Invertebrate Cell Volume Control Mechanisms

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Reference: Biol. BulL 163: 405—419.(December 1982)
INVERTEBRATE CELL VOLUME CONTROL MECHANISMS: A
COORDINATED USE OF INTRACELLULAR AMINO ACIDS
AND INORGANIC IONS AS OSMOTIC SOLUTE
SIDNEY K. PIERCE
Department ofZoology, University ofMaryland, College Park, MD 20742,
and Marine Biological Laboratory, Woods Hole, MA 02543
ABSTRACT
All cells have some capacity for cell volume regulation when confronted with
a hypoosmotic stress. The basis of this physiological response is an extrusion of
intracellular osmotic solute. The cells of euryhaline osmoconforming invertebrates
are capable of regulating volume over a wide range of external osmotic concentra
tions. Most of the existing data indicate that these cells utilize free amino acids from
a substantial intracellular pool as the solute source. However, recent studies indicate
that these invertebrate cells utilize inorganic ions as osmotic solute as well. The
relative contribution of each solute type varies from species to species and, perhaps,
from cell type to cell type. The two solute types are regulated by different mechanisms
and often with different time courses, but both solute control systems function in
a coordinated manner to regulate cell volume. In addition, evidence is appearing
demonstrating a role for organic solutes in the volume regulatory processes of ver
tebrate cells. At present, it seems that the volume regulatory mechanisms utilized
by all cells may be more similar than currently thought, differing in relative con
tributions of the two solute types rather than kind of solute utilized.
INTRODUCTION
The literature reporting on studies of osmotic control amongst the invertebrates
is vast. The water balance mechanisms utilized by invertebrate species have been
under some form of investigation for all of this century and even earlier. In company
with most trends in research of biological function, water balance studies have
proceeded from analysis of whole organism responses to the intricacies of cellular
physiology. The studies cited in the following pages do not constitute an encyclopedic
review, but rather point out some of the more recently discovered features of in
vertebrate cellular water balance systems and, where there are some data, the sim
ilarities of the cellular osmotic control mechanisms between species, the presence
or absence of a backbone notwithstanding. My remarks here are confined only to
the responses to hypoosmotic stress since that has been the most intensely studied.
Whole animal responses —¿�some generalities
It is now apparent that few, if any, invertebrates are isosmotic with their envi
ronment. Even the body fluids of marine osmoconformers are slightly hyperosmotic
to their environment (Remmert, 1969; Pierce, 1970; Oglesby, 1978, 1981, for ex
amples). Thus, most, if not all, animals have an osmotic gradient between the
environment and the extracellular fluid and some physiological capacity for handling
Received 9 July 1982; accepted 24 September 1982.
405
406 SIDNEY K. PIERCE
the water movement resulting from that gradient. This form of osmotic stress is
usually a modest one in an osmoconformer and, as Oglesby points out in his review
(1981), easily handled by excretory systems. The osmoregulators may have much
larger osmotic gradients between extracellular fluids and the environment, but in
the adapted state the water movements are dealt with by appropriate ionic transport
systems of excretory systems, gills, guts, and integument. Of greater physiological
consequence are changes in the osmotic concentration of the external environment,
for example an alteration in salinity, or the occurrence of some pathology causing
malfunction of the extracellular osmotic and ionic homeostatic mechanisms. The
physiological response by invertebrates to salinity change has been studied in great
detail. Pathological studies have been done only in higher vertebrates (see Pollock
and Arieff, 1980, for a review) for the most part, and that aspect will be touched
on only briefly below.
No animal appropriately tested behaves like a piece of dialysis tubing when
exposed to a hypoosmotic stress. The capacity for volume control under such a
stress may be limited and the range of tolerated osmotic concentrations narrow, but
some volume regulatory capacity is present nonetheless. Osmoconforming animals
rapidly swell in response to the osmotic influx of water produced by the hypoosmotic
20
00
90
0 2 4 6 8 0@ 24
Time (IT)
FIGURE 1. The pattern of whole animal volume regulation in Elysia chlorotica. At time 0, animals
acclimatedto 100%sea waterweretransferredto the salinitiesindicatedand weighedat intervalsfor 3
days.Eachpoint is a meanfrom6 animals.ErrorbarsindicateS.E.M.(FromPierceet al., 1982).
