High and low affinity binding sites for endothelin on cultured rat glomerular mesangial cells

Vol. 161, No. 2, 1989

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 776-781

June 15. 1989

HIGH

AND

LOW AFFINITY

F.

Badr*,

Karen

Kamal

Departments

Received

of

April

28,

renal

described

signal

transduction

marked and,

intravenous

infusion

alkalinization

rat peptide

these

*

correspondence

Abbreviations Endothelin: angiotensin (1,4,5)-trisphosphate:

Mindy

School

used atria1 A II;

cells

confirmed the the in

should in

this natriuretic intracellular InsPj.

0006-291x/89 $1.50 Copyright 0 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

which

of

responses

in

by

(2).

others

presence

present

of studies,

likely

mechanism

result

induce

rat

from

actions receptors

examined

its

intracellular

mesangial

These

specific we

in

ultrafiltration

which to

the

increases

capillary

found

through

concentrations.

the

rate was

cells

calcium is

renal

increases

include

free

glomerular

ET

and

Endothelin

mesangial

filtration

mitogenic

In

mesangial

ET; II:

the

addition,

been

cells.

to

whom

In

(1).

contraction

in

suggested

[1251]-ET

To

RAT

Snajdar,

glomerular,

kidney

and

cell

provoke since

cells

on

M.

University

cell,

rat glomerular

glomerular (1).

has

mesangial

Vanderbilt 37232-2312

pathways

reductions hence,

and which

the

contracts

mesangial the

in

trisphosphate

Endothelin-induced

finding

ET

and

coefficient

TN

mesangial

to

inositol

mediating

Sugiura, Rudolf Tadashi Inagami

Biochemistry, Nashville,

resistance of

activation intracellular

ON CULTURED

glomerular mesangial cells, thereby influencing filtration rate. Here, we demonstrate the presence of two sites on cultured rat mesangial cells with Kds of 0.76 and binding capacity (Bmax) values of 6.78~10~ and 27.60~10~ respectively. Binding of [12sI]-ET was maximal at 120 min the subsequent60 min, and selective. No competition for with >lOOO-fold concentrations of atria1 natriuretic II, arginine vasopressin, nicardipine, or nifedipine. The receptors for ET on glomerular mesangial cells suggests a peptide in the regulation of glomerular filtration rate.

responses

vascular

Masanori and

ENDOTHELIN CELLS

1989

recently

have

microcirculatory

SITES FOR MESANGIAL

A. Munger, Schwartzberg,

Medicine and Medicine,

Endothelin contracts glomerular size and ET-specific binding 44.70 nM, and maximal binding sites/cell, at 4OC, stable for binding was observed angiotensin peptide, presence of specific major role for this 0 1989 Academic Press. Inc.

We

BINDING GLOMERULAR

the

cells, of for binding

a ET

in

this of

culture.

be

addressed.

paper: factor: calcium

776

ANF; arginine concentration:

vasopressin: [Ca++li;

