Heterogeneity of pH in the aqueous cytoplasm

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FASEB

Heterogeneity of pH in the aqueous cytoplasm

of renal proxim al tubule cells

TA K YEE A W ’ AN D DEA N P. JON ES

D epartm ent o f B iochem istry, E mory U niversity School of M edicine, A tlanta , G eorgia 30322, U SA

ABSTRACT

Heterogeneity of cy toso lic pH w as studied w ith

com-

pounds that distribute between the cy toso l and m ito-

chondrial m atrix in fundam entally d ifferen t ways, i.e .,

according to th e

extent

of

ionization

or accord ing to

the function of H f-coupled transport systems. Resu lts

show that the

average

cy tosolic pH is considerably

more

alkaline than the

region

to w hich m itoch on dria

are

exposed. B ecause m itochondria

are

localized pre-

dom inan tly in the basal reg ion , the results are consis-

ten t w ith a transcellu lar pH gradient w ith in the cy toso l

of

proxim al tubule cells. Experim ents analyzing the

effects of inhibiting efilux of HC03 at the basal surface

and Na-H exchange at the apical surface support the

in terpretation that the function of these systems

con-

tributes to

the transcellular pH grad ient. The

existence

of a heterogeneity in pH w ith in the cytosol has

impor-

tant

im plications concern ing the function and regula-

tion of n um erou s c ell p ro ce ss es . Aw , T. Y .; JONES,

D . P . H eterogeneity of pH in the aqueous cytoplasm of

renal prox imal tubu le cells .

FASEB

J.

3:

52-58;

1989 .

Key

Wordc:

m itochondria kidney m icrocom partm entation

energy metabolism bicarbonate Na,H’ exchange proximal

tubule cell cytosolic pH

CONSIDERABLE EVIDENCE HAS ACCUM ULATED

tha t m icro-

compartm entation of m etabolites and ions can occur in

eukaryotic cells due to spatial separation of system s

responsib le for the ir introduction and removal. Such

heterogeneities occur as a consequence of a high flux

relative to the effective diffusion of the species in the

aqueous m edium and are typically detectab le under

conditions in which concentrations are low and flux is

m axim ally stim ulated , or w hen supply rate is lim ited

relative to consumption rate . For instance, heteroge-

neities in 02 and ATP concentration have been found

in adult rat hepatocytes under hypoxic conditions be-

cause of the heterogeneous distribution of m itochondria

w ithin the cells and the restricted supply of the respec-

tive species rela tive to its consumption rate (1-4).

S tudies w ith fluorescent ind ica tors show that concen-

tration grad ien ts of C a2 can also occur in cells during

signal transduction processes (5-9). Earlier stud ies

have show n that radio labeled glycine incorporated in to

hippurate in perfused kidney has the sam e specific ac-

tivity as that in the perfusion medium , whereas the

specific activity of the glycine in the tissues remains

considerably low er (10, 11). These resu lts indicate that

a m icrocompartm entation of glycine m etabolism occurs

in the k idney . In other radio tracer studies, Paul et al.

and Lynch and Paul documented the m icrocompart-

m entation of glucose m etabolism in smooth muscle

(12-14). In princip le, regional d ifferences in

pH could

also occur in cells, even though pro ton (H f) conductance

in aqueous system s is h igh , because W flux can be high

and the W

concentra tion is low (subm icrom olar).

The kidney proxim al tubule cell provides an ideal

model to exam ine regional differences in cytosolic

pH

w ith in m ammalian cells because its ro le in organism ic

acid/base balance resu lts in a high transcellular H flux

(15). In princip le, several system s could contribu te to

heterogeneity of pH in the kidney cell. B icarbonate

(HC03) efflux system s in the basal region function to

elim inate base equivalen ts, resu lting in acidification of

the cytoso l in the basal reg ion. O n the apical end of the

cell, W export mechanism s function to move H out of

the cytosol and resu lt in a re lative alkalinization of the

cytoso l. In addition , because hydrolysis of A TP results

in release of H , the high concentration of the

Na ,K -ATPase on the basal surface of the cell could

also result in a local acidification. F inally , ox idative

phosphory lation depends on the cycling of H through

a circuit associated w ith the m itochondrial inner m em-

brane. A lthough there is a controversy over whether the

principal

W

flux is localized betw een the m atrix and

inner m embrane prote ins (16) or occurs betw een the

m atrix and the bulk aqueous phase of cytosol (17), suffi-

cient flux of W , other charged species, and ionizable

species in to or out of the m itochondria m ay occur to

generate a pH gradien t rad iating from the m itochon-

dna. M itochondria are present in high density at the

basal region of the cells (18) and thus their function

could also contribu te to regional differences in pH .

