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Sodium fluxes in isolated body walls of the euryhaline polychaete, Nereis (Neanthes)succinea
Com& Biachem. Physiol., 1974, Vol. GA, pp. 221 to 228. Pergamon Prem. Printed in Great Britain
SODIUM FLUXES IN ISOLATED BODY WALLS OF THE EURYHALINE POLYCHAETE, NEREIS (NEANTHES)
SUCCINEA* BYRON A. DONEEN* and MARY E. CLARK Department of Biology, California State University, San Diego (Receive& 25 June 1973) Abstract-l. A method is described for isotopically measuring sodium fluxes in isolated body-wall preparations of the polychaete Nereis (Neanrhes) succirzea. 2. Body-wall potentials in the isolated preparations are similar to those reported for intact nereids. 3. *sNa influx is greater and Na+ permeability is lower in body walls from Nerek adapted to 20% sea water (osmoregulating) than in those adapted to 50% sea water (osmoconforming).
INTRODUCTION
EURYHALINEpolychaetes survive in dilute salinities by concentrating ions in the body fluids (Beadle, 1937; Smith, 1955). Previous studies employing radioactive isotopes of sodium and chloride have been carried out on intact NAY& divenicolor adapted to salinities in which hyperionic regulation occurs. The results of these experiments strongly suggest that regulation is accomplished by both a reduced body-wall permeability to ions (Oglesby, 1972), and by an ability to concentrate Cl- (Jorgensen & Dales, 1957; Smith, 1970) and Na+ (Fretter, 1955 ; Oglesby, 1972) against concentration gradients. We have extended these observations by examining sodium ion permeability and flux rates across isolated body-wall preparation of Noel {Ne~~~~es) ~ci~u. This report describes a method for studying isolated pieces of body wall removed from individual worms acchmated to various salinities. Based on the procedure commonly used to investigate ion transporting properties of frog skin (Ussing & Zerahn, 1951), the method allows a direct assessment of the physiological properties of tissues subjected to different osmotic conditions. Experiments comparing the Naf-permeability and saNa-transporting properties of isolated body walls from animals adapted to a salinity of isosmotic conformity * This investigation was supported by grant number 261-134 from the San Diego State University Foundation, and represents research completed by one of us (B. A. D.) in partial fulfillment of the Master of Science degree at California State University, San Diego. + Present address: Department of Zoology, University of California, Berkeley, California 94720. 221
222
BYRON A. DONEEN
AND
MARY E. CLARK
(50% SW) and to a salinity at which hyperionic regulation described. Preliminary accounts of the effects of potassium ouabain on Na+ movements are also presented. MATERIALS
AND
occurs (20% concentration
SW) are and of
METHODS
Animals N. sz~ccinea were collected throughout the year from a brackish water region in Alamitos Bay, Long Beach, California. Animals were maintained in the laboratory without feeding in small glass tubes in 100% SW (560 mM Cl-) at 15°C. Acclimation to the desired salinity was accomplished by stepwise transfer of worms at 2-S-day intervals through progressively more dilute salinities. Animals remained in the final adaptational salinity for 2-4 weeks before experimental use. Apparatus To prepare a tissue for flux measurements, the anterior segments housing the proboscis and the posterior half of a large (about 500 mg) worm were removed. A longitudinal cut through the parapodia of one side allowed the remaining body wall to be opened for removal of the gut. The body wall was placed between two Plexiglas slides each having two circular openings 0.645 cm in diameter (area = 0.32 cm2). These openings thus framed two adjacent regions of the tissue, exposing external and coelomic surfaces in opposite directions. The tissue in its Plexigias frame was then inserted between slotted halves of a modified Ussing apparatus (Ussing & Zerahn, 1951) consisting of two parallel sets of chambers. Upon clamping, the apparatus formed two identical chambers, each with a section of body wall at the center. Accordingly, two regions of the same tissue could be observed simultaneously. Alternatively, a single tissue could be observed while the parallel chamber, filled with the appropriate solution, was used to zero the potentiometer. Each half-chamber contained separate ports at either side of the tissue for insertion of salt bridges, for addition and removal of solutions, and for an air pump connection providing aeration and circulation of bathing media. The 2.5 M KC1 salt bridges placed near surfaces of the body wall were connected by calomel electrodes to a Cary Model 32 vibrating-reed electrometer (Applied Physics, Corp., Monrovia, California) with an input impedance of 1On s2. During flux determinations body-wall potentials were abolished by the application of a short circuit current through silver-silver-chloride electrodes.
