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Pardaxin increases solute permeability of gills and rectal gland in the dogfish shark (Squalus acanthias)
Camp. Biochem. Phwiol. Vol. 78C. No. 2. pp. 483-490, Printed in Great Britain
1984
0306~4492/84 $3.00 + 0.00
!Q 1984Pergamon Press Ltd
PARDAXIN INCREASES SOLUTE PERMEABILITY OF GILLS AND RECTAL GLAND IN THE DOGFISH SHARK (SQUALL8 ACANTHIAS)” NAFTALI PRIMOR,* JOSE A. ZADUNAISKY,~ H. VICTOR MURDAUGH JR.,$ JAMES L. BOYER~ and JOHN N. FORREST,JR.$ Laboratories of Marine Sciences, New York Aquarium, Coney Island, Brooklyn, NY 11224,
*Osborn USA, Telephone 266-8500, TDepartment of Physiology and Biophysics, New York University Medical Center, New York, NY 10016, USA, $Department of Medicine, University of South Carolina, Columbia, SC 29201. USA, $Department of Medicine, Yale University School of Medicine, New Haven, CN 06510, USA and Mount Desert Island Biological Laboratory, Salsbury Cove, ME 04672, USA (Received 5 May 1983)
Abstract-l. The action of the ichthyotoxic secretion of the Red Sea flatfish Pardachirus tnarmoratus and its derived toxin, pardaxin, was examined in the dogfish shark (Squalus_acanthias). 2. Pardaxin was more toxic when administered to the bathing medium than when injected into a dorsal artery and it transiently diminished the spiracular rate and caused a severe struggling response in the adult shark only when administered to the head region of the shark. 3. Pardaxin caused a transient leakage to urea and sodium between the shark and the seawater. 4. In the isolated perfused rectal gland pardaxin irreversibly reduced the rate of chloride secretion and concentration gradient of urea between perfusate and rectal gland fluid. In addition, ultrastructural studies on the rectal gland showed that ionic lanthanum penetrated the tight junctions and foci of cell necrosis were observed. 5. These studies indicate that in shark the gills are the most probable target of the toxicity of pardaxin.
Barnholz, 1983) and to cause lysis in cells (Primor and Lazarovici, 1981) and to display a surfactant-like action by decreasing surface tension (Primor et al., 1983a). Previous work suggests that in teleosts the gills are the primary site of the toxic effects of PX (Primor et a/., 1980). Furthermore, studies using the isolated gill-like opercular epithelium from the teleost Fundulus heteroclitus show a transient increase in gill permeability and production of Na+ influx (Primor, 1983). However, the effects of PX on osmoregulatory tissue in the elasmobranch shark have not been previously investigated. We studied the effects of PX on whole animals, using both adults and fetuses (pups) of the spiny dogfish shark, Squalus acanthius. Our studies determined its toxicity, its effect on spiracular rate and gill permeability to sodium and urea. In addition we studied the action of PX upon the isolated perfused rectal gland.
INTRODUCTION Pardachirus marmoratus, has hundred glands located along its fins (Clark and Chao, 1973). The
The Red Sea flatfish, more dorsal
than and
two anal
secretion of these glands had been found to be toxic to various teleosts (Clark and George, 1979) and to repel sharks (Clark, 1974) suggesting that the biological significance of the secretion is the defense of the flatfish against predators (Clark, 1983). Studies using a whole secretion (PMC) or isolated indicate multiple pharmafraction (pardaxin) cological effects. The ichthyotoxicity of Parduchirus secretion was enhanced in fish adapted to hyperosmotic media which suggests that it acts upon the ability of the fish to osmoregulate adequately (Primor et al., 1980). In other experiments Purduchirus secretion elicited transmitter release and histopathological changes in frog neuro-muscular junction (Spira et al., 1976) and produced a contraction in guinea-pig ileum (Parness and Zlotkin, 1976). It increases the electrical conductance of the lipid bilayer membrane in uniform steps indicating formation of transmembranal channels (Moran et al., 1978; Korchak, 1979). Toxicity has been attributed to a proteinaceous component in the secretion. This toxin, named pardaxin (PX), has been isolated and characterized. It is a protein composed of 160 amino acids cross-linked with four disulphide bridges (Primor et al., 1978; Primor and Tu, 1980). Pardaxin was shown to interact with bilayer membrane making it permeable to several proteins (Pal et al., 1981a,b; Zlotkin and *Part of this work was presented Desert Is/. hiol. Lab., Summer,
in an abstract 1980.
MATERIALS
AND METHODS
Pardachirus marmoratus fish (Pisces; Soleidae) were collected from the Red Sea (Eilat, Israel). P. murmoratus secretion (PMC) was obtained by the method described by Clark and Chao (1973). Pardaxin (PX), the ichthyotoxic component isolated from PMC, was prepared according to Primor et al. (1978) and stored in a lyophylized form. Toxicity was determined in dogfish fetuses (pups) which were removed from sacrificed females and maintained in aquaria with running seawater and used within two days. The lethality was determined in two different ways: (1) administration of pardaxin into a tank of 200 ml seawater with 5-6 pups; (2) injection via caudal artery using a syringe and needle gauge number 20. The lethal dose (LD,,) was
in BUM.Mt
483
484
NAFTALI PRIMORrr~1.
calculated by the method of Reed and Muench (1938) using 40 pups for each determination. Female dogfish sharks, Squalus ucanthcas, weighing 4-5 kg were taken from Frenchman’s Bay, Maine, for study at the Mount Desert Island Laboratory. They were maintained in marine live-cars in a fasted state and used within 3-4 days of capture. Isotope meusuremenls The effect of PX on gill permeability to urea and sodium was studied in adult dogfish positioned in an apparatus with a rubber diaphragm inserted close to the posterior gill cleft, separating the head region from the rest of the body. With the fish in position the head and rear chambers contained 8 and 12 I. of water respectively. During the experiments the fish were kept in the apparatus with running (5 I. min-‘) seawater (15 + 1 C). The dorsal aortae of the dogfish were catheterized via the cdudal artery (using polyethylene tubing PE 90, Clay Adams Co., Parsippany. NJ) for the purpose of injecting pardaxin and isotopes and collecting blood samples. The urinary papillae were also cdnnulated and urine was collected in a balloon. Three different experimental procedures were followed using this apparatus. (I) Determination of the effect of seawater-administered PX on the rate of appearance of plasma “Na + and [“C]urea from the fish to the bathing seawater (five experiments). Fish injected with 200 p Ci of “Na + or [“‘Clurea were kept in the diaphragm apparatus for 3 hr. Then the seawater flow was stopped and every 2 min blood and water samples from both sides of the diaphragm were taken and spiracular movements were counted. After 10 min the running seawater was restored for 3 hr. The same procedure was then repeated in the presence of PX. (2) Determination of the effect of seawater administered PX on the rate of appearance of [%Y]urea from the bathing seawater to the plasma (five experiments). [“‘CJUrea (25OpCi) was added to the bathing seawater in the head section of the apparatus while the flow of water was shut off. Every 2 min samples of blood and water from both sides of the diaphragm were taken and the spiracular movements were counted. Ten minutes after the water flow was shut off, the water from the chamber was removed and fresh running seawater was introduced for 3 hr. The procedure was then repeated with PX added together with the [lJC]urea. (3) Determination of the effect of PX administered by injection to the blood on the appearance of “Na’ and [“‘C]urea from the plasma to the seawater (three experiments). Fish injected with 200 FCi of isotope were kept in the diaphragm box for 3 hr. The water flow was shut otTand PX was then injected into the cannulated dorsal artery. For a IO min period blood and water samples from both sides of the diaphragm were taken every 2 min and the spiracular movements were counted. In order to determine rates of ‘?Na+ and [“Clurea appearance in the plasma or in the seawater, a time period of no water flow was required. The period of IOmin was chosen because the control fish easily recovered when running seawater was introduced after this interval. The blood PO? (partial oxygen pressure) recovered to normal levels and no change in gill urea permeability due to the lack of Rowing water was observed. Blood samples (I ml) were kept in glass syringes on ice. Blood PO, was determined within I hr using a Clark-type PO, electrode attached to a Radiometer pH meter gas monitor. To determine the leakage from seawater to the blood the red blood cells were centrifuged (800 x g) and [14C]urea activity in 0.05 ml of plasma was determined. To determine the leakage from the plasma to seawater. the isotopes ([‘+‘C]urea and “Na + ) were determined in 0.2 ml of seawater. “Na’ leakage from seawater to the blood was not determined to avoid a possible radiation hazard involved
in maintaining the fish in high ?‘Na * activity. Since. in fish. it is hard to estimate the gill area from where urea and sodium are transported it is inappropriate to express their transport in terms of flux (usually given per area). Instead, a leakage to urea and sodium was determined. The following calculation was applied: Change
in the cpm in the cold side
Specific activity of the isotope species
x
Sampling
time
(given in hr).