I
0
.@
280
240
a @ssw
•¿�25%sw
•¿�50%sw
•¿�Th%sw
220
200
80
36 48 72
160
40
407INVERTEBRATECELL VOLUME CONTROL
stress, but with time in the reduced salinity will at least partially recover the original
volume (Fig. 1). The recovery time course varies from species to species. In general,
the more euryhaline an animal the more rapid the recovery. Osmoregulators may
show a similar response or may simply swell less than predicted (see Oglesby, 1981,
for a thorough review). These whole animal responses are the result of water balance
mechanisms which function at two levels within an organism. First, the extracellular
systems mentioned above, bulk movement of extracellular water by the excretory
system, ion transport by various epithelia, and integumental water permeability all
function in some combination to remove the excess water (again, see Oglesby, 1981).
In addition, the osmotic influx of water into the extracellular compartments results,
perforce, in a dilution of the extracellular environment. This dilution places an
osmotic stress on the cells. Thus, although it can not be distinguished by whole
anmal measurements, the second level of response is at each cell.
All cells tested to date have some volume regulatory ability. Like the whole
animal, when the isolated cell is exposed to a hypoosmotic stress, it swells. With
time in the reduced osmotic concentration the cell returns toward its original volume
(Fig. 2a, b). Few cells, if any, are able to recover the exact original volume. Rather,
an incomplete volume regulation is the rule. The cells of euryhaline osmoconforming
invertebrates, often naturally exposed several times daily to wide and rapid osmotic
fluctuations, are excellent volume regulators, but the cells from invertebrate species
have no monopoly on this response. Vertebrate cells also regulate volume albeit
usually over comparatively narrow ranges of osmotic concentration (mammalian
o 30 60 90 20
Time(min) Time(min)
FIGURE 2. (A) Pattern of volume regulation by red blood cells isolated from Noetia ponderosa
adapted to full strength sea water and exposed to 50% sea water at time 0. Cell volume was determined
as packed cell hematocrits. (Data from Amende and Pierce, 1980). (B) Pattern of volume regulation by
red coelomocytesisolatedfrom Glyceradibranchiazaadaptedto 996 mosm and then exposedto the
osmotic concentration indicated at time 0. Cell volume was measured with a Coulter counter (Data from
Costa et aL, 1980).
996W592mosm
996 WS96mosm@
5,
E
a,
a,
C
0
-C
C)
a,
E
0
>
a,
0
50
I40@
4,
E
0
>
= 30-
4,
C)
0
C
0
o 120'
I I0-@
I00@
A B
0 0 20 30 40 50 60 20 30
408 SIDNEYK. PIERCE
and avian red blood cells [Kregenow, 197 1; Poznansky and Solomon, 1972; Schmidt
and McManus, 1974], Ehrlich ascites cells [Hendil and Hoffman, 1974; Hoffman,
1978], mammalian heart [Thurston et al., 1981], mammalian brain [Pollock and
Arieff, 1980; Thurston et al., 1980], flounder red cells [Fugelli, 1967; Cala, 1977],
Amphiuma blood cells [Cala, 1980], human lymphocytes [Bui and Wiley,1981],
and rat liver [van Rossum and Russo, 1981] have all been looked at in this regard).
No doubt a major reason for the weaker volume regulatory ability of these types
of cells is the evolution of vertebrate homeostatic mechanisms.
The volume regulating capacity of the cells of euryhaline osmoconforming in
vertebrates has attracted some experimental attention from investigators with one
of two points of view over the past decade. First, from the point of view of envi
ronmental physiology, these cells have been utilized in various attempts to under
stand the basis of salinity tolerance. Second, from the standpoint of cellular phys
iology, the mechanisms of cell volume regulation should be more obvious in a cell
type that functions over a wide range of osmotic concentrations since the responses
should be magnified. The utility of this last approach is only of value if all cells use
a mechanism of hydration control in common, at least in its general characteristics.
As the studies of invertebrate cell volume regulation unfolded, similarities with
vertebrate cell mechanisms were indeed found, but also some major differences.
More recent data indicate that the mechanisms may be more similar than we first
thought.
