143
Gen Physiol Biophys (1998), 1 7 , 1 4 3 - 156
Effect of Ouabain on t h e Breakdown of Adenine
Nucleotides in Glucose-Depleted Nucleated
Red Blood Cells. Characterization of ATPase
M
KAIOMANNI, O
TSIKRIKTSI AND P
TSIANOPOULOU
LaboratoT g of Animal Physiology, Zoology
Department
S(u rice School Anitotle
Umveisity of
Thessalonili
Tin ssalcmiki 54006 Greece
A b s t r a c t . The present repoit confirms the presence of N a + - K + - M g 2 + ATPase in
the eiythrocvte membianes of the frog Rana balcarnca (previously Rana ridibunda)
(Schneidei et al 1993, Sofiamdou et al 1994) T h e N a + - K + - M g 2 + ATPase activity
was 60% reduced lyy the piesence of ouabain The pH optimum was 8 0 the optimum M g 2 + A T P concentration ratio was 2 2 1 T h e existence of an ATPase with
a high Km for A T P (1 48 mmol/1) was postulated
At pH 7 4 and 8 0, the adenine nucleotide pattern of glucose-depleted e i \ t h i o c U c s showed a characteristic leduction m A T P contents \ d e n m e nucleotide
< onccntiations weie highei at pH 7 4 than at pH 8 0 Ouabain inhibited ATP breakdown at both pH \alues studied The strongest inhibition was o b s e n e d at pH 7 4
The decline of the total contents of adenine nucleotides appears to be determined
b\ the late of V \ I P bieakdown
K e y w o r d s : R ana baliamca
Red cells
ATPase — Adenine nucleotides
Introduction
4d< nine nucleotides aie known to be the principal nucleotides m m a t u r e human and
amphibian e i > t h i o c \ t e s (Jacobasch et al 1974, Kaloyianm-Dimitiiades and Beis
1984a), and to pla-\ an important role m the normal metabolism and functioning of
the led cell (Nakao et al 19C1) In contiast to the metabolic stabiht> of hemoglobin
(Shemm and Rittenbeig 1946) adenine nucleotides in mammalian eiythiocytes are
m a dynamic state (Shemm and Rittenberg 1946) The closely connected processes
of sMithesis and breakdown of adenine nucleotides lemams a majoi obstacle of a
Coircspondence to Di M Kaloviamu L a b o r a t o r \ of A.mmal Physiology, Zoolog\
D e p a r t m e n t Science School \ n s t o t l e U i m e r s i t v of Thessaloinki Thessalomki 54006
Gieece E-mail Kaloyian@bio a u t h g r
kalov ldimi et al
144
comprehensive a n a h s i s of then metabolism m the red cell
Even if the nnportaiic e of A T P bieakdown m led cells has long been iecogm/ed,
it has not \ e t been subject of systematic studies m nucleated erythrocytes ATPase is among the mechanisms involved m A T P consumption Aecoichng to Siems
et al (1983) 23% of ATP m human leticulocvtes is split by Na+-K+ \ T P a s c
ATP concentiations can also be affected by v a i \ m g concentiations of fiuctose-2 6bisphosphate and tnose phosphate m Rana balcanica erythrocytes (Kalovianm et
al 1994) On the othei hand, m frog eiythiocv tes ATP is lesponsible foi low (Ting of
H b - 0 2 affinity (Baitlett 1970) Consequently, maintenance of A T P concentiation
is ciucial foi piopei oxygenation of the1 fiog tissues In addition to the possible
íole in ATP consumption, ATPase p l a \ s a fundamental íole m ion tianspoit and
osmoiegulation m aquatic oigamsms (Epstein et al 1967)
To the best of om knowledge howevei, theie have not been airy reports on
ATPase ac tivitv m amphibian ei y throe vtes so fai As a fust step we studied ATPase m the membiane of led blood cells of the amphibia Rana balcanica and t n e d
to chaiactenze it Moieovei, pH-mduced (pH 7 4 and 8 0) changes of adenine nucleotides m glucose-depleted red cells of Rana balcanica were studied By eliminating the hexokmase-phosphofructokmase (HK-PFK) step and pieventmg A T P
íegeneiation via the mam pathway of glycolysis the piocess of ATP bieakdown
