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J A Wagner

Publications and source records attributed to J A Wagner.

At least 73 records · Page 4Linked to original sources
PubMed1993

Peptide growth factors protect against ischemia in culture by preventing nitric oxide toxicity.

Reduction or elimination of nitric oxide (NO) production in cortical neurons by NO synthase (NOS) inhibitors during glutamate toxicity in vitro or during focal cerebral ischemia in vivo can prevent neuronal cell death. In contrast, growth factors can prevent neuronal degeneration induced by treatment with glutamate or potassium cyanide. We have determined whether NO mediates hippocampal cell death during anoxia in vitro and whether the peptide growth factors basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) can prevent hippocampal neuronal death during anoxia or NO exposure. Both bFGF and EGF increased hippocampal neuronal survival from about 35% in anoxic cultures to about 65% in treated cultures during an 8 hr period of anoxia. Inhibition of NOS by NG-monomethyl-L-arginine, a competitive inhibitor of NOS, rescued 65-70% of the neurons that would normally die during an 8 hr anoxic incubation, and this effect was reversed by L-arginine, a precursor for NO. Thus, hippocampal neuronal death following anoxia is, at least in part, mediated by NO. NO, generated by either nitroprusside or 3-morpholino-sydnonimine, was toxic to hippocampal neurons. Pretreatment of cultures with either bFGF (10 ng/ml) or EGF (10 ng/ml) prior to NO exposure increased survival from approximately 40% in untreated cultures to 80% in treated cultures, yet the effect of combining bFGF and EGF was not greater than treatment with either of the growth factors alone. Knowledge that the growth factors bFGF and EGF are neuroprotective against NO toxicity provides insights into the mechanisms of ischemic neuronal death that may direct future therapeutic modalities for cerebrovascular disease and neurodegenerative disorders.

Amino Acid Oxidoreductases↗
PubMed1993

Growth factors protect PC12 cells against ischemia by a mechanism that is independent of PKA, PKC, and protein synthesis.

We have established an in vitro model of ischemia incorporating the combination of anoxia with glucose deprivation, which is toxic to PC12 cells. In this model, nerve growth factor (NGF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) improve PC12 cell survival. K252a, a specific inhibitor of NGF-induced trk p140 autophosphorylation, did not alter the neuroprotection provided by EGF or bFGF, yet it completely abolished the protection provided by NGF. Activation of protein kinase A (PKA) with dibutyryl-cAMP also protected during ischemia, although it was not additive with the effect provided by growth factors. Furthermore, growth factors protected a PKA-deficient mutant as effectively as the parental cell line; thus, activation of PKA is protective against ischemia but is not necessary for the action of peptide growth factors. Neither the stimulation of protein kinase C (PKC) with acute phorbol ester treatment nor the downregulation of PKC with chronic high-dose phorbol ester treatment resulted in an altered response to growth factors in either the PC12 wild type or PKA-deficient mutant. Thus, protection by peptide growth factors depends on neither PKA nor PKC. Furthermore, downregulation of PKC alone was protective, indicating that PKC may contribute to toxicity. Interestingly, treatment with the kinase inhibitor H-7 was neuroprotective and may have enhanced the neuroprotective effect of NGF. In contrast, staurosporine, a broadly acting kinase inhibitor, inhibited the neuroprotective effect of NGF, but not of EGF or FGF.(ABSTRACT TRUNCATED AT 250 WORDS)

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

Retinoic acid regulates both expression of the nerve growth factor receptor and sensitivity to nerve growth factor.

In PC12 cells, retinoic acid (RA) stimulates the expression of p75NGFR, a component of the nerve growth factor (NGF) receptor, as indicated by a rapid increase in p75NGFR mRNA, an increase in the binding of 125I-labeled NGF to p75NGFR, and an increase in the binding of NGF to low affinity sites. RA-treated cells are more sensitive to NGF, but not to either fibroblast growth factor or phorbol 12-myristate 13-acetate, showing that RA has a specific effect on the responsiveness of PC12 cells to NGF. Exposure to RA leads neither to an increase in the expression of mRNA for trk, another component of the NGF receptor, nor to an increase in binding to high affinity receptors, suggesting that an increase in the expression of p75NGFR is sufficient to make cells more sensitive to NGF. This work suggests that, in addition to having direct effects on gene expression, RA can indirectly modulate differentiation of neurons by modifying their expression of cell surface receptors to peptide growth factors.

