Oscillations of calcium ion concentrations in Physarum polycephalum.

Aequorin is a photoprotein which emits light in response to changes in free calcium concentration. When aequorin was microinjected into plasmodia of Physarum polycephalum, light emission varied in synchrony with the motile oscillations of the organisms. Therefore, movement is correlated which changes in the concentration of free calcium.

Control of chemotaxis in Physarum polycephalum.

Plasmodia migrate towards those situations which increase the frequency of their alternations in streaming, and away from those which decrease the frequency. Therefore peristalsis-like waves in Physarum move in the direction opposite from the net movement of the organism. The mechanism is fundamentally related to other known types of chemotaxis.

A free calcium wave traverses the activating egg of the medaka, Oryzias latipes.

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. Image intensification techniques now show a spreading wave of high free calcium during fertilization. The wave starts at the animal pole (where the sperm enters) and then traverses the egg as a shallow, roughly 20 degrees-wide band which vanishes at the antipode some minutes later. The peak free calcium concentration within this moving band is estimated to be about 30 microM (perhaps 100-1,000 times the resting level). Eggs activated by ionophore A23187 may show multiple initiation sites. The resulting multiple waves never spread through each other; rather, they fuse upon meeting so as to form spreading waves of compound origin. The fertilization wave is nearly independent of extracellular calcium because it is only slightly slowed (by perhaps 15%) in a medium containing 5 mM ethylene glycol-bis[beta-aminoethyl ether]N,N'-tetraacetic acid (EGTA) and no deliberately added calcium. It is also independent of the large cortical vesicles, which may be centrifugally displaced. Normally, however, it distinctly precedes the well-known wave of cortical vesicle exocytosis. We conclude that the fertilization wave in the medaka egg is propagated by calcium-stimulated calcium release, primarily from some internal sources other than the large cortical vesicles. A comparison of the characteristics of the exocytotic wave in the medaka with that in other eggs, particularly in echinoderm eggs, suggests that such a propagated calcium wave is a general feature of egg activation.

Hysteresis in the force-calcium relation in muscle.

Calcium ions activate muscle contraction. The mechanism depends on the calcium sensitivity of the proteins that regulate contraction. Evidence is presented for the reverse phenomenon, where contraction modulates calcium sensitivity. Increasing the force level increased calcium sensitivity in intact fibers showing that the relation between force and calcium is not unique. A particular calcium concentration can maintain a higher force level than it can create. The results were confirmed in skinned fiber experiments. Transient reduction of the force led to a transient reduction in calcium binding, suggesting a simple mechanism for the hysteresis.

Extra calcium on shortening in barnacle muscle. Is the decrease in calcium binding related to decreased cross-bridge attachment, force, or length?

Barnacle single muscle fibers were microinjected with the calcium-specific photoprotein aequorin. We have previously shown (Ridgway, E. B., and A. M. Gordon, 1984, Journal of General Physiology, 83:75-104) that when barnacle fibers are stimulated under voltage clamp and length control and allowed to shorten during the declining phase of the calcium transient, extra myoplasmic calcium is observed. The time course of the extra calcium for shortening steps at different times during the calcium transient is intermediate between those of free calcium and muscle force. Furthermore, the amplitude increases with an increased stimulus, calcium transient, and force. Therefore, the extra calcium probably comes from the activating sites on the myofilaments, possibly as a result of changes in calcium binding by the activating sites. The change in calcium binding may be due, in turn, to the change in muscle length and/or muscle force and/or cross-bridge attachment per se. In the present article, we show that the amount of the extra calcium depends on the initial muscle length, declining at shorter lengths. This suggests length-dependent calcium binding. The relation between initial length and extra calcium, however, parallels that between initial length and peak active force. The ratio of extra calcium to active force is therefore virtually independent of initial length. These data do not distinguish between a direct effect of length on calcium binding and an indirect effect owing to changes in cross-bridge attachment and force through some geometrical factor. The amount of extra calcium increases with the size of the shortening step, tending toward saturation for steps of greater than or equal to 10%. This experiment suggests that calcium binding depends on muscle force or cross-bridge attachment, not just length (if at all). There is much less extra calcium seen with shortening steps at high force when the high force results from stretch of the active muscle than when it results from increased stimulation of muscle.(ABSTRACT TRUNCATED AT 400 WORDS)

Length-dependent electromechanical coupling in single muscle fibers.

