Bending the MDCK Cell Primary Cilium Increases Intracellular Calcium

We tested the hypothesis that the primary cilium of renal epithelia is mechanically sensitive and serves as a flow sensor in MDCK cells using differential interference contrast and fluorescence microscopy. Bending the cilium, either by suction with a micropipette or by increasing the flow rate of perfusate, causes intracellular calcium to substantially increase as indicated by the fluorescent indicator, Fluo-4. This calcium signal is initiated by Ca²⁺-influx through mechanically sensitive channels that probably reside in the cilium or its base. The influx is followed by calcium release from IP₃-sensitive stores. The calcium signal then spreads as a wave from the perturbed cell to its neighbors by diffusion of a second messenger through gap junctions. This spreading of the calcium wave points to flow sensing as a coordinated event within the tissue, rather than an isolated phenomenon in a single cell. Measurement of the membrane potential difference by microelectrode during perfusate flow reveals a profound hyperpolarization during the period of elevated intracellular calcium. We conclude that the primary cilium in MDCK cells is mechanically sensitive and responds to flow by greatly increasing intracellular calcium. Received: 4 April 2001/Revised: 28 June 2001     

Increased intracellular calcium is required for neurite outgrowth induced by a synthetic peptide ligand of NCAM

We have recently identified a synthetic peptide, termed C3, capable of binding the first immunoglobulin-like module of neural cell adhesion molecule (NCAM) by means of combinatorial chemistry and shown that this NCAM ligand promotes neurite outgrowth. By means of single cell calcium imaging using the calcium-sensitive probe fura-2-acetomethyl ester, we here show that the C3-peptide induced an increase in intracellular calcium in primary hippocampal neurons and PC12-E2 cells, presumably requiring mobilization of calcium from both extracellular and intracellular stores. We further observed that C3-induced neurite outgrowth was inhibited by antagonists of voltage-dependent calcium channels as well as by an inhibitor of intracellular calcium mobilization, TMB-8. These findings demonstrate at the single cell level that a synthetic NCAM ligand directly can induce an increase in intracellular calcium and suggest that NCAM-dependent neurite outgrowth requires calcium mobilization from both extracellular and intracellular calcium stores. Thus, the C3-peptide may be regarded as a useful tool for the study of NCAM-dependent signal transduction. Furthermore, the peptide may be of considerable therapeutical interest for the treatment of neurodegenerative disorders.     

Influence of calcium and calcium-modulating agents on differentiation of murine erythroleukaemia cells

Since the involvement of calcium ions in the regulation of cell division and differentiation has been proposed, in this study we have examined the effect of extracellular calcium and of calcium-modulating agents on the DMSO-induced differentiation of murine erythroleukaemia cells. Neither proliferation nor differentiation of these cells was affected by calcium deprivation in the culture medium. Moreover, calcium-chelating agents or agents blocking intracellular calcium uptake induced a marked inhibition of cell differentiation. Intracellular calcium antagonists induced inhibition when cells were grown in a calcium-deprived medium. In contrast, murine erythroleukaemia cell differentiation was unaffected by agents that increased intracellular concentration of calcium. Our results indicate that a mobilization of calcium is indispensable for eliciting full cellular response, but the increase in intracellular level of this cation is not sufficient for complete signal transduction. It is likely that a marked alteration of the intracellular calcium system and availability could be responsible for the independence of our cell system from calcium modulation.     

Cytosolic calcium oscillators

Many cells display oscillations in intracellular calcium resulting from the periodic release of calcium from intracellular reservoirs. Frequencies are varied, but most oscillations have periods ranging from 5 to 60 s. For any given cell, frequency can vary depending on external conditions, particularly the concentration of natural stimuli or calcium. This cytosolic calcium oscillator is particularly sensitive to those stimuli (neurotransmitters, hormones, growth factors) that hydrolyze phosphoinositides to give diacylglycerol and inositol 1,4,5-trisphosphate (Ins1,4,5P3). The ability of Ins1,4,5P3 to mobilize intracellular calcium is a significant feature of many of the proposed models that are used to explain oscillatory activity. Receptor-controlled oscillator models propose that there are complex feedback mechanisms that generate oscillations in the level of Ins1,4,5P3. Second messenger-controlled oscillator models demonstrate that the oscillator is a component of the calcium reservoir, which is induced to release calcium by a constant input of either Ins1,4,5P3 or calcium itself. In the latter case, the process of calcium-induced calcium release might be the basis of oscillatory activity in many cell types. The function of calcium oscillations is still unknown. Because oscillator frequency can vary with agonist concentration, calcium transients might be part of a frequency-encoded signaling system. When an external stimulus arrives at the cell surface the information is translated into a train of calcium spikes, i.e., the signal is digitized. Certain cells may then convey information by varying the frequency of this digital signal.     