22Na Fluxes 22Na influx and efllux were measured on pieces of body wall from animals acclimated to either 50% SW or 20% SW. Experimental media bathing the tissues contained the Na+ concentration of the adaptational medium and the approximate K+ concentration of coelomic fluid in animals adapted to that salinity (Fletcher, 1970; Free1 et al., 1973). Composition of the bathing media is shown in Table 1. Tissues mounted in the chamber were allowed a 30-min equilibration period in unlabeled solution prior to flux experiments. During this period body-wall potentials were measured and short-circuited. Ionically and osmotically identical bathing solutions were then added to both sides of the preparations. To measure Na+ influx, 1.0-2-S &i 22Na (New England Nuclear, Inc., Boston, Mass.) was added to the external solution of one tissue piece; to determine efflux a similar amount of the isotope was added to the coelomic side of the adjacent body-wall section. Initial attempts to determine both influx and efflux on the same section of body wall were unsatisfactory (see Results). Na+ flux across the short-circuited tissue was determined by measuring the appearance of 22Na in the unlabeled solution in the opposite half-chamber. At the end of a given time
SODIUM FLUXES IN BODY WAtL
TABLE ~-COMPOSITION OF BATHINGMEDIA FOR AND50% SW-ADAPTED
223
OF A POLYCHARTR
STUDY OF
*rNa
FLUXES ACROSS
20% SW-
BODY WALLS
Ion
20% SW (mM)
50% SW (mM)
Na+ K+ Caa+ Mge+ Clso**HCOa-
97.4 3.0 1.8 97 111.2 6.3 0.4
243.6 45 46 24.2 278-l 12.8 1.1
interval the whole of the originally unlabeled solution was removed and replaced with fresh solution. The entire (2 ml) sample was counted in a Packard Tri-Carb Liquid Scintillation Spectrometer (Model 3375, Packard Instrument Co., LaGrange, Ill.). Each sample was counted to 1000 counts. The scintillation fluid was 15 ml of a toluene-based solution containing 98% PPG-2% Bis-MSB (0 mnifluor, New England Nuclear, Boston, Mass.: 4 g/500 ml toluene) diluted one-third with Triton X-100 (Atlas Chemical and Manufacturing Co., San Diego, Caliiornia). A 5- or lo-,ul sample of the labeled solution was counted simultaneously to determine its specific activity. All counts were corrected for quenching and background. Flux experiments were performed at 23-25°C. Upon completion of an experiment the circular area of tissue exposed to the label was cut out and weighed. Flux rates are expressed as micromolesjg wet wt. per hr. Descriptions of preliminary attempts to assess the effects of Kf concentration and of ouabain on body-wall Na+ fluxes are presented in the results. RESULTS
Body-wall potentials The body-wall potential, coelomic surface negative with respect to the external, reported by Smith (1970) and Fletcher (1970) for intact hyperionically regulating N. d~~~~col~ was also observed in isolated pieces of body wall removed from animals adapted to salinities of 20% SW or less (Table 2). The potentials shown in Table 2 were measured with body-wall preparations bathed in the adaptational TABLE
2-IsoLxrm
BODY-WALL
POTENTIALS
IN TISSUES REMOVED FROM ANIMALS
ADAPTED
TO VARIOUS SALINITIES AND MEASURED IN SE4 WATER OF THE SAME DILUTION
Adaptational salinity (% SW)
N
50 20 10
11 13 27
Potential* (mv) + O-34 f O-68 -3.18f0.66 - 7.39 + 0.49
* Sign of potential refers to the inner surface with respect to the outer. Data shown are means f S.E.
BYRONA. DONEENANDMARY E. CLARK
224
medium. A slight reduction in potential was observed when tissues were bathed in solutions having the elevated K+ concentration used for the 22Na flux determinations. Significant potentials were not observed in tissues from animals adapted to 50% SW. Potentials generated by the 20% SW-adapted tissues were short-circuited by currents of less than 1*5 PA.
22Na j?uxes The time required to reach a uniform rate of 22Na influx and efflux was measured for several body walls. Figure 1 shows one such influx experiment performed on a tissue taken from an animal adapted to 20% SW. Typically 40 min were required
0”- 1800 fa 150? a E z 120-
-g
z._
01 0
.-.
IO
20
30 Time,
40
50
60
min
FIG. 1. The time required to reach the steady-state 22Na influx rate in a 20% SW-adapted body wall. Solutions bathing the coelomic surface were counted at 10-min intervals.
to reach a steady influx rate in these preparations. Only about 20 min were required to attain the steady-state efflux rate in tissue adapted to both salinities. The Na+ influx and efflux rates for paired adjacent skins, therefore, are based on counts obtained during a 15-min period after the steady 22Na flux rate had been reached.
SODIUMFLUXESIN BODY WALL OF A POLYCHAETE
225
Na+ fluxes, the calculated net flux and the influx to efflux ratio (i/o) determined for several sets of paired body walls removed from animals adapted to 20 and to 50% SW are shown in Table 3. Since the precise surface areas of the body walls could not be determined, results are presented on a weight basis. TABLE 3-Na+
FLUXBSIN BODY WALLS ISOLATEDFROM ANIMALS ADAPTEDTO 20% TO 50% SW poles
SW AND
g-l hr-l
Adaptational salinity (%
SW) 20 50
* Influx.