‘Address

c or re sp on de nc e t o: D r. T ak Y ee

Aw , Department of B io-

chemistry, Em ory University School of Medicine, A tlanta, GA

30322, USA .



A

I

I

‘a

I

0.

5

V

C

0

1)

.

8. 0

7. 0

1.00

0.50

0.00

6.8 7 .0 7.2

7. 4 7. 6

EXPERIM ENTAL APPROACH

HETEROGENEITY OF pH IN ISOLATED KIDNEY CELLS 53

To study regional d ifferences in pH , w e analyzed pH

from the distribution of compounds that are d istributed

betw een the cytoso l and m itochondria l m atrix by two

fundamentally d ifferen t m echanism s. In one approach

we used a tracer concentration (20 nM ) of the weak

acid dim ethadione (DM 0),2 which distributes accord-

ing to the exten t of ionization . This occurs because the

non-ion ized species is perm eable to m embranes but the

ionized form is not. In each compartm ent, the acid dis-

sociates accord ing to its pK and the pH of that com -

partm ent. Thus, from the distribution of [14C}D M O

among compartments, one can readily calculate the pH

in the internal compartm ents for m itochondria or cells

in a medium of know n pH . Results from this approach

for iso lated renal cortex m itochondnia (19) from 180- to

25O -g male rats (Kng: [Sprague-D aw ley] barrier reared ,

K ing Laboratories, O regon, W is.) incubated at d ifferent

pH values show that k idney m itochondria can regulate

m atrix pH over a w ide range of medium pH (Fig . 1).

A second method used to study differences in pH

w ith in cells relies on the distribution of an ionic species

that are transported across the m itochondnial inner

m embrane by elec troneutral, H -compensated m echa-

nism s, accord ing to the equation (23):

pH = ---- lo g

[Aim

n

[A]C

where pH is the pH difference betw een the m atrix

and the medium to w hich the m itochondria are ex-

posed, n is the change on A , and the subscripts m and

c refer to the concentrations on the m atrix and cytoso lic

sides of the m embrane. For suspensions of isola ted

renal cortex m itochondria suspended at different pH

values identical to that for the D M 0 measurements

m entioned earlier, calcu lation of m atrix pH and trans-

membranal pH from the distribu tion of pyruvate gave

resu lts comparable to that for DM 0 (Fig . 1). Thus, the

tw o methods are comparable in their ab ility to provide

pH measurements although they are based on funda-

m entally different mechanism s.

For cellu lar studies, renal proxim al tubule cells w ere

prepared from 180- to 250-g male ra ts by collagenase

perfusion of the kidneys (24). Prev ious studies using

fluorescence m icroscopy for visualization of cells loaded

w ith rhodam ine 123 show ed that the in trinsic pattern of

m itochondrial d istribution in the cells, i.e., in pre-

dom inantly one region, is preserved despite the spheri-

ca l nature of the isola ted cells (25).

RESULTS

Analysis of intrace llu lar pH from weak

a cid d is tr ib uti on

M easurem ents of pH in the m itochondrial matrix and

cytoso lic compartm ents w ere obta ined from [‘4C]D M O

distribu tion by a combination of digiton in fractiona-

6.8 7.0 7 .2 7 .4 7.6

Extram itochondria l pH

Extram itochondria l pH

F igure 1. Com parison of m atrix pH

a nd m ito ch on dr ia l

ipH from

distribution of DM 0 and pyruvate

in

iso la te d k id ney m ito ch on dria

incubated at different m edium

pH.

F re sh ly is ola te d m ito ch on dria

were incubated in m itochondrial buffer (in

m M: su crose, 1 30 ; T ris-

H CI, 60; K C1, 60; K HP O4,

1;

MgCl2 ,

5;

E IJ TA , 0 .5 )

containing 0. 2

m M ADP, 5 m M each of succinate and glutamate, and either

[‘4C ]DM O 20 nM ) o r p yr uv at e

(0 .3 m M ) at the d ifferen t m edium

pH. Incubations were carried Out at

35 {176}Cor

10 m m, a

t ime

sufficient for equ ilib ration of DM0 and pyruvate. A t the indicated

times, m itochondria were collected after centrifugation through a

silicone oil: paraffin oil layer (6:1,

v/v (20 , 21). ‘4C label associated

with the m itochondria was determ ined by scintillation counting and

m atrix pyru vate

was m easured by the m ethod of Lamprecht and

Heinz

(22).

