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At least 703 records · Page 39Linked to original sources
PubMed1997

Palytoxin-induced channel formation within the Na+/K+-ATPase does not require a catalytically active enzyme.

It has been demonstrated that palytoxin binds to and forms a channel within the Na+/K+-ATPase. To investigate whether palytoxin-induced channel formation within the sodium pump can occur independently of ATP hydrolysis and phosphorylation of the enzyme, an Asp369-->Ala mutant of the alpha1 subunit of the sheep sodium pump was produced and coexpressed with beta subunits in the yeast Saccharomyces cerevisiae. This aspartic acid residue, which during ion transport becomes phosphorylated from ATP, is essential for the function of the sodium pump. Therefore, as expected, microsomes isolated from yeast expressing the mutant sodium pump do not exhibit any ouabain sensitive ATPase activity, whereas in microsomes from yeast expressing the wild-type sodium pump, 60% of the total ATPase activity is ouabain-sensitive. Ouabain binds to yeast membranes containing either wild-type or mutant sodium pumps with similar Bmax (1.45+/-0.05 versus 1.37+/-0.02 pmol/mg) and Kd values (27.7+/-0.91 versus 29.57+/-0.93 nM), thus indicating that the mutant sodium pumps are expressed in the yeast and that the mutation does not considerably affect the conformation of the enzyme. In the presence of phosphate ouabain binds to microsomes containing the wild-type sodium pump with a Kd of 3.62+/-0.34 nM, showing that, although not necessary, phosphoenzyme formation enhances binding of the steroid. Phosphate or ATP, however, inhibit binding of ouabain to microsomes containing the mutant sodium pump with IC50 values of 78+/-3 microM and 3.0+/-0.4 microM, respectively. Despite these radical changes in the interactions of the mutant enzyme with ouabain, the interactions with palytoxin are not affected by the mutation. Palytoxin causes K+ efflux from yeast cells expressing the wild-type or mutant sodium pumps with EC50 values of 3.5+/-0.4 nM and 6.2+/-0.9 nM, respectively. Palytoxin-induced efflux from cells expressing wild-type or mutant sodium pumps occurs with similar t1/2 values of 20.3+/-2.1 min and 22.2+/-3.1 min, respectively. Ouabain inhibits K+ efflux from both cell types with IC50 values of 28+/-2 microM and 210+/-15 microM, respectively. Cells expressing the Asp369-->Ala mutants have an IC50 7.5-fold higher than that obtained with cells expressing the wild-type sodium pumps, possibly because ATP or phosphate present in the cytosol of the yeast cells influence and decrease ouabain binding to the mutant sodium pump. Thus, while ouabain binding and the associated inhibition of ion fluxes is promoted by phosphorylation of the wild-type enzyme by phosphate or ATP, palytoxin-induced channel formation is independent of phosphorylation and can be separated from the ATPase function of the sodium pump. Since ion fluxes through the sodium pump protein do not depend on ATP hydrolysis, the results suggest that the ionophores of pumps and ion channels might share common structural features.

Acrylamides↗
PubMed1991

Mode of action of palytoxin on the release of acetylcholine from rat cerebrocortical synaptosomes.

Palytoxin (PTX; 10(-14)-10(-6) M) caused a dose-dependent increase in the release of [3H]acetylcholine ([3H]ACh), cytosolic free Ca2+ concentration ([Ca2+]i), and uptake of 22Na+ and decrease in membrane potential in rat cerebrocortical synaptosomes. The dose-response curves for the PTX-induced increases in [3H]ACh release and in [Ca2+]i were depressed by removing extracellular Ca2+ or by decreasing extracellular Na+ concentrations. The release of [3H]ACh induced by concentrations of PTX less than 10(-10) M was more dependent on the simultaneous presence of both Ca2+ and Na+ than the release induced by higher concentrations of PTX. The PTX-induced increase both in [3H]ACh release and in [Ca2+]i was almost completely abolished by the combination of Ca2+ deprivation and Na+ concentration reduction. All responses to PTX were highly resistant to 10(-6) M tetrodotoxin. These results suggest that low concentrations of PTX cause depolarization as a result of an increase in Na+ permeability through tetrodotoxin-insensitive channels. This, in turn, increases Ca2+ influx and leads to an increase in the release of ACh. It appears that at high concentrations PTX increases the release of [3H]ACh by directly increasing the influx of Ca2+ into synaptosomes and by releasing Ca2+ from intracellular storage sites via an Na(+)-Ca2+ exchange mechanism.