Animals↗
PubMed1992

Antisense oligodeoxynucleotides to the cystic fibrosis transmembrane conductance regulator inhibit cAMP-activated but not calcium-activated chloride currents.

Phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) by cAMP-dependent protein kinase leads to chloride flux in epithelial cells. Is CFTR also required for the calcium-dependent activation of chloride channels? We used antisense oligodeoxynucleotides to CFTR to reduce the expression of CFTR in colonic and tracheal epithelial cells. The antisense oligomers were a pair of adjacent 18-mers complementary to nucleotides 1-18 and 19-36 of CFTR mRNA. Sense and misantisense oligomers served as controls. A 48-h antisense treatment reduced the expression of CFTR protein as assayed by immunoprecipitation and autoradiography to 26% of the level in sense-treated T84 cells. Whole-cell patch clamp revealed that a 48-h antisense treatment of T84 and 56FHTE-8o- fetal tracheal epithelial cells reduced the cAMP-activated chloride current to approximately 10% of that in sense-treated cells. The half-life of functional CFTR is less than 24 h in these cells. In contrast, the calcium-activated chloride current was not affected by antisense treatment. Hence, the cAMP and calcium pathways are separate. CFTR is required for the cAMP pathway but not for the calcium pathway.

Base Sequence↗
PubMed1992

Characterization of immortal cystic fibrosis tracheobronchial gland epithelial cells.

Tracheobronchial glands were isolated and cultured from a patient with cystic fibrosis (CF). Cultured epithelial cells were transformed with pSVori-. All transformed cell lines express cytokeratin filaments and at early passages express the junctional complex molecule cell CAM 120/80, indicating their epithelial origin. Several gland cell lines express antigens that localize to secretory cells in vivo. Cl- transport measured by 36Cl efflux shows that CF gland epithelial cells, like CF surface airway and nasal polyp epithelial cells, are unable to respond to increases in intracellular cAMP. However, they do produce an increase in intracellular cAMP after treatment with isoproterenol or forskolin. One CF gland cell line shows increased intracellular calcium in response to a number of agents and increased Cl- efflux comparable to that observed in a non-CF airway surface epithelial cell line after addition of calcium ionophore. All cell lines express CF transmembrane conductance regulator mRNA, as measured by PCR amplification of first-strand cDNA. The CF tracheobronchial gland cell lines described here are compound heterozygotes, having a single copy of the delta F508 mutation.

Base Sequence↗
PubMed1992

Synergistic effects of nerve growth factor and phorbol 12-myristate 13-acetate on rapid motility and process formation in PC12 cells: the role of laminin.

A126-1B2 cells, a PKA (cAMP-dependent protein kinase)-deficient variant of PC12 cells, but not parental PC12 cells, form processes within 15-30 min of exposure to both nerve growth factor (NGF) and activators of protein kinase C when grown on tissue culture plastic (Glowacka and Wagner, J Neurosci Res 25: 453-462, 1990). Time-lapse microscopy has demonstrated that these processes are formed by a novel mechanism, i.e., rapid movement of the cell body away from a point of attachment, which morphologically resembles a growth cone. These "fast" neurites are attached to the substratum at a number of points, which display membrane activity in the form of active ruffling and the extension of filopodia and membrane pleats. Thus, these processes are formed by a mechanism distinct from that used by PC12 and other neuronal cells to form processes in culture. Wild-type PC12 cells also migrate and form fast neurites in response to a combination of NGF and phorbol 12-myristate 13-acetate (PMA), when they are grown in conditioned media or plates, suggesting that a secreted factor that can bind to the substratum is essential for the rapid formation of these neurites. Similarly, wild-type PC12 cells grown on a laminin-coated substratum also migrate and form "fast neurites" in response to a combination of NGF and PMA. This rapid migration is attenuated by an anti-alpha 1, beta 1-integrin antisera, implicating a laminin-integrin interaction; and it is inhibited by alpha-lactalbumin, suggesting an involvement of a beta 1,4 galactosyltransferase in the response. The formation of fast neurites is not dependent on concurrent protein synthesis, but it is inhibited by lithium, cytochalasin D, and methylthioadenosine or pretreatment of cells with NGF. Thus PC12 cells grown on the appropriate substrate have the ability to migrate rapidly and thereby form neuron-like processes within minutes of exposure to NGF and PMA. This morphological response to a combination of agents may provide an alternative means by which nerve cells form connections. Alternatively, it may reflect a mechanism that facilitates cellular migration during developmental processes.

