THE STEROIDOGENIC RESPONSE OF ADULT RAT ADRENOCORTICAL CELLS IN MONOLAYER CULTURE

1973 ◽  
Vol 56 (3) ◽  
pp. 537-549 ◽  
Author(s):  
M. J. O'HARE ◽  
A. MUNRO NEVILLE
Keyword(s):  
Cyclic Amp ◽  
Time Course ◽  
Monolayer Culture ◽  
Zona Fasciculata ◽  
Adrenocortical Cells ◽  
Similar Time ◽  
Adult Rat ◽  
Monolayer Cultures ◽  
Corticosterone Secretion ◽  
Steady Rate

SUMMARY Quantitative aspects of corticosteroidogenesis were examined in monolayer cultures of cells from the zona fasciculata and zona reticularis of the adult rat adrenal cortex, maintained for up to 4 months. Corticosterone secretion continued at a steady rate in cultures maintained with corticotrophin (ACTH), the output of maximally stimulated cultures being approximately 12 μg/106 adrenocortical cells/day. In the absence of ACTH, a small amount of 20α-hydroxypregn-4-en-3-one, but no detectable corticosterone, was secreted, resulting in a fluorogenic steroid output 1/125 th of that of maximally stimulated cultures. Restimulation with ACTH of cultures maintained for up to 2 months in its absence resulted in maximum levels of corticosterone secretion after 4–5 days of continuous ACTH treatment. The levels of corticosterone secretion attained on restimulation were similar to those observed in cultures maintained with ACTH from the out set. Withdrawal of ACTH resulted in a fall in steroid output which took 10 days to reach final unstimulated levels. Trophic stimulation of corticosteroidogenesis with a similar time-course was obtained with both cyclic AMP and dibutyryl cyclic AMP, the latter being the more effective.

MORPHOLOGICAL RESPONSES TO CORTICOTROPHIN AND CYCLIC AMP BY ADULT RAT ADRENOCORTICAL CELLS IN MONOLAYER CULTURE

1973 ◽  
Vol 56 (3) ◽  
pp. 529-NP ◽  
Author(s):  
M. J. O'HARE ◽  
A. MUNRO NEVILLE
Keyword(s):  
Cyclic Amp ◽  
Monolayer Culture ◽  
Cell Spreading ◽  
Zona Fasciculata ◽  
Adrenocortical Cells ◽  
Confluent Monolayer ◽  
Zona Reticularis ◽  
Adrenal Cells ◽  
Adult Rat ◽  
Monolayer Cultures

SUMMARY Confluent monolayer cultures of cells from the zona fasciculata and zona reticularis of the normal adult rat adrenal cortex were maintained with or without corticotrophin (ACTH) for up to 4 months, without proliferation of adrenal cells. Proliferating fibroblast-like cells, however, eventually overgrew the adrenal monolayer in cultures both with and without ACTH. Adrenocortical cells in culture, maintained without ACTH, spread rapidly to form a confluent monolayer, whereas cell spreading was markedly inhibited in the presence of ACTH. Exposure of previously unstimulated cells to ACTH or cyclic AMP caused the adrenal cells to retract with loss of confluence, the process being reversed when ACTH or cyclic AMP was withdrawn. Ultrastructural features of cells cultured with ACTH were typical of normal adrenocortical cells; in cultures without ACTH they were similar to those of adrenocortical cells found in the hypophysectomized rat.

PRELIMINARY OBSERVATIONS OF BOVINE ADRENAL FASCICULATA-RETICULARIS CELLS IN MONOLAYER CULTURE: STEROIDOGENESIS, EFFECT OF ACTH AND CYCLIC AMP

1976 ◽  
Vol 83 (2) ◽  
pp. 373-385 ◽  
Author(s):  
Cynthia G. Goodyer ◽  
John S. Torday ◽  
Barry T. Smith ◽  
C. J. P. Giroud
Keyword(s):  
Cyclic Amp ◽  
Monolayer Culture ◽  
Progressive Increase ◽  
Zona Fasciculata ◽  
Dibutyryl Cyclic Amp ◽  
Hydroxylase Activity ◽  
Short Term ◽  
Adrenocortical Cells ◽  
Apparent Loss ◽  
Order Of Magnitude

