Elevation Of Intracellular Free Calcium Research Articles

Ceramic 3D-Printed Titanium Cranioplasty.

Inlay cranioplasties following partial craniectomy in tumor or trauma cases and onlay cranioplasties for reconstructions of residual developmental skull anomalies are frequently performed using CAD-CAM techniques. In this case series, we present a novel cranial implant design, being a combination of 3D-printed titanium grade 23 and calcium phosphate paste (CeTi). The titanium patient-specific implant, manufactured using selective laser melting, has a latticed border with interconnected micropores. The cranioplasty is miniscrew fixed and its border zone subsequently partially filled with calcium phosphate paste to promote osteoinduction and osteoconduction. From April 2017 to April 2019, 8 patients have been treated with such a CeTi implant. The inlay cranioplasties were each time revision surgeries of complicated cases. All implants were successful after a limited follow-up time (range 18-42 months). There were no dehiscences and no infections, and no complaints of thermal conduction. The proposed CeTi cranial implant combines the strength of titanium implants with the biological integration potential of ceramic implants and seems particularly resistant to infection, probably due to the biofunctionalized titanium surface and the antimicrobial activity of elevated intracellular free calcium levels.

Activation of CaMKKβ-AMPK-mTOR pathway is required for autophagy induction by β,β-dimethylacrylshikonin against lung adenocarcinoma cells

β,β-Dimethylacrylshikonin (DMAS), an active ingredient of Lithospermum erythrorhizon and Arnebia euchroma, possess anti-neoplasm properties. Recently, DMAS was reported to stimulate autophagy in lung adenocarcinoma cells. However, the mechanisms by which DMAS modulates autophagy. have not yet been clearly elucidated. In this study, we found that DMAS significantly elevated intracellular free calcium accumulation. This activated the CaMKKβ-AMPK-mTOR pathway, subsequently inhibited mTOR and its substrate p70s6k and 4E-BP1, eventually leading to autophagy. In addition, we demonstrated that inhibition of autophagy by BAPTA-AM or STO-609 or compound C potently enhanced DMAS-induced lung adenocarcinoma cells apoptosis and growth inhibition. Overall, our results suggested that cytoprotective autophagy was triggered by DMAS via CaMKKβ-AMPK-mTOR signaling cascade in human lung adenocarcinoma cells, meaning that combining use of DMAS and autophagy inhibitors as a novel therapeutic option for lung adenocarcinoma will be very promising.

Calcium phosphate nanoparticles as intrinsic inorganic antimicrobials: In search of the key particle property.

One of the main goals of materials science in the 21st century is the development of materials with rationally designed properties as substitutes for traditional pharmacotherapies. At the same time, there is a lack of understanding of the exact material properties that induce therapeutic effects in biological systems, which limits their rational optimization for the related medical applications. This study sets the foundation for a general approach for elucidating nanoparticle properties as determinants of antibacterial activity, with a particular focus on calcium phosphate nanoparticles. To that end, nine physicochemical effects were studied and a number of them were refuted, thus putting an end to frequently erred hypotheses in the literature. Rather than having one key particle property responsible for eliciting the antibacterial effect, a complex synergy of factors is shown to be at work, including (a) nanoscopic size; (b) elevated intracellular free calcium levels due to nanoparticle solubility; (c) diffusivity and favorable electrostatic properties of the nanoparticle surface, primarily low net charge and high charge density; and (d) the dynamics of perpetual exchange of ultrafine clusters across the particle/solution interface. On the positive side, this multifaceted mechanism is less prone to induce bacterial resistance to the therapy and can be a gateway to the sphere of personalized medicine. On a more problematic side, it implies a less intense effect compared to single-target molecular therapies and a difficulty of elucidating the exact mechanisms of action, while also making the rational design of theirs for this type of medical application a challenge.

