The Influence of Ca Buffers on Free [Ca] Fluctuations and the Effective Volume of Ca Microdomains

Intracellular calcium (Ca²⁺) plays a significant role in many cell signaling pathways, some of which are localized to spatially restricted microdomains. Ca²⁺ binding proteins (Ca²⁺ buffers) play an important role in regulating Ca²⁺ concentration ([Ca²⁺]). Buffers typically slow [Ca²⁺] temporal dynamics and increase the effective volume of Ca²⁺ domains. Because fluctuations in [Ca²⁺] decrease in proportion to the square-root of a domain’s physical volume, one might conjecture that buffers decrease [Ca²⁺] fluctuations and, consequently, mitigate the significance of small domain volume concerning Ca²⁺ signaling. We test this hypothesis through mathematical and computational analysis of idealized buffer-containing domains and their stochastic dynamics during free Ca²⁺ influx with passive exchange of both Ca²⁺ and buffer with bulk concentrations. We derive Langevin equations for the fluctuating dynamics of Ca²⁺ and buffer and use these stochastic differential equations to determine the magnitude of [Ca²⁺] fluctuations for different buffer parameters (e.g., dissociation constant and concentration). In marked contrast to expectations based on a naive application of the principle of effective volume as employed in deterministic models of Ca²⁺ signaling, we find that mobile and rapid buffers typically increase the magnitude of domain [Ca²⁺] fluctuations during periods of Ca²⁺ influx, whereas stationary (immobile) Ca²⁺ buffers do not. Also contrary to expectations, we find that in the absence of Ca²⁺ influx, buffers influence the temporal characteristics, but not the magnitude, of [Ca²⁺] fluctuations. We derive an analytical formula describing the influence of rapid Ca²⁺ buffers on [Ca²⁺] fluctuations and, importantly, identify the stochastic analog of (deterministic) effective domain volume. Our results demonstrate that Ca²⁺ buffers alter the dynamics of [Ca²⁺] fluctuations in a nonintuitive manner. The finding that Ca²⁺ buffers do not suppress intrinsic domain [Ca²⁺] fluctuations raises the intriguing question of whether or not [Ca²⁺] fluctuations are a physiologically significant aspect of local Ca²⁺ signaling.     

An insulin-sensitive cation channel controls [Na+]i via [Ca2+]o-regulated Na+ and Ca2+ entry.

The insulin-stimulated cation channel previously identified in patch-clamped muscle preparations is here shown to be responsible for bulk Na+ entry into the cell. The mainly Na+ current of the channel was shown to be accompanied by an inhibitory Ca2+ component responsible for oscillations. Here, using quantitative fluorescence imaging of Fura-2- and SBFI-loaded soleus muscle, we measure changes in [Na+]i and [Ca2+]i related to channel function. Insulin increased [Na+]i and [Ca+]i in a transient spike of < 1-min duration. There was a momentary dip in [Na+]i related to inhibition of the channel by the Ca2+ spike, and changes in external Ca2+ were shown to alter [Na+]i via the cation channel, all effects being blocked by the specific channel inhibitor mu-conotoxin, but not by tetrodotoxin. The [Ca2+]i spike could also be induced by 8-bromo cyclic-guanosine 5'-monophosphate, an analogue of the channel-activator cyclic-guanosine 5'-monophosphate (cGMP). In addition it was noted that insulin reduced the [Ca2+]i rise upon subsequent muscle depolarization by a factor of 3.5. Insulin could be substituted with phorbol ester for the same effect and HA1004, a protein kinase inhibitor, blocked the reduction.     

