Apartado 1827, Caracas 10, Venezuela (Received 25 June 1975)

Transcription

1 J. Phy8iol. (1976), 255, pp With 1 plate and 9 text-ftgure8 Printed in Great Britain THE EFFECT OF CAFFEINE AND TETRACAINE ON THE TIME COURSE OF POTASSIUM CONTRACTURES OF SINGLE MUSCLE FIBRES BY CARLO CAPUTO From the Centro de Biofisica y Bioquimica, Instituto Venezolano de Investigaciones Cientificas (I VIC), Apartado 1827, Caracas 10, Venezuela (Received 25 June 1975) SUMMARY 1. The time course of potassium contractures can be significantly prolonged by low concentrations of caffeine. 2. This effect of caffeine is not due to impairment of the fibre relaxing system. 3. Under conditions where contractile repriming is delayed (low temperature) an extra amount of activator can be released by caffeine, in addition to that released by potassium. 4. The source of this extra amount of activator is intracellular since its release can be shown in a 0 calcium EGTA medium. 5. Local anaesthetics, tetracaine, and to a lesser extent procaine, affect the release of contractile activator, without impairing the contractile machinery itself. 6. The results of the present paper support the view that the time course of potassium contracture is controlled by a membrane mechanism which is activated upon depolarization and later inactivates with time. 7. The effect of caffeine and local anaesthetics can be explained by assuming that the former prolongs the inactivation time course while the latter shortens it. INTRODUCTION Since the work of Hodgkin & Horowicz (1960), potassium contractures have been used as a convenient tool for the study of some of the phenomena involved in excitation-contraction coupling of striated muscle. It has been shown (Caputo, 1972b) that during a potassium contracture, release of the contractile activator proceeds continuously, suggesting that the time course of these responses represents roughly the time course of release of the contractile activator. Thus, under normal conditions, it 7 PHY 255

2 192 C. CAPUTO appears that the spontaneous relaxation of the potassium contractures is due to the ceasing of the activator release. Therefore, the characteristic phasic shape of the potassium contractures can be explained by assuming, either that the intracellular activator store is depleted during these responses, or that the activator release mechanism is controlled by a membrane process which is potential and time dependent (Hodgkin & Horowicz, 1960; Caputo, 1972b). It is known that the time course of potassium contractures can be changed either way by different experimental procedures. It can be shortened by exposing the fibres to low calcium solutions (Frank, 1960; Liittgau, 1963; Caputo & Gimenez, 1967) to local anaesthetics (Caputo, 1972b), or following denervation (Stuesse, Lindley & Kirby, 1974), or on the contrary, it can be prolonged by exposing the fibres to high calcium (Frankenhauser & Lannergreen, 1967), to drugs like chlorpromazine and imiprami'ne (Andersson, 1972), or to relatively low concentrations of lanthanum (Andersson & Edman, 1974). It seems of interest to know the mechanism by which these changes occur. In principle, these changes may be due to an enhanced or diminished sequestering action of the contractile activator by the sarcoplasmic reticulum, to a change in the size of the calcium pool available for release, or to an effect on the mechanism that regulates the release of the contractile activator. Littgau & Oetliker (1968) have shown that caffeine at low concentrations shifts toward lower potassium concentration the curve relating peak contracture tension to external potassium, while the opposite effect is caused by procaine (Etzensperger, 1970). These results could mean that these drugs may change the amount of contractile activator released during a potassium contracture. It was, therefore, thought of interest to test the effect of these drugs on the time course of potassium contracture induced with maximal concentration of potassium. In this work, we have used caffeine to prolong the time course of potassium contractures, and local anaesthetics, tetracaine and procaine to shorten it. A preliminary account of some of the experiments reported here has appeared elsewhere (Caputo, 1973). METHODS Single muscle fibres dissected from the semitendinosus or ileofibularis muscles of Rana pipien8 and Leptodactylu& in&sulari were used in the experiments reported here. The dissection technique, handling of the fibres and experimental procedures were similar to those already described in detail (Caputo, 1972a). The use of the fibres dissected from the South American frog L. insulari8 was restricted to experiments carried out at room temperature (200 C) since at low temperatures (< 90 C) a marked decrease of contractile ability occurs in these fibres (C. Caputo, unpublished experiment). With the exception of this temperature's effect, the behaviour of the fibres

