Protective Effects of Paeoniflorin Against MPP<Superscript>+</Superscript>-induced Neurotoxicity in PC12 Cells

In the present study, we investigated the protective mechanism of paeoniflorin (PF), a monoterpene glycoside extracted from Radix Paeoniae alba roots, on MPP⁺-induced neurotoxicity in cultured rat pheochromocytoma cells (PC12). Our work included examination of cell viability assessment, amounts of released lactic dehydrogenase (LDH), intracellular Ca²⁺ concentration, cell apoptosis, mitochondrial membrane potential, caspase-3 activity, and expression profiling of two apoptosis-related genes (Bcl-2 and Bax). It was shown that, PF functioned as an MPP⁺ antagonist, being able to suppress apoptosis, decrease LDH release and Ca²⁺ concentration, attenuate membrane potential collapse and, inhibit caspase-3 activation, decrease in Bax/Bcl-2 ratio. These observations suggest that PF could protect PC12 cells against MPP⁺-induced injury and the mechanism PF’s neuroprotective effect was closely associated with Bcl-2 up-regulation and Bax down-regulation. PF has neuroprotective effects on MPP⁺-induced apoptosis in PC12 cells via regulating mitochondrial membrane potential and Bcl-2/Bax/caspase-3 signaling pathways, and this new insight will help develop a PF-based therapeutic strategy for treatmenting neurodegenerative diseases and injury.     

Role of mitochondrial calcium uniporter‐mediated Ca2+ and iron accumulation in traumatic brain injury

Previous studies have suggested that the cellular Ca²⁺ and iron homeostasis, which can be regulated by mitochondrial calcium uniporter (MCU), is associated with oxidative stress, apoptosis and many neurological diseases. However, little is known about the role of MCU‐mediated Ca²⁺ and iron accumulation in traumatic brain injury (TBI). Under physiological conditions, MCU can be inhibited by ruthenium red (RR) and activated by spermine (Sper). In the present study, we used RR and Sper to reveal the role of MCU in mouse and neuron TBI models. Our results suggested that the Ca²⁺ and iron concentrations were obviously increased after TBI. In addition, TBI models showed a significant generation of reactive oxygen species (ROS), decrease in adenosine triphosphate (ATP), deformation of mitochondria, up‐regulation of deoxyribonucleic acid (DNA) damage and increase in apoptosis. Blockage of MCU by RR prevented Ca²⁺ and iron accumulation, abated the level of oxidative stress, improved the energy supply, stabilized mitochondria, reduced DNA damage and decreased apoptosis both in vivo and in vitro. Interestingly, Sper did not increase cellular Ca²⁺ and iron concentrations, but suppressed the Ca²⁺ and iron accumulation to benefit the mice in vivo. However, Sper had no significant impact on TBI in vitro. Taken together, our data demonstrated for the first time that blockage of MCU‐mediated Ca²⁺ and iron accumulation was essential for TBI. These findings indicated that MCU could be a novel therapeutic target for treating TBI.     

Activation of the Human FPRL-1 Receptor Promotes Ca<Superscript>2+</Superscript> Mobilization in U87 Astrocytoma Cells

The human formyl peptide receptor like 1 (FPRL-1) is a variant of the G_i-coupled formyl-peptide receptor. Functional FPRL-1 is endogenously expressed in the U87 astrocytoma cell line and there is accumulating evidence to suggest that FPRL-1 may be involved in neuroinflammation associated with the pathogenesis of Alzheimer’s disease. In this study, we examined the ability of FPRL-1 to mobilize intracellular Ca²⁺ in U87 astrocytoma cells, as well as in Chinese hamster ovary (CHO) cells stably expressing FPRL-1. We showed that Trp–Lys–Tyr–Met–Val–Met–NH₂ (WKYMVM), a specific agonist for FPRL-1, stimulated Ca²⁺ influx in both U87 and FPRL-1/CHO cells. These effects can be inhibited by the FPRL-1 selective antagonist, WRW⁴. Involvement of G_i proteins was demonstrated with the use of pertussis toxin, while inhibitors of store-operated channels (SOC) including 1-[2-(4-methoxyphenyl)]-2-[3-(4-methpxyphenyl)propoxy]ethyl-1H-imidazole hydrochloride (SKF96365) and 2-aminoethoxydiphenyl borate (2-APB) were found to abolish the WKYMVM-induced Ca²⁺ increase. However, intracellular Ca²⁺ mobilization in both cell lines were unaffected by the phospholipase Cβ inhibitor U73122 or selective ryanodine receptor inhibitors. Our data demonstrated that activation of G_i-coupled FPRL-1 can lead to Ca²⁺ influx possibly via SOCs in U87 and FPRL-1/CHO cells.     

