Caterina Squillacioti

H2O2 activity on platelet adhesion to fibrinogen and protein tyrosine phosphorylation

Platelets represent a target of reactive oxygen species produced under oxidative stress conditions. Controversial data on the effect of these species on platelet functions have been reported so far. In this study we evaluated the effect of a wide range of H(2)O(2) concentrations on platelet adhesion to immobilized fibrinogen and on pp72(syk) and pp125(FAK) tyrosine phosphorylation. Our results demonstrate that: (1) H(2)O(2) does not affect the adhesion of unstimulated or apyrase-treated platelets to immobilized fibrinogen; (2) H(2)O(2) does not affect pp72(syk) phosphorylation induced by platelet adhesion to fibrinogen-coated dishes; (3) H(2)O(2) reduces, in a dose-dependent fashion, pp125(FAK) phosphorylation of fibrinogen-adherent platelets; (4) concentrations of H(2)O(2) near to physiological values (10-12 microM) are able to strengthen the subthreshold activation of pp125(FAK) induced by epinephrine in apyrase-treated platelets; (5) H(2)O(2) doses higher than 0.1 mM inhibit ADP-induced platelet aggregation and dense granule secretion. The ability of H(2)O(2) to modulate pp125(FAK) phosphorylation suggests a role of this molecule in physiological hemostasis as well as in thrombus generation.

Pituitary adenylate cyclase activating peptide (PACAP) immunoreactivity and mRNA expression in the duck gastrointestinal tract

Abstract. The presence and distribution of pituitary adenylate cyclase activating peptide (PACAP) immunoreactivity were studied in the duck gastrointestinal tract using immunohistochemistry and radioimmunoassays. Expression and distribution of PACAP mRNA were also studied using reverse transcriptase polymerase chain reaction (RT-PCR) and hybridization techniques. In addition, a partial coding sequence (cds) of the duck growth hormone-releasing hormone (GRF)/PACAP gene was identified. The presence of both PACAP-38 and PACAP-27 was demonstrated, the former being the predominant form. PACAP immunoreactivity was found in neurons and fibers of the enteric nervous system (ENS), in endocrine cells and in the gut associated lymphoid tissue (GALT). Double immunostaining showed that PACAP is almost completely colocalized with vasoactive intestinal peptide (VIP) in the ENS. Moreover, PACAP was also found in nitric oxide synthase (NOS)-containing neurons and nerve fibers. Radioimmunoassay (RIA) performed on denervated gut showed that more than one-half of the duodenal PACAP is extrinsic in origin. RT-PCR, Northern blot analysis and in situ hybridization confirmed the immunohistochemical data. The findings of the present study suggest that, in birds, PACAP may have multiple roles in regulating gastrointestinal functions.

Presence, distribution and steroidogenic effect of the peptides orexin A and receptor 1 for orexins in the testis of the South American camelid Alpaca (Vicugna pacos)

The orexins A (oxA) and B are peptides discovered in the rat hypothalamus and successively found in some peripheral organs of the mammalian body. They binds two protein G-coupled receptors defined receptor 1 (ox1r) and 2 for orexins, the first of which is highly specific for oxA while the second binds both the peptides with equal affinity. This work aimed to detect the presence of oxA and ox1r in the testis of the South American camelid alpaca (Vicugna pacos) and investigate the role played by them on Leydig cell steroidogenesis. The species alpaca acquired, in the last years, increasing zootechnical interest for the quality of the wool produced and its breeding spread from the country of origin to USA, Australia and Europe. Immunohistochemistry allowed us to detect oxA in Leydig and Sertoli cells, spermatogonia, resting spermatocytes, round and oval spermatids. Ox1r-immunoreactivity was found in Leydig cells and round, oval and elongated spermatids. The expression of the two peptides in tissue extracts was established by using Western blotting technique. Such results demonstrated that in the alpaca testis exists in a cellular complex able to produce and/or internalize oxA. Finally, the effect of oxA on steroidogenesis was investigated by means of in vitro cultured thin testis slices which were added with oxA or/and Müllerian Inhibiting Substance (MIS), a steroidolitic agent basally produced by the Sertoli cell. OxA evoked increase of testosterone production while MIS a decrease. The consecutive addition of oxA and MIS, or vice versa, highlighted an antagonistic interplay between the two substances which has been thought to be the main molecular event at the basis of the oxA-stimulated steroidogenesis mechanism.

Expression of urocortin and corticotropin-releasing hormone receptors in the bovine adrenal gland.

