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Swelling and shrinkage phenomena in liver mitochondria IV. Reversible swelling changes linked to transport of monovalent cations stimulated by valinomycin
BIOCHIMICA ET BIOPHYSICA ACTA BBA
445
65382
SWELLING AND SHRINKAGE PHENOMENA IN LIVER MITOCHONDRIA IV. REVERSIBLE SWELLING CHANGES LINKED TO TRANSPORT OF MONOVALENT CAnONS STIMULATED BY VALINOMYCIN
ANGELO AZZI AND GIOVANNI FELICE AZZONE Unit "G. Vernoni" for the Study of Physiopathology and Institute of General Pathology, University of Padova, (Italy) (Received June r oth, Ig65)
SUMMARY 1. Addition of valinomycin to liver mitochondria accelerates the rate of K+ uptake. The high rate of K + uptake caused by valinomycin is accompanied by increase of respiration, oxidation of mitochondrial pyridine nucleotide and stimulation of ATPase activity. The apparent K m for K+ uptake in presence of valinomycin is about 12mM. 2. Addition of Pi or acetate to liver mitochondria oxidizing succinate increases the rate but not the extent of K + uptake. Both in the presence and absence of Pi and acetate about 4 K+ are taken up per H+ ejected. 3. The K+ uptake is accompanied by a decrease of absorbance. The rate of swelling is accelerated by Pi, arsenate, acetate, lactate or formate. Interruption of energy supply causes release of K+ and shrinkage. Both swelling and shrinkage are dependent on the concentration of K+ in the medium. The stimulation of swelling by increasing concentrations of KCl is inhibited competitively by dinitrophenol. The stimulation of swelling by increasing Pi concentrations is inhibited by dinitrophenol in a non-competitive manner. 4. Mitochondria rapidly lose their K+ content in the presence of valinomycin and absence of energy supply. A parallel shrinkage is observed. The shrinkage is inhibited by increasing concentrations of cations in the medium. 5. Addition of divalent cations causes release of K+ and shrinkage. The rate of shrinkage is higher with dinitrophenol than with divalent cations. 6. The swelling and shrinkage processes and the K + uptake may be limited either by diffusion or by a chemical reaction.
INTRODUCTION
Isolated mitochondria can maintain a high internal concentration of K+ or accumulate K+ after depletion. These processes appear to be dependent on mitochondrial metabolismi-» Biochim. Biophys. J1 eta, II3 (I9 66) 445-45 6
A. AZZI,
G. F.
AZZONE
MOORE AND PRESSMAN 1 0 have reported that addition of valinomycin to liver mitochondria oxidizing succinate causes a rapid K+/H+ exchange. Respiration is also stimulated if an anion is present. CHAPPELL AND CROFTSll have observed that addition of gramicidin in the presence of monovalent cations and a permeable anion causes a rapid swelling phase parallel to the H+ ejection. It has been suggested that valinomycin and gramicidin alter the permeability of the mitochondrial membrane to cations and thus the process of cation uptake is accelerated. The presence of an anion increases the uptake of K + and causes swelling by means of an osmotic mechanism. In the present article, experiments will be reported concerning some properties of the cation transport in liver mitochondria after addition of valinomycin. The relationship between the process of ion transport and reversible mitochondrial swelling will be also considered. Preliminary accounts of the present work have already been reported12 ,13 . METHODS Rat-liver mitochondria were used in all the experiments described. Measurements of the absorbance were carried out with an Eppendorf Photometer equipped with a recording apparatus. Usually the amount of mitochondrial protein in the cuvette was in the range of 130 {-lg/ml with an initial absorbance of 0.6. When simultaneous measurements of absorbance and of cations or pH were carried out, the amount of mitochondrial protein was 2 mg/ml. pH and K+ were measured with a Beckman pH meter (expanded scale) equipped with a recording apparatus. The Beckman glass electrode and cationic electrode were used for measuring pH and K+, respectively. Oxygen uptake was recorded polarographically. Mitochondrial pyridine nucleotide were measured with an Eppendorf Fluorimeter equipped with a recording apparatus. Pi was measured as described by LINDBERG AND ERNSTER14 . All experiments were carried out at 30°. RESULTS f{+
uptake induced by valinomycin From the data reported by CHRISTIE et al.s it can be calculated that the rate of
[KCg(mMJ
Fig. 1. K+ uptake in the presence of valinomycin at various KCI concentrations. Composition of the incubation medium was as follows: 250 mM sucrose, 20 mM 'Iris buffer (pI-I 7.4), 5 mM succinate, I pM rotenone, 0-4 !!g valinomycin and amounts of KCI as indicated in the abscissae. Amount of mitochondrial protein was 2 mgjml. Final volume, :il ml. Temperature, 30°.
Biochi m, Biopbys. Acta. 1I3 (rg66) 445-466
REVERSIBLE SWELLING LINKED TO
K+
UPTAKE
447
TABLE I UPTAKE AND RELEASE OF K+ AND EJECTION OF AND IN THE PRESENCE OF PI AND ACETATE
H+ BY
LIVER MITOCHONDR1A IN THE ABSENCE
The medium contained 250 mM sucrose, 8 mM MgC12 , 20 mM Tris buffer (pH 7.4), 0.4/tg valinomycin, 5 mM succinate, I pM rotenone and when indicated I mM Pi or 3 mM acetate. Release of K+ was obtained with roo pM dinitrophenol. The amount of mitochondrial protein was 2.2 mgjml. Temperature, 30°. Rates were expressed in llffioles/g protein per min, and extents in Ilmoles/g protein. Additions
J(+
H+
Uptake
Release
Release
Uptake
-----
None PI
Acetate
E;t:tent
Rate
Rate
Extent
Rate
Rate
3 05 390 3I5
33 3°0 I09
255 I090 I630
7I 7I 73
20
380
43 34
73°
355
K+ uptake by intact liver mitochondria with 20 mM KCI in the medium is about 4 ftmoles/g protein per min. The rate of K + uptake by liver mitochondria supplemented with valinomycin is about 100 times higher (cf. Table I). The amount of K+ taken up by the mitochondria increased linearly with increasing amounts of K + in the medium up to 10 mM (Fig. I). Since the sensitivity of the K+ electrode varies inversely with the concentration of K +, accurate measurements of the K + uptake could not be made above this concentration. From the correlation between K + uptake and swelling it might be predicted, however, that higher concentrations of K + in the medium should not cause a further increase of K + uptake. The rates and amounts of K + uptake and H + ejection in the presence of 2 mM K + are given in Table 1. Addition of Pi or acetate considerably increased the rate of I{+ uptake but only slightly increased the amount of K+ uptake (about 20%). The ratio K+/H+ was about 4, both without or with Pi or acetate. The rates of release of K + and uptake of H + following the addition of dinitrophenol were much faster than the uptake of K+ and ejection of H+. MOORE AND PRESSMAN 10, and CHAPPELL AND CROFTS l l reported that addition of valinomycin or of gramicidin in the absence of anions did not stimulate the respira-
Fig. 2. Oxidation of pyridine nucleotide induced by valinomycin at various KCl concentrations. The medium contained 20 mM Tris buffer (pH 7.4), 2 mM ti-hydroxybutyrate, IO mM MgC12 , 0.2 I~g valinomycin and variable amounts of KCI and sucrose up to a final concentration of 0.25 osM. Amount of mitochondrial protein was 0.7 mgjrnl, Biochim, Bio-phys, Acta, II3 (I966) H5-456
