BIOLOGY OF REPRODUCTION 39, 1117-1128 (1988)

1117

Effects of Inhibition of Prostaglandin Synthesis on Uterine Oxytocin ReceptorConcentration and Myometrial Gap Junction Density in Parturient Rats’

W. Y. CHAN,2’3 IRENE BEREZIN,4 and E. E. DANIEL4

Department of Pharmacology3

Cornell University Medical College

New York, New York 10021

and

Department of Neurosciences4

McMaster University

Hamilton, Ontario

Canada L8N3Z5

ABSTRACT

The development of oxytocin (OT) sensitivity in the parturient uterus is associated with increases in myo-

metrial OT receptor concentration, gap junction formation, and prostaglandin (PG) production. To investigate

whether PGs mediate these responses, we measured OT responsiveness, OT receptor concentrations, and gap

junction formations in uteri of Day 19, 20, 21, 22, 23 pregnant and Day 2 postpartum rats. Inhibition of

endogenous PG synthesis was produced by infusion of naproxen sodium delivered by an implanted osmotic

pump. Naproxen treatment, but not placebo treatment, markedly attenuated in vitro uterine PGE2, PGF�,

and PGI2 releases, suppressed OT responsiveness, and prolonged gestation. The increase of OT receptor concen-

tration that normally occurred on Day 23 term pregnancy was delayed to Day 24. Co-administration of PGF,.�

reversed the suppressive effects of naproxen. Naproxen treatment did not significantly affect gap junction

formations on Day 23 but appeared to delay both the onset and disappearance of gap junction formations.

PGF,� co-administration with naproxen also had no apparent effect on gap junction development. The inhibition

of OT receptor formation but not gap junction formation on Day 23 in the presence of nap roxen indicates that

these two events are controlled independently. Furthermore, the failure of naproxen-treated rats to deliver at

term suggests that gap junction formation in the absence of an increase in OT receptors is insufficient to initiate

labor. It appears that increases in both OT receptor concentrations and gap junction densities may be required

for labor.

INTRODUCTION

During pregnancy until shortly before term, the

gravid uterus is relatively quiescent, with little spon-

taneous contractile activity and low responsiveness to

the hormone oxytocin (OT). However, just prior to

term labor, the parturient uterus becomes highly

active and responsive to OT. The abrupt alteration in

the contractile state of the myometrium is associated

with a marked increase in the myometrial concentra-

Accepted June 29, 1988.Received February 12, 1988.

‘Supported by NIH Grant HD-20839 and an NIH BRSG 507

RRO5 396 to W.Y.C.2 Reprint requests: Dr. W. V. Chan, Department of Pharmacology,

Cornell University Medical College, 1300 York Avenue, New York,

NY 10021.

non of OT receptors (Alexandrova and Soloff, 1980a;

Soloff, 1985) and the development of large numbers

of gap junctions between myometrial cells (Garfield

et al., 1980a; Verhoeff and Garfield, 1986). It is

believed that the developments of OT receptors and

gap junctions are crucial to the initiation and propaga-

tion of labor, and these two events are regulated by

the ovarian hormones, estrogen and progesterone

(Burghardt et al., 1984a,b, 1987; reviewed by Soloff,

1985 and Verhoeff and Garfield, 1986).

Prostaglandins (PGs) are also released in large

amounts by the parturient uterus (Aiken, 1972;

Williams et a!., 1974; Chan, 1977). There appears to

be a direct relationship between uterine PG produc-

tion and OT sensitivity (Roberts and McCracken,

1976; Dubin et al., 1979; Chan, 1983). It is plausible

that PGs mediate the effects of ovarian hormones on

uterine OT receptor formation and gap junction

1118 CHAN ET AL.

development. PGF2a -induced premature abortion in

rats also increased uterine OT receptor concentration

(Alexandrova and Soloff, 1980b). PGs have also been

shown to stimulate the development of gap junctions

in vitro in myometrial cells (Garfield et al., 1980a).

