IIB homepage| zurück zu |
back to IIB homepage |
Application of chemiluminescence and fluorescence
probes in the studying cell signaling
Xun Shen
Institute of Biophysics, Chinese Academy of Sciences
International Institute of Biophysics, Neuss, Germany
Chemiluminescence method have become a powerful method to record the kinetic process of the response of neutrophils to chemotactic agonists such as N-formylmethionylleucylphenylaline (fMLP), the anaphylatoxin C5a, platelet-activating factor (PAF) and leukotriene B4 or phorbol myristate (PMA). Those agonists and PMA initiate the respiratory burst of neutrophil either by binding to their receptors on cell surface or by directly activating protein kinase C. However, It has not been frequently seen the application of chemiluminescence in studying signal transduction pathways in such stimulated leukocytes. On the other hand, in the last decade, a vast variety of fluorescence probes were developed to study molecular events occurred in living cells. For instance, many proteins and enzymes can be labeled with fluorescence dyes to show their status or location within cells. Fluorescence indicator can indicate many metal ions, particularly the calcium ions within cells. Even membrane potential, cytoskeleton, expression of some particular genes in chromosomes, the movement of molecular motors can be clearly visualized by using either fluorescence probes or fluorescence protein. In our laboratory, chemiluminescence and fluorescence probes were used to investigate the signal transduction in fMLP, PMA and arachdonic acid stimulated neutrophils. Recently, a new method using fluorescence quenching of some fluoresin-based probes was developed to study kinetically the cellular uptake of iron in our laboratory.
1.The regulation of fMLP- and PMA-stimulated respiratory burst by extracellular
calcium
Chemiluminescence study showed that when extracellular free calcium were removed, the cell response stimulated with fMLP, a receptor agonist, was significantly reduced. However, if the cells were stimulated with PMA, a direct activator of PKC, almost no effect of the extracellular calcium can be found. The results suggest that extracellular calcium regulates the respiratory burst in the neutrophils stimulated by receptor binding agonist, but not in the cells stimulated by PMA.
2.The regulation of fMLP- and PMA-stimulated respiratory burst by intracellular
calcium
Thapsigargin, a potent endomembrane Ca2+ ATPase inhibitor, was used to release calcium from intracellular stores and induce a maintained elevation of the intracellular calcium concentration, [Ca2+]i , by store-depletion regulated Ca2+ influx. The fluorescence probe, fura-2, was used to determine [Ca2+]i . When concentration of intracellular free calcium is elevated by increasing the applied concentration of thapsigargin,. the respiratory activity of the fMLP-stimulated neutrophils is significantly enhanced. However, elevating intracellular calcium did not enhance the intensity of the respiratory burst in PMA-stimulated neutrophils. But, the speed at which the cells undergo respiratory burst became fast at higher [Ca2+]i. It was previously believed that the PMA-stimulated respiratory burst was calcium-independent. But, our work showed that the static intracellular calcium also regulates the PMA-stimulated respiratory burst in a way that to accelerate the onset of the cell response. This probably due to the fact that higher intracellular calcium will promote translocation of protein kinase from cytosol to plasma membrane, and make the activation of NADPH oxidase by PKC more effectively. It has been well established that when receptor agonist such as fMLP bind to its receptor on cell surface, the receptor coupled G-protein is activated, then activates a phosphatidylinositol-specific phospholipase C, which results in the generation of two second messengers, IP3 and DAG. IP3 induces the release of Ca2+ from intracellular store and results in a transient rise of [Ca2+]i , DAG remains associated with membrane and participates in the activation of PKC. However, why the onset time of the receptor mediated respiratory burst is much shorter than that of the PMA-stimulated one? In order to answer this question, a synthetic DAG was used to stimulate cells and thapsigargin was used to elevate intracellular calcium. The results showed that when cells are stimulated by fMLP, the onset time does not changed with increasing intracellular calcium. However, the onset of the DAG-stimulated respiratory burst became fast as [Ca2+]i is elevated. At highest [Ca2+]i , the synthetic DAG initiates respiratory burst as fast as fMLP does (about 10 seconds). Our results demonstrate that the reason why fMLP stimulate respiratory burst much faster than PMA is because DAG activates PKC much efficiently than PMA.
3. How the cytoskeleton regulates the fMLP-stimulated respiratory burst in
neutrophils?
