Tyrosine Hydroxylase Phosphorylation in Bovine Adrenal Chromaffin Cells: The Role of Intracellular Ca2+ in the Histamine H1 Receptor-Stimulated Phosphorylation of Ser8, Ser19, Ser31, and Ser40

Abstract: Tyrosine hydroxylase (TOH), the rate-limiting enzyme in catecholamine biosynthesis, is regulated by phosphorylation. Activation of histaminergic H₁ receptors on cultured bovine adrenal chromaffin cells stimulated a rapid increase in TOH phosphorylation (within 5 s) that was sustained for at least 5 min. The initial increase in TOH phosphorylation (up to 1 min) was essentially unchanged by the removal of extracellular Ca²⁺. In contrast, the H₁-mediated response was abolished by preloading the cells with BAPTA acetoxymethyl ester (50 µ M ) and significantly reduced by prior exposure to caffeine (10 m M for 10 min) to deplete intracellular Ca²⁺. Trypticphosphopeptide analysis by HPLC revealed that the H₁ response in the presence or absence of extracellular Ca²⁺ resulted in a major increase in the phosphorylation of Ser¹⁹ with smaller increases in that of Ser⁴⁰ and Ser³¹. In contrast, although a brief stimulation with nicotine (30 µ M for 60 s) also resulted in a major increase in Ser¹⁹ phosphorylation, this response was abolished in the absence of extracellular Ca²⁺. These data indicate that the mobilization of intracellular Ca²⁺ plays a crucial role in supporting H₁-mediated TOH phosphorylation and may thus have a potentially important role in regulating catecholamine synthesis.     

Endothelin Induces a Calcium-Dependent Phosphorylation of PEA-15 in Intact Astrocytes: Identification of Ser104 and Ser116 Phosphorylated, Respectively, by Protein Kinase C and Calcium/Calmodulin Kinase II In Vitro

Abstract: PEA-15 (phosphoprotein enriched in astrocytes, M_r = 15,000) is an acidic serine-phosphorylated protein highly expressed in the CNS, where it can play a protective role against cytokine-induced apoptosis. PEA-15 is a major substrate for protein kinase C. Endothelins, which are known to exert pleiotropic effects on astrocytes, were used to analyze further the processes involved in PEA-15 phosphorylation. Endothelin-1 or endothelin-3 (0.1 µ M ) induced a robust phosphorylation of PEA-15 that was abolished by the removal of extracellular calcium, but only diminished by inhibitors of protein kinase C. Microsequencing of phosphopeptides generated by digestion of PEA-15 following endothelin-1 treatment identified two phosphorylated residues: Ser¹⁰⁴, previously recognized as the protein kinase C site, and a novel phosphoserine, Ser¹¹⁶, located in a consensus motif for either protein kinase casein kinase II or calcium/calmodulin-dependent protein kinase II (CaMKII). Partly purified PEA-15 was a substrate in vitro for CaMKII, but not for casein kinase II. Two-dimensional phosphopeptide mapping demonstrated that the site phosphorylated in vitro by CaMKII was also phosphorylated in intact astrocytes in response to endothelin. CaMKII phosphorylated selectively Ser¹¹⁶ and had no effect on Ser¹⁰⁴, but in vitro phosphorylation by CaMKII appeared to facilitate further phosphorylation by protein kinase C. Treatment of intact astrocytes with okadaic acid enhanced the phosphorylation of the CaMKII site. These results demonstrate that PEA-15 is phosphorylated in astrocytes by CaMKII (or a related kinase) and by protein kinase C in response to endothelin.     

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.     