Urocortin (UCN), a 40 amino acid peptide, is a corticotrophin-releasing hormone (CRH)-related peptide. The biological actions of CRH family peptides are mediated via two types of G protein-coupled receptors, CRH type 1 receptor (CRHR1) and CRH type 2 receptor (CRHR2). The aim of the present study was to investigate the expression of UCN, CRHR1 and CRHR2 by immunohistochemistry, western blot and real-time RT-PCR in the bovine adrenal gland to clarify the mechanisms of the intra-adrenal CRH-based regulatory system. The results showed that UCN, CRHR1 and CRHR2 were expressed in both the cortex and medulla. Specifically, UCN-immunoreactivity (IR) was distributed in the outer part of the zona fasciculata and in the zona reticularis of the cortex and in the medulla. UCN and CRHR2 mRNA expression levels were higher in the cortex than in the medulla, while CRHR1 mRNA levels were undetectable in the cortex. These results suggest that UCN, CRHR1 and CRHR2 are expressed in the bovine adrenal gland and that UCN might play a role in the intra-adrenal CRH-based regulatory system through an autocrine mechanism.

THE MYCOTOXIN FUMONISIN B1 INHIBITS INTEGRIN-MEDIATED CELL-MATRIX ADHESION

Fumonisin B1 (FB1), a mycotoxin produced by the corn fungus Fusarium moniliforme, causes a variety of animal diseases and is a suspected human carcinogen. The FB1 molecule bears remarkable structural resemblance to the long-chain sphingoid base backbones of sphingolipids. The toxicity and carcinogenicity of FB1 has been ascribed to its ability to inhibit ceramide synthase, a key enzyme in the metabolism of complex sphingolipids. In this study we have investigated whether the exposure of B16-BL6 mouse melanoma cells to FB1 affects cell growth and integrin-mediated cell matrix adhesion. Cell treatment with the highest tested dose (75 microM) of FB1 for 72 h induced an about 20% inhibition of cell growth. FB1 strongly affected B16-BL6 cell adhesion to immobilized fibronectin, by causing a dose-dependent inhibition of cell attachment to this substrate. FB1 also inhibited in a dose-dependent manner the adhesion of B16-BL6 cells to the immobilized anti-fibronectin receptor antibody, whereas it affected only to a low extent cell attachment to concanavalin A. Our results demonstrate that FB1 treatment alters integrin adhesive activity, thus affecting all cellular integrin-dependent functions.

Aquaporins in Health and Disease: An Overview Focusing on the Gut of Different Species.

Aquaporins (AQPs) play a pivotal role in gut homeostasis since their distribution and function is modulated both in physiological and in pathophysiological conditions. The transport of water and solutes through gut epithelia is essential for osmoregulation and digestive and absorptive functions. This passage is regulated by different AQP isoforms and characterized by their peculiar distribution in the gastrointestinal tract. To date, AQP localization has been identified in the gut and associated organs of several mammalian species by different techniques (immunohistochemical, western blotting, and RT-PCR). The present review describes the modulation of AQP expression, distribution, and function in gut pathophysiology. At the same time, the comparative description of AQP in animal species sheds light on the full range of AQP functions and the screening of their activity as transport modulators, diagnostic biomarkers, and drug targets. Moreover, the phenotype of knockout mice for several AQPs and their compensatory role and the use of specific AQP inhibitors have been also reviewed. The reported data could be useful to design future research in both basic and clinical fields.

Immobilised echistatin promotes platelet adhesion and protein tyrosine phosphorylation

Echistatin, a 5000-Da disintegrin, is a strong competitive inhibitor of platelet alpha(IIb)beta(3) binding to fibrinogen. In addition to its antiplatelet activity, echistatin also exhibits activating properties by inducing a switch of alpha(IIb)beta(3) conformation towards an active state. However, soluble echistatin, which is a monomeric ligand, provides only receptor affinity modulation, but it is unable to activate integrin-dependent intracellular signals. Since proteins may exhibit a multivalent functionality as a result of their absorption to a substrate, in this study we evaluated whether immobilised echistatin is able to stimulate platelet adhesion and signalling. The immobilisation process led to an increase of echistatin affinity for integrin(s) expressed on resting platelets. Unlike the soluble form, immobilised echistatin bound at comparable extent either unstimulated or ADP-activated platelets. Furthermore, echistatin presented in this manner was effective in stimulating integrin-dependent protein tyrosine phosphorylation. Platelets adhering to immobilised echistatin showed a pattern of total tyrosine phosphorylated proteins resembling that of fibrinogen-attached platelets. In particular, solid-phase echistatin induced a strong phosphorylation of tyrosine kinases pp72(syk) and pp125(FAK). Inhibitors of platelet signalling, such as apyrase, prostaglandin E(1), cytochalasin D and bisindolylmaleimide, while not affecting platelet adhesion to immobilised echistatin, abolished pp125(FAK) phosphorylation. This suggests that signals activating protein kinase C function, dense granule secretion and cytoskeleton assembly might be involved in echistatin-induced pp125(FAK) phosphorylation.

