Binding of an aldose reductase inhibitor to renal glomeruli

Vol.

129,

June

14.

No. 2, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

1985

BINDINGOFANAIDQSEREDUCTASE

530-535

INHIBITORTCRENALGI0MERULI

Margo Panush Cohen and Henry Klepser University

of Medicine and Dentistry of New Jersey Newark, New Jersey 07103

Received May 7, 1985 The aldose reductase inhibitor Sorbinil affects several membranecanplexes including Na/K-ATPase activity, transport processes, and impulse propagation. These considerations, coupled with the drug's aromatic nature, suggested the possibility of direct interaction with cell membranes. In the present study, binding of [3H]-Sorbinil to isolated gloneruli was demonstrated. Binding is dose-dependent and saturable, and can be inhibited by increasing concentrations of unlabeled Sorbinil. These results may help explain the compound's diverse effects on membraneassociated processes. 0 1985 Academic Press, Inc. associated

The polyol

pathway

consists

sorbitol

dehydrogenase,

sorbitol

and then to the ketosugar

glycemia,

the flux

leading

to intracellular

elevated

in peripheral

and increased

pathy,

recent

that

lens,

of polyols.

and nephropathy

indicating

(2-B).

(9-12).

there

including

myo-inositol

triphosphatase 18).

depletion

activity,

acccnpny

These changes aay be linked

in diabetes aldose

is increasing

since,

reductase

the increased

inhibitor

Sorbinil

0 1985 by Academic Press, Inc. in any form reserved.

of reproduction

in the

is intense

interest

in view of

beneficial

that other

accumulation

sorbitol, (15,16,19).

530

animals,

neuro-

particularly

to the enhanced polyol

like

is

peripheral

effects

metabolic

and reduced sodium-potassium

0006-291X/85 $1.50 Copyripht All righIs

content

on

and basement membrane thickening

evidence

polyol

augmented,

of diabetic

Hence, there

such agents exert

motor nerve conduction,

of hyper-

has been implicated

activity,

cataractogenesis,

However,

Sorbitol

such as cataracts,

reductase that

is greatly

glcrneruli

pathway

ccmplications

aldose

In the presence

pathway

and renal

and

to the sugar alcohol

(1).

this

of the polyol

aldose reductase

first

fructose

through

nerve,

inhibit

findings

glucose

accumulation

of diabetic

retinopathy,

in drugs

convert

of glucose

activity

pathogenesis

that

of two enzymes,

changes,

adenosine

in these tissues pathway

they are prevented Reduced activity

(13-

activity by

the

of Na/K-

Vol.

129,

No. 2, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

ATPase, an enzyme complex intimately

associated

inositol

from decreased

(201, is believed

membrane phospholipids glycemic

(211.

invoked

Hlow polyol

not clear, tissue

to explain pathway

myo-inositol.

proposes,

glucose

prevent that

influences

increased

efflux

independent

In this

derivative,

suggested

membranes.

These considerations

renal

inhibits

flux

myo-inositol content

in diabetes

(18).

of its

as an aldose

action

the lipophilicity

reductase One postulate

inhibitor,

Alternatively,

interaction

the present

to crude membranes prepared

Sorbinil

reductase

of Sorbinil,

of direct

is

to the decreased aldose

reductase

uptake,

metabolism

contributes

from the cell

prompted

At hyper-

is not understood.

or the aldose

the possibility

binding

depletion.

the mechanism by which

pathway,

regard,

inositol-containing

on myo-inositol

polyol

depletion

inhibitor.

examined Sorbinil

impacts

COMMUNICATIONS

membrane phosphatidyl-

the reduced myo-inositol

myo-inositol

myo-inositol

have effects

ccmpetitively

Similarly,

the polyol

with

to myo-inositol

activity

but presumably

inhibitors

could

consequent

concentrations,

a finding

to derive

RESEARCH

study,

a spirohydantoin with

cell

in which

from isolated

we rat

qlomeruli.

MATERIAISANDMETHODS --___ Gltmeruli were isolated from adult male white rats by sequential sieving throuqh stainless steel meshes, yielding preparations virtually free of tubular elements (22). Follawinq hypotonic lysis in 10 mM Tris HCl, pH 7.4, the crude qlomerular membranes were collected by repeated washing and centrifuqation and stored frozen at -7O'C. For binding studies, the lysed qlcmerular preparations were thawed and suspended in buffer (100 mM NaCl, 10 Ml KCl, 5 mM EGTA, 3 mM MgCl2, and 100 mM Imidazole buffer pH 7.4) on the morning of the incubations. [3H]-Sorbinil (specific activity 5.4-8.4 Ci/mnoll for binding studies was obtained fran Pfizer Central Research (Groton, Conn.). Lysed qlomeruli (50-100 ug protein per assay tube) were incubated in assay buffer containing 40-700 ~1 [3H]-Sorbinil and varyinq concentrations of unlabeled Sorbinil in a total volume of 250 ul. After a fifteen minute preincubation period, incubations were initiated by the addition of radiolabel and were conducted for 60 minutes at 25OC. Binding was terminated by addition of 3 ml of a solution of 2 mM Sorbinil to the assay tubes and inmediate filtration through a millipore filter, followed by two additional washes of the tubes and the millipore filters with the same volume of unlabeled Sorbinil solution. The millipore filters with adherent glcmerular protein to which [3H]-Sorbinil was bound were then counted by liguid scintillation. All experiments were conducted in duplicate and were paired with sirrolltaneous incubations in the presence of 1 mM unlabeled Sorbinil to assess nonspecific binding. Specific binding was determined as the difference in 531

