Pertussis Toxin Modification of PC12 Cells Inhibits a Protein Phosphatase 2A-Like Phosphatase

Abstract: We have found that modification of rat PC12 cells with pertussis toxin resulted in an ∼50% inhibition of a protein phosphatase 2A-like phosphatase. Protein phosphatase 2A (PP2A) is a major cellular serine/threonine-specific protein phosphatase. Treatment of extracts from pertussis toxin-modified PC12 cells with either immobilized alkaline phosphatase or Ca²⁺ reversed this inhibition. Reactivation of the PP2A-like phosphatase in Ca²⁺ appears to result from the dephosphorylation of a protein by the Ca²⁺/calmodulin-dependent protein phosphatase calcineurin. The PP2A-like phosphatase in extracts from pertussis toxin-modified PC12 cells eluted from a Mono Q column at a higher ionic strength than did the PP2A-like phosphatase in extracts from control cells. After incubation in Ca²⁺, the PP2A-like phosphatase in extracts from pertussis toxin-modified cells eluted from a Mono Q column at the same ionic strength as did the PP2A-like phosphatase in extracts from control cells. These results indicate that the effect of pertussis toxin on this PP2A-like activity results from the phosphorylation of either one of the subunits of the PP2A-like phosphatase or a protein that when phosphorylated binds to and inhibits this phosphatase. Pertussis toxin modification did not result in the phosphorylation of the catalytic subunit of PP2A. Because phosphorylation regulates the activities of many enzymes and cell surface receptors, a pertussis toxin-induced decrease in PP2A activity could alter signaling pathways and other cellular processes in which G proteins are not directly involved.     

Molecular and immunochemical evidences demonstrate that endooligopeptidase A is the predominant cytosolic oligopeptidase of rabbit brain

Oligopeptidases are tissue endopeptidases that do not attack proteins and are likely to be involved in the maturation and degradation of peptide hormones and neuropeptides. The rabbit brain endooligopeptidase A and the rat testes soluble metallopeptidase (EC 3.4.24.15) are thiol-activated oligopeptidases which are able to generate enkephalin from a number of opioid peptides and to inactivate bradykinin and neurotensin by hydrolyzing the same peptide bonds. A monospecific antibody raised against the purified rabbit brain endooligopeptidase A allowed the identification of a 2. 3 kb cDNA coding for a truncated enzyme of 512 amino acids, displaying the same enzymatic features as endooligopeptidase A. In spite of all efforts, employing several strategies, the full-length cDNA could not be cloned until now. The analysis of the deduced amino acid sequence showed no similarity to the rat testes metalloendopeptidase sequence, except for the presence of the typical metalloprotease consensus sequence [HEXXH]. The antibody raised against recombinant endooligopeptidase A specifically inhibited its own activity and reduced the thiol-activated oligopeptidase activity of rabbit brain cytosol to less than 30%. Analysis of the endooligopeptidase A tissue distribution indicated that this enzyme is mainly expressed in the CNS, whereas the soluble metallo EC 3.4.24.15 is mainly expressed in peripheral tissues.     

Endo-Oligopeptidase A., a Putative Enkephalin-Generating Enzyme, in the Vertebrate Retina

Endo-oligopeptidase A, EC 3.4.22.19, converts small enkephalin-containing peptides into the corresponding enkephalins in vitro. We investigated the presence of endooligopeptidase A in the retina and its possible colocalization with enkephalins in retinal neurons. The specific activity of endo-oligopeptidase. A found in pigeon retinae (30.3 +/- 7.3 mU/mg, mean +/- standard deviation) was four times higher than in rabbit retinae (7.0 +/- 1.1 mU/mg). The enzyme activity was not modified by EDTA, but it was enhanced by dithiothreitol and inhibited by zinc and 5,5'-dithiobis(2-nitrobenzoic acid). Immunohistochemical experiments with a purified antiserum against rabbit endo-oligopeptidase A revealed labeled neurons in both the inner nuclear layer and the ganglion cell layer of pigeon and rabbit retinae. Double-labeling immunofluorescence experiments demonstrated that about 90% of neurons containing endo-oligopeptidase A-like immunoreactivity also contained [Leu5]-enkephalin-like immunoreactivity. These colocalization results may represent an important step toward the demonstration of the possible involvement of endo-oligopeptidase A in enkephalin generation in vivo.     