The Protective Effect of Apocynin on Cyclosporine A-Induced Hypertension and Nephrotoxicity in Rats

Cyclosporine A (CsA) is the prototype of immunosuppressant drugs that has provided new perspectives in human and veterinary medicine to prevent organ transplant rejection and to treat certain autoimmune diseases and dermatologic diseases. Unfortunately, the treatment with CSA is often limited by severe adverse effects such as hypertension and nephrotoxicity. Some data suggest that reactive oxygen species (ROS) and the oxidative stress play an important role in its pathogenesis, in particular the superoxide (O2−) that is the most powerful free radical generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase present mainly in the kidney. The present study has been designed to investigate the role of Apocynin a selective inhibitor of NADPH oxidase activity on cyclosporine‐induced adverse effect. In this study, we have evaluated the effect of CsA, used alone or in association with Apocynin on blood pressure (BP), on glomerular filtration rate (GFR), on absoluted fluid reabsorption (Jv) in proximal tubule (PT), on O2− concentration, and on nitric oxide (NO) production. We have demonstrated that CsA administration increases superoxide concentration in the aorta, decreases the NO concentration, reduces GFR and the Jv in PT, and induces a significant increase in BP. Moreover, we have shown that Apocynin treatment restores these hemodynamic alterations, as well as NO and superoxide productions. In conclusion, the reported data indicate that CsA induced nephrotoxicity and hypertension are related to NADPH oxidase activity, in fact Apocynin protects the kidney function and BP from toxic effects induced by CsA through the inhibition of NADPH oxidase activity. J. Cell. Biochem. 116: 1848–1856, 2015.

Innervation of vas deferens and accessory male genital glands in the water buffalo (Bubalus bubalis). Neurochemical characteristics and relationships to the reproductive activity.

Autonomic nerves supplying mammalian male internal genital organs have an important role in the regulation of reproductive function. To find out the relationships between the neurochemical content of these nerves and the reproductive activity, we performed a histochemical and immunohistochemical study in a species, the water buffalo, exhibiting a seasonal sexual behaviour. The distribution of noradrenergic and nitric oxide synthase (NOS)- and peptide-containing nerves was evaluated during the mating and non-mating periods. Fresh segments of vas deferens and accessory genital glands were collected immediately after slaughter and immersed in 4% paraformaldehyde. Frozen sections were obtained and processed according to single and double labelling immunofluorescent procedures or NADPH-diaphorase histochemistry. During the mating period, a dense noradrenergic innervation was observed to supply the vas deferens as well as the accessory genital glands. NOS- and peptide-containing nerves were also observed but with a lower density. During the non-mating period noradrenergic nerves dramatically reduced. In addition, neuropeptide Y (NPY)- and vasoactive intestinal peptide (VIP)-containing nerves were also reduced. These findings suggest the presence of complex interactions between androgen hormones and the autonomic nerve supply in the regulation of male water buffalo reproductive functions.

Expression of urocortin and its receptors in the rat epididymis

Urocortin (UCN; 40 aa) is a corticotrophin-releasing hormone (CRH)-related peptide. The biological actions of CRH family peptides are mediated by two types of G-protein-coupled receptors, CRH type 1 receptor (CRHR1) and CRH type 2 receptor (CRHR2). The biological effects of the peptides are mediated and modulated not only by CRH receptors but also by a highly conserved CRH-binding protein (CRHBP). The aim of the present study was to investigate the expression of UCN, CRHR1, CRHR2 and CRHBP by immunohistochemistry, Western blot, RT-PCR and real-time RT-PCR in the rat epididymis. Urocortin, CRHR1 and CRHR2, but not CRHBP, were expressed in all segments of the rat epididymis. Specifically, UCN- and CRHR2-immunoreactivities (IRs) were distributed in epididymal epithelial cells of the caput, corpus and cauda. CRHR1-IR was found in the fibromuscular cells surrounding the epididymal duct and in the smooth musculature of the blood vessels throughout the organ. UCN and CRHR2 mRNA expression levels were higher in the caput and corpus than in the cauda, while CRHR1 mRNA level was higher in the cauda than those in the caput and corpus. In summary, UCN, CRHR1 and CRHR2 are expressed in the rat epididymis. It is suggested that CRH-related peptides might play multiple roles in the maturation and storage of spermatozoa.