Influx
EfflUx
Net flux
i/o
3.68 f 0.32 (12) 2.95 _+0.83 (5)
1.06 f 0.13 (11) 3.64 z!z1.08 (5)
2*82* + 0.34 (9) 0+73+ + 0.28 (5)
3.59 f 0.71 (9) 0.79 f 0.07 (5)
f- Efllux.
Data shown are means f S.E.
Number of body walls given in parentheses.
A large variability in flux rates was obtained when a single piece of body wall was used sequentially to determine first 22Na influx and then efflux. During the initial me~urement the external surface was exposed to labeled solution for 1 hr. Although the outer tissue surface was then rinsed four or more times before the inside surface was exposed to the 22Na-containing solution, this procedure resulted in 22Na effluxes frequently larger than influx rates. The apparent source of this exaggerated efflux is shown in Fig. 2. In this experiment a 20% SW-adapted tissue was exposed to 22Na at its external surface for 1 hr. After removal of the labeled solution both sides of the tissue were rinsed five times in 10 min. Subsequently fresh unlabeled medium was reapplied to both surfaces and the external solution withdrawn for counting and replaced at 10”min intervals. Even in the absence of 22Na-containing solution and despite repeated rinsing, a large 22Na efflux from the external surface persisted. The apparent exponential decline of external 22Na levels with time suggests an exchange by some component of the external solution with 22Na retained near the outer surface of the tissue. Determining both influx and efflux on the same tissue section produced results consistent with paired tissue techniques only when efflux was measured first. 22Na activity in the bathing solution was reduced to background levels by rinsing the internal surface two or three times. The effect of Kf concentration on body-wall 22Na movements in animals acclimated to 20% SW was investigated by bathing tissues in media having K+ concentrations (as K,SOJ of 1.8, 3-O or 4.5 mM. These experiments were performed using the same tissue piece for determination of both influx and efflux. For the reasons discussed above, there was considerable variation in the results, but they do suggest that K+ is required to achieve a net Na+ influx. Influx of Na+ is enhanced by increasing the K+ concentration from 1.8 to 4.5 mM. Na+ efflux was unaffected by the Kf concentration of the bathing solutions.
BYRONA. DONE~ ANDMARY E. CLARK
226
;r
2 f
z”
Of 0
I
IO
,
,
i
40
30
20 Time,
,
50
t
60
min
FIG. 2. Efflux of 22Na from the outer surface of a 20% SW-adapted body wall previously exposed to label on the outer surface for 1 hr. Solutions bathing the external surface were counted at lo-min intervals. Sensitivity of Na+ fluxes to ouabain (G-strophanthin, Calbiochem Inc., Los Angeles, California), a specific inhibitor of K+-dependent sodium transport (e.g. Bonting, 1970) was tested by preparing desired concentrations of the glycoside solutions replaced internal or external in bathing media. Ouabain-containing solutions after the steady 22Na flux rate had been attained. Ouabain applied to the internal surface of tissues from animals adapted to 2Oo/o SW reduced Na+ influx by 57 to 85 per cent of the pretreatment rate in five of eight attempts. Ouabain concentrations of 5 x 10~~ M or greater were effective when treatment exceeded 40 min. In three experiments, however, ouabain in concentrations as high as 10-s M failed to reduce Na+ influx. Applied to external body-wall surfaces, ouabain either did not affect Na+ efflux or occasionally produced a slight stimulation of efflux. Both Na+ influx and efflux in the 50% SW-adapted tissues were increased by SO-100 per cent following application of 5 x lo-” M ouabain to internal and external surfaces, respectively. DISCUSSION
Characteristics of sodium movement in isolated body wall removed from individual N. succineu adapted to 20% SW and to 50% SW are clearly different.