A. AZZl,
G. F.
AZZONE
tion. CHAPPELL AND CROFTSll suggested that the J{+jH+ exchange occurring in the absence of an anion did not require energy. In our experiments addition of valinomycin resulted in oxidation of the mitochondrial pyridine nucleotide also in the absence of PI, and the extent of oxidation was dependent on the concentration of KCI (Fig. 2). The K+ uptake also in absence of PI was strictly dependent either on the presence of an oxidizable substrate or on the presence of external ATP (cj. ref. 13). Respiration was stimulated by valinomycin up to a concentration of 0.02 Itg/mg protein in the presence and 0.008 Itg/mg protein in the absence of PI (Fig. 3). The respiratory rate was higher in the presence than in the absence of Pi. with 3mM P,
360
without
02
fl
04
Valinomycin (pg)
Fig. 3. Stimulation of respiration by various valinomycin concentrations in the presence and absence of Pi. The medium contained 210 mM sucrose, 20 mM 1(CI, 20 mM Tris buffer (pH 7.4), 10 mM MgCI2 , 5 mM succinate and I ftM rotenone. The amount of mitochondrial protein was 2 mg/rnl. Temperature, 30°.
MOORE AND PRESSMAN 1 0 reported an apparent Em for K+ after addition of valinomycin of about 5 mM. CHAPPELL AND CROFTSl l reported an apparent K m for K+ after addition of gramicidin of about 2 mM. Under our experimental conditions, which usually included MgC1 2 in the medium, the apparent J(m for K + was about 12 mM. This figure was obtained from measurements of respiration, of ATPase activity (Fig. 4B and A), and of swelling (Fig. 9).
B
A
20
.40
60
[KeD (mfvl)
1
2
3
1/[5] x10~
Fig. 4. (A, B) Stimulation of ATPase activity and respiration induced by valinomycin at various KCI concentrations. The medium contained 20 mM tris buffer (pH 7-4), 8 mM MgC12 and variable amounts of KCI and sucrose up to a final concentration of 0.25 osM. Also added were: in Expt. A, S mM ATP and I fLg valinomycin; in Expt. B, 5 mM succinate, I ,uM rotenone and 0.2 fIg valinomycin. The amount of mitochondrial protein was I mgjrnl in Expt. A and 2 mgjrnl in Expt. B. Temperature, 30°. Time of incubation in Expt. A was 20 min. Final volume 2 ml.
Biochim. Biophys. A eta, 113 (1966) 445-456
REVERSIBLE SWELLING LINKED TO
K+
UPTAI{E
449
K+ uptake and swelling CHAPPELL AND CROFTS ll
have reported that addition of Pi or arsenate to liver mitochondria supplemented with gramicidin, resulted in a rapid swelling phase. In the absence of anions the swelling was not observed. They suggested that the swelling phase was caused by an increased uptake of cations induced by the anions. As we have shown in Table I, under our experimental conditions addition of Pi or acetate considerably increased the rate but not the extent of K + uptake. Simultaneous measurements of K+ concentration and absorbance revealed a close correlation between uptake of IU and swelling (Fig. 5). In fact the uptake of K+ was slow in the absence of Q2~g
valinomycin
I
I
100WM dinitrophenol
100
~M
dinitrophenol
10mV a2~g vQli()QmYCIn
I 1.2mM Pi
t
I
I
100~M
100~M
dinitrophenol
dinItrophenol
Fig. 5. Correlation between K+ uptake and swelling in the absence and presence of P j • The medium contained 250 mM sucrose, 20 mM Tris buffer (pH 7.4), 2 mM KCI, 5 mM succinate, I pM rotenone and o.4/-lg valinomycin. Pi when present was 1.5 rnM. The amount of mitochondrial protein was 2 mg/m!. Final volume 4 ml.
P l , as was the decrease of absorbance. Addition of Pi either at the beginning or at the end of the process increased neither the extent of uptake of K+ nor the extent of swelling. The rate of K+ uptake and of swelling were, however, considerably accelerated when Pi was added at the beginning of the incubation. Addition of dinitrophenol induced a fast release of K+ and shrinkage both in the presence and in the absence of Pi. Release of ]{+ and shrinkage CHI
450
A. AZZI, G. F. AZZONE 0.21-'9 valir.amycin 1mM succinate
ST
liiny
I ----t~
100r~·oPhehOl
1
~
, m M succinate
l
1
100f.JMdinitrophenol
I t
0.21-'9~VOilnomYCln, t
K uptoke
~
12mv ...-..-..-. 60sec
Fig. 6.(A and B) 1(+ release and mitochondrial shrinkage caused by valinomycin in a K'r-free medium. The medium contained 250 mM sucrose, 20 mM Tris buffer (pH 7.4),0.4 !Ig valinomycin and I pM rotenone. The amount of mitochondrial protein was 2 rng/ml. Final volume 4 ml. The lower curve is a K+ electrode trace; the downward deflection indicates K+ uptake.
chondria after addition of valinomycin and was taken up after addition of succinate. The K+ accumulated was again released by dinitrophenol. While the K+ uptake was accompanied by swelling, the efflux of K+ from the mitochondria was accompanied by shrinkage (Fig. 6B). Both efflux and shrinkage were gradually inhibited by increasing the concentration of cations in the medium (Fig. 7A and B). The inhibition of the shrinkage was complete at 80 mM K + in the medium. Replacement of K + with Rb r did not change the pattern of the process (Fig. 7B). A
B
o.oe
ODS
-e
0.04 •
0 - 40 SO [KCU(mM)
0.04
120
40 80 [RbCD(mM)
120
Fig. 7. (A and B) Inhibition of shrinkage by increasing concentrations of cations in the medium. The medium contained 20 mM Tris buffer (pH 7.4), 0.02 !Ig valinomycin, roo !tM dinitrophenol, and variable amounts of KCl 01' RbCl together with sucrose up to a final concentration of 0.25 osmolar. The amount of mitochondrial protein was I30 !tg/ml.
Effect of the K+ concentration on swelling and shrinkage Acceleration of the rate of swelling was observed not only after the addition of Pi and arsenate icf, ref. II) but also with organic anions such as, lactate, acetate or formate. The rate and extent of swelling and the extent of shrinkage were dependent on the external concentration of KCl with all the anions tested (Fig. 8A, B and C). The rate of swelling increased up to 20 mM KCl and then remained constant. On the other hand, the extent of swelling diminished at increasing concentrations of KCl. Also the extent of shrinkage diminished at higher KCl concentrations and was practically abolished above 80 mM KCl. With acetate, the apparent K m for K+, measured with respect to the rate of swelling, was higher than with Pi or arsenate. Biochirn: Biopbys, A eta, II3 (19 66) 445-456
REVERSIBLE SWELLING LINKED TO
451
K+ UPTAKE Arsenate
Rate of swelling
Rate of swelling
0.4
-~------
::3
0.2
~ 0.2
<,
°O~-'--,J~---L--,,!:"-....L--..!!;;-
40
80
Acetate
o
120
40
80
Rote of swell ing
120
120
[KCOCmMl
[KCOCmMl Extent of swelling
Extent of swell ing
Extent of swelling
02
0.2 I,------~-'-----
1
""
<1 0.1
,"
!
