Chapter 16
Mechanism of Pharmacological Action of Palytoxin Yasushi Ohizumi
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Mitsubishi Kasei Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo 194, Japan Palytoxin (PTX), isolated from the zanthid Palythoa species, caused a rapid contraction followed by a slow phasic contraction o f the guinea pig vas deferens. T h e second component o f PTX-induced contraction was markedly inhibited by adrenergic blocking agents, whereas the first component was blocked by ouabain. I n pheochromocytoma cells, P T X caused a dose-dependent release o f norepinephrine (NE). T h e N E release induced by lower concentrations o f P T X increased proportionately with increasing Na concentrations, but was not modified by tetrodotoxin. However, the NE-releasing action o f higher concentrations o f P T X was dependent o n external Ca , but not Na . Thus o u r experimental results suggest that i n adrenergic neurons the PTX-induced release o f NE by lower concentrations o f P T X is brought about by tetrodotoxin-insensitive Na permeability, whereas that induced by higher concentrations is mainly caused by a direct increase o f Ca influx into smooth muscle cells. +
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Palytoxin ( P T X ) is o n e o f the most potent marine toxins k n o w n and the lethal dose ( L D ) o f the toxin i n mice is 0.5 /ig/kg when injected i.v. T h e molecular structure o f the toxin has been determined fully (1,2). P T X causes contractions i n s m o o t h muscle (3) and has a positive i n o t r o p i c action i n cardiac muscle (4-6). P T X also induces membrane depolarization i n intestinal s m o o t h (3), skeletal (4), and heart muscles (5-7), myelinated fibers (8), spinal cord (9), and squid axons (10). P T X has been demonstrated to cause N E release from adrenergic neurons (11,12). B i o c h e m i c a l studies have indicated that P T X causes a release o f K from erythrocytes, which is followed by hemolysis (13-15). T h e P T X - i n d u c e d release o f K from erythrocytes is depressed by ouabain and that the b i n d i n g o f ouabain to the membrane fragments is inhibited by P T X (15). 5 Q
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Methods T h e vas deferens was removed from male guinea pigs (250-350 g). T h e preparat i o n o f vas deferentia was carried o u t as described previously (16).
Culture of Pheochromocytoma Cells (PC12 Cells). P C 1 2 cells, kindly supplied by D r . H . H a t a n a k a o f o u r institute, were maintained i n Dulbecco's modified Eagle's medium containing 5% heat-inactivated horse serum. Assay of Endogenous NE
Endogenous N E release from the guinea p i g vas deferens was measured by means o f a high pressure-liquid chromatograph w i t h an O D S c o l u m n and an electrochemical detector as described previously (16).
0097-6156/90/0418-0219$06.00/0 o 1990 American Chemical Society
Hall and Strichartz; Marine Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
MARINE TOXINS: ORIGIN, STRUCTURE, AND MOLECULAR PHARMACOLOGY
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Assay of H - N E . 3
Experiments to determine performed as described previously (26). 22
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H - N E released from P C 1 2 cells were
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Assay of Na and Ca Influxes. Experiments t o determine ^ N a and C a influxes 4 5
into P C 1 2 cells were done as reported previously (12). Tissue Na and K Content Rectangular strips about 20 m g each were made by cutting t h e vas deferens longitudinally. After 30 m i n incubation under the control or test conditions, t h e strip was blotted, weighed, and then digested by incubation i n 0.2 m L o f a mixture containing equal amounts o f H N O - (61%) and H C 1 0 (60%) for 1 h r at 6 0 ° C ; the remaining tissue was then ashed by an overnight heating at 180 ° C . T h e ashed sample was dissolved i n cesium solution a n d t h e amount o f N a o r K was determined using an atomic absorption spectrophotometer (Varian, A A - 1 7 5 ) . 4
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Assay of Ionophore Activity. Ionophoretic activities o n rat liver mitochondria and liposomes were performed as described previously (12).
Results Mechanical Response. P T X (5 x 1 0 "
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to 3 x 1 0 ' M ) caused a concentrationdependent contraction o f the guinea p i g vas deferens. T h e configuration o f contractile response indicated the presence o f two components, an initial rapid c o m ponent followed by a second slow component. T h e first component o f the response t o P T X was abolished after treatment with M g (10 m M ) , C a - f r e e medium, o r ouabain (10" M ) , but remained almost unaffected by phentolamine (10" M ) , reserpine, 6 - O H D A , atropine, o r mecamylamine (10" M ) . T h e second component o f t h e response to P T X was also completely inhibited after t h e incubat i o n i n t h e high Mg " " o r C a - f r e e medium. Phentolamine, reserpine, o r 6O H D A markedly reduced only the second component. T h e second component also was inhibited by verapamil o r low N a medium, but was potentiated after treatment with ouabain (10" M ) . 2 +
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Endogenous N E Release. T h e release o f N E from the vas deferens was markedly 8
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increased by treatment with P T X (10" to 10" M ) . T h e P T X - i n d u c e d release o f N E was abolished by M g (10 m M ) o r C a - f r e e medium, but was potentiated by ouabain (10" M ) . 2 +
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H - N E Release. P T X ( 1 0 ' to 1 0 ' M ) caused a concentration-dependent release o f H - N E from P C I 2 cells. This releasing action o f P T X was markedly inhibited o r abolished by C a - f r e e medium, but was not modified by tetrodotoxin. T h e release o f H - N E induced by a low concentration (3 x 10" M ) o f P T X was abolished i n N a - f r e e medium and increased with increasing external N a concentrations from 3 to 100 m M . B u t the release induced by a high concentration (10" M ) was n o t changed by varying the concentration o f external N a from 0 to 100 m M . T h e release o f H - N E induced by both concentrations o f P T X increased as the external Ca concentration was increased from 0 to 3 m M , revealing the dependence o n extracellular C a . 3
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Na and Ca Influx. P T X caused a concentration-dependent increase i n N a and 2 2
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C a influxes into P C 1 2 cells at concentrations o f 1 0 to 1 0 M and 1 0 t o 1 0 M , respectively. T h e PTX-induced C a influx was markedly inhibited by C o but not by verapamil o r nifedepine, whereas the PTX-induced N a influx was n o t affected by tetrodotoxin. 4 5
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Assay of Na and K Content Figure 1 shows the effects o f PTX ( 1 0 M ) o n the 8
tissue N a and K content o f the guinea pig vas deferens.
