Maternal perinatal undernutrition has long-term consequences on morphology, function and gene expression of the adrenal medulla in the adult male rat.

Epidemiological studies suggest that maternal undernutrition sensitises to the development of chronic adult diseases, such as type 2 diabetes, hypertension and obesity. Although the physiological mechanisms involved in this 'perinatal programming' remain largely unknown, alterations of stress neuroendocrine systems such as the hypothalamic-pituitary-adrenal (HPA) and sympathoadrenal axes might play a crucial role. Despite recent reports showing that maternal perinatal undernutrition disturbs chromaffin cells organisation and activity in male rats at weaning, its long-term effects on adrenal medulla in adult animals are unknown. Using a rat model of maternal perinatal 50% food restriction (FR50) from the second week of gestation until weaning, histochemistry approaches revealed alterations in noradrenergic chromaffin cells aggregation and in cholinergic innervation in the adrenal medulla of 8-month-old FR50 rats. Electron microscopy showed that chromaffin cell granules exhibited ultrastructural changes in FR50 rats. These morphological changes were associated with reduced circulating levels and excretion of catecholamines. By contrast, catecholamine plasma levels were significantly increased after a 16 or 72 h of fasting, indicating that the responsiveness of the sympathoadrenal system to food deprivation was accentuated in FR50 adult rats. Among 384 pituitary adenylate cyclase-activating polypeptide-sensitive genes, we identified 129 genes (33.6%) that were under expressed (ratio < 0.7) in FR50 animals. A large number of these genes are involved in cytoskeleton remodelling and vesicle trafficking. Taken together, our results show that maternal perinatal undernutrition programmes adrenomedullary function and gene expression in adult male rats. Because catecholamines contribute to metabolic homeostasis, as well as arterial blood pressure regulation, the alterations observed in the adrenal medulla of adult male FR50 rats may participate in the programming of chronic adult diseases.

Diverse mechanisms of Wnt activation and effects of pathway inhibition on proliferation of human gastric carcinoma cells.

Human gastric carcinomas are among the most treatment-refractory epithelial malignancies. Increased understanding of the underlying molecular aberrations in such tumors could provide insights leading to improved therapeutic approaches. In this study, we characterized diverse genetic aberrations leading to constitutive Wnt signaling activation in a series of human gastric carcinoma cell lines. Downregulation of TCF signaling by stable transduction of dominant negative TCF4 (DNTCF4) resulted in inhibition of proliferation in Wnt-activated AGS tumor cells. c-Myc downregulation and the associated upregulation of its repression target, p21 observed in these tumor cells, as well as the profound growth inhibition induced by c-Myc small hairpin RNA (shRNA) implied their c-Myc addiction. In striking contrast, Wnt-activated MKN-28 and MKN-74 tumor cells appeared refractory to DNTCF4 inhibition of proliferation despite comparably decreased c-Myc expression levels. The resistance of these same tumor cells to growth inhibition by c-Myc shRNA established that their refractoriness to DNTCF was because of their independence from c-Myc for proliferation. There was no correlation between this resistance phenotype and the presence or absence of constitutive mitogen-activated protein kinase (MAPK) and/or AKT pathway activation, commonly observed in gastrointestinal tumors. However, in both DNTCF-sensitive and -resistant tumor cells with MAPK and/or AKT pathway activation, the ability of small molecule antagonists directed against either pathway to inhibit tumor cell growth was enhanced by Wnt pathway inhibition. These findings support the concept that although certain Wnt-activated tumors may escape c-Myc dependence for proliferation, disruption of other oncogenic pathways can unmask cooperative antiproliferative effects for Wnt pathway downregulation.

Pituitary adenylate cyclase-activating polypeptide stimulates secretoneurin release and secretogranin II gene transcription in bovine adrenochromaffin cells through multiple signaling pathways and increased binding of pre-existing activator protein-1-like transcription factors.

