Sympathetic innervation regulates macrophage activity in rats with polycystic ovary.

Polycystic ovarian syndrome (PCOS) is a low-grade inflammatory disease characterized by hyperandrogenism and ovarian hyperinnervation. The aim of this work is to investigate whether in vivo bilateral superior ovarian nerve (SON) section in adult rats with estradiol valerate-induced PCOS (PCO rats) affects macrophage spleen cells (MФ) and modifies the steroidogenic ability of their secretions. Culture media of MФ from PCO rats and PCO rats with SON section (PCO-SON rats) were used to stimulate in vitro intact ovaries. Compared with macrophages PCO, macrophages from PCO-SON rats released less tumor necrosis factor-α and nitric oxide, expressed lower Bax and Nfkb mRNA and showed reduced TUNEL staining. Also, in PCO rats, the SON section decreased kisspeptin and nerve growth factor mRNA expressions, without changes in Trka receptor mRNA levels. Macrophage secretions from PCO-SON rats decreased androstenedione and stimulated progesterone release in PCO ovaries, compared to macrophage secretions from PCO rats. No changes were observed in ovarian estradiol response. These findings emphasize the importance of the SON in spleen MΦ, since its manipulation leads to secondary modifications of immunological and neural mediators, which might influence ovarian steroidogenesis. In PCO ovaries, the reduction of androstenedione and the improvement of progesterone release induced by PCO-SON MΦ secretion, might be beneficial considering the hormonal anomalies characteristic of PCOS. We present functional evidence that modulation of the immune-endocrine function by peripheral sympathetic nervous system might have implications for understanding the pathophysiology of PCOS.

Niveles de hierro en sangre según adherencia a la dieta libre de gluten en niños celiacos de edad escolar / Blood iron levels in accordance with adherence to a gluten-free diet in celiac school aged children

Introducción: la enfermedad celiaca (EC) provoca atrofia intestinal, trastornos en la absorción de nutrientes y desnutrición progresiva. La deficiencia de hierro es una carencia nutricional muy prevalente. Una alimentación estricta libre de gluten (LG) permite calidad de vida. Objetivo: evaluar la situación nutricional del hierro de niños celiacos escolares mediante la determinación de parámetros bioquímicos, su relación con el consumo del mineral y la adherencia a la dieta LG en San Luis. Métodos: estudio observacional, analítico y transversal. Fueron incluidos 44 niños con EC, de seis a diez años de edad, con diagnóstico de celiaquía y registrados en entidades públicas y privadas de San Luis (Argentina) durante 2011-2012. Mediante una encuesta cuali-cuantitativa se determinaron hábitos alimentarios y características sociodemográficas. Se evaluaron niveles de hierro y adherencia a una dieta LG. Se construyeron modelos de regresión lineal generalizados para verificar la asociación de ferritina con el consumo de hierro y adherencia a la dieta. Resultados: la mayoría de las familias tenían nivel socioeconómico bajo y eran numerosas. La alimentación no previno la anemia ferropénica según biodisponibilidad. La mayoría de los niños presentaron un estado inmunológico, anticuerpos antiendomisio y antitransglutaminas normales. El 7% presentó bajos niveles de hierro. La ferritina en condiciones de consumo adecuado de hierro se relacionó con los anticuerpos predictores y la presencia de ambos padres en el hogar. Conclusión: en condiciones de consumo adecuado de hierro, sus niveles en sangre se relacionan con adherencia al tratamiento libre de gluten (AU)

Introduction: Celiac disease (CD) causes intestinal damage, inability to absorb nutrients, and progressive malnutrition. Iron deficiency is one of the predominant nutritional problems. A strict gluten-free diet (GF) allows for an optimal quality of life. Objective: To assess the nutritional situation of iron in school-aged celiac children by determining biochemical parameters, their relation to the consumption of the mineral and adherence to gluten-free diets in San Luis. Methods: Observational, analytical and cross-sectional study. We included 44 children with CD, from 6-10 years of age, with diagnosis of celiac disease and registered in public and private entities of San Luis (Argentina) during 2011-2012. A qualitative-quantitative survey was used to determine dietary habits and sociodemographic characteristics. Iron levels and adherence to a GF diet were evaluated. Generalized linear regression models were constructed to verify the association of ferritin with iron consumption and adherence to diet. Results: Most families had low socioeconomic status and were large families. Current feeding did not prevent iron deficiency anemia. Most children had normal immune system, and normal antiendomysial and antitransglutaminase antibodies; 7% of the children showed low levels of iron. Under adequate iron consumption conditions, ferritin was associated to predictor antibodies and the presence of both parents in the home. Conclusion: Under adequate conditions of iron consumption, the levels of iron in blood were related to adherence to gluten-free diets (AU)

Blood iron levels in accordance with adherence to a gluten-free diet in celiac school aged children.