Glutamine Uptake at the Blood-Brain Barrier Is Mediated by N-System Transport

Abstract: The aim of this study was to determine the effect of angiotensin II (AII) on tyrosine hydroxylase (TOH) activity and phosphorylation in bovine adrenal chromaffin cells (BACCs). We report here that stimulation of BACCs with AII (100 n M ) produced a significant increase in both TOH activity and phosphorylation over a period of 10 min. The increase in TOH activity was receptor-mediated. Tryptic phosphopeptide analysis by HPLC revealed that AII stimulated an increase in phosphorylation of three sites on TOH, Ser¹⁹, Ser³¹, and Ser⁴⁰, with the largest increase being observed for Ser³¹ phosphorylation. Pretreatment of the cells with the protein kinase C inhibitor Ro 31-8220 (10 µ M , 15 min) did not affect TOH activity or phosphorylation produced by AII. The inhibitor also did not affect the TOH activity or Ser⁴⁰ phosphorylation produced by forskolin (10 µ M , 10 min). In contrast, Ro 31-8220 fully inhibited the TOH activation as well as Ser³¹ and Ser⁴⁰ phosphorylation of TOH produced by phorbol 12, 13-dibutyrate (500 n M , 10 min). Removal of extracellular Ca²⁺ from the incubation medium inhibited the AII-induced TOH activity by 50% and significantly blocked Ser¹⁹ and Ser³¹ phosphorylation but did not affect Ser⁴⁰ phosphorylation in response to AII. These results indicate that AII activates a complex and perhaps novel signaling pathway leading to the phosphorylation and activation of TOH. The TOH activation by AII appears to be partially independent of Ser⁴⁰ phosphorylation, suggesting a potentially important role for Ser³¹ phosphorylation.     

Protein Kinase FA/Glycogen Synthase Kinase 3α Predominantly Phosphorylates the In Vivo Sites of Ser502, Ser506, Ser603, and Ser666 in Neurofilament

Abstract: In this report, the phosphorylation sites of neurofilament protein of medium molecular mass (NF-M) by protein kinase F_A/glycogen synthase kinase 3α (kinase F_A/GSK-3α) were determined by two-dimensional electrophoresis/TLC, phosphoamino acid analysis, HPLC, Edman degradation, and peptide sequencing. Kinase F_A/GSK-3α phosphorylates NF-M predominantly on serine, residue. Three major tryptic phosphopeptide peaks were resolved by C₁₈ reverse-phase HPLC. Edman degradation and peptide sequence analysis revealed that AKS(p)PVSK is the phosphorylation site sequence for the first major peak. When mapping with the amino acid sequence of neurofilament, we finally demonstrate Ser⁶⁰³-Pro, one of the in vivo sites in NF-M, as the major site phosphorylated by kinase F_A/GSK-3α. By using the same approach, we also identified the in vivo sites of Ser⁵⁰²-Pro, Ser⁵⁰⁶-Pro, and Ser⁶⁶⁶-Pro as the other three major sites in NF-M phosphorylated by kinase F_A/GSK-3α. Taken together, the results provide initial evidence that kinase F_A/GSK-3α may represent a physiologically relevant protein kinase involved in the in vivo phosphorylation of NF-M. Because Ser⁵⁰², Ser⁵⁰⁶, Ser⁶⁰³, and Ser⁶⁶⁶ are all flanked by a carboxyl-terminal proline residue, the results provide further evidence that F_A/GSK-3α may represent a proline-directed protein kinase involved in the structure-function regulation of the neuronal cytoskeletal system.     

Activation of Cyclic AMP-Dependent Protein Kinase in Okadaic Acid-Treated Neurons Potentiates Neurofilament Fragmentation and Stimulates Phosphorylation of Ser2 in the Low-Molecular-Mass Neurofilament Subunit

Abstract: The activation of cyclic AMP-dependent protein kinase (PKA) in rat dorsal root ganglion (DRG) cultures increased phosphorylation of the low-molecular-mass neurofilament subunit (NFL) at a site previously identified as Ser⁵⁵ but had no effect on neurofilament integrity. When PKA was activated in DRG cultures treated with 20–250 n M okadaic acid, neurofilament fragmentation was enhanced, and there was a corresponding increase in phosphorylation of NFL at a novel site. This site was also phosphorylated by PKA in vitro and was determined to be Ser² by mass spectrometric analysis of the purified chymotryptic phosphopeptide. The PKA sites in NFL were dephosphorylated by the purified catalytic subunit of protein phosphatase-2A but not that of protein phosphatase-1, and phosphoserine-2 was a better substrate than phosphoserine-55. The phosphorylation and dephosphorylation of Ser² and Ser⁵⁵ in NFL may therefore be involved in the modulation of neurofilament dynamics through the antagonistic effects of PKA and protein phosphatase-2A.     