The rapid immunosuppression in phytohemagglutinin-activated human T cells is inhibited by the proliferative Ca2+ influx induced by progesterone and analogs

Progesterone, an endogenous immunomodulator, suppresses human T-cell activation during pregnancy. A sustained Ca2 + influx is an important signal for T-cell proliferation after crosslinking of T-cell receptor/CD3 complexes by anti-CD3 antibodies or phytohemagglutinin (PHA). Progesterone targets cell membrane sites inducing rapid responses including elevated intracellular free calcium concentration ([Ca2+]i) and suppressed T-cell PHA-activated proliferation. Interestingly, both PHA and progesterone induce [Ca2+]i elevation, but it remains unclear whether the PHA-induced Ca2+ influx is affected by progesterone leading to T-cell immunosuppression. Primary T-cells were isolated from human peripheral blood and the quench effect on intracellular fura-2 fluorescence of Mn2+ was used to explore the responses to Ca2+ influx with cell proliferation being determined by MTT assay. PHA-stimulated Ca2+ influx was dose-dependently suppressed by progesterone and its agonist R5020, which correlated with PHA-activated T-cell proliferation inhibition. A similar dose-dependent suppression effect on cellular Ca2+ influx and proliferation occurred with the TRPC channel inhibitor BTP2 and selective TRPC3 channel inhibitor Pyr3. In addition, two progesterone analogs, Org OD 02-0 and 20α-hydroxyprogesterone (20α-OHP), also produced dose-dependent suppression of Ca2+ influx, but had no effect on proliferation. Finally, inhibition of PHA-activated T-cell proliferation by progesterone is further suppressed by 20α-OHP, but not by Org OD 02-0. Overall, progesterone and R5020 are able to rapidly decrease PHA-stimulated sustained Ca2+ influx, probably via blockade of TRPC3 channels, which suppresses T-cell proliferation. Taken together, the roles of progesterone and its analogs regarding the rapid response Ca2+ influx need to be further explored in relation to cytokine secretion and proliferation in activated T-cells.

Intracellular Calcium Attenuates Late Current Conducted by Mutant Human Cardiac Sodium Channels

Mutations of the cardiac voltage-gated sodium channel (SCN5A gene encoding voltage-gated sodium channel [NaV1.5]) cause congenital long-QT syndrome type 3 (LQT3). Most NaV1.5 mutations associated with LQT3 promote a mode of sodium channel gating in which some channels fail to inactivate, contributing to increased late sodium current (INaL), which is directly responsible for delayed repolarization and prolongation of the QT interval. LQT3 patients have highest risk of arrhythmia during sleep or during periods of slow heart rate. During exercise (high heart rate), there is elevated steady-state intracellular free calcium (Ca(2+)) concentration. We hypothesized that higher levels of intracellular Ca(2+) may lower arrhythmia risk in LQT3 subjects through effects on INaL. We tested this idea by examining the effects of varying intracellular Ca(2+) concentrations on the level of INaL in cells expressing a typical LQT3 mutation, delKPQ, and another SCN5A mutation, R225P. We found that elevated intracellular Ca(2+) concentration significantly reduced INaL conducted by mutant channels but not wild-type channels. This attenuation of INaL in delKPQ expressing cells by Ca(2+) was not affected by the CaM kinase II inhibitor KN-93 but was partially attenuated by truncating the C-terminus of the channel. We conclude that intracellular Ca(2+) contributes to the regulation of INaL conducted by NaV1.5 mutants and propose that, during excitation-contraction coupling, elevated intracellular Ca(2+) suppresses mutant channel INaL and protects cells from delayed repolarization. These findings offer a plausible explanation for the lower arrhythmia risk in LQT3 subjects during fast heart rates.

Effects of crude water-soluble extract of Momordica charantia on viability, caspase activity, cytochrome c release and on cytosolic calcium levels in different cancer cell lines