INTRODUCTION: Celiac disease (CD) causes intestinal damage, inability to absorb nutrients, and progressive malnutrition. Iron deficiency is one of the predominant nutritional problems. A strict gluten-free diet (GF) allows for an optimal quality of life. OBJECTIVE: To assess the nutritional situation of iron in school-aged celiac children by determining biochemical parameters, their relation to the consumption of the mineral and adherence to gluten-free diets in San Luis. METHODS: Observational, analytical and cross-sectional study. We included 44 children with CD, from 6-10 years of age, with diagnosis of celiac disease and registered in public and private entities of San Luis (Argentina) during 2011-2012. A qualitative-quantitative survey was used to determine dietary habits and sociodemographic characteristics. Iron levels and adherence to a GF diet were evaluated. Generalized linear regression models were constructed to verify the association of ferritin with iron consumption and adherence to diet. RESULTS: Most families had low socioeconomic status and were large families. Current feeding did not prevent iron deficiency anemia. Most children had normal immune system, and normal antiendomysial and antitransglutaminase antibodies; 7% of the children showed low levels of iron. Under adequate iron consumption conditions, ferritin was associated to predictor antibodies and the presence of both parents in the home. CONCLUSION: Under adequate conditions of iron consumption, the levels of iron in blood were related to adherence to gluten-free diets.

INTRODUCCIÓN: la enfermedad celiaca (EC) provoca atrofia intestinal, trastornos en la absorción de nutrientes y desnutrición progresiva. La deficiencia de hierro es una carencia nutricional muy prevalente. Una alimentación estricta libre de gluten (LG) permite calidad de vida. OBJETIVO: evaluar la situación nutricional del hierro de niños celiacos escolares mediante la determinación de parámetros bioquímicos, su relación con el consumo del mineral y la adherencia a la dieta LG en San Luis. MÉTODOS: estudio observacional, analítico y transversal. Fueron incluidos 44 niños con EC, de seis a diez años de edad, con diagnóstico de celiaquía y registrados en entidades públicas y privadas de San Luis (Argentina) durante 2011-2012. Mediante una encuesta cuali-cuantitativa se determinaron hábitos alimentarios y características sociodemográficas. Se evaluaron niveles de hierro y adherencia a una dieta LG. Se construyeron modelos de regresión lineal generalizados para verificar la asociación de ferritina con el consumo de hierro y adherencia a la dieta. RESULTADOS: la mayoría de las familias tenían nivel socioeconómico bajo y eran numerosas. La alimentación no previno la anemia ferropénica según biodisponibilidad. La mayoría de los niños presentaron un estado inmunológico, anticuerpos antiendomisio y antitransglutaminas normales. El 7% presentó bajos niveles de hierro. La ferritina en condiciones de consumo adecuado de hierro se relacionó con los anticuerpos predictores y la presencia de ambos padres en el hogar. CONCLUSIÓN: en condiciones de consumo adecuado de hierro, sus niveles en sangre se relacionan con adherencia al tratamiento libre de gluten.

Role of macrophage secretions on rat polycystic ovary: its effect on apoptosis.

