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PubMed2004

Large diameter of palytoxin-induced Na/K pump channels and modulation of palytoxin interaction by Na/K pump ligands.

Palytoxin binds to Na/K pumps to generate nonselective cation channels whose pore likely comprises at least part of the pump's ion translocation pathway. We systematically analyzed palytoxin's interactions with native human Na/K pumps in outside-out patches from HEK293 cells over a broad range of ionic and nucleotide conditions, and with or without cardiotonic steroids. With 5 mM internal (pipette) [MgATP], palytoxin activated the conductance with an apparent affinity that was highest for Na(+)-containing (K(+)-free) external and internal solutions, lowest for K(+)-containing (Na(+)-free) external and internal solutions, and intermediate for the mixed external Na(+)/internal K(+), and external K(+)/internal Na(+) conditions; with Na(+) solutions and MgATP, the mean dwell time of palytoxin on the Na/K pump was about one day. With Na(+) solutions, the apparent affinity for palytoxin action was low after equilibration of patches with nucleotide-free pipette solution. That apparent affinity was increased in two phases as the equilibrating [MgATP] was raised over the submicromolar, and submillimolar, ranges, but was increased by pipette MgAMPPNP in a single phase, over the submillimolar range; the apparent affinity at saturating [MgAMPPNP] remained approximately 30-fold lower than at saturating [MgATP]. After palytoxin washout, the conductance decay that reflects palytoxin unbinding was accelerated by cardiotonic steroid. When Na/K pumps were preincubated with cardiotonic steroid, subsequent activation of palytoxin-induced conductance was greatly slowed, even after washout of the cardiotonic steroid, but activation could still be accelerated by increasing palytoxin concentration. These results indicate that palytoxin and a cardiotonic steroid can simultaneously occupy the same Na/K pump, each destabilizing the other. The palytoxin-induced channels were permeable to several large organic cations, including N-methyl-d-glucamine(+), suggesting that the narrowest section of the pore must be approximately 7.5 A wide. Enhanced understanding of palytoxin action now allows its use for examining the structures and mechanisms of the gates that occlude/deocclude transported ions during the normal Na/K pump cycle.

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PubMed1997

Endothelium inhibits the palytoxin-induced depolarization and Ca2+ mobilization in porcine coronary artery through endothelium-derived hyperpolarizing factor and nitric oxide released by palytoxin.

Palytoxin induced increases in cytosolic Ca2+ and tension, which were dependent on external Ca2+, and depolarized the membrane in endothelium-denuded porcine coronary arteries. When the endothelium was present, however, these effects were greatly inhibited, suggesting that some factors from endothelium inhibited the palytoxin-actions. Pretreatment with 100 microM N omega-nitro-L-arginine partially reversed the inhibitory effect of endothelium on the Ca2+ movement and the contraction but not that on the depolarization. Pretreatment with 10 microM indomethacin did not affect the inhibition. These results suggest that palytoxin released both nitric oxide and endothelium-derived hyperpolarizing factor (EDHF) from the endothelium, both of which counteracted the actions of palytoxin on smooth muscle cells. It is thought that the palytoxin-induced depolarization was attenuated by hyperpolarization due to EDHF.

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PubMed1987

Production of antibodies to palytoxin: neutralization of several biological properties of palytoxin.

Palytoxin stimulated arachidonic acid metabolism (in bovine aorta endothelial and smooth muscle cells, rat keratinocytes, porcine aorta endothelial cells and rat liver cells), hemolyzed rat erythrocytes and was lethal to mice when administered intraperitoneally. Serum from rabbits immunized with a conjugate in which palytoxin was covalently bound to bovine albumin through its free amino group neutralized these biologic activities of palytoxin. Ninety-nine per cent of the neutralizing activity of the immunized rabbit serum was removed after precipitation of the rabbit IgG with a goat anti-rabbit IgG.

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PubMed1992

N-(4'-hydroxyphenylacetyl)palytoxin: a palytoxin prodrug that can be activated by a monoclonal antibody-penicillin G amidase conjugate.