Invertebrate cell volume regulation in response to hypoosmotic stress —¿�the early
results
The general features of cell volume regulation are the same regardless of the
specific source of the cell type. A reduction in external osmotic concentration pro
duces cellular swelling due to osmotic influx of water. To counter the swelling and
prevent osmotic lysis, the cells expel osmotic solute together with osmotically ob
ligated water and cell volume recovers back toward, but usually not reaching, the
original level. Historically, the osmotic solute source utilized during this process by
marine invertebrate cells was presented as being small organic molecules, usually
free amino acids, occasionally quaternary ammonium compounds. Vertebrate cells
(more likely, terrestrial animals) or cells from freshwater invertebrates, on the other
hand, usually seemed to use inorganic ions as osmotic solute. Indeed there is con
siderable evidence in support of this dichotomy. Each species seems to have its own
unique extracellular osmotic concentration, but in general the bloods of marine
animals are very close to sea water in osmotic concentration (900—1000 mosm),
while the fluids of terrestrial and freshwater animals are much lower in concentra
tion. The osmotic equilibrium between the cells of these organisms and the respective
extracellular fluids is such that the osmotic gradient is minimized (although not
zero). Thus, the cells of marine invertebrates presumably have osmotic concentra
tions approximately that of sea water, while the intracellular osmotic concentrations
of terrestrial and freshwater animals are much lower. The total intracellular inorganic
ion composition of vertebrates seems to account for 60—70%of the intracellular
osmotic concentration (see the reviews of Conway, 1957, and Burton, 1968, and
the data in Prosser, 1973). On the other hand, in marine invertebrates the intra
cellular inorganic ion composition is only slightly higher than vertebrate levels
(again, see Prosser's [1973] tables). Thus, the total inorganic concentration inside
invertebrate cells is much lower than extracellular concentrations. The physiological
reason for this ionic discrepancy is not clear. There seems to be some deleterious
INVERTEBRATECELL VOLUME CONTROL 409
sensitivity of some enzymes to salt concentrations higher than those found inside
cells ofanimals adapted to sea water (Clark and Zounes, 1977; Bowlus and Somero,
1979; Yancey et al., 1982), but the data are limited at present. In any case, the
osmotic differential between the extracellular and intracellular fluids in marine in
vertebrates is made up by intracellular free amino acids. Furthermore, there are
many studies demonstrating that these free amino acids are utilized as osmotic
solute during salinity stress (see the reviews by Holden, 1962 for access to the early
literature, more recently, Gilles, 1978; Pierce and Amende, 1981).
The intracellular amino acid concentration in a euryhaline invertebrate adapted
to sea water can easily be 700—800mM (see for example Pierce, 1971; Costa et al.,
1980). The amino acid pool size alone does not always imply salinity tolerance. For
example, the ascoglossan opthistobranch Elysia chlorotica which has the widest
salinity tolerance yet discovered for an osmoconformer (24—2480mosm) has a tiny
amino acid pool (30 smoles/gm dry wt. in sea water) (Pierce et al., 1982). Still, in
most cell types a specific portion of the amino acid pool size declines drastically
with acclimation to a reduced salinity. The amino acids utilized vary from cell type
to cell type and from species to species but always seem to be some combination
of non-essential amino acids. Glycine, alanine, proline, glutamate, taurine, occa
sionally aspartate and glutamine are the usual amino acids involved (reviewed by
Gilles, 1978; Pierce and Amende, 1981).
Amino acid mediated volume regulatory mechanisms —¿�older studies
Various aspects of amino acid mediated volume regulation have been studied
in a variety of species and cell types. Of these, one of the more persistent investi
gations into the mechanisms involved in the regulatory process has been accom
plished using two molluscan tissues as model systems: the isolated myocardium of
the ribbed mussel, Modiolus demissus, and the red blood cell of the blood clam,
Noetia ponderosa. The results of these investigations have indicated that in response
to low salinity, cell volume regulation is accomplished by an efflux of specific amino
acids from the cell (Pierce and Greenberg, 1972, 1973, 1976; Amende and Pierce,
1980). The entire decrease in intracellular amino acid concentration is accounted
for by the efflux. Thus, there is little, if any, intracellular amino acid catabolism nor
protein synthesis which occurs as part of the volume regulatory event. There is some
evidence that the amino acids may be catabolized after release from the cells. This
is reflected by increases in both blood ammonia concentrations and external am
monia excretion rates (for example Bartberger and Pierce, 1976; Mangum et a!.,
1976) which follow the appearance of a pulse of amino acids following, in turn, an
external salinity decrease (Bartberger and Pierce, 1976). There is also some evidence
that the amino acids once released from the cells are sequestered in blood proteins
for future osmotic uses (Gilles, 1977; Boone and Schoffeniels, 1979; Péqueuxet a!.,
1979) although this may be a phenomenon peculiar to the arthropods.