Polycystic ovarian syndrome is the most common endocrine disorder among women of reproductive age. Little is known about its etiology, although the evidence suggests an intrinsic ovarian abnormality in which endocrine, metabolic, neural and immune factors would be involved. In this work, the effects of macrophage (MO) secretion on ovarian apoptosis in a polycystic ovary syndrome rat model (PCO rat) induced by estradiol valerate are studied. Spleen MO secretions were used to stimulate ovaries and ovarian interstitial and granulosa cells from both PCO and control rats. Ovarian hormones and prostaglandin E2 (PGE2) were measured by RIA; ovarian mRNA levels of Bax, Bcl2 and NFkB by RT-PCR; and ovarian inducible nitric oxide synthase (iNOS) by western blot. The number of apoptotic cells was evaluated by TUNEL. In the PCO ovary, the MO secretions from PCO rats increased the Bax and NFkB mRNA expressions and increased TUNEL staining in both granulosa and theca cells. In addition, the PCO MO secretions produced a decrease of nitric oxide release, iNOS protein level and PGE2 content in the PCO ovary, and it also induced an increase of androstenedione production by PCO interstitial cells, in comparison with control MO secretions. Considering these results and knowing that testosterone stimulates tumour necrosis factor-α production by PCO MO modifying ovarian response by increasing androstenedione, it is reasonable to suggest that the increase of androgens stimulated in ovarian cells by PCO MO secretions could in turn stimulate the cytokine production from MO, thus maintaining an apoptotic vicious cycle in the PCO ovary.

Macrophage secretions modulate the steroidogenesis of polycystic ovary in rats: effect of testosterone on macrophage pro-inflammatory cytokines.

AIMS: The macrophage secretions' effect on ovarian steroidogenesis is investigated in a polycystic ovary syndrome rat model (PCO rat). The influence of testosterone environment on the expression of macrophage pro-inflammatory cytokines that participate in ovarian steroidogenesis is studied. MAIN METHODS: PCO rats were induced by estradiol valerate. Spleen macrophages were cultured with and without testosterone (10(-6) M) and their secretions were used to stimulate ovaries from PCO and control rats. Ovarian hormones released and ovary mRNA levels of P450 aromatase and 3ß-hydroxysteroid dehydrogenase were measured by radioimmunoassay and RT-PCR, respectively. The tumor necrosis factor alpha (TNFα) and nitric oxide (NO) levels in macrophage culture medium, along with the TNFα, interleukin (IL)-6, IL-10 and androgen receptors (AR) mRNA levels in macrophage cells were determined. KEY FINDINGS: Macrophages from PCO rats released more TNFα and NO, expressed higher TNFα and IL-6, lower AR, and no change in IL-10 mRNA levels than control macrophages. TNFα, IL-6 and AR changes were greater after macrophage testosterone treatment. Macrophage secretions from PCO rats stimulated androstenedione and decreased estradiol release and ovarian mRNA P450 aromatase expression in PCO rats compared to macrophage secretions from control rats. These effects were greater when macrophages from PCO rats were treated with testosterone. Ovarian progesterone response was unchanged. SIGNIFICANCE: The differential steroidogenic ability of macrophage secretions from PCO rats is associated to the in vitro testosterone environment. Testosterone, probably acting on macrophage AR, induces a greater release of TNFα, modifying ovarian response by increasing androstenedione and slightly decreasing estradiol without affecting progesterone.

Norepinephrine modulates the effect of neuropeptides in coeliac ganglion on ovarian hormones release: its relationship with ovarian nitric oxide and nerve growth factor.

OBJECTIVE: Ovarian steroids are modulated by neural influences. In this work we investigate whether norepinephrine (NE) modifies the vasoactive intestinal peptide (VIP) or neuropeptide Y (NPY) actions in coeliac ganglion (CG) on the ovarian hormone release, and evaluate the participation of nitric oxide (NO), measured as nitrite, and of inducible nitric oxide synthetase (iNOS) protein, nerve growth factor (NGF) and its trkA receptor gene expression in the ovarian response. METHODS: The study was performed in the ex vivo CG-superior ovarian nerve (SON)-ovary system of rats on diestrus day 2 (D2). CG and ovary were placed in separate compartments connected by the SON and incubated with Krebs-Ringer buffer. After addition of 50 ng/ml VIP, 50 ng/ml NPY, 10-6 M NE, or a mix of VIP+NE or NPY+NE in ganglion, samples from the ovarian compartment were taken at different times throughout 180 minutes to measure progesterone, androstenedione and nitrite levels. RESULTS: VIP and NPY in ganglion induced an increase of progesterone release that was associated for VIP, but not NPY, with a decrease of ovarian nitrite levels, iNOS protein, and NGF/trkA receptor mRNA expression. By contrast, NE in ganglion decreased progesterone, an effect that was suppressed by addition of propranolol in ganglion, and increased nitrites/iNOS and NGF/trkA receptor expression in ovary. GABA A receptor antagonist bicuculline (20 muM) added in ovarian compartment prevented the inhibitory effect on progesterone caused by NE in CG. Androstenedione was not modified under neuropeptides or NE ganglionic stimulation. CONCLUSIONS: Finally, results from VIP+NE or NPY+NE in ganglion showed that ovarian response on D2 induced by VIP or NPY alone is moderated by the opposite action of NE, and occurs only on progesterone, the most sensitive steroid to neural action.

