Exocytosis in alveolar type II cells revealed by cell capacitance and fluorescence measurements

Abstract

Measurement of lamellar body (LB) exocytosis at high spatial and temporal resolution was recently enabled by fluorescence of the dye FM 1-43 (FFM1-43). Here, the capabilities of this method were further examined and extended by simultaneous measurement of the cell membrane capacitance ( C m) and laser-scanning confocal microscopy. Step increases in C m were evoked by extracellular ATP (20 μM) or an elevated pipette Ca2+ concentration (≥3 μM). The delay between the first C m step and the increase in FFM1-43 was <1 s, indicating ready access of FM 1-43 to exocytosed LB contents. A specific C m of 0.88 μF/cm2 for the membrane of an exocytosed LB was calculated. Compound exocytosis was occasionally observed. Decreases in C m, indicative of transient fusion or endocytosis, did not occur within 20 min of stimulation. Exocytosis was stimulated by 160 μM guanosine 5′- O-(3-thiotriphosphate) in the pipette, but compound exocytosis was unaffected. The comparison of methods revealed that FM 1-43 is ideally suited to measure the onset of exocytosis and amount of secretion. Patch clamp is superior in resolving fusion events with the plasma membrane.