Eduardo Spinedi

Cytokines Stimulate the CRH but Not the Vasopressin Neuronal System: Evidence for a Median Eminence Site of Interleukin-6 Action

Antigen-activated immune cells acutely release cytokines which, besides their effects on the immune system, increase hypothalamopituitary-adrenocortical (HPA) function to counteract the inflammatory process. The present study was designed to test, using in vitro paradigms, whether there exists a hypothalamic and/or a median eminence site of action, whereby different substances derived from the immune system could stimulate the CRH and/or the arginine-vasopressin (AVP) neuronal pathway. For this purpose, whole medial basal hypothalamus (containing the median eminence) were dissected from female rats and incubated in vitro with several concentrations of interleukin-1 (IL-1)beta, interleukin-6 (IL-6), tumor necrosis factor (TNF)-alpha, thymosin fraction 5 (TF5) or bacterial lipopolysaccharide (LPS). After a 40-min incubation period, the amounts of CRH and AVP released into the incubation medium were measured by specific radioimmunoassays (RIAs). Additional experiments were carried out by superfusing isolated rat median eminence fragments with the different test substances; CRH and AVP released into the medium were also measured by RIAs. The results indicated that IL-1 beta (10(-11) to 10(-7) M), IL-6 (0.06 x 10(-10) to 0.4 x 10(-10) M), TNF-alpha (6 x 10(-9) to 6 x 10(-7) M) and TF5 (5-500 micrograms/ml) but not LPS (1-100 ng/ml) significantly enhanced hypothalamic CRH secretion above baseline in a concentration-related fashion. Additionally, superfusion experiments demonstrated that, among all test substances, only IL-6 possesses a direct and dose-dependent CRH-releasing activity at the median eminence level. Conversely, no preparation enhanced basal AVP release in either in vitro design.(ABSTRACT TRUNCATED AT 250 WORDS)

Angiotensin II and ACTH Release:Site of Action and Potency Relative to Corticotropin Releasing Factor and Vasopressin

The present studies were designed to test, using a combination of in vivo and in vitro paradigms, the potency of angiotensin II (AII) to release ACTH, in relation to that of other neural peptides with corticotropin-releasing activity (CRA), such as synthetic corticotropin releasing factor (CRF), arginine vasopressin (AVP) and oxytocin (OXY); and to determine whether a central or peripheral locus is the primary site of action of the peptide. Injection of AII (1 and 5 micrograms, i.p.) in intact unanesthetized male rats, resulted in an increase in plasma ACTH and beta-endorphin-like immunoreactivity (beta-end-LI) levels after the largest dose. The responses, however, were not as pronounced as those elicited by 0.5 microgram of CRF or 1 micrograms of AVP. Injection of AII (i.v., 1 or 5 micrograms) in centrally blocked rats (pretreated with chlorpromazine-morphine-nembutal) failed to elicit any increase in either ACTH or bet a-end-LI levels, whereas 0.5 microgram of either CRF or AVP increased both hormones several fold. In vitro studies using dispersed anterior pituitary cells obtained from male donor rats showed that AII as well as AIII could increase ACTH release at doses of 10(-8) M or larger. Under the same conditions, CRF and AVP stimulated ACTH release to a greater degree at 10(-10) and 10(-9) M, respectively. On the other hand, OXY was stimulatory at 10(-8) M. Dose-response studies indicated a rank order of CRA as follows: CRF greater than AVP greater than OXY greater than AII = AIII. Both AII and AIII showed additive effects when coincubated with either CRF or AVP.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in the Hypothalamo-Corticotrope Axis after Bilateral Adrenalectomy: Evidence for a Median Eminence Site of Glucocorticoid Action