Distinctive characteristics and functions of multiple mitochondrial Ca<Superscript>2+</Superscript> influx mechanisms

Intracellular Ca²⁺ is vital for cell physiology. Disruption of Ca²⁺ homeostasis contributes to human diseases such as heart failure, neuron-degeneration, and diabetes. To ensure an effective intracellular Ca²⁺ dynamics, various Ca²⁺ transport proteins localized in different cellular regions have to work in coordination. The central role of mitochondrial Ca²⁺ transport mechanisms in responding to physiological Ca²⁺ pulses in cytosol is to take up Ca²⁺ for regulating energy production and shaping the amplitude and duration of Ca²⁺ transients in various micro-domains. Since the discovery that isolated mitochondria can take up large quantities of Ca²⁺ approximately 5 decades ago, extensive studies have been focused on the functional characterization and implication of ion channels that dictate Ca²⁺ transport across the inner mitochondrial membrane. The mitochondrial Ca²⁺ uptake sensitive to non-specific inhibitors ruthenium red and Ru360 has long been considered as the activity of mitochondrial Ca²⁺ uniporter (MCU). The general consensus is that MCU is dominantly or exclusively responsible for the mitochondrial Ca²⁺ influx. Since multiple Ca²⁺ influx mechanisms (e.g. L-, T-, and N-type Ca²⁺ channel) have their unique functions in the plasma membrane, it is plausible that mitochondrial inner membrane has more than just MCU to decode complex intracellular Ca²⁺ signaling in various cell types. During the last decade, four molecular identities related to mitochondrial Ca²⁺ influx mechanisms have been identified. These are mitochondrial ryanodine receptor, mitochondrial uncoupling proteins, LETM1 (Ca²⁺/H⁺ exchanger), and MCU and its Ca²⁺ sensing regulatory subunit MICU1. Here, we briefly review recent progress in these and other reported mitochondrial Ca²⁺ influx pathways and their differences in kinetics, Ca²⁺ dependence, and pharmacological characteristics. Their potential physiological and pathological implications are also discussed.     

Regulation of Ca<Superscript>2+</Superscript> influx in proliferating and differentiating murine myoblasts with participation of L-type Ca<Superscript>2+</Superscript> channels

The basic mechanisms of regulation of Ca²⁺ influx have been studied in murine myoblasts proliferating and differentiating in culture. The presence of L-type Ca²⁺ channels in proliferating myoblasts is shown for the first time. It is also shown that the influx of Ca²⁺ through these channels is regulated by the adrenergic system. The influx of Ca²⁺ after activation of the adrenergic system by addition of adrenaline has been estimated in comparison with the contribution of reticular stocks exhausted by ATP in calcium-free medium. The Ca²⁺ influx in proliferating myoblasts is regulated by β-2 adrenergic receptors whose action is mediated by adenylate cyclase through L-type calcium channels. In differentiating myoblasts, the adrenaline-induced Ca²⁺ influx is substantially lower than in proliferating cells, and maximal influx of Ca²⁺ may be reached only upon exhaustion of reticular stocks.     

