Inhibition of calmodulin-dependent enzymes in rat brain by hexachlorocyclohexane

Ca²⁺ homeostasis is one of the major regulatory mechanisms operating in the nervous system, with calmodulin translating the Ca²⁺ message into cellular response. To check if hexachlorocyclohexane (HCH) acts as a calmodulin antagonist in the nervous system of rats, the in-vitro effect of HCH on calmodulin-dependent Ca²⁺-ATPase and cAMP-phosphodiesterase (PDE) in rat brain has been studied. In the membrane fraction from rat brain, a basal activity of Ca²⁺-ATPase was obtained in the absence of Ca²⁺. Inclusion of Ca²⁺ (1 mM) increased the enzyme activity by 70%. Further, addition of fluphenazine, a potent calmodulin antagonist, inhibited the Ca²⁺-dependent enzyme activity (IC₅₀ = 85 μM), demonstrating the calmodulin dependence of the enzyme activity. The Ca²⁺- and calmodulin-dependent Ca²⁺-ATPase was inhibited by HCH in a dose-dependent manner (IC₅₀ = 80–90 μM). Ca²⁺- and calmodulin-dependent cAMP-PDE from the cytosolic fraction of rat brain was inhibited by HCH (340 μM) by 79%. Addition of excess calmodulin reversed the inhibitory effects of HCH or fluphenazine on Ca²⁺-ATPase and cAMP-PDE, suggesting their direct interaction with calmodulin. By fluorescence interaction studies it has been shown that HCH interacts directly with calmodulin. These studies show that HCH may modulate the intracellular concentration of Ca²⁺ and cAMP, by decreasing the effectiveness of calmodulin towards its effector enzymes, resulting in an altered signal transduction in the nervous system.