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Dissertations and Theses
Dissertations and Theses
1984
Effects of dehydration on hemoglobin oxygen affinity and blood
cell volume in two anurans
Andrew Christopher Zygmunt
Portland State University
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Recommended Citation
Zygmunt, Andrew Christopher, "Effects of dehydration on hemoglobin oxygen affinity and blood cell volume in two anurans" (1984).
Dissertations and Theses. Paper 3424.
10.15760/etd.5056
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AN ABSTRACT OF THE THESIS OF Andrew Christopher Zygmunt for
the Masters of Science In Blology presented August 3, 1984.
Title: Effects of Dehydration on Hemoglobin Oxygen Aff lnlty
and Red Blood Cel I Volume In Two Anurans.
APPROVED BY MEMBERS OF THE THESIS COMMITTEE:
Richard R. Petersen
The degree of terrestrial Ism In anurans Is correlated
with a differential tolerance to desiccation.
Cardiovascular insuff lclency and reduced oxygen del Ivery to
the tissues may be the mechanism of dehydratfonal death f n
amphibians.
2
Two aspects of posslble adaptation In cardlovascular
performance caused by Increased plasma electrolytes were
examined. Cel Is In anfsotonfc plasma may either act as
osmometers or volume regulate.
Blood flow rate Is
dependent upon eel I viscosity, which Jn turn Is a
consequence of eel I volume and membrane deformabl I Jty.
Cel I volume changes which Increase membrane deformabll lty
wll I thus potentially extend the I lmlts of dehydration
tolerance.
It was found In.&... catesbelana
and~
marlnus
that red blood eel Is maintain constant volume during
dehydration.
Cel Is In vitro Initially lose water, but then
sodium, potassium and water move Into the eel I.
Cel I
viscosity within the physiologic range of hematocrlts was
higher In salt loaded non-regulating eel Is
of~
marl nus
than In regulatlng Isotonic eel Is.
A consequence of water loss In non-regulating eel Is,
or uptake of Ions In regulating eel Is, Is an Increase of
lntracel lular Ion concentration. Hyperosmolaltty Influences
oxygen loading characteristics of blood.
Ionic
Interactions are known mediators of hemoglobln function.
It was found In B.a.rut and .B.u.f.Q that Increasing lntracel lular
Ionic concentration
dissociation curve.
falled to Influence the oxygen
Adaptation was therefore not made to
Increase oxygen del lvered to the tissues during a time of
general clrculatory lnsuff lclency.
EFFECTS OF DEHYDRATION ON HEMOGLOBIN
OXYGEN AFFINITY AND BLOOD CELL
VOLUME IN TWO ANURANS
by
ANDREW CHRISTOPHER ZYGMUNT
A thesis submitted In partlal fulfll lment of the
requirements for the degree of
MASTER OF SCIENCE
fn
BIOLOGY
Portland State University
1984
TO THE OFFICE OF GRADUATE STUDIES AND RESEARCH:
The members of the committee approve the thesis of
Andrew Christopher Zygmunt presented August 3, 1984.
Richard S. Petersen
Department of Biology
Dean of Graduate Studies and Research
ACKNOWLEDGEMENTS
I want to thank Dr. Stan Hll Iman for Introducing me
to physlology and for sharing his tequlla.
I have valued
the assistance and humor of Ors. Phi I Ip Withers, Richard
Petersen, and Larry Crawshaw; as wel I as that of my tel low
graduate students.
Most Importantly, without the love and
caring of Diane this work would have been lmposslble.
TABLE OF CONTENTS
PAGE
ACKNOWLEDGEMENTS
11 I
LIST OF TABLES •
v
LIST OF FIGURES.
vi
INTRODUCTION •
•
•
•
MATERIALS AND METHODS.
Anlmals •
8
•
Mean Cel I Volume.
•
8
•
8
Hemoglobin Oxygen Aff lnlty.
11
lntracel lular Sodium and Potassium.
12
Viscosity
•
RESULTS.
•
Mean Cel I Volume.
•
13
13
•
13
•
Hemoglobln Oxygen Affinity.
15
lntracel lular Sodium and Potassium.
15
Viscosity.
15
DISCUSSION
REFERENCES
• •
• •
26
• •
•
32
LIST OF TABLES
TABLE
PAGE
lntracel lular sodium and potassium
concentrations CmEq/Kg dry mass) In the
erythrocytes of .IL. marlnys Cn=9) and B.... catesbelana
Cn=9).
Values are means± standard error assuming
10% trapped plasma.
Asterisk Indicates slgnlf lcant
difference Cp<0.025)..........................
17
LI ST OF FIGURES
PAGE
FIGURE
1.
In vivo volume of erythrocytes of .fL. marfnus
(water losses 28-36% lnltlal body mass)
and .B.a. catesbelana (water losses 14-20% lnltlal
body mass) • • • •
2.
Temp~rature
.......
.....
18
dependence of RBC volume regulatfon
In .fL.. marlnus and .B... catesbelana.
Rana,
CA9oc lsotonrc, e2ooc Isotonic, Agoc hypertonlc,
920oC hypertonfc).
