Cytoplasmic Calcium Ion Concentration Research Articles

Nuclear-cytoplasmic asynchrony in oocyte maturation caused by TUBB8 variants via impairing microtubule function: a novel pathogenic mechanism

BackgroundTUBB8, a crucial gene encoding microtubule protein, plays a pivotal role in cellular processes. Deleterious TUBB8 variants have been shown to significantly hinder oocyte maturation. In this study, we conducted an in vitro investigation using TUBB8 mutant mouse oocytes to elucidate the pathogenic mechanisms of TUBB8 variants in oocyte nuclear and cytoplasmic maturation.MethodsA mutant model was successfully established in mouse oocytes via microinjection to further investigate the effects of four novel discovered TUBB8 mutations on the nuclear and cytoplasmic maturation of mouse oocytes. Immunofluorescence and confocal microscopy were performed to observe the cortical polarity and spindle and of mutant oocytes. Active mitochondrial staining was performed to analyze mitochondrial distribution patterns. Endoplasmic reticulum and Ca2+ staining were conducted to assess ER distribution and cytoplasmic calcium ion concentration in oocytes.ResultsIn mouse oocytes, TUBB8 variants (p.A313V, p.C239W, p.R251Q, and p.G96R) resulted in a reduction of the first polar body extrusion rate, disruption of spindle assembly, and abnormal chromosome distribution. Additionally, these variants induced oocyte organelle abnormalities, including anomalies in mitochondrial redistribution and endoplasmic reticulum stress compared to the wild-type.ConclusionDeleterious TUBB8 variants could disrupt microtubule function, affecting critical processes such as spindle assembly, chromosome distribution, and organelle rearrangement during oocyte meiosis. These disruptions culminate in compromised nuclear-cytoplasmic maturation, consequently giving rise to oocyte maturation defects.

Calcium signals are necessary to establish auxin transporter polarity in a plant stem cell niche

In plants mechanical signals pattern morphogenesis through the polar transport of the hormone auxin and through regulation of interphase microtubule (MT) orientation. To date, the mechanisms by which such signals induce changes in cell polarity remain unknown. Through a combination of time-lapse imaging, and chemical and mechanical perturbations, we show that mechanical stimulation of the SAM causes transient changes in cytoplasmic calcium ion concentration (Ca2+) and that transient Ca2+ response is required for downstream changes in PIN-FORMED 1 (PIN1) polarity. We also find that dynamic changes in Ca2+ occur during development of the SAM and this Ca2+ response is required for changes in PIN1 polarity, though not sufficient. In contrast, we find that Ca2+ is not necessary for the response of MTs to mechanical perturbations revealing that Ca2+ specifically acts downstream of mechanics to regulate PIN1 polarity response.

Transient elevation of cytoplasmic calcium ion concentration at a single cell level precedes morphological changes of epidermal keratinocytes during cornification

Epidermal keratinocytes achieve sequential differentiation from basal to granular layers, and undergo a specific programmed cell death, cornification, to form an indispensable barrier of the body. Although elevation of the cytoplasmic calcium ion concentration ([Ca2+]i) is one of the factors predicted to regulate cornification, the dynamics of [Ca2+]i in epidermal keratinocytes is largely unknown. Here using intravital imaging, we captured the dynamics of [Ca2+]i in mouse skin. [Ca2+]i was elevated in basal cells on the second time scale in three spatiotemporally distinct patterns. The transient elevation of [Ca2+]i also occurred at the most apical granular layer at a single cell level, and lasted for approximately 40 min. The transient elevation of [Ca2+]i at the granular layer was followed by cornification, which was completed within 10 min. This study demonstrates the tightly regulated elevation of [Ca2+]i preceding the cornification of epidermal keratinocytes, providing possible clues to the mechanisms of cornification.

Novel identification and characterisation of Transient receptor potential melastatin 3 ion channels on Natural Killer cells and B lymphocytes: effects on cell signalling in Chronic fatigue syndrome/Myalgic encephalomyelitis patients.

