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J A Wagner

Publications and source records attributed to J A Wagner.

At least 91 records · Page 5Linked to original sources
PubMed1990

Role of the cAMP-dependent protein kinase and protein kinase C in regulating the morphological differentiation of PC12 cells.

The cell line A126-1B2 is a PC12-derived mutant that is resistant to the toxic effects of dibutyryladenosine 3':5'-cyclic monophosphate (dbcAMP) and is deficient in adenosine 3':5'-cyclic monophosphate (cAMP)-dependent protein kinase II (PKAII). This mutant formed neurites in response to nerve growth factor (NGF), but not in response to dbcAMP; and dbcAMP did not increase the rate of NGF-dependent neurite formation. Thus, while PKAII is essential for process formation in response to agents that act through the cAMP-dependent pathway, activation of PKAII is not essential for NGF-dependent neurite formation. Unexpectedly, NGF and phorbol 12-myristate 13-acetate (PMA; 10-1,000 nM) synergistically stimulated the formation of short processes that were apparent within 30 min of NGF addition in 85% of these mutant cells. These processes were similar, but not identical, in appearance to the NGF-dependent neurites that formed only after a period of 24-48 hr. This effect is dependent on the activation of protein kinase C (PKC) because an inactive phorbol ester was without effect. In contrast, there was only a small effect of NGF and/or PMA on process formation in wild type cells within the first few hours. The effect of PMA is not augmented by dbcAMP in the A126-1B2 mutant cells. After several hours, PMA caused a concentration-dependent decrease in cell adhesion; and higher concentrations of PMA resulted in a transient detachment of the cells and a loss of neurites. These experiments suggest a role for PKC in the regulation of process formation.

Adrenal Gland Neoplasms↗
PubMed1990

NICER elements: a family of nerve growth factor-inducible cAMP-extinguishable retrovirus-like elements.

We have shown previously that the transcription of the gene designated d5 is induced by nerve growth factor (NGF) in rat adrenal pheochromocytoma PC-12 cells and that this NGF induction is repressed by cAMP. In this paper we demonstrate that d5 is a member of a gene family that contains several hundred members, which is closely related to retroviruses and retrotransposons, as demonstrated by the following observations: (i) the original d5 cDNA hybridized to numerous restriction fragments in genomic DNA; (ii) d5 cDNA hybridized to genomic clones with various intensities, and genomic clones can be isolated with a frequency suggesting that this family includes several hundred members; and (iii) there were minor sequence variations in four independently isolated cDNA clones that were homologous to d5 cDNA. Primer extension studies show that initiation of the 5.7-kilobase d5 mRNA(s) occurs at a unique site relative to a synthetic primer. The 5' end of the cDNA sequence was homologous to Rasheed rat sarcoma virus; and a genomic clone contained several elements that are typical of a long terminal repeat (LTR), including a CCAAT box, a TATA box, a primer binding site, a poly(A) addition signal, and a poly(A) addition site. Furthermore, there is a LTR at the 3' end of at least one of the genes in this family, and there appeared to be a four-base duplication at the probable site of integration into host DNA. Since several members of this family retain responses to NGF and cAMP, we conclude that the regulatory elements present in the LTR have been conserved in many members of this family. We have named this family of genes the NICER elements because they are a family of NGF-inducible cAMP-extinguishable retrovirus-like elements.

Adrenal Gland Neoplasms↗
PubMed1990

Nerve growth factor and fibroblast growth factor regulate neurite outgrowth and gene expression in PC12 cells via both protein kinase C- and cAMP-independent mechanisms.

