Growth Factor-differentiated PC12 Cells Research Articles

Physical mobilization of secretory vesicles facilitates neuropeptide release by nerve growth factor-differentiated PC12 cells.

It has been speculated that neurosecretion can be enhanced by increasing the motion, and hence, the availability of cytoplasmic secretory vesicles. However, facilitator-induced physical mobilization of secretory vesicles has not been observed directly in living cells, and recent experimental results call this hypothesis into question. Here, high resolution green fluorescent protein (GFP)-based measurements in nerve growth factor-differentiated PC12 cells are used to test whether altering dense core vesicle (DCV) motion affects neuropeptide release. Experiments with mycalolide B and jasplakinolide demonstrate that neuropeptidergic DCV motion at the ends of processes is proportional to F-actin. Furthermore, Ba2+ increases DCV mobility without detectably modifying F-actin. Finally, we show that altering DCV motion by changing F-actin or stimulating with Ba2+ proportionally changes sustained neuropeptide release. Therefore, increasing DCV mobility facilitates prolonged neuropeptide release.

Proton-gated channels in PC12 cells.

Acid-sensing ion channels (ASICs) are expressed in various sensory and central neurons. The functional role of these channels remains elusive. Complex subunit combinations and lack of specific blockers for native receptors are likely to contribute to the difficulty of resolving the function of ASICs. Finding a neuronal cell line, which expresses a single population of ASICs, should prove to be useful in delineating the function of individual ASICs. Using patch-clamp, Ca(2+)-imaging, and RT-PCR techniques, we have explored the existence of ASICs in PC12 cells, a clonal neuronal cell line. Fast drops of extracellular pH activated transient inward currents in PC12 cells with pH(0.5) at 6.0-6.2. The ASICs in PC12 cells were selective for Na(+) with significant Ca(2+) permeability. Currents in PC12 cells were blocked by the nonselective ASIC blocker amiloride. PcTX1, a specific homomeric ASIC1a blocker, also blocked the ASIC currents with an IC(50) of approximately 1.5 nM. RT-PCR demonstrated the existence of ASIC1a transcript in both undifferentiated and nerve growth factor-differentiated PC12 cells. Our data suggest that PC12 cells likely contain a single population of functional proton-gated channel-homomeric ASIC1a. It might be an ideal neuronal cell line for the study of physiological and potential pathological roles of this key subunit of ASICs.

Origin binding protein-containing protein-DNA complex formation at herpes simplex virus type 1 oriS: role in oriS-dependent DNA replication.

Initiation of herpes simplex virus type 1 (HSV-1) DNA replication during productive infection of fibroblasts and epithelial cells requires attachment of the origin binding protein (OBP), one of seven essential virus-encoded DNA replication proteins, to specific sequences within the two viral origins, oriL and oriS. Whether initiation of DNA replication during reactivation of HSV-1 from neuronal latency also requires OBP is not known. A truncated protein, consisting of the C-terminal 487 amino acids of OBP, termed OBPC, is the product of the HSV UL8.5 gene and binds to origin sequences, although OBPC's role in HSV DNA replication is not yet clear. To characterize protein-DNA complex formation at oriS in cells of neural and nonneural lineage, we used nuclear extracts of HSV-infected nerve growth factor-differentiated PC12 and Vero cells, respectively, as the source of protein in gel shift assays. In both cell types, three complexes (complexes A, B, and C) which contain either OBP or OBPC were shown to bind specifically to a probe which contains the highest-affinity OBP binding site in oriS, site 1. Complex A was shown to contain OBPC exclusively, whereas complexes B and C contained OBP and likely other cellular proteins. By fine-mapping the binding sites of these three complexes, we identified single nucleotides which, when mutated, eliminated formation of all three complexes, or complexes B and C, but not A. In transient DNA replication assays, both mutations significantly impaired oriS-dependent DNA replication, demonstrating that formation of OBP-containing complexes B and C is required for efficient initiation of oriS-dependent DNA replication, whereas formation of the OBPC-containing complex A is insufficient for efficient initiation.

