H+-exchanger by an antagonist of muscarinic acetylcholine receptors

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 157, No. 3, 1988

Pages 92]-929

December 30, 1988

INHIBITION BY

AN

AND

LABELING

ANTAGONIST

OF

OF

THE

RAT

MUSCARINIC

T. Friedrich*

Na÷/H+-EXCHANGER

RENAL

ACETYLCHOLINE

RECEPTORS

and G. Burckhardt**

*Department of Medical Chemistry,

Kyoto University Faculty of Medicine,

Kyoto 606, Japan **Max-Planck-Institut

f~r Biophysik,

D-6000 Frankfurt 70, FRG

Received November 4, 1988

A covalently binding label for muscarinic acetylcholine receptors, propylbenzilylcholine mustard (PrBCM), irreversibly inhibits the Na+/H + exchanger in rat renal brush-border membrane vesicles. Substrates of the antiporter, Na + and Li +, as well as inhibitors, amiloride, 5-(N-ethyl-N-isopropyl)amiloride (EIPA) and propranolol, protect the antiporter from inactivation by PrBCM. With [3H]PrBCM a band with an app. M r of 65 kDa is predominantly labeled. Amiloride protects this band from labeling with [3H]PrBCM and [14C]N,N'-dicyclohexylcarbodiimide (DCCD) proving its identity with the renal Na+/ H + exchanger. Our data reveal a specific interaction of PrBCM with the Na+/H + exchanger and suggest structural relations between antiporter and receptors. © 1988 Academic

Press,

A comparison ceptors

of amino acid sequences revealed

(adrenoceptors),

driven H+-pump, have arisen proteins

Inc.

muscarinic

rhodopsin,

belong

from a common

acetylcholine

to a family

ancestral

receptor

are sites for glycosylation,

tic residues

in helix

receptors,

of membrane gene.

Common

for signal

ular masses in SDS polyacrylamide

re-

and the light-

proteins

that may

features

of these

seven membrane-spanning

II and helix III as putative

interaction with G-proteins

B-adrenoceptors

that e- and B-adrenergic

helices,

aspar-

ligand binding sites, and

transduction

(I). The apparent molec-

gels of the fully glycosylated e- (2,3) and

(4-6) and of the muscarinic

acetylcholine

receptor

(7) range

between 62 and 70 kDa. Recently, and

amiloride

~-adrenoceptors

cells

has

from

shown

to decrease

vascular

smooth

(8,9), and to the purified e2-adrenoceptor

sides blocking epithelial in

been

liver,

a variety

of

cells

(11,12). The action e2-adrenoceptor are closely relation

Na + channels,

including

of amiloride

amiloride

proximal as well

has led to the speculation

related,

are data

if not

showing

identical

tubule

921

of ligands

to e-

platelets,

renal

from porcine brain inhibits

(10). Be-

Na+/H + exchangers

cells of mammalian

kidneys

as of Na + and H + with the purified that receptor and Na+/H + exchanger

proteins

an inhibition

binding muscles,

(10).

In

support

of Na+/H + exchanger

of

such

a

by an ~2 adren-

0006-291X/88 $1.50 Copyright © 1988 by Academic Press, ~c. All r~h~ of reproduction in any form reserved.

Vol. 157, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ergic agonist,

clonidine

(13),

and alprenolol

(14). Moreover,

and by B-adrenergic

antagonists,

the rat renal Na+/H + exchanger can be specifi-

cally labeled with a photolabile ligand for S-adrenoceptors, pindolol-diazirine acetylcholine

(14).

receptors

propranolol

Finally,

similar

to

[125I]iodocyano-

adrenoceptors

and

muscarinic

the rat renal Na+/H + exchanger has an app. molecular

mass of 65 kDa (15) and is glycosylated (Friedrich, unpublished). In

this

contribution

we wish

to

demonstrate

that

the

renal

Na+/H + ex-

changer reacts with propylbenzilylcholine mustard (PrBCM), a covalently binding ligand for the muscarinic

acetylcholine

receptor

changer and several members of the G protein-coupled number of ligands

suggesting

structural

relations.

(16). Thus, receptor

Na+/H + ex-

family

share a

Part of the results have

been published in abstract form (14).

