Gene Transfer of Calbindin D28k cDNA via Herpes Simplex Virus Amplicon Vector Decreases Cytoplasmic Calcium Ion Response and Enhances Neuronal Survival Following Glutamatergic Challenge but Not Following Cyanide

Abstract: Excitatory amino acid overstimulation of neurons can lead to a marked rise in cytoplasmic Ca²⁺ concentration ([Ca²⁺])_i) and be followed by neuron death from hours to days later. If the rise in [Ca²⁺]_i is prevented, either by removing Ca²⁺ from the extracellular environment or by placing Ca²⁺ chelators in the cytosol of the stimulated cells, the neurotoxicity associated with excitotoxins can be ameliorated. We have recently shown that neurons infected with a herpes simplex virus amplicon vector expressing cDNA for calbindin D_28k responded to hypoglycemia with decreased [Ca²⁺]_i and increased survival relative to controls. We now report that vector-infected neurons respond to glutamatergic insults with lower [Ca²⁺]_i than controls and with increased survival. Infected neurons exposed to sodium cyanide did not respond with lower [Ca²⁺]_i than controls, nor did they demonstrate increased survival postinsult. We examine these results in light of our earlier report and in the context of the potential of vectors like this for neuronal gene therapy.     

Ca2+ and Reactive Oxygen Species in Staurosporine-Induced Neuronal Apoptosis

Abstract: Staurosporine (0.03–0.5 µ M ) induced a dose-dependent, apoptotic degeneration in cultured rat hippocampal neurons that was sensitive to 24-h pretreatments with the protein synthesis inhibitor cycloheximide (1 µ M ) or the cell cycle inhibitor mimosine (100 µ M ). To investigate the role of Ca²⁺ and reactive oxygen species in staurosporine-induced neuronal apoptosis, we overexpressed calbindin D_28K, a Ca²⁺ binding protein, and Cu/Zn superoxide dismutase, an antioxidative enzyme, in the hippocampal neurons using adenovirus-mediated gene transfer. Infection of the cultures with the recombinant adenoviruses (100 multiplicity of infection) resulted in a stable expression of the respective proteins assessed 48 h later. Overexpression of both calbindin D_28K and Cu/Zn superoxide dismutase significantly reduced staurosporine neurotoxicity compared with control cultures infected with a β-galactosidase overexpressing adenovirus. Staurosporine-induced neuronal apoptosis was also significantly reduced when the culture medium was supplemented with 10 or 30 m M K⁺, suggesting that Ca²⁺ influx via voltage-sensitive Ca²⁺ channels reduces this apoptotic cell death. In contrast, neither the glutamate receptor agonist NMDA (1–10 µ M ) nor the NMDA receptor antagonist dizocilpine (MK-801; 1 µ M ) was able to reduce staurosporine neurotoxicity. Cultures treated with the antioxidants U-74500A (1–10 µ M ) and N -acetylcysteine (100 µ M ) also demonstrated reduced staurosporine neurotoxicity. These results suggest a fundamental role for both Ca²⁺ and reactive oxygen species in staurosporine-induced neuronal apoptosis.     

α-Synuclein modulation of Ca2+ signaling in human neuroblastoma (SH-SY5Y) cells

Parkinson's disease (PD) is characterized in part by the presence of α-synuclein (α-syn) rich intracellular inclusions (Lewy bodies). Mutations and multiplication of the α-synuclein gene ( SNCA ) are associated with familial PD. Since Ca²⁺ dyshomeostasis may play an important role in the pathogenesis of PD, we used fluorimetry in fura-2 loaded SH-SY5Y cells to monitor Ca²⁺ homeostasis in cells stably transfected with either wild-type α-syn, the A53T mutant form, the S129D phosphomimetic mutant or with empty vector (which served as control). Voltage-gated Ca²⁺ influx evoked by exposure of cells to 50 mM K⁺ was enhanced in cells expressing all three forms of α-syn, an effect which was due specifically to increased Ca²⁺ entry via L-type Ca²⁺ channels. Mobilization of Ca²⁺ by muscarine was not strikingly modified by any of the α-syn forms, but they all reduced capacitative Ca²⁺ entry following store depletion caused either by muscarine or thapsigargin. Emptying of stores with cyclopiazonic acid caused similar rises of [Ca²⁺]_i in all cells tested (with the exception of the S129D mutant), and mitochondrial Ca²⁺ content was unaffected by any form of α-synuclein. However, only WT α-syn transfected cells displayed significantly impaired viability. Our findings suggest that α-syn regulates Ca²⁺ entry pathways and, consequently, that abnormal α-syn levels may promote neuronal damage through dysregulation of Ca²⁺ homeostasis.     

