Classes and mechanisms of calcium waves

The best known calcium waves move at about 5–30 μm/s (at 20°C) and will be called fast waves to distinguish them from slow (contractile) ones which move at 0.1-1 μm/s as well as electrically propagated, ultrafast ones. Fast waves move deep within cells and seem to underlie most calcium signals. Their velocity and hence mechanism has been remarkably conserved among all or almost all eukaryotic cells. In fully active (but not overstimulated) cells of all sorts, their mean speeds lie between about 15–30 μm/s at 20°C. Their amplitudes usually lie between 3–30 μM and their frequencies from one per 10–300 s. They are propagated by a reaction diffusion mechanism governed by the Luther equation in which Ca²⁺ ions are the only diffusing propagators, and calcium induced calcium release, or CICR, the only reaction; although this reaction traverses various channels which are generally modulated by IP₃ or cADPR. However, they may be generally initiated by a second, lumenal mode of CICR which occurs within the ER. Moreover, they are propagated between cells by a variety of mechanisms. Slow intracellular waves, on the other hand, may be mechanically propagated via stretch sensitive calcium channels.     

On the conservation of fast calcium wave speeds

Calcium waves were first seen about 25 years ago as the giant, 10 micro m/s wave or tsunami which crosses the cytoplasm of an activating medaka fish egg [J Cell Biol 76 (1978) 448]. By 1991, reports of such waves with approximately 10 micro m/s velocities through diverse, activating eggs and with approximately 30 micro m/s velocities through diverse, fully active systems had been compiled to form a class of what are now called fast calcium waves [Proc Natl Acad Sci USA 88 (1991) 9883; Bioessays 21 (1999) 657].This compilation is now updated to include organisms from algae and sponges up to blowflies, squid and men and organizational levels from mammalian brains and hearts as well as chick embryos down to muscle, nerve, epithelial, blood and cancer cells and even cell-free extracts. Plots of these data confirm the narrow, 2-3-fold ranges of fast wave speeds through activating eggs and 3-4-fold ones through fully active systems at a given temperature. This also indicate Q(10)'s of 2.7-fold per 10 degrees C for both activating eggs and for fully activated cells.Speeds through some ultraflat preparations which are a few-fold above the conserved range are attributed to stretch propagated calcium entry (SPCE) rather than calcium-induced calcium release (CICR).     

Stretch-activated calcium channels relay fast calcium waves propagated by calcium-induced calcium influx

For nearly 30 years, fast calcium waves have been attributed to a regenerative process propagated by CICR (calcium-induced calcium release) from the endoplasmic reticulum. Here, I propose a model containing a new subclass of fast calcium waves which is propagated by CICI (calcium-induced calcium influx) through the plasma membrane. They are called fast CICI waves. These move at the order of 100 to 1000 microm/s (at 20 degrees C), rather than the order of 3 to 30 microm/s found for CICR. Moreover, in this proposed subclass, the calcium influx which drives calcium waves is relayed by stretch-activated calcium channels. This model is based upon reports from approx. 60 various systems. In seven of these reports, calcium waves were imaged, and, in five of these, evidence was presented that these waves were regenerated by CICI. Much of this model involves waves that move along functioning flagella and cilia. In these systems, waves of local calcium influx are thought to cause waves of local contraction by inducing the sliding of dynein or of kinesin past tubulin microtubules. Other cells which are reported to exhibit waves, which move at speeds in the fast CICI range, include ones from a dozen protozoa, three polychaete worms, three molluscs, a bryozoan, two sea urchins, one arthropod, four insects, Amphioxus, frogs, two fish and a vascular plant (Equisetum), together with numerous healthy, as well as cancerous, mammalian cells, including ones from human. In two of these systems, very gentle local mechanical stimulation is reported to initiate waves. In these non-flagellar systems, the calcium influxes are thought to speed the sliding of actinomyosin filaments past each other. Finally, I propose that this mechanochemical model could be tested by seeing if gentle mechanical stimulation induces waves in more of these systems and, more importantly, by imaging the predicted calcium waves in more of them.     