Ji.Llf.g,
CY 9oC Isotonic,
•2ooc lsotonlc,~goc hypertonlcAt2ooc hypertonlc>
•
3.
•
•
•
•
• •
•
•
Volume regulatlon
after 1.5 hours.
•
•
•
•
•
•
•
•
•
• •
•
•
19
of RBC's In .B... catesbelana
Top I lne Indicates behavior as
osmometer, y=71.5 (±0.4)x-0.1 (±0.1), r=1.00.
Bottom lfne RBC's In normal Ringer's,
y=21.0 (±5.3)x-0.4 (±2.0), r=.78.
C•
, potassium free media;
•
, normal Ringer's) • • •
Y
,
oubaln media;
..........
20
vii
4.
Volume regulatton of RBC's In
hours.
y=66.7
.a...
martnus after t.5
Top I tne ts behavior asosmometer,
C±0.5)x+0.1 (±0.1), r=1.00.
Bottom llne
RBC 1 s In normal Ringer's, y=26.9 C±2.8)x-0.3 C±t.t>
r=.94. CA , potassium free media;
media; •
5.
•
, oubaln
, normal Ringer's) • • • • • • • • • •
21
The relattonshlp between red eel I tntracel lular ton.
concentrations
and plasma electrolytes In
.B... catesbelana corrected for 10% trapped plasma.
cesodtum y=0.5x-21, r=.75; A potassium y=1.1x+97,
r=.93.) • • • • • • • •
6.
• • • • • • • • • • •
22
The relatfonshtp between red eel I tntracel lular Ion
concentrations
a~d
plasma electrolytes In B.marlnus
corrected for .10% trapped plasma.
cesodtum y=0.5x-14, r=.73; Apotasslum y=0.4x+190,
r=.79) • • • • • • • • • • • • • • • • • • • •
7.
The relattonshtp between
.a...
23
marlnys hematocrlt and
the In transformation of red eel I viscosity at
a shear rate of 450/sec and 20oC.
Sol Id I lne
Is salt loaded (180 mEq/L), In y=0.028C±.001)x+0.90
C±.04), r=.98.
Dashed I fne ts Isotonic plasma,
In y=0.035 C±.002)x+0.43 C±.07), r=.93. • • • •
24
INTRODUCTION
Anurans are a diverse group of some 3000 species
which occupy habitats ranging from rain forests to deserts.
Although the vast majority of species are freshwater, their
number Includes species adapted for marine environments.
Modern anurans are descendants of the f lrst vertebrates to
face the desiccating environment of a terrestrlal habitat.
The examination of the phystologlcal correlates associated
with their degree of terrestrial Ism has been a fruitful
Inquiry.
Water balance In anurans has been described by a
number of workers.
With a few exceptions, CPhyl
tom~~,
Cblcomantls, and Hypecol lys), frogs and toads have
cutaneous evaporative water loss rates equal to a free
water surface (Bentley 1966a).
Thorson (1955> found no
correlation between evaporative water loss and degree of
terrestrial ism, but did find a high correlatlon between
Increased tolerance to water loss and xerlc habitats.
Hll Iman (1980) found the aquatic species Xenopys
laeyl~
to
tolerate 34% loss of lnltlal body mass, whereas Scapbtopys
coychll, an animal
found In desert areas with access only
to temporary ponds and soll moisture, tolerated a loss of
45% before dehydratf onal death.
2
The phystologtcal basts for tnterspeclf lc dehydration
tolerance has recently begun to emerge.
A terrestrlal
vertebrate in negative water balance Is necessarf ly
dependent upon stored water.
A positive correlation exists
between Increased capacity of anuran urinary bladders and
terrestrlal Ism.
Bladder capacity ranges between 1% body
mass for X... laevLs to 44% In
.a..
cognatus (Bentley 1966b).
Rulbal (1962), Shoemaker (1964), and McClanahan (1967) have
shown that bufonlds and pelobatlds maintain normal body
flufd concentrations until bladder stores are depleted.
Since anurans lack salt glands and kidneys stop urine
formation during dehydration, the osmotic concentration of
body fluids must Increase after depletton of bladder water.
Shoemaker (1964) has formulated the fol I owing equation to
describe the Increase In plasma electrolytes with
dehydration:
Cf=Co*CBWo/CBWo-WD>
where
Cf=flnal plasma osmolarlty
Co=ortglnal plasma osmolarlty.
BWo= ortglnal body water content
WD=water def lclt
Further bases for graded dehydration tolerance were
found by Hf I Iman (1976, 1978).
Terrestrial species
3
a..
cognatys and .S.... coychll have Increased oxygen storage
capabll lttes and maximum oxygen consumptions CVo 2 max).
He
found positive correlations between ventricle mass and
Vo 2 max, a stgnlf lcant decrease In Vo 2 max with
dehydration, and an Increase In whole animal
lactate at
critical activity point Closs of righting response).
The
lmpl tcatlon Is that terrestrial Ism Is associated with
selection for Increased aerobic capacity and that
dehydratlonal death In the species Investigated Is brought
on by a reduction In circulatory oxygen del Ivery.
