Internalization of atrial natriuretic factor by AtT‐20 corticotropin‐secreting cells

Abstract

Because extended exposure of AtT-20 corticotropin-secreting cells to atrial natriuretic factor (ANF) results in a desensitization of ANF-induced cGMP synthesis, we sought to establish whether pretreatment of AtT-20 cells with the atrial peptide also led to an internalization process. In fact, by coupling an ultrastructural approach to cryoultramicrotomy, ANF-immunoreactivity was detected at both the plasma membrane level and at intracellular sites in AtT-20 cells. Internalization was observed within 5 min at which time labelling was observed in the plasma membrane level, in vacuole-like structures in close proximity to the plasma membrane, in cytoplasmic matrix and sometimes in mitochondria. After 30 min exposure Golgi apparatus, mitochondria and nuclear euchromatin were also labelled. Following 1-4 hr, labelling in other cell compartments, e.g. lysosomal, was increased, while it was reduced in plasma membranes and vacuole-like structures. Secretory granules and endoplasmic reticulum were not labelled throughout the time course. Extraction of a intracellular [125I] ANF from AtT-20 cells following 4 hr incubation suggested that about 90% of the peptide was intact. The data suggest that internalization of ANF may serve to terminate the biological response associated with ANF receptor activation; subcellular distribution of internalized, intact ANF suggests that the peptide may have other, as yet unidentified, intracellular actions.

Elevation by Atrial Natriuretic Factors of Cyclic GMP Levels in Astroglia‐Rich Cultures from Murine Brain

“…A similar ANF-elicited accumulation of cyclic GMP is observed in several tissues and cell types, including kidney, lung, fibroblasts, adrenal cortical cells, and aortic smooth muscle cells (for review, see Hamet et al, 1986;Leitman and Murad, 1987). Elevation of cyclic GMP levels by ANFs and the presence of ANF receptors have also been observed in other cultured cells of neural origin (Friedl et al, 1986;Fiscus et al, 1987;Morel et al, 1987;Pandey et al, 1987).…”

Structure, Expression, and Function of Atrial Natriuretic Factor in Extraatrial Tissues*

“…ANF binding sites have also been detected in the rat posterior (119) and anterior (Fig. 5, and Refs, 64 and 120) lobes including the corticotropin-secreting cell line AtT-20 (121). In the anterior lobe, autoradiographic studies reveal the presence of ANF binding sites on gonadotrophs, corticotrophs, and lactotrophs (120).…”

“…Furthermore, ANF (100 ^M) had no effect on cAMP production; however it induced a significant accumulation of cGMP in enriched anterior pituitary cell populations obtained by fractions of centrifugal elutriation (125). The stimulation of cGMP production by ANF in anterior pituitary cell culture is not due to contamination by fibroblasts or endothelial cells since it occurs in a pure population of ACTH-secreting mouse tumor cell line AtT-20 (121). Therefore, ANF can act as a potent stimulator of intracellular cGMP accumulation but most likely is not involved directly in the secretory function of the anterior pituitary cells.…”

Internalization of atrial natriuretic peptide by adrenal glomerulosa cells

“…With the use of radiolabelled atrial peptide, we have been unable to observe coated-pit or endosome formation, for example, in adrenal medullary cells [31] or pancreatic acinar cells [9]. On the other hand, when nonradioactive ANP was used in an internalization study in clonal corticotrophs (AtT-20 cells) by means of a peroxidase revelation technique, we did indeed observe small vacuolar structures which probably corresponded to endosomes [28]. Thus, while it is not proven, the most likely route of ANP internalization into glomerulosa cells is by receptor-mediated endocytosis,…”

“…(2) Extraction of radiolabel from whole adrenal glands and rocessing through Sep-pak columns [28] to separate free 5I from [nSI]ANP indicated that 30 rain after injection of radioactive AHP, 64 ± 7% (N= 5) of counts in the cell eluted as [12SI]ANP.…”