Palytoxin Isolated from Marine Coelenterates THE INHIBITORY ACTION ON (Na,K)-ATPase*

Palytoxin (PTX), C12~H223N3064, a highly toxic sub- stance isolated from zoanthids of Palythoa tubercu-Zosa, inhibited (Na,K)-ATPase (ATP phosphohydro- lase, EC 3.6.1.3) prepared from guinea pig heart and hog cerebral cortex in a dose-dependent manner at concentrations >lo” M. In the presence of Na (100 mM) and K (20 mM), PTX showed potency nearly equal to that of ouabain. When the ATPase was activated by the various Na concentrations at a constant K concentration, both PTX and ouabain inhibited the ATPase activity noncompetitively. On the other hand, when K concentration was changed at a constant Na concentra- tion, PTX caused a competitive inhibition in all ranges of K concentrations employed, whereas ouabain caused a competitive inhibition at low concentrations and a noncompetitive inhibition at high concentrations. intravenous

PTX' was reported to be an extremely poisonous substance from marine coelenterates of the genus Palythoa (1). It was isolated from the zoanthid "lirnumake-o-Hana" (Palythoa toxzca). The 50% lethal dose (LDSo) was 0.15 pg/kg in mice by intravenous injection. In monkeys, the value of LDSO was reported to be extremely small, 0.08 pg/kg intravenously (2). Extracts from Palythoa caribaeorum and Palythoa mamilosa, which inhabit shallow waters off the shores of the Caribbean Islands, also caused the toxic response (3). Further, toxic Palythoa tuberculosa was found in tropical and subtropical coral reefs at Okinawa, Amami-Oshima, Marcus, and Tahiti islands (4). The toxicity is strong in eggs and also in other tissues of the female during maturation of ovary and, at the peak of toxicity, 1 g of egg is able to kill two tons of mice (4). Pigs have been reported to die after eating filefish, Altura scripta, which have fed on the toxic zoanthid (4).
The structure of PTX has recently been determined (5-7). P T X is one of the most complicated and largest naturally occurring molecules known ( M , = 2660-2680) except for the naturally occurring polymers.
P T X was reported to have a powerful positive inotropic effect on heart muscle (8, 9). Cardiac glycosides such as * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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The abbreviation used is: PTX, palytoxin. ouabain are well known to cause a cardiotonic action and have been beneficial for patients having cardiac failure. The mechanism of action of the glycosides is thought to be by the inhibition of (Na,K)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity (10,11). Further, P T X was suggested to cause a contraction of intestinal smooth muscle, similar to ouabain (12). It has recently been reported that in the presence of ouabain the effect of P T X was inhibited in smooth muscles (13,14) and erythrocytes (15), indicating some interaction between ouabain and PTX. Therefore, in the present experiment, the effect of PTX on the (Na,K)-ATPase activity was investigated.

MATERIALS AND METHODS
(Na,K)-ATPase was prepared from guinea pig heart as described hy Pitts and Schwartz (16). (Na,K)-ATPase from hog cerebral cortex prepared by the method of Nakao et al. (17) was purchased from Sigma. Specific activities of (Na,K)-ATPase were 1.8-2.5 @mol of Pi released/mg of protein/min for the heart ATPase and 0.8-1.2 for the cerebral cortex at 37 "C. The enzyme preparation was incubated in a solution containing 100 mM NaC1, 20 mM KCl, 5 mM MgCl,, 5 mM ATP, and 50 mM Tris-HC1 at pH 7.4 (37 "C). The final volume of the solution was 0.5 ml. The enzyme preparation was preincubated in the absence of ATP for 10 min and then the reaction was started by addition of ATP. PTX or ouabain was added 5 min before the addition of ATP. The amount of Pi liberated during the 15-min incubation was determined by the method of Martin and Doty (18). Activity of (Na,K)-ATPase was calculated by the difference between the quantity of P, liberated in the presence and absence of Na and K. In the absence of Na and K, Mg-activated ATPase activity was about 5% of total activity. Ouabain, lo" M, almost completely inhibited (Na,K)-ATPase activity in both heart and cerebral cortex preparations. Protein was determined by the method of Lowry et al. (19) with bovine serum albumin as the standard.
Ouabain and Tris-ATP was purchased from Merck and Sigma, respectively. PTX isolated and purified as described by Hirata et al. (20) was donated by Dr. Y. Hirata of the Faculty of Pharmacy, Meijo University, Tempaku, Nagoya 468, Japan. PTX ( M) was stored in small amounts (0.2-0.5 ml) at -70 "C. Each small amount of solution was only once used for experiments since freezing and thawing reduced effectiveness of PTX by one third.