Vol.

129,

No. 2, 1985

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS

radioactivity bound to glorreruli incubated in the absence and presence of Incubations conducted without tissue served as excess unlabeled Sorbinil. blanks to control for [3H]-Sorbinil binding to the assay tubes or to the millipre filters.

RFLSULE AND DISCUSSION ___In preliminary with

fixed

of protein

and saturable

and between

crude glmrular

membrane protein

incubation

and that

Subsequent

studies

glomerular

protein.

Total

indicated similar

binding

was relatively

[3H]-Sorbinil

binding

for

binding

or cerebral

Sorbinil,

11.

of excess be

(23,241.

0.01 - 0.1 uM, suggesting

of Specific

binding

This contrasts wherein unlabeled in

inhibited

as shown in Table 1. occurs

This

for binding

tissue

microvessels,

can

50 ug of

about 80% of total (Figure

of

1 hour of

protein.

in the literature

tested

to glomeruli

with

at 22, 25 and 37OC.

1 hour at 25OC using

constituting

bound [3H]-Sorbinil

between

as a function

was maximal within

were obtained

bound in the presence

manner by unlabeled

Sorbinil

high,

to retinal

of specifically

binding

binding

to microvascular

of [3Hl-Sorbinil

remains

of [3H]-Sorbinil,

of [3H]-Sorbinil

examining

a

at all with

about 30-50% of insulin

(23,24).

dose-dependent

About

50% displacement

at concentrations

of unlabeled

that

glcmerular

the specific

is of high affinity.

It is unlikely represents preparations

simple

that

Sorbinil

trapping

were collapsed

binding

in glomerular and disrupted 532

to crude glcmerular vascular by

was

With 50 ug

when the concentration

to those reported

hormone such as insulin

the total

- 1.9x106 qm)

there

to the amount of

results

peptide

binding

drug.

was 13.12 + 2.14 pm1 [3H]-Sorbinil/mg

is comparable

that

was also proportional

that

were conducted

binding

concentrations

of the radiola&led

Otherexperiments

time and temperature

indicated

to a maximum of about 0.5% of added counts Binding

was held constant.

amounts of [3H]-Sorbinil

preparations

binding

rose from negligible

insulin

of variable

44-704 pm1 (1.2~10~

350-700 ~l/incubation.

value

incubation

amounts of lysed glomerular

dose-dependent

binding

studies,

lumens, since

freezing

at -70°C,

membranes the thus

Vol.

129,

No. 2. 1985

BIOCHEMICAL

3H

Figure

AND

Sorbinil

BIOPHYSICAL

Added

RESEARCH

COMMUNICATIONS

(pmol)

1:

Binding of [3H]-Sorbinil to isolated glcaneruli. Results represent mean t SFM of duplicate observations in four separate eqrin-ents. Assays were performed with 50 ug glcmerular protein in a total volume of 250 ul. All results corrected for background, determined in blank incubations performed without tissue. Nonspecific binding represents that occurring in the presence of 1 mM unlabeled Sorbinil, andwas subtracted frcxnthetotalto determine specific binding.total binding; specific binding.

eliminating

intact

open luminal spaces. Rather, this binding appears to

represent a chemical or electrostatic

interaction

with membranecanponents.

The nature of such components is unknown, but the cxxnpxnd's aromatic suggests that they are lipoproteinaceous

structure

bilayers.

Diphenylhydantoin,

Table Unlabeled Sorbinil nrrol/ml

a related

1 - [3H]-Sorbinil

residues of the lipid

ccxnpound, is believed to bind to

Binding

to Isolated

Specific Binding pool 3H-Sorbinil/mg protein

Glcmeruli %PEiXiIlWl Binding 100

0

11.03 -t .64

10

7.16 + .66

64.9

+

6.7

100

3.84

+

.32

34.8

+

3.2

200

2.02

+

.32

18.3

+

3.3

8.9

-t

1.1

400

.98 -f -10

Results represent mean t SEM of duplicate determinations in four lysed glomeruli (50 ug protein separate assays. After free;ing and thawing, per sample) were incubated for 60 minutes at 25'C with 704 pool of [3H]Sorbinil in the presence of varying concentrations of unlabeled Sorbinil. Total volume of incubations was 250 ul. Nonspecific binding, taken as radltoactivity remaining bound in the presence of 1 mM unlabeled Sorbinil, was subtracted frcan each value. 533

Vol.

