Oxygen-induced Regulation of Na/K ATPase in cerebellar granule cells

Abstract

Adjustment of the Na/K ATPase activity to changes in oxygen availability is a matter of survival for neuronal cells. We have used freshly isolated rat cerebellar granule cells to study oxygen sensitivity of the Na/K ATPase function. Along with transport and hydrolytic activity of the enzyme we have monitored alterations in free radical production, cellular reduced glutathione, and ATP levels. Both active K(+) influx and ouabain-sensitive inorganic phosphate production were maximal within the physiological pO(2) range of 3-5 kPa. Transport and hydrolytic activity of the Na/K ATPase was equally suppressed under hypoxic and hyperoxic conditions. The ATPase response to changes in oxygenation was isoform specific and limited to the alpha1-containing isozyme whereas alpha2/3-containing isozymes were oxygen insensitive. Rapid activation of the enzyme within a narrow window of oxygen concentrations did not correlate with alterations in the cellular ATP content or substantial shifts in redox potential but was completely abolished when NO production by the cells was blocked by l-NAME. Taken together our observations suggest that NO and its derivatives are involved in maintenance of high Na/K ATPase activity under physiological conditions.

Figure 1.

(A) Original trace of oxygen concentration recording in cerebellum of 12 d postnatal rat using Oxylite 4000 (Oxford Optronix). Arrow shows the moment of cessation of breathing. (B) The amount of dead (propidium iodide–positive) cells at the beginning of experiment (To) and after 60 min of incubation at pO₂ of 20, 5, or 0.5 kPa assayed by flow cytometry. The age of the animals varied from P10 to P12. Data are means of five independent experiments ± SEM. * denotes P < 0.05 between the selected values.

Figure 2.

Reduced glutathione levels in cells incubated for 7, 30, or 60 min at pO₂ of 20, 5, or 0.5. Data are means of five independent experiments ± SEM. * denotes P < 0.05 compared with the level at 20 kPa after 7 min of incubation. Inset represents the changes in GSH:GSSG half-reduction potential Ehc in cells exposed to pO₂ of 20, 5, or 0.5 kPa for 60 min. Ehc was calculated using the following equation (for details see Schafer and Buettner, 2001): Ehc = −240 – (59.55/2)*log([GSH]/[GSSG]) for the T 25°C, pH 7.0.

Figure 3.

(A) The rate of H₂-DCF oxidation in cerebellar granule cells incubated at pO₂ of 20, 5, or 0.5 kPa over 60 min detected by means of flow cytometry. Data are means of five independent experiments ± SEM normalized to the fluorescent signal immediately after loading. (B) Oxygen dose dependence of H₂-DCF oxidation after 15 and 60 min of incubation at fixed pO₂. Data are means of six independent experiments ± SEM normalized to the fluorescent intensity at the onset of incubation at fixed pO₂ following 30 min of preincubation of cells loaded with fluorescent dye at pO₂ of 20 kPa.

Figure 4.

(A) l-NAME–sensitive component of H₂-DCF oxidation after 15 or 60 min of incubation at fixed pO₂ measured by means of flow cytometry. Data are means of five independent experiments ± SEM. (B) Nitrite and nitrate production by cerebellar granule cells detected in cell suspension after 60 min of incubation at fixed pO₂ using chemiluminiscence techniques. Data are means of six independent experiments ± SEM.

Figure 5.

Cellular ATP levels as a function of oxygen concentration. Data are means of 6–9 experiments ± SEM. ** denotes P < 0.01 and * denotes P < 0.05 compared with the levels at 7 min for pO₂ of 20 kPa.

Figure 6.

(A) Unidirectional active K⁺ influx into the cerebellar granule cells as a function of oxygen concentration. Data are means of 6–8 independent experiments ± SEM. (B) Effect of inhibition of NO synthases on oxygen-induced regulation of the active K⁺ influx in cells incubated for 30 min at pO₂ of 10, 3, or 1 kPa. Data are means of five independent experiments ± SEM.

Figure 7.

Oxygen dependence of the Na/K ATPase hydrolytic activity in cerebellar granule cells incubated at fixed oxygen concentrations for 30 min in the presence or in the absence of 100 μmol of l-NAME. Data are means of six independent experiments ± SEM.