In single muscle fibers from the giant barnacle, a small decrease in muscle length decreases both the calcium activation and the peak isometric tension produced by a constant current stimulus. The effect is most pronounced if the length change immediately precedes the stimulation. In some cases, the decrease in tension with shortening can be accounted for almost entirely by a decrease in calcium release rather than changes in mechanical factors such as filament geometry. During the constant current stimulation the muscle membrane becomes more depolarized at longer muscle lengths than at the shorter muscle lengths. Under voltage clamp conditions, when the membrane potential is kept constant during stimulation, there is little length dependence of calcium release. Thus, the effect of length on calcium release is mediated through a change in membrane properties, rather than an effect on a subsequent step in excitation-contraction coupling. Stretch causes the unstimulated fiber membrane to depolarize by about l mV while release causes the fiber membrane to hyperpolarize by about the same amount. The process causing this change in potential has an equilibrium potential nearly 10 mV hyperpolarized from the resting level. This change in resting membrane potential with length may account for the length dependence of calcium release.

Muscle calcium transient. Effect of post-stimulus length changes in single fibers.

We examined the effects of post-stimulus length changes on voltage-clamped, aequorin-injected single muscle fibers from the barnacle Balanus nubilus. Extra light (extra calcium) is seen when the fiber is allowed to shorten (a small percentage) during the declining phase of the calcium transient. The opposite is observed when the fiber is stretched. Increasing the extent of shortening increases the amount of extra calcium, as does decreasing the temperature. The extra calcium probably comes from the myofilaments and not from the sarcoplasmic reticulum because (a) there is a strong correlation between the extra calcium and the level of activation; (b) there is a strong correlation between the extra calcium and the amount of force redeveloped after a length change; and (c) the time course of the appearance of the extra calcium is intermediate between that of the free calcium concentration and that of force. We suggest (a) that the calcium binding to the activating myofibrillar proteins is sensitive to muscle length or muscle force, and (b) that there is a pool of bound calcium (activating calcium) that waxes and wanes with a time course intermediate between the free calcium concentration and force.

Stretch of active muscle during the declining phase of the calcium transient produces biphasic changes in calcium binding to the activating sites.

In voltage-clamped barnacle single muscle fibers, muscle shortening during the declining phase of the calcium transient increases myoplasmic calcium. This extra calcium is probably released from the activating sites by a change in affinity when cross-bridges break (Gordon, A. M., and E. B. Ridgway, 1987. J. Gen. Physiol. 90:321-340). Stretching the muscle at similar times causes a more complex response, a rapid increase in intracellular calcium followed by a transient decrease. The amplitudes of both phases increase with the rate and amplitude of stretch. The rapid increase, however, appears only when the muscle is stretched more than approximately 0.4%. This is above the length change that produces the breakpoint in the force record during a ramp stretch. This positive phase in response to large stretches is similar to that seen on equivalent shortening at the same point in the contraction. For stretches at different times during the calcium transient, the peak amplitude of the positive phase has a time course that is delayed relative to the calcium transient, while the peak decrease during the negative phase has an earlier time course that is more similar to the calcium transient. The amplitudes of both phases increase with increasing strength of stimulation and consequent force. When the initial muscle the active force. A large decrease in length (which drops the active force to zero) decreases the extra calcium seen on a subsequent restretch. After such a shortening step, the extra calcium on stretch recovers (50 ms half time) toward the control level with the same time course as the redeveloped force. Conversely, stretching an active fiber decreases the extra calcium on a subsequent shortening step that is imposed shortly afterward. Enhanced calcium binding due to increased length alone cannot explain our data. We hypothesize that the calcium affinity of the activating sites increases with cross-bridge attachment and further with cross-bridge strain. This accounts for the biphasic response to stretch as follows: cross-bridges detached by stretch first decrease calcium affinity, then upon reattachment increase calcium affinity due to the strained configuration brought on by the stretch. The experiments suggest that cross-bridge attachment and strain can modify calcium binding to the activating sites in intact muscle.

Calcium fluxes in hydrozoan embryos depend, in part, on exocytosis and fluid phase endocytosis.