RESULTS
PTX and ouabain inhibited activities of (Na,K)-ATPases prepared from guinea pig heart and hog cerebral cortex (Fig.  1). The IDS0 (50% inhibition of the maximum activity stimulated by Na and K ) of P T X was 3.1 X (Na,K)-ATPase Inhibition by Palytoxin The experimental conditions were the same as described in Fig. 2. Enzyme preparation from hog cerebral cortex was used.
When K concentration was changed a t a constant Na concentration of 100 mM, the enzyme of the cortex was also activated in a concentration-dependent fashion. Hunter-Downs plots (24) were employed to examine inhibitory effects of PTX and ouabain (Fig. 3 ) . Fig. 3a shows that PTX, 5 x 10-~ and 1 x M, yields a straight line with an appreciable slope in the whole range of K concentrations employed, suggesting that PTX is competitive with K. On the other hand, Fig. 36 suggests that ouabain, 5 X and 1 X M, is competitive at low ( 4 0 mM) concentrations of K but approaches noncompetitive inhibition at higher concentrations of K. Apparent K, values for competitive inhibition were 0.027 p~ for PTX and 0.019 pM for ouabain.

DISCUSSION
Crude P T X from P. cari6aeorum has previously been reported to cause inhibition of (Na,K)-ATPase of electroplax due to a contamination by 5-hydroxytryptamine (25,26) and the purified P T X had no apparent effect on the ATPase activity of erythrocytes up to concentrations of 5 X M (15). 5-Hydroxytryptamine inhibited the ATPase activity at concentrations more than M with the IDso of about M (26), which values are about 1000 times greater than those of PTX from P. tuberculosa in the present experiment. Furthermore, the purity of P T X used in these experiments was verified through various analyses including that by nuclear magnetic resonance (20,27). Therefore, it is unlikely that the P T X used here contains 5-hydroxytryptamine. PTX was reported to be very unstable (1, 27). It was also reported that chemical modification of PTX to N-acetyl-PTX reduced the biological activity by one hundredth (28). Thus, the discrepancy between present and previous experiments might be attributed to the instability of P T X or to a small difference of the chemical structure of the preparations used.
The present work demonstrates that PTX inhibits the activity of (Na,K)-ATPase prepared from heart and cerebral cortex. In the presence of both Na and K, IDso values for PTX and ouabain were nearly equal, about M. Both PTX and ouabain caused a noncompetitive inhibitory effect on the Na activation of the ATPase at a constant K concentration with K; values of about 5 PM. Furthermore, PTX and ouabain caused competitive inhibition of the K activation induced by low K concentration at a constant Na concentration with similar K, values of about 0.02 pM. Therefore, it is indicated that PTX inhibits the activity of (Na,K)-ATPase with nearly equal potency to that of ouabain, although the chemical structure is quite different.
When the K concentration was changed at a constant Na concentration, P T X caused a competitive inhibition in all ranges of K concentrations employed. On the other hand, ouabain caused a competitive inhibition at low (<lo mM) concentrations and a noncompetitive inhibition at high concentrations as reported previously (29,30). The aglycone of ouabain, ouabagenin was also reported to cause the similar dual inhibitory effect on the K activation of the ATPase (31). Therefore, it is suggested that, compared with ouabain, P T X causes a simple competitive inhibition on the K-activated site of (Na,K)-ATPase.