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1.
High-resolution differential interference contrast microscopy and digital imaging of the fluorescent calcium indicator dye fura-2 were performed simultaneously in single rat salivary gland acinar cells to examine the effects of muscarinic stimulation on cell volume and cytoplasmic calcium concentration ([Ca2+]i). Agonist stimulation of fluid secretion is initially associated with a rapid tenfold increase in [Ca2+]i as well as a substantial cell shrinkage. Subsequent changes of cell volume in the continued presence of agonist are tightly coupled to dynamic levels of [Ca2+]i, even during [Ca2+]i oscillations. Experiments with Ca2+ chelators and ionophores showed that physiological elevations of [Ca2+]i are necessary and sufficient to cause changes in cell volume. The relation between [Ca2+]i and cell volume suggests that the latter reflects the secretory state of the acinar cell. Agonist-induced changes in [Ca2+]i, by modulating specific ion permeabilities, result in solute movement into or out of the cell. The resultant cell volume changes may be important in modulating salivary secretion.  相似文献   

2.
Calcium gradients underlying polarization and chemotaxis of eosinophils   总被引:20,自引:0,他引:20  
The concentration of intracellular free calcium ([Ca2+]i) in polarized eosinophils was imaged during chemotaxis by monitoring fluorescence of the calcium-sensitive dye Fura-2 with a modified digital imaging microscope. Chemotactic stimuli caused [Ca2+]i to increase in a nonuniform manner that was related to cell activity. In cells moving persistently in one direction, [Ca2+]i was highest at the rear and lowest at the front of the cell. Before cells turned, [Ca2+]i transiently increased. The region of the cell that became the new leading edge had the lowest [Ca2+]i. These changes in [Ca2+]i provide a basis for understanding the organization and local activity of cytoskeletal proteins thought to underlie the directed migration of many cells.  相似文献   

3.
Continuous measurement and imaging of the intracellular free calcium ion concentration ([Ca2+]i) of mitotic and interphase PtK1 cells was accomplished with the new fluorescent Ca2+ indicator fura-2. No statistically significant difference between basal [Ca2+]i of interphase and mitotic cells was detected. However, mitotic cells showed a rapid elevation of [Ca2+]i from basal levels of 130 nM to 500 to 800 nM at the metaphase-anaphase transition. The [Ca2+]i transient was brief, lasting approximately 20 seconds and the elevated [Ca2+]i appeared uniformly distributed over the entire spindle and central region of the cell. The close temporal association of the [Ca2+]i transient with the onset of anaphase suggests that calcium may have a signaling role in this event.  相似文献   

4.
Digital imaging of calcium indicator signals (fura-2 fluorescence) from single cardiac cells has revealed different subcellular patterns of cytoplasmic calcium ion concentration ([Ca2+]i) that are associated with different types of cellular appearance and behavior. In any population of enzymatically isolated rat heart cells, there are mechanically quiescent cells in which [Ca2+]i is spatially uniform, constant over time, and relatively low; spontaneously contracting cells, which have an increased [Ca2+]i, but in which the spatial uniformity of [Ca2+]i is interrupted periodically by spontaneous propagating waves of high [Ca2+]i; and cells that are hypercontracted (rounded up) and that have higher levels of [Ca2+]i than the other two types. The observed cellular and subcellular heterogeneity of [Ca2+]i in isolated cells indicates that experiments performed on suspensions of cells should be interpreted with caution. The spontaneous [Ca2+]i fluctuations previously observed without spatial resolution in multicellular preparations may actually be inhomogeneous at the subcellular level.  相似文献   

5.
Free calcium at rest during "catch" in single smooth muscle cells   总被引:2,自引:0,他引:2  
Tension and intracellular free calcium concentration [( Ca2+]i) were measured simultaneously in single smooth muscle cells isolated from the anterior byssus retractor muscle (ABRM) of Mytilus edulis that were loaded with the fluorescent Ca2+ indicator fura-2. Electrical stimulation evoked a transient elevation of [Ca2+]i associated with a "catch" contraction. During the catch state, however, [Ca2+]i was effectively at its resting level and was unaffected by 5-hydroxytryptamine, which induced a rapid relaxation from catch. The results indicate that a maintained high [Ca2+]i is not required for the maintenance of catch tension in intact ABRM and that there was no significant change in [Ca2+]i upon abolition of catch.  相似文献   

