Induction of increase in intracellular calcium concentration of embryonic cells and acceleration of morphogenetic cell movements during amphibian gastrulation by a 50-Hz magnetic field

The influence of an alternating electromagnetic field (EMF) on early development of amphibian embryos was examined. When the embryos developed under the influence of a low-frequency EMF (50 Hz, 5-30 mT), the rate of early development was accelerated. The effect of EMF was exerted preferentially at the gastrula stage, and the period of gastrulation was shortened. Histological observations showed that EMF promoted morphogenetic cell movements during the gastrulation. The concentration of intracellular free Ca2+ ([Ca2+]i) in the embryonic cells under the influence of EMF was analyzed using Fura-2, an indicator of the intracellular concentration of calcium ions. The influence of EMF on [Ca2+]i was analyzed in embryonic cells isolated from blastula, gastrula, and neurula, EMF increased a [Ca2+]i particularly in the cells isolated from gastrula. Our results suggest that EMF specifically increased the [Ca2+]i of gastrula cells, thereby, accelerating the rate of morphogenetic cell movements during gastrulation.     

Localised and rapid Ca2+ micro-events in human neutrophils: conventional Ca2+ puffs and global waves without peripheral-restriction or wave cycling

Ultra-localised and peripherally restricted zones of elevated Ca2+ (z-waves) have been reported to cycle around the periphery of neutrophils at low frequency (1/20s) in the absence of conventional localised Ca2+ (puffs) and global Ca2+ (waves) signals. However, we report here that fast confocal laser scanning of human neutrophils loaded with either cytosolic fluo4 or its membrane associated analogue, MOMO reports both "conventional" stationary Ca2+ "puffs" (diameter c.3 microm) and global Ca2+ waves that sweep across the cell. The Ca2+ puff size and frequency of detection suggests that each neutrophil contained only a single release site and that its detection was limited by the location of the confocal plane relative to the event. Both formylated peptide receptor stimulation and cytosolic IP3 uncaging generated Ca2+ puffs (c.6% of cells) and global Ca2+ signals (c.75% of cells). The Ca2+ puffs peaked at approx. 250 nM and had a duration of approx. 235 msec and remained at a single locus. This was similar to other Ca2+ events in other cell types but in direct contrast to the reported z-waves. It was concluded that the micro-events which underlie Ca2+ signalling in neutrophils are conventional and that the existence of novel Ca2+z-waves is doubtful.     

Role of Ca(2+)-ATPase in spontaneous oscillations of cytosolic free Ca2+ in GH3 rat pituitary cells.

The relative contribution of voltage-sensitive Ca2+ channels, Ca(2+)-ATPases, and Ca2+ release from intracellular stores to spontaneous oscillations in cytosolic free Ca2+ concentration ([Ca2+]i) observed in secretory cells is not well characterized owing to a lack of specific inhibitors for a novel thapsigargin (Tg)-insensitive Ca(2+)-ATPase expressed in these cells. We show that spontaneous [Ca2+]i oscillations in GH3 cells were unaffected by Ca2+ depletion in inositol-1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores by the treatment of Tg, but could be initiated by application of caffeine. Moreover, we demonstrate for the first time that these spontaneous [Ca2+]i oscillations were highly temperature dependent. Decreasing the temperature from 22 to 17 degrees C resulted in an increase in the frequency, a reduction in the amplitude, and large inhibition of [Ca2+]i oscillations. Furthermore, the rate of ATP-dependent 45Ca2+ uptake into GH3-derived microsomes was greatly reduced at 17 degrees C. The effect of decreased temperatures on extracellular Ca2+ influx was minor because the frequency and amplitude of spontaneous action potentials, which activate L-type Ca2+ channels, was relatively unchanged at 17 degrees C. These results suggest that in GH3 secretory cells, Ca2+ influx via L-type Ca2+ channels initiates spontaneous [Ca2+]i oscillations, which are then maintained by the combined activity of Ca(2+)-ATPase and Ca(2+)-induced Ca2+ release from Tg/IP3-insensitive intracellular stores.     

