A quantitative electron microscope autoradiographic study on [ 3H]prostaglandin E 1 and F 2α internalization in bovine luteal slices

Abstract

Very few silver grains were seen on the luteal cell surface and none intracellularly after incubation for 2 h at 4°C with 10 nM [ 3H]prostaglandin (PG) E 1 or [ 3H]PGF 2α. However, incubation at 22°C or 38°C for 2 h resulted in association of numerous grains on the luteal cell surface as well as in several intracellular organelles. Qualitatively, the grain distribution was similar at 22°C and 38 °C, but quantitatively there were fewer grains at 22°C than at 38 °C. In addition to luteal cells, the grains were also found on erythrocytes, platelets, fibroblasts and capillary endothelial cells in luteal tissue incubated with [ 3H]PGE 1. Grain association with non-luteal cells was never seen with [ 3H]PGF 2α. Coincubation of [ 3H]PGE 1 or [ 3H]PGF 2α with only corresponding excess unlabeled PGs resulted in complete disappearance of silver grains from all the subcellular organelles. In autoradiographs, grains were seen on plasma membranes, nuclei, lysosomes, mitochondria, rough and smooth endoplasmic reticulum and Golgi. Three-step mask analysis, which effectively corrects for radiation spread, revealed that the grains found on mitochondria and rough endoplasmic reticulum in the case of [ 3H]PGE 1, and those found on mitochondria, rough and smooth endoplasmic reticulum and Golgi in the case of [ 3H]PGF 2α, were due to the radiation spread. For both [ 3H]PGE 1 and [ 3H]PGF 2α the plasma membrane associated grains progressively decreased, while the intracellular organelle associated grains increased with time at 38 °C. There were no grains in nuclei at 5 min but they appeared at 10 min and increased until 60 or 120 min. The optimized source density following 2 h of incubation at 38 °C with [ 3 H] PGE 1 was Golgi > plasma membranes > lysosomes = nuclei , and for [ 3H]PGF 2α, lysosomes > plasma membranes > nuclei. In summary, the present studies demonstrate for the first time that exogenously added [3H]PGE 1 and [ 3H]PGF 2α internalize in bovine luteal cells in a ligand-specific, time- and temperature-dependent manner. The observations that the internalized [ 3H]PGE 1 does not associate with rough endoplasmic reticulum and that [ 3H]PGF 2α does not associate with rough endoplasmic reticulum and Golgi, even though they contain the binding sites, suggest the presence of mechanism(s) to direct the internalized [ 3H]PGs to only certain intracellular organelles of luteal cells.