Knockdown of Amyloid Precursor Protein Increases Ion Channel Expression and Alters Ca2+ Signaling Pathways.

Although the physiological role of the full-length Amyloid Precursor Protein (APP) and its proteolytic fragments remains unclear, they are definitively crucial for normal synaptic function. Herein, we report that the downregulation of APP in SH-SY5Y cells, using short hairpin RNA (shRNA), alters the expression pattern of several ion channels and signaling proteins that are involved in synaptic and Ca2+ signaling. Specifically, the levels of GluR2 and GluR4 subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptors (AMPAR) were significantly increased with APP knockdown. Similarly, the expression of the majority of endoplasmic reticulum (ER) residing proteins, such as the ER Ca2+ channels IP3R (Inositol 1,4,5-triphosphate Receptor) and RyR (Ryanodine Receptor), the Ca2+ pump SERCA2 (Sarco/endoplasmic reticulum Ca2+ ATPase 2) and the ER Ca2+ sensor STIM1 (Stromal Interaction Molecule 1) was upregulated. A shift towards the upregulation of p-AKT, p-PP2A, and p-CaMKIV and the downregulation of p-GSK, p-ERK1/2, p-CaMKII, and p-CREB was observed, interconnecting Ca2+ signal transduction from the plasma membrane and ER to the nucleus. Interestingly, we detected reduced responses to several physiological stimuli, with the most prominent being the ineffectiveness of SH-SY5Y/APP- cells to mobilize Ca2+ from the ER upon carbachol-induced Ca2+ release through IP3Rs and RyRs. Our data further support an emerging yet perplexing role of APP within a functional molecular network of membrane and cytoplasmic proteins implicated in Ca2+ signaling.

Pendimethalin induces apoptosis in testicular cells via hampering ER-mitochondrial function and autophagy.

Pendimethalin (PDM) is a dinitroaniline crop pesticide that is extensively utilized worldwide. However, the reproductive toxicity and cellular mechanisms of PDM have not been identified. Therefore, we elucidated the adverse effects of PDM on the reproductive system using mouse testicular Leydig and Sertoli cells (TM3 and TM4 cells, respectively). Our results demonstrated that PDM suppressed the viability and proliferation of TM3 and TM4 cells. Additionally, PDM induced cytosolic calcium upregulation and permeabilization of mitochondrial membrane potential in both TM3 and TM4 cells. We also verified that PDM activates the endoplasmic reticulum (ER) stress pathway and autophagy. Furthermore, we confirmed that activation of ER stress and autophagy were blocked by 2-aminoethoxydiphenyl borate (2-APB) treatment. Finally, we confirmed PDM-induced cell cycle arrest and apoptosis in TM3 and TM4 cells. Thus, we first demonstrated that PDM impedes the survival of testis cells, and further, their function.

Mechanism of beryllium sulfate-induced apoptosis in human embryonic lung fibroblast / 中国职业医学

OBJECTIVE: To investigate the effect of beryllium sulfate( BeSO_4) on apoptosis of human embryonic lung fibroblast( MRC-5 cell). METHODS: MRC-5 cells were cultured in vitro and randomly divided into 6 groups: a control group,a low-,medium- and high-dose BeSO_4 group,an antagonist group,and an activator group. The former 4 groups were given final concentrations of 0,1,10 and 100 μmol / L of BeSO_4,respectively. The combined treatment of BeSO_4and2-aminoethoxydiphenyl borate( 10 μmol / L final concentration) was used in the antagonist group. The combined treatment of BeSO_4 and inositol triphosphate( IP3)( 10 μmol / L final concentration) was used in the activator group. After 24 and48 hours of culture,the cells were harvested. The apoptosis of MRC-5 cells was detected by flow cytometry. The intracellular calcium ion( Ca~(2+)) was detected using laser scanning confocal microscope. Quantitative real-time polymerase chain reaction was used to detect the relative expression of IP_3RⅢ and B-cell lymphoma-2( BCL-2) mRNA and the protein expression of IP_3RⅢ and IP3 were detected by enzyme-linked immunosorbent assay. RESULTS: The apoptosis rates of cells in the 3 BeSO_4 dose groups at the time points of 24 and 48 hours were lower than those in the control group at the same time points( P < 0. 05). The apoptosis rate of the antagonist group was lower than those in medium-dose BeSO_4 group and control group at the same time points( P < 0. 05). At the time point of 48 hours,the apoptosis rate of the activator group was lower than that of control group( P < 0. 05) and higher than that of the medium-dose BeSO_4group( P < 0. 05). As for the Ca~(2+)concentration at time point of 24 hours,the low-dose BeSO_4 group was lower than the control group( P < 0. 05),and the high-dose BeSO_4 group was higher than the control group( P < 0. 05). The Ca~(2+)concentrations at time point of 48 hours in the medium- and high-dose BeSO_4 groups were lower than that in the control group( P < 0. 05). Compared with the medium-dose BeSO_4 group and control group at time points of 24 and 48 hours,the Ca~(2+)concentrations in the antagonist group decreased( P < 0. 05),while thoes of the activator group increased( P < 0. 05). The expression of BCL-2and IP_3RⅢmRNA in the 3 BeSO_4 groups,the activator and antagonist group were higher than those of the control group( P <0. 05). The expression of IP3 R Ⅲ protein at the time point of 24 hours in the medium-dose BeSO_4 group,the activator group and the antagonist group were lower than that of control group( P < 0. 05). The expression of IP_3RⅢ protein at the time point of 48 hours,the high-dose BeSO_4 group was lower than the control group( P < 0. 05); the activator and antagonist groups were higher than the medium-dose BeSO_4group( P < 0. 05). The expression of IP3 protein in the lowand medium-dose BeSO_4 groups and the activator group were higher than that in the control group( P < 0. 05). The expression of IP3 protein in activator group was higher than the medium-dose group( P < 0. 05). CONCLUSION: BeSO_4 might change the Ca~(2+)concentration and inhibite the apoptosis of MRC-5 cell through regulating the IP3 R / Ca~(2+)pathway,IP3 can improve the decrease of Ca~(2+)concentration in MRC-5 cells induced by BeSO_4.