L-type Ca2+ channels and K+ channels specifically modulate the frequency and amplitude of spontaneous Ca2+ oscillations and have distinct roles in prolactin release in GH3 cells

GH3 cells showed spontaneous rhythmic oscillations in intracellular calcium concentration ([Ca2+]i) and spontaneous prolactin release. The L-type Ca2+ channel inhibitor nimodipine reduced the frequency of Ca2+ oscillations at lower concentrations (100nM-1 microM), whereas at higher concentrations (10 microM), it completely abolished them. Ca2+ oscillations persisted following exposure to thapsigargin, indicating that inositol 1,4,5-trisphosphate-sensitive intracellular Ca2+ stores were not required for spontaneous activity. The K+ channel inhibitors Ba2+, Cs+, and tetraethylammonium (TEA) had distinct effects on different K+ currents, as well as on Ca2+ oscillations and prolactin release. Cs+ inhibited the inward rectifier K+ current (KIR) and increased the frequency of Ca2+ oscillations. TEA inhibited outward K+ currents activated at voltages above -40 mV (grouped within the category of Ca2+ and voltage-activated currents, KCa,V) and increased the amplitude of Ca2+ oscillations. Ba2+ inhibited both KIR and KCa,V and increased both the amplitude and the frequency of Ca2+ oscillations. Prolactin release was increased by Ba2+ and Cs+ but not by TEA. These results indicate that L-type Ca2+ channels and KIR channels modulate the frequency of Ca2+ oscillations and prolactin release, whereas TEA-sensitive KCa,V channels modulate the amplitude of Ca2+ oscillations without altering prolactin release. Differential regulation of these channels can produce frequency or amplitude modulation of calcium signaling that stimulates specific pituitary cell functions.     

Melatonin protects rat cerebellar granule cells against electromagnetic field‐induced increases in Na+ currents through intracellular Ca2+ release

Although melatonin (MT) has been reported to protect cells against oxidative damage induced by electromagnetic radiation, few reports have addressed whether there are other protective mechanisms. Here, we investigated the effects of MT on extremely low‐frequency electromagnetic field (ELF‐EMF)‐induced Na_v activity in rat cerebellar granule cells (GCs). Exposing cerebellar GCs to ELF‐EMF for 60 min. significantly increased the Na_v current (I_Na) densities by 62.5%. MT (5 μM) inhibited the ELF‐EMF‐induced I_Na increase. This inhibitory effect of MT is mimicked by an MT₂ receptor agonist and was eliminated by an MT₂ receptor antagonist. The Na_v channel steady‐state activation curve was significantly shifted towards hyperpolarization by ELF‐EMF stimulation but remained unchanged by MT in cerebellar GC that were either exposed or not exposed to ELF‐EMF. ELF‐EMF exposure significantly increased the intracellular levels of phosphorylated PKA in cerebellar GCs, and both MT and IIK‐7 did not reduce the ELF‐EMF‐induced increase in phosphorylated PKA. The inhibitory effects of MT on ELF‐EMF‐induced Na_v activity was greatly reduced by the calmodulin inhibitor KN93. Calcium imaging showed that MT did not increase the basal intracellular Ca²⁺ level, but it significantly elevated the intracellular Ca²⁺ level evoked by the high K⁺ stimulation in cerebellar GC that were either exposed or not exposed to ELF‐EMF. In the presence of ruthenium red, a ryanodine‐sensitive receptor blocker, the MT‐induced increase in intracellular calcium levels was reduced. Our data show for the first time that MT protects against neuronal I_Na that result from ELF‐EMF exposure through Ca²⁺ influx‐induced Ca²⁺ release.     

Cell-specific patterns of oscillating free Ca2+ in carbamylcholine-stimulated insulinoma cells

The effect of the muscarinic agonist carbamylcholine on cytoplasmic Ca2+ concentration ([Ca2+]i was examined at the single cell level in clonal pancreatic beta-cells (HIT). Cells were loaded with the indicator dye fura 2, and [Ca2+]i was measured by microfluorimetry. Carbamylcholine induced changes in Ca2+ that differed from cell to cell and provoked in some cells oscillatory Ca2+ fluctuations. During a transient, free Ca2+ rose to a peak within 1-3 s. The frequency of the oscillations increased with agonist concentration. Oscillations in [Ca2+]i occurred in the absence of external Ca2+. When cells were perifused for a sufficient period of time without carbamylcholine, near identical Ca2+ responses were elicited in each cell by successive applications of the agonist. Thus, individual cells displayed characteristic and reproducible Ca2+ responses with respect to amplitude, frequency, and shape of the transients as well as latency in onset of the initial Ca2+ rise. We propose that the biological response to a Ca2+ agonist in a given cell is not only determined by the frequency and amplitude of Ca2+ oscillations but is governed by the unique pattern of the Ca2+ signal of each cell, which may be termed "Ca2+ fingerprint."     