Acetylcholine↗
PubMed1980

The potent depolarizing action of palytoxin isolated from Palythoa tubercurosa on the isolated spinal cord of the frog.

1. The effects of palytoxin (PTX) isolated from Palythoa tubercurosa were tested on the isolated intra-arterially perfused spinal cord of the frog. The resting and evoked potentials were recorded by means of a sucrose-gap technique. 2. PTX caused a marked depolarization of both ventral and dorsal roots. The minimum effective concentration was extremely low, approximately 10(-11) M. During the depolarization the evoked ventral and dorsal root potentials were markedly reduced in amplitude. The ventral root reflex was first augmented and then decreased. 3. The depolarization caused by PTX was markedly reduced when the preparation was perfused with NaCl-deficient medium. However, tetrodotoxin (10(-7) M) only slightly inhibited the depolarization. 4. In a high Ca2+ medium (3.6 mM), the time required to reach the maximum depolarization evoked by PTX was significantly prolonged. In contrast, in a low Ca2+ medium (0.9 mM), PTX caused a marked depolarization soon after application. In a Ca2+-free, Mg2+ (9.0 mM) medium, PTX caused rhythmic oscillatory potentials in both ventral and dorsal roots. 5. The potency of N-acetyl PTX was one hundredth that of the parent compound. 6. It is suggested that PTX may interfere with the stabilizing action of Ca2+ on the neuronal membrane, consequently facilitating Na+ permeability. The primary amine in the PTX molecule may be important for its pharmacological action.

Acrylamides↗
PubMed1988

Single ionic channels induced by palytoxin in guinea-pig ventricular myocytes.

1. Mechanisms of palytoxin-induced ion permeability were examined in isolated single ventricular cells of guinea-pig under whole-cell-attached patch clamp conditions. 2. Palytoxin (1-2 x 10(-11) M, dissolved in Tyrode solution and put in the patch electrode) induced an elementary current flowing through single channels. Direction of the current was inward and the amplitude was 0.65 +/- 0.03 pA (mean +/- s.e. mean) at the resting membrane potential. The amplitude increased linearly with membrane hyperpolarization and decreased with depolarization; the single channel conductance was 9.5 +/- 0.5 pS. 3. Palytoxin-induced single channel current was resistant to tetrodotoxin (5 x 10(-5) M) or cobalt ions (2 x 10(-3) M) and was observed under Ca-free conditions. However, no channel current was induced by palytoxin (10(-11) - 10(-9) M) dissolved in Na+-free, choline-Tyrode solution. 4. Palytoxin also induced single channel currents in Na+-free, NH4+-, Li+- or Cs+-Tyrode solution, and the slope conductances were 16.5 +/- 1.6 pS, 9.2 +/- 0.7 pS and 11.0 +/- 0.7 pS, respectively. 5. These results indicate that palytoxin forms a new type of ionic channel with unique ion selectivity and gating behaviour.

Acrylamides↗
PubMed1991

Characterization of the palytoxin-induced sodium conductance in frog skeletal muscle.

1. The effects of palytoxin (PTX) on transmembrane potentials and currents of frog skeletal muscle were analyzed by intracellular microelectrode techniques and the double sucrose-gap voltage clamp method. 2. PTX irreversibly depolarized the membrane. The depolarization was Na-sensitive. 3. Under voltage clamp, PTX induced an inward resting current which did not inactivate, was inhibited by external Na+ removal and was a function of external Na concentration. 4. This resting current could be carried either by Na+, Li+, K+ or by guanidinium according to the permeability sequence K+ less than Li+ less than Na+ less than Gua+. 5. The PTX-induced current was only weakly sensitive to tetrodotoxin. It was reversibly and dose-dependently inhibited by amiloride with a one to one stoichiometry and a KD of 0.3 mM. 6. Acidic pH partially inhibited the current induced by PTX which was also highly sensitive to external Cd2+ and La3+. The inhibitory sequence for divalent cations was: Mg2+ less than Ca2+ = Ba2+ = Mn2+ less than Cd2+; with La3+ greater than Cd2+. 7. The amplitude of the PTX-induced I(rest) was markedly reduced in the absence of external Ca2+. 8. PTX induced a Na+ resting conductance in frog skeletal muscle. The size of the channel induced by PTX is larger than the guanidinium ion. External membrane Ca2+ might be a cofactor involved in the mode of action of PTX.