M atrix volum e was determ ined

by the

distribution of

3H20 with

[‘C ]inulin as the extram itochondrial m arker. V alues are

mean

± three separate determ inations on each of two m itochon-

drial preparations. DM 0 0 ; pyruvate

s) .

tion and centrifugation through a silicone/paraffin oil

layer (20, 21). From this d istribution the m itochondrial

m atrix pH was calcula ted to be 7 .63 (Table 1), a value

comparable to that obtained w ith renal cortex m ito-

chondnia (F ig . 1), sim ilar to that obtained by this tech-

2Abbrevia {128}ions:M 0, dim ethadione; FCCP, carbonyl cyanide-p-

triflu orom etho xy p he ny thy dra zon e; D ID S, 4 ,4’-d iiso thio cya no stilb ene -

2,2’-disulfonic

acid; BCECF, bis-carboxyethylcarboxyfluorescein.



54 Vol. 3 Jan. 1989

T he F AS EB J ou rn al

AW

AND JONES

TABLE

1. Cytosolic and m itochondrial matrix pH in isola ted renal

proxim al tubule cell?

Treatment

n

PHcyto

PHmito

Contro l

6 7.41 ± 0 .02

7.63 ± 0 .05

FCCP, 1 iM

6

7.31 ± 0.03 7.43 ± 0 .07

+Am iloride, 1 m M 5 6.98 ± 0.07

7.60 ± 0 .08

+DIDS, 50 sM

5

7.53 ± 0.04

7.73 ± 0 .10

Renal proxim al tubule cells were prepared from 180- to 25O-g male

rats by collagenase perfusion of the kidneys

(24).

Incubations w ere carried

ou t

with 106 cells/m I for

30 mm inmodifiedKrebs Henseleitbuffer pH 7.2

i n t he a bs en ce o r p re se nc e o f F CC P a mi lo ri de a nd D ID S a t t he

con-

centrations indicated. D igitonin fractionation to obtain the two compart-

ments was carried out as previously described

(20, 21)

an d

pH

m easurem ents w ere determ ined

by the in tracellu lar distribution of DM0

(20, 21). Values are means ± SEM of the number of cell preparation

indicated.

nique in hepatocytes (26 , 27), and in agreem ent w ith

that obta ined recently by using the fluorescence ratio

imaging technique after the loading of kidney cells w ith

the fluorescence pH indicator bis-carboxyethylcarboxy-

fluorescein (BCECF; T . Y . A w , D . J.

W illiford, S . -S.

Sheu, D . P. Jones, unpublished observations). A lthough

treatm ent of cells w ith am iloride or 4 ,4’-diiso thio-

cyanostilbene-2 ,2’ - disulfonic acid (D IDS) sign ifican tly

changed the cytosolic pH (described later), these agents

had little effect on the m atrix pH (Table 1). This is in

agreem ent w ith the data shown in Fig. I and demon-

strates that the function of the plasma membrane W

and H C0 transport system s has little effect on the

regulation of the m atrix pH . Treatment w ith the pro-

tonophore carbonyl cyanide-p-trifluorom ethoxy phenyl-

hydrazone (FCCP) caused a sm all bu t detectable de-

crease in m atrix pH (Table 1), w hich is consisten t w ith

an equilibration of the m atrix pH w ith that of the cyto-

solic pH .

The cytoso lic pH value obtained by the DM 0 method

was 7 .41 (Table 1). A ddition of FCCP had little effect

on the cytosolic pH w hen the extracellu lar pH was

7.3-7 .4 . Inh ibition of the Na-H exchanger w ith

am iloride (28-31) low ered the cytosolic pH to 6 .98,

whereas inhibition of HC03 efflux system s w ith D ID S

(32-37) raised the cytosolic pH to 7 .53 (Table 1). Thus,

the cytosolic pH as m easured by DM 0 distribution

changes as expected ow ing to perturbations of the

m echanism s for regulating intracellu lar pH . The data

further show that the cytoso lic pH as m easured by DM 0

distribu tion is sensitive to the function of the N a-H

exchanger but is less sensitive to the function of the

H C03 efflux systems.

