A Protein Titration Hypothesis for the Temperature-Dependence of Tissue CO₂ Content in Reptiles and Amphibians

Whole-body CO ₂ stores are known to increase with cooling in reptiles and amphibians (-Δ[CO ₂ ]/ΔT ). The aim of this study was to determine the mechanism(s) producing this inverse relationship. The Δ[CO ₂ ]/ΔT coefficients were determined for eight reptilian and one amphibian species and were found to differ by a factor of approximately 10, from -0.21 mmol kg ^-1 °C ^-1 in the Mediterranean spur-thighed tortoise Testudo graeca to -0.02 mmol kg ^-1 °C ^-1 in the bullfrog Rana catesbeiana . The Δ[CO2]/ΔT coefficients were correlated with values in the literature for in vivo plasma ΔpH/ΔT coefficients (Δ[CO ₂ ]/ΔT=-0.18--8.24ΔpH/ΔT; r ² =0.87). Plasma electrolyte concentrations (Na ⁺ , K ⁺ , Ca ²⁺ , Mg ²⁺ , Cl ^- , inorganic phosphate, SO ₄ ^2- and lactate), [protein], [CO ₂ ], P _CO2 and pH were measured in chronically cannulated resting black racer snakes Coluber constrictor . When the temperature was reduced from 30 to 10 °C, pH increased slightly (by -0.0028 pH units °C ^-1 ), P _CO2 decreased by 7 mmHg, [CO ₂ ] increased by 3.2 mmol l ^-1 and [HPO ₄ ^2- +H ₂ PO ₄ ^- ] increased by 0.7 mmol l ^-1 . Concentrations of protein and of the remaining electrolytes were not significantly different (P>0.05) at 30 and 10 °C. Net plasma protein charge, calculated from the principle of electroneutrality (the sum of the cations in mequiv = the sum of anions in mequiv), was -0.48 mequiv g ^-1 protein at 30 °C and -0.38 mequiv g ^-1 protein at 10 °C. This 21 % decrease was attributed to the increases in [CO ₂ ] (i.e. carbonic acid) and inorganic phosphate concentration. Between 30 and 10 °C, skeletal muscle pH and [CO ₂ ] in C. constrictor increased (by -0.009 units °C ^-1 and -0.125 mmol kg ^-1 °C ^-1 , respectively), [Na ⁺ ] and [Cl ^- ] each decreased by approximately 12 mequiv l ^-1 , and [K ⁺ ] and the percentage of water did not change significantly. It is concluded that the increase in whole-body CO ₂ stores with cooling in reptiles and amphibians results from the passive effects of temperature changes upon the ionization constants of proteins and the active adjustment of P _CO2 (ventilatory regulation), so that -ΔpK is greater than -ΔpH. Active transmembrane ion-exchange processes do not appear to be involved.