Nerve growth factor (NGF), acidic fibroblast growth factor (aFGF), and basic fibroblast growth factor (bFGF) promote the survival and differentiation of a variety of peripheral and central neurons. The signal transduction mechanisms that mediate the actions of these factors in neuronal cells are not well understood. We examined the effect of a deficiency in protein kinase C (PKC) and/or cAMP second messenger systems on the actions of NGF, aFGF, and bFGF in the pheochromocytoma (PC12) cell line. Activation of PKC was not required for NGF, aFGF, and bFGF to maximally induce ornithine decarboxylase (ODC), transcription of the early response genes, d2 and d5, or neurite outgrowth. In a PC12 cell mutant that is deficient in cAMP responsiveness (A126-1B2), all three growth factors maximally induced the transcription of d5 and neurite outgrowth, but aFGF and bFGF did not induce significant increases in ODC. NGF and aFGF maximally induced the transcription of d2 in A126-1B2 cells, but bFGF-induced d2 transcription was attenuated. NGF, aFGF, and bFGF maximally induced neurite outgrowth and d5 transcription in A126 cells that were made deficient in PKC. The d2 transcriptional response was substantially reduced in cells deficient in both PKC and cAMP responsiveness. These observations lead us to conclude that (a) cAMP- and PKC-dependent events are, at least in part, causally linked to NGF, aFGF, and bFGF induction of both ODC and transcription of d2 and may control functionally redundant pathways; (b) NGF, aFGF, and bFGF can elicit neurite outgrowth and increase transcription of d2 and d5 in PC12 cells via mechanisms that are independent of both PKC and cAMP; (c) NGF, aFGF, and bFGF can induce ODC in the absence of PKC; and (d) aFGF and bFGF require cAMP responsiveness to induce ODC in PC12 cells.

Adrenal Gland Neoplasms↗
PubMed1990

Differential effects of amiodarone and desethylamiodarone on calcium antagonist receptors.

Amiodarone and its pharmacologically active metabolite desethylamiodarone have a sodium channel blocking action that explains some of their antiarrhythmic efficacy. However, the well-documented depression of the calcium channel-dependent sinus node and atrioventricular node function that occurs with amiodarone therapy suggests that amiodarone also blocks calcium influx through voltage-dependent calcium channels. Recent electrophysiologic data support the notion that amiodarone, but not desethylamiodarone, acts as a calcium channel antagonist. In this study, the effects of amiodarone and desethylamiodarone on calcium antagonist receptors associated with the voltage-dependent calcium channels were characterized. Amiodarone, but not its active metabolite desethylamiodarone, was a potent competitor at dihydropyridine and phenylalkylamine (verapamil-like) calcium antagonist binding sites in rat heart, brain, and skeletal and smooth muscles. Substantial inhibition of calcium antagonist binding was retained even after extensive washing of membranes and 2 days after in vivo treatment of rats with amiodarone. The pattern of inhibition of calcium antagonist binding suggests that amiodarone acts at phenylalkylamine binding sites. It is suggested that the acute effects of amiodarone--sinus and atrioventricular node inhibition, vasodilatation, and negative inotropic actions--may reflect calcium antagonist influences of amiodarone itself. Chronic effects of drug therapy, such as inhibition of ventricular conduction by sodium channel blockade, may selectively involve desethylamiodarone.

Amiodarone↗
PubMed1990

Nerve growth factor-induced down-regulation of calmodulin-dependent protein kinase III in PC12 cells involves cyclic AMP-dependent protein kinase.

Treatment of PC12 cells with nerve growth factor (NGF), epidermal growth factor (EGF), or agents that raise intracellular cyclic AMP (cAMP) levels (e.g., forskolin) reduces the activity of calmodulin-dependent protein kinase III (CaM-PK III) over a period of 8 h. The mechanism of this effect of NGF has now been examined in more detail, making use of a mutant PC12 cell line (A126-1B2) that is deficient in cAMP-dependent protein kinase activity. Control experiments showed that A126-1B2 cells retain other NGF-mediated responses (e.g., the induction of ornithine decarboxylase, a cAMP-independent event) and contain a complement of CaM-PK III and its substrate, elongation factor-2, comparable to that of wild-type cells. The ability of NGF or forskolin, but not of EGF, to down-regulate CaM-PK III was markedly attenuated in A126-1B2 compared to wild-type cells. Treatment of wild-type cells with the cAMP phosphodiesterase inhibitor, isobutylmethylxanthine, enhanced the effects of NGF, but not of EGF. The possibility that NGF led to a stimulation of cAMP-dependent protein kinase activity in wild-type cells was assessed by measurement of the "activation ratio" (-cAMP/+cAMP) of this enzyme before and at various times after NGF addition. A small, but significant, increase in the activation ratio from 0.3 to 0.48 was observed, reaching a peak 5 min after NGF treatment. EGF had no effect on the activation ratio in wild-type cells.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1989

Calcium-antagonist receptors in the atrial tissue of patients with hypertrophic cardiomyopathy.