However, it is im portant to recognize that th is m ea-

surement w ith

DM0 gives only an estim ate of the aver-

age pH in the cytosolic compartment and does not pro-

vide inform ation about possible inhomogeneities in pH

w ithin the cell. This occurs because only a tracer con-

centration of D M 0 (20 nM ) is used; a higher concen-

tra tion is avoided because it could artifactually alter the

pH homeostasis. A t tracer concentrations, ([DM 0] <

[H J), a standing pH gradien t can cause a sign ifican t

grad ien t of the ionized form of DM 0 (Fig. 2) indepen-

dent of a m embrane barrier. Thus, the results w ith

DM 0 show that an average difference in pH of about

0.2 pH units occurs between the cytosol and the matrix

but the results do not provide inform ation on the exis-

tence of heterogeneities in pH w ith in these compart-

ments.

M easurem ent of m itochondrial m embrane

pH gradient

Because m itochondria contain W -coupled anion trans-

port system s, the distribution of anions across the

m itochondrial inner m embrane according to the func-

tion of these H -coupled mechanisms is a m eans to esti-

m ate the m itochondrial transmembranal pH . U sed in

conjunction w ith the m atrix pH measurem ents prev i-

ously m entioned , this provides a m eans to estim ate the

perim itochondrial pH . Pyruvate is an endogenous

anion suitable for use w ith th is approach because sig-

n ifican t concentration gradients of pyruvate are un-

likely to occur w ithin the cytoso l under normal aerobic

conditions. The cytoso lic pyruvate concentration in the

kidney cell is 0.2-0.3 mM , at least three orders of m ag-

n itude higher than the H concentra tion. The vect9rial

flux of pyruvate is also considerably less than that for

H , i.e., p robably less than 2% of the H ’ flux for

m itochondria, assum ing three H per ATP is synthe-

sized and 18 ATP is synthesized for each pyruvate con-

sum ed. Thus, even though a tracer molecule such as

D M 0 can exist w ith considerable heterogeneity in con-

centration w ith in the cytoso l, pyruvate concentration

can be essentially homogeneous (see F ig. 2). This diff-

erence betw een the nature of d istribution w ith in the

cytoso l of tracer amounts of D M 0 and endogenous

pyruvate allow s the use of pyruvate distribu tion to ob-

ta in additional inform ation about the cytoso lic pH ,

nam ely, the pH in the vicin ity of the m itochondria to

w hich the H -coupled pyruvate transporter responds.

Because m itochondria are present at a h igh density in

the basal region of cells, this also provides inform ation

on the poten tial occurrence of a transcellular cytosolic

pH gradient.

W e have previously found that the m itochondrial

transmembranal pyruvate distribu tion in liver cells can

be readily m easured by the digitonin fractionation

method as described previously for DM 0 (20, 21 , 26 ,

27). U sing th is separation technique to obtain the dis-

tribution of pyruvate

(Table 2), w e calculated from

Eq. 1 that the zpH across the m itochondrial inner

m embrane is 0.69

±

0.01. Given that the matrix pH

is

7.6 (Table 1), this means that the pH in the region im -

mediately around the m itochondria is about 6.9, even

though the distribu tion of D M 0 showed that the aver-

age pH of the cytoso l w as 7.4. Thus, the data are com -

patib le if a pH gradien t occurs from the perim itochon-

drial cy toso l to the cytosol distal to the m itochondria.

Several lines of ev idence support th is interpretation ..

F irst, in stud ies in w hich pyruvate and DM 0 are

directly compared as pH indica tors in iso lated kidney

m itochondria, the tw o compounds provide comparable

measures of m atrix pH and m itochondrial transm em -



A

cytosol

B

HDMO

1/

I1 DMO

I I I

0.2 0.6 1.0 1.4

mitochondrial

matrix

pyr

H

-

HDMO

E

rr

5-

>1

0.

LJ

c_I

0

a

0.25

0.20

0.15

20.0

17.5

15.0

I I

H

DM0 0.2 0.6 1.0

1. 4

C

HETEROGENE ITY OF pH IN ISOLATED K IDNEY CELLS 55

p y r

7.6

[H I x 10’ M

pH

-J

6. 8

Figure 2.

