Aiming to clarify the mechanism of inhibition of (Na
+
, K
+
)-ATPase activity by polyamines, we examined the effects of exogenous putrescine, spermidine, and spermine on the kinetic behavior of ...phosphoenzyme-linked partial reactions using a microsomal gill (Na
+
, K
+
)-ATPase from juvenile and adult
M. amazonicum
, a freshwater palaemonid shrimp. The time course of phosphointermediate formation is greater (0.089 ± 0.006 s
−1
) in adults than in juveniles (0.053 ± 0.003 s
−1
) for spermidine, but similar to juveniles (0.059 ± 0.004 s
−1
) for putrescine. Maximum phosphointermediate formation for the (Na
+
, K
+
)-ATPase from juveniles decreased by 46% and 32% with spermidine and putrescine, respectively. In adults, maximum phosphointermediate levels decreased by 50% and 8%, respectively. For both spermidine and putrescine, dephosphorylation rates were higher for adults than for juveniles, and were higher than in controls without polyamines. Spermine had a negligible effect (<10%) on phosphorylation/dephosphorylation rates of both juvenile and adult enzymes. This is the first report on the effects of polyamines on phosphoenzyme-linked partial reactions in juvenile and adult
M. amazonicum
gill (Na
+
, K
+
)-ATPases. Our findings suggest that the phosphorylation/dephosphorylation steps of this gill enzyme may be regulated by polyamines during ontogenetic development.
Cardiac glycosides (CGs) are useful drugs to treat cardiac illnesses and have potent cytotoxic and anticancer effects in cultured cells and animal models. Their receptor is the Na
+
,K
+
ATPase, but ...other plasma membrane proteins might bind CGs as well. Herein, we evaluated the short- and long-lasting cytotoxic effects of the natural cardenolide glucoevatromonoside (GEV) on non-small-cell lung cancer H460 cells. We also tested GEV effects on Na
+
,K
+
-ATPase activity and membrane currents, alone or in combination with selected chemotherapy drugs. GEV reduced viability, migration, and invasion of H460 cells spheroids. It also induced cell cycle arrest and death and reduced the clonogenic survival and cumulative population doubling. GEV inhibited Na
+
,K
+
-ATPase activity on A549 and H460 cells and purified pig kidney cells membrane. However, it showed no activity on the human red blood cell plasma membrane. Additionally, GEV triggered a Cl-mediated conductance on H460 cells without affecting the transient voltage-gated sodium current. The administration of GEV in combination with the chemotherapeutic drugs paclitaxel (PAC), cisplatin (CIS), irinotecan (IRI), and etoposide (ETO) showed synergistic antiproliferative effects, especially when combined with GEV + CIS and GEV + PAC. Taken together, our results demonstrate that GEV is a potential drug for cancer therapy because it reduces lung cancer H460 cell viability, migration, and invasion. Our results also reveal a link between the Na
+
,K
+
-ATPase and Cl
-
ion channels.
Graphic Abstract
Eight molecules, four peptides (SPs) and four lipopeptides (LPs) derived by rational design from surfactin, a well‐known secreted biosurfactant from Bacillus subtilis, were produced employing ...Fmoc‐based solid‐phase synthesis. These new peptides were tested to evaluate their potential biosurfactant and biological activities, aiming at possible applications in industrial, biological, pharmaceutical, and medical use. Five molecules (SP1, SP2, SP4, LP5, and LP8) presented potential for medical uses, mainly due to their drug delivery properties as suggested by their synergistic activity with the antibiotic vancomycin against Staphylococcus aureus. All synthetic peptides showed low toxicity against Vero cell cultures, in assays of hemolysis, and in different cytotoxicity assays. In addition, we found that three peptides (SP1, LP6, and LP7) had potential technological and industrial use because of their emulsifying capacity, low toxicity, and ability to physically stabilize solutions. These novel molecules retained some properties of the parental molecule (surfactin, which was originally obtained through nonribosomal synthesis in Bacillus subtilis) but have the advantage of being linear peptides, which can be produced at large scales through the use of conventional heterologous protein expression protocols.
We synthesized eight novel linear peptides derived from surfactin using classical Fmoc‐based solid‐phase synthesis method for their production. All of the peptides had surface tension reduction activity, and several of them facilitated the delivery of the antibiotic vancomycin, exhibiting synergism with its action against a virulent strain of S aureus (MU50). Cytotoxicity was evaluated through different assays; the promising results indicated that all of the peptides were poorly cytotoxic and that only one (LP8) had considerable hemolytic activity.