120
120
40
80
120
[KCOCmMl Extent of shrinkage in~. of swelling phase
100
40
4
80
(KCI] (mM)
Fig. 8. Swelling and shrinkage with different anions at various KCl concentrations. The medium contained 20 mM Tris buffer (p H 7.4), 0.02 ftg valinomycin, r ftg oligomycin, 15 mM MgCI 2, 5 mM succinate, I pM rotenone, and variable amounts of KCl and sucrose up to a final concentration of 0.25 osM. Anions wore: I mM PI. 1 mM arsenate and 5 mM acetate. The amount of mitochondrial protein was 130 fig/m!.
Dinitrophenol inhibited the swelling phase and the inhibition was competitive with respect to the concentration of ReI (Fig. gAl. The apparent /(rn for K+ in the presence of 0,10,20,4° anc150,uM dinitrophenol were 12.3, 14.5,3°, 83.5 and 145 mM, respectively. On the other hand, dinitrophenol inhibited in a non-competitive manner the increase of rate of swelling promoted by Pi (Fig. gB). The apparent /(1/1 for P, was about 300 ,uM. A
30
50IJM dinitrophenol 40IJM dinitrophenol
none
20
30
B
50
o
Fig. 9. (A and B) Inhibition of swelling by dinitrophenol at various 1(C\ and PI concentrations. Experimental conditions as in Fig. 8. Abscissae: A, reciprocal of the 1(Cl concentration, B, reciprocal of the PI concentration. Ordinates: the reciprocals of the rates of swelling. Biochirn. Biophys. A cia, 113 (1966) 445-45 6
452
A. AZZI, G. F. AZZONE
The shrinkage phase Addition of uncouplers or respiratory chain inhibitors reversed the swelling process linked to the transport of monovalent cations. Shrinkage followed also the addition of divalent cations, such as Ca2+ and Mn 2+. The shrinkage phase was accompanied by a release of l{+ from the mitochondria (Fig. 10) and lasted for the time 0.1 fJg valinomycin
J K' uptake
I
0.1~g
valinomycin
,J\
500~M
5mvl I/' ~~
100~M MnS04
MnS04
125fJM CoCI 2
~
60se~ Fig. 10. Release of K+ induced by divalent cations. Experimental conditions as in Fig. 5. 5 mM Mgel. and I rn M PI were also present. Final volume 2 ml.
required to remove the divalent cation from the medium. In this respect the shrinkage phase caused by divalent cations in valinomycin-supplemented mitochondria is similar to that caused by ADP in EDTA-supplemented mitochondria-". In Table II are reported the rates of shrinkage induced by uncouplers, respiratory chain inhibitors, cations and ADP in valinomycin-supplemented mitochondria. The highest rate of TABLE II RATES OF SHRINKAGE INDUCED BY UNCOUPT.ERS, RESPIRATORY CHAIN INHIBITORS, CATIONS AND
ADP The medium contained 20 mM Tris buffer (pH 7.4), 210 mM sucrase, 20 roM KCI, 2 mM PI, 0.02 fig valinomycin, 5 m'M succinate and 111M rotenone. The amount of mitochondrial protein was 130 flg/m!. Temperature, 30°.
Additions
Concentration
Rate of shrinkage in LlAlmin
Dinitrophenol
100 ftM Sao flM 1.4 ftg 1.7 mM 500 11M 330 flM
0.260
Ca H
Antimycin A
KeN Mn'+ ADP
0.215
0.r90 0.r60 0.Il5 0.05 0
shrinkage was that given by dinitrophenol, followed by that of Ca 2 +. This result is in agreement with the observation that dinitrophenol causes the highest rate of release of K+ and uptake of H+. Biochim. Biophys. .11 eta,
113 (1966)
445-45 6
REVERSIBLE SWELLING LINKED TO
K+
UPTAKE
453
Effect oj temperature on swelling and shrinkage and on K: uptake We have reported previously that the swelling and shrinkage phases of EDTAsupplemented mitochondria had a high QlO (see ref. IS) thus suggesting a chemical reaction as rate-limiting step in these processes. In the case of the swelling and shrinkage occurring in valinomycin-supplemented mitochondria it was found that the QI0 for both the swelling and the shrinkage phase were influenced by the presence of albumin in the medium. The values of QI0 obtained were, in the presence of serum albumin, r.8 for the swelling phase and 4.3 for the shrinkage phase. In the absence of serum albumin the values of QI0 were 3·7 for the swelling phase and 2.5 for the shrinkage phase. The value of QI0 for the respiration was r.6. In the case of the K+ uptake the value of QI0 varied greatly according to the range of temperature in which
2
0.0033
0.0034
1/r Fig, I!. Effect of temperature on I{+ uptake in the presence of valinomycin. The medium contained 250 mM sucrose, 20 mM Tris buffer (pH 7"1·), 2 mM ICCl, 5 mM MgCl 2 • 5 mM succinate, I ftM rotenone. I urM Pi and 0,4/tg valinomycin. The amount of mitochondrial protein was 2 mg/ml. The release of K+ was obtained by adding I ttg antimycin A,
it was measured (Fig. II). It is seen that the QIO was high at low temperature and low at higher temperature. It would thus seem that under most conditions diffusion of cations is rate limiting with respect to energy supply from the respiratory chain. DISCUSSION
The concentration gradient oj Kv Active transport has been defined by CONWAy16 as flow "dependent on the activity or energy change of another system". According to this definition the transport of monovalent and divalent cations inside the mitochondrion must be considered as "active" since it is inhibited by respiratory chain inhibitors or uncouplers which stop the energy supply for the cation uptake. Another criterion, however, defines a flow of ions or metabolites as "active transport", namely occurrence of a movement against concentration gradients. The transport of K + inside the mitochondrion, analyzed in the present and in previous papers1 2 ,13 , satisfies both criteria for an "active process". The cyclic changes of the intramitochondrial K+ content depending on the energy supply, the ratio between the final concentration of extra- and intramitochondrial cations, and the dependence of the release of the intramitochondrial K+ on the concentration of extramitochondrial cations, are all in agreement with this conclusion. Biochim, Biophys, Acta. II3 (Ig66) 445-45 6
454
A. AZZI, G. F. AZZONE:
Whether the transport of K + must be defined as " acti ve" also according to the criterion formul ated by USSIN G1 7, n amely movem ent aga inst an electrochemi cal gradien t , is be yond the available experimental dat a . The uptake of monovalent cati ons in the absence of P I or acetate is an en ergy requiring process as indicate d b y its dependence on t he presence of oxidi zable subst rate (or of ATP ), by the inhibi tory effect of dinitrophenol, and by the st imulat ion of respiration and of pyridine nucleotide oxidat ion accompa nyi ng the uptake of cations . It would seem, therefore, t ha t the lack of respiratory st imulat ion observed by M OORE AND PRESSMAN 10 and by C HA P PE L L AND CROFT Sll , afte r addition of valinomycin and gramicidin to liver mi t ochondria oxidizi ng succin ate in the ab sence of P I, was du e to the effectivene ss of the State 4 respiration to sup p ort the K+ uptake under the ir experimental condit ions. At variance with the results obtained by JU DAH et al», the uptake of K+ was not accompanied by an eje ction of H-I- in a r(I ratio. Under our experimental conditi ons, both in the pr esence and in the absence of PI or acetate, the ratio K-I-/H -I- was about 4. Theoretical explanat ions for this discrepancy cannot be formulated before more in format ion is obtaine d concern ing the molecula r mechanism of cation transp ort in mit ochondria.