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.     

Ca2+ and Phospholipid-Dependent Protein Kinase Activity and Phosphorylation of Endogenous Proteins in Bovine Adrenal Medulla

Abstract: Soluble and membrane fractions of bovine adrenal medulla contain several substrates for the Ca²⁺/ phospholipid-dependent and cyclic AMP-dependent protein kinases. The phosphorylation of soluble proteins (36 and 17.7 kilodaltons) and a membrane protein (22.5 kilo-daltons) showed an absolute requirement for the presence of both Ca²⁺ and phosphatidylserine; other substrates showed less stringent phosphorylation requirements and many of these proteins were specific for each of the protein kinases. The Ca²⁺/phospholipid-dependent phosphorylation was rapid, with effects seen as early as at 30 s of incubation. Measurement of enzyme activities with histone HI as an exogenous substrate demonstrated that the Ca²⁺/phospholipid-dependent protein kinase was equally distributed between the soluble and membrane fractions whereas the cyclic AMP-dependent enzyme was predominantly membrane-bound in adrenal medulla and chromaffin cells. The activity of the soluble Ca²⁺/phos-pholipid-dependent protein kinase of adrenal medulla was found to be about 50% of the enzyme level present in rat brain, a tissue previously shown to contain a very high enzyme activity. These results suggest a prominent role for the Ca²⁺/phospholipid-dependent protein kinase in chromaffin cell function.     

The State of Phosphorylation of Normal Adult Brain τ, Fetal τ, and τ from Alzheimer Paired Helical Filaments at Amino Acid Residue Ser262

Abstract: Antibody Ab262 was raised against a synthetic τ peptide (SKIGSTENLK, amino acids 258–267 of τ, termed Ser²⁶² peptide). The antibody was more reactive with Ser²⁶² peptide and unphosphorylated τ than a related phosphopeptide [SKIGS(P)TENLK, termed P-Ser²⁶² peptide] and τ phosphorylated by a partially purified kinase, glycogen synthase kinase (GSK) 3β. Ab262 reacted poorly with a peptide having the sequence DRVQSKIGSLD (amino acids 348–358). Treatment of P-Ser²⁶² peptide or GSK 3β phosphorylated τ with alkaline phosphatase increased Ab262 immunoreactivity, indicating that Ab262 is a reagent useful for studying τ phosphorylation at the Ser²⁶² residue. The Ab262 immunoreactivity was detected in τ from normal brains and Alzheimer paired helical filament (PHF-τ) and in PHFs. Alkaline phosphatase treatment had no effect on the Ab262 immunoreactivity of normal τ and PHF-τ but altered the Tau-1 and PHF-1 immunoreactivities. τ proteins from rat brains at 3 and 8 h postmortem exhibited 5 and 19%, respectively, more Ab262 immunoreactivity than τ from fresh tissues. In comparison, rat τ at 8 h postmortem was 40% more immunoreactive with Tau-1. The results suggest that Ser²⁶² is not a major phosphorylation site in vivo. Moreover, there is little or no difference between PHF-τ and normal τ in the extent of phosphorylation at Ser²⁶².     

Glutamate Induces a Calcineurin-Mediated Dephosphorylation of Na+,K+-ATPase that Results in Its Activation in Cerebellar Neurons in Culture

Abstract: In primary cultures of cerebellar neurons glutamate neurotoxicity is mainly mediated by activation of the NMDA receptor, which allows the entry of Ca²⁺ and Na⁺ into the neuron. To maintain Na⁺ homeostasis, the excess Na⁺ entering through the ion channel should be removed by Na⁺,K⁺-ATPase. It is shown that incubation of primary cultured cerebellar neurons with glutamate resulted in activation of the Na⁺,K⁺-ATPase. The effect was rapid, peaking between 5 and 15 min (85% activation), and was maintained for at least 2 h. Glutamate-induced activation of Na⁺,K⁺-ATPase was dose dependent: It was appreciable (37%) at 0.1 µ M and peaked (85%) at 100 µ M . The increase in Na⁺,K⁺-ATPase activity by glutamate was prevented by MK-801, indicating that it is mediated by activation of the NMDA receptor. Activation of the ATPase was reversed by phorbol 12-myristate 13-acetate, an activator of protein kinase C, indicating that activation of Na⁺,K⁺-ATPase is due to decreased phosphorylation by protein kinase C. W-7 or cyclosporin, both inhibitors of calcineurin, prevented the activation of Na⁺,K⁺-ATPase by glutamate. These results suggest that activation of NMDA receptors leads to activation of calcineurin, which dephosphorylates an amino acid residue of the Na⁺,K⁺-ATPase that was previously phosphorylated by protein kinase C. This dephosphorylation leads to activation of Na⁺,K⁺-ATPase.     