Acrylamides↗
PubMed1993

3,4 dichlorobenzamil-sensitive, monovalent cation channel induced by palytoxin in cultured aortic myocytes.

1. Smooth muscle cells were dispersed from rat aorta and then cultured. The action of palytoxin on rat aortic myocytes was analysed by measurement of 22Na+ uptake and single channel recording techniques. 2. Palytoxin induced an increase in 22Na+ uptake, with a concentration of 50 nM producing half-maximal activation. The action of palytoxin was inhibited by amiloride derivatives and by ouabain. The concentrations of inhibitor producing half-maximal inhibition were 10 microM for 3,4 dichlorobenzamil, 30 microM for benzamil, 100 microM for phenamil and 1 mM for ouabain. 3. In outside-out patches, palytoxin induced single channel currents that reversed near 0 mV with NaCl or KCl in the extracellular solution, but were outward with N-methyl-D-glucamine chloride or CaCl2 (110 mM), indicating that palytoxin induced a cation channel permeable to Na+ and K+ (PK/PNa = 1.2) but not to Ca2+ (PK/PCa > 30) or to N-methyl-D-glucamine (NMDG) (PK/PNMDG > 11) The unit channel conductance was 11-14 pS. 4. A high (> 0.1 mM) extracellular concentration of Ca2+ was necessary to observe channel activation by palytoxin. A high (150 mM) extracellular concentration of K+ partially prevented and reversed channel activation by palytoxin. 5. The channel activity was fully blocked by 3,4 dichlorobenzamil (20 microM) and partially blocked by phenamil (50 microM). It was not reduced by ouabain (200 microM).

Acrylamides↗
PubMed1997

Transposing results from an artificial minicell to a real cell. Experimental evidence for a working hypothesis linking Na,K-ATPase permeability states to specific alterations of cell life.

The present work sets in parallel data obtained with the Na,K-ATPase in artificial vesicular membranes (artificial minicells) and in peripheral human lymphocytes ex vivo. The Na,K-ATPase was purified and reconstituted into single-walled, tight liposomes filled with a reservoir of ATP and Na in which the Na,K-ATPase functions as in cells, that is, the receptor is accessible on the liposome surface (artificial minicells). In this system, an E2-ouabain state impermeable to Rb ions and an Na,K-ATPase-palytoxin state leaky for Rb ions were characterized. The tight E2-ouabain form preserves the viability of isolated lymphocytes, whereas the leaky Na,K-ATPase-palytoxin induces rapid cell bursting and death by a ouabain-sensitive mechanism. Thus, the effect of Na,K-ATPase inhibitors on lymphocyte survival can be predicted from permeability measurements in artificial minicells as verified also with the leak-inducing metals mercury and silver.

Acrylamides↗
PubMed2003

Cation stoichiometry and cation pathway in the Na,K-ATPase and nongastric H,K-ATPase.

The mechanism of cation translocation by the Na,K-ATPase was investigated by cysteine scanning mutagenesis and measurements of accessibility through exposure to cysteine reagents. In the native protein, accessible residues were found only at the most extracellular residues of the 5th and 6th transmembrane segments (TMS) and the short loop between them. However, after modification by palytoxin a number of residues became accessible along the whole length of the 5th TMS and in the outer half of the 6th TMS, showing the contribution of each of these segments to the "channel" formed by the palytoxin-transformed Na,K-pump. Assuming that this structure is similar in the native and the palytoxin-transformed pump, our data allow us to determine the residues lining the cation pathway from the extracellular solution to their binding sites. A critical position in the 5th TMS contains a lysine conserved in all known nonelectrogenic H,K-ATPases, and a serine in all known electrogenic Na,K-ATPase sequences. Wild-type or mutant Na,K-or H,K-ATPase a subunits were coinjected with the Bufo beta2 subunit in Xenopus oocytes and Rb(86) uptake and electrophysiological measurements were performed. An electrogenic activity was recorded for the H,K-ATPase mutants in which the positively charged lysine had been replaced by neutral or negatively charged residues, while nonelectrogenic transport was observed with the S(782)R mutant of the Na,K-ATPase. The presence or the absence of a positively charged residue at the S(782) position appears to be critical for the stoichiometry of cation exchange.

Acrylamides↗
PubMed1999

Unusual antimicrotubule activity of the antifungal agent spongistatin 1.

