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A crabtree effect in amytal- or progesterone-treated ascites tumor cells
ARC’IIIVES
OF
BIOCHEMISTRY
AKD
A Crabtree
BIOPHYSICS
98,
67-76 (1962)
Effect in AmytalAscites
Rcccivc4
or Progesterone-Treated
Tumor
Cells’
January
22. 1962
The effect of glucose on respiration associstcd with auccinate oxidation was studied with intact nscites tumor cells treated with nmytnl or progcsteronr whic~h inhibit the oxidation of reduced diphosphopyridinc nuclcotidc (DPNH) The addition of glucose produced a significant depression in the rate of oxygen consuml)tion associated with succinate oxidation. This inhibitory effect was observed in tlrc prcsonce of uncoupling agents but was partially rcleawd b>- the addition of Tri:: or l~hosphate in concentrations with high buffering capacity. Seither glucose in the prcscnec of iodoucetntc nor 2-dcosrglucosc t~rertcxl an a finding which is in contrast to the calassic inhibitory effect on this respiration, Crabtree effect. Furthermore, in the normal intact cells, glucose partiall!: released the pronounced inhibitory cffcct of 2-deosyglucaose and \-ice versa; it is therefore concluded that the inhibitory effect, produced by 2-deosyglucos(! differs from that produced by glucose. The data are compatible with the explanation that a factor in this dq)ression of respiration is a change in in~ramit,ochontlri31 pH as a conwlucnce of in~rawlluln~ acidification.
ridine nuclcotide (DPN)-cytochome c reduct,ase. The effect of hydrogen ions, which arc released when glucose is phosphorylated, was also examined since Santa10 and Park” found that the Crabt,ree effect observed in tumor homogenates is released by the addit’ion of efficient, buffers. It was of particular int,ercst to study the effect of glucose on succinate oxidation under conditions which modify pH or the rate of glycolysis in the presence of the above inhibitors since react’ions involving oxidation of reduced pyridint nuclcotidcs arc influcnccd by hydrogen ions i 1, 2j. Thus, the effects of glucose in the prcdcncc of iodoacetat~e, 2-dcoxyglucose, uncoupling agents, and phosphate were invcstigatcd. In addition, it was of interest to tictcrmine whether an inhibition of rcspira-
IX’RODUCTION
The mechanism by which glucose inhibits the respiration of ascites tumor cells is of inter& since the Crabt,ree effect has been considered to be a manifestation of respiratory control. In an attempt to determine the sites of inhibition by glucose, the effect of glucose on respirat.ion in which a portion of the respiratory chain is blocked was investigated. Specifically, the effect of glucose on succinoxidase respiration of intact ~11s was studied. The oxidation of reduced diphosphopyridine nucleotide (DPNH) by oxygen was prevented by the use of amytal or progesterone, inhibitors of diphosphopy’ This work was supported in part by rescnrch grants from the National Institutes of Health. U. S. Public Health Service, and an institutional grant from the American Cancer Society.
I’ R. (!rl,Pijo-Ssnt:~lo 67
and J. Park, in prelwxtion.
68
IsHIBITIoN
BY
(;I.r-wsr:
0F
S;Iv~~s.am
0xII~r\~Iox
IN
THE
PRESEXCE
OF
AMYTAI,
I’;hrlich I,ettr@ ascites tllmor cells equiwlent to 17 mg. dry tissue weight were incuhnted iu CCL++of 0.003 fret Iirebs Ringer phosphate buffer in :I total volume of 3.0 ml. Amytnl in :I final conccntmtion III when added was present in the center ronlpartnient , :rnd subst,rntes were tipped in after 10 min. of temperature equilihrnt ion at 38”. Succinaic-1 ,&CL4 and glucose, in a final concrnt ration of 0.01 :I/, were then tipped in from the side arm. The oxygen consumption wns linear throllghout the period of inculuriion 160 min.). t\dditions
Amytal
1. oxygen uptake Cwbtree effect Succinate-I Cl”Or to c;40z pmles
-
None
(;lucose
+
12.6 1.7
+
8.3 2.2
Succinatr-1
,-k-C’”
+
14.2 5.7
Succinate-1 glucose
,I-CL4 +
+
94 2.9
tion by glucose is observed with neoplastic cells having a low rate of glycolysis, e.g., the 6C3HED lymphoma and the MClM rhabdomyosarcoma. M.ITERIAI,S
-WI)
METHODS
The cletailr of the experimental prowdure arc ewentially the dame as described in the pwrious p:il)er (3). RESULTS
AND
DISCUSSIOS
EFFECT OF GLLCOSE ON SUCCINATE OXID.~TION IN THE PRE~E~UCE AND XssENcE OF INHIBITORS 0~ DPN-CYTOCHROME c REDUCTASE The effect of amytal on the respiration and glycolysis of Ehrlich Lettrk ascites tumor cells is demonstrated in Table I. The introduction of 3 x low3 A! amytal produced a marked inhibition of respiration which was largely restored upon addit,ion of succinate. The further addition of glucose produced a significant depression in the rate of oxygen consumption as it did in the absence of amytal. Thus a Crabtree effect is demonstrated in which the respiration does not involve the oxidation of reduced diphosphopyridine nucleotide.
pu retzt
pakms
Lactic acid accumulated /mdlPS
Glucoseutilized Jl,?ifJlC3
0 0.81 34
18.6 25.0 2.62 0.39
34 49
3.20 0.32
13.5 15.5
0.20 1.98 14.0 25.7
12.5 15.7
The addition of amytal increased glycolysis,” as measured by glucose utilization and lactate accumulation, and stimulated lactate accumulation to a greater extent than glucose disappearance which suggests that the increased level of DPNH promotes a more efficient utilization of glucose foi lactate production. The use of labeled succinate in this experiment permitted furt,her characterization of the respiratory substrate. Since little radioactivity was incorporat’ed into the respiratory CO2 in t,he presence of amytal, it appears that the major portion of oxygen consumption is derived via surcinoxidase. STIMULATION OF TIIE CRABTREE EFFTXT IS CELLS WITH Low R.~TES OF GLYCOLTSIS BP THE ADDITIOX OF INHIBITORS 01; DPK-CYTOCHROME c REDUCTASF: It is known that not all neoplast.ic cells exhibit a high rate of glycolysis, and we have observed that t.he 6C3HED lymphoma and MClM rhabdomyosarcoma cells have ” In stlldic.5 vurron tly rillcle~~~~, higher conwntrations of smytal or high conccntmtions of pl’ogesterone produce inhibitor)effects on glycolyeis which are probably related to an rxcessirc destrrlc~tion of ndc~nosinr~ I ril~hoq)h:ltc (.\TI’).
Experiment
1
if
Ihygcn uptake I,;tct ic acid
:tccrmul:ttetl
G~IICOSP utilized
0 5 x 10-J
8.1 0
0 5 x 10-a
0 0
7.8 (3.7) 0.6 6.3 12.6
10.3 1.0 0 0
6.0 11 .o
0 5 x IO-~
X.8 (15) 1.9 (52) 6.3 12.6 6.0 lo.!)
Amytal
kkperiment
osygen uptake
0 1 x lo-” 3 x 10-s
6.1 0.3 0.4
Lactic
0 1 x lo-” 3 x lo-” 0 1 x 10-a 3 x lo-”
0.01 0.1 0.1
Glllcose
acid
:uxurnulated
utilized
comparatively low rates. It was therefore of interest to determine whether a Crabtree effect is observed in these cells and to study the relationship of glycolysis to t,he respiratory inhibition in the presence of progesterone or amytal which increase glucose utilization. In the experiment with 6C3HED lymphoma cells reported in Table II, the Crabtree effect was hardly observable in the absence of progesterone. However, a Crabtree of significant magnitude was produced in the presence of succinate by the addit,ion of progesterone which doubled the rate of glycolysis.
