Real-time monitoring of intracellular signal transduction in PC12 cells by two-dimensional surface plasmon resonance imager

Real-time observation of intracellular process of signal transduction is very useful for biomedical and pharmaceutical applications as well as for basic research work of cell biology. The conventional methods used to observe intracellular reactions have not been convenient with several steps such as labeling and washing steps prior to the readout. Consequently, there is a critical need for label-free observation techniques for monitoring intracellular reactions. For feasible and reagentless observation of intracellular alterations in real time, we examined the use of a high-resolution two-dimensional surface plasmon resonance (2D–SPR) imager for monitoring of intracellular signal transduction that was mainly translocation of protein kinase C via local refractive index change in PC12 cells adhered on a gold sensor slide without any indicator reagent. PC12 cells were stimulated with KCl and phorbol-12-myristate-13-acetate (PMA, a protein kinase C [PKC] activator) at different concentrations in order to induce intracellular PKC translocation. 2D–SPR signal (reflection intensity change) is very consistent with the cellular response normally detected for these stimulants. Our results suggest that complex intracellular reactions could be real-time monitored and characterized by the 2D–SPR imager. It is further expected that signal transmission that was followed by the translocation of signaling proteins could be observed at the single cell level with the high-resolution 2D–SPR imager.     

Multiple ecto-nucleotidases in PC12 cells: identification and cellular distribution after heterologous expression

The physiological action of extracellular ATP and other nucleotides in the nervous system is controlled by surface-located enzymes (ecto-nucleotidases) of which several families with partially overlapping substrate specificities exist. In order to identify ecto-nucleotidases potentially associated with neural cells, we chose PC12 cells for analysis. PC12 cells revealed surface-located ATPase and ADPase activity with apparent K(m)-values of 283 microM and 243 microM, respectively. Using PCR we identified the mRNA of all members of the ecto-nucleoside triphosphate diphosphohydrolase family investigated (NTPDase1 to NTPDase3, NTPDase5/6), of ecto-nucleotide pyrophosphatase/phosphodiesterase3 (NPP3), tissue-non-specific alkaline phosphatase and ecto-5'-nucleotidase. The surface-located catalytic activity differed greatly between the various enzyme species. Our data suggest that hydrolysis of ATP and ADP is mainly due to members of the ecto-nucleoside triphosphate diphosphohydrolase family. Activity of ecto-5'-nucleotidase and alkaline phosphatase was very low and activity of NPP3 was absent. For a detailed analysis of the cellular distribution of ecto-nucleotidases single and double transfections of PC12 cells were performed, followed by fluorescence analysis. Ecto-nucleotidases were distributed over the entire cell surface and accumulated intracellularly in varicosities and neurite tips. PC12 cell ecto-nucleotidases are likely to play an important role in terminating autocrine functions of released nucleotides and in producing extracellular nucleosides supporting the survival and neuritic differentiation of PC12 cells.     

Inhibition of the canonical Wnt signaling pathway by apolipoprotein E4 in PC12 cells

We examined the effect of the three human isoforms of apolipoprotein E (ApoE2, ApoE3, and ApoE4) on the canonical Wnt signaling pathway in undifferentiated PC12 cells. Addition of recombinant ApoE4 reduced Wingless-Int7a-stimulated gene expression at concentrations of 80 and 500 nm. Recombinant ApoE2 and ApoE3 were virtually inactive. Recombinant ApoE4 also inhibited Wnt signaling when combined with very low density lipoproteins (VLDLs) or in cells over-expressing the low density lipoprotein receptor-related protein, LRP6. In contrast, the enforced expression of LRP5 unmasked an inhibition by ApoE2 and ApoE3, which, however, were less effective than ApoE4 in inhibiting Wnt signaling. We also transfected PC12 cells with constructs encoding for the three human ApoE isoforms to examine whether endogenously expressed ApoE isoforms could modulate the Wnt pathway. Under these conditions, all three ApoE isoforms were able to inhibit Wnt signaling, although ApoE4 showed the greatest efficacy. Only the conditioned medium collected from cultures transfected with ApoE4 induced a significant inhibition of Wnt7a-stimulated gene expression, confirming that ApoE4 has an extracellular action that is not shared by the other ApoE isoforms. We conclude that ApoE4 behaves as an inhibitor of the canonical Wnt pathway in a context-independent manner.     

