calcimycin and Pheochromocytoma

In an effort to identify genes involved in neuronal differentiation, we have used representational difference analysis (RDA) to clone cDNAs that are preferentially induced by nerve growth factor (NGF) vs. epidermal growth factor (EGF) in PC12 pheochromocytoma cells. We now report the cloning of a previously unknown primary response gene, NID67. In addition to a robust induction by NGF and FGF, both of which cause PC12 cells to differentiate, NID67 is strongly induced by forskolin, A23187 and ATP. EGF, TPA and KCl induce NID67 only weakly. NID67 mRNA is most abundant in heart, ovary and adrenal. Modest levels are present in most brain regions, testis, thyroid, thymus, pituitary, kidney and intestine; little NID67 is present in skeletal muscle and cerebellum. The NID67 cDNA contains a 180 bp open reading frame (ORF) that encodes a 60 amino acid protein. The central 29 amino acids are very hydrophobic and very likely comprise a transmembrane domain. Mouse and human NID67 cDNAs contain an ORF similar to NID67; the rat and human protein sequences are 85% identical whereas the rat and mouse sequences are 92% identical. In vitro transcription and translation reactions confirmed that the ORF we identified produces a 6000 Da protein product. Several small membrane proteins are similar to NID67; they contain a transmembrane domain and little more. All of these proteins participate in forming or regulating ion channels. NID67 may play a similar role in cellular physiology.

Topics: Adrenal Gland Neoplasms; Amino Acid Sequence; Animals; Base Sequence; Calcimycin; Calcium; Cell Differentiation; Chromosomes, Human, Pair 5; Colforsin; Culture Media, Serum-Free; DNA, Complementary; DNA, Neoplasm; Epidermal Growth Factor; Female; Fibroblast Growth Factors; Gene Expression Regulation, Neoplastic; Humans; Ion Channels; Ionophores; Membrane Proteins; Mice; Molecular Sequence Data; Myocardium; Neoplasm Proteins; Nerve Growth Factor; Nerve Tissue Proteins; Open Reading Frames; Organ Specificity; Ovary; PC12 Cells; Pheochromocytoma; Polymerase Chain Reaction; Potassium Chloride; Protein Structure, Tertiary; Rats; Second Messenger Systems; Sequence Alignment; Sequence Homology, Nucleic Acid; Tetradecanoylphorbol Acetate

The Bcl-2 family and the ICE family of cysteine proteases play important roles in regulating cell death. We show here that induction of cell death by a Ca2+ ionophore or hypoxia results in increased levels and activity of active ICE(-like) proteases and the subsequent activation of CPP32/Yama(-like) proteases, and that inhibition of these protease activities reduces the extent of cell death. Overexpression of the anti-apoptotic proteins Bcl-2 or Bcl-xL inhibits the cell death and the activation of ICE(-like) and CPP32/Yama(-like) proteases, indicating that Bcl-2 and Bcl-xL act upstream of these proteases. We also show that specific inhibition of ICE(-like) proteases in vivo prevents activation of CPP32/Yama(-like) proteases, whereas inhibition of CPP32/Yama(-like) proteases does not prevent activation of ICE(-like) proteases, suggesting the existence of a protease cascade in vivo that requires ICE(-like) proteases for activation of CPP32/Yama(-like) proteases. Induction of necrotic cell death by KCN also induces activation of ICE(-like) proteases but not of CPP32/Yama(-like) proteases, and Bcl-2 and Bcl-xL inhibit the activation and the cell death, suggesting that the functional site of Bcl-2 and Bcl-xL is also upstream of ICE(-like) proteases in at least some forms of necrosis.

Topics: Adrenal Gland Neoplasms; Animals; Apoptosis; Calcimycin; Caspase 1; Caspase 3; Caspases; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Enzyme Activation; Humans; Hypoxia; Ionophores; Oligopeptides; Pheochromocytoma; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Tumor Cells, Cultured

Incubation with a K+/H+ ionophore nigericin attenuated the nerve growth factor (NGF)-induced neurite outgrowth in rat pheochromocytoma PC12 cells. However, a Na+/H+ ionophore monensin and a Ca2+ ionophore A23187 did not inhibited the neurite outgrowth. Nigericin also inhibited the NGF-caused induction of acetylcholinesterase and suppression of cell proliferation. These changes were dependent on the amount of the ionophore added to the culture. In addition, a distinct K+ ionophore, valinomycin, similarly inhibited the NGF-induced neuronal differentiation. These results suggest the presence of the K+ ionophore-sensitive mechanism in the NGF-induced differentiation system in PC12 cells.