METHODS

Vesicle preparation. Renal brush-border membrane vesicles from male Wistar rats (180 g) were prepared according to Biber et al. (17) by a Mg2+-precipi tation method. The vesicles were taken up in 100 mM TMACI, 50 mM KCl, 5 mM Hepes/Tris, pH 7.0, and stored in liquid nitrogen at a protein concentration of 10 mg/ml. Protein was determined according to Bradford (18) using bovine serum albumin as a standard. Basolateral membrane vesicles were prepared by a Percoll density gradient separation technique (19).

Irreversible inhibition of Na+/H + exchange with PrBCM and DCCD. Brush-border vesicles were rapidly thawed and diluted to a final protein concentration of 3.33 mg/ml. The vesicle suspension was preincubated with buffers and inhibitors for 30 minutes and then PrBCM or DCCD were added from ethanol stocks for further 30 minutes. The reaction was stopped by dilution of the vesicle suspension into 35 ml of 100 mM NaCI, 50 mM KCI, 5mM Hepes/Tris, pH 7.0, followed by centrifugation in a Sorvall SS34 rotor for 20 min at 20,000 rpm. The pellet was resuspended in the same buffer used to stop the reaction.

Determination of Na+/H + exchanger activity. Brush-border membrane vesicles (25 ~g protein) loaded with 100 mM NaCI, 50 mM KCI, 5 mM Hepes/Tris, pH 7.0, were diluted into I ml of 100 mM TMACI, 50 mM KCI, 5 mM Hepes/Tris, pH 7.0, containing 2.5 ~M valinomycin (to clamp the membrane potential to zero) and 6 ~M acridine orange (to monitor intravesicular acidification). Na + efflux via the Na+/H + exchanger acidifies the vesicle interior causing intravesicular accumulation of acridine orange and a decrease in its absorption. The absorption decrease was monitored in a Shimadzu UV 300 dual wavelength spectrophotometer (Kyoto, Japan) at 492 nm using 540 nm as reference wavelength. Na+/H + exchanger activity is expressed as rates of absorption decrease recorded within the first 10 seconds.

Labeling of membrane proteins with [3H]PrBCM and [14C]DCCD. Aliquots of brush-border membrane vesicles and basolateral membrane vesicles (1 mg of protein each) were labeled with [3H]PrBCM (125 nmol = 0.42 ~ o i / i ) or [14C]DCCD (2.5 nmol = 0.0083 mmol/l) at a protein concentration of 3.33 mg/ml for 30 min at room temperature in the presence or absence of inhibitors as indicated in the legends of the figures. After labeling the vesicles were washed as indicated above and the pellet was left on ice for further 3 hours. Then, the proteins were dissolved by shaking for 30 min in O'Farrell's sample buffer (20). Portions containing 0.5 mg dissolved proteins were then directly applied to SDS-polyacrylamide gel electrophoresis.

922

Vol. 157, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and determination of the radioactivity. SDS-PAGE was performed in glas rods. Total acrylamide concentration was 8.5%. Molecular mass standards were phosphorylase b (94 kDa), albumin (67 kDa), ovalbumin (43 kDa) and carboanhydrase (30 kDa). The apparent molecular mass of labeled proteins was determined from plots of ig M r vs migration distance which were linear for the applied marker proteins (r 2 >0.98). The gels were fixed with 12.5% trichloroacetic acid, stained with Coomassie Brilliant Blue R 250, and destained in 250 ml ethanol, 50 ml acetic acid, 700 ml water. After scanning the gels were cut into 2 m m slices and treated with 0.5 ml Protosol overnight. Thereafter, scintillation fluid was added to determine the radioactivity contained in each sample. Materials. All chemicals were of analytical grade and obtained from commercial sources. PrBCM and [3H]PrBCM (60 Ci/mmol = 2220 GBq/mmol) were obtained from NEN. DCCD was obtained from Aldrich, [14C]DCCD (54 Ci/mol = 2 GBq/mol from Amersham. Amiloride and derivatives were synthetized as described by Cragoe et al. (21). Protosol was obtained from NEN. Before use, 75 ml water was added to 500 ml Protosol. RESULTS

AND

DISCUSSION

In order to test whether PrBCM interacts

with the renal Na+/H + exchanger,

brush-border membrane vesicles were preincubated without or with 0.5 mM PrBCM for 30 min at room temperature. Then, unreacted PrBCM was removed by washing and the vesicles were pending Na+-loaded

loaded with Na +. Na+/H + exchange was initiated by sus-

vesicles

in a Na+-free

as an indicator of intravesicular

buffer

the decrease of acridine orange absorbance cles as compared

to controls

containing

acidification.

