(GnRH) Binding to Pituitary Receptors in Goldfish

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of Gonadotropin-Releasing. Hormone. (GnRH). Binding to Pituitary. Receptors in Goldfish. (Carassiusauratus)1. HAMID. R. HABIBI,2'4. RICHARD. E. PETER,4.
BIOLOGY

36, 844-853

OF REPRODUCTION

(1987)

Characterization of Gonadotropin-Releasing to Pituitary Receptors in Goldfish HAMID

R. HABIBI,2’4

RICHARD

JEAN

E. PETER,4

E. RIVIER,5

and

Department University Edmonton, Alberta, The

Clayton

Foundation San

Hormone (GnRH) (Carassiusauratus)1 MIRKA

WYLIE

Binding

SOKOLOWSKA,3

W. VALE5

of Zoology4 of Alberta Canada T6G and

Laboratories Salk Institute

for

The Diego,

California

92037

2E9

Peptide

Biology5

ABSTRACT Goldfish agonist

pituitary

analog

‘251-tGnRH-A

gonadotropin-releasing GnRH

of

teleost

to

a goldfish

pituitary

binding was significantly was a function of tissue Incubation of the pituitary saturable

binding

after slower

tGnRH

after

than

than

the

native

observed

with

tGnRH-A

had

and

provided

the

of

presence

of site.

tGnRH-A, a greater

cold

of

indicating

characterization

of

the

GnRH

enhanced potency of gonadotropin-releasing

attributed pituitary

to a greater GnRH-receptors

Vale et al., degradation

1981), (Koch

GnRH.

presence

The

as well et al.,

the and

acid

the

indicating cold

that

analog

above.

the

The

analog

displaced

binding

is more

bound

23, 1986. 1986. supported

both

peptides than

receptors

the

in goldfish

peptides Catt,

to

the

tGnRH.

and

Fisheries,

ul.

incubation

at

is

of 4#{176}C;

radioligand

per tube. indicated was

two linear unlabeled

effective

in displacing and

sites

same

classes

of

These

results

demonstrated

the

Scatchard

label

analysis

site and in parallel

binding

found

a low-afto that

sites;

however,

the

presence

pituitary.

and replacement ethylamide are

of the glycinamid&#{176} thought to be the main

residue factors

by for

influencing the metabolic stability and enhanced affinity of GnRH-agonist analogs (Conn et a!., 1984). Although some information is available on the

for 1981;

at position

bind native

a superbinding

be resolved into addition of excess

of binding sites: a high-affinity/low-capacity was displaced by tGnRH from both

receptors

structure-activity ous nonmammalian

6

functional

relationships vertebrates

relationship

of GnRH in (Peter, 1986),

between

GnRH

(GtH) release has in mammals (Marian

varithe

receptors been studied et a!., 1981;

Vale et al., 1981; Loumaye et al., 1982) and, to some extent, in chickens (Millar and King, 1983). Teleosts would provide a particularly interesting by

grants

from

the

Natural

and Engineering Research Council of Canada to REP., Heritage Foundation for Medical Research Postdoctoral to H.R.H., and NIH Grant HD 13525 to W.W.V. and J.E.R. 2 Reprint requests. 3Present address: Academy of Agriculture, Department biology Poland.

using

of 1I-tGnRH-A

‘251-GnRH-A,

and gonadotropin extensively only

Accepted October Received July 29, ‘This work was

a 30-mm

and

as to a greater resistance to 1977) compared to native

of a D-amino

after

dissociation curve could were observed after the

in mammals of superactive hormone (GnRH) affinity of (Clayton

achieved

pM

INTRODUCTION

The analogs

was

by Equilibrium

the

that

for

characterized

of 470

at least two classes Bound ‘251-GnRH-A

affinity

were

Pro9-NHEt]-GnRH).

analog, and of 1251-tGnRH-A

tGnRH-A,

Addition

receptors Leu8,

preparation

concentrations

addition

Trp7,

increasing incubation temperature to 22#{176} C. Binding of the with a linear correlation over the range of 0.5-2 pituitary preparation with increasing concentrations of ‘25I-tGnRH-A

membrane

peptide.

suggested the finity/high-capacity

membrane

addition of the rates of dissociation

(GnRH)