3 CAFFEINE AND TETRACAINE ON K-CONTRACTURES 193 dissected from the two different species, in response to the experimental treatments described in this work, was similar. The composition of the normal Ringer and 190 mm potassium solutions was the same as used previously (Caputo, 1972a, Table 1). To these solutions caffeine, procaine or tetracaine was added. For the experiments carried out in low calcium, the same solutions were used except that calcium was not added. In this case, the contaminant calcium concentration was about 10 /M. When 1 mm-egta is added it can be estimated that the free calcium concentration is about 2 x 10-9 M (Armstrong, Bezanilla & Horowicz, 1972). For the experiments carried out with skinned fibres, a procedure similar to that described by Julian (1971) was followed. In short, the fibres were first exposed to the 190mm potassium contracture solution, then bathed for 60min in solution containing 47-5 % glycerol, EGTA 2 m-mole/l., and Tris buffer ph 7 10 m-mole/l. After this, they were exposed for 60 min to a solution containing 100 KCL m-mole/l., EGTA 2 m-mole/l., ATP 4 m-mole/l., MgC12 1 m-mole/l., and Lubrol W.X (Sigma) 5 g/l. From this solution the fibres were passed to a relaxing solution which had a similar composition to the preceding one, except that Lubrol was omitted. The contraction-inducing solution was prepared adding enough calcium to lower the pca to about 6*0. RESULTS The effect of caffeine Andersson (1972) has shown that compounds like chlorpromazine, imipramine and quinidine prolong the time course of potassium contractures. Text-fig. 1 shows that caffeine at 1 mm concentration, which by itself does not induce tension, also prolongs the duration of a contracture induced by 190 mm potassium. The increased duration of the response is mostly due to prolongation of the plateau phase of the contracture, while the fast relaxation phase is slowed down only to a small extent, thus contributing very little to the increase of the area under the tension curve, which is this case amounts to 40 % of the normal value. With higher caffeine concentration a greater effect on the rapid relaxation phase was observed. For four fibres similarly treated, the mean increase in the area under the tension curve amounted to 48+7 % (mean + S.E. of mean). It is possible that higher caffeine concentration could have produced a greater effect, but the lower concentration was chosen in order to be well below the threshold value for caffeine contracture. It is possible to explain the action of caffeine and of other agents that prolong the duration of the potassium contractures, if it is assumed that these agents inhibit the active calcium uptake by the sarcoplasmic reticulum (Weber & Hertz, 1968). However, a more likely explanation is that caffeine affects the mechanism of release of the contractile activator. Although it could be argued that more than one source of contractile activator is present inside the fibre (for instance, the mitochondrial system), most of the evidence available indicates that the caffeine releases calcium from the sarcoplasmic reticulum (Weber & Hertz, 1968; Ogawa, 7-2