Specific vulnerability of mouse spinal cord motoneurons to membrane depolarization

Intracellular calcium (Ca²⁺) concentration determines neuronal dependence on neurotrophic factors (NTFs) and susceptibility to cell death. Ca²⁺ overload induces neuronal death and the consequences are thought to be a probable cause of motoneuron (MN) degeneration in neurodegenerative diseases. In the present study, we show that membrane depolarization with elevated extracellular potassium (K⁺) was toxic to cultured embryonic mouse spinal cord MNs even in the presence of NTFs. Membrane depolarization induced an intracellular Ca²⁺ increase. Depolarization-induced toxicity and increased intracellular Ca²⁺ were blocked by treatment with antagonists to some of the voltage-gated Ca²⁺ channels (VGCCs), indicating that Ca²⁺ influx through these channels contributed to the toxic effect of depolarization. Ca²⁺ activates the calpains, cysteine proteases that degrade a variety of substrates, causing cell death. We investigated the functional involvement of calpain using a calpain inhibitor and calpain gene silencing. Pre-treatment of MNs with calpeptin (a cell-permeable calpain inhibitor) rescued MNs survival; calpain RNA interference had the same protective effect, indicating that endogenous calpain contributes to the cell death caused by membrane depolarization. These findings suggest that MNs are especially vulnerable to extracellular K⁺ concentration, which induces cell death by causing both intracellular Ca²⁺ increase and calpain activation.     

AMPA Receptor-Mediated Excitotoxicity in Human NT2-N Neurons Results from Loss of Intracellular Ca2+ Homeostasis Following Marked Elevation of Intracellular Na+

Abstract: Human NT2-N neurons express Ca²⁺-permeable α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid glutamate receptors (AMPA-GluRs) and become vulnerable to excitotoxicity when AMPA-GluR desensitization is blocked with cyclothiazide. Although the initial increase in intracellular Ca²⁺ levels ([Ca²⁺]_i) was 1.9-fold greater in the presence than in the absence of cyclothiazide, Ca²⁺ entry via AMPA-GluRs in an early phase of the exposure was not necessary to elicit excitotoxicity in these neurons. Rather, subsequent necrosis was caused by a >40-fold rise in [Na⁺]_i, which induced a delayed [Ca²⁺]_i rise. Transfer of the neurons to a 5 m M Na⁺ medium after AMPA-GluR activation accelerated the delayed [Ca²⁺]_i rise and intensified excitotoxicity. Low-Na⁺ medium-enhanced excitotoxicity was partially blocked by amiloride or dizocilpine (MK-801), and completely blocked by removal of extracellular Ca²⁺, suggesting that Ca²⁺ entry by reverse operation of Na⁺/Ca²⁺ exchangers and via NMDA glutamate receptors was responsible for the neuronal death after excessive Na⁺ loading. Our results serve to emphasize the central role of neuronal Na⁺ loading in AMPA-GluR-mediated excitotoxicity in human neurons.     

Endoplasmic reticulum Ca(2+) homeostasis and neuronal death

The endoplasmic reticulum (ER) is a universal signalling organelle, which regulates a wide range of neuronal functional responses. Calcium release from the ER underlies various forms of intracellular Ca²⁺ signalling by either amplifying Ca²⁺ entry through voltage-gated Ca²⁺ channels by Ca²⁺-induced Ca²⁺ release (CICR) or by producing local or global cytosolic calcium fluctuations following stimulation of metabotropic receptors through inositol-1,4,5-trisphosphate-induced Ca²⁺ release (IICR). The ER Ca²⁺ store emerges as a single interconnected pool, thus allowing for a long-range Ca²⁺ signalling via intra-ER tunnels. The fluctuations of intra-ER free Ca²⁺ concentration regulate the activity of numerous ER resident proteins responsible for post-translational protein folding and modification. Disruption of ER Ca²⁺ homeostasis results in the developing of ER stress response, which in turn controls neuronal survival. Altered ER Ca²⁺ handling may be involved in pathogenesis of various, neurodegenerative diseases including brain ischemia and Alzheimer dementia.     