In previous studies, we have demonstrated that

inhibition of endogenous PG synthesis prevented the

development of OT sensitivity in the term pergnant

rat uterus (Chan, 1983, 1987b). This suppressive

effect might be the result of a poor or failed develop-

ment of OT receptors and/or gap junctions consequent

to inhibition of PG synthesis and release. There-

fore, in this investigation, we sought to delineate the

role of PGs in the developments of myometrial OT

receptors and gap junctions. We studied the effects of

inhibition of PG synthesis on the concentration of

OTreceptors and density of gap junctions in myometria

of late to term pregnant rats. We also monitored the

uterine responsiveness to OT to provide the functional

correlates to the biochemical and cellular measure-

ments. A preliminary report on the effects of PGs on

OT receptor formations has been presented (Chan,

1 987a,b).

MATERIALS AND METHODS

Female Wistar rats were used in this investigation.

Dated pregnant rats were purchased from Hilltop Lab

Animals (Scottsdale, PA). Rats were mated in the

morning on days as specified on our orders. In the

afternoon, they were examined for vaginal plugs.

Those found with plugs were identified, separated,recorded as Day 1 pregnant on that day and delivered

to our animal care facilities on Day 13 of pregnancy.

Rats were used for experiments on Days 19, 20, 21,

22, 23 (term pregnant) and Day 2 postpartum.

Inhibition of Endogenous PG Synthesis

Naproxen sodium was administered s.c. to suppress

PG synthesis; 2.0 mg/day was infused via an Alzet

mini-osmotic pump, model 2001 (AIza, Palo Alto,

CA), implanted s.c. under inhalation anesthesia with

2% isoflurane (Anaquest, Madison, WI). The pump

releases 1.0 p1/h and has a delivery capacity for 7

days. The pump was filled with an appropriate

concentration of naproxen sodium and implanted at

least 3 days prior to the experimental day. A priming

dose of 1.0 mg of naproxen sodium was injected s.c.

on completion of the pump implantation. Rats that

served as the placebo-treated group were given the

pump implant filled with .09% NaC1. Rats that served

as the control group received no pump implants.

A group of naproxen-treated rats were also given a

single s.c. injection of 0.5 mg PGF2� (tromethamine

salt) on Day 21 and Day 22 of pregnancy.

Determination of OT Sensitivity

Uterine contractile responses to OT were measured

in Day 22 pregnant rats. The rats were anesthetized

with urethane i.p. and prepared for in vivo uterine

contractile recording according to a method described

previously (Chan et al., 1974). Essentially, the

technique consists of recording an isometric contract

of a segment of the pregnant uterus in situ. Uterine

contractions, spontaneous or OT-induced, were

expressed as contractile activity by the area under the

contractile curve, measured by a compensating polar

planimeter, for the 10-mm interval immediately prior

to OT injection and the 10-mm interval immediately

following OT injection. The difference of the two

measurements was the OT-induced contractile activity.

OT, 30 mU and 60 mU, was administered via a

jugular venous catheter. In the majority of prepara-

tions, the two doses were repeated at least once in the

same animal. The interval between injections was 30

mm.

At the conclusion of the contractile response

measurement, one segment of the uterine horn was

removed for OT receptor binding assays. The remain-

ing segments were fixed for gap junction measurement

by electron microscopy. Previous work by others had

shown that acute OT injections for a short period of

1-2 h had no effect on OT receptor concentrations

(Riemer et al., 1986) or gap junction formations

(Garfield et al., 1978). This was confirmed in the

present study. For other gestational groups, uterine

tissues were removed under isoflurane anesthesia for

OT receptor and gap junction measurements without

exposure to exogenous OT.

