María P. Alvarez

24‐Hour Changes in ACTH, Corticosterone, Growth Hormone, and Leptin Levels in Young Male Rats Subjected to Calorie Restriction

Calorie restriction of young male rats increases plasma prolactin, decreases luteinizing hormone (LH) and testosterone, and disrupts their 24 h secretory pattern. To study whether this could be the consequence of stress, we examined the 24 h variations of plasma adrenocorticotropic hormone (ACTH) corticosterone, growth hormone (GH), leptin, and adrenal corticosterone. Rats were submitted to a calorie restriction equivalent to a 66% of usual intake for 4 weeks, starting on day 35 of life. Controls were kept in individual cages and allowed to eat a normal calorie regimen. Significantly lower ACTH levels were detected in calorie‐restricted rats. Plasma corticosterone levels during the light phase of the daily cycle were significantly higher in calorie‐restricted rats. Time‐of‐day variation in plasma ACTH and corticosterone levels attained significance in calorie‐restricted rats only, with a maximum toward the end of the resting phase. The daily pattern of adrenal gland corticosterone mirrored that of circulating corticosterone; however, calorie restriction reduced its levels. Plasma ACTH and corticosterone correlated significantly in controls only. Calorie restriction decreased plasma GH and leptin, and it distorted 24 h rhythmicity. In a second study, plasma ACTH and corticosterone levels were measured in group‐caged rats, isolated control rats, and calorie‐restricted rats during the light phase of the daily cycle. Plasma ACTH of calorie‐restricted rats was lower, and plasma corticosterone was higher, compared with isolated or group‐caged controls. The changes in the secretory pattern of hormones hereby reported may be part of the neuroendocrine and metabolic mechanisms evolved to maximize survival during periods of food shortage.

24-hour pattern of circulating prolactin and growth hormone levels and submaxillary lymph node immune responses in growing male rats subjected to social isolation.

To assess the effect of social isolation of growing rats on 24-h rhythmicity of circulating prolactin and growth hormone (GH) levels and submaxillary lymph node immune responses, male Wistar rats were either individually caged or kept in groups (4–5 animals per cage) for 30 d starting on d 35 of life. Plasma prolactin and GH levels, and submaxillary lymph node lymphocyte subset populations, interferon (IFN)-γ release and mitogenic responses to concanavalin A (Con A) and lipopolysaccharide (LPS) were determined at six time intervals during the 24 h span. Social isolation brought about changes in mean values and 24-h pattern of plasma prolactin and GH levels and lymph node immune responses. After isolation, prolactin and GH mean values decreased, and lymph node T, B, non T-non B, CD8+, and CD4+-CD8+ cells augmented, whereas lymph node CD4+/CD8+ ratio, IFN-γ release and mitogenic responses decreased. Social isolation resulted in disruption of 24 h rhythmicity of every immune parameter tested. CD4+/CD8+ ratio, IFN-γ release and Concanavalin A (Con A) and lipopolysaccharide (LPS) responses correlated significantly with plasma prolactin or GH levels while T/B ratio correlated with plasma prolactin levels only. B, non T-non B, and CD4+-CD8+ cells correlated negatively with plasma prolactin. Modifications in mean value and 24-h rhythmicity of plasma prolactin and GH levels are presumably involved in the effect of social isolation on immune responsiveness.

Effect of Interferon-γ Treatment on 24-Hour Variations in Plasma ACTH, Growth Hormone, Prolactin, Luteinizing Hormone and Follicle-Stimulating Hormone of Male Rats