Inhibitory effects of endogenous dopaminergic neurotoxin, norsalsolinol on dopamine secretion in PC12 rat pheochromocytoma cells

Naturally occurring neurotoxins, 6,7-dihydroxy-1,2,3,4-tetrahydroisoquinolines (DHTIQs), thought to be the causative agents of Parkinsonism. DHTIQs including norsalsolinol have been found in the mammalian central nervous system. Norsalsolinol can be formed by a non-enzymatic Pictet–Spengler condensation reaction between dopamine and formaldehyde, and has been detected in the urine of Parkinsonian patients. However, the effects of DHTIQs on the secretion of dopamine, as well as other neurotransmitters, are not well understood. This study investigated the effects of norsalsolinol on dopamine secretion from nerve growth factor-differentiated PC12 cells. Norsalsolinol (1–100 μM) pretreatment suppressed both ATP (100 μM)- and K + (50 mM)-induced dopamine secretion from PC12 cells in a concentration-dependent fashion, but did not affect basal dopamine secretion. In β-escin-permeabilized PC12 cells, norsalsolinol pretreatment suppressed Ca 2+ (pCa=4–8)-induced dopamine secretion, but did not inhibit the secretagogue-induced change in intracellular Ca 2+ concentration. These results suggest that norsalsolinol causes the inhibition of secretagogue-induced dopamine secretion from PC12 cells without altering intracellular Ca 2+ concentration. Inhibition of dopamine secretion by norsalsolinol may also be involved in postural abnormality in Parkinson's disease.

Modulation of KCNQ2/3 potassium channels by the novel anticonvulsant retigabine.

Retigabine is a novel anticonvulsant with an unknown mechanism of action. It has recently been reported that retigabine modulates a potassium channel current in nerve growth factor-differentiated PC12 cells (), however, to date the molecular correlate of this current has not been identified. In the present study we have examined the effects of retigabine on recombinant human KCNQ2 and KCNQ3 potassium channels, expressed either alone or in combination in Xenopus oocytes. Application of 10 microM retigabine to oocytes expressing the KCNQ2/3 heteromeric channel shifted both the activation threshold and voltage for half-activation by approximately 20 mV in the hyperpolarizing direction, leading to an increase in current amplitude at test potentials between -80 mV and +20 mV. Retigabine also had a marked effect on KCNQ current kinetics, increasing the rate of channel activation but slowing deactivation at a given test potential. Similar effects of retigabine were observed in oocytes expressing KCNQ2 alone, suggesting that KCNQ2 may be the molecular target of retigabine. Membrane potential recordings in oocytes expressing the KCNQ2/3 heteromeric channel showed that application of retigabine leads to a concentration-dependent hyperpolarization of the oocyte, from a resting potential of -63 mV under control conditions to -85 mV in the presence of 100 microM retigabine (IC(50) = 5.2 microM). In control experiments retigabine had no effect on either resting membrane potential or endogenous oocyte membrane currents. In conclusion, we have shown that retigabine acts as a KCNQ potassium channel opener. Because the heteromeric KCNQ2/3 channel has recently been reported to underlie the M-current, it is likely that M-current modulation can explain the anticonvulsant actions of retigabine in animal models of epilepsy.

Hrs interacts with SNAP-25 and regulates Ca(2+)-dependent exocytosis.

Synaptosome-associated protein of 25 kDa (SNAP-25) is a neuronal membrane protein essential for synaptic vesicle exocytosis. To investigate the mechanisms by which SNAP-25 mediates neurosecretion, we performed a search for proteins that interact with SNAP-25 using a yeast two-hybrid screen. Here, we report the isolation and characterization of a SNAP-25-interacting protein that is the rat homologue of mouse hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs). Hrs specifically interacts with SNAP-25, but not SNAP-23/syndet. The association of Hrs and SNAP-25 is mediated via coiled-coil interactions. Using an Hrs-specific antibody, we have shown that Hrs is highly enriched in brain, where it codistributes with SNAP-25 in most brain regions. Subcellular fractionation studies demonstrate that in brain, Hrs exists in both cytosolic and membrane-associated pools. Studies using indirect immunofluorescence and confocal microscopy reveal that, in addition to early endosomes, Hrs is also localized to large dense-core secretory granules and synaptic-like microvesicles in nerve growth factor-differentiated PC12 cells. Moreover, overexpression of Hrs in PC12 cells inhibits Ca(2+)-dependent exocytosis. These results suggest that Hrs is involved in regulation of neurosecretion through interaction with SNAP-25.

