D-beta-hydroxybutyrate inhibits the apoptosis of PC12 cells induced by 6-OHDA in relation to up-regulating the ratio of Bcl-2/Bax mRNA

D-beta-hydroxybutyrate (DbetaHB) is a predominant member of ketone bodies produced by hepatocytes and, to a lesser extent, by astrocytes. It is an alternative source of energy in the brain when glucose supply is depleted such as during starvation. It has been reported that ketone bodies could protect dopaminergic culture. However, the biological function of DbetaHB in Parkinson disease (PD) is still unclear. In the present work, we investigated the role of DbetaHB in protecting rat pheochromocytoma (PC12) cells from apoptosis induced by 6-Hydroxydopamine (6-OHDA). DbetaHB rescued PC12 cells from apoptotic death induced by 6-OHDA by MTT assay, acridine orange (AO) staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining and the activity of caspase-3. DbetaHB prevented the decrease of cell viability and the increase of caspase-3 activity induced by 6-OHDA in a dose-dependent manner in PC12 cells. AO and TUNEL staining showed that DbetaHB prevented the apoptosis of PC12 cells induced by 6-OHDA. The ratio of Bcl-2/Bax at mRNA levels, which regulates the apoptosis of PC12 cells when exposed to 6-OHDA, increased when DbetaHB was preincubated. The data showed that DbetaHB inhibited the apoptosis of PC12 cells induced by 6-OHDA in relation to up-regulating the ratio of Bcl-2/Bax mRNA.     

尿酸减轻6-羟基多巴胺对PC12细胞的毒性作用

目的：评价尿酸减轻6．羟基多巴胺（6-OHDA）对PC12细胞的毒性作用。方法：应用PCI2细胞制作帕金森细胞模型,分为对照组、尿酸组、6-OHDA组、尿酸＋6-OHDA组。采用MTT测定各组PC12细胞活性,免疫荧光法观察各组PCI2细胞caspase-3激活情况,流式细胞术检测各组PC12细胞凋亡率。尿酸100～400μmol·L^-1不影响PCI2细胞生存率,尿酸100～400μmol·L-1可显著提高6-OHDA50gmol-L。作用6、12和24h造成的PCI2细胞生存率的下降（P〈0．01）;尿酸能减少6-OHDA导致的PCI2细胞caspase-3激活,降低6-OHDA导致的凋亡率（P〈0．05）。结论：尿酸具有减轻6-OHDA对PC12细胞的毒性作用。     

Enhanced cystatin C and lysosomal protease expression following 6-hydroxydopamine exposure

6-Hydroxydopamine (6-OHDA) is a selective neurotoxin used to induce apoptosis in catecholamine-containing neurons. Although biochemical products and reactive oxygen species (ROS) of 6-OHDA have been well documented, the activation of cellular pathways following exposure are not well understood. Apoptosis in PC12 (Pheochromocytoma) cells was induced by 6-OHDA in a dose (10-150 microM) and time-dependent (24-72 h) manner compared to experimental controls (no treatment). PC 12 cells exposed to 50 microM 6-OHDA demonstrated the involvement of caspase 3 and lysosomal protease alterations. Following 6-OHDA exposure, the caspase 3-like inhibitor Ac-DEVD-CHO significantly decreased 6-OHDA induced cell death. In addition, alterations in expression of the lysosomal cysteine and aspartic proteases, cathepsin B (CB) and cathepsin D (CD) and the endogenous cysteine protease inhibitor cystatin C were observed utilizing immunocytochemical analysis at 24, 48, and 72 h following 6-OHDA exposure. Furthermore, CB and CD and cystatin C immuno-like reactivity was more pronounced in TUNEL positive cells. Moreover, Western blot analysis confirmed a significant increase in protein expression for CB and CD at 72 h and a temporal and concentration dependent increase in cystatin C in response to 6-OHDA. Cells treated with pepstatin A, an inhibitor for CD, showed a significant decrease in cell death, however, CA-074ME, a specific inhibitor for CB, failed to protect cells from 6-OHDA induced cell death. Thus, these results suggest that apoptosis induced by 6-OHDA exposure is mediated in part through caspase 3 activation and lysosomal protease CD.     