Role of IRS-4 in PI3-K activation by insulin in HepG2 cells, modulation by Angiotensin II.

Insulin receptor substrate-4 (IRS-4) has a limited tissue expression and its modulation by tyr-phosphorylation is still controversial. We evaluated the participation of IRS-4 in the cross-talk between Angiotensin II (Ang II) and Insulin (Ins) receptors in HepG2 cells. Ins (10(-7)M) induced tyr-phosphorylation of IRS-4 (maximal at 5 min), an effect potentiated by Ang II AT(1) receptors. Phosphatydilinositol-3 kinase (PI3-K) inhibitors Wortmanin or LY294002 reduced Ang II effect on tyr-phosphorylation of IRS-4 to a level comparable to that of Ins alone. Physical association between IRS-4 substrate and PI3-K was demonstrated by co-immunoprecipitation. Recruitment of PI3-K by IRS-4 was induced by Ins (10(-7)M, 5 min) not by Ang II (10(-7)M) and this was inhibited by Wortmanin and LY294002. Ang II did not modify either the association or activation of PI3-K in immunocomplexes. The present data provide novel evidence of IRS-4 phosphorylation mediated by Ins, an effect modulated by Ang II. We report also Ins-induced PI3-K activation mediated by IRS-4. Our findings suggest a role for IRS-4 as a docking protein in the Ins signaling pathway that involves PI3-K association and activation. The present data suggest a possible participation of IRS-4 in cell proliferation Ins-induced.

Inhibition of Angiotensin II receptors during pregnancy induces malformations in developing rat kidney.

Evidence suggests that Angiotensin II plays an important role in the complex process of renal organogenesis. Rat kidney organogenesis starts between E13-14 and lasts up to 2 weeks after birth. The present study demonstrates histologic modifications and changes in receptor localisation in animals born from mothers treated with Angiotensin II, Losartan or PD123319 (1.0 mg/kg/day) during late pregnancy. Angiotensin II-treated animals exhibited very well developed tubules in the renal medulla in coincidence with higher AT(1) binding. Control animals exhibited angiotensin AT(2) binding in the outer stripe of the outer medulla, while in the Angiotensin II-treated animals binding was observed to the inner stripe. In Angiotensin II-treated 1-week-old animals, the nephrogenic zone contained fewer immature structures, and more developed collecting tubules than control animals. Treatment with Losartan resulted in severe renal abnormalities. For newborn and 1-week-old animals, glomeruli exhibited altered shape and enlarged Bowman spaces, in concordance with a loss of [(125)I]Angiotensin II binding in the cortex. Blockade with PD123319 led to an enlarged nephrogenic zone with increased number of immature glomeruli, and less glomeruli in the juxtamedullary area. Autoradiography showed a considerable loss of AT(1) binding in the kidney cortex of PD123319-treated animals at both ages. The present results show for the first time histomorphological and receptor localisation alterations following treatment with low doses of Losartan and PD123319 during pregnancy. These observations confirm previous assumptions that in the developing kidney Angiotensin II exerts stimulatory effects through AT(1) receptors that might be counterbalanced by angiotensin AT(2) receptors.

Morphological and endocrine study of the ovarian interstitial tissue of viscacha (Lagostomus maximus maximus).

The morphological and endocrine aspects of the ovarian interstitial tissue of adult female viscachas were investigated to establish the probable function and the biological significance of this compartment in this rodent. Pregnant and nonpregnant adult female viscachas were used. The histological characteristics, histochemical properties, and ultrastructural features of the interstitial tissue were studied. A morphometric study was carried out to measure the relative area of lipid droplets. The progesterone and androstenedione levels in ovarian tissue as well as in serum were determined by radioimmunoassay. In this species, the histological observations showed an abundant interstitial tissue that contained a large amount of lipids. The cholesterol and its esters were present in nonpregnant females and were scarce in pregnant animals. The most ultrastructural differences were observed at mid-pregnancy. At this stage, the interstitial cells showed features that suggested higher steroidogenic activity. Furthermore, during mid-pregnancy, the relative area of lipid droplets was smaller. Both progesterone and androstenedione levels in ovarian tissue and serum were higher during pregnancy. Our results suggest that the interstitial tissue may be storage of precursor substances for the steroidogenesis via. These precursors are probably used when the endocrine requirements are high, that is, during the pregnancy. Thus, this compartment may contribute to the normal gestation of Lagostomus. However, the relation between the interstitial tissue and the pregnancy is complex, and further studies are needed to clearly establish it.