K+ ~tp ta ke and s1J!)elling Variations of t he m itochondrial content of K -I- were alway s accompanied by changes of abs orban ces ; i .e. uptake was accompanied by swelling and release was accomp anied by shrink age. Furthermore, as the exte nt of uptake and of release of cations were independent of the addition of Pi or a cetate, so the extent of swelling and of shrinkage were also independent ofthe addition of Pi or acet ate. These obser vations indicate that the K -I- accumulated in the mitochondrion is always present as an ionized, osmotically acti ve species. The differen ces b etween th e behaviour of mon ovalent and divalent cations will be examine d in a subsequen t paper. R ecently PRESSMAN 18 has presented calculations which cast some doubt on the hypothesis that the volume changes obs erved during the uptake of K + are ind eed du e t o an osmotic mechanism . An osmot ic mech ani sm of water transp ort is considerably support ed by our da t a. First ly our experiments indicate always a close parallelism between extent and rate of K+ uptake and ext ent and rate of swelling. Secondly, according to the calculation of PRESSMAN 18 , the entrance of I It! of H 20/mg protein, corresponding to a 20% decrease of light scattering sh ould clemand a net uptake of 150 m,umoles K-I-/mg protein . The figure of K + uptake pr esented in Table I satisfy the requirement for an osm otic swelling both in the presence and in the absence of P, or acetate. Effect oj the anions C HAPPE LL AND C ROFT Sl l
explain the lack of swelling which they observe in the absence of Pi by suggesting th at the K + uptake is "limi ted by the net n egative charge withi n the m itoch ondria". When PI enters "it provides more H + to b e pumpe d out in exchange for K +; now there is an increase of inte rn al osmot ic pressure (due to accumu lation of K-I- and Pi ) and the m it ochondria swell" . In the me chanism propos ed here, en ergy is required for the dissociation of intramitoch ondrial an ions. The dissociati on provides H + whi ch is ejected in ex change for K -1- , and negati ve ch arges which B iochim, B iop hy s. Acta, II 3 (1966) 445- 45 6
REVERSIBLE SWELLING LINKED TO
K+
UPTAKE
455
maintain electrical neutrality during K + uptake. The internal osmotic pressure increases when undissociated mitochondrial anions are replaced by dissociated K+ salts of the anions. This mechanism is consistent with the observations reported above (cJ. also ref. 13) that the K+ uptake and the parallel swelling are independent of the presence of Pi but dependent on energy supply. The charge separation mechanism of MITCHELL19 offers an explanation for the stimulating effect of Pi. Owing to the dissociation of intramitochondrial anions, a high pH is reached inside the mitochondria when Pi is absent. The high pH inhibits electron transport and thereby also K+ uptake and swelling, The internal pH is lowered when H + is provided by dissociation of the second acidic group of intramitochonclrial PI, and respiration is stimulated. In steady-state conditions the presence of Pi causes an increased turnover of K+ through the mitochondria. The shrinkage phase In previous papers 1a ,15 , 2 0 we have proposed denoting as "irreversible", the swelling processes which require an external supply of energy for shrinkage, and "reversible", those which do not require an external supply of energy for shrinkage. The swelling linked to the transport of monovalent cations, occurring in the presence of valinomycin, gramicidin or EDTA, therefore fall in the category of the reversible swelling processes, The occurrence of these latter shrinkage phases in the presence of respiratory-chain inhibitors or of uncouplers, raises the question of the energy dependence of these processes. As we have discussed above, the swelling process follows the establishment of concentration gradients of cations, The shrinkage phase must be considered as due to the utilization of the energy of the concentration gradient which drives the efflux of the cation from the mitochondrion. The inhibition of the shrinkage phase at higher concentrations of Kel in the medium is due to a lowering of the concentration gradient which is dependent on the ratio between intra- and extramitochondrial concentrations of cations. If the concentration gradient of cations is lost, the mitochondria lose the capacity of shrinkage without external supply of energy. It is suggested that the occurrence of the cation gradient represent the basic distinction between reversible and irreversible swelling processes.
ACKNOWLEDGEMENTS
The skillful technical assistance of Miss B, BARBIN and Mr. L. AGOSTI is gratefully acknowledged. This investigation was aided in part by the Muscular Dystrophy Associations of America Incorporation. REFERENCES I V. G. SPECTOR, Pvoc, Roy. Soc., London, SBr. B, 14-1 (1953) 268. 2 C. A. PRICE, A. FONNESU AND R. E. DAVIES, Biochem. J., 64 (r95 6) 754· 3 J, A. AMOORE AND "V. BARTLEY, Biochem. J., 69 (195 8 ) 348 . 4 J. L. GAMBLE. JR., J. BioI. Chem., 228 (1957) 255· 5 R. L. SCOTT AND J. L. GAMBLE, JR.,]. Bioi. Chern" 23 6 (19 6 1) 57°· 6 L. SHARE, Am, j. Physiol., 194 (195 8) 47. 7 F. ULRICH, Am. j. Physiol., 198 (lg60) 846. 8 G, S. CHRISTIE, K. AHMED, A. E. M, McLEAN AND J. D. JUDAH, Biochim. Biopbys. Acta, 94 (19 6 5) 43 2.
Biochirn. Biophys. Acta, II3 (19 66) 445-45 6
A. AZZI , G. F. AZZONE
9
J . D . Jun AH, A. E . M. M cLEAN , K. A HMJ;:D AND G. S. CHRISTIE. B ioclii m , B ioPIIJ'S. Acta, 94 (19 6 5) 4 4 1 . 10 C. MOORE AND B. C . PRESSMAN , B iochem , B iop hys. R es. Com mltn ., IS ( 19 6 4 ) 562 . I I J. B. CHAPPELL AND A . R. CROFTS . Biochem , J., 95 (196 5) 393. 12 A. A ZZI AND G. F . AZZONE, B iochem , f. , 9 6 ( 1965) r c. 13 G. F . A ZZONE AND A. A ZZI, in ] . M. T AGER, S. PAPA , E . QUAGLIA R IELLO AN D E . C. S L AT ER .
