Effect of morphine on hypothalamic corticotropin-releasing factor (CRF) and pituitary-adrenocortical activity

The effects of intraperitoneal and intra-third ventricular administration of morphine on the hypothalamic corticotropin-releasing factor (CRF) and the pituitary-adrenocortical activity were examined in unanesthetized, freely moving rats. Hypothalamic CRF was measured by rat CRF radioimmunoassay. Intraperitoneal or intra-third ventricular administration of morphine increased blood concentrations of ACTH and corticosterone while intraperitoneal administration tended to increase CRF concentration in the whole hypothalamus including the median eminence and intra-third ventricular administration increased CRF concentration in the hypothalamus excluding the median eminence. However, morphine seemed to inhibit the increase in CRF concentration in the hypothalamus induced by the ether-laparotomy stress. The main site of morphine action on the hypothalamo-pituitary-adrenocortical system seemed to be in the hypothalamic area.     

Dual effects of (D-Ala2,Met5)-enkephalinamide on CRF and ACTH secretion

An intra-third ventricular administration of (D-Ala2,Met5)-enkephalinamide (DALA) did not elevate plasma ACTH and corticosterone levels in unanesthetized freely moving rats, but intra-third ventricular administration of DALA and methionine (Met)-enkephalin potentiated a mild stress (hanging for 10 or 30 sec)-induced plasma ACTH and corticosterone elevations in unanesthetized freely moving rats. DALA and Met-enkephalin seemed to stimulate CRF release from the median eminence to increase plasma ACTH, as the CRF concentration in the median eminence area was reduced after injection in these stressed rats. When hypothalamic tissues were perifused in vitro, DALA (1-100 ng/ml) reduced the release of CRF. These results suggest that the opiates seem to have a dual effect on the CRF-ACTH system depending on which action overrides the other.     

Corticotropin-releasing factor (CRF) and vasopressin concentration in major brain regions after adrenalectomy and their involvement in adrenalectomy-induced ACTH elevation

CRF and vasopressin concentrations in major brain regions after bilateral adrenalectomy and their involvement in adrenalectomy-induced ACTH secretion were investigated. At 5, 14 and 28 days after bilateral adrenalectomy, the plasma ACTH level was greatly elevated, whereas hypothalamic CRF content was reduced at 5 days and was not changed at 14 and 28 days after adrenalectomy. The CRF concentration in the medulla oblongata was reduced at 2-4 weeks after adrenalectomy. On the other hand, the arginine vasopressin (AVP) concentration was significantly elevated 2-4 weeks after adrenalectomy. An intrajugular administration of anti-ovine or anti-rat CRF serum significantly suppressed the elevated plasma ACTH level in adrenalectomized, freely moving rats, whereas anti-AVP serum or antipressor AVP antagonist, dpTyr(Me)AVP did not suppress the ACTH level. These results indicate that CRF played an important role in the adrenalectomy-induced ACTH elevation but that vasopressin was not involved.     

Influence of nitric oxide synthase inhibitors on the vasopressin-induced pituitary-adrenocortical activity and hypothalamic catecholamine levels.

In the present study the role of endogenous nitric oxide (NO) in the vasopressin-induced ACTH and corticosterone secretion was investigated in conscious rats. Vasopressin (AVP 5 microg/kg i.p.) considerably augmented ACTH and corticosterone secretion. L-arginine (120 and 300 mg/kg i.p.) did not significantly alter the AVP-induced secretion of those hormones. Nitric oxide synthase (NOS) blockers N(omega)-nitro-L-arginine (L-NNA) and its methyl ester (L-NAME) given i.p. 15 min before AVP markedly increased the AVP-induced ACTH secretion. L-NNA (2 mg/kg) more potently and significantly increased the AVP-induced ACTH secretion, whereas L-NAME elicited a weaker and not significant effect. Both those NOS antagonists intensified significantly and to a similar extent the AVP-induced corticosterone secretion. L-arginine (120 mg/kg i.p.) reversed the L-NNA-induced rise in the AVP-stimulated ACTH secretion and substantially diminished the accompanying corticosterone secretion. Neither vasopressin alone nor in combination with L-arginine and L-NAME evoked any significant alterations in the hypothalamic noradrenaline and dopamine levels. L-NNA (2 and 10 mg/kg i.p.) elicited a dose dependent and significant decrease in the hypothalamic noradrenaline level. The hypothalamic dopamine level was not significantly altered by any treatment. These results indicate that in conscious rats endogenous NO has an inhibitory influence on the AVP-induced increase in ACTH and corticosterone secretion. L-NNA is significantly more potent than L-NAME in increasing the AVP-induced ACTH secretion. This may be connected with a considerable increase by L-NNA of hypothalamic noradrenergic system activation which stimulates the pituitary-adrenal axis in addition to specific inhibition of NOS.     

