Cyclic AMP independent protein kinase activity in rat cerebral cortex synaptic vesicles—Partial characterization

The intrinsic protein kinase activity of a highly purified synaptic vesicle preparation was characterized. The time-course of the reaction was found to be rapid and linear for about 1 min, but plateaued after 30 min by which time approximately 1 nmol of³²P pering protein was incorporated into trichloroacetic acid precipitated vesicular protein. The enzyme was optimally active at pH 6.0 (37°C), and had apparentK _m values of 40 and 88 M for ATP and GTP respectively. The enzyme was not stimulated by cAMP or cGMP. Mg²⁺ was required for maximal activity. The reaction was inhibited by free Ca²⁺, and non-selectively by Na⁺, K⁺, and NH₄ ⁺.Deceased.     

ER Ca2+ release and store-operated Ca2+ entry – partners in crime or independent actors in oncogenic transformation?

Ca²⁺ is a pleiotropic messenger that controls life and death decisions from fertilisation until death. Cellular Ca²⁺ handling mechanisms show plasticity and are remodelled throughout life to meet the changing needs of the cell. In turn, as the demands on a cell alter, for example through a change in its niche environment or its functional requirements, Ca²⁺ handling systems may be targeted to sustain the remodelled cellular state. Nowhere is this more apparent than in cancer. Oncogenic transformation is a multi-stage process during which normal cells become progressively differentiated towards a cancerous state that is principally associated with enhanced proliferation and avoidance of death. Ca²⁺ signalling is intimately involved in almost all aspects of the life of a transformed cell and alterations in Ca²⁺ handling have been observed in cancer. Moreover, this remodelling of Ca²⁺ signalling pathways is also required in some cases to sustain the transformed phenotype. As such, Ca²⁺ handling is hijacked by oncogenic processes to deliver and maintain the transformed phenotype. Central to generation of intracellular Ca²⁺ signals is the release of Ca²⁺ from the endoplasmic reticulum intracellular (ER) Ca²⁺ store via inositol 1,4,5-trisphosphate receptors (InsP₃Rs). Upon depletion of ER Ca²⁺, store-operated Ca²⁺ entry (SOCE) across the plasma membrane occurs via STIM-gated Orai channels. SOCE serves to both replenish stores but also sustain Ca²⁺ signalling events. Here, we will discuss the role and regulation of these two signalling pathways and their interplay in oncogenic transformation.     

Ca2+-dependent and -independent release of neurotransmitters from PC12 cells: a role for protein kinase C activation?

The intracellular mechanisms regulating the process of evoked neurotransmitter release were studied in the cloned neurosecretory cell line PC12. Various agents were employed that were known, from previous studies in other systems, to stimulate release in a manner either strictly dependent or independent of the concentration of extracellular Ca2+, [Ca2+]o. Three parameters were investigated in cells suspended in either Ca2+-containing or Ca2+-free Krebs-Ringer media: release of previously accumulated [3H]dopamine; average free cytoplasmic Ca2+ concentration, [Ca2+]i (measured by the quin2 technique); and cell ultrastructure, with special reference to the number and structure of secretion granules. The release induced by the ionophores transporting monovalent cations, X537A and monensin, occurred concomitantly with profound alterations of secretory granule structure (swelling and dissolution of the dense core). These results suggest that the effect of these drugs is due primarily to leakage of dopamine from granules to the cytoplasm and extracellular space. In contrast, the changes induced by other stimulatory drugs used concerned not the structure but the number of secretory granules, indicating that with these drugs stimulation of exocytosis is the phenomenon underlying the increased transmitter release. The release response induced by the Ca2+-ionophore ionomycin was dependent on [Ca2+]o, occurred rapidly, was concomitant with a marked rise of [Ca2+]i, and ceased after 1-2 min even though [Ca2+]i remained elevated for many minutes. 12-O-tetradecanoylphorbol, 13-acetate and diacylglycerol (both of which are known as activators of protein kinase C) induced slow responses almost completely independent of [Ca2+]o and not accompanied by changes of [Ca2+]i. Combination of an activator of protein kinase C with a low concentration of ionomycin failed to modify the [Ca2+]i rise induced by the ionophore, but elicited a marked potentiation of the release response, which was two- to fourfold larger than the sum of the responses elicited separately by either drugs. Thus, activation of protein kinase C seems to play an important role in the regulation of exocytosis in neurosecretory cells, possibly by increasing and maintaining the sensitivity to Ca2+ of the intracellular apparatus regulating granule discharge by exocytosis.     