Repetitive calcium transients in hamster oocytes

Golden hamster oocytes show repetitive Ca2+ transients at fertilization: a propagating Ca2+ rise from the sperm attachment site in the first 2-3 responses and synchronous Ca2+ rise in the entire egg in the succeeding responses. Cyclic Ca2+ rises are produced in unfertilized eggs by an injection of GTP gamma S or continuous injection of inositol 1,4,5-trisphosphate (InsP3). Both InsP3-induced Ca2+ release (IICR) and Ca(2+)-induced Ca2+ release (CICR) are observed in hamster eggs, associated with a refractory period of 1-2 min after a Ca2+ release. In addition, external Ca2+ is a prerequisite for maintaining the repeated Ca2+ transients. The conditions that are expected to alter Ca2+ influx affect the frequency of Ca2+ transients with little effect on each response. The fertilizing sperm causes an increase in Ca2+ permeability of the egg plasma membrane and an increase in Ca2+ sensitivity of CICR. Feedback inhibition through protein kinase C is observed in G-protein-mediated Ca2+ transients but this inhibition seems to operate rather tonically. A model of Ca2+ oscillation is proposed: basically a second messenger-controlled oscillator model. InsP3 as the rigger of Ca2+ release is continuously supplied while an elevated basal [Ca2+]i level due to Ca2+ influx provides a favourable condition for IICR and CICR as well as for recharging the Ca2+ pools ready to release Ca2+ again.     

Muscarinic-receptor-mediated changes in intracellular Ca2+ and inositol 1,4,5-trisphosphate mass in a human neuroblastoma cell line, SH-SY5Y.

This study reports increased intracellular Ca2+ and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] in response to muscarinic-cholinergic stimulation of human neuroblastoma (SH-SY5Y) cells. Carbachol stimulation leads to a rapid increase in intracellular Ca2+ and Ins(1,4,5)P3 mass, both reaching a peak at around 10 s and then declining to a new maintained phase significantly above basal. Dose-response analysis of peak and plateau phases of intracellular Ca2+ shows different agonist potencies for both phases, carbachol being more potent for the plateau phase. The plateau-phase intracellular Ca2+ was dependent on extracellular Ca2+, which is admitted to the cell through a non-voltage-sensitive Ni2(+)-blockable Ca2+ channel. Using a Mn2+ quench protocol, we have shown that Ca2+ entry occurs early during the discharge of the internal stores. The plateau phase (Ca2(+)-channel opening) is dependent on the continued presence of agonist, since addition of atropine closes the Ca2+ channel and intracellular Ca2+ declines rapidly back to basal. We also failed to detect a refilling transient when we added back Ca2+ after intracellular Ca2+ had reached a peak and then declined in Ca2(+)-free conditions. These data strongly suggest that muscarinic stimulation of SH-SY5Y cells leads to a rapid release of Ca2+ from an Ins(1,4,5)P3-sensitive internal store and a parallel early entry of Ca2+ across the plasma membrane.     

Calcium signaling in non-excitable cells: Ca2+ release and influx are independent events linked to two plasma membrane Ca2+ entry channels

