV, but increases at more positive potentials. The time course of the inhibitory action of caffeine and the recovery from inhibition was studied with test pulses to + 2 0 mV applied once per minute. The time necessary for a 50 ~ block decreased with increasing caffeine concentrations and was 2.5 min in 1 m M and 1 min in 25 mM caffeine. Maximum effects were obtained after 5-15 min in caffeine solution. Recovery was slightly faster at low external concentrations, but was slower and often incomplete at higher concentrations. Caffeine is a neutral molecule which is known to diffuse through cell membranes 3 and it is possible that the site of action of caffeine is on the inner rather than the outer membrane surface. To test this possibility, caffeine was injected into cells and the delayed outward K ÷ current was measured with voltage-clamp pulses before, immediately after and at various times after caffeine injection (Fig. 2). Maximal reduction of the K ÷ current occurs immediately after injection. The amount of block and the time course of recovery depends on the quantity of caffeine injected but recovery was complete within at least 5 min. Methylxanthines are inhibitors of phosphodiesterase 5. It is possible therefore that the blocking action of caffeine on K ÷ currents is a result ofintracellular accumulation of cAMP. I B M X (isobuthylmethylxanthine) is a potent inhibitor of phosphodiesterase and increases cAMP in the Aplysia abdominal ganglion at concentrations between 10 -5 M and 3 × 10 -4 M 21. When I B M X was used up to 10 -3 M concentration it had, however, no significant effect on delayed K ÷ currents after test periods of 60 min. The results provide the first clear evidence that caffeine blocks the delayed K +
483
control
0
2
5
[
lpA
I
I
100 ms Fig. 2. Effect of internal caffeine on the delayed outward K + current. Outward K + current before (control), immediately after, 2 min and 5 min after electroosmotic injection of caffeine using an injection intensity of 1000 nA for 1 min. The cell was previously injected with EGTA (500 nA/5 min) and bathed in Caz÷ free ASW containing 5 × 10 ~ M TTX. The traces show the membrane currents measured with voltage pulses to +20 mV from a holding potential of 4 5 mV. RI5 cell.
conductance of the neuronal membrane. The relatively slow time course of external caffeine to achieve a maximum effect at a given concentration when contrasted to the immediate maximum effect obtained after injection of caffeine suggests that the drug may have to penetrate into or through the membrane to reach a blocking site which is at or close to the inner membrane surface, although it is not possible, using the present data, to exclude the possibility of separate receptor sites. The dependence of the block on membrane potential suggests further that this site may be within the membrane electrical field. IBMX alters bursting discharge of Aplysia R15 neurons and it has been suggested that an increase of cyclic nucleotide is involved in this action 21. There is, however, no effect of IBMX on K + outward current as reported here. This suggests that cAMP is not responsible for alternation of K ÷ conductance and that caffeine which is also known to increase intracellular cAMP but blocks K + current does not involve a cAMP mechanism of action. The results explain the prolongation of the molluscan pacemaker neuron action potential 11 which may have important consequences since an increase in action potential duration in these cells prolongs the influx of Ca 2+ ions and, thus causes a greater change in the intracellular Ca ~+ concentration 10. The results may also help to explain some of the effects of caffeine and other methylxanthines on excitable cells in other tissues. For example, in Limulus photoreceptors 7 the methylxanthines reduce voltage dependent outward currents, and in heart muscle8,13 caffeine increases action potential duration. Both effects could be caused by the inhibition of the delayed K + outward current. Caffeine and other methylxanthines appear to have multiple actions on K + conductance. In sympathetic ganglion cells there is evidence that caffeine 15 and the related methylxanthine, theophylline4,20, increase the Ca 2+ activated K + current. Caffeine has a similar effect on the Ca 2÷ activated K + current of molluscan cells, but the effect takes some time to developlL The voltage dependent and Ca 2+ activated potassium conductances are involved in the regulation of the excitability and the
484 r e p e t i t i v e d i s c h a r g e o f n e u r o n s . T h e effects o f caffeine o n t h e s e t w o systems is likely to p r o v i d e a basis f o r its c o m p l e x a c t i o n o n n e u r o n a l excitability in v e r t e b r a t e a n d i n v e r t e b r a t e n e r v o u s systems. T h i s w o r k was s u p p o r t e d by U S P H S G r a n t N S 11429 a n d D F G G r a n t S F B 138. ! a m g r a t e f u l to D r . A. L. F. G o r m a n f o r v a l u a b l e c o m m e n t s o n the m a n u s c r i p t .
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