Bilateral adrenalectomy (ADX) leads to increased ACTH synthesis and secretion. It is thought that endogenous glucocorticoids exert a feedback mechanism at both pituitary and brain levels. The present study has been performed in order to determine the effect of ADX on the release of hypothalamic neuropeptides with corticotropin-releasing activity (CRA) and if there exists a median eminence site of glucocorticoid action to regulate hypothalamic-pituitary-adrenal (HPA) function. Adrenalectomized and sham-operated male rats were killed at different periods after surgery (2, 5, 7 and 14 days) and trunk blood was collected for ACTH and corticosterone (B) concentrations measurement. Brain (median eminence, ME; and medial basal hypothalamus, MBH) and pituitary (anterior lobe, AP; and neurointermediate lobe, NIL) tissues were dissected in order to evaluate either peptide content or in vitro hormone release. The results indicate that ADX blunted plasma B levels and increased AP ACTH content and secretion in a time-related fashion up to the 14th day. ADX significantly decreased both CRF and CRA contents in the ME at all periods studied; ME arginine-vasopressin (AVP) increased 7 and 14 days after ADX. MBH CRF decreased after ADX, but returned to sham value 2 weeks later; similarly, MBH AVP decreased at all periods after ADX. Removal of endogenous glucocorticoids did not vary neither oxytocin (OXY) content in the ME and MBH nor AVP and OXY contents in the NIL. In our superfusion experiments, we found that ADX increased basal AVP release and did not change spontaneous CRF secretion from ME terminals. Dexamethasone (Dxm, 10 nM) diminished AVP but not CRF output by ME tissues from adrenalectomized rats. A direct relationship was found between ME CRF and 28 mM KCl (hK+)-induced CRF release by MEs from adrenalectomized rats. ME fragments from adrenalectomized rats were hyperresponsive to kH+ stimulation of AVP release. Dxm (10 nM) decreased the hK(+)-evoked CRF and AVP release by MEs from adrenalectomized rats. ADX and dexamethasone treatment did not influence basal and hK(+)-elicited ME OXY release. Additionally, a rapid glucocorticoid inhibitory effect on ACTH secretion by isolated AP cells from both sham and adrenalectomized rats was found, and an in vitro corticotrope hyporesponse to 0.63 nM CRF and 9.25 nM AVP stimulation during several days after ADX.(ABSTRACT TRUNCATED AT 400 WORDS)

Sex Differences in the Hypothalamo-Pituitary-Adrenal Axis Response to Inflammatory and Neuroendocrine Stressors

Susceptibility to inflammatory disease in infantile Lewis (LEW/N) female rats seems to be related to their impaired hypothalamo-pituitary-adrenal (HPA) axis response to different inflammatory stimuli, while the relative resistance to this type of disease in Fischer (F344/N) female rats is apparently due to their potent HPA axis response to the same stimuli. In the present study, we attempted to elucidate whether there is an impairment in the HPA axis response in the juvenile female LEW/N rat to inflammatory and noninflammatory stimuli, and also to determine whether the endogenous sex-steroid environment influences the HPA axis function in both strains of rats. For these purposes, juvenile F344/N and LEW/N rats of both sexes were submitted to different treatments: (a) inhalation of normal atmosphere or ether vapors for 1 min (Ether); (b) i.p. injection of vehicle alone or containing CRH (0.5 microgram/rat), arginine vasopressin (AVP; 5 micrograms/rat, angiotensin II (AII; 5 micrograms/rat), insulin (INS; 0.3 IU/rat), bacterial lipopolysaccharide (LPS; 100 micrograms/rat) or snake venom (SV; 100 micrograms/rat). Rats were then killed at different time intervals (in min) after treatments: 20 for Ether, AVP and CRH, 30 for AII, 45 for INS, 60 for SV and 120 for LPS.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of Glucocorticoids in the Response of the Hypothalamo-Corticotrope, Immune and Adipose Systems to Repeated Endotoxin Administration

The present study was designed to determine whether the glucocorticoid inhibitory feedback mechanism plays a role in the well-known tolerance of the neuroendocrine-immune axis response to repeated endotoxemia. Adult male rats underwent adrenalectomy (ADX) and were implanted with a subcutaneous corticosterone (compound B, CB, 75 mg) pellet, or sham operated and implanted with a placebo pellet. On the morning of day 8 after surgery (experimental day, D1), all rats received an intravenous injection of lipopolysaccharide (LPS) (25 µg/kg body weight) which was repeated daily until D5. Blood was drawn via intravenous indwelling catheters before (sample time zero) as well as 1, 2, 3 and 4 h after LPS treatment on D1, 3 and 5 for measurements of corticotropin (ACTH), CB, tumor necrosis factor-α (TNF-α) and leptin. In sham animals, tolerance to repeated LPS administration was complete by D5 for the corticotrope axis and the immune response. In addition, LPS was found to stimulate leptin secretion on day 1 in intact rats, an effect that also disappeared thereafter. ADX + CB rats showed only a partial tolerance of the corticotrope axis on D5, whereas tolerance of the immune response was similar to that found in sham animals. Interestingly, the acute stimulation of leptin secretion by LPS in ADX + CB rats was qualitatively similar to that of intact controls on D1, but plasma leptin levels were significantly reduced on D3 and 5 compared to controls. Our results demonstrate that the adrenal response tolerance of the hypothalamo-pituitary-adrenal axis to repeated endotoxemia. In addition, our finding that TNF-α secretion follows the same pattern in sham-operated and in adrenalectomized animals suggests that unlike the corticotrope axis, tolerance of the immune response does not depend upon stimulated CB levels. The decrease in circulating levels of leptin following ADX is consistent with the stimulatory effects of glucocorticoids on leptin secretion. However, our finding of an acute stimulation of leptin secretion by LPS in ADX + CB animals demonstrates that this effect of endotoxemia is at least partially glucocorticoid independent.