Sirtuin 1 alleviates neuroinflammation-induced apoptosis after traumatic brain injury

Sirtuin 1 (SIRT1) plays a very important role in a wide range of biological responses, such as metabolism, inflammation and cell apoptosis. Changes in the levels of SIRT1 have been detected in the brain after traumatic brain injury (TBI). Further, SIRT1 has shown a neuroprotective effect in some models of neuronal death; however, its role and working mechanisms are not well understood in the model of TBI. This study aimed to address this issue. SIRT1-specific inhibitor (sirtinol) and activator (A3) were introduced to explore the role of SIRT1 in cell apoptosis. Results of the study suggest that SIRT1 plays an important role in neuronal apoptosis after TBI by inhibiting NF-κB, IL-6 and TNF-α deacetylation and the apoptotic pathway sequentially, possibly by alleviating neuroinflammation.     

Ca<Superscript>2+</Superscript>, Mg<Superscript>2+</Superscript>-dependent DNase Involvement in Apoptotic Effects in Spermatozoa of Sea Urchin <Emphasis Type="Italic">Strongylocentrotus intermedius</Emphasis> Induced by Two-Headed Sphingolipid Rhizochalin

Previously, we have purified three distinct DNases from spermatozoa of sea urchin Strongylocentrotus intermedius and we suppose the role of Ca²⁺, Mg²⁺-dependent DNase (Ca, Mg-DNase) in apoptosis of spermatozoa. Two-headed sphingolipid rhizochalin (Rhz) induced characteristic apoptotic nuclear chromatin changes, internucleosomal DNA cleavage, and activation of caspase-9, caspase-8, and caspase-3 in spermatozoa as was shown by fluorescence Hoechst 33342/PI/FDA analysis, DNA fragmentation assay, and fluorescence caspase inhibitors FAM-LEHD-fmk, FAM-IETD-fmk, and FAM-DEVD-fmk, respectively. Inhibitor of caspase-3 z-DEVD-fmk subdued Rhz-induced internucleosomal ladder formation, which confirmed the major role of caspase-3 in apoptotic DNA cleavage probably through Ca, Mg-DNase activation. Participation of sea urchin Ca, Mg-DNase in apoptosis of spermatozoa was demonstrated by ions Zn²⁺ blocking of Rhz-induced DNA fragmentation due to direct inhibition of the Ca, Mg-DNase and internucleosomal cleavage of HeLa S and Vero E6 cell nuclei chromatin by highly purified Ca, Mg-DNase.     

Differential Modulation of 7-Ketocholesterol Toxicity Against PC12 Cells by Calmodulin Antagonists and Ca<Superscript>2+</Superscript> Channel Blockers

The present study assessed the influence of intracellular Ca²⁺ and calmodulin against the neurotoxicity of oxysterol 7-ketocholesterol in relation to the mitochondria-mediated cell death process and oxidative stress in PC12 cells. Calmodulin antagonists calmidazolium and W-7 prevented the 7-ketocholesterol-induced mitochondrial damage, leading to caspase-3 activation and cell death, whereas Ca²⁺ channel blocker nicardipine, mitochondrial Ca²⁺ uptake inhibitor ruthenium red, and cell permeable Ca²⁺ chelator BAPTA-AM did not reduce it. Exposure of PC12 cells to 7-ketocholesterol caused elevation of intracellular Ca²⁺ levels. Unlike cell injury, calmodulin antagonists, nicardipine, and BAPTA-AM prevented the 7-ketocholesterol-induced elevations of intracellular Ca²⁺ levels. The results show that the cytotoxicity of 7-ketocholesterol seems to be modulated by calmodulin rather than changes in intracellular Ca²⁺ levels. Calmodulin antagonists may prevent the cytotoxicity of 7-ketocholesterol by suppressing the mitochondrial permeability transition formation, which is associated with the increased formation of reactive oxygen species and the depletion of GSH.     