Hypertrophic cardiomyopathy is characterized by a nondilated, hypertrophied left ventricle in the absence of any overt cause. A possible role of adrenergic innervation or of cellular calcium regulation is suggested by the presence of hyperdynamic left ventricular function and by the clinical and symptomatic improvement seen in patients treated with beta-receptor antagonists or calcium antagonists. Therefore, we measured the density of calcium-antagonist receptors and beta-adrenoceptors in the atrial myocardium of 16 patients with hypertrophic cardiomyopathy and 19 patients with various other cardiac disorders. For comparison, we also measured the number of voltage-sensitive sodium channels. Calcium-antagonist binding sites, measured as the amount of dihydropyridine bound to atrial tissue, were increased by 33 percent in patients with hypertrophic cardiomyopathy (mean [+/- SD], 397 +/- 104 fmol per milligram of protein in patients with hypertrophic cardiomyopathy, as compared with 299 +/- 108 in patients with other cardiac disorders; P less than 0.01). The densities of saxitoxin-binding sites on voltage-sensitive sodium channels and beta-adrenoceptors were the same in the two groups, although the density of beta-adrenoceptors was higher in atrial samples from patients receiving beta-receptor antagonists (165 +/- 86 fmol per milligram of protein [patients receiving beta-blockers] vs. 85 +/- 60 [patients not receiving beta-blockers]; P less than 0.04). The increase in the number of calcium-antagonist receptors in hypertrophic cardiomyopathy suggests that abnormal calcium fluxes through voltage-sensitive calcium channels may play a pathophysiologic part in the disease.

Adolescent↗
PubMed1989

Heparin potentiates the action of acidic fibroblast growth factor by prolonging its biological half-life.

The mechanism(s) by which heparin influences the biological activities of acidic and basic fibroblast growth factors (aFGF and bFGF) is not completely understood. One mechanism by which heparin could alter the biological activities of aFGF and bFGF is by altering their biological half-lives. We investigated the possibility that heparin potentiates aFGF-induced neurite outgrowth from PC12 cells by prolonging its biological half-life. Under conditions where heparin potentiated aFGF-induced neurite outgrowth, we observed that heparin increased the biological half-life of aFGF from 7 to 39 hr. We determined that greater than 25 hr of exposure to active aFGF was required for induction of neurite outgrowth. If aFGF activity was maintained for greater than 25 hr by periodic readdition of factor, heparin no longer potentiated aFGF-induced neurite outgrowth. These observations strongly suggest that heparin potentiates the activity of aFGF by prolonging its biological half-life. The protease inhibitors hirudin, leupeptin, and pepstatin A did not potentiate aFGF-induced neurite outgrowth, indicating that proteases inhibited by these inhibitors are not responsible for the loss of aFGF activity that we observed. However, aprotinin potentiated aFGF neurite-promoting activity approximately sevenfold, indicating that proteases that are inhibited by aprotinin are at least partially responsible for aFGF inactivation. These observations suggest that heparin regulates the activity of aFGF by regulating its proteolytic degradation, thereby regulating its biological half-life.

Adrenal Gland Neoplasms↗
PubMed1989

Bone remodeling signaled by a dihydropyridine- and phenylalkylamine-sensitive calcium channel.

An osteoblast calcium channel demonstrated by single channel recordings is associated with calcium antagonist receptor binding sites in osteoblast-like osteosarcoma cells. By using whole cell current recordings we now show that this channel is stimulated by the dihydropyridine calcium agonist drug BAY K 8644. A physiological relevance of these channels is apparent from the stereoselective, potent inhibition of parathyroid hormone-stimulated calcium uptake into osteoblast-like cells in culture by desmethoxyverapamil, a phenylalkylamine calcium antagonist. Secretion by these cells of the bone matrix protein osteocalcin is stimulated by BAY K 8644 and blocked by desmethoxyverapamil and nitrendipine. Evidence for a role of this channel in bone remodeling in intact animals comes from enhanced bone resorption in fetal rat bones observed with BAY K 8644 and stereoselective, potent blockade of resorption by desmethoxyverapamil.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1989

Nerve growth factor regulates gene expression by several distinct mechanisms.

