Coupling of the Cloned μ-Opioid Receptor with the ω-Conotoxin-Sensitive Ca2+ Current in NG108-15 Cells

Abstract: Voltage-dependent Ca²⁺ currents were measured in NG108-15 neuroblastoma × glioma hybrid cells transformed to express the rat μ-opioid receptor by the whole-cell configuration of the patch-clamp technique with Ba²⁺ as charge carrier. A μ-opioid receptor-selective agonist, [ d -Ala², N -Me-Phe⁴,Gly⁵-ol]enkephalin caused significant inhibition of voltage-dependent Ca²⁺ currents in μ-receptor-transformed NG108-15 cells but not in nontransfected or vector-transformed control cells. On the other hand, a δ-opioid receptor-selective agonist, [ d -penicillamine², d -penicillamine⁵]enkephalin, induced inhibition of voltage-dependent Ca²⁺ currents in both control and μ-receptor-transformed cells, which is mediated by the δ-opioid receptor expressed endogenously in NG108-15 cells. The inhibition of voltage-dependent Ca²⁺ currents induced by [ d -Ala², N -Me-Phe⁴,Gly⁵-ol]enkephalin and [ d -penicillamine², d -penicillamine⁵]enkephalin was reduced by pretreatment of the cells with pertussis toxin or ω-conotoxin GVIA. These results indicate that the μ-opioid receptor expressed from cDNA functionally couples with ω-conotoxin-sensitive N-type Ca²⁺ channels through the action of pertussis toxin-sensitive G proteins in NG108-15 cells.     

Increase of brain-derived neurotrophic factor gene expression in NG108-15 cells by the nuclear isoforms of Ca2+/ calmodulin-dependent protein kinase II

We have reported that the delta3 isoform of Ca2+/ calmodulin-dependent protein kinase II (CaM kinase II) is abundant in the nucleus in cerebellar granule cells. To examine the possibility that the nuclear isoforms of CaM kinase II are involved in the expression of brain-derived neurotrophic factor (BDNF), we transiently overexpressed the delta3 isoform in NG108-15 cells. The quantitative RT-PCR analysis revealed that rat cerebellum and NG108-15 cells expressed the exon IV-containing mRNA of BDNF (exon IV-BDNF mRNA) more than the exon III-BDNF mRNA. Treatment of NG108-15 cells with Bay K 8644 increased both exon III- and exon IV-BDNF mRNAs, and overexpression of the 83 isoform potentiated the expression of the exon IV-BDNF mRNA. The potentiation was not observed in the cells that were overexpressed with either the 61 isoform, a nonnuclear isoform, or the inactive mutant of the delta3 isoform. We constructed the luciferase reporter gene following the promoter upstream of exon IV and confirmed that overexpression of the delta3 isoform increased luciferase gene expression. Double-immunostaining of NG108-15 cells with the antibodies to CaM kinase II and BDNF clearly showed that BDNF was highly expressed in the cells that were overexpressed with the delta3 isoform or the alphaB isoform, another nuclear isoform of CaM kinase II. These results suggest that the nuclear isoforms of CaM kinase II are involved in the expression of BDNF.     

Overexpression of Ca2+/Calmodulin-Dependent Protein Kinase II Inhibits Neurite Outgrowth of PC12 Cells

Abstract: To investigate the physiological role of Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II) in neuronal differentiation, we transfected the cDNA of the α subunit of mouse CaM kinase II (CaM kinase IIα) into PC12 cells and established clonal cell lines that constitutively express the transfected CaM kinase IIα gene. The expression of CaM kinase IIα was confirmed by northern blot and immunoblot analyses. Northern blot analysis showed that the γ and δ subunits of CaM kinase II are mainly expressed in PC12 cells. Treatment of the cells with ionomycin activated CaM kinase IIα through autophosphorylation and generation of the Ca²⁺/calmodulin-independent form. It is interesting that the neurite outgrowth induced by dibutyryl cyclic AMP was inhibited in these cell lines in accordance with the activities of overexpressed CaM kinase IIα. The activity of cyclic AMP-dependent protein kinase showed similar levels among these cell lines. These results suggest that CaM kinase II is involved in the modulation of the neurite outgrowth induced by activation of the cyclic AMP system.     

