Animals and Environmental Fitness: Physiological and Biochemical Aspects of Adaptation and Ecology. Abstracts

189 Pages·1980·28.986 MB·English

by  R. Gilles (Eds.)

This volume gathers the summaries of the communications
from those participants who wished to have an abstract
published.
The Organizing Committee of the Conference and the
editor have allowed the authors full latitude to express their
views, which remain under their entire responsibility.
Proceedings of the First Conference of the
European Society for Comparative Physiology and Biochemistry
27 to 31 August 1979 - Ltege - Belgium
ANIMALS AND
ENVIRONMENTAL FITNESS
Physiological and Biochemical Aspects
of Adaptation and Ecology
Volume 2
ABSTRACTS
Conference organizer and volume editor
R. GILLES
Laboratory of Animal Physiology
University of Li&ge, Belgium
Under the patronage of
The Commission of the European Communities
Le Fonds National de la Recherche Scientifique
Le Ministdre de PEducation Nationale et de la Culture Frangaise
L'Universite de Ltege
The European Society for Comparative Physiology and Biochemistry
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First edition 1980
British Library Cataloguing in Publication Data
European Society for Comparative Physiology
and Biochemistry (Conference), 1st, Liege, 1979
Animals and environmental fitness.
Vol. 2: Abstracts
1. Adaptation (Biology) - Congresses
I. Title II. Gilles, R.
591.5 QH546 80-40080
ISBN 0-08-024939-6
Published as Supplement No 2 1980 to the journal
Comparative Biochemistry and Physiology.
In order to make this volume available as economically
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AMINO ACID TRANSPORT AND
METABOLISM DURING OSMOTIC
SHOCK IN EHRLICH ASCITES
TUMOR CELLS
I. H. Lambert and E. K. Hoffmann
Inst. Biol. Chem. A, August Krogh Institute, University of Copenhagen 13,
Universitetsparken, DK-2100 Copenhagen 0, Denmark
The participation of the amino acid pool in the regulation of osmotic equi­
librium that the cell maintains with its surrounding fluid, is a well known
phenomenon (see Gilles 1979, Hoffmann 1977). Modification of the transport para­
meters and the intracellular metabolism of the amino acids have been proposed as
the two main mechanisms implicated in the regulation of the amino acid concentra­
tion that takes place during the cellular volume readjustment which follows an
osmotic stress (Gilles 1979). We have therefore undertaken a study on an effort
to characterize those mechanisms in EhrKch Asoites tumor cells where amino acids
are found to be active in the osmoregulation (Hoffmann and Hendil 1976).
The largest changes under osmotic stress were recorded in concentrations of small
nonessential amino acids and taurine, with the greatest changes found in alanine,
glycine and taurine (Hoffmann and Hendil 1976). This report therefore deals more
specifically with the transport and metabolism of these.
Metabolism. To distinguish the effect of alteration in extracellular osmolarity
from the effect of reduction in extracellular ionic concentration during hypoos-
motic stress, we compared results from cells in five different types of media:(A)
control medium with unchanged ion concentration and unchanged osmolarity.(B) & (C)
salt solution diluted with distilled water in order to lower both ion concentra­
tion and total osmolarity to respectively 75% and 50% of the control values.(D) &
(E) salt solution diluted with sucrose solution in order to decrease ion concen­
tration as in (B) and (C) respectively, but maintaining solutions isotonic with
the control medium.All measurements were made after the cells had reached their
new steady state volume. By comparing (B) with (D) and (C) with (E) we observed
the effect of osmotic changes. By comparing (A) with (D) or (E) we observed the
the effect of reduction in ion concentration.
The direct method of Warburg (Umbreit-Burris-Stauffer 1964) was used to estimate
the oxygen consumption and the total carbon dioxide production. The production
of 14C-C02 from labelled amino acids was measured by addition of the 14C-amino
acids to the cell suspension in Warburg vessels, and collecting all the produced
carbon dioxide on a filterpaper, moistened with KOH. The respiratory quotient (RQ)
is calculated as carbon dioxide-production divided by the oxygen-consumption.
l
? I. Lambert and E. K. Hoffmann
The Warburg experiments showed that:
(1) During hypoosmotic stress the RQ-values increase as a result of a reduction
in oxygen consumption (induced by reduction in ion concentration) and an in­
crease in carbon dioxide production (induced by reduction in osmolarity).
(2) Glycine and Alanine, but not taurine, can be oxidized to carbon dioxide by
Ehrlich Ascites cells.
(3) During hypoosmotic stress there is an increased 14C-C0 production from 14C-
2
alanine, while HC-CO^production from 14C-glycine is unchanged.
(4) The 14C-C0p specific activity for alanine increases significantly when the
medium is ailuted to 75% of its normal value (medium B).