Regu lati on of Metabolic Processes in M itochondri a, Elsev ier, Amsterdam , 19 66, P- 332. 14 O. L I NDBERG AND L. ERNSTER, Methods B iochem. Anal., 3 ( 1956) 1. 15 A. AZZ I AND G. F . AZZONE, B iochi m: B iopliy s, A cta, 10 5 ( 1965) 265 . 16 E . ]. CONWAY, Symp , S oc. Expti. Bio l., 8 ( 19 5 4) 2 97 · 17 H. H. USSING, T he A lkali Meta l. I ons in I solated Sy stem s and T i ssues, Springer, Berlin, 1 9 6 0 , p. I . 18 B. C. PRESSMAN. P roc. N ail. A cad, S ci. U.S ., 53 ( 1 9 65) 1°7 6 . 19 P. M ITCHELL, in J. M . T AGE R, S . P AP A, Eo Q UA GLI AR I ELLO AND E . C . SLATER, R egulati on of Me taboli c Processes in M itochondria, Elsevier, Amsterdam, 1966. 2 0 G. F . AZZONE AND A. AZZI , P roc. Notl, A cad. Sci. U.S ., 53 (19 6 5 ) 108 4. Biochdm, Bia phvs. Acta, II3 (1966)
445-456
445
65382
SWELLING AND SHRINKAGE PHENOMENA IN LIVER MITOCHONDRIA IV. REVERSIBLE SWELLING CHANGES LINKED TO TRANSPORT OF MONOVALENT CAnONS STIMULATED BY VALINOMYCIN
ANGELO AZZI AND GIOVANNI FELICE AZZONE Unit "G. Vernoni" for the Study of Physiopathology and Institute of General Pathology, University of Padova, (Italy) (Received June r oth, Ig65)
SUMMARY 1. Addition of valinomycin to liver mitochondria accelerates the rate of K+ uptake. The high rate of K + uptake caused by valinomycin is accompanied by increase of respiration, oxidation of mitochondrial pyridine nucleotide and stimulation of ATPase activity. The apparent K m for K+ uptake in presence of valinomycin is about 12mM. 2. Addition of Pi or acetate to liver mitochondria oxidizing succinate increases the rate but not the extent of K + uptake. Both in the presence and absence of Pi and acetate about 4 K+ are taken up per H+ ejected. 3. The K+ uptake is accompanied by a decrease of absorbance. The rate of swelling is accelerated by Pi, arsenate, acetate, lactate or formate. Interruption of energy supply causes release of K+ and shrinkage. Both swelling and shrinkage are dependent on the concentration of K+ in the medium. The stimulation of swelling by increasing concentrations of KCl is inhibited competitively by dinitrophenol. The stimulation of swelling by increasing Pi concentrations is inhibited by dinitrophenol in a non-competitive manner. 4. Mitochondria rapidly lose their K+ content in the presence of valinomycin and absence of energy supply. A parallel shrinkage is observed. The shrinkage is inhibited by increasing concentrations of cations in the medium. 5. Addition of divalent cations causes release of K+ and shrinkage. The rate of shrinkage is higher with dinitrophenol than with divalent cations. 6. The swelling and shrinkage processes and the K + uptake may be limited either by diffusion or by a chemical reaction.
INTRODUCTION
Isolated mitochondria can maintain a high internal concentration of K+ or accumulate K+ after depletion. These processes appear to be dependent on mitochondrial metabolismi-» Biochim. Biophys. J1 eta, II3 (I9 66) 445-45 6
A. AZZI,
G. F.
AZZONE
MOORE AND PRESSMAN 1 0 have reported that addition of valinomycin to liver mitochondria oxidizing succinate causes a rapid K+/H+ exchange. Respiration is also stimulated if an anion is present. CHAPPELL AND CROFTSll have observed that addition of gramicidin in the presence of monovalent cations and a permeable anion causes a rapid swelling phase parallel to the H+ ejection. It has been suggested that valinomycin and gramicidin alter the permeability of the mitochondrial membrane to cations and thus the process of cation uptake is accelerated. The presence of an anion increases the uptake of K + and causes swelling by means of an osmotic mechanism. In the present article, experiments will be reported concerning some properties of the cation transport in liver mitochondria after addition of valinomycin. The relationship between the process of ion transport and reversible mitochondrial swelling will be also considered. Preliminary accounts of the present work have already been reported12 ,13 . METHODS Rat-liver mitochondria were used in all the experiments described. Measurements of the absorbance were carried out with an Eppendorf Photometer equipped with a recording apparatus. Usually the amount of mitochondrial protein in the cuvette was in the range of 130 {-lg/ml with an initial absorbance of 0.6. When simultaneous measurements of absorbance and of cations or pH were carried out, the amount of mitochondrial protein was 2 mg/ml. pH and K+ were measured with a Beckman pH meter (expanded scale) equipped with a recording apparatus. The Beckman glass electrode and cationic electrode were used for measuring pH and K+, respectively. Oxygen uptake was recorded polarographically. Mitochondrial pyridine nucleotide were measured with an Eppendorf Fluorimeter equipped with a recording apparatus. Pi was measured as described by LINDBERG AND ERNSTER14 . All experiments were carried out at 30°. RESULTS f{+
uptake induced by valinomycin From the data reported by CHRISTIE et al.s it can be calculated that the rate of
[KCg(mMJ
Fig. 1. K+ uptake in the presence of valinomycin at various KCI concentrations. Composition of the incubation medium was as follows: 250 mM sucrose, 20 mM 'Iris buffer (pI-I 7.4), 5 mM succinate, I pM rotenone, 0-4 !!g valinomycin and amounts of KCI as indicated in the abscissae. Amount of mitochondrial protein was 2 mgjml. Final volume, :il ml. Temperature, 30°.
Biochi m, Biopbys. Acta. 1I3 (rg66) 445-466
REVERSIBLE SWELLING LINKED TO
K+
UPTAKE
447
TABLE I UPTAKE AND RELEASE OF K+ AND EJECTION OF AND IN THE PRESENCE OF PI AND ACETATE
H+ BY
LIVER MITOCHONDR1A IN THE ABSENCE
The medium contained 250 mM sucrose, 8 mM MgC12 , 20 mM Tris buffer (pH 7.4), 0.4/tg valinomycin, 5 mM succinate, I pM rotenone and when indicated I mM Pi or 3 mM acetate. Release of K+ was obtained with roo pM dinitrophenol. The amount of mitochondrial protein was 2.2 mgjml. Temperature, 30°. Rates were expressed in llffioles/g protein per min, and extents in Ilmoles/g protein. Additions
J(+
H+
Uptake
Release
Release
Uptake
-----
None PI
Acetate
E;t:tent
Rate
Rate
Extent
Rate
Rate
3 05 390 3I5
33 3°0 I09
255 I090 I630
7I 7I 73
20
380
43 34
73°
355
K+ uptake by intact liver mitochondria with 20 mM KCI in the medium is about 4 ftmoles/g protein per min. The rate of K + uptake by liver mitochondria supplemented with valinomycin is about 100 times higher (cf. Table I). The amount of K+ taken up by the mitochondria increased linearly with increasing amounts of K + in the medium up to 10 mM (Fig. I). Since the sensitivity of the K+ electrode varies inversely with the concentration of K +, accurate measurements of the K + uptake could not be made above this concentration. From the correlation between K + uptake and swelling it might be predicted, however, that higher concentrations of K + in the medium should not cause a further increase of K + uptake. The rates and amounts of K + uptake and H + ejection in the presence of 2 mM K + are given in Table 1. Addition of Pi or acetate considerably increased the rate of I{+ uptake but only slightly increased the amount of K+ uptake (about 20%). The ratio K+/H+ was about 4, both without or with Pi or acetate. The rates of release of K + and uptake of H + following the addition of dinitrophenol were much faster than the uptake of K+ and ejection of H+. MOORE AND PRESSMAN 10, and CHAPPELL AND CROFTS l l reported that addition of valinomycin or of gramicidin in the absence of anions did not stimulate the respira-
Fig. 2. Oxidation of pyridine nucleotide induced by valinomycin at various KCl concentrations. The medium contained 20 mM Tris buffer (pH 7.4), 2 mM ti-hydroxybutyrate, IO mM MgC12 , 0.2 I~g valinomycin and variable amounts of KCI and sucrose up to a final concentration of 0.25 osM. Amount of mitochondrial protein was 0.7 mgjrnl, Biochim, Bio-phys, Acta, II3 (I966) H5-456