[D-Arg6,

reduced after concentration,

at radioligand

to be reversible components;

hormone

(tGnRH-A;

Ambrosowa

6,

30-149

model for such studies, a hypothalamo-hypophysial the control of pituitary through GnRH delivered by nerve fibers (Kaul and

Sciences an Alberta Fellowship

of

Icthyo-

Krakow-Mydlniki,

Also, 844

dopamine

has

because

they do not have portal system, and GtH release is exerted directly to the pars distalis Vollrath, 1974; Ball, 1981).

a potent

GtH

release-inhibitory

GnRH

action teleosts

directly (Peter

on the GtH et al., 1986).

the predominant determined as wood

et a!.,

cells The

GnRH in [Trp7, Leu8]

1983).

This

of

release

in vivo

enzie Pro9

et al.,

tGnRH

in

(Peter

et a!.,

1984).

-NHEt]

The

-GnRH

significantly

more

that receptors

1985)

and

terms

[D-Arg6,

GtH

Trp7,

was

Leu8,

found to

to

tGnRH

receptors as labeled

-teleost

Labeled demonthe also

affinity

in

similar pg of

for

and

sexually

February

of

to those tGnRH-A

regressed

or

in

included

100

early

stages

that of

9.2,

gonadal

approach

and

its superactive

analog

according

to protocols

developed

with were

2% ethanedithiol for deprotection. mediated by dicyclohexylcarbodiimide.

was

the original Ammonolysis overnight at GnRH coupled

Merrifield with liquid 5#{176}C.Both to MBHA

chloromethylated ethylamine was protected [Trp7, and fully protected

Pro9-

1, 1% acetic

=

equilibrated

Couplings tGnRH resin on

radioactivity 1). The

-

M)

with

to

alter area

0.1

the mass

to

the

binding 125

specific

concentrations

I-tGnRH-A.

buffer,

0.02% and

sodium aliquots

the elution fraction was rabbit globulin

act.

of

peak

antiin Figure and was of

carried out on radioimmuno-

I-tGnRH

curves with in-

tGnRH-A was

1100

pCipg’. Membrane

Fish were methansulfonate,

Preparations

killed -

a

was estimated

displacement antibody

unlabeled 125

antibody E., un-

Determination

relationship

of

Sp.

R.

antiserum

tGnRH-A

I-tGnRH-A was a self-displacement from tGnRH-A

(bed

tGnRH-A was indicating iodination

studies.

radioactivity

labeled

M borate

using

shown within the main the highest immunoreactivity

of the basis by

the column

NY) as the second Y. P. and Peter,

binding

receptor

by addition and

determine of each

activity

creasing

Pituitary

(0.1

0.1% gelatin and 1 ml were collected,

binding level by incubating

1300

10 pg chloramine-

a QAE-Sephadex

at 50% obtained

125

et al. (1980); 5 M acetic acid) was

terminated

results). Immunoreactive in only the main peak,

for

assay;

was acid

resin. achieved Leu8 ] super-

15 ml)

125

by radioimmunoassay, serum and goat anti-rabbit

sp. act. routine

by Rivier

assembled on a methyl benzhydrylamine (MBHA), whereas the tGnRH-A was constructed

on

for (Fig.

not

used

were phase

et a!. (1986). In brief, t-Butyloxycarbonyl group used for a-amino protection and trifluoracetic

purified

body; the 1 displayed

[DArg6,

Pro9 -NHEt] -GnRH, tGnRH-A, by using the Merrifield solid

buffer

containing Fractions of

published detected did

GnRH,

acid GnRH

I-Na (dissolved using procedures

using

was

(Arnel Inc., Brooklyn, (validated by Huang,

were

Hormones Teleost

(c

with by

I-Na,

125

reaction

borate

estimated tGnRH-A

recrudescence.

Trp7, Leu8, synthesized

-21.3

=

was labeled buffer)

1 mCi The

pl

counted profile

between

fish

[a] D

acid)

by Piyachaturawata (dissolved in 0.1

with

azide.

in a 1800-! flow-through a simulated natural photoand fed a commercial fish

and

GnRH

1 mm.

pH

July

Peptides

tGnRH-A

iodinated

mainly

1, 1% acetic

=

Teleost GnRH-A 0.5 M phosphate

volume,

performed

(c

perliquid

HPLC. Amino in 4 N meth-

[DArg6,

-38.5

=

Goldfish, Carassius auratus, common or comet variety (mixed sex, ranging 8-13 cm in length) were purchased from Grassyfork Fisheries Co., Martinsville,

were

1984).