Animals↗
PubMed1992

Rapid fibroblast growth factor-induced increases in protein phosphorylation and ornithine decarboxylase activity: regulation by heparin and comparison to nerve growth factor-induced increases.

Fibroblast growth factors (FGFs), like nerve growth factor (NGF), induce morphological differentiation of PC12 cells. This activity of FGF is regulated by glycosaminoglycans. To further understand the mechanisms of FGF and glycosaminoglycan actions in PC12 cells, we studied the regulation of protein phosphorylation and ornithine decarboxylase (ODC) activity by FGF in the presence and absence of heparin. As with NGF, aFGF and bFGF increased the incorporation of radioactive phosphate into the protein tyrosine hydroxylase (TH). The increase in TH phosphorylation was localized to the tryptic peptide, T3. Both T3 and T1 phosphorylations occur in response to NGF, but there was no evidence that aFGF or bFGF stimulated the phosphorylation of the T1 peptide. This result suggests differential regulation of second messenger systems by NGF and FGF in PC12 cells. Heparin, at a concentration that potentiated aFGF-induced neurite outgrowth 100-fold (100 micrograms/ml), did not alter the ability of aFGF to increase S6 phosphorylation or ODC activity. One milligram per milliliter of heparin, a concentration that inhibited bFGF-induced neurite outgrowth, also inhibited bFGF-induced increases in S6 phosphorylation and ODC activity. These observations suggest (i) that acidic and basic FGF activate a protein kinase, possibly protein kinase C, resulting in the phosphorylation of peptide T3 of TH; (ii) that the FGFs and NGF share some but not all second messenger systems; (iii) that heparin potentiates aFGF actions and inhibits bFGF actions in PC12 cells via distinct mechanisms; (iv) that heparin does not potentiate the neurite outgrowth promoting activity of aFGF by enhancing binding to its PC12 cell surface receptor; and (v) that heparin may coordinately regulate several activities of bFGF (induction of protein phosphorylation, ODC and neurite outgrowth) via a common mechanism, most likely by inhibiting the productive binding of bFGF to its PC12 cell surface receptor.

Animals↗
PubMed1992

The activity of cAMP-dependent protein kinase is required at a posttranslational level for induction of voltage-dependent sodium channels by peptide growth factors in PC12 cells.

The synthesis and expression of voltage-dependent sodium (Na) channels is a crucial aspect of neuronal differentiation because of the central role these ion channels play in the generation of action potentials and the transfer of information in the nervous system. We have used rat pheochromocytoma (PC12) cell lines deficient in cAMP-dependent protein kinase (PKA) activity to examine the role of PKA in the induction of Na channel expression by nerve growth factor (NGF) and basic FGF (bFGF). In the parental PC12 cell line both NGF and bFGF elicit an increase in the density of functional Na channels, as determined from whole-cell patch clamp recordings. This increase does not occur in two PC12 cell lines deficient in both isozymes of PKA (PKAI and PKAII), and is strongly reduced in a third line deficient in PKAII, but not PKAI. Despite the inability of the neurotrophic factors to induce functional Na channel expression in the PKA-deficient cells, Northern blot hybridization studies and saxitoxin binding assays of intact cells indicate that NGF and bFGF are still capable of eliciting increases in both Na channel mRNA and Na channel protein in the membrane. Thus, PKA activity appears to be necessary at a posttranslational step in the synthesis and expression of functional Na channels, and thereby plays an important role in determining neuronal excitability.