N-Terminal-Specific Anti-B-50 (GAP-43) Antibodies Inhibit Ca2+-Induced Noradrenaline Release, B-50 Phosphorylation and Dephosphorylation, and Calmodulin Binding

Abstract: B-50 (GAP-43) is a presynaptic protein kinase C (PKC) substrate implicated in the molecular mechanism of noradrenaline release. To evaluate the importance of the PKC phosphorylation site and calmodulin-binding domain of B-50 in the regulation of neurotransmitter release, we introduced two monoclonal antibodies to B-50 into streptolysin O-permeated synaptosomes isolated from rat cerebral cortex. NM2 antibodies directed to the N-terminal residues 39–43 of rat B-50 dose-dependently inhibited Ca²⁺-induced radiolabeled and endogenous noradrenaline release from permeated synaptosomes. NM6 C-terminal-directed (residues 132–213) anti-B-50 antibodies were without effect in the same dose range. NM2 inhibited PKC-mediated B-50 phosphorylation at Ser⁴¹ in synaptosomal plasma membranes and permeated synaptosomes, inhibited ³²P-B-50 dephosphorylation by endogenous synaptosomal phosphatases, and inhibited the binding of calmodulin to synaptosomal B-50 in the absence of Ca²⁺. Similar concentrations of NM6 did not affect B-50 phosphorylation or dephosphorylation or B-50/calmodulin binding. We conclude that the N-terminal residues 39–43 of the rat B-50 protein play an important role in the process of Ca²⁺-induced noradrenaline release, presumably by serving as a local calmodulin store that is regulated in a Ca²⁺- and phosphorylation-dependent fashion.     

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.     

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.     

Ca2+ -dependent protein phosphorylation in germinated pollen of Nicotiana alata, an ornamental tobacco

The 40 000 g supernatant and 40 000 g pellet from extracts of germinated pollen of Nicotiana alata Link et Otto contain protein kinase activity which catalyzes the phosphorylation of histones, casein and a range of endogenous polypeptides. Phosphorylation of certain low-molecular-weight, casein-derived polypeptides is activated at low (12–37 μ M ) and partially inhibited at higher (540 μ M ) concentrations of free Ca²⁺. Histone phosphorylation is largely Ca²⁺-dependent and is activated by 540 μM free Ca²⁺. No activation of protein phosphorylation by micromolar concentrations of calmodulin is found, but phenothiazine-derived calmodulin antagonists markedly stimulate protein phosphorylation.     

Protein Kinase FA/GSK-3 Phosphorylates on Ser235-Pro and Ser404-Pro that Are Abnormally Phosphorylated in Alzheimer's Disease Brain

Abstract— Previously, we identified protein kinase F_A/gly-cogen synthase kinase-3 (GSK-3) as a microtubule-associated protein kinase that can incorporate 4 mol of phosphates into 1 mol of protein and cause its electrophoretic mobility shift in sodium dodecyl sulfate gels, a unique property characteristic of paired helical filament-associated pathological (PHF-) in Alzheimer's disease brains. In this report, we identified TPPKS(p)PSAAK and SPVVSGDTS(p)PR as two phosphorylation site sequences phosphorylated by kinase F_A/GSK-3 in using peptide sequence analysis and sequential manual Edman degradation for radiosequencing. When mapping with human brain sequence, we further identified Ser²³⁵-Pro and Ser⁴⁰⁴-Pro as the two major phosphorylation sites according to the numbering of the longest isoform. Ser²³⁵ and Ser⁴⁰⁴ have been reported as two of the major abnormal phosphorylation sites in PHF-. Taken together, the results provide initial evidence that protein kinase F_A/GSK-3 may represent one of the Ser-Pro motif-directed kinases involved in the abnormal phosphorylation of pathological PHF- in Alzheimer's disease brain.     

