- Email: [email protected]
Pathophysiology of Ca2+ transport
022 Ca2+ RESPONSES IN Fas-MEDIATED APOPTOSIS AND HUMAN NATURAL KILLER CELL CYTOTOXICITY S. Miyazaki, Y. Oshimi, Y. Honda, and S. Oda Department of Physiology, Tokyo Women’sMedical College, Tokyo, Japan
PATHOPHYSIOLOGY OF CA” TRANSPORT B. Khodorov institute of General Pathology and Pathophysiology, I2531 5, Moscow, Russia Ca2’ plays a critical role in cell signaling. However, intracellular Ca” overload occurring under pathological conditions may lead to development of irreversible cell injury. In mammalian central neurons a perturbation of Ca2’ homeostasis caused by overstimulation of glutamate receptors is due mainly to mitochondrial deenergization. The latter results from excessive mitochondrial Ca2+ uptake which in turn enhances generation of reactive oxygen species and triggers opening of the mitochondrial “pore”. Collaps of the mitochondrial potential inhibits the mitochondrial ATP synthesis and enhances its hydrolysis. A decrease in the cellular ATP level, accompanied by lowering of the cytoplasmic pH and Ir\rd], elevation suppress Ca2’ extrusion from the cell via both Ca2’ pump and Na’/Ca2’ exchanger of the neuronal membrane. The exact mechanism of this suppression is not yet elucidated. The existing data allow us to hypothesize that during a continuous glutamate exposure, depolymerization of the actin cytoskeleton by high [Ca”], and ATP depletion underlies a progressive run-down of both the Ca2’ pump and NdlCa’+ exchanger.
We have examined changes in intracellular Ca2+ concentration during cell death in single cells by Ca’+ imaging. We found that Fas receptor-mediated apoptosis is associated with an early Ca2+ rise followed by sustained Ca” elevation as a prerequisite for fragmentation of DNA, chromatin, and cells. -During interaction between human natural killer cells INKS) and tareet tumor cells a raoid Ca2+ or-rise occurred in NKs in 2 min after contact with targets, followed l-2 min later by an abrupt Ca2’ rise in targets. NKs induced apoptosis in cell lines expressing high levels of Fas, but necrosis in other cells. The two types of cell death were associated with distinct Ca’+ response patterns in both NK and target cells. The NK cytotoxicity has been thought to be mediated by the pathway dependent on perforin which is released from NKs and forms pores in the target cell membrane. However, we found that Fas ligand/Fas receptormediated pathway is also involved. Fas ligand mRNA was expressed in freshly isolated NKs. NK-induced apoptosis was inhibited by an anti-Fas monoclonal antibody in Ca2’free medium in which perforin pores are known not to be formed. Transfection of the Fas gene in a target cells facilitated the induction of anootosis. comoared with the parental cell line. Apoptoti’c ‘cells’ undergo secondary necrosis in culture, or fragmented apoptotic bodies are disposed by phagocytes in tissue. Peripheral blood monocytes that were placed around necrotic cells exhibited a marked Ca2+ rise. Macrophages as well as their precursor monocytes show the similar Ca rise in response to ATP. The cells may detect necrotic cells by ATP released during cell damage. I
,
CALCIUM SIGNALING AND THE CONTROL OF GENE EXPRESSION 0. H. Petersen, T. Takeo, M. Craske and A. V. Tepikin MRC Secretory Control Research Group, The Physiological Laboratory, The University of Liverpool, Liverpool, UK
ER Ca’+-STORE RELEASE INDUCED CELL SIGNALLING David H. Llewellyn, Jonathan M. Kendall, and Anthony K. Campbell Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, CARDIFF, CF4 4m, Wales The role of transient changes in cytosolic Ca2+ concentration ([Ca2’],) in triggering crucial cellular events such as movement, proliferation, fertilisation, cell defence and cell death has been well documented. However, it is increasingly ap arent that cell events can be signalled by the release of P+ Ca from the ER. These include; influx of Ca2+into cells via plasma membrane channels, induction of gene expression in the nucleus, changes in nuclear transport via regulation of complex formation and permeability, nuclear pore proteolysis, and inhibition of protein secretion. We have shown that depletion of ER Ca + induces the expression of genes encoding ER lumenal Ca2’ binding proteins that control Ca2’ signalling and protein folding e.g. calreticulin. Experiments using ER targeted apo-aequorin have also demonstrated the presence of a proteolytic mechanism that is signalled as a result of this process. These signalling systems involved are likely to play a central role in viral infection, as well as cellular stress and insults, such as hypoxia and ischaemia. We have developed a novel strategy to investigate this signalling pathway by measuring Ca2+, ATP, kinases and proteases in the ER, nucleus, cytosol and at the plasma membrane using targeted fluorescent proteins and multicoloured bioluminescent indicators that can change colour through chemiluminescent resonance energy transfer (CRET). These can be imaged ratiometrically enabling signals to be quantified in defined compartments of live cells.