2
4.9 (23) 0. i 0.3 4.2 1l.i 18.3 i.6 12.7 15.6
9 .5 4.6 3 5 0.02 0.2 0 .2
7.6 (22) 2.7 (11) 1.6 (54) 5.7 17.3 19.5 T.0 10.1 12.2
A Crabtree effect was observed in the MClNI cells which have a similar low rate of glycolysis, but it was less than half that observed in cells with a high rat’e of glycolysis. The respiratorv inhibition observed in the presence of succinate was also markedly increased upon amytal addition and was accompanied by an increased glycolysis as measured by lactate accumulation and glucose utilization. Thus the above experiments support the idea that t’he Crabtree effect is not a peculiarity of tumors but a property of a high ratmeof glycolysis. The mechanism for this inhibitory effect, would appear to require
'70
WEIV-NER
AND
an explanat,ion which is dependent the intensity of glycolpsis. COMPARISOS
OF
YSECDO-CRABTREE
ON RESPIRATIOS SUCCINATE
the presence of iodoacetatc reported by Racker (5) and by Chance (6) is obsei~vctl in the absence of progesterone. This inhibitory cffcct occurred in thus lmwncc of concentrations of iodoacctat~e which lmwntcd tlie accuiiiulation of lactat’c. In contra&. the adtlit,ion of glucose and iotloacctat,e to progctsterone-t.rcntctl cells proclucetl a stimulation of rwpimtion. In fact, at the higher concentration of iodoacctatc studied, the stiiilulat~ion of rcsl)irat,ion l)y glucose was over 2056. Although the iiit~clianisiii by which these coinpounds inhibit respiration iu not clear, our cxperiuients at’<’ in agrcenient nit11 the statement of (‘olowick that the (Irubt,rec effect, in the pwsenw of iodoacotatle is different from that of tlrc classic Crabtree effect. The diffcrencw obserrctl wit,11 2-tlcoxVglucose in the untrcatcd ascites cell and “in the amyt.al-t,rc~:lt’ecI cells arc rcportccl in Table IV. 2-l)coxyglucosc protluccd :L niarkccl inhil)ition of respiration in the at,-
upon
EFFECTS
ASSOCIATED AKD
CEREIJO-SANTALO
WITH
REDUCED
DIPIIOSPHOPYRIDINE NUCLEOTIDE OXIDATION
Glucose in the presence of iodoacetate and 2-deoxyglucose produced inhibit’ions of respiration which ham been considered by Papaconstantinou and Colowick as pseudoCrabtree effects (4). The effect.s of these compounds on the respirat.ion associated with succinnte oxidation in the presence of progesterone was therefore compared. Xs described in an expcriuicnt reported in Table III, the effect of glucose in the lmscncc of iodoacctat,e was stud&l with the two respiratory chains. The charactcristic inhibition of respiration by glucose in TAHI,I’ I+:FFECT
ofi'
I~IH).~(~~:T.~TE
III ON
'I'EIE
KFFE(-I
CRABTREE
Ichrlich LettrC ascites tumor cells eyuiv:&mt to 25 mg. dry tissue wright were inclll):tt,ed for 30 min. at 38°C. I’rogest,erone ant1 iodoacetale were present in the (aenter compxtment and srlhstratcs were :&led 10 min. after kmperature equilibration.
IAA 0
15.1 7.8
Craht,ree
15 X lOF.If
0
12.2 8.5
12.8 9.3
0 .3
18.7
3oL,;,
-
+ glucose Crabtree
x lo-’ .If
I- 28c,,, -l-
effect
.---~Succinate ,Succinate
11
effect
27’, ;,
1
I
I 1.2 O.fP 1.8 1 .R
‘L Results
obtained
in a separate
hut, similar
experiment
CRIBTREE
EFFECT
sence of amytal but only a slight or negligible effect in the presence of amytal. Thus it would appear that the mechanism by which 2-deoxyglucose inhibits respiration is different from the classic effect of glucose. The most striking evidence for a difference in the Crabtrcc effects procl~~e~l by glucose and 2-deoxyglucose is obtained from cxpcrimente with the normal intact cell. In the untreated cc’lls, the Crabtrce effect produced by glucose is 40% and that produced by 2-deoxyglucose is 48% !reported in Table IT). \\Xen both arc prcscnt, the inhibit,ion is only 230/A. The release of the glucose-induced Crabtree effect by the addition of 2-deoxyglucose has also been reported by Wick et al. (7). Our finding t’liat the reverse is true, that glucose relcases the 2-tleoxyglucosc-induced Crabtree effect, suggwts that the inhibitory mechanism of each is quite different. TIIE
EFFECT
OF UNCOUPLISG
ON THE
Two
71
ON SUCCI?;ATE
EFFECT
OF
2~1~~osuc:r~r-c~osr THE
PRESENCE
ox
OF
KES~IRATION
1s
;ZMYTAL
Ehrlich Lettri! uscites tumor cells equivalent to 24 mg. dry weight were incubated for :I period of 1 hr. under conditions similar to those dewrilwd in T:~l)le I.
None Glucose (0.01 31) 2-I)eosyglncose (0.02 31) (;lucose (0.01 31) + 2deosyglucose (0.02 111) Succiriate (0.01 111) Snccirmte (0.01 :\I) + I glncose (0.01 31) Succimtte (0.01 dl) + 2deosyglucose (O.o2 Jl) I
I 7.4
, 6.0 1 50 7.7 / 1
AGENTS
CHAISS
The succinoxidase chain differed wi-ith regard to the use of uncoupling agents. As seen in Table V, in the absence of amytal the inhibition of respiration by glucose was considerably less markctl in the presence of the uncoupling agent. However, in the prcsence of amytal the addition of dicumarol did not change the magnitude of the Crabtree cffert. This difference in the two respirat,ory chains st,udied was also noted when dinitrophenol served as the uncoupling agent. Thus, if uncoupling agents release t,he Crabtrec effect on the entire chain, it would appear that there is a difference in the Crabtree effects on the DPN-flavoprotein portion of the chain as compared t’o the cytochrome components, or a diff‘erence in the response of the flavoprotein to uncoupling agents. AMditional evidence t’hat the uncoupling agents aBcct DPNH oxidation diffcrcntly than succinatc oxidation is obtained from the data with labeled succinntc in this cxperimcnt. In the absence of amytal the uncoupling agents produced a marked stimulation in respirat,ion but did not increase the incorporation of succinatc carbon into the
respiratory CO2 .4 These findings with intact cells where problems of artifacts due to isolation techniques arc minimized arc in agreement with t,hc data of Siekevitz et nl. (8,9) who suggested that the dinitrophcnolactiwted adenosinetriphosphatase (XTPreflects only one l)liosase) mainly phorylation, that occurring in the dinphorase region, and t,hc findings of Hatefi et (11. (10) 1~7.110 suggested that the oxidation of pyridinoprotein substrates but not sucrinatc is strongly regulated by a respiratory control mechanism. T~IE EFFECT
OF BITTERS OS THE “SUCCINOXIDASE CRARTRW EFFECT”
The failure of uncoupling agents to release the Crabtrcc effect on the succinoxidase syst.em is also compatible with the ’ .\lthough this finding mny also be interpretal to indicate that, ox&cet;lte formation inhibit:: tlrc, rate of oxidation via suwinoxidnse so that further stimulation is prevented, this :t*qears lmlikcl? since, in preliminnry rsperiment~, xe hare failed to observe n stimulntion by dinitrol~henol at pH 6-6.5 in which incorporation of zuccinntc-I .4-P into tlrc> req)ir:Ltory CO2 :mtt prPrjumal)ly os;~lnwtate tlc~:~rhoryl:~l ion is :~l)prwial)lt~.
72
WEKNER
A?JD CEREIJO-SAXTAI,O TABLE
THE
EFFWT
OF
I'N(Y)LPIJSG
AGESTS PRESEN\‘CE
OS AK,)
V
THE
CRABTREE
;ZBSENCE
OF
I~~FFIKTS
OBSERVEI)
IS
THE
AMYTAI.
Ehrlich Lettrk ascites tumor cells equivalent to 24 mg. dry tisstle weight werr inrrltxlted for 50 min. Dicumarol (2 X lo-” M) and dinitrophenol (1 X 10e5 ;II) were lipped in from t,hc side arm. Sllwin:Ltp and glucose were in a final conccnt ration of 0.01 111.