Poss Ible mechanisms for reduct ton In
o2
del tvered to
the tissues Include the effects of hyperosmolal tty,
hypovolemta, and viscosity.
Investigations of the
contractile performance of cardiac muscle bathed In
hyperosmottc solutions have shown both postlve <Koch-Weser
1963; Atkins et al. 1973), and negative lnotroptc effects,
CWtldenthal 1975; Htl Iman 1984).
Ht II man (1978, 1980)
demonstrated Increased tolerance for plasma sodium tn
terrestrial species and suggested that a necessary
adaptation for extending deydratlon tolerance Is to extend
the Intrinsic osmotic I lmlt.
This effect of electrolytes
was found to be Independent of hematocrlt and thus
viscosity effects.
Compensation associated with hypovolemtc stress was
shown by Hf I Iman and Sommerfeldt (1981) to Include a
redistribution of blood flow to the head, enhanced return
4
of lymph to the plasma space, CHll Iman and Zygmunt,
unpubl I shed), and an Increase of resting heart rate
CHll Iman 1978).
In this manner brain function and cardiac
output Is maintained.
Blood flow rate Is Inversely proporttonal to blood
viscosity.
During dehydration, blood viscosity Increases
after bladder and lymph flutd volumes have been exhausted.
Any compensation In ctrculatory resistance tnvolvtng
dtlatton of pertpheral vessels ts counter-productive since
the appropriate response to hypovolemta ts constriction of
the perlpheral vessels.
To summarize, dehydrattonal death In anurans appears
to be the result of curtatlment of oxygen del Ivery to the
tissues, resulttng from hypovolemta, hypervtscostty, and
hyperosmolal lty. Consequently, adaptations which fact I ltate
oxygen del Ivery with dehydration should enhance tolerance.
The objectives of this study were to Investigate aspects of
the effects of Increased plasma electrolytes during
dehydration on RBC function In .B.... catesbelana and
~mart
nus.
Two aspects of red eel I function under hyperosmottc
stress are germane to the question of oxygen del Ivery.
first Is
wheth~r
The
the red blood eel Is volume regulate or not
with hyperosmolal lty.
Cel I deformabtl tty Is a functlonal
consequence of eel I volume.
Blood viscosity Is related to
eel I deformabll tty, hence eel I volume.
Mechanisms which
5
reduce blood viscosity with dehydration wtl I enhance oxygen
del Ivery and be adaptive relattve to dehydrattonal
tolerance.
The second question ts whether the
hyperosmolal tty Influences the oxygen loadtng
characteristics of the blood.
Ionic Interactions are known
to mediate hemoglobln oxygen loadlng.
During dehydration
tntracel lular tonic strength Increases, therefore the
effects of hyperosmolal tty on oxygen dissociation curves ts
central to understanding hyperosmottc stress on oxygen
del Ivery.
One aspect of the lnabll tty to regulate plasma
electrolytes ts that eel Is wll I be bathed In hyperosmotlc
flutds.
Net movement of water Is dependent upon
extracellular osmolal tty.
In hypertontc soluttons, eel I ·
volumes wll I decrease unless tntracel lular osmolyte
concentrations are Increased to match extracellular
osmolal tty.
A number of Investigators have examined volume
regulatton of eel Is In anlsotonlc media.
Shoemaker (1964)
found retention of water f n skeletal muscle, I Iver, kidney,
lung, and heart In the toad
80% of orlglnal mass.
.a....
marlnus after dehydration to
He associated this volume regulatlon
with an Increase In eel lular electrolytes, CNa+, K+, Cl-).
Katz (1978) found dlfferentlal volume regulatlon In tissues
of
.a....
ylrldls.
A reduction In water content between 15-30%
was found for erythrocytes, muscle, and I Iver.
Heart water
content changed very I lttle after adaptation to 500 mOsm
6
Oubaln C10- 3 M>, an active transport blocker,
NaCl.
affected tonic composition, but not eel lular water content.
Studies of erythrocytes In hypotontc media have shown
volume regulation to be the result of either reduction In
eel I free amino acids CFugel I I 1967; Costa and Pierce
1983), or eel I electrolytes CKregenow 1971a). Duck red
eel Is In hypertonlc media Increase eel lular Na+ and K+
content after an lnltlal period of eel I shrinking CKregenow
1971b).
Red eel Is In anlsotonlc solutlons change surface to
volume ratio and packing of eel I hemoglobin.
potent I ally affect blood viscosity.
reported confl feting results.
These factors
Investigators have
Melselman et al. (1967)
found hypertonlc suspensions of red eel Is to have Increased
viscosity, whereas eel Is In hypotontc solutton had reduced
viscosity relative to controls.
Rand and Burton (1964)
found red eel I membranes to be more easily deformed In
hypertonlc solutions.
Increased deformabtl lty should yleld
a reduced viscosity <Braasch 1971).
I have Investigated·
blood viscosity over a range of hematocrfts In salt loaded
red blood eel Is.