A. AZZl,
G. F.
AZZONE
tion. CHAPPELL AND CROFTSll suggested that the J{+jH+ exchange occurring in the absence of an anion did not require energy. In our experiments addition of valinomycin resulted in oxidation of the mitochondrial pyridine nucleotide also in the absence of PI, and the extent of oxidation was dependent on the concentration of KCI (Fig. 2). The K+ uptake also in absence of PI was strictly dependent either on the presence of an oxidizable substrate or on the presence of external ATP (cj. ref. 13). Respiration was stimulated by valinomycin up to a concentration of 0.02 Itg/mg protein in the presence and 0.008 Itg/mg protein in the absence of PI (Fig. 3). The respiratory rate was higher in the presence than in the absence of Pi. with 3mM P,
360
without
02
fl
04
Valinomycin (pg)
Fig. 3. Stimulation of respiration by various valinomycin concentrations in the presence and absence of Pi. The medium contained 210 mM sucrose, 20 mM 1(CI, 20 mM Tris buffer (pH 7.4), 10 mM MgCI2 , 5 mM succinate and I ftM rotenone. The amount of mitochondrial protein was 2 mg/rnl. Temperature, 30°.
MOORE AND PRESSMAN 1 0 reported an apparent Em for K+ after addition of valinomycin of about 5 mM. CHAPPELL AND CROFTSl l reported an apparent K m for K+ after addition of gramicidin of about 2 mM. Under our experimental conditions, which usually included MgC1 2 in the medium, the apparent J(m for K + was about 12 mM. This figure was obtained from measurements of respiration, of ATPase activity (Fig. 4B and A), and of swelling (Fig. 9).
B
A
20
.40
60
[KeD (mfvl)
1
2
3
1/[5] x10~
Fig. 4. (A, B) Stimulation of ATPase activity and respiration induced by valinomycin at various KCI concentrations. The medium contained 20 mM tris buffer (pH 7-4), 8 mM MgC12 and variable amounts of KCI and sucrose up to a final concentration of 0.25 osM. Also added were: in Expt. A, S mM ATP and I fLg valinomycin; in Expt. B, 5 mM succinate, I ,uM rotenone and 0.2 fIg valinomycin. The amount of mitochondrial protein was I mgjrnl in Expt. A and 2 mgjrnl in Expt. B. Temperature, 30°. Time of incubation in Expt. A was 20 min. Final volume 2 ml.
Biochim. Biophys. A eta, 113 (1966) 445-456
REVERSIBLE SWELLING LINKED TO
K+
UPTAI{E
449
K+ uptake and swelling CHAPPELL AND CROFTS ll
have reported that addition of Pi or arsenate to liver mitochondria supplemented with gramicidin, resulted in a rapid swelling phase. In the absence of anions the swelling was not observed. They suggested that the swelling phase was caused by an increased uptake of cations induced by the anions. As we have shown in Table I, under our experimental conditions addition of Pi or acetate considerably increased the rate but not the extent of K + uptake. Simultaneous measurements of K+ concentration and absorbance revealed a close correlation between uptake of IU and swelling (Fig. 5). In fact the uptake of K+ was slow in the absence of Q2~g
valinomycin
I
I
100WM dinitrophenol
100
~M
dinitrophenol
10mV a2~g vQli()QmYCIn
I 1.2mM Pi
t
I
I
100~M
100~M
dinitrophenol
dinItrophenol
Fig. 5. Correlation between K+ uptake and swelling in the absence and presence of P j • The medium contained 250 mM sucrose, 20 mM Tris buffer (pH 7.4), 2 mM KCI, 5 mM succinate, I pM rotenone and o.4/-lg valinomycin. Pi when present was 1.5 rnM. The amount of mitochondrial protein was 2 mg/m!. Final volume 4 ml.
P l , as was the decrease of absorbance. Addition of Pi either at the beginning or at the end of the process increased neither the extent of uptake of K+ nor the extent of swelling. The rate of K+ uptake and of swelling were, however, considerably accelerated when Pi was added at the beginning of the incubation. Addition of dinitrophenol induced a fast release of K+ and shrinkage both in the presence and in the absence of Pi. Release of ]{+ and shrinkage CHI
450
A. AZZI, G. F. AZZONE 0.21-'9 valir.amycin 1mM succinate
ST
liiny
I ----t~
100r~·oPhehOl
1
~
, m M succinate
l
1
100f.JMdinitrophenol
I t
0.21-'9~VOilnomYCln, t
K uptoke
~
12mv ...-..-..-. 60sec
Fig. 6.(A and B) 1(+ release and mitochondrial shrinkage caused by valinomycin in a K'r-free medium. The medium contained 250 mM sucrose, 20 mM Tris buffer (pH 7.4),0.4 !Ig valinomycin and I pM rotenone. The amount of mitochondrial protein was 2 rng/ml. Final volume 4 ml. The lower curve is a K+ electrode trace; the downward deflection indicates K+ uptake.
chondria after addition of valinomycin and was taken up after addition of succinate. The K+ accumulated was again released by dinitrophenol. While the K+ uptake was accompanied by swelling, the efflux of K+ from the mitochondria was accompanied by shrinkage (Fig. 6B). Both efflux and shrinkage were gradually inhibited by increasing the concentration of cations in the medium (Fig. 7A and B). The inhibition of the shrinkage was complete at 80 mM K + in the medium. Replacement of K + with Rb r did not change the pattern of the process (Fig. 7B). A
B
o.oe
ODS
-e
0.04 •
0 - 40 SO [KCU(mM)
0.04
120
40 80 [RbCD(mM)
120
Fig. 7. (A and B) Inhibition of shrinkage by increasing concentrations of cations in the medium. The medium contained 20 mM Tris buffer (pH 7.4), 0.02 !Ig valinomycin, roo !tM dinitrophenol, and variable amounts of KCl 01' RbCl together with sucrose up to a final concentration of 0.25 osmolar. The amount of mitochondrial protein was I30 !tg/ml.
Effect of the K+ concentration on swelling and shrinkage Acceleration of the rate of swelling was observed not only after the addition of Pi and arsenate icf, ref. II) but also with organic anions such as, lactate, acetate or formate. The rate and extent of swelling and the extent of shrinkage were dependent on the external concentration of KCl with all the anions tested (Fig. 8A, B and C). The rate of swelling increased up to 20 mM KCl and then remained constant. On the other hand, the extent of swelling diminished at increasing concentrations of KCl. Also the extent of shrinkage diminished at higher KCl concentrations and was practically abolished above 80 mM KCl. With acetate, the apparent K m for K+, measured with respect to the rate of swelling, was higher than with Pi or arsenate. Biochirn: Biopbys, A eta, II3 (19 66) 445-456
REVERSIBLE SWELLING LINKED TO
451
K+ UPTAKE Arsenate
Rate of swelling
Rate of swelling
0.4
-~------
::3
0.2
~ 0.2
<,
°O~-'--,J~---L--,,!:"-....L--..!!;;-
40
80
Acetate
o
120
40
80
Rote of swell ing
120
120
[KCOCmMl
[KCOCmMl Extent of swelling
Extent of swell ing
Extent of swelling
02
0.2 I,------~-'-----
1
""
<1 0.1
,"
!