The role of class II molecules in human B cell activation. Association with phosphatidyl inositol turnover, protein tyrosine phosphorylation, and proliferation.

Cross-linking class II molecules on resting human B cells can initiate phosphatidyl inositol turnover and an increase in intracellular calcium concentration levels comparable with that seen with the cross-linking of surface Ig receptors. The calcium response is most evident on dense B cell fractions: buoyant cells are less responsive, even though the levels of class II expression are similar on dense and buoyant tonsillar B cells. Human B cell lines exhibit the same absence of correlation between intensity of the calcium signal and levels of surface class II expression, indicating that responsiveness is related to the state of differentiation of the cell rather than the amount of class II expressed. Cross-linking class II on normal B cells or B cell lines caused accumulation of inositol phosphates, suggesting class II induces calcium release from intracellular stores, rather than through direct regulation of calcium channels. The calcium response mediated through class II was completely abolished by bringing the protein tyrosine phosphatase, CD45, into close proximity with surface class II. This result indicated that protein tyrosine phosphorylation might regulate the signal transduced through this molecule. In support of this notion we found that tyrosine phosphorylation is induced when small dense tonsillar B cells are stimulated with either anti-Ig or with antibodies to class II. Finally, in B cell proliferation assays we show that cross-linking class II molecules on dense tonsillar B cells synergize strongly with suboptimal concentrations of PMA or IL-4. The significance of these results is discussed with regard to the cognate signal between B and T lymphocytes.     

Calcium ionophore enhances the electron spin resonance signal from isolated skeletal muscle

Electron spin resonance studies of the free radical signal from isolated skeletal muscle during experimental damage have shown that elevation of muscle intracellular calcium with the calcium ionophore A23187 induced an average 61% increase in the amplitude of the signal of g value 2.0047 compared to paired, untreated control muscles, accompanied by a large efflux of intracellular creatine kinase to the external medium. Inhibitors of the calcium-induced loss of cell viability leading to enzyme efflux, i.e., chlorpromazine, phenidone and nordihydroguaiaretic acid had variable effects on the signal, suggesting that the free radical signal obtained from skeletal muscle by electron spin resonance techniques is stimulated by intracellular calcium overload, but is not directly related to the mechanisms by which calcium overload leads to a loss of cell viability leading to intracellular enzyme efflux.     

Casein binds to the cell membrane and induces intracellular calcium signals in the enteroendocrine cell: a brief communication

Dietary protein but not amino acids stimulates cholecystokinin (CCK) secretion in rat mucosal cells. However, the dietary protein sensory mechanisms and the intracellular signal pathway in the enteroendocrine cells have not yet been clarified. The relationship between dietary protein binding to cell membrane and intracellular calcium responses were examined in the CCK-producing enteroendocrine cell line STC-1. The binding of solubilized STC-1 cell membrane to proteins was analyzed using a surface plasmon resonance sensor. Intracellular calcium concentrations of STC-1 cell suspensions loaded with Fura-2 AM were measured using a spectrafluorophotometer system with continuous stirring. Intracellular calcium concentrations in STC-1 cells were increased by exposure to alpha-casein or casein sodium, but not to bovine serum albumin. Solubilized STC-1 membranes bound to alpha-casein and casein sodium but did not bind to bovine serum albumin. alpha-Casein demonstrated higher membrane binding and intracellular calcium stimulating activities than casein sodium. Thus, protein binding to the STC-1 cell membrane and intracellular calcium responses were correlated. Intracellular calcium responses to alpha-casein were suppressed by an L-type calcium channel blocker. These results suggest that casein, a dietary protein, binds to a putative receptor on the CCK-producing enteroendocrine cell membrane and elicits the subsequent intracellular calcium response via an L-type calcium channel.     