These experiments answer questions
regarding posslble viscosity advantages ln regulating vs
nonregulattng nucleated red eel Is. A reduction In viscosity
offers an obvious advantage In oxygen del.lvery.
If erythrocytes maintain volume as a consequence of
intracellular uptake of electrolytes, or if they simply
7
lose water, It would be expected that the oxygen
dissociation curve would be shifted to the right, due to an
Increase In lntracel lular Ion concentration C Rossl-Fanel I I
et al. 1961; Brunorl et al. 1975).
A rightward shift of
the curve would be a useful adaptation for promoting the
oxygen supply to the tissues by decreasing hemoglobin
affinity for oxygen Clenfant et al. 1970; Metcalfe and
Fact I ttatton of o 2 del Ivery at the tissues
would offset general clrculatory Insufficiency which
Dhtndsa 1970).
accompanies dehydration.
I have undertaken experiments to
determine the half saturation point of hemoglobin In
control and dehydrated animals.
The half saturation point
of hemoglobin, CP 50 >, Is a measure of the position of the
oxygen dtssoctatton curve.
·In summary, the thrust of this study ts to elucidate
factors for possible compensation of problems Involved with
q2
del Ivery durl ng dehydration In anurans.
These factors arise from reduced eel I volume In hypertonlc
media, or from Increased tntracel lular tonic concentration
with dehydration.
A comparison between species of
differing tolerance to dehydration, Cau.tg>.Ba.n.a>, may
Identify adaptations for terrestrial radiation In anurans.
8
MaterJals and Methods
An Ima ls
.BJLtg marlnys (mean mass=253g) and .B.A.n.A catesbelana
(mean mass•438g) were purchased from commercial suppl lers.
B... maclnys were maintained In the lab between 17-20oC on a
sand substrate.
Pans of dlstf I led water were Included
within the enclosure for rehydration •
.B... catesbelana were kept In a sheet metal enclosure with
avaf lable water and were used within two weeks of arrival
In the lab.
Mean Cel I Volume
Mean eel I volume Cmcv> determinations were made using
a Coulter Counter, (model Zbl), and Channel lzer, (model
C-1000), Interfaced with a Tektronix 4051 computer.
The
counter produces a pulse which Is In prf nclple proportfonal
to the volume of suspensfon electrolyte dfsplaced by a eel I
wlthfn the countf ng aperture.
The current pulse Is
theoretically assumed to be caused by an fnsulatlng
particle movf ng within a conducting medium, and this
predicts that mcv measurements wll I be affected by eel I
membrane charge. Adams and Gregg (1972) found that errors
f ntroduced by eel I charges are lnslgnlf lcant due to the
9
comparatively large resistive current of the electrolyte.
Additional errors due to eel I path, tumbl Ing of the eel I,
and adherent eel Is passing through the aperture as
doublets, were reduced by use of an electronic editor on
the model C-1000 Channel lzer.
Coulter Electronics suggest
eel I counts be less than 40,000 eel ls/ml for the 100 micron
aperture tube to reduce counting errors, a procedure always
fol lowed In these experiments.
Further correction for the
above anomal Jes was made by using human red blood eel Is In
Isotonic solutlon to cal lbrate the counter and channel lzer.
Since experiments required eel Is to be suspended In
electrolyte of Increasing tonlclty, mcv determinations for
a 23.2 micron diameter latex bead (Coulter Electronics)
were made In salt solutions covering the range experienced
by dehydrated individuals.
For sodium concentrations
between 100-250 mM, mcv as determined on the Coulter
counter was within 1% of Its calculated volume assuming the
latex particle to be a sphere.
Volume regulation experiments fel I Into two
catagorles.
Red blood eel I Crbc) volume was fol lowed
during dehydration In .B.a. marlnus Cn=6) and .B... catesbelana
(n=6).
Animals were weighed after their bladder was
drained by cloacal cannulatlon.
After control blood
samples were obtained by ventrlcular puncture, anlmals were
placed In screened piastre cages and subsequently lost 6-9%
of Initial body mass/day. Dally blood sampl Ing continued
10
untll water losses of 35% lnltial body mass In .BJU.g and 24%
tn .B.A.D.A were achieved.
Control and al I subsequent blood
sampl Ing was made by ventricle puncture.
Blood was
collected In heparlnlzed tubes and a quantity of whole
blood was centrifuged at 4,000 rpm for 2 min.
Hematocrlt
was recorded and plasma sodium and potassium determined on
an IL model 143 flame photometer.
Red eel Is which were to
be analysed by the Coulter counter were suspended In
Ringer's Isotonic for the nonpermeant sodium ton, and
containing 5 mM KHC0 , 1 mM CaCl , 5 mM glucose.
2
3
Addttlonal experiments were performed to describe the
effects of temperature, extracellular potassium, and the
function of the Na+/K+ pump on eel I volume regulation.
Blood obtained- by heart puncture from hydrated .fL. marlnus
and B.... catesbelana was suspended In Ringer's soluttons
ranging from Isotonic to soluttons containing 225 mM Na+.
These suspensions were maintained at either 20°c or 9°C and
mcv determinations made every 15 minutes for 2 hours.