Objective: Interferon-γ (IFN-γ) is a cytokine produced by T helper cells on antigenic challenge that may affect the release of several pituitary hormones. However, in vitro or in vivo studies have yielded disparate results with stimulatory, inhibitory or absent effects of IFN on pituitary hormone release. One of the reasons for these discrepancies could be that hormone changes were commonly assessed at a single time point in the day-night cycle. In this study we measured the circadian pattern of plasma ACTH, growth hormone (GH), prolactin, luteinizing hormone (LH) and follicle-stimulating hormone (FSH) at 6 different time points within a 24-hour cycle in adult male Wistar rats. Methods: Groups of 6–8 rats kept under light from 08:00 to 20:00 h daily received 5 daily injections intraperitoneally of human IFN-γ (105 IU/kg body weight) or saline at 08:30 h. Plasma ACTH, GH, prolactin, LH and FSH levels were measured by a homologous specific double antibody RIA. Results: A factorial ANOVA for main effects indicated a significant 43% increase of circulating prolactin in IFN-γ-treated rats. Time of day changes were significant for the five hormones examined and these diurnal variations became altered by IFN-γ administration, with a phase advance of ACTH peak, a suppression of the rest phase peak of GH, the appearance of a second peak of prolactin at an early phase of daily photoperiod, and the blunting of the 24-hour variations of plasma FSH. Conclusion: The data point out an effect of IFN-γ on the mechanisms responsible for the circadian organization of pituitary hormone release.

Effect of Chronic Ethanol Feeding on 24-Hour Rhythms of Mitogenic Responses and Lymphocyte Subset Populations in Thymus and Spleen of Peripubertal Male Rats

This work analyzes the effect of chronic ethanol feeding on the 24-hour variation of mitogenic responses and lymphocyte subset populations in thymus and spleen. Animals were maintained under a 12:12-hour light/dark photoperiod and they received a liquid diet for 4 weeks, starting on day 35 of life. The ethanol-fed group received a similar diet to controls except that maltose was isocalorically replaced by ethanol. Ethanol replacement provided 36% of the total caloric content of the diet. Rats were killed at 6 time intervals around the clock, beginning at Zeitgeber time (ZT) 1 (ZT 0 = lights on). Under ethanol intake the splenic and thymic weight decreased. In addition, mean values of the thymic, but not of the splenic T cell number decreased, and mean values of the thymic and splenic CD8+ and CD4+CD8+ number increased. Consequently, the thymic T/B ratio and the thymic and splenic CD4+/CD8+ ratio decreased in ethanol-fed rats. At the same time there was a significant increase in the response of the thymic cells to LPS. The ethanol diet modified the 24-hour rhythmicity of thymic and splenic T, B and CD4+CD8+ cells, thymic CD4+ and splenic CD8+ cells, thymic and splenic T/B and CD4+/CD8+ ratios, as well as of mitogenic responses in both tissues. Chronic ethanol administration presumably affects the endogenous clock that modulates the circadian variation of immune responsiveness in growing rats.

Effect of litter separation on 24-hour rhythmicity of plasma prolactin, follicle-stimulating hormone and luteinizing hormone levels in lactating rabbit does

Background This work describes the effect of a 48-h litter separation on 24-h patterns of plasma prolactin, FSH and LH concentration in female lactating rabbits kept under a 16:8 light-dark photoperiod (lights on at 0800 h). Methods Groups of 6–7 female lactating rabbits maintained with their litters or separated from them for 48 h were killed by decapitation on day 11 post-partum, at 6 different time points throughout a 24-h cycle, starting at 0900 h. Plasma levels of prolactin, FSH and LH were measured by specific double antibody radio-immunoassays. Results Plasma level of prolactin in control and separated does changed in a similar way throughout the day, showing two maxima, at 0500–0900 h and at 1700–2100 h, respectively. Litter separation significantly augmented plasma FSH and LH and disrupted their 24-h rhythmicity. Conclusion Since previous studies had shown that litter separation for short periods of time augmented sexual receptivity and fertility of the doe, the changes in FSH and LH reported may influence the massive release of gonadotropin releasing hormone, LH and FSH triggered by mating or artificial insemination in litter-separated mothers.

Glial fibrillary acidic protein-like immunoreactivity in cat satellite cells of sympathetic ganglia

The presence and distribution of glial fibrillary acidic protein (GFAP), an astrocytic marker protein associated with glial filaments in the sympathetic ganglia of adult cats, was investigated immunocytochemically. This study revealed GFAP plus cells throughout the ganglion. Immunopositive cells surround nerve cell bodies and separate them from the blood vessels.