The regulatory mechanism of Ca2+ influx into the cytosol from the extracellular space in non-excitable cells is not clear. The "capacitative calcium entry" (CCE) hypothesis suggested that Ca2+ influx is triggered by the IP(3)-mediated emptying of the intracellular Ca2+ stores. However, there is no clear evidence for CCE and its mechanism remains elusive. In the present work, we have provided the reported evidences to show that inhibition of IP(3)-dependent Ca2+ release does not affect Ca2+ influx, and the experimental protocols used to demonstrate CCE can stimulate Ca2+ influx by means other than emptying of the Ca2+ stores. In addition, we have presented the reports showing that IP(3)-mediated Ca2+ release is linked to a Ca2+ entry from the extracellular space, which does not increase cytosolic [Ca2+] prior to Ca2+ release. Based on these and other reports, we have provided a model of Ca2+ signaling in non-excitable cells, in which IP(3)-mediated emptying of the intracellular Ca2+ store triggers entry of Ca2+ directly into the store, through a plasma membrane TRPC channel. Thus, emptying and direct refilling of the Ca2+ stores are repeated in the presence of IP(3), giving rise to the transient phase of oscillatory Ca2+ release. Direct Ca2+ entry into the store is regulated by its filling status in a negative and positive manner through a Ca2+ -binding protein and Stim1/Orai complex, respectively. The sustained phase of Ca2+ influx is triggered by diacylglycerol (DAG) through the activation of another TRPC channel, independent of Ca2+ release. The plasma membrane IP(3) receptor (IP(3)R) plays an essential role in Ca2+ influx, by interacting with the DAG-activated TRPC, without the requirement of binding to IP(3).     

Calcium sparks in mouse ventricular myocytes at physiological temperature

In cardiac muscle, Ca2+ is released from the sarcoplasmic reticulum (SR) in units called Ca2+ sparks. Ca2+ spark characteristics have been studied almost entirely at room temperature. This study compares characteristics of spontaneous sparks detected with fluo 3 in resting mouse ventricular myocytes at 22 and 37 degrees C. The incidence and frequency of Ca2+ sparks decreased dramatically at 37 degrees C compared with 22 degrees C. Also, spark amplitudes and times to peak were significantly reduced at 37 degrees C. In contrast, spatial width and decay times were unchanged. During field stimulation, peak spatially averaged transients were similar at 22 and 37 degrees C, and experiments with fura 2 demonstrated that diastolic and systolic Ca2+ concentrations were unchanged. However, SR Ca2+ content decreased significantly at 37 degrees C. Restoration of SR Ca2+ by superfusion with 5 mM Ca2+ increased spark frequency but did not reverse the effects of temperature on spark parameters. Thus effects of temperature on spark frequency may reflect changes in SR stores, whereas changes in spark amplitude and rise time may reflect known effects of temperature on ryanodine receptor function.     

Inositol trisphosphate and calcium oscillations

InsP3 has two important functions in generating Ca2+ oscillations. It releases Ca2+ from the internal store and it can contribute to Ca2+ entry. A hypothesis has been developed to describe a mechanism for Ca2+ oscillations with particular emphasis on the way agonist concentration regulates oscillator frequency. The main idea is that the InsP3 receptors are sensitized to release Ca2+ periodically by cyclical fluctuations of Ca2+ within the lumen of the endoplasmic reticulum. Each time a pulse of Ca2+ is released, the luminal level of Ca2+ declines and has to be replenished before the InsP3 receptors are resensitized to deliver the next pulse of Ca2+. It is this loading of the internal store that explains why frequency is sensitive to external Ca2+ and may also account for how variations in agonist concentration are translated into changes in oscillation frequency. Variations in agonist-induced entry of external Ca2+, which can occur through different mechanisms, determine the variable rates of store loading responsible for adjusting the sensitivity of the InsP3 receptors to produce the periodic pulses of Ca2+. The Ca2+ oscillator is an effective analogue-to-digital converter in that variations in the concentration of the external stimulus are translated into a change in oscillator frequency.     

Effects of low frequency electromagnetic field on proliferation of human epidermal stem cells: An in vitro study

To investigate the effects of low frequency electromagnetic fields (EMF) on the proliferation of epidermal stem cells, human epidermal stem cells (hESC) were isolated, expanded ex vivo, and then exposed to a low frequency EMF. The test and control cells were placed under the same environment. The test cells were exposed for 30 min/day to a 5 mT low frequency EMF at 1, 10, and 50 Hz for 3, 5, or 7 days. The effects of low frequency EMF on cell proliferation, cell cycle, and cell‐surface antigen phenotype were investigated. Low frequency EMF significantly enhanced the proliferation of hESC in the culture medium in a frequency‐dependent manner, with the highest cell proliferation rate at 50 Hz (P < 0.05). Exposure to a low frequency EMF significantly increased the percentage of cells at the S phase of the cell cycle, coupled with a decrease in the percentage of cells in the G1 phase (P < 0.05) but the effect was not frequency dependent. The percentage of CD29⁺/CD71^? cells remained unchanged in the low frequency EMF‐exposed hESC. The results suggested that low frequency EMF influenced hESC proliferation in vitro, and this effect was related to the increased proportion of cells at the S phase. Bioelectromagnetics 34:74–80, 2013. © 2012 Wiley Periodicals, Inc.     