Topics: Acetylcholinesterase; Adrenal Gland Neoplasms; Animals; Calcimycin; Cell Differentiation; Dose-Response Relationship, Drug; Kinetics; Monensin; Nerve Growth Factors; Neurites; Neurons; Nigericin; PC12 Cells; Pheochromocytoma; Potassium; Rats; Valinomycin

The nature of second messengers involved in the nicotine-evoked release of dopamine from PC12 cells was examined. Calmidazolium, a calmodulin inhibitor, abolished the nicotine-evoked release. A23187, a Ca2+ ionophore, enhanced dopamine release, and this was inhibited by calmidazolium. Further, 2', 5'-dideoxyadenosine abolished both the nicotine- and A23187-evoked release. Forskolin, dibutyryl-cyclic AMP, and rolipram (a cyclic AMP phosphodiesterase inhibitor) all enhanced dopamine release. 1, 9-Dideoxyforskolin, a forskolin analog which does not activate adenylate cyclase, did not alter dopamine release. These results suggest an obligatory role for Ca2+ and calmodulin-sensitive adenylate cyclase in the nicotine-evoked release process.

Topics: Adenylyl Cyclases; Adrenal Gland Neoplasms; Animals; Calcimycin; Calcium; Colforsin; Cyclic AMP; Dideoxyadenosine; Dopamine; Intracellular Fluid; Nicotine; Pheochromocytoma; Rats; Receptors, Nicotinic; Tumor Cells, Cultured

Receptor mediated internalization of 125I-ANF (99-126) and the underlying mechanism was studied in PC12 cells. Phosphorylation of PC12 cell plasma membrane proteins at 0 degrees C or 37 degrees C was not altered in presence of ANF (99-126) or c-ANF (4-23). Exposure of cells to phorbol 12-myristate 13-acetate (PMA, 100 ng/ml) did not alter the endocytic rate or extent of 125I-ANF (99-126) internalization. When cells were treated with a combination of PMA and the calcium ionophore A23187, internalization was not stimulated. Incubation with A23187 (10 microM) alone decreased 125I-ANF (99-126) internalization by 22% in Ca2+ containing medium. Cell surface binding increased 10% in the presence of Ca2+ compared to Ca2+ free medium, irrespective of the presence of A23187. Ca2+ appears to play an important role in the binding of ANF to the receptor and initiation of ligand-receptor complex internalization. Activation of protein kinase C or receptor phosphorylation is not an essential step in initiating ANF receptor internalization.

Topics: Adrenal Gland Neoplasms; Animals; Atrial Natriuretic Factor; Calcimycin; Calcium; Cell Membrane; Kinetics; Membrane Proteins; Molecular Weight; Pheochromocytoma; Phosphoproteins; Phosphorylation; Rats; Receptors, Atrial Natriuretic Factor; Receptors, Cell Surface; Tetradecanoylphorbol Acetate

Membrane depolarization has been widely used to elucidate the response of the nervous system to prolonged neuronal activity or stress. We studied the effect of treating PC12 cells with membrane depolarizing stimuli, 50 mM KCl, or 150 microM veratridine, and the subsequent changes in the mRNA levels of the catecholamine biosynthetic enzymes, tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH). TH mRNA levels were found to increase 2- to 5-fold after continuous treatment for 1-12 h with 50 mM KCl. Depolarization with 150 microM veratridine had a similar effect on TH mRNA. In contrast, DBH mRNA levels were unchanged by either KCl or veratridine treatment. The role of calcium in the increase of TH mRNA levels elicited by depolarization was examined. The increase in TH mRNA was inhibited by the chelation of calcium with 3 mM EGTA. However, in contrast to their effect on phosphorylation of TH elicited by acute depolarization, the calcium channel blockers, nitrendipine and verapamil, and the calmodulin antagonists, W7 and trifluoperazine, did not prevent the increase in TH mRNA levels subsequent to several hours exposure to depolarizing stimuli. The calcium ionophore, A23187, alone was unable to induce TH mRNA levels. Thus, the increase in TH mRNA elicited by depolarization is mediated differently than the acute phosphorylation of the enzyme.