Inositol Stereoisomers Stabilize an Oligomeric Aggregate of Alzheimer Amyloid β Peptide and Inhibit Aβ-induced Toxicity

Inositol has 8 stereoisomers, four of which are physiologically active. myo-Inositol is the most abundant isomer in the brain and more recently shown that epi- and scyllo-inositol are also present. myo-Inositol complexes with Abeta42 in vitro to form a small stable micelle. The ability of inositol stereoisomers to interact with and stabilize small Abeta complexes was addressed. Circular dichroism spectroscopy demonstrated that epi- and scyllo- but not chiro-inositol were able to induce a structural transition from random to beta-structure in Abeta42. Alternatively, none of the stereoisomers were able to induce a structural transition in Abeta40. Electron microscopy demonstrated that inositol stabilizes small aggregates of Abeta42. We demonstrate that inositol-Abeta interactions result in a complex that is non-toxic to nerve growth factor-differentiated PC-12 cells and primary human neuronal cultures. The attenuation of toxicity is the result of Abeta-inositol interaction, as inositol uptake inhibitors had no effect on neuronal survival. The use of inositol stereoisomers allowed us to elucidate an important structure-activity relationship between Abeta and inositol. Inositol stereoisomers are naturally occurring molecules that readily cross the blood-brain barrier and may represent a viable treatment for AD through the complexation of Abeta and attenuation of Abeta neurotoxic effects.

Cyclooxygenase-2 Expression Inhibits Trophic Withdrawal Apoptosis in Nerve Growth Factor-differentiated PC12 Cells

Cyclooxygenase-2 (Cox-2), an enzyme responsible for catalyzing the committed step in prostanoid biosynthesis, is the product of an immediate early gene capable of being up-regulated by diverse stimuli. Significantly Cox-2 mRNA is absent from rat pheochromocytoma (PC12) cells, both basally and following stimulation with a range of agonists. Using PC12 cells engineered to stably express isopropyl-1-thio-beta-D-galactopyranoside-inducible Cox-2 (PCXII-4), we have investigated the putative effects of Cox-2 expression on differentiation, proliferation, and trophic withdrawal apoptosis. Cox-2 bioactivity had no effect on nerve growth factor-induced differentiation, epidermal growth factor-induced proliferation, or aromatic L-amino acid decarboxylase expression. However, trophic withdrawal apoptosis, induced by the removal of nerve growth factor following differentiation, was markedly reduced in the PCXII-4 when compared with control cells, as assessed by annexin V staining, DNA laddering, and Hoechst 33258 staining. The specificity of this effect was confirmed using two pharmacologically distinct nonsteroidal anti-inflammatory drugs, indomethacin and NS398. Investigations showed that the activity of the pro-apoptotic protease caspase-3 was reduced in PCXII cells. This study demonstrates that Cox-2-derived prostaglandins exert cytoprotective effects in trophic factor withdrawal apoptosis and provides evidence that this is, at least in part, due to suppression of caspase-3 activity.

Synaptotagmin IV Is Present at the Golgi and Distal Parts of Neurites

Synaptotagmin IV (SytIV) is an immediate early gene induced by membrane depolarization in PC12 cells and in rat brain. However, little is known about the function of SytIV or the functional relationship between SytIV and SytI, because SytIV has yet to be localized. Here we show that SytIV was localized at the Golgi and distal part of neurites in nerve growth factor-differentiated PC12 cells and cultured hippocampal neurons by immunocytochemistry using an isoform-specific antibody (anti-SytIV). These SytIV signals were not colocalized well with SytI signals. Upon membrane depolarization, SytIV signals were increased at both the Golgi and distal part of neurites within several hours in both types of cells. We further show that the increase of SytIV protein levels results from protein kinase A-dependent gene up-regulation. In hippocampal neurons, SytIV was developmentally regulated. These results suggest that SytIV may play a role at the Golgi and tips of neurites during development and synaptic plasticity.

Bcl-2 protects against apoptosis-related microtubule alterations in neuronal cells.

Bcl-2 is a gene with clear anti-apoptotic properties in neurodegenerative conditions. One of the earliest hallmarks of degeneration in neuronal cell cultures is the loss of neurite morphology. Therefore the effect of Bcl-2 on neuronal morphology and microtubule stability was studied in nerve growth factor differentiated PC12 cells. Microtubule dynamics were modulated using the microtubule stabilizer taxol and the microtubule destabilizer, okadaic acid, a protein phosphatase inhibitor. It was shown that Bcl-2 protects against both taxol- and okadaic acid induced neurite retraction. Bcl-2 overexpression also significantly reduced the increased ratio of acetylated tubulin over total tubulin induced by taxol treatment. Interestingly, Bcl-2 attenuates the decrease of the same ratio after exposure to okadaic acid, suggesting that Bcl-2 is able to normalize the level of acetylated tubulin. In addition, cell death and nuclear fragmentation, induced by okadaic acid, were reduced in Bcl-2 overexpressing cells. This protection is either downstream or independent of tau phosphorylation as quantitative immunocytochemistry with AT8 showed that Bcl-2 did not modify the level of tau phosphorylation. The data suggest that the protective effect of Bcl-2 on the neuronal cytoskeleton is probably linked to changes in the post-translational modification of tubulin.