Dependence of the resting [Ca2+]cyt of RBL-1 cells on Ca2+ entry

The cytoplasmic Ca²⁺ concentration ([Ca²⁺]_cyt) in resting cells in an equilibrium between several influx and efflux mechanisms. Here we address the question of whether capacitative Ca²⁺ entry to some extent is active at resting conditions and therefore is part of processes that guarantee a constant [Ca²⁺]_cyt. We measured changes of [Ca²⁺]_cyt in RBL-1 cells with fluorometric techniques. An increase of the extracellular [Ca²⁺] from 1.3 mM to 5 mM induced an incrase in [Ca²⁺]_cyt from 105±10 nM to 145±8.5 nM. This increase could be inhibited by 10 μM Gd³⁺, 10 μM La³⁺ or 50 μM 2-aminoethoxydiphenyl borate, blockers of capacitative Ca²⁺ entry. Application of those blockers to a resting cell in a standard extracellular solution (1.3 mM Ca²⁺) resulted in a decrease of [Ca²⁺]_cyt from 105±10 nM to 88.5±10 nM with La³⁺, from 103±12 to 89±12 nM with Gd³⁺ and from 102±12 nM to 89.5±5 nM with 2-aminoethoxydiphenyl borate. From these data, we conclude that capacitative Ca²⁺ entry beside its function in Ca²⁺ signaling contributes to the regulation of resting [Ca²⁺]_cyt.     

Rapidly exchanging Ca2+ stores: Role in the control of Ca2+ homeostasis and [Ca2+] in oscillations

The rapidly exchanging intracellular calcium stores play an important role in control of cytoplasmic calcium homeostasis and in generation of intracellular calcium signals. These stores are specific intracellular compartments which are able to accumulate and release calcium in response to appropriate stimuli. Two types of stores can be distinguished in nonmuscle cells based on substances discharging these stores: (1) Ca²⁺-sensitive and (2) inositol-1,4,5-trisphosphate-sensitive intracellular depots. These two depots can be either separate intracellular compartments or a single compartment that shares both releasing mechanisms. The state of the art of our understanding of the cytoplasmic calcium release is the focus of this review.Neirofiziologiya/Neurophysiology, Vol. 26, No. 1, pp. 9–15, January–February, 1994.     

Measurement of sub-membrane [Ca2+] in adult myofibers and cytosolic [Ca2+] in myotubes from normal and mdx mice using the Ca2+ indicator FFP-18

The hypothesis that intracellular Ca(2+) is elevated in dystrophic (mdx) skeletal muscle due to increased Ca(2+) influx is controversial. As the sub-sarcolemmal Ca(2+) ([Ca(2+)](mem)) should be even higher than the global cytosolic Ca(2+) in the presence of increased Ca(2+) influx, we investigated [Ca(2+)](mem) levels in collagenase-isolated adult flexor digitorum brevis (FDB) myofibres and myotubes of mdx and normal mice with the near-membrane Ca(2+) indicator FFP-18. Confocal imaging showed strong localization of FFP-18 to the sarcolemma only. No significant difference in [Ca(2+)](mem) was found in FDB myofibres of normal (77.3+/-3.8 nM, n=68) and mdx (79.3+/-5.6 nM, n=21, p=0.89) mice using FFP-18. Increasing external Ca(2+) to 18 mM did not significantly affect [Ca(2+)](mem) in either the normal or mdx myofibres. In the myotubes, the FFP-18 was non-selectively incorporated, distributing throughout the cytoplasm, and FFP-18-derived [Ca(2+)] values were similar to values obtained with Fura-2. Nevertheless, in the mdx myotubes, the [Ca(2+)] measured with FFP-18 increased linearly to a level approximately 2.75 times that of controls as the time of culture was prolonged. In older mdx myotubes (>or=8 days in culture), 18 mM extracellular Ca(2+) increased the steady state cytosolic [Ca(2+)] to approximately 22 times greater level than controls. This study suggests that the sub-sarcolemmal Ca(2+) homeostasis is well maintained in isolated adult mdx myofibers and also further supports the hypothesis that cytosolic Ca(2+) handling is compromised in mdx myotubes.     

