Morphological Responses to Corticotrophin and Cyclic Amp by Adult Rat Adrenocortical Cells in Monolayer Culture

O'Hare, MJ; Am, Neville

doi:10.1677/joe.0.0560529

Cited by 80 publications

(14 citation statements)

References 18 publications

(23 reference statements)

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“…1A is an example of a typical monolayer found in these cultures. Additions of ACTH to the culture medium resulted in a characteristic morphological response similar to that reported by O'Hare and Neville (10). There was a marked retraction of the epithelial cells of the culture, leaving intercellular spaces on the surface of the petri dish.…”

Section: Methodssupporting

confidence: 77%

Inhibition of replication of normal adrenocortical cells in culture by adrenocorticotropin.

Ramachandran¹,

Suyama²

1975

Proc. Natl. Acad. Sci. U.S.A.

141

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Adrenocorticotropic hormone (ACTH) 160-180 g) were anesthetized with Nembutal, and the adrenal glands were aseptically removed from surrounding fat, trimmed, bisected, and decapsulated. The tissue was then minced and collected in a sterile 50-ml capped conical polypropylene tube containing 15 ml of phosphate-buffered saline at room temperature. The phosphate-buffered saline solution was removed and replaced with 15 ml of a sterile solution of medium 199 with Earle's salts containing collagenase (4 mg/ml) and DNase (0.1 mg/ml). The tube was gassed with 95% 02-5%CO2, capped, and placed in a gyratory water bath at 370 for 1 hr. The tissue fragments were allowed to settle after this incubation. The medium was removed and replaced with 5 ml of a sterile solution of medium 199 containing no enzymes, and the tissue was dispersed mechanically by drawing it through a sterile siliconized pasteur pipet with a flamepolished tip. The fragments were allowed to settle, and the supernatant was removed and filtered through two layers of cheese cloth. Five milliliters of medium 199 were again added to the tissue fragments, and the procedure was repeated until the tissue was completely dissociated. The cells were then collected by centrifugation, and the pellet was washed twice with medium. Finally, the cells were suspended in medium 199 with 10% fetal calf serum, 100 IU/ml of penicillin, and 100 ug/ml of streptomycin.The cells were plated at a density of about 2.5 to 3.0 X 105 cells per cm2 on 10-cm2 plastic petri dishes. Staining with trypan blue showed that 80-90% of the cells were viable. The medium was removed and replaced with fresh medium after 48 hr of culture. At this time, about 30-50% of the cells had firmly attached and, thereafter, the medium was replaced every 2 days. These cultures were maintained for periods of up to 1 month.Adrenocortical cells from 17-to 20-day-old male SpragueDawley rats were also prepared in a similar manner. In addition, the capsular tissue of the adult rats (160-180 g) was also digested with collagenase and cells were prepared for culture. 113Abbreviations: ACTH, adrenocorticotropin; NPS-ACTH, o-nitrophenyl sulfenyl derivative of ACTH.

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Section: Methodssupporting

confidence: 77%

Inhibition of replication of normal adrenocortical cells in culture by adrenocorticotropin.

Ramachandran¹,

Suyama²

1975

Proc. Natl. Acad. Sci. U.S.A.

141

View full text Add to dashboard Cite

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“…In vitro experiments have given discordant results. In fact, while O'Hare and Neville (16) could not observe any proliferative activity, both in the presence and in the lack of ACTH, of rat adult adrenocortical cells in a different primary tissue culture system, we, in keeping with the present data, could report an early, significant (but transient) stimulation by ACTH of the entry of such cells into the S stage of mitotic cycle (1 ,4 ). It is undeniable that a stimulation of DNA synthesis and cell proliferation following ACTH administration is closer to the in vivo response of normal cortical cells (8,12).…”

Section: Discussionsupporting

confidence: 76%

“…Differences in tissue culture methodology may, therefore, deeply change the metabolic responses of cultured parenchymal cells. In fact, the cells of 0 ' Hare and Neville (16) were covered by a 2-mm thick layer of growth medium, whereas ours were separated from the oxygen of atmosphere only by the very thin (0.0375 mm) polyethylene disc (1 -4 ). It was previously shown that a fluid overlay thicker than 0.34 mm in air (the gas phase of both O ' Hare's and our cultures) and 1.7 mm in pure oxygen made impossible the normal aerobic respiration and endoergonic pathways of cultured mammalian parenchymal cells (17).…”

Section: Discussionmentioning

confidence: 98%

Primary Tissue Culture of Normal Adult Human Decapsulated Adrenal Cortex: Radioautographic Studies on the Metabolic Effects of ACTH<sub>1-2</sub><sub>4</sub>

Armato¹,

Andreis²,

Draghi³

et al. 1975

Horm Res

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ACTH-deprived primary normal adult human adrenocortical cells on the 23rd day in vitro were found to lack any DNA synthetic and proliferative activity, but incorporated uridine-³H and l-leucine-³H actively. In cortical cells of the same age treated for 2 and 7 days with ACTH_1-24 (3 X 10^-6M) the incorporation of the precursors for RNA and DNA synthesis and the cell proliferation were found to be markedly stimulated. The apparent uptake of leucine-³H was instead reduced in 2-day and, less still, in 7-day treated cells. In parallel with ultrastructural studies, these data suggest that ACTH orderly activates the template activity of chromatin while eliciting the differentiation and hypertrophy of human adrenocortical cells.

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“…Despite many in vivo studies in animal models which have shown that ACTH can stimulate adrenal growth, ACTH stimulation of adrenal cells in vitro, in most cases, results in a decrease in cell number (34)(35)(36)(37)(38)(39). This apparent antimitotic effect of ACTH has been demonstrated in a number of adrenal cell culture systems, including primary cultures of human fetal adrenal cells.…”

Section: Introductionmentioning

confidence: 99%

Basic fibroblast growth factor expression is regulated by corticotropin in the human fetal adrenal: a model for adrenal growth regulation.

Mesiano

Mellon

Gospodarowicz

et al. 1991

Proc. Natl. Acad. Sci. U.S.A.

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Human fetal adrenal growth after midgestation is very rapid and appears to be dependent upon pituitary adrenocorticotropin (ACTH) in vivo. We hypothesized that the regulation of fetal adrenal growth by ACTH is mediated by ACTH-stimulated local growth factor production. As we have found basic fibroblast growth factor (bFGF) to be a potent mitogen for human fetal adrenal cells in culture, we conducted studies to determine whether bFGF is synthesized by the human fetal adrenal gland and whether bFGF gene expression in primary cultures of human fetal adrenal cells is regulated by ACTH. Bioassayable bFGF-like activity was detected in ex-

show abstract

Morphological Responses to Corticotrophin and Cyclic Amp by Adult Rat Adrenocortical Cells in Monolayer Culture

Cited by 80 publications

References 18 publications

Inhibition of replication of normal adrenocortical cells in culture by adrenocorticotropin.

Inhibition of replication of normal adrenocortical cells in culture by adrenocorticotropin.

Primary Tissue Culture of Normal Adult Human Decapsulated Adrenal Cortex: Radioautographic Studies on the Metabolic Effects of ACTH<sub>1-2</sub><sub>4</sub>

Basic fibroblast growth factor expression is regulated by corticotropin in the human fetal adrenal: a model for adrenal growth regulation.

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