Uptake and incorporation of docosahexaenoic acid (DHA) into neuronal cell body and neurite/nerve growth cone lipids: evidence of compartmental DHA metabolism in nerve growth factor-differentiated PC12 cells.

Docosahexaenoic acid (DHA) accumulates in nerve endings of the brain during development. It is released from the membrane during ischemia and electroconvulsive shock. DHA optimizes neurologic development, it is neuroprotective, and rat adrenopheochromocytoma (PC12) cells have decreased PLA2 activity when DHA is present. To characterize DHA metabolism in PC12 cells, media were supplemented with [3H]DHA or [3H]glycerol. Fractions of nerve growth cone particles (NGC) and cell bodies were prepared and the metabolism of the radiolabeled substrates was determined by thin-layer chromatography. [3H]glycerol incorporation into phospholipids indicated de novo lipid synthesis. [3H]DHA uptake was more rapid in the cell bodies than in the NGC. [3H]DHA first esterified in neutral lipids and later in phospholipids (phosphatidylethanolamine). [3H]glycerol primarily labeled phosphatidylcholine. DHA uptake was compartmentalized between the cell body and the NGC. With metabolism similar to that seen in vivo, PC12 cells are an appropriate model to study DHA in neurons.

Inhibition of neutral sphingomyelinase activation and ceramide formation by glutathione in hypoxic PC12 cell death.

Reduced glutathione (GSH) and N-acetylcysteine (NAC), but not other antioxidative or reducing agents, were found to inhibit cell death, both apoptosis and necrosis, induced by hypoxia in naive and nerve growth factor-differentiated PC12 cells. The level of intracellular total GSH decreased time-dependently during hypoxia, but exogenously added GSH prevented such a decrease in GSH. Pretreatment of cells with exogenous GSH or NAC resulted in inhibition of both neutral sphingomyelinase (SMase) activation and ceramide formation during hypoxia. In the in vitro assay system, neutral SMase activity was inhibited dose-dependently by GSH and NAC. Activation of caspase-3 induced by hypoxia was also inhibited by either GSH or NAC. NAC but not GSH inhibited caspase-3 activation induced by C2-ceramide. These results suggest that GSH protects cells from hypoxic injury by direct inhibition of neutral SMase activity and ceramide formation, resulting in inhibition of caspase-3 activation, and that NAC exerts an additional inhibitory effect(s) downstream of ceramide.

Signal transduction pathway regulating prostaglandin EP3 receptor-induced neurite retraction: requirement for two different tyrosine kinases

We reported previously that activation of the prostaglandin E receptor EP3 subtype triggered neurite retraction through the small GTPase Rho-, and its target, RhoA-binding kinase α (ROKα)-, dependent pathway in EP3 receptor-expressing PC12 cells. Here we examined the involvement of tyrosine kinases in this pathway in nerve growth factor-differentiated PC12 cells. Tyrphostin A25, a tyrosine kinase inhibitor, blocked neurite retraction and cell rounding induced by activation of the EP3 receptor, however, it failed to block neurite retraction and cell rounding induced by microinjection of constitutively active RhoA, RhoAV14, indicating that a tyrphostin-sensitive tyrosine kinase was involved in the pathway from the EP3 receptor to Rho activation. On the other hand, genistein, another tyrosine kinase inhibitor, blocked neurite retraction and cell rounding induced by both activation of the EP3 receptor and microinjection of RhoAV14. However, genistein did not block neuronal morphological changes induced by microinjection of a constitutively active mutant of ROKα. These results indicate that two different tyrosine kinases, tyrphostin A25-sensitive and genistein-sensitive kinases, are involved in the EP3 receptor-mediated neurite retraction acting upstream and downstream of Rho, respectively.