Palytoxin (PTX), one of the most toxic nonprotein molecules known, is cytotoxic at picomolar concentrations against a wide variety of cell types. In contrast to most cytotoxins, PTX exerts its activity extracellularly. A method for targeting PTX to tumor cells is described in which a monoclonal antibody-enzyme conjugate activates a PTX prodrug at surfaces of tumor cells. The prodrug, N-(4'-hydroxyphenylacetyl)palytoxin (NHPAP), was prepared by reacting PTX with an active ester of 4-hydroxyphenylacetic acid. NHPAP was 1000 times less toxic than PTX to a panel of carcinoma and lymphoma cell lines. The cytotoxic activity of the combination of penicillin G amidase from Escherichia coli with NHPAP was equal to PTX. Two cell lines that were multidrug resistant showed no enhanced resistance to NHPAP +/- penicillin G amidase. Immunologically specific activation of NHPAP took place when H2981 cells (L6 antigen positive) were treated with the monoclonal antibody conjugate L6-penicillin G amidase followed by NHPAP. This system is distinguished from other prodrug activation schemes, since the released drug exerts its activity extracellularly, has high potency, and may be able to overcome the multidrug resistant phenotype.

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PubMed1989

Structure-activity studies of the nonphorbol tumor promoter palytoxin in Swiss 3T3 cells.

Derivatives of palytoxin have been prepared which are modified on either the hydroxyl terminus or the amino terminus of the molecule. Previously we have shown that palytoxin, a non-12-O-tetradecanoylphorbol-13-acetate-type tumor promoter, can inhibit epidermal growth factor binding in Swiss 3T3 cells through a pathway which is sodium dependent but not calcium or protein kinase C dependent. We used the epidermal growth factor receptor system to determine whether the specific chemical modifications of palytoxin present in these derivatives alter the cellular mechanism of action of the toxin. The dose response and ion dependence of palytoxin, the hydroxyl terminus derivative palytoxin-COOH, and the amino terminus derivatives N-acetylpalytoxin and N-(p-bromobenzoyl)palytoxin were compared with respect to inhibition of epidermal growth factor binding. The potency of palytoxin-COOH was similar to that of palytoxin. By contrast, N-acetylpalytoxin and N-(p-bromobenzoyl)palytoxin were approximately 1/100 as potent as palytoxin in this assay. All three derivatives were at least 100-fold less toxic than palytoxin. Like palytoxin, the activities of palytoxin-COOH, N-acetylpalytoxin and N-(p-bromobenzoyl)palytoxin were dependent upon the presence of extracellular sodium. However, there was a significant difference in the dependence of the derivatives on extracellular calcium. Our results suggest that the hydroxyl terminus is important for determining the calcium dependence of the molecule and the amino terminus is important for determining the biological potency of palytoxin. We conclude that modification of the hydroxyl terminus region is an effective means of reducing the toxicity of palytoxin while retaining the biological effects.

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PubMed1989

Palytoxin down-modulates the epidermal growth factor receptor through a sodium-dependent pathway.

Palytoxin, a non-12-O-tetradecanoylphorbol-13-acetate type tumor promoter, has been shown to inhibit epidermal growth factor (EGF) binding to both high and low affinity receptors through a protein kinase C-independent pathway. In the present paper, we have investigated the mechanism of palytoxin action in Swiss 3T3 cells. Two lines of evidence indicate that calcium is not required for palytoxin activity. First, palytoxin can induce the loss of EGF binding sites in the absence of external calcium. Second, studies with the photosensitive protein aequorin indicate that palytoxin does not cause the influx of external calcium or the release of calcium from internal stores under the conditions used in these studies. However, palytoxin action does appear to be dependent upon the presence of sodium. When extracellular sodium is replaced by either choline, Tris, or sucrose, palytoxin is unable to decrease EGF binding to either high or low affinity receptors. Studies of sodium influx indicate that palytoxin induces rapid sodium uptake and that the rate of sodium uptake is dose-dependent. Furthermore, there appears to be a direct correspondence between the extent of inhibition of EGF binding by palytoxin and the rate of sodium uptake. Finally, the palytoxin-induced inhibition of EGF binding can be mimicked by monensin, a sodium ionophore. The specificity of this sodium dependence was tested by substituting lithium, potassium, or cesium for sodium. Although lithium is an effective substitute for sodium, palytoxin can no longer inhibit EGF binding when sodium is replaced by either potassium or cesium. Marked inhibition of palytoxin action is also obtained when 5.4 mM potassium or 5.4 mM cesium are added to the sodium-containing medium. These studies suggest that palytoxin is able to down-modulate the EGF receptor through a novel mechanism involving the activation or formation of a sodium pump or channel.

