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Relaxing action of a holothurian toxin on mammalian smooth muscle
Camp Emcltem. Phys~ol Vol 94C. No
1. pp 165-I 67. 1989
0306-4492189 $3 00 + 0 00 fc 1989 Pergamon Press plc
Pnnted I” Great Rntam
RELAXING
ACTION OF A HOLOTHURIAN TOXIN ON MAMMALIAN SMOOTH MUSCLE DENISE E. DE VORE and Jose DEL CASTILLO
Institute of Neurobiology,
UPR-MSC.
Old San Juan,
Puerto
Rico, U.S.A.
Tel. (809) 7214149
Recerred 26 January 1989) Abstract-l. The crude epidermal mucous secretion produced by the sea cucumber Holofhuria mexicana Ludwig Inhibits the tonus and spontaneous mechanical activity of rabbit ileum at concentrations of 0.1 mgjml and higher. 2. This effect, similar to that of epinephrine, is attributed to a compound referred to as mucotoxm (MuTX) 3. MuTX also relaxes strips of the rabbit aorta contracted by 1O-8 M norepinephrine and exerts a similar. less marked, effect on the same strips contracted in high potassium solutions. 4. These observations suggest that the relaxing effect of MuTX on mammalian smooth muscle is not medlated by an adrenergic mechanism.
INTRODUCTION
secretlon, the debris which covers the skin of the sea cucumbers was washed off. Then the animals were taken out of the water and brushed with a laboratory bottle brush (nylon bristles) until foaming was induced. As soon as the secretion was stimulated, the animals were placed m 300 ml of distilled water where the mucus was colected for 5 mm. Batches of three animals were processed together. No attempt has been made, so far, to investigate possible sexual differences in the toxicity of the secretion; animals of both sexes were used. The mucus was lyophihzed and kept at room temperature. Preliminary characterization of MuTX indicates it IS non-proteinaceous. non-dlalizable, and heatstable. Before use. the lyophilized crude MuTX was dissolved in a 5% solution of Tween-40 in distilled water. 2. Mammalran preparations. Fifteen adult albino rabbits 1.5-2.5 kg m weight were used in this work. They were sacrificed by asphyxatlon in a CO? atmosphere followed by cervical dislocation. Both the ileum and the aorta were rapidly excised and placed in cold (4 C) saline solution. The ileum was washed repeatedly in cold saline and cut into segments about 2cm long. The fat and connective tissue adhering to the aorta were carefully removed and, following the technique of Ohizuml and Yasumoto (1983). the aortlc wall was cut into helical strips approximately 4mm wide and 2cm long. Although the experiments were started immediately after dissection. both ileum and aortlc strips kept at 4’C remained in good functional condition for at least 48 hours. 3. Mechanical recording. Both the lleal segments and the aortic strips were mounted in a thermostatically controlled vertical organ bath, 10 ml in capacity, and attached by a silk thread to an isometric transducer connected to a Grass Model 7D kymograph. After adJusting the preparations to their approximate resting length, a load of 1 g was applied to the ileum segments and 0.5 g to the aortic strips. Before each experiment the preparations were allowed to equilibrate in the bath for at least 30mm. 4. Physiological saline. The Locke’s salme solution had the followmg ionic composltion (m mM), Na+. 154; K+, 5.6, Ca2+ > 3.1 HCO; 2; Cl-, 166 and dextrose. 2 Its pH was adjusted to 7.3 with 1 N HCI. A mixture of 95% oxygen + 5% CO, was bubbled continuously through the bath solution which was kept at 37°C. 5. Drugs. Epmephrine and norepmephrine were obtained from Sigma Chemical Company, St. Louis. MO.
It has long been known that certain species of sea cucumbers synthesize substances that are toxic to fishes and other marine animals (Frey, 1951; Nigrelli, 1952; Yamanouchi, 1955; Ruggieri and Nigrelli, 1960). Recent work has shown that the crude mucous secretion of Holohuria mexicana Ludwig exerts several biological actions. Prominent among them is its ichthyotoxicity (De Vore, 1987). Indeed, specimens of the fish Atherinomorus stripes (Miiller and Troschel) exposed to crude mucus preparations at concentrations as low as 3 pg/ml died within twenty minutes. This effect was attributed to the presence, in the secretion, of a toxin which we shall refer to as mucotoxin (MuTX). In an attempt to elucidate the mechanism of action of MuTX we have screened its effects on several biological preparations and found that it produces a marked inhibitlon of both tonus and spontaneous activity of isolated segments of rabbit ileum in r!irro. This effect is similar to that elicited on the same preparations by epinephrine and related compounds. The possibility was considered that MuTX might behave as an adrenergic agonist. However, MuTX produced a slow relaxation, rather than a contracture, of strips of the wall of the rabbit thoracic aorta, which respond to the application of 10-8 M norepinephrine with a prolonged tonic shortening. In addition, MuTX was seen to relax aortic strips contracted by norepinephrine or by increasing the external concentration of K+ ions.