Signal transduction pathway regulating prostaglandin EP3 receptor-induced neurite retraction: requirement for two different tyrosine kinases

We reported previously that activation of the prostaglandin E receptor EP3 subtype triggered neurite retraction through the small GTPase Rho-, and its target, RhoA-binding kinase alpha (ROKalpha)-, dependent pathway in EP3 receptor-expressing PC12 cells. Here we examined the involvement of tyrosine kinases in this pathway in nerve growth factor-differentiated PC12 cells. Tyrphostin A25, a tyrosine kinase inhibitor, blocked neurite retraction and cell rounding induced by activation of the EP3 receptor, however, it failed to block neurite retraction and cell rounding induced by microinjection of constitutively active RhoA, RhoAV14, indicating that a tyrphostin-sensitive tyrosine kinase was involved in the pathway from the EP3 receptor to Rho activation. On the other hand, genistein, another tyrosine kinase inhibitor, blocked neurite retraction and cell rounding induced by both activation of the EP3 receptor and microinjection of RhoAV14. However, genistein did not block neuronal morphological changes induced by microinjection of a constitutively active mutant of ROKalpha. These results indicate that two different tyrosine kinases, tyrphostin A25-sensitive and genistein-sensitive kinases, are involved in the EP3 receptor-mediated neurite retraction acting upstream and downstream of Rho, respectively.

Inhibitors of V-type ATPases, bafilomycin A1 and concanamycin A, protect against beta-amyloid-mediated effects on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction.

The functional viability of cells can be evaluated using a number of different assay determinants. One common assay involves exposing cells to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), which is converted intracellularly to a colored formazan precipitate and often used to assess amyloid peptide-induced cytotoxic effects. The MTT assay was employed to evaluate the role of endosomal uptake and lysosomal acidification in amyloid peptide-treated differentiated PC12 cell cultures using selective vacuolar-type (V-type) ATPase inhibitors. The macrolides bafilomycin A1 (BAF) and concanamycin A (CON) block lysosomal acidification through selective inhibition of the V-type ATPase. Treating nerve growth factor-differentiated PC12 cells with nanomolar concentrations of BAF or CON provides complete protection against the effects of beta-amyloid peptides Abeta(1-42), Abeta(1-40), and Abeta(25-35) and of amylin on MTT dye conversion. These macrolides do not inhibit peptide aggregation, act as antioxidants, or inhibit Abeta uptake by cells. Measurements of lysosomal acidification reveal that the concentrations of BAF and CON effective in reversing Abeta-mediated MTT dye conversion also reverse lysosomal pH. These results suggest that lysosomal acidification is necessary for Abeta effects on MTT dye conversion.

Docosahexaenoic acid decreases phospholipase A2 activity in the neurites/nerve growth conesof PC12 cells

Docosahexaenoic acid (DHA) accumulates in nerve growth cones (NGC) during perinatal development and it is neuroprotective in ischemia. Because the phospholipases A2 (PLA2) are present in NGC and these enzymes function in both ischemia and long-term potentiation, the relationship between DHA and PLA2 was investigated in the NGC of nerve growth factor-differentiated PC12 cells. When PC12 cells were incubated with [3H]DHA, it primarily esterified in ethanolamine glycerolipids and concentrated initially in cell bodies with similar levels present in the neurite/nerve growth cone (N/NGC) fraction after 4 days. PLA2 activity in the N/NGC fraction was investigated using [14C]arachidonic acid-labeled phosphatidylinositol ([14C-AA]PI) as substrate. Heat denaturation and pharmacological inhibition showed that much of the PLA2 activity was calcium-independent and secretory rather than cytosolic. Supplementing the media with as little as 33 nM DHA significantly reduced PLA2 activity in the N/NGC fraction.

Lysophosphatidic acid and apoptosis of nerve growth factor-differentiated PC12 cells.

The lipid biomediator lysophosphatidic acid (LPA) elicits a unique response in hippocampal neurons, LPA induces neuronal apoptosis. This study explores the effects of LPA on cells with neuronal properties, nerve growth factor-differentiated PC6 cells, a clone of PC12 cells. LPA induced apoptosis in these cells as assessed by chromatin condensation, terminal dUTP nick end-labeling of DNA, protection against these nuclear alterations by a general caspase inhibitor and the lack of release of lactic dehydrogenase. LPA caused oxidative stress, namely a decreased reduction of MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. This oxidative stress appears to be of functional significance, since cells were protected by pretreatment with the antioxidant propyl gallate and by stable transfection with cDNA encoding the antioxidant enzyme, manganese superoxide dismutase. Mitochondrial and nitric oxide participation in LPA-induced apoptosis are suggested by the protection afforded by pretreatment with either cyclosporin A, an inhibitor of mitochondrial permeability transition, or nitric oxide synthase inhibitors. The nitric oxide synthase inhibitor findings are novel, since to our knowledge, LPA has not heretofore been associated with an increase in nitric oxide. In addition, as observed for many neurotoxic agents, insulin-like growth factor I protected against LPA-induced apoptosis of PC6 cells.