Calcium signalling in wound-responsive normal human urothelial cell monolayers

Epithelial tissue repair requires coordination of migratory and proliferative activity both adjacent to and remote from the wound edge. Although calcium signalling is implicated, the specific mechanisms are poorly understood. This study characterises the calcium signal invoked in response to scratch wounding of normal human urothelial (NHU) cells and relates it to the localised cellular response. Immediately after wounding of confluent NHU cell monolayers, cells adjacent to the wound edge showed a sustained (>30 min) rise in [Ca(2+)](i), while there was an independent, but simultaneous calcium wave that propagated out from the wound edge. The transient signal involved release of calcium from intracellular stores and was not mediated via gap junctions, but by diffusion of extracellular agonists. We demonstrated that ATP was partially responsible for the initiation and propagation of the calcium wave and showed that the calcium release mechanism was mediated in part via activation of inositol-1,4,5-triphosphate (IP(3)) receptors. By contrast, the sustained calcium signal originated from the extracellular milieu and correlated with an increased rate of migration by these cells. The work presented here provides supportive evidence that the calcium signature, defined by its temporal and amplitude characteristics, is important in co-ordinating the response of cells within an epithelial cell monolayer after wounding.     

Integrin-mediated calcium signaling and regulation of cell adhesion by intracellular calcium

Integrins are ubiquitous trans-membrane adhesion molecules that mediate the interaction of cells with the extracellular matrix (ECM). Integrins link cells to the ECM by interacting with the cell cytoskeleton. In cases such as leukocyte binding, integrins mediate cell-cell interactions and cell-ECM interactions. Recent research indicates that integrins also function as signal transduction receptors, triggering a number of intracellular signaling pathways that regulate cell behavior and development. A number of integrins are known to stimulate changes in intracellular calcium levels, resulting in integrin activation. Although changes in intracellular calcium regulate a vast number of cellular functions, this review will discuss the stimulation of calcium signaling by integrins and the role of intracellular calcium in the regulation of integrin-mediated adhesion.     

双歧杆菌粘附体外肠上皮细胞的钙信号传递的研究

本文采用钙荧光探剂 Ｆｌｕｏ—３／ＡＭ染色法,定量研究了双歧杆菌１０２７株、肠致病性大肠杆菌（ＥＰＥＣ）对体外肠上皮细胞Ｌｏｖｏ细胞株粘附的钙信号传递机制。结果表明,双歧杆菌１０２７株粘附可引起Ｌｏｖｏ细胞内Ｃａ~＋２随时间延长而梯度升高,但双歧杆菌１０２７株的作用远不如ＥＰＥＣ明显。同时发现双歧杆菌粘附引起Ｌｏｖｏ。细胞内Ｃａ~２＋升高主要源于细胞外Ｃａ~２＋内流所致,这与ＥＰＥＣ粘附引起宿主细胞内Ｃａ~２＋升高主要源于细胞内Ｃａ~２＋储池的Ｃａ~２＋释放不同。ＥＰＥＣ粘附引起宿主细胞内Ｃａ~２＋大幅度升高是其致病的重要信号传递基础;而双歧杆菌粘附仅引起宿主细胞内Ｃａ~２＋轻度升高,可能是其作为生理性细菌与肠上皮细胞和谐共生的信号传递基础。     

An epithelial cell destined for apoptosis signals its neighbors to extrude it by an actin- and myosin-dependent mechanism.

