275 research outputs found
“Affidabilità della flussimetria doppler nello studio dell’iperemia post-prandiale nell’uomo in corso di cirrosi epatica ed in soggetti di controllo”
No full text
H+/glycyl-glycine cotransport in eel intestinal brush border membrane vesicles: studies with the pH-sensitive dye acridine orange
No full textMonitoring the fluorescence quenching of the pH-sensitive dye Acridine orange, proton accumulation in the presence of an inside-negative transmembrane potential was measured in eel (Anguilla anguilla) intestinal brush-border membrane vesicles. It was demonstrated that the proton accumulation was specifically increased by the presence of the dipeptide glycyl-glycine in the extravesicular space, showing saturation kinetics at increasing dipeptide concentrations and was specifically inhibited by diethylpyrocarbonate. Data reported sugg
How many Na+-dependent carriers for l-alanine and l-proline in the eel intestine? Studies with brush-border membrane vesicles
No full textUsing brush-border membrane (BBM) vesicles prepared from the intestine of the European eel, the specificity of l-alanine and l-proline Na+-dependent transport was investigated by measuring the uptake of isotopically labelled subsrates. In the presence of Na+ ions, cross-inhibition between alanine and proline transports was observed; in addition α-(methylamino)isobutyric acid (MeAIB) inhibited proline but had no effect on alanine uptake. These results can be explained by the presence, in eel intestinal BBM vesicles, of at least two distinct agencies for Na+-dependent proline and alanine translocation. The first system is specific for alanine and short-chain neutral amino-acids; the second system, specific for imino acids and the N-methylated analogues, is regulated by alanine concentration
Atypical PKC-ζ and PKC-ι mediate opposing effects on MCF-7 Na+/K+ATPase activity
No full textWe demonstrated previously that in serum-starved MCF-7 breast cancer cell line, Ang II increased Na+/K(+)ATPase activity and activated the protein kinase C xi(PKC-xi) (Muscellaetal., 2002J Enclocrinol 173:315-323; 2003 J Cell Physiol 197:61 -68.). The aim of the present study was to investigate the modulation of the activity of the Na+/K(+)ATPase by PKC-xi in MCF-7 cells. Here, using serum-starved MCF-7cells, we have demonstrated that the effect of Ang II on the Na+/K(+)ATPase activity was inhibited by a synthetic myristoylated peptide with sequences based on the endogenous PKC-xi pseudosubstrate region (xi-PS) and by high doses of GF109203X, inhibitor of PKCs. When MCF-7 cells, grown in 10% fetal bovine serum (FBS), were stimulated with Ang II adose-and time-dependent inhibition of the Na+/K(+)ATPase activity was obtained. Under this growth condition we found that mRNAs for AT1, AT2, and for Na+/K(+)ATPase alpha(1) and alpha(3) subunits were unchanged; besides both the activity of the Na+/K(+)ATPase and the level of PKC-xi also were unaffected by the serum. The atypical PKC-xi level (present in very low abundance in serum-starved MCF-7) was increased and Ang II provoked its translocation from the cytosol to plasma membrane. PKC-xi was localized to the membrane, and upon Ang II treatment its cellular localization did not change. The Ang II-mediated decrease of the Na+/K(+)ATPase activity was inhibited by high doses of GF109203X but not by xi-PS, thus indicating that such effect was not due to PKC-xi activity. The treatment of cells with PKC-xi antisense oligodeoxynucleotides inhibited the effects of Ang II on the Na+/K(+)ATPase activity. Additionally, the effect of Ang II on Na+/K(+)ATPase activity was also blocked by the phosphatidylinositol 3-kinase (Pl3K) inhibitors, wortmannin and LY294002, and by the actin depolymerizing agents, cytochalasin D. In conclusion, in MCF-7 cells Ang II modulates the Na+/ K(+)ATPase activity by both atypical PKC-xi/-i. The effects of Ang II are opposite depending upon the presence of the serum-sensitive PKC-i, with the inhibitory effect possibly due to the redistribution of sodium pump from plasma membrane to the inactive intracellular pool
Studies of Sparus aurata sperm motility by computer-assisted sperm analysis (CASA).