97% pure on analytical after acid hydrolysis

[a]D

Animals

Experiments

et a!.,

was

amino teleost

peptide

diet.

Rivier

in the acetic

acid).

AND METHODS

IN. Fish were maintained aquarium at 16 ± 1#{176}C on period in Alberta, Canada,

HF dilute

anesulfonic acid, gave the expected ratios. Optical rotation was as follows: NHEt]

be

liquid with

Purification high-performance

(HPLC;

at least analysis,

T for

MATERIALS

treated with and extracted

and

in a teleost, ligand. We

has a higher native peptide.

NHEt were of anisole

chromatography were acid

(Mack-

(Peter et al., 1985). we describe the first

tGnRH-A than the

to

of

in vitro

compared

of pituitary GnRH using tGnRH-A

demonstrate pituitary

in

analog

GnRH in vivo in goldfish In the present study, stration goldfish,

goldfish

845

acid prior to lyophiization. formed by preparative

other teleost species et al., 1984; King and have demonstrated

(tGnRH-A) potent

is known

PITUITARY

agonist presence

salmon has been (tGnRH) (Sher-

of tGnRH

be present in a number of (Breton et a!., 1984; Sherwood Millar, 1985). Previous studies bioactivity

IN GOLDFISH

in goldfish and other primary structure of

chum -GnRH

form

RECEPTORS

after a light anesthesia 0.02%), pituitaries

(tricaine removed,

or -

HABIBI

846

ET

AL.

Binding

Studies

Incubations polypropylene

()20

coated

overnight

experiment, pmoles tGnRH-A

15

z

in

(in triplicate) microcentrifuge

the

C,

presence

and

(peptide

displacement

10

-

250

stant 22-29

at

20

30

40

lines)

profile (1-mi column. Fractions were pooled and

mm

fractions) of ‘25I-tGnRH-A 19, 20, 21, and 22 (area used for the receptor assay.

Intact

and

containing

serum 10-ml

1 mM

per

gradual

increase

100

(doubled

buffer

at 250 15-mi

and and

was

resuspended

buffer

initially,

to 5 ml). for 5 mm, polycarbonate

buffer, mm for mination,

unless

otherwise

receptor aliquots

binding of the

an of stated)

in pre-

in the

I-

M unlabeled maximum

assay

volume

of

experiments,

pituitary

pituitary incubated

equivalent with a con-

(3 0,000-40,000 presence and

cpm; absence

the

appropriate and

were washed twice by centrifugation with BSA-free Tris-HCI and assayed content (Bradford, 1976), using BSA

15,000 prepara-

volume per used

of

100

within

containing

the

binding membrane

labeled

peptide

binding

to tissue.

to

the in

for

of

tube and the presence

pellets

in a gamma carried out in

for determination absence of pituitary

nonspecific membrane

BSA, and of excess

determination

of

to

with un-

nonspecific

Analysis

A computerized program (LIGAND)

supernatant tube, diluted at

vessels

into tubes for counting Parallel incubations were

determine pituitary

Data

by aspiration of the supernatant. rinsed twice with 500 pl cold assay

by

homogenate

1 pituitary

studies. For crude membrane

in a (50

followed

The

-

bovine

homogenized homogenizer

X g

in

(equivalent

buffer [Tris]

0.5%

volume) and further centrifuged 30 mm at 4#{176}C. The crude membrane

x g for assay

pl assay

a

ice-cold assay aminomethane

dithiotheritol

of volume

was centrifuged transferred to

tion

ml

albumin EBSA] pH 7.6), glass-teflon (prechilled)

pituitary

10

in a final (one was

incubation

all experiments binding in the

HC!

saturation

at which

other

preparation or as indicated)

transferred spectrometer.

10

of

obtained)

4#{176}C.For

at 4#{176}C, followed pellets were

buffer,

washed (3 times) with (10 mM Tris[hydroxymethyl]

the

For

out

analog in a final volume of 250 p! at 4#{176}C otherwise stated). At appropriate intervals (30 for equilibrium binding studies), incubations termined by centrifugation (15,000 X g) for 5

mm were

FRACTION elution

absence

level of ‘25ItGnRHA fmoles per tube)

.

