Differential Inactivation of Postsynaptic Density-Associated and Soluble Ca2+/Calmodulin-Dependent Protein Kinase II by Protein Phosphatases 1 and 2A

Abstract: Autophosphorylation of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) at Thr²⁸⁶ generates Ca²⁺-independent activity. As an initial step toward understanding CaMKII inactivation, protein phosphatase classes (PP1, PP2A, PP2B, or PP2C) responsible for dephosphorylation of Thr²⁸⁶ in rat forebrain subcellular fractions were identified using phosphatase inhibitors/activators, by fractionation using ion exchange chromatography and by immunoblotting. PP2A-like enzymes account for >70% of activity toward exogenous soluble Thr²⁸⁶-autophosphorylated CaMKII in crude cytosol, membrane, and cytoskeletal extracts; PP1 and PP2C account for the remaining activity. CaMKII is present in particulate fractions, specifically associated with postsynaptic densities (PSDs); each protein phosphatase is also present in isolated PSDs, but only PP1 is enriched during PSD isolation. When isolated PSDs dephosphorylated exogenous soluble Thr²⁸⁶-autophosphorylated CaMKII, PP2A again made the major contribution. However, CaMKII endogenous to PSDs (³²P autophosphorylated in the presence of Ca²⁺/calmodulin) was predominantly dephosphorylated by PP1. In addition, dephosphorylation of soluble and PSD-associated CaMKII in whole forebrain extracts was catalyzed predominantly by PP2A and PP1, respectively. Thus, soluble and PSD-associated forms of CaMKII appear to be dephosphorylated by distinct enzymes, suggesting that Ca²⁺-independent activity of CaMKII is differentially regulated by protein phosphatases in distinct subcellular compartments.     

Calcium/Calmodulin-Dependent Kinase II Phosphorylates Drosophila Visual Arrestin

Abstract: Light activation of rhodopsin in the Drosophila photoreceptor induces a G protein-coupled signaling cascade that results in the influx of Ca²⁺ into the photoreceptor cells. Immediately following light activation, phosphorylation of a photoreceptor-specific protein, phosrestin I, is detected. Strong sequence similarity to mammalian arrestin and electroretinograms of phosrestin mutants suggest that phosrestin I is involved in light inactivation. We are interested in identifying the protein kinase responsible for the phosphorylation of phosrestin I to link the transmembrane signaling to the light-adaptive response. Type II Ca²⁺/calmodulin-dependent kinase is one of the major classes of protein kinases that regulate cellular responses to transmembrane signals. We show here that partially purified phosrestin I kinase activity can be immunodepleted and immunodetected with antibodies to Ca²⁺/calmodulin-dependent kinase II and that the kinase activity exhibits regulatory properties that are unique to Ca²⁺/calmodulin-dependent kinase II such as Ca²⁺ independence after autophosphorylation and inhibition by synthetic peptides containing the Ca²⁺/calmodulin-dependent kinase II autoinhibitory domain. We also show that Ca²⁺/calmodulin-dependent kinase II activity is present in Drosophila eye preparations. These results are consistent with our hypothesis that Ca²⁺/calmodulin-dependent kinase II phosphorylates phosrestin I. We suggest that Ca²⁺/calmodulin-dependent kinase II plays a regulatory role in Drosophila photoreceptor light adaptation.     

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.     

A calcium and calmodulin-dependent protein kinase present in differentiating Dictyostelium discoideum

Abstract A protein kinase from Dictyostelium discoideum which phosphorylates the synthetic peptide, calmodulin-dependent protein kinase substrate (CDPKS, amino acid sequence: PLRRTLSVAA) and is stimulated by Ca²⁺/calmodulin is described. This is the first report of a protein kinase with these characteristics in D. discoideum . The enzyme was partially purified by Q-Sepharose chromatography. The protein kinase is very labile, and rapidly loses Ca²⁺/calmodulin-dependence upon standing at 4°C, even in the presence of protease inhibitors, making further purification and characterisation difficult. In the active fractions, a 55 kDa polypeptide is labelled with [γ-³² P]ATP in vitro under conditions in which intramolecular rather than intermolecular reactions are favoured. The phosphorylation of this peptide is stimulated in the presence of Ca²⁺ and calmodulin but not Ca²⁺ alone. Ca²⁺/calmodulin-dependent stimulation is inhibited in the presence of the calmodulin antagonist, trifluoperazine (TFP). It is proposed that the 55 kDa polypeptide may represent the autophosphorylated form of the enzyme.     

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.     