OT Receptor Binding Assays

OT receptor binding assays were carried out on

crude plasma membrane fractions by the method

described by Soloff and Swartz (1974). Uterine

horns, after removal of fetal tissues, were homogenized

in 3 volumes of ice-cold 10 mM tris (hydroxymethyl)

aminomethane (Tris)-HC1 buffer, pH 7.4, containing

2 mM ethylene diaminetetraacetate (EDTA)-2Na,

and 0.5 mM dithiothreitol. The homogenate was

PG, OT RECEPTORS AND GAP JUNCTIONS 1119

centrifuged at 1000 X g for 20 mm at 4#{176}C.The

resultant supernatant was centrifuged at 100,000 X g

for 60 mm at 4#{176}C.The EDTA in the preparation

buffer was present to dissociate OT from its binding

sites and ensure the availability of all OT binding sites

for ligand-receptor binding in the binding assay

(Perimutter and Soloff, 1979). The pellet that

contained the plasma membrane fraction was washed

three times with assay buffer (50 mM Tris-maleate

buffer, pH 7.6, containing 10 mM MnCl2 and 0.1%

gelatin) to remove EDTA. This membrane-microsomal

fraction has been shown to be richer in OT binding

sites than the 10,000 X g and 20,000 X g frac-

tions. The washed pellet was then resuspended in

assay buffer to yield a protein concentration of

20-25 mg/ml, determined by the method of Lowry

et a!. (1951). The membrane-microsomal suspension

was stored in liquid nitrogen until used for receptor

binding assays.

For binding assays, the crude membrane prepara-

tion was diluted to 10 mg/ml. The radioligand-

receptor binding assay was carried out with 1.0

mg/ml of membrane protein, incubated with six

concentrations of [3HIOT, 0.5-10 nM, in a final

volume of 250 pl. The incubation was carried out at

22#{176}Cfor 60 mm and in the absence and presence of

excess unlabeled OT, 10 pM, to distinguish non-

specific and specific bindings. The incubation was

terminated by adding 5 ml of ice-cold gelatin-free

assay buffer. Free and bound [3H]OT was separated

by filtration with a Whatman GF/F glass microfiber

filter. The filter was rinsed twice with 1.0 ml of

ice-cold gelatin-free assay buffer. The filtration and

rinse were completed in 5 s. The filter was then dried

and the content of radioactivity was determined by

liquid scintillation spectrometry. All assays were

performed in duplicate.

The binding data were evaluated by Scatchard plot

analysis (Scatchard, 1949). A computer-assisted

ligand-binding data analysis program was used to

calculate the dissociation constant (Kd) and the

saturable binding sites (BM�).

Measurements of Gap Junctions by

Electron Microscopy

Electron microscopy. All uterine tissues were fixed

in situ by filling the peritoneal cavity and uterine

horns with the fixative (2% glutaraldehyde + 4.5%

sucrose + 1 mM CaCl in 0.075% M cacodylate buffer,

pH 7.4). After 5 mm initial fixation, uterine horns

were removed and pinned out in Petri dishes in the

�me fixative. After 20-30 mm of further fixation,

small longitudinal strips from the middle of each

uterine horn were cut and placed in vials with the

&me fixative for an additional 1.5 h. After fixation,

the pieces were washed overnight in cacodylate

buffer, containing 6% sucrose and 1.25 mM CaCl2,

pH 7.4 at 4#{176}C,postfixed in 2% #{176}s#{176}4(in 0.05 M

cacodylate buffer, pH 7.4) at room temperature

for 90 mm, stained en bloc with saturated uranyl

acetate for 60 mm, dehydrated in graded ethanol and

propylene oxide, and embedded in Spurr resin (Man-

vac, Halifax, Nova Scotia). Tissues were oriented in

molds to cut the longitudinal muscle layer in cross-

sections. Sections were cut on an Ultracut E ultramicro-

tome (Cambridge Reichert-Jung, Toronto, Ontario,

Canada), stained for 2 mm with lead citrate, and exam-

ined in a Phillips 301 electron microscope at 60 kv.Quantitative measurements. Cross-sections of longi-

tudinal muscle were examined under the scanning

mode of the microscope, and nonoverlapping areas of

grid squares covered by smooth muscle cell profiles

were photographed at 2830X magnification with

35-mm film. The lengths of smooth muscle plasma

membrane surveyed for each tissue were determined

on negatives by tracing, on a monitor, the images of

muscle profiles enlarged in a dissecting microscope.