Effect of Acute Ether Stress on Monoamine Metabolism in Median Eminence and Discrete Hypothalamic Nuclei of the Rat Brain and on Anterior Pituitary Hormone Secretion

This study was designed to correlate the endocrine responses elicited by acute ether stress with the changes in metabolism of several monoamines in discrete nuclei of the rat brain. Concentrations of norepinephrine (NE), dopamine (DA), and 5-hydroxytryptamine (5-HT) and also of the specific metabolites of NE, DA, and 5-HT, 3-methoxy-4-hydroxyphenylethylene glycol, 3,4-dihydroxyphenylacetic acid, and 5-hydroxyindole-3-acetic acid, respectively, were concurrently measured in microdissected nuclei using high-performance liquid chromatography with electrochemical detection. The ratio of the metabolites to their respective amines was used as an estimate of the metabolism of NE, DA, and 5-HT. Acute exposure to ether vapors induced, within 5-15 min, large increments in plasma levels of adrenocorticotropic hormone (ACTH), beta-endorphin, and prolactin (PRL), and decrements in the levels of plasma growth hormone (GH). Significant increases in NE metabolism were observed in the rostral (ANr) and caudal (ANc) divisions of the arcuate nucleus, as well as in the paraventricular (PVN) and dorsomedial nuclei, 15 min after ether stress. A significant decrease in 5-HT metabolism was observed in the PVN, supraoptic nucleus, and ANc, whereas significant increases in 5-HT metabolism were detected in the suprachiasmatic nucleus and ANr. DA metabolism selectively increased in the ANr. The present results indicate that the acute changes in ACTH, beta-endorphin, PRL, and GH release induced by ether exposure are temporally correlated with increases in NE metabolism in many hypothalamic nuclei; a selective increase in DA metabolism restricted to the ANr, and differential effects on 5-HT metabolism, probably reflecting selective activation or inhibition of different populations of 5-HT neurons.

Sexual Dimorphism in the Mouse Hypothalamic-Pituitary-Adrenal Axis Function after Endotoxin and Insulin Stresses during Development

Bidirectional communication between the immune and the endocrine systems is now widely accepted as essential for the survival of the organism. Since a classical nonresponsive period of the hypothalamic-pituitary-adrenal (HPA) axis takes place shortly after birth and because endogenous sex hormones modulate immune function, the aim of the present work was to determine whether sex steroids regulate the PHA axis response to immune (bacterial, lipopolysaccharide, LPS) and nonimmune (insulin, INS) stressors in mice during development. For this purpose 7-, 15-, 30-, 45- and 60-day-old mice of both sexes were intraperitoneally injected with either vehicle alone (basal) or containing LPS (2 mg/kg body weight) or INS (12 IU/kg body weight). The animals were then killed by decapitation, 2 h or 45 min after LPS or INS, respectively. Plasma samples were assayed to measure corticosterone concentrations. The results indicated that: (a) there was a transient increase in basal plasma corticosterone levels during development, with a peak value at the juvenile age, regardless of sex; (b) a higher basal plasma corticosterone concentration in females than in males characterized the adult age; (c) the infantile age is a period of the HPA axis function nonresponsive to purely neuroendocrine but not to inflammatory stimuli; (d) during the juvenile age, females showed a hyporesponsive HPA axis to neurendocrine and immune stress, whereas male mice were fully unresponsive to both challenges; (e) animals of both sexes showed a maximal HPA axis response to purely neuroendocrine stress at the prepubertal age; this response to the immune stimulus was also maximal in 30-day-old males, while it was found in females after puberty (45-day-old mice); (f) sexual dimorphism in the HPA axis response to a purely neuroendocrine stimulus was found at 30 days of age or later, while this characteristic of the response to endotoxin was not present until puberty. These data clearly suggest that these are gender-dependent characteristics of the ontogeny of the HPA and HP-gonadal axes that are responsible for the sexual dimorphism of HPA axis function in mice.

Direct effect of ghrelin on leptin production by cultured rat white adipocytes.

Objective: Because ghrelin is known to stimulate adipogenesis, we tested whether ghrelin could contribute to the maintenance of homeostasis, directly affecting rat white adipocyte leptin production.