BACKGROUND: Simple epithelia encase developing embryos and organs. Although these epithelia consist of only one or two layers of cells, they must provide tight barriers for the tissues that they envelop. Apoptosis occurring within these simple epithelia could compromise this barrier. How, then, does an epithelium remove apoptotic cells without disrupting its function as a barrier? RESULTS: We show that apoptotic cells are extruded from a simple epithelium by the concerted contraction of their neighbors. A ring of actin and myosin forms both within the apoptotic cell and in the cells surrounding it, and contraction of the ring formed in the live neighbors is required for apoptotic cell extrusion, as injection of a Rho GTPase inhibitor into these cells completely blocks extrusion. Addition of apoptotic MDCK cells to an intact monolayer induces the formation of actin cables in the cells contacted, suggesting that the signal to form the cable comes from the dying cell. The signal is produced very early in the apoptotic process, before procaspase activation, cell shrinkage, or phosphatidylserine exposure. Remarkably, electrical resistance studies show that epithelial barrier function is maintained, even when large numbers of dying cells are being extruded. CONCLUSIONS: We propose that apoptotic cell extrusion is important for the preservation of epithelial barrier function during cell death. Our results suggest that an early signal from the dying cell activates Rho in live neighbors to extrude the apoptotic cell out of the epithelium.     

Stimulation of capacitative calcium entry in HL-60 cells by nanosecond pulsed electric fields

Nanosecond pulsed electric fields (nsPEFs) are hypothesized to affect intracellular structures in living cells providing a new means to modulate cell signal transduction mechanisms. The effects of nsPEFs on the release of internal calcium and activation of calcium influx in HL-60 cells were investigated by using real time fluorescent microscopy with Fluo-3 and fluorometry with Fura-2. nsPEFs induced an increase in intracellular calcium levels that was seen in all cells. With pulses of 60 ns duration and electric fields between 4 and 15 kV/cm, intracellular calcium increased 200-700 nM, respectively, above basal levels (approximately 100 nM), while the uptake of propidium iodide was absent. This suggests that increases in intracellular calcium were not because of plasma membrane electroporation. nsPEF and the purinergic agonist UTP induced calcium mobilization in the presence and absence of extracellular calcium with similar kinetics and appeared to target the same inositol 1,4,5-trisphosphate- and thapsigargin-sensitive calcium pools in the endoplasmic reticulum. For cells exposed to either nsPEF or UTP in the absence of extracellular calcium, there was an electric field-dependent or UTP dose-dependent increase in capacitative calcium entry when calcium was added to the extracellular media. These findings suggest that nsPEFs, like ligand-mediated responses, release calcium from similar internal calcium pools and thus activate plasma membrane calcium influx channels or capacitative calcium entry.     

A mechanism for sensing noise damage in the inner ear

Our sense of hearing requires functional sensory hair cells. Throughout life those hair cells are subjected to various traumas, the most common being loud sound. The primary effect of acoustic trauma is manifested as damage to the delicate mechanosensory apparatus of the hair cell stereocilia. This may eventually lead to hair cell death and irreversible deafness. Little is known about the way in which noxious sound stimuli affect individual cellular components of the auditory sensory epithelium. However, studies in different types of cell cultures have shown that damage and mechanical stimulation can activate changes in intracellular free calcium concentration ([Ca(2+)](i)) and elicit intercellular Ca(2+) waves. Thus an attractive hypothesis is that changes in [Ca(2+)](i), propagating as a wave through support cells in the organ of Corti, may constitute a fundamental mechanism to signal the occurrence of hair cell damage. The mechanism we describe here exhibits nanomolar sensitivity to extracellular ATP, involves regenerative propagation of intercellular calcium waves due to ATP originating from hair cells, and depends on functional IP(3)-sensitive intracellular stores in support cells.     

The role of cytoplasmic free calcium concentration in B-cell tolerance

Calcium is an important factor in the immune response. Extracellular calcium is required for antibody production by B lymphocytes. Several investigators have demonstrated that crosslinking of receptors on B lymphocytes by anti-mu antibody induces an increase in intracellular calcium. There are few data on the role of intracellular calcium mobilization or calcium influx in tolerance induction in B cells. We studied changes in free intracellular calcium concentration ([Ca+2]i) induced by exposure of dinitrophenyl (DNP)-specific B cells to the tolerance-inducing conjugate DNP-murine IgG2a (DNP-MGG). Splenic B cells enriched for DNP-specific cells and DNP-specific continuous B-cell lines were used for the studies. Exposure of B cells to the tolerogen DNP-MGG, the antigen DNP-keyhole limpet hemocyanin (DNP-KLH), or the antigen DNP-Ficoll induced an increase in free [Ca+2]i which was due to both mobilization of Ca+2 from endoplasmic reticulum (ER) and influx of extracellular Ca+2. This increase was DNP specific since no significant change was seen with carriers alone and no change was seen in cells that were not DNP specific. The DNP-MGG and DNP-Ficoll induced the same amount of Ca+2 release from ER but the release induced by DNP-KLH was higher. When B cells, which were made tolerant by in vitro incubation with DNP-MGG, were incubated with antigens, a mobilization of Ca+2 from endoplasmic reticulum occurred that was the same as that of nontolerant B cells. Since Ca+2 mobilization is associated with Ig receptor-dependent early B-cell activation, it is likely that the tolerant B cell can still receive an activation signal through the Ig receptors.     