Figure

acridine

I, left,

orange

shows

is slower in PrBCM-treated

(pretreatment without PrBCM).

This

that vesi-

result dem-

onstrates the partial irreversible inhibition of the Na+/H + exchanger following treatment with PrBCM. The degree of irreversible (Na+/H +

exchange

is

faster)

presence of amiloride Figure

I, right,

when

vesicles

were

exhibits

the dependence

I mM PrPCM is required for half-maximal decreases

gesting Na+/H +

the inhibition

a competition exchanger.

carboxyl

with

PrBCM

in

the

on PrBCM concentration of irre-

These

group reagent,

At 30 min preincubation time,

inhibition

of the antiporter.

at 0.5 mM PrBCM more

for a common results

or

are

comparable

to

in

binding

data

twofold

higher

sites

obtained

at the

with

the

(DCCD), which inactiv-

an amiloride-protectable

approximately

Amilo-

than at I mM PrBCM sug-

closely related

N,N'-dicyclohexylcarbodiimide

ates the Na+/H + exchanger compared to DCCD,

treated

is decreased

(Figure I, left, middle trace).

versible inactivation of the Na+/H + exchanger.

ride

inhibition

manner

(15,22,23).

As

of PrBCM

are

concentrations

required to produce an equivalent degree of irreversible inhibition. Besides

amiloride

(0.5 mM,

ethyl-N-isoproyl)amiloride renal

Na+/H +

exchanger

the substrates

adrenergic

antagonist,

1) also the high-affinity

analog 5-(N-

(EIPA, 0.05 mM) affords some protection of the rat

from

Similarly,

Figure

irreversible

of the Na+/H +

propranolol,

inactivation

exchanger,

protect

the

by PrBCM

Na + and Li +,

antiporter

(Table

I).

and the B-

partially.

The

same compounds were effective in protecting the Na+/H + exchanger from inactivation

by DCCD

(14,15).

These results 923

suggest

that,

similar

to DCCD,

PrBCM

Vol. 157, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I

=

5

X I==

<~ ¢~

-0,02

v

~> 3

with

40 ,--

-0,04

; 0.5raM

30'

PIBCM

without

0 4,J

~

L.

g2 e-

30';0.5mM PIBCM + 0.5 mM Amiloride

AMILORIDE

e~ L) x

o -0,06

+=1

e~ • e o n t zol

+ Z

-0,08 0

o:s

1

Time

(mln)

i

PrBCM concentration

(ml)

Figure I . Irreversible inhibition of Na+/H + exchange by PrBCM and protection by amiloride. Left: Rat renal b r u s h - b o r d e r membrane vesicles were incubated without (control) or with 0.5 mM PrBCM for 30 min in the absence or presence of 0.5 mM amiloride. Thereafter, v e s i c l e s were washed, loaded with Na +, and suspended in a N a + - f r e e b u f f e r c o n t a i n i n g acridine orange. The absorption loss of acridine orange was measured at 492 nm as a function of time. Right: Vesicles were incubated for 30 min with different concentrations of P r B C M in the presence or absence of 0.5 mM amiloride. The initial rate of absorption loss is plotted as a function of the PrBCM concentration.

Table I Protection of Na+/H + exchange from irreversible inhibition by PrBCM

PrBCM

(mM)

cation

(mM)

inhibitor (mM)

activity

(AABS/min-mg)

0

TMA +

(100)

-

5.11

0.5

TMA +

(100)

-

3.09 + 0.1

+ 0.35

p

-

<0.001"

0.5

Na +

(100)

-

3.77 + 0.2

<0.001"*

0.5

Li +

(100)

-

3.94 + 0.3

<0.001"*

0.5

TMA +

(100)

propranolol

(0.5)

3.86 + 0.23

<0.001.*

0.5

TMA +

(100)

(0.05)

3.53 + 0.14

<0.001.*

EIPA

Rat renal b r u s h - b o r d e r membrane vesicles were incubated with 0.5 mM PrBCM for 30 min in the presence of the indicated cations and inhibitors. After w a s h i n g and Na + loading of the vesicles, Na+/H + e x c h a n g e r activity was d e t e r m i n e d by m e a s u r i n g the rate of acridine orange absorbance decrease, AABS/min.mg protein. The table shows means + s t a n d a r d deviations from 5 determinations. TMA +, tetramethylammonium; *, s i g n i f i c a n t l y d i f f e r e n t from control (0 mM PrBCM); **, s i g n i f i c a n t l y d i f f e r e n t from the value obtained w i t h 0.5 mM PrBCM, 100 mM TMA + (no inhibitors).