Topics: Adrenal Gland Neoplasms; Animals; Calcimycin; Calcium; Calcium Channel Blockers; Calmodulin; Dopamine beta-Hydroxylase; Enzyme Induction; Membrane Potentials; Neoplasm Proteins; Pheochromocytoma; Potassium Chloride; Rats; RNA, Messenger; RNA, Neoplasm; Second Messenger Systems; Tumor Cells, Cultured; Tyrosine 3-Monooxygenase; Veratridine

Maitotoxin (MTX) increases formation of [3H]inositol phosphates from phosphoinositides and release of [3H]arachidonic acid from phospholipids in pheochromocytoma PC12 cells. Formation of [3H]inositol phosphates is detected within 1 min of incubation even with concentrations as low as 0.3 ng/ml (90 pm) MTX, whereas release of [3H]arachidonic acid is not detected until 20 min even with concentrations as high as 1 ng/ml (300 pm) MTX. Stimulation of arachidonic acid release can be detected at 0.03 ng/ml (9 pm) MTX, whereas 0.1 ng/ml (30 pm) MTX is the threshold for detection of phosphoinositide breakdown. Organic and inorganic calcium channel blockers, except Cd2+ and a high concentration of Mn2+, have no effect on MTX-elicited phosphoinositide breakdown, whereas inorganic blockers (e.g., Co2+, Mn2+, Cd2+), but not organic blockers (nifedipine, verapamil, diltiazem), inhibit MTX-stimulated arachidonic acid release. All calcium channel blockers, however, inhibited MTX-elicited influx of 45Ca2+ and the MTX-elicited increase in internal Ca2+ measured with fura-2 was markedly reduced by nifedipine. MTX-elicited phosphoinositide breakdown and arachidonic acid release are abolished or reduced, respectively, in the absence of extracellular calcium plus chelating agent. The calcium ionophore A23187 has little or no effect alone but, in combination with MTX, A23187 inhibits MTX-elicited phosphoinositide breakdown and enhances arachidonic acid release, the latter even in the absence of extracellular calcium. The results suggest that different sites and/or mechanisms are involved in stimulation of calcium influx, breakdown of phosphoinositides, and release of arachidonic acid by MTX.

Topics: Adrenal Gland Neoplasms; Animals; Arachidonic Acid; Arachidonic Acids; Calcimycin; Calcium; Calcium Channel Blockers; Calcium Channels; Calcium Radioisotopes; Drug Interactions; Enzyme Activation; Marine Toxins; Oxocins; Pheochromocytoma; Phosphatidylinositols; Phospholipases; Phospholipases A; Rats; Tumor Cells, Cultured; Type C Phospholipases

1-[N,O-Bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpipera zine (KN-62), a selective inhibitor of rat brain Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM kinase II) was synthesized and its inhibitory properties in vitro and in vivo were investigated. KN-62 inhibited phosphorylation of exogenous substrate (chicken gizzard myosin 20-kDa light chain) by Ca2+/CaM kinase II with Ki value of 0.9 microM, but no significant effect up to 100 microM on activities of chicken gizzard myosin light chain kinase, rabbit brain protein kinase C, and bovine heart cAMP-dependent protein kinase type II. KN-62 also inhibited the Ca2+/calmodulin-dependent autophosphorylation of both alpha (50 kDa) and beta (60 kDa) subunits of Ca2+/CaM kinase II dose dependently in the presence or absence of exogenous substrate. Kinetic analysis indicated that this inhibitory effect of KN-62 was competitive with respect to calmodulin. However, KN-62 did not inhibit the activity of autophosphorylated Ca2+/CaM kinase II. Moreover, Ca2+/CaM kinase II bound to a KN-62-coupled Sepharose 4B column, but calmodulin did not. These results suggest that KN-62 affects the interaction between calmodulin and Ca2+/CaM kinase II following inhibition of this kinase activity by directly binding to the calmodulin binding site of the enzyme but does not affect the calmodulin-independent activity of already autophosphorylated (activated) enzyme. We examined the effect of KN-62 on cultured PC12 D pheochromocytoma cells. KN-62 suppressed the A23187 (0.5 microM)-induced autophosphorylation of the 53-kDa subunit of Ca2+/CaM kinase in PC12 D cells, which was immunoprecipitated with anti-rat forebrain Ca2+/CaM kinase II polypeptides antibodies coupled to Sepharose 4B, thereby suggesting that KN-62 could inhibit the Ca2+/CaM kinase II activity in vivo.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Animals; Binding Sites; Binding, Competitive; Calcimycin; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Chickens; Chromatography, Affinity; Cyclic AMP; Gizzard, Avian; Isoquinolines; Kinetics; Myosin-Light-Chain Kinase; Myosins; Pheochromocytoma; Phosphorylation; Piperazines; Protein Kinase C; Protein Kinase Inhibitors; Protein Kinases; Rats; Tumor Cells, Cultured

