Calcium influx in the sartorius muscle of the frog (~ ~) has been estimated from the rate of entry of Ca 46. In the unstimulated preparation it is about equal to what has been reported for squid giant axons, but that per impulse is at least 30 times greater than in nerve fibers. The enhanced twitch when NO~" replaces Cl-in Ringer's is associated with at least a 50 per cent increase in influx during activity, whereas this anion substitution does not affect the passive influx significantly. Calcium entry during potassium contracture is even more markedly augmented than during electrical stimulation, but only at the beginning of the contracture; thus, when a brief Ca 45 exposure precedes excess K + appUeation, Ca 45 uptake is increased three-to fivefold over the controls not subjected to K +, whereas when Ca 45 and K + are added together, no measurable increase in Ca 4s uptake occurs. These findings are in keeping with the brevity of potassium contracture in "fast (twitch)" fibers such as in sartorins muscle.

Caffeine increases resting calcium influx approximately threefold in normally polarized and in potassium-depolarized fibers of frog sartorius muscles. It does not affect the transient rapid increase in calcium influx that occurs at the beginning of a potassium depolarization. Calcium outflux in Ringer's solution, in zero calcium Ringer's solution, and in zero calcium Ringer's solution plus 0.004 M EDTA is also markedly increased by caffeine. The increased out-flux reaches a rate which is approximately the same as the increased calcium influx. One interpretation of the findings is that caffeine reduces the binding of calcium both in the membrane and in the myoplasm; this increases the "permeability" to calcium and the ionic activity of calcium in muscle. This interpretation is consistent with the view that the contractile state of muscle is dependent at least in part on the thermodynamic activity of calcium in the muscle fibers.Experiments by Axelsson and Thesleff (I) suggested that the alkaloid caffeine (free base) initiates a process in the muscle which leads to contraction, and that this process is not mediated by changes in resting potential or in ionic permeability of the muscle membrane. The addition of Ca up to five times its normal concentration or the depletion of Ca by soaking the muscle in 0.1 per cent ethylenediaminetetraacetic acid (EDTA) has no observable effect on the contracture produced by caffeine. Calcium has been suggested as a possible link between depolarization and contraction (6, 9, 13, 3), a suggestion supported by recent findings (2, 15). The following experiments were performed to determine whether caffeine might still be exerting its effects by way of the calcium in the muscle, especially that remaining after E D T A treatment (5).

The distribution of Ca 4s in frog (Rana pipiens) sartorius muscle, after 4 hours' exposure to Ringer's solution containing radiocalcium, has been analyzed by observing the kinetics of escape of the radioisotope into a non-radioactive Ringer's solution with caldum present or absent and by assuming that the tendon of Achilles is a satisfactory model of the extent of the uptake and rdease of Ca 46 by the interstitial connective tissue (c.t.). In a Ringer's solution containing 1 m~/liter calcium, the exchangeable calcium distribution in micromoles per gram wet weight is as follows: (a) Aqueous phase of c.t. space: 0.16; (b) bound to c.t.: 0.16; (c) bound to surface of fibers: 0.13, of which 0.03 is displaced only by self-exchange, whereas the rest, as in c.t., can be displaced by other ions; and (d) in myoplasm: 0.33. The kinetics of Ca 45 exit suggests that in infinite time of exposure to Ca 45 the myoplasmic component would rise to 0.85. In the muscles, the half-time of the quickly emerging Ca 41 averages about 3 minutes, whereas the time constant of the slowly released component is about 500 minutes. In the tendons the percentage rate of escape falls exponentially, the half-time of emergence being about 10 minutes.Direct evidence has been presented that augmented entry of calcium is intimately associated with contraction and contracture in frog sartorius muscle (1,3,17). These studies suggested that the additional calcium which enters is derived from readily accessible calcium-binding sites on the muscle fibers. Thus, since the calcium entry per twitch was not measurably changed by trebling the extraceUular concentration whereas the entry in unstimulated muscle increased proportionately, the sites concerned with contraction were saturated with calcium at the calcium concentration of the medium while those involved in "passive" calcium entry were not. Similarly, an increase in Ca ~ entry could not be demonstrated during potassium depolarization * Address for 1959: Office of Naval Research, Branch Office,