ABSTRACT Bovine adrenocortical cells dispersed by trypsin digestion of fasciculata-reticularis minces were maintained in monolayer culture for up to 6 weeks. During the first week cells grown in medium containing ACTH (1 mU/ml) secreted steroids at a rate 10 to 20-fold greater than control cultures, cortisol accounting for 80–90% of the corticotrophic response. Using tracer amounts of [3H]progesterone and [3H]pregnenolone, the major products were cortisol, corticosterone, 11-deoxycortisol and 11-deoxycorticosterone in decreasing order of magnitude. After 10 to 15 days in culture steroidogenesis was no longer enhanced by ACTH. This was concomitant with an apparent loss of 11β-hydroxylase activity which was mainly manifested by a sharp increase in the formation of 11-deoxycortisol. Short-term incubations of these cells during the first week in culture provided evidence that steroidogenesis was related to ACTH concentrations (from 0.1 to 100 μU/ml) and stimulated by dibutyryl cyclic AMP, the corticotrophic responses being further enhanced by theophylline (0.5 to 50 μmoles/5 ml). Exposure of the cells to ACTH (50 μU/ml) resulted in a rapid increase in intracellular cyclic AMP concentrations concomitant with a progressive increase in the corticosteroids released into the medium. The data are consistent with the conclusion that during the first week in culture these cells provide a valuable model for the study of factors regulating steroidogenesis in the zona fasciculata-reticularis.

STEROID METABOLISM BY ADULT RAT ADRENOCORTICAL CELLS IN MONOLAYER CULTURE

1973 ◽  
Vol 58 (3) ◽  
pp. 447-462 ◽  
Author(s):  
M. J. O'HARE ◽  
A. MUNRO NEVILLE
Keyword(s):  
Cyclic Amp ◽  
Culture Medium ◽  
Monolayer Culture ◽  
Cultured Cells ◽  
Steroid Metabolism ◽  
Zona Fasciculata ◽  
Adrenal Cells ◽  
Isomerase Activity ◽  
Adult Rat ◽  
Prolonged Absence

SUMMARY The patterns of exogenous steroid metabolism by rat adrenocortical zona fasciculata and zona reticularis cells cultured together as a monolayer have been examined. In the continued presence of corticotrophin (ACTH), the cultured cells synthesized corticosterone, 18-hydroxydeoxycorticosterone and deoxycorticosterone from [3H]pregnenolone. In the prolonged absence of ACTH, however, [3H]pregnenolone was metabolized mainly to progesterone, 20α-dihydroprogesterone, 20α-hydroxy-5α-pregnan-3-one and 5α-pregnane-3β,20α-diol with small amounts of 5α-pregnane-3,20-dione, 11β-hydroxyprogesterone and 11β-hydroxy-20α-dihydroprogesterone, while virtually no corticosterone, 18-hydroxydeoxycorticosterone or deoxycorticosterone were produced. While 5-ene-3β-hydroxysteroid dehydrogenase-isomerase activity persisted at a substantial level in unstimulated cells and the 11β- and 18-hydroxylases were reduced but still present, 21-hydroxylase activity was almost completely lost when ACTH was omitted from the culture medium. These changes in enzyme activities were completely reversed by the addition of ACTH, cyclic AMP or dibutyryl cyclic AMP to unstimulated cultures for 4–5 days, during which time no proliferation of adrenal cells was observed.

scholarly journals Calcium metabolism in rat hepatocytes

1978 ◽  
Vol 170 (3) ◽  
pp. 615-625 ◽  
Author(s):  
S Foden ◽  
P J Randle
Keyword(s):  
Cyclic Amp ◽  
Time Course ◽  
Rat Hepatocytes ◽  
Ionophore A23187 ◽  
Total Calcium ◽  
Free Medium ◽  
Adrenergic Agonists ◽  
Calcium Efflux ◽  
Similar Time ◽  
Carbonyl Cyanide