This indicates that the Ehrlioh Ascites cells withstand a hypoosmotic stress part­
ly by a modification in the mechanisms controlling the catabolic activity of ala­
nine and a general modification of the related oxidative metabolism.
Pump and leak parameters. Influx and efflux were measured as initial rate fluxes,
i.e. 6-8 samples were taken during the first minute after addition of the amino
acid by a filter technique (Hoffmann £t aj_ 1979)
Results showed that a decrease in ion concentration with unchanged osmolarity had
no significant effect on the leak permeability of either glycin or taurin. At 50%
the active uptake of glycine decreases whereas the active uptake of taurine in­
creases ionic concentration.
On the other hand, a decrease in osmolarity with unchanged ion concentration, in­
creased the permeability and decreased the active uptake of both glycine and tau­
rine.
Conclusion. This report shows that the decrease seen in the pool of nonessential
amino acids and taurine under hypoosmotic conditions is achieved partly by an in­
creased oxidation of e.g. alanine, and partly by an increased permeability to
some amino acids as well as an decreased pump rate for the same amino acids.
These selective changes in transport parameters are induced in Ehrlich Ascites
tumor cells by changes in the cell volume.
R.Gilles (1979), Mechanisms of Osmoregulation in Animals. John Wiley & Sons New
York
Else K. Hoffmann (1977), Control of Cell volume, from Transport of Ions and Water
in Animals. Academic Press Inc. (London).
Else K. Hoffmann and K. Hendil (1976), The Role of Amino Acids and Taurine in
Isosmotic Intracellular Regulation in
Ehrlich Ascites Mouse Tumor Cells.
J.comp.Physiol.108,289-286 (1976)
Else K. Hoffmann, Lars 0. Simonsen and Carsten Sjoholm (1979), Membrane Potential,
Chloride Exchange, and Chloride Conductance in Ehrlich Ascites
Tumor Cells. J.comp.Physiol. in print.
W.W. Umbreit, R.H. Burris, J.F. Stauffer, Manometric techniques, fourth edition
Burgess Publishing Company (1964).
CELL VOLUME REGULATION IN
ISOLATED HEART VENTRICLES FROM
THE FLOUNDER, PLATICHTHYS FLESUS,
PERFUSED WITH ANISOSMOTIC MEDIA
T. Vislie
Zoological Institute, University of Oslo, Blindern, Oslo 3, Norway
Adapting sea water flounders, Platiohthys flesus, to fresh
water the plasma osmolality decreases about 20% (Vislie & Fugelli,
1975) . By readapting these fresh water flounders to sea water
the original plasma osmolality is restored (Vislie, 1979). The
cell volume regulation mechanism (isosmotic intracellular
regulation) which counteracts volume changes during alterations in
plasma osmolality, has previously been found in heart ventricle
musculature in vivo. About 50°6 of the total free amino acid like
substances in flounder heart ventricle consists of taurine, which
was together with K , the dominating cellular osmo-effectors in
vivo (Vislie & Fugelli, 1975; Vislie, 1979).
The present report deals with the cell volume regulation
mechanism in isolated, beating heart ventricles from sea water
(S.W.) and fresh water (F.W.) adapted flounders perfused with a
hyposmotic (230 mOsm) and a hyperosmotic (380 mOsm) saline (fish
ringer), respectively. Hearts from both S.W. and F.W. adapted
flounders were as controls, perfused with salines isosmotic to the
plasma (330 mOsm and 280 mOsm, respectively).
In both these control groups the tissue water content (Fig. 1)
and the intracellular concentrations of solutes in heart ventricles
showed only small variations, and were consistent with the values
found in vivo (Vislie & Fugelli, 1975). Thus, the perfusion
procedure, per se> seemed to have no effect on the ventricle
musculature which could influence the results. However, the water
content of heart ventricles from S.W. flounders, submitted to the
hyposmotic saline (230 mOsm), increased to a maximal value within
6 0 min, which subsequently was followed by a shrinkage to a new
steady level within 6 hrs (Fig. 1). Correspondingly, when hearts
isolated from F.W. adapted flounders, were perfused with the hyper­
osmotic saline (380 mOsm), the ventricle tissue water content
initially decreased, but was subsequently readjusted back towards
the values in the control hearts (Fig. 1).
