Vol. 166, No. 3, 1990 February 14, 1990

BIOCHEMICAL

THE INFLUENCE

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1352-1357

OF pH ON Ca*+ EXCHANGE

IN FERRET HEART MTOCHONDRIA

C.H. Fry’ and J.A.S. McGuigan Department Department Received

of Physiology,

University of Bern, Biihlplatz

of Physiology, St Thomas’ December

22,

5, CH-3012 Bern, Switzerland

Hospital Medical School (U.M.D.S.),

London SE1 7EH, U.K.

1989

Summary: The effect of pH chanses on Ca2+ transport by isolated heart mitochondria was measured. Two components of Ca + transport were identified, an accumulation dependent on mitochondfial respiration and a Na+-dependent efflux. A decrease of pH over the range 7.7-6.7 reduced the initial rate and the total amount of respiration dependent Ca2+ accumulation. At pH 7.2 the [Na+] required to activate half-maximal efflux, k, 2, was 7.5fl .l mM. Decreasing the pH over the range 7.7 to 6.9 increased the kpz from 3. 6 to 11.6. The effect of acidosis was more profound on the respiration dependent Ca uptake than the Na+-dependent efflux. 0 19'30

Academic

Press,

Introduction: positive

Acidosis

on cardiac

effect (1) and the weight

largely

mediate

(e-c) coupling sensitivity under

Inc.

these phenomena. are attenuated conditions

intracellular

[Ca2’]

is negatively

Several

by hydrogen

of the myofibrils.

certain

muscle

of evidence

intracellular

Heart

appear

is high (2).

driving

mitochondrial

exhibit

force

to contribute

potential

transport.

Adequate

a

changes

current and the Ca2+

in e-c coupling

is debatable

especially

is often associated

but

when

the

with an intracellular

of the action of H+ and Na+ on mitochondrial

Ca2+

is supplied

and

Na+

of the conditions

succinate

be present

to the suspension

which can transport

gradients

that exists pathway

across

the

to generate

medium.

(3).

more accumulation

across

the inner

The mitochondrial by electron

The Na+-dependent

mitochondrial

these two processes conditions

the

it and in this paper

Ca2+ in either direction

alter the steady-state

in which

and is generated

is

pathway

depending

membrane.

on the

At certain

exist in a steady-state

will lead to Ca2+ flux one way or the other.

if H+ would

influence

by Crompton

by the potential

must therefore

Ca2+ and Na+ concentrations

to ascertain

one via a uniporter

on the inner side of the organelle

substrate

alteration

it would

transport,

of Ca2+

and the other via a Na+-dependent

is negative

therefore

Materials described

modes

counter-exchanger,

extramitochondrial

whether

two

by adding potassium

is via a NGa prevailing

exerts

in excitation-contraction

to the process,

The latter situation

for Ca2+

membrane

membrane supplied

alkalosis acid-base

would be appropriate.

mitochondria

inward

steps involved

The role of the mitochondria

it would

whilst

that intracellular

ions, such as the second inward

acidosis and a raised [Na+] so that an investigation Ca2+ exchange

inotropic,

suggests

but

It was of interest

to any great

extent

and

or release of Ca2+.

Mitochondria were prepared from ferret ventricles by the method et al. (3) but in which the initial homogenisation medium contained

0006-291x/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form resewed.

1352

Vol.

BIOCHEMICAL

166, No. 3, 1990

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

0.05% fatty acid free bovine serum albumin and O.lmM EGTA. Protein mitochondrial pellet was determined by a modified biuret method (4).

content

in the final

Ion exchange and 02 consumption was determined in a water-jacketted chamber, the floor of which was an 0, electrode and the roof modified to accept Ca2+ and H+-selective electrodes (2). An uptake by the preparation of an ion or 0, was recorded as a reduction of the suspension concentration. Measurements were made with a sample of the mitochondrial preparation (I-3mg protein) in a suspension medium containing 120mM KCI (or 240mM sucrose), 20mM HEPES, ImM MgC12, 0.2mM EGTA. pH was initially 7.2 titrated with KOH. In some experiments Smmol/l NaCl was added, but had no effect on the initial rate of Ca2+ accumulation. Ca2+ accumulation was initiated by addition of K-succinate (20mM) to the suspension medium. Na+-dependent Ca2+ release was initiated by addition of NaCl samples (3M stock solution) when the initial accumulation had achieved a steady-state or was inhibited by adddition of ruthenium red (2nM). Ca2+ electrode calibration was carried out by the method described by Bers (5). All experiments were performed at 22°C. Results

and Discussion;

cardiac

mitochondria;