SODIUM FLUXES IN BODY WALL OF A F’OLYCHAETS
227
The mean Naf influx in 20% SW-adapted tissue is 24 per cent greater than in preparations from 50% SW-adapted animals (Table 3). This larger influx occurs in bathing medium having a Na+ concentration 40 per cent lower than that in 50% SW-based solutions. Na+ influx and efflux are equivalent in 50% SWadapted preparations. The slightly larger mean efflux is not statistically significant. If it is assumed that Na+ movements across 50% SW-acclimated tissues can be accounted for on the basis of diffusion, then the Na+ permeability of 20% SW-adapted tissues may be contrasted with this “standard”. Setting the ratio of the mean Naf e&x in 50% SW-adapted tissues to the Naf concentration of the bathing medium (243.6 mM) equal to l-0, the ratio for the 20% SW-adapted preparations (Na+ concentration, 97.4 mM) is O-72. It maybe concluded that the 20% SW-acclimated body wall is about 30 per cent less permeable to Na+ than body wall from a 50% SW-adapted animal. Reduced body-wall Na’ permeability at low salinities has been suggested by Oglesby (1972) from whole animal isotopic studies on N. diverskolor. Na+ influx in 20% SW-adapted tissue is about threefold greater than expected from diffusion through body walls having permeability properties of 50% SWadapted tissues, and is 3-S-fold greater than Naf efflux in ZOojo SW-adapted preparations. Accelerated Na+ influx, it is h~othesized, is established by the activation of an inwardly directed sodium-tr~spo~ing system operating in 20% SW-adapted animals. The mechanism either does not operate in the body walls of animals adapted to 50% SW or functions at such a low level as to be undetectable relative to the total influx. The prolonged 22Na efflux from the external surface of 20% SW-adapted labeled preparations (Fig. 2) may represent 22Na-23Na exchange between sodium of the medium and a labeled pool within the tissue. This exchangeable pool is not readily accessible to the coelomic surface of the body wall. Exchange diffusion associated with sodium transport at low salinities has been suggested by Oglesby (1972) on the basis of 22Na efflux in intact N. diversicolor. Preliminary attempts to determine the conditions under which active sodium transport occurs were carried out both by varying the K+ concentration of the bathing media and by addition of ouabain. Although neither set of experiments provided conclusive data, the results obtained suggest the following possibilities. Sodium influx, but not efflux, appears to be dependent on Kf concentration at salinities in which the animal is osmoregulating. Ouabain in most cases reduced Nat influx when applied to the coelomic surface of 20% SW-adapted body walls. Interpretation of the effect of ouabain was complicated by its ability to stimulate et&x in both 20% SW- and 50% SW-adapted tissues when applied to the external body-wall surface. A similar stimulation of Na+ influx by coelomic side ouabain treatment of 50% SW-adapted tissues suggests that ouabain acts in these cases either by causing a non-specific increase of ion permeability or by a more specific interaction with ion-exchanging sites. The experiments reported here support the concept of passive diffusion of Na+ across the body wall at osmoconforming salinities and of a reduced passive
228
BYRON A. DONEENANDMARY
E. CLARK
sodium permeability at osmoregulating salinities, coupled with an active sodium influx. It is not yet clear whether this influx is caused by an accelerated inward sodium transport per se or, perhaps, to a separate chloride pump, which could account for the observed inside-negative potentials. The technique of studying fluxes across isolated body-wall preparations from euryhaline polychaetes may provide the answers to these questions. Ac~o~ledg~~~s-The authors express thanks to Professors H. A. Bern and R. I. Smith, Department of Zoology, University of California, Berkeley, for a critical reading of the manuscript. Figures were drawn by Emily Reid. REFERENCES BEADLE L. C. (1937) Adaptation to changes in salinity in the polychaetes. r. exp. Biol. 14, 56-70. BONTING S. L. (1970) Sodium-potassium activated adenosinetriphosphatase and cation transport. In Membranes and Ion Transport (Edited by BITTAR E. E.), Vol. 1, pp. 257363. Wiley-Interscience, New York. FLETCHER C. R. (1970) The regulation of calcium and magnesium in the brackish water polychaete, Nereis diversicolor, 0. F. M. J, exp. Biol. 53, 425-443. FREEL R. W,, MEDLER S. G. & CLARK M. E. (1973) Solute adjustments in the coelomic fluid and muscle fibers of a euryhaline polychaete, Neunthes succineu, adapted to various salinities. Biol. Bull., mar. biol. Lab., Woods Hole 144,289-303. FRETTER V. (1955) Uptake of radioactive sodium (Na2*f by Nereis d~v~sico~or Muefler and Perinereis czrltrifera Grube. J. mar. Biol. Ass. U.K. 34, 151-160. JL)RGENSONC. B. & DALES R. P. (1957) The regulation of volume and osmotic regulation in some nereid polychaetes. Physiol. camp. Oecol. 4, 357-374. OGLESBY L. C. (1972) Studies on the salt and water balance of Nereis diversicolor-II. Components of total sodium efflux. Comp. Biochem. Physiol. 41A, 756-790. SMITH R. I. (1955) Comparisons of the level of chloride regulation by Nereis diversicolor in different parts of its geographical range. Biol. Bull., mar. biol. Lab., Woods Hole 109, 453-474. SMITH R, I. (1970) Chloride regulation at low salinities by Nereis dive&color (Annelida, Polychaeta)-I. Uptake and exchanges of chloride. J. exp. Biol. 53, 75-92. USSING H. H. & ZERAHN K. (1951) Active transport of sodium as the source of electric current in the short-circuited isolated frog skin. Acta physiol. stand. 23, 110-127. Key Word Index-Body-wall polychaete; sodium flux.
