Pituitary adenylate cyclase-activating polypeptide induces translocation of its G-protein-coupled receptor into caveolin-enriched membrane microdomains, leading to enhanced cyclic AMP generation and neurite outgrowth in PC12 cells

Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the secretin/glucagon/vasoactive intestinal peptide family expressed throughout the nervous system, binds to the PACAP-specific G-protein-coupled receptor family members to promote both neuronal differentiation and survival. Although the PACAP receptor is known to activate its effector protein, adenylate cyclase (AC), and thus enhance cAMP generation, the molecular mechanism utilized by the receptor to activate AC is lacking. Here, we show that PACAP induces neurite outgrowth in PC12 cells by induction of translocation of the PACAP type 1 receptor (PAC1R) into caveolin-enriched Triton X-100-insoluble microdomains, leading to stronger PAC1R-AC interaction and elevated cAMP production. Moreover, we demonstrate that translocation of PAC1R is blocked by various treatments that selectively disrupt caveolae. As a result, intracellular cAMP level is decreased and consequently the PACAP-induced neurite outgrowth retarded. In contrast, addition of exogenous ganglioside GM1 to the cells shows the opposite effects. These results therefore identify the PACAP-induced translocation of its G-protein-coupled receptor into caveolae, where both AC and the regulating G-proteins reside, as the key molecular event in activating AC and inducing cAMP-mediated differentiation of PC12 cells.     

Dedifferentiated Schwann cells promote perineural invasion mediated by the PACAP paracrine signalling in cervical cancer

Perineural invasion (PNI) has emerged as a key pathological feature and be considered as a poor prognostic factor in cervical cancer. However, the underlying molecular mechanisms are largely unknown. Here, PNI status of 269 cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) samples were quantified by using whole-slide diagnostic images obtained from The Cancer Genome Atlas. Integrated analyses revealed that PNI was an indicative marker of poorer disease-free survival for CESC patients. Among the differentially expressed genes, ADCYAP1 were identified. Clinical specimens supported that high expression of PACAP (encoded by ADCYAP1) contributed to PNI in CESC. Mechanistically, PACAP, secreted from cervical cancer cells, reversed myelin differentiation of Schwann cells (SCs). Then, dedifferentiated SCs promoted PNI by producing chemokine FGF17 and by degrading extracellular matrix through secretion of Cathepsin S and MMP-12. In conclusion, this study identified PACAP was associated with PNI in cervical cancer and suggested that tumour-derived PACAP reversed myelin differentiation of SCs to aid PNI.     

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. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 537–549, 1998     

Ubiquitin and ubiquitin-protein conjugates in PC12h cells: Changes during neuronal differentiation

Ubiquitin and ubiquitin-protein conjugates in PC12h cells were detected with in vitro [¹²⁵I]ubiquitination, and quantified by immunoblotting. These levels were altered by nerve growth factor (NGF), which promotes neuronal differentiation. (i) Levels of high molecular weight (HMW) ubiquitin-protein conjugates ranging from 40 to 1,000kDa were increased by 2 days of NGF treatment, and remained high up to 10 days of NGF treatment. (ii) Ubiquitin and a 23-kDa conjugate tended to be decreased from days 2 to 10 of NGF treatment. 10-Day culture with 10 nM staurosporine, an protein kinase inhibitor, that blocks NGF-induced neurite outgrowth suppressed the NGF-induced increases in levels of HMW conjugates. Cyclic AMP and forskolin, both of which promote neurite outgrowth, mimicked the NGF-induced changes in ubiquitin and HMW conjugates, but phorbol ester and epidermal growth factor had little effect. These findings suggest that changes in ubiquitin-protein conjugates are closely coupled with neuronal differentiation.     

神经营养因子诱导分化的神经元样PC12细胞分裂的研究

神经营养因子（nerve growth factor,NGF）诱导PC12细胞分化产生的神经元样细胞一直被认为属于分裂后的细胞,没有分裂能力。然而在本研究中,我们观察了一些已经发生分化的PC12细胞,这些细胞长有很长的神经突起,在形态上属于神经元样细胞。在这些细胞中,我们不仅检测到DNA合成,而且观察到这些细胞的分裂现象。更令人感兴趣的是,除了胞体发生分裂外,位于胞体分裂位置的突起也一分为二,分别分配给两个子细胞。这些结果说明,形态发生分化的神经元样PC12细胞仍有分裂能力。本研究首次报道神经元样PC12细胞及其突起能发生分裂。     