BackgroundTransient receptor potential melastatin 3 (TRPM3) cation channels are ubiquitously expressed by multiple cells and have an important regulatory role in calcium-dependent cell signalling to help maintain cellular homeostasis. TRPM3 protein expression has yet to be determined on Natural Killer (NK) cells and B lymphocytes. Multiple single nucleotide polymorphisms have been reported in TRPM3 genes from isolated peripheral blood mononuclear cells, NK and B cells in Chronic fatigue syndrome/Myalgic encephalomyelitis (CFS/ME) patients and have been proposed to correlate with illness presentation. The object of the study was to assess TRPM3 surface expression on NK and B lymphocytes from healthy controls, followed by a comparative investigation examining TRPM3 surface expression, and cytoplasmic and mitochondrial calcium influx in CD19+ B cells, CD56bright and CD56dim cell populations from CFS/ME patients.ResultsTRPM3 cell surface expression was identified for NK and B lymphocytes in healthy controls (CD56bright TRPM3 35.72 % ± 7.37; CD56dim 5.74 % ± 2.00; B lymphocytes 2.05 % ± 0.19, respectively). There was a significant reduction of TRPM3 surface expression on CD19+ B cells (1.56 ± 0.191) and CD56bright NK cells (17.37 % ± 5.34) in CFS/ME compared with healthy controls. Anti-CD21 and anti-IgM conjugated biotin was cross-linked with streptavidin,and subsequently treatment with thapsigargin. This showed a significant reduction in cytoplasmic calcium ion concentration in CD19+ B lymphocytes. CD56bright NK cells also had a significant decrease in cytoplasmic calcium in the presence of 2-APB and thapsigargin in CFS/ME patients.ConclusionsThe results from this preliminary investigation identify, for the first time, TRPM3 surface expression on both NK and B lymphocytes in healthy controls. We also report for the first time, significant reduction in TRPM3 cell surface expression in NK and B lymphocytes, as well as decreased intracellular calcium within specific conditions in CFS/ME patients. This warrants further examination of these pathways to elucidate whether TRPM3 and impaired calcium mobilisation has a role in CFS/ME.Electronic supplementary materialThe online version of this article (doi:10.1186/s40659-016-0087-2) contains supplementary material, which is available to authorized users.

A flow cytometric approach for studying alterations in the cytoplasmic concentration of calcium ions in immune cells following stimulation with thymic peptides

[Ca2+]i alterations are vital in signaling pathways of cell activation. We tried to detect such changes, in intracellular signaling pathways downstream TLR4 in immune cells, following stimulation with prothymosin alpha (proTα) and its decapeptide proTα(100–109). Human leukocytes were activated with LPS, proTα or proTα(100–109), directly or after 24h stimulation, while neutrophils were directly challenged. Cells were loaded with Fluo-4 and cytoplasmic Ca2+ alterations were recorded by flow cytometry. Direct challenge with 20μg/mL LPS induced a measurable [Ca2+]i increase in macrophages and neutrophils. Monocytes and macrophages incubated for 24h with LPS, proTα or proTα(100–109) and challenged with LPS, displayed a robust response. Lymphocytes and iDCs exhibited no alterations. Conclusively, we assessed a flow cytometry-based method for monitoring Ca2+ ion influx changes in immune cells. Their stimulation with proTα or proTα(100–109) generates an activating background, similar to LPS, allowing for the detection of [Ca2+]i alterations induced upon subsequent challenge.

Evidence for the possible involvement of the P2Y6 receptor in Ca2+ mobilization and insulin secretion in mouse pancreatic islets

Subtypes of purinergic receptors involved in modulation of cytoplasmic calcium ion concentration ([Ca2+]i) and insulin release in mouse pancreatic β-cells were examined in two systems, pancreatic islets in primary culture and beta-TC6 insulinoma cells. Both systems exhibited some physiological responses such as acetylcholine-stimulated [Ca2+]i rise via cytoplasmic Ca2+ mobilization. Addition of ATP, ADP, and 2-MeSADP (each 100 µM) transiently increased [Ca2+]i in single islets cultured in the presence of 5.5 mM (normal) glucose. The potent P2Y1 receptor agonist 2-MeSADP reduced insulin secretion significantly in islets cultured in the presence of high glucose (16.7 mM), whereas a slight stimulation occurred at 5.5 mM glucose. The selective P2Y6 receptor agonist UDP (200 µM) transiently increased [Ca2+]i and reduced insulin secretion at high glucose, whereas the P2Y2/4 receptor agonist UTP and adenosine receptor agonist NECA were inactive. [Ca2+]i transients induced by 2-MeSADP and UDP were antagonized by suramin (100 µM), U73122 (2 µM, PLC inhibitor), and 2-APB (10 or 30 µM, IP3 receptor antagonist), but neither by staurosporine (1 µM, PKC inhibitor) nor depletion of extracellular Ca2+. The effect of 2-MeSADP on [Ca2+]i was also significantly inhibited by MRS2500, a P2Y1 receptor antagonist. These results suggested that P2Y1 and P2Y6 receptor subtypes are involved in Ca2+ mobilization from intracellular stores and insulin release in mouse islets. In beta-TC6 cells, ATP, ADP, 2-MeSADP, and UDP transiently elevated [Ca2+]i and slightly decreased insulin secretion at normal glucose, while UTP and NECA were inactive. RT-PCR analysis detected mRNAs of P2Y1 and P2Y6, but not P2Y2 and P2Y4 receptors.

