Nicotine regulates nicotinic cholinergic receptors and subunit mRNAs in PC 12 cells through protein kinase A

To understand the up-regulation of neuronal nicotinic cholinergic receptors (nAcChRs) that results from chronic in vivo treatment with nicotine, we studied the effect of nicotine on [3H]nicotine binding sites on PC 12 cells. PC 12 cells were grown in nicotine hemisulfate (10(-6) to 10(-3) M) or vehicle for 7 days, and specific [3H]nicotine binding was measured. Nicotine (10(-6) to 10(-4) M) dose-dependently increased specific binding by up to 2.6-fold over basal levels in 5-7 days, whereas a 10(-3) M concentration failed to do so. In contrast, [3H]nicotine binding to PC 12 cell mutants (A126.1B2 and A123.7), deficient in cAMP-responsive protein kinase A Types I and/or II, was unaffected by nicotine. Northern gel analysis of nAcChR subunit mRNAs from wild type PC 12 cells showed that the mRNA encoding the dominant agonist-binding subunit, alpha 3, was significantly reduced by nicotine, as early as 4 h after treatment, whereas mRNA for the structural beta 2 subunit was slightly increased. In contrast, the alpha 3 subunit mRNA from the PC 12 cell mutant A123.7 was not significantly decreased after 4 h and 7 days of nicotine treatment. These studies indicate that nicotine up-regulates expression of nAcChRs on wild type PC 12 cells and reduces the content of alpha 3 subunit mRNA; these effects require an intact protein kinase A system. The divergent effects of nicotine on the nAcChR compared to its alpha 3 subunit mRNA suggests that enhanced expression of nicotinic receptors may not involve synthesis of new receptor subunit proteins.     

ATP-activated chloride permeability in biliary epithelial cells is regulated by calmodulin-dependent protein kinase II

Previous studies in freshly isolated rat biliary epithelial cells and in the human cholangiocarcinoma cell line Mz-ChA-1 have demonstrated that ATP activates a calcium-dependent chloride conductance. The coupling between the rise in intracellular calcium and activation of chloride channels has not previously been investigated. In the present study, we evaluated the potential role of calmodulin-dependent protein kinase II (CaMKII) in ATP-activated chloride permeability in Mz-ChA-1 cells. ATP stimulated [125I] efflux, a marker for Cl- movement. Peak efflux rates were inhibited by approximately 60% in cells pretreated with the calmodulin antagonist, W-7. In whole-cell patch clamp recordings, ATP and ionomycin activated calcium-dependent Cl- currents. Pretreatment of cells with the CaMKII inhibitor KN-62 blocked activation by either agent. It is concluded that calcium-dependent activation of chloride currents in Mz-ChA-1 cells is coupled to a CaMKII-dependent process.     

Activation of a calcium-calmodulin-dependent protein kinase I cascade in PC12 cells

It has been observed that the activity of Ca2+-calmodulin (CaM)-dependent protein kinase I is enhanced up to 50-fold by its phosphorylation in vitro by a distinct CaM kinase I kinase (Lee, J. C., and Edelman, A. M. (1994) J. Biol. Chem. 269, 2158-2164). It has, however, been unclear whether this event represents an acute form of cellular regulation. We demonstrate here the phosphorylation and activation of CaM kinase I in PC12 pheochromocytoma cells in response to elevation of intracellular Ca2+. Treatment of PC12 cells with the Ca2+-ionophore, ionomycin, or with a depolarizing concentration of KCl, led to rapid, biphasic phosphorylation of CaM kinase I and to increases in CaM kinase I activity of 5.1- and 7. 3-fold, respectively. Depolarization-induced activation of CaM kinase I was reduced by approximately 80% by blockade of Ca2+ influx through L-type voltage-dependent Ca2+ channels and completely abolished by removal of extracellular Ca2+. The ability of PC12 cell CaM kinase I to be phosphorylated and activated by purified CaM kinase I kinase in vitro was markedly reduced by prior depolarization of the cells, consistent with intracellular phosphorylation and activation of CaM kinase I by CaM kinase I kinase. These results demonstrate the existence in PC12 cells of a CaM kinase I cascade, the function of which may be to sensitize cells to signal-induced elevations of intracellular Ca2+.     