From each tissue, 18 negatives were examined. To

estimate the lengths of smooth muscle membrane

on negatives, a BQ system IV (R&M Biometrics

Inc., Nashville, TN), in conjunction with standard

hardware (computer, digitizing board, monitor)

connected with a video camera attached to a dissecting

microscope, was used. The same negatives were used

to determine the number and length of gap junctions

for each tissue studied. Negatives were enlarged 10

times to 28,300X magnification by using a Durst

photoenlarger (Treck Hall, Toronto, Ontario, Canada),

and lengths of gap junctions were measured. Gap

junctions were identified as 5-7 lined structures. To

estimate the length of gap junction membrane for

each tissue, cross-sectioned lengths of each gap

junction were corrected (2X) because gap junctions

comprise components of two cells and, in determina-

tion of gap junction membrane length, both compo-

nents are considered.

For each tissue, the number of gap junctions per

1000 pm of membrane surveyed, the length of gap

junction in nm, and the gap junction density in

percentage of membrane surface were measured.

1120 CHAN ET AL.

Uterine PG Release and

Radioimmunoassays (RIAs) of PGs

Effectiveness of inhibition of PG biosynthesis was

verified in a separate group of placebo-treated and

naproxen-treated Day 22 pregnant rats by comparing

their rates of in vitro PG release from uterine horns

into the incubation medium.

The rats were killed by cervical dislocation. The

uterine horns of each rat were quickly removed and

placed in a beaker containing 100 ml Kreb’s-Ringer

bicarbonate solution, pH 7.4, at room temperature.

The uterine horns were opened and the fetuses and

placentae were removed and discarded. The open

horns were then transferred to 25 ml fresh Kreb’s-

Ringer solution and allowed to remain for 10 mm.

This period was the preincubation period. The uterine

tissue was then rinsed twice with 25 ml of Kreb’s-

Ringer solution. The three volumes of preincubation

incubate were pooled and extracted for PGs.

The uterine tissue was then incubated in 50 ml

Kreb’s-Ringer solution, with continuous aeration with

95% 02 and 5% CO2 at 37#{176}Cin a shaker bath for 30

mm. At the end of the incubation period, the uterine

tissue was removed and its wet weight was taken. The

incubate was extracted for PGs.

The preincubate and the incubate were acidified to

pH 4.0 with 10% formic acid and passed twice

through a Sep-Pak C18 cartridge (Waters, Milford,

MA). This was followed by a 5-ml water wash and a

2-mi hexane wash. The PGs were then eluted with S

ml of 80% ethanol. The alcoholic PG extract was

evaporated to dryness under a stream of N2 at

50#{176}C.The dry sample was stored at -20#{176}Cuntil used

for RIAs of PGs. The recoveries of PGE2, PGF2a,

6-keto-PGF1�, (stable metabolite of PGI2) and TXB2

(stable metabolite of TXA2) from this extraction

system were determined with tritiated isotopes. The

recovery ranged from 76% to 80%. Extraction loss

was corrected in each experiment.

RIAs were performed for PGE2, PGF2a, and

6-keto-PGF1a, in a phosphate-saline buffer system

containing 0.1% gelatin and with a dextran-charcoal

system for the separation of free and bound ligands.

Rabbit anti-PG sera were used. Cross-reactivities at

50% displacement of the standard curve were measured

in our assay system. For anti-PGE2, it was “�‘2% with

PGF2a and <1% with 6-keto-PGF1a and TXB2; for

anti-PGF2�, <0.5% with PGE2 and TXB2 and “.� 1.5%

with 6-keto-PGF1a; for anti-6-keto-PGF1�, “2% with

PGE2 and <0.5% with PGF2a and TXB2.

Each test sample was measured in duplicates and at

two different dilutions. The sensitivity of the assay

for PGE2 and PGF2a was 15 pg; for 6-keto-PGF1a, 25

pg. The intraassay coefficient of variation was <10%

and the interassay coefficient of variation was <15%.

Materials

The OT used was either synthetic OT (Peninsula

Labs., Belmont, CA) for receptor binding or Pitocin

(Parke-Davis, Morris Plains, NJ) for OT response

measurement. PGE2 and PGF2a (PGF2��THAM)

were generous gifts from Drs. Thomas J. Vecchio

and John Pike of the Upjohn Co., Midland, MI.