4 194 C. CAPUTO 1970). There are several reasons favouring the hypothesis that caffeine affects the mechanism of release of the contractile activator. One of these is the observation that most of the effect of caffeine at low concentrations is on the duration of the plateau phase, while the rapid relaxation phase is affected to a lesser degree. Another reason stems from the observation of Luttgau & Oetliker (1968) that short tetani can be superimposed upon caffeine contractures, showing that muscle fibres are capable of relaxation even when exposed to higher caffeine concentrations. _ NR li 100 mg +1 mm caf. 180" NR+1 mm caf. r.. 2 sec Text-fig. 1. Prolongation of the time course of potassium contracture of single muscle fibre by 1 mm caffeine. The upper record shows a normal response (NR) to 190 /tm potassium (). The lower record shows the response in the presence of 1 mm caffeine. Notice that the main effect of caffeine is on the plateau phase of the response. R. pipes fibre; diameter 70 AM. Temperature 200 C. Confirming these results of Luttgau & Oetliker (1968), Text-fig. 2 shows some experiments carried out superimposing potassium contractures upon caffeine contractures. Different runs are shown in which the fibres had reached different levels of contractile activation in response to caffeine. It is evident that the extra mount of tension due to increased potassium, depends on the level of activation due to caffeine alone. It is also clear, that the extra tension due to potassium is not maintained, but, as in the case of superimposed tetani, it is followed by partial relaxation. Finally, a third reason that supports the hypothesis that caffeine affects the release mechanism is also derived from the finding of Luttgau & Oetliker (1968) that caffeine contractures can be cut short by adding local anaesthetics to the contracture medium. It has been shown before

5 CAFFEINE AND TETRACAINE ON K-CONTRACTURES 195 (Caputo, 1972b) that local anaesthetics also cut potassium contractures short, presumably by interfering with the release ofthe contractile activator. (This finding will be corroborated later.) Text-fig. 3 and Table 1 show the effect of 0 5 mm tetracaine on potassium and caffeine contractures of different fibres. In order to facilitate the solution change (addition of tetracaine) while the contractures were in progress, these runs were carried out at 190 K Caffeinef 2F.5lk K 2-5 I5LL Caffeine <1 K~~~~~~~~~~9 0 Caffeine 190 fs1m0./--~~~~~~~~~ I~mg 10 sec Text-fig. 2. Superposition of potassium contracture upon caffeine contractures in three different fibres dissected from L. insularis. The records on the left show the response to 190 mm potassium. The records on the right show the response to caffeine and then to 190 mm potassium in the presence of caffeine. The fibre diameters were 80, 70, and 100,tm, respectively for the three runs starting from the top. Temperature 200 C. 40 C (Caputo, 1972a). From this Text-figure it appears that in some fibres, after the addition of tetracaine, there is a longer delay for the onset of relaxation in the case of caffeine contractures. However, as better shown in Table 1, once the rapid relaxation phase begins, it proceeds with similar rates for both potassium and caffeine contractures showing that the fibre's capacity to relax is the same in both cases. According to the evidence presented above, the effect of caffeine on the

6 196 C. CAPUTO flo0 mg 1r90 K -I. I100 mg Caffeine 4 m Caffeine 4 m Tetrac. 0-5m -- I I I I Sec Sec. -~~-I10~I00 mg Tetrac. 0-5 m- Tetrac. 0*5 m I100 mg Caffeine 4 m Tetrac. 0-5 m Caffeine 4 m Tetrac. 0-5 m 0 I I I I S Sec Sec. Text-fig. 3. Action of tetracaine on potassium (upper) and caffeine (lower) contractures in four different fibres from R. pipien8. Addition of tetracaine (Tetrac.) caused faster relaxation. Had tetracaine not been added, potassium contractures would have lasted up to 80 sec, and caffeine contractures much longer (see Caputo, 1972b). Temperature 30 C. TABLE 1. Rates of the terminal phase of relaxation of potassium and caffeine contractures at 40 C in the presence of tetracaine (0.5 mm) Rates of relaxation Fibre Mean K (190 mm) contracture *042 0* Caffeine (4 mm) contracture 0* * *048