Remodeling of the actin cytoskeleton is coordinately regulated by protein kinase C and the ADP-ribosylation factor nucleotide exchange factor ARNO

ARNO is a member of a family of guanine-nucleotide exchange factors with specificity for the ADP-ribosylation factor (ARF) GTPases. ARNO possesses a central catalytic domain with homology to yeast Sec7p and an adjacent C-terminal pleckstrin homology (PH) domain. We have previously shown that ARNO localizes to the plasma membrane in vivo and efficiently catalyzes ARF6 nucleotide exchange in vitro. In addition to a role in endocytosis, ARF6 has also been shown to regulate assembly of the actin cytoskeleton. To determine whether ARNO is an upstream regulator of ARF6 in vivo, we examined the distribution of actin in HeLa cells overexpressing ARNO. We found that, while expression of ARNO leads to disassembly of actin stress fibers, it does not result in obvious changes in cell morphology. However, treatment of ARNO transfectants with the PKC agonist phorbol 12-myristate 13-acetate results in the dramatic redistribution of ARNO, ARF6, and actin into membrane protrusions resembling lamellipodia. This process requires ARF activation, as actin rearrangement does not occur in cells expressing a catalytically inactive ARNO mutant. PKC phosphorylates ARNO at a site immediately C-terminal to its PH domain. However, mutation of this site had no effect on the ability of ARNO to regulate actin rearrangement, suggesting that phosphorylation of ARNO by PKC does not positively regulate its activity. Finally, we demonstrate that an ARNO mutant lacking the C-terminal PH domain no longer mediates cytoskeletal reorganization, indicating a role for this domain in appropriate membrane localization. Taken together, these data suggest that ARNO represents an important link between cell surface receptors, ARF6, and the actin cytoskeleton.     

Migration of retinal pigment epithelium cells in vitro is regulated by protein kinase C

The migration of retinal pigment epithelial (RPE) cells is an important step in various pathologic conditions, including subretinal neovascularization (SRN) and proliferative vitreoretinopathy (PVR). Therefore, elucidation of the mechanism of RPE migration may be useful in devising effective treatment for these disorders. Since protein kinase C (PKC) has been shown to regulate the migration of other cell types, we studied the effects of PKC agonists and antagonists on RPE migration. We used an in vitro wound healing model in which a small area of a confluent monolayer of bovine RPE cells was denuded with a razor blade. The cultures were subsequently incubated with agents known to stimulate [phorbol 12-myristate 13-acetate (PMA)] or inhibit (calphostin C, staurosporine) PKC. After 20 hr, migration was measured as the number of cells that had entered the denuded area. We also measured the translocation of PKC from the cytosol to the membrane in order to determine the activation or inhibition of PKC by PMA and calphostin C in the cells. The phorbol ester PMA stimulated migration by 41%, and calphostin C and staurosporine inhibited migration by 38% and 31%, respectively, in a medium supplemented with 10% serum. To determine the requirement for serum in this modulation, we also measured the effects of PMA and calphostin C on RPE migration in serum-free medium. Under these conditions, basal migration was greatly decreased, but PMA stimulated migration by 177% and calphostin C inhibited migration by 93%. Since PKC modulation is known to induce the proliferation of cells, we also tested the effects of these agents on growth-inhibited migration by pretreating the cells with the antiproliferative drug mitomycin C. We found that modulation of PKC under these conditions equally affected growth-inhibited and growth-dependent migration. Therefore, based on the increase in RPE migration induced by a PKC agonist, and the decrease in migration caused by PKC antagonists, it is suggested that PKC-mediated signal transduction plays a crucial role in RPE cell migration. This knowledge may be useful in devising effective treatments for SRN and PVR.     