There is no significant (less than 2”,,) difference in quench as ?‘Na’ or [‘%Z]urea were added to 0.05 ml of plasma or to 0.2ml of seawater. Therefore. quench corrections for plasma or seawater were not made in these experiments. Appearance (leak) of sodium and urea from the fish 10 the seawater (when injected to the blood) or from the seawater to the fish body (when administered via the bathing water) was determined by liquid scintillation spectrometry and expressed in pequivalents hr ’ 5 kg ’ weight and corrected for background.
The procedure for removing the rectal gland, subsequent catheterization and its in vitro perfusion was performed as described in detail by Staff et u/. (1977). After basal perfusion for 20-30 min, the rectal gland was stimulated by the addition of theophylline (0.25 mM) and dibutyryl cyclic AMP (0.05 mM) to the perfusatc for 50-60min. After reaching a constant rate of rectal gland fluid secretion. PX was injected via the catheterized artery in a volume 01 0.2-0.3 ml of elasmobranch Ringer solution over I@15 sec. Rectal gland Ruid secretion was collected at intervals of IO min and the rate of secretion was determined by volumetric measurement. Since the volume of the perfusatc in the individual rectal gland was not determined, the final concentration of PX in the rectal gland remains unknowjn.
In perfused rectal gland prepared for electron microscopy, bicarbonate and phosphate were removed from the Ringer and replaced with 3 mM Tris to avoid precipitation of ionic lanthanum. Following perfusion in the stimulated state (theophylline 0.25mM and dibutyryl cyclic AMP. 0.05 mM) in the presence or absence of PX. the perfusate was changed to an identical solution containing ionic lanthanum (3 mM) and the perfusion was continued for 5 min. These glands were then fixed by perfusion for 15 min at 4-5,-C with a Karnovsky-type fixative containing 3”, glutaraldehyde. 47, paraformaldehyde. 0.1 M cacodylate at pH 7.4. Following fixation by perfusion. the glands were sectioned at the level of the rectal gland artery and small pieces (l-2 mm’) were obtained from the outer one-third of rectal gland tissue. Sections were incubated for 2 hr m fixative and then washed three times with 0. I M cacodylate buffer and refrigerated. Sections were examined under low power electron microscopy as reported perviously (Forrest ef (II., 1982). Theophylline was purchased from Merck. dibutyryl cyclic AMP, urease from soya bean type IX. were obtained from Sigma Chemical Co. [‘%Z]Urea and ‘INa. were obtained from New England Nuclear, Boston, Mass. Samples of plasma and seawater were dissolved in a scintillation cocktail and counted in a liquid scintillation counter.
RESULTS Toxicity PX
and the response of’s, acanthias
to PMC
and
The toxicities (LD,,) PMC and PX in dogfish pups (46 g) was determined using 40 specimens for each LD,, and were found to be 8.0 and 5.1 mg I ‘, respectively, as determined 1 hr after administration into 250 ml of seawater. The LD,,, (determined after
Pardaxin
V-
A‘ECOVER’ I --f+ -S
increases
shark
EXPERIMENTAL
Fig. 1. The effect of pardaxin (PX) on opercular rate, blood pH and partial oxygen pressure (PO,) in the adult shark Squalus acanthius. The sharks were studied in an apparatus with a diaphragm separating the head section and body. Each fish was studied during a control period of IOmin, allowed to recover for 3 hr and then restudied for 10min with PX (25 mg I-‘) in the head section of the apparatus. During the 10 min of both the control and the PX studies, the flow of seawater through the apparatus was shut off. Blood samples were collected to determine pH and PO,, and spiracular movements were counted every 2 min for a period of 10min. (~-0) Spiracular movements per min; (0-O) PO, in the blood samples; (0-O) blood pH. All results shown are the means of four experiments.
1 hr) of PMC and PX injected into a dorsal artery was found to be approximately ten times higher, 90.0 and 54.0 mg kg-‘, respectively. At 10 mg 1-l body wt, PX administered into seawater within 5-10 set was observed to cause severe struggling of the pups and a profound decrease in the spiracular rate, from 50-55 min’ to 3-5 min’ during the first 4 min, with a gradual increase to 20-25 mini’ after 10 min. An effect of PMC on opercular rate in teleosts was previously reported by Clark and George (1979). However, no immediate change in the spiracular rate was observed when a higher dose of PX, 50 mg kg-~’ was injected into the dorsal artery. In order to determine if the toxicity was mediated via the head of the fish, an apparatus was constructed with a rubber diaphragm separating the head of the pup from the rest of the body. The volumes of seawater were 40ml (head section) and 200ml (rear section). The diaphragm was inserted close behind the pup’s posterior gill cleft. PX (10 mg I-‘) was administered to the head section of the apparatus and was found to have a toxicity similar to that without a diaphragm. In addition, struggling and a decreased spiracular rate were always observed. PX (20 mg I-‘) administered to the rear section of the apparatus apparently did not cause toxicity as no struggling or changes in the spiracular rate occurred. The response to PX was also examined in five adult dogfish (4.0-5.0 kg) positioned in a large apparatus with a rubber diaphragm inserted close behind the posterior gill cleft. It was demonstrated that PX (25 mg I- ‘) administered to the region caused a diminished spiracular rate, from 40-42 min-’ to 8-l 1 min -’ as well as behavioral changes, struggling and opening the mouth as if to gasp. None of these responses were observed when PX (25 mg 1-l) was administered to the rear part of the apparatus.
gill permeability
485
to solute
The effects of PX on spiracular rate, blood pH and PO, in adult fish are shown in Fig. 1. Whereas stopping the flow of seawater in the chamber did not affect the spiracular rate (40-42 min’) in control fish, the spiracular rate was radically diminished in the presence of PX. Blood PO, in control fish was observed to fall during the stoppage of seawater flow. However, the PO, was significantly lower in the PX treated fish at each time period compared to controls (Table 1). No apparent changes in blood pH were observed throughout both control and PX exposure periods of study. These observations demonstrate that in both pups and adult dogfish, the head region is the target for PX activity. Following the abrupt decrease in the spiracular rate from 40-41 min-’ to 8-11 min’ at 2 min despite the presence of PX, the spiracular rate partially recovered to 30 min-’ indicating a transient property of this effect. The efect of’ PX on gill permeability sodium in adult dogfish
to urea and
Because the head region of the dogfish was found to be the target of PX action and because the gills of the dogfish are known to be relatively impermeable to urea and sodium (Boylan, 1967) it was of interest to determine whether PX induced changes in gill permeability to these solutes. No measureable radioactivity was detected in either the anterior (head) or posterior (body) chambers during stoppage in the seawater flow in five control fish injected 3 hr previously with 200 PCi [14C]urea or **Na + However, PX (25 mg I-‘) administered via the head section of the apparatus elicited a transient but marked increase in the permeability of both [14C]urea and **Na+ from plasma to seawater and [14C]urea from seawater to plasma (Fig. 2). Permeabilities increased maximally at 2 min for [14C]urea (seawater to plasma) and at 4 and 6 min for [14C]urea and **Nat (plasma to seawater) (Fig. 2). The concentration of PX used in these experiments (25 mg 1-l) was not lethal as all tested fish survived. No measurable radioactivity in seawater was observed when 40 mg of PX was injected into the circulation of fish previously injected with [14C]urea or **Na + (see Materials and Methods for experiment group No. 3). In addition, in experiments when PX was injected into the circulation no struggling or mouth opening occurred and no change in the spiracular rate was observed.