924

Vol. 157, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

interacts with the antiporter directly

at, or close

to, the cation and inhi-

bitor (amiloride) binding site. Since PrBCM and DCCD are hydrophobic, sume

that both reagents

amino

acid

residues

in

inactivate

we as-

the Na+/H + exchanger by binding to acidic

a hydrophobic

molecular

environment.

Interestingly,

also in the muscarinic acetylcholine receptor PrBCM and DCCD interact with a carboxylic highly

group

embedded

deeply

conserved

aspartic

acid

located

within

the

in

the molecule

thought

hydrophobic,

(16).

to be involved

membrane

spanning

In

adrenoceptors

a

in ligand binding is

helices

(I).

Comparable

reactivity towards hydrophobic reagents

and overlapping

ligand specificities

suggest

protein-coupled

receptors

that

Na+/H +

exchanger

and

G

may

share

molecular properties within their ligand binding sites. After

having

demonstrated

a

covalent,

PrBCM to the rat renal Na+/H + exchanger, proteins

are

labeled

with

[3H]PrBCM.

amiloride-protectable

To

this

renal brush-border membrane vesicles with separated

thereafter

panel, open symbols)

the

membrane

binding

of

we next investigated which membrane end we

either

polyeptides

incubated aliquots of

[3H]PrBCM or

by

SDS-PAGE.

[14C]DCCD and Figure

2

(top

shows that [3H]PrBCM is mainly incorporated into a broad

band with an apparent molecular mass of 65 kDa. The same band is labeled with [14C]DCCD (closed symbols). other protein bands

[14C]DCCD, however, is less specific as it label~

as well

(Figure I, and ref. 15). Figure 1, bottom panel,

reveals that amiloride decreased the incorporation of [3H]PrBCM as well as of [14C]DCCD into the 65 kDa band. the

labeling

previous

of

notion

this

band

Sodium,

(data

not

EIPA and propranolol

shown)

which

is

also decreased

compatible

with

our

(15) that a membrane protein with an apparent mass of 65 kDa

is identical to the renal Na+/H + exchanger. As a control we also labeled basolateral membrane vesicles with [3H]PrBCM. These membrane vesicles proximal tubule cells opposed

to

the

originate

from the contraluminal

and are not equipped with

brush-border

membrane,

they

(blood)

side of the

a Na+/H + exchanger

possess

~-

and

(24). As

B-adrenoceptors

(25-28). Figure 3, top panel, shows that after affinity labeling of rat renal basolateral membranes

[3H]PrBCM is incorporated mainly into two bands corres-

ponding to apparent molecular masses

of 89 kDa and 67 kDa. Amiloride depres-

ses strongly the labeling of the 67 kDa band, but leaves the incorporation of radioactivity into the 89 kDa band virtually unchanged. [3H]PrBCM branes cross

labeling patterns of brush-border

(Figure

A comparison

of the

2) and basolateral

mem-

(Figure 3) reveals a clearly different pattern. This finding rules out contaminations

of

membranes

and

proves

that

the

labeled

originates from the brush-border membranes whereas the labeled

65

kDa band

89 kDa and 67

kDa bands are constituents of the basolateral membrane. The

[3H]PrBCM-labeled

89 kDa band in basolateral membranes may be identi-

cal to a band of similar mass labeled previously with

[14C]DCCD and thought

to be identical to the a-subunit of the (Na++K+)-ATPase

(15). Labeling of the

925

Vol. 157, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

94000 67000

8O0O

43~

14C]DCCD 500

-

Aml lorlde

[3HI PrBCM 6000 2

- 200

C~ -

bOO0

e~

-I00

2000

0

°

~

8000

wlth

[

300

•200

8 -

4000

-100 2000

t

i

0

i

i

2 4 Mlgratlon

i

i

6 8 Dlstance

0

I0 (cm)

12

Figure 2. Double-labeling of renal brush-border membrane proteins with [3H]PrBCM and [14C]DCCD and protection by amiloride. I mg vesicles were incubated with 0.125 nmol [3H]PrBCM and 2.5 nmol [14C]DCCD for 30 min in the absence (top panel)

or presence

of

0.5 mM

amiloride

(bottom panel).

