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Changes in coelomic fluid and intracellular ionic composition in holothurians exposed to diverse sea water concentrations
C
Pht.~+ol+ 1~7~. h d
54A, /+p Its7 r,J 174 Pt-r+l,tm,+a I+r~,s.+ Pri~tt++d i+t Gr+.,+t tiritail+
C H A N G E S IN C O E L O M I C F L U I D A N D I N T R A C E L L U L A R I O N I C C O M P O S I T I O N IN H O L O T H U R I A N S E X P O S E D T O D I V E R S E SEA WATER CONCENTRATIONS ERNESTO MADRID, I. PERCY ZANDERS AND FRANCL~O C. HERRERA Laboratorio de Eeofisiolo~a Animal, Centro de ~ioffsica y Bioquimica, instituto Venezolmao de lnvestigaciones Cientflicas, IVIC, Apdo. 1-827, Caracas, Venezuela
(Receiced 21 Alu.lust 1975) Abstract--I. Ionic coaceqtrations of coelomic fluid of l.w)s(ichol,US badionotu.~ equilib|a!cd with 80 and 120'!-~;sea water in 4-6hr. 2. With increasing external medit, m concentration, intestinal albumin space remained constalll whereas intracellular space decreased; in muscle total water and ialracellular space decreased a n d extraeellular space increased. 3. Non-albumin space ionic content per unit dry weight, with the exception of intestinal sodium, remains practically unchanged in intestine and muscle in passing from 80 to 120'.'¢~sea water. 4. lntracel|ular ionic concentrations rise proportionately to that of external medium. Adjustment of intracellular osmolality is due mainly to water movements into and out of the cells. INTRODUCTION tonic or hypotonic sea water and the variations of intraceltular electrolytes in intestine a n f f longitudinal THE ECHINODERMS and amongst them the holothurmuscle bands of the body wall caused by clmnges tans, are of exclusively marine habitat and the Phyin the concentration .of the medium were studied in lum is mainly c o m p o s e d o f stenohaline representatile holothurian Iso.stichopus badionotus. tives. Nevertheless, many echinoderms can tolerate Small changes in salinity of ambient sea water. In the last 20 years, echinoderm species living in waters differing widely in salinity from that 0 f s ~ v~atcr (35%3 MATERIALS AND METHODS have been reported. Thus, T h o m a s (1961) found Ophiofi'a.qmus filograneozls living in water of 7",,,, The animals used in the present experiments were colsalinity. Zenkevitch (1959) reported Cuctonaria oftenlected in Santa Fe Bay in the Eastern part of Venezuela. tails. Synapla hispida and Lxlhhlophtx diqitata in They were kept in reeirculated, filtered sea water. Salinity, pH and temperature were controlled and water losses comwaters o f 18",, salinity in the Black Sea. O n the other pensated by the addition of distilled water. The specimens hand. species tolerating" 46",,, salinity have been weighed between 500 and 700 g and they varied in length reported from the Red Sea (Binyon, 196I). Therefore. between 25 and 30 cm. The dissection of the intestine and although individual species of this Phylum m a y be longitudinal muscular bands of the body wall was perstenohaline, different species inhabiting a wide range formed as described previously (Zanders & Herrera, 1974). of salinities have been reported. In order to study the time course "of equilibration of The limited osmoregu latory powers of holothurians the coelomic fluid with the hypertonic or hypolonic exterhave confined them to the sea (Krogh, 1965). T h e nal media, the animals were exposed to 80, 100 and 120~ composition of their perivisceral or c o e l o m i c fluid is sea water for 24 hr. Samples of coelomic fluid were taken at hourly intervals during the first 8hr and subsequently quite close to that of the external m e d i u m (Zanders at I0, 12 and 24 hr of exposure by means of a hypodermic & Herrera, 1974). Q u i n t o n (1900) found that the syringe. Hypotonie sea water was prepared by diluting chloride concentration o f the perivisceral fluid of natural sea water with distilled water. Hypertonic sea Asterias rubens decreased with that of the external water was prepared by evaporating natural sea water to medium. Koizumi (1932) found that tiffs equilibrium 5/6 its initial volume. Concentrations differing only 20~,, was reached in a matter o f hours. T h e changes occurfrom the normal concentration were used in an effort to ring at the intraceltular level in response to modificaminimize possible damage to the animals. The sea waters tions of the concentration o f the external medium used and the coelomic fluid were analyzed for sodium, have not been extensively studied. Schoffeniels (1967) potassium, calcium and magnesium by flame and atomic absorption spectrophotometry (EEL 100 flame phot+ometer has shown in marine invertebrates that the total intraand Varian AA5 atomic absorption spectrophotometer). cellular inorganic electrolyte concentration is lower Chloride was determined amperometrically on a Buehlerthan that of the extracellular medium, osmotic equalCotlove Chloridometer. Osmolalities of sea waters and ity being maintained by the presence of small organic coelomic fluids were determined by freezing point depresmolecules (mainly a m i n o acids) in the intracellular sion on a Fiske osmometer (Model G-66). medium. The behavior of the intracellular inorganic electrolytes in relation to changes in the ionic concenTissue ionic content tration o f the external m e d i u m has been little studied Tissue sodium, potassium, chloride, magnesium and calin echinoderms. In the present paper, the time course cium content, total tissue water and extracellular space were determined on the intermediate intestine (Zone 5', o f equilibration o f the perivisceral fluid with hyper167
|-:,RNILSIO ~ l A l ) l , t l l ) t't ill.
IhN
Zanders & |lerrera, 1974) and muscle tissue of holothurhtns exposed for t 0 h r to 80, I 0 0 and 120'!;, sea water. This f~criod was stlllicicn! to allow cquilihralion o:f the c~)ehtmic Iluid with the snrronnding sea wilier. The tissues a|~d coelomic Iluitl were removed from the animals and the intestine and muscle were ineuhated in their respective coelomic fluid to which I"~t-lahclled human serum ;tlburain JR111SAh ohlidned from the Radiochemical C'entre. Arnersham, had hcen added. The tissues were further incubated during 3,5 hr in this medium Io :dlow the alhumin t~: equilibrate with the tissue space accessible to it, At the end of this period, the tissues were carefully blotted between two pieces of Whalman No. 54 filter paper and weighed to determine wcl weight. They were subsequcnlly dried ,,~vernight at 1(15 C and reweighed to determine dry weight. Tissue w:|ter was taken.as the difference between wet and th T weight. RIIISA space was dclermined by counting the dry tissue residues, along with .'|liquors of the bathing coelomi¢ Iluid. in a gamma spectrometer. The dry residues were subsequently extracted with 1 N nitric acid during 48 hr for eleclrolyle determination. Intracellular electrolytes were estirnaled by sublracting from lolat tissue electrolytes the contribution made by those in the extraeelh|lar fluid ~Zanders & Herrera, 1974). Calcium and magnesium, being rhoslly bound tap i~t the structure of the spicules, were no! includ¢
Stdtisth'~l tm.t~lmc.nt The signilic;mce of the dill;creates between the means of the dah~ at the 0.05 level of probabilily was (letermined by applyiug analysis of variance and the Student-Newmun-Kculs procedure tSoknl & Rohlf. 1969l. Least significant ranges were thel~ calculated to test for signilic'mce between the means of the results obtained in the tissues exposed to the different sczt walcr concentrations. "Fhc'orcth'al cuh'itkttion ty" the" time cour,~c" ~{/"eclldlihr~ttio~l *tf tire coehm~ic fltdd In order to arrive at a simple expression h~r the time cot,rse of equilibration of the coelomic fluid with the surrounding, sea water, several assumptions should be made: (1) the permeability of the body wall to ionic and water movement should be constanl; (2) the concentrations have been ttlken as equal to the activities of tile different ions; [3) the volume of the surrounding fluid should be snlticiently large so tlu*t the concentrations of the different ions remilin practically invariable despite exch~mge of'water and electrolytes belween the :mimals nnd the surrotmding sea
waler and (41 the volume anti composition o f the eoelomie fl~id arc not actively modified by lhe animals. T h e s e assumptions lead to ;he following expression for the rate of change of the difference of coneeatration.~ of a given ion between the coclotnic fluid and the external sea water as a function of time d f A C I/dt = -. k(ACI (II
~c=c-
c.
where C is tile conce/llration of a given ion in the coelomic fluid at any instant: C,. is the concentration of this ion in the surrounding sea water: k is the rate constant describing the appro.'|eh to equilibrium of the ioni¢coneentration in thc coelomie fluid with the external medium which may be estimated from the integrated form of equation (!): k = (D/fin(C,, -- Cm)..'(C -- Cm) where C,, is the concentration of the ion in the coelomie fluid at t = 0. When C,. > C. this expression yields C = C,,, - (C,,, - C,,)exp -- kt (2) When C'~, < C,, C = C~ + (C,. - C . l e x p - kt
R ESU LTS
O,smolali O" a n d Cmnl, o s i t h m o f t l w sea w a t e r x a m l c o e l o mh" Jhdd.~ T h e c o m p o s i t i o n o f the sea w a t e r s used, n o m i n a l l y 80, 100 a n d 120')i. a n d o f the c o e l o m i c fluids o f anim a l s e x p o s e d 1 0 h r to t h e m are s h o w n in T a b l e 1. T h e c o m p o s i t i o n o f n a t u r a l sen w a t e r a c c o r d i n g t o Barnes (1959} a n d SiliCa (196l) is a l s o i n c l u d e d for reference. Tht~ small dc;,,iations f r o m the e x p e c t e d values o b t a i n e d in thc sea w a t e r s "used for the experi m e n t s m a y be due to the fact that the s a m p l e s o f sen wate¥ were t a k e n at the end o f t h e 1 0 h r p e r i o d of e x p o s u r e o f the a n i m a l s which p r e c e d e d the r e m o val of the intestinal a n d muscle lissues. T h e c o n c e n " I r a | i o n s o f the different ions in the n a t u r a l sea w a t e r used in the p r e s c n l e x p e r i m e n t s a r c slightly higher t h a n t h o s e r e p o r t e d by B a r n e s (1959) a n d Sill6n (1961). T h e c o n c e n t r a t i o n s o f the different i o n s in the c o c l o m i c fluids c o r r e s p o n d within the e x p e r i m e n t a l e r r o r to t h o s e o f the c o r r e s p o n d i n g sea water. T h u s .