Gastrodin Inhibits Glutamate-Induced Apoptosis of PC12 Cells via Inhibition of CaMKII/ASK-1/p38 MAPK/p53 Signaling Cascade

Previous research demonstrated that glutamate induces neuronal injury partially by increasing intracellular Ca²⁺ concentrations ([Ca²⁺]_i), and inducing oxidative stress, leading to a neurodegenerative disorder. However, the mechanism of glutamate-induced injury remains elusive. Gastrodin, a major active component of the traditional herbal agent Gastrodia elata (GE) Blume, has been recognized as a potential neuroprotective drug. In the current study, a classical injury model based on glutamate-induced cell death of rat pheochromocytoma (PC12) cells was used to investigate the neuroprotective effect of gastrodin, and its potential mechanisms involved. In this paper, the presence of gastrodin inhibits glutamate-induced oxidative stress as measured by the formation of reactive oxygen species (ROS), the level of malondialdehyde (MDA), mitochondrial membrane potential (MMP), and superoxide dismutase (SOD); gastrodin also prevents glutamate-induced [Ca²⁺]_i influx, blocks the activation of the calmodulin-dependent kinase II (CaMKII) and the apoptosis signaling-regulating kinase-1 (ASK-1), inhibits phosphorylation of p38 mitogen-activated kinase (MAPK). Additionally, gastrodin blocked the expression of p53 phosphorylation, caspase-3 and cytochrome C, reduced bax/bcl-2 ratio induced by glutamate in PC12 cells. All these findings indicate that gastrodin protects PC12 cells from the apoptosis induced by glutamate through a new mechanism of the CaMKII/ASK-1/p38 MAPK/p53-signaling pathway.     

Nanosecond Electric Pulses: A Novel Stimulus for Triggering Ca<Superscript>2+</Superscript> Influx into Chromaffin Cells Via Voltage-Gated Ca<Superscript>2+</Superscript> Channels

Exposing bovine chromaffin cells to a single 5 ns, high-voltage (5 MV/m) electric pulse stimulates Ca²⁺ entry into the cells via L-type voltage-gated Ca²⁺ channels (VGCC), resulting in the release of catecholamine. In this study, fluorescence imaging was used to monitor nanosecond pulse-induced effects on intracellular Ca²⁺ level ([Ca²⁺]_i) to investigate the contribution of other types of VGCCs expressed in these cells in mediating Ca²⁺ entry. ω-Conotoxin GVIA and ω-agatoxin IVA, antagonists of N-type and P/Q-type VGCCs, respectively, reduced the magnitude of the rise in [Ca²⁺]_i elicited by a 5 ns pulse. ω-conotoxin MVIIC, which blocks N- and P/Q-type VGCCs, had a similar effect. Blocking L-, N-, and P\Q-type channels simultaneously with a cocktail of VGCC inhibitors abolished the pulse-induced [Ca²⁺]_i response of the cells, suggesting Ca²⁺ influx occurs only via VGCCs. Lowering extracellular K⁺ concentration from 5 to 2 mM or pulsing cells in Na⁺-free medium suppressed the pulse-induced rise in [Ca²⁺]_i in the majority of cells. Thus, both membrane potential and Na⁺ entry appear to play a role in the mechanism by which nanoelectropulses evoke Ca²⁺ influx. However, activation of voltage-gated Na⁺ channels (VGSC) is not involved since tetrodotoxin (TTX) failed to block the pulse-induced rise in [Ca²⁺]_i. These findings demonstrate that a single electric pulse of only 5 ns duration serves as a novel stimulus to open multiple types of VGCCs in chromaffin cells in a manner involving Na⁺ transport across the plasma membrane. Whether Na⁺ transport occurs via non-selective cation channels and/or through lipid nanopores remains to be determined.     