potential ; Nereis (Nea~thes) sllccikea ; osmoregulation ;
SODIUM FLUXES IN ISOLATED BODY WALLS OF THE EURYHALINE POLYCHAETE, NEREIS (NEANTHES)
SUCCINEA* BYRON A. DONEEN* and MARY E. CLARK Department of Biology, California State University, San Diego (Receive& 25 June 1973) Abstract-l. A method is described for isotopically measuring sodium fluxes in isolated body-wall preparations of the polychaete Nereis (Neanrhes) succirzea. 2. Body-wall potentials in the isolated preparations are similar to those reported for intact nereids. 3. *sNa influx is greater and Na+ permeability is lower in body walls from Nerek adapted to 20% sea water (osmoregulating) than in those adapted to 50% sea water (osmoconforming).
INTRODUCTION
EURYHALINEpolychaetes survive in dilute salinities by concentrating ions in the body fluids (Beadle, 1937; Smith, 1955). Previous studies employing radioactive isotopes of sodium and chloride have been carried out on intact NAY& divenicolor adapted to salinities in which hyperionic regulation occurs. The results of these experiments strongly suggest that regulation is accomplished by both a reduced body-wall permeability to ions (Oglesby, 1972), and by an ability to concentrate Cl- (Jorgensen & Dales, 1957; Smith, 1970) and Na+ (Fretter, 1955 ; Oglesby, 1972) against concentration gradients. We have extended these observations by examining sodium ion permeability and flux rates across isolated body-wall preparation of Noel {Ne~~~~es) ~ci~u. This report describes a method for studying isolated pieces of body wall removed from individual worms acchmated to various salinities. Based on the procedure commonly used to investigate ion transporting properties of frog skin (Ussing & Zerahn, 1951), the method allows a direct assessment of the physiological properties of tissues subjected to different osmotic conditions. Experiments comparing the Naf-permeability and saNa-transporting properties of isolated body walls from animals adapted to a salinity of isosmotic conformity * This investigation was supported by grant number 261-134 from the San Diego State University Foundation, and represents research completed by one of us (B. A. D.) in partial fulfillment of the Master of Science degree at California State University, San Diego. + Present address: Department of Zoology, University of California, Berkeley, California 94720. 221
222
BYRON A. DONEEN
AND
MARY E. CLARK
(50% SW) and to a salinity at which hyperionic regulation described. Preliminary accounts of the effects of potassium ouabain on Na+ movements are also presented. MATERIALS
AND
occurs (20% concentration
SW) are and of
METHODS
Animals N. sz~ccinea were collected throughout the year from a brackish water region in Alamitos Bay, Long Beach, California. Animals were maintained in the laboratory without feeding in small glass tubes in 100% SW (560 mM Cl-) at 15°C. Acclimation to the desired salinity was accomplished by stepwise transfer of worms at 2-S-day intervals through progressively more dilute salinities. Animals remained in the final adaptational salinity for 2-4 weeks before experimental use. Apparatus To prepare a tissue for flux measurements, the anterior segments housing the proboscis and the posterior half of a large (about 500 mg) worm were removed. A longitudinal cut through the parapodia of one side allowed the remaining body wall to be opened for removal of the gut. The body wall was placed between two Plexiglas slides each having two circular openings 0.645 cm in diameter (area = 0.32 cm2). These openings thus framed two adjacent regions of the tissue, exposing external and coelomic surfaces in opposite directions. The tissue in its Plexigias frame was then inserted between slotted halves of a modified Ussing apparatus (Ussing & Zerahn, 1951) consisting of two parallel sets of chambers. Upon clamping, the apparatus formed two identical chambers, each with a section of body wall at the center. Accordingly, two regions of the same tissue could be observed simultaneously. Alternatively, a single tissue could be observed while the parallel chamber, filled with the appropriate solution, was used to zero the potentiometer. Each half-chamber contained separate ports at either side of the tissue for insertion of salt bridges, for addition and removal of solutions, and for an air pump connection providing aeration and circulation of bathing media. The 2.5 M KC1 salt bridges placed near surfaces of the body wall were connected by calomel electrodes to a Cary Model 32 vibrating-reed electrometer (Applied Physics, Corp., Monrovia, California) with an input impedance of 1On s2. During flux determinations body-wall potentials were abolished by the application of a short circuit current through silver-silver-chloride electrodes.
