Ca2+ signalling during fertilization of echinoderm eggs

The Ca2+ rise at fertilization of echinoderm eggs is initiated by a process requiring the sequential activation of a Src family kinase, phospholipase C gamma, and the inositol trisphosphate receptor/channel in the endoplasmic reticulum. The consequences of the Ca2+ rise include exocytosis of cortical granules, which establishes a block to polyspermy, and inactivation of MAP kinase, which functions in linking the Ca2+ rise to the reinitiation of the cell cycle.     

Intracellular Ca2+ regulation in rat motoneurons during development

Changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) control the setting up of the neuro-muscular synapse in vitro and probably in vivo. Dissociated cultures of purified embryonic (E15) rat motoneurons were used to explore the molecular mechanisms by which endoplasmic reticulum Ca(2+) stores, via both ryanodine-sensitive and IP(3)-sensitive intracellular Ca(2+) channels control [Ca(2+)](i) homeostasis in these neurons during ontogenesis. Fura-2 microspectrofluorimetry monitorings in single neurons showed that caffeine-induced responses of [Ca(2+)](i) increased progressively from days 1-7 in culture. These responses were blocked by ryanodine and nicardipine but not by omega-conotoxin-GVIA or omega-conotoxin-MVIIC suggesting a close functional relationship between ryanodine-sensitive and L-type Ca(v)1 Ca(2+) channels. Moreover, after 6 days in vitro, neurons exhibited spontaneous or caffeine-induced Ca(2+) oscillations that were attenuated by nicardipine. In 1-day-old neurons, both thapsigargin or CPA, which deplete Ca(2+) stores from the endoplasmic reticulum, induced an increase in [Ca(2+)](i) in 75% of the neurons tested. The number of responding motoneurons declined to 25% at 5-6 days in vitro. Xestospongin-C, a membrane-permeable IP(3) receptor inhibitor blocked the CPA-induced [Ca(2+)](i) response in all stages. RT-PCR studies investigating the expression pattern of RYR and IP(3) Ca(2+) channels isoforms confirmed the presence of their different isoforms and provided evidence for a specific pattern of development for RYR channels during the first week in vitro. Taken together, present results show that the control of motoneuronal [Ca(2+)](i) homeostasis is developmentally regulated and suggest the presence of an intracellular ryanodine-sensitive Ca(2+) channel responsible for a Ca(2+)-induced Ca(2+) release in embryonic motoneurons following voltage-dependent Ca(2+) entry via L-type Ca(2+) channels.     

L-type Ca2+ currents in ascidian eggs.

We have studied Ca2+ currents in ascidian eggs using the whole-cell clamp technique. T and L components, as observed in somatic cells, are present and the L-type current predominates. Since the IV relationship for these inward currents overlap at -30 mV, separation of the two components using different voltage regimes is not feasible. Increasing external Ca2+ results in larger currents. The L-type current decreases in a dose-dependent fashion in the presence of Mn2+ and Nifedipine, while the T-type current is inhibited in Ni2+. When Ba2+ was used as the carrier ion, channel kinetics and conductance were completely altered. Considering the density and kinetics of L-type channels in unfertilized eggs it is probable they play an important role in regulating cytosolic Ca2+ during early developmental processes.     

Intracellular Ca2+ concentration and latency of light-induced Ca2+ changes in photoreceptors of the honeybee drone

We have measured Ca_i at rest and upon light stimulation in the photoreceptors of the honeybee drone microfluorometrically with the fluorescent Ca²⁺ indicator dyes fura-2, fluo-3 and Ca-green 5N.In darkness, Ca_i was 90 nM after 5 min of dark adaptation. A saturating light step caused Ca_i to rise in the bulk cytoplasm to 750 nM within 1 s. Our measurements with the low affinity dye Ca-green 5N showed that bright 1-s light flashes cause a rapid increase in Ca_i which was graded with stimulus intensity. Ca-green 5N fluorescence reached a peak in about 200 ms, and then decayed to a slightly lower sustained plateau. The fluorescence signal peaked, when the receptor potential was repolarizing from its peak to the plateau. This observation is in agreement with the proposal that the peak-to-plateau transition of the receptor potential is caused by the rise in Ca_i From our Fluo-3 measurements it appears that the latency of the Ca²⁺ increase is by 3–4 ms longer than the latency of the receptor potential elicited by bright 100-ms light flashes. This result provides no support for the proposal that Ca²⁺ mediates the opening of those membrane channels responsible for the upstroke of the receptor potential.Abbreviations ER endoplasmic reticulum - IP₃ Inositol 1,4,5-trisphosphate - SMC submicrovillar cisternae     

Unusual sensitivity of cytosolic free Ca2+ to changes in extracellular Ca2+ in rat C-cells

