In Aplysia californica enterocytes, alanine-stimulated Na+ absorption increases both apical membrane exocytosis and fractional capacitance (fCa; a measure of relative apical membrane surface area). These increases are thought to reduce membrane tension during periods of nutrient absorption that cause the enterocytes to swell osmotically. In the absence of alanine, exocytosis and fCa are constant. These findings imply equal rates of constitutive endocytosis and exocytosis and constitutive recycling of the apical plasma membrane. Thus, the purpose of this study was to confirm and determine the relative extent of constitutive apical membrane recycling in Aplysia enterocytes. Biotinylated lectins are commonly used to label plasma membranes and to investigate plasma membrane recycling. Of fourteen biotinylated lectins tested, biotinylated wheat germ agglutinin (bWGA) bound preferentially to the enterocytes apical surface. Therefore, we used bWGA, avidin D (which binds tightly to biotin), and the UV fluorophore 7-amino-4-methylcoumarin-3-acetic acid (AMCA)-conjugated avidin D to assess the extent of constitutive apical membrane recycling. A temperature-dependent (20 vs. 4 degrees C) experimental protocol employed the use of two tissues from each of five snails and resulted in a approximately 60% difference in apical surface fluorescence intensity. Because the extent of membrane recycling is proportional to the difference in surface fluorescence intensity, this difference reveals a relatively high rate of constitutive apical membrane recycling in Aplysia enterocytes.

We used the Aplysia californica intestinal epithelium to investigate the effect of alanine-stimulated Na+ absorption on apical membrane exocytosis and whether stimulated exocytosis requires intact actin filaments. The fluid-phase marker fluorescein dextran was used to determine rates of apical membrane exocytosis. L-alanine significantly increased apical exocytosis by approximately 30% compared to controls, and there is a modest, positive correlation between alanine-stimulated exocytosis and short-circuit current (ISC). Thus, apical exocytosis is modulated to some extent by the magnitude of Na+ and alanine entry across the apical membrane. Apical exocytosis is also responsive to virtually any increase in Na+ and alanine entry because increments in alanine-stimulated ISC as small as 1 microA/cm2 stimulated exocytosis. We used D-alanine to determine which parameter (sensitivity to transport vs. magnitude of transport) was most important in activation of apical exocytosis. D-alanine-stimulated ISC was one-sixth that of L-alanine, but stimulated exocytosis was only 29% less than that of L-alanine. Therefore, the apical exocytic system is more responsive to small increases in transport than to the magnitude of transport. Latrunculin A (Lat-A) disrupts the actin cytoskeleton and reduced constitutive apical exocytosis by approximately 65% and completely abolished alanine-stimulated exocytosis. Hence, constitutive exocytosis and alanine-stimulated exocytosis require actin filaments for recruitment of vesicles to the apical membrane. During nutrient absorption, actin filament-regulated apical exocytosis may represent a negative feedback system that modulates apical membrane tension.