Naproxen sodium (Anaprox) was provided by Dr.

John Nestor of Syntex Research (Palo Alto, CA).

Rabbit anti-PGE2 and anti-PGF2a were purchased

from Advanced Magnetics (Cambridge, MA). 6-keto-

PGFIa RIA kit, multilabeled [3H]PGE2 and [3HJ

PGF2� (150-200 Ci/mmol) and [3H]OT (37-59

Ci/mmol) were purchased from New England Nuclear

(Boston, MA).

Statistical Analysis

All data were expressed as sample mean ± SEM and

analyzed by analysis of variance. Significant difference

between sample means were analyzed by Students

t-test, paired or unpaired. Differences were considered

significant if p<0.05.

RESULTS

Effects of Naproxen Treatment on Uterine

PG Release and Contractile Response to OT

Naproxen infusion at 2.0 mg/day delivered for 3

days by an osmotic pump implanted s.c. markedly

attenuated the in vitro release of PGs from isolated,

Day 22 pregnant uteri. Table 1 shows the results of

four experiments. Each experiment consisted of one

placebo-treated and one naproxen-treated Day 22

pregnant rat, forming a matched pair. The major

prostanoid released into the incubation medium was

prostacyclin (PGI2), measured as 6-keto-PGF1a.

PGF2�, the next in order, was one-half to one-third

that of 6-keto-PGF1a. PGE2 was the least amount

released. Naproxen treatment significantly reduced

the in vitro release of both PGF2a and 6-keto-PGF1a

during the 10-mm preincubation period and the 30-mm

incubation period. PGE2 was significantly reduced

only during the preincubation period.

21-Day Pregnant Uteri 23-Day Pregnant Uteri300

250

200

150

A

B

A

C

2 4 6 8

13H10’r Concentration, nM

C

2 4 6 8 10

(�I4]OT Concentration, nM

PG, OT RECEPTORS AND GAP JUNCTIONS

TABLE 1. Inhibition of in vitro prostaglandin (PG) release from Day 22 pregnant rat uteri by naproxen sodium.

1121

Treatmenta

In vitro PG releas e, ng/min/g tissue

Preincubation release Incubation release

PGE2 PGF20 6-Keto-PGF10 PGE2 PGF� 6-Keto-PGF1n

Placebo-control 0.26 ± 0.02 0.79 ± 0.16 1.46 ± 0.20 0.13 ± 0.01 0.54 ± 0.07 1.70 ± 0.24Naproxen�treatedb 0.17 ± 0.03* 0.19 ± 0.10� 0.22 ± 0.06’ 0.09 ± 0.02 0.23 ± 0.07 0.61 ± 0.12**

= 4 for each treatment group.

bNaproxen treatment: 2.0 mg/day for 3 days delivered by osmotic pump implanted s.c.

* Significantly different from control, p<0.05, paired t-test.

s*significantly different from control, p<O.Ol, paired t-test.

In vivo contractile responses to 30 mU and 60 mU

of OT were measured in 5 control, Day 22 pregnant

rats and 3 naproxen-treated, Day 22 pregnant rats. In

confirmation of our earlier studies (Chan, 1983,

1987b), inhibition of endogenous PG synthesis

reduced OT responsiveness. Naproxen sodium treat-

ment reduced the response to the low dose of OT by

61% and to the high dose by 31%.

[3H]OT Binding in Rat Uterine Membrane

[3H]OT bound to crude uterine membranes to a

linear, nonsaturating binding site (interpreted as non-

specific binding) and to a high affinity, low capacity

300

C-4C

250

0.

- 150‘OC

0

100I.0

- 50

0

B

10

200

binding site (saturable specific binding). Figure 1

shows the representative binding curves from Day 21

and Day 23 pregnant uterine membranes.

[3H]OT ligand binding assays were performed in

uterine membranes prepared from uteri of Day 19,

20, 21, 22, 23 pregnant and Day 2 postpartum rats.