Additions I -~-
+ I - / +
-
N0Ile
1 12.8
(:lucose Succirinte Succinnte Succinate ~uccimtte phenol Succiriat e
1 6.4 14.0 7.2 17.5 1 15.1
Srlccinate
+ gIllrose + dinitrophenol + glucose +
+ dicumxrol + glucose + dicumwol
Dicurnarol (ilucose f
THE
dinitro-
EFFECT
ON
+
50
16
49
36
14
30
2.31 2.50 2 33
0.42 0.28 0.40 0.22
15 15
1.69
18.7
16.9 17.2 16.0
dicunlarol
OF ~'HOSPHSTE
0.9 1.3 i.2 4.6 8.3 5.1
-
THE
CRABTREE
EFFECT
C)BSERVEII
IN
THE
PRESENCE
OF ~'ROGESTI~:HOSE
Ehrlich Let,tri! ascites tumor cells equivalent t,o 25 mg. dry tissue weight were incrlkxtted for 30 mill. at 38”. Progesterone was present in the center compartment, and succinnt~e-1 ,4-C14 and glucose werr tipped in from the side arm 10 min. after temperature equilibration.
Additions
- I+ Succinate-1 Succinnte-1 phosphate ~moles) None Phosphate Phosphate Succin:tte-1 Succinate-1 phosphate Succinate-1 phosphate pmoles)
,-l-Cl1 ,4-C’” + (100
I 1
’ (50 pmoles) (100 mmoles) i ,-L-C” ,-l-C” + ’ (50 pmoles) ,4-C’” + (100 ,
-
-
1 7.25 1 6.65 1 8.4 7.2 1 7.1 8.4
‘+
,~ i.3
f
1 7.15 6.9 7.1 6.5 i.4 ~ 6.85
: +
i.35
;:‘:
i 7.0 ( I
-
+ i 1 5.6 5.3
33 ?J
+
0 (50 0.51
0.47
1.29
16.2 20.5
13.7 20 0
20.0 23, i 25.0 18.8
0.5 6.1 6.1
0.9 0 .9 1.0 2.8 1.3
54 30
0.50 0.56
16.9 19.8 ~ 24 .!) 0.36 li.0 0.14 19.8
6.2
5. 1
18
0 56
0.50
1.0
a. 9
CRABTREE
EFFECT
effect is exerted cannot be att,ributed to a decreased glucose utilization ; to the contrary, the decrease in the Crabtree effect occurred in the presence of an increased glycolysis as measured by glucose disappearance and lactic acid product,ion. Thv inhibitory effects of glucose in the presence and absence of progesterone were also Audied untlcr conditions in which (Tris) tris (hydroxymet,hyl) aminomethane served as buffer. As seen in an experiment described in Table YII, the Crabtree effect as well a:: the rate of glycolysis was obserwd to increaw by the addition of Tris buffer in the absence of progesterone. Thus, the effect of phosphate in 6nulating the Crwbt,rec cffcct’ in intact cells would also appear to be related to the increase in glycolysis producctl by it’s buffering effect. The simult,aneous measurement of PUCcinntc-1 .4-(‘11 into tlio CO- which pre-
idea that intracellular acidification may be responsible for the observed inhibit,ion produccd by glucose in the presence of progesterone. It was therefore of int,erest to determine whet2ler a Crabtrcc effect was observed in the presence of progesterone in highly buffered media. In an experiment described in Table VI, the effect of glucose on the inhibition of rcspirat,ion in the presence and absence of progesterone was studied at varied concentrations of phosphate buffer. As had been observed by Kvamme i 11) , added phosphate did not release but increased the classical Crabtree effect observed with intact, cells. However, t,he Crabtree effect obwrvcd in the presence of progesterone decreased as t,he concent,ration of phosphate was raised to levels which mew more cfficicnt in t,hcir buffering capacity. The diminution in the extent to which the Crabt,rcc
THE
1
OF
TRlS
I%I-FBER
OS
h;SI’IRATlOZI
IN
Fkrlich Let trC ascites tumor cells equivalent volume of 3.0 ml. with Ca ++-free Krehs-Ringer was in a final concentration of 5 X 1W4 J-1.
73
OS SUCCISATF:
THE
PRESENCE
AXIl
i\BSb:SC’E
OF
I’RO(:E:~TER()N~:
to 28 mg. dry tissue weight were inruhatcd in :I total phosphate for a prriod of 10 nlin. at 38”. Progesterone
-
+
-
-I-
i
+
-
-
C’ .r
‘rrioles
39
3.3 20.7
Succimtte + pmoles Tris
50
10.8 6.0
44
5.5 3 .:3
1.i 18.2
Succinatr + pmoles Tris
100
1O.T 5.x
46
5.0 3 .2
18.0
25.0 2.4 20.7
Succinate + 100 ~rnoics phosphnt e
4.x 3 4
~ 2.5 , 25.2
Y-one
0.T ~
0.X
palms
0.48 1.21
Lalolll.5
0.48 0.38 0.38 0.35
4.4 3.0
100 ~rnoles Tris
-
1.2
0.50 0.27
74
WEKKER
AND CEREIJO-SANTALO
sumably measures oxalacetat,c decarboxylation provided an index of the intracellular pH in this experiment. Thus, the incorporation of succinnt’e-1 ,4-C11 into the respiratory CO, observed principally with low intracellular pH was diminished as the concentration of Tris was elevated. The addition of Tris buffer in the presence of progesterone produced a decrease in the inhibition of respiration by glucose despite an increased glycolysis as measured by lactic acid accumulation. Furthermore, the partial release of the inhibition by glucose was as great with Tris as that observed with added phosphate. It also should be pointed out that the stimulation of lactate accumulation by the addition of Tris was equivalent to that observed with phosphate buffer. GENERAL
duced by glucose alone or that the inhibition produced by t,hesc compounds is based on critical pH changes associated with early phosphorylation. It is therefore of interest to examine the evidence reported hcrc which purport,s that there arc differences in the mechanism of the inhibitory effects observed in the presence of glucose and in the ot’her cases. The evidence that pseudo-Crabtree cffects are exerted by these compounds is based on the findings that neither glucose in t’he presence of iodoacetam nor 2-deoxyglucose exertecl an inhibitory effect on the “succinoxiclasc system.” The additional finding that, glucose released the 2-deoxyglucose-induced Crabtrec effect and vice versa can be considered as confirming the idea that 2-deoxyglucose exerts a pseudoCrabtree effect.