Role of adrenergic structures of the central nervous system in altering the function of the hypothalamo-hypophyseo-adrenal system in exogenous hyperthermia]

Subcutaneous injection of chlorpromazine to rats caused a lesser increase in body temperature and a higher content of the corticotropin-releasing factor (CRF) in the hypothalamus and the ACTH in the hypophysis under conditions of exogenous hyperthermia. The ascorbic acid content in the adrenal glands decreased in the same way as in the animals overheated without any chlorpromazine. There was no significant change in the weight of the adrenal gland. A conclusion was drawn on the participation of the central adrenergic structure in the regulation of the synthesis or the secretion of the CRF.     

Thymosin fraction 5 (TF5) stimulates secretion of adrenocorticotropic hormone (ACTH) from cultured rat pituitaries

In vivo administration of a partially purified thymic hormone-containing extract of the thymus gland, TF5, causes an increase in serum glucocorticoids. The lack of a direct effect of TF5 on adrenal corticosterone secretion suggests that it is mediated at the level of the pituitary. Cultured rat pituitary monolayers were used to determine if the effect is mediated by stimulation of ACTH secretion from the pituitary. Two lots of TF5, BPP100 and C114080-01, caused a dose dependent secretion of ACTH from cultured pituitary monolayers. There was a synergistic effect when the cells were treated with both TF5 and corticotropin-releasing factor (CRF). Immunoneutralization studies were done in which the cells were treated with TF5 or CRF and an antibody to CRF. The antibody completely blocked CRF induced ACTH release, but had no effect on TF5 stimulated ACTH release, suggesting that the activity is not due to a CRF-like peptide in TF5. A number of peptides isolated from TF5, and certain other peptides produced by the immune system were evaluated for their ability to stimulate ACTH secretion. These included thymosin (TSN) alpha 1, alpha 11, and beta 4, prothymosin alpha (PT alpha, thymopoeitin 5 (TP5), factuer thymique serique (FTS), interferon alpha (INF alpha), INF gamma, interleukin 1 (IL-1), and interleukin 2 (IL-2). None of these factors had any effect on pituitary ACTH secretion. These results demonstrate that some peptide component of TF5 causes an increase in serum corticosteroids by stimulating pituitary ACTH release.     

Blockade of nitric oxide formation impairs adrenergic-induced ACTH and corticosterone secretion.

It has been suggested that adrenergic agents might modulate the L-arginine-NO pathway. Sympathomimetic agonists enhance the basal release of NO, and noradrenaline increases the synthesis of nitric oxide synthase (NOS) in the medial basal hypothalamus in vitro. In the present study possible involvement of NO in central stimulation of the hypothalamic-pituitary-adrenal (HPA) axis by adrenergic agents was investigated in conscious rats. The nitric oxide synthase blocker N(omega)-nitro-L-arginine methyl ester (L-NAME 2 and 10 microg) was administered intracerebroventricularly (i.c.v.) 15 min before the adrenergic agonist given by the same route; 1 h later the rats were decapitated. Plasma levels of ACTH and corticosterone were measured. L-NAME significantly diminished the ACTH and corticosterone response to phenylephrine (30 microg), an alpha1-adrenergic receptor agonist. These hormone responses to clonidine (10 microg), an alpha2-receptor agonist, were dose-dependently suppressed or totally abolished by L-NAME. A significant rise in the ACTH and corticosterone secretion induced by isoprenaline (10 microg), a beta-adrenergic receptor agonist, was only moderately diminished by pretreatment with L-NAME. These results indicate that NOS is considerably involved in central stimulation of the HPA axis by alpha1- and alpha2-adrenergic receptor agonists, and that NO mediates the stimulatory action of these agonists on ACTH and corticosterone secretion. The stimulation induced by beta-adrenergic receptors is only moderately affected by endogenous NO.     