Six rats were assigned to each gestation group and 2

to the postpartum group. Ligand binding affinity and

concentration of saturable binding sites were estimated

by Scatchard plot analysis. Table 2 summarizes the

binding data obtained. The number of high affinity

OT binding sites were low in Days 19-22 pregnant

uterine membranes. The OT receptor concentration

increased abruptly and markedly in Day 23 (term)

FIG. 1. [3 Hloxytocin (OT) binding to pregnant rat uterine membrane fractions. Each binding curve represents the average of ± SEM six binding

experiments. A, Total binding; B, specific (saturable) binding; C, nonspecific binding. Scatchard plot analysis of specific binding data indicated a

single binding site. For Day 21 pregnant uteri: BM� = 36 fmol/mg membrane protein and Kd = 1.3 nM. For Day 23 pregnant uteri: BM� = 226fmol/mg membrane protein and Kd = 1.0 nM.

pregnant uteri. Compared to Day 19, OT receptor

concentration increased by 10-fold, 27 vs. 290

fmol/mg membrane protein. OT binding affinity

showed no significant changes at term. Scatchard

plots were linear, indicating a single, high affinity

binding site.

1122 CHAN ET AL.

TABLE 2. Effects of prostaglandin (PG) on oxytocin (OT) receptor concentrations in pregnant rat uteri.a

Treatment group

Maximal binding of 1’ H]OT in fmol/mg membrane proteint)(with dissociation constant, Kd, in nM)

Day 19 Day 20 Day 21 Day 22 Day 23 Day 24

Day 2

Postpartum

Control 27.5 ± 6.9

(1.57 ± 0.04)31.9 ± 5.4

(1.90 ± 0.38)

43.4 ± 8.2 83.0 ± 19.9 290 ± 21

(2.50 ± 0.35) (1.89 ± 0.50) (1.00 ± 0.05)

- 24

(0.99)

Placebo 35.0 ± 4.3

(1.98 ± 0.40)

28.7 ± 4.7

(1.45 ± 0.52)

36.1 ± 5.8 67.7 ± 13.4 242 ± 23

(1.35 ± 0.29) (1.23 ± 0.21) (1.37 ± 0.25)

- 38

(1.70)

Naproxenc 28.6 ± 3.2

(1.58 ± 0.36)30.0 ± 5.3

(1.00 ± 0.16)

35.1 ± 4.0 51.6 ± 6.4 79.7 ± 17.4*

(1.12 ± 0.13) (1.47 ± 0.22) (1.00 ± 0.16)

214

(1.00

20

0.09)

54

(1.47)

Naproxen + pGF�c - - 355 ± 35

(1.03 ± 0.13)

aTerm pregnancy for control group and placebo-treated group was 23 days.

bValues shown are means ± SEM, n = 6 except for naproxen + PGF�, group. n = 3 and postpartum group, n = 2.

cNaproxen treatment, 2.0 mg/day for 3 days; PGF2�, 0.5 mg/day for 2 days.

Significantly different from corresponding control and placebo groups, p<O.Ol. Difference in binding affinity, Kd, between groups and gestational

periods not statistically significant.

Effects of Inhibition of PG Synthesis on

Uterine OT Receptor Concentration

The effects of inhibition of endogenous PG synthesis

on the formation of uterine OT receptors were

studied in Day 19 to Day 23 pregnant rats. Six rats

were assigned to each gestational group. The treated

rats were given naproxen sodium, 2.0 mg/day, for 3

days via an osmotic pump implanted s.c. The placebo-

treated rats received pumps filled with saline. The

results are shown in Table 2. The placebo-treated

group yielded a similar pattern of OT receptor forma-

tion to the control group. There was an abrupt and

marked increase in OT receptor concentration at term

(Day 23). The increase in OT receptor concentration

subsided rapidly postpartum (2 rats). The dynamic

changes in OT receptor concentrations observed were

essentially the same as those originally reported by

Soloff (Soloff et al., 1979; Alexandrova and Soloff,

1 980a).

Inhibition of PG synthesis by naproxen prevented

the increase of OT receptor concentration on Day 23.

The difference, 79.7 vs. 242 fmol/mg membrane

protein of the placebo-treated group, was significant,

p<0.Oi. Inhibition of PG synthesis, however, did not

abolish but only delayed the increase of OT receptor

concentration to Day 24. Term pregnancy for the

control was Day 23. The naproxen-treated group did

not deliver on the morning of Day 24 when the rats

were killed for the experiment.