雌二醇对6-羟基多巴胺引起的大鼠嗜铬细胞瘤细胞损伤的影响

目的:研究雌激素对6-羟基多巴胺(6-OHDA)引起的大鼠嗜铬细胞瘤细胞(PC12)损伤的影响。方法:采用台盼蓝染料排除法和检测细胞乳酸脱氢酶(LDH)漏出量水平,估测PC12细胞的损伤程度。结果:同型异构体17β雌二醇和17α雌二醇均可明显减轻6-OHDA引起的PC12细胞存活率的降低,雌激素受体拮抗剂他莫昔芬对17β雌二醇和17α雌二醇的细胞保护作用无明显影响。结论:雌激素可能是通过非受体依赖的抗氧化机制对6-OHDA引起的PC12细胞损伤起保护作用。     

黄芪甲苷对PC12细胞氧化应激损伤的保护作用

目的 探讨黄芪甲苷对PC12细胞氧化应激损伤的作用。方法 体外培养PC12细胞，6-羟基多巴胺（6-OHDA）作用PC12细胞导致氧化应激损伤。根据细胞作用方法随机分为4组:①对照组，采用完全培养基正常培养；②6-OHDA组，给予终浓度为100 μmol/L的6-OHDA处理24 h；③低、中、高剂量黄芪甲苷组，分别给予终浓度为25、50、100 μmol/L的黄芪甲苷预处理24 h，然后给予终浓度为100 μmol/L 6-OHDA处理24 h；④AG490组，以20 μmol/L AG490（JAK2/STAT3信号通路抑制剂）预处理16 h，加入终浓度为100 μmol/L黄芪甲苷处理24 h，然后再给予终浓度为100 μmol/L 6-OHDA处理24 h。CCK8法检测细胞生存率，流式细胞仪检测细胞凋亡率，酶联免疫吸附实验法检测细胞上清液超氧化物歧化酶（SOD）和丙二醛（MDA）水平，免疫印迹法检测细胞p-JAK2和p-STAT3蛋白表达。结果 6-OHDA作用后，PC12细胞存活率明显降低，细胞凋亡率明显升高，细胞培养液SOD水平明显降低、MDA水平明显升高，细胞p-JAK2和p-STAT3蛋白表达水平明显降低；黄芪甲苷预处理明显逆转6-OHDA的作用，而且呈剂量依赖性；AG490预处理明显逆转黄芪甲苷的作用。结论 6-OHDA作用PC12细胞，可导致氧化应激损伤促使细胞凋亡；黄芪甲苷预处理能激活JAK2/STAT3信号通路，抑制6-OHDA对PC12细胞的损伤，对PC12细胞起保护作用。     

Ribozyme-mediated inhibition of caspase-3 activity reduces apoptosis induced by 6-hydroxydopamine in PC12 cells

6-Hydroxydopamine (6-OHDA) is a neurotoxin used in the induction of experimental Parkinson's disease in both animals and PC12 cells, which are derived from rat pheochromocytoma tumors and have many properties similar to dopamine neurons. Biochemical and molecular approaches have shown that low doses of 6-OHDA induce apoptosis in PC12 cells and, in the processing of apoptosis, caspases are crucial mediators, and caspase inhibition is sufficient to rescue PC12 cells from apoptosis induced by 6-OHDA. However, because this caspase inhibition targets multiple caspases, it is not known whether a single caspase is primarily responsible for effecting cell death in this model. To assess the particular member (caspase-3) of the ced-3 family relevant to cell death and to position their activation within the apoptotic pathway, we constructed a hammerhead ribozyme directed against rat caspase-3, which could downregulate the expression of caspase-3 in vitro and in vivo, and transfer to PC12 cells. The results show that the ribozymes against caspase-3 could protect PC12 cells from apoptosis induced by low doses of 6-OHDA. The PC12 cell transfected with the ribozymes shows a significant decrease in caspase-3 activity compared with control cells at various time points. Parallel to the reduced caspase-3 protease activity, similar decreased levels of apoptotic cells and DNA fragmentation were also assessed by staining with Hoechst 33258 and ELISA, respectively. Overexpression of p35, a general caspase inhibitor, also protected PC12 cells from apoptosis. These results confirm that caspases play an important role in 6-OHDA-induced PC12 cell apoptosis and indicate that caspase-3 itself is one of the crucial mediators of neurotoxin-induced PC12 cell apoptosis.     