Corticotropin releasing factor stimulates growth hormone secretion in neonatal rats

Corticotropin releasing factor (CRF) both stimulates ACTH secretion from the pituitary and inhibits secretion of growth hormone (GH) in adult rats through actions in the CNS. The purpose of the present study was to evaluate these pituitary and central actions of CRF in neonatal rats, in which the hypothalamo- pituitary adrenal (HPA) axis is relatively hypo-functional. The results of this study show that central or peripheral administration of CRF evokes a marked dose-related rise in serum corticosterone in 6-day old rats. The same doses of CRF stimulate, rather than inhibit GH secretion. These results suggest that CRF has unique central actions early in ontogeny.     

Inhibition of spontaneous ACTH release and improved response to CRF in sheep premedicated with the enkephalin analogue DAMME

The effect of IV injections of an enkephalin analogue (DAMME) in sheep prior to the administration of CRF has been compared with chlorpromazine-morphine (CPZ-M) injections as well as a chlorpromazine-morphine-nembutal (CPZ-M-N) regime. The results showed that the administration of DAMME 60 minutes prior to CRF injections, gave more consistent and more sustained inhibition of spontaneous plasma ACTH and plasma cortisol secretion than either CPZ-M or CPZ-M-N pre-treatment. Changes in plasma ACTH and plasma cortisol levels were more readily detectable in DAMME-treated sheep as compared with saline controls when 50 micrograms injections of CRF were given into the carotid artery. This suggests that DAMME may inhibit endogenous CRF release by an action at hypothalamic level or above.     

Effect of neuropeptide Y on the hypothalamic-pituitary-adrenal axis in the dog

There is increasing evidence that neuropeptide Y (NPY) affects the release of pituitary hormones, including adrenocorticotropic hormone (ACTH). The present study was designed to clarify the mechanism by which NPY activates the hypothalamic-pituitary-adrenal (HPA) axis in the dog. Mongrel dogs were equipped with a chronic cannula allowing intra-third (i.t.v.) or intra-lateral (i.l.v.) cerebroventricular administration. A 1.19 nmol, i.t.v. dose of NPY produced as great an ACTH and cortisol response as did equimolar ovine corticotropin releasing factor (CRF). This action of NPY was dose-dependent and shared by peptide YY (PYY) and pancreatic polypeptide (PP), other members of the PP family peptide. Intravenously (i.v.) administered NPY (1.19-11.9 nmol) was much less potent than i.v. CRF in stimulating ACTH and cortisol secretion. However, i.v. NPY significantly increased plasma ACTH and cortisol concentrations, raising the possibility that NPY may modulate the activity of corticotrophs. We have next investigated the possible relationship between NPY and CRF on the HPA axis. Pretreatment with a novel CRF antagonist, alpha-helical CRF9-41 (130.9 nmol i.t.v. or 261.8 nmol i.v.), partly but significantly attenuated the ACTH and cortisol responses to i.t.v. NPY (1.19 nmol). Furthermore, adding a subthreshold dose of i.t.v. NPY (0.119 nmol) to i.t.v. CRF (1.19 nmol) or i.v. NPY (2.38 nmol) to i.v. CRF (0.595 nmol) resulted in the potentiation of CRF-induced ACTH secretion. These results indicate that NPY may activate the HPA axis in concert with CRF probably at hypothalamic and/or pituitary levels. The present findings that NPY evokes ACTH secretion and potentiates the effectiveness of CRF as a secretagogue, together with high concentrations of NPY in the hypothalamus and pituitary portal blood, suggest that NPY is involved in the multihormonal control of ACTH release.     

Effect of L-NAME, a specific nitric oxide synthase inhibitor, on corticotropin-releasing hormone-elicited ACTH and corticosterone secretion.