COKSIDERATIONS
PSEUDO-CRABTREE EFFECTS OF %D~ox~~~ucos13 OR GLGCOSE IN THE PRESENCE OF IODOACETATE
The suggestion that the inhibition of respiration by glucose may arise from the release of hydrogen ions has been proposed by Tiedemann and considered by Chance, Racker and others [cf. review by Ibsen (1211, but this concept has not received much experimental support. The studies of Rosent,hal et al. (13)) McKee et al. (14)) Chance and Hess (15)) and Racker (5) establish that the inhibition is not due to an extracellular pH change. However, evidence that the pH change may be greater within the cell than in the medium (16) lends support’ to the possibility t’hat intracellular acidification, as a result of insufficient internal buffering capacity anal rapid glycolysis, plays a part in the Crabtrec cffeet,, as considered by Block-Frankenthal and Wcinhouse (2). Some of the objections to the idea that pH changes are in part responsible for the inhibition produced by glucose have been pointed out, by Ibsen (12)) and it becomes necessary to assume that, t,he mechanism by which glucose in the presence of iodoacetate or 2-deoxyglucose inl1ibit.n respirat,ion differs from that pro-
EVIDENCE FOR THE SUCCINOXIDASE CRABTREE EFFECT DUE TO INTRACELLULAR pH CHANGES
Prior to a consideration of the part which intracellular pH changes play in the classic Crabtree effect, it is pertinent to examine the evidence for an explanation of the “succinoxidase Crabtree effect” based on changes in intracellular pH. In support of the idea t’liat the “succinoxidasc Crabtret: effect,” arises principally from the release of hydrogen ions produced during glycolysis, phosphate in concentrations with high buffering capacity or Tris was found to almost, completely release t,hc inhibitory effect. Furthermore, uncoupling agents were not able to rclcase the inhibitory effect produced by glucose. In addition, neither glucose in the prescncc of iodoacctate nor 2-deoxyglucose exerted an inhitntory cffcct on t,he “succinoxidase system.” It is somewh:at difficult, to assess the loci of respiratory inhibition in the present stuclies although an obvious site is the sucrinic dehydrogcnasc reaction, which has been rcportctl to exhibit, a rather sharp pH optimuni bctwccn pH 7 and 8 (17). In atltlition, Cooper and Lchninger have rcportctl that the oxygen consumption associated with the span of cytochrome c to oxygen is markcclly dclu~~ctl by a rlccrrawc in pH
(18). Therefore, it, would uppar that the inhibitory effects produced by the lowering of intracellular pH on glucose addition may also be exerted on components of the respiratory chain other than succinic tlchydrogenase. RELATIONSHIP OF THE SUCCINOXIDASE CRABTREEEFFECT To THE CIJasslc CRABTREEEFFECT From the above considerations it is likely that. the terminal components of the cytochrome chain are inhibited by pH changes owing to intracellular acidification. Since the terminal electron-transport chain is presumed to be common to DPI%H oxidation as well, it appears reasonable to assume that intracellular pH changes are also involved in the classic Crabtree effect. Of course, the comparison of t’he succinoxidase Crabtrce effect with the classic Crabtree effect is made difficult since the effects of glucose on the DPN-flnvoprotein portion of t,he respiratory chain have not been studied, and the present study lends further support to the importance of the dinphornse region in respiratory control. The possibilit,y remains that the effects of 2-dcoxyglucose or glucose in the presence of iodoacetate are exerted on the flavoprotein portion of the chain. However, t’he mechanism by which respiration is inhibited by 2-cleoxyglucoscr or glucose in the prcsencc of iodoacctatc rcmains obscure. The observation that the inhibitory effects of glucose in neoplasms with low rates of glycolysis were significantly greater upon addition of inhibit~ors of DPh’-cytochromc c reductase suggests that the Crabt,rec cffeet is a property of a high rate of glycolysis, as has been reported by Rosenthal et ~(1. ( 13) and Luganova et (11. 119j. This evirlcnw is also compatible with the explanation that the initiation of a Crabtrw effect involves a rate of glucose phoq)horylation sufficiently great to release hydrogen ions by which the intracellular pH is loncrcrl. However, t,hcxe studies do not. rule out the involvement of a competition of coenzymcs of glycolysis and respiration in the classic C’rabtrec effect despite the occurrcncc of the r~nhanwtl inhibitory effects under csonditions
of a lowered respiration where the requircmcnts for t,he phosphorylating systems are considerably diminished. It, is of int,erest that t,he Crabtree effect is not as pronounced in the presence of bicarbonate (2). a buffer which we have obacrvcd maintains the pH of the external medium more effectively than phosphate buffer, and recently Zatti and Rossi (,20,1 have reported that glucose stimulates respiration in biwrbonate buffer but inhibits respiration in phosphate buffer, an obacrwt,ion that can be intcrprcted to indicate that bicarbonate releases the Crabtrer tlffcct.” STIMULATION OF RESPIRATIOS BY CLWOSE: IN THE PRESENCEOF IODOACETATEOR Low CONCESTRATIOKSOF G~ccom In contrast to the observed respiratory inhibition produced by glucose alone, a stimulation of succinoxidasc respiration by glucose was observed in t,lie presence of iodoacetate, and is analogous to the stimulation of respiration by low concent.rations of glucose report,ed first by RlcKec et nl. f 14). This enhancement in oxygen conwilption is attributed to the production of adcnosine diphosphate (ADP) marlc awilable by glucose phosphorylation via the hcxokinaac reaction. Unpublished work by one of t.hc authors (Santa10 IL’ with homogenates of Ehrlich :iscit,es cells or normal rat brain has shown that it, is possible to wry the rwpiratory response to glucose tq changing the ratio of supernatant to nlitocliondia. I3y increasing the mitoclioncfri:~ or decrrasing t.he supernatant, conditions under which the rate of glycolysis is low, glucow is found to exert :I stiniulntory effect
76
WENNER
-iND
on respiration; by decreasing the mitochondria or increasing t,he supernatant, conditions in which the rat,e of glycolysis is high, a Crabtree effect appears which can be reversed by the use of suitable buffers. These observations lead to the consideration that glucose may exert a biphasic response, namely a stimulation of respiration when the hexokinase reaction is slow; but when the hexokinase reaction is fast, a respiratory inhibition appears instead. In summary, it is concluded that the inhibition of succinate respiration by glucose is produced by intracellular acidification, and that whether an increase or decrease in respiration is observed upon glucose addit,ion depends on t,he rate of glycolysis. The authors are indebted to John Hackney and Lewis Keeler for t,heir very capable technical assistance, and to Dr. T. S. Hauschka for supplying the mice bearing ascitea t,umor cells used in these studies. REFEREXCES 1. RACKFX, E.. J. Bid.
Chcm. 184, 313 (1950). L., ASD WMSHOUSE, S., Cancer Research 17, 1082 (1957). 3. WENNER, C. E.. Arch. Biochem. Riophys. 98, 58 (1962). 4. P.~~.~cossTA~T~~-ou, J.. AND COI,OWICK. S. l’., J. Biol. Chem. 236, 278 (1961). 2. BLOCK-FRASPENTHAL,
CII:REIJO-S~~~T.I\I,O 5. RACKER, E., Ann. X. Y. Acd Sci. 63, 1017 (1956). 6. CHATCE, B., J. Bid. Chem. 234, 2416 (1959). 7. (:HRISTEXSES, E., BROOKS, J. L., STEWART, c. J., AND WICE~, .2. S., Biochcm. Wiophys. I<+ scmd~ Commurts. 5, 209 (1961). 8. SIEKEVITZ, I’., L,iiw, H.. ERWIXR, L., AYII LIXDBERC, O., Niochim. et Biophys. Actn 29, 378 (1958). 9. L6w, H., SIEKEVITZ, P., ERNSTER, L., AXD LINDHP:RG, O., niochim.
et Biophys.
ilcta
29,
392
( 1958). 1-‘., AND HAAWK, A. G., 10. HATEFI, Y., JVRTSHUK, Arch. Biochern. Riophys. 94, 148 (1961). 11. KVAMME, E.: Acta Physiol. Stand. 42, 204 (1958). 12. lesn-, Ii. II., Cancer Hvseurch 21, 829 (1961). 13. ROSESTHAI,, O., BOWIE, M. ii., AND WAQOXER, G., Sciemx 92, 382 (1940). 14. MCI&:, R. W., LOSBERG-HOLM, F., ASD J~rm.. J., Canw~~ 12esenrch 13, 537 (1953). 15. CHASCE, B., ASI) HESS, B., Arm. n;. Y. Acad. Sci. 63,100s ( 1956,. 16. DEWEY, I>. I,.. AID GREEK, F. O., Hiochcm. J. 72, 160 ( 1959). 17. BURRIS, 11. H. in ‘LManonletric Tcchniqucs and
Tissue Met~:~holism” (TYmbreit, Burris, :~ntl Stauffrr, Etls.). 2nd ed., p. 105. Burgess Pull. Co.. Minnwpolis, Minn., 1949. 18. Coor~rc, C., ASI) LEHSIXER, A. I,., J. Wid. Chcm. 219, 519 (1956). 19. I,UCASOY.A, I. ti., SliITZ, I. E’., ASD T~o~~o~or~c~, T’. I.. IMAI~/~/ Akwl. Xwk S.S.S.R. 112, 1082 (1957). 20. %.4TTI, 121., ASI, RosSI, Ii’., Ilt/h//r J. Nioc,/c?r,r 10, 19 (1961).