To help elucidate the mechanisms by which nerve growth factor (NGF) regulates gene expression, we have identified and studied four genes (a-2, d-2, d-4, and d-5) that are positively regulated by NGF in PC12 cells, including one (d-2) which has previously been identified as a putative transcription factor (NGF I-A). Three of these genes, including d-2, were induced very rapidly at the transcriptional level, but the relative time courses of transcription and mRNA accumulation of each of these three genes were distinct. The fourth gene (d-4) displayed no apparent increase in transcription that corresponded to the increase in its mRNA, suggesting that NGF may regulate its expression at a posttranscriptional level. Thus, NGF positively regulates gene expression by more than one mechanism. These genes could also be distinguished on the basis of their response to cyclic AMP. The expression of d-2 and a-2 was increased by cholera toxin and further augmented by NGF; however, cholera toxin not only failed to increase the levels of d-5 and d-4 mRNA but also actually inhibited the NGF-dependent increase. The expression of each of these genes, including d-2 (NGF I-A), was also increased by fibroblast growth factor, epidermal growth factor (EGF), phorbol myristate acetate, and in some cases insulin, showing that the regulation of these genes is not unique to NGF. Because each of these genes was expressed in response to phorbol myristate acetate and EGF, their expression may be necessary but is certainly not sufficient for neurite formation. The protein kinase inhibitor K-252a prevented the NGF-associated, but not the acidic FGF-associated, induction of d-2 and d-5 gene expression, suggesting that these two growth factors may regulate gene expression via different cellular pathways. The study of the regulation of the expression of these and other NGF-inducible genes should valuable new information concerning how NGF and other growth factors cause neural differentiation.

Animals↗
PubMed1989

Alterations in calcium antagonist receptors and sodium-calcium exchange in cardiomyopathic hamster tissues.

The Syrian cardiomyopathic (CM) hamster (BIO 14.6) develops a progressive cardiomyopathy characterized by cellular necrosis, hypertrophy, and, eventually, cardiac dilatation and congestive heart failure. Several lines of evidence implicate cellular calcium overload as an important etiologic factor. We previously reported an increased number of receptors for calcium antagonist drugs, which block voltage-dependent calcium channels, in heart, skeletal muscle, and brain tissue of these hamsters in the early necrotic stage of the disease. To better characterize the pathophysiological significance of this abnormality we evaluated calcium antagonist receptor binding and Na+-Ca2+ exchange in CM and control hamsters at different stages of disease as documented by quantitative histopathologic assessment. In CM hamsters as young as 10 days, an age previously thought to be before the onset of disease, we identified cardiac myocyte hypertrophy, a twofold increase in calcium antagonist receptor binding in heart and brain, and a 50% increase in skeletal muscle. Overt histological lesions were present in skeletal muscle at 25 days and in heart between 28-30 days. The size of cardiac lesions increased over time and changed from necrotic foci with cellular infiltration to fibrotic or calcified lesions by 360 days. Myocardial cellular hypertrophy persisted through the late stages of the disease (360 days), but increased calcium antagonist binding was present in heart only to 6 months of age, in skeletal muscle to 90 days, and in brain to 30 days. Na+-Ca2+ exchange in heart was normal until 15 days and then increased by 400% at 30 days suggesting that this augmentation might be a secondary response to the earlier increase in calcium antagonist receptors. At 360 days cardiac Na+-Ca2+ exchange was decreased by 50%, likely reflecting progressive cardiac damage. The increase in calcium antagonist receptors in CM animals as young as 10 days supports the hypothesis that abnormalities in voltage-dependent calcium channels play a role in the pathophysiology of CM hamsters.

Animals↗
PubMed1988

Phenylalkylamine-sensitive calcium channels in osteoblast-like osteosarcoma cells. Characterization by ligand binding and single channel recordings.