An essential function of C-cells is to monitor extracellular Ca2+ concentration ([Ca2+]e) and to respond to changes in [Ca2+]e by regulating hormone secretion. Using the calcitonin-secreting rat C-cell line rMTC 44-2, we have investigated a possible tight linkage between [Ca2+]e and cytosolic free Ca2+ ([Ca/+]i). We have demonstrated, using the Ca2+ indicator Quin 2, that the [Ca2+]i is particularly sensitive to changes in [Ca2+]e. Sequential increases in [Ca2+]e as small as 0.1 mM evoke clear elevations in [Ca2+]i. In contrast, other cell types tested did not alter their [Ca2+]i in response to increasing [Ca2+]e even to levels as high as 4.0 mM. Sequential 1.0 mM increments in [Ca2+]e caused the [Ca2+]i to rise from a base line of 357 +/- 20 nM Ca2+i at 1.0 mM Ca2+e to a maximum of 1066 +/- 149 nM Ca2+i at 5.0 mM Ca2+e. [Ca2+]e above 2.0 mM produced a biphasic response in [Ca2+]i consisting of an immediate (less than 5 s) spike followed by a decay to a new plateau. Treatment of rMTC 44-2 cells with either 50 mM K+ or 100 nM ionomycin at 1.0 mM Ca2+e caused an immediate spike in [Ca2+]i to micromolar levels. Pretreatment with EGTA or verapamil inhibited completely the increase in [Ca2+]i induced by 50 mM K+. However, pretreatment with EGTA only slightly attenuated the spike phase in [Ca2+]i produced by ionomycin, demonstrating that ionomycin released intracellular stores of calcium. We conclude that rMTC 44-2 cells regulate [Ca2+]i by monitoring small physiological changes in [Ca2+]e, the primary secretagogue for C-cells.     

Intracellular free Ca2+ and the hypercontracture of adult rat heart myocytes

The Ca2+ sensitivity of a population of isolated adult rat heart myocytes has been related to the Na+ content of the cells prior to Ca2+ exposure, and the intracellular free Ca2+ as reported by quin2 fluorescence when the cells are challenged with millimolar external Ca2+. Myocytes exposed to Ca2+ during quin2 loading show a resting intracellular free Ca2+ of 150 +/- 30 nM and retain the rod cell morphology of heart cells in situ. The myocytes take up Na+ and lose K+ when incubated in the cold in the absence of Ca2+. Large numbers of these rod-shaped, Na+-loaded myocytes hypercontract into grossly distorted round cell forms when exposed to physiological levels of Ca2+. The number of cells that hypercontract is proportional to the Na+ content of the cells prior to Ca2+ addition and can be directly related to the intracellular free Ca2+ concentration attained following Ca2+ addition. Fifty percent of the cells in a myocyte population hypercontract when the internal free Ca2+ concentration reported by quin2 reaches 400 nM and virtually all of the cells hypercontract when this value reaches 1 microM. The entry of Ca2+ into Na+-loaded myocytes is biphasic with one phase inhibited by Ca2+ channel blockade. This suggests that Ca2+ enters Na+-loaded myocytes by the Ca2+ channel as well as by Na+/Ca2+ exchange.     

Intracellular Ca2+ activity during slow synaptic hyperpolarizations in Helix pomatia

In the "bursting" cell RPal of Helix pomatia, intracellular Ca2+ activity increased during the slow synaptic hyperpolarization termed LLH. The Ca2+ increase had a double exponential time course and the kinetics provide evidence for an intracellularly located Ca2+ source. The Ca2+ wave could be mimicked by dopamine and inhibited by ergometrine. dB-cAMP also enhanced the Ca2+ activity. Intracellular K+ was measured during LLH. An appendix by J. D. C. Labmert demonstrates that a dominant mechanism underlying LLH is the increase of K+ permeability.     

Intracellular ionic changes in normal and transformed human fibroblasts after extracellular Ca2+ deprivation.

The lowering of extracellular Ca2+ concentration in the growth medium reversibly blocks normal, but not SV40-transformed WI38 diploid fibroblasts in the early G1/G0 phase of the cell cycle. This growth response is characterized by specific changes in ionic content and transport. Ca2+ deprivation (0.03 mM) has little effect on the K+ content of either normal or transformed cells. Na+ content, however, is increased nearly 2-fold in the normal cells. This increase is presumably due to a 3-fold increase in unidirectional Na+ influx in Ca2+-deprived cells. The increased intracellular Na+ also gives rise to a nearly 3-fold enhancement of the active (ouabain-sensitive) Na+ efflux. Ca2+ deprivation causes only slight increases in Na+ influx, ouabain-sensitive Na+ efflux and intracellular Na+ in the transformed cell. In contrast, the transformed cells lose nearly 60% of their intracellular Ca2+ on deprivation, whereas normal WI38 cells lose only 10%. The data suggest that the growth arrest exhibited by the normal cell but not the transformed cell may be related to different membrane-transport and permeability changes in response to Ca2+ deprivation.     