Mathematical modelling of the calcium–left ventricular pressure relationship in the intact diabetic rat heart

The objective was to characterize cross-bridge kinetics from the cytoplasmic calcium ion concentration ([Ca2+](i)) and the left ventricular pressure (LVP) in the early-stage diabetic rat heart under baseline conditions and upon beta-adrenergic stimulation. Four weeks after the induction of diabetes in rats by the injection of streptozotocin, the hearts were perfused according to Langendorff, and [Ca2+](i) was obtained by epifluorescence measurements using Indo-1 AM. [Ca2+](i) and LVP were measured simultaneously at a temporal resolution of 200 Hz. The input/output relationship between the Ca2+ and the pressure transients was described by a mathematical model representing the chemical binding of Ca2+ to troponin C on the actin myofilament (TnCA), and the subsequent cooperative force-producing cross-bridge formation of the Ca2+-TnCA complex with myosin. The kinetic parameters of this model were evaluated using a numerical optimization algorithm to fit the model equations to the experimental data. beta-adrenergic stimulation of the hearts with increasing doses of isoproterenol allowed quantification of the model parameters over an extended dynamic range, because isoproterenol administration increased developed pressure, heart rate, as well as [Ca2+](i) amplitude in a dose-dependent manner. Model analysis of the experimental data indicates that beta-adrenergic stimulation of healthy hearts resulted in a decreased sensitivity of TnCA for Ca2+, increased rates of cross-bridge cycling and decreased cooperativity. By contrast, the responses in cross-bridge kinetic parameters to isoproterenol stimulation were blunted in the 4-week diabetic heart. We conclude from our modelling results that myocardial cross-bridge cycling is impaired at the early stage of diabetes.

Calcium signalling in cardiac muscle cells.

In heart cells, several distinct kinds of transient spatial patterns of cytoplasmic calcium ion concentration ([Ca2+]i) can be observed: (1) [Ca2+]i waves, in which regions of spontaneously increased [Ca2+]i propagate at high velocity (100 microns/s) through the cell; (2) Ca2+ 'sparks', which are spontaneous, non-propagating changes in [Ca2+]i that are localized in small (approximately 2 microns) subcellular regions; and (3) evoked [Ca2+]i transients that are elicited by electrical depolarization, in association with normal excitation-contraction (E-C) coupling. In confocal [Ca2+]i images, evoked [Ca2+]i transients appear to be nearly spatially uniform throughout the cell, except during their rising phase or during small depolarizations. In contrast to [Ca2+]i waves and spontaneous Ca2+ sparks, evoked [Ca2+]i transients are triggered by L-type Ca2+ channel current and they are 'controlled', in the sense that stopping the L-type Ca2+ current stops them. Despite their different characteristics, all three types of Ca2+ transient involve Ca(2+)-induced release of Ca2+ from the sarcoplasmic reticulum. Here, we address the question of how the autocatalytic process of Ca(2+)-induced Ca2+ release, which can easily be understood to underlie spontaneous regenerative ('uncontrolled'), propagating [Ca2+]i waves, might be 'harnessed', under other circumstances, to produce controlled changes in [Ca2+]i, as during normal excitation-contraction coupling, or changes in [Ca2+]i that do not propagate. We discuss our observations of Ca2+ waves, Ca2+ sparks and normal Ca2+ transients in heart cells and review our results on the 'gain' of Ca(2+)-induced Ca2+ release. We discuss a model involving Ca2+ microdomains beneath L-type Ca2+ channels, and clusters of Ca(2+)-activated Ca2+ release channels in the sarcoplasmic reticulum which may form the basis of the answer to this question.