Spongistatin 1, a macrocyclic lactone from the marine sponge Hyrtios erecta, has broad-spectrum antifungal activity. Since this compound is a potent antimicrotubule agent in mammalian cells, we examined its effects on the filamentous fungus Aspergillus nidulans to determine if its antifungal effects are due to antimicrotubule activity. At 25 microg/ml (twice the MIC), spongistatin 1 caused a greater-than-twofold elevation of the chromosome and spindle mitotic indices. Immunofluorescence microscopy revealed that mitotic spindles were smaller and shorter than in control germlings. However, late-anaphase and telophase nuclei were seen occasionally, and this suggests that the spindles are capable of segregating chromosomes. Spongistatin 1 had more dramatic effects on cytoplasmic microtubules. At 30 min after initiation of treatment, 83% of germlings contained fragmented microtubules and after 2 h of treatment, microtubules had disappeared completely from 82% of germlings. In contrast, microtubules disappeared rapidly and completely from germlings treated with benomyl. We conclude that spongistatin 1 has antimicrotubule activity in A. nidulans and that its mechanism of action may involve a novel microtubule-severing activity.

Antifungal Agents↗
PubMed1995

Actions of palytoxin on Na+ and Ca2+ homeostasis in human osteoblast-like Saos-2 cells.

Palytoxin (PTx) is a potent membrane-active agent produced by marine coelenterates that acts to stimulate bone resorption in organ culture at nanomolar concentrations. We report here the actions of PTx on Na+ and Ca2+ homeostasis in human osteoblast-like Saos-2 cells. PTx induced a rise in the cytosolic free Na+ concentration ([Na]i) by causing entry of extracellular Na+ (Na(e)+). PTx also caused a concentration-dependent biphasic rise in the cytosolic free Ca2+ concentration ([Ca2+]i) by enhancing entry of extracellular Ca2+ (Ca(e)2+). Entry of Na+ was dependent on the presence of Ca(e)2+ and was prevented by the Na+/Ca2+ exchange antagonist 3,4-dichlorobenzamil (DCB). Entry of Ca2+ was dependent on the presence of Na(e)+ but was not prevented by DCB. The actions of PTx on [Na+]i and [Ca2+]i were completely inhibited by pretreatment of the cells with ouabain. Ouabain alone had no acute effect on [Na+]i or [Ca2+]i in Saos-2 cells. We propose that interaction of PTx with the Na+ pump created a channel that allowed influx of Na(e)+ and Ca(e)2+. The rise in [Ca2+]i then stimulated the activity of the plasma membrane Na+/Ca2+ exchanger, which further enhanced Na(e)+ entry.

Acrylamides↗
PubMed1996

Palytoxin modulates cytosolic pH in human osteoblast-like Saos-2 cells via an interaction with Na(+)-K(+)-ATPase.

Palytoxin (PTx) at nanomolar concentrations enhances the permeability of mammalian cell membranes to both Na+ and Ca2+. In basal human osteoblast-like Saos-2 cells, PTx (8 nM) caused a persistent decrease in cytosolic pH (pHi) of about 0.2 units, which required the presence of extracellular Ca2+ (Cae2+) and Na+ (Nae+). We acidified Saos-2 cells by incubation with nigericin to examine the action of PTx in cells with an activated Na+/H+ antiporter. Under these conditions, PTx increased the pHi without requiring Cae2+ or Nae+, and the alkalinization was unaffected by hexamethylene amiloride. We conclude that the PTx-induced rise in pHi did not involve the Na+/H+ antiporter. PTx increased the rate of 86Rb+ efflux. We propose that PTx induced alkalinization in nigericin-acidified cells by collapsing the K+ gradient. Exposure to ouabain had no effect on pHi, but it prevented the actions of PTx on PHi in both basal and nigericin-acidified cells. Ouabain-resistant mutant cells were less sensitive to PTx in extruding 86Rb+ than their ouabain-sensitive parents. We conclude that PTx interacts with the Na(+)-K(+)-adenosinetriphosphatase to regulate pHi in both basal and nigericin-acidified Saos-2 cells.

Acrylamides↗
PubMed1997

Amino acids Val115-Ile126 of rat gastric H(+)-K(+)-ATPase confer high affinity for Sch-28080 to Na(+)-K(+)-ATPase.