Neuronal calcium signaling via store-operated channels in health and disease

Store-operated calcium entry (SOCE) is the flow of calcium ions (Ca²⁺) into cells in response to the depletion of intracellular Ca²⁺ stores that reside predominantly in the endoplasmic reticulum (ER). The role of SOCE has been relatively well understood for non-excitable cells. It is mediated mostly by the ER Ca²⁺ sensor STIM1 and plasma membrane Ca²⁺ channel Orai1 and serves to sustain Ca²⁺ signaling and refill ER Ca²⁺ stores. In contrast, because of the complexity of Ca²⁺ influx mechanisms that are present in excitable cells, our knowledge about the function of neuronal SOCE (nSOCE) is still nascent. This review summarizes the available data on the molecular components of nSOCE and their relevance to neuronal signaling. We also present evidence of disturbances of nSOCE in neurodegenerative diseases (namely Alzheimer’s disease, Huntington’s disease, and Parkinson’s disease) and traumatic brain injury. The emerging important role of nSOCE in neuronal physiology and pathology makes it a possible clinical target.     

Mitochondrial Ca<Superscript>2+</Superscript> uptake pathways

Calcium (Ca²⁺) plays diverse roles in all living organisms ranging from bacteria to humans. It is a structural element for bones, an essential mediator of excitation-contraction coupling, and a universal second messenger in the regulation of ion channel, enzyme and gene expression activities. In mitochondria, Ca²⁺ is crucial for the control of energy production and cellular responses to metabolic stress. Ca²⁺ uptake by the mitochondria occurs by the uniporter mechanism. The Mitochondrial Ca2+ Uniporter (MCU) protein has recently been identified as a core component responsible for mitochondrial Ca²⁺ uptake. MCU knockout (MCU KO) studies have identified a number of important roles played by this high capacity uptake pathway. Interestingly, this work has also shown that MCU-mediated Ca²⁺ uptake is not essential for vital cell functions such as muscle contraction, energy metabolism and neurotransmission. Although mitochondrial Ca²⁺ uptake was markedly reduced, MCU KO mitochondria still contained low but detectable levels of Ca²⁺. In view of the fundamental importance of Ca²⁺ for basic cell signalling, this finding suggests the existence of other currently unrecognized pathways for Ca²⁺ entry. We review the experimental evidence for the existence of alternative Ca²⁺ influx mechanisms and propose how these mechanisms may play an integral role in mitochondrial Ca²⁺ signalling.     

Protective effect of leptin-mediated caveolin-1 expression on neurons after spinal cord injury

Spinal cord injury (SCI) causes long-term disability and has no effective clinical treatment. After SCI, extracellular adenosine triphosphate (ATP) leads to an influx of extracellular Ca²⁺, and this Ca²⁺ overload causes neuronal toxicosis and apoptosis. The biological functions of leptin have been widely investigated in the central nervous system. In this study, we discovered that the administration of leptin could improve locomotor recovery following SCI. The aim of this study was to determine the neuroprotective mechanism of leptin in vivo and in vitro. The neuronal apoptosis and Ca²⁺ imaging signal induced by ATP were suppressed by leptin, due to elevated caveolin-1 expression. In vivo two-photon observations revealed that leptin reduced the neuronal Ca²⁺ imaging signal in the exposed spinal cords of live Thy1-YFP mice. In conclusion, leptin promotes locomotor functional recovery and suppresses neuronal impairment after SCI, suggesting that leptin has a promising clinical therapeutic value for treatment of SCI.     

Intracellular Free Calcium Dynamics in Stretch-Injured Astrocytes

Abstract: We have previously developed an in vitro model for traumatic brain injury that simulates a major component of in vivo trauma, that being tissue strain or stretch. We have validated our model by demonstrating that it produces many of the posttraumatic responses observed in vivo. Sustained elevation of the intracellular free calcium concentration ([Ca²⁺]_i) has been hypothesized to be a primary biochemical mechanism inducing cell dysfunction after trauma. In the present report, we have examined this hypothesis in astrocytes using our in vitro injury model and fura-2 microphotometry. Our results indicate that astrocyte [Ca²⁺]_i is rapidly elevated after stretch injury, the magnitude of which is proportional to the degree of injury. However, the injury-induced [Ca²⁺]_i elevation is not sustained and returns to near-basal levels by 15 min postinjury and to basal levels between 3 and 24 h after injury. Although basal [Ca²⁺]_i returns to normal after injury, we have identified persistent injury-induced alterations in calcium-mediated signal transduction pathways. We report here, for the first time, that traumatic stretch injury causes release of calcium from inositol trisphosphate-sensitive intracellular calcium stores and may uncouple the stores from participation in metabotropic glutamate receptor-mediated signal transduction events. We found that for a prolonged period after trauma astrocytes no longer respond to thapsigargin, glutamate, or the inositol trisphosphate-linked metabotropic glutamate receptor agonist trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid with an elevation in [Ca²⁺]_i. We hypothesize that changes in calcium-mediated signaling pathways, rather than an absolute elevation in [Ca²⁺]_i, is responsible for some of the pathological consequences of traumatic brain injury.     