Decrease in acetylcholine-induced current by neomycin in PC12 cells

The effects of neomycin, one of the aminoglycoside antibiotics, on the acetylcholine (ACh)-induced current (I(ACh)) were studied in pheochromocytoma cells by using the whole-cell clamp technique. The I(ACh) proved to be generated through neuronal nicotinic receptor. ACh (30 microM) induced an inward current at a holding potential of -80 mV. When cells were treated with neomycin (0.01-1 mM) and ACh (30 microM) simultaneously, an inhibitory effect of neomycin on the peak of I(ACh) was found. This effect was fast, reversible, and concentration dependent. Pretreatment with neomycin for 3-8 min had no effect on the inhibition of I(ACh) induced by neomycin. External application of 0.1 mM neomycin neither shifted the dose-response curve of the peak I(ACh) to the right (dissociation constant (K(d)) = 16.5 microM) nor affected its coefficient (1.8) but inhibited the curve amplitudes by approximately 33%. Stimulated protein kinase C activation by using an exogenous activator produced inhibition of I(ACh), while using protein kinase C inhibitor (PKCI 19-31) had no effect on the inhibition of I(ACh) induced by neomycin. These results suggest that neomycin has an inhibitory effect on I(ACh) without the involvement of phospholipase C. It indicates that neomycin binds to a specific site on the cell membrane, probably on the neuronal nicotinic receptor-coupled channel, and inhibits the I(ACh) in a noncompetitive manner, thus controlling the immediate catecholamine release from the sympathetic cells.     

依达拉奉对MPP+处理的PC12细胞线粒体融合和分裂平衡的保护作用

本文旨在探讨依达拉奉(edaravone, Eda)对帕金森病细胞模型线粒体融合、分裂动态平衡的作用及机制。用500μmol/L1-甲基-4-苯基吡啶离子(1-methyl-4-phenylpyridinium, MPP^+)处理PC12细胞建立帕金森病细胞模型,采用噻唑蓝(MTT)比色法检测不同浓度Eda对MPP^+处理的PC12细胞存活率的影响,用激光共聚焦显微镜检测线粒体形态,用Western blot检测线粒体融合与分裂相关蛋白OPA1、MFN2、DRP1和Fis1的表达变化。结果显示,预先加入不同浓度的Eda能减轻MPP^+处理的PC12细胞损伤,作用呈一定的量效关系;经MPP^+处理48 h,PC12细胞线粒体出现碎片化,OPA1和MFN2蛋白表达下调,DRP1和Fis1蛋白表达上调,而Eda预处理能逆转PC12细胞的上述变化,但对Fis1的蛋白表达没有影响。以上结果提示,Eda可上调OPA1和MFN2的蛋白表达,下调DRP1的表达,从而抑制线粒体碎片化,发挥神经细胞线粒体保护作用。     

Nonylphenol enhances apoptosis induced by serum deprivation in PC12 cells

Although nonylphenol is well known as an endocrine disrupting chemical, there is little information concerning biological effect of nonylphenol. In this study, we investigated effect of nonylphenol on apoptosis induced by serum deprivation in PC12 cells using TUNEL and DNA fragmentation assays. In addition, changes in contents of proapoptotic factors, Bad and Bax, and antiapoptotic factor, Bcl-2, and enzyme activity of caspase-3 were studied. Below 100 ng/ml of nonylphenol increased TUNEL signals, DNA fragmentation and content of proapoptotic factor, Bad as compared to those by serum deprivation without nonylphenol. Furthermore, addition of nonylphenol enhanced caspase-3 activity and Z-VAD, caspase-3 inhibitor, diminished such effect. These results indicated that below 100 ng/ml of nonylphenol enhanced apoptosis induced by serum deprivation via caspase-3 activation in PC12 cell.     

Nongenomic inhibition of catecholamine secretion by 17beta-estradiol in PC12 cells