Characterization of the vitamin D-dependent Ca2+-binding sites in rat intestinal Golgi-enriched membrane fractions.

Rat intestinal Golgi-enriched membrane fractions take up Ca2+ by a vitamin D-dependent process that has been shown to recover within 15 min of repletion of vitamin D-deficient animals with intravenous 1,25-dihydroxycholecalciferol. The present paper reports studies characterizing the Ca2+-binding sites of these membrane fractions. Equilibrium binding of Ca2+ at concentrations between 5 and 400 microM showed significant decreases at all concentrations in membranes derived from vitamin D-deficient animals when compared with normal control-diet-fed animals. The predominant class of binding sites had a relatively high affinity for Ca2+ (KD approx. 3 microM). Vitamin D-deficiency did not change the affinity of this class of site, but decreased the number from 347 +/- 26 to 168 +/- 50 nmol of Ca2+ bound/mg of protein (means +/- S.D.). Mg2+ inhibited binding only at low Ca2+ concentrations, and the characteristics of this binding suggested positive co-operativity between two binding sites. Equimolar concentrations of Zn2+, La3+, Pb2+ and Mn2+ inhibited Ca2+ binding by over 50%. Increased ionic strength decreased Ca2+ binding by no more than half. Binding was maximal at pH 7.5 and half-maximal at pH 6.3. The large number of binding sites with relatively high affinity for Ca2+ suggests that it is unlikely that this binding is to any specific protein or to non-specific sites present on many proteins, and that the most likely sites are lipid molecules.     

Occluded calcium sites in soluble sarcoplasmic reticulum Ca2+-ATPase

Rabbit muscle sarcoplasmic reticulum Ca2+-ATPase has been shown to bind gadolinium ion (Gd3+) at two high affinity Ca2+ sites (Stephens, E. M., and Grisham, C. M. (1979) Biochemistry 18, 4876-4885). Gd3+ bound at these sites exhibits an unusually long electron spin relaxation time, consistent with occlusion of these sites and reduced contact with solvent H2O. In this report, the nature of the Gd3+ sites was examined in preparations of the enzyme solubilized with the detergent C12E8. The frequency dependence of water proton relaxation in solutions containing the solubilized Ca2+-ATPase yields dipolar correlation times, tau c, for the 1H-Gd3+ interaction of 1.04 X 10(-9) s for Gd3+ bound at site 1 and 1.98 X 10(-9) s for Gd3+ bound at site 2. The correlation time itself is frequency dependent below 30 MHz, indicating that the correlation time is dominated by the electron spin relaxation time of bound Gd3+. The long values of the correlation time found in the present study are consistent with a poor accessibility of these Gd3+ sites (particularly site 2) to solvent water molecules. Analytical ultracentrifugation and molecular sieve high performance liquid chromatography indicated that the active fraction of the soluble Ca2+-ATPase was monomeric. Thus occlusion of the Ca2+ sites in this enzyme is largely dependent on the tertiary structure of the monomeric ATPase and does not appear to depend on multimeric membrane structures.     

ET-1 activates Ca2+ sparks in PASMC: local Ca2+ signaling between inositol trisphosphate and ryanodine receptors