Identification of the isoforms of Ca(2+)/Calmodulin-dependent protein kinase II in rat astrocytes and their subcellular localization

Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) occurs in astrocytes as well as in neurons in brain. We have reported that CaM kinase II is involved in the regulation of cytoskeletal proteins and gene expression in astrocytes. In this study, we identified all isoforms of CaM kinase II in astrocytes and examined their subcellular localization. When we amplified the isoforms of four subunits by RT-PCR followed by the "nested" PCR, totally 10 isoforms were obtained. Immunoblot analyses with five types of antibodies against CaM kinase II indicated that the most abundant isoform was delta2. Immunostaining suggested that the delta2 isoform was localized predominantly at the Golgi apparatus. The localization of the delta2 isoform at the Golgi apparatus was also observed in NG108-15 cells. We overexpressed all isoforms that contained the nuclear localization signal to examine their nuclear targeting in NG108-15 cells. In contrast to the alphaB and delta3 isoforms that entered the nucleus, as reported, the gammaA isoform was excluded from the nucleus in the transfected NG108-15 cells. These results suggest that the 15-amino acid insertion following the nuclear localization signal inhibits the nuclear targeting of the gammaA isoform.     

Calmodulin/Ca2+-ATPase interaction at the Arabidopsis thaliana plasma membrane is dependent on calmodulin isoform showing isoform-specific Ca2+ dependencies

Arabidopsis thaliana plasma membrane (PM) Ca²⁺-ATPase is a type IIB P-type ATPase, which binds calmodulin (CaM) to an autoinhibitory N-terminal domain. Here, we took advantage of the fact that PM isolated from cultured cells mainly contains At -ACA8, the first cloned A. thaliana PM Ca²⁺-ATPase, to analyse its interaction with CaM in detail. Analysis of the ability of different peptides designed from At -ACA8 N-terminus to compete with the native protein for binding of bovine brain CaM (bbCaM) showed that peptide ⁴¹I-T⁶³ had the same affinity of the native protein [apparent dissociation constant (KD) at 10 µ M free Ca²⁺ about 25 n M ], thus localizing At -ACA8 CaM-binding site within this sequence. The interaction of At -ACA8 N-terminus with bbCaM, as determined by surface plasmon resonance, was rapid, and slowly but was fully reversible. Analysis of Ca²⁺-ATPase activation as a function of the concentration of different isoforms of A. thaliana CaM showed that Ca²⁺-ATPase is activated to similar extent by bbCaM and by different isoforms of homologous CaM. However, the affinity for the divergent A. thaliana isoform CaM8 was lower than that for canonical CaM isoforms such as A. thaliana CaM2, CaM4 and CaM6 or bbCaM. The apparent KD for CaM isoforms of the native enzyme increased with the decrease of free Ca²⁺ concentration, suggesting that enzyme conformation is affected by Ca²⁺. Binding of CaM isoforms to At -ACA8 N-terminus was affected differently by free Ca²⁺ concentration, suggesting that plant CaMs may have different affinities for Ca²⁺.     

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.     

Developmental regulation of type II calcium/calmodulin-dependent kinase isoforms in rat cerebellum

Two distinct isoforms of a Type II calcium/calmodulin-dependent protein kinase were separated from high-speed supernates (cytosol) of rat neonatal [postnatal day 10 (P10)] and adult [postnatal day 40 (P40)] cerebellum using cation-exchange chromatography. The isoenzymes contained variable amounts of three subunits of apparent Mr's of 50 kDa (alpha), 58 kDa (beta'), and 60 kDa (beta). The specific activity of calmodulin-dependent kinase (CaM kinase II) in crude homogenates increased sixfold between P10 and P40 using exogenous MAP 2 as substrate. Cytosol from cerebellum at P40 contained a predominant isoform (approximately 40% of total cytosolic activity) with a 1:5 molar ratio of alpha:beta',beta subunits that eluted with 150 mM NaCl (designated 150) and a less abundant isoform (approximately 20% of total cytosolic activity) containing a 1:8 molar ratio of alpha:beta',beta subunits that eluted with 350 mM NaCl (designated 350). In neonatal cerebellum at P10, the relative abundance of the two isoforms was reversed such that approximately 50% of the cytosolic calmodulin-dependent kinase activity was recovered in the 350 isoform, whereas only 20% of the total cytosolic kinase activity was recovered in the 150 isoform. Previous studies indicate that cerebellar granule cells may contain an all beta',beta isoform of CaM kinase II that lacks alpha subunit. Thus, to assess the cell-specific localization of kinase isoforms within cerebellum, cytosol prepared from primary cultures of rat cerebellar granule cells was applied to cation-exchange chromatography and analyzed for calmodulin-dependent kinase activity. The cells contained both isoforms of the kinase that were present in fresh tissue suggesting that granule cell-enriched cultures express all three kinase subunits. The data demonstrate that rat cerebellum contains unique mixtures of CaM kinase II isoenzymes and that their expression is developmentally regulated.     