Figure 1 illustrates

the three traces illustrate

The left side of the ion-selective addition

of KOH

accumulation represent

to maintain

was initiated

an 0, consumption.

the two modes of Ca2+ transport

electrode

to achieve

of K-succinate

When Ca2+ accumulation

a period of 3 minutes)

18mM in six equal steps resulted

addition

the

required

of CaC12 samples starting

[Ca2+].

where shown and was accompanied

achieved

in

from the O,, H+ and Ca2+ electrodes.

traces show the final addition

pH at 7.2)

by addition

outputs

demonstrable

steady-state

of NaCl samples

(and Ca2+

also by

(the break in the tracings

to raise the concentration

from zero to

in a graded efflux of Ca2+.

-

100

i 7.25 720

02 nmol /ml

pH

715 -57

- 56

-55

PC0

.5L

lmln

- 53

Figure 1. Ca2+ accumulation and relase by cardiac mitochondria. The three traces show, from the top down, outputs of 0 , H+ and Ca2+ electrodes. The initial [Ca2+J was achieved by addition of CaC12 samples, with p l-f corrected by addition of KOH. At the first break in the Ca2+ electrode trace the gain was increased ten-fold, the calibration curve refers to the high gain record. K-succinate was added to achieve a final concentration of 20mM. The second break in the traces represents a period of 3 minutes. The suspension [Na] was increased in steps of 3mmoVI from zero to 18mM. Further details are given in the text. 1.2mg protein/ml, 22°C.

1353

Vol.

166, No. 3, 1990

Increasing

BIOCHEMICAL

the suspension

and conversely accumulation performed,

pH from 7.2 up to 7.7 accelerated

decreasing was

AND BIOPHYSICAL

the initial rate of Ca2’

the pH to 6.7 reduced the initial rate.

strongly

dependent

on the

RESEARCH COMMUNICATIONS

starting

Because

[Ca2’],

dependence

dependent

on the initial [Ca2+] between

of this initial rate ratio (using a logarithmic

2. The data can be described from 7.2 to 7.54 (range (range

Alteration

7.47-7.65,

6.76-6.95,

to suspension

by a linear relationship

to achieve

.l mM

in figure

as pH is raised from 7.2

the Na+-dependent

such a curve to be characterised

half maximal

route of Ca2+ efflux. A plot of the

of [Na+], from data as in figure 1, yielded a sigmoid efflux, k,,2.

pH altered the Na+ dependence reduced

increased appear

representative

on test pH is shown

The

curve.

A

by a slope factor, s, and the [Na+]

At pH 7.2 the k,,,

value was calculated

to be

(7 determinations).

7.7 progressively an acidosis

ordinate)

and halved as the pH is reduced

The slope factor of the Hill plot was not significantly suspension

1 and 22uM for a given test pH.

10 determinations).

Hill plot of the data allowed

however

were

The Value of this ratio was

such that the rate is doubled

9 determinations)

pH also influenced

rate of Ca2+ efflux as a function required

of experiments

one at control pH and the other at a test pH, in which the initial [Ca2+] was the same.

not significantly

7.5fl

the initial rate of Ca2+

pairs

The ratio of initial rates (I.Rte,t PH/I.R. pH7.2) was then calculated.

to 6.86

accumulation

altered by suspension

of Ca2+ efflux. Alkalosis

the k,,2 value, that is efflux was enhanced

pH.

the

Na+-sensitivity

of Ca2+

by lower [Na].

efflux further.

-0.8

!

6.6

is plotted

I

6.8

as a function

I

of suspension

I

7.0

7.2

pH.

Conversely

acidosis did not

Figure

plots of the Na+ sensitivity of Ca2+ efflux at normal, alkaline

In figure 3b the k,,2 value

3a shows

and acid pH values. The

I

I

7.4

7.6

straight

line was

I

7.8

Test pH Figure 2. The effect of pH on the initial rate of Ca2+ accumulation. The initial rate is expressed as a ratio of that obtained at a test pH and the control (7.2) value. The ordinate is expressed as a logarithmic scale. 1354

of the

from the control pH of 7.2 to

the k,,2 value down to a pH of about 6.9; a more profound

to reduce

Variation

Vol.