Table I. Compositipn and osmolality of 80, 10<3 and 120';b sea waters and of the coelomic fluids of holothnrians exposed 10 hr to them 80~
t20¢
I00~
Sea ~ater
~ater
sea ~ater
Coel om|c Fluid
(6)
I7)
(6)
t7)
{6)
h06,9
409.7
514.1
*_s.~
._10,2
~9~.3
600~6 +6.7
*_11,1
Potass t um
8.6 +o. I
8.9 *o. I
11-3 t_0.1
11-1. +.p. +
13-6 +o. 2
13,2 +_o. 1
C~nloo ,-|de
t,~2°3
+~.~
k53.3 +5,7
$6~.7 *_9.1
594.0 +_.e.t~
712.9 +s.~
717.7 *6.8
5~8. jl
8.2 +_o.2
8.t~
10.7
10.9
|].1
13,1
10.2
10.8
,_o.z
+o. 2
+o.z
+0.z
+o. 2
59.~ +..o,~
58.9 +_o,s
71.9
70,!
53.57
50.5
+~.3
-._o.s
~e~ Veter
(7) Sodium
Se~
(ea~nes. 19~91
Coelo~|c F l u i d ~ ! le~sfs (i
CoelOrnt¢ rluld
co~1om{c Fluid
S|lt~n~ 1961)
Calcium
• 6.~
+84
t~mae-
s~6.~
47.5
,i,~
+._o.z
+o.9
0 ~,~',o-
855
B73
1137
1135
138;5
571.5
47G.2 9.96
460 11 J3 523
1382
~|eetfoly~e concentration i~ m M l l ° O s m o | a l i t y i n mOsm/K 9. Each f i g u r e represent~ t h e m e a n ~ S . E ° Number OF d e t e r m i n a t i o n s in p~rentheses.
Ionic changes in holothurian tissues
169 POTASSIUM
SODIUM
6600 00
_
.
T ;L~
,
-~.
.J
,
-
I
"
13
J J--
I
}
~
'
}
~
"
6
600
5
0
400 ~
0
t o~
L
"
z e
io
=
~
..'
'~
1
1
T
T
T
~
,
t
i20%
o
~
"
t 8'o'/. z4.
80 %
7 O
,z
. t~__
120%
2
4
G
63
~O
12
24 L
%
~ o -
~
'
~---
80%
Fig. t, The time-course of equilibration of the concentrations of sodium, potassiutn and chloride in the coelomie fluid with their respective concentrations in the exlernal medium. Each point represents a mean value from at least three animals: where indicated, the sizes of the vertical bars represent + one S,E. For further details see text. after l O h r of exposure, the c o e l o m i c fluid of the exp e r i m e n t a l a n i m a l s has practically e q u i l i b r a t e d with the s u r r o u n d i n g sea water. The c o m p o s i t i o n of the c o e l o m i c fluid of Cm,lintt chilensis r e p o r t e d by K o i z u m i (1935b) is included for c o m p a r i s o n . T h e time course o f the e q u i l i b r a t i o n o f the c o n c e n trations o f sodium, p o t a s s i u m and c h l o r i d e in the e o e l o m i c fluid with those o f the anabient sea water ark s h o w n in Fig. I. T h e rate coefficients, k ( r a i n - l ) , were calculated from the d a t a using e q u a t i o n s 3 and 4 and the calculated curves have been fitted to the points. T h e rate coefficients are all o f the o r d e r of 0.01 rain-~ and are a p p r o x i m a t e l y equal in animals exposed to h y p o t o n i e o r h y p e r t o n i c solutions. In anim a l s m a i n t a i n e d in I00% sea water, little o r no c h a n g e was seen in the sodium, p o t a s s i u m and c h i e f -
ide c o n c e n t r a t i o n s o f the c o e l o m i c fluid. It m a y be seen from the curves that after 6 hr the ionic conce~trations o f tile e o e l o m i c fluid c h a n g e very little. It will bc c o n s i d e r e d hereafter that the ionic c o m p o s i lion o f the c o e l o m i c lluid a n d the respective a m b i e n t sea w a t e r arc the s a m e and the results will be referred to in terms o f the ambient sea w a t e r e m p l o y c d . Cha/tges #~ tissue water T a b l e 2 s h o w s the effects o f exposure to the different a m b i e n t m e d i a (i.e. c o e l o m i c fluid o f animals equilibrated with 80, 100 a n d t 2 0 % sea water) on total tissue water, extraoellular space and intracellular water expressed in m l / g dry wt. T h e Student--Newm a n - K e u l s p r o c e d u r e was used to test for significance
Table 2, Tissue water Ambient 80~; Dlff,
I000
t'ledium Oiff.
120~
DiFf. 80-120g
$,t. Gr,~p flean
L,S,R, P~O 05K=3 (@) K=2
INTESTINE Te .~ Wate
5.32
0-39
~+93
0.27
~.66
0,66
0.23
0.68
0.8~
AlbL Sp~
b4
0.19
1.o3
O.tO
l.i3
0.29
o,lo
0.30
o.3&
.55
3.93
0.~0
3,53
0~5"
0.26
0.77
0,9~
0.64*
4.10
0.4)~
3.67
1.07 ~
0.05
0,16
0,I9
MU-. Tota I Water
~. 7h
A I btamlr~ Space
0.75
0.OZ
0.77
0.27.+
1.02
0,29~
O,O~
0.2t~
0,29
Intra¢:el l u l a r Space
3.9 8
0-654
).33
0.70 ~
2,6)
1.35~
0.0~
0.26
0.32
Water c~ntent and spaces in mIJg dry wet hi (1)Difference) b e t ~ e n consecut|ve ~ I n $ ~((] be )ign(61c)nt(~)at or bclo~ th~ 0 * 0 ) l e v e l o f p r o b a b i l i t y I f they a r e g r e a t e r than L.S.R.k~ 2 D i f f e r e n c e s b e t ~ e n means o f r e s u l t s in 804 and 120t amb|e~t m e d | ~ Will be s i g n i f i c a n t a t t h i s l e v e l i f they are g r e a [ e r than L.S,R.Rw 3
170
ERsF,sro ~vJAI)RII) et al. Table 3. Total tissue sodium, potassium and chloride content referred to total tissue water hmb 80~
i ent
~e
Diff.
IOO~
Cl t ur,~
Dtff,
120¢
Oi f f , 80-120~
S.E, Group P~e~n
Z38,E*
~5.5
L.S.R. , P-O.O5 k'2 k" 3 I t )
IHTESTtNE Sodium Potassium Chloride
3|2.~
IO5.5
~17,9
133.3
551,2
87,5
5.2
82.3
8,5
~0.8
3,3
~.'8
135.0 16~.0 17,3
21.0
368.7
120.9"
429.6
1OI.9"
531,5
222 B~
1~.5
~3.1
52.3
~9.5"
37.5
HUSCLE Sodium
|83.3
65.3*
2~8.6
8~,3 ~
332,9
|0,~
30,8
Potassium
133,3
26,8*
160.1
27,~ ~
187,~
5~.2"
5.2
tS,~
18,7
Chloride
2OG,~
89,3*
289,~
~9.3"
~S9,2
158.6"
10,1
29.9
36,k
[]ectro|yte
cont¢~t
l~ t.M/~ t i s s u e water
( ~ ) r o r e ~ p l a n a t l o n 0r s t a t i s t i c a l
tre~t~nt
between the means. In the intestine no significant difference could be observed in these parameters with the exception of the difference between the intr,'tcellular • spaces of tissues exposed to 80 and 120"~; sea water. In muscle, almost all changes arc significant with the exception of the change in extracellular space between 80 and 10CI"{, sea water. I1 may be observed in this tissue that with increasing ambient sea water concentration, total tissue water and intracellular space decrease, whereas a l b u m i n space increases slightly. Analysis of variance of the intracellular spaces in muscle indicated that the dil]~rences were highly signific~mt statistically and that the variation ~wnongst them could be due to a linear dependence of the intracellular space on sea water concentration. Therefore, an attempt was made to tit the data to a straight line relating intracellular space to the a m b i e n t sea water (or coelomic lluidl concentration. A very ~ t i s -
see Table 2
fitctory fit, over the range studied, was obtained for the line described by the equation IW --- 6.70 - (0.03382 __, 0.O0080)SW where IW is the intraccllular water in ml/g dr3' wt and SW the concentration of the sea water in per cent. The 95';,~, confidence limits for the regression coefficient are 0,04402 and 0.02362. It must be emphasized that this is an empirical line and that the intercept 6.70ml/g dry wt is probably meaningless. The decrease in total tissue water in muscle with increasing sea water concentration is completely accounted for by the decrease in intracellular water. Ti,ssue e l e c t r o l y t e content
Total tissue sodium, potassium a n d tent expressed in ima/ml tissue water Table 3. In the intestine tissue chloride increasing sea water concentration a n d
T~lblc 4. Total tissue electrolyte content expressed in terms of dry weight A~b
i e~
t
H e d | ur~
Dill, ~0-~ ZO~
S.E. group Hean
L.S.R P'O*05 k~2 k'~ it)
~O~
Diff.
lO0~
Diff.
|20~
1662
377*
2039
509~
25~8
8BS*
92
273
~3
4~
399
19
~18
2~
22
G6
8!