Neurotrophin-3 reduces apoptosis induced by 6-OHDA in PC12 cells through Akt signaling pathway

Previous work has demonstrated that 6-hydroxydopamine (6-OHDA) induces apoptosis in PC12 cells. The goal of the present study was to investigate the mechanisms underlying the protection by neurotrophin-3 (NT-3) against 6-OHDA-induced apoptosis in PC12 cells. Treatment of PC12 cells with 6-OHDA resulted in activation of caspase-3 and subsequent apoptosis, as detected by TUNEL staining. In addition, Akt phosphorylation was decreased following 6-OHDA treatment. Pretreatment with NT-3 reduced the percentage of apoptotic cells and caspase-3 activity induced by 6-OHDA and suppressed the cleavage of caspase-3 and Poly(ADP-ribose) polymerase (PARP) with a significant decrease in cell viability. Moreover, Akt phosphorylation was enhanced and 6-OHDA-induced chromatin condensation was suppressed by NT-3. Such NT-3-evoked suppression in chromatin condensation was reversed by anti-TrkA antibody receptor blockade. Further study revealed that LY294002, an inhibitor of PI3-kinase (a molecule upstream of Akt), enhanced 6-OHDA-induced apoptosis. These data indicate that NT-3 prevents 6-OHDA-induced apoptosis in PC12 cells via activation of PI3-kinase/Akt pathway.     

6-羟基多巴胺诱导PC12细胞帕金森模型中GRP78表达的研究

目的 探讨神经毒素6-羟基多巴胺（6-OHDA）诱导PC12细胞的帕金森（PD）模型中GRP78的表达.方法 在建立6-OHDA诱导PC12细胞帕金森模型的基础上,MTT法测细胞存活率和Hoechst33342染色检测细胞凋亡.分别提取6-OHDA处理组和对照组细胞总蛋白,应用荧光差异凝胶电泳（Differential Gel Electrophoresis,DIGE）技术获得蛋白点的差异表达信息,运用MALDI-TOF质谱鉴定出差异蛋白质.结果 实验组细胞存活率为60%±4.8%,与对照组比较显著下降（P＜0.05）.荧光染色可见细胞核呈固缩状或碎裂状的典型的凋亡形态学改变.DIGE分析发现6-OHDA组表达明显增高的一个蛋白质点,经质谱分析鉴定确认为GRP78.结论 6-OHDA能够诱导PC12细胞凋亡,凋亡过程中GRP78表达增高,提示GRP78增高可能与PD的发病机制有关.     

All‐trans retinoic acid inhibits cobalt chloride‐induced apoptosis in PC12 cells: Role of the dimethylarginine dimethylaminohydrolase/asymmetric dimethylarginine pathway

Previous studies have shown that the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) and its specific hydrolase dimethylarginine dimethylaminohydrolase (DDAH) are involved in the regulation of apoptosis in different cell types. In the present study, we investigated the role of the DDAH/ADMA pathway in cobalt chloride (CoCl₂)–induced apoptosis and the antiapoptotic effect of all‐trans retinoic acid (atRA) in undifferentiated pheochromocytoma (PC12) cells. Treatment of CoCl₂ (125 μM) for 48 hr significantly induced the apoptosis of PC12 cells, concomitantly with increased intracellular reactive oxygen species (ROS) production and caspase‐3 activity. CoCl₂ treatment also decreased the activity of DDAH and the expression of DDAH2 (mRNA and protein), resulting in an increased level of ADMA. All these alterations induced by CoCl₂ were attenuated by atRA (0.1, 1, or 10 μM). Interestingly, the antiapoptotic effects of atRA were inhibited by DDAH2 small RNA interference. In contrast, DDAH2 overexpression inhibited the proapoptotic effects of CoCl₂. We also found that treatment of exogenous ADMA (3, 10, or 30 μM) induced the apoptosis of PC12 cells in a concentration‐ and time‐dependent manner, which was inhibited by the antioxidant or the caspase‐3 inhibitor. These findings suggest that the modulation of the DDAH/ADMA/ROS pathway plays an important role in CoCl₂‐induced apoptosis and the antiapoptotic effects of atRA in undifferentiated PC12 cells. © 2009 Wiley‐Liss, Inc.     