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.     

Glutamate-Induced Loss of Ca2+/Calmodulin-Dependent Protein Kinase II Activity in Cultured Rat Hippocampal Neurons

Abstract: The exposure of cultured rat hippocampal neurons to 500 µ M glutamate for 20 min induced a 55% decrease in the total Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II) activity. The Ca²⁺-independent activity and autophosphorylation of CaM kinase II decreased to the same extent as the changes observed in total CaM kinase II activity, and these decreases in activities were prevented by pretreatment with MK-801, an N -methyl- d -aspartate (NMDA)-type receptor antagonist, and the removal of extracellular calcium but not by antagonists against other types of glutamate receptors and protease inhibitors. Similarly, the decrease in the CaM kinase II activity was induced by a Ca²⁺ ionophore, ionomycin. Immunoblot analysis with the anti-CaM kinase II antibody revealed a significant decrease in the amount of the enzyme in the soluble fraction, in contrast with the inverse increase in the insoluble fraction; thus, the translocation was probably induced during treatment of the cells with glutamate. These results suggest that glutamate released during brain ischemia induces a loss of CaM kinase II activity in hippocampal neurons, by stimulation of the NMDA receptor, and that inactivation of the enzyme may possibly be involved in the cascade of the glutamate neurotoxicity following brain ischemia.     

Light-Induced CREB Phosphorylation and Gene Expression in Rat Retinal Cells

Abstract: The signal pathway for light-induced expression of c- fos and the neuropeptide somatostatin (SS) in rat retinal cells was investigated. Flashing light induced c- fos and SS mRNA in the inner nuclear layer and the ganglion cell layer. As both c- fos and SS genes have a cyclic AMP response element (CRE) in their promoters, CRE-binding protein (CREB) phosphorylation in retinal cells was examined with a phospho-CREB-specific antibody. Both flashing light and administration of the L-type Ca²⁺ channel activator Bay K 8644 induced phosphorylation of CREB in the nuclei of the amacrine cells and the ganglion cells where c- fos /SS mRNAs were expressed. These cells could be double-stained with anti-calmodulin kinase II (anti-CaM kinase II) monoclonal antibody and phospho-CREB-specific polyclonal antiserum after Bay K 8644 administration, indicating the colocalization of phosphorylated CREB at Ser¹³³ and CaM kinase II in the neural retina.     

Ca2+/Calmodulin-Dependent Transcriptional Activation of δ-Opioid Receptor Gene Expression Induced by Membrane Depolarization in NG108-15 Cells

Abstract: Regulation of gene expression is one of the mechanisms by which neuronal activity elicits long-term changes in neuronal phenotype and function. Although activity-dependent induction of immediate-early genes has been extensively studied, much less is known about the late-response genes. We have investigated the activity-dependent regulation of δ-opioid receptor (DOR) mRNA levels in NG108-15 cells. Transsynaptic activation was mimicked by depolarization with 55 m M KCl or veratridine. Both treatments lead to a time-dependent increase of DOR mRNA levels. Ca²⁺ entry through L-type voltage-dependent Ca²⁺ channels activated by depolarization appears to be involved, because L-type channel blockers reduced the induction of DOR expression. Ca²⁺ binding to calmodulin is the next step in the signal transduction pathway, because a calmodulin antagonist, W7, reduced the effect of veratridine. A selective inhibitor of calmodulin kinases (KN-62) and cyclosporin, an inhibitor of calcineurin, also antagonized the depolarization-induced increase in DOR mRNA levels, which indicates that both calcium/calmodulin-dependent enzymes are involved in the activity-dependent induction of DOR gene expression. Induction of DOR gene expression by an activity-dependent increase in intracellular Ca²⁺ concentration may serve as a feedback regulatory mechanism because activation of DOR leads to hyperpolarization and lower excitability of neurons.     