AVP; inositol-

BIOCHEMICAL

Vol. 161, No. 2, 1989

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Mesangial

Cell Culture: Rat mesangial cells were isolated and cultured as described previously (1). Briefly, kidneys were excised from two young Sprague-Dawley rats under sterile conditions, and the cortices removed, minced, and washed several times in Hank's Balanced Salt Solution, containing 10 mM HEPES pH 7.4, amphotericin (0.25 pg/ml), and gentamicin (50 pg/ml). The tissue was then passed through consecutive sterilized stainless mesh filters with pore sizes of 212 p, 150 m, and then onto the final mesh of 75 pun. The isolated glomeruli were harvested from the surface of the 75 p filter and washed twice with the Hank's Media. The glomeruli were suspended in RPMI 1640 Medium with 15% Fetal Bovine Serum (Gibco), penicillin (100 U/ml) and streptomycin (100 pg/ml) (Gibco), plated into 100 mm cell culture petri dishes, and incubated at 31' C in a humidified atmosphere of 95% air and 5% C02. Mesangial cell colonies were subcultured in 24-well plates, and experiments were carried out on cells from passages 3 or 4. Criteria used to establish the identity of mesangial cells have been established previously The mesangial cells were routinely grown in RPMI-1640, supplemented with (1). 15% fetal calf serum, penicillin 100 U/ml, and streptomycin 100 pg/ml. Iodination of Endothelin: Synthetic porcine ET was radioiodinated by the lactoperoxidase method (3). ET (2.5 1.19 in 10 1.11 of 0.015% Triton-X305) was mixed with 30 1.11 of 0.5 M sodium phosphate, pH 1.0, and 0.7 mCi of carrier free [lz51]-NaI (4.8 wl). The reaction was started by adding lactoperoxidase pg/ml) and Hz02 (10 ~1, 0.003%). After I to 10 min at room (10 1.11, 17.5 temperature, the reaction was stopped by adding 100 ~1 of 1% BSA/O.l% trifluoroacetic acid. Iodinated ET was separated from ET and free iodide by HPLC on a Vydac Cl8 column with a linear gradient of acetonitrile (20-80s) containing 0.1% trifluoroacetic acid at a flow rate of 1 ml/min. Specific activity of iodinated ET used was 1263 Ci/rmnol or 1579 Ci/mmol. [lz51]-ET Binding Studies: Studies of [125I]-ET binding were performed on mesangial cells grown to confluence in 24-well cluster dishes. Experiments were performed in a buffer consisting of RPMI 1640 with 0.1% BSA. Binding studies were routinely performed at 4O C. Cells were washed once with buffer and then exposed to the appropriate concentration of L~~~II-ET in buffer. At the completion of the experiment, the experimental medium was removed and the cells were washed 5 times with ice cold buffer. The cells were then dissolved with 1.0 ml of 1.0 N NaOH overnight, then tranferred to small tubes for determination of the bound radioactivity using a gamma counter (Beckman Gamma 4000). Non-,specific binding was determined by measuring the amount of [125~]ET bound in the presence of 1000 fold excess of unlabeled ET. Cell density was determined by counting cells from replicate wells, USing a Coulter Counter ZBi (Coulter Electronics, Inc., Hialeah, Fla.) . The time course of specific [125I]-ET binding to mesangial cells was assessed in 2 experiments in which 25 pM [125I]-ET was incubated with mesangial cells as described above and specific binding assessed at 10, 30, 60, 90, 120 and 180 min following addition of the agonist. To dete.rmine the affinity and density of ET-binding sites, mesangial cells [125~]-~~ in were incubated with 25 pM the presence of increasing concentrations (0.01 to 500 nM) of cold ET (n=4 experiments, each concentration of which was performed in duplicate). Specific binding was determined for each concentration, and values for the dissociation constant (Kd) and maximal binding capacity (Bmax) calculated using Scatchard analysis (4). Competitive binding-inhibition studies were carried out by incubation of mesangial cells with 25 pM [125I]-ET and addition, at equilibrium, of lOOOfold concentrations of ANF, AVP, AII, nicardipine, and nifedipine.

Time-course As cells following

of

shown

in

increased addition

[~~~II-ET Figure

1,

Binding specific

progressively of

the

at binding

with agonist,

4OC: of

time, and

777

25

pM

[125I]-ET

reaching remained

a stable

to

rat

maximum for

mesangial at

up

to

120 180

min min.

Vol.

161,

No.

2, 1989

BIOCHEMICAL

0

AND

30

60

BIOPHYSICAL

90

RESEARCH

120

150

COMMUNICATIONS

180

Time (min) Figure mesangial Squares: specific

1:

Time cells. Total Binding.

Nonspecific

dependent Binding Binding; Values

binding

stable

reached

thereafter.

reversed

by

addition

2 depicts ET

increasing

by the

nM).

absence AVP,

of or

and

at

remained

relatively

equilibrium

of ET (Figure

excess

inhibition

Binding

of

of

cold

nM

each),

competitive

AI1

(25 pM) binding to rat 120 min. Symbols; Open Binding: Diamonds: Non-

10 min

binding

lOOO-fold

percent

concentrations

of ANF,

a

at

was

totally

2).

Parameters: the

(0.01-500

at

maximum

[12511-ET of

Binding

Figure

its

Specific

the

Equilibrium cold

increase in [12511-ET equilibrium was reached Closed Squares: Specific are means k SEM.

(500

ET

binding

of

[lz5

II-ET

and

was

upon nicardipine

binding

of

25

decreased highly

pM

specific,

addition

[12511-ET

by

progressively

with

as

of excess

demonstrated

concentrations

or nifedipine

(100

nM, each).

loo? 5 c

80-

z

60-

Li +

40-

ANF AVP All

g

20-

Nit Nit

8 .OOl 01

0

2

.I .T I 1

O-1

.