Table I. The effect of pardaxin (PX) on partial oxygen pressure (PO,) in the dogfish shark (S. aranlhias). The sharks were studied in a” apparatus with a diaphragm separating the head section and body. Each fish was studied during a control period of IOmin, allowed to recover for 3 hr and then restudied for 10 min with PX (25 mg I-‘) in the head section of the apparatus. During the IOmin of both the control and the PX studies the flow of seawater through the apparatus was shut ofI The data are expressed as mean ? SD of four experiments and statistical significance (P) between means was tested with Student’s I-test “sing paired data. Time (min) 2 4 6
8 10
Control 62.2 49.5 38.2 28.5 18.0
i f i + +
With pardaxin PO, CmmHg)
2.2 2.3 1.7 2.6 2.1
48.7 It 27.0 i II.71 5.5 f 1.7 +
1.8 2.1 1.7 13 1.5
Significance (P)
0.01f
486
NAFTALIPRIMORe/al.
TIME (mid Fig. 2. The effect of pardaxin (PX) on gill permeability to urea and sodium in adult shark Squalus acanthias. The sharks were studied in an apparatus with a diaphragm separating the head section from the body. Each fish was studied during a control period of 10 min, allowed to recover for 3 hr and restudied for 10min with PX. During the 10 min of both the control and PX studies, the flow of seawater through the apparatus was shut off. Every 2min during the 1Omin of each study, blood and seawater samples from both sides of the diaphragm was taken. In five experiments [“‘Clurea or “Na + (200 pCi) were injected into the circulation of fish and PX (25 mg I-‘) was administered in seawater in the head section of the apparatus (see experiment No. 1 in Materials and Methods). (0-O) Appearance of [r4C]urea in seawater in the head section of the apparatus; (O---O) [14C]urea in seawater in the rear section of the apparatus; (n-a) appearance of ‘?Na+ in seawater in the head section of the apparatus; (A-- -A) **Na+ in seawater in the rear section of the apparatus. In five experiments [“‘Clurea (25OpCi) and PX (25mgml ‘) were added to the seawater in the head section of the apparatus (see experiment No. 2 in Materials and Methods). (e-@) Appearance of [‘VZ]urea in the shark plasma. The data are expressed as means + SE for five fish. Note a transient increase in leak to sodium and urea.
gland secretion was typically low at 15 f 5 mM (mean + SE, n = 6). PX was administered by injection into the artery of the isolated perfused rectal gland during stimulation of chloride secretion by theophylline and dibutyryl cyclic AMP. The rate of secretion of urea in the gland fluid was found to be increased in a manner dependent on the PX dosage and no further effect was found above 400 ,ug. Moreover, the urea concentration gradient between the perfused Ringer in solution (350 mM) and the gland fluid (15 mM) was abolished while urea concentration in the perfusate approaches 280 mM (Fig. 3). While injection of 50 pg of PX had no effect on Cl secretion rate, 1000 p g inhibited within 45 min loo”/, of Cl secretion (Fig. 4). Furthermore, the chloride secretion rate was reduced in a dose-dependent manner. The injected PX was observed to result in the appearance of significant amounts of mucus in the gland fluid. In contrast to the effects on the gills, the increased permeability to urea and dissipation of chloride secretion in the gland were not transient at any dose and the function was not observed to be restored. This finding suggests permanent damage to the rectal gland tissue and this assumption was confirmed by ultrastructural studies. The results of ultrastructural studies are shown in Fig. 5. In control rectal glands perfused with ionic lanthanum during stimulation of secretion with theophylline and dibutyryl cyclic AMP (Fig. 5A) lanthanum was seen to fill the intracellular spaces up to the tight junctions but did not penetrate through to the lumen. These observations in control glands are identical to that reported previously by Forrest ef al. (1982). In contrast, in glands stimulated with
The effect of PX on the isolated perfused rectal gland As PX was observed to increase permeability to urea and sodium in the head region it was of interest to determine the effects of this agent on other osmoregulatory organs in the shark. It has been demonstrated that theophylline and dibutyryl cyclic AMP have a profound stimulatory effect on the flow rate in the isolated perfused rectal glands. These glands secrete chloride actively against its electrochemical gradient (Silva et al., 1977) and have ordinarily low permeability to urea (Burger, 1967). It was used to study the effects of drugs on active transport mechanisms (Epstein et al., 1981; 1983). The present study deals with the effects of PX on the performance of the rectal gland. In glands stimulated with theophylline (0.24 mM) and dibutyryl cyclic AMP (0.05 mM), prior to administration of PX, the rectal gland flow rate 2920~300~lhr’gwetwt ’ was averaging (mean f SE, n = 6) and the chloride secretion rate in the gland secretion was found to average (mean k SE, 1515+ 145pequiv.hr’gwetwtt n = 6). The urea concentration in the rectal
IO
30
50 TIME (min)
Fig. 3. Dose response of pardaxin (PX) on urea secretion rate in the perfused dogfish rectal gland fluid. The rectal gland was stimulated with dibutyryl cyclic AMP (0.05 mM) and theophylline (0.25 mM). After reaching a constant rate of secreted chloride in the control periods of 50-60 min. a single dose of PX was injected by the cannulated artery. The urea content in the rectal gland fluid was determined by phenolhypochloride method and expressed as percentage change in urea secretion rate as compared to mean control periods. For doses of PX (fig per rectal gland): (0~~0) 50; (e---0) 100; (n-/Q 200; (m---m) 400; and (0-n) 1000. The data are means of ten experiments, two experiments at each dosage with secretion rate of urea (percentage of control) given at IO min intervals for 60 min of perfusion with PX.
Pardaxin
IO
30
increases
shark
50 TIME
(mtn)
Fig. 4. Effect of pardaxin (PX) on the chloride secretion rate in the perfused dogfish rectal gland. The rectal gland was stimulated with dibutyryl cyclic AMP (0.05 mM) and theophylline (0.25 mM) for a control period of 50-60 min. After reaching a constant rate of secretion (1515 f 145 pequiv. hr-’ wet wt-‘) a single dose of PX was injected by the cannulated artery. Data are expressed as percentage inhibition of chloride secretion rate following injection of PX as compared to rate of chloride secretion (pequiv. Cl- secreted hr-’ g gland wet wt-‘) in control period. Symbols as in Fig. 3. The data are the means of ten experiments with two experiments of each dosage.
theophylline and dibutyryl cyclic AMP and exposed to PX, extensive lanthanum was seen in the lumen (Figs 5B and 5C, arrows) in association with disruption of the intracellular architecture. In some areas discrete foci of cellular necrosis were observed. DISCUSSION
In this study we determined that PX-induced toxicity struggling reaction, diminished spiracular rate, decrease in blood PO, and an increase in permeability of the gills to sodium and urea is mediated through the head region of the shark which is exposed to the outside bathing seawater. None of the above responses were produced by administration via the blood of 40 mg of PX. Based on the dogfish plasma volume of 6.6% body wt (Burger, 1967), one may estimate that the PX concentration in the plasma is approximately 121 mg 1-l in a 5.0 kg fish. However, when administered to the bathing water PX elicits responses at a dose of 25mg 1-l. Therefore it is unlikely that PX-induced responses are elicited by its penetration from the medium into the body and its action is elicited from the seawater side only. It is suggested that in elasmobranchs gills are the major organ for Na+ transport (Payan and Maetz, 1973; Evans, 1982; Evans et al., 1982). Therefore the leakage of sodium and urea between the shark’s body and the bathing seawater (Fig. 2) suggests that the gills are the primary site of PX action. It is unclear, however, how PX acts to inhibit the spiracular rate. Inhibition of spiracular rate may also contribute to the lower blood PO, in the presence of PX (Table 1). The pH of the blood is determined by the ratio of HCO,/CO, concentrations and will be
gill permeability
to solute
487
maintained as long as this ratio stays unchanged. The unchanged blood pH during control and in the presence of PX periods (Fig. 1) suggests that the ratio of concentrations HCO; /CO, was not changed while PO, values fall during control and in the presence of PX periods (Fig. 1). However, measurements of total COz concentrations are needed to support this assumption. In order to determine the effect of PX on shark tissues that actively transport chloride ions and are ordinarily impermeable to urea, we have studied in vivo the gills and the isolated perfused rectal gland. In both epithelial tissues, PX disrupts function in association with changes in solute permeability. However, the increased permeability of gills was shown to be transient even in the presence of PX (Fig. 2). The transient increase in permeability observed in whole fish resembles that observed in the isolated gill-like opercular epithelium of the teleost (Primor, 1983) indicating PX’s similarity in the mode of action in teleosts and elasmobranchs. However, irreversible damage was observed in rectal gland function while the toxin was washed out (Figs 3-5) and generalized cellular toxicity was observed. The significance of this difference is not clear. However, it may correlate with the proposed action to repel predators rather than to cause death. Measurements show PX’s ability to act as a surfactant by lowering the surface tension of aqueous solution (Primor et al., 1983). Furthermore, some commercial surfactants were shown to elicit repellent action in sharks (Gruber, 1982; Gruber and Zlotkin, 1982). The term “surfactant” generally refers to substances which reduce surface-tension. In biological systems, surfactants diminish the separation between water- and lipid-soluble substances and therefore allow interactions between water-soluble compounds and the cell membrane. It is not unique for biologically active substances to be armed with a surfactant quality. For example, potent detergents are generated from activation of membrane-bound phospholipase Az in mammalian sperm and it has been suggested that the significance of these detergents is to promote membrane fusion (Thakkar et al., 1983). It seems likely, then, that Pardachirus utilizes the detergent property of PX to facilitate interaction with biological membranes. The mechanism of PX’s action involves its insertion into the lipid bilayers as a functional transmembranal channel. Consequently, it causes an increase in their permeability to the seawater constituents. This sequence of events finally leads to its noxious effect in situ. It is suggested, here, that PX’s surfactant property promotes its spreading and adsorption from the water into which it is released by the flatfish and facilitates its noxious action upon the gill membranes of predators. It is noteworthy that substances capable of reducing surface-tension, in spite of their diverse chemical identity, such as dodecyl sodium sulphate-a derivative of lauryl alcohol (Gruber, 1982; Gruber and Zlotkin, 1982); holothurin-a toxic principle of a Bahamian holothurian of a lactone-like structure (Sobotka, 1965); and a proteinaceous PX were all toxic as well as producing a noxious and/or repellent response in sharks. A common action of such entirely different compounds suggests a similarity in their
488
NAFTALIPRIMORetal.