Effects of Calcium Channel Antagonists on Calcium Entry and Glutamate Release from Cultured Rat Cerebellar Granule Cells

Abstract: Using a range of Ca²⁺ channel blockers we have investigated the Ca²⁺ channel subtypes that mediate the depolarisation-induced elevation of the intracellular free Ca²⁺ concentration ([Ca²⁺]_i) and glutamate release from cultured rat cerebellar granule cells. ω-Conotoxin-GVIA had little effect on either the transient or plateau phase of the depolarisation-induced [Ca²⁺]_i rise or on glutamate release, ruling out a significant role for N-type Ca²⁺ channels. Nifedipine substantially inhibited the initial transient rise in [Ca²⁺]_i and the plateau phase of the [Ca²⁺]_i rise and glutamate release, suggesting the involvement of L-type Ca²⁺ channels. Both ω-agatoxin and ω-conotoxin-MVIIC also inhibited the transient rise in [Ca²⁺]_i and glutamate release but not the plateau phase of the [Ca²⁺]_i rise. The inhibitions by nifedipine were not increased by coaddition of ω-conotoxin-MVIIC, suggesting overlapping sensitivity to these channel blockers. These data show that glutamate release from granule cells in response to depolarisation with a high KCI level involves Ca²⁺ currents that are sensitive to nifedipine, ω-agatoxin-IVA, and also ω-conotoxin-MVIIC. The overlapping sensitivity of the channels to these toxins prevents attribution of any of the phases of the [Ca²⁺]_i rise or glutamate release to distinct P-, Q-, or O-type Ca²⁺ currents.     

Differential Expression and Subcellular Localization of Protein Kinase C α, β, γ, δ, and ɛ Isoforms in SH-SY5Y Neuroblastoma Cells: Modifications During Differentiation

Abstract: A decrease in protein kinase C activity caused either by treatment with inhibitors, such as staurosporine or H-7, or by prolonged exposure to phorbol diesters has been proposed to be involved in the early events of SH-SY5Y neuroblastoma cell differentiation. Because eight distinct isoforms of protein kinase C with discrete subcellular and tissue distributions have been described, we determined which isoforms are present in SH-SY5Y cells and studied their modifications during differentiation. The α, β, δ, and ɛ isoforms were present in SH-SY5Y cells, as well as in rat brain. Protein kinase C-α and -β₁ were the most abundant isoforms in SH-SY5Y cells, and immunoreactive protein kinase C-δ and -ɛ were present in much smaller amounts than in rat brain. Subcellular fractionation and immunocytochemistry demonstrated that all four isoforms are distributed bimodally in the cytoplasm and the membranes. Immunocytochemical analysis showed that the α isoform is associated predominantly with the plasma membrane and the processes extended during treatment with 12-tetradecanoyl-13-acetyl-β-phorbol or staurosporine, and that protein kinase C-ɛ is predominantly membrane-bound. Its localization did not change during differentiation. Western blots of total SH-SY5Y cell extracts and of subcellular fractions probed with isoform-specific polyclonal antibodies showed that when SH-SY5Y cells acquired a morphologically differentiated phenotype, protein kinase C-α and -ɛ decreased, and protein kinase C-β₁, did not change. These data suggest distinct roles for the different protein kinase C isoforms during neuronal differentiation, as well as possible involvement of protein kinase α and ɛ in neuritogenesis.     

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.     