Ch|or~de
165,
~2'
2094
387*
2481
829*
12h
369
~8
Calclu~
1035
83
1118
167
1285
250
1~5
43T
523
3~|
56
~17
77
~94
13~*
29
B5
103
129!
Z38
I~TESTINE Sodium Potassium
~gnes|um
331
MUSCLE Sodlum
86~
150
t01~
272 ~
422*
66
196
Potesslu~
630
26
65G
29
685
55
29
S&
69
Chloride
952
237*
1189
129
1318
366 ~
~7
1~0
170
Calcium
67
16
5!
5
56
t0
6
18
21
~gnesium
98
19
1t7
&
123
25
9
27
33
E | e c t r 0 1 y t e c o n t e n t expressed | n ~HI 9 d r y w e i g h t (t)
For e x p l a n a t | o n a f s t a t i s t i c a l
t r e a t m e n t see Table 2
chloride conare shown in increases with tissue sodium
Ionic changes in hololhurian tissues
171
Table 5. Intracellular sodium, potassium and chloride contenl expressed in terms of dry tissue weight Arab I e n t /4e d t u m ~Ol I) I f f , t0091 r,I f f . 1 2 0 ~ , 80_ D i f120~. f. G.E, Group I~ean
L.S.g* P~O,05 k-2 k-3 (~)
INTESTINE
$od~um
1318
221
t53~
1896
578 ~
lOB
320
~35
39
396
7
~O3
32
22
65
79
1272
184
1~56
Zl?
1673
601
1E0
416
505
~SO
Potassium
Chloride
3~ ~
389
M~SCLE Sodl~
560
77
G37
56
~93
13~
SO
I~9
Potassium
fi23
25
6~8
2~
672
~9
18
53
65
Chloride
61~
12~
738
;Oh
63~
20
SO
1~9
181
Electrolytes e ~ p r e s s e d i n ~M/g d r y w e i g h t (t)
For explanation Of s t a t i s t i c a l
treatment
increases when sea water is raised from 80 to 120%o. However tissue potassium does not vary significantly. In the case o f muscle, all three ions increase with increasing a m b i e n t medium concentration. These changes could be due to three factors: (1) increasing concentration of the extracellular fluid; (2) increased electrolyte content of the intracellular space and (3) loss o f tissue w a t e r from the intracellular a n d extracellular c o m p a r t m e n t s as the concentration of the ambient sea water increases. In order to determine whether there were net gains or losses in tissue electrolyte content, the results were expressed in terms of d r y weight thereby ruling out the effects of the changes in tissue hydration. Nevertheless, changes in tissue ion content caused b y m o d i fications of that o f the extracellular space will still contribute to the changes in tissue ion content. Therefore these data, which are shown in T a b l e 4, show increases in sodium and chloride content of the tissues with increasing sea water c o n c e n t r a t i o n as a result o f possible changes in intracellular and extracellular ion contents. The interest of the present investigation is centered on the changes in the electrolytes of the intracellular c o m p a r t m e n t caused by modification of the a m b i e n t sea water concentration. Therefore, an attempt was m a d e to estimate these quantities by subtracting from the total ion content the electroTable 6. lntracellular sodium, potassium and chloride expressed in terms of intraoellular water 80I
DIff.
100t
Meal DIff.
UI~ 120t
/~rab I en~
S.E. DIff. 80-1201
(;roup Nean
L,S*I~. P~D,95 k-2 ~'3 (~)
INTESTINE Sodium
29k
lOZ*
396
150"
545
ZSI*
2Z
6&
Potassium
97
7
104
Ik
118
21
8
23
28
Chlor|d~
280
98"
37B
B9%
467
187"
19
S6
68
see T a b l e Z
lyte content contributed by the a l b u m i n space (assumed to represent the extraoellular space): Intracellular ion contents were expressed p e r u n i t tissue dry weight in order to detect net changes in non-albumin space ion content and per unit intracellular wa~er to relate them to changes in n o n - a l b u m i n space watei" (taken as intracellular water). Table 5 summarizes intracellular ion. c o n t e n t s L~r unit d r y weight. It m a y be observed that m the intestine the intracellular s o d i u m c o n t e n t changes o n l y when passing from 109. to 120~ sea water. Since there is some question as to the eo(respondence Of extrace-l~ lular s p a c e and R I H S A gpace in this tissue (Zanders & Herrera, 1974)this is not Surprising. In longitutlinal muscle, where R I H S A spac~'seenls to be a better measure o f the extracellular space, sodium, p o t a s s i u m a n d chloride content per tinit d r y weight remain invariable in passing from one.Sea ~vater conceniration to a n o t h e r . T h i s indicates t h a t cell electrolyte content is independent of e x t r a c e l l u l a r c o n c e n t r a t i o n over the range studied. However, if the i n t r a c e l t u l a r ionic contents are expressed in terms o f intracellular water, as is done in T a b l e 6, a clear cut increase in ~ntracellular ionic concentration with increasing extracellular concentration is observed, specially in muscle. Since intracellular water decreases with increasing sea water concentration, increased intracellular ion concentration is d u e primarily to the toss of intracellular water rather than to any intracetlular ionic accumulation. Tissue calcium and magnesium content being closely related to spicule c o n t e n t were not studied systematically and are only shown for referehce in T a b l e 4.
BO
DISCUSSION
Electrolyte concentration of the coelomic fluid The coelomic fluid o f Isostichopus badionotus SQdlu~
140
57"
197
43*
Z40
I00"
12
37
qk
Potess]um
IS7
3B*
195
68*
262
106"
t0
3t
4S
Chloride
15Z
fi6 ¢
~lB
24
2kZ
90*
I!
3J
3~
E l e ~ t r o | y t ~ s e~re~$ed In t l n a s Of I~l./g I . t r Q c ~ 1 | ~ l | r ~ t t r
(~) F o r explanation o r s t a t l l t l ~ l
lreatmmRt see Table 2
rapidly a p p r o a c h e s the c o m p o s i t i o n and osmolality of the external m e d i u m when the latter is concentrated or diluted. This h a d been observed in other species of echinoderms by Henri & Lalou (1903); G a r r e y (1904) at the beginning of this century and m o r e recently by K o i z u m i (1932, 1935a, b). It has been postulated
172
EaNl:S-lo M^i)l,liD el at.
that this exchange takes place through the body wall and respiratory trees (Berger & Bethe, 1931; Delaunay, 1931; Koizumi, 1932, t935a; Robertson, 1953; Ahearn, 1968). More recently, Lawrence et al. (1967); Zanders & Herrera (1974) observed that the intestine may play tin important role as a site of high ionic permeability through which equilibration of the coelomie fluid with the external medium could take place. Nevertheless, the time course of equilibration has not been extensively studied. Botazzi (1906) observed that after 24 hr the freezing point depression and conductivity of c o d o m i c fluid were identical to those of the external sea water which had a freezing point of - 2 . 6 5 " C , approximately the same as that of the hypertonic sea water used in the present investigation. Dakin {190g) using hypotonic a n d hypertonic sea waiters with freezing point depressions of 0.76 and 2.98 respectively tried to measure the time of eqvilibration in A,~teria rubens and Echintts e.~euIcntus The time o f immersion (3.5 hr) was insulticient Ibr the freezing point depression of the pcrivisceral Ituid to reach that of the sea water. If immersion time wa,s increased the animals died before reaching cquilibrit,m, This is not surprising since these concentrations are far removed from the normal sea water concentration, corresponding to 36 and 142','., sea water. In order to minimize damage ttp the animals, tile hypotonic and hyperto,aic concentrations used in the present investigation were 855 a n d 1385 mOsm/kg, nominally 80 and 120';,,, sea water, ,'espectively. This allowed incubation periods of up to 24 hr without, obvious deterioration of the animals. The results reported by Koizumi ( 19321 working with (~tudina chih,lisis arc close to those found in the present in vcsiiga t ion. intracelhskn" spat'c, ~dlmmin sp~tce and tisstR, w~tter
Several organic molecules have been used to estimate e×tracellular space. Theoretically albumin, mannitol, rallinose and sorbitol should give a g o o d measure of this space because the large size o f their molecules would exclude them from the ceils. However, extraccilukir s p a t e could be overestimated if they entered the cells or were metabolized: on the other hand, if regions impermeable to these molecules exist in the extracellular space the latter would be underestimated. Albumin, because of its high molecular weight, is p r o b a b l y excluded from the cells but it may also be excluded from the hemal lacunae which abound in the intestinal tissue (Zanders & Herrera, 1974). Simon et al. (1964) found that h o l o t h u r i a n muscle does not present barriers to albumin diffusion in the extracellular space, albumin being, thus, a g o o d probing molecule for the latter. Inulin has been used also as a m a r k e r for extracellular space. However, inulin appears to enter the cells since inulin space increases with time (Conway, 1957: Tasker et al., 1959; Burnstock et al., 1963; Rapp, 1964). In the present work, the extracellular spaces found are lower than the inulin spaces found by Z a n d e r s & H e r r e r a (1974) in the same tissues of lsostichopus badionotus, This p r o b a b l y explains the lower intracellular potassium concentrations found in the present work since the intracellular spaces would be correspondingly larger. Hagemeijer el" aL (1965) have reviewed the methods for determination o f the extracellular space
and have pointed out the problems and pitfidls involved. The existence o f hemal lacunae, possibly inaccessible to RIHSA, probably hHroduccs a large degrce of uncertainty in thc mcasurcmcnt of tl~e cxtraccllular space of the intestine and consequently the measurement of the intracellular space woukl also be- in doubt. This would seem to bc contirmcd by lhe fact that the changes in tissue water., exlracellular space and intracellular space caused by increasing the concentration o f the sea water are not markedly significant in the intestine, whereas in muscle, where the hemal lacunae ;ire not as developed, ll{e changes determined in these spaces and particularly the mtracellulatr space are quite signiticant. Therefore. it woukt appear that the estimation of the intracellular space in musetc is subject to less uncertainty than in the intestine and furthermore, the fact that the losses in total muscle tissue water are fully accounted for by the losses in intracellular water tends to support this hypothesis, "H~tal tissue electrolyte content
