ENDOCYTOSIS AND UPTAKE OF LUCIFER YELLOW BY CULTURED ATRIAL MYOCYTES AND ISOLATED INTACT ATRIA FROM ADULT-RATS - REGULATION AND SUBCELLULAR-LOCALIZATION
E. Page et al., ENDOCYTOSIS AND UPTAKE OF LUCIFER YELLOW BY CULTURED ATRIAL MYOCYTES AND ISOLATED INTACT ATRIA FROM ADULT-RATS - REGULATION AND SUBCELLULAR-LOCALIZATION, Circulation research, 75(2), 1994, pp. 335-346
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.