Fibroblast growth factor 2 induces apoptosis in the early primary culture of rat cortical neurons

In the central nervous system, fibroblast growth factor 2 (FGF2) is known to have important functions in cell survival and differentiation. In addition to its roles as a neurotrophic factor, we found that FGF2 caused cell death in the early primary culture of cortical neurons. FGF2-induced neuronal cell death showed apoptotic characters, e.g., chromatin condensation and DNA fragmentation. The ultrastructural morphology of FGF2-treated neurons indicated apoptotic features such as progressive cell shrinkage, blebbing of the plasma membrane, loss of cytosolic organelles, clumping of chromatin, and fragmentation of DNA. Tyrosine kinase inhibitors significantly rescued neurons from FGF2-induced apoptosis. FGF2 potentiated a marked influx of Ca²⁺ into neurons before apoptosis. Both a calcium chelator and L-type voltage-sensitive Ca²⁺ channel (L-VSCC) blockers attenuated FGF2-induced apoptosis, whereas other blockers of VSCCs such as N-type and P/Q-types did not. Blockers of L-VSCCs significantly suppressed FGF2-enhanced Ca²⁺ influx into neurons. Moreover, FGF2 also generated reactive oxygen species (ROS) before apoptosis. Radical scavengers reduced not only the FGF2-generated ROS, but also the FGF2-induced Ca²⁺ influx and apoptosis. In conclusion, we demonstrated that FGF2 caused apoptosis via L-VSCCs in the early neuronal culture.     

Down-Modulation of Ca<Superscript>2+</Superscript> Channels by Endogenously Released ATP and Opioids: from the Isolated Chromaffin Cell to the Slice of Adrenal Medullae

Modifications in Ca²⁺ influx may lead to profound changes in the cell activity associated with Ca²⁺-dependent processes, from muscle contraction and neurotransmitter release to calcium-mediated cell death. Therefore, calcium entry into the cell requires fine regulation. In this context, understanding of the modulation of voltage-dependent Ca²⁺ channels seems to be critical. The modulatory process results in the enhancement or decrement of calcium influx that may regulate the local and global cytosolic Ca²⁺ concentrations. Here, we summarize the well-established data on this matter described in isolated chromaffin cells by our laboratory and others, and the new results we have obtained in a more physiological preparation: freshly isolated slices of mouse adrenal medullae.     

The mechanism of neuroprotection by positive modulation of Ca<Superscript>2+</Superscript>-activated K<Superscript>+</Superscript> channels of cerebellar neurons in primary culture

Here we show that positive modulators (CyPPA and NS309) of Ca²⁺-activated K⁺ channels of small (SK) and intermediate (IK) conductances in cerebellar neurons decrease glutamate-evoked Ca²⁺ entry into neurons independently on the presence of Mg²⁺ in extracellular media. An analysis of neuronal viability after long-term (240 min) glutamate treatments demonstrated neuroprotective action of CyPPA and NS309. Extracellular Mg²⁺ did not protect neurons from apoptosis during prolonged treatment with glutamate. Activation of SK and IK channels results in local membrane hyperpolarization, which enhances Mg²⁺ block of NMDA receptors and reduces activation of voltage-dependent Ca²⁺ channels, which can explain neuroprotection caused by CyPPA or NS309. The obtained results reveal an important role Ca²⁺-activated K⁺ channels of small and intermediate conductance in the regulation of Ca²⁺ entry into cerebellar neurons via NMDA receptors and voltage-gated Ca²⁺ channels.     

