Hormonal reactions to fighting in rat colonies: prolactin rises during defence, not during offence.

Male Wistar rats living in colonies of 4 males plus 4 females were compared to noncolony males, cohabitating with a female. Irreversible dominance relationships developed between one dominant male (D) and three subordinates (S). Dominants developed high basal testosterone levels and large preputial glands. Subordinates had reduced preputial glands despite normal testicle weights and normal basal testosterone levels. Basal corticosterone was elevated in both ranks, in S more so than in D, while in acute encounters both ranks showed a similar increase in the corticosterone-to-ACTH ratio. They also underwent a similar reduction in thymus weight, while an increase in adrenal weight was more pronounced in D. In D-S-I encounters, during which D simultaneously attacked S and an intruder (I) for 20 minutes, both defenders showed a 3-4 fold increase in plasma prolactin, while in the offensive dominant the level remained low. Similar, but weaker hormonal contrasts between offence and defence were found for beta-endorphin, ACTH, and corticosterone, while alpha-MSH and testosterone did not discriminate. In our view, the marked hyporesponsiveness of prolactin to acute offence may be associated with a specific offensive setting of dopaminergic inhibitory and beta-adrenergic stimulatory influences.

Sympathoadrenal activity facilitates beta-endorphin and alpha-MSH secretion but does not potentiate ACTH secretion during immobilization stress.

The potential involvement of the sympathoadrenal system in stress-induced secretion of peptides from the intermediate lobe of the pituitary gland and the activation of the pituitary-adrenal axis was studied. Male Wistar rats were subjected to control procedures, to sympathectomy by chronic administration (8 weeks) of guanethidine and/or to medullectomy by adrenal enucleation 9 weeks prior to exposure to forced immobilization stress for various periods of time. In intact or sham-operated rats, immobilization caused a prompt increase of circulating norepinephrine, epinephrine (EPI), corticosterone and of immunoreactive adrenocorticotropic hormone (ACTHi), alpha-melanocyte-stimulating hormone (alpha-MSHi) and beta-endorphin (beta-ENDi). Peak levels of pituitary hormones were found after 10 min of stress exposure, but fell to less than 30% of these levels after 2.5 h of immobilization. Adrenal medullectomy, which abolished the stress-induced release of EPI, reduced the acute increase of plasma alpha-MSHi and beta-ENDi, but did not influence the acute increase of plasma ACTHi during immobilization stress. Also in medullectomized plus sympathectomized rats, the initial stress response of circulating ACTHi was not different from that of controls. Adrenal medullectomy with or without additional sympathectomy caused a marked increase in plasma ACTHi concentrations after prolonged stress exposure. We conclude that: catecholamines originating from the adrenalmedulla facilitate the stress-induced secretion of intermediate lobe peptides (alpha-MSHi, beta-ENDi); catecholamines from the sympathoadrenomedullary system do not contribute to the acute release of ACTH during immobilization stress; the sympathoadrenomedullary system is involved in the secondary reduction of circulating ACTHi levels seen during prolonged stress.

Role of epinephrine and vasopressin in the control of the pituitary-adrenal response to stress.

In addition to corticotropin-releasing factor (CRF) and structurally related peptides, arginine vasopressin (AVP), oxytocin, angiotensin II, vasoactive intestinal polypeptide, peptide histidine isoleucinamide, epinephrine (E), and norepinephrine induce secretion of adrenocorticotropin (ACTH) from corticotropic cells in vitro. The apparent affinity and intrinsic ACTH-releasing activity of these substances are lower than those of CRF. These substances can also act synergistically with CRF. In this paper the role of catecholamines and AVP in the control of ACTH release is discussed. Infusion i.v. of E increases plasma ACTH and corticosterone to levels that are normally found during stress. E-induced stimulation of pituitary-adrenal activity is mediated by beta adrenoceptors and involves release of CRF, because it can be prevented by beta-adrenoceptor blockers and by destruction of CRF neurons (hypothalamic lesions), blockade of CRF release (chlorpromazine, morphine, and Nembutal), or administration of CRF antiserum. Although stress can cause a vast increase in plasma E, circulating E is not essential for the acute stress-induced release of ACTH because blockade of beta (or alpha) adrenoceptors, administration of chlorisondamine, or extirpation of the adrenal medulla and sympathectomy do not prevent the pituitary-adrenal response to stress. In contrast, circulating E plays a major role in the release of intermediate-lobe peptides during emotional stress. Studies of the role of AVP in pituitary-adrenal control by the use of pressor receptor (V1) antagonists are not valuable because of the ineffectiveness of such antagonists in blocking AVP-induced release of ACTH from corticotropic cells in vitro. Treatment of rats with an antiserum to AVP reduces the ACTH response to stress. We conclude that AVP has an important role in stress-induced activation of the pituitary-adrenal system, possibly by potentiating the effects of CRF.

Adrenergic mechanisms involved in the control of pituitary-adrenal activity in the rat: a beta-adrenergic stimulatory mechanism.

