Vacuolation of Isolated Amphibian Urocytes

Abstract

The epithelium of amphibian urinary bladder dissociated readily into single cells during exposure to solutions of low Ca²⁺ activity. The isolated urocytes rounded off and, within 2 h, developed large intracellular vacuoles derived from internalized basolateral membrane areas. Removal of Na⁺, K⁺ or Cl^– from the medium suppressed vacuolar expansion. Vacuolation was also inhibited by blocking apical Na⁺ channels with amiloride, basolateral Na⁺/K⁺ ATPases with ouabain or energy metabolism with 2-deoxy-<i>D</i>-glucose. Vacuolar expansion was enhanced by stimulation of apical Na⁺ transport with <i>p</i>-chloro mercuryphenyl sulfonic acid. Binding of fluorescent ouabain to the Na⁺/K⁺ ATPase showed the enzyme to be present first in the basolateral membrane, later in the membranes of the vacuoles. The appearance of the ATPase in the membranes of the vacuoles was also shown histochemically by the development of lead-phosphate deposits. Using X-ray microanalysis, we found a vacuolar Na⁺ concentration of some 80 mmol/kg wet weight and a K⁺ concentration near 30 mmol/kg wet weight. It appears that the internalization of basolateral membrane material firstly resulted in the formation of small vesicles, containing in their limiting membranes transporters typical of the previous basolateral membrane. The vesicles fused to vacuoles which slowly increased their volume, provided (a) the Na⁺ pump in the vacuolar membrane remained functional and (b) Na⁺ was made available to the pump by amiloride-blockable Na⁺ channels in the outward-facing membrane. Thus the vacuoles enlarged by the ‘pumping’ action of active Na⁺ transport. Their expansion was achieved by the transport machinery which normally provides for transepithelial movement of NaCl and water.