OF
BIOCHEMISTRY
AKD
A Crabtree
BIOPHYSICS
98,
67-76 (1962)
Effect in AmytalAscites
Rcccivc4
or Progesterone-Treated
Tumor
Cells’
January
22. 1962
The effect of glucose on respiration associstcd with auccinate oxidation was studied with intact nscites tumor cells treated with nmytnl or progcsteronr whic~h inhibit the oxidation of reduced diphosphopyridinc nuclcotidc (DPNH) The addition of glucose produced a significant depression in the rate of oxygen consuml)tion associated with succinate oxidation. This inhibitory effect was observed in tlrc prcsonce of uncoupling agents but was partially rcleawd b>- the addition of Tri:: or l~hosphate in concentrations with high buffering capacity. Seither glucose in the prcscnec of iodoucetntc nor 2-dcosrglucosc t~rertcxl an a finding which is in contrast to the calassic inhibitory effect on this respiration, Crabtree effect. Furthermore, in the normal intact cells, glucose partiall!: released the pronounced inhibitory cffcct of 2-deosyglucaose and \-ice versa; it is therefore concluded that the inhibitory effect, produced by 2-deosyglucos(! differs from that produced by glucose. The data are compatible with the explanation that a factor in this dq)ression of respiration is a change in in~ramit,ochontlri31 pH as a conwlucnce of in~rawlluln~ acidification.
ridine nuclcotide (DPN)-cytochome c reduct,ase. The effect of hydrogen ions, which arc released when glucose is phosphorylated, was also examined since Santa10 and Park” found that the Crabt,ree effect observed in tumor homogenates is released by the addit’ion of efficient, buffers. It was of particular int,ercst to study the effect of glucose on succinate oxidation under conditions which modify pH or the rate of glycolysis in the presence of the above inhibitors since react’ions involving oxidation of reduced pyridint nuclcotidcs arc influcnccd by hydrogen ions i 1, 2j. Thus, the effects of glucose in the prcdcncc of iodoacetat~e, 2-dcoxyglucose, uncoupling agents, and phosphate were invcstigatcd. In addition, it was of interest to tictcrmine whether an inhibition of rcspira-
IX’RODUCTION
The mechanism by which glucose inhibits the respiration of ascites tumor cells is of inter& since the Crabt,ree effect has been considered to be a manifestation of respiratory control. In an attempt to determine the sites of inhibition by glucose, the effect of glucose on respirat.ion in which a portion of the respiratory chain is blocked was investigated. Specifically, the effect of glucose on succinoxidase respiration of intact ~11s was studied. The oxidation of reduced diphosphopyridine nucleotide (DPNH) by oxygen was prevented by the use of amytal or progesterone, inhibitors of diphosphopy’ This work was supported in part by rescnrch grants from the National Institutes of Health. U. S. Public Health Service, and an institutional grant from the American Cancer Society.
I’ R. (!rl,Pijo-Ssnt:~lo 67
and J. Park, in prelwxtion.
68
IsHIBITIoN
BY
(;I.r-wsr:
0F
S;Iv~~s.am
0xII~r\~Iox
IN
THE
PRESEXCE
OF
AMYTAI,
I’;hrlich I,ettr@ ascites tllmor cells equiwlent to 17 mg. dry tissue weight were incuhnted iu CCL++of 0.003 fret Iirebs Ringer phosphate buffer in :I total volume of 3.0 ml. Amytnl in :I final conccntmtion III when added was present in the center ronlpartnient , :rnd subst,rntes were tipped in after 10 min. of temperature equilihrnt ion at 38”. Succinaic-1 ,&CL4 and glucose, in a final concrnt ration of 0.01 :I/, were then tipped in from the side arm. The oxygen consumption wns linear throllghout the period of inculuriion 160 min.). t\dditions
Amytal
1. oxygen uptake Cwbtree effect Succinate-I Cl”Or to c;40z pmles
-
None
(;lucose
+
12.6 1.7
+
8.3 2.2
Succinatr-1
,-k-C’”
+
14.2 5.7
Succinate-1 glucose
,I-CL4 +
+
94 2.9
tion by glucose is observed with neoplastic cells having a low rate of glycolysis, e.g., the 6C3HED lymphoma and the MClM rhabdomyosarcoma. M.ITERIAI,S
-WI)
METHODS
The cletailr of the experimental prowdure arc ewentially the dame as described in the pwrious p:il)er (3). RESULTS
AND
DISCUSSIOS
EFFECT OF GLLCOSE ON SUCCINATE OXID.~TION IN THE PRE~E~UCE AND XssENcE OF INHIBITORS 0~ DPN-CYTOCHROME c REDUCTASE The effect of amytal on the respiration and glycolysis of Ehrlich Lettrk ascites tumor cells is demonstrated in Table I. The introduction of 3 x low3 A! amytal produced a marked inhibition of respiration which was largely restored upon addit,ion of succinate. The further addition of glucose produced a significant depression in the rate of oxygen consumption as it did in the absence of amytal. Thus a Crabtree effect is demonstrated in which the respiration does not involve the oxidation of reduced diphosphopyridine nucleotide.
pu retzt
pakms
Lactic acid accumulated /mdlPS
Glucoseutilized Jl,?ifJlC3
0 0.81 34
18.6 25.0 2.62 0.39
34 49
3.20 0.32
13.5 15.5
0.20 1.98 14.0 25.7
12.5 15.7
The addition of amytal increased glycolysis,” as measured by glucose utilization and lactate accumulation, and stimulated lactate accumulation to a greater extent than glucose disappearance which suggests that the increased level of DPNH promotes a more efficient utilization of glucose foi lactate production. The use of labeled succinate in this experiment permitted furt,her characterization of the respiratory substrate. Since little radioactivity was incorporat’ed into the respiratory CO2 in t,he presence of amytal, it appears that the major portion of oxygen consumption is derived via surcinoxidase. STIMULATION OF TIIE CRABTREE EFFTXT IS CELLS WITH Low R.~TES OF GLYCOLTSIS BP THE ADDITIOX OF INHIBITORS 01; DPK-CYTOCHROME c REDUCTASF: It is known that not all neoplast.ic cells exhibit a high rate of glycolysis, and we have observed that t.he 6C3HED lymphoma and MClM rhabdomyosarcoma cells have ” In stlldic.5 vurron tly rillcle~~~~, higher conwntrations of smytal or high conccntmtions of pl’ogesterone produce inhibitor)effects on glycolyeis which are probably related to an rxcessirc destrrlc~tion of ndc~nosinr~ I ril~hoq)h:ltc (.\TI’).
Experiment
1
if
Ihygcn uptake I,;tct ic acid
:tccrmul:ttetl
G~IICOSP utilized
0 5 x 10-J
8.1 0
0 5 x 10-a
0 0
7.8 (3.7) 0.6 6.3 12.6
10.3 1.0 0 0
6.0 11 .o
0 5 x IO-~
X.8 (15) 1.9 (52) 6.3 12.6 6.0 lo.!)
Amytal
kkperiment
osygen uptake
0 1 x lo-” 3 x 10-s
6.1 0.3 0.4
Lactic
0 1 x lo-” 3 x lo-” 0 1 x 10-a 3 x lo-”
0.01 0.1 0.1
Glllcose
acid
:uxurnulated
utilized
comparatively low rates. It was therefore of interest to determine whether a Crabtree effect is observed in these cells and to study the relationship of glycolysis to t,he respiratory inhibition in the presence of progesterone or amytal which increase glucose utilization. In the experiment with 6C3HED lymphoma cells reported in Table II, the Crabtree effect was hardly observable in the absence of progesterone. However, a Crabtree of significant magnitude was produced in the presence of succinate by the addit,ion of progesterone which doubled the rate of glycolysis.