A
comparison of the degree and time course of volume
regulatton was made between eel Is maintained at the two
temperatures.
In order to Investigate posstble tonic contrfbuttons
to volume regulation, red eel Is from hydrated
and B.... catesbelana
a..
martnus
were placed In hypertontc media which
were either potassium free or contained oubain at a
concentration of 10- 3 M. At this concentration, oubaln
11
blocks the operation of the Na +/K + pump CKregenow 1971b).
Mean eel I volume comparisons were made between eel Is In
hypertontc normal, K+ free, and oubatn media.
Hemoglobin Oxygen Affinity
Hydrated .B.a. marl nus Cn=12) and .IL. catesbelana
Cn=12) were doubly pithed, after which the ventricle was
exposed.
A 4 ml sample of blood
containing heparin.
wa~
collected In a syringe
Blood from dehydrated
.BJll.Q
(35% loss
lnttlal body mass, n=9) and Rall (20% loss lnltlal body
mass, n=6) was slml I art ly collected.
Measurement of the half saturation point of
hemoglobln CP50 > was made using the technique of Edwards
and Martin, (1966). Four ml of blood was divided between 2
test tubes and each tube sealed by rubber stopper.
Two 18
gauge needles on which 3-way stopcocks had been mounted
al lowed ff I I Ing of each test tube with gas mixtures under a
positive pressure.
Within these equtl tbratlon tubes, whole
b I ood was f u I I y oxygenated .(gas mt xture 20% 02 , 5% C02 ,
balance N2 > or fully deoxygenated Cgas mixture 5% C02 ,
ba I a nc e N2
) •
Af t er a n e q u I I I b r at I on of 4 5 mI n , 15 0 u I
oxygenated blood was Injected In a 300 ul capll lary tube,
fol lowed by Immediate Injection of 150 ul deoxygenated
blood Into the column of saturated blood. This volume of
blood was mixed anaerobtcal ly and analyzed for Po 2 , pH, and
Pco 2 at 19°C using an IL model 313 blood gas analyzer.
12
Since equll lbrtum oxyhemoglobtn saturation Is dependent
upon the relatlve volume contribution of saturated Hb to
the total volume of mixed blood, the Po 2 of a mixture of
equal volumes of oxygenated and deoxygenated blood Is the
P50·
Determinations were made at a temperature of 19°c, pH
7.55, and Pco 2 of 40 mmHg.
lntracel lular Sod tum and Potassium
Approximately 2 ml of blood was collected by heart
puncture from fully hydrated .B... marl nus Cn=9) and
.B... catesbelana Cn=7).
lndtvtduals were subsequently
dehydrated untll they lost 35% of Initial body mass In
and 25% Jn .B.a.n.A.
.BJL.tg
Animals were doubly pithed and the
ventrlcle exposed for blood col lectlon In heparlnlzed
syringes.
Blood was centrifuged at 4,000 rpm for 10 min In
300 ul caplllary tubes.
Plasma and white eel I fraction
were removed and the red eel Is were blown Into test tubes
weighed to the nearest 0.1 mg, (Sauter model 414 balance).
Red eel Is were dried to constant mass at 40°c.
Two ml of
0.8N HN03 was used to extract Na+ and K+ from the eel Is.
Al lquots of 100 ul, 500 ul, and
ml were evaporated to
dryness In order to concentrate tons.
Lithium dlluent was
added and determinations made on an IL model 143 flame
photometer.
After correction for trapped plasma, C10% wet
13
mass), lntracel lular concentrations were expressed as
meQ/Kg dry mass eel Is.
Viscosity
au.t.g marlnys Cn=3) were doubly pithed and the
ventrlcle exposed.
Blood was collected Jn test tubes
containing ammonium heparin and centrifuged at 4,000 rpm
for 5 min.
Separated plasma was salt loaded to a
concentration of 180 meQ NaCl.
Red eel Is were then
suspended In salt loaded plasma In order to manufacture
hematocrtts between 0-77%.
A 200 ul sample of suspended
eel Is was lmmedlately analysed at 2ooc with a
Wei ls-Brookf teld cone/plate viscometer model LVTDCP.
Viscosity reported was for a shear rate of 450/sec.
RBC's
In hypertontc media tnftfal ly behave as osmometers before
gaining water <Figure 2).
Cel Is placed In salt loaded
plasma for Immediate viscosity determinations are thus
termed nonregulatlng.
Comparison was made with viscosity
of red eel Is In Isotonic plasma of hydrated au.t.g <Hedrick,
unpub I I shed).
RESULTS
Mean Cel I Volume
The results of In vivo regulation of red eel I volume
In .B... catesbelana
and~
marlnys are shown In Figure 1.
Mean eel I volume of hydrated .B... catesbelana Cn=4) was 770 ±
14
20 u3 <±se).
Loss of 14-20% of lnltlal body mass resulted
Mean eel I volume of a._ marl nus
In a mcv of 771 + 18 u3.
Cn=4) red eel Is was 460
±
11 u3, and 451 ± 11 u3 after
water losses ranging from 28-36% of lnltlal body mass.