The significance of physiological [Ca2+] and [Mg2+] for in vitro experiments on synaptic transmission

Since 1932 $\text{in}$$\text{vitro}$ physiological and pharmacological studies on neuromuscular and other types of synaptic transmission have been carried out usually in Krebs' of similar balanced electrolyte solutions. It has been disregarded, however, that although the total calcium [Ca_t] (2.5 mM) and [Mg_t] (1.2 mM), are about the same in human plasma and in Krebs' solution, the physiologically important [Ca²⁺] and [Mg²⁺], primarily because of binding to plasma proteins, are much lower in plasma (1.1 and 0.6 mM) than in Krebs' solution (2.0 and 1.1 mM). We observed that in a modified Krebs' solution in which the [Ca_t] and [Mg_t] are 1.4 and 0.9 mM respectively and the [Ca²⁺] and [Mg²⁺] are about the same as in human plasma, the Ca²⁺ dependent volley output of acetylcholine is less and the inhibition of the electrically induced isometric twitch tension of the rat phrenic nerve - hemidiaphragm preparation by nondepolarizing neuromuscular blocking agents and certain antibiotics is greater than in conventional Krebs' solution, in which the [Ca²⁺] and [Mg²⁺] are higher than $\text{in}$$\text{vivo}$. Similarly, during electrical field stimulation of the guinea-pig myenteric plexus - longitudinal muscle preparation volley output of acetylcholine is lower and the inhibition of the isometric contraction of the muscle by normophine is greater in modified than in conventional Krebs' solution. It is suggested that for greater relevance to $\text{in}$$\text{vivo}$ conditions the [Ca²⁺] and [Mg²⁺] of balanced electrolyte solutions used in in vitro experiments on synaptic transmission should be the same as in human plasma or in the plasma of the species of the experimental animal.     

Signaling between intracellular Ca2+ stores and depletion-activated Ca2+ channels generates [Ca2+]i oscillations in T lymphocytes

Stimulation through the antigen receptor (TCR) of T lymphocytes triggers cytosolic calcium ([Ca2+]i) oscillations that are critically dependent on Ca2+ entry across the plasma membrane. We have investigated the roles of Ca2+ influx and depletion of intracellular Ca2+ stores in the oscillation mechanism, using single-cell Ca2+ imaging techniques and agents that deplete the stores. Thapsigargin (TG; 5-25 nM), cyclopiazonic acid (CPA; 5-20 microM), and tert- butylhydroquinone (tBHQ; 80-200 microM), inhibitors of endoplasmic reticulum Ca(2+)-ATPases, as well as the Ca2+ ionophore ionomycin (5-40 nM), elicit [Ca2+]i oscillations in human T cells. The oscillation frequency is approximately 5 mHz (for ATPase inhibitors) to approximately 10 mHz (for ionomycin) at 22-24 degrees C. The [Ca2+]i oscillations resemble those evoked by TCR ligation in terms of their shape, amplitude, and an absolute dependence on Ca2+ influx. Ca(2+)- ATPase inhibitors and ionomycin induce oscillations only within a narrow range of drug concentrations that are expected to cause partial depletion of intracellular stores. Ca(2+)-induced Ca2+ release does not appear to be significantly involved, as rapid removal of extracellular Ca2+ elicits the same rate of [Ca2+]i decline during the rising and falling phases of the oscillation cycle. Both transmembrane Ca2+ influx and the content of ionomycin-releasable Ca2+ pools fluctuate in oscillating cells. From these data, we propose a model in which [Ca2+]i oscillations in T cells result from the interaction between intracellular Ca2+ stores and depletion-activated Ca2+ channels in the plasma membrane.     

Synaptosomal [Ca2+]i as influenced by Na+/Ca2+ exchange and K+ depolarization.