In the hydrozoan Phialidium gregarium, the constitutive calcium influx of cleavage stage embryos in sea water is 1.96 +/- 0.75 x 10(-15) moles/embryo/minute. Treating embryos with 227 mM KCl in seawater briefly increases the calcium influx more than 100-fold, to 3.9 x 10(-13) mol/embryo/min. About 62% of the KCl-induced calcium influx is due to calcium flowing through voltage-sensitive calcium channels. This causes a marked intracellular calcium transient and secretion of intracellular vesicles. The other component (approximately 38%) of the calcium influx occurs via fluid phase endocytosis of the extracellular medium (detected using extracellular 3H-sucrose). KCl-treatment of 45Ca loaded embryos induces a 45Ca efflux which can reach peak fractional rates of 0.98/min, during which 55-75% (mean 66%) of the total 45Ca is lost. The KCl-induced calcium efflux is due, in part, to secretion because loaded 3H-sucrose is effluxed simultaneously. This pathway may be important for the calcium efflux necessary for long-term calcium homeostasis in cells.

Cross-bridges affect both TnC structure and calcium affinity in muscle fibers.

In vertebrate striated muscle, calcium binding to troponin initiates contraction, a strong interaction of actin and myosin. In isolated proteins and skinned fibers, the strong interaction of myosin with actin also affects troponin. Fluorescent labels attached to troponin C show structural changes in the TnC environment with cross-bridge attachment and also with calcium binding. Evidence that this effect of crossbridges also occurs in intact striated muscle comes from studies in partially activated cardiac or skeletal muscle by others and in barnacle muscle by us. Length changes which detach myosin cross-bridges produce a brief burst of extra calcium that can be detected by aequorin in activated, voltage clamped single barnacle muscle fibers. That this calcium is coming from calcium bound to the activating site (troponin-C) is supported by several pieces of evidence. Studies on the dependence of the extra calcium on force and the time of the length change are consistent with the amplitude of the extra calcium being proportional to the bound calcium (CaTnC) and with increased cross-bridge attachment and force increasing calcium binding to troponin-C by up to a factor of 10. Importantly, stretch of active muscle (which first detaches cross-bridges and then enhances steady force) gives a biphasic response: first extra calcium (presumably due to cross-bridge detachment) and then, decreased calcium (presumably due to enhanced calcium binding to TnC). The enhanced calcium binding we see with elevated force (via strained cross-bridges) implies that calcium binding to TnC is enhanced not only be cross-bridge attachment but also by crossbridge (or thin filament) strain. This effect of cross-bridge attachment/force on calcium binding is consistent with a dual mechanism of calcium activation of contraction. First, calcium binds to troponin in the thin filament activating strong myosin binding to the thin filament. Then, strong myosin binding in turn provides additional activation either by increasing calcium binding or by changing the thin filament structure directly allowing additional cross-bridge attachment.

Muscle cross-bridge attachment: effects on calcium binding and calcium activation.

Data from intact and skinned muscle fibers support the hypothesis that cross-bridge interaction modifies TnC structure and calcium activation. Barnacle single muscle fibers microinjected with the calcium bioluminescent photoprotein, aequorin, show extra light (calcium) when shortened during the declining phase of the calcium transient. The extra calcium is increased by increases in muscle force, and its decline is delayed at higher forces. This extra calcium occurs probably because calcium binding to the activating sites is increased by cross-bridge interaction. In rabbit muscle, TnC structure is modified by cross-bridge interaction, since in skinned rabbit psoas muscle fibers TnC extraction is slower at shorter sarcomere lengths, where cross-bridge attachment is increased. Thus the rigor bridges formed in the extraction solution strengthen the attachment of TnC to the thin filament. Reintroduction of TnC, labeled with fluorescent probes near the Ca specific binding sites (Danzylaziridine-DANZ) and Ca-Mg sites (Rhodamine), into the partially TnC extracted fibers allows us to assess the structural changes (total fluorescence for the DANZ probes, linear dichroism for the RHOD probe) in response to calcium binding and cross-bridge attachment. At sarcomere lengths beyond overlap, calcium binding increases the DANZ-TnC fluorescence and disorders the RHOD-TnC label. At full overlap of filaments, rigor cross-bridges also increase the DANZ-TnC fluorescence and RHOD-TnC disorder. The addition of calcium in rigor increases the DANZ-TnC fluorescence little but causes additional RHOD-TnC disorder, although both fluorescence and disorder are increased further in the presence of calcium plus MgATP. In fibers containing DANZ-TnC, decreasing MgATP in the absence of calcium increases both the force and the fluorescence as rigor cross-bridges activate the muscle. In the presence of calcium, an increase in MgATP to 0.75 microM produces a small fluorescent enhancement, but an increase in MgATP to 10 microM and to 3 mM produces a substantial enhancement. The data imply that calcium activates the thin filament, but that the filament is activated further by rigor cross-bridges. Active cross-bridges activate the thin filament still further. Thus, cross-bridges modify TnC structure and calcium activation, with active cross-bridges being more effective than rigor cross-bridges.