The changes in tissue hydration caused by exposure to hypotonic and hyperlonic media precluded the expression of tissue electrolyte content in terms of tissue waiter as a measure of changes i,a net ionic content.-l'herefore, electrolyte content was referred to tissue dry weight which presumably is ,aot all;acted by changes in tissue hydration. The net changes of sodium and chloride in both intestine and muscle a n d the slight changes in intestinal magnesium, seen in Table 4, p r o b a b l y reflect changes iu the electrolyte content of the extracellular space. This assumption is strengthened by the observation that tissue potassium, which should be mainly intrac'elhllar, and tissue calcium, of which a substantial portion is b o u n d up in the spicules IZanders & Herrera, 1974}, d o not vary with changing sea water concentration. lntroct'lhtklr ionic" cont~'nt
The fraction of total ionic content regarded us i~traceilular d e p e n d s on the correct estimation of the cxtraeellular space. It would a p p e a r that albumin space is a good a p p r o x i m a t i o n of the extracellular space in muscle. However. in the intestine several facts suggest that this is not the ease: (a) n o n - a l b u m i n space in the intestine is at least as large as that o f muscle despite the fact thai the intestine is richer in connective tissue and hemal lacunae than muscle. This sugges~ts that albumin is excluded, in the intestine, from structures other than cells; these structures could ,,,,'ell be the hemal lacunae. Moreoever, the extraceiluktr space o f the intestine is not affected by the changes in concentration of the ambient medium. (b) n o n - a l b u m i n space sodium and chloride expressed both in terms o f dr2," weight a n d o f nonalbumin space water are considerably higher in the intestine than in muscle. This could be the result o f underestimating the extracellular space a n d ascribing the sodium and r t g r i d e c o n t e n t of the hemal lacunae to tt~e cell c o m b - , i m e n t . (c) Furthermore, the low non-albumin ("intracellufar") potassium seen in the intestine could be due to the same type of error. Total tissue sodium and chlor-
173
Ionic changes in holothurian tissues ide are much higher in the intestine than in muscle; this suggests that, if the intracellular sodium and chloride content of the intestine approaches that of the muscle tissue, the true ¢xtracellttkw space of intestine must be considerably higher than the measured albumin space, if an extracellul:lr sp.'lcc o f 3 m l / g dry wt is used to calcuktte the intraceltukar sodium, potassium and chloride concentrations of the intestine exposed to 100",, sea water the following values are obtained: sodium, 2 5 8 / t M / g intracellular water; potassium, 189/tM/g intracellular water and chloride, 199ttM/g intracelluhtr water. These results are quite close to those obtained in muscle. M o r e o e v e r an extracellular space of 3 ml/g dry wt corresponds to 61% of tissue water which agrees quite closely with inulin s p a ~ of intestine determined in a previous work (Zanders & Herrera, 1974). Despite the fact that extracelhdar space is underestimated in the intestine, the changes of ionic content in this space accounts for a large enough fraction of the changes in total ionic content to retader the changes of sodium and chloride content of the non-albumin space statistically non-significant. It is clear in muscle and less so in intestine thal the intracellular space loses water as the external concentration is raised from 80 through 100 to 120. T h u s intraeelluktr ionic concentration is raised with increasing external osmolality since the intracelluktr ionic content, in terms of dry weight, remains unchanged. This suggests that the adjustments of intraccllular osmolality are due mainly to water m o v e m e n t s in both tissues. Moreover. the changes of the concentrations of sodium, potassium and chloride of the muscle fibers is in good agreement whith those of the external medium: the intracellular concentrations o f the three monovaler~t ions is 1.5-1.6 times higher in tissues exposed to 120'?.o sea water than in those exposed to 80~,; sea water which corresponds to the ratio of the external concentrations. Thus the cells appear to maintain their ionic content within more or less n a r r o w limits. Muscles of certain cruslacea such as Ctdlinectes s a p i d , s when placed in hypotonic saline increase in volume: however, with time they decrease spontaneously towards their initial volume. W h e n returned to isotonic salines the muscles shrink below their initial volume {Lang & Gainer, 1969a). These responses of C. saphh~s muscle have been referred to as volume readjustment. This p h e n o m e n o n is reminiscent of the volume regulation seen in some annelid polychaetes (Skaer, 1974). However, the mechanisms are different in the two cases. In C. sapidus the secondary decrease in volume in hypotonic solutions is associated with a loss o f free intracellular a m i n o acids (Lang & Gainer, 1969b) whereas in polychaetes it has been ascribed to passive exchanges o f salts between the animal and its environment (Skaer, 1974). In lsostichopus b a d i o n o m s no volume readjustment was detected in muscle and intestine subjected to h y p o t o n i c solutions; intracellular water increased in proportion to the decreased external osmotic pressure showing no return to initial volumes. T h e lack of volume regulation in holothurian tissues, associated to the constancy o f the intracellular ionic c o m p o s i t i o n (referred to dry tissue residue), suggests that changes in intracellular free a m i n o acid pool are not involved in the adaptation o f the intracellular milieu to de-
creased osmolarity of the external mcdiun~. Luidia a euryhaline echinoderm, responds to reduced salinitics by isosmotie intracellu lar regulation and coelomie fluid reguhttion, After one day's exposure to hyposmotic sea water, ninhydrin positive substances of podia and pyloric caeca were lower than in tissues of c o n t r o l animals (Ellington & Lawrence, 1974). These results are in contrast with the reported lack o f c o e l o m i c fluid volume regulation in Stronayh)centrottts purpttrattts (Giese & F a r m a n f a n n a i a n , 1963) and in a stenohaline population of Asterias rubens (Binyon, 1961). lsostichoptts t)adionotus would therefore appear to behave as other stenohaline echinoderms. c'htthrata,
Ackm)wlcdttemems-This investigation has been supported in part by Grant No. DF-5il-047 from Cons~io National dc lnvcstigacioncs Cicntificas y Tccnol6gicas.
REFERENCES AHEARN G. A, (1968) A comparative study of p32 uptake by whole animals and isolated body regions of the sea cucumber ltoloturia atra. Biol. Bull. mar. biol. Lab., H'bods ttoh, 134, 367-381, BARNESn. (1959) Apparatus and Methods t ~ Oceano[iraph y, 1st Edn, p. 315. George Allen & Unwin. London. BERGER E. & BE'ritE A. (1931) Die Ourchl~issigkeit der K/.irperoberfl~ichen wirbclloser Tiere for Jodionen. Pfliiffers Arch. ges. Physiol 228, 769-789. BINVON J. (1961) Salinity tolerance and permeability to water of the starlish Asterias rtlbeas L d, mar. biol. A,~s., U.K. 41, 161-174. BINYON J. (1966) Salinity tolerance and ionic regulation. In Physiok)gy o f Echinodermata (Edited by BOt)LOOTIAN R.), pp. 359-377. Interscience, New York. BINYON J. (1972) The effects of diluted sea water upon podial tissues of the starfish Asterias rubens L. J. Comp. Biachem. Phrsiol. 41A, 1-6. BO'rTAZ~ F. (1906) Sulla regulazione della pressione osmotica negli organismi animali. Arch. Fisiol. 3, 416-446. BIJRNSTC~.K G., DEXVHURST D. J. & SIMON S. E. (1963) Sodium exchange in smooth muscle, d. PhysioL, Loml. 167, 210-228. CONWAV E. J. (1957) Nature and significance of concentration relations of potassium and sodium ions in skeletal muscle. PhysioL Rer. 37, 84-132. DAKIN N. J. (1908) Variations in the osmotic concentration of the blood and coelomic fluid of aquatic animals caused by changes in the external medium. Biochem. J. 3, 473-490. DELAUNAY H. (1931) L'Excretion azot6e des invert6br6s. Biol. Rer. 6, 265-301. Et.LINGTONW. R. & LAWRENCEJ. M. {1974) Coelomic fluid regulation and isosmotic intracellular regulation by Luidin elathrata (Echinodermata: Asteroidea) in response to hyposmotic stress. Biol. Bttll. mar. hioL Lab., Woods Hole 146, 20-31. G~REV W. E. 0904) The osmotic pressure of seawater and of the blood of marine animals. Biol. Bull mar. biol. Lab., Woods Hole 8, 257--270. HAG~MEIJER F., RORIVE G. & SCHOF*FENIELSE. (1965) The ionic composition of rat aortic smooth muscle fibres. Archs int. Physiol. Biochim. 721, 453-475. HENRI V. & LALOU S. (1903) R6gulation osmotique des liquides internes chez los holothuries. C. r. Sdanc. soc. Biol. Paris 55, 1244-1245. KOIZUMt T. {1932) Studies on the exchange and the equilibrium of water and electrolytes in a holothurian, Caudina ehilensis (J. Miifler)---[. Permeability of the animal surface to water and ions in sea water together with osmotic
174
t?RNI-STO MADRID el al.
and ionic equilibrium between the body fluid of the animal and its surrounding sea water, involving some corrections to our previous paper (1926). Sci. Rep. ~hoku Unit.,. IV, 7, 259--31I. KoJZUMI T. (1935a) Sttidies on the exchange and the equilibrium of water and eleclrolytes in a holothurian, C~ntdina chih'nsis (J. Mullet}--HI. (a) On the velocity of permeation of K+, Na +, Ca 2* and Mg 2÷ through the iso. lated body wall of Catalhta; (b) An acidimetric micromethod for the determination of Na + as triple acetate: (c) a volumetric micromethod for the determination of K ~ as iodoplatinate. Sci. Rep. "lfihoku Univ. IE i0, 269--2'/5. KolZUMI T. (1935b) Studies on the exchange and the equilibrium of water and electrolytes in a holothurian, Caudina chilensis (J. Muller)---IV. On the inorganic composition of the corpuscles of the body fluid. Sci. Rep. Tohoku Univ. IV, 10, 27%280. KRtX;Iq A. (1965) Osmotic retlulatiotl in Aquatic Aninutls, pp. 32-36. Dover Publications, New York. L^NG M. A. & GAINt~.R H. (1969a) Volume control by muscle fibers of the blue crab. Volume readjustment in hypotonic relines. J. oen. Physiol. 53, 323-341. LANG M. A. & GAtNt~g H. (1969b) Isosmotic intracellular reguhtion as a mechanism of volume control in crab muscle fibers. Comp. Biochem. Phy.siol. 30, 445-456. LASGE R. (1964) The osmotic adjustment in the echinoderm, Stron.qyocentrotus droe~achiensis. Comp. Biochem. Physiol. 13, 205--216. LAWRENC|-~ D. C., LAWRENCE A. L., GRf,F,R M. L. & MAILMaN D. (1967) Intestinal absorption in the sea cucumber Stichopus parvimensis. Comp. Bi(~'hem. PhyshJl. 20, 619- 627.