P160 RhoA-binding Kinase ROKα Induces Neurite Retraction

We previously reported that the activation of prostaglandin E receptor EP3 subtype caused neurite retraction via small GTPase Rho in the EP3B receptor-expressing PC12 cells (Katoh, H., Negishi, M., and Ichikawa, A. (1996) J. Biol. Chem. 271, 29780-29784). However, a potential downstream effector of Rho that induces neurite retraction was not identified. Here we examined the morphological effect of p160 RhoA-binding kinase ROKalpha, a target for RhoA recently identified, on the nerve growth factor-differentiated PC12 cells. Microinjection of the catalytic domain of ROKalpha rapidly induced neurite retraction similar to that induced by microinjection of a constitutively active Rho, RhoV14, whereas microinjection of the kinase-deficient catalytic domain of ROKalpha did not induce neurite retraction. This morphological change was observed even though C3 exoenzyme, which was known to inactivate Rho, had been preinjected. On the other hand, microinjection of the Rho-binding domain or the pleckstrin homology domain of ROKalpha inhibited the EP3 receptor-induced neurite retraction. These results demonstrate that ROKalpha induces neurite retraction acting downstream of Rho in neuronal cells.

Toxicity of cholesterol oxides on cultured neuroretinal cells

PURPOSE. By using nerve growth factor-differentiated PC12 cells as a model for sympathetic neurons, we have recently shown that cholesterol oxides are toxic to cells of neural origin. Since lipid metabolism is known to be involved in some pathological conditions associated with the visual system, we sought to extend this line of research by studying the potential cytotoxicity of cholesterol oxides on primary cultures derived from neuroretinas. METHODS. Dissociated cultures derived from neuroretinas of 1-day-old Sprague-Dawley rats were used in this series of studies. Immunohistochemical staining was used to identify neuronal and glial cell types in these cultures. MTT assay was used to determine the cytotoxicity of cholesterol oxides, including 7-beta-OH-, 7-keto-, 19-OH-, 22(R)-OH-, 22(S)-OH- and 25-OH-cholesterol. RESULTS. Among the cholesterol oxides tested, 7-beta-OH- and 7-keto-cholesterol were the most effective in causing cell death, such that 20 µg/ml (50 µM) of these agents killed approximately 80% of cells in 3 days. A time-dependent experiment indicated that 10 µg/ml of 7-beta-cholesterol was able to kill 50% of cells in approximately 5 h. A number of pharmacological agents were tested for their ability to prevent cell death induced by cholesterol oxides. Among them, vitamin E and methyl-beta-cyclodextrin were able to prevent cholesterol oxide-induced cell death in a dose-dependent manner. CONCLUSIONS. These results suggest that, in addition to causing pathological changes in cells directly involved in atherosclerosis, cholesterol oxides may be toxic to cells derived from neuroretinas.

Chronic exposure to inorganic lead increases high-threshold voltage-gated calcium currents in rat pheochromocytoma (PC12) cells

Rat pheochromocytoma (PC12) cells were exposed to lead acetate (0, 10, 25 and 50 μM) in their growth media for up to 12 weeks. High-threshold voltage-gated calcium currents were recorded each week from nerve growth factor-differentiated PC12 cells using the whole-cell patch-clamp technique. Chronic exposure for 1 month did not modify peak or sustained calcium current amplitudes in lead-treated cells when compared to sister control cultures. Two month exposure to 25 and 50 μM significantly increased peak and sustained calcium current amplitudes, while 10 μM had little effect. During the third month of exposure, peak and sustained calcium current amplitudes remained increased in the cells exposed to 25 and 50 μM lead acetate. By the end of the second month of exposure to 25 and 50 μM lead acetate, the voltage at which maximal current amplitude was attained shifted from +10 mV to 0 mV. The observed effects of toxicologically relevant lead concentrations on high-threshold calcium currents in chronically exposed mammalian cells provide further support for the notion that at least one cellular target of the heavy metal's neurotoxic action may be the voltage-gated calcium channel.