MATERIALS
AND METHODS
I. MuTX. The crude mucous secretion of H. mexicana was obtained and processed at the Marine Station of the U. of Puerto Rico, La Parguera, Lajas, P.R. Specimens of this sea cucumber were collected at the Caracoles Reef and transported to the laboratory where they were kept in running sea water tanks. To obtain the cutaneous mucous
165
166
DENISE E. DE VORE and Jo& DEL CASTILLO
Frg. I. Relaxing effect of MuTX (crude mucous preparation activtty
of the isolated
ileum of the rabbtt.
The two upper
dtssolved m 5% Tween-40) on the mechamcal records of column A rllustrate the inhibitory
effect of lo-’ M epinephrine on both tonus and spontaneous mechanical activrty of two segments of ileum After washing, the two preparations were exposed to 1 mg/ml MuTX (column B), which exerted effects qualitatively and quantitattvely simtlar to those of epmephrine. The bottom records of each column shovv the lack of effect of Tween-40 (5% solutron m Locke’s) on the same preparattons. Column C illustrates the effects of different concentrations of MuTX on another preparation A segment of Ileum was exposed (from top to bottom) to IO-’ M epinephrine, 1.0, 0.5. 0.25 and 0.1 mglml MuTX, and agam to IO--’ M epinephrine Cahbrations: vertical 1 g; hortzontal I mm. Note that, in this preparatton the effect of I mg/ml of MuTX 1s somewhat smaller than m the expertment of columns A and B.
RESULTS AND DISCUSSION
identical to that exerted by 10 -’ M epinephrine on these particular preparattons. The bottom records of columns A and B of Fig. 1 show that the Tween40 used to facilitate the solution of MuTX had, by itself. no appreciable effects on the motility of the ileum. The inhibitory effect of MuTX on the mechanical activity of the Intestine is concentration-dependent: this is illustrated by the records of column C of Fig. 1
Addition of 1 mg/ml of MuTX, dissolved in 5% Tween-40, to the saline solution bathing a segment of ileum induced a rapid inhibition of both tonus and spontaneous activity as illustrated in the two upper records of column B. Fig. 1. As seen by comparing these records with the two upper ones of column A in the same figure. this effect IS almost
J t
i
Fig. 2. Effects of MuTX on the mechanical activity of strrps of rabbit aorta. (A) Upper record tome contraction induced by lo-sM norepinephrine on an aortic strip. Lower record: after washing. norepinephrine was apphed agam (first arrow), followed, at the second arrow, by 1 mg/ml MuTX. A marked relaxation took place. (B) Upper and lower records show the sustained contraction induced m two different strtps by increasing the external potassium concentration from 5.6 to 102 mM (first arrows). At the second arrow in each record 1 mg/ml MuTX was added to the bath, producing a small and transient, but clear relaxation. Calibrations: vertical, 0.5 g: horizontal, 1min.
Relaxing
action
of mucotoxin
where a segment of ileum was exposed (from top to bottom) to epinephrine (lo-’ M), to 1.0, 0.5, 0.25 and 0.1 mg/ml of MuTX and again to lo-’ M epinephrine. The similarity of the actions of MuTX and epinephrine on the isolated segments of intestine suggested that MuTX might behave as an adrenergic agonist. This possibility was explored by testing the effects of MuTX on another tissue highly sensitive to catecholamines; namely, the thoracic aorta of the same rabbits used to obtain the ileum. Circular rings or helical strips of aortic wall respond with powerful tonic contractions to the application of epinephrine and norepinephrine at conentrations as low as lo-* M. A contraction of this preparation should be expected, therefore, following its exposure to MuTX, a substance which had been shown to exert. on the ileum, effects comparable to those of epinephrine. However, no shortening of the aortic strips was observed when MuTX (1 mg/ml) was added to the bath. Only a small, slow relaxation of the strips was observed in some preparations in response to the toxin. This relaxing action was confirmed by applying MuTX (1 mg/ml) to aortic strips contracted under the influence of either lOmEM norepinephrine or by increasing the external concentration of K+ ions by a factor of 10-20 times. The top record of column A of Fig. 2 illustrates the sustained tonic contraction of an aortic strip induced by lo-‘M norepinephrine. The lower record of the same column shows the marked relaxing effect of MuTX (1 mg/ml) on the same preparation. The two records of column B show the very prolonged contractions induced in two aortic strips by increasing the external concentration of K+ ions from 5.6 to 103 mM (first arrows). At the second arrows 1 mg/ml of MuTX was applied; a small and transient, but unequivocal relaxation was seen to follow. These results demonstrate that the crude epidermal mucous
secretion
which
normally
coats
the
body
on smooth
muscle
167
surface of the sea cucumber and is produced in much larger amounts as a response to stress, contains a compound which exerts a relaxing effect on both the intestinal longitudinal and the circular aortic mammalian smooth muscle. Although marine toxins with adrenergic activity have been described (cf. Kobayashi, Nakamura and Oihizumi, 1988) the observed actions of MuTX do not obviously involve adrenergic receptors. Yet, depending upon the particular type of smooth muscle used, MuTX behaves as a synergist (ileum) or a physiological antagonist (aorta) of catecholamines. Acknowledgemenls-We thank Dr T. Tosteson for valuable help and the use of the facilities at La Parguera, Dr G. Escalona de Motta for discussions and loan of equipment, Drs R. K. Orkand and A. Rivera for comments on the MS and Mr. F Mackenzre for help with the laboratory animals. This work was supported by NIH grants NS-07464 and NS-14938.