Differential Effects of Light/Dark Recombinant Human Prolactin Administration on the Submaxillary Lymph Nodes and Spleen Activity of Adult Male Mice

Objectives: Day/night variations in cellularity, percentage of CD4+, CD8+ and double-positive (CD4+–CD8+) lymphocytes, lipopolysaccharide (LPS)- and concanavalin A (Con A)-induced lymphocyte proliferation and natural killer (NK) activity, and the effect of timed administration of recombinant human prolactin (h-PRL) on the above-mentioned parameters were investigated in the submaxillary lymph nodes and spleen of adult male mice. Results: In controls, the percentage of CD4+, double-positive lymphocytes, LPS- or Con A-induced blastogenic proliferation and NK activity in the spleen differ during the dark phase as compared to the light phase. When administered during the dark period, h-PRL induced immunosuppresion in the percentage of CD4+, double-positive (CD4+–CD8+) lymphocytes. Con A- and LPS-induced lymphocyte proliferation and NK activity as compared to untreated controls. When h-PRL was administered during the light period, the cellularity increased, and h-PRL was immunosuppressive in Con A- and LPS-induced lymphcoyte proliferation and NK activity as compared to controls. Moreover, in control submaxillary lymph nodes the cellularity, percentage of CD8+, double-positive lymphocytes, blastogenic proliferation in the presence of Con A and LPS and NK activity differ when comparing the dark with the light phase. When administered during the dark period h-PRL induced immunosuppression in the percentage of double-positive (CD4+–CD8+) lymphocytes, Con A- and LPS-induced lymphocyte proliferation as compared to controls. When h-PRL is administered during the light period, no effects were observed. Conclusions: These results indicate the existence of differential day/night variations in the cellular immune response depending upon the lymphoid organ considered. Because of the administration of h-PRL a differential modulation of this circadian variation was also observed.

Effects of overexpression of growth hormone on T cell activity in transgenic mice

Growth hormone plays a key role in the maturation and maintenance of the immune response, however, the effects of chronic high circulating concentrations of the hormone on the immune system is poorly understood. Transgenic mice overex-pressing bovine growth hormone (b-GH) gene, fused to the rat phosphoenolpyruvate carboxykinase promoter (PEPCK), with very high plasma concentration of heterologous b-GH and their littermate normal siblings were used. Spleen cellularity, percentages of total T lymphocytes, CD4+ and CD8+ cells, ratio of T cell subpopulations, mitogen-induced lymphocyte proliferation and natural killer (NK) cell activity were examined in male transgenic mice and normal littermate mice at 2 and 6 months of age. The number of splenic lymphocytes was greater in transgenic mice than in matched normal littermates at both ages. The NK cell activity was lower in transgenic mice than in the matched normal littermates at both ages, with the lowest values found in older mice. The b-GH transgenic mice had lower percentages of T cells at both ages, however, in young transgenic mice, the percentage of CD4+ cells was reduced while percentage of CD8+ cells was increased in comparison to normal controls. Both basal and mitogen-induced proliferation capacity of splenocytes were reduced in PEPCK-b-GH-25 mice as compared to normal littermates of both ages. Proliferative indexes in response to concanavalin A and phytohemagglutinin were markedly decreased in 6 month old PEPCK-b-GH-25 mice as compared to littermate controls or younger mice. These results indicate that overexpression of b-GH in mice is associated with decreased T cell function and that these abnormalities are age-dependent.ResumenLa hormona de crecimiento juega un papel central en la maduración de la respuesta inmunológica, aunque los efectos de su elevación crónica no son bien conocidos en la actualidad. En este estudio se han utilizado ratones transgénicos que sobreexpresan el gen de la hormona de crecimiento bovina (b-GH), unido al promotor de la fosfoenolpiruvato carboxiquinasa de rata (PEPCK), y muestran niveles extremadamente incrementados de la hormona en plasma. Se analiza la celularidad esplénica, los porcentajes de linfocitos T totales, las subpoblaciones CD4+ y CD8+, el índice de subpoblaciones CD4+/CD8+, la proliferación blastogénica linfocitaria inducida por mitógenos, así como la actividad de células asesinas naturales (NK), en ratones transgénicos y sus correspondientes animales normales de las mismas camadas, a los 2 y 6 meses de edad. El número de linfocitos esplénicos está incrementado en los ratones transgénicos respecto de los controles independientemente de la edad considerada. La actividad NK está disminuida en los ratones transgénicos, y este efecto aumenta con la edad al compararla con la observada en los ratones controles. Los ratones transgénicos muestran valores disminuidos en los porcentajes de linfocitos T en las edades estudiadas. Sin embargo, el porcentaje de células CD4+disminuye en ratones transgénicos jóvenes, mientras que el porcentaje de células CD8+ aumenta. Tanto la capacidad proliferativa basal como la estimulada por mitógenos está reducida en los ratones transgénicos respecto de sus hermanos de camada normales en ambas edades. Los índices de proliferación en respuesta a la concanavalina A y fitohemaglutinina están muy reducidos en los ratones transgénicos de 6 meses, en comparación con los de 2 meses de edad y sus controles. Los resultados sugieren que la sobre-expresión del gen de b-GH en ratones se asocia con una función T reducida y que esta alteración es dependiente de la edad.