Qt~INTON R. 11900) Communication osmotique, chez rinvertebr6 marine normal entre le milieu int~rieur de rantreal et le milieu ext6rieur C. r. hebd. Sdmc. Acad. Sci., Paris 131,905-908. Stt.L~N L. G. (1961) The physical chemistry of sea water. In Ocetvu;~raphy (Edited by SI~AP,s M.L p. 549. A.A.A.S., Washington, D.C. RAt,r, J. P. (1964) Electrolyte and juxtaglomerular changes in adrenal regeneration hypertension. Am. J. Physiol. 206, 93-104. Roat~aTgm 3. D. (1953) Further studies on ionic regulation in marine invertebrates. J. exp. Biol. 30, 277-296. SCIIt)FFIi'.NIEI.SE. (1967) Celhdar Aspects ~" Men#brttne Per. meabilit.v. Perg~unon Pres~ New York. S~Mtm S. E., MULLt.~ J. & Dt:.WHURSTD. J. (1964) Ionic partition in holothuroidean muscle. J. cell. comp. PhysioL 63, 77--84. SKAt.;R H. LE B. (197,1) The water balance of a serpulid ~,,lyehaete, Mercierelkt eni.qmativa (Fauvel)--l. Osmotic :oncentration and volume regulation../, exp. Bhd. 60, 32 b-330. SOKAL R. R. & ROIILF F. J. (1969) Biometr); pp. 204--252. Freeman, San Francisco. TASKF.R P., SIMON S. E., JOIINSTONE B. M.. ~rtANKI.EY K. .I. & StrAW F. H~ (1959) The dimensions of the extracellubr space in muscle, d. ~]en. Phy.~iol. 43, 39-53. THoxlAs L. P. (1961) Distribution ~tnd gdinity tolerance in the amphiurid brittlestar OI,hiophra~lmJls filo~lrant't~s (Lyman IN75). B,ll. tour. Sci. Gulf Carihb. I!, 158-.160. ZaNt~l:.as I. P. & Hl'.'ggrRA F. C. (1974) Ionic distribution and fluxes in holothurian tissues. Comp. Bitx'hem. Physiol. 47A, 1153--1170. ZENKEVITCHL. A. (1959) Biolo~ly nf the Seas of lice U.S.S.R. George Allen & Unwin. London.
Pht.~+ol+ 1~7~. h d
54A, /+p Its7 r,J 174 Pt-r+l,tm,+a I+r~,s.+ Pri~tt++d i+t Gr+.,+t tiritail+
C H A N G E S IN C O E L O M I C F L U I D A N D I N T R A C E L L U L A R I O N I C C O M P O S I T I O N IN H O L O T H U R I A N S E X P O S E D T O D I V E R S E SEA WATER CONCENTRATIONS ERNESTO MADRID, I. PERCY ZANDERS AND FRANCL~O C. HERRERA Laboratorio de Eeofisiolo~a Animal, Centro de ~ioffsica y Bioquimica, instituto Venezolmao de lnvestigaciones Cientflicas, IVIC, Apdo. 1-827, Caracas, Venezuela
(Receiced 21 Alu.lust 1975) Abstract--I. Ionic coaceqtrations of coelomic fluid of l.w)s(ichol,US badionotu.~ equilib|a!cd with 80 and 120'!-~;sea water in 4-6hr. 2. With increasing external medit, m concentration, intestinal albumin space remained constalll whereas intracellular space decreased; in muscle total water and ialracellular space decreased a n d extraeellular space increased. 3. Non-albumin space ionic content per unit dry weight, with the exception of intestinal sodium, remains practically unchanged in intestine and muscle in passing from 80 to 120'.'¢~sea water. 4. lntracel|ular ionic concentrations rise proportionately to that of external medium. Adjustment of intracellular osmolality is due mainly to water movements into and out of the cells. INTRODUCTION tonic or hypotonic sea water and the variations of intraceltular electrolytes in intestine a n f f longitudinal THE ECHINODERMS and amongst them the holothurmuscle bands of the body wall caused by clmnges tans, are of exclusively marine habitat and the Phyin the concentration .of the medium were studied in lum is mainly c o m p o s e d o f stenohaline representatile holothurian Iso.stichopus badionotus. tives. Nevertheless, many echinoderms can tolerate Small changes in salinity of ambient sea water. In the last 20 years, echinoderm species living in waters differing widely in salinity from that 0 f s ~ v~atcr (35%3 MATERIALS AND METHODS have been reported. Thus, T h o m a s (1961) found Ophiofi'a.qmus filograneozls living in water of 7",,,, The animals used in the present experiments were colsalinity. Zenkevitch (1959) reported Cuctonaria oftenlected in Santa Fe Bay in the Eastern part of Venezuela. tails. Synapla hispida and Lxlhhlophtx diqitata in They were kept in reeirculated, filtered sea water. Salinity, pH and temperature were controlled and water losses comwaters o f 18",, salinity in the Black Sea. O n the other pensated by the addition of distilled water. The specimens hand. species tolerating" 46",,, salinity have been weighed between 500 and 700 g and they varied in length reported from the Red Sea (Binyon, 196I). Therefore. between 25 and 30 cm. The dissection of the intestine and although individual species of this Phylum m a y be longitudinal muscular bands of the body wall was perstenohaline, different species inhabiting a wide range formed as described previously (Zanders & Herrera, 1974). of salinities have been reported. In order to study the time course "of equilibration of The limited osmoregu latory powers of holothurians the coelomic fluid with the hypertonic or hypolonic exterhave confined them to the sea (Krogh, 1965). T h e nal media, the animals were exposed to 80, 100 and 120~ composition of their perivisceral or c o e l o m i c fluid is sea water for 24 hr. Samples of coelomic fluid were taken at hourly intervals during the first 8hr and subsequently quite close to that of the external m e d i u m (Zanders at I0, 12 and 24 hr of exposure by means of a hypodermic & Herrera, 1974). Q u i n t o n (1900) found that the syringe. Hypotonie sea water was prepared by diluting chloride concentration o f the perivisceral fluid of natural sea water with distilled water. Hypertonic sea Asterias rubens decreased with that of the external water was prepared by evaporating natural sea water to medium. Koizumi (1932) found that tiffs equilibrium 5/6 its initial volume. Concentrations differing only 20~,, was reached in a matter o f hours. T h e changes occurfrom the normal concentration were used in an effort to ring at the intraceltular level in response to modificaminimize possible damage to the animals. The sea waters tions of the concentration o f the external medium used and the coelomic fluid were analyzed for sodium, have not been extensively studied. Schoffeniels (1967) potassium, calcium and magnesium by flame and atomic absorption spectrophotometry (EEL 100 flame phot+ometer has shown in marine invertebrates that the total intraand Varian AA5 atomic absorption spectrophotometer). cellular inorganic electrolyte concentration is lower Chloride was determined amperometrically on a Buehlerthan that of the extracellular medium, osmotic equalCotlove Chloridometer. Osmolalities of sea waters and ity being maintained by the presence of small organic coelomic fluids were determined by freezing point depresmolecules (mainly a m i n o acids) in the intracellular sion on a Fiske osmometer (Model G-66). medium. The behavior of the intracellular inorganic electrolytes in relation to changes in the ionic concenTissue ionic content tration o f the external m e d i u m has been little studied Tissue sodium, potassium, chloride, magnesium and calin echinoderms. In the present paper, the time course cium content, total tissue water and extracellular space were determined on the intermediate intestine (Zone 5', o f equilibration o f the perivisceral fluid with hyper167
|-:,RNILSIO ~ l A l ) l , t l l ) t't ill.