Expression of type II cGMP-dependent protein kinase in rat kidney is regulated by dehydration and correlated with renin gene expression

To define the current indications for surgical management of pleuropulmonary tuberculosis and analyze the results of operative procedures, the records of 59 patients operated on between January 1987 and December 1993 were reviewed. Three patient categories were defined. Group I patients (n = 25) underwent operation for diagnostic purposes: solitary mediastinal node or mediastinal adenopathy associated with pulmonary lesions (n = 10), pulmonary infiltrates (n = 4), pulmonary nodules or masses (n = 10), or chronic pleurisy (n = 1). Postoperative mortality and morbidity rates in this group were both 4%. Group II patients (n = 18) underwent operation for active lesions: intrapulmonary cavity (n = 6), destroyed lung parenchyma (n = 6), or chronic loculated pleural effusion (n = 6). Postoperative morbidity and mortality rates were 16.6% and 5.5%, respectively. Group III patients (n = 16) underwent operation for a complication of therapy or for sequelae of previously "cured" tuberculosis: calcified pyothorax (n = 8), empyema (n = 2), fistulized nodes (n = 2), bronchiectasis (n = 3), or aspergilloma (n = 1). Morbidity and mortality rates in this group were 31.25% and 12.5%, respectively. Surgery continues to have both diagnostic and therapeutic indications for management of pleuropulmonary tuberculosis, despite the morbidity and mortality rates associated with operative procedures.     

Requirement of p38 mitogen-activated protein kinase for neuronal differentiation in PC12 cells

Nerve growth factor (NGF) induces sustained activation of classical MAP kinase (MAPK, also known as ERK) and neuronal differentiation in PC12 cells, whereas epidermal growth factor (EGF) induces transient activation of ERK/MAPK and stimulates proliferation of the cells. Although previous studies showed that sustained activation of ERK/MAPK is important for neuronal differentiation of the cells, a recent report revealed that inhibition of the sustained phase of ERK/MAPK activation alone does not block neurite outgrowth caused by NGF. These results suggest requirement for an additional signaling pathway(s) triggered by NGF in neuronal differentiation. Here we show that NGF induces sustained activation of p38, a subfamily member of the MAPK superfamily, and that inhibition of the p38 pathway blocks neurite outgrowth in PC12 cells. Surprisingly, expression of constitutively active MAPK/ERK kinase (MAPKK, also known as MEK) results in p38 activation as well as ERK/MAPK activation, and a p38 inhibitor blocks neurite outgrowth caused by the constitutively active MAPKK/MEK. Moreover, constitutive activation of p38 is able to induce neurite outgrowth when combined with EGF treatment. These results reveal an essential role of p38 in neuronal differentiation in PC12 cells.     

Calmodulin modulates mitogen-activated protein kinase activation in response to membrane depolarization in PC12 cells

In the absence of neurotrophic factors, chronic depolarization of plasma membrane has been shown to maintain several populations of primary neurons in culture. We report that in the PC12 cell line, depolarization causes Ca2+ influx through voltage-gated Ca2+ channels, which is able to stimulate extracellular-regulated kinase (ERK) activity. We studied which mediators were responsible for ERK activation resulting from increased levels of Ca2+ in the cytoplasm and found that calmodulin was involved in this process. The addition of W13, a calmodulin inhibitor, to the culture medium, prevented ERK activation when PC12 cells were depolarized. In addition, we show that high K+ treatment did not induce Trk A phosphorylation, thus excluding the possibility of Ca2+ operating through this receptor to activate the ERK signal transduction pathway. Moreover, although high K+ treatment is able to phosphorylate the epidermal growth factor receptor (EGFR) and thus to activate the ERK signal transduction pathway, we demonstrate that W13 did not alter the state of EGFR phosphorylation in conditions that almost completely blocked ERK activation. These data suggest that calmodulin mediates ERK activation induced by increases in intracellular Ca2+ concentration in PC12 cells by a mechanism that seems to be independent of Trk A and EGFR activation.     