Erythropoietin attenuates 6-hydroxydopamine-induced apoptosis via glycogen synthase kinase 3β-mediated mitochondrial translocation of Bax in PC12 cells

The aim of this study was to determine the mechanism by which erythropoietin (EPO) suppressed 6-hydroxydopamine (6-OHDA)-induced apoptosis. Our results showed that 6-OHDA remarkably decreased phosphorylation of glycogen synthase kinase 3β (GSK3β) as well as enhanced the level of Bax in the mitochondria. Besides, 6-OHDA decreased the mitochondrial expression of Bcl-2 without altering the cytoplasmic expression of Bcl-2. In line with these results, 6-OHDA treatment enhanced the apoptosis and caspase 3 activity in PC12 cells. These findings indicated that mitochondrial dysfunction was involved in the neurotoxicity of 6-OHDA and GSK3β might act upstream of Bax/Bcl-2 and the caspase 3 pathways in 6-OHDA-treated PC12 cells. Furthermore, EPO reduced 6-OHDA-induced growth inhibition. Western blot exhibited that GSK3β inhibitor 4-benzyl-2-methyl-1, 2,4-thiadiazolidine-3, 5-dione (TDZD8) and EPO not only increased the phosphorylation of GSK3β but also inhibited the mitochondrial translocation of Bax. In agreement with these results, EPO and TDZD8 obviously increased the mitochondrial expression of Bcl-2. Finally, TDZD-8 and EPO significantly suppressed the enhanced apoptosis and activity of caspase 3 induced by 6-OHDA. Taken together, GSK3β-mediated mitochondrial cell death pathway is involved in the neuroprotective effect of EPO against 6-OHDA-induced apoptosis.     

Olfactory ensheathing cells conditioned medium prevented apoptosis induced by 6-OHDA in PC12 cells through modulation of intrinsic apoptotic pathways

Olfactory ensheathing cells (OECs) express a high level of growth factors which play a very important role as neuronal support. Recent evidence in literatures showed that transplantation of OECs may improve functional restoration in 6-OHDA-induced rat model of Parkinson's disease (PD). However, the biological function of various factors released from OECs in Parkinson' disease is still unclear. In this study, we examined the effects of newborn rat OECs conditioned medium (CM) on PC12 cells. Cells treated with 6-OHDA underwent cytotoxicity and apoptotic death determined by MTT assay and Hoechst 33342/PI staining. OECs CM was able to reduce the cellular damage in PC12 cells. Further investigation results showed that CM inhibited the disruption of mitochondrial transmembrane potential, up-regulation of Bcl-2 and down-regulation of Bax. Taken together, this study indicates that CM has a neuroprotective effect on 6-OHDA induced apoptosis of PC12 cells, which is through up-regulation of the Bcl-2/Bax ratio and protection for mitochondrion.     

Ultrastructural confirmation of neuronal protection by melatonin against the neurotoxin 6-hydroxydopamine cell damage

6-Hydroxydopamine (6-OHDA) is a neurotoxin used in the induction of experimental Parkinson's disease in both animals and cultured neuronal cells. Biochemical and molecular approaches showed previously that low doses of 6-OHDA induced apoptosis in PC12 cells, while high doses of this neurotoxin induced necrosis. Melatonin has been shown to protect against the neuronal programmed cell death induced by 6-OHDA, although it was not able to prevent the massive necrotic cellular death occurring after the addition of high doses of the neurotoxin. In the present work, we demonstrate by ultrastructural analysis that although low doses of 6-OHDA induced apoptosis in PC12 cells, it also damaged the non-apoptotic cells, morphologically corresponding this damage to incipient and reversible necrotic lesions. When the doses of the neurotoxin increase, there are still apoptotic cells, although most of the cells show necrotic irreversible lesions. We also found that melatonin partially prevents the incipient necrotic lesions caused by low doses of 6-OHDA. The fact that melatonin was shown in previous work to prevent apoptosis caused by low doses of 6-OHDA, but not necrosis induced by high doses of the neurotoxin, seemed to indicate that this agent is only able to protect against apoptosis. However, our present results, melatonin preventing also the incipient necrotic neuronal lesions, suggest that this hormone may provide a general protection against cell death, suggesting that higher doses should be tried in order to prevent the necrotic cell death induced by high doses of the neurotoxin.     

Effects of CDNF on 6-OHDA-induced apoptosis in PC12 cells via modulation of Bcl-2/Bax and caspase-3 activation

Progressive dopamine neuron degeneration in the substantia nigra pars compacta is considered the most prominent pathological characteristic of Parkinson’s disease (PD). Currently, there is no cure, but only the capability to relieve the symptoms of PD. The conserved dopamine neurotrophic factor (CDNF) protects and rescues dopamine neurons in vivo. However, the molecular function of CDNF in PD remains unclear. In present study, we investigated the role and intrinsic mechanism of CDNF in preventing and reversing rat pheochromocytoma (PC12) cells from apoptosis induced by 6-hydroxydopamine (6-OHDA). We demonstrate that 6-OHDA induces cell death in PC12 cells, but that CDNF attenuates this effect in a dose-dependent manner. Further study shows that upregulation of the Bcl-2/Bax ratio and downregulation of caspase-3 activity are observed in a dose-dependent manner upon pre-treatment or post-treatment with CDNF, suggesting a pathway of regulation of apoptosis by CDNF. These data demonstrate that CDNF prevents the apoptosis of PC12 cells induced by 6-OHDA by modulating Bcl-2/Bax and caspase-3 activation.     