NMDA Receptor-Mediated Neurotoxicity: A Paradoxical Requirement for Extracellular Mg2+ in Na+/Ca2+-Free Solutions in Rat Cortical Neurons In Vitro

Abstract: Accumulation of intracellular Ca²⁺ is known to be critically important for the expression of NMDA receptor-mediated glutamate neurotoxicity. We have observed, however, that glutamate can also increase the neuronal intracellular Mg²⁺ concentration on activation of NMDA receptors. Here, we used conditions that elevate intracellular Mg²⁺ content independently of Ca²⁺ to investigate the potential role of Mg²⁺ in excitotoxicity in rat cortical neurons in vitro. In Ca²⁺-free solutions in which the Na⁺ was replaced by N -methyl- d -glucamine or Tris (but not choline), which also contained 9 m M Mg²⁺, exposure to 100 µ M glutamate or 200 µ M NMDA for 20 min produced delayed neuronal cell death. Neurotoxicity was correlated to the extracellular Mg²⁺ concentration and could be blocked by addition of NMDA receptor antagonists during, but not immediately following, agonist exposure. Finally, we observed that rat cortical neurons grown under different serum conditions develop an altered sensitivity to Mg²⁺-dependent NMDA receptor-mediated toxicity. Thus, the increase in intracellular Mg²⁺ concentration following NMDA receptor stimulation may be an underestimated component critical for the expression of certain forms of excitotoxic injury.     

Hypoxia Enhances [3H]Noradrenaline Release Evoked by Nicotinic Receptor Activation from the Human Neuroblastoma SH-SY5Y

Abstract: We have used the human sympathetic neuronal line SH-SY5Y to investigate the effects of hypoxia on noradrenaline (NA) release evoked by either raised [K⁺]_o (100 m M ) or the nicotinic acetylcholine receptor (nAChR) agonist dimethylphenylpiperazinium iodide (DMPP). NA release was monitored by loading cells with [³H]NA and collecting effluent fractions from perfused cells kept in a sealed perifusion chamber. Cells were challenged twice with either stimulus and release was expressed as that evoked by the second challenge as a fraction of that evoked by the first. K⁺-evoked release was unaffected by hypoxia (P o ₂≅ 30–38 mm Hg), but release evoked by DMPP was significantly increased. For both stimuli, replacement of Ca²⁺_o with 1 m M EGTA abolished NA release. K⁺-evoked release was also dramatically reduced in the presence of 200 µ M Cd²⁺ to block voltage-gated Ca²⁺ channels, but DMPP-evoked release was less affected. In hypoxia, DMPP-evoked Cd²⁺-resistant NA release was dramatically increased. Our findings indicate that hypoxia increases NA release evoked from SH-SY5Y cells in response to nAChR activation by increasing Ca²⁺ influx through the nAChR pore, or by activating an unidentified Cd²⁺-resistant Ca²⁺-influx pathway. As acetylcholine is the endogenous transmitter at sympathetic ganglia, these findings may have important implications for sympathetic activity under hypoxic conditions.     

Glutamate Receptor-Mediated Calcium Surges in Neurons Derived from P19 Cells

Abstract: Retinoic acid-treated murine P19 embryonal carcinoma cells differentiate into cells with neuronal morphology that display typical neuronal markers. In this study, the presence of glutamate receptors linked to Ca²⁺-signaling mechanisms on these neurons was demonstrated by testing the effects of glutamate agonists and antagonists on the intracellular calcium ion concentration ([Ca²⁺]_i). Glutamate (1 m M ) induced either sustained or transient increases in [Ca²⁺]_i. The sustained glutamate-induced increase in [Ca²⁺]_i was mimicked by NMDA (40 µ M ). The NMDA-triggered [Ca²⁺]_i response was abolished by incubating the cells in Ca²⁺-free medium or by pretreating them with Mg²⁺ (2 m M ) or MK-801 (0.1 µ M ). These responses were unaffected by the non-NMDA antagonist CNQX (10 µ M ), but they required glycine (3–30 µ M ). Kainate (40 µ M ) and AMPA (40 µ M ) did not affect [Ca²⁺]_i. Without external Ca²⁺, glutamate triggered transient, sometimes oscillating, increases in [Ca²⁺]_i. These responses were mimicked by the metabotropic agonist trans -(1 S ,3 R )-1-amino-1,3-cyclopentanedicarboxylic acid (300 µ M ). These results suggest that neurons derived from P19 embryonal carcinoma cells have NMDA and metabotropic, but not AMPA/kainate receptors, which are linked to Ca²⁺-signaling mechanisms. These cells could provide a consistent and reproducible model with which to study neuronal differentiation, neurotoxicity, and glutamate receptor-signaling mechanisms.     