22Na Fluxes 22Na influx and efllux were measured on pieces of body wall from animals acclimated to either 50% SW or 20% SW. Experimental media bathing the tissues contained the Na+ concentration of the adaptational medium and the approximate K+ concentration of coelomic fluid in animals adapted to that salinity (Fletcher, 1970; Free1 et al., 1973). Composition of the bathing media is shown in Table 1. Tissues mounted in the chamber were allowed a 30-min equilibration period in unlabeled solution prior to flux experiments. During this period body-wall potentials were measured and short-circuited. Ionically and osmotically identical bathing solutions were then added to both sides of the preparations. To measure Na+ influx, 1.0-2-S &i 22Na (New England Nuclear, Inc., Boston, Mass.) was added to the external solution of one tissue piece; to determine efflux a similar amount of the isotope was added to the coelomic side of the adjacent body-wall section. Initial attempts to determine both influx and efflux on the same section of body wall were unsatisfactory (see Results). Na+ flux across the short-circuited tissue was determined by measuring the appearance of 22Na in the unlabeled solution in the opposite half-chamber. At the end of a given time
SODIUM FLUXES IN BODY WAtL
TABLE ~-COMPOSITION OF BATHINGMEDIA FOR AND50% SW-ADAPTED
223
OF A POLYCHARTR
STUDY OF
*rNa
FLUXES ACROSS
20% SW-
BODY WALLS
Ion
20% SW (mM)
50% SW (mM)
Na+ K+ Caa+ Mge+ Clso**HCOa-
97.4 3.0 1.8 97 111.2 6.3 0.4
243.6 45 46 24.2 278-l 12.8 1.1
interval the whole of the originally unlabeled solution was removed and replaced with fresh solution. The entire (2 ml) sample was counted in a Packard Tri-Carb Liquid Scintillation Spectrometer (Model 3375, Packard Instrument Co., LaGrange, Ill.). Each sample was counted to 1000 counts. The scintillation fluid was 15 ml of a toluene-based solution containing 98% PPG-2% Bis-MSB (0 mnifluor, New England Nuclear, Boston, Mass.: 4 g/500 ml toluene) diluted one-third with Triton X-100 (Atlas Chemical and Manufacturing Co., San Diego, Caliiornia). A 5- or lo-,ul sample of the labeled solution was counted simultaneously to determine its specific activity. All counts were corrected for quenching and background. Flux experiments were performed at 23-25°C. Upon completion of an experiment the circular area of tissue exposed to the label was cut out and weighed. Flux rates are expressed as micromolesjg wet wt. per hr. Descriptions of preliminary attempts to assess the effects of Kf concentration and of ouabain on body-wall Na+ fluxes are presented in the results. RESULTS
Body-wall potentials The body-wall potential, coelomic surface negative with respect to the external, reported by Smith (1970) and Fletcher (1970) for intact hyperionically regulating N. d~~~~col~ was also observed in isolated pieces of body wall removed from animals adapted to salinities of 20% SW or less (Table 2). The potentials shown in Table 2 were measured with body-wall preparations bathed in the adaptational TABLE
2-IsoLxrm
BODY-WALL
POTENTIALS
IN TISSUES REMOVED FROM ANIMALS
ADAPTED
TO VARIOUS SALINITIES AND MEASURED IN SE4 WATER OF THE SAME DILUTION
Adaptational salinity (% SW)
N
50 20 10
11 13 27
Potential* (mv) + O-34 f O-68 -3.18f0.66 - 7.39 + 0.49
* Sign of potential refers to the inner surface with respect to the outer. Data shown are means f S.E.
BYRONA. DONEENANDMARY E. CLARK
224
medium. A slight reduction in potential was observed when tissues were bathed in solutions having the elevated K+ concentration used for the 22Na flux determinations. Significant potentials were not observed in tissues from animals adapted to 50% SW. Potentials generated by the 20% SW-adapted tissues were short-circuited by currents of less than 1*5 PA.
22Na j?uxes The time required to reach a uniform rate of 22Na influx and efflux was measured for several body walls. Figure 1 shows one such influx experiment performed on a tissue taken from an animal adapted to 20% SW. Typically 40 min were required
0”- 1800 fa 150? a E z 120-
-g
z._
01 0
.-.
IO
20
30 Time,
40
50
60
min
FIG. 1. The time required to reach the steady-state 22Na influx rate in a 20% SW-adapted body wall. Solutions bathing the coelomic surface were counted at 10-min intervals.
to reach a steady influx rate in these preparations. Only about 20 min were required to attain the steady-state efflux rate in tissue adapted to both salinities. The Na+ influx and efflux rates for paired adjacent skins, therefore, are based on counts obtained during a 15-min period after the steady 22Na flux rate had been reached.
SODIUMFLUXESIN BODY WALL OF A POLYCHAETE
225
Na+ fluxes, the calculated net flux and the influx to efflux ratio (i/o) determined for several sets of paired body walls removed from animals adapted to 20 and to 50% SW are shown in Table 3. Since the precise surface areas of the body walls could not be determined, results are presented on a weight basis. TABLE 3-Na+
FLUXBSIN BODY WALLS ISOLATEDFROM ANIMALS ADAPTEDTO 20% TO 50% SW poles
SW AND
g-l hr-l
Adaptational salinity (%
SW) 20 50
* Influx.