7 CAFFEINE AND TETRACAINE ON K-CONTRACTURES 197 time course of the potassium contracture can be explained, either by assuming that more activator is released, or that the same amount of activator is released in a more efficient way. Implicit in the first alternative is the assumption that the store of contractile activator available for release is not completely depleted during a normal potassium contracture. The availability of extra amounts of contractile activator in the fibre can be evidenced by experiments of the type shown in Text-fig. 4. It has been shown before (Caputo, 1972b) that, at low temperature potassium contractures can be interrupted for reasonably long time intervals and then resumed again by appropriate changes in the external potassium concentration. The upper record of Text-fig. 4 shows a potassium P_.C K 2b K 2- r-'-j-~ fioo mg K 25J Caffeine S 2L I a I I I I jii I m Sec Text-fig. 4. Interposition of a caffeine contracture between the two portions of an interrupted potassium contracture. The upper record shows an uninterrupted potassium contracture at 4 C. The centre record shows a response interrupted and resumed again by proper changes in the external potassium concentration. The lower record shows that a caffeine contracture, interposed between the two portions of the interrupted response does not affect the tension and the time course of the second portion of the interrupted contracture. R. pipiens fibre; diameter 80,m. Temperature 40 C.

8 19 8 C. CAPUTO contracture induced at 4 C. The second record shows an interrupted potassium contracture. Finally, the third record shows that exposing the fibre to 2 5 mm caffeine during the interval between the two portions of the interrupted potassium contracture causes the fibre to develop tension and that after withdrawal of the drug, the ability of the fibre to respond to the high potassium solution is not impaired. In fact, in this case, the ratios of the areas under the tension due to potassium for the three cases is :1: 1 1: 1. From the third record of Text-fig. 4, it is evident that in addition to the activator released by membrane depolarization, there is plenty more that can be released by the action of caffeine. The effect of caffeine in the absence of external calcium The increased duration of potassium contractures in the presence of caffeine could be alternatively explained if it is assumed that extra activator is made available to the fibre from the external medium. In fact, it is known that caffeine increases the fibre membrane permeability to calcium (Bianchi, 1961). However, this explanation is not correct since this particular effect of caffeine is maintained in the absence of external calcium, as shown below. The time course of potassium contractures can be shortened by exposure of the fibres to media prepared without calcium (Frank, 1960; Littgau, 1963; Caputo & Gimenez, 1967). It is possible to discard the idea that this shortening of the response may be due to reduced calcium entry during membrane depolarization. This can be done by considering the fact that the original time course of these responses can be partially restored by several experimental procedures (Caputo, 1968, 1972b) and taking into account the observation that the contractile response to electrical stimulation can be maintained for a relatively long time in media in which the external calcium has been reduced to very low values (10-9 M) by adding EGTA (Armstrong et al. 1972). Tension development in response to increased potassium can also be maintained in media prepared with 0 calcium and 1 mm-egta. As shown in Text-fig. 5, the effect of this drastic calcium reduction is mainly on the time course of the contractures, confirming earlier results obtained without adding EGTA (Caputo & Gimenez, 1967). As can be observed in the records, a partial recovery of the time course of the response can be obtained by adding caffeine at low concentrations (0 5 mm). The extent of restoration depends on the period of exposure of the fibre to the low calcium medium. Probably a more complete restoration could be achieved using higher caffeine concentrations; however, for the purpose of this work, it was preferred to maintain the drug concentration well below the threshold value for caffeine contractures. What is important to notice is that caffeine greatly

9 CAFFEINE AND TETRACAINE ON K-CONTRACTURES 199 increases the area under the tension curve of potassium contractures induced in the virtual absence of external calcium. The results of these experiments, confirmed with several other fibres, can be utilized to stress two points: the first deals with the effect of caffeine on the time course of potassium contractures, which is shown to be independent of the presence --Y-~%%%%~--~-- I100 mg L I100 mg Ca I mm-egta 60" 0 Ca 1 mm -EGTA OCa1 mm-egta ~~~~ 1C 05mMcaf. - 60" 0 Ca 1 mm -EGTA 0 5 mm caf. I I I I I Sec I I I I I Sec Text-fig. 5. Action of caffeine on potassium contractures of two different fibres in the virtual absence of external calcium. In the experiment shown on the left side of the Text-fig., the fibre was not pre-exposed to 0 calcium EGTA or to 0 calcium EGTA caffeine media before inducing the potassium contracture. In the experiment shown on the right, the fibre was exposed for 60 sec to those solutions before inducing the potassium contracture. Fibres from L. insularis. Fibre diameters 75 and 110 Iam, for the runs on the left and right side respectively. Temperature 200 C. of external calcium; the second confirms the earlier conclusion that the action of low calcium on the time course of potassium contractures is most probably due to an effect upon the mechanism regulating the release of contractile activator and not due to depletion of an intracellular store of calcium ions. The maintenance of the fibre's ability to develop tension in response to potassium in media with no calcium and with EGTA is apparently contrary to the conclusion of Stefani & Chiarandini (1973). Although their own records (their Fig. 1) show that even their fibres had partially retained the ability to develop tension, it was thought of interest to investigate this matter further. To explain the discrepancy between

