Role of mitochondrial oxidative phosphorylation in the maintenance of intracellular pH

3ournal of Molecular

andCellular

Cardiology

(1979)

11,933-940

Role of Mitochondrial Oxidative Phosphorylation Maintenance of Intracellular pH*

in the

of Pharmacology, University Medical School of Sreged, 6701 Szeged, P.O. Box 115, Hungary

Defiartment

P. L. V~GHY. Role of Mitochondrial Oxidative Phosphorylation in the Maintenance of Intracellular pH. Journal of Molecular and Cellular Cardiology ( 1979) 11,933-948. The oxidative phosphorylation of isolated rabbit heart mitochondria and the accompanying pH changes were simultaneously measured in the same sample. The nearly constant extramitochondrial proton concentration during State 4 respiration decreased considerably under conditions of oxidativephosphorylation.Ontheotherhand, theprotonconcentrationincreasedwhenATP was hydrolysed by the mitochondrial ATPase. Thelysis of mitochondria by Triton X-100 equilibrating the pH difference across the inner membrane did not cause significant change in the proton concentration of the sample neither before nor after State 3 respiration. The accumulationof protons during the extramitochondrial hexokinase reaction was reduced by the mitochondrial oxidative phosphorylation. It is suggested that in the normoxic myocardial cell, the protons generated by the extramitochondrial hydrolysis of ATP are utilized during mitochondrial oxidative phosphorylation. KEY

cardial

WORDS: Heart ischemia.

mitochondria;

Oxidative

phosphorylation;

Intracellular

pH;

Myo-

1. Introduction The cytoplasmic metabolic processes requiring the hydrolysis of ATP as energy sources have been shown to be accompanied by generation of protons [ 8,171. All of them may contribute to a continuous increase in the intracellular proton concentration. On the contrary, little is known about the intracellular processes being able to counterbalance the proton-generating reactions, keeping thereby the pH of the cytoplasm at a constant level in normoxic conditions. The splitting of ATP by the mitochondrial ATPase has also been found to be a proton-generating process [3, 10, 191. At the same time, the question has not been settled whether the mitochondrial oxidative phosphorylation is a net proton-consuming reaction or not [8]. It has recently been suggested that there is no net production or utilization of protons under conditions of oxidative phosphorylation [a, lo]. On the other hand, consumption of protons coupled to the bacterial photophosphorylation and mitochondrial oxidative phosphorylation has previously been demonstrated [4, 161. In *Winner of the first Bing Young Investigator 1978. Presented at the Ninth Congress, India, Dedicated 00222828/79/100933+08$02.00/0

to Richard

Prize of the International 1 October 1978. J. Bing M.D. 0

Society

for Heart

Research,

(London)

Limited

on his 70th birthday. 1979 Academic

Press Inc.

934

P.

I..

VxkGHY

this paper experiments are presented showing tightly coupled isolated rabbit heart mitochondria of protons from the extramitochondrial space.

2. Materials

that oxidative phosphorylation of is accompanied by net utilization

and Methods

Mitochondria were isolated from the rabbit heart according to the method of Sordahl el al. [ZU]. The isolation medium contained 0.18 M KCI, 0.01 M EDTA and 0.5% bovine serum albumin; its pH was adjusted to 7.40 at 4°C by addition of Tris base. The mitochondrial pellet obtained was resuspended in the same medium and the protein concentration was determined by the biuret reagent [9]. The proton concentration changes and the oxygen consumption of mitochondria were simultaneously measured in the same sample. Oxygen electrode of Clarke type and glass as well as reference electrodes were inserted into a water-jacketed cuvette developed in our laboratory. The content of the cuvette was thermostabilized at 37°C and magnetically stirred. The electrodes were attached to an Oxygraph (Gilson Medical Electronics) and to a Radiometer PHM 64 Research pH meter, repectively, and the changes were simultaneously recorded by a two channels Micrograph (BD6 Kipp and Zonen) . The experimental medium consisted of 0.25 M sucrose, 1 mM sodium-pyruvate and 5 mM potassium-phosphate buffer, pH 7.00 at 37°C. In a 4 ml sample about 4 to 5 mg mitochondrial protein was used. Other additions are indicated on the figures. The pH of the solutions used was carefully adjusted to the pH of the medium. The exact concentration of the ADP solution was enzymatically determined using Boehringer ADP/AMP test-combinations. The ADP:O ratio was calculated according to Estabrook [7]. The determination of H+: ATP ratio is described in the results The chemicals used were: sucrose, potassium dihydrogen phosphate tryst , di-potassium hydrogen phosphate anhydrous, sodium pyruvate (Merck), bovine serum albumin (Phylaxia Hungary), adenosine-5’-triphosphoric acid disodium salt, magnesium chloride, 2-4-dinitrophenol, ethylene diaminetetracetic acid di-sodium salt and Tris-(hydroxymethyl)-amino-methan (Reanal), adenosine-5’diphosphoric acid tryst. and ADP/AMP test-combination (Boehringer), Triton X-100 and hexokinase from yeast tryst. (Serva), atractyloside (Calbiochem), oligomycin (Sigma). All reagents were of analytical grade.