Fig. 5. Low power (x 12,150) cross section of tubules from isolated perfused rectal glands (Syuahs acanthias) during stimulation of chloride secretion with theophylline and dibutyryl cyclic AMP in the presence and absence of PX. A: Control gland demonstrating presence of electron dense lanthanum in the lateral intracellular space (arrow) up to but not through the tight junction and absence of lanthanum in the lumen; B and C: in PX perfused glands lanthanum appears within the intracellular space and extensive lanthanum is present in the lumen (see arrows). In the presence of pX there is partial loss of cellular architecture and vesiculation of the endoplasmic re+iculum.
mechanism. In rat ileum, synthetic anionic surfactants given to the mucosal (luminal) side elicited a net sodium secretion (Sund, 1975; Sund and Olsen, 198 1); a toxic, noxious and surface-active polypeptide
derived from the dart frog skin increases the rate of sodium entry when given to the outer face of the frog skin (Greenwell and Low, 1981; Montecucchi and Henschen, 1981). PX Jicited a net sodium influx in
Pardaxin increases shark gill permeability to solute the fish gill-like operculum skin, also acting from the outerface side only (Primor, 1983). Therefore it is possible that some of those compounds capable of producing a net sodium entry upon the outface of ion- and water-transporting epithelia could also exert a noxious and/or repellent effect in sharks. The remaining possibility to attribute the noxious effect to a general surfactant quality looks less valid because the cationic surfactants did not repel sharks (Gruber and Zlotkin, 1982) and the pulmonary surfactants secreted upon physiological conditions (Dobbs et al., 1982) are without a noxious action. This hypothesis, however. should be submitted to a controlled test. Acknowledgements-This work was performed during the tenure of J. N. Forrest, Jr. as an established investigator of the American Heart Association (77-228). The authors wish to acknowledge Dr. Barbara Kent for making the PO, measurements and Oi Cheng Ng for technical assistance with the electron micrographs, and Esta Van Tuyl for excellent typing of the manuscript. This research was supported by NIH Research Grants AM-17433 and PHS/EY 01340, NIH Research Grant PHS/GM 25002, NIH Research Grant PHS/EY 03151, The Department of Defense, Office of Naval Research, Research Grant No. N00014-81-K-0667 and N00014-82-C-0435 and National Science Foundation Grant BG-28139 to the Mount Desert Island Biological Laboratory.
REFERENCES Boylan J. W. (I 967) Gill permeability in Squalus acanthias. In Sharks, Skates and Rays (Edited by Gilbert P. W., Mathewson R. F. and Rail D. P.), pp. 197-205. Johns Hopkins Press, Baltimore. Burger J. W. (1967) Problems in the electrolyte economy of the spiny dogfish Squalus acanthias. In Sharks, Skates and Rays (Edited by Gilbert P. W., Mathewson R. F. and Rail D. P.), pp. 177-185. Johns Hopkins Press, Baltimore. Burger J. W. and Hess W. N. (1960) Function of the rectal gland in the spiny dogfish. Science 131, 670-671. Clark E. (1974) The Red Sea’s shark proof fish. Nat. geogr. Msg. 146, 718-728. Clark E. (1983) Repellent effect of the Red Sea moses sole. In Shark Repellents from the Sea: New Perspectives (Edited by Zahuranec B. J.), pp. 135~150. American Association for the Advancement of Science, Washington, DC. Clark E. and Chao S. (1973) A toxic secretion from the Red Sea flatfish Pardachirus marmoratus (Lacepede). Sea Fish Res. Stn Haifa Bull. 60, 53-56. Clark E. and George A. (1979) Toxic soles, Pardachirus marmoratus, from Red Sea and P. pavoninus from Japan, with notes on other species. Em. Viol. Fish. 4, 103-123. Dobbs L. G.. Mason R. J.. Williams M. C.. Benson B. J. and Sueishi K. (1982) Secretion of surfactant by primary cultures of alveolar type II cells isolated from rats. Biochim. biophys. Acta 713, 118-127. Epstein F. H., Stoff J. S. and Silva P. (1981) In Hormonal -Regulation of Epithelial Transport of Ions and Water (Edited bv Scott W. N. and Goodman D. B. P.), Vol. 372, ;tr6f3-625. The New York Academy of Sciences, New Epstein F. H., Stoff J. S. and Silva P. (1983) Mechanism and control of hyperosmotic NaCI-rich secretion by the rectal gland of Squalus acanthias. J. exp. Biol. 106, 25-41. Evans D. H. (1982) Mechanisms of acid extrusion by two marine fishes: the teleosts, Opsunus beta and the elasmobranch, Squalus acanthias. J. exp. Biol. 97, 289-299. Evans D. H., Aimo O., Kormanik G. A. and Mansberger L. (1982) Osmoregulation by the prenatal spiny dogfish, Squalus acanthias. J. exp. Biol. 101, 295-306.
