Preproorexin and orexin receptors are expressed in cortisol-secreting adrenocortical adenomas, and orexins stimulate in vitro cortisol secretion and growth of tumor cells.

Orexins A and B are hypothalamic peptides that originate from the proteolytic cleavage of preproorexin and act through two subtypes of receptors, named OX1-R and OX2-R. OX1-R almost exclusively binds orexin-A, whereas OX2-R is nonselective for both orexins. We previously found that orexin-A, via the OX1-R, stimulates cortisol secretion from dispersed human adrenocortical cells. In this study, we demonstrate that six of eight cortisol-secreting adenomas expressed preproorexin mRNA, and seven of 10 adenomas contained measurable amounts of orexin-A but not orexin-B. Normal adrenal cortexes neither expressed preproorexin nor contained orexins. All adenomas expressed OX1-R and OX2-R mRNAs, and real-time PCR showed that the expression of both receptors was up-regulated in adenomas, compared with normal adrenal cortex. Orexin-A concentration-dependently raised basal cortisol secretion from freshly dispersed normal and adenomatous cells, minimal and maximal effective concentrations being 10(-10) and 10(-8) m, and the peptide efficacy (percent increase elicited by 10(-8) m orexin-A) was significantly higher in adenomas than in the normal adrenal cortex. Orexin-B was ineffective, thereby indicating that orexin secretagogue action is mediated by the OX1-R. In contrast, both orexins (10(-8) m) raised the proliferative activity of cultured normal and adenomatous cells, suggesting that this effect is mediated by OX2-R or both receptor subtypes. Collectively, our findings allow us to conclude that the orexin system is overexpressed in cortisol-secreting adenomas and suggest that orexin-A may act as an autocrine-paracrine regulator of the secretory activity and growth of some of these adrenal tumors.

Cholecystokinin (CCK) stimulates aldosterone secretion from human adrenocortical cells via CCK2 receptors coupled to the adenylate cyclase/protein kinase A signaling cascade.

Cholecystokinin (CCK) IS a regulatory peptide that acts via two receptor subtypes, CCK1-R and CCK2-R. RT-PCR demonstrated the expression of both CCK1-R and CCK2-R in the zona glomerulosa (ZG), but not zona fasciculata-reticularis cells of the human adrenal cortex. CCK and the CCK2-R agonist pentagastrin enhanced basal aldosterone secretion from ZG cells without affecting cortisol production from zona fasciculata-reticularis cells. The aldosterone response to CCK and pentagastrin was suppressed by a CCK2-R antagonist, but not by a CCK1-R antagonist. Pentagastrin evoked a sizeable cAMP, but not inositol triphosphate, response from ZG cells, whereas CCK plus CCK2-R antagonist was ineffective. The cAMP response to pentagastrin was abrogated by CCK2-R antagonist or the adenylate cyclase inhibitor SQ-22536, and the aldosterone response was abolished by both SQ-22536 and the protein kinase A inhibitor H-89. Both CCK and pentagastrin increased steroidogenic acute regulatory protein mRNA expression in ZG cells; the effect was abrogated by CCK2-R antagonist. We conclude that CCK exerts secretagogue action on human ZG cells, acting through CCK2-Rs coupled to the adenylate cyclase/protein kinase A signaling cascade, which, in turn, stimulates the expression of steroidogenic acute regulatory protein, the rate-limiting step of steroidogenesis.

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.

Experimental abdominal wall defect repaired with acellular matrix.

In the surgical repair of congenital abdominal-wall defects (AWD), the ready availability of a non-immunogenic and non-prosthetic biomaterial that could guide the regeneration of normal tissue is a fascinating possibility. Biomaterials are already in use, but in our experience, an acellular matrix (ACM) can stimulate exact regeneration of the absent tissue. We explored the possibility of using an ACM to repair a muscular AWD in an animal model. Male New Zealand white rabbits (3-4 kg, n = 18) were anesthetized and the abdominal wall was shaved and scrubbed; a vertical incision was made in the left lower quadrant and a large patch of external-oblique muscle was resected (3 x 3 cm). The animals underwent reconstruction with homologous diaphragm acellular matrix (HDAM) grafts that were previously prepared using a detergent enzymatic method. The patches were evaluated histologically at 9 (n = 6), 40 (n = 6), and 90 (n = 6) days post-surgery in each group; moreover, 90 days post-surgery an electromyogram (EMG) (n = 6) of the implanted matrix was recorded. Histologic analysis demonstrated that the HDAM supported fibroblast migration, deposition of newly-formed collagen, and neovascularization. No signs of necrosis, or evidence of skeletal-muscle-cell ingrowth were detected. The EMG revealed minimum muscular electrophysiologic activity, probably due to muscle underlying the patch. The HDAM we employed was thus not able to produce reconstruction of the skeletal muscle, and was progressively remodeled into fibrous tissue. Since the ultimate reason for failure of muscle regeneration is a lack of myogenesis, future studies will use ACMs preconditioned by various regulators of myoblast proliferation and differentiation.

Prostaglandin F2 alpha can modulate the growth and the differentiation of bovine corneal epithelial cells cultured in vitro.

The effects of PGF2 alpha on the growth, morphology, morphometry and keratinization pattern of bovine corneal epithelial cells cultured in vitro were studied. The cells were grown with a basal medium or, in the presence of keratocytes and/or their products, using a keratocyte-conditioning medium. Cell growth was evaluated by MTT assay. Daily treatments with exogenous PGF2 alpha at concentrations equal to or lower than 10(-6) M induced significant increases in cell proliferation when the epithelial cells were cultured on a keratocyte feeder-layer or with the conditioning medium. No variations were observed in cultures grown with the basal medium. 10(-5) M PGF2 alpha induced a decrease in cell growth under all culturing conditions. PGF2 alpha did not affect cell morphology and modified only nuclear dimensions among the cells grown under different culturing conditions. No variations of any parameters were observed between cells cultured on feeder-layer, with conditioning or basal medium and the corresponding cultures supplemented with the autacoid. Moreover, PGF2 alpha induced only the disappearance of 43 kDa keratin in cells grown on basal medium, while the keratin pattern of epithelial cells cultured on feeder-layer or with the conditioning medium was not modified by the autacoid. From these data we can suppose that a cooperation could exist between PGF2 alpha and fibroblasts and their products for the modulation of cell growth. Finally, it was observed that the autacoid had no effect on cell morphology and morphometry, except for nuclear dimensions, despite the presence of other prostaglandins, such as PGE2.