Cellular calcium and atherosclerosis: A brief review

Evidence for and against the theory that cell calcium is causally involved in the pathogenesis of atherosclerosis is presented and evaluated. In particular, it is argued that: (1) arterial calcium is increased in atherosclerosis; (2) this increase in tissue calcium content is largely intracellular; (3) this increased intracellular calcium content is caused by increased plasma membrane calcium permeability; (4) the increased calcium content is causally related to atherogenesis; (5) many of the cell physiological, cell biological, biochemical, and molecular biological processes, known to function abnormally in atherosclerosis, are also known to be calcium regulated; and (6) these processes are activated or inactivated in atherosclerosis in a manner consistent with increased cell calcium. It is concluded that the calcium-atherogenesis hypothesis has the potential to unify macroscopic clinical risk factors in terms of intracellular mechanisms that are controlled by cell calcium, and that this hypothesis deserves further experimental tests.     

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca²⁺ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.     

Changes of Plasma Membrane Properties in a Human Pre-T Cell Line Undergoing Apoptosis

A variety of cellular functions are modulated by the physical properties of the cell membrane, and the modification of intracellular transfer, resulting from loss of membrane integrity, may contribute toward setting the cell onto the pathway of apoptosis. Apoptosis in lymphoid cells can be induced in different ways and biochemical modifications occur at an early phase of cell death, while the morphological features of apoptosis are evident later. We previously reported that DMSO is an efficient apoptosis-inducing factor in the human RPMI-8402 pre-T cell line. The aim of the present study was to verify the effect of DMSO on the plasma membrane fluidity, the intracellular calcium concentration and the phosphodiesterase activity in DMSO-induced apoptosis. Our results show a modification of membrane fluidity associated with an increase of intracellular Ca²⁺ concentration. Moreover, we demonstrate that these modifications are related to a decrease in the phosphodiesterase (PDE) activity. The correlation between the proceedings of added DMSO and the induction of apoptosis will provide significant information regarding the first part of the apoptotic process.     

Overexpression of Trpp5 contributes to cell proliferation and apoptosis probably through involving calcium homeostasis

Trpp5 is one member of the polycystic kidney disease (PKD) family, which belongs to transient receptor potential (TRP) superfamily. Our previous study has shown that Trpp5 is developmentally expressed in mouse testis and overexpression of Trpp5 increases intracellular free calcium concentration in MDCK cells. However, the roles of this protein in cellular processes are largely unknown. Here, we demonstrated that Trpp5 resided in both cytoplasm and cell membrane of HEK293 cells. We found that overexpression of Trpp5 slightly increased the calcium current amplitude of HEK293 cells and shifted the reversal potential to a more negative value. Meanwhile, overexpression of Trpp5 suppressed proliferation of Hela cells via inhibiting DNA replication and induced apoptosis of Hela cells with morphological changes and accumulation of fragmented DNA. Collectively, these findings suggest that Trpp5 might involve calcium homeostasis contributing to cell proliferation and apoptosis.     

Calcium entry through receptor-operated channels in bovine pulmonary artery endothelial cells

The activation of endothelial cells by endothelium-dependent vasodilators has been investigated using bioassay, patch clamp and 45Ca flux methods. Cultured pulmonary artery endothelial cells have been demonstrated to release EDRF in response to thrombin, bradykinin, ATP and the calcium ionophore A23187. The resting membrane potential of the endothelial cells was -56 mV and the cells were depolarized by increasing extracellular K+ or by the addition of (0.1-1.0 mM)Ba2+ to the bathing solution. The electrophysiological properties of the cultured endothelial cells suggest that the membrane potential is maintained by an inward rectifying K+ channel with a mean single channel conductance of 35.6 pS. The absence of a depolarization-activated inward current and the reduction of 45Ca influx with high K+ solution suggests that there are no functional voltage-dependent calcium or sodium channels. Thrombin and bradykinin were shown to evoke not only an inward current (carried by Na+ and Ca2+) but also an increase in 45Ca influx suggesting that the increase in intracellular calcium necessary for EDRF release is mediated by an opening of a receptor operated channel. High doses of thrombin and bradykinin induced intracellular calcium release, however, at low doses of thrombin no intracellular calcium release was observed. We propose that the increased cytosolic calcium concentration in endothelial cells induced by endothelium dependent vasodilators is due to the influx of Ca2+ through a receptor operated ion channel and to a lesser degree to intracellular release of calcium from a yet undefined intracellular store.     