Spinal morphine/clonidine antinociceptive synergism is regulated by protein kinase C, but not protein kinase A activity

When coadministered spinally, morphine and clonidine interact synergistically to produce antinociception. The mechanism for the synergism is unknown, but may depend on intracellular messenger systems. Agents that alter the activities of protein kinases alter antinociception produced by opioids, but their effects on clonidine-induced antinociception or the morphine/clonidine interaction are not known. In these studies, mice were pretreated intrathecally with inhibitors or activators of protein kinase C and cyclic AMP-dependent protein kinase (protein kinase A). Antinociceptive responses to intrathecally administered morphine, clonidine and morphine/clonidine combinations were then measured in the radiant heat tail flick test. Inhibition of protein kinase C activity with chelerythrine or calphostin C changed the morphine/clonidine interaction from synergistic to additive. Inhibition of protein kinase A activity with H-89 did not alter the morphine/clonidine interaction, it remained synergistic. Stimulation of protein kinase C activity with phorbol 12,13-dibutyrate attenuated morphine antinociception, but did not alter the synergistic interaction. Increasing spinal cyclic AMP concentrations with either forskolin or rolipram attenuated the antinociception produced by separately administered morphine and clonidine, but had no effect on the morphine/clonidine interaction. These results suggest that protein kinase C activity may regulate the interaction between spinal opioid and alpha-2 receptors, stimulated by morphine and clonidine.     

Physiological stress and nerve growth factor treatment regulate stress-activated protein kinase activity in PC12 cells

The stress-activated protein kinases (SAPKs) are differentially activated by a variety of cellular stressors in PC12 cells. SAPK activation has been linked to the induction of apoptotic cell death upon serum withdrawal from undifferentiated cells or following nerve growth factor (NGF) withdrawal of neuronally differentiated PC12 cells. However, withdrawal of trophic support from differentiated cells led to only a very modest elevation of SAPK activity and led us to investigate the basis of the relative insensitivity of these enzymes to stressors. NGF-stimulated differentiation of the cells resulted in the elevation of basal SAPK activity to levels four- to sevenfold greater than in untreated cells, which was correlated with an approximate fivefold increase in SAPK protein levels. Paradoxically, in NGF-differentiated PC12 cells, exposure to cellular stressors provoked a proportionately smaller stimulation of SAPK activity than that observed in naive cells, despite the presence of much higher levels of SAPK protein. The insensitivity of SAPK to activation by stressors was reflective of the activity of the SAPK activator SEK, whose activation was also diminished following NGF differentiation of the cells. The data demonstrate that SAPKs are subject to complex controls through both induction of SAPK expression and the regulation mediated by upstream elements within this pathway.     

Adenylyl cyclase 6 is selectively regulated by protein kinase A phosphorylation in a region involved in Galphas stimulation

Receptors activate adenylyl cyclases through the Galphas subunit. Previous studies from our laboratory have shown in certain cell types that express adenylyl cyclase 6 (AC6), heterologous desensitization included reduction of the capability of adenylyl cyclases to be stimulated by Galphas. Here we further analyze protein kinase A (PKA) effects on adenylyl cyclases. PKA treatment of recombinant AC6 in insect cell membranes results in a selective loss of stimulation by high (>10 nM) concentrations of Galphas. Similar treatment of AC1 or AC2 did not affect Galphas stimulation. Conversion of Ser-674 in AC6 to an Ala blocks PKA phosphorylation and PKA-mediated loss of Galphas stimulation. A peptide encoding the region 660-682 of AC6 blocks stimulation of AC6 and AC2 by high concentrations of Galphas. Substitution of Ser-674 to Asp in the peptide renders the peptide ineffective, indicating that the region 660-682 of AC6 is involved in regulation of signal transfer from Galphas. This region contains a conserved motif present in most adenylyl cyclases; however, the PKA phosphorylation site is unique to members of the AC6 family. These observations suggest a mechanism of how isoform selective regulatory diversity can be obtained within conserved regions involved in signal communication.     