This study was designed to determine the role of endogenous nitric oxide (NO) in the corticotropin-releasing hormone (CRH)-induced ACTH and corticosterone secretion, as well as possible involvement of hypothalamic dopamine and noradrenaline in that secretion in conscious rats. CRH given i.p. stimulated dose-dependently the pituitary-adrenocortical activity measured 1 h later. Dexamethasone (0.2 mg/kg i.p.) injected 1 h before CRH (1 microg/kg i.p.) totally abolished the CRH-elicited ACTH and corticosterone secretion, indicating a predominantly pituitary site of CRH-evoked stimulation. L-arginine (120 mg/kg i.p.) and N(omega)-nitro-L-arginine methyl ester (L-NAME 5-10 mg/kg i.p.) did not markedly affect the basal plasma ACTH and corticosterone levels. L-NAME given 15 min before CRH markedly, but not significantly, augmented the CRH-induced ACTH response, and enhanced more potently and significantly the corticosterone response. Pretreatment with L-arginine, a substrate for NOS, slightly diminished the CRH-induced ACTH response and considerably reduced the corticosterone response. L-arginine also significantly reversed the L-NAME-evoked increase in the CRH-induced ACTH and corticosterone secretion. L-NAME did not markedly alter the CRH-induced hypothalamic dopamine and noradrenaline levels, while L-arginine significantly increased noradrenaline level. However, those alterations were not directly correlated with the observed changes in ACTH and corticosterone secretion. These results indicate that in conscious rats NO plays a marked inhibitory role in the CRH-induced ACTH secretion and inhibits more potently corticosterone secretion. Hypothalamic dopamine and noradrenaline do not seem to be directly involved in the observed alterations in ACTH and corticosterone secretion.     

Cocaine induced secretion of ACTH, beta-endorphin, and corticosterone

The effect of intraperitoneal administration of cocaine on the concentrations of hypothalamic corticotropin releasing factor like-immunoreactivity (CRF-LI), plasma ACTH, beta-endorphin, and corticosterone was investigated. Groups of rats were injected with 20 mg/kg cocaine HCI or 0.9% NaCl and then killed 0, 10, 20, 30 or 60 minutes later. Hypothalamic CRF-LI, plasma ACTH, beta-endorphin, and corticosterone concentrations were determined by radioimmunoassay. A significant increase in plasma ACTH, beta-endorphin, and corticosterone concentrations was observed after cocaine administration. In contrast, cocaine had no significant effect on hypothalamic CRF-LI concentrations. Intravenous administration of 0.5 and 2.0 mg/kg cocaine to rats in which the endogenous release of CRF was blocked by chlorpromazine, morphine, and pentobarbital elicited a significant increase in plasma corticosterone concentrations. These results demonstrate that cocaine induces the release of ACTH, beta-endorphin, and corticosterone and suggest that this response is mediated at the pituitary level.     

Inhibitory effect of ouabain on epinephrine-induced stimulation of adrenal corticosterone secretion in rats.

Chronic administration of ouabain (3 mg/Kg body weight, subcutaneously, once daily for consecutive 15 days) definitely inhibited epinephrine-induced increase of adrenal corticosterone secretion. The inhibition rate increased along with frequency of ouabain administration. Increase in adrenal corticosterone synthesis and secretion by ACTH (20-50 mU/rat) administration was partially suppressed by pretreatment with chronic ouabain administration. A slight but significant increase of adrenal corticosterone secretion caused by epinephrine administration in hypophysectomized rats was also inhibited by pretreatment with ouabain administration. Chronic administration of neither phentolamine (1 mg/rat, intraperitoneally, once daily for consecutive 15 days) nor propranolol (3 mg/Kg body weight, subcutaneously, once daily for consecutive 15 days) caused significant changes in adrenal corticosterone secretion in response to ACTH as well as to epinephrine. Chronic administration of ouabain in rats causes not only elevated secretion of ACTH from anterior pituitary but also functional change in adrenals leading to suppression of corticosterone secretion in response to ACTH or epinephrine administration.     