The amino acid efflux is initiated by the osmotic pressure change rather than
the concomitant external ionic concentration decrease. On the other hand, the re
establishment of normal membrane permeability to amino acids (hence, the mag
nitude and duration of the efflux) is dependent upon external divalent cation con
centrations, ATP concentration, and temperatures and is independent of mono
valent cation concentrations (Pierce and Greenberg, 1973, 1976; Watts and Pierce,
1978a; Amende and Pierce, 1980; Otto and Pierce, l98lb). Thus, both an ionic and
metabolic component of the efflux control mechanism have been demonstrated.
Further, the M. demissus myocardial sarcolemma contains substantial divalent cat
410 SIDNEY K. PIERCE
ion requiring adenosine triphosphatase (ATPase) activity (Watts and Pierce, 1978b).
Inhibition or potentiation ofthis ATPase activity produced a correlative potentiation
or inhibition respectively of the amino acid efflux from the intact heart (Watts and
Pierce, 1978c). These results led to the hypothesis that the physiological basis of cell
volume regulation, and thereby oflow salinity tolerance, in osmoconforming marine
invertebrates rests witha membrane bound divalent ATPase which controls amino
acid permeability over a wide range of external divalent ion concentrations (Pierce
and Greenberg, 1973; Watts and Pierce, 1978c; Amende and Pierce, 1980; Pierce
and Amende, 198 1). Although no other invertebrate cell type had been studied in
this detail up to that point, these results were generally confirmed by others (Gilles
and Péqueux,198 1; Pierce and Amende, 1981). At present there are still no data
establishing cause and effect between the divalent ATPase and amino acid efflux
control, only correlations are established. Furthermore the mechanism of ATPase
action is unknown, but most hypotheses suggest a chemo-mechanical system of
permeability control such as that found by earlier studies of mammalian cells (Wins
and Schoffeniels, 1966; Bowler and Duncan, 1967; Rosenthal et a!., 1970; Palek et
al., 1971; Rorive and Kleinzeller, 1972; Quist and Roufogalis, 1976).
As the evaluation of these ideas for generality began, some important results
appeared. First, a comparison of the two molluscan cellular responses to hypoos
motic stress indeed indicates similar characteristics (ATP and divalent cation re
quirements for example), but also some interesting differences. The amino acid
efflux from the M. demissus myocardial cells involves only certain of the many
available intracellular amino acids. In these cells the permeability change seems to
be quite specific. In contrast, the efflux from the N. ponderosa blood cells is similar
in composition to the intracellular pool. The significance of this difference between
the two species is not clear although it presents the possibility that extremely eu
ryhaline animals (such as M. demissus) are so as a result of a highly selective per
meability system allowing for both specific solute efflux and intracellular solute
conservation. Results with cell types from other euryhaline animals (for example,
red coelomocytes from the polychaete Glycera dibranchiata [Costa et a!., 1980], M.
demissus myocardium [Pierce and Greenberg, 1972], Rangia cuneata myocardium
[Otto and Pierce, 198 la]) indicate a selectively permeable volume control system.
Second, and of greater importance, is that recent studies clearly indicate the in
volvement of inorganic ions in cell volume regulation by invertebrate cell types. In
addition, in some extremely euryhaline species this ionic component plays a major
role in the regulation. The majority of studies which have produced these results
have been done on invertebrate neurons.