3. Results Under our experimental conditions, during heart mitochondria, the proton concentration

State 4 respiration of isolated rabbit of the medium was nearly constant.

MITOCHONDRIAL

OXIDATIVE

PHOSPHORYLATION

935

Oxidative phosphorylation, i.e. the State 3 respiration, was induced by the addition of ADP to the sample. The rapid rate of oxygen consumption during oxidative phosphorylation was accompanied by a considerable decrease in the proton concentration of the medium. When all the added ADP was phosphorylated to ATP, the mitochondria returned to the State 4 respiration, i.e. the oxygen consumption was slowed down and the proton concentration of the medium remained constant [Figure 1 (a), solid line]. The ADP :0 ratio and the respiratory control index (RCI) were characteristics of tightly coupled mitochondria. Oligomycin, an inhibitor of the mitochondrial ATPase [19] or atractyloside, and agent blocking adenine nucleotide translocation [22], were found to be capable of preventing the effect of ADP both on the oxygen consumption and the change in the proton concentration when added before ADP [Figure 1 (a), broken line]. These results suggest that the change in the proton concentration of the medium is somehow related to the oxidative phosphorylation. When DNP an uncoupling agent and stimulator of mitochondrial ATPase was given after the transition of State 3 to State 4, the respiration was stimulated and the proton concentration of the medium was increased approximately to the level measured before the addition of ADP. Oligomycin or atractyloside given prior to the addition of DNP, inhibited the increase of the proton concentration but the repiration was stimulated by the uncoupling agent [Figure 1 (a), broken line]. Accordingly, the amount of protons disappearing during oxidative phosphorylation, appeared during hydrolysis of ATP by the mitochondrial ATPase. The next question was whether the proton consumption during oxidative phosphorylation was due to equilibration of the proton concentration gradient developed by the respiratory chain between the two sides of the inner membrane or it was a net proton utilization from the extramitochondrial space. Triton X-100 known to equilibrate the pH gradient by lysis of the mitochondrial membranes was used before and after addition of ADP. Neither the Triton X-100 treatment, nor the subsequent addition of ADP influenced the pH of the sample [Figure 1 (b)] . Triton X-100 added after the complete phosphorylation of ADP induced only a moderate increase of the proton concentration. After equilibration of pH between the extramitochondrial and matrix spaces, the buffering capacity of the sample was determined by the addition of 0. I N HCI [Figure 1 (c)l. From the amount of HCl needed for the restoration of the pH of the medium to the level measured at the end of the experiment performed with addition of ADP to Triton X-100 treated mitochondria [Figure 1 (b)], the net amount of protons disappeared during oxidative phosphorylation was determined. Furthermore, the H+: ATP ratio was calculated from the amount of ADP added and the amount of protons disappeared during the State 3 respiration (see Table 1). To study the relationship between the extramitochondrial ATP hydrolysis and the mitochondrial ATP synthesis, the oxidative phosphorylation was measured in the presence of glucose and hexokinase. In these experiments a reaction medium

processes

1. Net proton generation

phosphorylation

during

the metabolic

1.35

Glucose + Pi

ADP + Pi 2.35 1.35 --3

----f

Glucose + ATP ----f 3.14

+ ADP 2.35

1.76

Glucose-6-P

+ HsO

ATP + Hz0 3.14

Glucose-6-P 1.76

0.41

-0.56

0.97

Calculated mol of proton generated by the reactions

processes studied

Interaction ofreactants and the mol of proton dissociated by 1 mol of reactant at pH 7.0

and consumption

and that

-0.52 (-0.019)

Measured H+ : ATP ratio

The mol of proton dissociated by 1 mol of reactant at pH 7.0 was calculated from the pK value(s) of the reactant. The mol of proton generated by the reactions means the difference between the mol of proton dissociated from 1 mol of product(s) of produced by 1 mol of substrate(s). The measured value was the mean ( f S.E.) obtained from five different experiments.

oxidative

Hexokinase reaction plus

Oxidative phosphorylation

Hexokinase reaction

Metabolic

TABLE

MITOCHONDRIAL Olig :r

ADP: RCI

.

OXIDATIVE Triton X-100

ADP .

