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SUMMARY
The effect of ACTH on cortisol concentration in the plasma of the carp has been studied; cortisol was determined using a competitive protein-binding radioassay. Blood was sampled at 12.00 h (the diurnal minimum) by cardiac puncture. The plasma cortisol level of undisturbed carp (blood obtained 1–3 min after netting) was 46 ± 14 (s.e.m.) ng/ml.
One hour after injection of ACTH (1·0 i.u./100 g body wt., i.m.) the level of cortisol increased to 415 ± 62 ng/ml and reached a maximum of 656 ± 62 ng/ml after 2 h. The level of cortisol in saline-injected control carp after 2 h was 72 ± 33 ng/ml. A transient rise of cortisol was noted in both groups 30 min after injection (315 ± 51, 315 ± 62 ng/ml). This was attributed to a surge of endogenous ACTH, presumably due to handling.
Carp head kidneys were superfused in vitro. They released cortisol spontaneously, but the releasing rate declined exponentially and reached a minimum after 135 min. Addition of ACTH to the medium was followed by a prompt increase in cortisol release. The rate of cortisol release was maximal 30–45 min after addition of ACTH and was identical to the rate at the beginning of the superfusion.
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A superfusion system was developed in which secretion of cortisol from the interrenal tissue of Sarotherodon aureus could be stimulated by ACTH. Head kidneys from four fish were cut into fragments and superfused with Eagle's basal medium containing 4 mm-NaHCO3 and bovine serum albumin (50 mg/100 ml). The superfused medium was collected every 15 min before stimulation and every 5 min thereafter. Cortisol was measured in the medium by radioimmunoassay. The rate of cortisol secretion increased considerably after a 5 min pulse of 0·1 mu. porcine ACTH/ml, reaching a peak of 11·64 ± 2·40 (s.e.m.) ng/min (n = 5) after 20 min. In the subsequent 35 min the secretion rate decreased to a level of 0·49 ± 0·21 ng/min which was within the range of the baseline (0·2–0·7 ng/min). The amount of cortisol secreted by the superfused tissue in response to the corticotrophin, calculated from the area under the peak, was dose-dependent at the range of 0·06–273 mu. ACTH.
Cortisol secretion in this system could also be stimulated by a crude pituitary extract from the same fish. Using the dose–response line, the adrenocorticotrophic activity in the pituitary gland of S. aureus was estimated as 0·2 i.u./g or 1·87 mu. per gland, in porcine ACTH equivalents.
It was possible to substitute ACTH with dibutyryl cyclic AMP. The response of the interrenal tissue, i.e. the increase in secretion of cortisol, was dose-dependent at the range of 2–20 mmol/l. The response of the superfused interrenal tissue to ACTH could be extended by the addition of the synthetic phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (0·1 mmol/l), to the medium. These results indicated that the stimulation of cortisol secretion from the interrenal tissue of this fish by ACTH is, most probably, mediated by cyclic AMP as a second messenger.
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Superfused interrenal tissue of Sarotherodon aureus responded to ACTH with increased release of cortisol. The response to ACTH of interrenal tissue taken from fish treated with the organochlorine insecticide 1,1-dichloro-2-(o-chlorophenyl)-2-(p-chlorophenyl) ethane (o,p-DDD; 50 mg/kg) in vivo was almost totally abolished. Lower doses were less effective. The response was also suppressed by exposure in vitro of the interrenal tissue to the organochlorine; there seemed to be a dose–response to o,p-DDD over the range of 0·023 to 1 mg/l. Also 1,1-dichloro-2,2-bis (p-chlorophenyl) ethylene (p,p′-DDE) was effective in suppressing the response to ACTH at concentrations of 50–150 mg/l; 1,1,1-trichloro-2,2-bis (p-chlorophenyl) ethane (p,p′-DDT) or the polychlorinated biphenyl, Aroclor 1254, were ineffective at 50 mg/l.
No suppressive effect of o,p-DDD on output of cortisol could be detected in superfusion when N6,O2′-dibutyryl cyclic AMP (20 mmol/l) was substituted for ACTH. This may indicate that the interference of o,p-DDD with the interrenal response to ACTH results from an interruption in the generation of cyclic AMP.