was expected to become moie tiansparent Additionally, ouabain added to glucose
depleted cells was hoped to help elucidate the role of ATPase m Rana balcanica
e n thioc ytes
Materials and M e t h o d s
Animah
Fiogs (Rana balcanica) weighing 50 120 g weie supplied Ivy a local dealei aftei
having been caught m the viamtv of Lausa The v weit kept m containers m fiesh
water and used foi experiments within a week
Eipenmcntal
procedure s
Cells weie sepaiated fiom hepainn/ed fiog blood In c entnfugation at 3000 l p m foi
10 mm at 4°C Plasma and buffv coat weie chscaided All fuithei picpaiation and
< entzifugation steps weie earned out at 25°C
Puparation
of crythioc ute membráne
foi ATPase
actimty
measure merit1*
Hemoglobin-ftee membianes foi ATPase measurements weie piepaicd In, modification of the method of Buigei et al (1968)
The led cells weie washed thiee times m ice-cold 0 002 mol/1 Tns-HCl, 0 10
mol/l NaCl pH 7 4 The buffv coat was removed aftei each c entnfugation
I
1
Adenine Nucleotides m Nucleated Red Cells Chaiac teri/ation of ATPase
145
The washed red cells were then pooled hemoly/ed m 30 1 vol/vol hypotonic
solution (0 005 mol/1 Tns-HCl, 0 005 mol/1 \ I g C l 2 , pH 7 65), and centiifuged at
27,000 x c] at 4°C for 15 mm The supernatant was decanted and the mcTnbrancs
weie washed 6 to 8 times with the same volume of ice-cold hemolv/mg solution
until the s u p e i n a t a n t s wcjie almost coloiless The lesultmg fluffv niembianes weie
stoied m an equal volume of fresh hemolvzmg solution All ATPase assays weie
peiformed on the same day when the niembianes weie piepaied
P l o t em concentiations m e n throe y te niembianes weie determined m triplicate
aceoidmg to the method of Low n et al (1951)
ATPase activitv m the niembianes of the led blood cells was measuied aceoidmg to the method desciibed by Keeton and Kaneko (1972) The activitv was
expiessed as pmols P i / n n u / m g of membiane protein
Piepaiation
of glue osc-depleted
cells
The procedure to piepaie glucose-depleted e ells was c a m e d out aceoidmg to a
modification of the method desciibed by Tu et al (1979) The eiy tlnoe vtes weie
suspended m a 5-fold exc ess of an isotonic solution containing 2 minol/1 glucose 06
mmol/1 tiietlianolamine buffei 1 minol/1 \ a j H P O i and 100 nnnol/1 NaC'l, pH 7 4
Aftei 10 m m incubation with glucose the eivtlnocvtes weie centiifuged at 1500
x ej foi 10 m m The glucose1 depletion piexoss was staited at the second washing
step with the1 addition of a 10-fold excess of isotonic solution containing 33 mmol/1
tiietlianolamine 1 mniol/1 \a_>HPO[ and 100 mmol/1 NaCl pH 7 i
The t h u d washing was < a m e d out without bufíei m a 10-fold excess of a solution containing 100 mmol/1 NaCl, 5 mmol/1 KC1 1 mmol/1 MgCl_> and 0 5 mmol/1
N a i t l P O ] pH 7 4 Subsequently, the eivtlnocvtes weie sepaiated In (entiiiugation
at 0500 x c/ foi 10 mm
The packed c (Tis weie suspended 111 the indicated intubation medium ( V i C l
KC1 N a j H P O i ) to make a homatociit value of about 30% Antimycm (4 nig/100
ml) was added to pievcnt bactciial (ontammation duimg the incubations T h e pH
of the led cell suspensions was adjusted l a p i d h bv addition of 0 3 mol/1 NaOH 01
0 3 mol/1 HC1 and collected as neccssaiv (± 0 08 j)H) with the same1 solutions
Ouabain 1 0 _ t mol/1 was added to the incubation medium if indicated
De tc 1 rnrnation of adenine nucleotide s
1 01 the detcimmation of adenine nucleotides the samples weie added to one1 volume
of chilli d peichlorie acid nuclei stiong stilling Aftei e entnfugation the supernatant