We investigated the effects of 17beta-estradiol, an estrogen, on [(3)H]norepinephrine ([(3)H]NE) secretion in PC12 cells. Pretreatment with 17beta-estradiol reduced 70 mM K(+)-induced [(3)H]NE secretion in a concentration-dependent manner with a half-maximal inhibitory concentration (IC(50)) of 2 +/- 1 microM. The 70 mM K(+)-induced cytosolic free Ca(2+) concentration ([Ca(2+)](i)) rise was also reduced when the cells were treated with 17beta-estradiol (IC(50) = 15 +/- 2 microM). Studies with voltage-sensitive calcium channel (VSCC) antagonists such as nifedipine and omega-conotoxin GVIA revealed that both L- and N-type VSCCs were affected by 17beta-estradiol treatment. The 17beta-estradiol effect was not changed by pretreatment of the cells with actinomycin D and cycloheximide for 5 h. In addition, treatment with pertussis or cholera toxin did not affect the inhibitory effect of 17beta-estradiol. 17beta-Estradiol also inhibited the ATP-induced [(3)H]NE secretion and [Ca(2+)](i) rise. In PC12 cells, the ATP-induced [Ca(2+)](i) rise is known to occur through P2X(2) receptors, the P2Y(2)-mediated phospholipase C (PLC) pathway, and VSCCs. 17beta-Estradiol pretreatment during complete inhibition of the PLC pathway and VSCCs inhibited the ATP-induced [Ca(2+)](i) rise. Our results suggest that 17beta-estradiol inhibits catecholamine secretion by inhibiting L- and N-type Ca(2+) channels and P2X(2) receptors in a nongenomic manner.     

Bradykinin stimulates phospholipase D in PC12 cells by a mechanism which is independent of increases in intracellular Ca2+

These experiments were designed to learn the role of bradykinin induced changes in intracellular Ca²⁺ in the activation of phospholipase D activity in PC12 cells. Ionomycin at a concentration of 0.1M caused an increase in intracellular Ca²⁺ comparable to bradykinin, but had no effect on phospholipase D activity. Carbachol, ATP, and thapsigargin also increased intracellular Ca²⁺ but had no effect on phospholipase D activity. Increases in intracellular Ca²⁺ may be a necessary but not a sufficient factor in the activation of phospholipase D. To investigate this issue, the bradykinin induced increase in intracellular Ca²⁺ was blocked by preincubating the cells in Ca²⁺-free media plus EGTA or in media containing the intracellular Ca²⁺ chelator BAPTA/AM. These preincubations completely blocked the bradykinin induced increase in intracellular Ca²⁺ but only attenuated the bradykinin mediated activation of phospholipase D. Physiological increases in intracellular Ca²⁺ apparently do not mediate the effect of bradykinin on phospholipase D.     

N-CAM expression and localization in PC12 cells modulated by extracellular peptides

The neural cell adhesion molecules (N-CAMs) play an important role in mediating cell–cell interactions in the nervous system. Different isoforms of these membrane proteins are involved in the formation of the neuronal network and in the dynamic phases of neuronal plasticity.

We studied the early stages of the pseudo neuronal differentiation of PC12 cells induced by a class of small acidic peptides capable of modulating gene expression in these cells.

The data presented here indicate that peptides with specific sequences induce an increase in N-CAM mRNA expression and protein translocation to the plasma membrane to a comparable degree as NGF.     

Intracellular signaling pathways involved in post-mitotic dopaminergic PC12 cell death induced by 6-hydroxydopamine

Oxidative stress has been shown to mediate neuron damage in Parkinson's disease (PD). In the present report, we intend to clarify the intracellular pathways mediating dopaminergic neuron death after oxidative stress production using post-mitotic PC12 cells treated with the neurotoxin 6-hydroxydopamine (6-OHDA). The use of post-mitotic cells is crucial, because one of the suggested intracellular pathways implicated in neuron death relates to the re-entry of neurons (post-mitotic cells) in the cell cycle. We find that 6-OHDA sequentially increases intracellular oxidants, functional cell damage and caspase-3 activation, leading to cell death after 12 h of incubation. Prevention of cell damage by different antioxidants supports the implication of oxidative stress in the observed neurotoxicity. Oxidative stress-dependent phosphorylation of the MAPK JNK and oxidative stress-independent PKB/Akt dephosphorylation are involved in 6-OHDA neurotoxicity. Decrease in p21(WAF1/CIP1) and cyclin-D1 expression, disappearance of the non-phosphorylated band of retinoblastoma protein (pRb), and expression of proliferating cell nuclear antigen, not present in PC12 post-mitotic cells, suggest a re-entry of differentiated cells into cell cycle. Our results indicate that such a re-entry is mediated by oxidative stress and is involved in 6-OHDA-induced cell death. We conclude that at least three intracellular pathways are involved in 6-OHDA-induced cell death in differentiated PC12 cells: JNK activation, cell cycle progression (both oxidative stress-dependent), and Akt dephosphorylation (not related to the increase of oxidants); the three pathways are necessary for the cells to die, since blocking one of them is sufficient to keep the cells alive.     