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PubMed1999

Cell-type-specific activation of p38 protein kinase cascades by the novel tumor promoter palytoxin.

Palytoxin is a potent non-12-O-tetradecanoylphorbol-13-acetate (TPA)-type skin tumor promoter. We used COS7 and HeLa cells to investigate the protein kinase cascades by which palytoxin activates the mitogen-activated protein kinase (MAPK) p38. Three p38 kinases have been identified: stress-activated protein kinase/extracellular signal-regulated kinase kinase-1 (SEK1), MAPK kinase 3 (MKK3), and MKK6. SEK1 phosphorylates and activates both p38 and c-Jun NH(2)-terminal kinase (JNK), whereas MKK3 and MKK6 selectively phosphorylate and activate p38. Although transiently overexpressed SEK1 activates p38 in cells, the importance of endogenous SEK1 for the activation of p38 by specific types of stimuli is unclear because some agents, such as sorbitol, can activate p38 in cells derived from SEK1 knockout mice. Because we previously showed that palytoxin activates JNK through an SEK1-dependent pathway, we investigated whether SEK1 also mediates the activation of p38 by palytoxin. The results presented here demonstrate that endogenous SEK1 does play an important role in the activation of p38 by palytoxin in specific cell types. In COS7 cells, palytoxin stimulated the phosphorylation of SEK1 and MKK6, and expression of dominant negative mutants of either SEK1 or MKK6 inhibited palytoxin-stimulated p38 activation. In HeLa cells, palytoxin stimulated the phosphorylation of MKK3 in addition to SEK1 and MKK6. In contrast to COS7 cells, in HeLa cells expression of a dominant negative mutant of SEK1 did not inhibit palytoxin-stimulated activation of p38, although expression of dominant negative mutants of either MKK3 or MKK6 did inhibit palytoxin-stimulated p38 activation in this cell type. These studies indicate that the importance of SEK1 in the activation of p38 by palytoxin depends on the ability of palytoxin to activate MKK3 and MKK6.

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PubMed1995

Involvement of the Na,K-ATPase in the induction of ion channels by palytoxin.

The effects of ouabain, ATP, and vanadate on palytoxin induction of ion channels were examined with the aim of elucidating the role of Na,K-ATPase in palytoxin action. Palytoxin-induced membrane depolarization of crayfish giant axons and single channel currents of frog erythrocytes and mouse neuroblastoma N1E-115 cells were examined using the intracellular microelectrode and patch-clamp techniques. External application of palytoxin in nanomolar concentrations induced depolarization in the crayfish giant axons, and the depolarization was inhibited by pretreatment of the axon with ouabain (10 microM). Internally perfused axons were less sensitive to palytoxin unless ATP (6 mM) was added internally. In patch-clamp experiments, picomolar palytoxin in the patch electrode induced single channels in both cell-attached and inside-out patches of erythrocytes and neuroblastoma cells. The induced channels had a conductance of about 10 pS, reversed near 0 mV in physiological saline solution, and was permeable to Na+, K+, Cs+, and NH4+, but not to choline. Single channel activities induced by palytoxin were inhibited by ouabain (10 microM) and vanadate (1 mM), but promoted by ATP (1 mM). The modulating effects of ouabain, vanadate, and ATP on palytoxin action suggest that the Na,K-ATPase is involved in the induction of single channels by palytoxin. Palytoxin-induced and ouabain-inhibitable single channels were observed in planar lipid bilayer incorporated with purified Na,K-ATPase. The results indicate that an interaction between palytoxin and Na,K-ATPase leads to opening of a 10-pS ion channel. They further raise the possibility that a channel structure may exist in the sodium pump which is uncovered by the action of palytoxin.

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PubMed2003

Mitogen activated protein kinases selectively regulate palytoxin-stimulated gene expression in mouse keratinocytes.