Colocalization of protein kinase C ⊺-subtype and calcitonin gene-related peptide in rat spinal cord

Summary Colocalization of calcitonin gene-related peptide (CGRP) and protein kinase C -subtype (PKC-) like immunoreactivities (LI) and the subcellular localization of CGRP-LI were studied in the ventral horn of rat spinal cord. Ultrastructurally CGRP-LI was localized on the membranes of the Golgi-complexes, in multivesicular bodies and in vesicles adjacent to the Golgi-complex in motoneuron perikarya. The colocalization of PKC- and CGRP-LI was detected in most of the ventral horn motoneurons. However, few motoneurons were only PKC--immunoreactive. These results suggest that PKC- may be involved in the regulation of CGRP release from motoric axon terminals.     

Regulation of Ca2+ release by cAMP-dependent protein kinase. A mechanism for agonist-specific calcium signaling?

Calcium is an ubiquitous second messenger that is involved in the regulation of a number of cell functions. The mechanism by which the specificity of calcium signaling is achieved is not well understood. We suggest that calcium release from the ER can occur selectively at different spatial locations in response to different extracellular stimuli. We discuss a possible mechanism for such selectivity and present a model based on this mechanism. The suggested mechanism is based on the regulation of local Ca2+ release by cyclic AMP-dependent protein kinase (PKA) and relies upon two experimental observations: first, some G-protein coupled signaling pathways activate PLC and regulate adenylate cyclase at the same time, leading to IP3 production and altering PKA activity via changes in cAMP level; second, phosphorylation by PKA alters the properties of IP3 receptor (IP3R). In our model we consider allosteric regulation of IP3Rs by IP3 and cAMP-dependent phosphorylation. The differences in IP3Rs and PKA densities at different spatial locations within the cell allow the release of calcium selectively at each location in response to certain combination of IP3 and cAMP concentration. Specificity of agonist-response coupling is achieved if different combinations in the levels of these second messengers are specific for different extracellular stimuli.     

Lobe-specific functions of Ca2+·calmodulin in alphaCa2+·calmodulin-dependent protein kinase II activation

N-methyl-D-aspartic acid receptor-dependent long term potentiation (LTP), a model of memory formation, requires Ca2+·calmodulin-dependent protein kinase II (αCaMKII) activity and Thr286 autophosphorylation via both global and local Ca2+ signaling, but the mechanisms of signal transduction are not understood. We tested the hypothesis that the Ca2+-binding activator protein calmodulin (CaM) is the primary decoder of Ca2+ signals, thereby determining the output, e.g. LTP. Thus, we investigated the function of CaM mutants, deficient in Ca2+ binding at sites 1 and 2 of the N-terminal lobe or sites 3 and 4 of the C-terminal CaM lobe, in the activation of αCaMKII. Occupancy of CaM Ca2+ binding sites 1, 3, and 4 is necessary and sufficient for full activation. Moreover, the N- and C-terminal CaM lobes have distinct functions. Ca2+ binding to N lobe Ca2+ binding site 1 increases the turnover rate of the enzyme 5-fold, whereas the C lobe plays a dual role; it is required for full activity, but in addition, via Ca2+ binding site 3, it stabilizes ATP binding to αCaMKII 4-fold. Thr286 autophosphorylation is also dependent on Ca2+ binding sites on both the N and the C lobes of CaM. As the CaM C lobe sites are populated by low amplitude/low frequency (global) Ca2+ signals, but occupancy of N lobe site 1 and thus activation of αCaMKII requires high amplitude/high frequency (local) Ca2+ signals, lobe-specific sensing of Ca2+-signaling patterns by CaM is proposed to explain the requirement for both global and local Ca2+ signaling in the induction of LTP via αCaMKII.     

Localization of Ca2+-binding proteins of rat cortex on two-dimensional gels—II. analysis of Ca2+ binding proteins in ammonium sulfate fractions of rat brain

A total of seven high-affinity calcium-binding proteins have been detected in rat brain. This was accomplished using a combination of ammonium sulfate fractionation, two-dimensional gel electrophoresis, western blotting and ⁴⁵Ca²⁺-autoradiography. Of these seven proteins, three are detectable in a crude tissue punch of rat cortex while four are seen only after protein enrichment with ammonium sulfate. Four of the seven proteins detected in this study have been identified: calmodulin, the B subunit of calcineurin, the intestinal vitamin D-dependent calcium-binding protein and parvalbumin. The identities of the other three proteins visualized by ⁴⁵Ca²⁺-autoradiography in this study are unknown.     

