BIOLOGY

OF

REPRODUCTION

13,

Prostaglandin FRANCES

482-489

(1975)

E1 Specific

A. KIMBALL2,

Binding

KENNETH and LILLIAN

Fertility

J.

Research,

The

Kalamazoo,

in Human

T. KIRTON, WYNGARDEN

Myometrium1

CHARLES

Upjohn

H. SPILMAN

Company,

Michigan

ABSTRACT

The equilibrium binding constant and the concentration of specific prostaglandin (PG)E, binding sites was determined in the low speed supematant fraction of human myometrial homogenates. The apparent dissociation constant for 7 different preparations was 2.38 ± 0.38 X 10-9M. PGE, specific binding site concentration did not appear to vary during the menstrual cycle. The relative affinity of various prostaglandins, PGE, metabolites and non-prostaglandin combinding sites in human myometrium was determined. The relative affinities prostaglandins, PGE, ‘PGE3 >PGF20>PGB2 PGA, PGA2 >PGB, >PGD2; for the PGE, metabolites, PGE, >13,14-dihydro-PGE, >13,14-dihydro-15-keto-PGE1 >15-ketoPCE,. The prostaglandin precursors arachidonic acid and bis-homo--y-linolenic acid exhibited PGE, PGE2 >(1SS)15-methyl PGE2 methyl ester>PGF2 >(1 SS)-i 5-methyl PGF2 THAM>(1 5S)-1 5-methyl PGF2 , methyl ester. These results were compared with the relative uterine stimulating potency of several natural prostaglandins and the 15-methylated prostaglandin analogs in the rhesus monkey in vivo. pounds

for

H-PGE1

were: for the natural

INTRODUCTION

Exogenous labor-inducing (Karim,

mans 1971).

In

tion

of

1971;

general,

sensitivity

smooth

to

apparent

agents

and in

muscle

and

of interaction

with

Daniel,

1967).

Ramwell (1971)

et

indicate

taglandins changes

al.

are in

the

Studies (1969),

extreme

this paper

and the neurotrans-

E1

that

Kirton

responses

uterine

sensitive

to

prostaglandin

Earlier

reports prostaglandin

with

Accepted August 6, 1975. Received June 4, 1975. ‘Partially supported by Health Contract No. NIH-69-2208. 2Ad& reprint requests Fertility Research, The Upjohn Michigan 49001.

using

and

human

uterus. These intact

extended

tissue

to investigate

we

report

specific binding

studies

in human

on

prostaglandin

myometrial

prostaglandins

fracspethe for

the specific PGE1 binding site compared to the relative uterine stimulating potencies of these compounds

structural

vivo

further

tested

in the

rhesus

AND

METHODS

monkey

in

-

a specific bindMATERIALS

demonstrated binding

conducted

studies were

relative affinities of several

Forbes

to pros-

small molecule,

implicating an interaction ing site in uterine tissue. specific

and

1974)

tions, quantitative measurements of PGE1 cific binding in human myometrium, and

Angg#{176}ard (1966),

by and

were

monkey

Wyngarden,

the nature of specific high affinity prostaglandin binding in hamster myometrial low speed supernatant (Kimball and Wyngarden, 1975). In

in

mitters suggests that prostaglandins act on a specific uterine binding site or ‘receptor’ (Paton and

and

slices. The

Wiqvist,

vitro

stimulation,

studies

hu-

contrac-

exhibits

prostaglandin

lack

in

cause

both

(Wakeling

are effective

Bygdeman

muscle,

Uterine

vivo.

(PGs)

prostaglandins

smooth

et al., 1973),

(Wakeling

prostaglandins and abortifacient

the sites

National to: Dr. F. Company,

presence in

of

Human

hamster

Institutes

Myometrial

Human

PG Binding

uteri were

obtained from non-pregnant, pre-menopausal patient donors who had previously been scheduled for hysterectomy by their local physidan. After inspecting and obtaining representative tissue sections, the hospital pathologist released the specimens for assay purposes. None of the pre-surgery diagnoses was for gross tissue disease; this was confirmed by subsequent pathological examination of the tissue. By prescheduling with the pathologist, specimens could be processed from excision to placement

of

A. Kimball, Kalamazoo,

482

MYOMETRIAL

HUMAN

in iced

ug/ml were

saline-indomethacin

(0.9

indomethacin)

within

immediately

4#{176}C-room where saline-indomethacin.

