Rapid communication Comparison of Na-K ATPase activity in rat brain synaptosome under various conditions

doi:10.1016/S0197-0186(98)00043-6

Neurochemistry International

Volume 33, Issue 4, 1 October 1998, Pages 283-286

https://doi.org/10.1016/S0197-0186(98)00043-6 Get rights and content

Abstract

Rapid eye movement (REM) sleep deprivation alters neuronal excitability possibly by increasing Na-K ATPase activity. The enzyme activity is known to be affected by norepinephrine as well as calcium (Ca⁺⁺) and both are affected by REM sleep deprivation. Before studying its molecular mechanism of action, synaptosomal Na-K ATPase activity was estimated under various conditions. The enzyme activity in synaptosome increased after lysis and in the presence of EDTA. The increase in the lysed preparation was possibly because almost all the active sites of the enzyme molecules were exposed to express their activity, unlike unlysed preparation where half are likely to be inside out. EDTA possibly increased the enzyme activity by chelating the Ca⁺⁺ which is known to have an inhibitory effect on the enzyme activity. Also, the REM sleep deprivation induced increase in the enzyme activity was observed in lysed preparations and in the presence of EDTA only. These observations fit with the existing knowledge, however, the molecular mechanism of the increase needs to be investigated.

Introduction

Rapid eye movement (REM) sleep deprivation alters brain (Cohen and Dement, 1965) and neuronal excitability (Mallick et al., 1989, Mallick et al., 1991). The deprivation induced increase in brain excitability could be due, in some part, to increased Na-K ATPase [EC 3.6.1.3] activity in the rat brain (Gulyani and Mallick, 1993). This increase in the enzyme activity may be mediated by norepinephrine (NE) acting on α-1 adrenoceptor (Gulyani and Mallick, 1995) and changes in Ca⁺⁺ levels (Mallick and Gulyani, 1996). The molecular mechanism of REM sleep deprivation induced increase in Na-K ATPase activity is yet to be elucidated. Earlier, REM sleep deprivation induced increase in Na-K ATPase activity was studied in microsomal preparation. Since the synaptosome is rich in nerve endings, it is likely to be richer in Na-K ATPase than the microsome (Hokin and Hexum, 1972). Hence, synaptosome was preferred for studying the molecular mechanism of action of REM sleep deprivation induced increase in Na-K ATPase activity. However, the effect of REM sleep deprivation on synaptosomal Na-K ATPase activity was unknown. Besides, Ca⁺⁺ is an inhibitor of Na-K ATPase (Davis and Vincenzi, 1971) and its level changed after REM sleep deprivation (Mallick and Gulyani, 1996). Therefore, before proceeding further, the synaptosomal Na-K ATPase activities from free-moving and REM sleep deprived rat brains were estimated under various conditions.

Section snippets

Experimental procedures

Experiments were conducted on male Wistar rats (250–280 g), maintained at 12\12 h light\dark cycle and were supplied with food and water ad libitum. Standard flower-pot method (diameter 6.5 cm) was used for four days REM sleep deprivation, while large platform (diameter 13 cm) controls were carried out to rule out non-specific effects (Gulyani and Mallick, 1993; Jouvet et al., 1964).

Effect of lysis on synaptosomal Na-K ATPase activity

In non-EDTA preparation, the Na-K ATPase activity in unlysed preparation of synaptosome was 15.943±1.04 μmol Pi\mg prot\h (Fig. 1). The same preparation on lysis showed a significantly increased activity of 48.964±2.61 μmol Pi\mg prot\h (F(1,10) = 137.84, P < 0.001) (Fig. 1).

Effect of EDTA on synaptosomal Na-K ATPase activity

In the presence of EDTA, the enzyme activity in the unlysed preparation of synaptosome increased from 15.943±1.04 to 26.852±2.72 (F(1,12) = 10.981, P < 0.01) (Fig. 1), while in the lysed preparation, the activity increased

Discussion

The unlysed synaptosome showed lower activity while the activity in the lysed preparation was significantly higher. The enzyme activity in the lysed preparation was significantly higher possibly because most of the enzyme molecules were exposed to express their activities, unlike in the unlysed vesicle form where about 50% of the vesicles are likely to be inside-out membrane preparations. This is because the Na⁺ and ATP binding sites of the enzyme are located intracellularly (Albers et al., 1963

Acknowledgements

The financial support received from Indian Council of Medical Research is duly acknowledged.

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Rapid communication Comparison of Na-K ATPase activity in rat brain synaptosome under various conditions

Abstract

Introduction

Section snippets

Experimental procedures

Effect of lysis on synaptosomal Na-K ATPase activity

Effect of EDTA on synaptosomal Na-K ATPase activity

Discussion

Acknowledgements

Life Sci.

J. Biol. Chem.

Neuroscience

J. Biol. Chem.

Brain Res.

Neuroscience

J. Biol. Chem.

J. Neurochem.