10 200 C. CAPUTO their results and those of Armstrong et al. (1972), Stefani & Chiarandini (1973) postulated that even in the presence of 1 mm-egta, there might be a small amount of calcium bound to the fibie membrane, which could be involved in the contractile response in the experiments of Armstrong et al. (1972). Since in their own experiments the low calcium solutions contained magnesium, they assumed that this cation could displace the residual calcium bound to the membrane, thus causing the loss of the contractile response. 100 mg K K K 190 3jj-i 60' 0 Ca 1 mm-egta K "0 Ca5 mm-mg 1 mm-egta I 190 I I I I I Sec I I I I I Sec Text-fig. 6. Effect of 0 calcium 1 mm-egta on potassium contracture in the absence and presence of 5 mm magnesium. Fibre from R. pipiens, diameter 80 jam. Temperature 200 C. In view of the importance of this point in relation to the current ideas on the mechanism involved in contractile activation, it was decided to test the effect of 0 calcium EGTA solutions on the fibre's ability to develop potassium contractures in the presence and in the absence of 5 mm magnesium. Text-fig. 6 shows one of these experiments. Two contractures are shown on the left side: the upper one obtained under normal conditions, the lower one obtained after 60 see exposure to the low calcium medium. On the iight side, two contractures from the same fibre are also shown: the upper one as a control and the lower one obtained after 60 see exposure to the low calcium 5 mm magnesium medium. It is evident that magnesium does not impair tension development. On the contrary, it partially reverses the effect of low calcium, prolonging the response duration. This effect repeated with several other fibres is similar to that obtained with other divalent cations used as substitutes for calcium ions in the absence of EGTA (Frank, 1962).

11 CAFFEINE AND TETRACAINE ON K-CONTRACTURES 201 The effect of local anaesthetics It has been shown above that tetracaine at low concentrations induces prompt relaxation of potassium and caffeine contractures at low temperature. Here we present additional information on the mode of action of local anaesthetics. Text-fig. 7 shows the effect of tetracaine on the time course and of tension development of potassium contractures at 40 C. The left side of the 190'K 1100 mg 190K A>~T I100 mg NR 05 mm Tetrac. NR1 mmtetrac. NR 0 5 mm Tetrac. NR 1 mm Tetrac. I I I I Sec VI Sec I I Text-fig. 7. Effect of tetracaine on the time course of potassium contracture of two fibres from R. pipien8. Fibre diameter 70 and 80,sm for the runs at the left and right side respectively. Temperature 70 C. NR, normal Ringer. Text-figure shows the effect of exposing the fibre to 0 5 mm tetracaine for about 10 see before inducing a contracture. This treatment shortens the duration of the contracture without affecting tension development. It is also shown that the tetracaine effect disappears when the drug is washed out about 15 see before inducing the contracture. On the right side of the Text-fig., the effect of 1 mm tetracaine on another fibre is shown. In this case, tension development is practically abolished, although the effect is equally reversible after a short recovery time in normal Ringer. In other similar experiments, it was found that longer exposure to the drug affected tension development even at a lower concentration.