Nitroprusside and Cyclic GMP Stimulate Na+-Ca2+ Exchange Activity in Neuronal Preparations and Cultured Rat Astrocytes

Abstract: The effects of nitric oxide (NO)-generating agents on ⁴⁵Ca²⁺ uptake in rat brain slices and cultured rat astrocytes were studied in the presence of monensin, which is considered to drive the Na⁺-Ca²⁺ exchanger in the reverse mode. Sodium nitroprusside (SNP) at >10 µ M increased monensin-stimulated Ca²⁺ uptake in the slices, although it did not affect high K⁺-stimulated Ca²⁺ uptake. Another NO donor, 3-morpholinosydnonimine, was effective. The effect of SNP was antagonized by hemoglobin (50 µ M ), a NO scavenger, and mimicked by 8-bromo-cyclic GMP (100 µ M ). In rat brain synaptosomes, SNP increased monensin-stimulated Ca²⁺ uptake, but it did not affect high K⁺-stimulated Ca²⁺ uptake. 8-Bromocyclic GMP, but not SNP, increased Na⁺-dependent Ca²⁺ uptake significantly in synaptic membrane vesicles in the absence of monensin. In cultured rat astrocytes, SNP and 8-bromo-cyclic GMP increased Ca²⁺ uptake in the presence of ouabain and monensin, which were required for the Ca²⁺ uptake in the cells. These findings suggest that NO stimulates the Na⁺-Ca²⁺ exchanger in neuronal preparations and astrocytes in a cyclic GMP-dependent mechanism.     

Catecholamine Secretion from Bovine Adrenal Chromaffin Cells: The Role of the Na+/Ca2+ Exchanger and the Intracellular Ca2+ Pool

Abstract: The role of the Na⁺/Ca²⁺ exchanger and intracellular nonmitochondrial Ca²⁺ pool in the regulation of cytosolic free calcium concentration ([Ca²⁺]_i) during catecholamine secretion was investigated. Catecholamine secretion and [Ca²⁺]_i were simultaneously monitored in a single chromaffin cell. After high-K⁺ stimulation, control cells and cells in which the Na⁺/Ca²⁺ exchange activity was inhibited showed similar rates of [Ca²⁺]_i elevation. However, the recovery of [Ca²⁺]_i to resting levels was slower in the inhibited cells. Inhibition of the exchanger increased the total catecholamine secretion by prolonging the secretion. Inhibition of the Ca²⁺ pump of the intracellular Ca²⁺ pool with thapsigargin caused a significant delay in the recovery of [Ca²⁺]_i and greatly enhanced the secretory events. These data suggest that both the Na⁺/Ca²⁺ exchanger and the thapsigargin-sensitive Ca²⁺ pool are important in the regulation of [Ca²⁺]_i and, by modulating the time course of secretion, are important in determining the extent of secretion.     

Involvement of Calcineurin in Ca2+ Paradox-Like Injury of Cultured Rat Astrocytes