The modulation of the intrasynaptosomal concentration of Ca2+, [Ca2+]i, by Na+/Ca2+ exchange was studied using Indo-1 fluorescence. The electrochemical gradient of Na+ was manipulated by substituting Li+ or choline for Na+ in the external medium and, then, the influx of 45Ca2+ and the [Ca2+]i were measured. It was found that the increase in [Ca2+]i induced by K+ depolarization is lower if the value of [Ca2+]i has been previously raised by Na+/Ca2+ exchange, suggesting that Ca2+ entering by Na+/Ca2+ exchange reduces the Ca2+ entering by voltage-dependent calcium channels. Our results show that a value of [Ca2+]i of about 650 nM induced by Na+/Ca2+ exchange reduces by 50% the Ca2+ entering due to K+ depolarization and no Ca2+ enters through the channels if the [Ca2+]i is previously raised above about 800 nM. Furthermore, predepolarization of the synaptosomes in a Ca-free medium also inhibits by at least 40% the [Ca2+]i rise through Ca2+ channels. Thus, the results suggest that both predepolarization and [Ca2+]i rise due to Na+/Ca2+ exchange decrease the Ca2+ entering by voltage-sensitive Ca2+ channels. The Ca2+ entering by Na+/Ca2+ exchange might contribute to the regulation of neurotransmitter release. Our results also show that the presence of Li+ in the external medium decreases the buffering capacity of synaptosomes, probably by releasing Ca2+ from mitochondria by Li+/Ca2+ exchange.     

Buffers and oscillations in intracellular Ca2+ dynamics

I model the behavior of intracellular Ca(2+) release with high buffer concentrations. The model uses a spatially discrete array of channel clusters. The channel subunit dynamics is a stochastic representation of the DeYoung-Keizer model. The calculations show that the concentration profile of fast buffer around an open channel is more localized than that of slow buffers. Slow buffers allow for release of larger amounts of Ca(2+) from the endoplasmic reticulum and hence bind more Ca(2+) than fast buffers with the same dissociation constant and concentration. I find oscillation-like behavior for high slow buffer concentration and low Ca(2+) content of the endoplasmic reticulum. High concentration of slow buffer leads to oscillation-like behavior by repetitive wave nucleation for high Ca(2+) content of the endoplasmic reticulum. Localization of Ca(2+) release by slow buffer, as used in experiments, can be reproduced by the modeling approach.     

How source content determines intracellular Ca2+ release kinetics. Simultaneous measurement of [Ca2+] transients and [H+] displacement in skeletal muscle

In skeletal muscle, the waveform of Ca(2+) release under clamp depolarization exhibits an early peak. Its decay reflects an inactivation, which locally corresponds to the termination of Ca(2+) sparks, and is crucial for rapid control. In cardiac muscle, both the frequency of spontaneous sparks (i.e., their activation) and their termination appear to be strongly dependent on the Ca(2+) content in the sarcoplasmic reticulum (SR). In skeletal muscle, no such role is established. Seeking a robust measurement of Ca(2+) release and a way to reliably modify the SR content, we combined in the same cells the "EGTA/phenol red" method (Pape et al., 1995) to evaluate Ca(2+) release, with the "removal" method (Melzer et al., 1987) to evaluate release flux. The cytosol of voltage-clamped frog fibers was equilibrated with EGTA (36 mM), antipyrylazo III, and phenol red, and absorbance changes were monitored simultaneously at three wavelengths, affording largely independent evaluations of Delta[H(+)] and Delta[Ca(2+)] from which the amount of released Ca(2+) and the release flux were independently derived. Both methods yielded mutually consistent evaluations of flux. While the removal method gave a better kinetic picture of the release waveform, EGTA/phenol red provided continuous reproducible measures of calcium in the SR (Ca(SR)). Steady release permeability (P), reached at the end of a 120-ms pulse, increased as Ca(SR) was progressively reduced by a prior conditioning pulse, reaching 2.34-fold at 25% of resting Ca(SR) (four cells). Peak P, reached early during a pulse, increased proportionally much less with SR depletion, decreasing at very low Ca(SR). The increase in steady P upon depletion was associated with a slowing of the rate of decay of P after the peak (i.e., a slower inactivation of Ca(2+) release). These results are consistent with a major inhibitory effect of cytosolic (rather than intra-SR) Ca(2+) on the activity of Ca(2+) release channels.     