12 202 C. CAPUTO Text-fig. 8 shows that procaine at much higher concentrations (10 mm) exerts similar effects shortening the time course of potassium contractures induced with either 190 or 40 mm potassium at 200 C. Although these experiments appear to indicate that the main effect of these local anaesthetics is to reduce the amount of contractile activator liberated during a response, it is necessary to prove that these drugs do not directly affect the contractile machinery itself. J I100 mg K 2 - K 2-5 K " 10 mm Procaine 60" 10 mm Procaine Sec Sec Text-fig. 8. Effect of procaine on the time course of contractures induced with 40 or 190 um potassium. Fibre from R. pipien8. Diameter 70,tm. Temperature 200C. Ford & Podolsky (1972) have reported that 2 mm procaine does not affect tension development in mechanically skinned muscle fibres exposed to a solution with a pca of 6-5. Text-fig. 9 shows that tetracaine is similarly ineffective in blocking tension development in chemically skinned fibres in response to a low pca solution. For these experiments a procedure similar to that described by Julian (1971) was used since it appears that in chemically skinned fibres the sarcoplasmic reticulum capacity for storing calcium is abolished (Julian, 1971) contrary to what seems to be the case for mechanically skinned fibres (Hellam & Podolsky, 1969). The first record of each row in Text-fig. 9 shows a potassium contracture obtained with the intact fibre. Each row corresponds to a different fibre. After the potassium contracture, each fibre was exposed to the skinning solution for about 60 min and then bathed in the relaxing solution with a

13 CAFFEINE AND TETRACAINE ON K-CONTRACTURES 203 pca of about 9. The second record in the first row shows tension development of the corresponding fibre when the pca was lowered to 5-5. The second record in the second row shows a similar run carried out in the presence of 1 mm tetracaine. This drug was added to the relaxing solution 2 min before lowering the pca. Finally, the second and third record of the third row shows two runs, carried out with the same fibre in the absence and in the presence of 1 mm tetracaine respectively. It may be 190 IC 5.5 _ a_ 8. K Tetracaine 1 mm 10 sec Ki p C p~a a5 Tetracaine 1 mm Text-fig. 9. Tetracaine does not impair tension development in chemically skinned fibres exposed to low pca solutions. See text for further details. Temperature 20 C. observed that with this saturating calcium concentration, tensions are obtained which are similar to those obtained with the intact fibre in response to raised potassium; furthermore, it is clear that tetracaine does not abolish the capacity of the contractile machinery to develop tension in response to raised calcium. DISCUSSION The results of the experiments reported here indicate that the amount of contractile activator released during a potassium contracture can be increased or diminished by caffeine and local anaesthetics respectively. In the result section, several reasons have been given against the possibility that the increased duration of potassium contractures in the presence of low concentrations of caffeine could be due to reduced uptake of