Are calcium-dependent protein kinases involved in the regulation of glycolytic/gluconeogenetic enzymes? Studies with Ca2+/calmodulin-dependent protein kinase and protein kinase C

Changes in glycolytic flux have been observed in liver under conditions where effects of cAMP seem unlikely. We have, therefore, studied the phosphorylation of four enzymes involved in the regulation of glycolysis and gluconeogenesis (6-phosphofructo-1-kinase from rat liver and rabbit muscle; pyruvate kinase, 6-phosphofructo-2-kinase and fructose-1,6-bisphosphatase from rat liver) by defined concentrations of two cAMP-independent protein kinases: Ca2+/calmodulin-dependent protein kinase and Ca2+/phospholipid-dependent protein kinase (protein kinase C). The results were compared with those obtained with the catalytic subunit of cAMP-dependent protein kinase. The following results were obtained. 1. Ca2+/calmodulin-dependent protein kinase phosphorylates 6-phosphofructo-1-kinase and L-type pyruvate kinase at a slightly lower rate as compared to cAMP-dependent protein kinase. 2. 6-Phosphofructo-1-kinase is phosphorylated by the two kinases at a single identical position. There is no additive phosphorylation. The final stoichiometry is 2 mol phosphate/mol tetramer. The same holds for L-type pyruvate kinase except that the stoichiometry with either kinase or both kinases together is 4 mol phosphate/mol tetramer. 3. Rabbit muscle 6-phosphofructo-1-kinase is phosphorylated by cAMP-dependent protein kinase but not by Ca2+/calmodulin-dependent protein kinase. 4. Fructose-1,6-bisphosphatase from rat but not from rabbit liver is phosphorylated at the same position but at a markedly lower rate by Ca2+/calmodulin-dependent protein kinase when compared to the phosphorylation by cAMP-dependent protein kinase. 5. 6-Phosphofructo-2-kinase is phosphorylated by Ca2+/calmodulin-dependent protein kinase only at a negligible rate. 6. Protein kinase C does not seem to be involved in the regulation of the enzymes examined: only 6-phosphofructo-2-kinase became phosphorylated to a significant degree. In contrast to the phosphorylation by cAMP-dependent protein kinase, this phosphorylation is not associated with a change of enzyme activity. This agrees with our observation that the sites of phosphorylation by the two kinases are different. The results indicate that Ca2+/calmodulin-dependent protein kinase but not protein kinase C could be involved in the regulation of hepatic glycolytic flux under conditions where changes in the activity of cAMP-dependent protein kinase seem unlikely.     

An allosteric modulator of metabotropic glutamate receptors (mGluR₂), (+)-TFMPIP, inhibits restraint stress-induced phasic glutamate release in rat prefrontal cortex

The potential anxiolytic effects of a novel positive allosteric modulator (PAM) of the metabotropic glutamate receptor subgroup 2 (mGluR?) were investigated using a self-referencing recording technique with enzyme-based microelectrode arrays (MEAs) that reliably measures tonic and phasic changes in extracellular glutamate levels in awake rats. Studies involved glutamate measures in the rat prefrontal cortex during subcutaneous injections of the following: vehicle, a mGluR?/? agonist, LY354740 (10?mg/kg), or a mGluR? PAM, 1-Methyl-2-((cis-(R,R)-3-methyl-4-(4-trifluoromethoxy-2-fluoro)phenyl)piperidin-1-yl)methyl)-1H-imidazo[4,5-b]pyridine ((+)-TFMPIP; 1.0 or 17.8?mg/kg). Studies assessed changes in tonic glutamate levels and the glutamatergic responses to a 5-min restraint stress. Subcutaneous injection of (+)-TFMPIP at a dose of 1.0?mg/kg (day 3: -7.1?±?15.1 net AUC; day 5: -24.8?±?24.9 net AUC) and 17.8?mg/kg (day 3: -46.5?±?33.0 net AUC; day 5: 34.6?±?36.8 net AUC) significantly attenuated the stress-evoked glutamate release compared to vehicle controls (day 3: 134.7?±?50.6 net AUC; day 5: 286.6?±?104.5 net?AUC), whereas the mGluR?/? agonist LY354740 had no effect. None of the compounds significantly affected resting glutamate levels, which we have recently shown to be extensively derived from neurons. Taken together, these data support that systemic administration of (+)-TFMPIP produces phasic rather than tonic release of glutamate that may play a major role in the effects of stress on glutamate neuronal systems in the prefrontal cortex.     