REFERENCES
De Vore D. E. (1987) Ichthyotoxicity of Holothuriu mexicana Ludwig mucous secretion. Toxicon 25, 139. Abstract. Frey D. G. (1951) The use of sea cucumbers in poisoning fishes. Copera 1951(2), 175-176. Kobayashi J., Nakamura H. and Ohizumr Y. (1988) a-Adrenoceptor blocking action of hymenin, a novel marine alkaloid. Experientia 44, 86-87. Nigrelh R. F. (1972) The effects of holothurin on fish and mace with Sarcoma 180. Zoologica 37, 89-90. Ohtzumi Y. and Yasumoto T. (1983) Contractrle response of the rabbit aorta to mattotoxin, the most potent marine toxin. J. Physlol. 337, 71 I-721. Ruggieri G. D. and Nigrelli R. F. (1960) The effects of holothurin. a steroid saponin from the sea cucumber, on the development of the sea urchin. Zoologrca 45, 1-16. Yamanouchi T. (1955) On the poisonous substance contamed in holothunans. Publ. Seto. Mar. Biol. Lab. IV (2-3). 183-202.
1. pp 165-I 67. 1989
0306-4492189 $3 00 + 0 00 fc 1989 Pergamon Press plc
Pnnted I” Great Rntam
RELAXING
ACTION OF A HOLOTHURIAN TOXIN ON MAMMALIAN SMOOTH MUSCLE DENISE E. DE VORE and Jose DEL CASTILLO
Institute of Neurobiology,
UPR-MSC.
Old San Juan,
Puerto
Rico, U.S.A.
Tel. (809) 7214149
Recerred 26 January 1989) Abstract-l. The crude epidermal mucous secretion produced by the sea cucumber Holofhuria mexicana Ludwig Inhibits the tonus and spontaneous mechanical activity of rabbit ileum at concentrations of 0.1 mgjml and higher. 2. This effect, similar to that of epinephrine, is attributed to a compound referred to as mucotoxm (MuTX) 3. MuTX also relaxes strips of the rabbit aorta contracted by 1O-8 M norepinephrine and exerts a similar. less marked, effect on the same strips contracted in high potassium solutions. 4. These observations suggest that the relaxing effect of MuTX on mammalian smooth muscle is not medlated by an adrenergic mechanism.