Age-dependent decrease in the ghrelin gene expression in the human adrenal cortex: a real-time PCR study.

Numerous lines of evidence indicate that ghrelin, an endogenous ligand of the growth hormone-secretagogue receptor, is expressed in the human and rat adrenal cortex. In this study, we examined whether ghrelin gene expression undergoes changes in the human adrenal cortex during aging. Semi-quantitative real-time reverse transcription-polymerase chain reaction demonstrated a highly significant negative correlation between ghrelin mRNA and age in adrenal cortex of 27 patients (aged from 33 to 82 years), who underwent unilateral adrenalectomy/nephrectomy for kidney cancer. No significant differences in the level of adrenal ghrelin expression were observed between males and females. Since it has been previously shown that ghrelin exerts a marked growth-stimulating action on cultured adrenocortical cells, we hypothesize that the down-regulation of ghrelin gene transcription in adrenals could be associated with the reported decrease in adrenal DNA synthesis and mitogenic activity during aging.

Adrenomedullin and vascular endothelium growth factor genes are overexpressed in the regenerating rat adrenal cortex, and AM and VEGF reciprocally enhance their mRNA expression in cultured rat adrenocortical cells.

Adrenomedullin (AM) is an endogenous regulatory peptide, which exerts growth promoting and neoangiogenic actions in several normal and neoplastic tissues. Evidence has been provided that AM and the pro-angiogenic peptide vascular endothelium growth factor (VEGF) are expressed in the adrenal gland, and we investigated whether their gene transcription is modified in a model of rapid rat adrenal growth, namely regeneration after enucleation and contralateral adrenalectomy. Real-time polymerase chain reaction (PCR) showed that AM and VEGF mRNAs were significantly increased in regenerating adrenals with respect to the intact adrenocortical tissue of sham-operated control rats. Subsequent studies were carried out on dispersed rat adrenocortical cells cultured in vitro for 72 h. Conventional PCR demonstrated that cultured cells expressed AM and VEGF mRNAs. Moreover, real-time PCR showed that 24 h exposure to 10-8 M AM or 50 ng/ml VEGF significantly raised the expression of VEGF and AM, respectively, without affecting that of their own mRNA, suggesting the occurence of autocrine-paracrine up-regulatory mechanisms. Taken together, our findings allow us to conceive that the coordinate up-regulation of AM and VEGF expression may favor cell proliferation and neoangiogenesis, thereby leading to a rapid restoration of morphology and function of regenerating adrenals.

Similar sequence-free amplification of human glyceraldehyde-3-phosphate dehydrogenase for real time RT-PCR applications.

One of the major applications of real time polymerase chain reaction (PCR) is relative quantification, where the expression of a target gene is determined as a ratio to a stably expressed reference gene, the so-called housekeeping gene. Glyceraldehyde-3-phosphate dehydrogenase (GAPD) is a glycolytic enzyme, which is active in all mammalian tissues and is frequently used as housekeeping gene in expression studies. The functional locus maps to human chromosome 12p13, but several GAPD-related sequences, including processed pseudogenes, GenBank homologous sequences and computationally predicted sequences are present along the human genome. Due to the high level of GAPD-related sequences it is very important to avoid genomic DNA amplification when GAPD is used as endogenous control in mRNA quantification. We have outlined a GAPD couple of primers that avoid any genomic DNA amplification for real time reverse transcription PCR applications by SYBR-Green Dye. These new designed primers are an useful and chip alternative to probe technologies, and can carry out specific and reproducible data in mRNA expression studies.

Neonatal superior ovarian nerve transection disturbs the cyclic activity of the female rats.