~

”

0

I

[&d

1’0

1 do

A$onisf] (nM)

Id00

.-

. 2

I 3

lob00

B (fmol/lO

Figure 2: Percent binding of 25 pM [ 1251]-ET in the presence concentrations (on log scale) of cold ET (closed squares) agents. Note the reversibility of binding by ET and its total presence of other tested agents. Values are means f SEM. Figure presence of 0.76 binding

.

3:

Scatchard transformation of the data in Figure 2, of two separate binding sites for ET on rat mesangial (ER-1) and 44.70 nM (ER-21, and Bmax values of 6.713~10~ sites/cell, respectively. 778

6cells)

of increasing (n=41 and other absence in the revealing the cells with Kds and 27.60~102

L 4

Vol. 161, No. 2, 1989 Transformation

of

presence

of

Bmax Kd

of

44.70

(ER-2)

nM

data

classes

of

binding

sites

and

(Figure

In

the

two

6.8~10~

of

BIOCHEMICAL

The

described

The

physiologic

close

to

membranes

was

concentrations

by

to

EGTA.

colleagues

seems

of reasonable

ET

the

a

studies, in

less to

than advance

the

papillary

our

whole

779

stores

followed

by

to

1 nM,

together that

however, by

maximal by in

was

higher rat

as

of well was

ET

mesangial

with mesangial

of

our

for

whereas [Ca++]i as not present cell

a

values

maintained

(EGTA),

contraction

Taken

by

characterized

spike (l),

to was

concentrations

which

studies

cells generation

lower

picomolar

inositol

followed

subsequently

transient

hypothesis

of

elicited

Ca"

cell 1 nM.

is

(spike

was with

[Ca+']i

onset in

nM)

from

InsP3

0.1

signal

extracellular

mesangial

renal

mesangial

The

course

response reported

Furthermore,

nM.

plateau,

were

rapid

the

0.76

stimulation

concentrations

those

of

are

addition,

determined

intracellular

calcium

increase

in

(5),

ET

spike

In

removal

resulted

concentrations it

(4).

which

(1.2,s)

In

rat

of

10 time

of

and

sustained

abolished

Its

i.

a sustained

results al

putative at

(ET-~,

that

exposure than

At

the

of

bound

these

cells

for

The

membrane

here.

(61

the

cells.

of

these

ET-induced

greater

(1).

al

half

release

in

et

et

demonstrate these

site

of

nM).

[Cat+1

noted,

Similar

slow

Martin

upon

or

to

in

the

presence

concentrations

reported

demonstrated

to

we

that

the

in

intracellular

presence

observed

approximately

Ca++

Simonson a

by

in

obtained

insensitive

a

phosphate

the

on

binding

(1.3

equal

increase

those

and

and site

endothelin inositol

suggests

constants

suggested

gradual

been

for

Here,

the

affinity

we

extracellular

stimulated

a

transduction

concentration,

since

generation)

ET

generation

by

further

have

and

increases

concentrations.

and

nM)

plateau)

cells,

binding

reported

(InsPs

by

and

(ER-1)

and

affinity

signal

[12511-ET

suggest

high

studies,

of

InsP3

the

nM

0.76

low

include

cells.

binding

preparations

previous

turnover

than

min

the

(0.66

accompanied

slow

ET

that

membrane

of

the

actions

for

dissociation

the

close

influx

of

suggesting

these

relevant,

the

for

concentrations

no

of

to

to

Kd

sustained

with

site

for

cells

sites

biologically

responses

lipid

Kd

responses,

contraction,

on

[ 12511-ET

affinity

are

our

consistent

affinity

cell

calcium

and

binding

of

glomerular

high

per

mesangial

receptors

strikingly

the

was

apparent

systemic

intracellular

specific

receptors.

In

in

in

characteristics

of

an

cellular and

responses

of

remarkably

for sites

glomerular,

mitogenesis,

receptors

method with

cell

we

endothelin-specific

cell

per

(l),

elevations

presence

sites

27.6~10'

cellular

alkalinization,

value

binding

of

renovascular,

rat.

Scatchard

a Bmax

studies

turnover,

the

3)

recent

pathways,

by

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

ET 20

higher which

was

of

Dunn

those observed

findings, contraction

at

Vol. 161, No. 2, 1989

in

response

to

resulting

from

affinity,

higher

presence

of

ET,

extracellular

nature

of

(ER-1)

ER-1,

in

may

responses

to blockers

(1,2,5,7,8).

findings

is

identify the

three

human

ET

subtypes.