Reversible effect of calcium-binding protein regucalcin on the Ca2+-induced inhibition of deoxyuridine 5′-triphosphatase activity in rat liver cytosol

The effect of regucalcin, a calcium-binding protein isolated from rat liver cytosol, on deoxyuridine 5′-triphosphatase (dUTPase) in the cytosol of rat liver was investigated. Addition of Ca²⁺ up to 5.0 μM to the enzyme reaction mixture caused a significant decrease of dUTPase activity, while Zn²⁺, Cd²⁺, Co²⁺, Al³⁺, Mn²⁺ and Ni²⁺ (10 μM) did not have an appreciable effect. The Ca²⁺-induced decrease of dUTPase activity was reversed by the presence of regucalcin; the effect was complete at 1.0 μM of the protein. Regucalcin had no effect on the basal activity of the enzyme. Meanwhile, the reversible effect of regucalcin on the Ca²⁺ (10 μM)-induced decrease of dUTPase activity was not altered by the coexistence of Cd²⁺ or Zn²⁺ (10 μM). The present data suggest that liver cytosolic dUTPase is uniquely regulated by Ca²⁺ of various metals, and that the Ca²⁺ effect is reversed by regucalcin.     

Design and synthesis of protein kinase Cα activators based on 'out of pocket' interactions

Novel types of PKCα activators based on isobenzofuranone bearing a myo-inositol moiety were designed and synthesized. The derivatives with bulky substituents on the myo-inositol moiety significantly activated PKCα, but their binding sites were not the same as that of phorbol ester.     

Biochemical characterization of tau protein and its associated syndapin 1 and protein kinase Cɛ for their functional regulation in rat brain

Background

We recently reported that both sulfatide and cholesterol-3-sulfate (SCS) function as potent stimulators for the GSK-3β-mediated phosphorylation of tau protein (TP) in vitro [J. Biochem. 143 (2008) 359–367].

Methods

By means of successive gel filtration on a Superdex 200 pg column and three distinct ion-exchange column chromatographies, TP and its associated proteins were highly purified from the extract of rat brain.

Results

We found that (i) syndapin 1 and novel protein kinase C? (nPKC?) were identified as the TP-associated proteins; (ii) SCS highly stimulated the phosphorylation of TP and syndapin 1 by nPKC? as well as CK1; (iii) the full phosphorylation of TP and syndapin 1 by nPKC? in the presence of sulfatide resulted in their dissociation; (iv) TP primed by CK1 functioned as an effective phosphate acceptor for GSK-3β; (v) syndapin 1 highly stimulated the GSK-3β-mediated phosphorylation of TP; and (vi) TP isoforms were highly expressed in aged brain, whereas syndapin 1 was consistently detected in adult brain, but not in newborn brain.

General significance

These results provided here suggest that (i) TP-associated nPKC? suppresses the GSK-3β-mediated phosphorylation of TP through the phosphorylation of GSK-3β by the kinase in vitro; and (ii) SCS act as effective sole mediators to induce the GSK-3β-mediated high phosphorylation of both TP and its associated syndapin 1 involved in the biochemical processes of neuronal diseases, including Alzheimer's disease.     

Impaired TNF-α control of IP3R-mediated Ca2+ release in Alzheimer's disease mouse neurons

The misguided control of inflammatory signaling has been previously implicated in the pathogenesis of several neurological disorders, including Alzheimer's disease (AD). Induction of tumor necrosis factor-alpha (TNF-α), a central mediator of neuroinflammation, occurs commensurate with the onset of early disease in 3xTg-AD mice, which develop both amyloid plaque and neurofibrillary tangle pathologies in an age- and region-dependent pattern. Herein, we describe regulation inherent to 3xTg-AD neurons, which results in the loss of TNF-α mediated enhancement of inositol 1,4,5 trisphosphate (IP₃R)-mediated Ca²⁺ release. This modulation also leads to significant down-regulation of IP₃R signaling following protracted cytokine exposure. Through the experimental isolation of each AD-related transgene, it was determined that expression of the PS1^M146V transgene product is responsible for the loss of the TNF-α effect on IP₃R-mediated Ca²⁺ release. Furthermore, it was determined that the suppression of TNF-α receptor expression occurred in the presence of the presenilin transgene. Our findings attribute this familial AD mutation to suppressing a Ca²⁺-regulated signal cascade potentially intended to “inform” neurons of proximal neuroinflammatory events and trigger compensatory responses for protection of neural transmission.     

