ENDOCYTOSIS AND UPTAKE OF LUCIFER YELLOW BY CULTURED ATRIAL MYOCYTES AND ISOLATED INTACT ATRIA FROM ADULT-RATS - REGULATION AND SUBCELLULAR-LOCALIZATION

The time course of endocytic uptake of Lucifer yellow (LY) was followe d by fluorescence and electron microscopy after exposure of primary cu ltures of atrial myocytes from adult rats to LY under conditions that prevented transplasmalemmal LY entry via channels or carriers. After a 2-minute exposure to LY at 37 degrees C, electron microscopy revealed classic clathrin-coated vesicles fused to endosomes, which were absen t in LY-free medium or at 2 degrees C, suggesting that LY turns on end ocytosis or accelerates a slow constitutive endocytosis. Fluorescence microscopy, which detected no LY entry at 2 minutes in LY, showed punc tate cytoplasmic fluorescent densities after 10 minutes, which were re adily distinguishable from intrinsic perinuclear fluorescence. Fluores cence microscopy after immunostaining with antibodies against clathrin , vacuolar H+-ATPase, atrial peptide, or a marker for acidified compar tments suggested LY sorting into an acidified prelysosomal pathway. Us ing absence of punctate fluorescence after 10 minutes in LY as a crite rion for inhibition of endocytosis, we showed that endocytosis was inh ibited by inhibitors of protein phosphatases 1 and 2A or inhibitors of cAMP-dependent protein kinases 1 and 2, by effects of caffeine on sar coplasmic reticulum Ca2+ release, and by temperatures below 18 degrees C, but not by staurosporine, phorbol esters, pertussis toxin, thapsig argin, preventing contractions with nifedipine, ryanodine and low [Ca2 +](o), or raising cytosolic cAMP concentrations. Both phosphatase inhi bitors and caffeine also inhibited a fraction of LY uptake by intact r at atria. We conclude that endocytic uptake of LY is an energy-depende nt, specifically regulated process, whose understanding and control ar e potentially important for the medically relevant problem of introduc ing drugs and macromolecules into atrial heart muscle cells.