Quantitative localization of Na-K pump sites in the frog sacculus

The Na-K pump site distribution within the macula, perimacula, and wall epithelia of the sacculus in the frog inner ear was examined with quantitative [3H]ouabain autoradiography. Excised tissue was incubated for 10-30 min (23 degrees C) in micromolar concentrations of high specific activity [3H]ouabain (14-70 muCi ml-1, 5-15 Ci mmol-1), washed for 30 min (4 degrees C), then rapidly frozen (-175 degrees C) and processed for light and electron microscope autoradiography. Control experiments based on (1) high K+ (50 mM) in the incubation and (2) low specific activity [3H]ouabain (1 mM, 0.013-0.025 Ci mmol-1) indicated negligible nonspecific binding of the [3H]ouabain. Measurable levels of specific [3H]ouabain binding occurred in all saccular regions examined. Binding was localized to the basolateral cell membranes with no detectable binding to the apical membranes. [3H]ouabain binding across the apical-basal axis of the saccule macular epithelium was nonuniform. Binding was low in the apical region, rose to a peak in the middle two-thirds, and then fell again close to the basement membrane. Electron microscope autoradiography suggested that this peak was due to ouabain binding to nerve terminals. Denervation of the sacculus eliminated the peak in [3H]ouabain binding and quantitative grain density analysis revealed that 45% of the Na-K pumps within the saccule macula were located on the nerve terminals. Na-K pump site density per unit volume was estimated by quantitative grain density analysis and the following values were obtained (sites micron-3 X 10(3), means +/- S.E.M.): saccule macula, 1.9 +/- 0.2; saccule perimacula, 1.1 +/- 0.1; saccule wall, 2.3 +/- 0.3. Stereological analysis of conventionally fixed tissue was used to estimate overall plasma membrane surface area per unit volume (Sv). Na-K pump site densities per unit membrane area for the various regions were calculated by combining the autoradiographical and stereological data. The following values were obtained (sites micron-2 +/- 25%): saccule macula, 2500; saccule perimacula, 2500. Values for individual cells within the macula (sites micron-2 +/- 25%) were: hair cells, 3000; nerve terminals, 3000; supporting cells, 1500.