Prior to the availability of chemotherapeutic agents, dietary measures, including traditional medicines derived from plants, were the major forms of cancer treatment. One such plant is Momrdica charantia (Family: Cucurbitaceae), whose fruit is known as corilla or bitter gourd/melon. M. charantia possesses anti-carcinogenic properties and it can modulate its effect via xenobiotic metabolism and oxidative stress. This study investigated the anti-cancer effect of an active water soluble extract (s) of M. charantia on cell viability and its cellular mechanism of action in inducing cell death. The fruit was washed and cut in to small pieces, liquidised in deionised water using a blender and subsequently, dried using a rota evaporator and oven. Both time course (800 µg/ml) and dose- dependent (200 µg/ml-800 µg/ml) experiments were performed treating six different cancer cell lines, namely 1321N1, Gos-3, U87-MG, Sk Mel, Corl -23, Weri Rb-1 and normal L6 muscle cell line with the crude fruit extract for 24 hours at 37 °C. Cell viability was measured using the MTT assay. The results show that the crude water soluble extract of M. charantia can evoke both time-course at (800 µg/ml) and dose-dependent (20 µg/ml - 800 µg/ml) decreases in cell viability (cell death) with maximal increases in cell death with 800 µg/ml over a period of 24 hours following incubation. The results of this study have also shown that the crude water-soluble extract of M. charantia (800 µg/ml) can elicit marked and significant (p < 0.050) increases in the activities of caspase - 3 and caspase - 9 in all the cell lines. The crude water soluble extract of M. charantia can stimulate the release of cytochrome-c and elevated intracellular free calcium concentrations [Ca 2+ ] i in the different cancer cell lines compared to untreated cell lines. These results clearly show that M. charantia is exerting its anti- cancer effect via an insult to the mitochondria resulting in apoptosis, calcium overloading and subsequently, cell death.

Abstract 22: Calpain-Dependent Induction of Endoplasmic Reticulum Stress in Cardiomyocyte Apoptosis and Post--Myocardial Infarction Remodeling

Background: Calpain is up-regulated and implicated in cardiomyocyte apoptosis and myocardial remodelling post myocardial infarction. Endoplasmic reticulum (ER) stress is induced and contributes to myocardial injury in a variety of cardiac diseases. This study was to investigate whether calpain plays a role in ER stress, thereby mediating apoptosis in cardiomyocytes and myocardial remodelling after infarction. Methods and Results: In rat cardiomyoblasts H9C2 cells, over-expression of calpain-1 increased the protein levels of Bip and CHOP, indicative of ER stress, and induced apoptosis determined by a decrease in Bcl-2 and increases in caspase-3 activation and DNA fragmentation. Inhibition of calpain activity or ER stress prevented apoptosis induced by calpain-1 over-expression. In contrast, over-expression of calpain-2 failed to induce apoptosis. The induction of ER stress by calpain-1 might be mediated through SERCA2a/Calcium signaling as up-regulation of calpain-1 reduced SERCA2a protein and elevated intracellular free Calcium in H9C2 cells. In a mouse model of myocardial infarction induced by coronary artery ligation, calpain activity was increased and ER stress was induced in the infracted heart. Up-regulation of calpastatin, the endogenous calpain inhibitor, inhibited calpain activation, prevented ER stress and apoptosis, reduced myocardial remodelling and improved myocardial function after infarction in calpastatin transgenic mice compared with their wild-type littermates. Conclusion: Calpain-1 induces ER stress through the proteolysis of SERCA2a, thereby mediating apoptosis in cardiomyocytes. Inhibition of calpain prevents ER stress and apoptosis, reduces myocardial remodelling and dysfunction after infarction. Thus, ER stress may represent a novel mechanism by which calpain mediates myocardial injury in the infracted heart.

Elevated BCR signaling and decreased survival of Lyn‐deficient transitional and follicular B cells

The Src-family tyrosine kinase Lyn negatively regulates BCR signaling and also myeloid cell activity. Mice deficient in Lyn have substantially decreased numbers of peripheral B cells, despite spontaneously producing IgG anti-DNA antibodies. Here, we examine the mechanism underlying the B-cell depletion in these mice. Lyn-deficient B cells were out-competed by WT B cells in mixed BM chimeras at two steps, at the T1 to T2 transitional maturation stage in the spleen and again between the T2 or T3 stage and the mature follicular B-cell population. Lyn-deficient T2 and follicular B cells expressed elevated levels of the pro-apoptotic factor Bim and deletion of Bim restored splenic B cells of Lyn-deficient mice to close to WT numbers. Lyn-deficient T2 and later stage B cells also had changes in cell surface phenotype consistent with increased in vivo BCR signaling. Similarly, an increased proportion of T2 and follicular B cells had elevated basal intracellular free calcium levels. Overall, these observations suggest that increased BCR signaling is responsible for increased death of weakly self-reactive Lyn-deficient B cells both at the T2 stage and additionally as these cells mature to follicular B cells.