Abstract: The Ca²⁺/calmodulin-dependent phosphatase calcineurin may have physiological and pathological roles in neurons, but little is known about the roles of the enzyme in glial cells. We have previously reported that reperfusion of cultured astrocytes in Ca²⁺-containing medium after exposure to Ca²⁺-free medium caused Ca²⁺ influx followed by delayed cell death. In this study, we examined if calcineurin is involved in this Ca²⁺-mediated astrocytic injury. FK506, an inhibitor of calcineurin, protected cultured rat astrocytes against paradoxical Ca²⁺ challenge-induced injury in a dose-dependent manner (10⁻¹⁰–10⁻⁸ M ). Cyclosporin A at 1 µ M mimicked the effect of FK506. Rapamycin (1 µ M ) did not affect astrocyte injury, but it blocked the protective effect of FK506. Deltamethrin (20 n M ), another calcineurin inhibitor, had a similar protective effect, whereas okadaic acid did not. FK506 affected neither paradoxical Ca²⁺ challenge-induced increase in cytosolic Ca²⁺ level nor Na⁺-Ca²⁺ exchange activity in the cells, suggesting that the calcineurin is involved in processes downstream of increased cytosolic Ca²⁺ level. Immunochemical studies showed that both calcineurin A (probably the Aβ2 isoform) and B subunits were expressed in the cells. It is concluded that calcineurin is present in cultured astrocytes and it has a pathological role in the cells.     

Nitric Oxide Disrupts Ca2+ Homeostasis in Hippocampal Neurons

Abstract: Nitric oxide has been recognized in recent years as an important mediator of neuronal toxicity, which in many cases involves alterations of the cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i). In [Ca²⁺]_i fluorimetric experiments on cultured hippocampal neurons, the nitric oxide-releasing agent S -nitrosocysteine produced a delayed rise in [Ca²⁺]_i over a 20-min exposure, which was accompanied by a progressive slowing of the kinetics of recovery from depolarization-induced [Ca²⁺]_i transients. These effects were blocked by oxyhemoglobin and by superoxide dismutase, confirming nitric oxide as the responsible agent, and suggesting that they involved peroxynitrite formation. Similar alterations of [Ca²⁺]_i homeostasis were produced by the mitochondrial ATP synthase inhibitor oligomycin, and when an ATP-regenerating system was supplied via the patch pipette in combined whole-cell patch-clamp-[Ca²⁺]_i fluorimetry experiments, S -nitrosocysteine had no effect on the resting [Ca²⁺]_i or on the recovery kinetics of [Ca²⁺]_i transients induced by direct depolarization. We conclude that prolonged exposure to nitric oxide disrupts [Ca²⁺]_i homeostasis in hippocampal neurons by impairing Ca²⁺ removal from the cytoplasm, possibly as a result of ATP depletion. The resulting persistent alterations in [Ca²⁺]_i may contribute to the delayed neurotoxicity of nitric oxide.     

Role of Ca2+ in Differentiation Mediated by Nerve Growth Factor and Dibutyryl Cyclic AMP in PC12 Cells

Abstract: Nerve growth factor (NGF) and dibutyryl cyclic AMP (dbcAMP) have synergistic effects on the neurite outgrowth of rat pheochromocytoma PC12 cells. The sites of interaction between NGF and dbcAMP have been studied extensively; however, the role of Ca²⁺ in differentiation induced by the two agents remains unclear. To understand whether intracellular Ca²⁺ is involved in the differentiation induced by the two agents, PC12 cells were treated with NGF, dbcAMP, or NGF plus dbcAMP for 2 days, and then effects on neurite outgrowth, ATP-induced Ca²⁺ influx, and Ca²⁺ mobilization from intracellular Ca²⁺ pools were examined. NGF or dbcAMP alone enhanced neurite outgrowth and Ca²⁺ accumulation by nonmitochondrial Ca²⁺ pools or the thapsigargin (TG)-sensitive Ca²⁺ pool. The dbcAMP acted synergistically with NGF to increase neurite outgrowth and to enlarge the TG-sensitive Ca²⁺ pool. The synergistic effect occurred within the first hour of treatment with dbcAMP plus NGF. On the other hand, dbcAMP abolished NGF's ability to enhance ATP-induced influx of extracellular Ca²⁺. Therefore, NGF and dbcAMP induced different effects on Ca²⁺ signaling pathways through two different but interacting pathways. In PC12 cells pretreated with TG to deplete the TG-sensitive Ca²⁺ pool, the dbcAMP- or dbcAMP plus NGF-mediated neurite outgrowth was significantly inhibited, whereas NGF-mediated neurite outgrowth was not affected by TG pretreatment. Our results suggest that the intracellular nonmitochondrial Ca²⁺ pools were changed in the differentiation process and were necessary for the synergistic effect of NGF and dbcAMP.     