K and taurine were important osmo-effectors in the cell volume
regulation also in vitro. In heart ventricles from S.W. flounders,
4 T. Vislie
perfused with the hyposmotic saline (230 mOsm), the intracellular
K concentration was after 6 hrs of perfusion, reduced from about
125 mmoles/kg cell water (mean value in hearts perfused with the
isosmotic saline, 330 mOsm) to about 90 mmoles/kg cell water, and
the taurine concentration from about 65 mmoles/kg cell water to
about 35 mmoles/kg cell water. When hearts from F.W. adapted
flounders were perfused with the hyperosmotic saline (380 mOsm),
the cellular concentrations of K+ and taurine increased during the
volume readjustment. Thus, the concentration of K+ increased from
about 115 mmoles/kg cell water (mean value in hearts perfused with
the isosmotic saline, 280 mOsm) to about 135 mmoles/kg cell water,
while the taurine concentration increased from about 45 mmoles/kg
cell water to about 75 mmoles/kg cell water.
The perfusion salines did not contain taurine, and the only way
to explain the gain in cellular taurine is by assuming a net
synthesis of the compound in the heart ventricle tissue. On the
other hand, the reduction in cellular taurine during the volume
readjustment in heart ventricles from S.W. flounders, perfused
with the hyposmotic saline, appeared to be the result of a net
release into the perfusion saline. Thus, the magnitude of the
cellular taurine reduction could reasonably well be explained by
the increase in taurine content of the perfusion saline.
85, S.W. flounder (-
Fig. 1. Tissue water content
FW. flounder (-
of isolated, perfused heart
.'*^. ventricles from the flounder,
a; 83\ - • Platiohthys flesus^ as a
function of perfusion time.
Hearts from sea water (S.W.)
>>« flounders perfused with
isosmotic (330 mOsm) (0) or
hyposmotic (230 mOsm) (#)
saline. Hearts from fresh water
c79 (F.W.) adapted flounders
perfused with isosmotic
(280 mOsm) (D) or hyperosmotic
(380 mOsm) (f) saline. Each
£77 point represents result from
£ J one heart.
Time (hours)
References
Vislie, T. (1979). In preparation.
Vislie, T. and K. Fugelli (1975). Comp. Biochem. Physiol., 5 2A,
415-418.
EFFECTS OF CHOLINERGIC DRUGS
ON THE OSMOTIC FRAGILITY
OF ERYTHROCYTES
B. Soria
Department of Biochemistry and Physiology, Faculty of Medicine,
Avda. Blasco Ibanez-17, Valencia, Spain
Detection of subtle changes in fluidity of the membrane hydrocarbon regions
in presence of cholinergic drugs (1) and high affinity of membranes to quinucli-
dinyl benzilate (2) allow to the authors describe a functional acetylcholine re­
ceptor (Ach-R) in human erythrocyte,although its physiological role remains un­
clear.Ach-R have been demonstrated in other non-synaptic regions,i.e. in lobster
axon (3),frog sciatic nerve (4),spermatozoa (5),etc.A local function controlling
ionic permeability has been proposed by Nachmansohn and Neumann (6) for explain
bioexcitability.Osmotic fragility of red blood cells has been extensively inves­
tigated,particulary as a model system for the study of the mode of action of va­
rious drugs (7).Thus,we decide study the effect of cholinergic drugs on the osmo­
tic fragility of the erythrocytes.
Materials and methods:Blood was obtained from adult human volonteers (Homo sapiens)
and used within three days of withdeawal.Red cells were washed two times with bu­
ffer containing 136 mM ClNa,5.3 mM C1K.0.8 mM S0 Mg,0.33 mM P0 HNa ,0.4A mM PO^K,
4 4 2
0.414 mM CC~HNa and 0.55 mM glucose and a third time with thw same solution and
1.67 mM ClpCa.Washed erythrocytes at a hematocrit of about 1.5% were incubated
with isotonic (146 mM CINa,phosphate buffer 0.2 M pH:7.4) solutions of a drug for
5 min. at 10-15°C,centrifuged (2000 x g for 5 min.) and reincubated into a hypoto-
nic solution (85 mM CINa,phosphate buffer 0.2 M pH:7.4) at the same concentration
of the drug.Concentration of drug larger than 1 mM were adjusted for a 146 mM CINa,
or 85 mM CINa,similar final tonicity.The problem and control samples were centri-
fuged (2000 x g for 5 min.) and the absorbances of the supernatans were measured
at 543 nm.Each experiment was repeated 8-10 times with essentially identical results
Results and discussion:At isotonic solution,no hemolysis was observed in controls
and drug samples.The results with hypotonic solutions (85 mM CINa) are showed in
5
6 B. Soria
*0x (1) Ach 10 (l)Ach
(2) Proc (2)Proc + DTT
s
(3) Nict \ (3)Ach + DTT
(4) Atrp 2* \ (4)Proc
.;: 200- >i 200- M5)DTT
>>
'o o
E E
3 ,
> +++ +
QJ 100—J 2x. 4X-... v 4
4-> . 1 ^1 «
3*_ • ••' _j.+..t..t|j-..t..t|
CU
TT 1$ d
10 io3 10 10 To6 10 16' io* 10 10
Concentration (M) Concentration (M)
l)Acetylcholine (Ach) enhances osmotic fragility of erythrocyte showing a sigmoid,
not linear,action (n of Hill coefficient aprox. eoual to 1.88).Dithio threitol (DTT)
at the same concentration of Ach,diminishes it action.2)Atropine (Atrp),nicotine
(Nict),procaine (Proc) and DTT stabilize red blood cells against hypotonic hemoly-
sis.The mechanism of this action are not clear,probably,while atropine,procaine and
nicotine partially blocks Ach-R,DTT splits a specific disulphide bond situated near_
ly the anionic site of the receptor,similar to the S-S bond described at the synap-
tic level (8,9).3)Procaine and DTT,two stabilizing factors of membranes,do not show
additive effects.When procaine,that acts at other levels by competitive inhibition
of Ach-R (3,6) is added together with DTT,an increase of osmotic fragility of red
cells is observed.4)If we suppose that Ach system controls ionic permeability in
erythrocyte,a group of proteins (Ach-R,acetylcholinesterase,cholineacetyltranferase,
and a storage protein) with very similar active sites can be involved and the drugs
cited before can interact with any of them.