Life after the birth of the mitochondrial Na<Superscript>+</Superscript>/Ca<Superscript>2+</Superscript> exchanger,NCLX

Powered by the mitochondrial membrane potential, Ca²⁺ permeates the mitochondria via a Ca²⁺ channel termed Ca²⁺ uniporter and is pumped out by a Na⁺/Ca²⁺ exchanger, both of which are located on the inner mitochondrial membrane. Mitochondrial Ca²⁺ transients are critical for metabolic activity and regulating global Ca²⁺ responses. On the other hand, failure to control mitochondrial Ca²⁺ is a hallmark of ischemic and neurodegenerative diseases. Despite their importance, identifying the uniporter and exchanger remains elusive and their inhibitors are non-specific. This review will focus on the mitochondrial exchanger, initially describing how it was molecularly identified and linked to a novel member of the Na⁺/Ca²⁺ exchanger superfamily termed NCLX. Molecular control of NCLX expression provides a selective tool to determine its physiological role in a variety of cell types. In lymphocytes, NCLX is essential for refilling the endoplasmic reticulum Ca²⁺ stores required for antigendependent signaling. Communication of NCLX with the store-operated channel in astroglia controls Ca²⁺ influx and thereby neuro-transmitter release and cell proliferation. The refilling of the Ca²⁺ stores in the sarcoplasmic reticulum, which is controlled by NCLX, determines the frequency of action potential and Ca²⁺ transients in cardiomyocytes. NCLX is emerging as a hub for integrating glucose-dependent Na⁺ and Ca²⁺ signaling in pancreatic β cells, and the specific molecular control of NCLX expression resolved the controversy regarding its role in neurons and β cells. Future studies on an NCLX knockdown mouse model and identification of human NCLX mutations are expected to determine the role of mitochondrial Ca²⁺ efflux in organ activity and whether NCLX inactivation is linked to ischemic and/or neurodegenerative syndromes. Structure-function analysis and protein analysis will identify the NCLX mode of regulation and its partners in the inner membrane of the mitochondria.     

Mechanisms of Extracellular NO and Ca<Superscript>2+</Superscript> Regulating the Growth of Wheat Seedling Roots

Our previous studies suggested the cross talk of nitric oxide (NO) with Ca²⁺ in regulating stomatal movement. However, its mechanism of action is not well defined in plant roots. In this study, sodium nitroprusside (SNP, a NO donor) showed an inhibitory effect on the growth of wheat seedling roots in a dose-dependent manner, which was alleviated through reducing extracellular Ca²⁺ concentration. Analyzing the content of Ca²⁺ and K⁺ in wheat seedling roots showed that SNP significantly promoted Ca²⁺ accumulation and inhibited K⁺ accumulation at a higher concentration of extracellular Ca²⁺, but SNP promoted K⁺ accumulation in the absence of extracellular Ca²⁺. To gain further insights into Ca²⁺ function in the NO-regulated growth of wheat seedling roots, we conducted the patch-clamped protoplasts of wheat seedling roots in a whole cell configuration. In the absence of extracellular Ca²⁺, NO activated inward-rectifying K⁺ channels, but had little effects on outward-rectifying K⁺ channels. In the presence of 2 mmol L⁻¹ CaCl₂ in the bath solution, NO significantly activated outward-rectifying K⁺ channels, which was partially alleviated by LaCl₃ (a Ca²⁺ channel inhibitor). In contrast, 2 mmol L⁻¹ CaCl₂ alone had little effect on inward or outward-rectifying K⁺ channels. Thus, NO inhibits the growth of wheat seedling roots likely by promoting extracellular Ca²⁺ influx excessively. The increase in cytosolic Ca²⁺ appears to inhibit K⁺ influx, promotes K⁺ outflux across the plasma membrane, and finally reduces the content of K⁺ in root cells.