Contributions of Ca2+ and Zn2+ to spreading depression-like events and neuronal injury

The phenomenon of spreading depression (SD) involves waves of profound neuronal and glial depolarization that spread throughout brain tissue. Under many conditions, tissue recovers full function after SD has occurred, but SD-like events are also associated with spread of injury following ischemia or trauma. Initial large cytosolic Ca²⁺ increases accompany all forms of SD, but persistently elevated Ca²⁺ loading is likely responsible for neuronal injury following SD in tissues where metabolic capacity is insufficient to restore ionic gradients. Ca²⁺ channels are also involved in the propagation of SD, but the channel subtypes and cation fluxes differ significantly when SD is triggered by different types of stimuli. Ca²⁺ influx via P/Q type channels is important for SD generated by localized application of high K⁺ solutions. In contrast, SD-like events recorded in in vitro ischemia models are not usually prevented by Ca²⁺ removal, but under some conditions, Zn²⁺ influx via L-type channels contributes to SD initiation. This review addresses different roles of Ca²⁺ in the initiation and consequences of SD, and discusses recent evidence that selective chelation of Zn²⁺ can be sufficient to prevent SD under circumstances that may have relevance for ischemic injury.     

Agents that Promote Protein Phosphorylation Inhibit the Activity of the Na+/Ca2+ Exchanger and Prolong Ca2+ Transients in Bovine Chromaffin Cells

Abstract: The Na⁺/Ca²⁺ exchanger is an important element in the maintenance of calcium homeostasis in bovine chromaffin cells. The Na⁺/Ca²⁺ exchanger from other cell types has been extensively studied, but little is known about its regulation in the cell. We have investigated the role of reversible protein phosphorylation in the activity of the Na⁺/Ca²⁺ exchanger of these cells. Cells treated with 1 m M dibutyryl cyclic AMP (dbcAMP), 1 µ M phorbol 12,13-dibutyrate, 1 µ M okadaic acid, or 100 n M calyculin A showed lowered Na⁺/Ca²⁺ exchange activity and prolonged cytosolic Ca²⁺ transients caused by depolarization. A combination of 10 n M okadaic acid and 1 µ M dbcAMP synergistically inhibited Na⁺/Ca²⁺ exchange activity. Conversely, 50 µ M 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a protein kinase inhibitor, enhanced Na⁺/Ca²⁺ exchange activity. Moreover, we used cyclic AMP-dependent protein kinase and calcium phospholipid-dependent protein kinase catalytic subunits to phosphorylate isolated membrane vesicles and found that the Na⁺/Ca²⁺ exchange activity was inhibited by this treatment. These results indicate that reversible protein phosphorylation modulates the activity of the Na⁺/Ca²⁺ exchanger and suggest that modulation of the exchanger may play a role in the regulation of secretion.     

Involvement of Intracellular Stores in the Ca2+ Responses to N-Methyl-D-Aspartate and Depolarization in Cerebellar Granule Cells

Abstract: The [Ca²⁺]₁ of cerebellar granule cells can be increased in a biphasic manner by addition of NMDA or by depolarization (induced by elevating the extracellular K⁺ level), which both activate Ca²⁺ influx. The possibility that these stimuli activate Ca²⁺-induced Ca²⁺ release was investigated using granule cells loaded with fura 2-AM. Dantrolene, perfused onto groups of cells during the sustained plateau phase of the [Ca²⁺]₁ response to K⁺ or NMDA, was found to reduce the response to both agents in a concentration-dependent manner. Preincubation with thapsigargm (10 μ M ) substantially reduced the plateau phase of the [Ca²⁺], response to K⁺ and both the peak and plateau phases of the NMDA response. Preincubation with ryanodine (10 μ M ) also reduced both the K⁺-evoked plateau response and both phases of the NMDA response. Neither had a consistent effect on the peak response to K⁺. The effects of thapsigargin and ryanodine on the NMDA response were partially additive. These results demonstrate that in cerebellar granule cells a major component of both K⁺- and NMDA-induced elevation of [Ca²⁺]₁ appears to be due to release from intracellular stores. The partial additivity of the effects of thapsigargin and ryanodine suggests that these agents affect two overlapping but nonidentical Ca²⁺ pools.     