In spite of the intensity with which nervous function has been investigated, there
are surprisingly few data on the effects of osmotic variation on neurons. The volume
regulatory response of axons to hypoosmotic stress seems to be quite similar to that
found in other cell types (see above). For example, isolated Callinectes axons swell
during exposure to a hypoosmotic stress and then return toward the initial volume
utilizing a mechanism that requires Ca2@ and ATP (Gerard, 1975). Axons from
other euryhaline crustacea also show similar patterns of volume changes (Gilles,
1973; Kéverset a!., 1979a). Although electrical recordings have not accompanied
the above studies, neuronal cell volume regulation is accompanied by an adaptation
in the electrical properties of the cells. Usually a rapid hyperpolarization of the
membrane followed by a slower depolarization and reduction in excitability occurs
following a hypoosmotic stress (Maia axons [Pichon and Treherne, 1976], Sabella
giant axons [Treherne and Pichon, 1978], Mercierella axons [Benson and Treherne,
1978a, b; Skaer et a!., 1978], Mytilus cerebro-visceral connective [Willmer, 1978],
Mya cell bodies [Beres and Pierce, 1979, 1981]).
INVERTEBRATE CELL VOLUME CONTROL 411
Many ofthese studies were conducted over short time courses. Recordings made
for longer intervals after the salinity decrease indicated that the depolarization and
loss of excitability is transient, the time course depending upon the magnitude of
the salinity change and presumably the time course of volume regulation. For ex
ample, the spontaneous burst frequency, spike pattern within the burst, and resting
potential of follower cells in the isolated Limulus cardiac ganglion all returned to
control levels within 2—3hours following a salinity change from 100% sea water
(SW) to 50% SW (Prior and Pierce, 198 1). Similar results occurred with the cell
bodies ofneurons in the visceral ganglion ofthe bivalve ofMya arenaria (Beres and
Pierce, 198 1) and the salivary burstor neuron in Limax (Prior, 198 1). Finally, all
of these responses are due to the osmotic rather than ionic change that accompanies
the sea water dilution. None of the electrical changes occur if only the ionic con
centration is reduced (osmolality maintained with sucrose) (Beres and Pierce, 1981;
Prior, 198 1; Prior and Pierce, 1981).
Invertebrate neurons —¿�cell volume regulation mediated by inorganic solutes
Only a few studies have examined neurons in this connection. Blue crab axons
(Callinectes sapidus) volume regulate during hypoosmotic stress using intracellular
amino acids from a substantial amino acid pool (Gerard, 1975; Gerard and Gilles,
1972). Osmotic adaptation of other neurons involves at least a partial role of in
organic ions as osmotic solute. For example, the hypoosmotic adaptation of Sabella
penicillus axons includes a loss of intracellular K'1'(estimated from resting potential
changes in response to external K' variation) (Treherne and Pichon, 1978). Treherne
(1980) has proposed that the K@is lost as osmotic solute. Somewhat similar ionic
responses occur during the adaptation of both Mytilus cerebro-visceral connective
axons (Willmer, 1978) and Mercierella enigmatica giant axons (Benson and Treh
erne, l978b) to hypoosmotic stress. In these two cases, however, the ionic changes
alone cannot account for the entire adaptation (Treherne, 1980), and Mytilus, at
least, has a substantial free amino acid pool (amino acids have not been measured
in Mercierella). Finally, axons isolated from Carcinus lose Na'1',K'1',and Cl―at least
transiently during volume regulation to a hypoosmotic stress (Kéverset a!., 1979b).
Carcinus axons also have a substantial intracellular amino acid pool (Evans, 1973)
which is apparently utilized during volume regulation (Kéverset al., 1979a). Taken
together, these studies indicate that amino acid regulation is not the entire story to
invertebrate cell volume regulation. There are two other invertebrate cell types that
have been studied in some detail with respect to inorganic solute utilization during
volume control: red coelomocytes from the blood worm Glycera dibranchiata and
the isolated myocardium from Limulus polyphemus. Both cell types have produced
some intriguing results.