Secretoneurin (SN) is a novel bioactive peptide that derives from the neuroendocrine protein secretogranin II (SgII) by proteolytic processing and participates in neuro-immune communication. The neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP-38) dose-dependently stimulates (EC(50) approximately 3 nM) SN release (up to 4-fold) and SgII gene expression (up to 60-fold) in cultured bovine adrenochromaffin cells. The effect of PACAP on both SN secretion and SgII mRNA levels is rapid and long lasting. We analyzed in this neuroendocrine cell model the transduction pathways involved in both SN secretion and SgII gene transcription in response to PACAP. The cytosolic calcium chelator BAPTA-AM and the nonselective calcium channel antagonist NiCl(2) equally inhibited both secretion of the peptide and transcription of the SgII gene, indicating a major contribution of calcium influx in PACAP-induced SN biosynthesis and release in chromaffin cells. Inhibition of protein kinase A (PKA) or C (PKC) also reduced PACAP-evoked SN release but did not alter the stimulatory effect of PACAP on SgII mRNA levels. Conversely, application of mitogen-activated protein kinase inhibitors suppressed PACAP-induced SgII gene expression. The effect of PACAP on SgII mRNA levels, like the effect of the PKC stimulator 12-O-tetradecanoylphorbol-13-acetate (TPA), was not affected by cycloheximide, whereas the effects of the PKA stimulator forskolin or cell-depolarization by high K(+) were significantly reduced by the protein synthesis inhibitor. PACAP and TPA both increased the binding activity of the SgII cAMP response element to trans-acting factors present in chromaffin cell nuclear extracts, which are recognized by antibodies to activator protein-1-related proteins. These data indicate that SN biosynthesis is regulated by PACAP in chromaffin cells through complex signaling cascades, suggesting that SN may play a function during trans-synaptic stimulation of the adrenal medulla.

Pharmacological and molecular characterization of 5-hydroxytryptamine(7) receptors in the rat adrenal gland.

Serotonin (5-hydroxytryptamine; 5-HT) is a potent stimulator of aldosterone secretion in the rat adrenal gland but the type of receptor involved in the mechanism of action of 5-HT remains unknown. The aim of the present study was to determine the pharmacological profile and to clone the receptor responsible for the corticotropic effect of 5-HT in rat glomerulosa cells. A series of 10 serotonergic receptor agonists and 12 receptor antagonists was used to characterize the receptor mediating the effect of 5-HT on aldosterone secretion from perifused rat adrenocortical slices. Correlation analysis between the potencies of the different compounds in our model and those previously reported for various recombinant 5-HT receptors showed that the rat adrenal 5-HT receptor exhibits the same pharmacological profile as the 5-HT(7) receptor transiently expressed in COS-7 cells (r = 0.82 for agonists, p <.05; r = 0.83 for antagonists, p <.01). Polymerase chain reaction with specific primers revealed the expression of 5-HT(7) receptor mRNA in the rat adrenal gland. Cloning of the polymerase chain reaction product confirmed that the amplified DNA corresponded to the 5-HT(7) receptor cDNA sequence. Western blot analysis showed the presence of a protein with an apparent molecular mass of 66 kDa in the adrenal cortex but not in the medulla. Taken together, these data demonstrate that the rat adrenal glomerulosa expresses functional 5-HT(7) receptors. Rat glomerulosa cells will thus provide a robust and sensitive bioassay for future studies on native 5-HT(7) receptors.

Pharmacological profile of serotonergic receptors in the adrenal gland.

The secretory activity of the adrenal gland is mainly regulated by peptidergic hormones (ACTH, angiotensin II) and ions. However, there is now increasing evidence that local factors, including neuropeptides and neurotransmitters, can also participate in the control of adrenocortical cells. In particular, serotonin (5-HT), produced by adrenochromaffin cells in frog and rat as well as by mast cells in the adrenal gland of rat and human, stimulates corticosteroid secretion. In both frog and human adrenal gland, the benzamide derivative (R,S)-zacopride induces a robust increase in corticosteroid release suggesting that the effect of 5-HT on steroidogenesis is mediated through activation of 5-HT4 receptors. In contrast, in rat, the stimulatory effect of 5-HT on aldosterone secretion is clearly not mediated by 5-HT4 receptors. In all three species, incubation of adrenocortical fragments with 5-HT induces a significant increase in cAMP formation. Our data suggest that 5-HT, released within the adrenal cortex, may act as a paracrine factor to stimulate steroid secretion. Although the corticotropic effect of 5-HT has been conserved from amphibians to primates, the type of receptors involved in the action of 5-HT markedly differs across species.