Tanshinone IIa Alleviates the Biochemical Changes Associated with Hypoxic Ischemic Brain Damage in a Rat Model

This study aimed to investigate the effects of Tanshinone IIa (TanIIa) on the biochemical changes associated with hypoxic ischemic brain damage (HIBD) in a rat model. Neonatal SD rats were randomized into normal control, HIBD and TanIIa + HIBD groups. At different time points after HIBD, TanIIa was given at 1 µg/g. The intracellular free calcium concentration and the expression of phospho-NR1 S897 was determined. The intracellular free calcium concentration in the HIBD group was significantly increased. The induction of intracellular free calcium concentration in the TanIIa + HIBD group was less than that in the HIBD group. Large amounts of phospho-NR1 S897 positive cells were distributed in the cortex in the normal control group; the number of phospho-NR1 S897 positive cells in the ipsilateral cortex was dramatically decreased at 24 h after HIBD. Both the number of phospho-NR1 S897 positive cells and the FITC fluorescent density in the HIBD + TanIIa group were less than those in the normal control group at every time point after HIBD, but more than those in the HIBD group. TanIIa alleviated the down-regulation of phospho-NR1 S897 and the elevated intracellular free calcium concentration in the cerebral cortex in the HIBD model. TanIIa could exert a neuroprotective effect through affecting NMDA receptor expression, inhibiting calcium transportation and decreasing the intracellular free calcium concentration.

Calcium-mediated Stress Kinase Activation by DMP1 Promotes Osteoblast Differentiation

Calcium signaling and calcium transport play a key role during osteoblast differentiation and bone formation. Here, we demonstrate that DMP1 mediated calcium signaling, and its downstream effectors play an essential role in the differentiation of preosteoblasts to fully functional osteoblasts. DMP1, a key regulatory bone matrix protein, can be endocytosed by preosteoblasts, triggering a rise in cytosolic levels of calcium that initiates a series of downstream events leading to cellular stress. These events include release of store-operated calcium that facilitates the activation of stress-induced p38 MAPK leading to osteoblast differentiation. However, chelation of intracellular calcium and inhibition of the p38 signaling pathway by specific pharmacological inhibitors and dominant negative plasmid suppressed this activation. Interestingly, activated p38 MAPK can translocate to the nucleus to phosphorylate transcription factors that coordinate the expression of downstream target genes such as Runx 2, a key modulator of osteoblast differentiation. These studies suggest a novel paradigm by which DMP1-mediated release of intracellular calcium activates p38 MAPK signaling cascade to regulate gene expression and osteoblast differentiation.

Diethylstilbestrol and Other Nonsteroidal Estrogens: Novel Class of Store-Operated Calcium Channel Modulators

Compounds with the stilbene pharmacophore and other nonsteroidal estrogens have previously been shown to inhibit thrombin-induced elevation of intracellular free calcium ([Ca]i) in human platelets. Thrombin elevates [Ca]i in platelets predominantly by activating a store-operated Ca entry (SOCE) mechanism, probably involving STIM1 and Orai1 although other components may be involved. Human platelets were loaded with the Ca sensitive indicator fura-2, various concentrations of stilbene compounds and other nonsteroidal estrogens were added to the platelets, and thrombin was added to elevate [Ca]i. The degree of inhibition by each compound was determined at the peak increase in [Ca]i induced by thrombin. The additional compounds that were examined in the present study were analogs of diethylstilbestrol (DES), namely trans-resveratrol, hexestrol, tetrahydrochrysene (THC), indenestrol, isoflavones, flavones, and flavanones. DES, indenestrols, and substituted THC diols had the highest inhibitory activity. Dietary polyphenols were less active, and isoflavones were more active than flavones. Glycosides of flavones, flavanones, and isoflavones were inactive. Equol (a product of isoflavone metabolism) had low activity. Among the compounds with a stilbene moiety the presence of unsubstituted phenyl hydroxyls in the para- position were required for activity. Esterification of hydroxyls and bulky substituents at a hydroxyl group diminished or abolished activity. Presence of the ethyl side chains enhanced activity, and shortening or removal of these side chains was detrimental to activity. Presence of the conjugated double bound was necessary for activity. Reduction of the double bond (in fused rings such as equol, dihydrogenistein, indanestrol, or in open chain stilbene derivatives) or repositioning of this double bond outside the stilbene moiety was detrimental to activity, because phenyl rings are not co-planar and have to be at a certain angle to each other. DES likely represents the pharmacophore of this group of nonsteroidal estrogens as an inhibitor of SOCE in platelets.