Interplay between Ca2+ release and Ca2+ influx underlies localized hyperpolarization-induced [Ca2+]i waves in prostatic cells.

Calcium seems to be a major second messenger involved in the regulation of prostatic cell functions, but the mechanisms underlying its control are poorly understood. We investigated spatiotemporal aspects of Ca2+ signals in the LNCaP cell line, a model of androgen-dependent prostatic cells, by using non-invasive external electric field pulses that hyperpolarize the anode facing membrane and depolarize the membrane facing the cathode. Using high-speed fluo-3 confocal imaging, we found that an electric field pulse (10-15 V/cm, 1-5 mA, 5 ms) initiated rapidly, at the hyperpolarized end of the cell, a propagated [Ca2+]i wave which spread through the cell with a constant amplitude and an average velocity of about 20 microns/s. As evidenced by the total wave inhibition either by the block of Ca2+ entry or the depletion of Ca2+ stores by thapsigargin, a specific Ca(2+)-ATPase inhibitor, the [Ca2+]i wave initiation may imply a localized Ca2+ influx linked to a focal auto-regenerative process of Ca2+ release. Using different external Ca2+ and Ca2+ entry blockers concentrations, Mn2+ quenching of fluo-3 and fura-2 fluorescence and inhibitors of InsP3 production, we found evidence that the [Ca2+]i wave progression required, in the presence of basal levels of InsP3, an interplay between Ca2+ release from InsP3-sensitive Ca2+ stores and Ca2+ influx through channels possibly activated by the [Ca2+]i rise.     

激发子诱发的小麦叶肉细胞原生质体[Ca2+]cyt升高主要源于胞外钙离子内流

以小麦叶肉细胞原生质体-激发子互作为研究体系,借助共聚焦激光扫描显微镜观察结合药物学试验,对激发子刺激后不同抗叶锈性小麦品种原生质体[Ca2+]cyt的动态变化和[Ca2+]cyt升高的钙来源进行了研究。结果表明：抗叶锈小麦品种‘洛夫林10’的原生质体在激发子处理后,[Ca2+]cyt明显升高,随后有所下降,但在试验检测时间范围内仍保持较高浓度水平;而感病品种‘郑州5389’经激发子处理后,[Ca2+]cyt只发生轻微的波动。使用质膜钙通道抑制剂抑制胞外钙离子流入胞内,再经激发子处理,原生质体[Ca2+]cyt虽也有升高,但升高幅度大大降低。这一结果表明,激发子刺激诱发的[Ca2+]cyt升高主要源于胞外钙离子内流。     

A [Na+]o-independent, pHo-dependent mechanism for reduction of intracellular [Ca2+] after influx through Ca2+ channels in mouse pituitary cells

The effect of extracellular pH (pHo) on the duration of calcium-dependent chloride currents (ICl(Ca] was studied in voltage clamped AtT-20 pituitary cells. ICl(Ca) was activated by Ca2+ influx through plasma membrane Ca2+ channels, which were opened by step depolarization to voltages between -20 and +60 mV. Increasing pHo from 7.3 to 8.0 reversibly prolonged ICl(Ca) tail currents in perforated patch recordings from cells bathed in both Na(+)-containing and Na(+)-free solutions. This prolongation was prevented in standard whole cell recordings when the pipette solution contained 0.5 mM EGTA. The effects of raised pHo were not due to alteration of intracellular pH, since tail current prolongation still occurred when intracellular pH was buffered at 7.3 with 80 mM HEPES. The prolongation of ICl(Ca) at pHo 8 could not be accounted for by a direct action on Ca2+ channels, since tail currents were prolonged when pHo was changed rapidly during the tail current, after all Ca2+ channels were closed. The effects of increasing pHo on ICl(Ca) also could not be explained by a direct action on Cl- channels, since changing to pHo 8 did not prolong Cl- tail currents when intracellular Ca2+ concentration [( Ca2+]i) was fixed by EGTA in whole cell recordings. Raising pHo did, however, prolong depolarization-evoked [Ca2+]i transients, measured directly with the Ca2+ indicator dye, fura-2. Taken together, these data demonstrate the presence of a Na(+)-independent, pHo-sensitive mechanism for reduction of [Ca2+]i after influx through Ca2+ channels. This mechanism is associated with the plasma membrane, and is active on a time scale that is relevant to the duration of single action potentials in these cells. We suggest that this mechanism is the plasma membrane Ca2+ ATPase.     