The role of calcium ion in pain and antinociception

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Effects of Selective Protein Kinase C Isozymes in Prostaglandin2α-Induced Ca2+ Signaling and Luteinizing Hormone-Induced Progesterone Accumulation in the Mid-Phase Bovine Corpus Luteum1

A single-cell approach for measuring the concentration of cytoplasmic calcium ions ([Ca(2+)](i)) and a protein kinase C-epsilon (PKCepsilon)-specific inhibitor were used to investigate the developmental role of PKCepsilon in the prostaglandin F(2alpha)(PGF(2alpha))-induced rise in [Ca(2+)](i) and the induced decline in progesterone accumulation in cultures of cells isolated from the bovine corpus luteum. PGF(2alpha) increased [Ca(2+)](i) in Day 4 large luteal cells (LLCs), but the response was significantly lower than in Day 10 LLCs (4.3 +/- 0.6, n = 116 vs. 21.3 +/- 2.3, n = 110). Similarly, the fold increase in the PGF(2alpha)-induced rise in [Ca(2+)](i) in Day 4 small luteal cells (SLCs) was lower than in Day 10 SLCs (1.6 +/- 0.2, n = 198 vs. 2.7 +/- 0.1, n = 95). A PKCepsilon inhibitor reduced the PGF(2alpha)-elicited calcium responses in both Day 10 LLCs and SLCs to 3.5 +/- 0.3 (n = 217) and 1.3 +/- 0.1 (n = 205), respectively. PGF(2alpha) inhibited LH-stimulated progesterone (P(4)) accumulation only in the incubation medium of Day 10 luteal cells. Both conventional and PKCepsilon-specific inhibitors reversed the ability of PGF(2alpha) to decrease LH-stimulated P(4) accumulation, and the PKCepsilon inhibitor was more effective at this than the conventional PKC inhibitor. In conclusion, the evidence indicates that PKCepsilon, an isozyme expressed in corpora lutea with acquired PGF(2alpha) luteolytic capacity, has a regulatory role in the PGF(2alpha)-induced Ca(2+) signaling in luteal steroidogenic cells, and that this in turn may have consequences (at least in part) on the ability of PGF(2alpha) to inhibit LH-stimulated P(4) synthesis at this developmental stage.

Developmental Sensitivity of the Bovine Corpus Luteum to Prostaglandin F2α (PGF2α) and Endothelin-1 (ET-1): Is ET-1 a Mediator of the Luteolytic Actions of PGF2α or a Tonic Inhibitor of Progesterone Secretion?1

We examined the responsiveness of large luteal cells (LLC), small luteal cells (SLC), and endothelial cells of the Day 4 and Day 10 bovine corpus luteum (CL) to prostaglandin (PG) F2alpha and endothelin (ET)-1. Using a single-cell approach, we tested the ability of each agonist to increase the cytoplasmic concentration of calcium ions ([Ca2+]i) as function of luteal development. All tested concentrations of agonists significantly (P = 0.05) increased [Ca2+]i in all cell populations isolated from Day 4 and Day 10 CL. Day 10 steroidogenic cells were more responsive than Day 4 cells to PGF2alpha and ET-1. Response amplitudes and number of responding cells were affected significantly by agonist concentration, luteal development, and cell type. Response amplitudes were greater in LLC than in SLC; responses of maximal amplitude were elicited with lower agonist concentrations in Day 10 cells than in Day 4 cells. Furthermore, on Day 10, as the concentration of PGF2alpha increased, larger percentages of SLC responded. Endothelial cells responded maximally, regardless of agonist concentration and luteal development. In experiment 2, we tested the developmental responsiveness of total dispersed and steroidogenic-enriched cells to the inhibitory actions of PGF2alpha and ET-1 on basal and LH-stimulated progesterone accumulation. The potency of PGF2alpha steroidogenic-enriched cells on Day 4 was lower than on Day 10; in contrast, the potency of ET-1 was not different. Therefore, ET-1 was a tonic inhibitor of progesterone accumulation rather than a mediator of PGF2alpha action. The lower efficacy of PGF2alpha in the early CL more likely is related to signal transduction differences associated with its receptor at these two developmental stages than to the inability of PGF2alpha to up-regulate ET-1.

Live-cell dynamics and the role of costimulation in immunological synapse formation.