Systematic Characterization of Initial Calcium Signaling in T Cells

Elevation of intracellular free Calcium is part of the key signals during T-cell activation by antigens. Following activation a remarkable variety of this signals - ranging from infrequent spikes to sustained oscillations and plateaus - is shaped by the interactions of the different Calcium sources and sinks in the cell.We present an approach to study calcium signalling in T-cells at a large scale in parallel fashion that allow to extract proteins and their interactions involved in generating this Calcium signals. Briefly T cells, knock out T cell lines and T cells with proteins knocked down by siRNA were synchronized and exposed to surfaces, coated with stimulatory and non stimulatory antibodys. The assay yielded a data set of several thousand calcium traces from which parameters like number of spikes or length of plateaus were extracted and used to cluster the data hypothesis free. Based on this similarities and differences between signalling pathways are inferred, which may provide the basis to systematically explore proteins and their interactions governing calcium signalling pathways in T-cells.

Integration of Single and Multicellular Wound Responses

Single cells and multicellular tissues rapidly heal wounds. These processes are considered distinct, but one mode of healing--Rho GTPase-dependent formation and closure of a purse string of actin filaments (F-actin) and myosin-2 around wounds--occurs in single cells and in epithelia. Here, we show that wounding of one cell in Xenopus embryos elicits Rho GTPase activation around the wound and at the nearest cell-cell junctions in the neighbor cells. F-actin and myosin-2 accumulate at the junctions and around the wound itself, and as the resultant actomyosin array closes over the wound site, junctional F-actin and myosin-2 become mechanically integrated with the actin and myosin-2 around the wound, forming a hybrid purse string. When cells are ablated rather than wounded, Rho GTPase activation and F-actin accumulation occur at cell-cell junctions surrounding the ablated cell, and the purse string closes the hole in the epithelium. Elevation of intracellular free calcium, an essential upstream signal for the single-cell wound response, also occurs at the cell-cell contacts and in neighbor cells. Thus, the single and multicellular purse string wound responses represent points on a signaling and mechanical continuum that are integrated by cell-cell junctions.

Structurally Distinct Phosphatases CD45 and CD148 Both Regulate B Cell and Macrophage Immunoreceptor Signaling

The receptor-type protein tyrosine phosphatase (RPTP) CD148 is thought to have an inhibitory function in signaling and proliferation in nonhematopoietic cells. However, its role in the immune system has not been thoroughly studied. Our analysis of CD148 loss-of-function mice showed that CD148 has a positive regulatory function in B cells and macrophages, similar to the role of CD45 as a positive regulator of Src family kinases (SFKs). Analysis of CD148 and CD45 doubly deficient B cells and macrophages revealed hyperphosphorylation of the C-terminal inhibitory tyrosine of SFKs accompanied by substantial alterations in B and myeloid lineage development and defective immunoreceptor signaling. Because these findings suggest the C-terminal tyrosine of SFKs is a common substrate for both CD148 and CD45 phosphatases and imply a level of redundancy not previously appreciated, a reassessment of the function of CD45 in the B and myeloid lineages based on prior data from the CD45-deficient mouse is warranted.