Ca+ sparks originating from ryanodine receptors (RyRs) are known to cause membrane hyperpolarization and vasorelaxation in systemic arterial myocytes. By contrast, we have found that Ca2+ sparks of pulmonary arterial smooth muscle cells (PASMCs) are associated with membrane depolarization and activated by endothelin-1 (ET-1), a potent vasoconstrictor that mediates/modulates acute and chronic hypoxic pulmonary vasoconstriction. In this study, we characterized the effects of ET-1 on the physical properties of Ca2+ sparks and probed the signal transduction mechanism for spark activation in rat intralobar PASMCs. Application of ET-1 at 0.1-10 nM caused concentration-dependent increases in frequency, duration, and amplitude of Ca2+ sparks. The ET-1-induced increase in spark frequency was inhibited by BQ-123, an ETA-receptor antagonist; by U-73122, a PLC inhibitor; and by xestospongin C and 2-aminoethyl diphenylborate, antagonists of inositol trisphosphate (IP3) receptors (IP3Rs). However, it was unrelated to sarcoplasmic reticulum Ca2+ content, activation of L-type Ca2+ channels, PKC, or cADP ribose. Photorelease of caged-IP3 indicated that Ca2+ release from IP3R could cross-activate RyRs to generate Ca2+ sparks. Immunocytochemistry showed that the distributions of IP3Rs and RyRs were similar in PASMCs. Moreover, inhibition of Ca2+ sparks with ryanodine caused a significant rightward shift in the ET-1 concentration-tension relationship in pulmonary arteries. These results suggest that ET-1 activation of Ca2+ sparks is mediated via the ETA receptor-PLC-IP3 pathway and local Ca2+ cross-signaling between IP3Rs and RyRs; in addition, this novel signaling mechanism contributes significantly to the ET-1-induced vasoconstriction in pulmonary arteries.     

Effects of radiation emanating from base transceiver station and mobile phone on sleep,circadian rhythm and cognition in humans – a review

The electromagnetic fields (EMF) are ubiquitous. The base transceiver station (BTS) and mobile phones (MPs) contribute to the generation of EMF around their locations and are regarded as important sources of non-ionizing radiations. The use of mobile phone has increased dramatically in recent years so also the skepticism regarding its effects. In this review, we have made an attempt to scan the key research papers those aimed at elucidating the effects of EMF starting from extreme low frequency (ELF) to radio frequency (RF) through low frequency (LF). We have selected papers that dealt with the effects of radiations emanating from the BTS and MPs on human sleep, circadian rhythm, and cognition. Mostly, we have concentrated on papers published in the last 15 years. We came across conflicting reports. The findings reported in many papers suggest that the exposure to EMF has potentiality to compromise parameters related to sleep quality; in contrast, there are several reports those have given a clean sheet to the EMF exposure. The effects of EMF on circadian rhythms also remain inconclusive. The EMF exposure while did not produce any effect on circadian rhythm of heart rate and blood chemistry, it modulated the rhythms in cortisol and melatonin characterized by a decline in their 24-h circulating levels. The effects of exposure to EMF on cognitive parameters, like performance and memory, are also equivocal. The existing contradictory findings could be attributed to inter-individual variability in tolerance, gender-, and age-dependent differences in response, latitudinal differences in efficacy, variability among employed methodologies and differences in specific absorption rate, frequency of the mobile phone usage, and interaction of EMF with other physiological and environmental factors, among others. The future research should be carried out with added focus on elucidating the modulatory effects of these factors to put an end to the existing controversies on the biological effects of low/RF EMF radiations.     

Dual effects of cyclic ADP-ribose on sarcoplasmic reticulum Ca2+ release and storage in cardiac myocytes isolated from guinea-pig and rat ventricle

The actions of cyclic ADP-ribose (cADPR), a regulator of Ca2+-induced Ca2+ release (CICR), were investigated on Ca2+ release and sarcoplasmic reticulum (SR) Ca2+ loading in cardiac myocytes at physiological temperature. In guinea-pig ventricular cells, cADPR, applied via patch pipette or from photorelease of its caged derivative, increased contraction amplitude and whole-cell Ca2+ transients, without affecting SR Ca2+ load (measured in response to rapid caffeine application). Under voltage-clamp conditions, photorelease of caged cADPR enhanced Ca2+ transient magnitude without affecting the peak amplitude of L-type Ca2+ current or its rate of decay, indicative of an increase in CICR gain. In rat permeabilised ventricular myocytes, rapid application of cADPR increased Ca2+ spark frequency within 30 s, and this effect was maintained over a 10 min exposure. Enhancement of spark frequency was not associated with changes in SR Ca2+ load at 30 s and 3 min of exposure to cADPR; however, prolonged exposure (10 min) was associated with an increased SR Ca2+ load (32+/-7%). The observations are consistent with dual actions of cADPR: a rapid effect on CICR that does not depend on an increased SR Ca2+ load, and an additional slower effect that is associated with enhanced SR Ca2+ levels.     