No full textAim: Sperm of Sparus aurata, like those of other teleosts, become activated when spawned into the external medium. Several extracellular factors (ions, osmotic pressure, O2/CO2, sperm activating peptides) have been reported to control sperm motility activation. These factors act on the flagellar motile apparatus, the axoneme, through signal transduction across the plasma membrane and determining the dynein-mediated sliding of the axonemal outer-doublet microtubules through protein phosphorylation. In the present study we have investigated the role of different proteins involved in this signal transduction cascade using cryopreserved sperm of Sparus aurata.
Methods: Studies have been performed by using cryopreserved sperm (Fabbrocini et al., Cryobiology 40:46-53, 2000). To investigate the role played by different proteins involved in the signal transduction cascade we evaluated: (1) the effect of specific protein inhibitors on sperm motility (assessed by CASA), (2) quantitative and qualitative evaluation of phosphorylated proteins (tyrosine-phosphorylated, serine-phosphorylated and threonine-phosphorylated) by western blot analysis in activated and non-activated sperm.
Results: Results obtained indicated that: (1) calcium and potassium were not involved in sperm motility activation; (2) osmolality values <1,000 mOsm/Kg inhibited the sperm motility activation; (3) inhibition of Adenylyl Cyclase by 500 μM MDL-12330A hydrochloride and Protein-Kinase A (PKA) by 50 μM U73122 prevented the sperm activation; (4) inhibition of Tyrosine-kinase (by 100 μM AG18) and CAM-Kinase (by 50 μM KN93) were ineffective on sperm activation, but determined changes in sperm motility parameters. Particularly, AG18 significantly decreased the Curvilinear Velocity (VCL), while KN93 significantly decreased both Curvilinear Velocity (VCL) and Straight Line Velocity (VSL).
Conclusion: In Sparus aurata sperm, an important factor controlling sperm motility activation was the osmolality of the external medium. The osmotic shock induces sperm activation by cAMP/PKA signaling pathway. In addition also Tyrosine-kinases and CAMkinases seems to be involved in the control of sperm motility
Protein kinase C (PKC)-delta/-epsilon mediate the PKC/Akt-dependent phosphorylation of extracellular signal-regulated kinases 1 and 2 in MCF-7 cells stimulated by bradykinin
No full textIn this paper the signal transduction pathways evoked by bradykinin (BK) in MCF-7 breast cancer cells were investigated. BK activation of the B, receptor provoked: (a) the phosphorylation of the extracellular signal-regulated kinases 1 and 2 (ERK1/2); (b) the translocation from the cytosol to the membrane of the conventional protein kinase C-alpha (PKC-alpha,) and novel PKC-delta and PKC-epsilon; (c) the phosphorylation of protein kinase B (PKB/ Akt); (d) the proliferation of MCF-7 cells. The BK-induced ERK1/2 phosphorylation was completely blocked by PD98059 (an inhibitor of the mitogen-activated protein kinase kinase (MAPKK or MEK)) and by LY294002 (an inhibitor of phosphomositide 3-kinase (PI3K)), and was reduced by GF109203X (an inhibitor of both novel and conventional PKCs); G66976, a conventional PKCs inhibitor, did not have any effect. The BK-induced phosphorylation of PKB/Akt was blocked by LY294002 but not by PD98059. Furthermore, LY294002 inhibited the BK-provoked translocation of PKC-delta and PKC-epsilon suggesting that PI3K may be upstream to PKCs. Finally, the proliferative effects of BK were blocked by PD98059, GF109203X and LY294002. These observations demonstrate that BK acts as a proliferative agent in MCF-7 cells activating intracellular pathways involving novel PKC-6/-E, PKB/Akt and EKK1/2
Molecular mechanism regulating axoneme activation in marine fish: a review
No full textIn many marine fish species, the spermatozoa are immotile in the testis and seminal plasma, and motility is induced when they are released in the aqueous environment. It is well known that the extracellular factors (hyperosmolality or sperm-activating peptides), controlling sperm motility in marine fish, act on the axonemal apparatus through signal transduction across the plasma membrane. To better understand the molecular mechanism regulating axoneme activation in marine fish, the present review examines the existing literature, with particular emphasis on protein phosphorylation/dephosphorylation process.
