Physiological And Pathological Roles Of NCX1.1, NCX1.4and NCX1.5Isoforms In Stably Transfected CHO Cells

The Na+/Ca2+exchanger (NCX) is an ion transporter that expressed in the plasma membrane of almost every cell type. It contains nine transmembrane segments and works bi-directionally depending on membrane potential or transmembrane ion gradients. Under physiological condition, NCX not only plays a pivotal role in cardiac myocytes during excitation contraction coupling but also participates in neurotransmitter realse in neurons. Besides, NCX is involved in regulating the homeostasis of intracellular Ca2+concentration ([Ca2+]i) in resting condition of almost every cell. Under pathological condition, NCX may cause Ca2+influx in the reverse operation mode. It has been reported that intracellular Ca2+overload via NCX reverse activity is commonly observed in various diseases. Therefore, there is a phathophysiological meaning with the bidirectional sodium calcium exchange activity. To date, there are considerable researches focused on the function of NCX1exerting in ischemic related diseases. Unfortunately, the results obtained even under the same experimental condition are not always comparable, there are still some conflicting conclusions. Based on this issue, we express3NCX1isoforms, one presented in heart (NCX1.1) and other two (NCX1.4and NCX1.5) are typical isoforms distributed in brain in CHO cells with the method of Lipofectamine transfection, to determine whether NCX1reverse activity is modulated by protein kinase phosphorylation and investigate functional differences among alternatively spliced isoforms of NCX1in ischemic related diseases, or screening potential NCX modulators.Part Ⅰ. Construction of stable transfected cell lines with NCX1.1, NCX1.4and NCX1.5isoformsThe NCX1.1, NCX1.4and NCX1.5genes which are integreted into pcDNA3.1+plasmid are identified with two restriction enzymes, BamH Ⅰ and Hind Ⅲ. The results show that the number of digestion products is consistent with the expected number, their locations on1%ethidium bromide-stained agarose gel are also correct, which means the targeted NCX genes are inserted accurately. In addition, the above plasmids are sent to Invitrogen Corporation or Takara Bio Inc. for sequencing, results indicate that the DNA array of inserted NCX genes are the same as the theoretical sequences enclosed in GenBank.Using Lipofectamine transfection reagent, CHO-K1cells expressing the NCX1isoforms are generated by transfection of the pcDNA3.1+plasmid carrying the rat cDNA of cardiac NCX1.1, brain NCX1.4or NCX1.5. To select the transfected cells, different concentrations of G418are applied to the transfected and non-transfected cells for about2-3weeks. Single-cell clones that expressed similar levels of NCX1.1, NCX1.4and NCX1.5proteins are chosen for the study. Western blotting analysis demonstrates that there is no NCX proteins detected in wide type cells, whereas a high expression of NCX proteins is detected in three stably transfected CHO cells, besides, the protein levels of three NCX isoforms are very similar. For measurement of the bioactivity of expressed NCX proteins, the fluorescent Ca2+indicator Fura-2is chosen for monitoring intracellular Ca2+concentration when altering the extrecellular Na2+gradient. To elicit reverse mode NCX activity, the bath buffer is switched to Na+-deficient NMDG+medium. As a matter of fact, CHO wide type cells, which lack all three NCX isoform expressions, do not respond to Na+-free solution, while the [Ca2+]i of NCX1.1, NCX1.4and NCX1.5elicited by Na+-free solution are significantly increased, suggesting that the NCX reverse activity is induced after the replacement of NaCl with equimolar N-methyl-D-glucamine, which means the NCX proteins expressed in CHO cells are functional.A potent inhibitor of the Na+/Ca2+exchanger, KB-R7943, is also an isothiourea derivatives, is widely used as a tool in studying the roles of NCX played under physiological and pathological conditions. The effects of different concentrations of KB-R7943, the NCX inhibitor, on NCX current are also investigated in our experiments. Results show that both the outward and inward NCX currents are significantly decreased during KB-R7943exposure. There is a dose dependent inhibitory effects observed, but this inhibition is lack of isoform selectivity.Our results illustrate that the CHO cell lines stably expressed rat cardiac NCX1.1, rat brain NCX1.4and rat brain NCX1.5isoforms are successfully constructed. Thery are very useful in studying the physiological modulators of cardiac and brain NCX1isoforms, moreover, they are also eligible