Fast calcium waves

Calcium waves are propagated in five main speed ranges which cover a billion-fold range of speeds. We define the fast speed range as 3-30μm/s after correction to a standard temperature of 20°C. Only waves which are not fertilization waves are considered here. 181 such cases are listed here. These are through organisms in all major taxa from cyanobacteria through mammals including human beings except for those through other bacteria, higher plants and fungi. Nearly two-thirds of these speeds lie between 12 and 24μm/s. We argue that their common mechanism in eukaryotes is a reaction-diffusion one involving calcium-induced calcium release, in which calcium waves are propagated along the endoplasmic reticulum. We propose that the gliding movements of some cyanobacteria are driven by fast calcium waves which are propagated along their plasma membranes. Fast calcium waves may drive materials to one end of developing embryos by cellular peristalsis, help coordinate complex cell movements during development and underlie brain injury waves. Moreover, we continue to argue that such waves greatly increase the likelihood that chronic injuries will initiate tumors and cancers before genetic damage occurs. Finally we propose numerous further studies.     

Slow calcium waves accompany cytokinesis in medaka fish eggs

Animal cells are cleaved by the formation and contraction of an extremely thin actomyosin band. In most cases this contractile band seems to form synchronously around the whole equator of the cleaving cell; however in giant cells it first forms near the mitotic apparatus and then slowly grows outwards over the cell. We studied the relationship of calcium to such contractile band growth using aequorin injected medaka fish eggs: we see two successive waves of faint luminescence moving along each of the first three cleavage furrows at approximately 0.5 micron/s. The first, narrower waves accompany furrow extension, while the second, broader ones, accompany the subsequent apposition or slow zipping together of the separating cells. If the first waves travel within the assembling contractile band, they would indicate local increases of free calcium to concentrations of about five to eight micromolar. This is the first report to visualize high free calcium within cleavage furrows. Moreover, this is also the first report to visualize slow (0.3-1.0 micron/s) as opposed to fast (10-100 microns/s) calcium waves. We suggest that these first waves are needed for furrow growth; that in part they further furrow growth by speeding actomyosin filament shortening, while such shortening in turn acts to mechanically release calcium and thus propagates these waves as well as furrow growth. We also suggest that the second waves act to induce the exocytosis which provides new furrow membrane.     

Temporal and spatial correlation of fertilization current, calcium waves and cytoplasmic contraction in eggs of Ciona intestinalis

Eggs of the ascidian Ciona intestinalis were loaded with the calcium indicator fura-2 via whole-cell clamp electrodes and changes in cytoplasmic calcium and cell currents were monitored during fertilization either in separate eggs or simultaneously in the same egg. The first indication of egg activation was the fertilization current; which reached peak values around 1 nA after 30 s. A wave of elevated calcium was detectable between 5 s and 30 s (mean = 21 s) after the start of the fertilization current. This wave spread across the egg increasing cytoplasmic calcium levels to at least 10 microM. When the fertilization current and calcium wave were complete and cytoplasmic calcium levels were decreasing to prefertilization levels, a cortical contraction wave spread across the egg surface. In eggs showing normal fertilization current, the calcium wave and the contraction wave were in the same direction. A region of elevated calcium persisted at the animal pole. Changing cytoplasmic calcium levels locally by local application of ionophore A23187 caused a contraction wave originating at the site of ionophore application. Increasing cytoplasmic calcium uniformly by facilitating calcium entry through voltage-regulated channels did not result in a contraction wave.     

Periodic calcium waves cross ascidian eggs after fertilization

Ascidian eggs respond to fertilization with one to two dozen periodic calcium pulses (J.E. Speksnijder, D.W. Corson, C. Sardet, and L.F. Jaffe, 1989a, Dev. Biol. 135, 182-190). We examined the spatial pattern of these pulses and found that they are initiated in discrete regions from which they propagate as waves. The first few pulses start in the animal hemisphere, whereas the later ones are mostly initiated near the vegetal pole. Such vegetal waves are often followed by a contraction of the egg surface. Since these waves are attenuated as they spread, they repeatedly expose the vegetal pole region to more calcium. The mechanism of these repetitive calcium waves and their possible role in establishing pattern or completing meiosis is discussed.     