Glycera red coelomocytes —¿�volume control by amino acids and K'1'
The isolated, hemoglobin-containing coelomocytes of Glycera rapidly volume
regulate in response to a hypoosmotic stress (Costa et a!., 1980) (Fig. 2b). The amino
acid pool size is large and decreases in content as the cells volume regulate. Fur
thermore, volume regulation by the isolated coelomocytes is accompanied by an
effiux of free amino acids from the cells (Fig. 3) (Costa et al., 1980). The volume
regulatory process in these cells requires the presence of extracellular divalent cations
but is not specific; either Ca2'1'or Mg2―will suffice. The volume regulatory process
appears to also be dependent upon the metabolic production of ATP (Costa and
Pierce, 1982). None of these results is particularly surprising based on previous
412 SIDNEY K. PIERCE
20
2 600-
a-
0@@ @! 500
•¿�,I0@ —¿�400@ ii Ii ii
r;.fl@T@[:@:1.;l 300
C
30:@@ 20:
__________ .1 1_I 1_i_I Li@i I__LI
996 0 0 20 60
m0sm/Kg H20 Time (mm)
FIGURE3. Amino acid effluxes from Glycera red coelomocytes isolated from worms adaptedto
996 mosm and exposed to the osmotic concentration indicated for 40 mm (From Costa et aL, 1980).
FIGURE 4. Intracellular K@content in Glycera red coelomocytes isolated from worms adapted to
996 mosm and then exposed to 996 or 498 mosm at time 0 (From Costa and Pierce, 1982).
studies, but intracellular K@ content also changes during volume control in
these cells.
A rapid decrease in intracellular K@content occurred in hypoosmotically stressed
G!ycera coelomocytes. Within 10 mm of exposure to dilute media intracellular K@
content declined by 10% (Fig. 4). Further, incubation of the cells in Ca2'- Mg@
free media appears to disrupt cellular control of K4 content as well as volume
regulation. Coelomocytes incubated in Ca2@-Mg24-free media lose K4 steadily
regardless of the external osmotic concentration. The K@changes observed were
unaffected by ouabain and could be potentiated by incubation in 2-4 dinitrophenol
(DNP). This last result is of particular interest because in Glycera cells cellular amino
acid content was unaffected by DNP not only indicating that K4 and the amino
acids leave the cells by different means, but also that the two types of solute are
responding to at least some factors not held in common. Finally, the use of K' as
an osmotic solute seems to be only transitory. While intracellular amino acid content
is markedly reduced in coelomocytes taken from low salinity adapted worms, K@
content in these coelomocytes is not different from that in cells taken from high
salinity adapted animals (Costa and Pierce, 1982) (Table I).
Limulus myocardial cell volume regulation —¿�Na4, C@, and g!ycine betaine
The remarkable euryhalinity of Limulus suggests an abundant amino acid pool.
However, while the intracellular free amino acid pool of Limu!us declines with
adaptation to low salinity, it is only a small amino acid pool (total = 100 @mole/
gm dry wt. in 100% SW adapted crabs) (Robertson, 1970; Warren and Pierce, 1982).
Furthermore, amino acids efflux from that pool in response to a salinity decrease,
but the efflux is much too small to account for volume regulation (Prior and Pierce,
1981). Instead Limu!us can tolerate a wide osmotic coocentration range without a
C
‘¿�4
UI@
592
Acclimation osmotic concentration
(mosm/Kg H2O)K@content**1000417
(±24.8)18750456
(±22.7)500413
(±23.7)
S
INVERTEBRATECELL VOLUMECONTROL 413
TABLE I
The intracellular K@content of red coelomocytes taken from Glycera dibranchiata acclimated
to various salinities.
* nmoles/l06 cells (±S.E.).
@ Intracellular K@ content is the same for all treatments, and thus the use of K@ as osmotic solute
in these cells is only transient (from Costa and Pierce, 1982).
large amino acid pool because the cells regulate volume with a mechanism that
relies on inorganic ions and the quaternary ammonium compound, glycine betaine.
The role of quaternary ammonium compounds as osmotic solute was occa
sionally pointed out in the older literature (for example, Bricteux-Grégoireet al.,
1964). More recent investigators have tended to ignore these potentially important
compounds largely because their identification and quantification was difficult and
rather imprecise. Recently a high performance liquid chromatographic analysis has
been developed which solves these analytical problems (Warren and Pierce, 1982).
The major quaternary ammonium compounds in Limu!us cardiac tissue are glycine
betaine and homarmne. Of these glycine betaine is quite high in concentration in
tissue taken from Limu!us adapted to full strength sea water and declines in con
centration in Limulus adapted to lower salinities (Fig. 5) (Warren and Pierce, 1982).