to examine the roles of NCX isoforms played in various pathological conditions.Part Ⅱ. NCX1.1, NCX1.4and NCX1.5reverse activity regulated by protein kinase phosphorylationThere are numerous factors involved in regulating NCX activity, including monovalent, divalent and trivalent cations, neurotransmitters, hormones and peptides. Among these factors, the relationship between protein kinase phosphorylation and NCX activity are still uncertain. In order to verify the regulation of protein phosphorylation on NCX activity independently, the stably transfected CHO cells with single NCX isoform are utilized to investigate the effects of protein kinase C and protein kinase A on NCX reverse operation mode.In the present study, the effect of PKC activator Phorbol12-myristate13-acetate (PMA) and NCX inhibitor KB-R7943on NCX activity in wide type CHO cells and in stably transfected NCX cells are evaluated. Results show that there is no notable effect of PKC activator PMA on [Ca2+]i in wide type CHO cells. However, the reverse operation mode of cardiac NCX1.1is strikingly upregulated by PKC activator PMA, inducing a dramatical calcium transient in NCX1.1cells, and reach maximum at the concentration of3μM PMA with an [Ca2+]i increase rate of23.82nM/sec. Preincubation of NCX inhibitor KB-R7943could effectively block this rise, suggesting the Ca2+increase is mediated by NCX. Further, when the Ca2+chelator EGTA is added into extracellular solution, the calcium transient observed in stable NCX1.1cells is abolished, thus suggesting the Ca2+rise induced by NCX is closely correlated with extracellular Ca2+. Although the activity of brain NCX1.4and NCx1.5isoforms are also increased during PKC activator PMA application, the brain NCX isoforms are at least10times less sensitive to PMA compared with cardiac NCX1.1. Only10μM PMA could significantly activate the NCX1.5reverse activity, and Ca2+rise rate is20.64nM/sec, with a dramatical increase compared with control cells.In addition, the effects of PKA activator8-Bromoadenosine3',5'-cyclic momophoshate (8-Br cAMP) and NCX inhibitor KB-R7943on NCX activity in wide type CHO cells and in stably transfected NCX cells are also examined. Results indicate that there is no notable effect of PKA activator8-Br cAMP on [Ca2+]i in wide type CHO cells. However, the reverse operation mode of cardiac NCX1.1is strikingly upregulated by PKA activator8-Br cAMP, inducing a dramatical calcium oscillation in NCX1.1cells. Preincubation of NCX inhibitor KB-R7943could effectively block this rise, suggesting the Ca2+increase is mediated by NCX. Further, when the Ca2+chelator EGTA is added into extracellular solution, the calcium oscillation observed in stable NCX1.1cells is eliminated, thus suggesting the Ca2+rise induced by NCX is closely correlated with extracellular Ca2+. The PKA phosphorylated regulations of brain NCX1.4and NCX1.5isoforms are similar to cardiac NCX1.1.8-Br cAMP at10μM could cause a dramatic calcium oscillation in the above NCX transefected cells.Our results demonstrate that both PKC and PKA are involved in regulation of cardiac NCX1.1, brain NCX1.4and NCX1.5reverse activities. Contrast to cardiac NCX1.1, brain NCX1.4and NCX1.5isoforms are at least10times less sensitive to PKC activator PMA, thus suggesting the regulation of PKC phosphorylatoin is different in tissue specific NCX variants.Part Ⅲ. Investigation of the pathological functions of NCX1.1, NCX1.4and NCX1.5in ischemic-related diseases1. Role of NCX in H2O2-induced oxidative stessThe effect of H2O2-induced oxidative stess on wide type CHO cells and NCX transfected cells is evaluated.24hours after H2O2exposure, the cell viabilities of both wide type CHO cells and NCX transfected cells decrease dose dependently. There is a significant reduction of cell survival in both NCX1.4and NCX1.5cells compared with wide type CHO cells after exposure to70μM H2O2for24h. The cell viabilities of the above cells are45.1%,44.0%and75.3%, respectively. Hoechst33342and PI double staining results show that the ratio of cell death in NCX1.1, NCX1.4and NCX1.5are 32.7%,49.5%and52.7%, with a significant increase in contrast to24.2%in wide type CHO cells. Further, calcium imaging is applied in monitoring the dynamic [Ca2+]i level in the above cells during H2O2exposure. Results show that the extent of [Ca2+]i changed in wide type CHO cells is less significant compared with NCX transfected cells, and the Ca2+rise rate is much slower. In the present part, oxygen free radicals could destroy the plasma membrane calcium extrusion systems, leading the [Ca2+], rise, while H2O2could activate the NCX reverse activity and