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  • In many cells AR mediated activation of

    2024-04-13

    In many CVT 10216 α1-AR mediated activation of PLC has been shown to be the downstream process for NA-induced signalling. The activated PLC initiates subsequent signalling by breakdown of PIP2 to IP3 and DAG (Fisher and Agranoff, 1987) and the process continues. To confirm if such mechanism exists in regulating Na-K ATPase expressions, in this study the cells were treated with U73122, a PLC specific antagonist. Our results showed that the U73122 efficiently prevented the NA-induced increase in α1- and α3-Na-K ATPase expression but not that of β1-Na-K ATPase expression. Thus, the findings confirmed the role of PLC in NA-induced downstream signalling in the expression of α1- and α3-subunits of Na-K ATPase. The [Ca++]i plays a major role in the regulation of several neuronal gene expressions (Bito et al., 1997). The role of [Ca++]i in REMSD and allosteric modulation of neuronal Na-K ATPase activity has been studied in detail (Das et al., 2008; Mallick et al., 2000; Mallick and Singh, 2011). However, the role of [Ca++]i in NA-induced transcriptional regulation of CVT 10216 neuronal Na-K ATPase expression was not investigated. The [Ca++]i concentration can be modulated by several mechanisms including quality and quantity of stimulus and its downstream signalling. One of such signalling process involves activation of IP3 mediated release of Ca++ from endoplasmic reticulum and intracellular stores, while another mechanism is influx of extracellular Ca++ through different voltage- and ligand-gated Ca++ channels. The specific role of the extracellular Ca++ due to its influx, or [Ca++]i due to its non-sequestration on NA induced expression of neuronal Na-K ATPase subunits was evaluated by treating the cultured Neuro-2a cells with Nif or CyA, respectively. It is known that Nif decreases Ca++-influx by blocking the L-type Ca++-channels and thus, reduces [Ca++]i level (Ng and Gurney, 2001); while, CyA inhibits SERCA and prevents reuptake of intracellular Ca++ from the cytoplasm into the Ca++-stores and thus, elevates the level of [Ca++]i. In this study, Nif could not prevent NA action, while CyA inhibited the NA-induced expression of Na-K-ATPase subunits (Fig. 6). Thus, the findings suggest that the NA by acting on α1-AR inhibited the elevation of [Ca++]i, which in turn increased expression of Na-K ATPase subunits. It may be supported by the fact that treatment of Neuro-2a cells with Nif alone induced increased expression of α1- and β1-subunits of Na-K ATPase. This is probably because Nif alone (i.e in the absence of NA) prevented Ca++ influx and elevated the mRNA expression of the subunits. Our views can be supported by our in vivo and in vitro findings that synaptosomal Ca++ levels decreased after REMSD (Mallick and Gulyani, 1996), while NA chelates Ca++ (Mallick and Adya, 1999), and in both the conditions the Na-K ATPase activity was increased. The Sp1 is the major transcription factor involved in positive regulation of various isoforms of Na-K ATPase (Kawakami et al., 1996; Wang et al., 2007; Wendt et al., 2000). The transcription factors ATF1/CREB and Sp1/Sp3 have been shown to be involved in the regulation of α1-Na-K ATPase expression (Kobayashi et al., 1997). We observed peak expressions of pCREB-Serine133, the activated phosphorylated form of CREB, and α1-Na-K ATPase mRNA in cells treated with 50 μM of NA; the expression was sustained by treatment with 100 μM NA as well. This observation supports the involvement of CREB in NA-mediated stimulation of α1-Na-K ATPase expression in Neuro-2a cells. On the other hand, the expression of Sp1 was relatively higher at 100 μM NA and that could be well correlated with the increased α3-Na-K ATPase mRNA expression in Neuro-2a cells after NA treatment; however, these findings need to be confirmed with EMSA or CHIP-assay. Besides, NF-y and Sp3 have also been shown to be involved in the regulation of ATPase subunit expressions under various conditions (Pathak et al., 1994), which we have not studied and require further investigation.