These data Indicate volume regulation since predicted
reduction of Tnltlal volumes should equal 15% In Ra.
catesbelana and 28% in lL. marl nus if these eel Is acted as
perfect osmometers in hypertonic plasma.
The
mechanism of volume regulation is temperature
dependent as shown in Figure 2 for both species.
Recovery
of eel I volume after challenge by hypertonic medium, (180
mEq Na+), occurs within 45 minutes at 2ooc, whereas at goc,
recovery Ts incomplete after 2.5 hours.
The results of In vitro experiments involving
regulation In potassium free solutions and normal Ringer's
containing oubaln c10-3 M> ls shown for
(Figure 3) and lL. marinus (Figure 4).
Ra. catesbeiana
The relationships
between percent decrease of Initial eel I volume and media
toniclty were similar in both species.
Cel Is placed in K+
free media behaved as simple osmometers and did not volume
regulate Cp>0.05).
In a similar fashion, slopes of
regression I Ines for eel Is in normal and oubaln Ringer's
were not different Cp>0.05).
A significant decrease in
slopes -was found between predicted osmometer and eel Is In
normal Ringer's Cp<0.01>, or eel Is in Ringer's plus oubain.
15
Evidently, extracellular potassium Is required for the
observed volume regulation, but otibaln has no effect.
Hemoglobin Oxygen Aff lnlty
No difference Cp>0.05) was found between hydrated and
dehydrated hemoglobin oxygen affinities In either species.
P5 0 was deter ml ne d to be 4 8 • 8 ± 1 • 4 torr Cn= 1 2 > a n d 5 0 • 6
± 1 .6 torr Cn=6), respectively, tn hydrated and dehydrated
.B.a. catesbelana.
Oxygen aff lnlty was greater tn
.a....
marlnus;
41 .6 ± 1.4 torr Cn=12) In hydrated animals and 43.0 ± 0.5
torr Cn=9) tn dehydrated Individuals.
lntracel lular Sodium and Potassium
The relationships between plasma sodium and
tntracel lular ton concentration were similar for both
species <Figures 5 and 6).
A significant Increase of
lntracel lular K+ and Na+ concentrations accompanied
dehydration In both species Cp<0.05).
Results are I lsted
In Table 1.
Viscosity
Figure 7 shows the viscosity relatlonshlps for
.a....
marl nus red eel Is In Isotonic and hypertonlc (180 mEq
Na+) solutions.
The slope ts significantly lower Cp<0.005)
and the Intercept higher Cp<0.001) In salt stressed eel Is.
A higher Intercept may be the result of salt Interactions
16
with plasma proteins.
Control plasma viscosity (1.8
±
0.04cP) was lower than viscosity of plasma loaded to 180
mEq Na+ C2.6 ± 0.07cP>.
17
TABLE I
INTRACELLULAR SODIUM AND POTASSIUM CONCENTRATIONS
CMEQ/KG DRY MASS) IN THE ERYTHROCYTES OF
.6.&,
MARINUS CN=9) AND B--. CATESBEIANA CN=9).
VALUES ARE MEANS ± STANDARD ERROR ASSUMING
10% TRAPPED PLASMA.
ASTERISK INDICATES SIGNIFICANT DIFFERENCE CP<0.025).
SPECIES
.6.&,
marl nus
B--. catesbelana
CONTROL
DEHYDRATED
40.4 ± 1 .7
73.2 ± 8.7*
K+
234.8 ± 2.3
260.6 ± 6.0*
Na+
34.1 ± 2.3
58.3 ± 3.7*
K+
229.0 ± 3.4
269.o ± a.a*
Na+
18
RANA
825
775
BUFO
450
400
100
140
180
220
PLASMA SODIUM (mEq/L)
Figure 1.
In vivo volume of erythrocytes of .a.a, marlnus
(water losses 28-36% lnltlal body mass)
and .B... catesbelana (water losses 14-20% tnltlal
body mass).
19
800
RANA
•
750
700
.........
C')
:a.
.........
rzl
650
~
::J
~
BUFO
0
>
800
~
~
400
rzl
0
Q
r:l
~
~
<
r:l
~
350
300
0
120
80
TIME IN MINUTES
Figure 2.
Temperature· dependence of RBC volume
regulatlon In .IL. marlnus and .B.a. catesbelana.
Rana,
<•9°C Isotonic,• 20°c lsotonrc,&9°C hypertonrc,
e20°c hyperton r c).
.EW.f.g,
(.
9°C r soton I c,
e20°c lsotonlc~9°C hypertonlc~20°c hypertonlc).
20
40
ra1
:a
30
=
.>
..:I
0
..:I
....<
~
....
:z:
.....
20
pa
Cl2
<
~
=
0
~
Q
~
10
o.o
a.a
1.0
o.e
.
.
0.4
CQAJll2l.t fl.ASMA Na
· AL MEDIA Na
Figure 3.
Volume regulation
of RBC's In .B.a.
catesbe I ana after 1 .5 hours.