percent

NaCI

PGE,

plus

10

Competition

mm). The samples

10-30

carried, on ice, to the laboratory they

were rinsed After removing

several times the serosa

in and

endometrium, the myometrium was minced, rinsed in S-P-I-Ca buffer (0.2 SM sucrose-O.02M phosphate0.5 mM CaCI2 buffer, pH 7.4 pIus 10 Mg/mI indomethacm), blotted and weighed. The tissue was homogenized in 5 volumes of buffer in a Waring blender with repeated, 1 5-sec power surges. The homogenate was filtered through 2 layers of cheesecloth and the filtrate, or in some cases the crude homogenate, centrifuged 15 mm at 3000g at 4#{176}C.Bead shaped frozen particles were formed by slowly pouring the supernatant fraction (S) into liquid nitrogen. The frozen particles were stored in closed containers at -80#{176}C until use. Equilibrium binding constants for PGE, were determined for each sample using methodology developed earlier (Kimball and Wyngarden, 1975). Aliquots (200 MI) of freshly thawed S were incubated in triplicate with 9 levels (0.05-2.5 pmole) of 3H-PGE, (New England Nuclear, Boston, Mass., 87 Ci/mM) or 3H-PGE, plus excess unlabelled PGE, in 200 isl S-P-I-Ca terminated (2.5 percent

buffer for 1 hr at 37#{176}C.Incubation was by adding 1.0 ml cold charcoal suspension Norit A in 0.1 percent gelatin-0.02M

phosphate water

for

buffer, 10

mm.

pH 7.4), and The

tubes

placing were

the

tubes

centrifuged

in ice 15 mm

at 4#{176}C and the supernatant quantitated for The binding in the presence of excess unlabelled PGE, was subtracted from the total 3I-I-PGE, bound and expressed as specific binding. Protein concentration of S was determined by the method of Lowry et al. (1931). The binding data were transformed and plotted according to Scatchard (1949). The dissociation constant, Kd, was calculated by dividing unity by the negative slope of the best straight line determined by linear regression analysis. Binding-site concentration, Bmax, was derived from the X-axis line intercept. at

2200g

tritium.

used:

3The following prostaglandin

semi-systematic names have (PG)E, = (lla,13E,15S)-11,15-di-

been

hydroxy-9-oxoprost-13-en-1-oic acid; PGE2 (5 Z,1 1 a,1 3E,1 SS)-1 1,15-dihydroxy-9-oxoprosta-5 ,13dien-1-oic

acid;

PGF20

=

(5Z,9a,lla,13E,15S)-

9,11-1 5-trihydroxyprosta-5,1 3-dien-1-oic acid; PGA, = (1 3E,1 5S)-1 5-hydroxy-9-oxoprosta-10,1 3-dien-1-oic acid;

PGA2

=

(SZ,

13E,15S)-15-hydroxy-9-oxoprosta-

5,10, 13-trien-1-oic acid; PGB, (13E,15S)-15-hydroxy-9-oxoprosta-8( 1 2),1 3-dien-1-oic acid; PGB3 (5Z,

1 3E,

1 5S)-1

5-hydroxy-9-oxoprosta-5,8(1

2), 13-

trien-1 -oic acid; (5Z,9o, 1 3E, 1 5S)-9,1 5-dihydroxy-1 oxoprostra-5, 1 3-dien-1-oic acid; 1 3,14-dihydro PGE, (5 Z,9cs, 1 3E,15S)-9,1 5-dihydroxy-1 1-oxoprosta-5,1 dien-1-oic (1 la,1 5S)-1 1 3,14-dihydro-1

acid; 13,14-dihydro 1,1 5-dihydroxy-9-oxoprostan-1-oic 5-keto PGE, =

1=

3-

PGE, acid;

(1 la)-1 1-hydroxy15-keto PGE, =

9,1 5-dioxoprostan-1-oic acid; (11 a, 1 3 E)-1 1 -hydroxy-9,15-dioxoprost-13-en-1-oic acid; (15S)-15-methyl PGE2 = (5Z,lla,13E,15S)11,15 -dihydroxy-1 5-methyl-9-oxoprosta-5,1 3-dien-1oic acid; (1 5S)-15-methyl PGF2 (5Z,9a,lla,13E,15S)-9,11,15-trihydroxy-15-methylprosta-5,13-dien-1-oic acid (Nelson, 1974).