Involvement of Ca2+ and cGMP in bacterial taxis

Abstract The level of cGMP in a suspension of Escherichia coli cells increased transiently upon the addition of chemoattractants. Ca²⁺ (1 mM), but not Mg²⁺, produced constant tumbling of cells in the presence of the ionophore A23187. The effect was observed either in stationary-state cells, or in a logarithmic culture treated with EDTA to increase permeability by A23187. Under the same conditions, Ca²⁺ decreased the cytoplasmic level of cGMP. In Phormidium uncinatum , rapid ⁴⁵Ca²⁺ accumulation followed a light-dark stimulus, or the addition of tetramethylquinone (TMQ), a chemorepellent. La³⁺, which increases the reversal rate, also enhanced the level of cytoplasmic Ca²⁺, presumably by blocking the outward Ca²⁺ flux. In both E. coli and P. uncinatum Ca²⁺ inhibited methylaccepting chemotaxis protein (MCP) methylation. It is concluded that cGMP and Ca²⁺ are secondary messengers in taxis information-processing.     

Arginine-Modulated Receptor-Activated Calcium Influx via a NO/Cyclic GMP Pathway in Human SK-N-SH Neuroblastoma Cells

Abstract: The effects of arginine on calcium mobilization in human SK-N-SH neuroblastoma cells were examined. It was found that arginine potentiated an increase in carbachol-induced Ca²⁺ from the external Ca²⁺ influx as opposed to an internal Ca²⁺ release from intracellular pools. The potentiation effect of arginine on carbachol-induced calcium mobilization was mimicked by either 8-bromo cyclic GMP or sodium nitroprusside. In addition, it was found that arginine induced NO production and an increase in cyclic GMP. Moreover, arginine-induced potentiation, NO production, and cyclic GMP increases were all suppressed after the preincubation of cells with N -methyl- l -arginine or N -nitro- l -arginine, nitric oxide synthase inhibitor. It is suggested that the NO production and subsequent cyclic GMP elevation induced by arginine are responsible for the potentiation of carbachol-induced Ca²⁺ increase. Our results show the existence of a NO/cyclic GMP pathway and an interconnection of NO and Ca²⁺ signaling pathways in human SK-N-SH neuroblastoma cells. We also observed that NO, which is produced by endothelial CPAE cells, has a modulating effect on cyclic GMP elevation in human SK-N-SH neuroblastoma cells. The intercellular communication role of NO and its cell-diffusing character may also affect the regulation of nonneuronal cells in their interactions with neuronal cells.     

Involvement of intracellular Ca2+ in blue light-dependent proton pumping in guard cell protoplasts from Vicia faba

Recent studies have suggested that Ca²⁺/calmodulin (CaM) or CaM-like proteins may be involved in blue light (BL)-dependent proton pumping in guard cells. As the increase in cytosolic concentration of Ca²⁺ is required for the activation of CaM and CaM-like proteins, the origin of the Ca²⁺ was investigated by measuring BL-dependent proton pumping with various treatments using guard cell protoplasts (GCPs) from Vicia faba . BL-dependent proton pumping was affected neither by Ca²⁺ channel blockers nor by changes of Ca²⁺ concentration in the medium used for the GCPs. Addition of Ca²⁺ ionophores and an agonist to GCPs did not induce proton pumping. However, BL-dependent proton pumping was inhibited by 10 m M caffeine, which releases Ca²⁺ from the intracellular stores, and by 10 μ M 2,5-di-( tert -butyl)-1,4-benzohydroquinone (BHQ) and 10 μ M cyclopiazonic acid (CPA), inhibitors of Ca²⁺-ATPase in the sarcoplasmic and endoplasmic reticulum (ER). By contrast, the inhibitions were not observed by 10 μ M thapsigargin, an inhibitor of animal ER-type Ca²⁺-ATPase. The inhibitions by caffeine and BHQ were reversible. Light-dependent stomatal opening in the epidermis of Vicia was inhibited by caffeine, BHQ, and CPA. From these results, we conclude that the Ca²⁺ thought to be required for BL-dependent proton pumping may originate from intracellular Ca²⁺ stores, most likely from ER in guard cells, and that this origin of Ca²⁺ may generate a stimulus-specific Ca²⁺ signal for stomatal opening.