Systemic Imbalance of Essential Metals and Cardiac Gene Expression in Rats Following Acute Pulmonary Zinc Exposure

It was recently demonstrated that particulate matter (PM) containing water-soluble zinc produces cardiac injury following pulmonary exposure. To investigate whether pulmonary zinc exposure produces systemic metal imbalance and direct cardiac effects, male Wistar Kyoto (WKY) rats (12–14 wk age) were intratracheally (IT) instilled with saline or 2 μmol/kg zinc sulfate. Temporal analysis was performed for systemic levels of essential metals (zinc, copper, and selenium), and induction of zinc transporter-2 (ZT-2) and metallothionein-1 (MT-1) mRNA in the lung, heart, and liver. Additionally, cardiac gene expression profile was evaluated using Affymetrix GeneChips (rat 230A) arrays to identify zinc-specific effects. Pulmonary zinc instillation produced an increase in plasma zinc to ∼20% at 1 and 4 h postexposure with concomitant decline in the lung levels. At 24 and 48 h postexposure, zinc levels rose significantly (∼35%) in the liver. At these time points, plasma and liver levels of copper and selenium also increased significantly, suggesting systemic disturbance in essential metals. Zinc exposure was associated with marked induction of MT-1 and ZT-2 mRNA in lung, heart, and liver, suggesting systemic metal sequestration response. Given the functional role of zinc in hundreds of proteins, the gene expression profiles demonstrated changes that are expected based on its physiological role. Zinc exposure produced an increase in expression of kinases and inhibition of expression of phosphatases; up- or downregulation of genes involved in mitochondrial function; changes in calcium regulatory proteins suggestive of elevated intracellular free calcium and increases in sulfotransferases; upregulation of potassium channel genes; and changes in free radical-sensitive proteins. Some of these expression changes are reflective of a direct effect of zinc on myocardium following pulmonary exposure, which may result in impaired mitochondrial respiration, stimulated cell signaling, altered Ca2+ homeostasis, and increased transcription of sulfotransferases. Cardiotoxicity may be an outcome of acute zinc toxicosis and occupational exposures to metal fumes containing soluble zinc. Imbalance of systemic metal homeostasis as a result of pulmonary zinc exposure may underlie the cause of extrapulmonary effects.

Mode of action of taurine as a neuroprotector

Previously, it has been shown that taurine exerts its protective function against glutamate-induced neuronal excitotoxicity through its action in reducing glutamate-induced elevation of intracellular free calcium, [Ca 2+] i. Here, we report the mechanism underlying the effect of taurine in reducing [Ca 2+] i. We found that taurine inhibited glutamate-induced calcium influx through L-, P/Q-, N-type voltage-gated calcium channels (VGCCs) and NMDA receptor calcium channel. Surprisingly, taurine had no effect on calcium influx through NMDA receptor calcium channel when cultured neurons were treated with NMDA in Mg 2+-free medium. Since taurine was found to prevent glutamate-induced membrane depolarization, we propose that taurine protects neurons against glutamate excitotoxicity by preventing glutamate-induced membrane depolarization, probably through its effect in opening of chloride channels and, therefore, preventing the glutamate-induced increase in calcium influx and other downstream events.

How adhesion, migration, and cytoplasmic calcium transients influence interleukin-1beta mRNA stabilization in human monocytes.

We investigated the mechanisms by which primary human monocyte migration and the production of important cytokines are co-regulated. Motile monocytes underwent cyclic morphologic and adhesive changes that were associated with intracellular free calcium changes; in such cells, cytokine transcripts were unstable and translationally repressed. Agents that activate monocytes, including lipopolysacharrides (LPS), cytomegalovirus (CMV), and tumor necrosis factor (TNFalpha), have been shown to de-repress translation and these agents stabilize adhesion-induced transcripts for IL-lbeta and IL-8 and markedly diminish cell migration in the presence of autologous serum. LPS suppressed Rho A activity and either this agent or C3 transferase elevated intracellular free calcium, stabilized transcripts, and, in tandem, inhibited cell migration by preventing tail retraction, a prerequisite for cell translocation. These results, therefore, suggest that monocyte activating agents inhibit the RhoA pathway and continuously elevate intracellular calcium leading to a concomitant decrease in monocyte migration and stabilization of cytokine transcripts prior to translation.