We have been investigating how the novel skin tumor promoter palytoxin transmits signals through mitogen activated protein kinases (MAPKs). Palytoxin activates three major MAPKs, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38, in a keratinocyte cell line derived from initiated mouse skin (308). We previously showed that palytoxin requires ERK to increase matrix metalloproteinase-13 (MMP-13) gene expression, an enzyme implicated in carcinogenesis. Diverse stimuli require JNK and p38 to increase MMP-13 gene expression, however. We therefore used the JNK and p38 inhibitors SP 600125 and SB 202190, respectively, to investigate the role of these MAPKs in palytoxin-induced MMP-13 gene expression. Surprisingly, palytoxin does not require JNK and p38 to increase MMP-13 gene expression. Accordingly, ERK activation, independent of palytoxin and in the absence of JNK and p38 activation, is sufficient to induce MMP-13 gene expression in 308 keratinocytes. Dexamethasone, a synthetic glucocorticoid that inhibits activator protein-1 (AP-1), blocked palytoxin-stimulated MMP-13 gene expression. Therefore, the AP-1 site present in the promoter of the MMP-13 gene appears to be functional and to play a key role in palytoxin-stimulated gene expression. Previous studies showed that palytoxin simulates an ERK-dependent selective increase in the c-Fos content of AP-1 complexes that bind to the promoter of the MMP-13 gene. JNK and p38 can also modulate c-Fos. Palytoxin does not require JNK or p38 to increase c-Fos binding, however. Altogether, these studies indicate that ERK plays a distinctly essential role in transmitting palytoxin-stimulated signals to specific nuclear targets in keratinocytes derived from initiated mouse skin.

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PubMed1988

Effects of palytoxin or ouabain on growth and squamous differentiation of human bronchial epithelial cells in vitro.

The effects of the non-12-O-tetradecanoylphorbol-13-acetate type tumor promoter palytoxin on human bronchial epithelial cells was studied in an in vitro serum-free culture system. Unlike the results of previous studies with another tumor promoter, 12-O-tetradecanoylphorbol-13-acetate, palytoxin did not induce squamous differentiation of normal bronchial epithelial cells and was equally cytotoxic for normal human bronchial epithelial cells, a human lung tumor cell line, and human bronchial epithelial cells immortalized by infection with adenovirus 12-SV40 hybrid virus (BEAS-2B cells). Palytoxin did not induce a change in free cytosolic Ca2+ concentration of BEAS-2B cells. The effect of palytoxin on the c-myc mRNA steady state level in BEAS-2B cells was studied: 1 pM palytoxin increased the steady-state level at 12 and 18 h. Furthermore, the induction was accompanied by an increase in [3H]thymidine uptake. Because palytoxin binds to (Na+ + K+)ATPase, the effects of ouabain were compared to the effects of palytoxin. A ouabain-resistant cell line was as sensitive to the growth inhibitory effect of palytoxin as the parent ouabain-sensitive cell line, suggesting different binding sites to the (Na+ + K+)-ATPase for palytoxin and ouabain. Ouabain also increased the steady-state level of c-myc gene expression, but earlier than palytoxin, and the increase in the level of c-myc mRNA was accompanied by a drop in DNA synthesis. These results suggest that palytoxin does not act by growth stimulation, differential cytotoxicity or terminal differentiation of normal versus neoplastic cells which are proposed mechanisms of tumor promotion.

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PubMed1991

Effects of apical vs. basolateral palytoxin on LLC-PK1 renal epithelia.