Epinephrine or isoproterenol was infused into a lateral tail vein of female Wistar rats under Nembutal anesthesia. After 20 min of diffusion, trunk blood was collected for the determination of plasma corticosterone (B) and ACTH immunoreactivity (ACTHi). Infusion of l-epinephrine resulted in a dose-related increase in plasma ACTHi and B. Maximal levels were similar to those observed during ether stress. The pituitary-adrenal system appeared more sensitive than the cardiovascular system to epinephrine, since the ED50 values of epinephrine for its effects on ACTHi and heart rate were 165 and 840 ng/kg . min, respectively. The effect of epinephrine on pituitary-adrenal activity could be mimicked by the beta-adrenergic agonist l-isoproterenol and could be blocked by the beta-adrenergic antagonist l-propranolol, whereas d-propranolol was ineffective. The response of the pituitary-adrenal system to epinephrine was not caused by effects on peripheral parameters such as the distribution or clearance of ACTH or B but was mediated by an increase in ACTH release. The pituitary-adrenal response to epinephrine and isoproterenol was not related to changes in heart rate, blood pressure, or vasopressin secretion. Infusion of epinephrine at a dose that induced a maximal increase in plasma ACTHi and B (1000 ng/kg . min) resulted in a circulating epinephrine concentration of 11 pmol/ml, which is within the physiological range. From these data we conclude that 1) circulating epinephrine can stimulate pituitary-adrenocortical activity, 2) this action is mediated by a beta-adrenergic receptor mechanism, and 3) such a mechanism may be involved in the response of the pituitary-adrenal axis during certain forms of stress.

Hypothalamic deafferentation in the rat appears to discriminate between the anterior lobe and intermediate lobe response to stress.

Anterolateral deafferentation of the mediobasal hypothalamus prevented the increase of the plasma corticosterone concentration induced by ether, histamine and electric footshock. Hypothalamus deafferentation also prevented the ether stress-induced increase of the plasma levels of ACTH and beta-endorphin immunoreactivity (ACTHi, beta-ENDi). Infusion of isoproterenol evoked an increase of the plasma levels of corticosterone, ACTHi, beta-ENDi and alpha-MSH immunoreactivity (alpha-MSHi) in sham-operated rats. In rats with a deafferented hypothalamus, the responses of plasma corticosterone and ACTHi to isoproterenol were blocked but the responses of plasma beta-ENDi and alpha-MSHi remained intact. We conclude that circulating beta-ENDi after exposure to ether is of anterior lobe origin while circulating beta-ENDi after infusion of isoproterenol is of intermediate lobe origin.

beta-Adrenergic stimulation of the release of ACTH- and LPH-related peptides from the pars intermedia of the rat pituitary gland.

The intermediate lobe of the rat pituitary gland produces a series of peptides related to ACTH and LPH. The spontaneous and isoproterenol-stimulated release of such peptides was studied during in vitro superfusion of rat neurointermediate lobes with Krebs-Ringer medium. Products released into the superfusion medium were quantified by direct measurement or after chromatography on Sephadex G-50. ACTH bioactivity was determined by use of adrenal cortical cell suspension assay. In addition, NH2-terminal ACTH, CO2H-terminal ACTH, alpha-MSH and beta-endorphin radioimmunoassays were used. The results show that 1. neurointermediate lobes of rats secret spontaneously various ACTH- and LPH-related peptides in amounts proportional to the amounts in which these peptides are found in extracts of the neurointermediate lobe; 2. the beta-adrenergic agonist, isoproterenol, stimulated the spontaneous release of various peptides, including alpha-MSH, ACTH, CLIP, glycosylated CLIP, and beta-endorphin-like peptides; 3. isoproterenol induced a dose-dependent (10(-9)-10(-7) M), parallel increase in the release of alpha-MSH and ACTH following similar time courses and showing identical EC50 values (about 10(-8)M). Although the spontaneous release of alpha-MSH and ACTH from rat neurointermediate lobes is not strictly coupled under the conditions used in this study, isoproterenol seems to affect the spontaneous release of these peptides to the same relative extent.

Control of intermediate lobe peptide secretion.

It appears that the intermediate lobe of the rat is able to process alpha-MSH, ACTH and endorphins from the precursor pre-opio-corticotrophin (31K). The release of these hormones is under a neural dopaminergic inhibitory control. A beta-adrenergic receptor mediating a stimulatory control, also appears to play a role in this release. The possible implications are discussed.

Effects of hypothalamic lesions and drugs interfering with dopaminergic transmission on pituitary MSH content of rats.