129,

BIOCHEMICAL

No. 2, 1985

membrane sites

associated

Sorbinil

contains

(25).

reductase

inhibitory

S'-dione).

in vivo,

group,

which

aldose

reductase

Sorbinil

aldose

(7).

ccmpound's

diverse

membrane-associated dependent metabolism, impulse

Pfizer,

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

inhibiting

in this

canplexes.

transport

propagation,

in part

necessary

nervous for

through

its

system aldose

myllbranes for

enccmpass a reactive

attack

binds to isolated

structures

Supported Inc.

regions

an interaction

effects,

believed

at the inhibitor

Thus, Sorbinil

reductase

specifically

COMMUNICATIONS

in the central

penetration

nucleophilic

protein

RESEARCH

(S-6-fluorospirochrcman-4,4'-imidazolidin-2',

the hydrophobic

membranes could represent the molecule's

sodium channels

to allming

undergoes

BIOPHYSICAL

a chrcman ring,

activity

In addition

activity

with

AND

binding

conccznitant property. glcmeruli

carbonyl site

of the

to glcmerular to, or distinct

frcnn,

The demonstration may help explain

and other

tissues,

These include

effects

and cycles,

such as Na/K-ATPase

processes,

such as myo-inositol

that

relate

that

the to

on phospholipidand phosphoinositide influx

or efflux,

and

such as nerve conduction.

by NIH Grant No. AM32447 and by a research

grant

from

Gabbay, K.H. (1973) N. Engl. J. Med. 288:831-836 Kinoshita, J.H., Fukushi, S., Kador, P. and Merola, L-0. (1979) Metabolism 28:462-469 Kinoshita, J.H. (19651 Gpthalmol 4:786-799 Gabbay, K.H. (1973) Adv. Metab. Disord. (Suppl) (19731 2:417-424 Yue, D.K., Hanwell, M.A., Satchell, P.M. and Turtle, J.R. (1982) Diabetes 31:789-794 Buzney, S-M., Frank, R.N., Vanna, S.D., Tanishine, T. and Gabbay, K.H. (1977) Invest. Gphthalmol. Vis. Sci. 16:392-396 Cqan, D-G., Kinoshita, J.H., Kador, P.F., Robinson, G., Datilis, M.G., Cobo, M. and Kupfer, C. (19841 Ann. Int. Med. 101:82-91 Beyer-Mears, A., KU, L. and Cohen, M.P. (1984) Diabetes 33:604-607 Fukushi, S., Msrola, L.O. and Kinoshita, J.H. (19801 Invest. Gpthalmol. Vis. Sci. 19:313-315 Beyer-Mears, A. and Cruz, E. (1985) Diabetes 34:15-21 Judzewitsch, R.G., Jaspan, J-B., Polonsky, K.S. et al (1983) N. Engl. J. Med. 308:119-125 Robinson, W-G., Kador, P.F. and Kinoshita, J.H. (19831 Science 221:11771179 Greene, D.A., DeJesus, D.V. and Winegrad, A-1. (1975) J. Clin. Invest. 55:1326-1336 Greene, D.A. and Lattimer, S.A. (1983) J. Clin. Invest. 72:1058-1063 534

Vol.

129,

No. 2, 1985

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RESEARCH

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15. Cohen, M.P. and Dasmahapatra, A. (1984) Clin. Res. 32:796A 16. Cohen, M.P., Dasr@ahapatra, A. and Shapiro, E. (1985) Manuscript Subnitted 17. MacGregor, L.C., Rosecan, L.R., Laties, A.M. and Matschinsky, F.M. (1984:) Diabetes 89:354A 18. Finegold, D., Lattimre, S.A. Nolle, S., Bernstein, M. and Greene, D.A. (1983) Diabetes 32:988-992 19. Greene, D.A. and I.&timer, S.A. (1984) Diabetes 33:712-716 20. Roelofsen, B. and Trip, M.V.L-S (1981) Biochim. Biophys. Acta 647:302306 21. Greene, D.A. and Lattimer, S.A. (1982) J. Clin. Invest. 70:1009-1018 22. Cohen, M.P. and Klein, C.V. (1979) J. Exp. Med. 149:623-631 23. Baskell, J.F., i%ezan, E. and Pillion, D.J. (1984) Endocrinol. 115:698704 24. Raskell, J.F., Naezan, E. and Pillion, D.J. (1985) Am. J. Physiol. 248: Ells--El25 25. Willcw, M., Kuenzel, E.A. and Catteral, W.A. (1984) ml. Phamacol. 25:228-234

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