Top I I ne Ind I cates
behavior as osmometer, y=71.5 <±0.4)x-0.1 <±0.1),
r=1.00.
y=2 1 • 0
Bottom I lne RBC 1 s In normal Ringer's,
C±5
• 3 ) x - 0 • 4 ( ±2 • 0 ) , r = • 7 8 •
CA , potassium free media;
• , normal Ringer's)
Y , oubaln media;
21
3
~
::a
=
~
0
·>
~
<
....e..
....
.....:z
20
Pa
Cll
<
~
g::
c
~
·Q
. 'if.
.10
o.e.
o.a
1.0
0.4
CONTROL PLASMA Na
FINAL MEDIA Na
Figure 4.
Volume regulatton of RBC's Jn .6.a. martnus
after 1 .5 hours.
y=66.7 <±0.5)x+0.1
Top I lne ts behavior as osmometer,
<±0.1), r=1.00.
Bottom ltne
RBC's In normal Ringer's, y=26.9 <±2.B>x-0.3 <±1.1),
r=.94.
C~, potassium free media;
media;
. , normal Ringer's)
9 ,
ouba f n
22
_.....
.a
·~
280
....~
"tS
...
bl
'Ci:lct
a
aJ
z0
-
240
......
~
<
~
200
:.:::>
~
~
rzl
80
•
0
<
~
E-t
z.....
40
•
0
140
100
180
PLASMA SODIUM (mEq/L)
Figure 5.
The relatlonshtp between red eel I
lntracel lular ton
electrolytes
In~
concentrations
and plasma
catesbelana corrected for 10%
trapped plasma.
(~sodium
r=.93).
y=O.Sx-21, r=.75; •potassium y=1.1x+97,
23
-+a
~
280
••
•
•
•
.,,...
~
bf)
::id
':
s
a.l
z
240
~
0
. 1-4
~
<
~
200
::::>
~
...:i
•
r.J:l
0
<
~
80
E-4
z
1-4
40
•
0
140
100
180
PLASMA SODIUM (mEq/L)
Figure 6.
The relatlonshlp between red eel I
lntracel lular Ion concentrations and plasma
electrolytes In .£L.. marlnus corrected for 10% trapped
plasma.
ce sodium
r=.79).
y=0.5x-14, r=~73; ~potassium y=0.4x+190,
24
Figure 7.
The relationship between .B.a. marlnus
hematocrlt and the In transformation of red eel I
viscosity at a shear rate of 450/sec and
20°c.
Sol Id
I lne Is salt loaded (180 mEq/L), In y=0.028
<±.001 )x+0.90 <±.04), r=.98.
Isotonic plasma.
r=.93.
Dashed I lne Is
In y=0.035 <±.002)x+0.43 <±.07),
25
0
co
0
co
-
r/.l
...:I
...:I
rzl
0
~
(/e.
..__
.
\
0
~
\
\
.
E-4
<
:il
rzl
~
0
~
\
\
\
\
\
0
c.j
0
·~
~
0
0
\
0
E-4
.....
...
~
(d:>). A..LISOOSIA '1'13:0. 111
0
=
26
DISCUSSION
.B... catesbelana·and .B... marlnys regulate red blood eel I
volume during dehydration, (Figure 1).
In vitro
experiments In hypertonlc media Indicate slmllar responses
for red blood eel Is of both species and a common mechanism
for volume regulatlon.
Figure 2 shows the biphaslc nature
of volume regulation In RBC 1 s.
lnitlal ly, eel Is In
hypertonlc media shrink, fol lowed by volume recovery within
45 min for eel Is maintained at 2ooc.
The lnltlal shrinkage
Is not slgnlf lcantly different from eel I behavior as an
osmometer.
Kregenow (1971b), Cala (1977), and Amende and
Pierce (1980),
demonstrated a slmllar blphaslc volume
regulation for red eel Is In ducks, flounder, and mol I uses.
Shown by Figure 2 Is the temperature dependence of volume
regulatlon, since cells maintained at 90C require a longer
time to regain volume In hypertonlc media than eel Is at
2ooc.
Temperature dependence Indicates a mechanism
lnvolvlng an active uptake of plasma osmolytes.
Addltlonal
experiments were designed to test volume regulatlon In the
face of a Na+/K+ pump blockade by oubaln c10-3M), as wel I
as eel I osmotic behavior In potassium free Ringer's •
catesbelana (Figure 3) and
.a..
.B...
marlnus (Figure 4) show a
regulatory behavior In K+ free solution which Is not
27
stgntftcantly different from an osmometer Cp>0.05).
Oubatn
falls to affect volume regulatton since this treatmen--t ts
not slgnlf fcantly different from eel I behavior In normal
media.
These results are In agreement with data presented
for red eel Is of ducks and polychaete worms by Kregenow
C1971b) and Costa and Pierce (1983).
Figure 5 <.B.a.n.A> and Figure 6
(BJL!.s2)
demonstrate
sodium and potassium uptake during dehydration, Indicating
the Importance of these cations In maintenance of RBC
volume.
Schmidt-Nielsen (1975) found sodium permeabll tty
Increased for flounder red blood eel Is In hypertontc media.
Kregenow C1971b) showed an Increase of tntracel lular
potassium In duck red eel Is exposed to hyperosmottc shock.