The NGFI-B gene is rapidly activated by a variety of stimuli that induce cells to differentiate or proliferate. It encodes a protein with a predicted molecular mass of congruent to 61 kDa and is a member of the thyroid/steroid hormone receptor gene family. To characterize this protein, monoclonal antibodies were raised against a bacterial TrpE-NGFI-B fusion protein that encompasses a large portion (Glu-410 to Leu-527) of the carboxy-terminal domain of NGFI-B. These antibodies detected a protein that was rapidly synthesized in response to nerve growth factor (NGF) and migrated as a broad band on sodium dodecyl sulfate-polyacrylamide gels with an apparent molecular mass that ranged from 63 to 88 kDa. Pulse-chase analysis demonstrated that NGFI-B was rapidly posttranslationally modified and was a short-lived protein. NGFI-B was found to be a phosphorylated protein, and the multiple NGFI-B species coalesced into a single, more rapidly migrating species when treated with alkaline phosphatase. PC12 cells grown in the absence of NGF contained low levels of NGFI-B that was underphosphorylated. Epidermal growth factor, phorbol ester, and the calcium ionophore A23187 stimulated the synthesis of NGFI-B that was composed largely of underphosphorylated, rapidly migrating species. In contrast, basic fibroblast growth factor, which promotes differentiation of PC12 cells, induced the synthesis of NGFI-B species similar to those synthesized in response to NGF treatment. The underphosphorylated NGFI-B found in uninduced PC12 cells was found only in the nucleus, whereas NGFI-B in NGF-stimulated PC12 cells was present in approximately equal quantities in the cytoplasm and nucleus. Consistent with the cellular distribution observed in nonstimulated PC12 cells, the highly phosphorylated species were predominantly cytoplasmic whereas the more rapidly migrating forms were nuclear.

Topics: Adrenal Gland Neoplasms; Animals; Calcimycin; Cell Line; DNA-Binding Proteins; Epidermal Growth Factor; Fibroblast Growth Factor 2; Multigene Family; Nerve Growth Factors; Nuclear Receptor Subfamily 4, Group A, Member 1; Pheochromocytoma; Phosphorylation; Protein Processing, Post-Translational; Rats; Receptors, Cytoplasmic and Nuclear; Receptors, Steroid; Receptors, Thyroid Hormone; Tetradecanoylphorbol Acetate; Transcription Factors

Depolarization of plasma membrane potential has a potent inhibitory effect on divalent cation influx catalyzed by the carboxylic ionophores ionomycin and A23187. This effect is observed in different cell models and does not depend on either inhibition of Ca2+-activated cation channels or activation of Ca2+ extrusion mechanisms as suggested previously. A dependence of divalent cation influx on the magnitude of membrane potential is observed also in artificial liposomes. The inhibition of ionophore-dependent divalent cation transport by membrane potential depolarization can be modified varying the ionophore concentration and the external pH. These findings suggest that both neutral and positively charged ionophore-cation complexes can cross the plasma membrane and that their contribution to the overall transport process can be varied according to the experimental conditions.

Topics: Adrenal Gland Neoplasms; Animals; Benzofurans; Calcimycin; Calcium; Calcium Channels; Cations, Divalent; Cations, Monovalent; Cell Line; Chlorides; Fluorescent Dyes; Fura-2; Gramicidin; Ionomycin; Kinetics; Liposomes; Manganese; Manganese Compounds; Mathematics; Membrane Potentials; Models, Theoretical; Pheochromocytoma; Rats

Botulinum C2 toxin is known to ADP-ribosylate actin. The toxin effect was studied on [3H]noradrenaline secretion of PC12 cells. [3H]Noradrenaline release was stimulated five- to 15-fold by carbachol (100 microM) or K+ (50 mM) and 10-30-fold by the ionophore A23187 (5 microM). Pretreatment of PC12 cells with botulinum C2 toxin for 4-8 h at 20 degrees C, increased carbachol-, K+-, and A23187-induced, but not basal, [3H]noradrenaline release maximally 1.5-to three-fold, whereas approximately 75% of the cellular actin pool was ADP-ribosylated. Treatment of PC12 cells with botulinum C2 toxin for up to 1 h at 37 degrees C also increased stimulated [3H]noradrenaline secretion, whereas toxin treatment for greater than 1 h decreased the enhanced [3H]noradrenaline release stimulated by carbachol and K+ but not by A23187. Concomitantly with toxin-induced stimulation of secretion, 20-50% of the cellular actin was ADP-ribosylated, whereas greater than 60% of actin was modified when exocytosis was attenuated. The data indicate that ADP-ribosylation of actin by botulinum C2 toxin largely modulates stimulation of [3H]noradrenaline release. Moreover, the biphasic toxin effects suggest that distinct mechanisms are involved in the role of actin in secretion.