Treatment of PC12 cells by nerve growth factor,dexamethasone, and forskolin

Several lines of anatomical, neurochemical, electrophysiological, and behavioral evidence suggest the existence of physiological interactions between neurotensin (NT) and the brain dopaminergic systems. Thus, NT has been shown to exert a neuroleptic-like action and could be implicated in the pathogenesis and treatment of schizophrenia. It is thus of particular importance to develop in vitro cell culture systems as models to study such interactions. Rat adrenal pheochromocytoma PC12 cells, which expressed high levels of tyrosine hydroxylase, were used in the present study. In contrast to rat brain cells in primary cultures, PC12 cells did not express functional NT receptors. However, they were able to express both NTmRNA and NT in response to NGF, forskolin, and dexamethasone. Those neurochemical modifications furthermore may be related to changes in the morphology of the PC12 cells in response to NGF, forskolin, and dexamethasone alone or in combination. These data suggest that PC12 cells may provide a useful model to study in vitro the regulation of both catecholamine and neurotensin phenotypes.     

Leumorphin has an anti-apoptotic effect by activating epidermal growth factor receptor kinase in rat pheochromocytoma PC12 cells

Endogenous opioid peptides, found in the central and peripheral nervous systems, perform neuromodulatory roles, and display a wide range of functional and pharmacological properties in vitro and in vivo. In this study, we investigated the effects of prodynorphin gene products on intracellular signaling events and cell survival in rat pheochromocytoma PC12 cells. Leumorphin, but not other prodynorphin gene products including dynorphin A, beta-neoendorphin and rimorphin (dynorphin B), increased cell viability in PC12 cells. The cytoprotective effect of leumorphin was dependent on the phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways, but was insensitive to both naloxone, a general antagonist of the opioid receptor, and nor-binaltorphimine, a specific antagonist of the kappa opioid receptor. Moreover, a competition-binding assay clearly revealed that leumorphin had another binding site(s) in addition to that for the kappa opioid receptor. Interestingly, leumorphin induced activation of the epidermal growth factor receptor via a Src-dependent mechanism, which was proved to be responsible for the increased survival response. Flow cytometric and microscopic analysis showed that leumorphin rescued cells from serum deprivation-induced apoptosis. Collectively, we suggest that leumorphin prevents apoptosis via epidermal growth factor receptor-mediated activation of the phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways, which occur independent of the kappa opioid receptor.     

Nicotine-induced phosphorylation of Akt through epidermal growth factor receptor and Src in PC12h cells

Nicotine treatment triggers calcium influx into neuronal cells, which promotes cell survival in a number of neuronal cells. Phosphoinositide (PI) 3-kinase and downstream PI3-kinase target Akt have been reported to be important in the calcium-mediated promotion of survival in a wide variety of cells. We investigated the mechanisms of nicotine-induced phosphorylation of Akt in PC12h cells, in comparison with nicotine-induced ERK phosphorylation. Nicotine induced Akt phosphorylation in a dose-dependent manner. A nicotinic acetylcholine receptor (nAChR) alpha7 subunit-selective inhibitor had no significant effect on nicotine-induced Akt phosphorylation, while a non-selective nAChR antagonist inhibited the phosphorylation. L-type voltage-sensitive calcium channel (VSCC) antagonists, calmodulin antagonist, and Ca2+/calmudulin-dependent protein kinase (CaM kinase) inhibitor prevented the nicotine-induced Akt phosphorylation. Three epidermal growth factor receptor (EGFR) inhibitors prevented the nicotine-induced phosphorylation of both extracellular signal-regulated protein kinase (p42/44 MAP kinase, ERK) and Akt. In contrast, an inhibitor of the Src family tyrosine kinase prevented the nicotine-induced Akt phosphorylation but not ERK phosphorylation. These results suggested that nicotine induces the activation of both PI3-kinase/Akt and ERK pathways via common pathways including non-alpha7-nAChRs, L-type VSCC, CaM kinase II and EGFR in PC12h cells, but Src family tyrosine kinases only participate in the pathway to activate Akt.     