aggravate cell damage.2. Role of NCX in extracellular acidosisThe effect of moderate acidosis (pH6.4) on wide type CHO cells and NCX transfected cells is evaluated. After incubation with an extracellular solution of pH6.4, the cell viabilities of both wide type CHO cells and NCX transfected cells decreased time dependently. There is a significant reduction of cell survival in wide type CHO cells compared with NCX transfected cells after exposure to an extracellular solution of pH6.4for24h. The cell viabilities of NCX1.1, NCX1.4, NCX1.5and wide type CHO cells are55.2%,62.5%,61.3%and48.2%, respectively. Hoechst33342and PI double staining results show that the ratio of cell death in NCX1.1, NCX1.4and NCX1.5are20.7%,22.7%and23.2%, with a significant decrease in contrast to33.3%in wide type CHO cells. Further, calcium imaging is applied in monitoring the dynamic [Ca2+]i level in the above cells during acidosis. Results show that the [Ca2+]i levels in wide type CHO cells significantly increase,while the [Ca2+]i levels in NCX transfected cells decrease mildly or unchanged. In the present part, glucose is not removed from extracellular solution, therefore, the NCX forward operation mode is preserved with a normal ATP supplying, thus could play a protectivel role by extruding the intracellular Ca2+3. Role of NCX in calcium paradoxThe wide type CHO cells and NCX transfected cells are incubated with a calcium free solution for1h, and then changing into either standard extracellular solution or extracellular solution with2.5mM Ca2+or extracellular solution with3.8mM Ca2+, then evaluate the effect of calcium paradox on wide type CHO cells and NCX transfected cells. Results show that the cell viabilities of both wide type CHO cells and NCX transfected cells decreased time dependently.There is a significant reduction of cell survival in wide type CHO cells compared with NCX transfected cells after exposure to a standard extracellular solution for24h. The cell viabilities of NCX1.1, NCX1.4, NCX1.5and wide type CHO cells are79.2%,80.3%,81.6%and64.7%, respectively. Hoechst33342and PI double staining results show that the ratio of cell death in NCX1.1, NCX1.4and NCX1.5are19.4%,25.4%and21.4%, with a significant decrease in contrast to29.9%in wide type CHO cells.There is also a significant reduction of cell survival in wide type CHO cells compared with NCX transfected cells after exposure to an extracellular solution with2.5mM Ca2+for24h. The cell viabilities of NCX1.1, NCX1.4, NCX1.5and wide type CHO cells are83.0%,83.3%,80.9%and68.0%, respectively. Hoechst33342and PI double staining results show that the ratio of cell death in NCX1.1, NCX1.4and NCX1.5are23.2%,28.6%and23.8%, with a significant decrease in contrast to29.4%in wide type CHO cells.In addition, there is also a significant reduction of cell survival in wide type CHO cells compared with NCX transfected cells after exposure to an extracellular solution with3.8mM Ca2+for24h. The cell viabilities of NCX1.1, NCX1.4, NCX1.5and wide type CHO cells are77.4%,75.2%,73.2%and54.90%, respectively. Hoechst33342and PI double staining results show that the ratio of cell death in NCX1.1, NCX1.4and NCX1.5are31.2%,35.8%and32.8%, with a significant decrease in contrast to51.3%in wide type CHO cells.Further, calcium imaging is utilizd in monitoring the dynamic [Ca2+]i level in the above cells during calcium paradox. Results show that during perfusion of Ca2+free solution, the [Ca2+]i levels in wide type CHO cells rarely change, while the [Ca2+]i levels in NCX transfected cells decrease mildly but sigmificantly. This [Ca2+], decrease is mediated by NCX forward operation mode.1hour after reperfusion of Ca2+containing solution, the [Ca2+]i levels in wide type CHO cells gradually increase, while the [Ca2+]i levels in NCX transfected cells maintain in a relative low levels. In the present part, during reperfusion of Ca2+containing solution, there is a rapid Ca2+rise in all the above cells, therefore, this rapid [Ca+]i elevation induces the NCX forward acitivity, thus could play a protectivel role by extruding the intracellular Ca2+In the present study, the closely interacted respects of oxidative stress, acidosis and calcium overload are mimicked by H2O2, extracellular solution acidification to pH6.4and calcium paradox models, with the help of successfully constructed stable transfected NCX cell lines, we investigate the functions of cardiac NCX1.1, brain NCX1.4and NCX1.5exterting in the ischemic related diseases. Our results demonstrate that the NCX transport mode could change under distinct circumstances, thus aggravate or alleviate the cell damage.