Specific adhesion between pheochromocytoma (PC12) cells and adrenal medullary endothelial cells in co-culture

Summary Chromaffin cells in the adrenal medulla are found in close proximity to capillary endothelial cells, thereby forming the classical endocrine complex. To examine the possible chemical basis of their interaction in more detail, we have grown bovine adrenal medullary endothelial (BAME) cells in monolayer cultures and added to them pheochromocytoma (PC12) cells, a chromaffin tumor cell line of rats. The PC12 cells were chosen because of the similarities they share with adrenal medullary chromaffin cells. PC12 cells rapidly attached to BAME cells cultures, their rate of adhesion being significantly enhanced over binding of PC12 cells to either uncoated plates or to monolayers of unrelated cell cultures. Consistent with this observation, we noted that the extracellular matrix (ECM) derived from the BAME cells did not enhance PC12 cell adhesion and did not promote neurite sprouting as previously described for ECM derived from corneal endothelial cells. The specific adhesion between PC12 and BAME cells could be abolished by cell surface extracts derived from these two cells but not by extracts derived from unrelated cell types. This activity was heat-labile, sensitive to trypsin and, to a lesser extent, to neuraminidase. We therefore conclude that PC12 cells may interact with BAME cells by specific proteinaceous adhesive factors associated with their plasma membranes. These interactions might represent the formative role of cell-cell contacts in the organization of the developing adrenal gland.Abbreviations BAME bovine adrenal medullary endothelial cells - DMEM Dulbecco's modified essential medium - ECM extracellular matrix - EMEM Eagle's modified essential medium - FCS fetal calf serum - PBS phosphate-buffered saline - PC12 rat pheochromocytoma cells     

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.     

Increase in β-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.     

Receptors for the vasodilator maxadilan are expressed on selected neural crest and smooth muscle-derived cells

Maxadilan is a potent vasodilator peptide isolated from salivary glands of the blood feeding sand fly Lutzomyia longipalpis. The peptide relaxes rabbit aortic rings in an endothelium independent manner while elevating levels of cAMP and has been found to bind to membrane homogenates from brain. These studies on tissues have now been expanded with an examination of binding and signaling of maxadilan to a number of established cell lines and primary cultures. The data reveal that maxadilan binds to and stimulates the accumulation of cAMP in the rat pheochromocytoma line PC12 and the human neuroblastoma line NBfl. Accumulation of cAMP occurred in a transformed mouse pancreatic smooth muscle line (MILE) and primary rabbit aorta smooth muscle cells. The peptide did not bind to or induce cAMP formation in the rat thoracic aorta line L6. Scatchard analysis of binding to the PC12 and NBfl lines indicates that maxadilan binds to a single class of high-affinity receptors. Similar pharmacologic actions and possible structural homologies between maxadilan and calcitonin generelated peptide (CGRP) suggested the possibility that they shared receptors. However, competition studies and comparative second messenger analysis reveal that maxadilan does not interact with receptors for CGRP, amylin or adrenomedullin and suggest that this peptide may bind to a novel receptor whose endogenous ligand remains unknown.     

Endooligopeptidase A activity in rabbit heart: Generation of enkephalin from enkephalin containing peptides

Two endopeptidases displaying similar specificities towards peptide hormone substrates but differing in molecular size have been identified in rabbit heart and isolated by a combination of ion-exchange chromatography, gel filtration and preparative gel electrophoresis. These two enzymes share several properties with the previously described rabbit brain endooligopeptidase A. They were shown to produce, by a single peptide bond cleavage, [Met⁵] enkephalin and [Leu⁵]enkephalin from small enkephalin containing peptides. They also hydrolyze the Phe⁵-Ser⁵ bond of bradykinin and the Arg⁸-Arg⁹ bond of neurotensin. Characteristically, the activity of both low and high Mr enzymes is restricted to oligopeptides. Both forms of heart endooligopeptidase A are inhibited by antibodies raised against the brain enzyme. When electrophoresed in SDS-polyacrylamide gel under denaturing conditions, the low Mr heart enzyme shows a major band of Mr=73,000, comparable in size to the brain enzyme. The SDS-PAGE of the high and low Mr enzymes analyzed by immunoblotting with an antibody raised against low Mr brain endooligopeptidase A, showed a major antigen band corresponding to Mr=72,000. In addition, immunoblotting has also demonstrated that a monoclonal antibody antitubulin reacts with a polypeptide corresponding to Mr=50,000 present in the purified high Mr endooligopeptidase A. Both enzymes are activated by dithiothreitol and inhibited by thiol reagents, but are not affected by leupeptin, DFP or EDTA, thus indicating that they should be classified as nonlysosomal cysteinyl-endooligopeptidase A.     