Control of cell division: a unifying hypothesis.

A constant feature of the initiation of cell division in a number of different cells is a rise in the intracellular level of calcium. The importance of cyclic nucleotides may depend on the way they interact with calcium. Cyclic AMP is apparently not an essential regulator of cell division but through its ability to modulate the intracellular level of calcium this cyclic nucleotide can exert profound effects on cell growth. In some systems (liver and salivary glands) cyclic AMP seems to augment the calcium signal whereas in others (lymphocytes and fibroblasts) it opposes calcium and can thus inhibit cell division. A rise in the level of calcium may be responsible for the parallel increase in cyclic GMP level which is usually associated with the stimulus to divide. An appealing feature of this calcium hypothesis is that it can account for the growth characteristics revealed by fibroblasts in tissue culture or embryonic cells during development. In both cases there is an initial phase of exponential growth during which I have proposed that the high level of calcium at mitosis persists into early G1 to provide the signal for the next division. In order to account for the sudden cessation of cell division at confluency, or at a specific stage during development, it is necessary to postulate that there is something different about the final mitosis which sets it apart from earlier mitoses. It is proposed that as the cells leave the last mitosis the level of calcium falls much more rapidly than it did during preceeding mitoses perhaps as a result of a more rapid rise in the level of cyclic AMP. This rapid rise in cyclic AMP level may have a dual function. Not only will it lower the level of calcium thus preventing further division, but it may also stimulate differentiation. Many of the embryonic cells which differentiate into specialized cells (lymphocytes, liver, salivary gland) retain the ability to divide if provided with appropriate stimuli. Although the nature of these stimuli vary considerably, they all seem to act by elevating the intracellular level of calcium.     

Investigation of signal transduction pathways involved in melanoma cell spreading

Integrins are a major family of heterodimeric adhesion receptors that are responsible for anchoring cells to extracellular matrix and they also can initiate intracellular signal pathways. Here parental and alpha 4-expressing human malignant melanoma cell lines were used to study the effect of protein kinase C (PKC), protein tyrosine kinases (PTKs) and intracellular Ca2+ on alpha 4 beta 1-mediated cell spreading on VCAM-1. Incubation of melanoma cells with PKC inhibitor inhibited alpha 4 beta 1-mediated melanoma cell spreading completely. Effect of intracellular Ca2+ on melanoma cell spreading was also investigated by non-phorbol ester tumor promotor, thapsigargin, which blocks the ability of the endoplasmic reticulum to replenish stocks of calcium which naturally leak out into the cytosol leading to a transient increase in concentration of intracellular calcium. The results showed that alpha 4 beta 1-mediated spreading was also required intracellular calcium involvement. However, in the presence of PTKs inhibitor melanoma cells showed long, thin dendiritic projections compared to control cells. Previously, data was obtained from immunofluorescense experiments showed that after genistein treatment, alpha 4-expressing cells exhibited considerable amounts of alpha 4 integrin and PTKs in both the focal contact points as well as over the whole cell. PTKs inhibitor did not have any effect on alpha 4-expressing cells spreading. This could be related to the amount of the PTKs present in these cells.     