Calcium sensitivity is modified by contraction.

This paper summarizes three lines of experimental evidence showing that crossbridge interaction affects calcium sensitivity and probably also affects calcium binding. Evidence is presented that this is a true hysteresis, not just a slow approach to equilibrium. In barnacle single muscle fibers injected with aequorin to monitor intracellular Ca, a long duration stimulus under voltage clamp conditions can produce a long duration calcium transient and force record which both approach steady levels. However, if the stimulus is briefly elevated to transiently produce a higher force early in the contraction, the same steady state Ca level can eventually maintain a higher steady force. Thus Ca sensitivity is modified. In "skinned" barnacle muscle activated by Ca in the presence of buffered Ca, MgATP, and pH, force was measured in split, detergent treated fiber while it was transferred consecutively between solutions which were relaxing, submaximal contracting, maximal contracting, the same submaximal contracting, and finally relaxing. The submaximal contracting solution produced more force when stepping down in Ca concentration (as in relaxation) than when stepping up. Extending the time in the initial submaximal contracting solution did not result in more force. Thus, the force-pCa relationship shows marked hysteresis. The same phenomenon was seen in frog and mammalian muscle fibers. These experiments confirm the findings that contraction modifies Ca sensitivity. In the barnacle single muscle fiber preparation (under both voltage clamp and controlled length conditions), phasic (400 msec) depolarization leads to a calcium transient and a twitch contraction. Releasing the muscle to allow it to shorten rapidly during the declining phase of the calcium transient causes the force to fall and leads to extra Ca in the sarcoplasm. Rapidly stretching the muscle produces the opposite effect. The extra Ca probably comes from a myofilament Ca activating site. Thus, a length change (force change) affects Ca binding.

The role of intracellular calcium and pH during fertilization and egg activation in the hydrozoan Phialidium.

A calcium transient occurs at fertilization in the eggs of the hydrozoans Mitrocomella and Phialidium. The eggs of Phialidium have an intracellular pH (pHi) of 7.6-7.95. There is no increase in pHi following fertilization. Both calcium ionophore and ammonia treatments activate Phialidium eggs. Calcium ionophore causes a calcium transient without changing pHi. Ammonia concentrations of 10-20 mM at pH 8 cause a rise in pHi but no detectable calcium transient. Both activating agents can (1) block subsequent fertilization, (2) initiate cell cycle events such as DNA synthesis, nuclear envelope breakdown, chromosome condensation, and cleavage-related events, and (3) initiate voltage-dependent calcium channel function. While calcium ionophore treatment invariably elicits all of these manifestations of egg activation, ammonia sometimes induces only the induction of voltage-dependent calcium channel function. Oocytes were also treated with calcium ionophore or ammonia at different stages during their maturation. Ammonia treatment did not induce egg activation when applied at any stage during maturation; however, calcium ionophore initiated voltage-dependent calcium channel function when oocytes were treated after germinal vesicle breakdown. Ionophore treatment during maturation did not render these eggs unfertilizable or initiate cell cycle events. These experiments show that elevation of [Ca2+] with ionophore and increasing pHi with ammonia may activate these eggs through different pathways, that specific maturational events must occur before they can do so, and that the activation of voltage-dependent calcium channel function can be dissociated from other egg activation events.

Determination of resting free calcium in barnacle muscle using modified aequorins, buffered calcium injections, and simultaneous image-intensified video microscopy.