In 4 naproxen-treated rats, 0.5 mg PGF2a was

injected s.c. daily beginning on Day 21. One rat

delivered on the evening of Day 22. [3H]OT binding

assays on Day 23 membranes of the remaining 3

rats showed that PGF2� co-administration not only

overcame the inhibitory effect of naproxen on OT

receptor formation but in fact stimulated OTreceptor

formation (Table 2).

Effects of Inhibition of PG Synthesis on

Development of Myometrial Cell Gap Junctions

Myometnial cells were surveyed for gap junctions.

For each sample, 2000-3000pm of plasma membrane

were surveyed. Tables 3 and 4 show the gap junction

frequency and density in control, placebo-treated,

naproxen-treated, and naproxen-treated with PGF2a

co-administration animals at different gestational

ages.

Gap junctions developed in the normal pattern in

myometria of placebo-treated and control animals,

i.e. they appeared in very small numbers prior to Day

22, rapidly increased in number and reached a maxi-

mum on Day 23 (term), and disappeared afterwards

within 48 h. Naproxen treatment did not markedly

affect gap junction formations and a similar maximum

was reached on Day 23. The onset of gap junction

PG, OT RECEPTORS AND GAP JUNCTIONS 1123

TABLE 3. Frequency of gap junctions in pregnant rat myometrium at different gestational ages and effects of suppression of prostaglandin (PG)synthesis on gap junction formations.a

Treatment

Number of g ap junctions/i 000 �i m membraneb

Day 19 Day 20 Day 21 Day 22 Day 23 Day 24

Day 2Postpartum

Control 0

(1)

0.85

(1)

1.38

(2)

2.48 ± 0.60

(4)

3.54 ± 0.67*

(5)

Placebo 0

(2)

0.42 ± 0.05

(3)

0.54 ± 0.16

(3)

1.23 ± 0.51

(3)

3.17

(2)

- 0

(2)

Naproxen 0

(1)

0.67 ± 0.12

(3)

0.81

(2)

0.77 ± O.i4�

(4)

3.20 ± 0.97”

(4)

6.40

(2)

0

(1)

Naproxen+PGF20 - - - - 3.12±0.95*8

(3)

aTerm pregnancy for control and placebo-treated animals was Day 23.

bValues shown are means ± SEM when available; numbers in parentheses give the number of animals studied in each gestation group; 2000 3000

�im of plasma membrane was surveyed for gap junctions in the myometrium of each animal studied.

* Significantly different from Day 22 values for placebo and naproxen group (p<0.O5), but not Day 22 controls.

**significantly different from Day 22 values for placebo group (p<0.05) and naproxen group (p<0.0i).

�Significantly different from Day 22 values for controls (p<0.05), but not placebo group.

formations in naproxen-treated rats appeared to have

been delayed. Day 22 gap junction values for naproxen-

treated rats were lower than those for control rats

and placebo-treated rats but were statistically signifi-

cant only against controls (p<0.05). Naproxen-treated

rats that had not delivered on Day 23 when studied

on Day 24 had the highest gap junction density. As

in control rats, gap junctions disappeared rapidly

postpartum.

Rats that received naproxen and PGF2� together

likewise had a normal complement of gap junctions

on Day 23. Figure 2 shows an electron micrograph of

gap junctions in myometrial cells of a Day 23 pregnant,

naproxen-treated rat.