6-羟基多巴胺诱导的PC12细胞的凋亡和谷氨酸的释放不依赖蛋白激酶C

目的:了解6-羟基多巴胺(6-OHDA)诱导的PC12细胞凋亡及谷氨酸(Glu)的释放与蛋白激酶C(PKC)的关系,为探讨6-OHDA的毒性作用机制及诱导Glu释放的信号传递途径提供实验依据。方法:培养PC12细胞,以特定浓度6-OHDA刺激24h,用缺口末端标记法(TUNEL)测定细胞凋亡,用噻唑蓝(MTT)法检测细胞存活率,用高效液相色谱法(HPLC)检测Glu的释放量。提前1h加入蛋白激酶C抑制剂HA-100,观察细胞活力和Glu水平的变化。结果:HA-100对6-OHDA诱导的PC12细胞外Glu水平无明显影响,且不能减少6-OHDA诱导的细胞凋亡。结论:6-羟基多巴胺诱导的PC12细胞凋亡和谷氨酸的释放是PKC非依耐性的。     

EPO-Dependent Activation of PI3K/Akt/FoxO3a Signalling Mediates Neuroprotection in In Vitro and In Vivo Models of Parkinson’s Disease

Erythropoietin (EPO) may become a potential therapeutic candidate for the treatment of the neurodegenerative disorder — Parkinson’s disease (PD), since EPO has been found to prevent neuron apoptosis through the activation of cell survival signalling. However, the underlying mechanisms of how EPO exerts its neuroprotective effect are not fully elucidated. Here we investigated the mechanism by which EPO suppressed 6-hydroxydopamine (6-OHDA)-induced neuron death in in vitro and in vivo models of PD. EPO knockdown conferred 6-OHDA-induced cytotoxicity. This effect was reversed by EPO administration. Treatment of PC12 cells with EPO greatly diminished the toxicity induced by 6-OHDA in a dose- and time-dependent manner. EPO effectively reduced apoptosis of striatal neurons and induced a significant improvement on the neurological function score in the rat models of PD. Furthermore, EPO increased the expression of phosphorylated Akt and phosphorylated FoxO3a, and abrogated the 6-OHDA-induced dysregulation of Bcl-2, Bax and Caspase-3 in PC12 cells and in striatal neurons. Meanwhile, the EPO-dependent neuroprotection was notably reversed by pretreatment with LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K). Our data suggest that PI3K/Akt/FoxO3a signalling pathway may be a possible mechanism involved in the neuroprotective effect of EPO in PD.     

褐藻多糖硫酸酯对6-羟基多巴胺所致MN9D细胞损伤的保护作用及其机制

目的 以6-羟基多巴胺(6-OHDA)为工具药,建立帕金森病(PD)的细胞模型,观察褐藻多糖硫酸酯(Fuc)对细胞模型的保护作用并初步探讨其作用机制.方法 用不同浓度(12.5、25、50、100、200 μmol/L)的6-0HDA处理MN9D细胞24 h,挑选出合适浓度50 μmol/L.再以50μmol/L 6-OHDA处理MN9D细胞不同时间(6、12、24及48 h),建立细胞损伤模型,挑选出合适时间24h.用0.01、0.1、1.0mg/mL Fuc预孵育MN9D细胞1 h后加入50μmol/L 6-OHDA共同作用24h,以探讨Fuc的保护作用.MTT法检测细胞存活率,生化法测定乳酸脱氢酶(LDH)释放量,二氯荧光索乙二酯(DCF-DA)染色法检测细胞内的氧化应激水平.结果 随着6-OHDA浓度增加或作用时间延长,MN9D细胞MTT值逐渐降低.50 μmol/L6-OHDA处理细胞24 h,MTT值明显下降,LDH释放量增加.而0.1、1.0mg/mL的Fuc预孵育1 h可明显减轻MN9D细胞的损伤,提高MTT值并降低LDH释放量,细胞形态学改变与生化实验结果一致.50 μmol/L6-OHDA作用8h可明显升高MN9D细胞内的氧化应激水平,而1.0mg/mL的Fuc预处理1 h可以拮抗6-OHDA引起的细胞内氧化应激水平增高.结论 Fuc可以有效的拮抗6-OHDA对MN9D细胞的损伤作用,其作用机制可能与抗氧化活性有关.     