After washing,

membrane proteins were separated by SDS-gelelectrophoresis. The gels were cut into 2 mm slices and radioactivity was determined. Closed symbols and left ordinate, incorporation of [3H]PrBCM; open symbols, incorporation of [14C]DCCD.

purified

~-subunit

with

PrBCM

been

tested

ments

are n e c e s s a r y

PrBCM-labeled The

to be

that

of

similarly when

the

irine

to d e f i n i t e l y

the

labeled

clarified.

the

Na+/H +

small,

Its

branes

of the prove

kDa

apparent

exchanger

or d i s p r o v e

the e - s u b u n i t 67

band

of the in

molecular

labeled

photolabile

S-adrenergic

antagonist,

is not i d e n t i c a l

identical

was used

to

the

we

to

feel

label that

exchanger.

926

Thus,

more

nor

has

experi-

of the [3H]-

(Na++K+)-ATPase.

is

membranes

slightly membranes

in m o l e c u l a r

renal the

to the 65 kDa band Na+/H +

demonstrated

the identity

in b r u s h - b o r d e r

difference

Therefore,

been

basolateral mass

reproducible,

(14).

not

(Na++K+)-ATPase.

but

(ICYP-diazirine),

membranes

not

of

has h i t h e r t o

an i n h i b i t o r

89 kDa band with

nature

also

as

[3H]PrBCM

remains

higher

than

(65 kDa).

masses

was

A

found

[125I]iodocyanopindolol-diazbrush-border

67 k D a

band

in b r u s h - b o r d e r Different

and

basolateral

in b a s o l a t e r a l membranes

amount

of

mem-

and thus

incorporated

Vol. 157, No. 3, 1988

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

,

i

i

94000

67000

, 43000

without

,C

~ ~

10~-

with Amllorlde

51100-

0

~

,

0

t

2 Rlgratlo

l

i

l

6

8

10

12

(cm)

Distance

Figure 3. Labeling of renal basolateral membrane proteins with [3H]PrBCM and protection by amiloride. I mg vesicles were labeled with 0.125 nmol [3H]PrBCM for 30 min. They were further treated as described in the legend to Figure 2.

radioactivity labeling

of

amiloride,

and

different

the

67

kDa

to

be

reflects

whether

and

greater

specific

labeling

[3H]PrBCM

as

67 kDa

to compare labels

and

specificity

radioactivity

the

reflect band

[14C]DCCD,

for

the

rat

label

than

[14C]DCCD.

does

of [14C]DCCD,

the

much

(Figure

I):

amounts

to approximately

The

are found with useful

maximal

incorporation 300 cpm/gel

[3H]PrBCM. for

Thereby,

further

(the

0.5

mM

also suggest that

distinct

proteins.

It

membranes

(25-28).

Na+/H + exchanger.

renal In

Na+/H +

addition,

less counts

of

[14C]DCCD whereas

exchanger due

with

to the low

are incorporated

into close

[125I]ICYP-diazirine and

Both,

into

(not shown) and [3H]PrBCM

slice,

characterization

by

[125I]iodocyanopindolol-di-

renal

[3H]PrBCM,

amiloride

depressed

in basolateral

the 65 kDa band as compared to [125I]ICYP-diazirine

prove

towards

completely

located in this membrane

it is worthwhile

[125I]ICYP-diazirine much

of

nearly

67 kDa and 65 kDa bands

established

the adrenoceptors

Finally azirine

being

that of the 65 kDa band only partially reduced)

both [3H]PrBCM-labeled needs

sensitivity

band

the

65

kDa

band

to 5000 cpm/slice

and [3H]PrBCM should

purification

of

the

renal

Na+/H + exchanger. Taken choline

together, receptor,

our

data

rat renal Na+/H + exchanger ing site,

and specifically

an inhibitor

show

that

a ligand

propylbenzilylcholine

of the

Na+/H +

probably labels

mustard

close

for

the

(PrBCM),

to or directly

the related membrane

exchanger,

amiloride,

927

with

muscarinic interacts

acetylwith

the

at the cation bindprotein.

Reaction

adrenoceptors

of

(8-I 0)

Vol. 157, No. 3, 1988

and,

conversely,

acetylcholine structural

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of ligands

receptor

features

of

with

the

antiporter

the

ligand

binding

Na+/H + exchanger

has

smilarly

to

receptors

Based

the

on

the

number

for adrenoceptors

a comparable

of

molecular

(Friedrich,

similarities

exchanger belongs to the family

Determination

Na+/H + exchanger

will

of

the

provide

(this

be

(15)

amino

basis

the

and

speculated

acid

common

rat

renal

is glycosylated

Burckhardt:

are comparable

primary the

indicates

Moreover,

mass

Sablotni,

it may

study)

sites.

of G protein-coupled

ly, parts of the molecular architecture proteins.

