Binding of palytoxin

Figure 4. Specific binding of palytoxin to anti-palytoxin (Rabbit 633D-24, iy25,000) at 35 C (o) and O C (A).

The Na-K ATPase is the known receptor to palytoxin and ostreocins. The interaction shows lower potency for ostreocins, the natnre and site of the binding of palytoxin is unknown, it acts as a functional antagonist of ouabain, and their binding sites on the receptor are probably different. ... 

To ensure that the inhibition of EGF binding by palytoxin was not a consequence of cell toxicity, the effect of palytoxin on DNA synthesis in Swiss 3T3 cells was monitored. When cells were incubated in the presence of palytoxin, 10% fetal calf serum, and H-thymidine for 19.5 hr, no depression in the extent of H-thymidine incorporation into DNA was detected up to 3.7 pM palytoxin (Table I). Although 11 pM palytoxin was toxic when present for a prolonged period, under the conditions of the assays described above no toxicity was detected (Table I). When cells were incubated in the presence of palytoxin, 0.1% fetal calf serum, and H-thymidine, palytoxin did not stimulate significant incorporation of H-thymidine into DNA. Thus, although it can modulate the EGF receptor system under these conditions, palytoxin alone does not appear to be mitogenic for Swiss 3T3 cells. 

Inhibition of EGF binding by palytoxin could be due to a decrease in receptor affinity, as in the case of TPA-type tumor promoters, and/or a decrease in receptor number. In Swiss 3T3 cells there are two classes of EGF receptors. The dissociation constants for the two EGF receptor classes were determined to be approximately 2 X 10 M and 2 x 10" M, corresponding to approximately 1 x 10 and 1 X 10 receptor molecules per cell, respectively (33). Scatchard analysis revealed that treatment of Swiss 3T3 cells with palytoxin, like PDBu, caused an apparent loss in high-affinity binding (Figure 2). However, in contrast to PDBu, palytoxin also caused a significant (approximately 50%) loss of low affinity EGF binding. 

The differences between palytoxin and PDBu with respect to kinetics, temperature dependence, and effect on low affinity binding suggest that these two different types of tumor promoters may be acting through different mechanisms. Further, in contrast to PDBu, the effect of palytoxin is not readily reversible (33). To determine where the two pathways differ, we compared the relative ability of palytoxin and PDBu to inhibit EGF binding in protein kinase C depleted cells. Swiss 3T3 cells were depleted of protein kinase C to different extents by exposing confluent quiescent cells to 0, 20, 200, or 2000 nM PDBu for 72 hr. Previous results indicate that this treatment depletes cells of protein kinase C activity in a dose-dependent manner (31). 

Figure 1. Effect of tumor promoter treatment on binding of I-EGF to Swiss 3T3 cells. Confluent quiescent Swiss 3T3 cells (A and B) were treated for the indicated times at 37 C with PDBu [A 2 nM (o), 20 nM (A), 200 nM ( )] or palytoxin [B 1.1 pM ( ), 3.7 pM...

Because these results suggest that extracellular Na is required for inhibition of EGF binding by palytoxin in these cells, we determined if palytoxin caused Na influx in Swiss 3T3 cells. When Na influx was monitored at an early time point (7 min), it was found that palytoxin causes an influx of Na and that the rate of Na influx is dose dependent (Figure 6). In parallel with its effect on EGF binding, palytoxin at different doses increases intracellular Na to the same final level (42). Although Na influx occurs prior to the inhibition of EGF binding, these results and the apparent Na dependence of the palytoxin effect suggest a role for Na in the action of palytoxin on the EGF receptor. 

Figure 7. Comparison of effects of palytoxin and phorbol dibutyrate on EGF receptor binding.

Palytoxin (PTX) is one of the most potent marine toxins known and the lethal dose (LD q) of the toxin in mice is 0.5 Mg/kg when injected i.v. The molecular structure of the toxin has been determined fully (1,2). PTX causes contractions in smooth muscle (i) and has a positive inotropic action in cardiac muscle (4-6). PTX also induces membrane depolarization in intestinal smooth (i), skeletal (4), and heart muscles (5-7), myelinated fibers (8), spinal cord (9), and squid axons (10). PTX has been demonstrated to cause NE release from adrenergic neurons (11,12). Biochemical studies have indicated that PTX causes a release of K from erythrocytes, which is followed by hemolysis (13-15). The PTX-induced release of K from erythrocytes is depress by ouabain and that the binding of ouabain to the membrane fragments is inhibited by PTX (15). 