Topics: Actins; Adenosine Diphosphate Ribose; Adrenal Gland Neoplasms; Animals; Botulinum Toxins; Calcimycin; Carbachol; Exocytosis; Norepinephrine; Pheochromocytoma; Potassium; Rats; Tumor Cells, Cultured

Heat shock has a profound influence on the metabolism and behavior of eukaryotic cells. We have examined the effects of heat shock on the release from cells of arachidonic acid and its bioactive eicosanoid metabolites, the prostaglandins and leukotrienes. Heat shock (42-45 degrees) increased the rate of arachidonic acid release from human, rat, murine, and hamster cells. Arachidonate accumulation appeared to be due, at least partially, to stimulation of a phospholipase A2 activity by heat shock and was accompanied by the accumulation of lysophosphatidyl-inositol and lysophosphatidylcholine in membranes. Induction of arachidonate release by heat did not appear to be mediated by an increase in cell Ca++. Stimulation of arachidonate release by heat shock in hamster fibroblasts was quantitatively similar to the receptor-mediated effects of alpha thrombin and bradykinin. The effects of heat shock and alpha thrombin on arachidonate release were inhibited by glucocorticoids. Increased arachidonate release in heat-shocked cells was accompanied by the accelerated accumulation of cyclooxygenase products prostaglandin E2 and prostaglandin F2 alpha and by 5-lipoxygenase metabolite leukotriene B4. Elevated concentrations of arachidonic acid and metabolites may be involved in the cytotoxic effects of hyperthermia, in homeostatic responses to heat shock, and in vascular and inflammatory reactions to stress.

Topics: Animals; Arachidonic Acids; Bradykinin; Calcimycin; Cell Line; Cricetinae; Cricetulus; Eicosanoids; Fibroblasts; Glucocorticoids; HeLa Cells; Hot Temperature; Leukotriene B4; Mice; Mice, Inbred BALB C; Pheochromocytoma; Phospholipases A; Phospholipases A2; Prostaglandins; Rats; Thrombin; Tumor Cells, Cultured

The effects of bradykinin (BK) and lithium on the phosphatidylinositol cycle were examined in PC12 cells cultured for 20 h in the presence [PC12(+)] or in the absence [PC12(-)] of nerve growth factor (NGF). BK (1 microM) induced a small stimulation of the incorporation of myo-[2-3H]inositol into the lipids of PC12(-) cells and a three- to fourfold stimulation of such incorporation into the lipids of PC12 (+) cells. About 15 h of incubation with NGF and greater than 10 min of incubation with BK were needed for maximal stimulation of inositol incorporation by BK. In the presence of 25 mM LiCl, BK stimulated the inositol monophosphate levels nine-fold in PC12 (-) and 30-fold in PC12 (+) cells. After incubation for 20 h with NGF, an increased binding of [3H]BK to the PC12 (+) cells was observed at 4 degrees C. Exposure of the cells for 30 min to 25 mM LiCl enhanced the effect of BK on the inositol incorporation into total inositol lipids, especially in PC12(+) cells. In these cells, LiCl in the presence of BK also increased several-fold the intracellular levels of inositol bisphosphate and inositol trisphosphate.

Topics: Adrenal Gland Neoplasms; Animals; Bradykinin; Calcimycin; Chlorides; Inositol; Inositol Phosphates; Lipid Metabolism; Lithium; Lithium Chloride; Nerve Growth Factors; Pheochromocytoma; Phosphatidylinositols; Rats; Tumor Cells, Cultured

Rat hepatocytes contain several types of Ca2+-linked receptors, all of which stimulate glycogen breakdown by increasing cytosolic free Ca2+ concentration [( Ca2+]c). In vivo desensitization of this Ca2+ messenger system was studied in hepatocytes isolated from either pheochromocytoma (PHEO)-harboring and chronically norepinephrine (NE)-infused rats. Homologous desensitization for alpha 1-adrenergic receptor-mediated phosphorylase activation developed in the early stage of PHEO rats (3-4 wk after implantation), whereas, in the later stage of tumor development or in the NE-infused rats, phosphorylase responses to all Ca2+-mobilizing stimulations were subsensitive (heterologous desensitization). In the homologous desensitization, the [Ca2+]c response to alpha 1-adrenergic stimulation was selectively reduced. We found, using the phenoxybenzamine inactivation method, that there was a linear relationship between alpha 1 receptor density and the [Ca2+]c response; consequently, the blunted [Ca2+]c response to alpha 1-adrenergic stimulation could not be explained by the 34% downregulation of alpha 1 receptors seen in these rats. These results indicated that uncoupling at a step proximal to alpha 1 receptor-stimulated [Ca2+]c increase is also of primary importance in homologous desensitization of phosphorylase activation. On the other hand, heterologous desensitization also involved alteration(s) at steps distal to the rise in [Ca2+]c. Our data demonstrate that prolonged exposure to catecholamines results in desensitization of the [Ca2+]c mobilization pathway and may involve multiple mechanisms.