神经生长因子分化后的PC12 细胞对乙酰胆碱的敏感性及其特性

目的和方法：采用全细胞膜片钳技术观察神经生长因子（NGF）分化后的PC12细胞对乙酰胆碱（ACh)的敏感性,并对ACh诱发电流（IACh)的特性进行分析。结果：NGF处理后的PC12乐仅形态上向交感神经元分化,而且具有电学兴奋性,它对ACh敏感性比未分化前显著提高。药理学鉴定表明PC12上的IACh是由烟碱受体（nAChR)引起的,具有明显的失敏特性。宏观IACh呈内向整流和浓度依赖性。结论：PC12细胞培养方便,同源性好,加入NGF后向交感神经元分化,且其具有神经元烟碱受体,可以作为交感神经元烟碱受体研究的很好的模型系统。     

Neurite outgrowth in PC12 cells stimulated by acetyl-l-carnitine arginine amide

Senescence of the central nervous system is characterized by a progressive loss of neurons that can result in physiological and behavioral impairments. Reduction in the levels of central neurotrophic factors or of neurotrophin receptors may be one of the causes of the onset of these degenerative events. Thus, a proper therapeutic approach would be to increase support to degenerating neurons with trophic factors or to stimulate endogenous neurotrophic activity. Here we report that acetyl-l-carnitine arginine amide (ST-857) is able to stimulate neurite outgrowth in rat pheochromocytoma PC12 cells in a manner similar to that elicited by nerve growth factor (NGF). Neurite induction by ST-857 requires de novo mRNA synthesis and is independent of the action of several common trophic factors. The integrity of the molecular structure of ST-857 is essential for its activity, as the single moieties of the molecule have no effect on PC12 cells, whether they are tested separately or together. Also, minor chemical modifications of ST-857, such as the presence of the arginine moiety at a position other than the amino one, completely abolish its neuritogenic effect. Lastly, the presence of ST-857 in the culture medium competes with the high affinity NGF binding in a dose dependent fashion. These results, although preliminary, are suggestive of a possible role for ST-857 in the development of therapeutic strategies to counteract degenerative diseases of the CNS.     

Pituitary adenylate cyclase-activating polypeptide (PACAP) stimulates the expression and the release of tissue plasminogen activator (tPA) in neuronal cells: involvement of tPA in the neuroprotective effect of PACAP

Pituitary adenylate cyclase-activating polypeptide (PACAP) and tissue plasminogen activator (tPA) play important roles in neuronal migration and survival. However, a direct link between the neurotrophic effects of PACAP and tPA has never been investigated. In this study, we show that, in PC12 cells, PACAP induced a 9.85-fold increase in tPA gene expression through activation of the protein kinase A- and protein kinase C-dependent signaling pathways. In immature cerebellar granule neurons (CGN), PACAP stimulated tPA mRNA expression and release of proteolytically active tPA. Immunocytochemical labeling revealed the presence of tPA in the cytoplasm and processes of cultured CGN. The inhibitory effect of PACAP on CGN motility was not affected by the tPA substrate plasminogen or the tPA inhibitor plasminogen activator inhibitor-1. In contrast, plasminogen activator inhibitor-1 significantly reduced the stimulatory effect of PACAP on CGN survival. Altogether, these data indicate that tPA gene expression is activated by PACAP in both tumoral and normal neuronal cells. The present study also demonstrates that PACAP stimulates the release of tPA which promotes CGN survival by a mechanism dependent of its proteolytic activity.     

蛇毒神经生长因子诱导PC12细胞分化超微结构的观察

目的观察蛇毒神经生长因子（Nerve growth factor,NGF）诱导大鼠肾上腺嗜铬细胞瘤细胞系（Pheo—chromocytoma cells。PC12）细胞分化后,细胞超微结构的改变。方法取对数生长期PC12细胞接种24孔板,设200ng／ml NGF实验组和对照组。培养72h,离心,分别收集细胞制成电镜标本,镜下观察各组细胞超微结构的改变。结果与对照组细胞相比,实验组细胞长出大量突起．并且胞质的细胞器逐渐消失．出现较多的脂滴。结论广西眼镜蛇毒神经生长因子可以促进PC12细胞增殖．并诱导其向神经样细胞分化,长出突触。     

Influence of pulsed electromagnetic field with different pulse duty cycles on neurite outgrowth in PC12 rat pheochromocytoma cells

The influence of low frequency (50 Hz repetition rate) pulsed electromagnetic field (EMF) on PC12 cell neurite outgrowth in vitro was investigated in this study. We studied the percentage of neurite bearing cells, average length of neurites, and directivity of neurite outgrowth in PC12 cells cultured for 96 h in the presence of nerve growth factor (NGF). PC12 cells were exposed in one incubator to pulsed EMF at 1.36 mT (peak value) generated by a pair of Helmholtz coils, and the control samples were placed in another identical incubator. We found that the pulse duty cycle had significant effect on neurite outgrowth. Low (10%) pulse on-time significantly inhibited the percentage of neurite bearing cells, but at the same time increased the average length of neurites, while 100% on-time (DC) had exactly the opposite effects. Furthermore, we found that neurites were prone to extend along the direction of pulsed EMF with 10% pulse on-time. Our studies show that neurite outgrowth in PC12 cells is sensitive to the pulse duty and this sensitivity was associated with NGF concentration.     