(13,14) and for the muscarinic

unpublished).

that

receptors.

the Na+/H + Alternative-

in otherwise unrelated sequence

to distinguish

of

between

the these

renal pos-

sibilities.

ACKNOWLEDGEMENTS

The

authors

thank

Drs.

Y. Mori

and K.

J. Ullrich

for valuable

This work was supported by the Deutsche Forschungsgemeinschaft

discussions.

(SFB169/A2).

REFERENCES

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17. 18. 19. 20.

Dohlman, H. G., Caron, M. G., and Lefkovitz, R. (1987) Biochemistry 26, 2657-2664 Repaske, M. G., Nunnari, J. M., and Limbird, L. E. (1987) J. Biol. Chem. 262, 12381-12386 Convents, A., DeBaker, J.-P., Van Driesche, E., Convents, D., Beekmans, S., and Vauquelin, G. (1988) FEBS Letters 234, 480-484 Bahout, S. W., and Malbon, C. C. (1987) Biochem. J. 248, 557-566 Kaveri, S. V., Cervantes-Olivier, P., Delavier-Klutchko, C. and Strosberg, A. (1987) Eur. J. Biochem. 167, 449-456 Stiles, G. L. (1985) Arch. Biochem. Biophys. 237, 65-71 Haga, K., and Haga, T. (1985) J. Biol. Chem. 260, 7927-7935 Hiussinger, D., Brodde, O.-E., and Starke, K. (1987) Biochem. Pharmacol. 36, 3509-3515 Howard, M. J., Mullen, M. D., and Insel, P. A, (1987) Am. J. Physiol. 253, F21-F25 Nunnari, J. M., Repaske, M. G., Brandon, S., Cragoe, E. J. Jr., and Limbird, L. E. (1987) J. Biol. Chem. 262, 12387-12392 Frelin, C., Vigne, P., Barbry, P., and Lazdunski, M. (1987) Kidney Int. 32, 785-793 Aronson, P. S. (1983) Am. J. Physiol. 245, F647-F659 Ganapathy, M. E., Leibach, F. H., Mahesh, V. B., Devoe, L. D., and Ganapathy, V. (1986) Biochem. Pharmacol. 35, 3989-3994 Friedrich, T., and Burckhardt, G. (1988) Biol. Chem. Hoppe-Seyler 369, 817 (Abstract) Friedrich, T., Sablotni, J., and Burckhardt, G. (1986) J. Membrane Biol. 94, 253-266 Wheatley, M., Hulme, E. C., Birdsall, N. J. M., Curtis, C. A. M., Eveleigh, P., Pedder, E. K., and Poyer, D. (1988) TIPS February Suppl. 19-24 Biber, J., Stieger, B., Haase, W., and Murer, H. (1981) Biochim. Biophys. Acta 647, 169-176 Bradford, M. M. (1976) Anal. Biochem. 72, 248-254 L6w, I., Friedrich, T., and Burckhardt, G. (1984) Am. J. Physiol. 246, F334-F342 O'Farrell, P. H. D. (1975) J. Biol. Chem. 250, 4007-4021

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21. Cragoe, E. J. Jr., Woltersdorf, O. W., Bicking, J. B., Kwong, S. F., and Jones, J. H. (1967) J. Med. Chem. 10, 66-75 22. Igarashi, P., and Aronson, P. S. (1987) J. Biol. Chem. 262, 860-868 23. Kinsella, J. L., Wehrle, J., Wilkins, N., and Sacktor, B. (1987) J. Biol. Chem. 262, 7092-7097 24. Sabolic, I., and Burckhardt, G. (1983) Biochim. Biophys. Acta 734, 210220 25. Matsushima, Y., Akanabe, S., and Ito, K. (1986) Biochem. Pharamacol. 35, 2593-2600 26. Matsushima, Y., Akabane, S., Kawamura, M., and Ito, K. (1987) Japan. J. Pharmacol. 45, 284-287 27. Sundaresan, P. R., Fortin, T. L., and Kelvic, S. L. (1987) Am. J. Physiol. 253, F848-F856 28. Sundaresan, P. R., Guarnaccia, M. M., and Izzo, J. L. Jr. (1987) Am. J. Physiol. 253, F1063-FI067

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