Na(+)-K(+)-ATPase is inhibited by cardiac glycosides and is insensitive to Sch-28080, an inhibitor of gastric H(+)-K(+)-ATPase. Gastric H(+)-K(+)-ATPase is not inhibited by cardiac glycosides. Both ouabain and, Sch-28080 binding are inhibited by K+, and it has been suggested that the inhibitors bind to corresponding regions on the alpha-subunit of each ion pump. For identification of regions of each pump that interact with the specific inhibitors, chimeric alpha-subunits consisting of selected regions from Na(+)-K(+)-ATPase and gastric H(+)-K(+)-ATPase have been prepared. One chimera (gM1/2) has been constructed from cDNA of the sheep alpha1-subunit of Na(+)-K(+)-ATPase by replacement of the last 12 amino acids of the first predicted transmembrane region (Ile99-Ile110) with corresponding amino acids from rat gastric H(+)-K(+)-ATPase. gM1/2 was expressed in yeast cells together with either the rat Na(+)-K(+)-ATPase beta 1-subunit (NK beta 1) or rat gastric H(+)-K(+)-ATPase beta-subunit (HK beta). Western blots show that the expression level of the chimeric alpha-subunit was comparable to the Na(+)-K(+)-ATPase alpha 1. Ouabain binds with high affinity to gM1/2+NK beta 1 [ouabain binding affinity (Kd) = 9.5 nM] but not to gM1/2+HK beta. The Kd for ouabain binding to Na(+)-K(+)-ATPase was 7.8 nM. Na(+)-K(+)-ATPase activity of gM1/2+NK beta 1 was inhibited both by ouabain and Sch-28080. The 50% inhibition concentration for Sch-28080 was 20-60 nM. Sch-28080 at 10 microM did not inhibit Mg(2+)- and Pi-dependent ouabain binding to gM1/2+NK beta 1. Ouabain (0.75 mM) inhibited palytoxin-induced K+ efflux from yeast cells expressing either gM1/2+NK beta 1 or gM1/2+NK beta, and Sch-28080 increased the palytoxin-induced K+ efflux from yeast cells expressing gM1/2+NK beta 1 or gM1/2+HK beta. These results implicate a small number of amino acids in the first transmembrane part of gastric H(+)-K(+)-ATPase as partial determinants of the sensitivity to Sch-28080. The data also suggest that ouabain and Sch-28080 do not bind to the same site on the chimera.

Acrylamides↗
PubMed1982

Palytoxin both induces and inhibits the release of histamine from rat mast cells.

Palytoxin (PTX) is a potent releaser of histamine from rat mast cells. The concentration response curve is bell-shaped with its range between 0.05 ng/ml and 5 micrograms/ml and its maximum at 50 ng/ml. The release of histamine by PTX is specific because (a) heat-inactivated (50 degrees C) mast cells are insensitive to PTX, and (b) extracellular calcium is essential. Removal of extracellular potassium as well as the presence of borate shifts the curve to the left by factor of 10. At higher concentrations (5 micrograms/ml), PTX inhibits the release of histamine due to compound 48/80, MCD-peptide, Con-A, ionophore A-23187, and PTX itself.

Acrylamides↗
PubMed1987

Palytoxin: an extraordinarily potent stimulator of prostaglandin production and bone resorption in cultured mouse calvariae.

Palytoxin, a nonphorbol ester-type tumor promoter, stimulated the production of prostaglandin E2 (PGE2) and bone resorption in neonatal mouse calvariae in organ culture. The action of palytoxin on bone resorption occurred at extraordinarily low concentrations; enhanced resorption was regularly observed at 0.5 pg/ml, and the ED50 was 1-2 pg/ml (approximately 3 X 10(-13) M). Palytoxin-induced formation of PGE2 and bone resorption were inhibited completely by indomethacin (200 ng/ml). Concentrations of palytoxin above 10 pg/ml led to progressively decreasing enhancement of bone resorption; by 100-250 pg/ml no stimulation of resorption was observed despite continued high production of PGE2. Treatment with high concentrations of palytoxin (100 or 250 pg/ml) for 24-72 h inhibited cAMP accumulation stimulated by exogenous PGE2 or PTH and inhibited bone resorption induced by PGE2, PTH, or an analog of cAMP. Thus, palytoxin exhibited a biphasic dose-response curve for enhanced bone resorption, with stimulation at low concentrations (0.5-10 pg/ml) and toxic inhibition at high concentrations (greater than 50 pg/ml). Palytoxin is one of the most potent stimulators of bone resorption yet identified.

1-Methyl-3-isobutylxanthine↗