6.
Receptors for immunoglobulin G immune complexes (Fc gamma RII and Fc gamma RIII) are expressed on most hematopoietic cells and show much structural and functional diversity. In order to determine the genetic basis for this diversity, a family of genes encoding the human and mouse receptors was isolated and characterized. Humans have five distinct genes for low-affinity Fc gamma Rs, in contrast to two in the mouse. With the use of yeast artificial chromosomes, the genes encoding the human receptors were oriented and linked, which established the structure of this complex locus. Comparison of the human and mouse genes generated a model for the evolutionary amplification of this locus.  相似文献   

7.
Various signaling pathways rely on changes in cytosolic calcium ion concentration ([Ca2+]i). In plants, resting [Ca2+]i oscillates diurnally. We show that in Arabidopsis thaliana, [Ca2+]i oscillations are synchronized to extracellular Ca2+ concentration ([Ca2+]o) oscillations largely through the Ca2+-sensing receptor CAS. CAS regulates concentrations of inositol 1,4,5-trisphosphate (IP3), which in turn directs release of Ca2+ from internal stores. The oscillating amplitudes of [Ca2+]o and [Ca2+]i are controlled by soil Ca2+ concentrations and transpiration rates. The phase and period of oscillations are likely determined by stomatal conductance. Thus, the internal concentration of Ca2+ in plant cells is constantly being actively revised.  相似文献   

8.
Sodium-calcium exchange in heart: membrane currents and changes in [Ca2+]i   总被引:10,自引:0,他引:10  
Recordings have been made of changes in intracellular calcium ion concentration ([Ca2+]i) that can be attributed to the operation of an electrogenic, voltage-dependent sodium-calcium (Na-Ca) exchanger in mammalian heart cells. Guinea pig ventricular myocytes under voltage clamp were perfused internally with fura-2, a fluorescent Ca2+-indicator, and changes in [Ca2+]i and membrane current that resulted from Na-Ca exchange were identified through the use of various organic channel blockers and impermeant ions. Depolarization of cells elicited slow increases in [Ca2+]i, with the maximum increase depending on internal [Na+], external [Ca2+], and membrane voltage. Repolarization was associated with net Ca2+ efflux and a decline in the inward current that developed instantaneously upon repolarization. The relation between [Ca2+]i and current was linear, and the slope was made steeper by hyperpolarization.  相似文献   

9.
Inositol 1,4,5-trisphosphate [I(1,4,5)P3] is a second messenger generated along with diacylglycerol upon the binding of various physiological agents with their cell surface receptors. I(1,4,5)P3 mobilizes Ca2+ from intracellular storage sites through a receptor-coupled mechanism, and the subsequent increased intracellular free calcium ion concentration [( Ca2+]i) activates a multitude of cellular responses. Electropermeabilized neoplastic rat liver epithelial (261B) cells were used to study Ca2+ sequestration, a process that reverses the elevated [Ca2+]i to resting levels and replenishes intracellular Ca2+ pools. Although I (1,4,5)P3-mobilized Ca2+ is readily sequestered into storage pools by the action of Ca2+-adenosine triphosphatases, Ca2+ mobilized by addition of the nonmetabolized inositol trisphosphate isomer I(2,4,5)P3 is not sequestered, suggesting that metabolism is necessary to eliminate the stimulus for Ca2+ release. Several inositol phosphate compounds were examined for their ability to lower the buffer [Ca2+] to determine if a specific I(1,4,5)P3 metabolite might be involved in stimulating Ca2+ sequestration; of these, I(1,3,4,5)P4 alone was found to induce Ca2+ sequestration, demonstrating a physiological role for this inositol trisphosphate metabolite.  相似文献   