Approaches to measure regional heterogeneities in

pH

based on different characteristics of pyruvate

an d

[‘4C]DMO distribu-

tion.

A)

Th e m itochondrial inner mem brane is im permeable to free diffusion of

both the ionized form of pyruvic acid (pyr) and the free

acid form (Hpyr), bu t pyf is transported across by an electroneutral transport system that can b e f orm all y

described as an H ’,pyr sym -

porter. Function of th is system resu lts in distribution of pyr across

the membrane according to the pH, and the measurement of pyr

concen trations in the cytoso l and matrix provide a means to estim ate the pH . B)

M embranes are im permeable to th e ionic form of DM 0

(DMO) but permeable to the uncharged form (HDM O). The pK value is 6.13, and tracer concentrations of DM 0 e qu ilibra te throu gh ou t

the cells according to the exten t of ionization at the pH

in different sites. D igitonin fractionation allows distinction between th e

cytosolic

and m itochondrial con ten t of

DM0, and from the accumulation ratio of DM 0 concentration in one of these com partments relative to

the ex tracellular DM 0 concentration , one can calcu late the pH

of

that compartm ent at a known extracellular pH .

C)

T he d iff ere nc e

be -

tween

the

inform ation provided

by these two m ethods is dependent on the relative

concentrations of th e in dicator sp ecies. A s an ex og eno us

indicator, DM 0

is added

at a tracer concentration to avoid artifactual effects on th e

pH

in th e com partments of interest. At a tracer con-

centration such as

20

nM , the rate of diffusion of DM 0 is slow relative to its rate of ionization so that a standing gradient of DM O

will occur

if a g rad ien t

in H concentration ex ists. The m easurement ob tained w ith DM 0 distribution from dig iton in-fractionated cells

d oes n ot giv e in fo rm atio n on whether such standing gradients occur,

but gives only an average for all o f the cytoso lic vo lum e. Thus, the

local

pH w ithin different regions of the cytosol can vary and not be detected by this m easurem ent. Such variations have no effect on the

mitochondrial content of DM0 because this is determ ined by the matrix pH and the pK of the acid. In contrast to

DM 0, pyruvate is

present at m illim olar concentrations in the cytosol. W ith a relatively low pK value,

it

is present alm ost

totally in the

io nized form . At

a high concentrat ion

relative to

H*, only insign ifican t gradien ts of pyruvate can occur as a resu lt of a cytoso lic

H’ gradient. Thus, the

concentration of pyr to

which the

mitochondria are exposed can

be

assumed to be the same as

the average cytoso lic concentration . This

allows use of

the accum ulation ratio of pyruvate (m ito :cyto) to estim ate the pH between the m itochondrial m atrix and the perim itochon-

drial space. Therefore, w e have relied on the pyruvate data

to give information on

the pH only in the v icinity of the m itochondria and

we have used the DM0 data to give information on the average cytosolic pH .

branal pH (see Fig. 1). Thus, d ifferen tial b ind ing of the

indicators by m itochondria cannot account for the ob-

served differences in the pH measurem ents. Second,

these two indicators gave the sam e value for m itochon-

drial ipH in isolated hepatocytes (26, 27). Thus,

differen tia l cytosolic binding of one of the indicators is

unlikely to account for the differences seen in the kid-

ney cell. This is supported by studies w ith FCCP,

w hich elim inates pH gradien ts and resu lts in uniform

concentrations of both indicators in the tw o compart-

m ents (Table 1 and Table 2). Third, o ther m easures of

tubule cell cytosolic pH give values comparable to those

obtained w ith D M 0. A lthough differen t prepara tions

and techniques reveal variab ility in cytosolic pH mea-

surem ents, a varie ty of techniques, v iz., pH -sensitive

dyes (32-36), m icroelectrodes (36), [31P]N M R (37,

40), and distribu tion of w eak acids (41-43), show that

the renal cell cytosolic pH is typically in the range of



TABLE

2.

M itochondria l transm e,nbranal distribution of pyruvate, citra te, and phosphate in renal proximal tubule cells’

56 Vol. 3

Jan.

