Asymmetry of salivary cortisol and α-amylase responses to psychosocial stress in anorexia nervosa but not in bulimia nervosa.

BACKGROUND: The stress response involves the activation of the hypothalamic-pituitary-adrenal (HPA) axis and the sympathetic nervous system (SNS). As a role for stress in determining of the onset and the natural course of eating disorders (EDs) has been proposed, the study of the psychobiology of the stress response in patients with anorexia nervosa (AN) and bulimia nervosa (BN) should be helpful in understanding the pathophysiology of these disorders. The two neurobiological components of the stress response can be easily explored in humans by the measurement of salivary cortisol and α-amylase response to a stressor. Therefore, we assessed salivary cortisol and α-amylase responses to the Trier Social Stress Test (TSST) in symptomatic patients with AN and BN compared to healthy controls. METHOD: Seven AN women, eight BN women and eight age-matched healthy females underwent the TSST between 1530 and 1700 h. Salivary cortisol and α-amylase levels were measured by an enzyme-linked immunosorbent assay (ELISA). RESULTS: Compared to healthy women, AN patients showed a normal cortisol response to the TSST, although this occurred at significantly increased hormone levels, and an almost complete absence of response of α-amylase. BN women, however, exhibited enhanced pre-stress levels of salivary α-amylase but a normal response of the enzyme and cortisol to the TSST. CONCLUSIONS: These findings demonstrate, for the first time, the occurrence of an asymmetry between the HPA axis and SNS components of the stress response in the acute phase of AN but not in BN. The pathophysiological significance of this asymmetry remains to be determined.

Connexin 43 expression in the testis of the frog Rana esculenta.

Testicular cell-to-cell interactions play a key role in the regulation of spermatogenesis. In the testis, cell contacts are mediated through several mechanisms, including paracrine and direct contacts depending on gap junctional pathways. Gap junctions require connexin (Cx) channels and connexin-43 (Cx43) represent the most abundant Cx found in mammalian testis. Little is known about Cx expression in non-mammalian testis. Here we report the partial cloning of a Cx43 transcript of 381 bp from Rana esculenta testis. We also demonstrate that, in the frog testis, Cx43 transcript and protein show a parallel temporal and spatial pattern of expression throughout the reproductive annual cycle, with higher levels from September to January (when spermatogenesis is at a maximum level). In situ hybridization, carried out on testis collected in October, indicated that Leydig cells (LC) and Sertoli cells express Cx43 transcript, while the hybridization signal was less intense in germ cells. To obtain more information on Cx43 expression in the frog testis, we have used ethane-dimethane sulphonate (EDS), a toxin that specifically destroys LC. RT-PCR analysis shows a progressive decrease in Cx43 expression in EDS-treated testis from day 1 to day 4 after the injection, associated with LC destruction. Moreover, Cx43 expression returns to normal on day 28, when a new population of LC reappear in the interstitium, indicating that Cx43 is mainly expressed by LC. Taken together our data provide evidence that Cx43 is present in the frog testis with an important role in spermatogenesis.

Inhibition of the basal and oestradiol-stimulated mitotic activity of primary spermatogonia by melatonin in the testis of the frog, Rana esculenta, in vivo and in vitro.

Melatonin has a direct inhibitory effect on the basal and oestradiol-stimulated mitotic activity of primary spermatogonia in the testis of the frog, Rana esculenta. In this study oestradiol was used to induce spermatogonial proliferation to verify the anti-proliferative effect of melatonin. The colchicine metaphase arrest technique was used. The results obtained from in vivo experiments confirm that oestradiol increases the mitotic index of primary spermatogonia and, for the first time, indicate that melatonin has an inhibitory role on the proliferation of primary spermatogonia in the frog testis. Similar results were obtained from testes of melatonin-injected frogs that were exposed to oestradiol in vitro; in fact spermatogonia were unresponsive to hormonal stimulation. In addition, in short-term cultured testes, melatonin (at physiological concentration) interferes with the effects of oestradiol on spermatogonial proliferation, supporting the hypothesis that melatonin exerts the inhibitory effect directly via its local action on the frog gonads. Morphological observation after in vivo or in vitro melatonin treatments indicates that Leydig cells display degenerative features, whereas in adjacent germinal tubules, Sertoli cells show heterochromatic nuclei. These results indicate that melatonin may act on Leydig cells and confirm that there is a paracrine interaction between interstitial and germinal compartments. The results of the present study indicate, for the first time, that melatonin may be directly involved in the inhibitory control of spermatogonial proliferation in the testis of the frog, R. esculenta.

Aggressive behavioral characteristics and endogenous hormones in women with Bulimia nervosa.