Research on palytoxin focuses on its action as a tumor promoter and its ionophoretic action in cell membranes. The first property is unusual because palytoxin is not a protein kinase C activator. The second property is remarkable in that it may require interaction with the Na(+)-K(+)-adenosinetriphosphatase (ATPase). Our studies here with palytoxin exposure to the LLC-PK1 renal epithelial cells have yielded the following results: 1) unlike protein kinase C-activating tumor promoters (tetradecanoylphorbol 12,13-acetate or teleocidin), palytoxin does not produce a specific effect on the tight junctions between epithelia; 2) palytoxin instead produces an irreversible cytotoxic effect characterized by a pronounced cell swelling associated with sharply elevated levels of intracellular Na+ and decreased levels of intracellular K+; 3) these fluctuations in intracellular Na+ and K+ levels are explained by marked elevations in the membrane flux of 22Na+ and 86Rb+; 4) the electrophysiological reflection of these altered ion fluxes is a pronounced depolarization of the cell sheet if palytoxin is presented to the basolateral cell surface and a pronounced hyperpolarization (due to sharply elevated apical Na+ flux and transepithelial short-circuit current) if palytoxin is administered apically; 5) the apical effect of palytoxin can be blocked by apical ouabain; and 6) this apical effect of palytoxin decreases as a function of the age of the cell sheet. This first report of palytoxin action in a polar epithelial cell system provides additional evidence for palytoxin effects being mediated by contact with the Na(+)-K(+)-ATPase. It also adds to a growing literature suggesting the existence of Na(+)-K(+)-ATPase in the apical cell surface of epithelia under certain conditions.

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PubMed1996

Activation of stress-activator protein kinase/c-Jun N-terminal kinase by the non-TPA-type tumor promoter palytoxin.

Previous studies have shown that structurally diverse tumor promoters can modulate protein kinases involved in signal transduction. In this study, we show that palytoxin, a potent non-12-O-tetradecanoylphorbol-13-acetate (TPA)-type skin tumor promoter, induces a signaling pathway leading to the activation of the stress-activated protein kinases/c-Jun N-terminal kinases (JNK) in Swiss 3T3 fibroblasts. Treatment of cells with doses as low as 0.1 mN palytoxin results in significant activation of JNK. In contrast to epidermal growth factor, which induces a transient activation of JNK in Swiss 3T3 cells, palytoxin causes prolonged enzyme activation. Since stimulation of ion flux appears to play an important role in the mechanism of action of palytoxin in other systems, we investigated the role of sodium and calcium in the activation of JNK: (a) our results show that incubation of Swiss 3T3 cells in a sodium-free medium dramatically reduced the magnitude of JNK activation by palytoxin; and (b) we found that the sodium ionophore gramicidin activates JNK. Together, these results suggest that sodium influx, which is a hallmark of palytoxin action, may play a key role in the activation of JNK by palytoxin. Our results indicate that calcium influx is not necessary or sufficient for palytoxin-induced activation of JNK. In contrast to palytoxin, the TPA-type tumor promoter phorbol 12,13-dibutyrate and the non-TPA-type tumor promoters thapsigargin and okadaic acid do not appear to activate JNK in this system. In contrast to phorbol 12,13-dibutyrate, palytoxin does not activate the p42/p44 mitogen-activated protein kinases. Our results demonstrate that Swiss 3T3 fibroblasts, palytoxin can activate a protein kinase signaling pathway that is distinct from that activated by the prototypical phorbol ester tumor promoters and other potent skin tumor promoters.

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PubMed1996

Palytoxin-induced Na+ influx into yeast cells expressing the mammalian sodium pump is due to the formation of a channel within the enzyme.

Palytoxin forms ionic channels in animal cell membranes but does not have similar effects on bacteria or yeast cells. These channels appear to be associated with the sodium pump. Using a heterologous expression system for the mammalian sodium pump in the yeast Saccharomyces cerevisiae, we recently demonstrated palytoxin-induced K+ efflux from yeast cells. Using the same system, we now show that the palytoxin-induced Na+ influx measured by others in animal cells is also directly associated with the sodium pump. Under the influence of palytoxin, yeast cells that express the mammalian sodium pump exchange extracellular Na+ ions for intracellular K+ ions with a stoichiometry of approximately 1:1. Both fluxes can be inhibited by ouabain. K+ efflux can also be observed when extracellular Na+ is replaced by Li+, Cs+, or NH4+. These data suggest that all palytoxin-induced ion fluxes measured so far in various cell systems are directly associated with the sodium pump. Palytoxin-induced Na+ influx or K+ efflux does not occur with yeast cells that express a truncated form of the sodium pump that is missing 44 of the carboxyl-terminal amino acids of the alpha 1 subunit. Scatchard analysis reveals only a slightly lower affinity of the truncated form for [3H]ouabain compared with the affinity of the native enzyme. Yeast cells expressing the truncated enzyme can bind [3H]ouabain, which can be displaced by palytoxin. Therefore, the inability of the truncated form to conduct ions under the influence of palytoxin is not due to the removal of the palytoxin binding site but rather to the removal of a part of the enzyme that participates in a direct or indirect way in the formation of the palytoxin-induced channel. Based on these findings, we conclude that palytoxin opens a channel within and not merely in the vicinity of the sodium pump. This might be the same channel that under normal conditions actively transports Na+ and K+ ions.