Possible involvement of nitric oxide in the modulation of photolytic flash‐induced intercellular calcium waves in cultured astrocytes

AbstractWaves of elevated intracellular free calcium that propagate between neighboring astrocytes are important for the intercellular communication between astrocytes as well as between neurons and astrocytes. In this study, intercellular calcium waves were evoked by focal photolysis of a caged calcium inophore in cultured astrocytes. The focal photolysis of the caged compound resulted in an increase of intracellular calcium in the flashed cells, and this increase then propagated to neighboring astrocytes. The evoked calcium increase was inhibited by incubating cells with an inhibitor of nitric oxide synthase as well as with a scavenger of nitric oxide (NO). In addition, treatment of cultures with an NO donor resulted in the marked enhancement of the photolytic flash‐induced calcium rise in astrocytes. This enhancement was reversed by treatment with inhibitors of soluble guanylyl cyclase as well as of protein kinase G.

Calcium responses to caffeine and muscarinic receptor agonists are altered in traumatically injured neurons.

A fundamental mechanism that is believed to contribute to neuronal injury and death following traumatic brain injury (TBI) is a disruption in cellular calcium homeostasis. Of primary importance to these homeostatic mechanisms are intracellular calcium stores located on the endoplasmic reticulum. These intracellular stores play an important role in maintaining normal levels of calcium and calcium-mediated signaling through these stores is critical to several physiological processes in neurons. Using an in vitro model of stretch-induced traumatic injury and fura-2 digital calcium imaging, we investigated alterations in calcium-induced calcium release (CICR) and inositol (1,4,5)-trisphosphate (IP(3))-linked signaling through intracellular calcium stores in populations of cultured rat cortical neurons. Caffeine, which stimulates CICR, produced a rapid elevation of intracellular free calcium ([Ca(2+)](i)) in 70% of uninjured neurons. Fifteen min after injury the population of caffeine-responsive neurons was reduced to 30%. The IP(3)-linked muscarinic acetylcholine receptor agonists, CDD-0097 HCl and McN-A-343, produced elevations in [Ca(2+)](i) in 91% and 70% of uninjured neurons, respectively. Following injury the population of responders was reduced to 19% and 26%, respectively. Differential responses to agonists were also noted after injury, in which the majority of neurons within a given culture well were unresponsive to agonists while others elicited a normal elevation of calcium. These results suggest disruptions in intracellular calcium store-mediated signaling and altered calcium signaling population dynamics following injury. These alterations could affect normal neurotransmission in the brain and may contribute to some of the pathology of TBI.

Quantification and distribution of equine oocyte cortical granules during meiotic maturation and after activation.

In vitro fertilization (IVF) is being routinely used in humans and several domestic species, however, limited success has been achieved in the horse. Although immature equine oocytes are capable of completing meiosis in vitro, subsequent fertilization, and embryonic development of those oocytes are questionable. The lack of development of these oocytes could be attributed to an impaired cytoplasmic maturation. In the horse, the study of oocyte cytoplasmic maturation and post-fertilization development has been hindered by the lack of progress in IVF. In mammalian oocytes, migration of cortical granules (CG) has been used as an important criterion to evaluate cytoplasmic maturation. The aim of this study was to describe and quantify the CG distribution of equine oocytes during in vitro meiotic maturation and to assess activation of oocytes with calcium ionophore based upon fluorescein isothiocyanate (FITC)-labeled Lens culinaris agglutinin (LCA) and laser confocal microscopy. The results of this study indicate that CG are distributed throughout the cytoplasm of oocytes at the germinal vesicle (GV) stage (immature). As maturation proceeds, a progressive centripetal migration of CG to the oocyte cortex occurs with the formation of a monolayer adjacent to the plasma membrane starting by the end of a 30 hr incubation period and increasing significantly after 36 hr. After activation, significant reduction in the number of CG was observed (P < 0.001) suggesting that oocytes cultured under the present conditions possess the ability to release CG in response to the elevation of intracellular free calcium.