Acute effects of orexins A and B on the rat pituitary-adrenocortical axis

Orexins A and B are hypothalamic peptides, which act through two receptor subtypes, called OX1R and OX2R. They belong to a group of neuropeptides involved in the central regulation of food intake, members of which (neuropeptide Y and leptin) are known to modulate the function of the pituitary-adrenocortical axis (PAA). We examined the effects at 60 and 120 min of a subcutaneous injection of 5 or 10 nmol/kg of orexins on the function of the rat PAA. Orexin-A raised plasma concentrations of ACTH, aldosterone and corticosterone at both 60 and 120 min, corticosterone response being the most intense one. Orexin-B evoked a sizeable decrease in the plasma level of ACTH, without changing that of corticosterone. The effect of orexin-B on aldosterone plasma concentration was biphasic, the lower dose decreasing and the higher one increasing it at both 60 and 120 min. Evidence indicates that OX1R binds both orexins, while OX2R is selective for orexin-B, and that only OX2R is present in the hypothalamic nucleus paraventricularis. On these grounds, our findings allow us to conclude: (i) OX1R stimulates and OX2R inhibits rat PAA; (ii) orexin-A stimulates PAA, the activation of OX1R prevailing over that of OX2R, while orexin-B suppresses PAA function; and (iii) the aldosterone-secreting response to the higher dose of orexin-B may probably be ascribed to the activation of one or more extra-PAA mechanisms enhancing secretory activity of the zona glomerulosa

PANCREATIC-POLYPEPTIDE STIMULATES CORTICOSTERONE SECRETION BY ISOLATED RAT ADRENOCORTICAL-CELLS

Pancreatic polypeptide (PP) dose-dependently enhanced both basal and submaximally ACTH-stimulated corticosterone production by dispersed zona fasciculata/reticularis cells of the rat adrenal gland. Conversely PP did not affect either basal or ACTH- and angiotensin-II-stimulated aldosterone and corticosterone secretion of zona glomerulosa cells. These findings could throw light on the physiological significance of the marked increase in the pancreatic release of PP during stresses

Up-regulation of adrenomedullin receptor gene expression in activated local stem cells during rat adrenal regeneration.

Previous studies showed that adrenomedullin (AM) gene expression was up-regulated in the regenerating rat adrenal cortex after enucleation and contra-lateral adrenalectomy, the effect being significant at day 1 after surgery and peaking between days 3 and 7. Using the same experimental model, we investigated by real time-polymerase chain reaction the mRNA expression of the AM receptor components: calcitonin receptor-like receptor (CRLR) and receptor activity-modifying proteins (RAMP)2 and 3. At time 0 (60 min after enucleation; control group), the CRLR mRNA content was approximately 2- and 5-fold higher than that of RAMP2 and RAMP3, respectively. No significant changes in CRLR mRNA expression were observed in relation to the time elapsed from enucleation. RAMP2 and RAMP3 mRNAs did not exhibit significant changes at day 1 after surgery, but underwent a marked increase between days 3 and 7. The mRNA content of the two RAMPs decreased at days 14 and 28, although remaining significantly higher than that of the controls. These findings indicate that the AM receptor subtypes AM1-R (CRLR-RAMP2) and AM2-R (CRLR-RAMP3) are up-regulated in enucleated adrenals, and the hypothesis is advanced that this effect depends on the increased local production of AM. The concerted increase in AM and its receptor expression would greatly improve the autocrine-paracrine mechanism(s) by which AM favors proliferation of zona glomerulosa stem cells during adrenal regeneration

A new image analysis method based on topological and fractal parameters to evaluate the angiostatic activity of docetaxel by using the matrigel assay in vitro

Angiogenesis is regulated both by positive and negative factors that modulate the migration, proliferation, proteolytic activity, and differentiation of endothelial cells. Various in vivo and in vitro models have been developed to test the effect of angiogenic agonists or antagonists. Until very recently, morphometric methods to evaluate the effects of angiogenic inducers and inhibitors were mostly descriptive. Here, we have described a new, automatic, image analysis method to evaluate the angiostatic activity of docetaxel by using the in vitro Matrigel assay. This method allows to establish several parameters, such as dimensional (area % covered by endothelial cells and the total length of the cellular network per field), topological (the number of meshes and the number of branching points per field), and fractal (fractal dimension, lacunarity) of the capillary-like network. Following docetaxel treatment, a reduction of about 25% in the size parameters of cell network, of about 45% in branching points per field, and more 50% in mesh number were observed. The resulting fractal analysis was consistent with these findings and fractal dimension significantly correlated with both the number of branching points and the number of meshes. These results indicate that both topological and fractal parameters allowed a characterization of the spatial texture generated by endothelial cells during in vitro angiogenesis, the former being easier to implement in a computer program and faster to calculate. Finally, topological parameters seem to exhibit a wider dynamic range than the fractal ones