BIOCHEMICAL

166, No. 3, 1990

/

pH 7.20 /

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

pH s 752

1

0

3

6

9

I

12

15

1

21

18

24

z

E

9 -cu . Y

fG

I

6.6

617

;8

619

710

712

771

713

714

715

716

717

718

PH

Figure 3. Part a. The Nadependence of Ca ‘+ efflux from cardiac mitochondria; efflux rates are expressed as a proportion of the maximal rate. Curves were obtained at pH 7.68, 7.20 and 7.02. and reducing the pH shifted the curves to the right. Part b. The dependence of the [Na] necessary for half maximal efflux, k,,2 The straight line was fitted through all data points except the two at pH = 6.7. The value (&.D.) at 7.2 was obtained from 9 determinations.

obtained

by linear regression

[Na]/pH

unit.

The

normal

intracellular conditions

an alkalosis

also enhance opposite

intracellular pH about Ca2+

argument

of the data points between

pH 7.7 and 6.9 and has a slope of 7.6mM

[Na+]

at 22°C

7.2 (6). would

increase

efflux through would

effect on Ca2+ transport

in cardiac The

pertain

muscle

previous

experiments

is about

have

lo-14mM

indicated

Ca2’ uptake via the respiration-dependent

the Na+-dependent for an acidosis.

pathway It is unclear,

at steady state, the result depending 1355

and the

that under

these

mechanism

and

as the k1,2 value is reduced

- the

therefore,

what would

on the predominant

be the net route.

An

Vol.

166, No. 3, 1990

BIOCHEMICAL

AND SIOPHYSICAL

RESEARCH COMMUNICATIONS

1 min 4.7

Fiaure 5 The influence of suspension pH on steady-state Ca 2+ accumulation. Outputs from H+ and Ca + electrodes are shown. Ca2+ accumaulation was initiated by addition of K-succinate. At steady state suspension pH was altered by addition of KOH and then returned to the control value by addition of HCI. See text for details of the solutions. 1 .l mg prouml, 22°C.

experiment

designed

to answer

in a medium containing on contaminant

this question

is shown in figure 4. Mitochondria

no EGTA or added Ca2+, so that the initial [Ca2’],

levels, the pH was 7.21 and the [Na] 10mM.

by addition

of K-succinate.

suspension

and a further small uptake of Ca2+ was observed.

7.22 this additional of the uptake Similarly,

uptake was attenuated.

pathway

by alkalosis

but not shown, reduction

that depression

of the uptake

Na+-dependent

indicate

Ca2+ exchange accumulating

capacity

and the opposite

i.e. pHt 7.0-7.2

the uniporter

Ca2+

greater

is balanced

of the pH back to

of Na+-dependent

a net Ca2+ release

than

the reduced

Alteration

(7).

Ca2+

efflux.

suggesting

again

efflux

through

the

would

for mitochondrial

at which

be appropriate involvement

the intracellular would

agree

[Na+]

myocyte

1pM because

minor

relaxation

pathway.

processes

is low has been

with these findings.

shown

In addition, 1356

muscle

Opposite

of [Na+].

concentrations,

intracellular

exceeds

of both ions will raise

are in steady-state.

and reduction

in intact

the organelle

[Ca2+]

the [Ca2+] at which the

of the concentration

at diastolic

uptake through

However,

such that an increase

alkalosis

in

normal

is not seen under

if the intracellular

pH and [Na+] will change

accumulation

under

at this concentration

the Na+-dependent

to an intracellular

is to reduce Ca2’

The role of mitochondria

is probably

the two mitochondrial in Ca2+

affect both routes of

Net Ca2+ accumulation

of contractile

to the intracellular

are at steady-state

the Ca2+ concentration

pH can profoundly

with an alkalosis.

the

IO-14mM.

by efflux through

this value

two processes

observed within

when the [Ca2+] falls below to the initial phase

experiments

was

of KOH to the

that augmentation

such that the net effect of an acidosis

and [Na+]t

may contribute

when

the increase

to extamitochondrial

mitochondria

of intracellular

these conditions

arguments

Upon correction suggests therefore

of pH below 7.2 induced

that changes

in cardiac

regulation

conditions,

outweighs

solely

of Ca2+ was initiated

the pH was raised to 7.55 by addition This experiment

pathway

Accumulation

depended

pathway.

The results

the

At steady-state,

were suspended

17uM,

Evidence

i.e