Cyclothiazide Modulates AMPA Receptor-Mediated Increases in Intracellular Free Ca2+ and Mg2+ in Cultured Neurons from Rat Brain

Abstract: We investigated the modulation of (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-induced increases in intracellular free Ca²⁺ ([Ca²⁺]_i) and intracellular free Mg²⁺ ([Mg²⁺]_i) by cyclothiazide and GYKI 52466 using microspectrofluorimetry in single cultured rat brain neurons. AMPA-induced changes in [Ca²⁺]_i were increased by 0.3–100 µ M cyclothiazide, with an EC₅₀ value of 2.40 µ M and a maximum potentiation of 428% of control values. [Ca²⁺]_i responses to glutamate in the presence of N -methyl- d -aspartate (NMDA) receptor antagonists were also potentiated by 10 µ M cyclothiazide. The response to NMDA was not affected, demonstrating specificity of cyclothiazide for non-NMDA receptors. Almost all neurons responded with an increase in [Ca²⁺]_i to both kainate and AMPA in the absence of extracellular Na⁺, and these Na⁺-free responses were also potentiated by cyclothiazide. GYKI 52466 inhibited responses to AMPA with an IC₅₀ value of 12.0 µ M . Ten micromolar cyclothiazide significantly decreased the potency of GYKI 52466. However, the magnitude of this decrease in potency was not consistent with a competitive interaction between the two ligands. Cyclothiazide also potentiated AMPA- and glutamate-induced increases in [Mg²⁺]_i. These results are consistent with the ability of cyclothiazide to decrease desensitization of non-NMDA glutamate receptors and may provide the basis for the increase in non-NMDA receptor-mediated excitotoxicity produced by cyclothiazide.     

Alkalinization Prolongs Recovery from Glutamate-Induced Increases in Intracellular Ca2+ Concentration by Enhancing Ca2+ Efflux Through the Mitochondrial Na+/Ca2+ Exchanger in Cultured Rat Forebrain Neurons

Abstract: Increasing extracellular pH from 7.4 to 8.5 caused a dramatic increase in the time required to recover from a glutamate (3 µ M , for 15 s)-induced increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) in indo-1-loaded cultured cortical neurons. Recovery time in pH 7.4 HEPES-buffered saline solution (HBSS) was 126 ± 30 s, whereas recovery time was 216 ± 19 s when the pH was increased to 8.5. Removal of extracellular Ca²⁺ did not inhibit the prolongation of recovery caused by increasing pH. Extracellular alkalinization caused rapid intracellular alkalinization following glutamate exposure, suggesting that pH 8.5 HBSS may delay Ca²⁺ recovery by affecting intraneuronal Ca²⁺ buffering mechanisms, rather than an exclusively extracellular effect. The effect of pH 8.5 HBSS on Ca²⁺ recovery was similar to the effect of the mitochondrial uncoupler carbonyl cyanide p -(trifluoromethoxyphenyl)hydrazone (FCCP; 750 n M ). However, pH 8.5 HBSS did not have a quantitative effect on mitochondrial membrane potential comparable to that of FCCP in neurons loaded with a potential-sensitive fluorescent indicator, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide (JC-1). We found that the effect of pH 8.5 HBSS on Ca²⁺ recovery was completely inhibited by the mitochondrial Na⁺/Ca²⁺ exchange inhibitor CGP-37157 (25 µ M ). This suggests that increased mitochondrial Ca²⁺ efflux via the mitochondrial Na²⁺/Ca²⁺ exchanger is responsible for the prolongation of [Ca²⁺]_i recovery caused by alkaline pH following glutamate exposure.     