was taken up and neutiali/ed with 0 25 volume of a solution containing 0 5 mol/1
tiiethanolaimne hydioc hlonde and 1 3 mol/1 K2CO3 The KC10i-pi(?upitate was
K moved by c entnfugation The samples weie then fiozen and kept at —120°C until
the assay The concentiations of A T P ADP and AMP weie deteimined e n / y m a t i callv aceoidmg to Boiginevei (1970)
146
Kalov íanin et al
Statistic al
evahition
Student's /-test (;; < 0 005) foi u n p a n e d giouped samples was used
Results
1
1
Tabic 1 shows the (fleets of extiacellulaily
ATPase activity
less a(tivity
added M g
1
ATPase was dependent upon N a
than when M g 2 + was only piesent
+
2+
. Na
oi K
+
+
and K
+
ions on
alone but it showed
A combination of sodium and
potassium without magnesium pioduced significant activation compaied to t h a t
m the absence of the ions m extiacellulai medium
Also a dependence of ATP­
ase on \ I g 2 + was suggested by a use m aítivitv compaied with that measured m
the piesencc of sodium only
In the absence of extiacellulai ions ATPase activity
íeachc d half the values measured in the presence of N a + and K +
1
lose significantly
ATPase activity
to 51 //moles P i / m m / g p i o t e m when all t h e h e e ions tested weie
piesent m the incubation medium T h e maximum activity of membiane A T P a s e
was measured at 2 87 mmol/1 M g 2 +
120 mmol/1 N a + and 5 mmol/1 K +
Table 1. Effects of magnesium sodium and potassium on ATPase activity of Rana bal
came a led blood cell membranes
Ionic composition of the
incubation medium mmol/1
Mg > + (2 87) Na+ (120) K+ (5)
M g 2 + (2 87)
\ a + (120)
K + (120)
\ a + (120) K+ (120)
Na+ (120) K+ (5)
No ions piesent
//mols Pi/mm/g ])rotem
51 9 ± 4
40 2 ± 3
33 5 ± 2
373±2
41 8 ± 3
41 8 ± 3
19 3 ± 2
3
7
1
4
8
8
3
Eadi value íeprescnts mean ± S E M of 4 diffeient expennients Thiee to hve animals
vuie used foi each ox])Pimient the blood cells of which were pooled
When the led cell niembianes weie incubated at v a n o u s pH, total ATPase
activity giew at pH 8 0 indicating a pH maximum in this vicinity (Table 2) Fig 1
shows t h e effect of ouabain on t h e ATPase activity of Rana balcanica e i y t h r o c y t e
membranes A 50% inhibition was achieved at ouabain cone initiation of 1 0 "
+
l
mol/1
+
As shown in Fig 2, t h e t o t a l ATPase and ouabam-msensitive ( N a - K - M g 2 f ATPase) activities peaked at M g 2 + concentration of 8 mmol/1 On t h e othei h a n d
the ouabam-sensitivo Na + -K + -ľvIg 2 + -ATPase
mciement showed a m a x i m u m at
147
Adenine Nucleotirles m Nucleatetl R e d Cells Chaiac t e n / a t i o n of ATPase
T a b l e 2. Effect of p H on total ATPase activity
pH
En/vmati< activ ltv
(//mols P i / m m / g / j j r o t e m )
20
39
18
45
9
3
0
4
±
±
±
±
2
3
4
4
7
8
5
3
Each value l e p i e s e n t s m e a n ± S E M of 4 different experiments T h r e e to ftve animals
weie used foi each e x p e i i m e n t t h e blood cells of which were polled
T h e conditions of t h e e x p e i i m e n t were as follows M g 2 + 2 87 m m o l / 1 N a + 0 12 mol/1
K + 0 005 mol/1
55-
50-
45
35-
30-
s,
25-
0 00000
I
0 00005
0 00010
-/h
1—
i
i
~~I—
02
04
06
08
10
mmols/l ouabain
F i g u r e 1. Effect of o u a b a i n on t h e N a + K + M g ' + - A T P a s e activity T h e following ex
p e n m e n t a l conditions weie employed t e m p 25°C pH 8 0 MgCl> 3 m m o l / 1 , NaCl 112
miiiol/ KC1 5 m m o l / 1 t i m e 135 m m Each value rejnesents m e a n ± S E M of 4 different
e x p e n m e n t s T h r e e to five animals were used foi each expeiiment and t h e n blood cells
weie jjooled
Kalov íanni et al
148
50'
c
a)
without ouabain
40
C)
o.
m
c
30
F
a.
ouabain sensitive
05
O
E
3.