The palytoxin-anti-palytoxin reaction is unique in that its binding increases with increasing temperature (Figure 4). The apparent association constant of the palytoxin to anti-palytoxin was 4.9 x 10 M at 0 C and 1.1 x 10 M" at 35 C, suggesting that H2O must be displaced from some of palytoxin s epitopes before they can bind to their antibody combining sites. 

Figure 2. Binding of [ IJpalytoxin to anti-palytoxin (Rabbit 633) before ( ) and after primary (o), first boost (A), second boost (A), and third boost ( , Rabbit 633D-24) of immunization and after absorption of IgG in Rabbit 633D-24 with goat anti-rabbit IgG ( ).

The postulated mechanism of action of palytoxin is to bind to the mammalian Na-K-ATPase (or sodium pump) and convert this enzyme into an open channel. However, the ubiquity of pumps and channels in the living tissues and the high diversity of preparations nsed to study the mechanism of action of palytoxin make it difficult to rule out the possibility that another site may be involved. In this sense, recent evidences indicate that the Na-K-ATPase may not be the only target of the toxin. It must be pointed out that the vast majority of work performed to test the mechanism of action of palytoxin had relied on the initial experiments indicating palytoxin binding to Na-K-ATPase (Bottinger and Habermaim 1984 Bottinger et al. 1986). 

In conclusion, what seems clear to the moment is that normally low concentrations of palytoxin are sufficient to produce a massive increase in the permeability of cells to cations. On the basis of experimental evidence, palytoxin appears to bind to the Na-K-ATPase at the cell membrane, open up a yet unidentified pathway for cations, and inhibit the sodium pump. Experiments with a truncated a subunit of the sodium pump that can still bind palytoxin without allowing the formation of a channel, indicating that the palytoxin-induced channel is not formed between the palytoxin molecnle and the enzyme but rather within the protein of the sodium pump (Redondo et al. 1996). Whether the high cytotoxicity of palytoxin is merely a consequence of its disruption of the ionic environment of the cell remains to be elncidated. 

Bottinger, H., Beress, L., and Habermann, E. 1986. Involvement of (Na+ + K+)-ATEase in binding and actions of palytoxin on human erythrocytes. Biochim BiophysActa 861, 165-176. 

Very few data are available with regard to metabolism of palytoxin in the animal/human body and its connection with toxicity. The main mode of palytoxin action (which is presented in detail in Chapter 34) is related to disruption of the mammahan cell sodium pump functionality. Targeting the Na, K -adenosine triphosphatase (ATPase) pump, palytoxin binds to the ATPase and converts the pump into a nonspecific ion channel, thereby, short-circuiting membrane function of the cell and eventually causing cell lysis [64]. 

Research on palytoxin effects generally relates to two major areas. A broad range of studies focuses on how palytoxin regulates ion flux, which is the first effect of this toxin on the cell. Another broad class of studies concentrates on several cellular effects elicited by the toxin such as tumor promotion. The vast majority of work performed to test the mechanism of action of palytoxin has relied on the initial experiments indicating PTX binding to Na , K -ATPase therefore, this section will examine the main evidences supporting an action of the toxin in the sodium pump and continue with the effects of the toxin in other cellular targets. 

Another common cellular target of palytoxin and TPA is the EGF receptor. It has been described that palytoxin inhibited the binding of EGF to low- and high-affinity receptors in a manner independent of protein kinase C. Moreover, picomolar concentrations of palytoxin inhibited EGF binding in the absence of cytosolic calcium increase. Further studies indicated that the palytoxin effects on EGF receptor were not due to common secondary effects of sodium influx, including membrane depolarization, changes in intracellular pH, or inhibition of protein synthesis. ... 

Ito, S. Ohta, T. Kadota, H. Kitamura, N. Nakazato, Y. Measurement of intracellular Na" " concentration by a Na -sensitive fluorescent dye, sodium-binding benzofuran isophthalate, in porcine adrenal chromaffin cells—usage of palytoxin as a Na ionophore. J. Neurosci. Methods 1997, 75, 21-27. 

By interfering with any one of the many phases associated with these second messenger pathways, toxins may alter channel gating. For example, the blue green algal toxins, aplysiatoxin, and lyngbyatoxin bind to and activate protein kinase C in a manner similar to phorbol esters (73). They also stimulate arachidonic acid metabolism (74). The coral toxin, palytoxin, also stimulates arachidonic acid breakdown albeit by an unknown mechanism (74) and affects other biochemical activities of the cell (see chapters by Fujiki et al., Wattenberg et al., and Levine et al., this volume). 

One fact does emerge from the assembled literature, despite its disarray. Palytoxin does not appear to act by binding to one single cellular component to trigger a cascade of responses. Original speculations that it might activate voltage-... 

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