INTRODUCTION
secretlon, the debris which covers the skin of the sea cucumbers was washed off. Then the animals were taken out of the water and brushed with a laboratory bottle brush (nylon bristles) until foaming was induced. As soon as the secretion was stimulated, the animals were placed m 300 ml of distilled water where the mucus was colected for 5 mm. Batches of three animals were processed together. No attempt has been made, so far, to investigate possible sexual differences in the toxicity of the secretion; animals of both sexes were used. The mucus was lyophihzed and kept at room temperature. Preliminary characterization of MuTX indicates it IS non-proteinaceous. non-dlalizable, and heatstable. Before use. the lyophilized crude MuTX was dissolved in a 5% solution of Tween-40 in distilled water. 2. Mammalran preparations. Fifteen adult albino rabbits 1.5-2.5 kg m weight were used in this work. They were sacrificed by asphyxatlon in a CO? atmosphere followed by cervical dislocation. Both the ileum and the aorta were rapidly excised and placed in cold (4 C) saline solution. The ileum was washed repeatedly in cold saline and cut into segments about 2cm long. The fat and connective tissue adhering to the aorta were carefully removed and, following the technique of Ohizuml and Yasumoto (1983). the aortlc wall was cut into helical strips approximately 4mm wide and 2cm long. Although the experiments were started immediately after dissection. both ileum and aortlc strips kept at 4’C remained in good functional condition for at least 48 hours. 3. Mechanical recording. Both the lleal segments and the aortic strips were mounted in a thermostatically controlled vertical organ bath, 10 ml in capacity, and attached by a silk thread to an isometric transducer connected to a Grass Model 7D kymograph. After adJusting the preparations to their approximate resting length, a load of 1 g was applied to the ileum segments and 0.5 g to the aortic strips. Before each experiment the preparations were allowed to equilibrate in the bath for at least 30mm. 4. Physiological saline. The Locke’s salme solution had the followmg ionic composltion (m mM), Na+. 154; K+, 5.6, Ca2+ > 3.1 HCO; 2; Cl-, 166 and dextrose. 2 Its pH was adjusted to 7.3 with 1 N HCI. A mixture of 95% oxygen + 5% CO, was bubbled continuously through the bath solution which was kept at 37°C. 5. Drugs. Epmephrine and norepmephrine were obtained from Sigma Chemical Company, St. Louis. MO.
It has long been known that certain species of sea cucumbers synthesize substances that are toxic to fishes and other marine animals (Frey, 1951; Nigrelli, 1952; Yamanouchi, 1955; Ruggieri and Nigrelli, 1960). Recent work has shown that the crude mucous secretion of Holohuria mexicana Ludwig exerts several biological actions. Prominent among them is its ichthyotoxicity (De Vore, 1987). Indeed, specimens of the fish Atherinomorus stripes (Miiller and Troschel) exposed to crude mucus preparations at concentrations as low as 3 pg/ml died within twenty minutes. This effect was attributed to the presence, in the secretion, of a toxin which we shall refer to as mucotoxin (MuTX). In an attempt to elucidate the mechanism of action of MuTX we have screened its effects on several biological preparations and found that it produces a marked inhibitlon of both tonus and spontaneous activity of isolated segments of rabbit ileum in r!irro. This effect is similar to that elicited on the same preparations by epinephrine and related compounds. The possibility was considered that MuTX might behave as an adrenergic agonist. However, MuTX produced a slow relaxation, rather than a contracture, of strips of the wall of the rabbit thoracic aorta, which respond to the application of 10-8 M norepinephrine with a prolonged tonic shortening. In addition, MuTX was seen to relax aortic strips contracted by norepinephrine or by increasing the external concentration of K+ ions.
MATERIALS
AND METHODS
I. MuTX. The crude mucous secretion of H. mexicana was obtained and processed at the Marine Station of the U. of Puerto Rico, La Parguera, Lajas, P.R. Specimens of this sea cucumber were collected at the Caracoles Reef and transported to the laboratory where they were kept in running sea water tanks. To obtain the cutaneous mucous
165
166
DENISE E. DE VORE and Jo& DEL CASTILLO
Frg. I. Relaxing effect of MuTX (crude mucous preparation activtty
of the isolated
ileum of the rabbtt.
The two upper
dtssolved m 5% Tween-40) on the mechamcal records of column A rllustrate the inhibitory
effect of lo-’ M epinephrine on both tonus and spontaneous mechanical activrty of two segments of ileum After washing, the two preparations were exposed to 1 mg/ml MuTX (column B), which exerted effects qualitatively and quantitattvely simtlar to those of epmephrine. The bottom records of each column shovv the lack of effect of Tween-40 (5% solutron m Locke’s) on the same preparattons. Column C illustrates the effects of different concentrations of MuTX on another preparation A segment of Ileum was exposed (from top to bottom) to IO-’ M epinephrine, 1.0, 0.5. 0.25 and 0.1 mglml MuTX, and agam to IO--’ M epinephrine Cahbrations: vertical 1 g; hortzontal I mm. Note that, in this preparatton the effect of I mg/ml of MuTX 1s somewhat smaller than m the expertment of columns A and B.