Volume regulatlon requires extracellular K+ avallabll lty.
Potassium Is accumulated against a concentration gradlant
In both species.
Schmidt and McManus (1974) describe a Na+
and K+ uptake which Is oubatn Insensitive and fact I ltates
cation movement during volume regulatlon.
Nakao et al.
(1963) Isolated from human red blood eel Is a Na+, K+ ATPase
which was not Inhibited by oubaln.
To summarize, volume regulatlon of RBC's In the two
anurans studied rs achieved by active uptake of plasma
potassium via an oubaln Insensitive pump and movement of
plasma sodium down a concentration gradient.
are In agreement with previous studies.
·These results
In addition, this
study demonstrates volume regulation and Ionic uptake
28
during dehydration.
Previous studies have largely Involved
mammal Jan or avian eel Is which do not normally experience
markedly Increased plasma electrolytes with dehydration.
Volume regulatton achieved by uptake of Ions offers
an opportunity to offset general clrculatory lnsuff Jclency
accompantng dehydration.
The effect of Ionic mediators of
hemoglobf n function Is to reduce Hb oxygen aff lnlty.
The
effect of salts Jn lowering oxygen aff tnfty ts thought to
reflect preferential binding of salts by deoxygenated as
opposed to the oxygenated form of hemoglobin CTyuma 1974).
A shift of the oxygen dissociation curve to the right has
been tmpl lcated In the adaptation to anemia and hypoxic
hypoxia In sheep Clenfant et al. 1970).
In this manner,
Increased tntracel lular electrolytes could substantially
Increase del Ivery of oxygen to the tissues and be adaptive
In terrestrtal species to extend tolerance to dehydration.
Control
0 determinations for both species are In
agreement with publ I shed values establ tshed under similar
~
conditions (Hal I, 1968; Tazawa et al., 1979).
Dehydrated Individuals do not have a slgnlftcantly
different oxygen aff Jnlty.
Increased lntracel lular
concentrations do not prove to be adaptive for del Ivery of
oxygen during dehydration In either species.
This
seemingly anomalous finding may be explained In terms of
Increased oxygen affinity caused by other modlf ters of Hb
29
function such as organic phosphates which override the
effects of salts.
Blood flow rate Is Inversely proportlonal to blood
viscosity.
Blood viscosity ts a combined term which
Includes the viscosity of the two componants of whole
blood, blood plasma and red blood eel Is.
The non-Newtonian
behavior of whole blood ts associated with substanttal
protein concentrations and suspended red eel Is.
~
martnus plasma viscosity Increased with Increased plasma
sodium.
Ionic Interactions with plasma protein were
presumably responstble for the slgnlf tcantly higher
Intercept for salt loaded blood CFtgure 7).
Red blood eel I viscosity ts dependent on shape,
volume, membrane rfgfdtty, and mean corpuscular hemoglobtn.
concentration CMCHC).
Erslev and Atwater (1963) found
nearly a doubl Ing of viscosity as MCHC Increased from
24%-38%.
In~
marl nus, the slope of the I tne tn Figure 7
for salt loaded eel Is, Cnon-regulatlng), Is slgnlftcantly
lower than the slope of the I tne for eel Is tn normal
plasma.
These slopes represent eel I viscosity st nee the
second component of the slope, plasma viscosity, remains
constant.
I therefore argue that nonregulattng red blood
eel Is tn hypertontc plasma are more dtstenstble and
therefore less viscous than normal eel Is In Isotonic
plasma, (dashed I fne, Figure 7).
Rand and Burton (1964)
found human red eel I membranes tn hypertonfc solutfon C1.2%
30
NaCl), to be more dlstenslble than membranes In Isotonic or
hypotonlc media.
Melselman et al. (1967) found human red
eel Is In hypertonlc plasma to be more viscous than eel Is In
hypotonlc plasma.
Their reported values lack estimates of
varlabll lty and may merely reflect Increased plasma
viscosity.
Within a physlologlc range of hematocrlts, blood
viscosity In
.a...
marl nus Is lower for red blood eel Is which
regulate volume.
This may not represent a true viscosity
advantage for regulatlng eel Is, since If correction Is made
for plasma viscosity, the previous advantage disappears.
Constraints for regulatlon of eel lular volume In red blood
eel Is may therefore Include factors other than viscosity.
One such selective pressure for maintenance of eel I volume
might be the proper function of membrane bound enzyme
systems.
In summary,
~
catesbelana and
.a...
marlnus maintain
red blood eel I volume during dehydratlonal stress by uptake
of sodium and potassium.
Increased lntracel lular Ionic
concentrations do not alter oxygen del Ivery by shifting the
oxygen dissociation curve to the right.
Hypertonlc plasma
does not appear to Increase red eel I viscosity although
whole blood viscosity Is higher for hematocrlts less than
70%.
Increased viscosity In salt loaded blood may be
largely attributed to Increased plasma viscosity.
31
Volume regulatlon of red eel Is occurs but Is Insufficient
to cancel the effect of Increasing plasma viscosity.
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