a

SPECIFIC

BINDING

for

3H-PGE,

483

binding

sites

was

deter-

mined by incubating 200 p1 S in tubes containing 200 p1 3H-PGE, (2.3 ng/ml) in S-P-I-Ca buffer with or without added unlabelled test compound for 1 hr at 37#{176}C. The prostaglandin synthetase inhibitor, indomethacin, was tested in the absence of indomethacin in the buffer. Several prostaglandins3 were tested at 6 levels in a concentration range of 1.0-300.0 ng/ml. Non-prostaglandin compounds were tested at 5 levels at concentrations ranging from 0.001-10.0 pg/mI. All compounds were prepared and diluted with absolute ethanol and stored at -20#{176}C. The alcoholic addition comprised PGF2a>PGB2PGAi’PGA2>

PGB1>PGD2.

metabolites affinities 13,

status

Binding

the

amounts

are given in Table

PGE1

phase.

cycle.

analysis of these data and for these

to the

luteal

no clear grouping of these binding site concentration

was

PGE1

would

H-PGE1.

5

early

of the samples,

prostaglandins

4

dominated

ovulation

steroid levels it is not possible to

There

Fig.

3

estrogen

through

endocrine

increasing

M x lO

12-17).

the

Competition

2

samples

(day

two

menstrual

BOUND

constant

about

by

which

1

are

7, were collected early in the menstrual patient number 7 was reported as men-

samples

.01

type

Most

However,

struating.

.02

dissociation

at mid-cycle

includes

precise

donors.

not

the

collected

sample.

women

blood

and

cycle, as determined the binding site concen-

dominated

Without

in the

(data

diagnosis

respective

progesterone

.03

incubated

specimen

late follicular phase

.04

3H-

1, as well as the age of the

and

the

free

of unincu-

protein

each

(Bmax) for

incubation.

those

3H-PGE1

patient, day of menstrual by a pathologist, with (Kd)

protein

undergo

and

myometrial

in Table

tration

the

with

pre-surgery

10-9M.

not

membrane-bound

surgery

included

did

identically

x

concentration

pmole/mg

3H-PGE1

metabolism

Extracts

5

dissociation

± 0.38 site

0.02-0.0

The

Also

of the spereveals a high The

2.38

binding

1.

7 different myo-

on

was

PGE1

pre-

Fig.

in

protein.

preparations,

(Table

I

3H-PGE1

myometrial

determined

specific

ranged

1

specific

human

binding

(Kd),

metrial

0

is

PGE1

The

z

and the

is a Scatchard transformation binding. The plot

constant

3-

Binding

3H-PGE1

affinity

w

of

tested

cific

5

PGE1

non-specific,

binding

shown

6

of

Similar

results

are given in Table for the metabolites

14-dihydro-PGE1

for 2B.

the The

were:

>13,14-dihydro-15-keto-

PGE1

relative PGE1>

HUMAN

MYOMETRIAL

PGE1

SPECIFIC

BINDING

485

HUMAN MYDMETNIUM

r-.

-

u

-

c’. r-.

-

US

N. ID

Pal,.

.2

,, 40

14

.PGE,

a

X0

14

IOU 0001

as

.i/.,1I

tODD

r.UI’OE-N ©.

#{176}. #{176}

.

#{176}#{176} #{176}#{176} ©

FIG. 2. Competition of several unlabelled natural for 3H-PGE1 binding sites in human low speed supernatant.

#{176}.

prostaglandins myometrial

#{176}

-u V V

a

PGE1>15-keto-PGE1.

o

did

..

,,.,

*

,,

not

even

..

>‘

for

the

the highest

at

ng/ml).

o

PGD2

compete

Table

and

15-keto-PGE1

3H-PGE1

2 also

site

binding

concentration

tested

contains

an

(300

index

of

precision (X) for each of the compounds The constancy of this term indicates

9 C

,o

9

mogeneity

of the assay

assay

tested. the ho-

over the period

of the

-C

2

study. The

competition

PGE2

and

binding

C

indicated

that

had a higher methyl

V

that

-.

-

99 >

9 9 9 o22

0,

u

‘

,-

.

ojvuaavE ,-

a

‘.

2 2 2 .c .c

C .

.

9

.

CCCOO

9

.

-u