489
Forrest J. N., Boyer J. L., Ardito T. A., Murdaugh V. A. Jr. and Wade J. B. (1982) Structure of tight junctions during Cl secretion in the perfused rectal gland of the dogfish shark. Am. J. Physiol. 242, C388-392. Greenwell J. R. and Low H. S. (1981) Action of caerulein, gastrin 17, pentagastrin, and secretin on the active transport of sodium by the frog skin. J. Membrane Biol. 61, 91-96. Gruber S. H. (1982) Shark repellents: perspectives for the future. Oceanus 24, 72-76. Gruber S. H. and Zlotkin E. (1982) Bioassay of surfactants as shark repellents. Nav. Res. Rev. 34, 18-27. Korchak Z. (1979) The electrochemical characteristics of lipid bilaye; modified with Pardachirus toxin. MSc Thesis, Technion-Israel Institute of Technology, Haifa, Israel. Montecucchi P. C. and Henschen A. (1981) Amino acid composition and sequence analysis of sauvagine, a new active peptide from the skin of Phyllomedusa sauvugei. Int. J. Peptide Protein Res. 18, 113-120. Moran A., Primor N. and Zlotkin E. (1978) The effect of Purduchirus venom on the conductance of lipid bilayer. Isr. J. Med. Sci. 14, 499-500. Pal R., Bamholz Y. and Wagner R. R. (1981a) Pardaxin, a hydrophobic toxin of Red Sea flatfish, disassembles the intact membrane of vesicular stomatis virus. J. biol. Chem. 256, 10,209-10,212. Pal R., Bamholz Y. and Wagner R. R. (1981b) Transcription of vesicular stomatitus virus activated by pardaxin, a fish toxin that permeabilized the viron membrane. J. Viral. 39, 64-645. Parness J. and Zlotkin E. (1976) Action of the toxic secretion of the flatfish Pardachirus marmoratus on the guinea pig ileum. Toxicon 14, 85-91. Payan P. and Maetz J. (1973) Branchial sodium transport mechanisms in Scyliothinum caniculu: Evidence for Na + /NH,’ and Na+/H+ exchanges and for a role of carbonic anhydrase. J. exp. Biol. 58, 487-502. Primor N. (1983) Pardaxin produces sodium influx in the teleost gill-like opercular epithelia J. exp. Biol. 105,83-94. Primor N. and Lazarovici P. (1981) Purdachirus marmoratus (Red Sea flatfish) secretion and its isolated toxic fraction pardaxin: the relationship between hemolysis and ATPase inhibition. Toxicon 19, 573-578. Primor N. and Tu A. T. (1980) Conformation of pardaxin, the toxin of the flatfish Pardachirus marmoratus. Biochim. biophys. Acta 626, 299-306. Primor N., Parness J. and Zlotkin E. (1978) Pardaxin: The toxic factor from the skin secretion of the flatfish Purdachirus marmoratus (Soleidae). In Toxins: Animal, Plant and Microbial (Edited by Rosenberg P.), pp. 539-547. Pergamon Press, New York. Primor N., Sabnay I., Lavie V. and Zlotkin E. (1980) Toxicity to fish, effect of gill ATPase and gill ultrastructural changes induced by Parduchirus secretion and its derived toxin pardaxin. J: exp. Zool. 211, 33-43. Primor N.. Zadunaiskv J. A. and Tu A. T. (1983a) Pardaxin. its mode of actio;, toxicity and str&ture.’ In Shark Repellents from the Sea: New Perspectives (Edited by Zahuranec B. J.), pp. 173-184. American Association for the Advancement of Science, Washington, D.C. Primor N., Bonaventura E. and Bonaventura J. (1983b) Effect of Purduchirus marmoratus secretion and its active factor, pardaxin, on elasmobranchs. In Shark Repellents from the Sea: New perspectives (Edited by Zahuranec B. J.), pp. 151-156. American Association for the Advancement of Science, Washington, D.C. Reed L. Y. and Muench H. (1938) A simple method for estimation fifty percent end points. Am. J. Hyg. 27, 493-497.. Silva O., Staff T., Field M., Fine L., Forrest J. N. and Epstein F. H. (1977) Mechanism of active chloride secretion by shark rectal gland: Role of Na, K-ATPase in chloride transport. Am. J. Physiol. 233, R298-306.
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Sobotka H. (1965) Comparative biochemistry of marine animals. BioScience 15, 583-584. Spira M. E., Klein M., Hochner B., Yarom Y. and Caste1 M. (1976) Ultrastructural changes accompanying the disturbances of neuromuscular transmission caused by Pardaxhirus toxin. Neuroscience 1, 11 I- 124. Stoff J. S., Silva P., Field A., Forrest J. N., Stevens A. and Epstein F. (1977) Cyclic AMP regulation of chloride transport in rectal gland of marine elasmobranchs. J. eq. Zool. 199, 443-448. Sund R. B. (1975) The effect of dodecyl sulphate upon net sodium and water transport from tied jejunal loops in anaesthetized rats. Actu pharmacol. 10.x. 31, 282-296.
Sund R. B. and Olsen G. (1981) Net sodium and glucose transport in the jejunum, ileum and colon of anaesthetized rats in response to intraluminal theophylline and anionic surfactants. Acta pharmacol. tax. 49, 6571. Thakkar J. K., East J., Seyler D. and Franson R. C. (1983) Surface-active phospholipase A, in mouse spermatozoa. Biochim. biophys. Acta 754, 44-50. Zlotkin E. and Bamholz Y. (1983) Studies on the mode of action of the ichthyotoxin pardaxin. In Shark Repel1ent.r from the Sea: New Perspectives (Edited by Zahuranec B. J.), pp. 157-171. American Association for the Advancement of Science, Washington, DC.
1984
0306~4492/84 $3.00 + 0.00
!Q 1984Pergamon Press Ltd
PARDAXIN INCREASES SOLUTE PERMEABILITY OF GILLS AND RECTAL GLAND IN THE DOGFISH SHARK (SQUALL8 ACANTHIAS)” NAFTALI PRIMOR,* JOSE A. ZADUNAISKY,~ H. VICTOR MURDAUGH JR.,$ JAMES L. BOYER~ and JOHN N. FORREST,JR.$ Laboratories of Marine Sciences, New York Aquarium, Coney Island, Brooklyn, NY 11224,
*Osborn USA, Telephone 266-8500, TDepartment of Physiology and Biophysics, New York University Medical Center, New York, NY 10016, USA, $Department of Medicine, University of South Carolina, Columbia, SC 29201. USA, $Department of Medicine, Yale University School of Medicine, New Haven, CN 06510, USA and Mount Desert Island Biological Laboratory, Salsbury Cove, ME 04672, USA (Received 5 May 1983)
Abstract-l. The action of the ichthyotoxic secretion of the Red Sea flatfish Pardachirus tnarmoratus and its derived toxin, pardaxin, was examined in the dogfish shark (Squalus_acanthias). 2. Pardaxin was more toxic when administered to the bathing medium than when injected into a dorsal artery and it transiently diminished the spiracular rate and caused a severe struggling response in the adult shark only when administered to the head region of the shark. 3. Pardaxin caused a transient leakage to urea and sodium between the shark and the seawater. 4. In the isolated perfused rectal gland pardaxin irreversibly reduced the rate of chloride secretion and concentration gradient of urea between perfusate and rectal gland fluid. In addition, ultrastructural studies on the rectal gland showed that ionic lanthanum penetrated the tight junctions and foci of cell necrosis were observed. 5. These studies indicate that in shark the gills are the most probable target of the toxicity of pardaxin.
Barnholz, 1983) and to cause lysis in cells (Primor and Lazarovici, 1981) and to display a surfactant-like action by decreasing surface tension (Primor et al., 1983a). Previous work suggests that in teleosts the gills are the primary site of the toxic effects of PX (Primor et a/., 1980). Furthermore, studies using the isolated gill-like opercular epithelium from the teleost Fundulus heteroclitus show a transient increase in gill permeability and production of Na+ influx (Primor, 1983). However, the effects of PX on osmoregulatory tissue in the elasmobranch shark have not been previously investigated. We studied the effects of PX on whole animals, using both adults and fetuses (pups) of the spiny dogfish shark, Squalus acanthius. Our studies determined its toxicity, its effect on spiracular rate and gill permeability to sodium and urea. In addition we studied the action of PX upon the isolated perfused rectal gland.
INTRODUCTION Pardachirus marmoratus, has hundred glands located along its fins (Clark and Chao, 1973). The
The Red Sea flatfish, more dorsal
than and
two anal
secretion of these glands had been found to be toxic to various teleosts (Clark and George, 1979) and to repel sharks (Clark, 1974) suggesting that the biological significance of the secretion is the defense of the flatfish against predators (Clark, 1983). Studies using a whole secretion (PMC) or isolated indicate multiple pharmafraction (pardaxin) cological effects. The ichthyotoxicity of Parduchirus secretion was enhanced in fish adapted to hyperosmotic media which suggests that it acts upon the ability of the fish to osmoregulate adequately (Primor et al., 1980). In other experiments Purduchirus secretion elicited transmitter release and histopathological changes in frog neuro-muscular junction (Spira et al., 1976) and produced a contraction in guinea-pig ileum (Parness and Zlotkin, 1976). It increases the electrical conductance of the lipid bilayer membrane in uniform steps indicating formation of transmembranal channels (Moran et al., 1978; Korchak, 1979). Toxicity has been attributed to a proteinaceous component in the secretion. This toxin, named pardaxin (PX), has been isolated and characterized. It is a protein composed of 160 amino acids cross-linked with four disulphide bridges (Primor et al., 1978; Primor and Tu, 1980). Pardaxin was shown to interact with bilayer membrane making it permeable to several proteins (Pal et al., 1981a,b; Zlotkin and *Part of this work was presented Desert Is/. hiol. Lab., Summer,
in an abstract 1980.