2
4.9 (23) 0. i 0.3 4.2 1l.i 18.3 i.6 12.7 15.6
9 .5 4.6 3 5 0.02 0.2 0 .2
7.6 (22) 2.7 (11) 1.6 (54) 5.7 17.3 19.5 T.0 10.1 12.2
A Crabtree effect was observed in the MClNI cells which have a similar low rate of glycolysis, but it was less than half that observed in cells with a high rat’e of glycolysis. The respiratorv inhibition observed in the presence of succinate was also markedly increased upon amytal addition and was accompanied by an increased glycolysis as measured by lactate accumulation and glucose utilization. Thus the above experiments support the idea that t’he Crabtree effect is not a peculiarity of tumors but a property of a high ratmeof glycolysis. The mechanism for this inhibitory effect, would appear to require
'70
WEIV-NER
AND
an explanat,ion which is dependent the intensity of glycolpsis. COMPARISOS
OF
YSECDO-CRABTREE
ON RESPIRATIOS SUCCINATE
the presence of iodoacetatc reported by Racker (5) and by Chance (6) is obsei~vctl in the absence of progesterone. This inhibitory cffcct occurred in thus lmwncc of concentrations of iodoacctat~e which lmwntcd tlie accuiiiulation of lactat’c. In contra&. the adtlit,ion of glucose and iotloacctat,e to progctsterone-t.rcntctl cells proclucetl a stimulation of rwpimtion. In fact, at the higher concentration of iodoacctatc studied, the stiiilulat~ion of rcsl)irat,ion l)y glucose was over 2056. Although the iiit~clianisiii by which these coinpounds inhibit respiration iu not clear, our cxperiuients at’<’ in agrcenient nit11 the statement of (‘olowick that the (Irubt,rec effect, in the pwsenw of iodoacotatle is different from that of tlrc classic Crabtree effect. The diffcrencw obserrctl wit,11 2-tlcoxVglucose in the untrcatcd ascites cell and “in the amyt.al-t,rc~:lt’ecI cells arc rcportccl in Table IV. 2-l)coxyglucosc protluccd :L niarkccl inhil)ition of respiration in the at,-
upon
EFFECTS
ASSOCIATED AKD
CEREIJO-SANTALO
WITH
REDUCED
DIPIIOSPHOPYRIDINE NUCLEOTIDE OXIDATION
Glucose in the presence of iodoacetate and 2-deoxyglucose produced inhibit’ions of respiration which ham been considered by Papaconstantinou and Colowick as pseudoCrabtree effects (4). The effect.s of these compounds on the respirat.ion associated with succinnte oxidation in the presence of progesterone was therefore compared. Xs described in an expcriuicnt reported in Table III, the effect of glucose in the lmscncc of iodoacctat,e was stud&l with the two respiratory chains. The charactcristic inhibition of respiration by glucose in TAHI,I’ I+:FFECT
ofi'
I~IH).~(~~:T.~TE
III ON
'I'EIE
KFFE(-I
CRABTREE
Ichrlich LettrC ascites tumor cells eyuiv:&mt to 25 mg. dry tissue wright were inclll):tt,ed for 30 min. at 38°C. I’rogest,erone ant1 iodoacetale were present in the (aenter compxtment and srlhstratcs were :&led 10 min. after kmperature equilibration.
IAA 0
15.1 7.8
Craht,ree
15 X lOF.If
0
12.2 8.5
12.8 9.3
0 .3
18.7
3oL,;,
-
+ glucose Crabtree
x lo-’ .If
I- 28c,,, -l-
effect
.---~Succinate ,Succinate
11
effect
27’, ;,
1
I
I 1.2 O.fP 1.8 1 .R
‘L Results
obtained
in a separate
hut, similar
experiment
CRIBTREE
EFFECT
sence of amytal but only a slight or negligible effect in the presence of amytal. Thus it would appear that the mechanism by which 2-deoxyglucose inhibits respiration is different from the classic effect of glucose. The most striking evidence for a difference in the Crabtrcc effects procl~~e~l by glucose and 2-deoxyglucose is obtained from cxpcrimente with the normal intact cell. In the untreated cc’lls, the Crabtrce effect produced by glucose is 40% and that produced by 2-deoxyglucose is 48% !reported in Table IT). \\Xen both arc prcscnt, the inhibit,ion is only 230/A. The release of the glucose-induced Crabtree effect by the addition of 2-deoxyglucose has also been reported by Wick et al. (7). Our finding t’liat the reverse is true, that glucose relcases the 2-tleoxyglucosc-induced Crabtree effect, suggwts that the inhibitory mechanism of each is quite different. TIIE
EFFECT
OF UNCOUPLISG
ON THE
Two
71
ON SUCCI?;ATE
EFFECT
OF
2~1~~osuc:r~r-c~osr THE
PRESENCE
ox
OF
KES~IRATION
1s
;ZMYTAL
Ehrlich Lettri! uscites tumor cells equivalent to 24 mg. dry weight were incubated for :I period of 1 hr. under conditions similar to those dewrilwd in T:~l)le I.
None Glucose (0.01 31) 2-I)eosyglncose (0.02 31) (;lucose (0.01 31) + 2deosyglucose (0.02 111) Succiriate (0.01 111) Snccirmte (0.01 :\I) + I glncose (0.01 31) Succimtte (0.01 dl) + 2deosyglucose (O.o2 Jl) I
I 7.4
, 6.0 1 50 7.7 / 1
AGENTS
CHAISS
The succinoxidase chain differed wi-ith regard to the use of uncoupling agents. As seen in Table V, in the absence of amytal the inhibition of respiration by glucose was considerably less markctl in the presence of the uncoupling agent. However, in the prcsence of amytal the addition of dicumarol did not change the magnitude of the Crabtree cffert. This difference in the two respirat,ory chains st,udied was also noted when dinitrophenol served as the uncoupling agent. Thus, if uncoupling agents release t,he Crabtrec effect on the entire chain, it would appear that there is a difference in the Crabtree effects on the DPN-flavoprotein portion of the chain as compared t’o the cytochrome components, or a diff‘erence in the response of the flavoprotein to uncoupling agents. AMditional evidence t’hat the uncoupling agents aBcct DPNH oxidation diffcrcntly than succinatc oxidation is obtained from the data with labeled succinntc in this cxperimcnt. In the absence of amytal the uncoupling agents produced a marked stimulation in respirat,ion but did not increase the incorporation of succinatc carbon into the
respiratory CO2 .4 These findings with intact cells where problems of artifacts due to isolation techniques arc minimized arc in agreement with t,hc data of Siekevitz et nl. (8,9) who suggested that the dinitrophcnolactiwted adenosinetriphosphatase (XTPreflects only one l)liosase) mainly phorylation, that occurring in the dinphorase region, and t,hc findings of Hatefi et (11. (10) 1~7.110 suggested that the oxidation of pyridinoprotein substrates but not sucrinatc is strongly regulated by a respiratory control mechanism. T~IE EFFECT
OF BITTERS OS THE “SUCCINOXIDASE CRARTRW EFFECT”
The failure of uncoupling agents to release the Crabtrcc effect on the succinoxidase syst.em is also compatible with the ’ .\lthough this finding mny also be interpretal to indicate that, ox&cet;lte formation inhibit:: tlrc, rate of oxidation via suwinoxidnse so that further stimulation is prevented, this :t*qears lmlikcl? since, in preliminnry rsperiment~, xe hare failed to observe n stimulntion by dinitrol~henol at pH 6-6.5 in which incorporation of zuccinntc-I .4-P into tlrc> req)ir:Ltory CO2 :mtt prPrjumal)ly os;~lnwtate tlc~:~rhoryl:~l ion is :~l)prwial)lt~.
72
WEKNER
A?JD CEREIJO-SAXTAI,O TABLE
THE
EFFWT
OF
I'N(Y)LPIJSG
AGESTS PRESEN\‘CE
OS AK,)
V
THE
CRABTREE
;ZBSENCE
OF
I~~FFIKTS
OBSERVEI)
IS
THE
AMYTAI.
Ehrlich Lettrk ascites tumor cells equivalent to 24 mg. dry tisstle weight werr inrrltxlted for 50 min. Dicumarol (2 X lo-” M) and dinitrophenol (1 X 10e5 ;II) were lipped in from t,hc side arm. Sllwin:Ltp and glucose were in a final conccnt ration of 0.01 111.