Using transfected fibroblasts expressing both wild-type I-E(k) and green fluorescent protein-tagged I-E(k) with covalently attached antigenic peptide, we have monitored movement of specific MHC:peptide complexes during CD4(+) T cell-APC interactions by live-cell video microscopy. Ag recognition occurs within 30 s of T cell-APC contact, as shown by a sharp increase in cytoplasmic calcium ion concentration. Within 1 min, small MHC:peptide clusters form in the contact zone that coalesce into an immunological synapse over 3-20 min. When T cells conjugated to APC move across the APC surface, they appear to drag the synapse with them. This system was used to examine the role of costimulation in the formation of the immunological synapse. Blocking CD80/CD28 or ICAM-1/LFA-1 interactions alters synapse morphology and reduces the area and density of accumulated complexes. These reductions correlate with reduced T cell proliferation, while CD69 and CD25 expression and TCR down-modulation remain unaffected. Thus, costimulation is essential for normal mature immunological synapse formation.

Cancer cell contact causes oscillatory and polarized rise of cytoplasmic ionized calcium concentration in human umbilical vein endothelial cells

The rise of cytoplasmic ionized Ca2+ concentration ([Ca2+]i) is an important factor in cellular signal transduction, which regulates a variety of cellular functions in vascular endothelial cells (vECs) such as morphological change, secretion of vaso-active molecules, or endothelial permeability. It is also known that the loss of vEC integrity plays pivotal roles in several physiological or pathological phenomena, such as inflammation or cancer metastasis. The aim of this study was to clarify the mode of cancer cell induced vEC retraction by cell to cell contact. Digital imaging analysis using Ca2+ indicator, fluo-3, clearly demonstrated an oscillatory intracellular and intercellular [Ca2+]i wave originated from the site of human breast cancer cell line MCF-7 cell contact in HUVECs, which was not inhibited by extracellular Ni2+ and was inhibited by pretreating the cells by paraformaldehyde. Although this phenomenon could be observed in malignant cells including murine malignant melanoma cell line, BL6, as well as benign cells including human fibroblast cell line, WI38, inert neutrophils did not cause vEC retraction nor [Ca2+]i oscillation. Thus, cancer cells could activate vECs by a cell to cell contact to cause retraction via common property of non-blood cells.

Involvement of calcium in pain and antinociception.

Calcium ions are widely recognized to play a fundamental role in the regulation of several biological processes. Transient changes in cytoplasmic calcium ion concentration represent a key step for neurotransmitter release and the modulation of cell membrane excitability. Evidence has accumulated for the involvement of calcium ions also in nociception and antinociception, including the analgesic effects produced by opioids. The combination of opioids with drugs able to interfere with calcium ion functions in neurons has been pointed out as a useful alternative for safer clinical pain management. Alternatively, drugs that reduce the flux of calcium ions into neurons have been indicated as analgesic alternatives to opioids. This article reviews the manners by which calcium ions penetrate cell membranes and the changes in these mechanisms caused by opioids and calcium antagonists regarding nociceptive and antinociceptive events.

Calcium oscillations and Nod signal transduction in Rhizobium-legume symbiosis

Rhizobium nodulation (Nod) factors are lipo-chitooligosaccharides that act as symbiotic signals, eliciting a number of key developmental responses in the roots of legume hosts. One of the earliest responses of root hairs to Nod factors is the induction of sharp oscillations of cytoplasmic calcium ion concentration ("calcium spiking"). This response was first characterised in Medicago sativa and Nod factors were found to be unable to induce calcium spiking in a nodulation-defective mutant of M. sativa. The fact that this mutant lacked any morphological response to Nod factors raised the question of whether calcium spiking could be part of a Nod factor-induced signal transduction pathway leading to nodulation. More recently, calcium spiking has been described in a model legume, Medicago truncatula, and in pea. When nodulation-defective mutants were tested for the induction of calcium spiking in response to Nod factors, three loci of pea and two of M. truncatula were found to be necessary for Nod factor-induced calcium spiking. These loci are also known to be necessary for Nod factor-induction of symbiotic responses such as root hair deformation, nodulin gene expression and cortical cell division. These results therefore constitute strong genetic evidence for the role of calcium spiking in Nod factor transduction. This system provides an opportunity to use genetics to study ligand-stimulated calcium spiking as a signal transduction event.

Congenital disorders of platelet signal transduction.