(-)-[3H]Desmethoxyverapamil ((-)-DMV) binds saturably to homogenates of the osteoblast-like cell lines UMR 106 and ROS 17/2.8 with KD values of 45 and 61 nM and Bmax values of 6.0 and 5 pmol/mg protein, respectively. Binding is stereoselective with (-)-DMV 8-10 times more potent than (+)-DMV. None of the dihydropyridine or benzothiazepine Ca2+ antagonists examined affect (-)-[3H]DMV binding. Monovalent cations such as Li+, Na+, and K+ inhibit (-)[3H]DMV binding in the 100-400 mM range. Divalent cations such as Ba2+, Sr2+, Ca2+, and Mg2+ are effective binding inhibitors in the 2-5 mM range. ROS 17/2.8 cells express a channel on the apical plasma membrane which conducts Ba2+ and Ca2+. With 110 mM BaCl2 or CaCl2 as charge carriers the single channel conductance is 3-5 picosiemens. In cell-excised patches the channel selects for Ba2+ over Na+ 3.3:1. In the absence of divalent ions the channel conducts Na+ ions with a single channel conductance of 13 picosiemens. This Na+ conductance decreases with physiological levels of Ca2+. The channel appears related to the (-)-[3H]DMV binding site, since its conductance is blocked by verapamil in a dose-dependent manner. Moreover, DMV blocks the channel stereoselectively with relative potencies of the isomers corresponding to their affinities for the binding site. The dihydropyridine drugs BAY K 8644 or (+)-202-791 do not affect channel opening. These binding and biophysical data indicate that osteoblast cells have a phenylalkylamine receptor associated with a Ca2+ channel.

Animals↗
PubMed1988

Sulfated glycosaminoglycans modify growth factor-induced neurite outgrowth in PC12 cells.

Glycosaminoglycans (GAGs), localized on the surfaces of cells and in the basement membrane, modulate the growth and differentiation of many cell types. Recent studies have shown that heparin, a GAG found in mast cells, potentiates the ability of acidic fibroblast growth factor (aFGF) to induce neurite outgrowth in pheochromocytoma (PC12) cells. We examined the effect of a variety of GAGs on aFGF, basic fibroblast growth factor (bFGF), and nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. The effects observed were dependent upon the specific GAG, the concentration of the GAG, and the growth factor. Heparin potentiated aFGF-induced neurite outgrowth in a concentration-dependent fashion; potentiation increased with increasing heparin concentrations of 0.01-100 micrograms/ml. At concentrations greater than 100 micrograms/ml, heparin potentiation decreased. The maximally active concentration of heparin (100 micrograms/ml) increased the potency of aFGF 102-fold. Increasing concentrations of heparan sulfate, dermatan sulfate, and chondroitin sulfate correlated with increasing aFGF potentiation. The maximally active concentrations of heparan sulfate (100 micrograms/ml), dermatan sulfate (10 mg/ml), and chondroitin sulfate (1 mg/ml) increased the activity of aFGF 11-, 110-, and 11-fold, respectively. Hyaluronic acid did not affect the neurite outgrowth-promoting activity of aFGF. Heparin also altered the activity of bFGF; increasing concentrations of heparin (0.01-1 micrograms/ml) correlated with increased potentiation. At concentrations greater than 1 microgram/ml, heparin concentration was inversely correlated with potentiation. Chondroitin sulfate only increased the percentage of neurite-bearing cells at concentrations greater than 10 micrograms/ml. Maximally active concentrations of heparin (1 microgram/ml) and chondroitin sulfate (1 mg/ml) increased the potency of bFGF 5-fold. The highest concentration of heparan sulfate studied (1 mg/ml) inhibited the activity of bFGF. Dermatan sulfate and hyaluronic acid (0.01-1000 micrograms/ml) had no effect on bFGF activity. Heparan sulfate and chondroitin sulfate showed concentration-dependent potentiation of NGF; maximally active concentrations of heparan sulfate (100 micrograms/ml) and chondroitin sulfate (1 mg/ml) increased the potency of NGF 3-fold, whereas heparin, dermatan sulfate and hyaluronic acid had no effect. None of the GAGs had any effect on PC12 neurite outgrowth when added alone. The specificity of the activity of the GAGs was verified by selective enzyme degradation.(ABSTRACT TRUNCATED AT 400 WORDS)

Adrenal Gland Neoplasms↗
PubMed1988

Acidic fibroblast growth factor stimulates adrenal chromaffin cells to proliferate and to extend neurites, but is not a long-term survival factor.