Influx
EfflUx
Net flux
i/o
3.68 f 0.32 (12) 2.95 _+0.83 (5)
1.06 f 0.13 (11) 3.64 z!z1.08 (5)
2*82* + 0.34 (9) 0+73+ + 0.28 (5)
3.59 f 0.71 (9) 0.79 f 0.07 (5)
f- Efllux.
Data shown are means f S.E.
Number of body walls given in parentheses.
A large variability in flux rates was obtained when a single piece of body wall was used sequentially to determine first 22Na influx and then efflux. During the initial me~urement the external surface was exposed to labeled solution for 1 hr. Although the outer tissue surface was then rinsed four or more times before the inside surface was exposed to the 22Na-containing solution, this procedure resulted in 22Na effluxes frequently larger than influx rates. The apparent source of this exaggerated efflux is shown in Fig. 2. In this experiment a 20% SW-adapted tissue was exposed to 22Na at its external surface for 1 hr. After removal of the labeled solution both sides of the tissue were rinsed five times in 10 min. Subsequently fresh unlabeled medium was reapplied to both surfaces and the external solution withdrawn for counting and replaced at 10”min intervals. Even in the absence of 22Na-containing solution and despite repeated rinsing, a large 22Na efflux from the external surface persisted. The apparent exponential decline of external 22Na levels with time suggests an exchange by some component of the external solution with 22Na retained near the outer surface of the tissue. Determining both influx and efflux on the same tissue section produced results consistent with paired tissue techniques only when efflux was measured first. 22Na activity in the bathing solution was reduced to background levels by rinsing the internal surface two or three times. The effect of Kf concentration on body-wall 22Na movements in animals acclimated to 20% SW was investigated by bathing tissues in media having K+ concentrations (as K,SOJ of 1.8, 3-O or 4.5 mM. These experiments were performed using the same tissue piece for determination of both influx and efflux. For the reasons discussed above, there was considerable variation in the results, but they do suggest that K+ is required to achieve a net Na+ influx. Influx of Na+ is enhanced by increasing the K+ concentration from 1.8 to 4.5 mM. Na+ efflux was unaffected by the Kf concentration of the bathing solutions.
BYRONA. DONE~ ANDMARY E. CLARK
226
;r
2 f
z”
Of 0
I
IO
,
,
i
40
30
20 Time,
,
50
t
60
min
FIG. 2. Efflux of 22Na from the outer surface of a 20% SW-adapted body wall previously exposed to label on the outer surface for 1 hr. Solutions bathing the external surface were counted at lo-min intervals. Sensitivity of Na+ fluxes to ouabain (G-strophanthin, Calbiochem Inc., Los Angeles, California), a specific inhibitor of K+-dependent sodium transport (e.g. Bonting, 1970) was tested by preparing desired concentrations of the glycoside solutions replaced internal or external in bathing media. Ouabain-containing solutions after the steady 22Na flux rate had been attained. Ouabain applied to the internal surface of tissues from animals adapted to 2Oo/o SW reduced Na+ influx by 57 to 85 per cent of the pretreatment rate in five of eight attempts. Ouabain concentrations of 5 x 10~~ M or greater were effective when treatment exceeded 40 min. In three experiments, however, ouabain in concentrations as high as 10-s M failed to reduce Na+ influx. Applied to external body-wall surfaces, ouabain either did not affect Na+ efflux or occasionally produced a slight stimulation of efflux. Both Na+ influx and efflux in the 50% SW-adapted tissues were increased by SO-100 per cent following application of 5 x lo-” M ouabain to internal and external surfaces, respectively. DISCUSSION
Characteristics of sodium movement in isolated body wall removed from individual N. succineu adapted to 20% SW and to 50% SW are clearly different.