1. The total calcium concentration in rat hepatocytes was 7.9 microgram-atoms/g dry wt.; 77% of this was mitochondrial. Approx. 20% of cell calcium exchanged with 45Ca within 2 min. Thereafter incorporation proceeded at a low rate to reach 28% of total calcium after 60 min. Incorporation into mitochondria showed a similar time course and accounted for 20% of mitochondrial total calcium after 60 min. 2. The alpha-adrenergic agonists phenylephrine and adrenaline + propranolol stimulated incorporation of 45Ca into hepatocytes. Phenylephrine was shown to increase total calcium in hepatocytes. Phenylephrine inhibited efflux fo 45Ca from hepatocytes perifused with calcium-free medium. 3. Glucagon, dibutryl cyclic AMP and beta-adrenergic agonists adrenaline and 3-isobutyl-1-methyl-xanthine stimulated calcium efflux from hepatocytes perifused with calcium-free medium. The effect of glucagon was blocked by insulin. Insulin itself had no effect on calcium efflux and it did not affect the response to dibutyryl cyclic AMP. 4. Incorporation of 45Ca into mitochondria in hepatocytes was stimulated by phenylephrine and inhibited by glucagon and by carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The effect of glucagon was blocked by insulin. 5. Ionophore A23187 stimulated hepatocyte uptake of 45Ca, uptake of 45Ca into mitochondria in hepatocytes and efflux of 45Ca into a calcium-free medium.

scholarly journals Ultrastructural changes induced by ACTH in normal adrenocortical cells in culture.

1977 ◽  
Vol 72 (3) ◽  
pp. 757-763 ◽  
Author(s):  
A T Suyama ◽  
J A Long ◽  
J Ramachandran
Keyword(s):  
Cyclic Amp ◽  
Calcium Ions ◽  
Monolayer Culture ◽  
Ultrastructural Changes ◽  
Dibutyryl Cyclic Amp ◽  
Adrenocortical Cells ◽  
Adult Rats ◽  
Adrenal Cells ◽  
Camp Synthesis ◽  
High Concentrations

The effects of ACTH, its o-nitrophenyl sulfenyl derivative (NPS-ACTH) and dibutyryl cyclic AMP (dbc AMP) on the ultrastructural morphology of adrenocortical cells of adult rats in monolayer culture have been investigated. NPS-ACTH, which has previously been shown to stimulate steroidogenesis but not cAMP synthesis in adrenal cells, induced the same characteristic transformation of mitochondrial architecture as produced by ACTH or high concentrations of dbcAMP. All three agents caused the disappearance of electron-opaque granules present in the mitochondria of unstimulated cells. It was found that these granules could be extracted with EGTA (ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetate). These results are discussed in the light of the known importance of calcium ions in the actions of ACTH.

Development of muscarinic potassium current in fetal and neonatal rat heart

1997 ◽  
Vol 272 (3) ◽  
pp. H1188-H1195 ◽  
Author(s):  
M. Takano ◽  
A. Noma
Keyword(s):  
Time Course ◽  
Single Channel ◽  
Cell Voltage ◽  
Neonatal Rat ◽  
Receptor Subtypes ◽  
Similar Time ◽  
Adult Rat ◽  
Functional Development ◽  
K Current ◽  
Underlying Mechanisms

Single atrial myocytes were isolated from fetal, neonatal, and adult rat hearts. The muscarinic K+ current activated by rapid application of acetylcholine (ACh) and adenosine (Ado) was recorded under the whole cell voltage clamp. The current density (pA/pF) of ACh-induced K+ current increased from gestation day 12 to the maximum on neonatal day 20 and decreased in the adult myocytes due to greater increase of the membrane capacitance. The development of Ado-induced K+ current followed a similar time course except for a remarkable decrease after neonatal day 10. No significant change was found in single-channel properties during the development. Receptor subtypes were M2 and A1 receptors for ACh and Ado, respectively. In the dose-response relationship, the half-maximal concentration for ACh-induced current markedly decreased with age, from 1.44 (fetus) to 0.17 microM (adult), whereas that for Ado increased from 0.45 (fetus) to 0.99 microM (adult). These changes of the muscarinic K+ current were discussed in relation to the functional development of cardiac myocytes and underlying mechanisms.