By Regulating Mitochondrial Ca2+-Uptake UCP2 Modulates Intracellular Ca2+

Introduction

The possible role of UCP2 in modulating mitochondrial Ca²⁺-uptake (mCa²⁺-uptake) via the mitochondrial calcium uniporter (MCU) is highly controversial.

Methods

Thus, we analyzed mCa²⁺-uptake in isolated cardiac mitochondria, MCU single-channel activity in cardiac mitoplasts, dual Ca²⁺-transients from mitochondrial ((Ca²⁺)m) and intracellular compartment ((Ca²⁺)c) in the whole-cell configuration in cardiomyocytes of wild-type (WT) and UCP2^-/- mice.

Results

Isolated mitochondria showed a Ru360 sensitive mCa²⁺-uptake, which was significantly decreased in UCP2^-/- (229.4±30.8 FU vs. 146.3±23.4 FU, P<0.05). Single-channel registrations confirmed a Ru360 sensitive voltage-gated Ca²⁺-channel in mitoplasts, i.e. mCa1, showing a reduced single-channel activity in UCP2^-/- (Po,total: 0.34±0.05% vs. 0.07±0.01%, P<0.05). In UCP2^-/- cardiomyocytes (Ca²⁺)m was decreased (0.050±0.009 FU vs. 0.021±0.005 FU, P<0.05) while (Ca²⁺)c was unchanged (0.032±0.002 FU vs. 0.028±0.004 FU, P>0.05) and transsarcolemmal Ca²⁺-influx was inhibited suggesting a possible compensatory mechanism. Additionally, we observed an inhibitory effect of ATP on mCa²⁺-uptake in WT mitoplasts and (Ca²⁺)m of cardiomyocytes leading to an increase of (Ca²⁺)c while no ATP dependent effect was observed in UCP2^-/-.

Conclusion

Our results indicate regulatory effects of UCP2 on mCa²⁺-uptake. Furthermore, we propose, that previously described inhibitory effects on MCU by ATP may be mediated via UCP2 resulting in changes of excitation contraction coupling.     

Synchronous free Ca2+ changes in individual neutrophils stimulated by leukotriene B4.

Calcium (Ca2+) signals were monitored in individual neutrophils using ratio imaging of fura-2. In contrast to N-formyl-L-leucyl-L-phenylalanine (f-met-leu-phe), which produced grossly asynchronous Ca2+ signals with delays in response (up to 60 s), leukotriene B4 (LTB4) provoked synchronous and immediate elevations in cytosolic free Ca2+. Some individual neutrophils which responded immediately to LTB4, subsequently displayed delayed Ca2+ signals in response to f-met-leu-phe. A sub-population of neutrophils failed to respond to both LTB4 and f-met-leu-phe. The asynchrony of the Ca2+ signalling to f-met-leu-phe is not, therefore, an obligatory property of signal transduction in neutrophils.     

Intracellular localization and physiological function of a rice Ca2+-permeable channel OsTPC1

Two-pore channels (TPCs) are cation channels with a voltage-sensor domain conserved in plants and animals. Rice OsTPC1 is predominantly localized to the plasma membrane (PM), and assumed to play an important role as a Ca²⁺-permeable cation channel in the regulation of cytosolic Ca²⁺ rise and innate immune responses including hypersensitive cell death and phytoalexin biosynthesis in cultured rice cells triggered by a fungal elicitor, xylanase from Trichoderma viride. In contrast, Arabidopsis AtTPC1 is localized to the vacuolar membrane (VM). To gain further insights into the intracellular localization of OsTPC1, we stably expressed OsTPC1-GFP in tobacco BY-2 cells. Confocal imaging and membrane fractionation revealed that, unlike in rice cells, the majority of OsTPC1-GFP fusion protein was targeted to the VM in tobacco BY-2 cells. Intracellular localization and functions of the plant TPC family is discussed.     

Predicted changes in concentrations of free and bound ATP and ADP during intracellular Ca2+ signaling

High Ca²⁺ concentrationscan develop near Ca²⁺ sourcesduring intracellular signaling and might lead to localized regulationof Ca²⁺-dependent processes. Byshifting the amount of Ca²⁺ andother cations associated with ATP, local highCa²⁺ concentrations might alsoalter the substrate available for membrane-associated and cytoplasmicenzymes. To study this, simultaneous equations were solved over a rangeof Ca²⁺ andMg²⁺ concentrations to determinethe general effects of Ca²⁺ on theconcentrations of free and Ca²⁺-and Mg²⁺-bound forms of ATP. Toobtain a more specific picture of the changes that might occur insmooth muscle cells, mathematical models ofCa²⁺ diffusion and regulation wereused to predict the magnitude and time course of near-membraneCa²⁺ transients and their effectson the free and bound forms of ATP near the membrane. The results ofthis work indicate that changes in freeCa²⁺ concentration over the rangeof 50 nM-100 µM would result in significant changes in free ATPconcentration, MgATP concentration, and the CaATP-to-MgATPconcentration ratio.