20
in the presence of 10"4 mmol/I ouabain
10
8
—r—
10
mmols/l Mg
12
15
2+
Figure 2. Eftee t of Mg >+ on total ATPase ae tiv ltv ouabam-sensitive ATPase and ouabain
insensitive ATPase aitivitv The e one c ntiation of NaCl was 114mmol/l that of KCl was
12 mmol/1 j)H 8 0 and time 120 mm The activity m the absence of ouabain lepiesents
the total ATPase activity The activity m the piesence of ouabain at 10 ' mol/1 lepiesents the ouabam-mseiisitive activity The difference between the two curves lepiesents
the ouabam-sensitive activity Each value lepiesents the mean of 4 different experiments
Tlucc to five animals woe used foi each expeiiment and then blood cells were pooled
\ l g ' 2 e one d u r a t i o n of 5 mmol/1 The ae tiv ltv peaks o b s e n e d foi the1 total ATPase
and foi ouabain-sensitive activities coiiespouded to a M g 2 + ATP concentration
i alios of 3 2 1 and 2 1 iospectivrelv The 2 1 eoneentiation latio íeciuned foi I\Ig,_+
and ATP suggests that the actual substiate of the en/vine is M g 2 + - A T P complex
Since the ouabam-sensitive and ouabam-msensitive activity nicienients b o t h decieased as the M g 2 + e oneontiation was raised above 8 mmol/1, fiee Mg2"1 might
have mteifcied by competing with ]\Ig 2 + -ATP (oinplex foi the en/yme oi might
have alteied the eii/vmo by binding at anothei site independent of the active substiate site
Figs 3, 4 5 show data on the changes of A T P ADP and A M P at pH 7 4 and pH
8 0 The same blood samples weie used to perfonn identical irieasumirents at pH
7 4 and at pH 8 0 It may be> seen that eluimg an initial p e n o d of 30 mm a decline
Adenine Nucleotides m Nucleated Red Cells Characterization of ATPase
149
o
o
.a
•a
o
E
oľ
i-
<
o
F i g u r e 3. Time couise of ATP breakdown m glucose depleted led blood cells and in
glucose depleted led blood cells treated with 10 ' mol/1 ouabain at j>H 7 4 and 8 0
Indicates statistical sigmfic anc e of the diffeience between the respective values at pH 7 1
and ])H 8 0 ** Indicates statistical significance of the difference between the lespective
v,lines m the absence and m the presence of ouabain at pH 7 4 All value's obtained fiom
the same experiment weie statistically significant from of values obtained at time 0
of
ATP and nicleases of A D P and AMP weie obseived which may accompany
the ae c umulation of glycolytic mtei mediates D u i m g the subsecment 30 m m (at
60 nun)
AMP and A D P lose and ATP dec leased to values similai as measuiecl
at pH 8 0 D u i m g this jiciiod the sum of nucleotides lemameel constant (Fig 6)
Theicartel
ATP concentiation decieased at a íapid l a t e winch declined with time
both at pH 7 4 and pH 8 0 A D P and A M P showed tiansient peaks after 3 h
incubation to fall slowly theieaftei
The sum of nucleotides decieased at a neailv
c c instant i a t e of about 0 3 0 4 /miols/ml cells/h d u i m g the 2 h of incubation T h e
mam diffeience between the values measrued at the two pH values is a greater
dec lease of the sum at t h e lowei pH duimg the hist 2 h of incubation Also, A T P
declined stiogei at pH 7 4 t h a n at pH 8 0 (Fig 3)
Ouabain did not affect t h e mass action íatios of adenylate kinase at pH 8 0
wheieas at pH 7 4 the mass ac tion latio of adenylate kinase mcieased significantly
aftei 3 h incubation of the led cells
Figs 3, 4, 5 and 6 also show the effect of ouabain on the e hanges m ATP, A D P .
AMP and t h e sum of adenine nuc leotidcs at pH 7 4 and pH 8 0 m glucose depleted
cells The adenine nucleotides exhibited a ehaiacteiistic p a t t e r n as desciibed eaihei
Kalov íanm et al
150
- • —D—
-A—A—
Control al pH 8
In the presence of ouaban at pH 8
Control at pH 74
In the presence of ouaban at pH 7 4
0 14
o
T>
0 12
XI
"O
010
o
o
Q)
0 08
0_
o
<
0 06-
O
E
n.
