Endocytotic uptake of cationized ferritin tracer into glomus cells dissociated from the adult rat carotid body

Abstract

Glomus cells from carotid bodies of adult rats dissociated by means of collagenase or collagenase + trypsin were used to study by electron microscopy the endocytotic uptake of cationized ferritin (CF) tracer into subcellular compartments. The glomus cells were incubated with the tracer (1) in a basic salt medium (BM), or (2) in the BM into which calcium ionophore A23187 had been added, or (3) in a potassium-rich medium. Incubation of the cells in BM containing CF for 30 min resulted in attachment of the tracer to the cell membrane and uptake of a few solitary tracer particles into small vesicles and multivesicular bodies. No uptake into the cisternae of the Golgi apparatus was observed. Further incubation in BM containing CF for another 30 min resulted in increased uptake of the tracer into small vesicles and multivesicular bodies. A similar pattern of uptake was observed when the dissociated glomus cells were first preincubated in BM with CF for 30 min and then incubated for 1 min or 30 min in the BM solution containing both the ionophore and CF. Upon such incubation, CF particles were seen to penetrate into coated pits and sites of exocytosis at the cell surface. When the 30-min preincubation in BM was followed by incubation in a CF-containing potassium-rich medium for 15-30 min, uptake into vesicles, small lysosomes and occasionally also into profiles of the smooth endoplasmic reticulum was seen. Endocytotic mechanisms of the glomus cells are outlined.

Endocytosis of cationized ferritin to vesicles in the Golgi region of the glomus cells dissociated from the adult rat carotid body

“…Twenty-two carotid bodies were pooled in an oxygenated (100% 0 2 ) calcium-free medium (Gronblad et al, 1982) of the following composition (in mM): NaCll20, KC1 4, MgC12 1.5, glucose 10, Hepes 20, KHP04 0.5, Na-HC03 5, brought to pH 7.4 with NaOH. The organs were then transferred to a dissociation medium otherwise similar but containing, in addition, 100 pM CaC12 and 0.25% collagenase Type I1 (Sigma, St. Louis, MO).…”

“…The cells were then further dissociated by triturations in micropipettes with tips of decreasing diameter. This method results in solitary cells and larger groups of cells, allowing extracellular tracer access to the membrane of the glomus cells (Gronblad et al, 1982).…”

“…Following the dissociation, the collagenase was carefully rinsed from the tissue using the calcium-free medium. Glomus cell viability after the dissociation procedure was approximately 85-90% as estimated by the exclusion of the dye trypan blue (Gronblad et al, 1982).…”

“…After centrifugation at 600g for 2-3 minutes in a cold-room, the rinsed cells were resuspended in 1 ml of the incubation medium containing an additional 1 mM CaCl2 and 0.5 mg/ml CF (10.4 mg/ml; Sigma). Incubation with CF was then carried out in a 37°C water bath with light shaking and continuous bubbling of 100% 0 2 into the medium (Gronblad et al, 1982). The incubation time was varied from 30 minutes to 2-3 hours, with one change of the medium with the longer incubation times.…”

“…In glomus cells of the carotid body, cationized ferritin (CF) taken up by endocytotic vesicles, after incubation of the cells with CF for up to 1 hour, was predominantly transported into lysosomal structures in the cell, notably to multivesicular bodies (Gronblad et al, 1982). However, the role of the Golgi apparatus in glomus cell endocytosis remained unsolved.…”