120
120
40
80
120
[KCOCmMl Extent of shrinkage in~. of swelling phase
100
40
4
80
(KCI] (mM)
Fig. 8. Swelling and shrinkage with different anions at various KCl concentrations. The medium contained 20 mM Tris buffer (p H 7.4), 0.02 ftg valinomycin, r ftg oligomycin, 15 mM MgCI 2, 5 mM succinate, I pM rotenone, and variable amounts of KCl and sucrose up to a final concentration of 0.25 osM. Anions wore: I mM PI. 1 mM arsenate and 5 mM acetate. The amount of mitochondrial protein was 130 fig/m!.
Dinitrophenol inhibited the swelling phase and the inhibition was competitive with respect to the concentration of ReI (Fig. gAl. The apparent /(rn for K+ in the presence of 0,10,20,4° anc150,uM dinitrophenol were 12.3, 14.5,3°, 83.5 and 145 mM, respectively. On the other hand, dinitrophenol inhibited in a non-competitive manner the increase of rate of swelling promoted by Pi (Fig. gB). The apparent /(1/1 for P, was about 300 ,uM. A
30
50IJM dinitrophenol 40IJM dinitrophenol
none
20
30
B
50
o
Fig. 9. (A and B) Inhibition of swelling by dinitrophenol at various 1(C\ and PI concentrations. Experimental conditions as in Fig. 8. Abscissae: A, reciprocal of the 1(Cl concentration, B, reciprocal of the PI concentration. Ordinates: the reciprocals of the rates of swelling. Biochirn. Biophys. A cia, 113 (1966) 445-45 6
452
A. AZZI, G. F. AZZONE
The shrinkage phase Addition of uncouplers or respiratory chain inhibitors reversed the swelling process linked to the transport of monovalent cations. Shrinkage followed also the addition of divalent cations, such as Ca2+ and Mn 2+. The shrinkage phase was accompanied by a release of l{+ from the mitochondria (Fig. 10) and lasted for the time 0.1 fJg valinomycin
J K' uptake
I
0.1~g
valinomycin
,J\
500~M
5mvl I/' ~~
100~M MnS04
MnS04
125fJM CoCI 2
~
60se~ Fig. 10. Release of K+ induced by divalent cations. Experimental conditions as in Fig. 5. 5 mM Mgel. and I rn M PI were also present. Final volume 2 ml.
required to remove the divalent cation from the medium. In this respect the shrinkage phase caused by divalent cations in valinomycin-supplemented mitochondria is similar to that caused by ADP in EDTA-supplemented mitochondria-". In Table II are reported the rates of shrinkage induced by uncouplers, respiratory chain inhibitors, cations and ADP in valinomycin-supplemented mitochondria. The highest rate of TABLE II RATES OF SHRINKAGE INDUCED BY UNCOUPT.ERS, RESPIRATORY CHAIN INHIBITORS, CATIONS AND
ADP The medium contained 20 mM Tris buffer (pH 7.4), 210 mM sucrase, 20 roM KCI, 2 mM PI, 0.02 fig valinomycin, 5 m'M succinate and 111M rotenone. The amount of mitochondrial protein was 130 flg/m!. Temperature, 30°.
Additions
Concentration
Rate of shrinkage in LlAlmin
Dinitrophenol
100 ftM Sao flM 1.4 ftg 1.7 mM 500 11M 330 flM
0.260
Ca H
Antimycin A
KeN Mn'+ ADP
0.215
0.r90 0.r60 0.Il5 0.05 0
shrinkage was that given by dinitrophenol, followed by that of Ca 2 +. This result is in agreement with the observation that dinitrophenol causes the highest rate of release of K+ and uptake of H+. Biochim. Biophys. .11 eta,
113 (1966)
445-45 6
REVERSIBLE SWELLING LINKED TO
K+
UPTAKE
453
Effect oj temperature on swelling and shrinkage and on K: uptake We have reported previously that the swelling and shrinkage phases of EDTAsupplemented mitochondria had a high QlO (see ref. IS) thus suggesting a chemical reaction as rate-limiting step in these processes. In the case of the swelling and shrinkage occurring in valinomycin-supplemented mitochondria it was found that the QI0 for both the swelling and the shrinkage phase were influenced by the presence of albumin in the medium. The values of QI0 obtained were, in the presence of serum albumin, r.8 for the swelling phase and 4.3 for the shrinkage phase. In the absence of serum albumin the values of QI0 were 3·7 for the swelling phase and 2.5 for the shrinkage phase. The value of QI0 for the respiration was r.6. In the case of the K+ uptake the value of QI0 varied greatly according to the range of temperature in which
2
0.0033
0.0034
1/r Fig, I!. Effect of temperature on I{+ uptake in the presence of valinomycin. The medium contained 250 mM sucrose, 20 mM Tris buffer (pH 7"1·), 2 mM ICCl, 5 mM MgCl 2 • 5 mM succinate, I ftM rotenone. I urM Pi and 0,4/tg valinomycin. The amount of mitochondrial protein was 2 mg/ml. The release of K+ was obtained by adding I ttg antimycin A,
it was measured (Fig. II). It is seen that the QIO was high at low temperature and low at higher temperature. It would thus seem that under most conditions diffusion of cations is rate limiting with respect to energy supply from the respiratory chain. DISCUSSION
The concentration gradient oj Kv Active transport has been defined by CONWAy16 as flow "dependent on the activity or energy change of another system". According to this definition the transport of monovalent and divalent cations inside the mitochondrion must be considered as "active" since it is inhibited by respiratory chain inhibitors or uncouplers which stop the energy supply for the cation uptake. Another criterion, however, defines a flow of ions or metabolites as "active transport", namely occurrence of a movement against concentration gradients. The transport of K + inside the mitochondrion, analyzed in the present and in previous papers1 2 ,13 , satisfies both criteria for an "active process". The cyclic changes of the intramitochondrial K+ content depending on the energy supply, the ratio between the final concentration of extra- and intramitochondrial cations, and the dependence of the release of the intramitochondrial K+ on the concentration of extramitochondrial cations, are all in agreement with this conclusion. Biochim, Biophys, Acta. II3 (Ig66) 445-45 6
454
A. AZZI, G. F. AZZONE:
Whether the transport of K + must be defined as " acti ve" also according to the criterion formul ated by USSIN G1 7, n amely movem ent aga inst an electrochemi cal gradien t , is be yond the available experimental dat a . The uptake of monovalent cati ons in the absence of P I or acetate is an en ergy requiring process as indicate d b y its dependence on t he presence of oxidi zable subst rate (or of ATP ), by the inhibi tory effect of dinitrophenol, and by the st imulat ion of respiration and of pyridine nucleotide oxidat ion accompa nyi ng the uptake of cations . It would seem, therefore, t ha t the lack of respiratory st imulat ion observed by M OORE AND PRESSMAN 10 and by C HA P PE L L AND CROFT Sll , afte r addition of valinomycin and gramicidin to liver mi t ochondria oxidizi ng succin ate in the ab sence of P I, was du e to the effectivene ss of the State 4 respiration to sup p ort the K+ uptake under the ir experimental condit ions. At variance with the results obtained by JU DAH et al», the uptake of K+ was not accompanied by an eje ction of H-I- in a r(I ratio. Under our experimental conditi ons, both in the pr esence and in the absence of PI or acetate, the ratio K-I-/H -I- was about 4. Theoretical explanat ions for this discrepancy cannot be formulated before more in format ion is obtaine d concern ing the molecula r mechanism of cation transp ort in mit ochondria.