Neuroendocrine and immunological mechanisms in stress-induced immunomodulation

Here, we report that emotional stressors (restraint, footshock) can affect humoral immune responses as well as the capacity of immune and accessory cells to secrete interleukins. Acute restraint stress (5 min) caused a 4- to 6-fold enhancement of splenic antibody responses to sheep red blood cells. In an attempt to study endocrine mechanisms, we administered antibodies raised in rats to corticotropin releasing factor (CRF). Intravenous administration of these antibodies prior to stress-exposure and immunization prevented the stress-induced increase in the humoral response. In a parallel experiment, we observed that CRF-immunoneutralization prevented the restraint stress-induced increase in plasma ACTH concentrations, but was without effect on plasma prolactin, melanocyte stimulating hormone, adrenaline and noradrenaline responses. These data suggest the presence of an indirect pathway involving ACTH and related peptides by which CRF controls humoral responses to stress. A pathway involving a direct mechanism of CRF at the level of the immune cells will be discussed. In a set of other experiments, we addressed the question of whether interleukin-1 and interleukin-6 plasma levels induced by injection of endotoxin could be modulated by emotional stress. Exposure to prolonged footshock stress (20 min) prior to endotoxin injection resulted in a blunted plasma ACTH and interleukin-1 response, without affecting the endotoxin-induced plasma interleukin-6 respose. These data suggest that at least one level at which emotional stress may influence immune function is by changing the capacity of immune cells to produce and/or secrete immune regulatory interleukins.     

A single corticosterone pretreatment inhibits the hypothalamic-pituitary-adrenal responses to adrenergic and cholinergic stimulation.

The purpose of the present study was to determine whether an increased plasma corticosterone or dexamethasone levels induced by a single corticosterone or dexamethasone injection to conscious rats affects the hypothalamic-pituitary-adrenocortical (HPA) activity induced by adrenergic and cholinergic agonists. Male Wistar rats were pretreated subcutaneously (s.c.) with a single dose of dexamethasone (5 mg/kg) or corticosterone (25 mg/kg) 24 or 48 h before intraperitoneal (i.p.) administration of adrenergic agonists: phenylephrine, an alpha1-adrenergic receptor agonist, clenbuterol, a beta2-adrenergic agonist and noradrenaline acting predominantly on alpha1-adrenoreceptors, and cholinergic agonists: carbachol, a predominant muscarinic receptor agonist and nicotine, a nicotinic receptor agonist. Dexamethasone profoundly decreased the resting ACTH levels in control rats and given 24 h before each of the stimulatory agonist abolished the adrenergic- and cholinergic agonists-induced ACTH and corticosterone responses. Pretreatment with corticosterone of control rats did not substantially alter the resting plasma ACTH and serum corticosterone levels measured 24 and 48 h later. A single pretreatment with corticosterone abolished or powerfully inhibited, perhaps by a feedback mechanism, the ACTH and corticosterone responses induced 24 and 48 h later by all adrenergic and cholinergic agonists used in this study. These results indicate that prolonged administration of corticosterone is not necessary to induce almost complete suppression of the HPA responsiveness to adrenergic or cholinergic stimulation. Chronic treatment with corticosteroids to achieve glucocorticoid receptors desensitization does not seem to be required.     

Different acute effects of the tyrosine hydroxylase inhibitors alpha-methyl-p-tyrosine and 3-iodo-L-tyrosine on hypothalamic noradrenaline activity and adrenocorticotrophin release in the rat

Computerized gas chromatography-mass spectrometry techniques using selected ion monitoring and deuterated internal standards were used to assay simultaneously the medial basal hypothalamic concentrations of dopamine (DA) and noradrenaline (NA) and their major metabolites in individual rats 30 min after the administration of two different inhibitors of tyrosine hydroxylase, alpha-methyl-p-tyrosine (alpha-MT) and 3-iodo-L-tyrosine (MIT). Consistent with inhibition of DA synthesis, administration of both alpha-MT and MIT resulted in marked reductions (P less than 0.005) in the hypothalamic concentrations of DA and its metabolite homovanillic acid as well as in highly significant increases in prolactin secretion. alpha-MT administration, but not MIT, resulted in a highly significant decrease in NA concentration and a highly significant increase in the concentration of the NA metabolite 3,4-dihydroxyphenylethyleneglycol (DHPG). The hypothalamic ratio DHPG/NA was thus markedly increased (P less than 0.005) by alpha-MT indicating increased NA neuronal activity. alpha-MT administration also resulted in increased ACTH secretion (P less than 0.0005), an effect not observed following MIT. It is proposed that the effects on hypothalamic NA activity and ACTH secretion caused by alpha-MT are stress-mediated and unrelated to tyrosine hydroxylase inhibition. MIT is devoid of these effects but exhibits blockade activity, thus indicating it to be a preferable drug for the acute inhibition of tyrosine hydroxylase in neuroendocrine investigations.     