Ei24-deficiency attenuates protein kinase Cα signaling and skin carcinogenesis in mice

Etoposide-induced gene 24 (Ei24) is a p53 target gene that inhibits growth, induces apoptosis and autophagy, as well as suppresses breast cancer. To evaluate the role of Ei24 in in vivo tumorigenesis, we generated an Ei24-deficient mouse model. Here, we report that, although Ei24 homozygous knockout mice are embryonic lethal, Ei24 heterozygous null mice are attenuated to DMBA/TPA-induced carcinogenesis with regard to the number and size of tumors but not the incidence. Ei24 contains a functional consensus motif, named as an R motif that is highly analogous to amino acids 105-110 of RINCK1, an E3 ligase for protein kinase C (PKC) proteins. We found that Ei24 stabilizes PKCαvia RINCK degradation and competition with RINCK for binding with the C1a domain of PKCα. We also found that Ei24 contributes to PKCα-mediated transactivation of EGFR by promoting PKCα membrane localization and interaction with EGFR. Finally, using Oncomine database we show that Ei24 and EGFR are upregulated in some subsets of human HNSCC. These results suggest that Ei24 is a regulator of the RINCK1-PKCα-EGFR signaling pathway in the development of skin-cancer.     

Teratogenicity of retinoic acid and its effects on TGF-β2 expression in the developing cerebral cortex of the rat

Vitamin A metabolites are potent teratogens in a wide variety of species, including man. Transforming growth factor betas (TGF-s) are involved in several mammalian prenatal developmental processes. The aim of this study was to determine the effects of exogenous and excessive all-trans retinoic acid on TGF2 expression in the developing cerebral cortex of the rat. Many of the malformations including exencephaly, exophtalmus, abdominal wall defects, extremity reduction defects observed in this study were dependent on the time of administration of retinoic acid. TGF-2 was diversely expressed, as revealed immunohistochemically, in the cerebral cortex and plexus choroideus. The diversity depended on the gestational day and the was affected by the administration of retinoic acid. In the 15-day-old fetus from mothers who had been fed by gavage a single dose of 60mg/kg body weight of all-trans retinoic acid on the 8th day of gestation, TGF-2 immunoreactivity in the brain was decreased. However, by the 18th day of gestation, TGF-2 expression increased. The expression of TGF-2 in fetuses whose mothers had been given all-trans retinoic acid after the neurulation period (on day 12 of gestation) was generally similar to that in a control group. We conclude that all-trans retinoic acid leads to severe congenital malformations if administered before neurulation whereas if given after neurulation, it is not so teratogenic. Further, retinoic acid has a variable effect on the expression of TGF-2.     

Convulsions and inhibition of glutamate decarboxylase by pyridoxal phosphate-γ-glutamyl hydrazone in the developing rat

We have previously shown that in the adult rat the inhibition of brain glutamate decarboxylase (GAD) activity by pyridoxal phosphate--glutamyl hydrazone (PLPGH) administration does not result in convulsions, whereas in the adult mouse intense convulsions invariably occur. In the present study we report that, surprisingly, immature rats from 2 to 20 days of age treated with PLPGH (80 mg/kg) showed generalized tonic-clonic convulsions, whereas no convulsions at all were present in 30 days-old or older rats. GAD activity, measured by enzymic determination of GABA formed in forebrain homogenates, was inhibited by about 60% at the time of convulsions in 15 days-old and younger rats, whereas the inhibition was between 40 and 50% in older animals. The addition of the coenzyme pyridoxal 5-phosphate to the incubation medium completely reversed this inhibition. In all treated animals GABA levels were lower compared to controls. The results indicate that the susceptibility of GAD in vivo to a diminished cofactor concentration decreases with age. It seems possible that changes in the expression of enzyme forms are reflected in developmental variations in the susceptibility to seizures induced by vitamin B₆ depletion, but alterations of other B₆-dependent biochemical pathways cannot be discarded.     