If

mesangial

cells

report

structurally

may

in

to

fact

Inoue

The

existence

discrepancy contractile

with et

al

the

of

in

we

for

to

which

our these

isopeptides

multiple

subtypes

receptors

respect

(9) distinct

existence

rat,

represent

affinity

apparent

interest

the

the

higher

ET-induced

pharmacologically

suggest

applies

(1,2,5).

dihydroperidine-sensitive-calcium-

by

and

and

same

the of

particular

recent

family

the

for

to

ET-stimulated

defined.

sensitivity

the and

cells

the

incompletely

lower on

nicardipine

these

by

the

i

ET

have

receptor

identified

distinct

forms

on of

ET

in

species. The

by

Of

the

in

[Cat+1

dependent

transduce

evoked

and

of

not by

to

explanation

in

activation

hence

responses

the

calcium

present authors

an

the

appears

remains

regarding

increases

inhibitable

responses

provide

channel

not

also

cells

extracellular

of and

mitogenic

biologic

reports

rapid

(ER-2)

and

and

mesangial

however, published

site

site

the

through

a consequence

Ca++

alkalinization

receptor

this

is binding

receptor

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

mediated

release,

This

The

for

is

density

intracellular

among

which

InsPs

nifedipine.

of

BIOCHEMICAL

total

ANF,

absence

A II,

during

and

nicardipine

fact

identical In

a

a calcium

we

have

transduces

including

These glomerular

We

described

function, may

evidence

be

of

a

of of

for

(ER-2)

cells.

suggest

their

lack

either

to

mesangial

cells

respective

competition

the

ET

receptors by

binding

nifedipine sites

is

in

channel. the

binding

propose

that

functional

regulatory

and relevance

the responses

of

for low

for

affinity to

model

endothelin

provide

a

in

endothelin-responsive

for

a high

affinity

endothelin

on site

endothelin

alkalinization,

role

other

existence sites

intracellular

contraction,

data

binding of

the that

provided

previously

cells,

activation

unlikely

affinity

mesangial

[ 1251]-ET

Furthermore, it

to

low

for

against

responses.

and

glomerular

competition

argues

makes

summary,

(ER-1)

which

AVP

ET-evoked

and

of

in

(ER-2) in

these

and

mitogenesis.

the

control

agonist-receptor

rat

of

interactions cells.

ACKNOWLEDGMENTS

This

work

and

HL35323

was

supported

by

NIH

Grants

DK38667

and

DK39261

(KFB)

and

HL14192

(TI).

REFERENCES 1. Badr, K. F., Murray, J. J. Breyer, M. D., Takahashi, K., Inagami, T. and Harris, R. C. (1989) J. Clin. Invest. 83:336-342. 2. Simonson, M. S., Wann, S. Mene, P., Dubyak, G. R., Kester, M., Nakazato, Sedor, J. R., and Dunn, M. J. (1989). J. Clin. Invest. 83:708-712. Y., 3. Hirata, Y. Yoshimi, H., Takata, S., Watanabe, T. X., Kumagi, S., Nakajima, K ., and Sakakibara, S. (1988) Biochem. Biophys. Res. Commun. 154:868-875.

780

Vol. 161, No. 2, 1989

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

4. Scatchard, G. (1949) Ann. N.Y. Acad. Sci. 51:660-672. 5. Dunn, M. J., Wolfe, J. A., and Simonson, M. S. (1989) Clin. Res. 37:489A. 6. Martin, E. R., Brenner, B. M., and Ballermann, B. J. (1989) Kidney Int. 35:316. 7.

Yanigasawa, M., Kurihara, H., Kimura, S., Tomobe,Y., Koboyashi,M., Mitsui,Y., Goto, K. and Masaki,T. (1988) Nature. 332:411-415. 8. Brain, S. D., Tippins, J. R., and Williams, T. J. (1988) Br. J. Pharmacol. 95: 1005-1007. 9. Inoue, A., Yanigasawa, M., Kimura, S., Kasuya, Y., Miyauchi, T., Goto, K., Proc. Nat'l. Acad. Sci. U.S.A. 86:2863-2867. and Masaki, T. (1989).

781