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The time course of endocytic uptake of Lucifer yellow (LY) was followed by fluorescence and electron microscopy after exposure of primary cultures 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 absent in LY-free medium or at 2 degrees C, suggesting that LY turns on endocytosis or accelerates a slow constitutive endocytosis. Fluorescence microscopy, which detected no LY entry at 2 minutes in LY, showed punctate cytoplasmic fluorescent densities after 10 minutes, which were readily distinguishable from intrinsic perinuclear fluorescence. Fluorescence microscopy after immunostaining with antibodies against clathrin, vacuolar H(+)-ATPase, atrial peptide, or a marker for acidified compartments suggested LY sorting into an acidified prelysosomal pathway. Using absence of punctate fluorescence after 10 minutes in LY as a criterion for inhibition of endocytosis, we showed that endocytosis was inhibited by inhibitors of protein phosphatases 1 and 2A or inhibitors of cAMP-dependent protein kinases 1 and 2, by effects of caffeine on sarcoplasmic reticulum Ca2+ release, and by temperatures below 18 degrees C, but not by staurosporine, phorbol esters, pertussis toxin, thapsigargin, preventing contractions with nifedipine, ryanodine and low [Ca2+]o, or raising cytosolic cAMP concentrations. Both phosphatase inhibitors and caffeine also inhibited a fraction of LY uptake by intact rat atria. We conclude that endocytic uptake of LY is an energy-dependent, specifically regulated process, whose understanding and control are potentially important for the medically relevant problem of introducing drugs and macromolecules into atrial heart muscle cells.

circulation research

Endocytosis and uptake of lucifer yellow by cultured atrial myocytes and isolated intact atria from adult rats. Regulation and subcellular localization.