The present review suggests that: (1) there is no single model that can explain the molecular activation and regulation of sperm motility of the marine fish; (2) only in some species (puffer fish, tilapia, gilthead sea bream, and striped sea bream) protein phosphorylation/dephosphorylation has been shown to be involved in flagellar motility regulation; (3) only a few proteins were identified, which show a change in their state of phosphorylation following sperm activation.
A model of molecular mechanism controlling the activation of sperm motility in gilthead sea bream is being proposed here, which could be a useful model to clarify the sperm motility activation process in other species
In vitro methods and results of ascorbic acid absorption in epithelial tissues of fish
No full textL-ascorbic acid (AA) is an essential nutrient for several teleost species. It plays an important role in many enzymatic reactions to maintain prosthetic metal ions in their reduced form (for example, Fe2+, Cu+) and for scavenging free radicals to protect tissues from oxidative damage. In mammals, it has recently been shown that the facilitative sugar transporters of the GLUT type (GLUT1 and GLUT3) can transport the oxidized form of the vitamin dehydro-L-ascorbic acid. However, the bulk of the vitamin, which is present in the plasma essentially in its reduced form, is carried by the Na+-dependent AA transporters SVCT1 (sodium-dependent vitamin C transporter 1) and SVCT2 (sodium-dependent vitamin C transporter 2), which have recently been functionally expressed in Xenopus oocytes, cloned and sequenced. SVCT1 is mainly confined to epithelial tissues, such as intestine, kidney and liver. In fish, many results, as obtained by different in vitro techniques, have detailed the presence of an electrogenic, Na+-coupled AA transport mechanism at the brush-border membrane of enterocytes, with kinetic characteristics similar to those found in mammals (apparent Km ranging between 0.22 and 0.75 mM), when measured using the same experimental approaches. At the basolateral level of fish intestinal absorbing epithelial cells, transport of dehydro-L-ascorbic acid (DHA) is mediated by Na+-independent transport pathway(s), presumably belonging to the GLUT type family as found in mammals, although this has not been demonstrated so far. We report data on both the kinetic characteristics of vitamin C transport through biological membranes of epithelial cells and the experimental approaches used over the time to study AA absorption in fish. The possibility of getting new theoretical information on ascorbic acid absorption and metabolism in fish by using the Xenopus laevis expression system and the perspective to develop new biotechnological applications in aquaculture by gene transfer are also pointed out
Angiotensin II stimulates the Na+/H+ exchanger in human umbilical vein endothelial cells via AT1 receptor
No full textAngiotensin II (Ang II) has an important role in cardiovascular regulation and in the control of electrolyte balance, and its role in the regulation of Na+ transcellular movements through its actions on the activity of Na+/K+ ATPase is well documented. We showed previously that human umbilical vein endothelial cells (HUVEC) express the Ang II type 1 (AT1) receptor, which mediates Ang II modulation of Na+/K(+)ATPase activity (1). We here investigate the effects of Ang II on the activity of the Na+/H+ exchanger in HUVEC. When compared with controls, incubation of HUVEC for 20 min with different concentrations of Ang II provoked significant increases in Na+/H+ activity. The stimulation was dose dependent between 1 and 10 nM Ang II and varied with time of incubation up to 20 min. The maximal response, obtained with 10 nM Ang II after 20 min treatment, resulted in a 65% increment in Na+/H+ activity. Preincubation of HUVEC with 10 mu M DuP753 blocked Na+/H+ activation by Ang II. These results suggest that the effects of Ang II on both the Na+/K(+)ATPase and the Na+/H+ exchanger may increase the transendothelial flux of Na+ and are mediated by the AT1 receptor
- …