Regulation of alpha3 nicotinic acetylcholine receptor subunit mRNA levels by nerve growth factor and cyclic AMP in PC12 cells

To investigate the effects of nerve growth factor (NGF) and cyclic AMP (cAMP) on the level of the nicotinic acetylcholine receptor subunit alpha3 mRNA, we used PC12h cells, PC12 cells expressing dominant-negative Ras protein, and the parental PC12 cells. PC12h cells have NGF-responsive tyrosine hydroxylase activity. Expression of dominant-negative Ras protein prevents the signaling through the Ras-mitogen-activated protein kinase cascade. The morphological changes of the parental PC12 cells in response to NGF and 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPTcAMP), a cell-penetrating cAMP analogue, were similar to those of PC12h cells. NGF up-regulated the alpha3 mRNA level in PC12h cells and down-regulated the alpha3 mRNA level in the parental PC12 cells. Expression of dominant-negative Ras protein and an inhibitor of mitogen-activated protein kinase kinase inhibited the effects of NGF on alpha3 mRNA level. CPTcAMP down-regulated the alpha3 mRNA level in all three PC12 cell lines. An inhibitor of protein kinase A inhibited the CPTcAMP-induced down-regulation of alpha3 mRNA. The alpha3 mRNA down-regulation required prolonged treatment with CPTcAMP even after cAMP response element binding protein phosphorylation was decreased. Membrane depolarization with high K+ had no effect on the alpha3 mRNA level in PC12h cells. Based on these results, we propose that at least two unknown effectors regulate alpha3 mRNA levels in PC12 cells.     

Generation of inositol phosphates, cytosolic Ca and secretion of noradrenaline in PC12 cells treated with glutamate

Glutamate transiently stimulated rat pheochromocytoma PC12 cells and caused an inositol trisphosphate formation and an increase in levels of Ca⁺ in the cytosol. The rank order of potency of glutamate> N-methyl-D-aspartate (NMDA) > KAINATE = quisqualate is characteristic of an interaction with NMDA receptors. The effect of glutamate on inositol trisphosphate formation disappeared in a low Mg²⁺ buffer and was not blocked by DL-2-amino-5-phosphonovalerate, an antagonist for NMDA receptors coupled to ion channels. Although glutamate failed to stimulate noradrenaline secretion, glutamate enhanced the effect of bradykinin, but not of Ca ionophore A23187, or KC1. These results suggest the existence of metabotropic glutamate receptors, different from previously reported receptors, in PC12 cells.     

低频脉冲电场对PC12细胞突起生长和NGF受体分布的影响

以PC12细胞为实验材料,研究低频脉冲电场(f=50Hz,τ=20ms,Epp=1V／m)对神经细胞突起生长及膜受体聚簇的影响。结果显示,该电场能促进NGF受体的聚簇。电场处理15min使PC12细胞表面的NGF受体发生明显的聚簇,30min组次之,5min组聚簇效果较弱。这表明细胞膜受体可能是电磁场与细胞相互作用的位点之一。运用细胞突起图形处理软件,追踪测定经电场处理后PC12细胞的突起数量和长度,发现该电场能显著促进细胞突起的生长,但对突起数量没有明显的影响。     

Chronic hypoxia leads to a glycolytic phenotype and suppressed HIF-2 signaling in PC12 cells

Background

Along with other regulators of cell metabolism, hypoxia-inducible factors HIF-1 and HIF-2 differentially regulate cell adaptation to hypoxia. Switches in HIF-1/HIF-2 signaling in chronic hypoxia have not been fully investigated.

Methods

Proliferation, viability, apoptosis, neuronal and bioenergetic markers, mitochondrial function, respiration, glycolysis, HIF signalling, responses to O₂ and glucose deprivation (OGD) were examined using tumor PC12 and SH-SY5Y cells continuously grown at 3% O₂.

Results

Hypoxic PC12 cells (H-cells) exhibit reduced proliferation and histone H4 acetylation, NGF-independent differentiation, activation of AMPK, inhibition of Akt, altered mitochondria and response to NGF. Cellular cytochrome c is increased with no effect on apoptosis. Reduction in respiration has minor effect on cellular ATP which is maintained through activated uptake (GLUT1) and utilization (HK2, PFK2) of glucose. H-cells exhibit resistance to OGD linked to increased glycogen stores. HIF-2alpha protein is decreased without changes in mRNA. Unlike HIF-1alpha, HIF-2alpha is not stabilized pharmacologically or by O₂ deprivation. Capacity for HIF-2alpha stabilization is partly restored when H-cells are cultured at normoxia. In low-respiring SH-SY5Y cells cultured under the same conditions HIF-2alpha stabilization and energy budget are not affected.

Conclusions

In chronically hypoxic PC12 cells glycolytic energy budget, increased energy preservation and low susceptibility to OGD are observed. HIF-2alpha no longer orchestrates adaptive responses to anoxia.

General significance

Demonstrated switch in HIF-1/HIF-2 signaling upon chronic hypoxia can facilitate cell survival in energy crisis, by regulating balance between energy saving and decrease in proliferation, on one hand and active cell growth and tumor expansion, on the other.     