References:l)Huestis,W.H.and H.M.McConnell(1974).Biochem.Biophys.Res.Commun.57,726-
732.2)Aronstan,R.S.,L.G.Aboud and M.K.McNeil (1977)l_ife Sci .20,1175-1180.3)Marauis,
J.K.,D.C.Hilt,V.A.Papadeas and H.G.Mautner (1977) Proc.Mat.Acad.Sci.USA 74,2278-
-2282.4)Soria,B.(1978)Ph.D.Thesis.University of Valencia.Spain.5)Nelson,L.(1973)
Mature 242,401-402.6)Nachmansohn,D.(1955) and Machmansohn,D. and Neumann,E.(1975,
revised)Chemical and molecular basis of nerve activity.Academic Press.New York.
7)Seeman P.(1966)Int.Rev.Neurobiol.9,245-264.8)Karl in,A.(1969) J.Gen.Physiol.54,
>
245-264.9)Steinacker,A.(1979) Nature,278,358-360
EFFECT OF VARIOUS HYPO- AND
ISOTONIC SALINES ON THE K +
INTRACELLULAR CONTENT OF
CARCINUS MAENAS ISOLATED AXONS
C. Kevers, A. Pequeux and R. Gilles
Laboratory of Animal Physiology, University of Liege,
22, Quai Van Beneden, Liege, Belgium
Isolated axons of crustaceans undergo a process of cell volume
regulation when submitted to hypo-osmotic conditions; the tissue in­
deed first swells and then progressively resumes a volume close to
control. Volume regulation can be dissociated into a rapid phase of
"volume swelling limitation" and a much slower phase of "volume read­
justment", both phases implicating decrease in the intracellular con­
tent of various osmotic effectors. It has been shown recently that K
is playing an important part in the volume swelling limitation pro­
cess at work in isolated axons of Caroinus maenas (Kevers, Pequeux
and Gilles, 1979a,b). The decrease in K is most likely due to.an
increase in permeability related to changes in plasma membrane con­
formation induced by the swelling per se and/or to changes in the
ionic content of the environmental medium. In order to bring some
light on this problem, the intracellular K+ amount has been measured
in C.maenas isolated axons undergoing volume regulation in hypo-osmo­
tic media of different ionic composition or in media kept isotonic
to the intracellular fluid by addition of sucrose.
Salines preparation, incubation procedure-, ionic concentrations
measurments and extracellular space determinations are achieved as
previously described (Kevers, Pequeux and Gilles, 1979b). The control
saline contains NaCl : 494 mM; KC1 : 11.3 mM, CaCl : 7.5 mM, MgSO,:
2
20 mM. All salines are buffered at pH 7.1 with a 2.5 mM bicarbonate
buffer.
Application of a hypo-osmotic shock by use of a twice diluted
control saline induces an important decrease in the intracellular
amount of K+(fig.l, A). When the K content of the hypo-osmotic sali­
ne is kept at control level, the observed decrease becomes slightly
less important. On the other hand, there is no significant difference
between the decreases observed in salines containing either control
levels of Ca2+ and Mg or half the control amount of these ions. It
thus appears that the modification in the intracellular content of K+
observed in hypo-osmotic conditions cannot be related to changes in
the saline concentration of Ca and Mg
If now the hypo-osmotic salines are supplemented with sucrose
280 mM, an amount shown to be necessary to avoid volume modifications
due to water osmotic movements, a significant decrease in the K+
7