The role of IP3 receptor channel clustering in Ca2+ wave propagation during oocyte maturation.

During oocyte maturation, the calcium-signaling machinery undergoes a dramatic remodeling resulting in distinctly different calcium-release patterns on all organizational scales from puffs to waves. The dynamics of the Ca(2+) release wave in mature as compared to immature oocytes are defined by a slower propagation speed and longer duration of the high Ca(2+) plateau. In this chapter, we use computational modeling to identify the changes in the signaling machinery, which contribute most significantly to the alterations observed in Ca(2+) wave propagation during Xenopus oocyte maturation. In addition to loss of store-operated calcium entry and internalization of plasma membrane pumps, we propose that spatial reorganization of the IP3 receptors in the plane of the ER membrane is a key factor for the observed signaling changes in Ca(2+) wave propagation.

Distinct Cellular Calcium Metabolism in Radiation-sensitive RKO Human Colorectal Cancer Cells.

Radiation therapy for variety of human solid tumors utilizes mechanism of cell death after DNA damage caused by radiation. In response to DNA damage, cytochrome c was released from mitochondria by activation of pro-apoptotic Bcl-2 family proteins, and then elicits massive Ca(2+) release from the ER that lead to cell death. It was also suggested that irradiation may cause the deregulation of Ca(2+) homeostasis and trigger programmed cell death and regulate death specific enzymes. Thus, in this study, we investigated how cellular Ca(2+) metabolism in RKO cells, in comparison to radiation-resistant A549 cells, was altered by gamma (γ)-irradiation. In irradiated RKO cells, Ca(2+) influx via activation of NCX reverse mode was enhanced and a decline of [Ca(2+)]i via forward mode was accelerated. The amount of Ca(2+) released from the ER in RKO cells by the activation of IP3 receptor was also enhanced by irradiation. An increase in [Ca(2+)]i via SOCI was enhanced in irradiated RKO cells, while that in A549 cells was depressed. These results suggest that γ-irradiation elicits enhancement of cellular Ca(2+) metabolism in radiation-sensitive RKO cells yielding programmed cell death.

Distinct Cellular Calcium Metabolism in Radiation-sensitive RKO Human Colorectal Cancer Cells

Radiation therapy for variety of human solid tumors utilizes mechanism of cell death after DNA damage caused by radiation. In response to DNA damage, cytochrome c was released from mitochondria by activation of pro-apoptotic Bcl-2 family proteins, and then elicits massive Ca2+ release from the ER that lead to cell death. It was also suggested that irradiation may cause the deregulation of Ca2+ homeostasis and trigger programmed cell death and regulate death specific enzymes. Thus, in this study, we investigated how cellular Ca2+ metabolism in RKO cells, in comparison to radiation-resistant A549 cells, was altered by gamma (gamma)-irradiation. In irradiated RKO cells, Ca2+ influx via activation of NCX reverse mode was enhanced and a decline of [Ca2+]i via forward mode was accelerated. The amount of Ca2+ released from the ER in RKO cells by the activation of IP3 receptor was also enhanced by irradiation. An increase in [Ca2+]i via SOCI was enhanced in irradiated RKO cells, while that in A549 cells was depressed. These results suggest that gamma-irradiation elicits enhancement of cellular Ca2+ metabolism in radiation-sensitive RKO cells yielding programmed cell death.

T-type calcium channel effects on RyR and IP_3R of sarcoplasmic reticulum in atrial myocytes during atrial fibrillation / 中国实用内科杂志

Objective To inquire into T-type calcium channel effects on RyR and IP 3R of sarcoplasmic reticulum in atrial myocytes during atrial fibrillation.Methods Ten dogs underwent continuous rapid atrial pacing (500 beats/min) to create persistent atrial fibrillation.In five rapidly-paced dogs,mibefradil dihydrochloride was given beginning 2 days after pacemaker implantation,continuing until the twenty-fourth week.A group of size-matched dogs (n=5) without given mibefradil was used as a pure atrial fibrillation group.Another group of size-matched dogs (n=5) without pacemaker implantation was used as a control group.Canine atrial myocytes isolated by enzymatic dissociation were used to study T-type Ca 2+ channel blocker effects on expression and function changes of RyR and IP 3R in atrial myocytes by confocal microscopy.Results RyR/IP 3R ratio (0.2965?0.01812) was markedly lower than that of control group (2.7043?0.2293),but there was no difference compared with that of atrial fibrillation group (0.2472?0.1355).Ca 2+transient of atrial myocytes was nremarkably changed (1.3031?0.1056)(P