14 204 C. CAPUTO calcium by the sarcoplasmic reticulum. In short, in the presence of caffeine at low concentrations, the relaxing ability of the frog muscle fibres is not impaired (LUttgau & Oetliker, 1968; Foulks, Perry & Sanders, 1971), and furthermore, it is known that caffeine at low concentrations causes release of calcium from isolated vesicles of the sarcoplasmic reticulum, without impairing its uptake capacity (Ogawa, 1970). It also seems difficult to explain the present results assuming that the relaxation from potassium contractures under the present condition is due to depletion of the activator store. In fact, one should assume that the store can be increased or diminished by caffeine and local anaesthetics respectively. It could be argued that a normal structural compartamentalization of the activator store (presumably the sarcoplasmic reticulum) is altered by caffeine, thus virtually enlarging the store of activator available for release during a potassium contracture. However, freeze fracture electron microscopy failed to show signs of structural compartamentalization at the level of the membrane system of the sarcoplasmic reticulum as can be observed in the picture of P1. 1, kindly provided by Dr Franzini- Armstrong. This evidence, however, does not rule out the possibility of functional compartamentalization of the contractile activator. Thus caffeine could release activator by acting at sites of the membrane of the sarcoplasmic reticulum not normally involved in excitation-contraction coupling. In this context, structural differences between non-functional and functional regions of the sarcoplasmic reticulum have been recently described (Franzini-Armstrong, 1975). Another possibility to be considered is that caffeine at low concentrations induces an independent release of subthreshold amounts of activator which added to the amount released during the potassium contracture could cause the effects described here. Some support for this explanation could be found in the finding that caffeine shifts the threshold for potassium contractures (LUttgau & Oetliker, 1968) and the observation of Marco & Nastuk (1968) that caffeine at low concentrations induces sarcomeric oscillations in frog skeletal muscle without tension development. However, one might wonder whether the amount of contractile activator responsible for these effects is sufficient to produce the observed increases in the area under the contracture curve. A more plausible explanation for the effects described in this paper is that during a potassium contracture the activator is not depleted, and that the amount of activator released is controlled by a process that is not only potential but also time dependent (Caputo, 1972b), and is affected by drugs like caffeine and local anaesthetics. Although there are several possible alternatives for explaining the effect of caffeine, the effects of local anaesthetics appear to be easier to explain. In fact, the only possible

15 AFFEINE AND TETRACAINE ON K-CONTRACTURES 205 explanation for this effect is that the amount of contractile activator released in the presence of these agents is reduced. Under conditions when the time course of the contracture is clearly shortened, the initial phase of tension development of the contracture is not affected. This was also shown to be the case for the contracture induced in the absence of calcium. These results suggest that immediately after membrane depolarization the rate of activator release is not diminished. In a previous work a schematic model for explaining the time course of potassium contractures was proposed (Caputo, 1972b) involving a membrane mechanism which is activated upon depolarization and later inactivates with time. The results of the present experiments can be interpreted in terms of the same model, assuming that inactivation time course is altered by drugs like caffeine and local anaesthetics. Recent work (Schneider & Chandler, 1973) on charge movement in voltage-clamped muscle fibres give support to the idea that a voltage- and time-dependent membrane mechanism might be involved in contractile activation. It would be of interest to test the effect of caffeine and local anaesthetics on the time course of the charge movement phenomenon described by Schneider & Chandler (1973). Reduction of calcium in the external medium appears to have a similar effect as that of local anaesthetics. The experiments carried out with very low external calcium concentrations ( < 10-8 M) support the idea of Armstrong et al. (1972) that a calcium-induced calcium release is not involved in contractile activation of skeletal muscle fibres under normal conditions. The author is greatly indebted to Dr Clara Franzini-Armstrong for providing the electron microscopy picture of P1. 1 and for helpful commentaries. This work was partially supported by a Research Fund ( S1-0296) of Consejonacional de Investigaciones Cientificas y Tecnologicas (Conicit). REFERENCES ANDERSSON, K. E. (1972). Effects of chlorpromazine, imipramine and quinidine on the mechanical activity of single skeletal muscle fibres of the frog. Acta physiol. scand. 85, ANDERSON, K. E. & EDMAN, K. A. P. (1974). Effects of lanthanum on potassium contractures of isolated twitch muscle fibres of the frog. Acta physiol. scand. 90, ARMSTRONG, C. M., BEZANILLA, F. B. & HOROWICZ, P. (1972). Twitches in the presence of ethylene glycol bis-(fi-aminoethyl ether)-n,n' tetraacetic acid. Biochim. biophys. acta 267, BIANCHI, C. P. (1961). The effect of caffeine on radiocalcium movement in frog sartorius. J. yen. Physiol. 44, CAPUTO, C. (1968). The role of calcium in the processes of excitation and contraction in skeletal muscle. J. gen. Physiol. 51, s.