Effect of thyroid hormones on angiotensinogen production in the rat in vivo and in vitro

1987 ◽  
Vol 115 (2) ◽  
pp. 311-315 ◽  
Author(s):  
M. Ruiz ◽  
M. Montiel ◽  
E. Jimenez ◽  
M. Morell
Keyword(s):  
Thyroid Hormones ◽  
Time Course ◽  
Plasma Renin ◽  
Rat Hepatocytes ◽  
In Vitro System ◽  
Adult Rat ◽  
Monolayer Cultures ◽  
Thyroid Dysfunctions

ABSTRACT The influence of thyroid hormones on angiotensinogen production was studied in vitro and in vivo. In the in-vitro system, angiotensinogen production rate (APR) of monolayer cultures of rat hepatocytes in response to tri-iodothyronine (T3) and thyroxine (T4) was assayed. In the in-vivo system, plasma angiotensinogen concentration (PAC) and liver angiotensinogen content (LAC) were measured in hyper- and hypothyroid rats. In both thyroid dysfunctions, a significant decrease of PAC was found compared with that in control animals; however, LAC showed a significant increase in hyperthyroidism and a marked decrease in hypothyroidism. As PAC is dependent upon both angiotensinogen production by the liver and angiotensinogen degradation by renin, the decrease in PAC observed in hyperthyroidism could be due to an increase in plasma renin concentration, which would overcome the increased synthesis of liver angiotensinogen observed in these animals. In fact, addition of various concentrations of T4 or T3 to monolayer cultures of adult rat hepatocytes significantly enhanced APR. This increase was greater and started earlier with T3 (1196·1 ± 143·7 (s.d.) pg/mg protein per 6-h incubation; significant differences at the third hour of incubation) than with T4 (858·3 ± 88·2 pg/mg protein per 6-h incubation; significant differences at the sixth hour of incubation). In addition, a close dose–response relationship was found in the cultures supplemented with T3. The different time-course in the response elicited by T3 and T4 on APR could be a consequence of the necessary transformation of T4 into T3 to acquire biological activity. J. Endocr. (1987) 115, 311–315

Protein kinase C signal transduction pathway in ACTH-induced growth effect of rat adrenocortical cells in primary culture

1994 ◽  
Vol 141 (2) ◽  
pp. 285-293 ◽  
Author(s):  
J Arola ◽  
P Heikkilä ◽  
R Voutilainen ◽  
A I Kahri
Keyword(s):  
Protein Kinase C ◽  
Inhibitory Effect ◽  
Zona Glomerulosa ◽  
Growth Effect ◽  
Zona Fasciculata ◽  
Adrenocortical Cells ◽  
Steroid Secretion ◽  
Kinase C ◽  
Corticosterone Secretion ◽  
Fetal Rat

Abstract ACTH exerts a biphasic effect on the growth of fetal rat adrenocortical cells in primary culture when bromodeoxyuridine (BrdU) incorporation is used as an indicator of proliferation. The immediate inhibitory effect during the first 24 h of ACTH stimulation is not dependent on cyclic AMP (cAMP). Protein kinase C (PKC) inhibitors H-7 and staurosporine blocked this inhibitory effect of ACTH, whereas 12-0-tetradecanoyl phorbol-13-acetate (TPA; a PKC activator) mimicked the ACTH-induced antimitogenic effect. The stimulatory growth effect of ACTH appears after 72 h of treatment. A similar mitogenic effect is also achieved with cAMP derivative 8-bromo cAMP (8-Br cAMP). However, both ACTH- and 8-Br cAMP-induced proliferations could be reduced with H-7. ACTH-induced corticosterone secretion was inhibited 50% with H-7 after 24 h, but 8-Br cAMP-induced secretion was unaffected. However, if the treatments were continued for 72 h, H-7 no longer reduced the steroid secretions. Reduction (50–75%) of cholesterol side-chain cleavage enzyme (P450scc) mRNA expression was also noted with H-7 in ACTH-treated cultures after 6 and 24 h. In contrast, TPA doubled the corticosterone secretion induced by 8-Br cAMP, but did not further increase the ACTH-induced secretion after 24 h. TPA alone, however, was not able to induce steroid secretion or P450scc mRNA expression. The morphological differentiation of fetal rat adrenocortical cells with ACTH or 8-Br cAMP from zona glomerulosa-like cells into zona fasciculata-like cells was not disturbed by H-7 nor was it induced by TPA alone. These results therefore suggest that PKC- and cAMP-dependent signal transductions are involved in the ACTH-induced biphasic growth effect of fetal rat adrenocortical cells. PKC plays a role in the inhibitory growth effect, and both PKC and cAMP are involved in the stimulatory growth phase of ACTH. Both PKC and cAMP are also involved in the steroid secretion of zona glomerulosa-type cells, but differentiation into zona fasciculata-type cells and their steroid production is transduced through cAMP. Journal of Endocrinology (1994) 141, 285–293

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