Superior Cervical Ganglionectomy Effects on Median Eminence and Anterior and Mediobasal Hypothalamic Taurine Content in Male Rats: Effects of Hyperprolactinemia

The neuroendocrine sequelae of acute or chronic superior cervical ganglionectomy in control or pituitary-grafted rats were studied by analyzing both plasma prolactin, growth hormone (GH) and ACTH levels, and taurine (TAU) content in the hypophysiotropic area of the hypothalamus or the median eminence. As expected, after either acute or chronic ganglionectomy, norepinephrine (NE) content decreased in the brain areas studied, although the values remained higher in hyperprolactinemic rats. TAU content was differentially modified by acute vs. chronic surgeries, thus indicating the possible existence of hypothalamic interactions between TAU and NE to regulate pituitary hormone secretion. Indeed, associated differential changes in plasma prolactin, GH and ACTH levels may be due to the observed TAU changes. As expected, pituitary grafting increased plasma prolactin, GH and ACTH levels, so that the presence of a pituitary graft differentially interferes with the effects of either surgery not only on TAU content but also on the plasma levels of the hormone studied. Globally, ongoing studies confirm the differential effects of acute and chronic superior cervical ganglionectomy on plasma prolactin, GH and ACTH levels, and provide new evidence about its effects on TAU content in the hypophysiotropic area of the hypothalamus and the median eminence that may partially explain the changes observed in the pituitary hormones studied.

Superior cervical ganglionectomy differentially modifies median eminence and anterior and mediobasal hypothalamic GABA content in male rats: effects of hyperprolactinemia.

This work was undertaken to analyze the changes in GABA concentrations in the anterior and mediobasal hypothalamus and in the median eminence after acute or chronic superior cervical ganglionectomy (SCGx), and whether high prolactin levels interfere with the effects of SCGx on GABA content. Acute but not chronic SCGx increased GABA content in all the areas studied, as compared to controls. The presence of a pituitary graft abolished the effects of acute SCGx in the median eminence and anterior hypothalamus, as compared to controls, but potentiated its effects in the mediobasal hypothalamus. Chronic SCGx increased GABA content in the mediobasal and anterior hypothalami, as compared to pituitary grafted controls. Acute SCGx decreased plasma prolactin and GH levels, but chronic surgery did not modify these hormone plasma levels. Acute SCGx increased plasma ACTH levels, whereas chronic SCGx did not modify them. Pituitary grafting increased circulating values of prolactin, ACTH and GH, as compared to controls. Acute SCGx did not modify plasma prolactin levels in grafted animals, although it increased plasma GH levels and decreased those of ACTH in this experimental group. Chronic SCGx further increased both plasma prolactin and GH levels, without modifying plasma ACTH levels. These results suggest that SCGx differentially modifies GABA content within the hypothalamus and median eminence. Induction of hyperprolactinemia in the neonatal age interferes with SCGx effects on both GABA content within the hypothalamus and median eminence and the secretory patterns of the pituitary hormones studied.