The Time Profile of the Effects of Moderate Hypothermia on Synaptic Acetylcholinesterase in Rat Brain.

Hypothermia in homeotherms significantly affects the neurotransmitter systems of the brain, including the cholinergic system. The function of the brain cholinergic system during prolonged moderate hypothermia is not known yet. We studied the effects of moderate hypothermia of various durations on the activity and kinetic parameters of synaptic acetylcholinesterase in rat brain. Immediately after body temperature decrease to 30°C, the efficiency of synaptic acetylcholinesterase catalysis significantly increases due to changes in both the maximum rate of reaction (Vmax; the rate of reaction when the enzyme is saturated with substrate) and Michaelis constant (Km). However, in the dynamics of prolonged hypothermia (1-3 h), it decreases to a level of intact animals, which was associated with normalization of the kinetic parameters of the enzyme. The detected changes in the kinetic parameters of the enzyme are compensatory and can be associated with both its reversible post-translational modifications and changes in the annular lipids.

Antioxidant Status of Rat Liver Mitochondria under Conditions of Moderate Hypothermia of Different Duration.

For evaluation of the contribution of the antioxidant system of mitochondria into the dynamics of changes in the prooxidant status, the content and activity of some of its components were studied under conditions of moderate hypothermia of varying duration. It was found that short-term hypothermia significantly increased superoxide dismutase activity and decreased the levels of low-molecular-weight antioxidants. Increasing the duration of hypothermia to 1 h led to suppression of activities of superoxide dismutase, glutathione reductase, and glutathione peroxidase and a decrease in glutathione content. Further prolongation of hypothermia (to 3 h) was associated with a significant increase in superoxide dismutase and glutathione peroxidase activities and normalization of the rate of glutathione reductase catalysis; the concentration of glutathione increased significantly.

Respiratory Characteristics of Rat Liver Mitochondria Depend on the Duration of Moderate Hypothermia.

The development of pathological or compensatory-and-adaptive reactions in homoeothermic animals during various periods of hypothermia can be caused by shifts in the respiratory functions of the mitochondria. Short-term hypothermia promoted an increase in the rates of the glutamate- and succinate-dependent respiration of mitochondria. Phosphorylation rate increased as well, while oxidative phosphorylation coefficient (P/O), respiratory control, and 2,4-DNP sensitivity depended. Changes in respiratory characteristics in the dynamics of prolonged hypothermia depends on the type of substrate. Prolongation of hypothermia to 1 h was associated with further intensification of succinate-dependent respiration, decrease in P/O and respiratory control, while prolongation of hypothermia to 3 h led to their normalization. The majority of respiratory characteristics of glutamate-dependent respiration did not change under these conditions and their levels were the same as during short-term hypothermia.

Thermostability of Lactate Dehydrogenase in Rat Brain under Conditions of Short-Term Moderate Hypothermia.

Thermostability of rat brain lactate dehydrogenase (LDH) was studied in intact animals and animals subjected to moderate short-term hypothermia. Two exponential stages, rapid and slow, were distinguished in the thermodenaturation kinetics. The contribution of the rapid phase to the lactate dehydrogenase denaturation kinetics was more significant: the energy of activation for this phase was 2.33 times lower than that for the slow phase. Moderate shortterm hypothermia led to a significant decrease of lactate dehydrogenase thermostability: thermodenaturation rate constants for the rapid (k1) and slow (k2) phases increased. Significant changes in parameters a and b reflecting the initial proportion of the two native forms of the enzyme developed only at 40°C. As hypothermia caused no appreciable changes in the energy of activation of lactate dehydrogenase denaturation, a significant contribution of the entropic factor to the decrease of free energy of enzyme denaturation was hypothesized. The data indicated significant labilization of lactate dehydrogenase structure under conditions of moderate hypothermia.

A New Tellurium- and Selenoorganic Compound as an Inhibitor of Acetylcholinesterase in Brain.

We studied the effect of a new cyanine dye containing selenium and tellurium on acetylcholinesterase activity in synaptic membrane in rat brain. The cyanine dye dose-dependently inhibits activity of this enzyme, and the concentration of half-maximal inhibition of acetylcholinesterase activity was 20.46 µM. The cyanine dye instantly inhibits the enzyme; the degree of inhibition depends on acetylthiocholine concentration: the lower is acetylthiocholine concentration, the higher is the degree of inhibition. On the Lineweaver-Burk plot, the concentration dependence curves of acetylcholinesterase with and without cyanine dye intersect in one point on the abscissa axis. In this case, the cyanine dye reduces the maximum inhibition rate (Vmax) and does not affect Michaelis constant (Km). The calculated inhibition constant Ki for the cyanine dye is 7.74 µM. Thus, the cyanine dye is a non-competitive inhibitor of acetylcholinesterase.

Effect of Hypothermia on Kinetic Characteristics of Lactate Dehydrogenase in Rat Brain under Conditions of Global Ischemia and Reperfusion.

We studied activity and kinetic characteristics of lactate dehydrogenase (LDH) in rat brain under conditions of incomplete global ischemia followed by reperfusion against the background of mild hypothermia. It was found that hypothermia leads to a decrease in LDH activity in the ischemic brain; the maximum velocity of the enzyme-catalyzed activity decreased and Michaelis constant increased, due to which the efficiency of catalysis decreased to the level observed in control rats. Ischemia against the background of hypothermia was accompanied by a decrease in the inhibition constant and narrowing of effective pyruvate concentration range. Blood flow resumption in the ischemic brain against the background of mild hypothermia led to an increase in LDH activity, the maximum reaction velocity increased, and Michaelis constant decreased, which lead to a significant increase in the efficiency of catalysis. This was accompanied by an increase in enzyme inhibition constant and a shift of the optimum on the concentration curve towards lower pyruvate concentrations.

[Temperature dependence of the acetylcholinesterase activity in synaptic membranes of the rat brain].

The temperature dependence (5-40 degrees C) of the acetylcholinesterase activity in synaptic membranes of the rat brain at different substrate concentrations was studied. At low substrate concentrations, the Arrhenius plot has two linear sections. At high concentration, there is one linear section throughout the temperature range. The addition of glycerol to incubation medium to final concentrations of 1 and 2% (w/v) increases the Michaelis constant, without affecting the maximal rate and the inhibition constant. The role of diffusion in the temperature dependence of the acetylcholinesterase activity is discussed.