Topics: Adrenal Gland Neoplasms; Angiotensin II; Animals; Arginine Vasopressin; Bucladesine; Calcimycin; Calcium; Colforsin; Glucagon; Hormones; Liver Glycogen; Male; Norepinephrine; Phenoxybenzamine; Phenylephrine; Pheochromocytoma; Phosphorylases; Prazosin; Rats; Receptors, Adrenergic, alpha; Second Messenger Systems

Stimulation of rat pheochromocytoma PC12 cells with ionophore A23187, carbachol, or high K+ medium, agents which increase intracellular Ca2+, results in the phosphorylation and activation of tyrosine hydroxylase (Nose, P., Griffith, L. C., and Schulman, H. (1985) J. Cell Biol. 101, 1182-1190). We have identified three major protein kinases in PC12 cells and investigated their roles in the Ca2+-dependent phosphorylation of tyrosine hydroxylase and other cytosolic proteins. A set of PC12 proteins were phosphorylated in response to both elevation of intracellular Ca2+ and to protein kinase C (Ca2+/phospholipid-dependent protein kinase) activators. In addition, distinct sets of proteins responded to either one or the other stimulus. The three major regulatory kinases, the multifunctional Ca2+/calmodulin-dependent protein kinase, the cAMP-dependent protein kinase, and protein kinase C all phosphorylate tyrosine hydroxylase in vitro. Neither the agents which increase Ca2+ nor the agents which directly activate kinase C (12-O-tetradecanoylphorbol-13-acetate or 1-oleyl-2-acetylglycerol) increase cAMP or activate the cAMP-dependent protein kinase, thereby excluding this pathway as a mediator of these stimuli. The role of protein kinase C was assessed by long term treatment of PC12 cells with 12-O-tetradecanoylphorbol-13-acetate, which causes its "desensitization." In cells pretreated in this manner, agents which increase Ca2+ influx continue to stimulate tyrosine hydroxylase phosphorylation maximally, while protein kinase C activators are completely ineffective. Comparison of tryptic peptide maps of tyrosine hydroxylase phosphorylated by the three protein kinases in vitro with phosphopeptide maps generated from tyrosine hydroxylase phosphorylated in vivo indicates that phosphorylation by the Ca2+/calmodulin-dependent kinase most closely mirrors the in vivo phosphorylation pattern. These results indicate that the multifunctional Ca2+/calmodulin-dependent protein kinase mediates phosphorylation of tyrosine hydroxylase by hormonal and electrical stimuli which elevate intracellular Ca2+ in PC12 cells.

Topics: Adenosine; Adrenal Gland Neoplasms; Animals; Calcimycin; Calcium; Calcium-Calmodulin-Dependent Protein Kinases; Carbachol; Cell Line; Cyclic AMP; Kinetics; Pheochromocytoma; Phosphorylation; Potassium; Protein Kinases; Tetradecanoylphorbol Acetate; Tyrosine 3-Monooxygenase

The addition of nerve growth factor (NGF) to PC12 cells induces an approximate doubling in the cell surface expression of the Thy-1 glycoprotein and the neural cell adhesion molecule (N-CAM) after 24 h of culture. Although both responses are measured at the same time point, their sensitivity to NGF differed with half-maximal induction of Thy-1 apparent at NGF concentrations (approximately 0.1 ng/ml NGF) that had little effect on N-CAM expression. Phorbol ester derivatives capable of activating Ca2+/phospholipid-dependent protein kinase (protein kinase C) and the calcium ionophore A23187 were found to mimic the NGF induction of Thy-1, but not N-CAM. Similar results were observed when a synthetic diacylglycerol was added to PC12 cell cultures. Increased expression of Thy-1 consequent to phorbol ester, calcium ionophore, or NGF treatment was associated with an increase in the expression of the mRNA species that encodes Thy-1. Increased expression of Thy-1 consequent to all three treatments was also reduced by treatment with the transcription inhibitor cordycepin. Treatment of PC12 cells with high concentrations of phorbol esters was found to inhibit the NGF induction of Thy-1, but not N-CAM. Whereas the above results are consistent with activation of protein kinase C underlying the NGF induction of Thy-1, the same data are not consistent with this pathway being important in the N-CAM response.