SODIUM FLUXES IN BODY WALL OF A F’OLYCHAETS
227
The mean Naf influx in 20% SW-adapted tissue is 24 per cent greater than in preparations from 50% SW-adapted animals (Table 3). This larger influx occurs in bathing medium having a Na+ concentration 40 per cent lower than that in 50% SW-based solutions. Na+ influx and efflux are equivalent in 50% SWadapted preparations. The slightly larger mean efflux is not statistically significant. If it is assumed that Na+ movements across 50% SW-acclimated tissues can be accounted for on the basis of diffusion, then the Na+ permeability of 20% SW-adapted tissues may be contrasted with this “standard”. Setting the ratio of the mean Naf e&x in 50% SW-adapted tissues to the Naf concentration of the bathing medium (243.6 mM) equal to l-0, the ratio for the 20% SW-adapted preparations (Na+ concentration, 97.4 mM) is O-72. It maybe concluded that the 20% SW-acclimated body wall is about 30 per cent less permeable to Na+ than body wall from a 50% SW-adapted animal. Reduced body-wall Na’ permeability at low salinities has been suggested by Oglesby (1972) from whole animal isotopic studies on N. diverskolor. Na+ influx in 20% SW-adapted tissue is about threefold greater than expected from diffusion through body walls having permeability properties of 50% SWadapted tissues, and is 3-S-fold greater than Naf efflux in ZOojo SW-adapted preparations. Accelerated Na+ influx, it is h~othesized, is established by the activation of an inwardly directed sodium-tr~spo~ing system operating in 20% SW-adapted animals. The mechanism either does not operate in the body walls of animals adapted to 50% SW or functions at such a low level as to be undetectable relative to the total influx. The prolonged 22Na efflux from the external surface of 20% SW-adapted labeled preparations (Fig. 2) may represent 22Na-23Na exchange between sodium of the medium and a labeled pool within the tissue. This exchangeable pool is not readily accessible to the coelomic surface of the body wall. Exchange diffusion associated with sodium transport at low salinities has been suggested by Oglesby (1972) on the basis of 22Na efflux in intact N. diversicolor. Preliminary attempts to determine the conditions under which active sodium transport occurs were carried out both by varying the K+ concentration of the bathing media and by addition of ouabain. Although neither set of experiments provided conclusive data, the results obtained suggest the following possibilities. Sodium influx, but not efflux, appears to be dependent on Kf concentration at salinities in which the animal is osmoregulating. Ouabain in most cases reduced Nat influx when applied to the coelomic surface of 20% SW-adapted body walls. Interpretation of the effect of ouabain was complicated by its ability to stimulate et&x in both 20% SW- and 50% SW-adapted tissues when applied to the external body-wall surface. A similar stimulation of Na+ influx by coelomic side ouabain treatment of 50% SW-adapted tissues suggests that ouabain acts in these cases either by causing a non-specific increase of ion permeability or by a more specific interaction with ion-exchanging sites. The experiments reported here support the concept of passive diffusion of Na+ across the body wall at osmoconforming salinities and of a reduced passive
228
BYRON A. DONEENANDMARY
E. CLARK
sodium permeability at osmoregulating salinities, coupled with an active sodium influx. It is not yet clear whether this influx is caused by an accelerated inward sodium transport per se or, perhaps, to a separate chloride pump, which could account for the observed inside-negative potentials. The technique of studying fluxes across isolated body-wall preparations from euryhaline polychaetes may provide the answers to these questions. Ac~o~ledg~~~s-The authors express thanks to Professors H. A. Bern and R. I. Smith, Department of Zoology, University of California, Berkeley, for a critical reading of the manuscript. Figures were drawn by Emily Reid. REFERENCES BEADLE L. C. (1937) Adaptation to changes in salinity in the polychaetes. r. exp. Biol. 14, 56-70. BONTING S. L. (1970) Sodium-potassium activated adenosinetriphosphatase and cation transport. In Membranes and Ion Transport (Edited by BITTAR E. E.), Vol. 1, pp. 257363. Wiley-Interscience, New York. FLETCHER C. R. (1970) The regulation of calcium and magnesium in the brackish water polychaete, Nereis diversicolor, 0. F. M. J, exp. Biol. 53, 425-443. FREEL R. W,, MEDLER S. G. & CLARK M. E. (1973) Solute adjustments in the coelomic fluid and muscle fibers of a euryhaline polychaete, Neunthes succineu, adapted to various salinities. Biol. Bull., mar. biol. Lab., Woods Hole 144,289-303. FRETTER V. (1955) Uptake of radioactive sodium (Na2*f by Nereis d~v~sico~or Muefler and Perinereis czrltrifera Grube. J. mar. Biol. Ass. U.K. 34, 151-160. JL)RGENSONC. B. & DALES R. P. (1957) The regulation of volume and osmotic regulation in some nereid polychaetes. Physiol. camp. Oecol. 4, 357-374. OGLESBY L. C. (1972) Studies on the salt and water balance of Nereis diversicolor-II. Components of total sodium efflux. Comp. Biochem. Physiol. 41A, 756-790. SMITH R. I. (1955) Comparisons of the level of chloride regulation by Nereis diversicolor in different parts of its geographical range. Biol. Bull., mar. biol. Lab., Woods Hole 109, 453-474. SMITH R, I. (1970) Chloride regulation at low salinities by Nereis dive&color (Annelida, Polychaeta)-I. Uptake and exchanges of chloride. J. exp. Biol. 53, 75-92. USSING H. H. & ZERAHN K. (1951) Active transport of sodium as the source of electric current in the short-circuited isolated frog skin. Acta physiol. stand. 23, 110-127. Key Word Index-Body-wall polychaete; sodium flux.
potential ; Nereis (Nea~thes) sllccikea ; osmoregulation ;