Calcium and plant organelles

Abstract. The role of intracellular organelles in the regulation of cytosolic Ca²⁺ levels and whether changes in these levels affect organelle metabolism is considered. We have assessed the biochemical properties of the Ca²⁺ transporting systems in mitochondrial, chloroplast and microsomal fractions. It is proposed that although all of these organelles can transport Ca²⁺ to varying extents it would appear that in some tissues at least mitochondria do not play a significant role in the maintenance of cytosolic Ca²⁺. The most important Ca²⁺ transporting systems are probably the ATP dependent Ca²⁺ extrusion across the plasma membrane and Ca²⁺ uptake by endoplasmic reticulum, as well as light driven Ca²⁺ uptake by chloroplasts. Changes in cytoplasmic [Ca²⁺] do appear to regulate the activity of NAD kinase in chloroplasts, the mitochondrial external NADH dehydrogenase and intra-mitochondrial glutamate dehydrogenase, all of which play a key role in plant cell metabolism. Since some of these enzymes are affected by primary stimuli such as light or hormones, it is concluded that Ca²⁺ may act as a second messenger mediating some of the primary responses.     

M1 Muscarinic Receptors Stimulate Rapid and Prolonged Phases of Neuronal Nitric Oxide Synthase Activity: Involvement of Different Calcium Pools

Abstract: This study shows that activation of M₁ muscarinic receptors, when coexpressed in Chinese hamster ovary (CHO)-K1 cells with neuronal nitric oxide (NO) synthase (nNOS), produces early and late phases of elevation of both intracellular Ca²⁺ concentration and nNOS activity. We examined the relationship between receptor-mediated increases in intracellular Ca²⁺ concentration and activation of nNOS over both short and long intervals using guanosine 3',5'-cyclic monophosphate (cGMP) formation as a measure of nNOS activity. The rapid phase of nNOS activation was dependent on release of Ca²⁺ from intracellular stores in both the CHO M₁/nNOS transfected cells and in neuroblastoma (N1E-115) cells, in which muscarinic receptors and nNOS are endogenously expressed. Two single point mutations in the M₁ muscarinic receptor that have previously been shown to uncouple differentially the receptor from phosphoinositide hydrolysis produced parallel attenuation of the rapid phase of nNOS activation. Characterization of the prolonged phase of nNOS activation was done using the conversion of l -[³H]arginine to l -[³H]citrulline as well as cGMP formation following stimulation of M₁ muscarinic receptors for 60 min. Both responses were dependent on influx of extracellular Ca²⁺ and were accompanied by prolonged formation of NO at functionally effective levels as late as 60 min following receptor activation. Therefore, this study demonstrates for the first time the existence of two mechanistically distinct phases of nNOS activation that are dependent on different sources of Ca²⁺.     

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.     

Annexin VI Binds to a Synaptic Vesicle Protein, Synapsin I

Abstract: Annexin VI bound to >14 species of proteins in the whole homogenate of rat forebrain in a Ca²⁺/phosphatidylserine- or phosphatidic acid-dependent manner. When the subcellular fractions of rat forebrain were examined with a blot from a sodium dodecyl sulfate-polyacrylamide gel, each annexin VI-binding protein showed a different distribution, suggesting that annexin VI is a multifunctional protein. Of these proteins, the doublets of M_r 80,000 were enriched in the purified synaptic vesicles and were identified as synapsin I. Annexin VI bound to the head domain of synapsin I. When the binding of annexin VI to synapsin I was characterized in the native state, the affinity of the binding for Ca²⁺ ( K _Ca) was 12.6 µ M , and the affinity for annexin VI ( K _D) was ∼270 n M . Phosphorylation of synapsin I by cyclic AMP-dependent protein kinase and by Ca²⁺/calmodulin-dependent protein kinase II inhibited the annexin VI binding. The mode of the inhibition was different between the two kinases. These results indicate that annexin VI may modulate the function of synapsin I in a Ca²⁺- and phospholipid-dependent manner.     

