SUMMARY1. Changes in ionized calcium in giant axons were followed by recording the light produced by injected aequorin.2. From the effect of injecting calcium buffers the internal concentration of ionized calcium was found to be about the same as in a mixture of 45 Ca EGTA: 55 free EGTA, i.e. about 04a1sM.3. After an axon had been exposedwto cyanide for 50-100 min the velocity of the aequorin reaction increased about 500 times. This effect, which could be reversed rapidly by removing cyanide, was probably brought about by release of calcium from an internal store.4. Injecting 30 tumole ATP per litre of axoplasm into a cyanide-poisoned axon caused a transient lowering of light intensity; oligomycin blocked the effect.5. Raising external cqicium or replacing external sodium by choline or lithium reversible increased the light produced by axons injected with aequorin.

Aequorin-injected eggs of the medaka (a fresh water fish) show an explosive rise in free calcium during fertilization, which is followed by a slow return to the resting level.

We have used the calcium-specific lightemitting protein aequorin to follow changes in free calcium concentration during fertilization and cleavage of eggs from medaka, a fresh-water fish. Aequorin-injected meda a eggs show a very low resting glow before they are fertilized, indicating a low calcium concentration in the resting state. Upon activation by sperm, the calcium-mediated light emission increases to a level some 10,000 times the resting level with a 1 to 2 sec time constant for an e-fold increase, and then slowly returns to the resting level. Upon activation by the ionophore A23187, the early rise in luminescence is much slower, but once a threshold has been reached the subsequent rise becomes as rapid as the normal sperm-induced response. We infer that the explosive rise in calcium involves calcium-stimulated calcium release, and that a sperm normally triggers this rise by somehow inducing-a more modest and localized rise in calcium.One of the most important aspects of fertilization is the activation of the previously resting egg by the entering sperm. This normal activation response of the egg is dramatic and selfsustaining, and has long been thought to involve and require an increase of free calcium in the cytoplasm. Early workers conceived of a calcium theory of activation because a variety of eggs can be activated parthenogenetically (i.e., without sperm) simply by exposure to calcium-rich solutions, or by various disturbances, e.g., pricking the egg, in (and only in) a calcium-bearing medium (ref. 1; also compare refs. 2-5). More recently, the calcium theory has been supported by experiments showing that the calcium-specific ionophore A23187 can activate eggs (6-9).The results of previous efforts to measure a change in free calcium during activation have surely been consistent with a large increase, but are rather easily interpretable in other ways (2,8,10,11). For example, the 10-to 100-fold increase in 45Ca efflux during activation of sea urchin eggs (7, 11) might well be due to an increase in free cytoplasmic calcium, but might also represent the release of calcium from cortical vesicles.In this paper, we demonstrate a dramatic rise in free calcium concentration within the cytoplasm of activating medaka eggs by injecting them with the calcium-specific photoprotein aequorin. This calcium indicator has been previously used to detect calcium transients in muscle (12) and nerve (13), and is presently, we believe, the most sensitive and reliable method for detecting changes in free calcium within cells. The egg we have used is from the medaka, a fresh-water fish. The advantages of this~egg are: first, it is large (1.2 mm diameter); second, it can be injected without blocking subsequent fertilization (3); and third, it is transparent. A measurement was first made of the egg's "resting glow." Then the photomultiplier was turned off, the chamber was opened, sperm were added about 5 mm away from the egg, the chamber was closed, and the tube was turned back on. The calcium-mediated light emission ...

SUMMARY1. The calcium-sensitive photoprotein aequorin has been used to follow the rapid changes in intracellular calcium concentration that occur during the contraction of single muscle fibres from the barnacle Balanus nubilus, Darwin. 2. The transient change in calcium-mediated light emission (calcium transient) and the changes in membrane potential and tension were recorded simultaneously, thus permitting an examination of the relationships between the chemical, electrical, and mechanical events ofexcitationcontraction coupling.3. With short-duration stimuli (< 200 msec), the calcium transient shows an S-shaped rising phase reaching a maximum soon after the cessation of the stimulus pulse. During membrane repolarization the calcium transient begins an exponential falling phase which has a time constant of 50-80 msec at 11-12°C.4. The shape of the calcium transient resembles the first derivative of the rising phase of the isometric tension response, thus suggesting that calcium controls the rate of tension development.5. There is no detectable increase of the light emission above resting values, during the falling phase of isometric tension.6. A plot of the calcium transient area (lumen x see) versus peak isometric force (g. cm-2) is linear over, at least, a range of forces from ca. 50-400 g. cm-2.7. When the fibre is capable of producing an active membrane response following the intracellular injection of potassium citrate, the onset and cessation of the calcium transient follow closely the onset and cessation * Present address. C. C. ASHLEY AND E. B. BIDG WA Y of the active membrane response. Tension responses under these conditions are much suppressed, suggesting that excitation-contraction coupling may be partially blocked between calcium release and the development of tension.8. Hypertonic salines (1 M sucrose or 1 M glycerol) cause little change in the membrane response, but greatly suppress the calcium transient and completely abolish the tension responses. These effects are readily reversible when normal saline is reintroduced, suggesting that excitationcontraction coupling may be temporarily blocked between the membrane response and calcium release.9. If the stimulus is prolonged (> 250-300 msec), the calcium transient falls slowly from its maximum value despite continued membrane depolarization, suggesting a time-dependent change in the ratio of the rate of release of calcium to the rate of calcium binding. The results from brief tetanic stimulation also support this suggestion.

4. Mn2+, Co2+, Ni2+ and the drug D-600 all reduce the Na currents, but have little effect on either outward or inward K currents. Tetraethylammonium blocks the outward K current but has no appreciable effect on the tetrodotoxin-insensitive entry of Ca.5. Concentrations of Mn between 5 and 50 mm substantially reduce the light output during a train of action potentials; they also slightly reduce the rate of rise of the action potential.6. On pharmacological grounds it is concluded that the tetrodotoxininsensitive component of Ca entry does not represent Ca ions passing through the K permeability channels. There must exist a potentialdependent late Ca channel that is distinct from the well known Na and K channels ofthe action potential. A possible function for this late Ca channel in the coupling of excitation to secretion is discussed.