The neural pathway most related with ovarian steroidogenesis has been identified as the superior ovarian nerve (SON). This work constitutes the first study of the effects of early ovarian SON transection, which was performed in rats of 4 days of age (SON-t rats) to magnify the effects of the denervation. The rats were studied at the prepubertal (30 days), peripubertal (41 days) and adult cyclic in dioestrus (60 days) reproductive stages. The SON-t rats showed a delay of vaginal opening, a notable disruption of oestrous cyclicity, and a large number of corpora lutea. In all the stages, the circulating levels of FSH, prolactin and growth hormone were lower in SON-t rats than in controls, whereas LH did not vary. Serum androstenedione levels were higher in SON-t rats at 30 days and lower at 41 days, compared with control animals while no difference was observed at 60 days. Serum progesterone levels did not differ between control and SON-t, but serum oestradiol concentrations were higher in SON-t rats in all of the stages. At the peripubertal stage, there were fewer ovarian beta-adrenergic receptors in SON-t ovaries, associated with a rise in the ovarian content of norepinephrine, but no changes were observed in SON-t rats at 30 and 60 days with respect to the controls. The release of progesterone in vitro from luteal cell in SON-t rats at 60 days was reduced in basal condition and under ovine LH or FSH stimulation, when compared with control animals; while no difference was observed in presence of isoproterenol or androstenedione in the culture medium. In corpora lutea of SON-t rats at 60 days, no change was observed in the activity of 3beta-hydroxysteroid dehydrogenase (3beta-HSD), but the activity of 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD) was reduced, suggesting abnormal luteolysis in spite of the large number of corpora lutea. The interruption of innervation at an early age by SON transection is very important in the regulation of ovarian development in prepubertal and cyclic rats. The functional changes observed in the ovary suggest a possible alteration in the hypothalamic-hypophyseal axis.

Adrenomedullin and its receptors are expressed in the zona glomerulosa of immature and adult rat adrenals: evidences of upregulation of ADM system in immature glands.

Evidence has been provided that adrenomedullin (ADM) stimulates the proliferative activity of adult rat adrenal zona glomerulosa (ZG). However, the selective ADM receptor antagonist ADM(22-52), although being able to block ADM effect, was per se ineffective. In contrast, in the companion paper, we showed that ADM(22-52) depresses the proliferation rate of ZG in 20-day-old rats, suggesting the involvement of endogenous ADM system in adrenal maturation. Hence, we investigated by semiquantitative reverse transcription-polymerase chain reaction and radioimmune assay the expression of ADM system in adult and immature rat ZG. ProADM mRNA and ADM-immunoreactivity were both more elevated in immature- than adult-rat ZG. Plasma ADM concentration did not show significant age-related differences. ADM acts via two subtypes of ADM(22-52)-sensitive receptors: the L1 receptor (L1-R) and the calcitonin-receptor-like-receptor (CRLR), the latter behaves as selective ADM receptor only in the presence of the receptor-activity-modifying proteins (RAMPs)2 and 3. L1-R expression was enhanced in immature rat ZG, while CRLR and RAMP(2,3) expression did not display significant differences. It is concluded that the endogenous ADM system located in the ZG is upregulated in immature rats, and plays an important autocrine-paracrine role in the maintenance of the elevated growth rate during adrenal maturation.

Adrenomedullin and its receptors are expressed in the zona glomerulosa of human adrenal gland: evidence that ADM enhances proliferation and decreases apoptosis in cultured ZG cells.

Adrenomedullin (ADM) is a hypotensive peptide, that derives from the proteolytic cleavage of pro(p)ADM and acts through two subtypes of receptors, called L1-receptor (L1-R) and calcitonin receptor-like receptor (CRLR). CRLR may function as a calcitonin gene-related peptide or a selective ADM receptor depending on the expression of the subtype 1 or the subtypes 2 and 3 of a family of proteins, named receptor-activity-modifying protein (RAMPs). Reverse transcription (RT)-polymerase chain reaction (PCR) consistently allowed the detection of pADM mRNA in zona glomerulosa (ZG), but not zona fasciculata-reticularis (ZF/R) cells of eight human adrenal cortexes. Despite the rather high level of pADM mRNA, immunocytochemistry and radioimmunoassay showed that the expression of ADM as protein in the ZG was weak. However, ZG cells expressed peptidyl-glycine alpha-amidating monooxigenase, the enzyme converting immature ADM to the mature peptide, thereby suggesting their potential ability to produce active ADM. RT-PCR demonstrated the presence in ZG, but not ZF/R cells of the specific mRNAs of L1-R, CRLR and RAMPs1-3. Semiquantitative PCR showed that L1-R expression was higher than that of CRLR, while the level of expression of the three RAMPs was nearly the same. ADM (10(-8) M) inhibited both angiotensin-II (10(-9) M)- and K(+) (10(-2) M)-stimulated aldosterone secretion from cultured ZG cells, without affecting basal production. ADM (10(-8) M) also increased proliferative activity and lowered apoptotic deletion of cultured cells. All the effect of ADM were reversed by the ADM-receptor antagonist ADM(22-52). In conclusion our study provides evidence that i) human ZG cells express ADM and ADM receptors of both L1 and CRLR/PAMP2,3 subtypes; and ii) through the activation of these receptors, ADM exerts an aldosterone antisecretagogue action and a growth promoting effect on cultured ZG cells, the latter an effect which includes both stimulation of proliferation and inhibition of apoptosis. Taken together, these findings make it likely that endogenous ADM system plays a potentially important role in the paracrine/ autocrine regulation of human adrenal cortex.