After injury to the blood vessel, platelets adhere to the exposed subendothelium by a process (adhesion) that involves the interaction of a plasma protein, von Willebrand factor (vWF), and a specific protein on the platelet surface, glycoprotein Ib (GPIb; the Figure⇓). Adhesion is followed by recruitment of additional platelets that form clumps, a process called aggregation (cohesion). This involves binding of fibrinogen to specific platelet surface receptors—a complex comprising glycoproteins IIb-IIIa (GPIIb-IIIa). Activated platelets release the contents of their granules (secretion or release reaction), such as ADP and serotonin from dense granules, which subsequently cause recruitment of additional platelets. In addition, platelets play a major role in coagulation mechanisms; several key enzymatic reactions occur on the platelet membrane–lipoprotein surface. A number of physiological agonists interact with specific receptors on the platelet surface to induce responses, including a change in platelet shape from discoid to spherical, aggregation, secretion, and thromboxane A2 (TxA2) production. Other agonists such as prostacyclin inhibit these responses. Ligation of the platelet receptors initiates the production or release of several intracellular messenger molecules, including Ca2+ ions, products of phosphoinositide (PI) hydrolysis by phospholipase C (PLC; diacylglycerol [DG] and inositol 1,4,5-triphosphate [InsP3]), TxA2, and cyclic nucleotides (cAMP; the Figure⇓). These subsequently induce or modulate the various platelet responses of Ca2+ mobilization, protein phosphorylation, aggregation, secretion, and liberation of arachidonic acid. The interaction between the agonist receptors and the key intracellular effector enzymes (eg, PLA2, PLC, adenylyl cyclase) is mediated by a group of GTP-binding proteins that are modulated by GTP. As in most secretory cells, platelet activation results in …

Behavior of blood cells in contact with water-soluble phospholipid polymer.

Omega-Methacryloyloxyalkyl phosphorylcholine (MAPC) polymer, which has various methylene chain lengths between the phosphorylcholine group and the backbone, was synthesized with attention to formation of the biomembrane. The effect of water-soluble poly(MAPC) on the function and activation of blood cells was evaluated to determine the interaction between blood cells and the MAPC polymer. The poly(MAPC) and the MAPC copolymer with a small amount of fluorescent units were synthesized by a conventional radical polymerization technique. Using a fluorescence spectrometer, it was determined that the MAPC polymer was adsorbed on the plasma membrane of platelets when the platelets were suspended in an aqueous solution of the MAPC copolymer. The hemolytic activity of poly(MAPC) was less than that of other water-soluble polymers, such as poly(ethylene glycol) and poly(1-vinyl-2-pyrrolidone) (PVPy). The change in the plasma membrane fluidity of platelets on contact with poly(MAPC) was determined with 1,6-diphenyl-1,3,5,-hexatriene. The plasma membrane fluidity of platelets decreased with an increase in the methylene chain length of the MAPC unit. The aggregation activity of platelets after contact with poly(MAPC) was also evaluated, but no significant difference between that of polymer-contacted platelets and native platelets was observed. Finally, the activity of platelets on contact with poly(MAPC) was determined by measuring the cytoplasmic calcium ion concentration ([Ca2+]i) in platelets. The increase in [Ca2+]i in the platelets after contact with poly(MAPC) was similar to that of native platelets. We conclude that the poly(MAPC) reduced platelet activation even though the poly(MAPC) adsorbed on the membrane surface of the platelets. In particular, poly(10-methacryloyloxydecyl phosphorylcholine) significantly reduced platelet activation compared with PVPy.

Activation of human T lymphocytes by ganglioside-containing liposomes.

We investigated the in vitro stimulatory effect of ganglioside (GM3, GD1a, GD1b, GT1b, or GQ1b)-containing liposomes on human immune cells. The effect of ganglioside-containing liposomes on the concentration of cytoplasmic free calcium ions ([Ca2+]i) in human immunocytes was examined using the confocal laser fluorescence microscopic method. The GD1a- and GT1b-containing liposomes significantly increased [Ca2+]i of human T lymphocytes compared with the GM3-, GD1b- and GQ1b-containing ones. The response of CD8+ and CD4+ cells was significantly higher than that of CD20+ cells. Our results show that the increase in [Ca2+]i may be caused by not the number of sialic acids contained in the gangliosides but the conformation of the sialic acid moiety to protrude exteriorly from the liposomal membrane surface, and that a sort of receptor recognizing the sialic acid moiety exists on human T lymphocytes (both CD8+ and CD4+ cells), which may be involved in the activation of the cells. The present results are almost the same as those obtained for the rat T lymphocyte system previously reported. This clearly confirms that a sort of ganglioside surely stimulates T lymphocytes directly, which is not species-specific but conserved in humans and rats among animal species.