The induction of apoptosis by daunorubicin and idarubicin in human trisomic and diabetic fibroblasts

In this study, we investigated apoptosis induced in human trisomic and diabetic fibroblasts by daunorubicin (DNR) and its derivative, idarubicin (IDA). The cells were incubated with DNR or IDA for 2 h and then cultured in a drug-free medium for a further 2–48 h. The apoptosis in the cultured cell lines was assessed by biochemical analysis. We found that both drugs induced a timedependent loss of mitochondrial membrane potential, and a significant increase in intracellular calcium and caspase-3 activity. Mitochondrial polarization and changes in the level of intracellular calcium were observed during the first 2–6 h after drug treatment. Caspase-3 activation occurred in the late stages of the apoptotic pathway. Our findings also demonstrated that idarubicin was more cytotoxic and more effective than daunorubicin in inducing apoptosis in trisomic and diabetic fibroblasts.     

Stabilizing role of calcium store-dependent plasma membrane calcium channels in action-potential firing and intracellular calcium oscillations

In many biological systems, cells display spontaneous calcium oscillations (CaOs) and repetitive action-potential firing. These phenomena have been described separately by models for intracellular inositol trisphosphate (IP3)-mediated CaOs and for plasma membrane excitability. In this study, we present an integrated model that combines an excitable membrane with an IP3-mediated intracellular calcium oscillator. The IP3 receptor is described as an endoplasmic reticulum (ER) calcium channel with open and close probabilities that depend on the cytoplasmic concentration of IP3 and Ca2+. We show that simply combining this ER model for intracellular CaOs with a model for membrane excitability of normal rat kidney (NRK) fibroblasts leads to instability of intracellular calcium dynamics. To ensure stable long-term periodic firing of action potentials and CaOs, it is essential to incorporate calcium transporters controlled by feedback of the ER store filling, for example, store-operated calcium channels in the plasma membrane. For low IP3 concentrations, our integrated NRK cell model is at rest at -70 mV. For higher IP3 concentrations, the CaOs become activated and trigger repetitive firing of action potentials. At high IP3 concentrations, the basal intracellular calcium concentration becomes elevated and the cell is depolarized near -20 mV. These predictions are in agreement with the different proliferative states of cultures of NRK fibroblasts. We postulate that the stabilizing role of calcium channels and/or other calcium transporters controlled by feedback from the ER store is essential for any cell in which calcium signaling by intracellular CaOs involves both ER and plasma membrane calcium fluxes.     

Chemico-genetic identification of drebrin as a regulator of calcium responses

Store-operated calcium channels are plasma membrane Ca²⁺ channels that are activated by depletion of intracellular Ca²⁺ stores, resulting in an increase in intracellular Ca²⁺ concentration, which is maintained for prolonged periods in some cell types. Increases in intracellular Ca²⁺ concentration serve as signals that activate a number of cellular processes, however, little is known about the regulation of these channels. We have characterized the immuno-suppressant compound BTP, which blocks store-operated channel mediated calcium influx into cells. Using an affinity purification scheme to identify potential targets of BTP, we identified the actin reorganizing protein, drebrin, and demonstrated that loss of drebrin protein expression prevents store-operated channel mediated Ca²⁺ entry, similar to BTP treatment. BTP also blocks actin rearrangements induced by drebrin. While actin cytoskeletal reorganization has been implicated in store-operated calcium channel regulation, little is known about actin-binding proteins that are involved in this process, or how actin regulates channel function. The identification of drebrin as a mediator of this process should provide new insight into the interaction between actin rearrangement and store-operated channel mediated calcium influx.     

Increased intracellular calcium activates serum and glucocorticoid-inducible kinase 1 (SGK1) through a calmodulin-calcium calmodulin dependent kinase kinase pathway in Chinese hamster ovary cells

SGK1 is one of the protein-serine/threonine kinases that is activated by insulin in a PI3K-dependent manner. Although SGK1 mediates a variety of biological activities, the mechanisms regulating its activity remain unclear. In this study, we examined the potential roles of calcium signaling in the activation of SGK1. Treatment of CHO-IR cells with a cell-permeable calcium chelator, BAPTA-AM, abolished the insulin-induced activation of SGK1. Increasing intracellular calcium concentration by treating cells with thapsigargin or ionomycin induced a 6-8 fold increase in SGK1 activation. This was not affected by a PI3K inhibitor, wortmannin, but was completely inhibited by the calmodulin inhibitors, W 7 and W 5. Co-transfection of CHO cells with FLAG-SGK1 and CaMKK revealed the direct association of CaMKK with SGK1. These results suggest a calcium-triggered signaling cascade in which an increase in intracellular calcium concentration directly stimulates SGK1 through CaMKK.     

