The effect of EGTA and Ca++ in regulation of the brain Na/K-ATPase by noradrenaline

Abstract

Background: The Na/K-ATPase activity of the brain synaptic plasma membranes (SPM) is regulated by noradrenaline (NA) and the synaptosomal factor SF (soluble protein obtained from the synaptosome cytosol). In the absence of SF, NA inhibits Na/K-ATPase, while, on addition of SF to the reaction medium, there is a NA-dependent activation of Na/K-ATPase. On the other hand, EGTA augments the Na/K-ATPase activity and attenuates the ability of NA to inhibit Na/K-ATPase.

Results: Considering that Ca2+ ion is a Na/K-ATPase modifier, it can be assumed that the effect of NA and SF is a Ca2+-dependent process. However, in the presence of 0.3 mM EGTA and 0.1 mM NA, the apparent inhibition constant for Ca2+ (at [Ca2+] > 0.3 mM) is not SF dependent, while the apparent activation constant for SF does not change at increasing Ca2+ concentration ([Ca2+] < 0.3 mM). At various Ca2+ concentrations (0.06, 0.35 and 0.6 mM), no significant changes occur in the mode of action of NA on the Na/K-ATPase activity in the presence of 5 microg/ml SF. EGTA also has no effect on the NA-independent activation of Na/K-ATPase evoked by high SF concentrations.

Conclusions: Taking into account that in the absence of EGTA similar results have been obtained, it can be concluded that the effect of NA and SF on brain Na/K-ATPase is a Ca2+-independent process.

Figure 1

Dependence of inverse values of Na/K-ATPase activity (μmol P_i/hour × mg protein) on [Ca²⁺] concentration (mM), in the presence of 0.3 mM EGTA. Broken line shows inverse value of Na/K-ATPase activity (l/V0) in the absence of EGTA and Ca²⁺ ions.

Figure 2

Dependence of inverse values Na/K-ATPase activity (μmol P_i/hour × mg protein) on [Ca²⁺] concentration (mM), in the presence of 0.3 mM EGTA, 0.1 mM NA and at various concentrations of SF. 1 – 20 μg/ml SF; 2 – 30 μg/ml SF; 3 – 40 μg/ml SF; 4 – 80 μg/ml SF.

Figure 3

Dependence of Na/K-ATPase activity (μmol P_i/hour × mg protein) on SF concentration (μg/ml) in inverse values and in the presence the of 0.3 mM EGTA, 0.1 mM NA and various concentrations of the Ca²⁺-ions. 1 – no Ca²⁺-ions added; 2 – added [Ca²⁺] = 0.1 mM.