The nucleus contains many Ca2’-calmodulin (CaM) regulated enzymes. In recent years there has been a particular interest in Ca2’-CaM kinase triggered phosphorylation of transcription factors (Malviya and Rogue, Cell 92, 17-23, 1998). We have previously demonstrated that the nuclear envelope is a Ca2’ store from which Ca2’ can be released into the nucleoplasm. In addition, Ca*+ can easily permeate the nuclear pore complex (Gerasimenko, Gerasimenko, Tepikin and Petersen, Cell 80, 439-444, 1995). We have now investigated Ca*+-induced CaM movements inside single cells. We used fluorescent CaM derivatives injected intracellularly by the patch clamp technique and monitored movement of CaM by confocal microscopy. In pancreatic acinar cells, cholecystokinin (50 PM) evokes regular global cytosolic Ca” spikes and these induce pulses of CaM movement into the nucleoplasm. The nucleus integrates this into a sustained rise in the nucleoplasmic CaM concentration. About 50% of the CaM that binds Ca2+ is tmnslocated. This major CaM translocation provides an important stimulus for the nuclear protein kinases involved in the control of gene expression.
133
PATHOPHYSIOLOGY OF CA” TRANSPORT B. Khodorov institute of General Pathology and Pathophysiology, I2531 5, Moscow, Russia Ca2’ plays a critical role in cell signaling. However, intracellular Ca” overload occurring under pathological conditions may lead to development of irreversible cell injury. In mammalian central neurons a perturbation of Ca2’ homeostasis caused by overstimulation of glutamate receptors is due mainly to mitochondrial deenergization. The latter results from excessive mitochondrial Ca2+ uptake which in turn enhances generation of reactive oxygen species and triggers opening of the mitochondrial “pore”. Collaps of the mitochondrial potential inhibits the mitochondrial ATP synthesis and enhances its hydrolysis. A decrease in the cellular ATP level, accompanied by lowering of the cytoplasmic pH and Ir\rd], elevation suppress Ca2’ extrusion from the cell via both Ca2’ pump and Na’/Ca2’ exchanger of the neuronal membrane. The exact mechanism of this suppression is not yet elucidated. The existing data allow us to hypothesize that during a continuous glutamate exposure, depolymerization of the actin cytoskeleton by high [Ca”], and ATP depletion underlies a progressive run-down of both the Ca2’ pump and NdlCa’+ exchanger.