ADP

,I

937

PHOSPHORYLATION

ADP

I

0 ~2.30 = 14.5

- O2 = zero

(0)

(b)

(cl

FIGURE 1. Changes of extramitochondrial pH during mitochondrial respiration. In 4 ml medium 4.5 mg mitochondrial protein was used. Eight micrograms oligomycin (Olig), 100 pg atractyloside (Atr), 0.4 pmol Z-4-dinitrophenol (DNP), 4 mg Triton X-100 and 1.38 pmol HCl were added. Additions are indicated by the arrows. The amount of ADP used was 1.78 and 2.67 qol in experiment (a) and experiments (b) and (c), respectively. Upper curves represent the oxygen consumption of mitochondria and the lower ones indicate the proton concentration changes in the medium. The results obtained after oligomycin or atractyloside treatment are indicated by the broken lines. Olig or

;

-

I

lpmol

-

0,

q

zero

H+

2min

FIGURE 2. Inhibition of extramitochondrial accumulation of protons by mitochondrial phosphorylation. The experimental medium was the same as described in the methods exception that 2 rn~ ATP, 10 VP/ml hexokinase and 0.2 mr.s MgCls were also present. 4.1 chondrial protein (M) was used in 4 ml medium. Forty micromol glucose, 8 pg oligomycin atractyloside were added. For symbols and other details see Figure 1.

oxidative with the mg mitoor 100 pg

938

P. L. VAGHY

being appropriate both for the mitochondrial oxidative phosphorylation and the hexokinase reaction was chosen. After addition of mitochondria to the medium containing ATP, MgCla and hexokinase in addition to pyruvate and phosphate, State 4 respiration and a constant pH in the medium was measured (Figure 2). Subsequent addition of glucose increased the rate of oxygen consumption of mitochondria and resulted in only a moderate increase of the proton concentration. When the oxygen content of the system was exhausted, the proton concentration was sharply elevated (Figure 2, solid line). Oligomycin or atractyloside added prior to glucose prevented the stimulation of the oxygen consumption, and immediately induced a considerable increase in the proton concentration (Figure 2, broken line).

4. Discussion The experimental results reported here show that State 3 respiration of isolated heart mitochondria was accompanied by the disappearance of protons from the extramitochondrial space. Oligomycin and atractyloside diminished the utilization of protons by inhibition of the oxidative phosphorylation. The same amount of protons that were consumed during synthesis of ATP appeared after hydrolysis of ATP by the DNP-stimulated mitochondrial ATPase. According to the widely propagated chemo-osmotic coupling theory of oxidative phosphorylation, the respiratory chain is able to produce a pH and/or an electrochemical gradient of protons (alkalic and negative inside, respectively) across the inner membrane of mitochondria. This latter is supposed to be the driving force of the diffusion of protons backward through the Fo component of the mitochondrial ATP-ase and the coupled synthesis of ATP [12-15,181. In this case there would be neither net production nor net utilization of protons during synthesis of ATP by the oxidative phosphorylation [Z, lo]. According to another opinion, however, the membrane potential does not play any role in the oxidative phosphorylation [21]. The current theories of oxidative and photosynthetic phosphorylation have recently been reviewed [I]. In our studies the lysis by Triton X-100 of mitochondria respiring in State 4, either before or after State 3 respiration, produced considerably smaller changes in the pH of the sample than the oxidative phosphorylation. We assume that the consumption of scalar protons is responsible for the decrease in the extramitochondrial proton concentration during State 3 repiration. The H+: ATP ratio was determined quantitatively at pH 7.0 which was shown to be equivalent with the intracellular pH [5, 61. Theoretically, the synthesis of ATP is coupled with the consumption of 0.56 mol proton per mol ATP. This is due to the smaller ability of ATP to dissociate protons at the same pH than ADP plus orthophosphoric acids. The measured and calculated H+: ATP ratios were found to be similar (Table 1). For calculations the pK values reported by Johnson were used [lq. The

MITOCHONDRIAL

OXIDATIVE

PHOSPHORYLATION

939

extramitochondrial proton concentration was increased considerably by the protongenerating hexokinase reaction even in the presence of mitochondria when the oxidative phosphorylation was blocked either by inhibitors or by the absence of oxygen. Theoretically, about 1 mol proton was produced during synthesis of 1 mol glucose-6-phosphate by the hexokinase reaction. When the oxidative phosphorylation was coupled with the extramitochondrial hydrolysis of ATP, only a moderate increase in the extramitochondrial proton concentration was measured. In this case, the generation of protons by the hexokinase reaction was at least partly compensated by the mitochondrial ATP formation. According to the calculations, the net synthesis of 1 mol glucose-6-phosphate from glucose and orthophosphoric acid was accompanied by the generation of 0.4 mol proton. These results provided additional evidences in favour of the assumption that oxidative phosphorylation is coupled to the decrease in the scalar proton concentration of the extramitochondrial space. These in vitro results suggest that in the normoxic myocardial cell, the protonconsuming mitochondrial oxidative phosphorylation is able to counterbalance the extramitochondrial proton-generating processes being coupled to the hydrolysis of ATP. In this respect the mitochondrial oxidative phosphorylation is responsible not only for the maintenance of the intracellular ATP concentration at a constant high level, but also for the maintenance of the intracellular proton concentration at a constant low level in normoxic conditions. According to our suggestion, the failure of the net proton-consuming oxidative phosphorylation plays an important role in the development of the ischemic acidification of the cytoplasm.

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P.L.V.kIHY GEVERS. W. Generation Mollecular

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