Ca2+-Dependent Enhancement of [3H]Dopamine Uptake in Rat Striatum: Possible Involvement of Calmodulin-Dependent Kinases

Abstract: We studied effects of Ca²⁺ in the incubation medium on [³H]dopamine ([³H]DA) uptake by rat striatal synaptosomes. Both the duration of the preincubation period with Ca²⁺ (0–30 min) and Ca²⁺ concentration (0–10 m M ) in Krebs-Ringer medium affected [³H]DA uptake by the synaptosomes. The increase was maximal at a concentration of 1 m M Ca²⁺ after a 10-min preincubation (2.4 times larger than the uptake measured without preincubation), which reflected an increase in V _max of the [³H]DA uptake process. On the other hand, [³H]DA uptake decreased rapidly after addition of ionomycin in the presence of 1 m M Ca²⁺. The Ca²⁺-dependent enhancement of the uptake was still maintained after washing synaptosomes with Ca²⁺-free medium following preincubation with 1 m M Ca²⁺. Protein kinase C inhibitors did not affect apparently Ca²⁺-dependent enhancement of the uptake, whereas 1-[ N,O -bis(1,5-isoquinolinesulfonyl)- N -methyl- l -tyrosyl]-4-phenylpiperazine (KN-62; a Ca²⁺/calmodulin-dependent kinase II inhibitor) and wortmannin (a myosin light chain kinase inhibitor) significantly reduced it. Inhibitory effects of KN-62 and wortmannin appeared to be additive. N -(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7; a calmodulin antagonist) also remarkably inhibited the enhancement. These results suggest that Ca²⁺-dependent enhancement of [³H]DA uptake is mediated by activation of calmodulin-dependent protein kinases.     

Regulation of the L-Type Ca2+ Channel Current in Rat Pinealocytes

Abstract : In the present study, the role of phosphoprotein phosphatase in the regulation of L-type Ca²⁺ channel currents in rat pinealocytes was investigated using the whole-cell version of the patch-clamp technique. The effects of three phosphatase inhibitors, calyculin A, tautomycin, and okadaic acid, were compared. Although all three inhibitors were effective in inhibiting the L-type Ca²⁺ channel current, calyculin A was more potent than either tautomycin or okadaic acid, suggesting the involvement of phosphoprotein phosphatase-1. To determine the kinase involved in the regulation of these channels, cells were pretreated with H7 (a nonspecific kinase inhibitor), H89 (a specific inhibitor of cyclic AMP-dependent kinase), KT5823 (a specific inhibitor of cyclic GMP-dependent kinase), or calphostin C (a specific inhibitor of protein kinase C). Pretreatment with either H7 or calphostin C decreased the inhibitory effect of calyculin A on the L-type Ca²⁺ channel current. In contrast, pretreatment with H89 or KT5823 had no effect on the inhibition caused by calyculin A. Based on these observations, we conclude that basal phosphatase activity, probably phosphoprotein phosphatase-1, plays an important role in the regulation of L-type Ca²⁺ channel currents in rat pinealocytes by counteracting protein kinase C-mediated phosphorylation.     

Assembly Properties of Neurofilament Light Chain Ser55 Mutants in Transfected Mammalian Cells

Abstract: Ser⁵⁵ within the head domain of neurofilament light chain (NF-L) is transiently phosphorylated by protein kinase A, and phosphorylation of this residue is thought to regulate assembly of neurofilaments. To understand how Ser⁵⁵ phosphorylation influences NF-L assembly, wild-type and mutant NF-L genes in which Ser⁵⁵ was mutated to alanine, so as to prevent phosphorylation, or to aspartate, so as to mimic permanent phosphorylation, were transfected into mammalian cells that contain or do not contain an endogenous intermediate filament network. Wild-type and mutant NF-Ls localised to the Triton X-100-insoluble fraction, which suggests that phosphorylation of Ser⁵⁵ does not inhibit assembly of NF-L and NF-L/vimentin polymers at or below the tetrameric stage. Immunofluorescence microscopy of transfected cells demonstrated that the wild-type and mutant NF-Ls all colocalised with vimentin to produce similar filamentous arrays. However, in cells lacking an endogenous intermediate filament network, the aspartate mutant produced a pattern of staining different from that of the wild-type or alanine mutant. These results suggest that phosphorylation of NF-L Ser⁵⁵ is not a mechanism that precludes assembly of neurofilaments from monomers into intermediate filament structures but that phosphorylation/dephosphorylation of this residue might confer more subtle characteristics on neurofilament assembly properties and architecture.