Absence of blocking effects on cardiac slow calcium channels by the intracellular calcium antagonist 2-n-propyl-3-dimethylamino-5,6-methylenedioxyindene

Extensive pharmacological evidence supports the contention that 2-n-propyl-3-dimethylamino-5,6-methylenedioxyindene hydrochloride (pr-MDI) is a calcium antagonist with a predominantly intracellular site of action. On the other hand, electro-physiological evidence points to a possible membrane slow inward calcium channel blocking property of this agent. To gain further insight as to the site of action of pr-MDI, the interactions between the negative inotropic action of this agent and the positive inotropic actions of excess extracellular calcium (which directly penetrates the myocardial cells through the slow calcium channels), isoproterenol (which indirectly augments calcium influx through the slow calcium channels), and ouabain (which enhances calcium influx through membrane calcium entry routes distinct from the slow calcium channels) were investigated in the isolated, electrically drive guinea pig left atrium. Although excess extracellular calcium, isoproterenol, and ouabain reversed the negative inotropic effect of pr-MDI, an analysis of the concentration-response relationships to all three positive inotropic agents in the presence and the absence of pr-MDI demonstrated that this agent did not significantly inhibit the contractile effects of calcium, isoproterenol, or ouabain, at pr-MDI concentrations which exhibit intrinsic negative inotropic effects. It is concluded that pr-MDI does not block the membrane slow inward calcium channel nor other presumptive membrane routes of calcium entry into myocardial cells at concentrations of 10(-4) M or less. At very high concentrations (3 X 10(-4) M) some inhibition of slow channel calcium influx may occur.     

A role for voltage gated T-type calcium channels in mediating "capacitative" calcium entry?

Calcium entry through plasma membrane calcium channels is one of the most important cell signaling mechanism involved in such diverse functions as secretion, contraction and cell growth by regulating gene expression, proliferation and apoptosis. The identity of plasma membrane calcium channels, the main regulators of calcium entry, involved in cell proliferation has been thus extensively sought. Among these, a calcium entry pathway called capacitative calcium entry (CCE), activated by calcium store depletion, is particularly important in non-excitable cells. Though this capacitative calcium entry is generally supposed to occur through TRP channels there is some evidence that voltage-dependent T-type calcium channels may contribute to calcium entry after store depletion. Here we show that though mibefradil, a T-type calcium channel blocker, is able to reduce capacitative calcium entry induced by either thapsigargin or ATP, this was not mimicked by any other T-type calcium channel inhibitors even in cells overexpressing alpha(1H) T-type calcium channels, leading us to conclude that T-type calcium channels are not responsible for the capacitative calcium entry observed in different cancer cell lines. On the contrary, we show that the action of mibefradil on capacitative calcium entry is due to an action on store-operated calcium channels.     

The propagation speeds of calcium action potentials are remarkably invariant

This paper critically compiles all published cases of established or putative calcium action potentials (or ultrafast calcium waves) where their speeds are known and are not limited by intercellular delays. The 127 cases include data from neurons or nerve nets within systems that range from cnidaria, ctenophores, molluscs, crustaceans, worms, echinoderms and tunicates up to mammalian brains; from muscle cells within organisms that range from Beröe, Cestum, moths, a crab, molluscs, a tunicate, frogs, chick embryos and turtles up to mammalian hearts; from epithelia in cnidaria and tunicates; even from a dinoflagellate and an insectivorous plant as well as reconstituted heart strands. They reveal a restriction to values of about 10–40 cm/sec at 20 degrees C and comparable restrictions at other temperatures. Moreover — unlike the speeds of sodium action potentials — the speeds of calcium ones are unrelated to cell diameter, at least over the available range of about 0.1 to 30 microns. Why do calcium action potentials have such fixed propagation speeds? Perhaps evolution has driven them to be the fastest waves of calcium influx which avoid subsurface poisoning.     