Luteinizing hormone (LH) stimulates both intracellular calcium ion ([Ca2+]i) mobilization and transmembrane cation influx in single ovarian (granulosa) cells: recruitment as a cellular mechanism of LH-[Ca2+]i dose response.

The gonadotropic hormones, LH and FSH, activate adenylyl cyclase in their respective target cells and thereby initiate many biochemical responses. In addition to stimulating cAMP production, both LH and FSH promote agonist-specific increases in the cytoplasmic concentration of free calcium ions ([Ca2+]i) in gonadal cells. Here, we have applied single cell fluorescence video microscopy with the Ca2+-sensitive dye fura-2 to investigate the mechanism(s) by which LH induces a rise in the [Ca2+]i in individual (swine) granulosa cells collected from single Graafian follicles. Stimulation with LH induced a rapid onset, biphasic, spike- and plateau-like [Ca2+]i signal in responsive granulosa cells. The cellular mechanisms mediating this biphasic LH-stimulated increase in [Ca2+]i were examined by external Ca2+ removal and via the manganese (Mn2+) quench technique, which showed that LH triggers initial intracellular Ca2+ mobilization followed by delayed transmembrane Ca2+ influx. Single cell Ca2+ assessment of the LH dose-response mechanism(s) revealed that higher concentrations of LH progressively recruit a larger number of responding individual granulosa cells. Further analyses disclosed a marked [Ca2+]i response heterogeneity among individual granulosa cells harvested from the same Graafian follicle. In addition, the percentage of cells responding to LH [but not to an alternative putative agonist of the phospholipase C (PLC) pathway, viz. endothelin-1] with a biphasic [Ca2+]i rise increased with maturational development of the follicle. Pretreatment of granulosa cells with a specific PLC inhibitor, U-73122 (but not with its inactive congener U-73343), significantly reduced the percentage of cells responding to a LH challenge from 78% to 25% (P < 0.0001) and prolonged the time required to achieve a half-maximal value of the [Ca2+]i transient, viz. from 22 +/- 1.5 sec (n = 27 cells) to 39 +/- 7.2 sec (n = 12 cells; P = 0.002). In cell population studies, LH stimulated in a concentration- and time-dependent manner the accumulation of inositol phosphate in porcine granulosa cells. In summary, the present single cell investigations in mature granulosa cells demonstrate that LH drives initial intracellular Ca2+ mobilization followed by transmembrane divalent cation influx. The PLC inhibitor U-73122 antagonizes this action of LH. By analyzing [Ca2+]i responses in individual living granulosa cells, we further show that, despite within-follicle diversity, the LH dose biphasic [Ca2+]i response arises via the recruitment of a larger number of responding gonadal cells rather than by increased [Ca2+]i signal amplitude. Finally, the percentage of individual LH (but not endothelin-1)-responding granulosa cells increases with follicular maturation. Collectively, these data highlight the potential importance of the LH-stimulatable, PLC-transduced [Ca2+]i signaling mechanism in the later stages of granulosa cell differentiation.

Localization of cytoplasmic free calcium ions in PC12 cells with varicose fibers.

Differentiated PC12 cells with varicose fibers were used as a model of sympathetic neurons to demonstrate the intracellular localization of cytoplasmic free calcium ions. Changes in the concentration of cytoplasmic free calcium ions were analyzed at individual varicosities upon stimulation with acetylcholine by laser scanning confocal microscopy. Transient increases in cytoplasmic free calcium ion concentration were localized in the varicosities and recognized in both the absence and presence of extracellular Ca2+. Immunocytochemical analysis of intracellular calcium channels, 1, 4, 5-trisphosphate receptors and ryanodine receptors, by electron microscopy demonstrated that immunoreactive sites were mainly localized in large dense core vesicles in the varicosities and neurites. These results suggest that the exocytosis of large dense core vesicles is regulated by an increase in cytoplasmic free calcium ion concentration from an intracellular Ca2+ store.