MATERIALS
AND METHODS
Pardachirus marmoratus fish (Pisces; Soleidae) were collected from the Red Sea (Eilat, Israel). P. murmoratus secretion (PMC) was obtained by the method described by Clark and Chao (1973). Pardaxin (PX), the ichthyotoxic component isolated from PMC, was prepared according to Primor et al. (1978) and stored in a lyophylized form. Toxicity was determined in dogfish fetuses (pups) which were removed from sacrificed females and maintained in aquaria with running seawater and used within two days. The lethality was determined in two different ways: (1) administration of pardaxin into a tank of 200 ml seawater with 5-6 pups; (2) injection via caudal artery using a syringe and needle gauge number 20. The lethal dose (LD,,) was
in BUM.Mt
483
484
NAFTALI PRIMORrr~1.
calculated by the method of Reed and Muench (1938) using 40 pups for each determination. Female dogfish sharks, Squalus ucanthcas, weighing 4-5 kg were taken from Frenchman’s Bay, Maine, for study at the Mount Desert Island Laboratory. They were maintained in marine live-cars in a fasted state and used within 3-4 days of capture. Isotope meusuremenls The effect of PX on gill permeability to urea and sodium was studied in adult dogfish positioned in an apparatus with a rubber diaphragm inserted close to the posterior gill cleft, separating the head region from the rest of the body. With the fish in position the head and rear chambers contained 8 and 12 I. of water respectively. During the experiments the fish were kept in the apparatus with running (5 I. min-‘) seawater (15 + 1 C). The dorsal aortae of the dogfish were catheterized via the cdudal artery (using polyethylene tubing PE 90, Clay Adams Co., Parsippany. NJ) for the purpose of injecting pardaxin and isotopes and collecting blood samples. The urinary papillae were also cdnnulated and urine was collected in a balloon. Three different experimental procedures were followed using this apparatus. (I) Determination of the effect of seawater-administered PX on the rate of appearance of plasma “Na + and [“C]urea from the fish to the bathing seawater (five experiments). Fish injected with 200 p Ci of “Na + or [“‘Clurea were kept in the diaphragm apparatus for 3 hr. Then the seawater flow was stopped and every 2 min blood and water samples from both sides of the diaphragm were taken and spiracular movements were counted. After 10 min the running seawater was restored for 3 hr. The same procedure was then repeated in the presence of PX. (2) Determination of the effect of seawater administered PX on the rate of appearance of [%Y]urea from the bathing seawater to the plasma (five experiments). [“‘CJUrea (25OpCi) was added to the bathing seawater in the head section of the apparatus while the flow of water was shut off. Every 2 min samples of blood and water from both sides of the diaphragm were taken and the spiracular movements were counted. Ten minutes after the water flow was shut off, the water from the chamber was removed and fresh running seawater was introduced for 3 hr. The procedure was then repeated with PX added together with the [lJC]urea. (3) Determination of the effect of PX administered by injection to the blood on the appearance of “Na’ and [“‘C]urea from the plasma to the seawater (three experiments). Fish injected with 200 FCi of isotope were kept in the diaphragm box for 3 hr. The water flow was shut otTand PX was then injected into the cannulated dorsal artery. For a IO min period blood and water samples from both sides of the diaphragm were taken every 2 min and the spiracular movements were counted. In order to determine rates of ‘?Na+ and [“Clurea appearance in the plasma or in the seawater, a time period of no water flow was required. The period of IOmin was chosen because the control fish easily recovered when running seawater was introduced after this interval. The blood PO? (partial oxygen pressure) recovered to normal levels and no change in gill urea permeability due to the lack of Rowing water was observed. Blood samples (I ml) were kept in glass syringes on ice. Blood PO, was determined within I hr using a Clark-type PO, electrode attached to a Radiometer pH meter gas monitor. To determine the leakage from seawater to the blood the red blood cells were centrifuged (800 x g) and [14C]urea activity in 0.05 ml of plasma was determined. To determine the leakage from the plasma to seawater. the isotopes ([‘+‘C]urea and “Na + ) were determined in 0.2 ml of seawater. “Na’ leakage from seawater to the blood was not determined to avoid a possible radiation hazard involved
in maintaining the fish in high ?‘Na * activity. Since. in fish. it is hard to estimate the gill area from where urea and sodium are transported it is inappropriate to express their transport in terms of flux (usually given per area). Instead, a leakage to urea and sodium was determined. The following calculation was applied: Change
in the cpm in the cold side
Specific activity of the isotope species
x
Sampling
time
(given in hr).
There is no significant (less than 2”,,) difference in quench as ?‘Na’ or [‘%Z]urea were added to 0.05 ml of plasma or to 0.2ml of seawater. Therefore. quench corrections for plasma or seawater were not made in these experiments. Appearance (leak) of sodium and urea from the fish 10 the seawater (when injected to the blood) or from the seawater to the fish body (when administered via the bathing water) was determined by liquid scintillation spectrometry and expressed in pequivalents hr ’ 5 kg ’ weight and corrected for background.
The procedure for removing the rectal gland, subsequent catheterization and its in vitro perfusion was performed as described in detail by Staff et u/. (1977). After basal perfusion for 20-30 min, the rectal gland was stimulated by the addition of theophylline (0.25 mM) and dibutyryl cyclic AMP (0.05 mM) to the perfusatc for 50-60min. After reaching a constant rate of rectal gland fluid secretion. PX was injected via the catheterized artery in a volume 01 0.2-0.3 ml of elasmobranch Ringer solution over I@15 sec. Rectal gland Ruid secretion was collected at intervals of IO min and the rate of secretion was determined by volumetric measurement. Since the volume of the perfusatc in the individual rectal gland was not determined, the final concentration of PX in the rectal gland remains unknowjn.
In perfused rectal gland prepared for electron microscopy, bicarbonate and phosphate were removed from the Ringer and replaced with 3 mM Tris to avoid precipitation of ionic lanthanum. Following perfusion in the stimulated state (theophylline 0.25mM and dibutyryl cyclic AMP. 0.05 mM) in the presence or absence of PX. the perfusate was changed to an identical solution containing ionic lanthanum (3 mM) and the perfusion was continued for 5 min. These glands were then fixed by perfusion for 15 min at 4-5,-C with a Karnovsky-type fixative containing 3”, glutaraldehyde. 47, paraformaldehyde. 0.1 M cacodylate at pH 7.4. Following fixation by perfusion. the glands were sectioned at the level of the rectal gland artery and small pieces (l-2 mm’) were obtained from the outer one-third of rectal gland tissue. Sections were incubated for 2 hr m fixative and then washed three times with 0. I M cacodylate buffer and refrigerated. Sections were examined under low power electron microscopy as reported perviously (Forrest ef (II., 1982). Theophylline was purchased from Merck. dibutyryl cyclic AMP, urease from soya bean type IX. were obtained from Sigma Chemical Co. [‘%Z]Urea and ‘INa. were obtained from New England Nuclear, Boston, Mass. Samples of plasma and seawater were dissolved in a scintillation cocktail and counted in a liquid scintillation counter.
RESULTS Toxicity PX
and the response of’s, acanthias
to PMC
and
The toxicities (LD,,) PMC and PX in dogfish pups (46 g) was determined using 40 specimens for each LD,, and were found to be 8.0 and 5.1 mg I ‘, respectively, as determined 1 hr after administration into 250 ml of seawater. The LD,,, (determined after
Pardaxin
V-
A‘ECOVER’ I --f+ -S
increases
shark
EXPERIMENTAL
Fig. 1. The effect of pardaxin (PX) on opercular rate, blood pH and partial oxygen pressure (PO,) in the adult shark Squalus acanthius. The sharks were studied in an apparatus with a diaphragm separating the head section and body. Each fish was studied during a control period of IOmin, allowed to recover for 3 hr and then restudied for 10min with PX (25 mg I-‘) in the head section of the apparatus. During the 10 min of both the control and the PX studies, the flow of seawater through the apparatus was shut off. Blood samples were collected to determine pH and PO,, and spiracular movements were counted every 2 min for a period of 10min. (~-0) Spiracular movements per min; (0-O) PO, in the blood samples; (0-O) blood pH. All results shown are the means of four experiments.
