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  • The activity of an enzyme may

    2023-02-25

    The activity of an enzyme may be regulated allosterically as well as transcriptionally. We have consistently studied REMS deprivation (REMSD) associated NA mediated allosteric regulation of Na-K ATPase activity in neurons and glia in vivo and in vitro (Amar and Mallick, 2015; Amar et al., 2016; Baskey et al., 2009; Mallick and Singh, 2011). However, for sustained effects, including upon REMSD, the synthesis of the enzyme is likely to be modulated. As the 2211 mg contains neurons and glia, we needed to study the effect of NA on both those cells. We have recently reported the effect of NA on glial Na-K ATPase activity and mRNA expressions (Amar et al., 2017). For a better understanding of REMSD associated NA-mediated altered functioning of the brain and behaviour, in this study we explored the molecular mechanism of NA-induced regulation of neuronal Na-K ATPase subunit expression. We have analysed the involvement of adrenergic receptors (AR), second messenger(s) and specific transcription factors in NA-induced regulation of Na-K ATPase subunits expression. We used Neuro-2a cells as a model system as a practical approach for convenience to exclude the influence of obvious, unavoidable confounding factors encountered while conducting in-vivo studies. The use of Neuro-2a in this study may be supported by the facts that these are rodent (mouse) c1300-neuroblastoma derived cells (Klebe and Ruddle 1969), possess Na-K ATPase and AR (Baskey et al., 2009; Kimelberg, 1974; Manthey et al., 2010). Also, due to the presence of several neuronal properties, Neuro-2a cells have been extensively used for studying neuronal biology including differentiation, axonal growth, signalling and functions (Dasgupta and Milbrandt, 2007; Evangelopoulos et al., 2005; Furmanski et al., 1971).
    Material and methods
    Results
    Discussion REMS plays an essential role in maintaining normal brain functions. Extensive research has shown that during REMS NA-ergic neurons must cease activity (Mallick et al., 2012). Also, loss of REMS resulted in increased NA-level in the brain (Mallick et al., 2002; Mehta et al., 2017), which then induces many of the REMS-loss associated acute and chronic effects (Mallick and Singh, 2011). In previous in vivo and ex vivo studies we have shown that REMSD-associated elevated NA acting on α1-AR increased activity as well as expression of various subunits of Na-K ATPase (Amar and Mallick, 2015; Baskey et al., 2009). However, the mechanism of NA-induced intracellular regulation of the expression of Na-K ATPase subunits remained unknown, which we have explored in this study. Using Neuro-2a cell line as a model system we have traced the intracellular pathway of NA-induced transcriptional regulation of neuronal Na-K ATPase subunit expression. We observed that NA acting on α1-AR increased Na-K ATPase activity in Neuro-2a cells. As the Na-K ATPase activity remained elevated after prolonged treatment of NA, it was likely to be due to increased transcription of α- and β-subunits of neuron-specific Na-K ATPase. The NA effect might be mediated by its action on either or both, α1- or β-ARs, which are present on the Neuro-2a cells (Katsouri et al., 2013; Manthey et al., 2010). We observed that NA acting on α1-AR induced PLC/PKC and CREB/Sp1 downstream targets to increase the expressions of α1- and α3-Na K ATPase subunits. On the other hand, NA acting on β-AR modulated the β1-subunit expression; further studies are needed to understand the detailed downstream signalling events involved in the process. We observed that NA (100 μM) acting on α1-AR stimulated the Na-K ATPase activity in Neuro-2a and this was partly due to increased expression (synthesis) of Na-K ATPase subunits. The peak expression of α1-subunit of Na-K ATPase was induced at a relatively lower dose of NA (50 μM), whereas, the expression of α3-isoform peaked at relatively higher dose (100 μM) of NA. The difference could be due to differential sensitivity (or threshold) of NA on the expression of Na-K ATPase subunits. This may be explained by the fact that the later (α3-subunit of Na-K ATPase) acts as a spare pump in restoring the resting membrane potential of a neuron and is activated only when the α1-subunit containing pumps are saturated (Blanco and Mercer, 1998). The effects of both the concentrations of NA were comparable in significantly stimulating the mRNA expression of regulatory β1-subunit of Na-K ATPase.