Increased aggressiveness frequently occurs in patients with bulimia nervosa (BN), but its neurobiological correlates have been poorly investigated. In this study, we investigated possible relationships between such clinical measure and blood levels of endogenous hormones in patients with BN. Morning plasma levels of testosterone, 17beta-estradiol, prolactin (PRL) and cortisol were measured in 33 bulimic women and 22 healthy female controls. The eating-related psychopathology, depression and aggressiveness were rated by specific psychometric scales. Bulimic patients showed decreased plasma levels of PRL and 17beta-estradiol, and increased concentrations of cortisol and testosterone. Moreover, patients scored higher than healthy controls on rating scales assessing eating-related psychopathology, depressive symptoms and aggressiveness. A significant positive correlation was found between testosterone plasma levels and aggressiveness in patients but not in controls. These findings suggest that in BN, increased plasma levels of testosterone may play a role in the modulation of aggressiveness.

Differences between morning and afternoon hormonal responses to D-fenfluramine in healthy humans.

The prolactin (PRL) and cortisol responses to oral D-fenfluramine (30 mg) and placebo were measured in seven healthy subjects (two women and five men) in the morning and in the afternoon. As compared to placebo, D-fenfluramine significantly increased plasma PRL levels in both the morning and the afternoon, with no significant circadian difference. On the contrary, D-fenfluramine significantly enhanced plasma cortisol levels in the afternoon, but not in the morning. These data suggest that the time of the day at which the D-fenfluramine challenge test is carried out may be an important variable in determining the glucocorticoid response to the 5-HT releasing agent in humans.

Sigma receptor modulation of noradrenergic-stimulated pineal melatonin biosynthesis in rats.

Because sigma receptors are richly concentrated in the rat pineal gland, the present study was performed to investigate their possible role in the modulation of melatonin production. To this purpose, we assessed in vivo the effects of the sigma-receptor ligands 1,3-di(2-tolyl)guanidine and (+)-N-allylnormetazocine on the rat pineal gland activity during either the daytime or the nighttime. Compared with vehicle, 1,3-di(2-tolyl)guanidine and (+)-N-allylnormetazocine potentiated the enhancement of N-acetyltransferase activity and pineal melatonin content induced by isoproterenol administration during the daytime, whereas they did not affect the diurnal basal biosynthetic activity of the gland. Conversely, at night, 1,3-di(2-tolyl)guanidine and (+)-N-allylnormetazocine enhanced significantly the physiological increases in both pineal N-acetyltransferase activity and melatonin levels. This enhancement was prevented by pretreatment with rimcazole, a specific sigma-receptor antagonist. These findings suggest that, in rats, the activation of pineal sigma-receptor sites does not affect the biosynthetic activity of the pineal gland during daytime, whereas it potentiates the production of melatonin when the gland is noradrenergically stimulated either by isoproterenol administration or by the endogenously released norepinephrine at nighttime.

(+)-N-allylnormetazocine enhances N-acetyltransferase activity and melatonin synthesis: preliminary evidence for a functional role of sigma receptors in the rat pineal gland.

In the present study, to evaluate the role that sigma receptors play in the physiology of the pineal gland, we assessed the effects of the sigma receptor ligand (+)-N-allylnormetazocine on the gland activity during either the day or the night. As compared to saline, (+)-N-allylnormetazocine enhanced the physiological increases in both pineal N-acetyltransferase (NAT) activity and melatonin content at night, but it did not affect the biosynthetic activity of the gland during the day. Moreover, (+)-N-allylnormetazocine potentiated the enhancement of NAT activity and pineal melatonin content induced by isoproterenol administration during the day. The nocturnal stimulation of pineal NAT activity and melatonin levels by (+)-N-allylnormetazocine was prevented by pretreatment with rimcazole, a specific sigma receptor antagonist. These results demonstrate that sigma receptor activation by (+)-N-allylnormetazocine is not able, by itself, to stimulate pineal melatonin production, whereas it potentiates the biosynthetic activity of the pineal gland when this is stimulated noradrenergically.

Effects of single and repeated electroconvulsive shock on isoproterenol-stimulated pineal N-acetyltransferase activity and melatonin production in rats.

The response of the pineal gland to acute isoproterenol administration represents a useful tool to investigate beta 1-adrenoceptor function, because the production of melatonin and the activity of its main synthesizing enzyme, N-acetyltransferase (NAT), are regulated by beta 1-adrenergic receptors. In the present study, rats underwent single electroconvulsive shock (ECS) administration (0.80 mA, 0.5 s, at midday), chronic ECS treatment (0.80 mA, 0.5 s, once daily for 8 days), or sham treatments. On the day after the last ECS or sham ECS, animals were injected with isoproterenol hydrochloride (1 mg.kg-1 SC) or volume-matched saline at 1600 h. After single ECS, isoproterenol injection induced a clear-cut increase in both pineal NAT activity and melatonin levels with no significant differences between ECS-treated rats and the sham-treated ones. In rats chronically treated with ECS, the isoproterenol-induced increases in both pineal NAT activity and melatonin content were significantly lower than in sham-treated animals (p < 0.001 for NAT activity; p < 0.005 for melatonin levels; Turkey's test). These data show that the pinealocyte beta-adrenoceptor function is reduced by chronic, but not acute ECS administration, and that this change is not due to the nonspecific stress effect of animal handling or to the acute effects of the last of a series of ECS.