Topics: Antigens, Surface; Calcimycin; Cell Adhesion Molecules; Diglycerides; Humans; Membrane Glycoproteins; Nerve Growth Factors; Pheochromocytoma; Phorbol Esters; Protein Kinase C; Thy-1 Antigens; Tumor Cells, Cultured

Previous work from these laboratories has shown that in PC12 cells the phosphorylation of a specific soluble protein is decreased by treatment with nerve growth factor. This protein, designated Nsp100, and its kinase have been separated and partially purified from PC12 cells. The present studies have been designed to investigate the role of calcium in this action of nerve growth factor. It is shown here, using PC12h cells, that A23187, a calcium ionophore, and high levels of K+, a depolarizing stimulus, also decrease phosphorylation of Nsp100. Furthermore, the actions of nerve growth factor as well as those of A23187 and high levels of K+ are prevented by treatment of the cells with the calcium chelator EGTA. It is also shown that agents that raise levels of cyclic AMP in the cells, specifically dibutyryl cyclic AMP and cholera toxin, also decrease phosphorylation of Nsp100 but, in addition, increase phosphorylation of tyrosine hydroxylase. The action of these latter agents on Nsp100 is blocked by EGTA, but their action on tyrosine hydroxylase is not, indicating that even agents such as cholera toxin act on Nsp100 through a Ca2+-dependent mechanism.

Topics: Animals; Bucladesine; Calcimycin; Calcium; Cell Line; Cholera Toxin; Cyclic AMP; Egtazic Acid; Nerve Growth Factors; Nerve Tissue Proteins; Pheochromocytoma; Phosphorylation; Potassium; Rats; Tyrosine 3-Monooxygenase

Incubation of rat pheochromocytoma PC12 cells with the calcium ionophore, A23187 (10(-5) M), 56 mM K+, or dibutyryl cAMP (2 mM) is associated with increased activity and enhanced phosphorylation of tyrosine hydroxylase in the cells. Both the activation and the increased phosphorylation of tyrosine hydroxylase produced by A23187 and 56 mM K+ are dependent on the presence of extracellular calcium, whereas similar effects produced by dibutyryl cAMP are independent of calcium. The effects of 56 mM K+ plus dibutyryl cAMP or A23187 plus dibutyryl cAMP on the activation and phosphorylation of tyrosine hydroxylase are additive. In contrast, the effects of 56 mM K+ plus A23187 on either the activation or the phosphorylation of the enzyme are not additive. Following stimulation of intact PC12 cells with 32Pi, in order to label ATP stores, and tryptic digestion of the phosphorylated enzyme, separation of the tryptic phosphopeptides by high pressure liquid chromatography yields four distinct 32P-peptide peaks. Incubation of the cells in the presence of either 56 mM K+ or A23187 is associated with increased 32Pi incorporation into three peptides whereas, in the presence of dibutyryl cAMP, increased 32Pi incorporation is observed in only one of these peptides. When tyrosine hydroxylase purified from rat pheochromocytoma tumor is incubated in vitro with [gamma-32P]ATP and either cAMP-dependent or calcium/calmodulin-dependent protein kinase under appropriate conditions, increased phosphorylation of tyrosine hydroxylase is observed. However, even though in vitro phosphorylation by cAMP-dependent protein kinase is associated with activation of tyrosine hydroxylase, in vitro phosphorylation by calcium/calmodulin-dependent protein kinase does not lead to activation of the enzyme. Tryptic digestion of tyrosine hydroxylase phosphorylated by calcium/calmodulin-dependent protein kinase yields three distinct 32P-peptide peaks, which are identical to those phosphorylated by treatment of intact PC12 cells with either high K+ or A23187. In contrast, cAMP-dependent protein kinase phosphorylates only one peptide, which is identical to that phosphorylated by treatment of the intact cells with dibutyryl cAMP. These results indicate that tyrosine hydroxylase is activated and phosphorylated at multiple sites in PC12 cells exposed to 56 mM K+ or A23187. The results suggests that the in situ phosphorylation of these sites is catalyzed by calcium/calmodulin-dependent protein kinase; however,

Topics: Adrenal Gland Neoplasms; Animals; Bucladesine; Calcimycin; Calmodulin; Cell Line; Cyclic AMP; Pheochromocytoma; Phosphorylation; Potassium; Protein Kinases; Rats; Tyrosine 3-Monooxygenase