Depolarization of Cerebellar Granule Cells Increases Phosphorylation of Rabphilin-3A

Abstract: Studies performed over the past several years have provided evidence that phosphorylation of proteins is important in the regulation of neurotransmitter release. In this study, it is shown that rabphilin-3A is present in cerebellar granule cells as a phosphoprotein, by using ³²P-labeling of cerebellar granule cells, immunoprecipitation, phosphoamino acid analysis, and phosphopeptide mapping. The level of phosphorylation was increased (224 ± 13%) (mean ± SEM) on depolarization of the cells with K⁺ (56 m M ) in the presence of external Ca²⁺ (1 m M ). Stimulation of protein kinase C with a phorbol ester (phorbol 12,13-dibutyrate) also enhanced the phosphorylation of rabphilin-3A (217 ± 21%). Inhibitors of Ca²⁺/calmodulin-stimulated protein kinases or protein kinase C reduced the depolarization-enhanced phosphorylation of rabphilin-3A, indicating that rabphilin-3A is one of the targets for Ca²⁺-activated protein kinases in the nerve terminal. Costimulation of cells with phorbol 12,13-dibutyrate and K⁺ depolarization produced an increased level of phosphorylation of rabphilin-3A compared with either stimulus alone (287 ± 61%). Phosphoamino acid analysis showed that serine was the main phosphorylated residue. A slight increase in the threonine phosphorylation could also be detected, whereas tyrosine phosphorylation could not be detected at all. These results suggest that rabphilin-3A is phosphorylated in vivo and undergoes synaptic activity-dependent phosphorylation during Ca²⁺-activated K⁺ depolarization.     

Hydrogen sulfide evokes neurite outgrowth and expression of high-voltage-activated Ca2+ currents in NG108-15 cells: involvement of T-type Ca2+ channels

We investigated if stimulation of T-type Ca²⁺ channels with sodium hydrosulfide (NaHS), a donor of hydrogen sulfide (H₂S), could cause neuronal differentiation of NG108-15 cells. Like dibutyryl cyclic AMP (db-cAMP), treatment with NaHS at 1.5–13.5 mM for 16 h enhanced neurite outgrowth in a concentration-dependent manner. Synergistic neuritogenic effect was obtained in the cells stimulated with NaHS in combination with db-cAMP at subeffective concentrations. Exposure to NaHS or db-cAMP for 2 days resulted in enhancement of expression of high-voltage-activated currents consisting of N-, P/Q-, L- and also other types, but not of T-type currents. Mibefradil, a pan-T-type channel blocker, abolished the neuritogenesis induced by NaHS, but not by db-cAMP. The NaHS-evoked neuritogenesis was also completely blocked by pretreatment with BAPTA/AM, a chelator of intracellular Ca²⁺, and by zinc chloride at a concentration known to selectively inhibit Ca_v3.2 isoform of T-type Ca²⁺ channels, but not Ca_v3.1 or Ca_v3.3. Further, l -ascorbate, recently proven to selectively inhibit Ca_v3.2, abolished the neuritogenic effect of NaHS, but not db-cAMP. Our data thus demonstrate that NaHS/H₂S is a novel inducer of neuronal differentiation in NG108-15 cells, as characterized by neuritogenesis and expression of high-voltage-activated currents, and suggest the involvement of T-type Ca²⁺ channels, especially Ca_v3.2.     

Sea urchin fertilization stimulates CaM kinase-II (multifunctional [type II] Ca2+/CaM kinase) activity and association with p34cdc2

Upon fertilization, the sea urchin egg synthesizes proteins which impart a Ca²⁺ dependence to M-phase onset. A potential target of this Ca²⁺ dependence may be CaM kinase-II (the multifunctional [type II] Ca²⁺/calmodulin [CaM]-dependent protein kinase) which is necessary for nuclear envelope breakdown in fertilized sea urchin eggs. This study was intended to determine whether sea urchin CaMK-II is activated after fertilization and whether it interacts with other known M-phase regulators, such as p34^cdc2. We report that total CaMK-II activity, measured by solution assays, increases after fertilization, peaking just prior to cleavage. Interestingly, total CaMK-II activity continues to fluctuate, peaking again prior to second and third cleavage. Gel assays also reveal enhanced levels of the 56 and 62 kDa potential CaMK-II phosphoproteins after fertilization. Finally, CaMK-II activity and only the 62 kDa phosphoprotein physically associate with p34^cdc2, but again only after fertilization. These changes in CaMK-II activity and p34^cdc2-association after fertilization may ensure that Ca²⁺ signals are targeted to the M-phase machinery at the appropriate developmental times.