T h e N a content increased
Hall and Strichartz; Marine Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
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16.
OHIZUMI
Mechanism of Pharmacological Action of Palytoxin
Control TTX 10~ M 6
Na cn
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O Treatment
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10" M Figure 1. Effects o f P T X o n the tissue N a and K content o f the vas deferens i n the presence o r absence o f tetrodotoxin ( T T X ) . P T X was added 30 m i n after the application o f T T X . T h e i o n content was measured 30 m i n after the application o f PTX.
Hall and Strichartz; Marine Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
222
MARINE TOXINS: ORIGIN, STRUCTURE, AND MOLECULAR PHARMACOLOGY
markedly 30 m i n after application o f P T X , whereas the K content fell. o f P T X was not affected by tetrodotoxin ( 1 0 M ) .
T h e effect
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Ionophoretic Activity. P T X , even at high concentrations, activity o n membranes o f mitochondria and liposomes.
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Discussion P T X caused a contraction o f the guinea pig vas deferens. T h e configuration o f the contractile response indicates the presence o f two components, an initial rapid component followed by a second slow component (72). T h e second component o f P T X - i n d u c e d contraction, but not the initial component, was markedly inhibited by alpha adrenoceptor blocking agents, reserpine, and 6 - O H D A , but was unaffected by cholinergic receptor blocking agents. B o t h components were abolished by M g containing o r C a - f r e e medium. P T X markedly increased the N a content o f the vas deferens. These data support the view that the first component is caused by a direct action o f P T X o n smooth muscle, possibly due to an increase i n N a influx, whereas the second component is the result o f an indirect action mediated through N E release from the adrenergic nerve ending o f the vas deferens. T h e initial component o f the P T X - i n d u c e d contraction is selectively inhibited by ouabain, whereas the second component is rather potentiated by it (27). A similar inhibitory effect o f ouabain o n the contractile response to P T X is observed i n the umbilical artery, which is devoid o f adrenergic innervation (18). Furthermore, it has been found that ouabain, convallatoxin, and cymarin inhibit the P T X - i n d u c e d contraction o f rabbit aortic vascular smooth muscle (19). These observations have also provided evidence for the involvement o f N a , K - A T P a s e o n the contractile effect o f P T X i n smooth muscle. P T X caused a release o f N E from adrenergic nerve endings o f the vas deferens and from P C 1 2 cells. In P C 1 2 cells, the release o f N E induced by a l o w concentration o f P T X was not modified by tetrodotoxin, but was abolished i n N a - f r e e m e d i u m . However, the release induced by a high concentration is not affected by variations i n the concentration o f external N a . T h e release o f N E induced by b o t h concentrations o f P T X is increased with raised external C a concentration. T h e above findings suggest that the P T X - i n d u c e d release o f N E by lower concentrations o f P T X is primarily brought about by an increased N a permeability, whereas that induced by higher concentrations is mainly caused by a direct increase i n C a influx. Electrophysiological studies o n neuronal tissues have indicated that P T X causes a depolarization o f the membrane o f myelinated fibers (8), spinal cord (9), and squid axons (10). T h e P T X - i n d u c e d depolarization is suppressed by removal o f N a from the external medium, but only slightly diminished i n the presence o f tetrodotoxin. P T X causes an increase i n the resting N a permeability and changes the current-voltage characteristics o f myelinated fibers (8,10). T h e P T X - p o i s o n e d m e m brane is also permeable to other cations such as C s , and N H ^ (10). In addition, P T X even at high concentrations does not exhibit any ionophoretic activity. Furthermore, it was reported that P T X triggered the formation o f small pores linked with N a , K - A T P a s e i n resealed ghosts (20). These observations taken together suggest that P T X creates a novel channel i n the membrane, thereby causing an increase i n cation permeability and depolarization and thus inducing a release o f N E from the adrenergic nerve. 2 +
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Acknowledgments I am greatly indebted to Prof. Y . H i r a t a o f M e i j o University and D r . D . U e m u r a o f S h i z u o k a University for kindly supplying palytoxin. I am grateful to M s . Y . M u r a k a m i o f this institute for secretarial assistance.
Hall and Strichartz; Marine Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
16. OHIZUMI
Mechanism ofPharmacological Action ofPalytoxin
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Hall and Strichartz; Marine Toxins ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