0 04
0 02
Time (h)
Figure 4. Time couise of ADP bieakdown m glucose depleted led blood cells and m
glucose depleted led blood cells treated with 10 ' mol/1 ouabain at pH 7 4 and 8 0
Indicates statistical significance of the diffeience between the íesppctive values at ]»H 7 4
and pH 8 0 ** Indicates statistical significance of the diffeience between the íespective
values m the absence and m the presence of ouabain at j)H 7 4 O Indicates statistical significance of the diffeience between the íespettive values m the piesencc and in
the absence of ouabain at pH 8 0 All values obtained fiom the same expeiiment weie
statistically significant fiom values obtained at time 0
with a dee line' of VTP wheieas ADP mc leased to flat maximum and declined
thoieaftei
The (>liects of ouabain at different pH values on the bieakdown of ATP niche ate
a 14 4 6 ' / inhibition of this piotess at j)H 7 1 At pH 8 0 wheie ATPase activity
is highei than at pH 7 4 (Table 2) ouabain seems to exeit significant effects on
A1P degiadation and on the bieakdown of A D P at the 3id, 1th, and 5th horn of
incubation (Fig 4) It seems that ATPase is i ('sponsible foi ATP bieakdown both
at pH 7 4 and pH 8 0 (Table 3)
Discussion
This is the hist ATPase study in amphibian eiytluocytes ATPase jilavs a fundamental íole in ion t i a n s p o i t and osmoiegulation in aquatic oigamsnis Theiefoie
Adenine Nucleotides in Nucleated Red Cells Chaiac ten/ation of ATPase
- • — Control at pH 8
—•— In the presence of ouabain at pH 8
- A - C o n t r o l at pH 74
—A— In the presence of ouaban at pH 74
-I
0
1
1
1
1
2
1
1
151
,„
1
3
1
1
4
1
r
5
Time (h)
Figure 5. Time couise of AMP bieakdown m ghuose depleted led bool <ells and in
glucose d( pletí d led blood cells treated with 10 ' mol/1 ouabain at pH 7 4 and 8 0
Indií ate s statistical significance of the diffeienc e betv\( en the lisppc tiv e values at pH 7 4
anil j)H 8 0 " Indicates statistical significance of the diffociue between the rtspeitivi
values m the absence and in the presence of ouabain at pH 7 4 All values obtained from
the same expeiiment were statistically significant from values obtained at time 0
this study is i m p o i t a n t foi the elucidation of metabolic processes within the led
cell The experiments piesented heiem yield interesting insights into the dynamics
of the adenine nucleotides m Rana balcanica eivtlnocvtes The data on the t i a n siloiv peiiods duimg glucose depletion show eleaily that the decline of ATP and
the e o n e s p o n d m g use of ADP and A M P at pH 7 4 and 8 0 are piobablv due to a
íelative excess of the H K - P F K system activitv as (ompaied with pyruvate kmasi
m human led cells (Rapopoit et al 1979)
Scheme 1 illustrates the glycolytic pathway and adenylate kinase that yield
ATP and the two pathways of AMP bieakdown, one via adenylate deaminase and
the othei one phosphatase-ielated followed by adenosine deaminase and nucleoside
phosphoivlase Uneloi conditions of glucose depletion the bieakdown of total nucleotides pioeeeds at a slowly dee leasing late which is less íapid at the alkaline ])H
It amounted to 0 3 /iinol/inl/h at pH 8 and to about 0 4 /miol/ml led cells/h at
pH 7 4 (Fig 6) T h e l a t e does not appeal ( o n e l a t e d to A M P coneentiation which
152
Kalov íanni et al
0
1
2
3
4
5
Time (h)
F i g u r e 6. Changes m total levels of adenine nucleotides d u i m g gliuose (lej)letion of Rana
baliarne u led blood cells at p H 7 4 and 8 0
T a b l e 3 . Inhibition of A T P - d e g i a d a t i o n by ouabain
I>H
Inhibition ('/)
7 1
11 4G
88
< 1
vanes ovei the pH lange studied This would indicate that the en/vine systems
involved m th( AMP bieakdown an s a t m a t e d with íespect to then substiatcs
Howevei when fiog eivtlnocvtes weie me ubated m the pie sence of glue osc to
tal nucleotides seemed to mciease (Kalovlanm-Dinntiiades and Beis 1981a) Two
explanations may be consideied foi this fact Foi one some unknown mechanism
may be postulated by winch degiading enzymes aie inhibited Such an explanation
appeals most unlikely as it has not been suppoited bv leseaieh ev idem es It is
much moie hkelv that the liKiease of total adenine nucleotides is a u suit of t h e n
e ontinuous lesynthesis which pioceeds as long as glucose is available Toi sue h a
lesynthesis m fiog erythrocytes an intact adenine moiety is a necossiv piecon
chtion (Miya/aki et al 1975) AMP may then be pioduced eithei by adenosine
kinase fiom adenosine oi via a specific nbosyl phosphate tiansfeiase fiom adenine
(Scheme 1) It has been repotted foi Rana balcanica eiy tlnoc v tes that the high
Adenine Nucleotides m Nucleated Red Cells Chaiae ten/ation of ATPase
NAD(P)H
/r>»
153
NAD(P)
«*Lac
23P,G
.ATP.