Effect of angiotensin II on ACTH release in vivo: role of corticotropin-releasing factor

The ability of angiotensin II (ANG II) to release ACTH was studied in vivo. ANG II elicited dose-related increases in plasma ACTH levels in freely-moving non-anesthetized rats. The observation that this effect was totally abolished by immunoneutralization of endogenous CRF, as well as the lack of stimulatory activity of ANG II in pharmacologically blocked animals, suggest that the induction of CRF secretion by ANG II represents an essential modulator of the ACTH-releasing potency of this peptide.     

Pressor, tachycardic and behavioral excitatory responses in conscious rats following ICV administration of ACTH and CRF are blocked by naloxone pretreatment

Experiments were conducted to compare the blood pressure and heart rate responses of conscious rats given intracerebroventricular (ICV) injections of adrenocorticotropin (ACTH 1-24) and corticotropin releasing factor (CRF). Under sodium pentobarbital anaesthesia, rats were implanted with a stainless-steel cannula into the lateral cerebral ventricle and had their right femoral artery and vein cannulated. Upon recovery (24-48 hr later) conscious, unrestrained rats were given ICV injections (total volume 5 microliter by gravity flow) of sterile saline, ACTH (1-24) (0.85 and 1.7 nmoles) or CRF (0.55 and 1.1 nmoles) and blood pressure and heart rate were monitored over the next 2 hr (from the abdominal aorta via the femoral arterial catheter). Both ACTH and CRF caused mean arterial pressure (MAP) to increase, which was paralleled with increases in mean heart rate (MHR). Moreover, these elevations in MAP and MHR were temporally associated with excessive grooming (for ACTH) and locomotor activity (for CRF), which occurred before and lasted as long as MAP and MHR were enhanced. Intravenous (IV) pretreatment whereby naloxone was given 10 min before ICV administration of ACTH (1.7 nmoles) or CRF (1.1 nmoles), showed that naloxone blocked the behavioral, pressor and tachycardic effects of both ACTH and CRF. The results demonstrate that the pressor, tachycardic and locomotor effects evoked in conscious rats by ICV administration of ACTH or CRF are antagonized by naloxone and that their hemodynamic changes may, in part, be mediated by prior behavioral activation.     

Starvation-induced changes in rat brain corticotropin-releasing factor (CRF) and pituitary-adrenocortical response

Starvation-induced changes in CRF concentration in major brain regions and abnormalities in the pituitary-adrenal axis were examined in rats using rat CRF radioimmunoassay. The CRF concentrations in the hypothalamus and cerebellum were significantly reduced in the completely starved rats, while those in the midbrain, thalamus and neurointermediate lobe of the pituitary were significantly increased in the semi-starved or completely starved rats. No significant changes in the CRF concentrations were found in the pons, medulla oblongata and cerebral cortex. In the completely starved rats, the serum ACTH level was significantly reduced, whereas the serum corticosterone level was markedly elevated. These observations suggest that starvation may stimulate the CRF-ACTH-corticosterone system and that not only hypothalamic CRF but also extrahypothalamic CRF may be discretely related to feeding behavior or starvation. The reduced serum ACTH level in starved rats may be ascribed to the negative feedback effect of the elevated serum corticosterone.     

Oxytocin potentiates the ACTH-releasing activity of CRF(41) but not vasopressin

The effects of CRF(41), oxytocin (OT), and arginine vasopressin (AVP) on ACTH secretion were studied alone and in combination in an in vitro system of superfused rat hemipituitaries. CRF(41) (10(-9)M) and AVP (10(-8)M) alone produced a significant increase in ACTH secretion while OT (10(-8)M) alone had no effect. However the same concentration of OT markedly potentiated the ACTH response to CRF(41) while having no effect on the ACTH response to AVP. The data support a physiologic role for OT in the regulation of ACTH secretion.