RESULTS AND DISCUSSION
identical to that exerted by 10 -’ M epinephrine on these particular preparattons. The bottom records of columns A and B of Fig. 1 show that the Tween40 used to facilitate the solution of MuTX had, by itself. no appreciable effects on the motility of the ileum. The inhibitory effect of MuTX on the mechanical activity of the Intestine is concentration-dependent: this is illustrated by the records of column C of Fig. 1
Addition of 1 mg/ml of MuTX, dissolved in 5% Tween-40, to the saline solution bathing a segment of ileum induced a rapid inhibition of both tonus and spontaneous activity as illustrated in the two upper records of column B. Fig. 1. As seen by comparing these records with the two upper ones of column A in the same figure. this effect IS almost
J t
i
Fig. 2. Effects of MuTX on the mechanical activity of strrps of rabbit aorta. (A) Upper record tome contraction induced by lo-sM norepinephrine on an aortic strip. Lower record: after washing. norepinephrine was apphed agam (first arrow), followed, at the second arrow, by 1 mg/ml MuTX. A marked relaxation took place. (B) Upper and lower records show the sustained contraction induced m two different strtps by increasing the external potassium concentration from 5.6 to 102 mM (first arrows). At the second arrow in each record 1 mg/ml MuTX was added to the bath, producing a small and transient, but clear relaxation. Calibrations: vertical, 0.5 g: horizontal, 1min.
Relaxing
action
of mucotoxin
where a segment of ileum was exposed (from top to bottom) to epinephrine (lo-’ M), to 1.0, 0.5, 0.25 and 0.1 mg/ml of MuTX and again to lo-’ M epinephrine. The similarity of the actions of MuTX and epinephrine on the isolated segments of intestine suggested that MuTX might behave as an adrenergic agonist. This possibility was explored by testing the effects of MuTX on another tissue highly sensitive to catecholamines; namely, the thoracic aorta of the same rabbits used to obtain the ileum. Circular rings or helical strips of aortic wall respond with powerful tonic contractions to the application of epinephrine and norepinephrine at conentrations as low as lo-* M. A contraction of this preparation should be expected, therefore, following its exposure to MuTX, a substance which had been shown to exert. on the ileum, effects comparable to those of epinephrine. However, no shortening of the aortic strips was observed when MuTX (1 mg/ml) was added to the bath. Only a small, slow relaxation of the strips was observed in some preparations in response to the toxin. This relaxing action was confirmed by applying MuTX (1 mg/ml) to aortic strips contracted under the influence of either lOmEM norepinephrine or by increasing the external concentration of K+ ions by a factor of 10-20 times. The top record of column A of Fig. 2 illustrates the sustained tonic contraction of an aortic strip induced by lo-‘M norepinephrine. The lower record of the same column shows the marked relaxing effect of MuTX (1 mg/ml) on the same preparation. The two records of column B show the very prolonged contractions induced in two aortic strips by increasing the external concentration of K+ ions from 5.6 to 103 mM (first arrows). At the second arrows 1 mg/ml of MuTX was applied; a small and transient, but unequivocal relaxation was seen to follow. These results demonstrate that the crude epidermal mucous
secretion
which
normally
coats
the
body
on smooth
muscle
167
surface of the sea cucumber and is produced in much larger amounts as a response to stress, contains a compound which exerts a relaxing effect on both the intestinal longitudinal and the circular aortic mammalian smooth muscle. Although marine toxins with adrenergic activity have been described (cf. Kobayashi, Nakamura and Oihizumi, 1988) the observed actions of MuTX do not obviously involve adrenergic receptors. Yet, depending upon the particular type of smooth muscle used, MuTX behaves as a synergist (ileum) or a physiological antagonist (aorta) of catecholamines. Acknowledgemenls-We thank Dr T. Tosteson for valuable help and the use of the facilities at La Parguera, Dr G. Escalona de Motta for discussions and loan of equipment, Drs R. K. Orkand and A. Rivera for comments on the MS and Mr. F Mackenzre for help with the laboratory animals. This work was supported by NIH grants NS-07464 and NS-14938.
REFERENCES
De Vore D. E. (1987) Ichthyotoxicity of Holothuriu mexicana Ludwig mucous secretion. Toxicon 25, 139. Abstract. Frey D. G. (1951) The use of sea cucumbers in poisoning fishes. Copera 1951(2), 175-176. Kobayashi J., Nakamura H. and Ohizumr Y. (1988) a-Adrenoceptor blocking action of hymenin, a novel marine alkaloid. Experientia 44, 86-87. Nigrelh R. F. (1972) The effects of holothurin on fish and mace with Sarcoma 180. Zoologica 37, 89-90. Ohtzumi Y. and Yasumoto T. (1983) Contractrle response of the rabbit aorta to mattotoxin, the most potent marine toxin. J. Physlol. 337, 71 I-721. Ruggieri G. D. and Nigrelli R. F. (1960) The effects of holothurin. a steroid saponin from the sea cucumber, on the development of the sea urchin. Zoologrca 45, 1-16. Yamanouchi T. (1955) On the poisonous substance contamed in holothunans. Publ. Seto. Mar. Biol. Lab. IV (2-3). 183-202.