IhN
Zanders & |lerrera, 1974) and muscle tissue of holothurhtns exposed for t 0 h r to 80, I 0 0 and 120'!;, sea water. This f~criod was stlllicicn! to allow cquilihralion o:f the c~)ehtmic Iluid with the snrronnding sea wilier. The tissues a|~d coelomic Iluitl were removed from the animals and the intestine and muscle were ineuhated in their respective coelomic fluid to which I"~t-lahclled human serum ;tlburain JR111SAh ohlidned from the Radiochemical C'entre. Arnersham, had hcen added. The tissues were further incubated during 3,5 hr in this medium Io :dlow the alhumin t~: equilibrate with the tissue space accessible to it, At the end of this period, the tissues were carefully blotted between two pieces of Whalman No. 54 filter paper and weighed to determine wcl weight. They were subsequcnlly dried ,,~vernight at 1(15 C and reweighed to determine dry weight. Tissue w:|ter was taken.as the difference between wet and th T weight. RIIISA space was dclermined by counting the dry tissue residues, along with .'|liquors of the bathing coelomi¢ Iluid. in a gamma spectrometer. The dry residues were subsequently extracted with 1 N nitric acid during 48 hr for eleclrolyle determination. Intracellular electrolytes were estirnaled by sublracting from lolat tissue electrolytes the contribution made by those in the extraeelh|lar fluid ~Zanders & Herrera, 1974). Calcium and magnesium, being rhoslly bound tap i~t the structure of the spicules, were no! includ¢
Stdtisth'~l tm.t~lmc.nt The signilic;mce of the dill;creates between the means of the dah~ at the 0.05 level of probabilily was (letermined by applyiug analysis of variance and the Student-Newmun-Kculs procedure tSoknl & Rohlf. 1969l. Least significant ranges were thel~ calculated to test for signilic'mce between the means of the results obtained in the tissues exposed to the different sczt walcr concentrations. "Fhc'orcth'al cuh'itkttion ty" the" time cour,~c" ~{/"eclldlihr~ttio~l *tf tire coehm~ic fltdd In order to arrive at a simple expression h~r the time cot,rse of equilibration of the coelomic fluid with the surrounding, sea water, several assumptions should be made: (1) the permeability of the body wall to ionic and water movement should be constanl; (2) the concentrations have been ttlken as equal to the activities of tile different ions; [3) the volume of the surrounding fluid should be snlticiently large so tlu*t the concentrations of the different ions remilin practically invariable despite exch~mge of'water and electrolytes belween the :mimals nnd the surrotmding sea
waler and (41 the volume anti composition o f the eoelomie fl~id arc not actively modified by lhe animals. T h e s e assumptions lead to ;he following expression for the rate of change of the difference of coneeatration.~ of a given ion between the coclotnic fluid and the external sea water as a function of time d f A C I/dt = -. k(ACI (II
~c=c-
c.
where C is tile conce/llration of a given ion in the coelomic fluid at any instant: C,. is the concentration of this ion in the surrounding sea water: k is the rate constant describing the appro.'|eh to equilibrium of the ioni¢coneentration in thc coelomie fluid with the external medium which may be estimated from the integrated form of equation (!): k = (D/fin(C,, -- Cm)..'(C -- Cm) where C,, is the concentration of the ion in the coelomie fluid at t = 0. When C,. > C. this expression yields C = C,,, - (C,,, - C,,)exp -- kt (2) When C'~, < C,, C = C~ + (C,. - C . l e x p - kt
R ESU LTS
O,smolali O" a n d Cmnl, o s i t h m o f t l w sea w a t e r x a m l c o e l o mh" Jhdd.~ T h e c o m p o s i t i o n o f the sea w a t e r s used, n o m i n a l l y 80, 100 a n d 120')i. a n d o f the c o e l o m i c fluids o f anim a l s e x p o s e d 1 0 h r to t h e m are s h o w n in T a b l e 1. T h e c o m p o s i t i o n o f n a t u r a l sen w a t e r a c c o r d i n g t o Barnes (1959} a n d SiliCa (196l) is a l s o i n c l u d e d for reference. Tht~ small dc;,,iations f r o m the e x p e c t e d values o b t a i n e d in thc sea w a t e r s "used for the experi m e n t s m a y be due to the fact that the s a m p l e s o f sen wate¥ were t a k e n at the end o f t h e 1 0 h r p e r i o d of e x p o s u r e o f the a n i m a l s which p r e c e d e d the r e m o val of the intestinal a n d muscle lissues. T h e c o n c e n " I r a | i o n s o f the different ions in the n a t u r a l sea w a t e r used in the p r e s c n l e x p e r i m e n t s a r c slightly higher t h a n t h o s e r e p o r t e d by B a r n e s (1959) a n d Sill6n (1961). T h e c o n c e n t r a t i o n s o f the different i o n s in the c o c l o m i c fluids c o r r e s p o n d within the e x p e r i m e n t a l e r r o r to t h o s e o f the c o r r e s p o n d i n g sea water. T h u s .
Table I. Compositipn and osmolality of 80, 10<3 and 120';b sea waters and of the coelomic fluids of holothnrians exposed 10 hr to them 80~
t20¢
I00~
Sea ~ater
~ater
sea ~ater
Coel om|c Fluid
(6)
I7)
(6)
t7)
{6)
h06,9
409.7
514.1
*_s.~
._10,2
~9~.3
600~6 +6.7
*_11,1
Potass t um
8.6 +o. I
8.9 *o. I
11-3 t_0.1
11-1. +.p. +
13-6 +o. 2
13,2 +_o. 1
C~nloo ,-|de
t,~2°3
+~.~
k53.3 +5,7
$6~.7 *_9.1
594.0 +_.e.t~
712.9 +s.~
717.7 *6.8
5~8. jl
8.2 +_o.2
8.t~
10.7
10.9
|].1
13,1
10.2
10.8
,_o.z
+o. 2
+o.z
+0.z
+o. 2
59.~ +..o,~
58.9 +_o,s
71.9
70,!
53.57
50.5
+~.3
-._o.s
~e~ Veter
(7) Sodium
Se~
(ea~nes. 19~91
Coelo~|c F l u i d ~ ! le~sfs (i
CoelOrnt¢ rluld
co~1om{c Fluid
S|lt~n~ 1961)
Calcium
• 6.~
+84
t~mae-
s~6.~
47.5
,i,~
+._o.z
+o.9
0 ~,~',o-
855
B73
1137
1135
138;5
571.5
47G.2 9.96
460 11 J3 523
1382
~|eetfoly~e concentration i~ m M l l ° O s m o | a l i t y i n mOsm/K 9. Each f i g u r e represent~ t h e m e a n ~ S . E ° Number OF d e t e r m i n a t i o n s in p~rentheses.
Ionic changes in holothurian tissues
169 POTASSIUM
SODIUM
6600 00
_
.
T ;L~
,
-~.
.J
,
-
I
"
13
J J--
I
}
~
'
}
~
"
6
600
5
0
400 ~
0
t o~
L
"
z e
io
=
~
..'
'~
1
1
T
T
T
~
,
t
i20%
o
~
"
t 8'o'/. z4.
80 %
7 O
,z
. t~__
120%
2
4
G
63
~O
12
24 L
%
~ o -
~
'
~---
80%
Fig. t, The time-course of equilibration of the concentrations of sodium, potassiutn and chloride in the coelomie fluid with their respective concentrations in the exlernal medium. Each point represents a mean value from at least three animals: where indicated, the sizes of the vertical bars represent + one S,E. For further details see text. after l O h r of exposure, the c o e l o m i c fluid of the exp e r i m e n t a l a n i m a l s has practically e q u i l i b r a t e d with the s u r r o u n d i n g sea water. The c o m p o s i t i o n of the c o e l o m i c fluid of Cm,lintt chilensis r e p o r t e d by K o i z u m i (1935b) is included for c o m p a r i s o n . T h e time course o f the e q u i l i b r a t i o n o f the c o n c e n trations o f sodium, p o t a s s i u m and c h l o r i d e in the e o e l o m i c fluid with those o f the anabient sea water ark s h o w n in Fig. I. T h e rate coefficients, k ( r a i n - l ) , were calculated from the d a t a using e q u a t i o n s 3 and 4 and the calculated curves have been fitted to the points. T h e rate coefficients are all o f the o r d e r of 0.01 rain-~ and are a p p r o x i m a t e l y equal in animals exposed to h y p o t o n i e o r h y p e r t o n i c solutions. In anim a l s m a i n t a i n e d in I00% sea water, little o r no c h a n g e was seen in the sodium, p o t a s s i u m and c h i e f -
ide c o n c e n t r a t i o n s o f the c o e l o m i c fluid. It m a y be seen from the curves that after 6 hr the ionic conce~trations o f tile e o e l o m i c fluid c h a n g e very little. It will bc c o n s i d e r e d hereafter that the ionic c o m p o s i lion o f the c o e l o m i c lluid a n d the respective a m b i e n t sea w a t e r arc the s a m e and the results will be referred to in terms o f the ambient sea w a t e r e m p l o y c d . Cha/tges #~ tissue water T a b l e 2 s h o w s the effects o f exposure to the different a m b i e n t m e d i a (i.e. c o e l o m i c fluid o f animals equilibrated with 80, 100 a n d t 2 0 % sea water) on total tissue water, extraoellular space and intracellular water expressed in m l / g dry wt. T h e Student--Newm a n - K e u l s p r o c e d u r e was used to test for significance
Table 2, Tissue water Ambient 80~; Dlff,
I000
t'ledium Oiff.
120~
DiFf. 80-120g
$,t. Gr,~p flean
L,S,R, P~O 05K=3 (@) K=2
INTESTINE Te .~ Wate
5.32
0-39
~+93
0.27
~.66
0,66
0.23
0.68
0.8~
AlbL Sp~
b4
0.19
1.o3
O.tO
l.i3
0.29
o,lo
0.30
o.3&
.55
3.93
0.~0
3,53
0~5"
0.26
0.77
0,9~
0.64*
4.10
0.4)~
3.67
1.07 ~
0.05
0,16
0,I9
MU-. Tota I Water
~. 7h
A I btamlr~ Space
0.75
0.OZ
0.77
0.27.+
1.02
0,29~
O,O~
0.2t~
0,29
Intra¢:el l u l a r Space
3.9 8
0-654
).33
0.70 ~
2,6)
1.35~
0.0~
0.26
0.32
Water c~ntent and spaces in mIJg dry wet hi (1)Difference) b e t ~ e n consecut|ve ~ I n $ ~((] be )ign(61c)nt(~)at or bclo~ th~ 0 * 0 ) l e v e l o f p r o b a b i l i t y I f they a r e g r e a t e r than L.S.R.k~ 2 D i f f e r e n c e s b e t ~ e n means o f r e s u l t s in 804 and 120t amb|e~t m e d | ~ Will be s i g n i f i c a n t a t t h i s l e v e l i f they are g r e a [ e r than L.S,R.Rw 3
170
ERsF,sro ~vJAI)RII) et al. Table 3. Total tissue sodium, potassium and chloride content referred to total tissue water hmb 80~
i ent
~e
Diff.