Neuronal differentiation of PC12 cells abolishes the expression of membrane androgen receptors

Sex steroids affect adrenal chromaffin cell function. In the present work, we have examined the expression and functional significance of membrane androgen receptor sites in normal rat adrenal chromaffin cells and in the PC12 rat pheochromocytoma cell line which can differentiate to either a neuronal or to an epithelial phenotype and expresses membrane estrogen receptor sites. Our data are as follows: (a) no cytosolic androgen receptors were found in both normal chromaffin and PC12 cells; (b) both types of chromaffin cells expressed high affinity membrane testosterone binding sites; (c) activation of these sites increased cytosolic Ca²⁺, decreased catecholamine secretion and induced apoptosis; (d) NGF-induced neuronal differentiation of PC12 cells resulted in the suppression of the number of membrane testosterone sites. In conclusion, our data provide evidence for the existence of specific membrane testosterone receptors on adrenal chromaffin cells via which androgens, (some of them originating in the cortex) modulate their function. Neuronal differentiation of chromaffin cells results in a significant attenuation of these effects, via suppression of the expression of membrane androgen receptors suggesting, that the latter are specific for epithelioid chromaffin cells.     

Comparative study on glucose-6-phosphate dehydrogenase from rabbit tissues

The activity of glucose-6-phosphate dehydrogenase (G6PD) was measured in bone marrow, spleen, lung, liver, kidney, adipose tissue, brain, heart, muscle, and in the erythroid cell line of rabbit. In tissues, the activity ranged from 6.87 to 0.09 U/g wet tissue, found in bone marrow and muscles, respectively, whereas in the erythroid cell line it ranged from 14.3 to 2.4 U/g cells for erythroblasts and erythrocytes, respectively. The electrophoretic patterns of the tissue crude extracts showed an identical set of three activity bands, and the immunotitration curves obtained with rat antirabbit erythrocyte G6PD antibodies shared the same equivalence point. The enzyme, purified to homogeneity from different tissues, showed no significant differences among the Km values for NADP and G6P. The results give a picture of the variability of the G6PD activity in rabbit tissues and suggest the presence of the same enzyme molecule in each tissue.     

Clonal variants of PC12 pheochromocytoma cells with defects in cAMP-dependent protein kinases induce ornithine decarboxylase in response to nerve growth factor but not to adenosine agonists.

We have isolated and partially characterized three mutants of the pheochromocytoma line PC12 by using dibutyryl cyclic AMP (cAMP) as a selective agent. Each of these variants, A126-1B2, A208-4, and A208-7, was resistant to both dibutyryl cAMP and cholera toxin when cell growth was measured. In comparison to wild-type PC12 cells, each of these mutants was deficient in the ability to induce ornithine decarboxylase (ODC) in response to agents that act via a cAMP-dependent pathway. In contrast, each of these mutants induced ODC in response to nerve growth factor. To understand the nature of the mutations, the cAMP-dependent protein kinases of the wild type and of each of these mutants were studied by measuring both histone kinase activity and 8-N3-[32P]cAMP labeling. Wild-type PC12 cells contained both cAMP-dependent protein kinase type I (cAMP-PKI) and cAMP-dependent protein kinase type II (cAMP-PKII). Regulatory subunits were detected in both soluble and particulate fractions. The mutant A126-1B2 contained near wild-type PC12 levels of cAMP-PKI but greatly reduced levels of cAMP-PKII. Furthermore, when compared with wild-type PC12 cells, this cell line had an altered distribution in ion-exchange chromatography of regulatory subunits of cAMP-PKI and cAMP-PKII. The mutant A208-4 demonstrated wild-type-level binding of 8-N3-[32P]cAMP to both type I and type II regulatory subunits, but only half the wild-type level of type II catalytic activity. The mutant A208-7 had type I and type II catalytic activities equivalent to those in wild-type cells. However, the regulatory subunit of cAMP-PKI occurring in A208-7 demonstrated decreased levels of binding 8-N3-[32P]cAMP in comparison with the wild type. Furthermore, all mutants were defective in their abilities to bind 8-N3-[32P]cAMP to the type II regulatory protein in the particulate fraction. Thus, cAMP-PK was altered in each of these mutants. We conclude that both cAMP-PKI and cAMP-PKII are apparently required to induce ODC in response to increases in cAMP. Finally, since all three mutants induced ODC in response to nerve growth factor, the nerve growth factor-dependent induction of OCD was not mediated by an increase in cAMP that led to an activation of cAMP-PK. These mutants will be useful in the elucidation of the many functions controlled by cAMP and nerve growth factor.     