α-tocopherol modulates tyrosine phosphorylation in human neutrophils by inhibition of protein kinase C activity and activation of tyrosine phosphatases

α-Tocopherol augmentation in human neutrophils was investigated for effects on neutrophil activation and tyrosine phosphorylation of proteins, through its modulation of protein kinase C (PKC) and tyrosine phosphatase activities. Incubation of neutrophils with α-tocopherol succinate (TS) resulted in a dose-dependent incorporation into cell membranes, up to 2.5 nmol/2 × 10⁶ cells. A saturating dose of TS (40 μmol/l) inhibited oxidant production by neutrophils stimulated with phorbol myristate acetate (PMA) or opsonized zymosan (OZ) by 86 and 57%, as measured by luminol-amplified chemiluminescence (CL). With PMA, TS inhibited CL generation to a similar extent to staurosporine (10 nmol/l) or genistein (100 μmol/l), and much more than Trolox (40 μmol/l). With OZ, TS inhibited CL to a similar extent to Trolox. Neutrophil PKC activity was inhibited 50% or more by TS or staurosporine. The enzyme activity was unaffected by genistein or Trolox, indicating a specific interaction of α-tocopherol. TS or Trolox increased protein tyrosine phosphorylation in resting neutrophils, and as with staurosporine further increased tyrosine phosphorylation in PMA-stimulated neutrophils, while the tyrosine kinase (TK) inhibitor genistein diminished phosphorylation. These effects in resting or PMA-stimulated neutrophils were unrelated to protein tyrosine phosphatase (PTP) activities, which were maintained or increased by TS or Trolox. In OZ-stimulated neutrophils, on the other hand, all four compounds inhibited the increase in tyrosine-phosphorylated proteins. In this case, the effects of pre-incubation with TS or Trolox corresponded with partial inhibition of the marked (85%) decrease in PTP activity induced by OZ. These results indicate that α-tocopherol inhibits PMA-activation of human neutrophils by inhibition of PKC activity, and inhibits tyrosine phosphorylation and activation of OZ-stimulated neutrophils also through inhibition of phosphatase inactivation.     

Hypoxia-inducible factor-1α regulates the expression of L-type voltage-dependent Ca2+ channels in PC12 cells under hypoxia

Hypoxia is an important factor in regulation of cell behavior both under physiological and pathological conditions. The mechanisms of hypoxia-induced cell death have not been completely elucidated yet. It is well known that Ca²⁺ is critically related to cell survival. Hypoxia-inducible factor-1α (HIF-1α) is a core regulatory factor during hypoxia, and L-type voltage-dependent Ca²⁺ channels (L-VDCCs) have been reported to play a critical role in cell survival. This study was conducted to explore the relationship between L-VDCC expression and HIF-1α regulation in PC12 cells under hypoxia. PC12 cells were treated at 20 or 3 % O₂ to observe its proliferation and the intracellular calcium concentration. Then, we detected the protein expression of HIF-1α and L-VDCCs subtypes, Ca_v1.2 and Ca_v1.3. At last, to verify the relationship between HIF-1α and Ca_v1.2 and Ca_v1.3, we got the expression of Ca_v1.2 and Ca_v1.3 with Western blot and luciferase report gene assays after PC12 cells were treated by echinomycin, which is an HIF-1α inhibitor. Compared with 20 % O₂ (normoxia), 3 % O₂ (hypoxia) inhibited cell proliferation, increased the intracellular calcium concentration, and induced protein expression of HIF-1α. The protein expression of two L-VDCCs subtypes expressed in the nervous system, Ca_v1.2 and Ca_v1.3, was upregulated by hypoxia and reduced dose dependently by treatment with echinomycin, a HIF-1α inhibitor. Luciferase report gene assays showed that the expression of Ca_v1.2 and Ca_v1.3 genes was augmented under 3 % O₂. However, echinomycin only slightly and dose dependently decreased expression of the Ca_v1.2 gene, but not that of the Ca_v1.3 gene. These data indicated that Ca_v1.2 might be regulated by HIF-1α as one of its downstream target genes and involved in regulation of PC12 cells death under hypoxia.     

Morphological changes and expression of protein kinase CK2 β subunit in the microglia after hypoglossal nerve transection

Following hypoglossal nerve transection, the microglia of the rat hypoglossal nucleus expressed protein kinase CK2 β subunit immunoreactivity. CK2 β immunostaining occurred on the operated side from postoperative day 3; on day 5 we observed strong immunoreactivity and the immunopositive microglial cell processes surrounded the injured neurones. Thereafter, the immunoreactivity decreased gradually and on day 10 the immunopositive cells surrounded only a few injured neurones. Electron microscopic observations on the hypoglossal nucleus revealed microglia-neuronal contact within 3 hours of nerve injury, and by day 3 all the injured neurones were in contact with microglial cells. These observations indicated that microglia-neuronal contact occurred earlier than the CK2 β subunit immunoreactivity. CK2 may not be implicated during the initial migration of the microglia to the injured neurones; however, it may enhance the growth and elongation of the microglial cell processes around the injured neurones.