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PubMed1992

Effects of palytoxin on porcine coronary artery rings.

Palytoxin, in concentrations as low as 100 fM, caused contractions of porcine coronary artery rings. Palytoxin concentrations of less than 1 nM caused slowly developing contractions which were not maximal even after 2 h. Rings contracted by 100 nM palytoxin achieved maximal tension by 10 min and relaxed to 53% of that maximum after 2 h. Verapamil (1 microM) reduced the rate of contractions induced by 10 nM palytoxin. Exposure of rings to greater than 10 nM palytoxin for 1-2 h reduced contractions to potassium 18 h later to 61% of the expected contraction and abolished those to palytoxin administered later. Both 10 and 100 nM palytoxin depleted potassium from coronary artery rings. Verapamil (10 microM) prevented potassium depletion by 10 nM palytoxin, but neither 10 microM verapamil nor 1 microM nifedipine prevented potassium depletion in rings exposed to 100 nM palytoxin. Thus, the contractile action and the potassium depleting action of palytoxin on the porcine coronary artery involve mobilization of nifedipine- and verapamil-sensitive calcium. Verapamil- and nifedipine-sensitive calcium was not required for depletion of potassium by the highest PTX concentration (100 nM), however.

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PubMed1998

Differential activation of mitogen-activated protein kinases by palytoxin and ouabain, two ligands for the Na+,K+-ATPase.

We previously demonstrated that the marine toxin and skin tumor promoter palytoxin activates the stress-activated protein kinase/c-Jun N-terminal kinase (JNK), but not the extracellular signal-regulated kinase (ERK), which is typically activated by mitogenic agents. JNK, ERK, and p38, another stress-activated protein kinase, are members of the mitogen-activated protein (MAP) kinase family of serine/threonine kinases, which coordinate the transmission of various signals through the cell. The Na+,K+-ATPase is the putative palytoxin receptor. Therefore, we hypothesized that the Na+,K+-ATPase inhibitor ouabain might also stimulate signaling pathways that activate MAP kinases. Using HeLa and COS7 cells, we found that, although there are similarities between the protein kinase cascades by which palytoxin and ouabain activate JNK, there are also significant differences between the activation of specific MAP kinases by palytoxin and ouabain. Transient expression of dominant negative mutants indicates that ouabain, like palytoxin, activates JNK through a protein kinase cascade that involves the JNK kinase SEK1 but does not require the GTPase Ras. Palytoxin activates JNK and p38 to a greater extent than ouabain. By contrast, ouabain activates ERK to a greater extent than palytoxin. Ouabain blocked palytoxin-stimulated activation of JNK and p38, but not anisomycin-stimulated activation of these kinases, supporting the conclusion that ouabain and palytoxin bind to the same site on the Na+,K+-ATPase. These results suggest that the Na+,K+-ATPase can differentially mediate the activation of MAP kinases by two diverse ligands, palytoxin and ouabain.

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PubMed2002

Extracellular signal-regulated kinase transmits palytoxin-stimulated signals leading to altered gene expression in mouse keratinocytes.