It was reported that a calmodulin antagonist,W-7, inhibits the fMLP-stimulated respiratory burst at higher concentration but primes the burst at lower concentration. However, the mechanism was not clear. We investigated the effect of W-7 on fMLP-receptor. A fluorescence-labeled formyl peptide (formyt-NleLeu-phe-Nle-Tyr-Lys Bodipy FL) was used to detect the numbers of the receptor on cell surface. Flow cytometric measurement shows that the cell number distribution in cells treated with low concentration of W-7 is shifted to the right, while the distribution in the cells treated with higher concentration ofW-7 is moved to the left. Obviously, right shift means more available receptors on cell surface, while left shift means less receptor on the surface. Since the expression of the receptors is regulated by cytoskeleton, we also investigated the effect of cytochalasin B, a blocker of the assembly of microfilaments, on the receptor expression. Furthermore, a fluorescence indicator of actin, Bodipy FL phallacidin, was used to observe possible effect of W-7 on actin polymerization in cells. The flow cytometric measurement showed that the cell number distribution against the fluorescence of the probe in the cells treated with lower concentration of W-7 is shifted to the left with respect too the control, while a right shift was observed in the cells treated with higher concentration of W-7. The results suggest that W-7 inhibits the polymerization of actin at lower concentration, but promotes assembly of actin into microfilaments at higher concentration. To further confirm the correlation between the enhancement of the expression of the fMLP receptors on cell surface and the depolymerization of actin, the effect of cytochalasin B on the polymerization of actin in neutrophils was also studied. The result indicates that cytochalasin B, which can increase expression of the formyl peptide receptor on membrane, also promotes depolymerization of actin. Our study clearly demonstrates that the ternary complex of formyl peptide receptor and cytoskeleton plays an important role in the stimulation of respiratory burst. W-7 primes the fMLP stimulation by direct action on actin leading to breakdown of micrfilaments and more expression of formyl peptide receptors, and inhibits the stimulation by indirect action on actin through inactivation of some Ca2+-dependent proteins resulting in assembly of actin into microfilaments. Which action is favorable depends on the drug concentration.
4.The role of phosphatidylinositol 4-kinase in the fMLP-stimulated respiratory burst
of neutrophil
It has been known that two signaling pathways mediated by PLC and phosphatidylinositol 3-kinase ( it is simply called PI3K) are activated by chemoattractant receptors. The PI3K pathway involves the activation of some membrane-trafficking proteins and enzymes such as the phosphoinositide-dependent kinase (PDKs), protein kinase B (PKB) andS6 kinase by the key second-messenger phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P3). The PIP3 is generated from phosphatidylinositol 4,5-biphosphate (PI(4,5)P2) through phosphorelation at the 3´ position of inositol ring by PI3K. However, PI(4,5)P2 is the product of phosphatidylinositol 4-kinase (PI4K). Recently, we investigated the role of PI4K in the respiratory burst of neutrophil stimulated by fMLP utilizing chemiluminescence method. Two inhibitors of PI4K, phenylarsine oxide (PAO) and the monoclonal antibody of PI4K were used. The results showed that both PI4K inhibitors depressed the fMLP-stimulated respiratory. Since a certain amount of PI(4,5)P2 is present on the membrane for the substrate of PI3K, even complete inactivation of PI4K by the antibody can not completely depress the respiratory burst. In order to confirm that PI4K is really inhibited by these two inhibitors, the activity of PI4K was also measured by catalyzing the 32P-incorporation in phospatidylinositol 4-monophosphate using Phosphatidylinositol as the substrate.
5. Kinetic study on the transferrin-receptor mediated cellular uptake of iron.
It has been know that iron is a ‘double sides sward’. It is a necessary metal ions for synthesis of hemoglobin and many important enzymes, and even DAN synthesis. However, too much iron in the tissue and cells would cause cell damage or death. An abnormal higher accumulation of iron are found in the brains in the patients of Alzheimer decease and Pakinson´s decease show. So, to study the signal transduction in cellular uptake of iron may be potentially important for human health. Recently, we used fluorescence probe called calcein to investigate kinetic process of the uptake of transferrin-bound iron by K562 cells, the erythroleukemic cells. When iron ions is taken into the cells by transferrin-receptor mediated endocytosis and then released to cytoplasm, the fluorescence of the probe is quenched by binding to iron ions. The results showed that as intracellular calcium is elevated, more iron is taken up and the speed of the uptake becomes faster. When either intracellular or extracellular calcium is chelated, cells will reduce or stop taking up the iron.