10.
Calcium-induced calcium release (CICR) may function widely in calcium-mediated cell signaling, but has been most thoroughly characterized in muscle cells. In a homogenate of sea urchin eggs, which display transients in the intracellular free calcium concentration ([Ca2+]i) during fertilization and anaphase, addition of Ca2+ triggered CICR. Ca2+ release was also induced by the CICR modulators ryanodine and caffeine. Responses to both Ca2+ and CICR modulators (but not Ca2+ release mediated by inositol 1,4,5-trisphosphate) were inhibited by procaine and ruthenium red, inhibitors of CICR. Intact eggs also displayed transients of [Ca2+]i when microinjected with ryanodine. Cyclic ADP-ribose, a metabolite with potent Ca(2+)-releasing properties, appears to act by way of the CICR mechanism and may thus be an endogenous modulator of CICR. A CICR mechanism is present in these nonmuscle cells as is assumed in various models of intracellular Ca2+ wave propagation.  相似文献   

11.
A wide variety of nonexcitable cells generate repetitive transient increases in cytosolic calcium ion concentration ([Ca2+]i) when stimulated with agonists that engage the phosphoinositide signalling pathway. Current theories regarding the mechanisms of oscillation disagree on whether Ca2+ inhibits or stimulates its own release from internal stores and whether inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DG) also undergo oscillations linked to the Ca2+ spikes. In this study, Ca2+ was found to stimulate its own release in REF52 fibroblasts primed by mitogens plus depolarization. However, unlike Ca2+ release in muscle and nerve cells, this amplification was insensitive to caffeine or ryanodine and required hormone receptor occupancy and functional IP3 receptors. Oscillations in [Ca2+]i were accompanied by oscillations in IP3 concentration but did not require functional protein kinase C. Therefore, the dominant feedback mechanism in this cell type appears to be Ca2+ stimulation of phospholipase C once this enzyme has been activated by hormone receptors.  相似文献   

12.
Synaptic efficacy critically depends on the presynaptic intracellular calcium concentration ([Ca2+]i). We measured the calcium sensitivity of glutamate release in a rat auditory brainstem synapse by laser photolysis of caged calcium. A rise in [Ca2+]i to 1 micromolar readily evoked release. An increase to >30 micromolar depleted the releasable vesicle pool in <0.5 millisecond. A comparison with action potential-evoked release suggested that a brief increase of [Ca2+]i to approximately 10 micromolar would be sufficient to reproduce the physiological release pattern. Thus, the calcium sensitivity of release at this synapse is high, and the distinction between phasic and delayed release is less pronounced than previously thought.  相似文献   

13.
TRPM4 has recently been described as a calcium-activated nonselective (CAN) cation channel that mediates membrane depolarization. However, the functional importance of TRPM4 in the context of calcium (Ca2+) signaling and its effect on cellular responses are not known. Here, the molecular inhibition of endogenous TRPM4 in T cells was shown to suppress TRPM4 currents, with a profound influence on receptor-mediated Ca2+ mobilization. Agonist-mediated oscillations in intracellular Ca2+ concentration ([Ca2+]i), which are driven by store-operated Ca2+ influx, were transformed into a sustained elevation in [Ca2+]i. This increase in Ca2+ influx enhanced interleukin-2 production. Thus, TRPM4-mediated depolarization modulates Ca2+ oscillations, with downstream effects on cytokine production in T lymphocytes.  相似文献   

14.
为探讨姬松茸多糖诱导HepG2细胞凋亡过程中线粒体膜电位和细胞内Ca2+浓度([Ca2+]i)的变化.以人肝癌细胞系HepG2细胞为对象,使用流式细胞仪检测姬松茸多糖对HepG2细胞凋亡及线粒体膜电位的影响,激光共聚焦显微镜检测HepG2细胞[Ca2+]i的变化.结果表明姬松茸多糖能显著诱导HePG2细胞凋亡,并不同程度降低HepG2细胞线粒体跨膜电位,升高细胞[Ca2+]i,造成钙稳态失衡.姬松茸多糖诱导HepG2细胞凋亡的机制与降低线粒体膜电位,造成细胞内Ca2+超载有关.  相似文献   