Internalization of Voltage-Dependent Sodium Channels in Fetal Rat Brain Neurons: A Study of the Regulation of Endocytosis

Abstract: In fetal rat brain neurons, activation of voltage-dependent Na⁺ channels induced their own internalization, probably triggered by an increase in intracellular Na⁺ level. To investigate the role of phosphorylation in internalization, neurons were exposed to either activators or inhibitors of cyclic AMP- and cyclic GMP-dependent protein kinases, protein kinase C, and tyrosine kinase. None of the tested compounds mimicked or inhibited the effect of Na⁺ channel activation. An increase in intracellular Ca²⁺ concentration induced either by thapsigargin, a Ca²⁺-ATPase blocker, or by A23187, a Ca²⁺ ionophore, was unable to provoke Na⁺ channel internalization. However, Ca²⁺ seems to be necessary because both neurotoxin- and amphotericin B-induced Na⁺ channel internalizations were partially inhibited by BAPTA-AM. The selective inhibitor of Ca²⁺/calmodulin-dependent protein kinase II, KN-62, caused a dose-dependent inhibition of neurotoxin-induced internalization due to a blockade of channel activity but did not prevent amphotericin B-induced internalization. The rate of increase in Na⁺ channel density at the neuronal cell surface was similar before and after channel internalization, suggesting that recycling of internalized Na⁺ channels back to the cell surface was almost negligible. Pretreatment of the cells with an acidotropic agent such as chloroquine prevented Na⁺ channel internalization, indicating that an acidic endosomal/lysosomal compartment is involved in Na⁺ channel internalization in neurons.     

Ca2+/Calmodulin Kinase II Translocates in a Hippocampal Slice Model of Ischemia

Abstract: Rat hippocampal slices were exposed to conditions that simulate an ischemic insult, and the subcellular distribution and the enzymatic activity of Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase) were monitored. Semiquantitative western blots using a monoclonal antibody to the 50-kDa α subunit showed that there was a significant redistribution of the enzyme from a supernatant to a pellet fraction after 10 min of an anoxic/aglycemic insult. No significant change in the total amount of CaM kinase enzyme was detected in the homogenates for up to 20 min of exposure to the insult. Ca²⁺/CaM-dependent enzyme activity did not significantly change in the pellet during the 20-min insult. Supernatant activity decreased throughout the insult. The persistence of Ca²⁺/CaM-dependent CaM kinase activity in the pellet fraction and the detected movement of enzyme from the supernatant to the pellet indicate that redistribution may be an important mechanism in regulating the cellular location of CaM kinase activity.     

Dynamic Regulation of the Activated, Autophosphorylated State of Ca2+/Calmodulin-Dependent Protein Kinase II by Acute Neuronal Excitation In Vivo

Abstract: Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) has been implicated in various neuronal functions, including synaptic plasticity. To examine the physiological regulation of its activated, autophosphorylated state in relation to acute neuronal excitation in vivo, we studied the effect of electroconvulsive treatment in rats on CaMKII activity and in situ autophosphorylation levels. As early as 30 s after the electrical stimulation, a profound but transient decrease in its Ca²⁺/calmodulin-independent activity, as well as in the level of its autophosphorylation at Thr²⁸⁶ (α)/Thr²⁸⁷ (β) measured by using phosphorylation state-specific antibodies, was observed in homogenate from hippocampus and parietal cortex, which was reversible in 5 min. In the later time course, a moderate, reversible increase, which peaked at around 60 min after the electrical stimulation, was observed in parietal cortex but not in hippocampus. The early-phase decrease was found to occur exclusively in the soluble fraction. In addition, partial translocation of CaMKII from the soluble to the particulate fraction seems to have occurred in this early phase. Thus, the activated, autophosphorylated state of CaMKII is under dynamic and precise regulation in vivo, and its regulatory mechanisms seem to have regional specificity.     

Persistent Translocation of Ca2+/Calmodulin-Dependent Protein Kinase II to Synaptic Junctions in the Vulnerable Hippocampal CA1 Region Following Transient Ischemia

Abstract: The influence of brain ischemia on the subcellular distribution and activity of Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II) was studied in various cortical rat brain regions during and after cerebral ischemia. Total CaM kinase II immunoreactivity (IR) and calmodulin binding in the crude synaptosomal fraction of all regions studied increase but decrease in the microsomal and cytosolic fractions, indicative of a translocation of CaM kinase II to synaptosomes. The translocation of CaM kinase II to synaptic junctions occurs but not to synaptic vesicles. The translocation in neocortex and CA3/DG (dentate gyrus) is transient, whereas in the hippocampal CA1 region, it persists for at least 1 day of reperfusion. The Ca²⁺/calmodulin-dependent activity of CaM kinase II in the subsynaptosomal fractions of neocortex is persistently decreased by up to 85%, despite the increase in CaM kinase II IR. The decrease in activity is more pronounced than the decline in IR, suggesting that CaM kinase II is covalently modified in the postischemic phase. The persistent translocation of CaM kinase II in the vulnerable ischemic CA1 region indicates that a pathological process is sustained in the area after the reperfusion phase and this may be of significance for ischemic brain injury.     