Localised MPF regulation in eggs

In this review we discuss the evidence that activation and inactivation of M-phase promoting factor (MPF), the universal mitotic activator, are regulated locally within the cell, and consider the mechanisms that might be responsible. Localised initiation of MPF activation has been demonstrated in Xenopus eggs and egg fragments by examination of the timing of surface contraction waves (SCWs), indicators of MPF activity, and confirmed by direct measurement of MPF in such fragments. Both the timing and the site of SCW initiation relate to the presence of nuclei and of associated centriole-nucleated microtubules. Localised MPF activation is likely to occur in the perinuclear cytoplasm as well as within the nucleus. Studies in a number of cell types show that the perinuclear/centrosomal region is the site of accumulation of MPF itself (the cyclin B-Cdc2 kinase complex) and of many of its molecular regulators. It also harbours calcium-regulating machinery, and in sea urchin eggs is the site of transient calcium release at the onset of mitosis. During mitosis MPF, regulatory molecules and calcium signalling components associate with spindle structures. Inactivation of MPF to end mitosis has been shown to be initiated locally at the mitoic spindle in Drosophila embryos. In sea urchin and frog eggs, calcium transients are required for both mitotic entry and exit and in mouse eggs, MPF inactivation requires both a calcium signal and an intact spindle. It thus appears that calcium signals coinciding with localised accumulation of MPF regulators are required first to set off and/or amplify the MPF activation process around the nucleus, and later to promote MPF inactivation via cyclin B destruction. Calcium release from sequestering machinery organised around nuclear and astral structures may act co-operatively with localised MPF regulatory molecules to trigger both mitotic entry and exit.     

Dependence of Ca outflow and depression of frog myocardium contraction on ryodipine concentration

The effect of ryodipine on calcium outflow from tissues, on contraction force, the duration of action potentials and the relaxation phase time-constant in the contraction cycles of myocardial strips was studied using frog heart preparations. It was found that calcium outflow (delta Ca) as a function on ryodipine concentration can be represented as: (formula; see text) A linear correlation exists between Ca2+, contraction blocking and the shortening of the action potential in the presence of various ryodipine concentrations. Ryodipine (10(-5) mol/l) decreased the relaxation time-constant by about 20% as compared to controls. It was concluded that calcium outflow from myocardial tissues in response to ryodipine is due to blockade of calcium entry into the cells and their output through the Na+--Ca2+ exchange system. Frog heart myocardial contractions are essentially under the control of calcium entry through sarcolemmal calcium channels.     

Intracellular calcium gradients in cultured human uterine smooth muscle: a functionally important subplasmalemmal space

The plasma membrane contains the key elements for the control of coupling excitation to contraction in smooth muscle. The superficial calcium buffer barrier, initially proposed by van Breemen for vascular smooth muscle, may participate in the regulation of calcium entry in other smooth muscle types. To investigate the relationship between the sarcoplasmic reticulum (SR) and the plasma membrane in myometrial smooth muscle cells, we performed experiments using videofluorescence imaging and cell-attached electrophysiology. The cell-attached patch was used as a reporter for the free calcium in the subplasmalemmal space by monitoring openings of the Maxi-K channel. Calcium green-1 was used to simultaneously monitor changes of the deep cytosolic calcium concentrations. The cell with the patch attached was stimulated via an intercellular calcium wave from an adjacent cell. In this fashion, release of SR calcium was accomplished with minimal disturbance of the plasma membrane and the subplasmalemmal space of the cell studied. With physiological bathing solution, six of seven calcium waves activated Maxi-K channels. Surprisingly, the Maxi-K channels began opening 6.3 +/- 4.7s (range 2.6-15.0s) after the wave passed the pipette location. When plasma membrane calcium fluxes were inhibited with 100 microM lanthanum, no Maxi-K channel openings were observed in six of seven experiments. These results are best explained by a subplasmalemmal space in which the calcium concentration is largely controlled by store-operated channels. These results suggest the superficial buffer barrier as merely one aspect of subplasmalemmal regulation of calcium dynamics, and emphasize the importance of store-operated calcium channels during dynamic calcium changes.     