Contribution of intracellular calcium stores to an increase in cytosolic calcium concentration induced by Mannheimia haemolytica leukotoxin

The contribution of intracellular calcium stores to Mannheimia haemolytica leukotoxin (LKT)-induced increase in cytosolic calcium concentration was studied by pharmacologically inhibiting transport of calcium across the plasma and endoplasmic reticulum membranes of bovine neutrophils exposed to LKT. Active intracellular storage of calcium by sarcoplasmic/endoplasmic reticulum calcium ATPase, influx of extracellular calcium across the plasma membrane, and release of stored calcium via inositol triphosphate receptors and ryanodine-sensitive calcium channels were inhibited using thapsigargin, lanthanum chloride, xestospongin C, and magnesium chloride, respectively. Pre-incubation with thapsigargin attenuated the increase in cytosolic calcium concentration produced by LKT, thus confirming the involvement of intracellular calcium stores. Inhibitory effects of lanthanum chloride, xestospongin C, and magnesium chloride indicated that the increase in cytosolic calcium concentration induced by LKT resulted from both influx of calcium across the plasma membrane and release of calcium from intracellular stores.     

Mechanism of intracellular calcium transients.

Calcium ions mediate extracellular signals on intracellular processes. The signalling system based on transient rises or oscillations of the cytoplasmic calcium concentration has potential advantages. The relevant mechanisms of intracellular concentration changes include calcium-induced calcium release and calcium dependent inactivation of calcium release. A model has been devised based on these processes to generate repetitive transients of the cytoplasmic calcium concentration.     

Induction of differentiation by radiation and hyperthermia in neuroblastoma—glioma hybrid cells

The effects of either radiation or hyperthermia on the differentiation potential of NG108-15, a neuroblastoma-glioma hybrid cell line, were studied. After radiation and hyperthermia, the outgrowth of neurites from NG108-15 cells was potentiated, and polarizing current and voltage pulses induced a distinct action potential and a diphasic (inward following outward) current, respectively. An increase in the specific activity of acetylcholinesterase was also observed. In addition, both treatments induced an elevation of the concentration of intracellular calcium in some cells. The increase in intracellular calcium concentration caused by applying the calcium ionophore, A23187, induced differentiation. It is suggested that both the radiation- and the hyperthermia-induced increases of electrical excitability and acetylcholinesterase activity may have originated from an increase in intracellular Ca²⁺ concentration.     

Membrane depolarization selectively inhibits receptor-operated calcium channels in human T (Jurkat) lymphoblasts

Jurkat lymphoblasts were stimulated by a monoclonal antibody against the CD3 membrane antigen and the evoked calcium signal was followed by the intracellular fluorescent calcium indicator indo-1. The technique applied allowed us to separately investigate the stimulus-induced intracellular calcium release and the calcium-influx pathways, respectively. In the same cells membrane potential was estimated by the fluorescent dye diS-C3-(5). The resting membrane potential of Jurkat lymphoblasts under normal conditions was between -55 and -60 mV. Membrane depolarization, obtained by increasing external K+ concentration, removing external Cl-, or by increasing the Na+/K+ leak permeability with gramicidin or PCMBS, did not induce calcium influx in the resting cells and did not influence the CD3 receptor-mediated internal calcium release, while strongly inhibited the receptor-mediated calcium influx pathway. Half-maximum inhibition of this calcium influx was observed at membrane potential values of about -35 to -40 mV and this inhibition did not depend on the external calcium concentration varied between 5 and 2500 microM. Membrane hyperpolarization by valinomycin did not affect either component of the calcium signal. The observed selective inhibition of the receptor-operated calcium influx pathway by membrane depolarization is probably an important modulator of calcium-dependent cell stimulation.     

^19FNMR在生物医学研究中的应用

核磁共振（ＮＭＲ）是一种无创伤的物理测试方法,它可以直接用于体内和体外的生物样品测定,提供分子水平的信息。正常体内含氟成分很少,测定进无本底信号干扰,因此在体内研究中引进氟代指示剂进行＾１９ＦＮＭＲ研究是目前普遍采用的方法。＾１９ＦＮＭＲ可可以用来测定药物在体内代谢过程、胸内游离的离子如Ｃａ＾２＋和Ｍｇ＾２＋、胞内ｐＨ、氧浓度或氧压力（ｐＯ２）、膜电位、组织温度、血液容积和细胞容积等多项生理生化指     

Regulation of the sodium permeability of the luminal border of toad bladder by intracellular sodium and calcium: role of sodium-calcium exchange in the basolateral membrane

Sodium movement across the luminal membrane of the toad bladder is the rate-limiting step for active transepithelial transport. Recent studies suggest that changes in intracellular sodium regulate the Na permeability of the luminal border, either directly or indirectly via increases in cell calcium induced by the high intracellular sodium. To test these proposals, we measured Na movement across the luminal membrane (th Na influx) and found that it is reduced when intracellular Na is increased by ouabain or by removal of external potassium. Removal of serosal sodium also reduced the influx, suggesting that the Na gradient across the serosal border rather than the cell Na concentration is the critical factor. Because in tissues such as muscle and nerve a steep transmembrane sodium gradient is necessary to maintain low cytosolic calcium, it is possible that a reduction in the sodium gradient in the toad bladder reduces luminal permeability by increasing the cell calcium activity. We found that the inhibition of the influx by ouabain or low serosal Na was prevented, in part, by removal of serosal calcium. To test for the existence of a sodium- calcium exchanger, we studied calcium transport in isolated basolateral membrane vesicles and found that calcium uptake was proportional to the outward directed sodium gradient. Uptake was not the result of a sodium diffusion potential. Calcium efflux from preloaded vesicles was accelerated by an inward directed sodium gradient. Preliminary kinetic analysis showed that the sodium gradient changes the Vmax but not the Km of calcium transport. These results suggest that the effect of intracellular sodium on the luminal sodium permeability is due to changes in intracellular calcium.     