The isolated Limu!us heart volume regulates in response to a hypoosmotic stress.
The tissue shows a pattern of incomplete volume recovery quite typical of the pattern
exhibited by most cell types (Fig. 6). However, no betaine appeared in the media
surrounding the volume regulating hearts and, indeed the betaine content in the
tissue was unchanged (Table II) (Warren and Pierce, 1982). Volume regulation by
the isolated heart was accomplished without utilizing this major osmotic solute.
600
500
400
300
200
00
•¿�Glycao belotne
•¿�Homorine
0 200 400 600 800 000
Salinity (mosm)
FIGURES. Concentrations of the quaternary ammonium compounds glycine betaine and homarine
in cardiac tissue of Limulus adapted for at least two weeks to the salinities indicated (From Warren and
Pierce, 1982).
940 mosm400mosm6h599±24w633±15l2h621±16631±2724h585±21620±21
0 2 4 6 8 10 12
Hours
414
a,
18
a
SIDNEY K. PIERCE
FIGURE 6. Volume regulation by isolated Limulus hearts. The hearts were removed from crabs
adapted to 940 mosm and then exposed directly to either 940 or 400 mosm at time 0. The hearts were
weighed at the time points indicated (From Warren and Pierce, 1982).
Similarly, K―concentration of the isolated cardiac tissue changed only as predicted
by hydration changes (Table III). On the other hand, cellular Na―'and Cl levels
decreased far more than cell hydration changes could account for during the hy
poosmotic stress (Table IV and V) (Warren and Pierce, 1982). Furthermore, these
ionic changes are ouabain independent. These results clearly show that Na―'and Cl
are utilized to regulate volume early on by the Limulus cells and glycine betaine
much later. Indeed, evidence from whole animal experiments indicates that Na―'
and Cl- partially return toward initial concentrations as glycine betaine declines in
the Limulus heart cells (Warren and Pierce, 1982).
Volume regulation may result from coordination of permeability control systems
—¿� conclusions
The results of the Glycera and Limulus studies taken together indicate that two
quite distinct solute permeability control mechanisms are utilized by these cells
during volume regulation. The ions involved (Na―‘,K―',or Cl depending upon the
cell type) respond to the decrease in external ionic concentrations which accompany
the salinity decrease. The amino acid efflux is triggered by the osmotic change. The
ionic movements are not affected by ouabain indicating that the Na―'pump is not
TABLE II
•¿�940—940 mosm
•¿�940—400mosm
24
Glycine betaine concentrations in hearts isolated from Limulus adapted to 940 mosm
and exposed to 400 mosm.
* mmoles/g dry wt ± S.E.
The lowsalinityvaluesarenotsignificantlydifferentfromthehighsalinitycontrols(fromWarren
and Pierce, 1982).
Intracellular K@in hearts isolatedfrom Limulus adapted to 940 mosm and exposed to 400mosm.Salinitymmoles/kg
H2Ommoles/kg drywt940
mosm400 mosmPredicted18940 mosm 400mosm6h
12h
24 h112.6±
7.5
113.0±5.7
114.5±5.374.7
±2.9
83.2 ±3.2
88.6±3.575.0±
5.2
73.6 ±4.3
79.3 ±4.6458±29
432±14
432 ±28 453 ±11
458 ±20 511 ±13
Intracellular Na@in hearts iso/atedfrom Limulus adapted to 940 mosmand exposed to 400mosm.Salinitymmoles/kg
H20mmoles/kg drywt940
mosm400 mosmPredicted18940 mosm 400mosm6
h237.7 ±13.079.0 ±16.9153.1 ± 8.9913 ±51 437 ±8912
h228.9 ±17.646.3 ± 6.3144.5 ±10.6905 ±72 273 ±34
INVERTEBRATE CELL VOLUME CONTROL 415
TABLE III
18 Calculated according to Fred et al., 1973.
The data are presented in two ways. First, as concentration (mmoles/kg H2O). A concentration
decrease of K@does occur, but only as much as is predicted by changes in tissue hydration. Second, as
content (mmoles/kg dry wt). There is no significant change in K'@content indicating that K'@is not used
as osmotic solute (from Warren and Pierce, 1982).
involved in the process. It is clear from the data cited above that the two types of
permeability systems can be made to operate independently of one another and that
they often function with very different time courses in the cell. Nonetheless, it is
also clear that both solute control systemsoperate in concert to control cell volume.