3PG
NADH«
NAD
GAP
• R 5P «-•• R 1 P
pool of the hexose
monophosphate shunt
hypoxanthme
NADPH2
NADP
S c h e m e 1. Sources of ATP and bieakdown of adenine nucleotide-
concentiations of adenosine may piomote deammation of adenosine lathei t h a n
jihosphoiv lation upon its entiv into the eivthiocytes (Kalovianm et al 1993) At
low concentiations of adenosine, fiog e n thioe v tes mcoipointed adenosine both v la
adenosine km<ise and via its conversion to the hypoxantlnne pathway (Miva/aki
et al 1975) E x p e n m e n t s on the utilization of adenosine in human led cells indicate that the metabolic fate of adenosine also depends on its concentiation in
the medium At low adenosine concentiations it is converted to ATP whereas
at extiae (llnlai e one entrations highei than 10 //mol/1 all aeknosine t i a n s p o i t e d
into the cells is deammated (Plagemann et al 1985) Furthermoie it has been
suggested that since A M P deaminase is stionglv inhibited m the cell mostly by
2 3 dijihosphoglvcerate oi bv unknown factois the m a m AMP bieakdown pathway
pioceeds via phosphatase (Rapopoit et al 1990)
\ M P is an aetivatoi of Rana balcanica crvthiocyte P F K (Kaloyianm et al
1991) Therefore, the lowering of the AMP concentiation at pH 8 0 compaied to
jiH 7 4 caused an inhibition of phosphohuc tokinase (PFK) This jiH dependence of
PTK activity (weaker activity at pH 8 0 t h a n at pH 7 4) has been icpoited eaihcr
foi Rana balcanica ciythiocvtes (Kaloyianm et al 1994) Thus, the glycolytic l a t e
of Rana balcanica cry thioevtes is affected both by pH (Kalov lanm-Diimtiiades
154
Kaloyianm et al
1983) as well as by the activatory action of AMP on P F K Nevertheless aceoidmg
to Rapopoit et al (1981) about one half of the usual glycolysis inclease is due to
the effec ts of adenine nucleotides
Rana balcanica ciythiocvtes aie nucleated and show high aetivitv of N a + - K + ATPase m i elation to non-nucleated mammalian eivthiocytes (Palma et al 1991
Rvbakowski and Lelmiann 1994) At j)H 8 0 the A T P concentiation significantly
diminished fiom 0 9 to 0 06 /miol/nil led e ells after 5 h incubation (Fig 3) which
may have been related to the enhanced activity of ATPase at this pH (Table 2) The
exponential decline of the ATP concentrations in the absence of glucose indicates
a stiong e oncentiation dependence of the ATP degiading piocesscs Rapopoit c t
al (1979) icpoited that when human led (ells were incubated nuclei the same
expeiiment al conditions, m the absence of glucose foi 3 h the concentiation of a
ledueing substance glutathione met eased and that of pyruvate decieased
The high mtiae ellulai K + concentiation as well the high K + / N a + iatio m
Rana balcanica eiy tlnoe v tes (7 34) (Kaloyianm et al 1997) show a positive coiíelation with the high ATP contents of the eivthiocytes (Kalovíanin-Diinitiiades
and Bers 1984a) the same also has been suggested foi sheep erythrocytes (Miseta et al 1993) It is possible therefore that the high mtiacellulai K + concentiations inciease the activity of the K + -sensitive íegulatoiv en/vinc p v i m a t e kinase m Rana balcanica erythrocytes (Kaloy lanm-Dimitiiades arrd Beis 1984b) In
Rana balcanica eivtlnocvtes the Embdcn-Mcyeihof pathway is the mam SOUK e
of ATP (Kalovlanm-Dimitiiades and Beis 1984a) Therefoie K + tianspoited by
ATPase may be late limiting with legaid to the geneiation of ATP molecules via
the Embden-Meyerhof pathway m Rana balcanica eivthiocytes
Ouabain inhibits the bieakdown of ATP m Rana balcanica led cells (Fig 3)
Ouabain also inhibits N a + - K + - M g 2 + - A T P a s e isolated from Rana balcanica led cell
membiane (Fig 1) The ouabam-sensitive ATPase makes up SO'/ of the total A T P
ase activity It has been postulated that lactate pioduction m human eivtlnocvtes
may be controlled by the membiane adenosine t u p h o s p h a t a s e activity (\\ liittam