CA2+ BINDING PROTEIN CALRETICULIN IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA): BIOCHEMICAL CHARACTERIZATION, DIFFERENTIAL EXPRESSION DURING SEXUAL REPRODUCTION, AND PHYLOGENETIC ANALYSIS

The occurrence of calreticulin, the main Ca²⁺ binding protein in the endoplasmic reticulum of eukaryotic cells, was investigated in the unicellular green alga Chlamydomonas reinhardtii Dangeard. The biochemical characterization of a diethylaminoethyl purified extract highlighted the presence, on SDS-PAGE, of a 55-kDa protein that stained blue with the Stains All dye, a diagnostic feature of acidic Ca²⁺ binding proteins. Immunoblot analyses revealed a strong cross-reaction of the Chlamydomonas reinhardtii protein with antibodies to plant calreticulins and the endoplasmic reticulum retention signal HDEL. Furthermore, the 55-kDa protein bound [⁴⁵Ca²⁺] and had an acidic isoelectric point (pI = 4.9) but was neither glycosylated nor phosphorylated. N-terminal sequencing revealed strong amino acid sequence similarity to calreticulin from other sources. The presence of calreticulin in Chlamydomonas reinhardtii suggested that an endoplasmic reticulum Ca²⁺ buffering mechanism was present in this unicellular chlorophyte. The data suggest an early origin and high conservation of endoplasmic-reticulum-mediated Ca²⁺ functions in eukaryotes, whereby specific posttranslational modifications of the proteinhave been specifically acquired in different lineages of photosynthetic eukaryotes. Moreover, northern and western blot analysis experiments showed a regulation of calreticulin expression during Chlamydomonas sexual reproduction with a high abundance of calreticulin mRNA and protein in reproductive cells.     

Effect of Hypothyroidism on the Subcellular Distribution of Ca2+/Calmodulin-Stimulated Protein Kinase II in Chicken Brain During Posthatch Development

Abstract: In developing chicken brain Ca²⁺/calmodulin-stimulated protein kinase II (CaMPK-II) changes from being primarily cytosolic to being primarily particulate during the protracted maturation period. To investigate whether thyroid hormone levels may be involved in regulating this subcellular redistribution, we raised chickens from 1 day posthatching on food soaked in 0.15% (wt/vol) propylthiouracil (PTU) plus 0.05% (wt/vol) methimazole (MMI). This produced a mild hypothyroidism specifically during the maturation period and resulted in a 67% reduction in the levels of free triiodothyronine (T₃) at 42 days. The concentrations of α- and β-CaMPK-II in cytosol (S3) and crude synaptic membrane (P2M) fractions from forebrain were measured by three methods: Ca²⁺/calmodulin- or Zn²⁺-stimulated autophosphorylation or binding of biotinylated calmodulin. By all three methods hypothyroid animals showed a marked retardation of the redistribution of both subunits of CaMPK-II: an increase in the concentration of the enzyme in S3 and a corresponding decrease in P2M with no overall change in the total amount of enzyme and little apparent change in the concentration of other proteins. In both fractions, there was a parallel change in the Ca²⁺/calmodulin-stimulated phosphorylation of endogenous protein substrates but no change in the basal or cyclic AMP-stimulated protein phosphorylation. Supplementing the PTU/MMI-treated diet with thyroxine (0.5 ppm) prevented all of the observed changes.     

Persistent Translocation and Inhibition of Ca2+/Calmodulin-Dependent Protein Kinase II in the Crude Synaptosomal Fraction of the Vulnerable Hippocampus Following Hypoglycemia

Abstract: Alterations in the levels and activity of Ca²⁺/calmodulin-dependent protein kinase II (CaM-kinase II) were studied in the rat hippocampus during and after insulin-induced hypoglycemic coma. A permanent loss of CaM-kinase II immunohistostaining in the neuronal layer begins at 10 min of isoelectricity in the tip of the dentate gyrus and at 30-min isoelectricity in the CA1 region. The reduction in immunohistostaining in the neurites is less pronounced. Immunoreactivity of CaM-kinase II on western blots increases in the crude synaptosomal fractions and decreases in cytosolic fraction, indicative of a translocation of CaM-kinase II. The translocation persists for at least 1 day of recovery after 30 min of isoelectricity in the vulnerable hippocampus (dorsomedial hippocampus) but not in the resistant hippocampus (dorsolateral hippocampus). Calmodulin binding to western blots shows changes similar to the immunoblots. Ca²⁺/calmodulin-dependent activity of CaM-kinase II in the crude synaptosomal fraction is elevated immediately before isoelectricity and is then inhibited during and after 30 min of isoelectricity, despite the increase of CaM-kinase II immunoreactivity. This was seen in the vulnerable hippocampus. The data indicate that stimulus of translocation and inhibition of CaM-kinase II persist during the recovery phase, preceding neuronal degeneration in the vulnerable hippocampus. This may be of significance for hypoglycemia-induced neuronal death.     