The mechanism underlying this remarkable coordination is unknown at present,
but may be Ca2―‘related. The amino acid efflux control mechanism requires Ca2@
(see above), and normal K―'permeability in the G!ycera cells is lost if Ca2―'is removed
(Costa and Pierce, 1982). However, at present little else is known about the char
acteristics of the ionic regulatory systems.
Finally, some comparisons to volume regulatory systems found in vertebrate
cells may be instructive. There is no doubt that a substantial inorganic ionic com
ponent is responsible for volume regulation in vertebrate cells. Usually Na―‘or K―'
or both are utilized in a ouabain insensitive volume regulatory process that occurs
following an osmotic alteration (as opposed to steady state osmotic balance which
is usually ouabain sensitive) (reviewed by Rorive and Gilles, 1979). Occasionally
other ions are involved. For example, Necturus gall bladder epithelial cells require
bicarbonate for volume regulation (Fisher et a!., 1981). In addition, although there
are not yet a lot of data, it seems that vertebrate cells also have an organic solute
component to the cell volume regulatory mechanism. This component utilizes
amino acids or quaternary ammonium compounds and is particularly obvious in
TABLE IV
18 Calculated according to Freel et aL, 1973.
These data are also presented two ways (see Table III). There is a substantial change in Na@con
centration which is greater than that which can be accounted for by hydration changes. This is verified
by the Na@content data which also shows very significant decreases during hypoosmotic stress. Therefore,
Na@ is used as osmotic solute (from Warren and Pierce, 1982).
Intracellular Cl in hearts isolatedfrom Limulus adapted to 940 mosmand exposed to 400mosm.Salinitymmoles/kg
H20mmoles/kg drywt940
mosm400 mosmPredicted18940 mosm 400mosm6
h
12h
24 h.199.9
±16.0
195.8±13.5
201.6±15.360.3
±8.7
39.5±4.6
38.8 ±5.1124.7
± 9.4
135.8 ±12.8
141.0 ± 8.3780
±52 352 ±56
762 ±48 221 ±26
834 ±76 213 ±24
416 SIDNEYK. PIERCE
TABLE V
18 Calculated according to Freel et aL, 1973.
a concentrationdecreasedmuchmorethanhydrationchangescouldaccountforandCl content
also showed large, significant decreases. Therefore a-, like Na@(Table IV), is regulated in the heart cells
in response to hypoosmotic stress (from Warren and Pierce, 1982).
vertebrate species that spend all or part of their lives in water (for example marine
toad [Bufo viridis] skeletal muscle [Gordon, 1965], flounder [Pleuronectes flesus]
red cells [Fugeffi, 1967], Myxine muscle cells [Cholette and Gagnon, 1973], skate
[Raja erinacea] and stingray [Dasyatis sabina] tissues [Boyd et al., 1977], skate
[Raja erinacea] erythrocytes and muscle [Goldstein, 198 1]). Other studies have
demonstrated utilization of organic osmotic solute, primarily taurine, in higher ter
restrial vertebrates including humans. For example, intracellular taurine concentra
tions respond to the plasma osmolality changes that occur during hypo- or hyper
natremia in both mammalian brain and heart cells (Thurston et a!., 1980, 1981;
also reviewed by Pollock and Arieff, 1980). Ehrlich ascites cells also utilize taurine
for volume control (Hendil and Hoffman, 1974; Hoffman, 1978). At present little
is known about the mechanisms utilized to control the organic solutes in these cell
types. Nonetheless, the historic intracellular osmotic solute differences held to occur
between vertebrates and invertebrates may be a strawman. There is a growing body
of information indicating that both types of solute are utilized by all cells, and
differences are in magnitude rather than kind. If this turns out to be true, then the
cells of euryhaline invertebrates may become important as well as interesting models
of osmotic function as a consequence of their remakable abilities of cell hydration
control.
ACKNOWLEDGMENTS
Some of the studies reported here were supported by N.I.H. Grant # GM-2373 1.
This paper is Contribution No. 190 from the Tallahassee, Sopchoppy & Gulf Coast
Marine Biological Association, Inc.
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