Fast Feedback Inhibition Research Articles

Glucocorticoid Effects on the Molecular Forms of Adrenocorticotrophic Hormone Secreted by Cultures of Rat Anterior Pituitary Cell Monolayers

The effects of glucocorticoids and corticotrophin-releasing factor (CRF) on the release of various molecular forms of adrenocorticotrophic hormone (ACTH) have been investigated in primary cultures of rat anterior pituitary. The rat cells responded to a 30 min challenge with CRF by secreting increased amounts of ACTH, as assessed both by bioassay, using rat adrenocortical cells, and by radioimmunoassay. Inclusion of a synthetic glucocorticoid, such as dexamethasone (DEX), in the incubation for 5 min prior to, and during the CRF challenge, had no effect on the response as measured by radioimmunoassay. Bioassay, however, indicated profound suppression of the response to CRF. This discrepancy between ACTH immuno- and bioactivity was investigated by fractionating the immunoreactive ACTH species using high-performance liquid chromatography. The lower molecular weight (<15kd) forms (ACTH(1-39) , phosphorylated ACTH(1-39) and glycosylated ACTH(1±39) ) were separated from higher molecular weight (>15kd) forms (i.e. ACTH biosynthetic intermediate and proopiomelanocortin) using C-18 Sep-Pak. The lower molecular weight molecules were further resolved into glycosylated and non-glycosylated ACTH, using an acetonitrile gradient high-performance liquid chromatography with trifluoroacetic acid as an ion-pairer. Neither the proportion of low molecular weight forms of ACTH, nor that of glycosylated ACTH(1-39) secreted in response to CRF, were affected by DEX. Further fractionation of non-glycosylated ACTH, also using acetonitrile gradient high-performance liquid chromatography but with heptafluorobutyric acid as the ion-pairer, yielded peaks corresponding to phosphorylated and non-phosphorylated ACTH(1-39) . DEX significantly increased the proportion of phosphorylated ACTH secreted in response to CRF by 18%. An additional effect of DEX was revealed when Sep-Pak extracts were treated with alkaline phosphatase, prior to analysis. After dephosphorylation, it became clear that the peptides released by CRF-stimulated cells were different if DEX was present in the medium. The peptide released in the presence of DEX (ACTH-S) had a slightly, but consistently, different retention time on high-performance liquid chromatography and very little biological activity. Antibody cross-reactivity studies suggested that ACTH-S was modified in the 1-24 region of the peptide. It is concluded that challenge of anterior pituitary cells in primary culture with CRF, following 5 min previous exposure to DEX, results in a molecular change. The consequence of this is that ACTH immunoreactivity is released, but the molecule has reduced biological activity. This may be part of the mechanism by which fast feedback inhibition of ACTH secretion is effected.

Loss of glucocorticoid fast feedback in depression.

A rate-sensitive fast-feedback inhibition of stress-induced corticotropin secretion by glucocorticoids is well documented in rats. Studies in patients with Cushing's disease or adrenal insufficiency have also supported the existence of fast feedback in humans. However, few studies exist in normal healthy subjects or depressed patients. This study compared fast-feedback inhibition of beta-endorphin/beta-lipotropin secretion by hydrocortisone in 16 control subjects and 16 depressed patients. A fast-feedback effect of hydrocortisone on beta-endorphin/beta-lipotropin secretion during the hour of the hydrocortisone infusion was demonstrated in control subjects. Depressed patients demonstrated no increase in beta-endorphin/beta-lipotropin concentrations during the infusion. These data suggest a decreased sensitivity to glucocorticoid fast feedback in depressed patients and complement existing studies demonstrating decreased sensitivity to proportional feedback by dexamethasone in depressed patients. We believe the data presented herein are the first demonstration that abnormal feedback occurs at the level of the brain rather than pituitary in depressed patients.