1 hr) of PMC and PX injected into a dorsal artery was found to be approximately ten times higher, 90.0 and 54.0 mg kg-‘, respectively. At 10 mg 1-l body wt, PX administered into seawater within 5-10 set was observed to cause severe struggling of the pups and a profound decrease in the spiracular rate, from 50-55 min’ to 3-5 min’ during the first 4 min, with a gradual increase to 20-25 mini’ after 10 min. An effect of PMC on opercular rate in teleosts was previously reported by Clark and George (1979). However, no immediate change in the spiracular rate was observed when a higher dose of PX, 50 mg kg-~’ was injected into the dorsal artery. In order to determine if the toxicity was mediated via the head of the fish, an apparatus was constructed with a rubber diaphragm separating the head of the pup from the rest of the body. The volumes of seawater were 40ml (head section) and 200ml (rear section). The diaphragm was inserted close behind the pup’s posterior gill cleft. PX (10 mg I-‘) was administered to the head section of the apparatus and was found to have a toxicity similar to that without a diaphragm. In addition, struggling and a decreased spiracular rate were always observed. PX (20 mg I-‘) administered to the rear section of the apparatus apparently did not cause toxicity as no struggling or changes in the spiracular rate occurred. The response to PX was also examined in five adult dogfish (4.0-5.0 kg) positioned in a large apparatus with a rubber diaphragm inserted close behind the posterior gill cleft. It was demonstrated that PX (25 mg I- ‘) administered to the region caused a diminished spiracular rate, from 40-42 min-’ to 8-l 1 min -’ as well as behavioral changes, struggling and opening the mouth as if to gasp. None of these responses were observed when PX (25 mg 1-l) was administered to the rear part of the apparatus.
gill permeability
485
to solute
The effects of PX on spiracular rate, blood pH and PO, in adult fish are shown in Fig. 1. Whereas stopping the flow of seawater in the chamber did not affect the spiracular rate (40-42 min’) in control fish, the spiracular rate was radically diminished in the presence of PX. Blood PO, in control fish was observed to fall during the stoppage of seawater flow. However, the PO, was significantly lower in the PX treated fish at each time period compared to controls (Table 1). No apparent changes in blood pH were observed throughout both control and PX exposure periods of study. These observations demonstrate that in both pups and adult dogfish, the head region is the target for PX activity. Following the abrupt decrease in the spiracular rate from 40-41 min-’ to 8-11 min’ at 2 min despite the presence of PX, the spiracular rate partially recovered to 30 min-’ indicating a transient property of this effect. The efect of’ PX on gill permeability sodium in adult dogfish
to urea and
Because the head region of the dogfish was found to be the target of PX action and because the gills of the dogfish are known to be relatively impermeable to urea and sodium (Boylan, 1967) it was of interest to determine whether PX induced changes in gill permeability to these solutes. No measureable radioactivity was detected in either the anterior (head) or posterior (body) chambers during stoppage in the seawater flow in five control fish injected 3 hr previously with 200 PCi [14C]urea or **Na + However, PX (25 mg I-‘) administered via the head section of the apparatus elicited a transient but marked increase in the permeability of both [14C]urea and **Na+ from plasma to seawater and [14C]urea from seawater to plasma (Fig. 2). Permeabilities increased maximally at 2 min for [14C]urea (seawater to plasma) and at 4 and 6 min for [14C]urea and **Nat (plasma to seawater) (Fig. 2). The concentration of PX used in these experiments (25 mg 1-l) was not lethal as all tested fish survived. No measurable radioactivity in seawater was observed when 40 mg of PX was injected into the circulation of fish previously injected with [14C]urea or **Na + (see Materials and Methods for experiment group No. 3). In addition, in experiments when PX was injected into the circulation no struggling or mouth opening occurred and no change in the spiracular rate was observed.
Table I. The effect of pardaxin (PX) on partial oxygen pressure (PO,) in the dogfish shark (S. aranlhias). The sharks were studied in a” apparatus with a diaphragm separating the head section and body. Each fish was studied during a control period of IOmin, allowed to recover for 3 hr and then restudied for 10 min with PX (25 mg I-‘) in the head section of the apparatus. During the IOmin of both the control and the PX studies the flow of seawater through the apparatus was shut ofI The data are expressed as mean ? SD of four experiments and statistical significance (P) between means was tested with Student’s I-test “sing paired data. Time (min) 2 4 6
8 10
Control 62.2 49.5 38.2 28.5 18.0
i f i + +
With pardaxin PO, CmmHg)
2.2 2.3 1.7 2.6 2.1
48.7 It 27.0 i II.71 5.5 f 1.7 +
1.8 2.1 1.7 13 1.5
Significance (P)
0.01f
486
NAFTALIPRIMORe/al.
TIME (mid Fig. 2. The effect of pardaxin (PX) on gill permeability to urea and sodium in adult shark Squalus acanthias. The sharks were studied in an apparatus with a diaphragm separating the head section from the body. Each fish was studied during a control period of 10 min, allowed to recover for 3 hr and restudied for 10min with PX. During the 10 min of both the control and PX studies, the flow of seawater through the apparatus was shut off. Every 2min during the 1Omin of each study, blood and seawater samples from both sides of the diaphragm was taken. In five experiments [“‘Clurea or “Na + (200 pCi) were injected into the circulation of fish and PX (25 mg I-‘) was administered in seawater in the head section of the apparatus (see experiment No. 1 in Materials and Methods). (0-O) Appearance of [r4C]urea in seawater in the head section of the apparatus; (O---O) [14C]urea in seawater in the rear section of the apparatus; (n-a) appearance of ‘?Na+ in seawater in the head section of the apparatus; (A-- -A) **Na+ in seawater in the rear section of the apparatus. In five experiments [“‘Clurea (25OpCi) and PX (25mgml ‘) were added to the seawater in the head section of the apparatus (see experiment No. 2 in Materials and Methods). (e-@) Appearance of [‘VZ]urea in the shark plasma. The data are expressed as means + SE for five fish. Note a transient increase in leak to sodium and urea.
gland secretion was typically low at 15 f 5 mM (mean + SE, n = 6). PX was administered by injection into the artery of the isolated perfused rectal gland during stimulation of chloride secretion by theophylline and dibutyryl cyclic AMP. The rate of secretion of urea in the gland fluid was found to be increased in a manner dependent on the PX dosage and no further effect was found above 400 ,ug. Moreover, the urea concentration gradient between the perfused Ringer in solution (350 mM) and the gland fluid (15 mM) was abolished while urea concentration in the perfusate approaches 280 mM (Fig. 3). While injection of 50 pg of PX had no effect on Cl secretion rate, 1000 p g inhibited within 45 min loo”/, of Cl secretion (Fig. 4). Furthermore, the chloride secretion rate was reduced in a dose-dependent manner. The injected PX was observed to result in the appearance of significant amounts of mucus in the gland fluid. In contrast to the effects on the gills, the increased permeability to urea and dissipation of chloride secretion in the gland were not transient at any dose and the function was not observed to be restored. This finding suggests permanent damage to the rectal gland tissue and this assumption was confirmed by ultrastructural studies. The results of ultrastructural studies are shown in Fig. 5. In control rectal glands perfused with ionic lanthanum during stimulation of secretion with theophylline and dibutyryl cyclic AMP (Fig. 5A) lanthanum was seen to fill the intracellular spaces up to the tight junctions but did not penetrate through to the lumen. These observations in control glands are identical to that reported previously by Forrest ef al. (1982). In contrast, in glands stimulated with
The effect of PX on the isolated perfused rectal gland As PX was observed to increase permeability to urea and sodium in the head region it was of interest to determine the effects of this agent on other osmoregulatory organs in the shark. It has been demonstrated that theophylline and dibutyryl cyclic AMP have a profound stimulatory effect on the flow rate in the isolated perfused rectal glands. These glands secrete chloride actively against its electrochemical gradient (Silva et al., 1977) and have ordinarily low permeability to urea (Burger, 1967). It was used to study the effects of drugs on active transport mechanisms (Epstein et al., 1981; 1983). The present study deals with the effects of PX on the performance of the rectal gland. In glands stimulated with theophylline (0.24 mM) and dibutyryl cyclic AMP (0.05 mM), prior to administration of PX, the rectal gland flow rate 2920~300~lhr’gwetwt ’ was averaging (mean f SE, n = 6) and the chloride secretion rate in the gland secretion was found to average (mean k SE, 1515+ 145pequiv.hr’gwetwtt n = 6). The urea concentration in the rectal
IO
30
50 TIME (min)
Fig. 3. Dose response of pardaxin (PX) on urea secretion rate in the perfused dogfish rectal gland fluid. The rectal gland was stimulated with dibutyryl cyclic AMP (0.05 mM) and theophylline (0.25 mM). After reaching a constant rate of secreted chloride in the control periods of 50-60 min. a single dose of PX was injected by the cannulated artery. The urea content in the rectal gland fluid was determined by phenolhypochloride method and expressed as percentage change in urea secretion rate as compared to mean control periods. For doses of PX (fig per rectal gland): (0~~0) 50; (e---0) 100; (n-/Q 200; (m---m) 400; and (0-n) 1000. The data are means of ten experiments, two experiments at each dosage with secretion rate of urea (percentage of control) given at IO min intervals for 60 min of perfusion with PX.