Normal stimulation of the synthesis,phosphorylation and DNA binding activity of c-Fos and Zif268 proteins by nerve growth factor is not sufficient to mediate neuronal differentiation of PC12 cells

Early-response genes (ERGs) are rapidly induced by nerve growth factor (NGF) in the PC12 rat pheochromocytoma cell line. To analyze the possible role of Ras and ERGs in neuronal differentiation, experiments were carried out to study the involvement of Ras proteins in the NGF-stimulated expression of two ERG-coded proteins (c-Fos and Zif268) implicated in NGF signaling. Using PC12 subclones expressing the dominant negative Ha-Ras Asn-17 protein, NGF-induced expression, phosphorylation and DNA-binding of these ERG products were found to be not sufficient to convey the biological response of PC12 cells to NGF.     

Effect of thiopental, propofol, and etomidate on vincristine toxicity in PC12 cells

Neurotoxicity is the dose-limiting side-effect of vincristine in cancer therapy. Using the nerve growth factor (NGF)-dependent neurite outgrowth and cell proliferation of the PC12 pheochromocytoma cell line as an in vitroassay, the protective effect of different intravenous anesthetics was assessed. Vincristine (1 nmol/L) significantly decreased the percentage of neurite-forming cells from 68%±9% to 27%±7% within a 3-day incubation period. The longer neurites (>2× cell body) in particular proved to be extremely sensitive to vincristine (from 17%±4% to 0% of total neurite-expressing cells). Flow cytometry results revealed an S-phase percentage of 15.85%±3.25% after NGF induction, with vincristine reducing this percentage to 0.68%±0.38%. Reversal of the inhibitory effect of vincristine was noted in the cells treated with thiopental or propofol but not etomidate. Bicuculline partially antagonized the protective effect of thiopental and propofol in both studies. We conclude that thiopental and propofol, but not etomidate, have a protective effect in vincristine-induced neurotoxicity. The protective effect produced by thiopental and propofol is probably secondary to activation of GABA_Areceptors. This revised version was published online in August 2006 with corrections to the Cover Date.     

Daily rhythms in the somatotropic axis of Senegalese sole (Solea senegalensis): The time of day influences the response to GH administration

Growth factors in vertebrates display daily rhythms, which, while widely described in mammals, are still poorly understood in teleost fish. Here, we investigated the existence of daily rhythms in the somatotropic axis of the flatfish Solea senegalensis. In a first experiment, daily rhythms of the expression of pituitary adenylate cyclase–activating polypeptide (pacap), growth hormone (gh), insulin-like growth factor 1 (igf1) and its receptor (igf1r) were analyzed under a 12:12 h light:dark cycle. All genes displayed daily rhythms with the acrophases of pacap, gh and igf1 located in the second half of the dark phase (ZT 20:28–0:04 h), whereas the acrophase of igf1r was located around mid-light (ZT 5:33 h). In a second experiment, the influence of the time of day (mid-light, ML, versus mid-darkness, MD) of GH administration on the expression of these factors and on plasma glucose levels was tested. The response observed depended on the time of injection: the strongest effects were observed at MD, when GH administration significantly reduced pituitary gh and enhanced liver igf1 expression. These results provide the first evidence of daily rhythms and differential day/night effects in growth factors in S. senegalensis, suggesting new insights for investigating the physiology of growth and possible applications to improve fish aquaculture.     

垂体腺苷酸环化酶激活肽基因的合成表达与活性研究

根据文献报道垂体腺苷酸环化酶激活肽(pituitary adenylate cyclase activating polypeptide, PACAP)的氨基酸序列,推导出其核苷酸序列并设计成部分互补的6条寡核苷酸片段,利用DNA合成仪人工合成、纯化这6条寡核苷酸片段,通过片段退火、连接、克隆及测序鉴定获得了PACAP基因.将PACAP基因克隆至pGEX-4T-3质粒中转化BL21进行表达分析,融合蛋白约占细胞总蛋白的30％,其中部分为可溶性,部分以包涵体形式存在.通过亲和层析纯化GST-PACAP融合蛋白,该蛋白质能促进PC12细胞轴突生长及脊髓神经元存活.