We have been probing the molecular mechanisms of tumor promoters that stimulate distinct initial signals to define critical downstream biochemical events in carcinogenesis. The action of the novel skin tumor promoter palytoxin on signaling and gene expression in keratinocytes, the primary target cells of tumor promoters, was therefore investigated. Palytoxin stimulated an increase in mRNA for matrix metalloproteinase-13 (MMP-13), an enzyme implicated in carcinogenesis, in a keratinocyte cell line derived from initiated mouse skin (308). Palytoxin stimulated an increase in c-Fos binding to the activator protein-1 (AP-1) site present in the promoter of the mouse MMP-13 gene. This effect was specific because palytoxin had little effect on c-Jun, JunB, JunD, FosB, Fra-1, or Fra-2 binding or on overall levels of transcription factor binding. The increase in c-Fos binding corresponded to a palytoxin-stimulated increase in c-Fos protein levels. Palytoxin stimulated the activation of the mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase, and p38. The MAPK kinase inhibitor PD 98059 blocked palytoxin-stimulated ERK activation. PD 98059 also blocked the palytoxin-stimulated increases in c-Fos protein levels, c-Fos binding to the AP-1 site, and MMP-13 mRNA. These studies identify important differences between palytoxin-stimulated signaling in keratinocytes derived from initiated mouse skin, the biologically relevant cell type, and other cell lines. Specifically, our data suggest that, in keratinocytes derived from initiated mouse skin, ERK plays an important role in transmitting palytoxin-stimulated signals to three downstream targets that are likely to affect carcinogenesis: c-Fos, AP-1, and MMP-13.

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PubMed1990

Characterization of palytoxin-induced channels in mouse neuroblastoma cells.

Palytoxin-induced whole-cell and single channel currents were recorded in mouse neuroblastoma cells. Palytoxin-induced single channel currents had a slope conductance of 26 pS (20-22 degrees C). Palytoxin-induced channels were permeable to sodium and potassium and slightly permeable to calcium, choline and tetramethylammonium. They did not seem to be significantly permeable to chloride or protons. Both the steady-state and the rate of the dose-dependent effects of palytoxin could be accounted for if one assumed that a palytoxin-induced channel resulted from the binding of two palytoxin molecules to a membrane receptor with respective dissociation constants of 5 nM and 10 pM. In the continued presence of low palytoxin concentrations (less than 1 nM) the effect was maintained. Higher palytoxin concentrations induced a transient and irreproducible effect. The effect of palytoxin was decreased when either external sodium was replaced by potassium or in the absence of calcium in external and/or internal media. The results suggest that ionic currents result from the binding of palytoxin molecules to a membrane receptor and that receptor-toxin complexes can be internalized.

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PubMed1998

Loss of cellular K+ mimics ribotoxic stress. Inhibition of protein synthesis and activation of the stress kinases SEK1/MKK4, stress-activated protein kinase/c-Jun NH2-terminal kinase 1, and p38/HOG1 by palytoxin.

The tumor promoter palytoxin has been found to activate the stress-activated protein kinase/c-Jun NH2-terminal kinase 1 (SAPK/JNK1), and it also potentiates, as demonstrated here, the p38/HOG1 mitogen-activated protein kinase and the upstream activator of SAPK/JNK1, SEK1/MKK4. In search of possible mechanisms for both the cytotoxicity and the activation of stress kinases by palytoxin, we found that palytoxin is a potent inhibitor of cellular protein synthesis. The inhibition of translation by palytoxin does not result from its direct binding to the translational apparatus. We have previously demonstrated that ribotoxic stressors (Iordanov, M. S., Pribnow, D., Magun, J. L., Dinh, T.-H., Pearson, J. A., Chen, S. L.-Y., and Magun, B. E. (1997) Mol. Cell. Biol. 17, 3373-3381) signal the activation of SAPK/JNK1 by binding to or covalently modifying 28 S rRNA in ribosomes that are active at the time of exposure to the stressor. Palytoxin acted as a ribotoxic stressor, inasmuch as it required actively translating ribosomes at the time of exposure to activate SAPK/JNK1. Palytoxin has been shown to augment ion fluxes by binding to the Na+/K+-ATPase in the plasma membrane of cells. To determine whether altered fluxes of either Na+ or K+ could be responsible for the effects of palytoxin on translation and on activation of SAPK/JNK1, cells were exposed to palytoxin in modified culture medium in which a major portion of the Na+ was replaced by either K+ or by choline+. The substitution of Na+ by K+ strongly inhibited the ability of palytoxin both to inhibit protein translation and to activate SAPK/JNK1, whereas the substitution of Na+ by choline+ did not. These results suggest that palytoxin-induced efflux of cellular K+ mimics ribotoxic stress by provoking both translational inhibition and activation of protein kinases associated with cellular defense against stress.

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