FIG. 1. A typical from the QAE-Sephadex indicated by vertical

p!)

of cold _______________ (unless

0

10

BSA.

concentration

was

pl

membrane per tube

5

2.5%

carried (500

3.6-300 fmoles per tube (14-1200 per liter; 5,000-400,000 cpm) of were incubated with membrane preparation

tGnRH-A

C

with

were tubes

pl 30

nonlinear least-square curve-fitting was used for Scatchard analysis

and determination of the best fit through the data points, as applied previously to GnRH binding data (Munson and Rodbard, 1980). A four-parameter logistic curve-fitting program (ALLFIT) was also used for

analysis

(50%

of

effective

curves dose)

protein deterpreparation (15,000 X g) for protein as a standard.

Fresh membrane preparations were used at all times for the radioreceptor assays, as significantly lower total and specific binding levels were obtained using membranes prepared from frozen goldfish pituitaries (results not shown).

and

determination

(DeLean

et a!.,

of

ED50

1978).

RESULTS

Equilibrium

Binding

Time-dependence studied preparation

during

of tGnRH-A of

125

incubation (one pituitary

I-tGnRH-A

binding

with pituitary equivalent per

and 22#{176}Cfor a total period of 180 mm Experiments were carried out in October, goldfish

that

were

sexually

regressed.

was

membrane tube) at 4 (Fig.

2). using

Maximum

GnRH

RECEPTORS

IN

GOLDFISH

847

PITUITARY

#{163}

-J-

12 I-

-j-

0

I-

I-

-S

10

I’.

U 61

-S

a

9

6,

AU.

-I

0

I-

0 I-

TIME

binding of the radioligand was achieved after 30 mm incubation at 4#{176}C, and both total and specific binding were stable for the following 30 mm. Increasing incubation

(pO.05) between total binding or specific binding for the 10-vs. 80-mm preincubations.

-J

I-

condition

at 4#{176}C and condition

0 I-

was for

-S.

0

z D

to

membrane 125

I-tGnRH-A

Binding in May, using goldfish study the correlation concentration binding

and was

deter-

mined in the presence and absence of 10_6 M cold tGnRH-A (Fig. 4). Less than 1% of the total added radioligand was found to bind to the tube as determined

10

Pre-

significantly I-tGnRH-A

binding

tGnRH-A

Experiments were performed were sexually matured,

tGnRH-A

80

10

0

Concentration

pituitary

ITT

r

binding of 22#{176}Cwas

studies.

between

T

T

recrudescing receptors

the

was observed after 30 mm incubation therefore used as standard experimental the

3

specific binding experiment was

sexually membrane

absence

80 mm specific

or

In

the

-6

#{149}lOnj

22#{176}Csignificantly

and specific binding at

out in January, using test the stability of

incubated incubation reduce

to

both Equilibrium

0

6.

(mln)

FIG. 2. Time and temperature dependence of ‘25l-tGnRH-A binding to crude goldfish pituitary membrane preparations (15,000 X g pellet, 30 g protein per pituitary equivalent, October). Data show fraction bound (BIT, percentage of total counts) corrected for nonspecific binding measured in the presence of 10-6 M cold tGnRH-A. The inset shows binding in the absence of cold peptide at 4#{176} and 22#{176}C. Values are mean ± SE of six observations (two experiments, each carried out in triplicate).

the

8

incubation

membrane. binding pituitary

From these of the radioligand membrane was

binding,

as determined

in

the

absence

of

results, maximum in the presence 11% of total counts. in the

presence

pituitary specific of excess Specific

of 106

M of

0.5

1

2

PITUITARY

3

4

5

EQUIVALENT/TUBE

FIG. 4. Equilibrium ‘25l-GnRH-A binding to increasing concentrations of crude goldfish pituitary membrane preparation, equivalent to 0.5 to 5 pituitaries per tube (20-200 g protein per tube, May). Parallel incubations were also carried out in the presence of 10-6 M tGnRH-A and the results used for correction of nonspecific binding. Values are mean ± SEM of three observations. Experiment was carried out in May, using 8- to 12-cm fish.