The physiological significance of the dopaminergic innervation of the pars intermedia was studied in rats. Electrothermic lesions were made in the mediobasal hypothalamus in order to destroy all hypothalamo-hypophysial connections. Pituitary MSH content decreased to 25% of control values 8 h after lesioning. From the 1st day, however, pituitary MSH content gradually increased, reaching control levels after about 1 week. Haloperidol and pimozide (both neuroleptics) were used to block specifically dopamine (DA)-receptors. Single administration of these drugs induced a time and dose dependent reduction in pituitary MSH. A maximal decrease to about 60% was found 4-5 h after i.p. administration of 2.5 mg/kg haloperidol and 1.0 mg/kg pimozide. Apomorphine and 2-Br-alpha-ergocryptine (DA-receptor stimulating drugs), had no effect on pituitary MSH stores of intact rats, but completely prevented the lesion-induced fall in pituitary MSH content. The results show that DA-receptors located within the pituitary itself are involved in the control of MSH release indicating that the effect of hypothalamic lesions on pituitary MSH content is primarily caused by interruption of dopaminergic neurotransmission in the hypophysis. We conclude therefore that the dopaminergic arcuato-hypophysial neurones innervating the pars intermedia tonically inhibit the release of MSH in intact rats.

A superfusion system technique for the study of the sites of action of glucocorticoids in the rat hypothalamus-pituitary-adrenal system in vitro. I. Pituitary cell superfusion.

Rat anterior pituitary cells were placed in a superfusion column and were stimulated by rat hypothalamic extract. The rate of ACTH secretion was monitored by collecting fractions eluting from the column every 2 min. Dispersed rat adrenocortical cells were used to determine the amounts of ACTH in the superfusate. ACTH appeared in the medium 6-12 sec after stimulation of the cells. A linear log dose-response relationship existed between 1/8 to 1 rat hypothalamus extract and the amounts of ACTH released. Repeated pulses of CRF stimulation of the same column of pituitary cells resulted in repeated, identical peaks of ACTH. Constant stimulation caused a plateau in hormone release that lasted as long as the stimulus was present. The stimulated, but not the basal ACTH release, could be inhibited by superfusion with corticosterone in vitro (0.2 mug/ml), or by treating the pituitary donor animal in vivo with corticosterone (5 mg/100g BW), cortisol or dexamethasone. Adrenalectomy of the donor animal caused increases in the basal and the stimulated ACTH release.

A superfusion system technique for the study of the sites of action of glucocorticoids in the rat hypothalamus-pituitary-adrenal system in vitro. II. Hypothalamus-pituitary cell-adrenal cell superfusion.

Basal and stimulated CRF release by hypothalamic blocks was studied by coupling the effluent of superfused hypothalamus tissue to a joint pituitary cell-adrenal cell superfusion system and measuring corticosterone production. Log dose-response curves of the adrenal cells for ACTH and of the pituitary cell-adrenal cell system for CRF were linear over the ranges used. Ca++-independent basal CRF release by the hypothalamus could be blocked in vitro by 0.2 mug/ml dexamethasone in the medium, or in vivo by treating the hypothalamus donor rats with corticosterone, 1 mg/rat ip 30 min before decapitation. These treatments did not impair CRF release caused by Veratridine (5 x 10(-6)M or by electrical stimulation. Adrenalectomy increased only basal but not stimulated CRF release. These results indicate that glucocorticoids have a hypothalamic site of action.

A combined cell column superfusion technique to study hypothalamus-pituitary-adrenal interactions in vitro.

Rat anterior pituitaries and rat adrenals were dispersed into cell suspensions. In a cell column superfusion system the pituitary cells were used to study ACTH release and the adrenal cells served as a means to detect that hormone. Linear log-dose-response relationships were exhibited by the adrenal cells for ACTH and by the pituitary cell-adrenal cell system for corticotropin-releasing factor (CRF). A combined system, constructed of a superfused hypothalamus, leading into a column of pituitary cells which was connected to a column of adrenal cells, was used to study the characteristics of CRF release from the hypothalamic block. Basal and stimulated CRF release were both Ca(2+)-dependent.

Re-introduction and evaluation of an accurate, high capacity bioassay for melanocyte-stimulating hormone using the skin of Anolis carolinensis in vitro.

A simple and rapid assay for the quantitative determination of melanocyte-stimulating hormone (MSH) activity, using skin fragments of the lizard Anolis carolinensis in vitro, is described in detail. This assay, in which a quantal response (e.g. induction of a brownish-green colour) can be detected by visual observation, allows determination of MSH activity in up to 50 samples/day by one person, using the skin of one lizard. The mean dose found to induce this colour change was 0-15 ng synthetic alpha-MSH/ml (range 0-02-0-5 ng/ml). The assays shows high accuracy, reproducibility and specificity. Anterior and posterior lobe hormones as well as pituitary catecholamines do not interfere with the determination of pituitary MSH activitymthe method is compared with the assay using hypophysectomized frogs (Rana pipiens) in vivo. Determinations of MSH in pituitary extracts by both methods gave quantitatively similar results when determined with alpha-MSH as the reference substance. However, when beta-seryl MSH was used as a reference, the two assays gave different results for the MSH activity of the pituitary extractsmthis indicates that MSH from the rat pituitary gland has a biological activity similar to that of alpha-MSH rather than to that of beta-seryl MSH.