Effects of gonadectomy and temperature on the N-acetyltransferase activity in the harderian gland of the green frog Rana esculenta.

Since, in the Harderian gland (HG) of the hamster, the N-acetyltransferase (NAT), the specific enzyme involved in the biosynthesis of melatonin, exhibits a sexual dimorphism, in the present study, we investigated whether such a dimorphism is present also in the HG of the green frog Rana esculenta. In intact frogs, no significant differences emerged between males and females in the HG NAT activity under both cold (10 degrees C) and warm (22 degrees C) temperature conditions. In female frogs, the HG NAT activity was significantly decreased by both gonadectomy (P < 0.001) and warm temperature (P < 0.001), the two effects being not additive. In male animals, neither gonadectomy nor temperature alone significantly affected the activity of the NAT enzyme in the HG. However, gonadectomized male frogs exposed to warm temperature exhibited a significant drop in the HG NAT activity (P < 0.005). These data show that, in Rana esculenta, although no sexual dimorphism exists in the HG NAT activity, a sex difference is evident in the modulation of the enzyme activity by gonads and temperature, the female frogs being more sensitive to the impairing effects of both gonadectomy and higher temperature.

The androgen receptor mRNA is up-regulated by testosterone in both the Harderian gland and thumb pad of the frog, Rana esculenta.

Alpha 32P-labelled cDNA probe from plasmid containing rat androgen receptor (rAR) has been tested in hybridization experiments using RNAs from the Harderian gland and thumb pad of the edible frog, Rana esculenta. Northern blot analysis has shown a high degree of homology between the rAR cDNA and the frog androgen receptor mRNA (fAR mRNA); this has been supported by both the hybridization conditions (high stringency) and the molecular size of fAR mRNA which is quite similar to those described in mammals (9.4 kb). The role of androgens has been further investigated with respect to the kinetics of expression of fAR mRNA in in vivo experiments. In both the Harderian gland and thumb pad, testosterone has increased the levels of fAR mRNA as compared with the untreated groups. The use of cyproterone acetate (CPA) in combination with testosterone has resulted in a loss of the increase in fAR mRNA as compared to testosterone-treated groups, while CPA alone has resembled the control group. In primary cultures of frog Harderian gland and thumb pad cells, the steady-state levels of fAR mRNA have been increased in the cells exposed to testosterone as compared to those not exposed. These findings confirm that, in these androgen target tissues, testosterone exerts an up-regulation on its own receptors, increasing the accumulation of fAR mRNA in the same way as oestrogens up-regulate the expression of their own receptors in Xenopus liver and oviduct cells.

Seasonal variations in the daily rhythm of melatonin and NAT activity in the Harderian gland, retina, pineal gland, and serum of the green frog, Rana esculenta.

Day-night variations of melatonin content and N-acetyltransferase (NAT) activity were studied in the Harderian gland (HG), retina, pineal gland, and serum of the green frog Rana esculenta. Throughout the year the retinal melatonin content was correlated with retinal NAT activity and was always higher than those in the pineal gland and HG. On the other hand, in these structures diurnal fluctuations in NAT activity were observed. There were clear seasonal differences in the magnitude of the nocturnal increase of retinal melatonin levels as well as in the nocturnal pattern of retinal NAT activity. In summer day-night variations of melatonin and NAT are absent. The prevailing photoperiod seems to affect melatonin and NAT circadian rhythms in R. esculenta.

Role of seizure activity in the decreased pineal response to isoproterenol in rats chronically treated with electroconvulsive shock.

Chronic electroconvulsive shock (ECS) has been previously reported to blunt the melatonin response to acute isoproterenol administration in rats. To assess whether electrically induced seizures are indeed required for the appearance of the blunted pineal response to isoproterenol, pineal and serum melatonin levels were measured after isoproterenol stimulation in rats treated with ECS (80 mA, 0.5 sec), subconvulsive shock (15 mA, 0.5 sec), or sham-ECS once per day at 11:30-12:00 h for 8 days. In ECS-treated rats, both pineal and serum melatonin levels after isoproterenol administration were significantly lower than those in sham-treated animals and in rats receiving subconvulsive shock. Moreover, as compared with sham treatment, chronic subconvulsive shock did not affect the melatonin response to isoproterenol. These data show that seizure activity is indeed required for the ECS-induced decrease in the pineal response to acute beta-adrenergic stimulation.

