1,721,161 research outputs found
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
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
H+-glycyl-L-proline cotransport by brush-border membrane vesicles of eel intestinal epithelium
No full textDexamethasone modulates the activity of the eel branchial Na+/K+ATPase in both chloride and pavement cells
No full textIn fish, gills actively accumulate ions in freshwater (FW) with Na+ absorption taking place at the level of pavement cells, and excrete monovalent ions, mainly Na+ and Cl-, through the chloride cells in sea water (SW). The Na+/K(+)ATPase plays a crucial role in the functionality of osmoregulatory cells and we showed previously that angiotensin II modulates its activity in the eel gill (1). We here show the effects of synthetic steroid dexamethasone (DEX) on the activity of Na+/K+ATPase in both gill pavement and chloride cells from FW- and SW-adapted animals. Results showed that in the chloride cells 100 nM DEX provoked a significant increment in the activity of Na+/K(+)ATPase in both SW- and FW-adapted animals. This effect was greatest at 2 hours in SW, and at 6 hours in FW. The increment in the activity of the Na+/K(+)ATPase was dose-dependent in both environmental adaptations, Conversely, in pavement cells from FW-adapted eels 100 nM DEX decremented the activity of Na+/K(+)ATPase (4-fold reduction after 6 hour incubations), while in SW, DEX increased the enzyme activity of about 25% at 2 hours, and of about 55% at 6 hours. These results are consistent with the different physiological roles that pavement and chloride cells have in the two different adaptive conditions
ANALYSIS OF COPPER TRANSPORT IN B104 NEUROBLASTOMACELLS
No full textCopper is an essential trace metal required as a cofactor in a broad range
of enzymatic functions including cellular antioxidant activity, oxidative
phosphorylation and neurological function. If in excess, it can initiate a
cascade of events leading to cell disruption. Consequently, cells have
developed sophisticated homeostatic mechanisms to maintain adequate
intracellular copper concentrations (Pena et al., J. Nutr. 129(7): 1251-60,
1999). In the attempt to characterise the components of brain copper
homeostasis, we studied the expression and function of copper transport
systems in a rat neuroblastoma cell line (B104). In our cell model copper
fluxes could be related to the functional expression of a member of the
Ctr family named rat Ctr1 and to the primary active copper transport
MNK protein. In both cases we demonstrated their mRNA expression in
B104 cells using specific primers and the RT-PCR amplification method.
Transmembrane copper fluxes were measured by loading cells with the
Cu2+-sensitive fluorophore Phen Green SK. Copper addition in the extra
cellular medium induced a rapid fluorescence quenching of the entrapped
probe, probably due to copper entry via Ctr1 protein, followed by a fast
recovery of fluorescence related to a putative MNK-mediated copper
efflux. The initial rate of copper uptake was dependent on external ion
concentrations and saturated at approximately 1 5M Cu2+. Furthermore,
we found that zinc can affect copper transport by partially inhibiting the
uptake. In conclusion, our results support the hypothesis that copper
transport in neuroblastoma cells is a complex phenomenon presumably
mediated by more than a single carrier process
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