et al 1964) In Rana balcanica led cells ouabain inhibited lactate pioduction bv
eivtlnocvtes suspended in adenosine suggesting that lactate pioduction m Rana
balcanica icel cells is íelated to active cation tianspoit and jiossiby to N a + / K 4 ATPase (Kaloyianm et al 1993)
The fact that ouabain is stiongei in glucose depleted cells incubated foi 30
nun at alkaline pH suppoits the notion that the i eduction of A T P concentiation at
pH 8 0 might be due to enhanced activity of the N a + - K + - M g 2 + - A T P a s e at this pH
(Fig 3) Accoidmgly the element of the contiol matiix (Su ) which gives the relative
dependence of the metabolite concentiation of the e n / y m e activity (Heinnch and
Rapopoit 1974) indicates t h a t activation of fiog ATPase at JDH 8 0 results m a
deciease of ATP (S,, = - 0 03) Thus, changes of ATPase activity would explain
the pattern of adenine nucleotide levels at alkaline j)H values On the othei hand, it
Adenine Nucleotides m Nucleated R e d Cells Chaiae t e n / a t i o n of ATPase
seems conceivable that the high A l u value foi the Rana balcanica
155
Na+-K+-ATPase
of 1 48 ± 0 84 mmol/1 may not account foi the bieakdown of A T P obseived aftei
2 h incubation of glucose depleted cells Aceoidmg to Seliner (1966) A T P may be
supplied t o ATPase m t h e mu room lionnient of the inembiane Hence mtiacellulai
ATP might not play a dneet role m legulatmg the ATPase activity
T h e piesent i e p o i t confiims t h e jnesenee of N a + - K + - M g 2 + - A T P a s e m
haleanica
eivthiocytes
Rana
T h e observations concerning t h e m e m b i a n e A T P a s e sug-
gested t h a t t h e en/vine is involved m t h e active t i a n s p o i t of elec tiolytes aeioss
biological niembianes. and t h a t the led blood cell maintains its cation composition
and volume by the action of a K + - N a + exchange p u m p
T h e p i e p o n d e i a n e e of
ATPase activity m iclation to A T P pioduction may be lesponsible foi changes m
adenine nucleotides o c c m i m g in glue osc depleted led blood cells of Rana
balcanica
Both pH aird ouabain aie effective modulators of adenine nucleotide breakdown
Noveltheless the exact physiological loles of the enzymes involved m adenine nu(leotide bieakdown will have t o be levealed by futuie studies
References
B a i t l e t t G R (1970) P a t t e r n s of p h o s p h a t e c o m p o u n d s m i e d blood cells of m a n a n d
animals Adv E x p Med Biol 6, 245 256
Beigmevei H U (1970) M e t h o d s of Enzv matíc Analysis Akadémie Ver lag, Berlin
B m g e i S P Fujn T H a n a h a n I F (1968) Stability of bovme erv t i n o c v t e membiane
Release of enzymes of lipid c o m p o n e n t s B i o i h e m i s t i v USA 7, 3682
Epstein F H K a t / A ] P i e k f o r d G E (1967) Sodium a n d p o t a s s i u m adenosine triphos
p h a t a s e of gills Role in a d a p t a t i o n of teleosts to salt water Science 1 5 6 , 1245
1247
Hermit h R R a p o p o i t T A (1974) Lmeai ste adv-state t i e a t m e n t of e n z y m a t i c chains
Geneial piopeities, control a n d effectoi strength E m 1 Biochem 4 2 , 89 95
Tacobasíh G M m a k a m i S R a p o p o r t S M (1974) Glycolysis of t h e e i v t h i o e v t e In
Cellular a n d Molec ulai Biology of E i v t l n o c v t e s ( E d s H \ o s h i k a w a a n d S M
R a p o p o i t ) University of Tokyo Piess
Kaloyianm M Mithaeliihs B M o u t o u K (1993) Effect of adenosine on glucose m e t a
b o h s m of Rana rielibunda e i v t h i o c y t e s I E\ji Biol 1 7 7 , 41 50
Kaloyianm M K o t m i s K G o u n a n s E G (1994) Purine ation a n d kinetic propel ties of
phosphofruc tokmasc from Rana iidibunda e i v t l n o c v t e s Com]) Biochem Physiol
1 0 7 B , 479 487
Kaloyianm M , Giannisis G G a v i n l P Boukla A (1997) Metabolic effects a n d c ellulai
volume responses induced by lioiadienalme m nucle ated eiv thiocv tes I Exj) Zool
2 7 9 , 337 - 3 4 6