Interplay between ER Ca2+ uptake and release fluxes in neurons and its impact on [Ca2+] dynamics

In neurons, depolarizing stimuli open voltage-gated Ca2+ channels, leading to Ca2+ entry and a rise in the cytoplasmic free Ca2+ concentration ([Ca2+]i). While such [Ca2+]i elevations are initiated by Ca2+ entry, they are also influenced by Ca2+ transporting organelles such as mitochondria and the endoplasmic reticulum (ER). This review summarizes contributions from the ER to depolarization-evoked [Ca2+]i responses in sympathetic neurons. As in other neurons, ER Ca2+ uptake depends on SERCAs, while passive Ca2+ release depends on ryanodine receptors (RyRs). RyRs are Ca2+ permeable channels that open in response to increases in [Ca2+]i, thereby permitting [Ca2+]i elevations to trigger Ca2+ release through Ca(2+)-induced Ca2+ release (CICR). However, whether this leads to net Ca2+ release from the ER critically depends upon the relative rates of Ca2+ uptake and release. We found that when RyRs are sensitized with caffeine, small evoked [Ca2+]i elevations do trigger net Ca2+ release, but in the absence of caffeine, net Ca2+ uptake occurs, indicating that Ca2+ uptake is stronger than Ca2+ release under these conditions. Nevertheless, by increasing ER Ca2+ permeability, RyRs reduce the strength of Ca2+ buffering by the ER in a [Ca2+](I)-dependent manner, providing a novel mechanism for [Ca2+]i response acceleration. Analysis of the underlying Ca2+ fluxes provides an explanation of this and two other modes of CICR that are revealed as [Ca2+]i elevations become progressively larger.     

Phase-dependent contributions from Ca2+ entry and Ca2+ release to caffeine-induced [Ca2+]i oscillations in bullfrog sympathetic neurons.

Sympathetic neurons display robust [Ca2+]i oscillations in response to caffeine and mild depolarization. Oscillations occur at constant membrane potential, ruling out voltage-dependent changes in plasma membrane conductance. They are terminated by ryanodine, implicating Ca(2+)-induced Ca2+ release. Ca2+ entry is necessary for sustained oscillatory activity, but its importance varies within the oscillatory cycle: the slow interspike rise in [Ca2+]i requires Ca2+ entry, but the rapid upstroke does not, indicating that it reflects internal Ca2+ release. Sudden alterations in [Ca2+]o, [K+]o, or [caffeine]o produce immediate changes in d[Ca2+]i/dt and provide information about the relative rates of surface membrane Ca2+ transport as well as uptake and release by internal stores. Based on our results, [Ca2+]i oscillations can be explained in terms of coordinated changes in Ca2+ fluxes across surface and store membranes.     