IOO~
Cl t ur,~
Dtff,
120¢
Oi f f , 80-120~
S.E, Group P~e~n
Z38,E*
~5.5
L.S.R. , P-O.O5 k'2 k" 3 I t )
IHTESTtNE Sodium Potassium Chloride
3|2.~
IO5.5
~17,9
133.3
551,2
87,5
5.2
82.3
8,5
~0.8
3,3
~.'8
135.0 16~.0 17,3
21.0
368.7
120.9"
429.6
1OI.9"
531,5
222 B~
1~.5
~3.1
52.3
~9.5"
37.5
HUSCLE Sodium
|83.3
65.3*
2~8.6
8~,3 ~
332,9
|0,~
30,8
Potassium
133,3
26,8*
160.1
27,~ ~
187,~
5~.2"
5.2
tS,~
18,7
Chloride
2OG,~
89,3*
289,~
~9.3"
~S9,2
158.6"
10,1
29.9
36,k
[]ectro|yte
cont¢~t
l~ t.M/~ t i s s u e water
( ~ ) r o r e ~ p l a n a t l o n 0r s t a t i s t i c a l
tre~t~nt
between the means. In the intestine no significant difference could be observed in these parameters with the exception of the difference between the intr,'tcellular • spaces of tissues exposed to 80 and 120"~; sea water. In muscle, almost all changes arc significant with the exception of the change in extracellular space between 80 and 10CI"{, sea water. I1 may be observed in this tissue that with increasing ambient sea water concentration, total tissue water and intracellular space decrease, whereas a l b u m i n space increases slightly. Analysis of variance of the intracellular spaces in muscle indicated that the dil]~rences were highly signific~mt statistically and that the variation ~wnongst them could be due to a linear dependence of the intracellular space on sea water concentration. Therefore, an attempt was made to tit the data to a straight line relating intracellular space to the a m b i e n t sea water (or coelomic lluidl concentration. A very ~ t i s -
see Table 2
fitctory fit, over the range studied, was obtained for the line described by the equation IW --- 6.70 - (0.03382 __, 0.O0080)SW where IW is the intraccllular water in ml/g dr3' wt and SW the concentration of the sea water in per cent. The 95';,~, confidence limits for the regression coefficient are 0,04402 and 0.02362. It must be emphasized that this is an empirical line and that the intercept 6.70ml/g dry wt is probably meaningless. The decrease in total tissue water in muscle with increasing sea water concentration is completely accounted for by the decrease in intracellular water. Ti,ssue e l e c t r o l y t e content
Total tissue sodium, potassium a n d tent expressed in ima/ml tissue water Table 3. In the intestine tissue chloride increasing sea water concentration a n d
T~lblc 4. Total tissue electrolyte content expressed in terms of dry weight A~b
i e~
t
H e d | ur~
Dill, ~0-~ ZO~
S.E. group Hean
L.S.R P'O*05 k~2 k'~ it)
~O~
Diff.
lO0~
Diff.
|20~
1662
377*
2039
509~
25~8
8BS*
92
273
~3
4~
399
19
~18
2~
22
G6
8!
Ch|or~de
165,
~2'
2094
387*
2481
829*
12h
369
~8
Calclu~
1035
83
1118
167
1285
250
1~5
43T
523
3~|
56
~17
77
~94
13~*
29
B5
103
129!
Z38
I~TESTINE Sodium Potassium
~gnes|um
331
MUSCLE Sodlum
86~
150
t01~
272 ~
422*
66
196
Potesslu~
630
26
65G
29
685
55
29
S&
69
Chloride
952
237*
1189
129
1318
366 ~
~7
1~0
170
Calcium
67
16
5!
5
56
t0
6
18
21
~gnesium
98
19
1t7
&
123
25
9
27
33
E | e c t r 0 1 y t e c o n t e n t expressed | n ~HI 9 d r y w e i g h t (t)
For e x p l a n a t | o n a f s t a t i s t i c a l
t r e a t m e n t see Table 2
chloride conare shown in increases with tissue sodium
Ionic changes in hololhurian tissues
171
Table 5. Intracellular sodium, potassium and chloride contenl expressed in terms of dry tissue weight Arab I e n t /4e d t u m ~Ol I) I f f , t0091 r,I f f . 1 2 0 ~ , 80_ D i f120~. f. G.E, Group I~ean
L.S.g* P~O,05 k-2 k-3 (~)
INTESTINE
$od~um
1318
221
t53~
1896
578 ~
lOB
320
~35
39
396
7
~O3
32
22
65
79
1272
184
1~56
Zl?
1673
601
1E0
416
505
~SO
Potassium
Chloride
3~ ~
389
M~SCLE Sodl~
560
77
G37
56
~93
13~
SO
I~9
Potassium
fi23
25
6~8
2~
672
~9
18
53
65
Chloride
61~
12~
738
;Oh
63~
20
SO
1~9
181
Electrolytes e ~ p r e s s e d i n ~M/g d r y w e i g h t (t)
For explanation Of s t a t i s t i c a l
treatment
increases when sea water is raised from 80 to 120%o. However tissue potassium does not vary significantly. In the case o f muscle, all three ions increase with increasing a m b i e n t medium concentration. These changes could be due to three factors: (1) increasing concentration of the extracellular fluid; (2) increased electrolyte content of the intracellular space and (3) loss o f tissue w a t e r from the intracellular a n d extracellular c o m p a r t m e n t s as the concentration of the ambient sea water increases. In order to determine whether there were net gains or losses in tissue electrolyte content, the results were expressed in terms of d r y weight thereby ruling out the effects of the changes in tissue hydration. Nevertheless, changes in tissue ion content caused b y m o d i fications of that o f the extracellular space will still contribute to the changes in tissue ion content. Therefore these data, which are shown in T a b l e 4, show increases in sodium and chloride content of the tissues with increasing sea water c o n c e n t r a t i o n as a result o f possible changes in intracellular and extracellular ion contents. The interest of the present investigation is centered on the changes in the electrolytes of the intracellular c o m p a r t m e n t caused by modification of the a m b i e n t sea water concentration. Therefore, an attempt was m a d e to estimate these quantities by subtracting from the total ion content the electroTable 6. lntracellular sodium, potassium and chloride expressed in terms of intraoellular water 80I
DIff.
100t
Meal DIff.
UI~ 120t
/~rab I en~
S.E. DIff. 80-1201
(;roup Nean
L,S*I~. P~D,95 k-2 ~'3 (~)
INTESTINE Sodium
29k
lOZ*
396
150"
545
ZSI*
2Z
6&
Potassium
97
7
104
Ik
118
21
8
23
28
Chlor|d~
280
98"
37B
B9%
467
187"
19
S6
68
see T a b l e Z
lyte content contributed by the a l b u m i n space (assumed to represent the extraoellular space): Intracellular ion contents were expressed p e r u n i t tissue dry weight in order to detect net changes in non-albumin space ion content and per unit intracellular wa~er to relate them to changes in n o n - a l b u m i n space watei" (taken as intracellular water). Table 5 summarizes intracellular ion. c o n t e n t s L~r unit d r y weight. It m a y be observed that m the intestine the intracellular s o d i u m c o n t e n t changes o n l y when passing from 109. to 120~ sea water. Since there is some question as to the eo(respondence Of extrace-l~ lular s p a c e and R I H S A gpace in this tissue (Zanders & Herrera, 1974)this is not Surprising. In longitutlinal muscle, where R I H S A spac~'seenls to be a better measure o f the extracellular space, sodium, p o t a s s i u m a n d chloride content per tinit d r y weight remain invariable in passing from one.Sea ~vater conceniration to a n o t h e r . T h i s indicates t h a t cell electrolyte content is independent of e x t r a c e l l u l a r c o n c e n t r a t i o n over the range studied. However, if the i n t r a c e l t u l a r ionic contents are expressed in terms o f intracellular water, as is done in T a b l e 6, a clear cut increase in ~ntracellular ionic concentration with increasing extracellular concentration is observed, specially in muscle. Since intracellular water decreases with increasing sea water concentration, increased intracellular ion concentration is d u e primarily to the toss of intracellular water rather than to any intracetlular ionic accumulation. Tissue calcium and magnesium content being closely related to spicule c o n t e n t were not studied systematically and are only shown for referehce in T a b l e 4.
BO
DISCUSSION
Electrolyte concentration of the coelomic fluid The coelomic fluid o f Isostichopus badionotus SQdlu~
140
57"
197
43*
Z40
I00"
12
37
qk
Potess]um
IS7
3B*
195
68*
262
106"
t0
3t
4S
Chloride
15Z
fi6 ¢
~lB
24
2kZ
90*
I!