Rate-Sensitive Glucocorticoid Feedback Inhibition of Adrenocorticotropin and β-Endorphin/β-Lipotropin Secretion in Rats*

In the present study, we present physiological evidence for rate-sensitive, fast feedback inhibition of secretion of ACTH and beta-endorphin (beta END)-related peptides. We used a 2 min restraint stress to physiologically increase plasma corticosterone, then examined the plasma responses of immunoreactive ACTH and beta END plus beta-lipotropin (beta END/beta LPH) to a subsequent restraint stress. After onset of this stress, plasma corticosterone increased from 2.5-10 min at a rate of 120 nM min-1, then remained at a peak from 10-15 min. A single 2 min restraint stress produced peak plasma levels of ACTH and beta END/beta LPH 2.5 min after onset of the stress, and these plasma concentrations declined after this initial stress at rates of 2.7 and 7.4 pM min-1, respectively. Application of a second restraint stress at the time of the peak corticosterone response produced plasma ACTH and beta END/beta LPH responses similar to those after the first stress. Application of a second stress during the period of significant rate-rise of corticosterone in plasma did not result in decreased incremental responses of plasma ACTH or beta END/beta LPH. However, the rates of decline of plasma ACTH and beta END/beta LPH of 7.6 and 32 pM min-1, respectively, from peak levels, were significantly greater after this second stress applied during the period of significant increase in plasma corticosterone concentration than the corresponding rates of decline observed after the initial stress or after a subsequent stress applied at the peak of plasma corticosterone. These differences in rates of decline of plasma ACTH or beta END/beta LPH appear to reflect differences in secretion rate rather than clearance, since disappearance of [125I]ACTH1-24 was not different after an initial vs. subsequent stress. In contrast to these data from intact rats, initial and subsequent stresses did not show different rates of decline of plasma ACTH or beta END/beta LPH in adrenalectomized rats. In conclusion, the stress-induced rate rise of glucocorticoid provides a negative feedback signal which serves to terminate and limit the duration, but not the peak, of the responses of POMC-derived peptides to subsequent stress.

Temporal Changes in Plasma Adrenocorticotropin Concentration after Repeated Neurotropic Stress in Male and Female Rats

To test the ability of the pituitary gland to secrete ACTH in response to repeated stress, 90-day-old male and female rats were subjected first to a 3-min psychological stress and then to a second identical stress 5, 10, or 15 min after the onset of the first stress. After decapitation 5, 10, 15, 20, 25, or 35 min after the onset of the second stress, plasma ACTH concentration was determined by bioassay, and plasma corticosterone was measured by a fluorometric method. Although the rate of increase in plasma corticosterone levels after the first stress was about 1.4 μg/100 ml-min in males and 2.1 μg/100 ml-min in females, 5-15 min after the onset of the first stress, no rapid inhibition of the second stress response appeared. Gradual hypersecretion of ACTH occurred in males after exposure to the second stress, and the resultant peak increment in plasma ACTH concentration was 2–3 times greater than the one observed in once-stressed rats. In females, a rapid and significant increase in plasma ACTH concentration occurred, but the ratio was only 0.6:0.9 compared to the control group. These results suggest that the fast feedback inhibition mechanism by endogenous corticosterone is overcome by a more powerful facilitatory effect of the first stress on subsequent ACTH release and that these two antagonistic systems which regulate ACTH release in response to subsequent stress may be sex-related in their onset and magnitude.

The effect of intracerebroventricular administration of methionine-enkephalin on the stress-induced secretion of corticosterone in mice.

Ether stress or intracerebroventricular (i.c.v.) administration of saline, met-enkephalin, or naltrexone raised plasma corticosterone levels in mice. The response to ether stress was abolished by a preceding ether stress or by pretreatment with i.c.v. saline or naltrexone. However, following i.c.v. met-enkephalin, plasma corticosterone was significantly elevated by ether stress, the effect being blocked by simultaneous injection of met-enkephalin and naltrexone. Met-enkephalin appears to prevent fast-feedback inhibition of the hypothalamus-pituitary-adrenal system.