Additions I -~-
+ I - / +
-
N0Ile
1 12.8
(:lucose Succirinte Succinnte Succinate ~uccimtte phenol Succiriat e
1 6.4 14.0 7.2 17.5 1 15.1
Srlccinate
+ gIllrose + dinitrophenol + glucose +
+ dicumxrol + glucose + dicumwol
Dicurnarol (ilucose f
THE
dinitro-
EFFECT
ON
+
50
16
49
36
14
30
2.31 2.50 2 33
0.42 0.28 0.40 0.22
15 15
1.69
18.7
16.9 17.2 16.0
dicunlarol
OF ~'HOSPHSTE
0.9 1.3 i.2 4.6 8.3 5.1
-
THE
CRABTREE
EFFECT
C)BSERVEII
IN
THE
PRESENCE
OF ~'ROGESTI~:HOSE
Ehrlich Let,tri! ascites tumor cells equivalent t,o 25 mg. dry tissue weight were incrlkxtted for 30 mill. at 38”. Progesterone was present in the center compartment, and succinnt~e-1 ,4-C14 and glucose werr tipped in from the side arm 10 min. after temperature equilibration.
Additions
- I+ Succinate-1 Succinnte-1 phosphate ~moles) None Phosphate Phosphate Succin:tte-1 Succinate-1 phosphate Succinate-1 phosphate pmoles)
,-l-Cl1 ,4-C’” + (100
I 1
’ (50 pmoles) (100 mmoles) i ,-L-C” ,-l-C” + ’ (50 pmoles) ,4-C’” + (100 ,
-
-
1 7.25 1 6.65 1 8.4 7.2 1 7.1 8.4
‘+
,~ i.3
f
1 7.15 6.9 7.1 6.5 i.4 ~ 6.85
: +
i.35
;:‘:
i 7.0 ( I
-
+ i 1 5.6 5.3
33 ?J
+
0 (50 0.51
0.47
1.29
16.2 20.5
13.7 20 0
20.0 23, i 25.0 18.8
0.5 6.1 6.1
0.9 0 .9 1.0 2.8 1.3
54 30
0.50 0.56
16.9 19.8 ~ 24 .!) 0.36 li.0 0.14 19.8
6.2
5. 1
18
0 56
0.50
1.0
a. 9
CRABTREE
EFFECT
effect is exerted cannot be att,ributed to a decreased glucose utilization ; to the contrary, the decrease in the Crabtree effect occurred in the presence of an increased glycolysis as measured by glucose disappearance and lactic acid product,ion. Thv inhibitory effects of glucose in the presence and absence of progesterone were also Audied untlcr conditions in which (Tris) tris (hydroxymet,hyl) aminomethane served as buffer. As seen in an experiment described in Table YII, the Crabtree effect as well a:: the rate of glycolysis was obserwd to increaw by the addition of Tris buffer in the absence of progesterone. Thus, the effect of phosphate in 6nulating the Crwbt,rec cffcct’ in intact cells would also appear to be related to the increase in glycolysis producctl by it’s buffering effect. The simult,aneous measurement of PUCcinntc-1 .4-(‘11 into tlio CO- which pre-
idea that intracellular acidification may be responsible for the observed inhibit,ion produccd by glucose in the presence of progesterone. It was therefore of int,erest to determine whet2ler a Crabtrcc effect was observed in the presence of progesterone in highly buffered media. In an experiment described in Table VI, the effect of glucose on the inhibition of rcspirat,ion in the presence and absence of progesterone was studied at varied concentrations of phosphate buffer. As had been observed by Kvamme i 11) , added phosphate did not release but increased the classical Crabtree effect observed with intact, cells. However, t,he Crabtree effect obwrvcd in the presence of progesterone decreased as t,he concent,ration of phosphate was raised to levels which mew more cfficicnt in t,hcir buffering capacity. The diminution in the extent to which the Crabt,rcc
THE
1
OF
TRlS
I%I-FBER
OS
h;SI’IRATlOZI
IN
Fkrlich Let trC ascites tumor cells equivalent volume of 3.0 ml. with Ca ++-free Krehs-Ringer was in a final concentration of 5 X 1W4 J-1.
73
OS SUCCISATF:
THE
PRESENCE
AXIl
i\BSb:SC’E
OF
I’RO(:E:~TER()N~:
to 28 mg. dry tissue weight were inruhatcd in :I total phosphate for a prriod of 10 nlin. at 38”. Progesterone
-
+
-
-I-
i
+
-
-
C’ .r
‘rrioles
39
3.3 20.7
Succimtte + pmoles Tris
50
10.8 6.0
44
5.5 3 .:3
1.i 18.2
Succinatr + pmoles Tris
100
1O.T 5.x
46
5.0 3 .2
18.0
25.0 2.4 20.7
Succinate + 100 ~rnoics phosphnt e
4.x 3 4
~ 2.5 , 25.2
Y-one
0.T ~
0.X
palms
0.48 1.21
Lalolll.5
0.48 0.38 0.38 0.35
4.4 3.0
100 ~rnoles Tris
-
1.2
0.50 0.27
74
WEKKER
AND CEREIJO-SANTALO
sumably measures oxalacetat,c decarboxylation provided an index of the intracellular pH in this experiment. Thus, the incorporation of succinnt’e-1 ,4-C11 into the respiratory CO, observed principally with low intracellular pH was diminished as the concentration of Tris was elevated. The addition of Tris buffer in the presence of progesterone produced a decrease in the inhibition of respiration by glucose despite an increased glycolysis as measured by lactic acid accumulation. Furthermore, the partial release of the inhibition by glucose was as great with Tris as that observed with added phosphate. It also should be pointed out that the stimulation of lactate accumulation by the addition of Tris was equivalent to that observed with phosphate buffer. GENERAL
duced by glucose alone or that the inhibition produced by t,hesc compounds is based on critical pH changes associated with early phosphorylation. It is therefore of interest to examine the evidence reported hcrc which purport,s that there arc differences in the mechanism of the inhibitory effects observed in the presence of glucose and in the ot’her cases. The evidence that pseudo-Crabtree cffects are exerted by these compounds is based on the findings that neither glucose in t’he presence of iodoacetam nor 2-deoxyglucose exertecl an inhibitory effect on the “succinoxiclasc system.” The additional finding that, glucose released the 2-deoxyglucose-induced Crabtrec effect and vice versa can be considered as confirming the idea that 2-deoxyglucose exerts a pseudoCrabtree effect.