Animals↗
PubMed1992

Activation of protein kinase A is necessary but not sufficient for ethanol-induced desensitization of cyclic AMP production.

Acute addition of EtOH to PC 12 pheochromocytoma cells increases cyclic AMP production, whereas chronic exposure to EtOH results in a decrease in the stimulation of cyclic AMP production in response to 2-chloroadenosine and forskolin. This EtOH-induced desensitization was not observed after chronic EtOH treatment of A126-1B2-1 cells which are a protein kinase A-deficient mutant cell line derived from PC 12 cells. Furthermore, in the parental PC 12 cell line the cell-permeable protein kinase A inhibitor, Rp-isomer of adenosine 3',5'-monophosphorothioate, blocked the development of EtOH-induced desensitization. Thus, activation of protein kinase A is apparently necessary for EtOH-induced desensitization of cyclic AMP production. Chronic treatment of PC 12 cells with forskolin qualitatively mimicked the desensitization observed with chronic EtOH exposure. However, the degree of desensitization induced by forskolin was significantly less than that caused by EtOH even though the acute addition of forskolin caused a greater increase in cyclic AMP production. Furthermore, the acute addition of EtOH inhibited forskolin-stimulated cyclic AMP production, yet inclusion of EtOH during the chronic forskolin treatment of PC 12 cells resulted in a greater degree of desensitization. These findings indicate an obligatory role of protein kinase A in EtOH-induced desensitization of cyclic AMP production in PC 12 cells. However, because protein kinase A activation alone is not sufficient to account for the degree of desensitization, EtOH probably also acts through a mechanism in addition to activation of protein kinase A.

2-Chloroadenosine↗
PubMed1992

NGF induces the expression of the VGF gene through a cAMP response element.

NGF is a peptide growth factor that plays a key role in the differentiation and survival of neurons in both the PNS and CNS. NGF acts through both transcription-dependent and transcription-independent mechanisms to regulate the differentiation of PC12 cells. To better understand the regulation of gene expression by NGF, we have defined a cis-acting sequence that is immediately upstream of the transcription start site of the VGF (a2/NGF33.1) gene that is required for induction by NGF. Within this sequence is a consensus cAMP response element (CRE) embedded in a 14 base pair palindrome. Mutations in this CRE eliminate induction of the VGF gene both by NGF and by agents that act via cAMP. Although this sequence confers transcriptional induction by both NGF and cAMP, it is not sufficient to allow induction by epidermal growth factor, acidic or basic fibroblast growth factor, or phorbol 12-myristate 13-acetate (PMA). Thus, this sequence defines an element that is selectively activated by NGF and cAMP. Promoter fragments from the VGF gene that include the core CRE efficiently bind the inducible transcription factor CREB, while fragments bearing mutations that eliminate NGF and cAMP inducibility fail to do so. Sequence comparisons and hybridization studies indicate that there are at least two alternatively spliced forms of VGF mRNA, and the accumulation of both of these forms is similarly regulated by NGF and cAMP.

Animals↗
PubMed1991

Induction of immediate early genes by Ca2+ influx requires cAMP-dependent protein kinase in PC12 cells.

Agents that activate cAMP-dependent protein kinase (PKA) as well as agents that increase intracellular calcium induce the expression of certain immediate early genes (IEGs). Recently, it has been demonstrated that the same cis-acting element in the 5' region of the c-fos gene has the ability to mediate both cAMP- and calcium-induced c-fos expression in PC12 cells (Sheng, M., McFadden, G., and Greenberg, M. (1990) Neuron 4, 571-582). Here we demonstrate that both cAMP- and calcium-mediated induction of c-fos and egr1 are dependent on PKA activity. Addition of either depolarizing concentrations of KCl or the calcium ionophore, ionomycin, to PC12 cells increased the expression of both c-fos and egr1, but these inductions were dramatically reduced in three PKA-deficient cell lines, 123.7, AB.11, and A126-1B2. Furthermore, pretreatment of PC12 cells with 20 microM H89, a specific inhibitor of PKA, inhibited forskolin, dibutyryl cAMP, and KCl-induced c-fos and egr1 induction, while having no effect on NGF induction. Likewise, in the PKA-deficient cells, NGF or an activator of protein kinase C induced c-fos and egr1 normally. To determine if PKA deficiency modifies the ability of Ca2+ to activate calcium-dependent kinases, autophosphorylation of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase) in response to Ca2+ influx was determined. In parental PC12 cells, PC12 cells pretreated with H89, and PKA-deficient cell lines, CaM kinase was activated equivalently in response to KCl depolarization. These results suggest that PKA is not required for Ca(2+)-induced increase in CaM kinase activity and that the induction of IEGs in response to Ca2+ influx is PKA-dependent. Thus, the requirement for PKA resides at a point distal to the activation of calmodulin-dependent processes.

