Quantification of Rat Cerebral Cortex Na⁺,K⁺‐ATPase: Effect of Age and Potassium Depletion

Abstract: Na⁺,K⁺‐ATPase concentration in rat cerebral cortex was studied by vanadate‐facilitated [³H]ouabain binding to intact samples and by K⁺‐dependent 3‐O‐methylfluorescein phosphatase activity determinations in crude homogenates. Methodological errors of both methods were evaluated. [³H]Ouabain binding to cerebral cortex obtained from 12‐week‐old rats measured incubating samples in buffer containing [³H]ouabain, and ouabain at a final concentration of 1 × 10^–6 mol/L gave a value of 11,351 ± 177 (n = 5) pmol/g wet weight (mean ± SEM) without any significant variation between the lobes. Evaluation of affinity for ouabain was in agreement with a heterogeneous population of [³H]ouabain binding sites. K⁺‐dependent 3‐O‐methylfluorescein phosphatase activity in crude cerebral homogenates of age‐matched rats was 7.24 ± 0.14 (n = 5) μmol/min/g wet weight, corresponding to a Na⁺,K⁺‐ATPase concentration of 12,209 ± 236 pmol/g wet weight. It was concluded that the present methods were suitable for quantitative studies of cerebral cortex Na⁺,K⁺‐ATPase. The concentration of rat cerebral cortex Na⁺,K⁺‐ATPase showed ∼10‐fold increase within the first 4 weeks of life to reach a plateau of ∼11,000–12,000 pmol/g wet weight, indicating a larger synthesis of Na⁺,K⁺ pumps than tissue mass in rat cerebral cortex during the first 4 weeks of development. K⁺ depletion induced by K⁺‐deficient fodder for 2 weeks resulted in a slight tendency toward a reduction in K⁺ content (6%, p > 0.5) and Na⁺,K⁺‐ATPase concentration (3%, p > 0.4) in cerebral cortex, whereas soleus muscle K⁺ content and Na⁺,K⁺‐ATPase concentration were decreased by 30 (p < 0.02) and 32% (p < 0.001), respectively. Hence, during K⁺ depletion, cerebral cortex can maintain almost normal K⁺ homeostasis, whereas K⁺ as well as Na⁺,K⁺ pumps are lost from skeletal muscles.