The wave of activation current in the Xenopus egg

A ring-shaped wave of inward current, the activation current, propagates across the Xenopus egg from the site of activation during the positive phase of the activation or fertilization potential. This activation current wave is due to an increased chloride conductance and reflects the propagated of the ionic channels responsible for the fertilization potential. These channels are present in the animal and vegetal hemispheres; however, the magnitude of the activation current is 6-7 times greater in the animal hemisphere. Outward current of a smaller magnitude and spread out over a larger area precedes and follows the inward current except at the point of activation where the current is first inward. The inward current wave is detected in all eggs activated by sperm and in eggs activated by pricking with a sharp needle, by application of the Ca2+ ionophore, A23187, and by intracellular iontophoresis of Ca2+ or inositol 1,4,5-trisphosphate. Reduction of the inward current by TMB-8, which blocks intracellular calcium release in some cells, suggests that the activation current channels are calcium sensitive and that the current wave is concomitant with a wave of increased intracellular calcium initiated by sperm-egg interaction. The wave of cortical granule exocytosis and two or more contraction waves follow the current wave.     

Furrow-related contractions are inhibited but furrow-unrelated contractions are not affected in af mutant eggs of Xenopus laevis

In embryos from af mutant females of Xenopus laevis, the cleavage furrows stayed on the surface and cytoplasmic divisions did not take place at all, while nuclear divisions continued (Kubota et al., 1991). To gain insights into the roles of the normal product of af on early development, contractile events which have been observed in the period from fertilization until first cleavage in wild-type eggs were examined in af mutant eggs. Activation waves, activation contraction, and surface contraction waves which were identical to those in wild-type eggs were observed in af eggs by time-lapse video recording. However, second polar body elimination was inhibited in af eggs, although a sign of the polar body formation was indicated by the cytoplasmic bulge of the egg surface as seen by light and electron microscopy. These results indicate that the normal product of af regulates furrow-related contractile events which involve formation of the contractile ring, but exerts no effects on furrow-unrelated contractions in early Xenopus eggs.     

Expression of apo-aequorin during embryonic development; how much is needed for calcium imaging?

Aequorin is a bioluminescent calcium indicator consisting of a 21 kDa protein (apo-aequorin) that is covalently linked to a lipophilic cofactor (coelenterazine). The aequorin gene can be expressed in a variety of cell lines and tissues, allowing non-invasive calcium imaging of specific cell types. In the present paper, we describe the possibilities and limitations of calcium imaging with genetically introduced apo-aequorin during embryonic development. By injecting aequorin into sea urchin, Drosophila and zebrafish eggs, we found that higher aequorin concentrations are needed in smaller eggs. Our results suggest that for measuring resting levels of free cytosolic calcium, one needs aequorin concentrations of at least 40 μM in sea urchin eggs, 2 μM in Drosophila eggs, and only 0.11 μM in zebrafish eggs. A simple assay was used to determine the absolute concentrations of expressed apo-aequorin and the percentage of aequorin formation in vivo. The use of this assay is illustrated by expression of the aequorin gene in Drosophila oocytes. These oocytes form up to 1 μM apo-aequorin. In our hands, only 0.3% of this apo-aequorin combined with coelenterazine entering from the medium to form aequorin, which was not enough for calcium imaging of the oocytes, but did allow in vivo imaging of the ovaries. From these studies, we conclude that coelenterazine entry into the cell is the rate limiting step in aequorin formation. Based on the rate of coelenterazine uptake in Drosophila, we estimate that complete conversion of 1 μM apo-aequorin would take 50 days in zebrafish eggs, 19 days in Drosophila eggs, 7 days in sea urchin eggs or 18 h in a 10 gm tissue culture cell. Our results suggest that work based on genetically introduced apo-aequorin will be most successful when large amounts of small cells can be incubated in coelenterazine. During embryonic development this would involve introducing coelenterazine into the circulatory system of late stage embryos. Calcium imaging in early stage embryos may be best done by injecting aequorin, which circumvents the slow process of coelenterazine entry.     