COKSIDERATIONS
PSEUDO-CRABTREE EFFECTS OF %D~ox~~~ucos13 OR GLGCOSE IN THE PRESENCE OF IODOACETATE
The suggestion that the inhibition of respiration by glucose may arise from the release of hydrogen ions has been proposed by Tiedemann and considered by Chance, Racker and others [cf. review by Ibsen (1211, but this concept has not received much experimental support. The studies of Rosent,hal et al. (13)) McKee et al. (14)) Chance and Hess (15)) and Racker (5) establish that the inhibition is not due to an extracellular pH change. However, evidence that the pH change may be greater within the cell than in the medium (16) lends support’ to the possibility t’hat intracellular acidification, as a result of insufficient internal buffering capacity anal rapid glycolysis, plays a part in the Crabtrec cffeet,, as considered by Block-Frankenthal and Wcinhouse (2). Some of the objections to the idea that pH changes are in part responsible for the inhibition produced by glucose have been pointed out, by Ibsen (12)) and it becomes necessary to assume that, t,he mechanism by which glucose in the presence of iodoacetate or 2-deoxyglucose inl1ibit.n respirat,ion differs from that pro-
EVIDENCE FOR THE SUCCINOXIDASE CRABTREE EFFECT DUE TO INTRACELLULAR pH CHANGES
Prior to a consideration of the part which intracellular pH changes play in the classic Crabtree effect, it is pertinent to examine the evidence for an explanation of the “succinoxidase Crabtree effect” based on changes in intracellular pH. In support of the idea t’liat the “succinoxidasc Crabtret: effect,” arises principally from the release of hydrogen ions produced during glycolysis, phosphate in concentrations with high buffering capacity or Tris was found to almost, completely release t,hc inhibitory effect. Furthermore, uncoupling agents were not able to rclcase the inhibitory effect produced by glucose. In addition, neither glucose in the prescncc of iodoacctate nor 2-deoxyglucose exerted an inhitntory cffcct on t,he “succinoxidase system.” It is somewh:at difficult, to assess the loci of respiratory inhibition in the present stuclies although an obvious site is the sucrinic dehydrogcnasc reaction, which has been rcportctl to exhibit, a rather sharp pH optimuni bctwccn pH 7 and 8 (17). In atltlition, Cooper and Lchninger have rcportctl that the oxygen consumption associated with the span of cytochrome c to oxygen is markcclly dclu~~ctl by a rlccrrawc in pH
(18). Therefore, it, would uppar that the inhibitory effects produced by the lowering of intracellular pH on glucose addition may also be exerted on components of the respiratory chain other than succinic tlchydrogenase. RELATIONSHIP OF THE SUCCINOXIDASE CRABTREEEFFECT To THE CIJasslc CRABTREEEFFECT From the above considerations it is likely that. the terminal components of the cytochrome chain are inhibited by pH changes owing to intracellular acidification. Since the terminal electron-transport chain is presumed to be common to DPI%H oxidation as well, it appears reasonable to assume that intracellular pH changes are also involved in the classic Crabtree effect. Of course, the comparison of t’he succinoxidase Crabtrce effect with the classic Crabtree effect is made difficult since the effects of glucose on the DPN-flnvoprotein portion of t,he respiratory chain have not been studied, and the present study lends further support to the importance of the dinphornse region in respiratory control. The possibilit,y remains that the effects of 2-dcoxyglucose or glucose in the presence of iodoacetate are exerted on the flavoprotein portion of the chain. However, t’he mechanism by which respiration is inhibited by 2-cleoxyglucoscr or glucose in the prcsencc of iodoacctatc rcmains obscure. The observation that the inhibitory effects of glucose in neoplasms with low rates of glycolysis were significantly greater upon addition of inhibit~ors of DPh’-cytochromc c reductase suggests that the Crabt,rec cffeet is a property of a high rate of glycolysis, as has been reported by Rosenthal et ~(1. ( 13) and Luganova et (11. 119j. This evirlcnw is also compatible with the explanation that the initiation of a Crabtrw effect involves a rate of glucose phoq)horylation sufficiently great to release hydrogen ions by which the intracellular pH is loncrcrl. However, t,hcxe studies do not. rule out the involvement of a competition of coenzymcs of glycolysis and respiration in the classic C’rabtrec effect despite the occurrcncc of the r~nhanwtl inhibitory effects under csonditions
of a lowered respiration where the requircmcnts for t,he phosphorylating systems are considerably diminished. It, is of int,erest that t,he Crabtree effect is not as pronounced in the presence of bicarbonate (2). a buffer which we have obacrvcd maintains the pH of the external medium more effectively than phosphate buffer, and recently Zatti and Rossi (,20,1 have reported that glucose stimulates respiration in biwrbonate buffer but inhibits respiration in phosphate buffer, an obacrwt,ion that can be intcrprcted to indicate that bicarbonate releases the Crabtrer tlffcct.” STIMULATION OF RESPIRATIOS BY CLWOSE: IN THE PRESENCEOF IODOACETATEOR Low CONCESTRATIOKSOF G~ccom In contrast to the observed respiratory inhibition produced by glucose alone, a stimulation of succinoxidasc respiration by glucose was observed in t,lie presence of iodoacetate, and is analogous to the stimulation of respiration by low concent.rations of glucose report,ed first by RlcKec et nl. f 14). This enhancement in oxygen conwilption is attributed to the production of adcnosine diphosphate (ADP) marlc awilable by glucose phosphorylation via the hcxokinaac reaction. Unpublished work by one of t.hc authors (Santa10 IL’ with homogenates of Ehrlich :iscit,es cells or normal rat brain has shown that it, is possible to wry the rwpiratory response to glucose tq changing the ratio of supernatant to nlitocliondia. I3y increasing the mitoclioncfri:~ or decrrasing t.he supernatant, conditions under which the rate of glycolysis is low, glucow is found to exert :I stiniulntory effect
76
WENNER
-iND
on respiration; by decreasing the mitochondria or increasing t,he supernatant, conditions in which the rat,e of glycolysis is high, a Crabtree effect appears which can be reversed by the use of suitable buffers. These observations lead to the consideration that glucose may exert a biphasic response, namely a stimulation of respiration when the hexokinase reaction is slow; but when the hexokinase reaction is fast, a respiratory inhibition appears instead. In summary, it is concluded that the inhibition of succinate respiration by glucose is produced by intracellular acidification, and that whether an increase or decrease in respiration is observed upon glucose addit,ion depends on t,he rate of glycolysis. The authors are indebted to John Hackney and Lewis Keeler for t,heir very capable technical assistance, and to Dr. T. S. Hauschka for supplying the mice bearing ascitea t,umor cells used in these studies. REFEREXCES 1. RACKFX, E.. J. Bid.
Chcm. 184, 313 (1950). L., ASD WMSHOUSE, S., Cancer Research 17, 1082 (1957). 3. WENNER, C. E.. Arch. Biochem. Riophys. 98, 58 (1962). 4. P.~~.~cossTA~T~~-ou, J.. AND COI,OWICK. S. l’., J. Biol. Chem. 236, 278 (1961). 2. BLOCK-FRASPENTHAL,
CII:REIJO-S~~~T.I\I,O 5. RACKER, E., Ann. X. Y. Acd Sci. 63, 1017 (1956). 6. CHATCE, B., J. Bid. Chem. 234, 2416 (1959). 7. (:HRISTEXSES, E., BROOKS, J. L., STEWART, c. J., AND WICE~, .2. S., Biochcm. Wiophys. I<+ scmd~ Commurts. 5, 209 (1961). 8. SIEKEVITZ, I’., L,iiw, H.. ERWIXR, L., AYII LIXDBERC, O., Niochim. et Biophys. Actn 29, 378 (1958). 9. L6w, H., SIEKEVITZ, P., ERNSTER, L., AXD LINDHP:RG, O., niochim.
et Biophys.
ilcta
29,
392
( 1958). 1-‘., AND HAAWK, A. G., 10. HATEFI, Y., JVRTSHUK, Arch. Biochern. Riophys. 94, 148 (1961). 11. KVAMME, E.: Acta Physiol. Stand. 42, 204 (1958). 12. lesn-, Ii. II., Cancer Hvseurch 21, 829 (1961). 13. ROSESTHAI,, O., BOWIE, M. ii., AND WAQOXER, G., Sciemx 92, 382 (1940). 14. MCI&:, R. W., LOSBERG-HOLM, F., ASD J~rm.. J., Canw~~ 12esenrch 13, 537 (1953). 15. CHASCE, B., ASI) HESS, B., Arm. n;. Y. Acad. Sci. 63,100s ( 1956,. 16. DEWEY, I>. I,.. AID GREEK, F. O., Hiochcm. J. 72, 160 ( 1959). 17. BURRIS, 11. H. in ‘LManonletric Tcchniqucs and
Tissue Met~:~holism” (TYmbreit, Burris, :~ntl Stauffrr, Etls.). 2nd ed., p. 105. Burgess Pull. Co.. Minnwpolis, Minn., 1949. 18. Coor~rc, C., ASI) LEHSIXER, A. I,., J. Wid. Chcm. 219, 519 (1956). 19. I,UCASOY.A, I. ti., SliITZ, I. E’., ASD T~o~~o~or~c~, T’. I.. IMAI~/~/ Akwl. Xwk S.S.S.R. 112, 1082 (1957). 20. %.4TTI, 121., ASI, RosSI, Ii’., Ilt/h//r J. Nioc,/c?r,r 10, 19 (1961).