Pardaxin
IO
30
increases
shark
50 TIME
(mtn)
Fig. 4. Effect of pardaxin (PX) on the chloride secretion rate in the perfused dogfish rectal gland. The rectal gland was stimulated with dibutyryl cyclic AMP (0.05 mM) and theophylline (0.25 mM) for a control period of 50-60 min. After reaching a constant rate of secretion (1515 f 145 pequiv. hr-’ wet wt-‘) a single dose of PX was injected by the cannulated artery. Data are expressed as percentage inhibition of chloride secretion rate following injection of PX as compared to rate of chloride secretion (pequiv. Cl- secreted hr-’ g gland wet wt-‘) in control period. Symbols as in Fig. 3. The data are the means of ten experiments with two experiments of each dosage.
theophylline and dibutyryl cyclic AMP and exposed to PX, extensive lanthanum was seen in the lumen (Figs 5B and 5C, arrows) in association with disruption of the intracellular architecture. In some areas discrete foci of cellular necrosis were observed. DISCUSSION
In this study we determined that PX-induced toxicity struggling reaction, diminished spiracular rate, decrease in blood PO, and an increase in permeability of the gills to sodium and urea is mediated through the head region of the shark which is exposed to the outside bathing seawater. None of the above responses were produced by administration via the blood of 40 mg of PX. Based on the dogfish plasma volume of 6.6% body wt (Burger, 1967), one may estimate that the PX concentration in the plasma is approximately 121 mg 1-l in a 5.0 kg fish. However, when administered to the bathing water PX elicits responses at a dose of 25mg 1-l. Therefore it is unlikely that PX-induced responses are elicited by its penetration from the medium into the body and its action is elicited from the seawater side only. It is suggested that in elasmobranchs gills are the major organ for Na+ transport (Payan and Maetz, 1973; Evans, 1982; Evans et al., 1982). Therefore the leakage of sodium and urea between the shark’s body and the bathing seawater (Fig. 2) suggests that the gills are the primary site of PX action. It is unclear, however, how PX acts to inhibit the spiracular rate. Inhibition of spiracular rate may also contribute to the lower blood PO, in the presence of PX (Table 1). The pH of the blood is determined by the ratio of HCO,/CO, concentrations and will be
gill permeability
to solute
487
maintained as long as this ratio stays unchanged. The unchanged blood pH during control and in the presence of PX periods (Fig. 1) suggests that the ratio of concentrations HCO; /CO, was not changed while PO, values fall during control and in the presence of PX periods (Fig. 1). However, measurements of total COz concentrations are needed to support this assumption. In order to determine the effect of PX on shark tissues that actively transport chloride ions and are ordinarily impermeable to urea, we have studied in vivo the gills and the isolated perfused rectal gland. In both epithelial tissues, PX disrupts function in association with changes in solute permeability. However, the increased permeability of gills was shown to be transient even in the presence of PX (Fig. 2). The transient increase in permeability observed in whole fish resembles that observed in the isolated gill-like opercular epithelium of the teleost (Primor, 1983) indicating PX’s similarity in the mode of action in teleosts and elasmobranchs. However, irreversible damage was observed in rectal gland function while the toxin was washed out (Figs 3-5) and generalized cellular toxicity was observed. The significance of this difference is not clear. However, it may correlate with the proposed action to repel predators rather than to cause death. Measurements show PX’s ability to act as a surfactant by lowering the surface tension of aqueous solution (Primor et al., 1983). Furthermore, some commercial surfactants were shown to elicit repellent action in sharks (Gruber, 1982; Gruber and Zlotkin, 1982). The term “surfactant” generally refers to substances which reduce surface-tension. In biological systems, surfactants diminish the separation between water- and lipid-soluble substances and therefore allow interactions between water-soluble compounds and the cell membrane. It is not unique for biologically active substances to be armed with a surfactant quality. For example, potent detergents are generated from activation of membrane-bound phospholipase Az in mammalian sperm and it has been suggested that the significance of these detergents is to promote membrane fusion (Thakkar et al., 1983). It seems likely, then, that Pardachirus utilizes the detergent property of PX to facilitate interaction with biological membranes. The mechanism of PX’s action involves its insertion into the lipid bilayers as a functional transmembranal channel. Consequently, it causes an increase in their permeability to the seawater constituents. This sequence of events finally leads to its noxious effect in situ. It is suggested, here, that PX’s surfactant property promotes its spreading and adsorption from the water into which it is released by the flatfish and facilitates its noxious action upon the gill membranes of predators. It is noteworthy that substances capable of reducing surface-tension, in spite of their diverse chemical identity, such as dodecyl sodium sulphate-a derivative of lauryl alcohol (Gruber, 1982; Gruber and Zlotkin, 1982); holothurin-a toxic principle of a Bahamian holothurian of a lactone-like structure (Sobotka, 1965); and a proteinaceous PX were all toxic as well as producing a noxious and/or repellent response in sharks. A common action of such entirely different compounds suggests a similarity in their
488
NAFTALIPRIMORetal.
Fig. 5. Low power (x 12,150) cross section of tubules from isolated perfused rectal glands (Syuahs acanthias) during stimulation of chloride secretion with theophylline and dibutyryl cyclic AMP in the presence and absence of PX. A: Control gland demonstrating presence of electron dense lanthanum in the lateral intracellular space (arrow) up to but not through the tight junction and absence of lanthanum in the lumen; B and C: in PX perfused glands lanthanum appears within the intracellular space and extensive lanthanum is present in the lumen (see arrows). In the presence of pX there is partial loss of cellular architecture and vesiculation of the endoplasmic re+iculum.
mechanism. In rat ileum, synthetic anionic surfactants given to the mucosal (luminal) side elicited a net sodium secretion (Sund, 1975; Sund and Olsen, 198 1); a toxic, noxious and surface-active polypeptide
derived from the dart frog skin increases the rate of sodium entry when given to the outer face of the frog skin (Greenwell and Low, 1981; Montecucchi and Henschen, 1981). PX Jicited a net sodium influx in
Pardaxin increases shark gill permeability to solute the fish gill-like operculum skin, also acting from the outerface side only (Primor, 1983). Therefore it is possible that some of those compounds capable of producing a net sodium entry upon the outface of ion- and water-transporting epithelia could also exert a noxious and/or repellent effect in sharks. The remaining possibility to attribute the noxious effect to a general surfactant quality looks less valid because the cationic surfactants did not repel sharks (Gruber and Zlotkin, 1982) and the pulmonary surfactants secreted upon physiological conditions (Dobbs et al., 1982) are without a noxious action. This hypothesis, however. should be submitted to a controlled test. Acknowledgements-This work was performed during the tenure of J. N. Forrest, Jr. as an established investigator of the American Heart Association (77-228). The authors wish to acknowledge Dr. Barbara Kent for making the PO, measurements and Oi Cheng Ng for technical assistance with the electron micrographs, and Esta Van Tuyl for excellent typing of the manuscript. This research was supported by NIH Research Grants AM-17433 and PHS/EY 01340, NIH Research Grant PHS/GM 25002, NIH Research Grant PHS/EY 03151, The Department of Defense, Office of Naval Research, Research Grant No. N00014-81-K-0667 and N00014-82-C-0435 and National Science Foundation Grant BG-28139 to the Mount Desert Island Biological Laboratory.
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