Characterization of the Increase in [Ca2+] i During Hypotonic Shock and the Involvement of Ca2+-activated K+ Channels in the Regulatory Volume Decrease in Human Osteoblast-like Cells

The calcium indicator fura-2 was used to study the effect of hypotonic solutions on the intracellular calcium concentration, [Ca²⁺] _i , in a human osteoblast-like cell line. Decreasing the tonicity of the extracellular solution to 50% leads to an increase in [Ca²⁺] _i from ∼150 nm up to 1.3 μm. This increase in [Ca²⁺] _i was mainly due to an influx of extracellular Ca²⁺ since removing of extracellular Ca²⁺ reduced this increase to ∼250 nm. After cell swelling most of the cells were able to regulate their volume to the initial level within 800 sec. The whole-cell recording mode of the patch-clamp technique was also used to study the effect of an increase in [Ca²⁺] _i on membrane currents in these cells. An increase in [Ca²⁺] _i revealed two types of Ca²⁺-activated K⁺ channels, K(Ca) channels. Current through both channel types could not be observed below voltage of +80 mV with [Ca²⁺] _i buffered to 100 nm or less. With patch-electrodes filled with solutions buffering [Ca²⁺] _i to 10 μm both channels types could be readily observed. The activation of the first type was apparently voltage-independent since current could be observed over the entire voltage range used from −160 to +100 mV. In addition, the current was also blocked by charybdotoxin (CTX). The second type of K(Ca) channels in these cells could be activated with depolarizations more positive than −40 mV from a holding potential of −80 mV. This type was blocked by CTX and paxilline. Adding paxilline to the extracellular solution inhibited regulatory volume decrease (RVD), but could not abolish RVD. We conclude that two K(Ca) channel types exist in human osteoblasts, an intermediate conductance K(Ca) channel and a MaxiK-like K(Ca) channel. MaxiK channels might get activated either directly or by an increase in [Ca²⁺] _i elicited through hypotonic solutions. In combination with the volume-regulated Cl⁻ conductance in the same cells this K⁺ channel seems to play a vital role in volume regulation in human osteoblasts. Received: 8 February 2000/Revised: 13 July 2000     

卵细胞受精相关胞质钙离子波、钙离子震荡信号特征及其形成机制

胞质[Ca2 ]i震荡的动力学变化在哺乳动物早期胚胎发育中发挥重要作用。卵母细胞的成熟伴随间断的、快速的[Ca2 ]i震荡的时空表达;在受精过程中精子因子诱导的反复[Ca2 ]i震荡的振幅和持续时间是卵细胞最有效的激活信号,这种信号形成自然连续的受精[Ca2 ]i波,并以长时持续[Ca2 ]i震荡形式在受精卵空间传递并持续数小时,直至受精完成;受精卵内源性的Ca2 释放所引起的[Ca2 ]i震荡形成第一次卵裂信号,启动早期胚胎的发育。精子PLCζ和cPKCs是形成受精卵[Ca2 ]波、[Ca2 ]震荡的重要因素。     

The [Ca2+ + Mg2+]-dependent adenosine triphosphatase of SV40 transformed WI38 lung fibroblasts

The Ca2+-stimulated, Mg2+-dependent ATPase of SV40 transformed WI38 lung fibroblast homogenates exhibits a high affinity for Ca2+ (K0.5 = 0.20 microM) and moderately high affinity for ATP (Km = 28.6 microM) and Mg2+ (K0.5 = 138.5 microM). This activity was NaN3, KCN and oligomycin insensitive but very sensitive to vanadate (I50 = 0.5 microM) suggesting its being neither mitochondrial or microsomal but plasma membrane in origin. Under optimal conditions of protein, hydrogen ion and substrate concentration, 16-19 nmoles phosphate was released per min per mg protein. Hill plot analysis indicated no cooperativity to occur between Ca2+ binding sites. Nucleotides other than ATP and dATP were ineffective as substrates. The trivalent cation, lanthanum (La3+) completely inhibited hydrolysis of ATP at approximately 70 microM (I50 = 25 microM). Calmodulin antagonists trifluoperazine and calmidazolium inhibited ATP hydrolysis in a dose dependent fashion.