A comparative study of melatonin production in the retina, pineal gland and harderian gland of Bufo viridis and Rana esculenta.

1. The circadian patterns of melatonin and of its synthesizing enzyme N-acetyltransferase (NAT) were investigated in the serum, retina, pineal gland and Harderian gland (HG) of two amphibian species, Bufo viridis and Rana esculenta. 2. Serum melatonin levels showed no diurnal fluctuations in Bufo viridis, whereas, in Rana esculenta, they exhibited a circadian rhythm, with the highest values occurring during the night. Retina melatonin exhibited characteristic circadian patterns in both species, with the highest values occurring during the day, in Bufo, and the highest concentrations occurring at night in Rana. 3. In the retina, NAT activity peaked at night in both amphibians, but in Bufo the levels were up to 30 times higher than in Rana. In the HG and in the pineal gland, NAT activity showed different patterns in the two species with no diurnal variations in Bufo, and characteristic circadian rhythms in Rana. 4. In the HG and pineal gland of both species, melatonin was only occasionally detectable over the 24-hr period. 5. This is the first report exploring melatonin production in Bufo viridis and Rana esculenta. In our experimental conditions, marked differences emerged between the two species.

The effects of gonadectomy and testosterone treatment on the Harderian gland of the green frog, Rana esculenta.

The effects of gonadectomy and testosterone treatment on the fine structure of the Harderian gland in male and female green frogs were investigated in different periods of the year. Gonadectomy, carried out when the glands are in the lowest secretory phase (September), causes degenerative changes consisting of a reduction of the rough endoplasmic reticulum, the appearance of autolysosomes, and an increase of nuclear heterochromatin. These effects can be prevented by testosterone treatment. No castration effects are found during the recovery (November) and enhancement (April-May) phases of secretory activity. The results suggest that the frog Harderian gland's sensitivity to testosterone changes during the annual cycle. The androgen dependence of the Harderian gland is correlated with the presence of androgen receptors in both male and female frogs.

Pineal response to isoproterenol in rats chronically treated with electroconvulsive shock.

Chronic electroconvulsive shock (ECS) has been shown to induce a downregulation of beta 1-adrenergic receptors in the rat cerebral cortex. Because the secretion of melatonin in the pineal gland is regulated primarily by beta 1-adrenoceptors, in the present study we investigated the effect of chronic administration of ECS on pineal beta-adrenergic responsiveness to isoproterenol. To this purpose, young adult male rats received once daily for 8 days ECS (80 mA, 0.5 s) or sham ECS. On the day after the last ECS or sham treatment, they were injected with isoproterenol hydrochloride (1 mg/kg SC) or volume-matched saline at 1600 h. Two hours later they were killed by decapitation. Results showed that the isoproterenol-induced increase in the pineal melatonin content was blunted in rats treated with ECS as compared to sham-treated animals (shock x drug interaction = p < 0.01). These data indicate that chronic ECS treatment affects beta 1 receptor-mediated melatonin production in the pineal gland. Further studies need to elucidate whether the blunted melatonin response to isoproterenol in ECS-treated rats is due to a downregulation of pinealocyte beta-adrenergic receptors.

Effect of castration and testosterone therapy on harderian gland protein patterns of the golden hamster (Mesocricetus auratus).

1. Sodium dodecyl sulphate 7-12% gradient polyacrylamide gel electrophoresis of male and female hamster Harderian gland whole homogenate shows a clear-cut sexual dimorphism, which consists of the presence of two male-specific glycoproteins (168 and 116 kDa) and two specific female proteins (210 and 190 kDa). 2. In the male, castration causes a significant decrease in the concentration of the two glycoprotein fractions. 3. Replacement therapy with testosterone propionate (T) restores the intact male pattern.

Testosterone induction of poly(A)(+)-RNA synthesis and [35S]methionine incorporation into proteins of Rana esculenta Harderian gland.

The role of androgens in the cyclic secretory activity of the Rana esculenta Harderian gland (HG) was studied. Total RNA showed a dramatic increase in October and May when the nuclear androgen receptors peak. During the resumption of the secretory activity a gradual increase of poly(A)(+)-RNA was detected; during the enhancement phase (May) a peak of the poly(A)(+)-RNA fraction was found. In in vitro experiments testosterone increased the incorporation of [3H]uridine into the poly(A)(+)-RNA fraction and also that of [35S]methionine into a newly synthesized protein fraction (100 kDa). The latter effect is prevented by the exposure of the cells to the antiandrogen, cyproterone acetate (CPA). These findings reveal that, besides hamsters, the HG is a target for androgens in the frog.