Conventional PKCs regulate the temporal pattern of Ca2+ oscillations at fertilization in mouse eggs

In mammalian eggs, sperm-induced Ca2+ oscillations at fertilization are the primary trigger for egg activation and initiation of embryonic development. Identifying the downstream effectors that decode this unique Ca2+ signal is essential to understand how the transition from egg to embryo is coordinated. Here, we investigated whether conventional PKCs (cPKCs) can decode Ca2+ oscillations at fertilization. By monitoring the dynamics of GFP-labeled PKCalpha and PKCgamma in living mouse eggs, we demonstrate that cPKCs translocate to the egg membrane at fertilization following a pattern that is shaped by the amplitude, duration, and frequency of the Ca2+ transients. In addition, we show that cPKC translocation is driven by the C2 domain when Ca2+ concentration reaches 1-3 microM. Finally, we present evidence that one physiological function of activated cPKCs in fertilized eggs is to sustain long-lasting Ca2+ oscillations, presumably via the regulation of store-operated Ca2+ entry.     

Synchronized oscillation of Ca2+ entry and Ca2+ release in agonist-stimulated AR42J cells

Oscillation in [Ca2+]i induced by agonists has been described in many cell types and is thought to reflect Ca2+ release from and uptake into internal stores. We measured [Ca2+]i and Mn2+ entry in single cells of the pancreatic acinar cell line AR42J loaded with Fura 2 to examine the behavior of Ca2+ influx across the plasma membrane (Ca2+ entry) during agonist-evoked [Ca2+]i oscillation. Addition of extracellular Ca2+ (Ca2+out) to agonist-stimulated cells bathed in Ca2(+)-free medium resulted in a marked [Ca2+]i increase blocked by La3+. The use of Mn2+ as a congener of Ca2+ to follow unidirectional Ca2+ movement reveals an oscillatory activation of Ca2+ entry by Ca2(+)-mobilizing agonists. The frequency at which Ca2+ entry oscillated matched the frequency of Ca2+ release from intracellular stores. Ca2+ entry is activated after completion of Ca2+ release and is inactivated within the time span of each [Ca2+]i spike. These studies reveal a new aspect of [Ca2+]i oscillation in agonist-stimulated cells, that is the oscillatory activation of [Ca2+]i entry during [Ca2+]i oscillation.     

KCa channel insensitivity to Ca2+ sparks underlies fractional uncoupling in newborn cerebral artery smooth muscle cells

In smooth muscle cells, localized intracellular Ca2+ transients, termed "Ca2+ sparks," activate several large-conductance Ca2+-activated K+ (KCa) channels, resulting in a transient KCa current. In some smooth muscle cell types, a significant proportion of Ca2+ sparks do not activate KCa channels. The goal of this study was to explore mechanisms that underlie fractional Ca2+ spark-KCa channel coupling. We investigated whether membrane depolarization or ryanodine-sensitive Ca2+ release (RyR) channel activation modulates coupling in newborn (1- to 3-day-old) porcine cerebral artery myocytes. At steady membrane potentials of -40, 0, and +40 mV, mean transient KCa current frequency was approximately 0.18, 0.43, and 0.26 Hz and KCa channel activity [number of KCa channels activated by Ca2+ sparksxopen probability of KCa channels at peak of Ca2+ sparks (NPo)] at the transient KCa current peak was approximately 4, 12, and 24, respectively. Depolarization between -40 and +40 mV increased KCa channel sensitivity to Ca2+ sparks and elevated the percentage of Ca2+ sparks that activated a transient KCa current from 59 to 86%. In a Ca2+-free bath solution or in diltiazem, a voltage-dependent Ca2+ channel blocker, steady membrane depolarization between -40 and +40 mV increased transient KCa current frequency up to approximately 1.6-fold. In contrast, caffeine (10 microM), an RyR channel activator, increased mean transient KCa current frequency but did not alter Ca2+ spark-KCa channel coupling. These data indicate that coupling is increased by mechanisms that elevate KCa channel sensitivity to Ca2+ sparks, but not by RyR channel activation. Overall, KCa channel insensitivity to Ca2+ sparks is a prominent factor underlying fractional Ca2+ spark uncoupling in newborn cerebral artery myocytes.