Knowing the resting free calcium is important in understanding the role of calcium as an intracellular second messenger. We used a bracketing (null) technique with a luminescent calcium indicator, aequorin, microinjection and image-intensification to measure free calcium in single muscle fibres from the barnacle, Balanus nubilus. We injected modified aequorins (recombinant, and hch-) which after a 30 min diffusion gave reasonable resting glows. Subsequent injection of calcium (strongly buffered with either EGTA or BAPTA, 10 mM) increased or decreased the resting glow depending on the free calcium level in the injected buffer solution. This bracketing (null) method is inherently accurate, but mechanical artifacts on calcium injection reduce the accuracy when total light emission is measured. We therefore used image-intensified video-microscopy of the injected region and video processing (Image-1) of artifact-free regions, to greatly improve the consistency. The luminescence in a pre-selected region of the muscle fibre was measured as a function of time during the injection. Solution calciums were chosen so that if the first injection decreased the resting glow, the second increased it, or vice versa, thus bracketing the true resting value. We used two methods to determine the true value bracketed by our injections: (1) a linear interpolation using the fractional changes in luminescence or (2) a power law interpolation assuming a 2.2 or 2.5 power relationship between luminescence and free calcium. Using these methods, we estimated the free calcium level in the lateral depressor fibres of freshly dredged barnacles to be 279 +/- 36 nM (+/- SD), 339 +/- 42 nM, or 352 +/- 45 nM for the linear, 2.2 and 2.5 powers respectively under the conditions of hch-aequorin and BAPTA buffers (using a K'Ca for BAPTA of 3.0 x 10(6) M-1 for our conditions). Recombinant-aequorin gave essentially the same result while EGTA buffers yielded a somewhat higher value but because of influences of pH on the K'Ca for EGTA (taken as 6.7 x 10(6) M-1 for our conditions) was considered less reliable. Minor changes in [Mg2+] upon buffer injection can lead to underestimates of the true resting [Ca2+] by at most 10%. Thus, we estimate the resting free calcium in barnacle muscle fibres to be 300-380 nM.

On the relationships between membrane potential, calcium transient and tension in single barnacle muscle fibres.

1. 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 of excitation-contraction 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 degrees 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 sec) 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 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 excitation-contraction 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.

Calcium transients and relaxation in single muscle fibers.

Muscle contraction is initiated by an elevation in intracellular calcium. The transient change in free calcium to a brief depolarization, the calcium transient, can be recorded using a calcium luminescent protein, aequorin. The calcium transient precedes force, peaking while force is rising and returning to the resting level as peak force is achieved. In single barnacle muscle fibers microinjected with aequorin, shortening the muscle during the declining phase of the calcium transient produces an addition light signal, indicating extra free calcium in the sarcoplasm. The amount of additional light is larger with larger length changes. It is also larger if the shortening occurs early in the calcium transient rather than later. The amount of this extra calcium correlates well with the instantaneous level of the calcium transient and not with the instantaneous force level. It is argued in a speculative manner that this extra calcium is coming from the myofilaments. This supports the hypothesis that calcium binding to the myofilaments is rapid and reversible, that reaccumulation of calcium into the sarcoplasmic reticulum (SR) could occur long before relaxation begins and that relaxation of tension could occur by some process other than the mere removal of calcium from the myofilaments.

Depolarization and calcium entry in squid giant axons.