16 206 C. CAPUTO CAPUTO, C. (1972a). The effect of low temperature on the excitation-contraction coupling phenomena of frog single muscle fibres. J. Phy8iol. 223, CAPUTO, C. (1972b). The time course of potassium contractures of single muscle fibres. J. Phy8iol. 223, CAPUTO, C. (1973). Release mechanism of the contractile activator during potassium contractures. Acta phy8iol. latinoam. 23, CAPUTO, C. & GIMENEZ, M. (1967). Effect of external calcium deprivation on single muscle fibers. J. gen. Phy8iol. 50, ETZENSPERGER, J. (1970). Effets des an6sth6siques locaux sur le potential d'action et la secousse de la fibre musculaire squel6tique de la grenouille. J. Physiol., Paris 62, FORD, L. E. & PODOLSKY, R. J. (1972). Calcium uptake and force development by skinned muscle fibres in EGTA buffered solutions. J. Physiol. 223, FoULKS, J. G., PERRY, F. A. & SANDERS, H. D. (1971). Effect of external potassium concentration on caffeine contractures in frog toe muscles. Can. Jnl Physiol. & Pharmacol. 49, FRANK, G. B. (1960). Effects of changes in extracellular calcium concentration on the potassium induced contracture of frog's skeletal muscle. J. Physiol. 151, FRANK, G. B. (1962). Utilization of bound calcium in the action of caffeine and certain multivalent cations on skeletal muscle. J. Physiol. 163, FRANKENHAEUSER, B. & LANNERGREN, J. (1967). The effect of calcium on the mechanical response of single twitch muscle fibres of Xenopus laevi8. Acta physiol. scand. 69, FRANZINI-ARMSTRONG, C. (1975). Membrane particles and transmissions at the triad. Fedn Proc. 34, HELLAM, D. C. & PODOLSKY, R. J. (1969). Force measurements in skinned muscle fibres. J. Physiol. 200, HODGKIN, A. L. & HOROWICZ, P. (1960). Potassium contractures in single muscle fibres. J. Physiol. 153, JULIAN', F. J. (1971). The effect of calcium on the force-velocity relation of briefly glycerinated frog muscle fibres. J. Physiol. 218, LUTTGAU, H. C. (1963). The action of calcium ions on potassium contractures of single muscle fibres. J. Physiol. 168, LUTTGAU, H. C. & OETLIKER, H. (1968). The action of caffeine on the activation of the contractile mechanism in striated muscle fibres. J. Physiol. 194, MARCO, L. A. & NASTUK, W. L. (1968). Sarcomeric oscillations in frog skeletal muscle. Science, N.Y. 161, OGAWA, Y. (1970). Some properties of fragment frog sarcoplasmic reticulum with particular reference to its response to caffeine. J. Biochem., Tokyo 67, SCHNEIDER, M. F. & CHANDLER, W. K. (1973). Voltage dependent charge movement in skeletal muscle: a possible step in excitation-contraction coupling. Nature, Lond. 242, STEFANI, E. & CHIARANDINI, D. J. (1973). Skeletal muscle: dependence of potassium contractures on extracellular calcium. Pfliigers Arch. ges. Physiol. 343, STUESSE, S. C., LINDLEY, B. D. & KIRBY, A. C. (1974). Potassium contractures of frog single denervated muscle fibers: time course and central spread. Am. J. Phy.iol. 227, WEBER, A. & HERZ, R. (1968). The relationship between caffeine contracture of intact muscle and the effect of caffeine on reticulum. J. gen. Physiol. 52,

17 The Journal of Physiology, Vol. 255, No. 1 Plate I CARLO CAPUTO (Pacing p. 207)

18 CAFFEINE AND TETRACAINE ON K-CONTRACTURES 207 EXPLANATION OF PLATE Freeze fracture across a frog sartorius muscle fibre showing the cytoplasmic leaflet of the sarcoplasmic reticulum covering the length of one sarcomere. The arrow indicates the direction of shadowing. x This picture was kindly provided by Dr Clara Franzini-Armstrong.