Neuronal Cell Death and Reactive Oxygen Species

1. We have investigated the role of reactive oxygen species (ROS) in cell death induced by ischemia or application of the excitatory amino acid agonist, N-methyl-D-aspartate (NMDA) or kainate (KA), in acutely isolated rat cerebellar granule cell neurons, studied by flow cytometry. Various fluorescent dyes were used to monitor intracellular calcium concentration, ROS concentration, membrane potential, and viability in acutely dissociated neurons subjected to ischemia and reoxygenation alone, NMDA or kainate alone, and ischemia and reoxygenation plus NMDA or kainate.2. With ischemia followed by reoxygenation, ROS concentrations rose slightly and there was only a modest increase in cell death after 60 min.3. When NMDA or kainate alone was applied to the cells there was a large increase in ROS and in intracellular calcium concentration but only a small loss of cellular viability. However, when NMDA or kainate was applied during the reoxygenation period there was a large loss of viability, accompanied by membrane depolarization, but the elevations of ROS and intracellular calcium concentration were not greater than seen with the excitatory amino acids alone.4. These observations indicate that other factors beyond ROS and intracellular calcium concentration contribute to cell death in cerebellar granule cell neurons.     

Cryopreservation induces mitochondrial permeability transition in a bovine sperm model

When the mitochondria of somatic cells are exposed to pathological calcium overload, these trigger mitochondrial permeability transition (MPT) leading to mitochondrial dysfunction and cell death. Cryopreservation procedures expose mammalian spermatozoa to physical and chemical stressors, which affect plasma membrane integrity and induce a pathological calcium overload that gradually promotes loss of sperm quality and ultimately function. Although several studies highlight the role of calcium in many physiological and pathological processes, the MPT induced by an intracellular calcium increase and its effect on the cell quality of mammalian spermatozoa are unknown. The aim of this study was to evaluate the effects of cryopreservation on MPT and its relationship with the deterioration of sperm quality in a bovine model. To do this, frozen bovine spermatozoa were thawed and adjusted to 2 × 10⁶ mL⁻¹ and incubated for 4 h at 38 °C. Using flow cytometry, we evaluated MPT by the calcein-AM and cobalt chloride method, intracellular Ca²⁺ level using FLUO3-AM, plasma membrane integrity by exclusion of propidium iodide, mitochondrial membrane potential (ΔΨm) with tetramethylrhodamine methyl ester perchlorate and intracellular ROS production with dihydroethidium. ATP levels were assessed by a chemiluminiscent method. The results showed that thawed spermatozoa trigger MPT associated with an intracellular calcium increase and that this was accompanied by ΔΨm dissipation, decrease of ATP levels and ROS production, and deterioration of plasma membrane integrity. In conclusion, cryopreservation induces MPT and this is associated with a loss of sperm quality.     

Nitrendipine-induced inhibition of calcium influx in a human T-cell clone: role of cell depolarization.

An increase in intracellular calcium concentration stimulated by anti-CD2 or CD3 antibodies has been measured with Fura-2 in P28 cells, a human CD4+ T cell clone. This intracellular calcium increase was sensitive to membrane potential changes, being increased when the cells were hyperpolarized and decreased when they were depolarized. The intracellular calcium increase was inhibited by nitrendipine (1-50 microM). Nitrendipine also induced a depolarization of the cells, due to the blockade of a potassium conductance. The inhibition of the calcium increase caused by nitrendipine could be partially reversed by hyperpolarizing the cells with valinomycin. It is concluded that the effects of nitrendipine on potassium channels may account for a large part of the inhibition that nitrendipine exerts on the calcium increase elicited by CD2 or CD3 stimulation.     

Studies of macrophage cellular response to the extracellular hydrogen peroxide by tilapia model

Reactive oxygen species (ROS) may act as signaling molecules in the physiology responses and the present study aims to investigate the effect of extracellular hydrogen peroxide on macrophages cellular response. The results obtained in the present study showed that the extracellular hydrogen peroxide affectively alter the membrane potential of the cell membrane and ion exchange channels in the cell membrane through intracellular NAD turnover that may lead to an intracellular calcium ion concentration alteration and subsequently induce the downstream signal activation.