We have examined changes in intracellular Ca2+ concentration during cell death in single cells by Ca’+ imaging. We found that Fas receptor-mediated apoptosis is associated with an early Ca2+ rise followed by sustained Ca” elevation as a prerequisite for fragmentation of DNA, chromatin, and cells. -During interaction between human natural killer cells INKS) and tareet tumor cells a raoid Ca2+ or-rise occurred in NKs in 2 min after contact with targets, followed l-2 min later by an abrupt Ca2’ rise in targets. NKs induced apoptosis in cell lines expressing high levels of Fas, but necrosis in other cells. The two types of cell death were associated with distinct Ca’+ response patterns in both NK and target cells. The NK cytotoxicity has been thought to be mediated by the pathway dependent on perforin which is released from NKs and forms pores in the target cell membrane. However, we found that Fas ligand/Fas receptormediated pathway is also involved. Fas ligand mRNA was expressed in freshly isolated NKs. NK-induced apoptosis was inhibited by an anti-Fas monoclonal antibody in Ca2’free medium in which perforin pores are known not to be formed. Transfection of the Fas gene in a target cells facilitated the induction of anootosis. comoared with the parental cell line. Apoptoti’c ‘cells’ undergo secondary necrosis in culture, or fragmented apoptotic bodies are disposed by phagocytes in tissue. Peripheral blood monocytes that were placed around necrotic cells exhibited a marked Ca2+ rise. Macrophages as well as their precursor monocytes show the similar Ca rise in response to ATP. The cells may detect necrotic cells by ATP released during cell damage. I
,
CALCIUM SIGNALING AND THE CONTROL OF GENE EXPRESSION 0. H. Petersen, T. Takeo, M. Craske and A. V. Tepikin MRC Secretory Control Research Group, The Physiological Laboratory, The University of Liverpool, Liverpool, UK
ER Ca’+-STORE RELEASE INDUCED CELL SIGNALLING David H. Llewellyn, Jonathan M. Kendall, and Anthony K. Campbell Department of Medical Biochemistry, University of Wales College of Medicine, Heath Park, CARDIFF, CF4 4m, Wales The role of transient changes in cytosolic Ca2+ concentration ([Ca2’],) in triggering crucial cellular events such as movement, proliferation, fertilisation, cell defence and cell death has been well documented. However, it is increasingly ap arent that cell events can be signalled by the release of P+ Ca from the ER. These include; influx of Ca2+into cells via plasma membrane channels, induction of gene expression in the nucleus, changes in nuclear transport via regulation of complex formation and permeability, nuclear pore proteolysis, and inhibition of protein secretion. We have shown that depletion of ER Ca + induces the expression of genes encoding ER lumenal Ca2’ binding proteins that control Ca2’ signalling and protein folding e.g. calreticulin. Experiments using ER targeted apo-aequorin have also demonstrated the presence of a proteolytic mechanism that is signalled as a result of this process. These signalling systems involved are likely to play a central role in viral infection, as well as cellular stress and insults, such as hypoxia and ischaemia. We have developed a novel strategy to investigate this signalling pathway by measuring Ca2+, ATP, kinases and proteases in the ER, nucleus, cytosol and at the plasma membrane using targeted fluorescent proteins and multicoloured bioluminescent indicators that can change colour through chemiluminescent resonance energy transfer (CRET). These can be imaged ratiometrically enabling signals to be quantified in defined compartments of live cells.
The nucleus contains many Ca2’-calmodulin (CaM) regulated enzymes. In recent years there has been a particular interest in Ca2’-CaM kinase triggered phosphorylation of transcription factors (Malviya and Rogue, Cell 92, 17-23, 1998). We have previously demonstrated that the nuclear envelope is a Ca2’ store from which Ca2’ can be released into the nucleoplasm. In addition, Ca*+ can easily permeate the nuclear pore complex (Gerasimenko, Gerasimenko, Tepikin and Petersen, Cell 80, 439-444, 1995). We have now investigated Ca*+-induced CaM movements inside single cells. We used fluorescent CaM derivatives injected intracellularly by the patch clamp technique and monitored movement of CaM by confocal microscopy. In pancreatic acinar cells, cholecystokinin (50 PM) evokes regular global cytosolic Ca” spikes and these induce pulses of CaM movement into the nucleoplasm. The nucleus integrates this into a sustained rise in the nucleoplasmic CaM concentration. About 50% of the CaM that binds Ca2+ is tmnslocated. This major CaM translocation provides an important stimulus for the nuclear protein kinases involved in the control of gene expression.
133