PC5-A-mediated processing of pro-neurotensin in early compartments of the regulated secretory pathway of PC5-transfected PC12 cells

Among the members of the proprotein convertase (PC) family, PC1 and PC2 have well established roles as prohormone convertases. Another good candidate for this role is PC5-A that has been shown to be present in the regulated secretory pathway of certain neuroendocrine tissues, but evidence that it can process prohormones is lacking. To determine whether PC5-A could function as a prohormone convertase and to compare its cleavage specificity with that of PC1 and PC2, we stably transfected the rat pheochromocytoma PC12 cell line with PC5-A and analyzed the biosynthesis and subcellular localization of the enzyme, as well as its ability to process pro-neurotensin/neuromedin N (pro-NT/NN) into active peptides. Our data showed that in transfected PC12 cells, PC5-A was converted from its 126-kDa precursor form into a 117-kDa mature form and, to a lesser extent, into a C-terminally truncated 65-kDa form of the 117-kDa product. Metabolic and immunochemical studies showed that PC5-A was sorted to early compartments of the regulated secretory pathway where it colocalized with immunoreactive NT. Furthermore, pro-NT/NN was processed in these compartments according to a pattern that differed from that previously described in PC1- and PC2-transfected PC12 cells. This pattern resembled that previously reported for pro-NT/NN processing in the adrenal medulla, a tissue known to express high levels of PC5-A. Altogether, these data demonstrate for the first time the ability of PC5-A to function as a prohormone convertase in the regulated secretory pathway and suggest a role for this enzyme in the physiological processing of pro-NT/NN.     

Physiological phosphorylation of protein kinase A at Thr-197 is by a protein kinase A kinase

Phosphorylation of the catalytic subunit of cyclic AMP-dependent protein kinase, or protein kinase A, on Thr-197 is required for optimal enzyme activity, and enzyme isolated from either animal sources or bacterial expression strains is found phosphorylated at this site. Autophosphorylation of Thr-197 occurs in Escherichia coli and in vitro but is an inefficient intermolecular reaction catalyzed primarily by active, previously phosphorylated molecules. In contrast, the Thr-197 phosphorylation of newly synthesized protein kinase A in intact S49 mouse lymphoma cells is both efficient and insensitive to activators or inhibitors of intracellular protein kinase A. Using [35S]methionine-labeled, nonphosphorylated, recombinant catalytic subunit as the substrate in a gel mobility shift assay, we have identified an activity in extracts of protein kinase A-deficient S49 cells that phosphorylates catalytic subunit on Thr-197. The protein kinase A kinase activity partially purified by anion-exchange and hydroxylapatite chromatography is an efficient catalyst of protein kinase A phosphorylation in terms of both a low Km for ATP and a rapid time course. Phosphorylation of wild-type catalytic subunit by the kinase kinase activates the subunit for binding to a pseudosubstrate peptide inhibitor of protein kinase A. By both the gel shift assay and a [gamma-32P]ATP incorporation assay, the enzyme is active on wild-type catalytic subunit and on an inactive mutant with Met substituted for Lys-72 but inactive on a mutant with Ala substituted for Thr-197. Combined with the results from mutant subunits, phosphoamino acid analysis suggests that the enzyme is specific for phosphorylation of Thr-197.     