     

Membrane permeability changes in amphibian eggs at ovulation

Electropotential differences between the cytoplasm and external medium have been compared in the mature R. pipiens occyte and the ovulated unfertilized egg as a function of [Na]_o, [K]_o, [Ca]_o and [Cl]_o. In solutions containing 1.0 mM Ca⁺⁺ the oocyte behaved as though it were predominantly permeable to K⁺ and Cl^?, i.e., like a KCl electrode. However, the steady potential decreased with decreasing [Ca]_o and in 5 × 10^?4 mM [Ca]_o the oocyte membrane behaved like a NaCl electrode. Studies on the steady potential as a function of [Na]_o, [K]_o and [Cl]_o in 1.0 mM Ca⁺⁺ or Ca-free solutions suggest that Ca⁺⁺ controls the passive permeability of the oocyte membrane to Na⁺ and Cl^?. In the ovulated unfertilized egg the K⁺ selectivity of the cell membrane disappeared and the system behaved like a NaCl electrode. No effect of external Ca⁺⁺ or K⁺ concentration changes on the steady potential was observed. These results indicate that the ion permeability properties of the ovulated egg are similar to that of the ovarian oocyte in Ca-deficient medium, and suggests that the mechanism of ovulation may involve the removal of Ca⁺⁺ regulation of ion permeability of the egg cell membrane.     

Intracellular free Ca2+ concentration in fowl spermatozoa and its relationship to motility and respiration in spermatozoa.

The ability of fowl spermatozoa to accumulate and de-esterify the intracellular fluorescent Ca2+ indicator fura-2 was established. The cytosolic Ca2+ concentrations, measured by this technique, did not change after the addition of 1 mmol EGTA l-1. Subsequently, addition of the calcium ionophore A23187 caused a reduction in cytosolic Ca2+ concentrations, presumably by efflux of Ca2+ from the spermatozoa. Intracellular free Ca2+ concentrations were then significantly increased by the addition of 1 mmol CaCl2 l-1. The motility of demembranated spermatozoa gradually decreased after the addition of EGTA alone or EGTA with A23187, but was instantly restored by the addition of CaCl2 in the presence of both EGTA and A23187. Unlike demembranated spermatozoa, intact spermatozoa maintained their motility, even after the addition of EGTA, but their motility was reduced by the addition of A23187 in the presence of EGTA. The addition of A23187 also reduced the rate of oxygen consumption, but not the ATP concentrations in intact spermatozoa. These results demonstrate that the motility and respiration of fowl spermatozoa are strongly influenced by their intracellular Ca2+ concentrations.     

川楝素对K+、Ca2+通道活动及细胞内Ca2+浓度的调控

川楝素是我国学者从驱蛔中药中分离、鉴定的一个三萜化合物,已证明具选择地影响神经递质释放,有效地对抗肉毒中毒,促进细胞分化、凋亡,抑制肿瘤增殖,抑制昆虫发育和取食,影响K 、Ca2 通道活动等多种生物效应.综述了证明川楝素抑制多种K 通道,选择地易化L型Ca2 通道和进而升高胞内Ca 浓度的研究资料,并对川楝素产生这些生物效应的机制进行了讨论.     

Intracellular Ca2+ stores in neurons. Identification and functional aspects.

Various aspects of the rapidly exchanging intracellular Ca2+ stores of neurons and nerve cells are reviewed: their multiplicity, with separate sensitivity to either the second messenger, inositol 1,4,5-trisphosphate, or ryanodine-caffeine (the latter stores are probably activated via Ca(2+)-induced Ca2+ release); their control of the plasma membrane Ca2+ permeability, via the activation of a peculiar type of cation channels; their ability to sustain localized heterogeneities of the [Ca2+]i that could be of physiological key-importance. Finally, the molecular composition of these stores is discussed. They are shown (by high resolution immunocytochemistry and subcellular fractionation) to express: i) a Ca2+ ATPase responsible for the accumulation of the cation; ii) Ca2+ binding protein(s) of low affinity and high capacity to keep Ca2+ stored; and iii) a Ca2+ channel, activated by either one of the mechanisms mentioned above, to release Ca2+ to the cytosol. Results obtained in Purkinje neurons document the heterogeneity of the stores and the strategical distribution of the corresponding organelles (calciosomes; specialized portions of the ER) within the cell body, dendrites and dendritic spines.