Acidic fibroblast growth factor (aFGF) is a heparin-binding polypeptide that is a mitogen for endothelial cells and glial cells, as well as a differentiation factor for PC12 cells and certain neurons. We show here that aFGF is as potent as nerve growth factor (NGF) in stimulating both neuritic outgrowth and proliferation in adrenal chromaffin cells from young rats, but it fails to support long-term survival. Heparin strongly potentiates aFGF-dependent neuritic outgrowth but not aFGF-dependent proliferation. As is the case with NGF, phorbol myristate acetate depresses aFGF-induced cell division and increases the outgrowth of neurites. On the other hand, dexamethasone antagonizes neuritic outgrowth elicited by both NGF and aFGF but inhibits only proliferation induced by NGF. The effects of basic FGF (bFGF) are similar but not identical to those of aFGF. Thus the regulatory pathways controlled by aFGF, bFGF, and NGF are partially distinct.

Adrenal Medulla↗
PubMed1988

Acidic fibroblast growth factor enhances regeneration of processes by postnatal mammalian retinal ganglion cells in culture.

Postnatal rat retinal ganglion cells (RGCs) were identified with specific fluorescent labels and placed in culture. Under these conditions, the outgrowth of processes by RGCs was found to be promoted to a far greater degree by acidic fibroblast growth factor (aFGF) than by basic fibroblast growth factor (bFGF). The effect of aFGF and bFGF on process extension by solitary RGCs was quantified after 24 hr in culture, a time when neither a FGF nor bFGF enhanced RGC survival. The action of aFGF on process outgrowth was markedly potentiated by the addition of heparin (10 micrograms/ml) to the medium, but heparin alone had no effect. In the presence of heparin, half-maximal process outgrowth occurred at an aFGF concentration of less than 20 pg/ml (1 pM). Since all of the centrally projecting processes have already been formed in the living animal prior to use (at 7-12 days of age), at least a portion of the process outgrowth in culture appears to represent a regenerative phenomenon. Statistical analysis of the increase in process growth revealed that aFGF with heparin contributed to both neurite initiation and elongation. The mean number of glial cells, identified with polyclonal antiserum against glial fibrillary acidic protein, was slightly increased in cultures receiving aFGF plus heparin, but this effect was variable, and these glial cells were not in contact with the solitary RGCs that were scored for regeneration of processes. Thus, glial cells probably did not exert a direct physical influence on the degree of process outgrowth observed in the solitary RGCs, although a humoral effect cannot be totally excluded. These results suggest that aFGF has a potent influence on the outgrowth of processes by a neuron in the mammalian central nervous system. The potentiation of this effect by heparin leads us to speculate that the interaction of aFGF with a heparin-like molecule located in the extracellular matrix (such as heparan sulfate proteoglycan) may produce physiological effects in vivo. Furthermore, the lack of a substantial effect of bFGF in this system under these conditions shows that a specific population of mammalian central neurons may be differentially influenced by these two closely related peptide growth factors.

Animals↗
PubMed1988

Induction of process outgrowth in vertebrate and invertebrate cell lines by a 2-pyridinyl thiosemicarbazone.

Regulation of differentiation in cells of disparate origin is often mediated by widely differing molecular signals and receptor mechanisms. For example, two neuron-like cell lines used extensively as models for molecular control of differentiation, the steroid-sensitive Kc line from Drosophila and the polypeptide- and cyclic nucleotide-sensitive PC12 line from rat, share no obvious growth factor or hormone receptors. However, we have found that a thiosemicarbazone, 1-pyrrolidinecarbothioic acid [1-(2-pyridinyl)ethylidene] hydrazide, one of a class of synthetic antineoplastic agents, induces process outgrowth - a marker of cellular differentiation - in cells of both of these lines. Moreover, the thiosemicarbazone induces process outgrowth in cells of mutant clones of these lines that are refractory to treatment with growth factors or hormones. Activity of the thiosemicarbazone is dependent upon the alpha-(N)-heterocyclic ring. These findings show that the 2-pyridinyl thiosemicarbazone mimics the effects of diverse epigenetic factors in inducing process outgrowth similar to that seen in cellular differentiation of these cell lines induced by natural regulators. Regulation may be by a mechanism, common to both invertebrate and vertebrate cells, which occurs downstream from the receptors that have been previously shown to mediate epigenetically induced differentiation.

Animals↗