Blotting, Northern↗
PubMed1991

Nerve growth factor-induced neuronal differentiation after dominant repression of both type I and type II cAMP-dependent protein kinase activities.

Clonal PC12 lines deficient in cAMP-dependent protein kinase (PKA) were made by stably expressing mutant regulatory subunits (RI) of PKA that are deficient in cAMP binding (Correll, L. A., Woodford, T. A., Corbin, J. D., Mellon, P. L., and McKnight, G. S. (1989) J. Biol. Chem. 264, 16672-16678). Expression of the mutant RIs repressed cAMP-dependent activation of both PKAI and PKAII while having no effects on the cAMP binding to either free RI or RII or the level of catalytic subunit protein. These data suggest that RI and RII compete for the same pool of catalytic subunit and that the level of PKAI and PKAII are interdependent. We have used these cell lines to examine the requirement for PKA in mediating the effects of nerve growth factor (NGF) and agents that are thought to act exclusively via cAMP-dependent pathways. While several responses to cAMP were strongly compromised in these lines, NGF-dependent responses were comparable in parental and PKA-deficient cells, including: 1) protein phosphorylation, 2) transcriptional induction of the immediate early gene egr1, 3) expression of the gene for GAP-43, 4) induction of ornithine decarboxylase activity, and 5) formation of neurites. Furthermore, transient expression of the cAMP-dependent protein kinase inhibitor (RSVPKI; Day, R. N., Walder, J. A., and Maurer, R. A. (1989) J. Biol. Chem. 264, 431-436) blocked cAMP, but not NGF, induction of regulatory elements derived from the gene for egr1. These experiments support the idea that NGF can regulate neuronal differentiation by pathways that are independent of cAMP-activatable PKA.

Amino Acid Sequence↗
PubMed1991

Activation of chloride channels in normal and cystic fibrosis airway epithelial cells by multifunctional calcium/calmodulin-dependent protein kinase.

Cystic fibrosis is associated with defective regulation of apical membrane chloride channels in airway epithelial cells. These channels in normal cells are activated by cyclic AMP-dependent protein kinase and protein kinase C. In cystic fibrosis these kinases fail to activate otherwise normal Cl- channels. But Cl- flux in cystic fibrosis cells, as in normal cells, can be activated by raising intracellular Ca2+ (refs 5-10). We report here whole-cell patch clamp studies of normal and cystic fibrosis-derived airway epithelial cells showing that Cl- channel activation by Ca2+ is mediated by multifunctional Ca2+/calmodulin-dependent protein kinase. We find that intracellular application of activated kinase and ATP activates a Cl- current similar to that activated by a Ca2+ ionophore, that peptide inhibitors of either the kinase or calmodulin block Ca2(+)-dependent activation of Cl- channels, and that a peptide inhibitor of protein kinase C does not block Ca2(+)-dependent activation. Ca2+/calmodulin activation of Cl- channels presents a pathway with therapeutic potential for circumventing defective regulation of Cl- channels in cystic fibrosis.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1991

Regulation of Cl- channels by multifunctional CaM kinase.

cAMP kinase has been shown to mediate the cAMP pathway for regulation of Cl- channels in lymphocytes, but the mediator of an alternative, Ca2+ pathway has not been identified. We show here that Ca2+ ionophore activates Cl- currents in cell-attached and whole-cell patch-clamp recordings of Jurkat T lymphocytes, but this activation is not direct. The effect of Ca2+ ionophore on whole-cell Cl- currents is inhibited by a specific peptide inhibitor of multifunctional Ca2+/calmodulin-dependent protein kinase (CaM kinase). Furthermore, Cl- channels are activated in excised patches by purified CaM kinase in a fashion that mimics the effect of Ca2+ ionophore in cell-attached recordings. These results suggest that CaM kinase mediates the Ca2+ pathway of Cl- channel activation.