Disrupted [Ca2+]i Homeostasis Contributes to the Toxicity of Nitric Oxide in Cultured Hippocampal Neurons

Abstract: Nitric oxide (NO) has been shown to be an important mediator in several forms of neurotoxicity. We previously reported that NO alters intracellular Ca²⁺ concentration ([Ca²⁺]_i) homeostasis in cultured hippocampal neurons during 20-min exposures. In this study, we examine the relationship between late alterations of [Ca²⁺]_i homeostasis and the delayed toxicity produced by NO. The NO-releasing agent S -nitrosocysteine (SNOC; 300 µ M ) reduced survival by about one half 1 day after 20-min exposures, as did other NO-releasing agents. SNOC also was found to produce prolonged elevations of [Ca²⁺]_i, persisting at 2 and 6 h. Hemoglobin, a scavenger of NO, blocked both the late [Ca²⁺]_i elevation and the delayed toxicity of SNOC. Removal of extracellular Ca²⁺ during the 20-min SNOC treatment failed to prevent the late [Ca²⁺]_i elevations and did not prevent the delayed toxicity, but removal of extracellular Ca²⁺ for the 6 h after exposure as well blocked most of the toxicity. Western blots showed that SNOC exposure resulted in an increased proteolytic breakdown of the structural protein spectrin, generating a fragment with immunoreactivity suggesting activity of the Ca²⁺-activated protease calpain. The spectrin breakdown and the toxicity of SNOC were inhibited by treatment with calpain antagonists. We conclude that exposures to toxic levels of NO cause prolonged disruption of [Ca²⁺]_i homeostatic mechanisms, and that the resulting persistent [Ca²⁺]_i elevations contribute to the delayed neurotoxicity of NO.     

The role of calcium ions in gravity singal perception and transduction

Ca²⁺ is implicated as a messenger in coupling various environmental stimuli, such as gravity and light, to response. In recent years, it has become evident that Ca²⁺ plays a central role in all three phases of gravitropism – perception, transduction and response. The root cap, which is known to contain high amounts of Ca²⁺ and calmoduin, is the primary site of gravity preeception. The possible role of phosphoinositide turnovr and Ca²⁺ and Ca²⁺ calmodulin-dependent enzymes such as Ca²⁺– ATPase and protein kinases in gravitropsim is discussed. A model is proposed to describe the role of Ca²⁺ in both normal and light-dependnt gravity response in roots.     

Differential Regulation of Intracellular Signaling Systems by Sodium Fluoride in Rat Glioma Cells

Abstract: We investigated the rapid and slow effects of NaF on intracellular signaling systems such as Ca²⁺ homeostasis and cyclic GMP (cGMP) generation in rat glioma C6 cells, using the Ca²⁺-sensitive dye fura-2 and cGMP enzyme immunoassay. We found that the following: (a) NaF enhanced cGMP generation in a concentration-dependent manner. This enhancement was abolished by pretreatment with 100 µ M BAPTA tetraacetoxymethyl ester or in the presence of W-7 in a concentration-dependent manner. N ^G-Monomethyl- l -arginine (NMMA), a competitive inhibitor of nitric oxide synthase (NOS), also inhibited the NaF-induced generation of cGMP. These results suggest that NaF-induced cGMP generation occurs via a calcium/calmodulin- and NOS-dependent pathway. (b) The basal intracellular Ca²⁺ concentration ([Ca²⁺]_i) was transiently greater at 1 and 3 h after pretreatment with NaF. W-7 and W-13 antagonized the increase in [Ca²⁺]_i, whereas NMMA had little effect. This suggests that the NaF-induced change in basal [Ca²⁺]_i was mediated by a calmodulin-dependent pathway but was independent of a NOS-sensitive pathway. (c) The serotonin (5-HT)-induced intracellular mobilization of Ca²⁺ was reduced by pretreating the cells with NaF. The reduction in Ca²⁺ mobilization was antagonized by genistein, a tyrosine kinase inhibitor. W-7, W-5, and H-8 had no effect. Results suggest that NaF differentially regulates the cGMP generation, basal [Ca²⁺]_i, and 5-HT_2A receptor function in C6 glioma 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.