1989 T he F AS EB J ou rn al AW AND JONES

M etabolite concentration, mM

Mitochondrial

Treatment

n

Total M itochondria Cytosol

cytosol

Pyruvate

Control 5 0 .38 ± 0 .02

0.92

± 0.08

0.19 ±

0.01 4.90 ± 0 .10

{247}FCCP , I

cM

3 0 .41 ± 0.01 0 .42 ± 0.02 0.41 ± 0 .01 1 .01 ± 0.02

i-Am iloride, 1

m M

5 0 .24 ± 0.02 0 .46 ± 0.04 0.10 ± 0.01 4.50 ± 0.15

+DIDS, 50 sM

5 0 .39 ± 0.02 0 .51

± 0.03

0.36 ± 0.02 1.40 ± 0.06

Citrate

Control 3 1.01

± 0.04 2.91

±

0.23

0.28 ± 0.02 10.50 ± 1.48

+FCCP,

1

ILM

3 0 .63 ± 0.04 0 .69

± 0.05

0.60 ± 0.04 1 .15 ± 0.03

Pi

Control 4 3 .20 ± 0 .37 6 .68 ± 0.43 1 .90 ± 0.33 3 .71 ± 0 .38

FCCP,

1 ltM

4

6 .00 ± 0.77

5.25

± 0.81

6.28 ± 0.83 0 .84 ± 0 .09

‘Incubations were carried out

with 106 cells/m l in the absence or presence of inhibitors, an d

com partments were separated by

th e

digitonin

fractionation

(20 , 21 ).

Pyruvate and citratewere determ ined spectrophotometrically by pyridine nucleotide-linked enzyme coupled assays of Lamprecht

an d H ein z (22) a nd M olle rin g (38), respectively, and total noncovalently bound inorganic phosphate was measured by the colorim etric assay of Fiske and

Subbarow

(39).

Values are mean ± SEM of the number of cell preparations indicated.

7.1-7 .5. A lthough th is range is more alkaline than some

other cell types, e.g., liver, pH -driven transport

m echanism s in the m itochondria are frequently the

same in differen t cell types (23). Further, m easure-

m ents of the steady-state matrix and cytoso lic concen-

trations of phosphate and citrate , which are also trans-

ported by H f-coupled mechanism s (Table 2), support

the data obtained w ith pyruvate , i.e ., that m itochondrial

pH in kidney cells is at least

0.49 . Precise calculation

of the pH from these species is not possible because

they are transported by multiple

systems;

how ever, the

distribu tions of phosphate and citrate betw een cytoso l

and matrix in k idney cells (m atrix :cytoso lic [m :c] ratios

of 3.7 and 10.5 , respective ly, Table 2) are comparable to

that presen t in liver cells (m :c ratios of 4.5 and 12.3 ,

respectively) w here the m itochondrial zpH is 0.78 (26 ,

27). Thus, w e conclude that a sign ifican t pH heter-

ogeneity occurs in kidney cells that specifica lly involves

a relatively low pH in the reg ion around m itochondria.

Factors affecting regional cy tosolic pH heterogeneity

To further exam ine factors contributing to reg ional

variations in pH as calcula ted from the pyruvate and

D M 0 distribu tions, effec ts of a protonophore (FCCP)

and inhibitors of the N a:H antiporter (am iloride) and

CV :HC03 exchange (D IDS) w ere used . FCCP caused

a large decrease in the transm embranal pyruvate d istri-

bu tion (Table 2), which is consisten t w ith a collapse of

the m itochondrial zpH , but had little effect on the pH

as measured from the DM 0 distribution (Table 1).

T reatm ent of cells w ith am ioride had no effect on the

pyruvate distribu tion but caused an acidification of the

average cytoso l as m easured by D M 0 distribution

(Table 1). T reatm ent w ith D IDS resu lted in a loss of the

pyruvate gradient (Table 2) w ith little effect on the aver-

age cytosolic pH as m easured by DM 0 distribution .

S tudies w ith iso lated m itochondria showed that D ID S

itself did not affect the m itochondrial m atrix pH or

resp iratory rate (data not shown), ind ica ting that loss of

the pyruvate grad ien t in cells in the presence of D IDS

was caused by a loss of pH as a result of alkalinization

of the perim itochondrial (basal) region. Thus, the

resu lts obtained w ith FCCP, am iloride , and D IDS sup-

port the interpre tation that the H f-coupled pyruvate

transporter responds to an aqueous volum e in w hich

the pH is low er than the average cytosolic pH .

