A Simple Method for the Preparation of ACTH Responsive Isolated Cells from Rat Adrenals

Nakamura, Masuhisa

doi:10.1507/endocrj1954.18.291

Cited by 5 publications

(3 citation statements)

References 5 publications

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“…Characterization of lsolated Bovine Adrenal Medullary Cells the whole adrenal gland. In these studies, cells were obtained by incubating pieces of adrenal gland with trypsin (31,43), coUagenase (25,27,34), coUagenase with DNAse (18,24), and a combination of five enzymes (23).…”

Section: Discussionmentioning

confidence: 99%

Functional and morphological characterization of isolated bovine adrenal medullary cells.

Fenwick¹,

Fajdiga²,

Howe³

et al. 1978

The Journal of Cell Biology

229

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Single bovine adrenal medullary cells have been obtained by retrograde perfusion of adrenal medullae with a solution of 0.05% coUagenase in Ca §247 free Krebs Henseleit buffer. Chromaffin cells were obtained in high yield (5 x 106 cells/g medulla), and more than 95% of these were viable as shown by exclusion of trypan blue. The isolated cells were capable of respiring at a linear rate for a minimum of 120 min.Ultrastructural examination revealed that the cells were morphologically intact, and two distinct types of adrenal medullary cells were identified, on the basis of the morphology of their electron-dense vesicles, as (a) adrenalinecontaining and (b) noradrenaline-containing cells.Biochemical analysis showed that the cells contained catecholamines and dopamine-/3-hydroxylase (DBH). The cells released catecholamines and DBH in response to acetylcholine (ACh), and this release was accompanied by changes in the vesicular and surface membranes observed at the ultrastructural level. The time-course of ACh-stimulated catecholamine and DBH release, and the dependence of this release on the concentration of ACh and extracellular Ca + § have been investigated. The isolated cells were pharmacologically sensitive to the action of the cholinergic blocking agents, atropine and hexamethonium.

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Section: Discussionmentioning

confidence: 99%

Functional and morphological characterization of isolated bovine adrenal medullary cells.

Fenwick¹,

Fajdiga²,

Howe³

et al. 1978

The Journal of Cell Biology

229

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“…A wide range of ED50 values for the complete ACTH molecule has been described in the literature. The values ranged from about 10 fmol/ml (Lowry et al 1973;Sayers et al 1971) to far more than 200 fmol/ml (Richardson 8c Schulster 1972; Nakamura & Tanaka 1971;Moyle et al 1973). Values reported for the ED50 of ACTH1"24 also vary rather dramatically, ranging from 2.4 fmol/ml (Lowry et al 1973) to far more than 100 fmol/ml (Finn et al 1976).…”

Section: Discussionmentioning

confidence: 99%

“…However, suspensions obtained by this technique generally appear to be less sensitive to ACTH than suspen¬ sions which can be produced by a trypsin treatment (Sayers et al 1971). Nevertheless, assays with the trypsin technique (Kitabchi & Sharma 1971;Naka-mura & Tanaka 1971; Lowry et al 1973;Fehm et al 1973;Gewirtz et al 1974;Liotta & Krieger 1975;Finn et al 1976) vary greatly in sensitivity to ACTH : the ED50 values range from about 10 fmol (Sayers et al 1971) to about 400 fmol/ml (Nakamura & Tanaka 1971). Also, a ra¬ ther broad range of potency values has been de¬ scribed for the various ACTH-peptides, particular¬ ly ACTH1"24 (Schwyzer et al 1971;Lowry et al 1973).…”

mentioning

confidence: 99%

Improved sensitivity to adrenocorticotrophin after purification and pre-incubation of isolated rat adrenal cells

Goverde

Pesman

Benraad

1980

Acta Endocrinologica

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Abstract. A highly sensitive bioassay for adrenocorticotrophin (ACTH) has been developed, in which isolated adrenal cells, purified previously by passage through a 5% BSA layer were pre-incubated for 1 h. The detection limit for hACTH1-39 in this system is 0.19 ± 0.10 (sd) fmol/ml. As a result of the purification step the ED50 for hACTH1-39 decreased from 14.1 ± 4.8 to 6.9 ± 1.2 fmol/ml. Pre-incubation of the purified cells decreased this value significantly to 4.0 ± 1.5 fmol/ml. For ACTH1-24 the increase of sensitivity after purification was even higher, the ED50 changing from 7.1 ± 4.2 to 1.6 ± 0.2 fmol/ml. After pre-incubation of the purified cells this value amounted to 0.8 ± 0.3 fmol/ml. After purification and pre-incubation of the adrenal cells the changes observed for the ED50's of hACTH1-32 and of a highly purified human hypophyseal extract paralleled exactly the change observed for hACTH1-39. The improved sensitivity of the purified cell suspension might in fact represent a decrease in ACTH degradation. Both purification and pre-incubation could lead to removal or inactivation of enzymes which attach the polypeptide hormone. This hypothesis is supported by experiments in which the cell suspension has been contaminated with an adrenal homogenated prior to the purification and pre-incubation steps. The present results indicate that pre-incubation of purified isolated adrenal cells has increased the sensitivity and that the amino acid sequence 33–39 is not involved in protecting the molecule from proteolytic attack.

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Primary tissue culture of human adult adrenocortical cells. Methodology and electron microscopic observations on ACTH-deprived and ACTH-treated cortical cells

et al. 1974

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A Simple Method for the Preparation of ACTH Responsive Isolated Cells from Rat Adrenals

Cited by 5 publications

References 5 publications

Functional and morphological characterization of isolated bovine adrenal medullary cells.

Functional and morphological characterization of isolated bovine adrenal medullary cells.

Improved sensitivity to adrenocorticotrophin after purification and pre-incubation of isolated rat adrenal cells

Primary tissue culture of human adult adrenocortical cells. Methodology and electron microscopic observations on ACTH-deprived and ACTH-treated cortical cells

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