Stability of nucleoside triphosphate levels in the red cells of the snake

Abstract

Nucleated red cells in the nonpregnant garter snake (Thamnophis elegans) contain relatively high concentrations of nucleoside triphosphate (NTP), largely in the form of ATP, which is found at concentrations of approximately 10 mmol l-1 relative to cell volume and 15 mmol l-1 relative to cell water. During pregnancy, levels of NTP in adult red cells rise by approximately 50 % concomitant with an increase in blood progesterone level. The stability of the NTP pool within these red cells was assessed by maintaining cells in vitro at 20 °C, without exogenous nutrients, and in the presence and absence of the metabolic inhibitors iodoacetate and/or cyanide. After 96 h, NTP levels in adult red cells not exposed to the inhibitors had decreased by only approximately 10 %, while in the presence of both inhibitors NTP levels had fallen by less than 50 %. Red cell NTP levels were not affected by acute exposure to high concentrations of progesterone either in vivo or in vitro. NTP levels were much more labile when the cells were maintained in vitro at either low or high pH. Maintenance of red cells at pH 6.0 for 24 h resulted in a decrease in NTP levels of approximately 50 % and at pH 10.0 the levels fell by approximately 80 %, while buffers containing only ATP caused no detectable degradation. Incubation at low or high pH promoted some cell swelling; however, the magnitude of the decreases in intracellular NTP concentration prompted by these pH levels could not be mimicked by incubation of red cells in hypotonic buffer. Total nonspecific ATPase activity at pH 6.0 was approximately 55 % greater than that at pH 7.4, while at pH 10.0 it was approximately 6 % of that at pH 7.4. The pH-dependent decrease in intracellular NTP levels cannot, therefore, be due to stimulation of ATPase activity, at least not at high pH. Overall, the data are consistent with the hypothesis that an appreciable portion of the NTP within these cells is compartmentalized in a stable, but pH-sensitive, pool sequestered from intracellular ATP-hydrolyzing processes.