1. 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 0.3 muM.3. After an axon had been exposed to 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 mumole ATP per litre of axoplasm into a cyanide-poisoned axon caused a transient lowering of light intensity; oligomycin blocked the effect.5. Raising external calcium or replacing external sodium by choline or lithium reversibly increased the light produced by axons injected with aequorin.6. Stimulation at 50-200 impulses/sec in a solution containing 112 mM-Ca caused the light intensity to increase to a new steady level; after stimulation the light intensity returned to its original level with a time constant of 10-30 sec. Similar but smaller effects were seen in solutions containing less external calcium. The recovery after stimulation is probably due to uptake of calcium by the internal store.7. Injecting 3 m-mole EGTA per litre axoplasm lowered the resting glow and abolished the aequorin response to stimulation.8. There was no light response to stimulation immediately after an axial injection of aequorin and the effect increased to a ;steady' level with a half-time of about 5 min. The conclusion is that the rise in calcium concentration resulting from stimulation is confined to the peripheral part of the axon and that the diffusion coefficient of aequorin in axoplasm is about 4 x 10(-7) cm(2)/sec.9. The increment in light per impulse often increased markedly during the course of a long experiment and there was also considerable variation between axons.10. If the light response to stimulation was small it was proportional to the frequency of stimulation; if large to the square of the frequency.11. Voltage-clamp experiments showed that the calcium entry associated with a depolarizing pulse could be divided into an early component which was abolished by tetrodotoxin (TTX), and a late component which was unaffected by this inhibitor.12. The time relations of the early calcium entry were consistent with its being a leak of calcium ions through the sodium channel; the permeability of the sodium channel to calcium was about 1% of the permeability to sodium.13. The late entry of calcium was little changed by injecting enough tetraethylammonium (TEA) to block the outward potassium current; it was greatly reduced by external concentrations of manganese which had little effect on the maximum potassium conductance.14. The voltage-response curve for the late entry of calcium had a well defined maximum and was similar in shape to the curve relating calcium entry to depolarization at the presynaptic ending (Katz & Miledi, 1969, 1970).

Effects of manganese and other agents on the calcium uptake that follows depolarization of squid axons.

1. The Ca-sensitive photoprotein aequorin was injected into squid axons and the light response to stimulation or depolarizing voltage clamp pulses recorded.2. The effects of Mn(2+), Co(2+), Ni(2+), La(3+) and of the organic Ca antagonists D-600 and iproveratril on the early tetrodotoxin-sensitive and late tetrodotoxin-insensitive components of the light response were studied.3. The late tetrodotoxin-insensitive component can be blocked, reversibly, by concentrations of Mn, Co and Ni that reduce but do not block the tetrodotoxin-sensitive component. The late component can also be blocked by La(3+) and the organic Ca antagonists D-600 and iproveratril.4. Mn(2+), Co(2+), Ni(2+) 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 tetrodotoxin-insensitive component of Ca entry does not represent Ca ions passing through the K permeability channels. There must exist a potential-dependent late Ca channel that is distinct from the well known Na and K channels of the action potential. A possible function for this late Ca channel in the coupling of excitation to secretion is discussed.

Calcium entry in response to maintained depolarization of squid axons.

1. Intracellular aequorin was used to monitor changes in Ca entry in response to maintained depolarization either produced electrically or by exposure to K-rich solutions.2. External K concentrations greater than 50 mM produce a phasic light response. The light rises to a peak in a few sec and then falls in 0.5-5 min to a new steady level that is always greater than the level in the absence of K.3. The phasic light response does not result from depletion of available aequorin at the periphery of the axon, but rather seems to reflect a phasic entry of Ca in response to depolarization.4. Similar phasic responses are produced by prolonged electrical depolarization. These results are consistent with depolarization serving both to activate and also to inactivate Ca entry.5. Following inactivation and after return to normal sea-water, there is an appreciable relative refractory period during which the response both to K-rich sea-water and electrical depolarization is reduced in size. Complete recovery takes 10-15 min.6. The response to 410 mM-KCl is dependent on the previous treatment of the preparation. Pre-treatment with 100 or 200 mM-KCl reduced the response to 410 mM-KCl. The potential for half inactivation was about -25 mV in 112 mM-Ca and -40 mV in 20 mM-Ca.7. The rate of onset of inactivation is potential dependent and is faster for depolarizations to zero potential than for smaller ones.8. The phasic Ca entry produced by K-rich solutions is insensitive to external tetrodotoxin and internal tetraethylammonium ions, but is blocked by external Mn(2+), Co(2+) and Ni(2+) ions and by the drugs D-600 and iproveratril. This suggests that the phasic Ca entry involves the late Ca channel.9. Recovery of the outward K current after a long depolarization is much faster than recovery of the late Ca entry system. This provides further support for the view that the late Ca channel and the K channel are distinct.

Free calcium increases explosively in activating medaka eggs.

We have used the calcium-specific light-emitting protein aequorin to follow changes in free calcium concentration during fertilization and cleavage of eggs from medaka, a fresh-water fish. Aequorin-injected medaka 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 retruns 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.