Baculovirus p35 gene greatly enhances PC12 cell's resistance against oxidative stress

Oxidative stress is thought to be a major contributor to the progress of the Parkinson's Disease (PD) because of the high vulnerability of dopaminergic cells against oxidative stress. The present work demonstrates that with the expression of the baculovirus p35 gene, PC12 cells could gain a high resistance against oxidative toxicants, hydrogen peroxide (H(2)O(2)) and 6-hydroxydopamine (6-OHDA). The DNA fragmentation analysis showed that PC12 cells underwent apoptosis after exposure to H(2)O(2) or 6-OHDA, while PP35 cells, a p35-expressing PC12 cell line, did not. Flow cytometric analysis showed that treatment with 150 microM H(2)O(2) or 120 microM 6-OHDA for 24 h caused 52.86% or 66.36% apoptotic cell, respectively, in PC 12 cells, but only 4.26% or 5.80% in PP35 cells. The cell viability measured by 3-(4,5-dimethylthiazal-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay indicated that H(2)O(2) and 6-OHDA induced a dose-dependent cell death on PC12 cells that were greatly remitted on PP35 cells. The viability of PP35 cells was even stronger than that of PC12 cells protected by glial cell line deprived neurotrophic factor (GDNF). The surviving PP35 cells remained normal cell morphology and showed positive with tyrosine hydroxylase (TH) immunocytochemical staining. These results indicate that baculovirus p35 gene possesses remarkable ability to rescue PC12 cells from death in experimental paradigms associated with oxidative stress.     

Rise in intracellular calcium concentration by propylene glycol in PC12 cells.

In order to elucidate the mode of excitatory actions of propylene glycol (PG) on nervous tissues, intracellular calcium concentration ([Ca2+]i) of PC12 cells was measured on the video-imaging analysis system with fura-2. PG concentration-dependently (0.1-20%v/v) raised the [Ca2+]i. Hexamethonium and d-tubocurarine inhibited the carbamylcholine-induced [Ca2+]i rise, but these blockers had no effect on PG. High K+ potentiated the action of PG. The extent of the rise induced by PG in the differentiated cells was larger than that in the undifferentiated ones. The findings suggest that the rise in [Ca2+]i is involved in the excitatory effects of PG.     

Potassium depolarization-induced cAMP stimulates somatostatin mRNA levels in cultured diencephalic neurons

In the nervous system, signals transmitted across synapses are known to regulate gene expression in the postsynaptic cells. This process often involves membrane depolarization and subsequent elevation of intracellular Ca(2+). We have previously demonstrated in fetal cerebrocortical cells, that somatostatin (SS) mRNA levels can be induced by depolarizing agents such as high potassium concentrations and veratridine (VTD), and that these effects are calcium dependent. SS expression is regulated by cAMP, and in the cerebral cortex adenylate cyclase activity is regulated through fluctuations in intracellular Ca(2+) concentrations.The present experiments were undertaken to determine the mechanism by which calcium upregulates the levels of SS mRNA. Cerebrocortical cells from 17-day-old fetuses were exposed to the different agents for 24 h and the levels of SS mRNA were measured by Northern blot. Incubation of cells with the calcium channel antagonist nifedipine (Nf), the calcium chelating agent EGTA, calcium free KRB and the calcium calmodulin inhibitors trifluoroperazine (TFP) and the napthelene sulfonamide, W7, resulted in the inhibition of K(+)-induced SS mRNA levels. K(+)-evoked depolarization increased the intracellular concentration of cAMP and this effect was antagonized by verapamil (VPM). Forskolin (Fk) provoked a higher increment in cAMP concentration than potassium, although the induction of SS mRNA was more evident following K(+) depolarization indicating a lack of correlation between levels of cAMP and induction of SS mRNA. The role of K(+)-induced cAMP on the increment of SS mRNA that occurred upon membrane depolarization was further explored with the inhibitor of protein kinase A (PKA), Rp cAMP whose presence significantly reduced depolarization-induced SS mRNA levels. This study confirms that Ca(2+) influx is required for K(+)depolarization-induced stimulation of cAMP whereby the increment of SS mRNA is partly produced.