Metabolic, Neuroendocrine and Immune Functions in Basal Conditions and during the Acute-Phase Response to Endotoxic Shock in Undernourished Rats

Chronic malnutrition is one of the most important causes of several metabolic, immune and neuroendocrine dysfunctions. The aim of the present study was to determine the influence of chronic food restriction on basal neuroendocrine, immune and adipocyte functions and during the acute-phase response to endotoxic shock in female rats. The effect of refeeding of undernourished rats on the above-mentioned functions was also investigated. For these purposes, plasma total protein, glucose, triglycerides, ACTH, corticosterone, tumor necrosis factor-α (TNF) and leptin (LEP) levels were determined in basal condition and 2 h after endotoxin (LPS; 180 µg/kg body weight, i.p.) administration in 3 different groups: (1) well-nourished (WN) controls; (2) undernourished (UN) rats as a consequence of chronic food restriction, and (3) UN rats re-fed to restoration of their body weights in the WN rat range. The results indicate that UN rats, in comparison with WN controls, developed an arrest in body weight gain as well as in basal hypoglycemia, hypotriglyceridemia, hypoleptinemia, hypercorticosteronemia and enhanced adrenal glucocorticoid content; however, no changes in basal total protein, ACTH and TNF plasma levels and in anterior pituitary ACTH concentrations were found. When endotoxic shock was induced, the LPS-induced hypoglycemia developed in WN rats was abolished in UN animals, and both ACTH and TNF plasma concentrations after endotoxin, albeit significantly (p < 0.05) higher than the respective basal values, were significantly (p < 0.05) lower in UN than in WN control rats. Despite the high basal plasma corticosterone concentration in UN vs. WN rats, the LPS-induced glucocorticoid release was similar in WN and UN rats. Additionally, LPS treatment did not modify basal plasma LEP levels, regardless of the group. Interestingly, UN rats fed ad libitum for 15 days restored their body weight to WN rat range values, and the various metabolic dysfunctions seen in UN rats in both basal and post-LPS conditions were fully normalized. Our results clearly indicate that chronic undernutrition not only affects, as earlier described, reproductive function but also metabolic, neuroendocrine, immune and adipocyte functions, and that the effects induced by undernutrition can be fully reversed after recovery of normal body weight. The present study strongly supports the involvement of the metabolic status in the effectiveness of the defense mechanisms developed in patients in inflammatory stress conditions.

Role of Several Mediators of Inflammation on the Mouse Hypothalamo-Pituitary-Adrenal Axis Response during Acute Endotoxemia

Cytokines secreted by bacterial endotoxin-activated immune cells are substances known to stimulate the hypothalamo-pituitary-adrenal (HPA) axis function. The present study was designed to better understand the effect of different mediators of inflammation, such as cytokines and histamine, on the acute HPA axis response induced by administration of a single dose of bacterial lipopolysaccharide (LPS) in adult, male, BALB/c mice. Two different experimental designs were set up. In the first design, mice (n = 8–11 per group) were injected i.p. with LPS (90 µg/kg body weight) and killed by decapitation 2 or 6 h after treatment. Additional groups of mice were pretreated i.p. 12 h before LPS treatment with: (a) 3–4 mg IgG/kg body weight of either an anti-tumor necrosis factor-α (TNF)-α, anti-interleukin (IL)-1β- or IL-6 serum; (b) IL-1 receptor antagonist (IL-1ra) (120 µg/kg body weight) immediately before LPS and also 3 h later (when animals were killed 6 h after LPS injection), or (c) 182 µg/kg body weight of clemastine, an antagonist of H1 histaminergic receptors, 2 h before LPS treatment; animals were killed in a similar fashion to that described for treatment with LPS alone. In the second experimental design, mice were pretreated (i.p., 10 mg/kg body weight, 30 min before administration of a similar dose of LPS) with different blockers of histaminergic pathway function such as: (a) mepyramine, another anti-H1, (b) cimetidine, an H2 receptor blocker, and (c) Rα-methylhistamine dihydrochloride, an H3 presynaptic receptor agonist which inhibits histamine synthesis and output. These animals were then killed by decapitation 40 min after endotoxin treatment. After decapitation, trunk blood was collected for further determination of plasma levels of both ACTH and corticosterone (B) by specific assays. The results indicate that plasma levels of both ACTH and B were several-fold increased over baseline, 2 and 6 h after LPS administration. Two hours, the effect of LPS on ACTH output was not modified by pretreatment with anti-IL-1β IgG, anti-IL-6 IgG, anti-TNF-α IgG nor with IL-1ra, although IL-1ra treatment was able to fully block the IL-1β (35 µg/kg body weight)-stimulated HPA axis function, 1 and 2 h after cytokine administration. Six hours after LPS administration, anti-IL-1β and anti-TNF-α IgGs were both able to significantly reduce HPA axis response to the endotoxins, whereas anti-IL6 IgG had no effect. Anti-IL-1β IgG reduced only B secretion, whereas anti-TNF-α IgG decreased both ACTH and B secretion. The blockade of histaminergic pathway functions did not impede the LPS-induced ACTH and B release regardless of the product employed. The present results indicate that TNF-α, and to a lesser extent IL-1β, are the most relevant cytokines involved in HPA axis response to endotoxin administration. Our data also suggest that, in mice, HPA axis activation after infection appeared to be independent of stimualtion of the histaminergic pathway.