3J
3~
E l e ~ t r o | y t ~ s e~re~$ed In t l n a s Of I~l./g I . t r Q c ~ 1 | ~ l | r ~ t t r
(~) F o r explanation o r s t a t l l t l ~ l
lreatmmRt see Table 2
rapidly a p p r o a c h e s the c o m p o s i t i o n and osmolality of the external m e d i u m when the latter is concentrated or diluted. This h a d been observed in other species of echinoderms by Henri & Lalou (1903); G a r r e y (1904) at the beginning of this century and m o r e recently by K o i z u m i (1932, 1935a, b). It has been postulated
172
EaNl:S-lo M^i)l,liD el at.
that this exchange takes place through the body wall and respiratory trees (Berger & Bethe, 1931; Delaunay, 1931; Koizumi, 1932, t935a; Robertson, 1953; Ahearn, 1968). More recently, Lawrence et al. (1967); Zanders & Herrera (1974) observed that the intestine may play tin important role as a site of high ionic permeability through which equilibration of the coelomie fluid with the external medium could take place. Nevertheless, the time course of equilibration has not been extensively studied. Botazzi (1906) observed that after 24 hr the freezing point depression and conductivity of c o d o m i c fluid were identical to those of the external sea water which had a freezing point of - 2 . 6 5 " C , approximately the same as that of the hypertonic sea water used in the present investigation. Dakin {190g) using hypotonic a n d hypertonic sea waiters with freezing point depressions of 0.76 and 2.98 respectively tried to measure the time of eqvilibration in A,~teria rubens and Echintts e.~euIcntus The time o f immersion (3.5 hr) was insulticient Ibr the freezing point depression of the pcrivisceral Ituid to reach that of the sea water. If immersion time wa,s increased the animals died before reaching cquilibrit,m, This is not surprising since these concentrations are far removed from the normal sea water concentration, corresponding to 36 and 142','., sea water. In order to minimize damage ttp the animals, tile hypotonic and hyperto,aic concentrations used in the present investigation were 855 a n d 1385 mOsm/kg, nominally 80 and 120';,,, sea water, ,'espectively. This allowed incubation periods of up to 24 hr without, obvious deterioration of the animals. The results reported by Koizumi ( 19321 working with (~tudina chih,lisis arc close to those found in the present in vcsiiga t ion. intracelhskn" spat'c, ~dlmmin sp~tce and tisstR, w~tter
Several organic molecules have been used to estimate e×tracellular space. Theoretically albumin, mannitol, rallinose and sorbitol should give a g o o d measure of this space because the large size o f their molecules would exclude them from the ceils. However, extraccilukir s p a t e could be overestimated if they entered the cells or were metabolized: on the other hand, if regions impermeable to these molecules exist in the extracellular space the latter would be underestimated. Albumin, because of its high molecular weight, is p r o b a b l y excluded from the cells but it may also be excluded from the hemal lacunae which abound in the intestinal tissue (Zanders & Herrera, 1974). Simon et al. (1964) found that h o l o t h u r i a n muscle does not present barriers to albumin diffusion in the extracellular space, albumin being, thus, a g o o d probing molecule for the latter. Inulin has been used also as a m a r k e r for extracellular space. However, inulin appears to enter the cells since inulin space increases with time (Conway, 1957: Tasker et al., 1959; Burnstock et al., 1963; Rapp, 1964). In the present work, the extracellular spaces found are lower than the inulin spaces found by Z a n d e r s & H e r r e r a (1974) in the same tissues of lsostichopus badionotus, This p r o b a b l y explains the lower intracellular potassium concentrations found in the present work since the intracellular spaces would be correspondingly larger. Hagemeijer el" aL (1965) have reviewed the methods for determination o f the extracellular space
and have pointed out the problems and pitfidls involved. The existence o f hemal lacunae, possibly inaccessible to RIHSA, probably hHroduccs a large degrce of uncertainty in thc mcasurcmcnt of tl~e cxtraccllular space of the intestine and consequently the measurement of the intracellular space woukl also be- in doubt. This would seem to bc contirmcd by lhe fact that the changes in tissue water., exlracellular space and intracellular space caused by increasing the concentration o f the sea water are not markedly significant in the intestine, whereas in muscle, where the hemal lacunae ;ire not as developed, ll{e changes determined in these spaces and particularly the mtracellulatr space are quite signiticant. Therefore. it woukt appear that the estimation of the intracellular space in musetc is subject to less uncertainty than in the intestine and furthermore, the fact that the losses in total muscle tissue water are fully accounted for by the losses in intracellular water tends to support this hypothesis, "H~tal tissue electrolyte content
The changes in tissue hydration caused by exposure to hypotonic and hyperlonic media precluded the expression of tissue electrolyte content in terms of tissue waiter as a measure of changes i,a net ionic content.-l'herefore, electrolyte content was referred to tissue dry weight which presumably is ,aot all;acted by changes in tissue hydration. The net changes of sodium and chloride in both intestine and muscle a n d the slight changes in intestinal magnesium, seen in Table 4, p r o b a b l y reflect changes iu the electrolyte content of the extracellular space. This assumption is strengthened by the observation that tissue potassium, which should be mainly intrac'elhllar, and tissue calcium, of which a substantial portion is b o u n d up in the spicules IZanders & Herrera, 1974}, d o not vary with changing sea water concentration. lntroct'lhtklr ionic" cont~'nt
The fraction of total ionic content regarded us i~traceilular d e p e n d s on the correct estimation of the cxtraeellular space. It would a p p e a r that albumin space is a good a p p r o x i m a t i o n of the extracellular space in muscle. However. in the intestine several facts suggest that this is not the ease: (a) n o n - a l b u m i n space in the intestine is at least as large as that o f muscle despite the fact thai the intestine is richer in connective tissue and hemal lacunae than muscle. This sugges~ts that albumin is excluded, in the intestine, from structures other than cells; these structures could ,,,,'ell be the hemal lacunae. Moreoever, the extraceiluktr space o f the intestine is not affected by the changes in concentration of the ambient medium. (b) n o n - a l b u m i n space sodium and chloride expressed both in terms o f dr2," weight a n d o f nonalbumin space water are considerably higher in the intestine than in muscle. This could be the result o f underestimating the extracellular space a n d ascribing the sodium and r t g r i d e c o n t e n t of the hemal lacunae to tt~e cell c o m b - , i m e n t . (c) Furthermore, the low non-albumin ("intracellufar") potassium seen in the intestine could be due to the same type of error. Total tissue sodium and chlor-
173
Ionic changes in holothurian tissues ide are much higher in the intestine than in muscle; this suggests that, if the intracellular sodium and chloride content of the intestine approaches that of the muscle tissue, the true ¢xtracellttkw space of intestine must be considerably higher than the measured albumin space, if an extracellul:lr sp.'lcc o f 3 m l / g dry wt is used to calcuktte the intraceltukar sodium, potassium and chloride concentrations of the intestine exposed to 100",, sea water the following values are obtained: sodium, 2 5 8 / t M / g intracellular water; potassium, 189/tM/g intracellular water and chloride, 199ttM/g intracelluhtr water. These results are quite close to those obtained in muscle. M o r e o e v e r an extracellular space of 3 ml/g dry wt corresponds to 61% of tissue water which agrees quite closely with inulin s p a ~ of intestine determined in a previous work (Zanders & Herrera, 1974). Despite the fact that extracelhdar space is underestimated in the intestine, the changes of ionic content in this space accounts for a large enough fraction of the changes in total ionic content to retader the changes of sodium and chloride content of the non-albumin space statistically non-significant. It is clear in muscle and less so in intestine thal the intracellular space loses water as the external concentration is raised from 80 through 100 to 120. T h u s intraeelluktr ionic concentration is raised with increasing external osmolality since the intracelluktr ionic content, in terms of dry weight, remains unchanged. This suggests that the adjustments of intraccllular osmolality are due mainly to water m o v e m e n t s in both tissues. Moreover. the changes of the concentrations of sodium, potassium and chloride of the muscle fibers is in good agreement whith those of the external medium: the intracellular concentrations o f the three monovaler~t ions is 1.5-1.6 times higher in tissues exposed to 120'?.o sea water than in those exposed to 80~,; sea water which corresponds to the ratio of the external concentrations. Thus the cells appear to maintain their ionic content within more or less n a r r o w limits. Muscles of certain cruslacea such as Ctdlinectes s a p i d , s when placed in hypotonic saline increase in volume: however, with time they decrease spontaneously towards their initial volume. W h e n returned to isotonic salines the muscles shrink below their initial volume {Lang & Gainer, 1969a). These responses of C. saphh~s muscle have been referred to as volume readjustment. This p h e n o m e n o n is reminiscent of the volume regulation seen in some annelid polychaetes (Skaer, 1974). However, the mechanisms are different in the two cases. In C. sapidus the secondary decrease in volume in hypotonic solutions is associated with a loss o f free intracellular a m i n o acids (Lang & Gainer, 1969b) whereas in polychaetes it has been ascribed to passive exchanges o f salts between the animal and its environment (Skaer, 1974). In lsostichopus b a d i o n o m s no volume readjustment was detected in muscle and intestine subjected to h y p o t o n i c solutions; intracellular water increased in proportion to the decreased external osmotic pressure showing no return to initial volumes. T h e lack of volume regulation in holothurian tissues, associated to the constancy o f the intracellular ionic c o m p o s i t i o n (referred to dry tissue residue), suggests that changes in intracellular free a m i n o acid pool are not involved in the adaptation o f the intracellular milieu to de-
creased osmolarity of the external mcdiun~. Luidia a euryhaline echinoderm, responds to reduced salinitics by isosmotie intracellu lar regulation and coelomie fluid reguhttion, After one day's exposure to hyposmotic sea water, ninhydrin positive substances of podia and pyloric caeca were lower than in tissues of c o n t r o l animals (Ellington & Lawrence, 1974). These results are in contrast with the reported lack o f c o e l o m i c fluid volume regulation in Stronayh)centrottts purpttrattts (Giese & F a r m a n f a n n a i a n , 1963) and in a stenohaline population of Asterias rubens (Binyon, 1961). lsostichoptts t)adionotus would therefore appear to behave as other stenohaline echinoderms. c'htthrata,
Ackm)wlcdttemems-This investigation has been supported in part by Grant No. DF-5il-047 from Cons~io National dc lnvcstigacioncs Cicntificas y Tccnol6gicas.
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