Role of nuclear material in the early cell cycle of Xenopus embryos

Activated Xenopus eggs show periodic surface contraction waves and oscillations in endogenous protein phosphorylation, MPF, and kinase activities timed with the cleavage cycle of control fertilized eggs. In this paper, we show that in activated eggs lacking the material that originates from the oocyte nucleus, MPF and kinase oscillations occur in the absence of surface contraction waves. Two mitotic phosphoproteins (M116 and M46), previously described by 32P labeling in nucleated eggs, are no longer detected in the enucleated eggs. We conclude that a cytoplasmic temporal control of MPF and kinase activities is likely to be the essential cell cycle oscillator. The oocyte nuclear components normally stored in the cytoplasm of the embryos are not involved in the clock although they appear to be required for the generation of surface contraction waves.     

A comparative study of changes in the fine structure of avian eggshells during incubation

A scanning electron microscope study of the morphological changes which occur in shells of hen and quail eggs during incubation is described. The results are compared with observations on the shells of hatched eggs taken from a range of species. It was found that less change occurred in the shells of altricial species than precocial ones, the difference being associated presumably with a smaller calcium requirement for developing altricial embryos. The cores rather than the sides of mammillae appeared to be the major sites of erosion.     

The activation wave of calcium in the ascidian egg and its role in ooplasmic segregation

We have studied egg activation and ooplasmic segregation in the ascidian Phallusia mammillata using an imaging system that let us simultaneously monitor egg morphology and calcium-dependent aequorin luminescence. After insemination, a wave of highly elevated free calcium crosses the egg with a peak velocity of 8-9 microns/s. A similar wave is seen in egg fertilized in the absence of external calcium. Artificial activation via incubation with WGA also results in a calcium wave, albeit with different temporal and spatial characteristics than in sperm-activated eggs. In eggs in which movement of the sperm nucleus after entry is blocked with cytochalasin D, the sperm aster is formed at the site where the calcium wave had previously started. This indicates that the calcium wave starts where the sperm enters. In 70% of the eggs, the calcium wave starts in the animal hemisphere, which confirms previous observations that there is a preference for sperm to enter this part of the egg (Speksnijder, J. E., L. F. Jaffe, and C. Sardet. 1989. Dev. Biol. 133:180-184). About 30-40 s after the calcium wave starts, a slower (1.4 microns/s) wave of cortical contraction starts near the animal pole. It carries the subcortical cytoplasm to a contraction pole, which forms away from the side of sperm entry and up to 50 degrees away from the vegetal pole. We propose that the point of sperm entry may affect the direction of ooplasmic segregation by causing it to tilt away from the vegetal pole, presumably via some action of the calcium wave.     

The repetitive calcium waves in the fertilized ascidian egg are initiated near the vegetal pole by a cortical pacemaker.

Ascidian eggs respond to fertilization with a series of repetitive calcium waves that originate mostly from the vegetal/contraction pole region (J. E. Speksnijder, C. Sardet, and L. F. Jaffe, 1990, Dev. Biol. 142, 246-249), where the myoplasm is concentrated during the first phase of ooplasmic segregation. This suggests that the myoplasm may be involved in initiating these calcium waves. To test this possibility, the starting position of the calcium waves was determined in eggs that had the subcortical, mitochondria-rich part of the myoplasm displaced by centrifugation. Such centrifuged eggs display four cytoplasmic layers: a large centrifugal yolk zone, a narrow clear zone, a mitochondria-rich layer, and a small clear zone at the centripetal pole. Imaging of the cytosolic calcium in centrifuged eggs that were injected with the calcium-specific photoprotein aequorin reveals a series of repetitive calcium waves after fertilization. About 70% of these waves start in the vegetal/contraction pole area, which is similar to the number of waves previously found to start in this area in uncentrifuged eggs. In contrast, only about 25% of the waves start close to the displaced mitochondria-rich layer. From this result it is concluded that the main wave initiation site is not displaced by the centrifugal forces that displace the subcortical, mitochondria-rich part of the myoplasm. Moreover, the observation that the animal-vegetal polarity of cortical components such as actin filaments and the endoplasmic reticulum has been retained after centrifugation further suggests that a cortical component located in the vegetal hemisphere--most likely the endoplasmic reticulum network in the cortical region of the myoplasm--is involved in initiating the repetitive calcium waves in the fertilized ascidian egg.