Examination of the Role of ADP-Ribosylation Factor and Phospholipase D Activation in Regulated Exocytosis in Chromaffin and PC12 Cells

Abstract: The possible role of ADP-ribosylation factor (ARF)-activated and constitutive phospholipase D (PLD) activity in regulated exocytosis of preformed secretory granules in adrenal chromaffin and PC12 cells was examined. With use of digitonin-permeabilised cells, the effect of GTP analogues and exogenous ARF1 on PLD activity was determined. No evidence was seen for ARF-stimulated PLD activity in these cell types. Exocytosis from cytosol-depleted permeabilised chromaffin cells was not increased by adding recombinant nonmyristoylated or myristoylated ARF1, and exocytosis from both cell types was resistant to brefeldin A (BFA). Addition of bacterial PLD with demonstrably high activity in permeabilised chromaffin cells did not increase exocytosis in cytosol-depleted chromaffin cells. Diversion of PLD activity from production of phosphatidic acid (PA) due to the presence of 4% ethanol did not inhibit exocytosis triggered by Ca²⁺ or poorly hydrolysable GTP analogues in permeabilised chromaffin or PC12 cells. These results indicate that exocytosis in these cell types does not appear to require a BFA-sensitive ARF and the triggering of exocytosis does not require PLD activity and formation of PA. These findings rule out a general requirement for PLD activity during regulated exocytosis.     

Complex ganglioside expression and tetanus toxin binding by PC12 pheochromocytoma cells

Ganglioside expression and tetanus toxin binding were studied in the rat pheochromocytoma cell line PC12. Seven ganglioside species were readily detected in extracts of PC12 cells; two were identified as tri- and tetrasialogangliosides, which are common brain constituents but unusual components of neuronal cell lines. Carbohydrate composition, acid and enzyme hydrolyses, and mass spectral analysis revealed that the major species is GT 1b, a predominant mammalian brain ganglioside previously reported to support high affinity tetanus toxin binding (Rogers, T. B., and Snyder, S. H. (1981) J. Biol. Chem. 256, 2402-2407). Direct binding of 125I-tetanus toxin to PC12 gangliosides on TLC plates revealed selective binding to the tri- and tetrasialogangliosides. Radioiodinated toxin also bound with high affinity to intact PC12 cells or their isolated membranes. The binding affinity (Kd = 1.25 nM), density of receptors (Bmax = 238 pmol/mg of membrane protein), and dependence on pH, ionic strength, and temperature were similar to those previously reported for toxin binding to rat brain synaptic membranes. Differentiation of PC12 cells caused an increase in expression of the tri- and tetrasialogangliosides and a closely matched increase in tetanus toxin binding to cell membranes. These data provide evidence that complex gangliosides may act as tetanus toxin receptors, and demonstrate the utility of the PC12 cell line for studies of tetanus toxicity and complex ganglioside expression.     

A Nerve Growth Factor-Sensitive S6 Kinase in Cell-Free Extracts from PC 12 Cells

Soluble extracts from nerve growth factor (NGF)-stimulated PC12 cells prepared by alkaline lysis show a two- to 10-fold greater ability to phosphorylate the 40S ribosomal protein S6 than do extracts from control cells. The alkaline lysis method yields a preparation of much higher specific activity than does sonication. Half-maximal incorporation of 32P from [32P]ATP into S6 occurred after 4-7 min of NGF treatment. The partially purified NGF-sensitive S6 kinase has a molecular weight of 45,000. It is not inhibited by NaCl, chlorpromazine, or the specific inhibitor of cyclic AMP (cAMP)-dependent protein kinase, nor is it activated by addition of diolein plus phosphatidylserine. Trypsin treatment of either crude extracts or partially purified S6 kinase from control or NGF-treated cells was without effect. These data suggest that the S6 kinase stimulated by NGF is neither cAMP-dependent protein kinase or protein kinase C nor the result of tryptic activation of an inactive proenzyme. Treatment of intact cells with dibutyryl cAMP or 5'-N-ethylcarboxamideadenosine also increases the subsequent cell-free phosphorylation of S6. This observation suggests that cAMP-dependent protein kinase may be involved in the phosphorylation of S6 kinase.