Adenosine Triphosphate↗
PubMed1991

Retinoic acid stimulates the differentiation of PC12 cells that are deficient in cAMP-dependent protein kinase.

Retinoic acid (RA) induced neuronal differentiation in A126-1B2 cells and 123.7 cells, two mutant lines of PC12 that are deficient in cAMP-dependent protein kinase, but not in the parental PC12 cell line. A single exposure to RA was sufficient to cause neurite formation and inhibit cell division for a period of greater than 3 wk, suggesting that RA may cause a long-term, stable change in the state of these cells. In A126-1B2 cells, RA also induced the expression of other markers of differentiation including acetylcholinesterase and the mRNAs for neurofilament (NF-M) and GAP-43 as effectively as nerve growth factor (NGF). Neither NGF nor RA stimulated an increase in the expression of smg-25A in A126-1B2 cells, suggesting that the cAMP-dependent protein kinases may be required for an increase in the expression of this marker. RA also caused a rapid increase in the expression of the early response gene, c-fos, but did not effect the expression of egr-1. RA equivalently inhibited the division of A126-1B2 cells, 123.7 cells and parental PC12 cells, so RA induced differentiation is not an indirect response to growth arrest. In contrast, the levels of retinoic acid receptors (RAR alpha and RAR beta), and retinoic acid binding protein (CRABP) mRNA were strikingly higher in both A126-1B2 cells and 123.7 cells than in the parental PC12 cells. The deficiencies in cAMP-dependent protein kinase may increase the expression of CRABP and the RARs; and, thus, cAMP may indirectly regulate the ability of RA to control neurite formation and neural differentiation. Thus, RA appears to regulate division and differentiation of PC12 cells by a biochemical mechanism that is quite distinct from those used by peptide growth factors.

Acetylcholinesterase↗
PubMed1991

Increase in beta-1,4-galactosyltransferase activity during PC12 cell differentiation induced by forskolin and 2-chloroadenosine.

Galactosyltransferase (GALTase) activity was measured in differentiating PC12 cells induced by either forskolin or 2-chloroadenosine. The specific activity of GALTase in whole cells and isolated Golgi membranes increased as early as 3 h after initiating treatment with 2-chloroadenosine, and maximal activity was reached at approximately 12 h. In two mutant PC12 cell lines deficient in protein kinase A, both forskolin and 2-chloroadenosine failed to increase GALTase activity. The adenosine A2 receptor antagonist, xanthine amine congener, prevented 2-chloroadenosine stimulation of GALTase, demonstrating that this adenosine derivative was mediating its effect via the A2 receptor. These data suggest that GALTase activity during PC12 cell differentiation is regulated by cyclic AMP (cAMP)- and protein kinase A-dependent processes. In support of the role of cAMP in regulating GALTase activity were studies with murine PC carcinoma cells demonstrating that the greatest stimulation of GALTase activity occurred with cells treated with both retinoic acid and dibutyryl cAMP.

2-Chloroadenosine↗
PubMed1991

A computerized representation of a medical school curriculum: integration of relational and text management software in database design.

We describe the development of a computer-based representation of the medical school curriculum at the University of North Carolina at Chapel Hill (UNC-CH). Over the past seven years the Medical School's Office of Academic Affairs has employed both relational database and text management software to design an integrated curriculum database system. Depending on the function selected--exploring the curriculum, searching through course outlines, retrieving elective descriptions, identifying teaching faculty, or searching for specific topics--either text management or relational database management routines are activated in a manner transparent to the user. Initial evaluation of the system has been positive but highlights the need for a more robust biomedical language for use as a controlled vocabulary to index content. Efforts are now underway, with support from the Association of American Medical Colleges (AAMC), to engage other interested schools in the U.S. and Canada in collaborating on further development of a system.

Abstracting and Indexing↗