15.
Elevated free Ca2+ concentrations found in adult dystrophic muscle fibers result in enhanced protein degradation. Since the difference in concentrations may reflect differences in entry, Ca2+ leak channels in cultures of normal and Duchenne human myotubes, and normal and mdx murine myotubes, have been identified and characterized. The open probability of leak channels is markedly increased in dystrophic myotubes. Other channel properties, such as mean open times, single channel conductance, ion selectivity, and behavior in the presence of pharmacological agents, were similar among myotube types. Compared to the Ca2+ concentrations in normal human and normal mouse myotubes, intracellular resting free Ca2+ concentrations ([Ca2+]i) in myotubes of Duchenne and mdx origin were significantly higher at a time when dystrophin is first expressed in normal tissue. Taken together, these findings suggest that the increased open probability of Ca2+ leak channels contributes to the elevated free intracellular Ca2+ concentration in Duchenne human and mdx mouse myotubes.  相似文献   

16.
Immunoglobulin G (IgG) mediates pro- and anti-inflammatory activities through the engagement of its Fc fragment (Fc) with distinct Fcg receptors (FcgRs). One class of Fc-FcgR interactions generates pro-inflammatory effects of immune complexes and cytotoxic antibodies. In contrast, therapeutic intravenous gamma globulin and its Fc fragments are anti-inflammatory. We show here that these distinct properties of the IgG Fc result from differential sialylation of the Fc core polysaccharide. IgG acquires anti-inflammatory properties upon Fc sialylation, which is reduced upon the induction of an antigen-specific immune response. This differential sialylation may provide a switch from innate anti-inflammatory activity in the steady state to generating adaptive pro-inflammatory effects upon antigenic challenge.  相似文献   

17.
Granulocyte and natural killer (NK) cell Fc receptors for immunoglobulin G (CD16) differ in only a few amino acids, yet have phosphatidylinositol glycan (PIG) or polypeptide membrane anchors, respectively. Mutagenesis shows that anchoring is regulated by a serine residue near the PIG anchor attachment site in the extracellular domain. The NK cell isoform was not expressed on the surface of COS cells unless cotransfected with a subunit that was expressed in NK cells and that was identical to the gamma subunit of the high affinity IgE Fc receptor (Fc epsilon RI). However, the CD16 sequence and not expression of the gamma subunit is dominant in regulating PIG reanchoring.  相似文献   

18.
19.
In order to observe the role of calcium ion in apoptosis of MD cancer cells induced by arsenic trioxide, inhibition percentage was detected by MTT assay; morphology changes were examined by fluorescence microscope; apoptosis was examined by DNA Ladder; [Ca^2+]i was investigated by spectrofluorimeter in vitro on MDCC-MSB 1 cells. The results showed that As2O3 inhibited the proliferation of MDCC-MSB1 cells in concentration dependent manner (P〈0.05 or P〈0.01); typical apoptosis character was observed by fluorescence microscope; DNA Ladder was observed; the [Ca^2+]i was elevated significantly after the treatment of As203 (P〈0.05 or P〈0.01) and showed a dose-dependent manner. It is concluded that the calcium may play an important role in apoptosis of MD cancer cells induced by arsenic trioxide.  相似文献   

20.
Antibodies that enhance human immunodeficiency virus (HIV) infectivity have been found in the blood of infected individuals and in infected or immunized animals. These findings raise serious concern for the development of a safe vaccine against acquired immunodeficiency syndrome. To address the in vivo relevance and mechanism of this phenomenon, antibody-dependent enhancement of HIV infectivity in peripheral blood macrophages, lymphocytes, and human fibroblastoid cells was studied. Neither Leu3a, a monoclonal antibody directed against the CD4 receptor, nor soluble recombinant CD4 even at high concentrations prevented this enhancement. The addition of monoclonal antibody to the Fc receptor III (anti-FcRIII), but not of antibodies that react with FcRI or FcRII, inhibited HIV type 1 and HIV type 2 enhancement in peripheral blood macrophages. Although enhancement of HIV infection in CD4+ lymphocytes could not be blocked by anti-FcRIII, it was inhibited by the addition of human immunoglobulin G aggregates. The results indicate that the FcRIII receptor on human macrophages and possibly another Fc receptor on human CD4+ lymphocytes mediate antibody-dependent enhancement of HIV infectivity and that this phenomenon proceeds through a mechanism independent of the CD4 protein.  相似文献   

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