Molecular mechanism of 2-APB-induced Ca influx in external acidification in PC12

2-Aminoethoxydiphenyl borate (2-APB) is used as a pharmacological tool because it antagonizes inositol 1,4,5-trisphosphate receptors and store-operated Ca²⁺ (SOC) channels, and activates some TRP channels. Recently, we reported that 2-APB enhanced the increase in cytotoxic [Ca²⁺]_i, resulting in cell death under external acidic conditions in rat pheochromocytoma cell line PC12. However, the molecular mechanism and functional role of the 2-APB-induced Ca²⁺ influx in PC12 have not been clarified. In this study, to identify the possible target for the action of 2-APB we examined the pharmacological and molecular properties of [Ca²⁺]_i and secretory responses to 2-APB under extracellular low pH conditions. 2-APB dose-dependently induced a [Ca²⁺]_i increase and dopamine release, which were greatly enhanced by the external acidification (pH 6.5). [Ca²⁺]_i and secretory responses to 2-APB at pH 6.5 were inhibited by the removal of extracellular Ca²⁺ and SOC channel blockers such as SK&F96365, La³⁺ and Gd³⁺. PC12 expressed all SOC channel molecules, Orai 1, Orai 2 and Orai 3. When we used an siRNA system, downregulation of Orai 3, but not Orai 1 and Orai 2, attenuated both [Ca²⁺]_i and secretory responses to 2-APB. These results suggest that 2-APB evokes external acid-dependent increases of [Ca²⁺]_i and dopamine release in PC12 through the activation of Orai 3. The present results indicate that 2-APB may be a useful pharmacological tool for Orai channel-related signaling.     

Nicotine-induced phosphorylation of extracellular signal-regulated protein kinase and CREB in PC12h cells

We have investigated mechanisms of nicotine-induced phosphorylation of extracellular signal-regulated protein kinase (p42/44 MAP kinase, ERK) and cAMP response element binding protein (CREB) in PC12h cells. Nicotine transiently induced ERK phosphorylation at more than 1 microM. The maximal level of nicotine-induced ERK phosphorylation was lower than that of the membrane depolarization induced and, to a great extent, the nerve growth factor (NGF)-induced ERK phosphorylation. Nicotinic acetylcholine receptor (nAChR) alpha7 subunit-selective inhibitors had no significant effect on nicotine-induced ERK phosphorylation. L-Type voltage-sensitive calcium channel antagonists inhibited nicotine-induced ERK phosphorylation. Calcium imaging experiments showed that alpha7-containing nAChR subtypes were functional at 1 microM of nicotine in the nicotine-induced calcium influx, and non-alpha7 nAChRs were prominent in the Ca(2+) influx at 50 microM of nicotine. An expression of dominant inhibitory Ras inhibited nicotine-induced ERK phosphorylation. A calmodulin antagonist, a CaM kinase inhibitor, a MAP kinase kinase inhibitor inhibited nicotine-induced ERK and CREB phosphorylation. The time course of the phosphorylation of CREB induced by nicotine was similar to that of ERK induced by nicotine. These results suggest that non-alpha7 nAChRs are involved in nicotine-induced ERK phosphorylation through CaM kinase and the Ras-MAP kinase cascade and most of the nicotine-induced CREB phosphorylation is mediated by the ERK phosphorylation in PC12h cells.     

Depletion of intracellular Ca2+ store itself may be a major factor in thapsigargin-induced ER stress and apoptosis in PC12 cells

The mechanisms of intracellular calcium store depletion and store-related Ca²⁺ dysregulation in relation to apoptotic cell death in PC12 cells were investigated at physiological temperatures with a leak-resistant fluorescent indicator dye Fura-PE3/AM by a cooled CCD imaging analysis system. Electron microscopic observations have shown thapsigargin (TG; 100 nM)-induced apoptosis in PC12 cells. Thorough starvation of stored Ca²⁺ by BAPTA/AM (50 μM), or La³⁺ (100 μM) enhanced while dantrolene (100 μM) attenuated the TG-induced apoptosis by preventing a calcium release from internal stores. An immunoblotting analysis revealed an enhanced expression of GRP78, the hallmark of endoplasmic reticulum (ER) stress when cells were treated by TG along with BAPTA/AM. These results indicate that the depletion of the intracellular Ca²⁺ stores itself induces the ER stress and apoptosis in PC12 cells without any involvement of the capacitative calcium entry (CCE) or a sustained elevation of intracellular Ca²⁺ concentrations ([Ca²⁺]_i).