The nerve growth factor (NGF)-mediated increase in c-fos gene expression in the rat pheochromocytoma PC12 cell line has been investigated. NGF treatment of PC12 cells results in an increased level of c-fos mRNA within 15 min. An approximately 100-fold increase in the level of c-fos mRNA occurs 30-45 min after exposure to NGF and the c-fos mRNA concentration returns to its basal level 2 hr after NGF treatment. Thus, the half-life of this RNA transcript is extremely short. In the presence of cycloheximide, the c-fos gene is superinduced and the increased level of c-fos mRNA persists for at least 24 hr. The induction of c-fos gene expression was further studied by utilizing a monoclonal antibody (mAb-192) that is directed against the NGF receptor but does not compete with NGF for binding to the receptor. Treatment of the cells with mAb-192 inhibits the NGF-stimulated elevation of c-fos mRNA, suggesting that the antibody may interfere with the receptor's ability to generate the signal required to stimulate the transcription of this gene. NGF is not the only agent capable of inducing c-fos gene expression in these cells; epidermal growth factor, the tumor promoter phorbol 12-myristate 13-acetate, and the calcium ionophore A23187, agents that induce the c-fos gene in other cell lines, are also effective in PC12 cells. The mRNA for the nuclear protein fos is rapidly induced by NGF and other agents to which PC12 cells respond. This supports the hypothesis that the fos gene product may play a role in signal transduction.

Topics: Animals; Antibodies, Monoclonal; Calcimycin; Cell Differentiation; Cell Line; Cycloheximide; Gene Expression Regulation; Kinetics; Nerve Growth Factors; Pheochromocytoma; Proto-Oncogene Proteins; Rats; Receptors, Cell Surface; Receptors, Nerve Growth Factor; RNA, Messenger; Tetradecanoylphorbol Acetate

Ca2+-dependent protein phosphorylation has been detected in numerous tissues and may mediate some of the effects of hormones and other extracellular stimuli on cell function. In this paper we demonstrate that a Ca2+/calmodulin-dependent protein kinase similar to the enzyme previously purified and characterized from rat brain is present in PC12, a rat pheochromocytoma cell line. We show that Ca2+ influx elicited by various forms of cell stimulation leads to increased 32P incorporation into tyrosine hydroxylase (TH), a major phosphoprotein in these cells. Several other unidentified proteins are either phosphorylated or dephosphorylated as a result of Ca2+ influx. Acetylcholine stimulates TH phosphorylation by activation of nicotinic receptors. K+-induced depolarization stimulates TH phosphorylation in a Ca2+-dependent manner, presumably by opening voltage-dependent Ca2+ channels. Ca2+ influx that results from the direct effects of the ionophore A23187 also leads to TH phosphorylation. Phosphorylation of TH is accompanied by an activation of the enzyme. These Ca2+-dependent effects are independent of cyclic AMP and thus implicate a Ca2+-dependent protein kinase as a mediator of both hormonal and electrical stimulation of PC12 cells.

Topics: Animals; Calcimycin; Calcium; Carbachol; Catecholamines; Cell Line; Cyclic AMP; Enzyme Activation; Ion Channels; Membrane Potentials; Pheochromocytoma; Phosphoproteins; Phosphorylation; Potassium; Protein Kinases; Rats; Receptors, Nicotinic; Tyrosine 3-Monooxygenase

Cultures of neurite-bearing pheochromocytoma (PC12) cells and of sympathetic neurons have been examined by time-lapse video microscopy. In the presence of nerve growth factor (NGF), the neurites of such cultures elongated and their growth cones changed geometry, via microspike and lamellipodial motion, on a time scale of minutes. Withdrawal of NGF caused process extension to cease and a progressive reduction in growth-cone area as a result of retraction of lamellipodia and microspikes. By approximately equal to 4 hr after NGF withdrawal, most neurite tips were smooth sided, devoid of conical expansions at their termini, and virtually immobile. Addition of NGF to cultures from which it had been withdrawn induced motion of microspikes and projections from the upper surface of growth cones within 2 min, while lamellipodial spreading and neurite reextension were induced after approximately equal to 20 min. For PC12 cells, these responses to replacement of NGF could not be mimicked by addition of dibutyryl cAMP (less than or equal to 2 mM) or the Ca2+ ionophore A23187 (less than or equal to 5 microM) to NGF-deprived cultures nor inhibited by the presence of EGTA (less than or equal to 2 mM) or calcium antagonists in the culture medium. Since neurite fragments formed by transection of processes of PC12 cells deprived of NGF responded to its replacement in a manner similar to intact neurites, it is concluded that the effects are focal to the neurite and growth cone and independent of the cell body. This influence of NGF on growth-cone shape and motility represents short-term local activation of this structure and has significance for control of neurite extension.

Topics: Adrenal Gland Neoplasms; Animals; Bucladesine; Calcimycin; Calcium; Cell Division; Ganglia, Sympathetic; Male; Mice; Nerve Growth Factors; Neurons; Pheochromocytoma; Rats; Rats, Inbred Strains; Time Factors