189403a0Nature1894762196102044034040028-0836196110.1038/189403a0ukNatureNatureNATUREnatureNature is a weekly international journal publishing the finest peer-reviewed research in all fields of science and technology on the basis of its originality, importance, interdisciplinary interest, timeliness, accessibility, elegance and surprising conclusions. Nature also provides rapid, authoritative, insightful and arresting news and interpretation of topical and coming trends affecting science, scientists and the wider public./nature/journal/v189/n4762issueJournal homeArchiveCurrent issueAdvance online publicationPrivacy policySubscribeNature Publishing GroupCurrent issue189403a0Significance of Membrane Calcium in Calcium-free and Potassium-rich Media
AU  - KOKETSU, K.
AU  - MIYAMOTO, S.Research Laboratories, Department of Psychiatry, University of Illinois College of Medicine, Chicago.FROG Ranvier's nodes immersed in potassium-rich media are capable of producing action potentials when the membrane is hyperpolarized by anodal currents1. This observation was confirmed in frog sartorius muscles by one of us, who found that action potentials were restored by anodal polarization in frog sartorius muscles which depolarized in calcium-free media2. The so-called /`hyper-polarizing response/' was also observed in muscle fibres soaked in both potassium-rich and calcium-free media. This experimental evidence seems to suggest that removal of calcium from the membrane in these two solutions might be responsible for the loss of membrane excitability, as suggested in a previous communication2. The rate of output of calcium-45 loaded on muscles, therefore, was studied in both calcium-free and potassium-rich solutions.Isolated frog sartorius muscle was soaked in Ringer containing calcium-45 for 2 hr. at room temperature. After washing the muscles for 20 sec. in Ringer's solution, the time course of the output of calcium-45 into normal Ringer and the test solutions was observed for 4 hr. During this procedure, the muscle was transferred successively into beakers containing 2 c.c. Ringer every 3-20 min. for 2 hr., and the same procedure was continued for another 2 hr. using beakers containing test solutions. The radioactivity in each beaker and muscle was measured, and the time course of the output was plotted (Fig. 1).
The output of calcium-45 markedly increased when the solution was changed from Ringer to either calcium-free (particularly with ethylenediamine tetra-acetic acid3) or potassium-rich (5-30 mM) Ringer4 (Fig. 1). Such increase was precipitous and transient, suggesting the membrane phenomenon. Furthermore, the increase of the outflux was observed without detectable muscle contraction. The total amount of calcium was slightly, but apparently, reduced in these muscles (Miyamoto and Koketsu, unpublished work). If such loss of calcium is responsible for inexcitability and also for depolarization, reduction of membrane potentials in these two solutions would be less pronounced when the release of calcium is prevented in some way. Actually, no appreciable depolarization was observed when cocaine (8 X 10~4 gm./c.c.) was added to the calcium-free Ringer in which the release of calcium-45 was apparently inhibited as shown in Fig. 1. Similar results were obtained by adding magnesium3, strontium, cobalt, or nickel ions to the calcium-free solution. Depolarization in potassium-rich solution could also be prevented by increasing the external calcium or adding these divalent cations ; the increased release of calcium-45 observed in potassium-rich solution was inhibited in these cases. Similar results were also obtained by adding cocaine (1 x 10~3 gm./c.c.) to the potassium-rich solution (Fig. 1). These experiments demonstrated clearly the close parallelism between the drop of resting potential and the output of calcium-45 in both calcium-free and potassium-rich solutions.
Fig. 1. Time-course of decline of calcium-45 content of individual muscles. Time-course during initial 120 min. was obtained in Ringer. After 120 min. bathing solutions were changed to test solutions : (1) A, calcium-free Ringer ; JBt calcium-free ethylenediamine tetraacetic acid (4 mAf) Ringer ; C, calcium-free Ringer containing cocaine (8 x 10~3 gm./c.c.). (2) A, potassium-rich (20 IQ.M) Ringer; JB, potassium-rich Ringer containing 50 mM calcium chloride; C, potassium-rich Ringer containing cocaine (1 x 10~3 gm./c.c.)
The observation4 that the efflux of radiocalcium increases during muscle activity has been confirmed by us. The problem whether removal of calcium is consequential4 or actually the cause of depolarization, in calcium-free and potassium-rich media or during stimulation, seems to be crucial.
This work was supported by the Institute of Neurological Diseases and Blindness, National Institutes of Health Grant 1650.Muller, , P., J. Gen. Physiol., 42, 137 (1958).PubMedKoketsu, , K., and Noda, , K., Nature, 187, 243 (1960).ArticlePubMedISIChemPortHarris, , E. J., Biochim. Biophys. Acta, 23, 80 (1957).ArticlePubMedISIChemPortShanes, , A. M., and Bianchi, , C. P., J. Gen. Physiol., 43, 481 (1960).ArticlePubMedISIChemPort