Kalov l a n m - D i m i t n a d e s M (1983) Studies on t h e encigv metabolism of Rana nclibunila
e i v t l n o c v t e s Dissertation Anstotle Umvíveisitv of Thessalomki Greece
Kalovlanm-Dimitriades M Beis I (1984a) Studies on t h e energy m e t a b o l i s m of Runa
iichlninda erv thioc v tes I C o m p Physiol B 1 5 5 , 109 115
Kalov laiini-Diimtiiades M Beis I (1981b) P u i i h t a t i o n catalytic a n d íegulatoiv piopeities of Rana rielibunda e r y t h r o c y t e pyruvate kinase Conij) Biochem Physiol
7 9 B , 245 250
156
K a l o y i a n m et al
Keeton K S , Kaneko I I (1972) C h a i a t t e i i / a t i o n of adenosine t r i p h o s p h a t e m e i v t h r o (v te n i e m b i a n e s of t h e cow Proe Soc Exj) Biol Med 1 4 0 , 3 0 — 3 5
Lowiv O H Rosebiough N 1 , F a n A L Randall R I (1951) P i o t e m m e a s u r e m e n t
with t h e Folm phenol leagent I Biol C h e m 1 9 3 , 2 6 5
275
Miseta A , Bogner P Beienvi E Kellcrmaver M Galambos C Wheatlcy D N , Camel on I L (1993) Relationshij) between cellular A T P , potassium s o d i u m a n d
m a g n e s i u m concentiations in m a m m a l i a n and avian e r y t h r o c y t e s B i o d n m Biophvs Acta 1 1 7 5 , 133 139
M n a / a k i H \ a m b u K H a s h i m o t o M (1975) Utilization of adenosine foi nucleotide
synthesis in t h e e i v t h i o c y t e s of some animals I Biochem 7 8 , 1075 1078
Nakao M Nakao T V i m a / o e S Aoshikawa H (1961) Aclenosme t i i p h o s p h a t e a n d
s h a p e of e i v t l n o c v t e s I Biochem 4 9 , 187 492
P a l m a F Ligi T S o v e i c h i a C (1994) C o m p a i a t i v e aspects of N a + - K + and C a 2 + - M g ' + ATPase m c r y t h i o c v t e n i e m b i a n e s of v a n o u s m a m m a l s C o m p Biochem Physiol
1 0 8 A , 609 617
Plage m a i m P G W Y\ ohlhue t ei R M K i a u p p M (1985) Adenosine uptake t i a n s p o r t
and metabolism m h u m a n eiv throe vtes J Cell Physiol 1 2 5 , 3 3 0 336
Raj)0])oit I R a p o p o i t S M a i e t / k i D Eisner R (1979) T h e bieakdown of a d e n i n e
nue 1( otides in glucose-depleted h u m a n l e d tells Acta Biol Med G e i m 3 8 , 1 4 1 9
1429
R a p o p o i t 1 R a p o p o i t S Eisner R (1981) Vce umulation of p h o s p h a t e esters a n d decline
of ATP m ice! cells incubated in intra is caused by lack of jiviuvatc Ac t a Biol Med
G e n u 4 0 , 115 121
R a p o p o i t I D i u n g I Rajxipoit S M (1990) C a t a b o h s m of adenine nucleotides in r a b b i t
blood (ells Biomeel Biochim A c t a 4 9 , 11 10
Rvbakowski I K Lehmaim W (1994) Decreased activity of e r y t h r o c y t e m e m b i a n e
VI Pases m dcpiession and schizophrenia Neuropsvehobiology 3 0 , 11 14
Schneidci H Smsch U Sofiamdou T (1993) T h e watei frogs of Gieece bioacoustic
evidence foi a new species Z Zool Svst Evol Foisch , 3 1 , 36 47
S c h n e r S L (1966) O i g a m z a t i o u of enzymes m h u m a n cry throe vtes m e m b r a n e s A m e i
1 Phvsiol 2 1 0 , 139
She m m D R i U e n b e i g D (1946) T h e life s p a n of the h u m a n red blood cells I Biol
C h e m 1 6 6 , 627 636
Siems W Dubiel W Dumdev R Mullei M R a p o p o r t S (1983) Balance of ATP e ons u m p t i o n of íetie uloc y tes Biomed Bioc him Acta 4 2 , S218 S222
Sofiamdou T Schneider H Smsch I (1994) Comparative electrophoietic investigation
on Rana bale ami a and Rana ruhhunda from noithcrii &iee( e Alvtes 1 2 , 9 3
108
Tu L D Maietzki D R a p o p o r t S R a p o p o i t I Scliewe Cn Lange I Eisner R
(1979) T h e effect of t e m p e i a t u i e a n d ouabain on the b i e a k d o w n of A T P a n d
2 3-bisphosphoglye crate in glucose-depleted h u m a n tells A c t a Biol Med G e n u
3 8 , 1413 1417
\\ h i t t a m R Agai M E Wiley I S (1964) C o n t i o l of lactate p i o d u c t i o n by m e m b r a n e
adenosine t i p h o s p h a t a s e activity m h u m a n erythrocytes N a t u r e 1 3 , 1111 1112
Final version accepted April 23 1998