Energetics, Thermal Sensitivity and Tolerance Mechanisms of Brain Synaptosomes: A Comparative Study

Abstract

In an attempt to correlate neuronal energy metabolism with thermal tolerance by means of a comparative approach, the rate of D-[¹⁴C]glucose conversion to ¹⁴CO₂ has been studied as a function of temperature (38–0°C) in brain synaptosomes from animal species characterized by different tolerances. The rat served as a reference homeotherm species; the heat-sensitive but cold-tolerant dogfish shark (Scyliorhinus canicula) represented a poikilotherm model of temperature in lower vertebrates, and was also chosen because of its known refractoriness to edema-inducing agents peculiar to the shark brain; the ground squirrel (Citellus citellus) was chosen for its coupled tolerance to hypothermia and anoxia, peculiar to hibernating mammals. In order to contribute by thermodynamic considerations to the understanding of molecular mechanisms underlying tolerance, apparent energies of activation (E), temperature coefficients (Q₁₀), transition or critical temperatures (T), and apparent dissociation constants have been derived from comparative experiments with synaptosomes in standard incubation media, and in media altered by the elimination of Na or the addition of ouabain. The consequences of uncoupling oxidations by means of 2,4-dinitrophenol (2,4-DNP) were also investigated. In addition, the effects of reduced concentrations of the substrate (D-glucose) and of the competitive inhibition of its utilization by 2-deoxy-D-glucose (2-DG) were explored.

Sharp discontinuities in Arrhenius plots of glucose oxidation rates occurred at critical temperatures (T) coinciding with thermal limits of survival in hypothermia (rat) or with upper tolerance (dogfish). Such discontinuities were absent in the case of synaptosomes from hibernating ground squirrels known to tolerate the entire range of temperatures explored. These results suggest a limiting role of neuronal energy metabolism in determining tolerance to extreme body temperatures. The comparison of E and Q₁₀ values obtained with synaptosomes of different animal origin and in particular thermal ranges also revealed a close correlation with the general thermo-physiological features of the respective species.

Particularly pertinent to the understanding of underlying mechanisms were the results concerning the Na⁺ -stimulated fraction of oxidations linked to active cation transport. Resistance to the inhibitory effects of cooling, indicated by the maintenance of relatively low E_a values, appeared in all our animal models to be due to an increasing contribution of Na -stimulated oxidations with falling temperature. The transition to greatly elevated Q₁₀ values below T appeared, on the other hand, to be a corollary of the failure at sub-critical temperatures of this sodium-stimulated and transport-linked fraction of oxidations. Increased E_a values were also recorded after the addition of 2,4-Bnp, which is known to interfere with active cation transport. However, the dose and temperature dependence of the effect of uncoupling appeared in addition to be a correlate of the glycolytic capacity of brain tissue and of tolerance to hypothermia and anoxia within the comparative animal series. The known effect of ouabain in preventing Wa stimulation in rat synaptosomes, which emphasizes the responsibility of the transport ATPase for the sodium-stimulation phenomenon, has been confirmed in experiments with shark synaptosomes. In rat brain synaptosomes, thermal sensitivity parameters and thermodynamic functions showed a close relationship to the temperature-dependence characteristics of an isolated rat-brain transport ATPase preparation described by other authors. Reduced concentrations of the substrate, as well as inhibition by 2-DG markedly depressed D-glucose oxidation rates in rat synaptosomes irrespective of temperature, but had comparatively little effect on thermal sensitivity. However, apparent dissociation constants derived from Dixon plots, as well as Q₁₀ and E_a values obtained with reduced substrate concentrations, indicated an increase with falling temperature, down to the T_C level, of the underlying enzyme-substrate affinities. In contrast, decreasing affinities were indicated below T_C. Coupled to the reduction of available thermal energy, they suggest a rapidly increasing incompatibility with the maintenance of efficient energy-yielding metabolic processes at subcritical temperatures.