We describe in this paper that the alkaloid 4-methylaaptamine, isolated from the marine sponge Aaptos aaptos, inhibited HSV-1 infection. We initially observed that 4-methylaaptamine inhibited HSV-1 ...replication in Vero cells in a dose-dependent manner with an EC50 value of 2.4 microM. Moreover, the concentration required to inhibit HSV-1 replication was not cytotoxic, since the CC50 value of 4-methylaaptamine was equal to 72 microM. Next, we found that 4-methylaaptamine sustained antiherpetic activity even when added to HSV-1-infected Vero cells at 4 h after infection, suggesting that this compound inhibits initial events during HSV-1 replication. We observed that 4-methylaaptamine impaired HSV-1 penetration without affecting viral adsorption. In addition, the tested compound could inhibit, in an MOI-dependent manner, the expression of an HSV-1 immediate-early protein, ICP27, thus preventing the inhibition of macromolecular synthesis induced by this virus. Our results warrant further investigation on the pharmacokinetics of 4-methylaaptamine and propose that this alkaloid could be considered as a potential compound for HSV-1 therapy.
•The sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) is inhibited by chelerythrine.•The inhibition is noncompetitive with ATP and shows high and low affinity sites.•The high affinity component has a ...Ki near to that described for PKC inhibition.•The E2 conformer of SERCA is favored by chelerythrine binding.•PBMC calcium levels increased after 30min when exposed to 1μM chelerythrine.
The isoquinoline alkaloid chelerythrine is described as an inhibitor of SERCA. The ATPase inhibition presented two non-competitive components, Ki1=1, 2μM and Ki2=26μM. Conversely, chelerythrine presented a dual effect on the p-nitrophenylphosphatase (pNPPase) of SERCA. Ca2+-dependent pNPPase was activated up to ∼5μM chelerythrine with inhibition thereafter. Ca2+-independent pNPPase was solely inhibited. The phosphorylation of SERCA with ATP reached half-inhibition with 10μM chelerythrine and did not parallel the decrease of ATPase activity. In contrast, chelerythrine up to 50μM increased the phosphorylation by Pi. Cross-linking of SERCA with glutaraldehyde was counteracted by high concentrations of chelerythrine. The controlled tryptic digestion of SERCA shows that the low-affinity binding of chelerythrine evoked an E2-like pattern. Our data indicate a non-competitive inhibition of ATP hydrolysis that favors buildup of the E2-conformers of the enzyme. Chelerythrine as low as 0.5–1.5μM resulted in an increase of intracellular Ca2+ on cultured PBMC cells. The inhibition of SERCA and the loss of cell Ca2+ homeostasis could in part be responsible for some described cytotoxic effects of the alkaloid. Thus, the choice of chelerythrine as a PKC-inhibitor should consider its potential cytotoxicity due to the alkaloid’s effects on SERCA.
In developing countries, the access to red blood cell (RBC) irradiators is restricted. Thus, it is a common practice in blood banks to stock irradiated RBC units until they expire. The aim of this ...work is to elucidate the involvement of Na,K-ATPase in potassium leakage from prophylactically irradiated RBCs.
Whole blood was collected from healthy donors, and blood concentrates were irradiated with 25Gy of γ-radiation within 24h of collection. At days 3, 5, 7, 9, 11, 14 and 28 post-irradiation, fractions were removed and centrifuged and Na,K-ATPase activity from ghost membranes was determined.
The inhibition of Na,K-ATPase activity in RBCs reached 12.6% by day 7 of storage and up to 50% by day 14 of storage. The addition of vitamin C prevented the irradiation-induced loss of Na,K-ATPase activity. The irradiation of RBCs provoked an increase in potassium plasma levels and a decrease in sodium plasma levels. The incubation of RBCs with ouabain did not change the sodium or potassium levels in the plasma, and the addition of vitamin C only partially prevented a decrease in sodium levels caused by irradiation.
Because the inhibition of Na,K-ATPase by ouabain did not cause potassium accumulation in the plasma, we conclude that the irradiation-induced inhibition of the pump is not a key factor driving this effect.
•We verify the effect of gamma irradiation on red blood cell and Na,K-ATPase effect.•The effect we measured at days 3, 5, 7, 9, 11, 14 and 28 post-irradiation.•We analyzed the effect of vitamin C on prevent membrane damages.•We reported that Na,K-ATPase is not the key role for potassium leakage effect of irradiation.•The addition of vitamin C prevent the effect of irradiation on Na,K-ATPase activity but not in potassium leakage.
