Pharmacological potency of the order exhibited by ryanodine is usually associated with a highly specific mechanism of action. The great variety of effects observed and the differences between the responses in different muscles make it difficult to suggest a single underlying mechanism for this drug, or even to distinguish between its primary action and secondary phenomena that represent degenerative or compensatory changes in a self-regulating cellular system. From the evidence available, it seems probable that the increased oxygen consumption, and perhaps some changes in the electrical properties of the cell membrane, may fall into the latter category.
In the case of vertebrate skeletal muscle, the evidence favors the view that ryanodine specifically interferes with relaxation, which is generally thought to be effected by sequestration of calcium ions by the sarcoplasmic reticulum. Ryanodine has been shown to obstruct both active binding and active uptake of Ca++ by particulate fractions of muscle homogenates, and to result in an efflux of Ca++ from skeletal muscle. It seems reasonable to conclude that ryanodine interferes with intracellular Ca++ translocation mechanisms that normally lower the sarcoplasmic concentration and thereby effect relaxation. The efflux of calcium could be accounted for by a compensatory increase in the rate of active transport across the cell membrane, either directly or through the T-tubular lumen. Although such a sarcolemmal transport system has not been unequivocally demonstrated, its existence can be deduced from the very large electrochemical gradient for Ca++ which is normally maintained across the resting muscle cell membrane. The increased oxygen consumption observed after ryanodine treatment could result from an elevated metabolic requirement presented by 1) uncoupling of intracellular Ca++ translocation mechanisms, 2) activation of a sarcolemmal Ca++ transport system, and 3) increased myofibrillar ATPase activity resulting from elevated sarcoplasmic Ca++ concentrations. Changes in the electrical properties of muscle cell membranes could also be secondary to alterations in calcium balance, since calcium concentration is well known to have a profound influence on alkali metal ion permeability, threshold, and other electrical characteristics of the membrane.
Although this proposal provides a reasonable explanation for many of the effects observed after ryanodine treatment, other experimental data are more difficult to account for. Perhaps the most important of these are the difference between the responses shown by skeletal and cardiac muscle and the apparent lack of effect on some types of smooth muscle. Hajdu (54,55) has drawn attention to the apparently intimate relationship between the action of ryanodine and the "reverse staircase effect" in both cardiac and skeletal muscle, and has presented data (55) showing that skeletal muscle also responds to ryanodine with progressive contractile failure in a calcium-free solution. This suggests that the different effects normally produced by ryanodine in cardiac and skeletal muscle may depend upon a quantitative rather than a qualitative difference in the mechanisms underlying the contraction-relaxation cycle. One possibility consistent with the proposal made here is a difference in the relative importance of intracellular translocation and transmembrane flux of Ca++ in regulating the contraction relaxation cycle. It is well established that cardiac muscle is dependent on extracellular calcium for its contractility, while skeletal muscle may retain its ability to contract for prolonged periods in the absence of extracellular calcium. If extrusion of calcium from the cell is relatively more efficient and important in effecting relaxation in cardiac muscle while intracellular translocation of calcium is the principle factor terminating the active state in skeletal muscle, interference with intracellular Ca++ transport by ryanodine might be insufficient to cause a rise in sarcoplasmic Ca++ above the critical level for contraction in the myocardium, so that the net effect of the drug would be to cause a progressive depletion of calcium [or at least the "contraction pool" (53, 101)] and hence contractile failure. In skeletal muscle, on the other hand, outward transport through the cell membrane may be inadequate to remove the calcium as it is liberated from an intracellular site by stimulation, and inhibition of intracellular uptake mechanisms would result in a progressive failure to relax and eventually in contracture. The contracture should be inhibited either by reducing the rate of intracellular Ca++ liberation (less frequent stimulation and perhaps cooling) or by favoring the operation of an outward sarcolemmal transport system (reducing extracellular Ca++ concentration or improving conditions for oxidative metabolism).
This explanation predicts a decrease in cellular calcium levels in both cardiac and skeletal muscle. The available data are consistent with this in the case of skeletal muscle (11, 54, 55) but conflicting in cardiac muscle (53-55). However, it seems significant (102) that the effects of ryanodine on cardiac muscle are antagonized by caffeine, digitalis glycosides, increased stimulation rate and elevated extracellular Ca++ concentrations; all of these have been shown to result in Ca++ accumulation by the myocardium (92, 96, 100, 149, 151), although toxic amounts of the drugs or Ca++ may be required. As others have pointed out, only a fraction of the total calcium appears to be involved in excitation-contraction coupling in cardiac muscle (95), and changes in it may be difficult to detect (53). Flux studies in contracting cardiac muscle are also complicated by changes in the duration of the action potential induced by drugs (53, 130).
The total lack of response to ryanodine in certain types of smooth muscle (61) could be simply explained if in these muscles intracellular calcium transport systems were of negligible importance compared to outward transport across the cell membrane. Information on this point is lacking, but the small cell dimensions in these muscles and the relatively slow contraction and relaxation that they normally exhibit would seem to make an intracellular calcium transport system less necessary for their normal function.
Studies on particulate components of vertebrate skeletal muscle have demonstrated heterogeneity in the ryanodine sensitivity of different fractions (41). The sensitive heavy fraction may be derived from a specialized intracellular uptake site, while the insensitive lighter fractions could represent resealed fragments of T-tubules and cell membrane. Investigation of this possibility will require considerable refinement in particle fractionation techniques and detailed correlation of biochemical properties with ultrastructure.
Postulation of an intracellular calcium uptake mechanism as the only site of action of ryanodine leaves unexplained a series of experiments in which effects of the drug have been found in model contractile systems free of functional remnants of the sarcoplasmic reticulum (35, 36, 37), and these must tentatively be accepted as indicating a second discrete action of the drug. Ryanodine has been shown to influence both a contractile protein complex and calcium binding by a reticular fraction under conditions in which the other sensitive system is absent or has been inactivated (37). However, both actions are demonstrable only when Ca++ is present in subsaturating amounts, and are discernible as an increase in Mg++-activated ATPase, which appears to be sensitized to calcium ions. It is tempting to speculate that a common molecular mechanism may underlie both effects. One possible mechanism which remains to be investigated is modification of the capacity or affinity of a key protein or proteins for complexing Ca++. Troponin, a protein which in association with tropomyosin has been shown to cause a calcium-reversible inhibition of actomyosin (i.e., to sensitize actomyosin to calcium) (28), has recently been reported to have a high capacity and affinity for calcium (27, 155), and binding of calcium to troponin in the myofibril may account for activation of myofibrillar contraction (48). This or a similar protein could serve as a sink in the intact sarcoplasmic reticulum [the granular material seen in terminal cisternae (104)?] in the same manner that oxalate is thought to function in vitro. Interference with the ability of such a protein to complex Ca++ could reduce the speed and efficiency of the reticular calcium pump and might also reduce the calcium requirement for activation of the contractile protein complex.
- 1969 by The Williams & Wilkins Co.