Gene silencing of human RAMP2 mediated by short-interfering RNA

Adrenomedullin (AM) is a regulatory peptide widely expressed, along its receptors, in cells and tissues, of which it controls many basic and specific functions acting in an autocrine-paracrine manner. However, the unequivocal demonstration of the physiological relevance of the regulatory role of AM would require the study of cells where the endogenous AM system has been suppressed. For this task we developed a technique to silence the AM gene in human umbilical vein endothelial cells (HUVECs) and the human embryonal kidney cell line (HEK-293). AM acts via two subtypes of receptor, named AM1 and AM2, which derive from the interaction of the calcitonin receptor-like receptors with two chaperones, called receptor activity modifying proteins (RAMP2 and RAMP3). Hence, we developed a protocol to suppress the human AM1 receptor by silencing the RAMP2 gene by transfection with short interfering RNAs (siRNAs). HUVECs were transfected using a new Ambion transfection reagent. RAMP2 gene silencing was determined in HUVECs by measuring RAMP2 mRNA levels in transfected and control cells by real-time polymerase chain reaction. The RAMP2 gene silencing was approximately 60% and was observed 48 h after transfection. Matrigel assay in vitro was carried out to evaluate the effects of siRNA sequences. HUVECs cells were plated on matrigel and the analysis of capillary-like tubule formation showed that the cells were viable. The knockdown of the RAMP2 gene decreased the formation of tubes in response to 10(-8) M AM. The conclusion is drawn that siRNA technology can be successfully used in the investigations on AM and AM receptor functions

Two selective rat adrenomedullin (AM)-receptor antagonists: AM20-50 and AM24-50

Adrenomedullin (AM) is a hypotensive peptide, which is produced in several organs and tissues, the functions of which it regulates in a autocrine-paracrine manner. Rat (r) and human (h) AM are 50- and 52-amino acid peptides, which differ for 2-amino acid deletions and six substitutions and contain a disulfide bridge-formed six-membered ring between adjacent cysteine residues in the 14 and 19 and 16 and 21 positions, respectively. The amidated C-terminal sequence is needed for AM to bind its receptors, and the ring structure (but not t he N-terminal sequence) seems to be required for AM to activate its receptors. Hence, we examined the effectiveness of some N-terminus and ring-lackingAM fragments as AM-receptor antagonists in the rat zona glomerulosa (ZG), whose cells are provided with abundant AM binding sites and display an AM-induced inhibition of K+-stimulated aldosterone secretion. Quantitative autoradiographic studies showed that cold rAMI-50, rAM20-50 and rAM24-50 displaced [125I]AM1-50 binding from rat ZG with the same potency and efficacy, which were significantly higher than those of hAM1-52, hAM22-52 and hAM26-52. Accordingly, rAM20-50 and rAM24-50 reversed the inhibitory effect of 10(-8) M rAMI-50 on aldosterone response of dispersed rat ZG cells to 10(-2) M K+ with significantly higher potency and efficacy than hAM22-52 and hAM26-52. Taken together, our findings confirm that CONH2-terminal AM fragments, lacking the six-membered ring structure, act as antagonists of AM receptors in the rat ZG. Moreover, they provide the first evidence that rAMI-50 and its fragments should be used in the investigations carried out in the rat

Two selective rat adrenomedullin (AM)-receptor antagonists: AM20-50 and AM24-50

Adrenomedullin (AM) is a hypotensive peptide, which is produced in several organs and tissues, the functions of which it regulates in a autocrine-paracrine manner. Rat (r) and human (h) AM are 50- and 52-amino acid peptides, which differ for 2-amino acid deletions and six substitutions and contain a disulfide bridge-formed six-membered ring between adjacent cysteine residues in the 14 and 19 and 16 and 21 positions, respectively. The amidated C-terminal sequence is needed for AM to bind its receptors, and the ring structure (but not t he N-terminal sequence) seems to be required for AM to activate its receptors. Hence, we examined the effectiveness of some N-terminus and ring-lackingAM fragments as AM-receptor antagonists in the rat zona glomerulosa (ZG), whose cells are provided with abundant AM binding sites and display an AM-induced inhibition of K+-stimulated aldosterone secretion. Quantitative autoradiographic studies showed that cold rAMI-50, rAM20-50 and rAM24-50 displaced [125I]AM1-50 binding from rat ZG with the same potency and efficacy, which were significantly higher than those of hAM1-52, hAM22-52 and hAM26-52. Accordingly, rAM20-50 and rAM24-50 reversed the inhibitory effect of 10(-8) M rAMI-50 on aldosterone response of dispersed rat ZG cells to 10(-2) M K+ with significantly higher potency and efficacy than hAM22-52 and hAM26-52. Taken together, our findings confirm that CONH2-terminal AM fragments, lacking the six-membered ring structure, act as antagonists of AM receptors in the rat ZG. Moreover, they provide the first evidence that rAMI-50 and its fragments should be used in the investigations carried out in the rat

ADRENAL-MEDULLA IS INVOLVED IN THE ALDOSTERONE SECRETAGOGUE EFFECT OF SUBSTANCE-P

Substance P (SP) increased aldosterone secretion of rat adrenal slices, but not of isolated zona glomerulosa cells, and this effect was annulled by two specific antagonist of SP (SP-A). Both tissue preparations displayed an aldosterone secretory response to isoprenaline (IP) that was blocked by l-alprenolol (AL). AL reversed the aldosterone response of adrenal slices to IP, SP, or IP plus SP, whereas SP-A only suppressed that to SP. Quarters of adrenocortical autotransplants, which are completely deprived of chromaffin cells, showed an aldosterone response to IP, but not to SP. These findings suggest that the mechanism underlying the aldosterone secretagogue action of SP probably involves the stimulation of catecholamine release by adrenal medulla chromaffin cells