► FXYD2 is phosphorylated by PKC, by PKA or both simultaneously, modulating Na,K-ATPase and PMCA. ► PKC phosphorylates a threonine residue and PKA marks a serine residue of FXYD2. ► Regulatory ...phosphorylation of the FXYD2 has differential effects on calcium uptake of the PMCA. ► Basal and calmodulin-stimulated activities of PMCA were regulated by phosphoforms of FXYD2. ► Conservation of FXYD2 anchoring site among P-type ATPases permits cross-regulation.
FXYD2 is a regulatory peptide associated with the α-subunit of the kidney Na,K-ATPase. FXYD2 can be phosphorylated by PKA, and its phosphorylation activates Na,K-ATPase. Here we show that FXYD2 is phosphorylated by PKC (PKC-FXYD2-P), by PKA (PKA-FXYD2-P) or by PKA and PKC simultaneously (FXYD2-P
2) modulating both the erythrocyte Na,K-ATPase and the plasma membrane Ca
2+-ATPase (PMCA). In erythrocyte ghosts, the addition of PKA-FXYD2-P activated Na,K-ATPase by 80%, while non-phosphorylated FXYD2 (np) activated only 55%. The addition of np FXYD2 did not affect PMCA basal activity, but FXYD2-P
2 increased the basal PMCA activity by up to 200%. Calmodulin-activated PMCA activity was increased by np FXYD2 (3-fold) or FXYD2-P
2 (2.5-fold). However, PKC-FXYD2-P increased PMCA activity only by 50%. In contrast, when PMCA was treated with PKA-FXYD2-P, the ATPase activity was inhibited by 50%. The effect of all forms of FXYD2-P on calcium uptake from PMCA resembled the pattern observed in ATP hydrolysis. Our results suggest that the FXYD2 anchoring site could be conserved among the P-ATPase family permitting cross regulation. The effects of FXYD2 on calcium uptake and calcium-stimulated ATP hydrolysis suggest a novel role for FXYD2 on PMCA.
Ouabain is a steroid derivative that can regulate many cellular events such as growth and proliferation. It modulates Na
+,K
+-ATPase activity leading to the activation of different intracellular ...pathways through protein–protein interactions that have been characterized during the last few years. The aim of this work was to study the role of ouabain in rat retinal ganglion cell survival after 48 h in culture. Our results demonstrated that ouabain significantly induced an increase in retinal ganglion cell survival. The effect was dose-dependent and was maximal with 3.0 nM. The blockade of protein kinase C activity by 1.25 μM chelerythrine chloride abolished the ouabain effect, indicating an involvement of this intracellular pathway. None of the protein kinase inhibitors usually employed in the study of ouabain-driven intracellular pathways (PD98059, Ly294002, herbimycin, and genistein) was able to influence neuronal survival induced by ouabain. The data presented suggest that ouabain may be the trigger of an intracellular pathway responsible for neuronal survival.
Aiming to clarify the mechanism of inhibition of (Na super(+), K super(+))-ATPase activity by polyamines, we examined the effects of exogenous putrescine, spermidine, and spermine on the kinetic ...behavior of phosphoenzyme-linked partial reactions using a microsomal gill (Na super(+), K super(+))-ATPase from juvenile and adult M. amazonicum, a freshwater palaemonid shrimp. The time course of phosphointermediate formation is greater (0.089 plus or minus 0.006 s super(-1)) in adults than in juveniles (0.053 plus or minus 0.003 s super(-1)) for spermidine, but similar to juveniles (0.059 plus or minus 0.004 s super(-1)) for putrescine. Maximum phosphointermediate formation for the (Na super(+), K super(+))-ATPase from juveniles decreased by 46% and 32% with spermidine and putrescine, respectively. In adults, maximum phosphointermediate levels decreased by 50% and 8%, respectively. For both spermidine and putrescine, dephosphorylation rates were higher for adults than for juveniles, and were higher than in controls without polyamines. Spermine had a negligible effect (<10%) on phosphorylation/dephosphorylation rates of both juvenile and adult enzymes. This is the first report on the effects of polyamines on phosphoenzyme-linked partial reactions in juvenile and adult M. amazonicum gill (Na super(+), K super(+))-ATPases. Our findings suggest that the phosphorylation/dephosphorylation steps of this gill enzyme may be regulated by polyamines during ontogenetic development.
The meroditerpenoids atomaric acid (1), epitaondiol (2) and the peroxylactone of 5'a-desmethyl-5'-acetylatomaric acid (3) were isolated from the Brazilian brown alga Stypopodium zonale collected in ...two localities (Búzios and Marataízes, RJ and ES States). These compounds showed strong anti-HSV-1 activity in vitro but neither of them inhibited the transcriptase reverse enzyme of HIV-1.
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