DISCUSSION

The volume surrounding the m itochondria, w hich has

a relatively low pH , is likely to be sm all rela tive to the

volume of the rest of the cellbecause DIDS increases

the estimated perim itochondrial pH to 7.58 w ith

only

an 0.1 pH unit increase in the average cytosolic pH

(Table I and Table 2). Initia l attempts to see this rela-

tive ly acidic reg ion w ith the use of BCECF w ere un-

successfu l (T . Y . A w , D .

J.

W illiford, S . - S . Sheu , D . P .

Jones, unpublished observations) because w e w ere able

to observe the signal only from the matrix volum e in

the basal region of the cells. E lectron m icrographs of

kidney cells show that the m itochondria are densely

packed, w ith little in term itochondrial space (18), and

th is suggests that it w ill no t be possib le to see th is region

directly unless a fluorescent pH indica tor is developed

that is comple tely excluded from the m itochondrial

matrix.

Earlier stud ies of the kidney w ith [31P}N M R revealed

a broad signal for phosphate that w as interpreted as

evidence for a heterogeneity in pH of about

0.5 pH

units (37 , 40). A lthough this heterogeneity m ay be due

partly to the perim itochondrial cy toso lic pH gradient

described here , m easurem ent by N M R cannot d istin-

guish betw een in tracellular and intercellular pH gra-

dients.

The system s responsib le for m ainta ining intracellu-

lar pH in the cytoso l of the proxim al tubule cells (the

H C03 effiux system s of the basolateral surface (32-35)

and the Na-H antiporter of the brush border (28-31))

also function in net transcellu lar movement of H as the

kidney contributes to the control of organism ic pH

balance. D ID S elim inated the m itochondrial zpH but



HETEROGENEITY OF pH IN ISOLATED KIDNEY CELLS 57

had no effect on the cytoso lic pH (as measured by

DM 0) w hereas am iloride decreased cytosolic pH (as

measured by D M 0) but had no effect on the m itochon-

drial pH . Thus, these transport system s contribute

sign ifican tly in determ ining the pH of the differen t

reg ions of the cell under the conditions of these experi-

ments .

Because m itochondria retain a clustered distribu tion

in the iso lated proximal tubule cells (25) and , under in

v ivo conditions are clustered in the basal reg ion of the

cells (18), the current results ind icate that the pH heter-

ogeneity reflects a normal transcellu lar cy toso lic pH

gradien t. The simplest in terpreta tion is that the pola-

rized effiux of W on the brush border surface and of

HC03 on the basal surface contribute to maintain ing

a grad ient of

pH between the basal and apical regions.

This does not exclude the possibility that diverse other

factors, such as the release of W by A TPases or cycling

of ions across the m itochondrial inner m embrane, also

are important in determ ining the perim itochondrial

pH . In addition, ex trapolation of the current resu lts to

cells in vivo must be done with caution because sig-

nificant differences in CO2 and HC03 distribution can

occur that w ould have an important effect on the distri-

bution and magnitude of pH gradien ts. Recent stud ies

of isolated renal cortex m itochondria show ed that the

m atrix pH and the m itochondrial pH were m arkedly

influenced by a bicarbonate buffer system (44, 45), but

in our studies, inh ibition of HC03 effiux by D IDS in

the isolated cell did not appear to significantly affec t the

m atrix pH (Table 1).

pH has long been considered an important factor

governing activities of numerous bio logica l system s (see

ref 46 for rev iew ). These diversified pH -dependent cel-

lular processes include activ ities of m etabolic enzymes,

synthesis of macromolecules, contro l of contractility

and ion conductivities in excitable cells, and several

regulatory

events of the cell cycle and cell division (46).

The relative ly low H concentration in cells m akes the

in tracellu lar pH responsive to transport system s and

chem ical reactions that result in perturbations in the

local steady-state H concentrations. Thus, reg ional

d ifferences in pH could be of great im portance in cell

b io logy by providing a mechanism for sem iautonomous

regulation of functions w ith in different regions of the

aqueous cytoplasm . In addition, such heterogeneities

could provide an elegantly sim ple m eans for sorting of

organelles and macromolecules in polarized cells.

H owever, the extent to which such heterogeneities of

pH occur in o ther cells and the m agnitude and distri-

bution of these heterogeneities are not know n. E il

Supported by National Institu tes of Health gran t GM 36538.

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