Secretion from neuropeptide-treated splenocytes modifies ovarian steroidogenesis

There are evidences for modulation of immune function by the sympathetic nervous system and its principal neurotransmitter norepinephrine (NE) throgugh superior ovarian nerve (SON)-coeliac ganglionnoradrenergic postganglionic innervation of the spleen. Seven days after SON transection at 53 days of age, the rat splenocytes were isolated and then cultured for 48h. These culture media, used to simulate ovaries from 60-day- old intact rats (neither SON-transected nor sham-operated) at diestrus 2 stage, in in vitro incubations, showed adecrease in progesterone release, an increase in estradiol release and no change in androstenedione release in relation to splenocyte culture media from control (sham-operated) rats.When esplenocytes from SON transected (SON-t) rats were treated with vasoactive intestinal peptide (VIP) or neuropeptide Y (NPY), both at 16-6M for 24h, their secretions increased the progesterone release while decreasing the estradiol release from the intact ovaries, compared with the secretions of untreated splenocytes from SON-t rats. Although the secretions of splenocytes treated with VIP decrease the androstenedione release from de ovaries, the treatment with NPY produced no change in hormone release. In the present paper the ovarian steroidogenic response, which was modified by the effects of an in vivo SON transection on spleen cells, was reverted by an in vitro system in which the splenocytes were treated with VIP or NPY. This could indicate that the spleen of SON-t rats does not receive those neuropeptides by neural route however, when they are added to splenocyte culture in vitro, the cell secretions revert the profile of steroid hormones released from the intact ovary. We also present functional evidence for modulation of the immune function by sympathetic nervous system and neurotransmitters other than NE.

Secretion from neuropeptide-treated splenocytes modifies ovarian steroidogenesis

There are evidences for modulation of immune function by the sympathetic nervous system and its principal neurotransmitter norepinephrine (NE) throgugh superior ovarian nerve (SON)-coeliac ganglionnoradrenergic postganglionic innervation of the spleen. Seven days after SON transection at 53 days of age, the rat splenocytes were isolated and then cultured for 48h. These culture media, used to simulate ovaries from 60-day- old intact rats (neither SON-transected nor sham-operated) at diestrus 2 stage, in in vitro incubations, showed adecrease in progesterone release, an increase in estradiol release and no change in androstenedione release in relation to splenocyte culture media from control (sham-operated) rats.When esplenocytes from SON transected (SON-t) rats were treated with vasoactive intestinal peptide (VIP) or neuropeptide Y (NPY), both at 16-6M for 24h, their secretions increased the progesterone release while decreasing the estradiol release from the intact ovaries, compared with the secretions of untreated splenocytes from SON-t rats. Although the secretions of splenocytes treated with VIP decrease the androstenedione release from de ovaries, the treatment with NPY produced no change in hormone release. In the present paper the ovarian steroidogenic response, which was modified by the effects of an in vivo SON transection on spleen cells, was reverted by an in vitro system in which the splenocytes were treated with VIP or NPY. This could indicate that the spleen of SON-t rats does not receive those neuropeptides by neural route however, when they are added to splenocyte culture in vitro, the cell secretions revert the profile of steroid hormones released from the intact ovary. We also present functional evidence for modulation of the immune function by sympathetic nervous system and neurotransmitters other than NE. (AU)