细胞凋亡中的钙离子调控

凋亡是细胞的一种生理性、主动性的自杀行为,它使机体能够有效清除多余或病态的细胞。作为细胞内普遍存在的第二信使,Ca2 在信号转导过程中发挥重要作用。它能够将细胞感受的刺激转化为其在不同细胞组分间的分布差异及自身浓度的振荡,这种在细胞内和细胞间的波动协调了细胞生命活动的各个方面。以往的研究认为细胞内Ca2 浓度的升高是凋亡进行到后期的结果,而最近的研究发现Ca2 也可以在凋亡通路的各个层次,通过不同的方式精细调控凋亡的进程,这构成了凋亡中复杂的钙调控网络。现对钙离子和线粒体凋亡途径中分子间的复杂联系以及钙调控细胞凋亡研究的最新进展进行综述。     

Macrophage migration inhibitory factor-induced Ca(2+) response in rat testicular peritubular cells

Macrophage migration inhibitory factor (MIF), originally described as a T-cell product, has recently been identified in several endocrine organs. In the rat testis, MIF is secreted by the Leydig cells into testicular interstitial fluid that directly contacts Sertoli and peritubular cells. To investigate whether MIF is involved in calcium-dependent signal transduction, we have isolated rat Sertoli and peritubular cells. Despite progress in understanding functional properties of MIF, the molecular mechanism of MIF action in target cells is almost completely unknown. Here we find that recombinant MIF evokes a transient increase in calcium levels in peritubular cells but not in Sertoli cells from dissociated rat testis. Concentrations in the range between 12.5 ng/ml and 120 ng/ml of recombinant MIF were found to be effective, with 50 ng/ml yielding the largest increase in intracellular calcium. Preincubation of MIF with a neutralizing monoclonal antibody specifically blocked the response. Incubation of the peritubular cells in calcium-free buffer clearly decreased the evoked response in intracellular calcium concentration. However, the calcium response was greatly decreased by thapsigargin, an inhibitor of the Ca(2+) ATPase of the endoplasmic reticulum. The results strongly indicate that calcium is mobilized from reticulum stores during MIF-mediated signal transduction in the testis. In conclusion, our results provide the first characterization of MIF signal transduction in the testis and suggest that signaling from Leydig cells to peritubular cells through MIF is mediated by receptors coupled to release of intracellular calcium.     

容量性钙内流通道的分子代表

细胞内钙库排空产生一种信号,诱导细胞膜上的钙库操纵的钙通道（SOC）开放,使Ca^2 由细胞外进入细胞内,称为容量性钙内流（CCE）,或钙释放激活的钙通道（CRAC）,可能由果蝇一过性受体电位（trp)和trp样（trpl)基因编码,钙库排空和通道开放之间的偶联机制不清,目前主要提出三种机制：（1）弥散信使;（2）蛋白质－蛋白质之间的相互作用;（3）囊泡分泌。本文综述了CCE的分子代表 ,可能机制及电生理表型。     

Pseudomonas aeruginosa autoinducer modulates host cell responses through calcium signalling

The opportunistic pathogen Pseudomonas aeruginosa utilizes a cell density-dependent signalling phenomenon known as quorum sensing (QS) to regulate several virulence factors needed for infection. Acylated homoserine lactones, or autoinducers, are the primary signal molecules that mediate QS in P. aeruginosa. The autoinducer N-3O-dodecanoyl-homoserine lactone (3O-C12) exerts effects on mammalian cells, including upregulation of pro-inflammatory mediators and induction of apoptosis. However, the mechanism(s) by which 3O-C12 affects mammalian cell responses is unknown. Here we report that 3O-C12 induces apoptosis and modulates the expression of immune mediators in murine fibroblasts and human vascular endothelial cells (HUVEC). The effects of 3O-C12 were accompanied by increases in cytosolic calcium levels that were mobilized from intracellular stores in the endoplasmic reticulum (ER). Calcium release was blocked by an inhibitor of phospholipase C, suggesting that release occurred through inositol triphosphate (IP3) receptors in the ER. Apoptosis, but not immunodulatory gene activation, was blocked when 3O-C12-exposed cells were co-incubated with inhibitors of calcium signalling. This study indicates that 3O-C12 can activate at least two independent signal transduction pathways in mammalian cells, one that involves increases in intracellular calcium levels and leads to apoptosis, and a second pathway that results in modulation of the inflammatory response.