K+ ~tp ta ke and s1J!)elling Variations of t he m itochondrial content of K -I- were alway s accompanied by changes of abs orban ces ; i .e. uptake was accompanied by swelling and release was accomp anied by shrink age. Furthermore, as the exte nt of uptake and of release of cations were independent of the addition of Pi or a cetate, so the extent of swelling and of shrinkage were also independent ofthe addition of Pi or acet ate. These obser vations indicate that the K -I- accumulated in the mitochondrion is always present as an ionized, osmotically acti ve species. The differen ces b etween th e behaviour of mon ovalent and divalent cations will be examine d in a subsequen t paper. R ecently PRESSMAN 18 has presented calculations which cast some doubt on the hypothesis that the volume changes obs erved during the uptake of K + are ind eed du e t o an osmotic mechanism . An osmot ic mech ani sm of water transp ort is considerably support ed by our da t a. First ly our experiments indicate always a close parallelism between extent and rate of K+ uptake and ext ent and rate of swelling. Secondly, according to the calculation of PRESSMAN 18 , the entrance of I It! of H 20/mg protein, corresponding to a 20% decrease of light scattering sh ould clemand a net uptake of 150 m,umoles K-I-/mg protein . The figure of K + uptake pr esented in Table I satisfy the requirement for an osm otic swelling both in the presence and in the absence of P, or acetate. Effect oj the anions C HAPPE LL AND C ROFT Sl l
explain the lack of swelling which they observe in the absence of Pi by suggesting th at the K + uptake is "limi ted by the net n egative charge withi n the m itoch ondria". When PI enters "it provides more H + to b e pumpe d out in exchange for K +; now there is an increase of inte rn al osmot ic pressure (due to accumu lation of K-I- and Pi ) and the m it ochondria swell" . In the me chanism propos ed here, en ergy is required for the dissociation of intramitoch ondrial an ions. The dissociati on provides H + whi ch is ejected in ex change for K -1- , and negati ve ch arges which B iochim, B iop hy s. Acta, II 3 (1966) 445- 45 6
REVERSIBLE SWELLING LINKED TO
K+
UPTAKE
455
maintain electrical neutrality during K + uptake. The internal osmotic pressure increases when undissociated mitochondrial anions are replaced by dissociated K+ salts of the anions. This mechanism is consistent with the observations reported above (cJ. also ref. 13) that the K+ uptake and the parallel swelling are independent of the presence of Pi but dependent on energy supply. The charge separation mechanism of MITCHELL19 offers an explanation for the stimulating effect of Pi. Owing to the dissociation of intramitochondrial anions, a high pH is reached inside the mitochondria when Pi is absent. The high pH inhibits electron transport and thereby also K+ uptake and swelling, The internal pH is lowered when H + is provided by dissociation of the second acidic group of intramitochonclrial PI, and respiration is stimulated. In steady-state conditions the presence of Pi causes an increased turnover of K+ through the mitochondria. The shrinkage phase In previous papers 1a ,15 , 2 0 we have proposed denoting as "irreversible", the swelling processes which require an external supply of energy for shrinkage, and "reversible", those which do not require an external supply of energy for shrinkage. The swelling linked to the transport of monovalent cations, occurring in the presence of valinomycin, gramicidin or EDTA, therefore fall in the category of the reversible swelling processes, The occurrence of these latter shrinkage phases in the presence of respiratory-chain inhibitors or of uncouplers, raises the question of the energy dependence of these processes. As we have discussed above, the swelling process follows the establishment of concentration gradients of cations, The shrinkage phase must be considered as due to the utilization of the energy of the concentration gradient which drives the efflux of the cation from the mitochondrion. The inhibition of the shrinkage phase at higher concentrations of Kel in the medium is due to a lowering of the concentration gradient which is dependent on the ratio between intra- and extramitochondrial concentrations of cations. If the concentration gradient of cations is lost, the mitochondria lose the capacity of shrinkage without external supply of energy. It is suggested that the occurrence of the cation gradient represent the basic distinction between reversible and irreversible swelling processes.
ACKNOWLEDGEMENTS
The skillful technical assistance of Miss B, BARBIN and Mr. L. AGOSTI is gratefully acknowledged. This investigation was aided in part by the Muscular Dystrophy Associations of America Incorporation. REFERENCES I V. G. SPECTOR, Pvoc, Roy. Soc., London, SBr. B, 14-1 (1953) 268. 2 C. A. PRICE, A. FONNESU AND R. E. DAVIES, Biochem. J., 64 (r95 6) 754· 3 J, A. AMOORE AND "V. BARTLEY, Biochem. J., 69 (195 8 ) 348 . 4 J. L. GAMBLE. JR., J. BioI. Chem., 228 (1957) 255· 5 R. L. SCOTT AND J. L. GAMBLE, JR.,]. Bioi. Chern" 23 6 (19 6 1) 57°· 6 L. SHARE, Am, j. Physiol., 194 (195 8) 47. 7 F. ULRICH, Am. j. Physiol., 198 (lg60) 846. 8 G, S. CHRISTIE, K. AHMED, A. E. M, McLEAN AND J. D. JUDAH, Biochim. Biopbys. Acta, 94 (19 6 5) 43 2.
Biochirn. Biophys. Acta, II3 (19 66) 445-45 6
A. AZZI , G. F. AZZONE
9
J . D . Jun AH, A. E . M. M cLEAN , K. A HMJ;:D AND G. S. CHRISTIE. B ioclii m , B ioPIIJ'S. Acta, 94 (19 6 5) 4 4 1 . 10 C. MOORE AND B. C . PRESSMAN , B iochem , B iop hys. R es. Com mltn ., IS ( 19 6 4 ) 562 . I I J. B. CHAPPELL AND A . R. CROFTS . Biochem , J., 95 (196 5) 393. 12 A. A ZZI AND G. F . AZZONE, B iochem , f. , 9 6 ( 1965) r c. 13 G. F . A ZZONE AND A. A ZZI, in ] . M. T AGER, S. PAPA , E . QUAGLIA R IELLO AN D E . C. S L AT ER .
Regu lati on of Metabolic Processes in M itochondri a, Elsev ier, Amsterdam , 19 66, P- 332. 14 O. L I NDBERG AND L. ERNSTER, Methods B iochem. Anal., 3 ( 1956) 1. 15 A. AZZ I AND G. F . AZZONE, B iochi m: B iopliy s, A cta, 10 5 ( 1965) 265 . 16 E . ]. CONWAY, Symp , S oc. Expti. Bio l., 8 ( 19 5 4) 2 97 · 17 H. H. USSING, T he A lkali Meta l. I ons in I solated Sy stem s and T i ssues, Springer, Berlin, 1 9 6 0 , p. I . 18 B. C. PRESSMAN. P roc. N ail. A cad, S ci. U.S ., 53 ( 1 9 65) 1°7 6 . 19 P. M ITCHELL, in J. M . T AGE R, S . P AP A, Eo Q UA GLI AR I ELLO AND E . C . SLATER, R egulati on of Me taboli c Processes in M itochondria, Elsevier, Amsterdam, 1966. 2 0 G. F . AZZONE AND A. AZZI , P roc. Notl, A cad. Sci. U.S ., 53 (19 6 5 ) 108 4. Biochdm, Bia phvs. Acta, II3 (1966)
445-456