Neurotoxin-induced cell death in neuronal PC12 cells is mediated by induction of apoptosis

Death of neuronal cells during development and following deprivation of trophic factors is known to occur via an active mechanism requiring RNA and protein synthesis, known as apoptosis. Apoptosis is a form of cell "suicide" whereby the cell decides its own fate by activating a genetic programme of cell death. In contrast, necrosis is a passive uncontrolled form of cell death often observed in response to a toxic insult. Although it is known that neuronal cell death during development occurs by apoptosis, the mechanisms underlying neurotoxin-induced neuronal cell death remain poorly understood. In this study we have examined the mechanism by which 6-hydroxydopamine, a specific neurotoxin for catecholaminergic cells, induces neuronal cell death in vitro. We report that 6-hydroxydopamine induces cell death in the neuronal PC12 cell line via a mechanism which has the characteristic morphological and biochemical hallmarks of apoptosis. PC12 cells induced to die by 6-hydroxydopamine treatment exhibited cell shrinkage, classical chromatin condensation and membrane blebbing. Analysis of DNA integrity from 6-hydroxydopamine-treated cells revealed cleavage of DNA into regular sized fragments, a biochemical hallmark of apoptosis. 6-Hydroxydopamine-induced apoptosis of PC12 cells was suppressed by desipramine, a monoamine uptake inhibitor, suggesting that 6-hydroxydopamine is initiating apoptosis via a specific intracellular mechanism. Aurintricarboxylic acid, a general inhibitor of nucleases, also suppressed 6-hydroxydopamine-induced apoptosis, suggesting the involvement of an endonuclease in the death pathway. The aetiology of idiopathic Parkinson's disease remains uncertain, although evidence suggests that endogenous and/or exogenous toxins may initiate neuronal cell death in this disease. The dopaminergic neurotoxin 6-hydroxydopamine is used to generate animal models of Parkinson's disease in vivo. We have demonstrated that this neurotoxin kills neuronal cells in vitro by an active process of apoptosis. Thus, the possibility exists that cell death in neurodegenerative diseases such as Parkinsonism also occurs in an active manner initiated by as yet unidentified environmental or metabolic toxins. Cell death that involves activation of an apoptotic programme can be modulated by addition of extracellular trophic factors, and is also controlled by the levels of intracellular factors. If neurotoxin-induced apoptosis plays a role in Parkinson's disease the implication is that the neuronal degeneration may be prevented by pharmacological manipulations.     

Expression of protein kinase C gene family members is temporally and spatially regulated during neural development in vitro

The dog mastocytoma BR cell line provides us with a permanent source of canine mast cells, allowing a characterization of secretory mediators that exert important effects in canine allergic and nonallergic diseases and in physiological processes. We studied the ultrastructural characteristics and histamine releasing activity after immunological and non-immunological stimuli of the dog mastocytoma BR cell line, and compared the cell line to normal skin mast cells enzymatically isolated from healthy dogs. The histamine content of BR cells was 0.04 +/- 0.002 pg/cell, approximately 100-fold less than that found in canine skin mast cells. Non-immunologic stimuli induced similar concentration-dependent histamine release from skin mast cells and BR cells: 29.3 +/- 0.9% vs. 12.7 +/- 0.7% (calcium ionophore A23187), 23.3 +/- 0.7% vs. 18.8 +/- 0.7% (substance P) and 12.5 +/- 0.3% vs. 12.1 +/- 0.9% (compound 48/80), respectively. Immunologic stimulation, however, was only effective on canine skin mast cells, causing 30.9 +/- 1.7%, 27.7 +/- 0.6% and 12.2 +/- 0.9% histamine release in response to anti-canine IgE, concanavalin A, and antigen Asc S 1, respectively. The absence of functional IgE receptors in BR cells was confirmed by the lack of response to anti-IgE and antigen Asc S 1 following passive sensitization with dog atopic serum and dog antigen sensitized serum. We conclude that BR cells are able to release histamine after non-immunologic stimulation in a similar manner to canine skin mast cells, but that there are morphological and functional differences possibly due to different states of maturity or differentiation. For this reason the study of the highly homogeneous BR cells could offer insights into dog mast cell biology in contexts where freshly isolated cells cannot be used because of low purity and recovery.