Correlation between 0 T Receptor Concentration

and Frequency of Gap Junctions in Myometria

In this experimental protocol, OT receptor concen-

trations and gap junction developments were deter-

TABLE 4. Gap junction density in pregnant rat myometrium at different gestational ages and effects of suppression of prostaglandin (PG) synthesis

on gap junction formations.a

Gap junction memb rane/Non-gap junctio n membrane (%)b

Day 2

Treatment Day 19 Day 20 Day 21 Day 22 Day 23 Day 24 Postpartum

Control 0

(1)

0.001

(1)

0.006

(2)

0.101 ± 0.034

(4)

0.261 ± 0.051’

(5)

Placebo 0

(2)

0.011 ± 0.001

(3)

0.011 ± 0.003

(3)

0.042 ± 0.014

(3)

0.180

(2)

0

(2)

Naproxen 0

(1)

0.016 ± 0.002

(3)

0.02 1

(2)

0.019 ± 0.003�

(4)

0.236 ± 0.097’

(4)

0.365

(2)

0

(1)

Naproxen+PGF2a 0.212±0.071’

(3)

aTerm pregnancy for control and placebo-treated animals was Day 23.

bValues shown are means ± SEM when available; numbers in parentheses give the number of animals studied in each gestational group; 2000 3000

�im of plasma membrane was surveyed for gap junctions in the myometrium of each animal studied.

8Significantly different from Day 22 values between groups and within group (p<0.O5).

�SignificantIy different from Day 22 value for controls (p<O.O5), but not placebo group.

1124 CHAN ET AL.

FIG. 2. Electron micrograph of a cross-section through the longitudinal muscle layer of myometrium from naproxen-treated rat, fixed on Day 23

of gestation, showing gap junctions (arrows) between smooth muscle cells (X 34,230).

mined in myometrial specimens from the same

animal. This allowed direct correlation of OT receptor

and gap junction formations. Although both OTreceptor concentration and gap junction frequency

increased abruptly at term pregnancy (Tables 2 and

3), the correlation between OT receptor concentra-

tions and gap junction frequencies was poor. Figure 3

shows the correlation analysis. The linear regression

coefficient of determination, r2, was 0.303, and a

correlation coefficient, r, was 0.55 1.

DISCUSSION

In this study, we have investigated the role of PGs

(cyclooxygenase products) in the development of

uterine OT receptors and myometnial gap junctions in

the partunient rat from Day 19 to Day 23. However,

we had not measured OT receptors and gap junctions

in rats during labor when both of these two parameters

are known to be highest. Term pregnancy for the

Wistar rat used in our experiments was 23 days.

Occasionally, delivery occurred prior to 0900 h of

Day 23. In the experiments here, all uterine tissues,

except some Day 22 tissues in which OT responsiveness

was measured, were removed between 1000 and 1200

h on the designated day.

Endogenous uterine PG synthesis was inhibited

by the administration of naproxen sodium, an inhibi-

tor of PG cyclooxygenase (via an osmotic pump

implanted s.c.) as shown by the suppression of in

vitro PG release by the treated Day 22 pregnant uteri

during the preincubation period and after incubation.

PGs are not stored in cells but are synthesized and

released de novo. In in vitro studies, large amounts of

PGs are released during the preincubation phase-

probably caused by mechanical agitation or injury to

the tissue. Although this preincubation release of

PGs is nonspecifically stimulated, it is of interest

because it is indicative of the intrinsic potential of

the tissue for synthesizing PGs. In the experiment

S

.

.

3.0

r2 = 0.303

S

#{149} S

S

S

S

S.

300

PG, OT RECEPTORS AND GAP JUNCTIONS 1125

6.0

C‘a44

.0C‘az

000‘-4

CnC0

.4.’oC

‘-3

0�

44

I

4.5

100 200

S

OT Receptor Conc., frnol/mg protein

FIG. 3. Correlation analysis between oxytocin (OT) receptor con-

centrations and gap junction frequencies in pregnant rat uteri. Each

point represents one rat. Day 21-23 pregnant uteri, n = 19. Linear

regression analysis, r2 = coefficient of determination. The correlation

coefficient was 0.5 51.

here, it also more accurately reflects the inhibitory

action of naproxen sodium (given in vivo) on the

cyclooxygenase since there is less wash-out of the

drug compared to the incubation phase.

The major prostanoid released by the pregnant

uterus was PG!2 (measured as its stable metabolite,

6-keto-PGF1a). PGF2�, the next in order of magnitude,

was only one-half to one-third that of 6-keto-PGF1�.

Others also have found 6-keto-PGF1a as the major

PG of the pregnant or pseudopregnant rat uterus

(Fenwick et a!., 1977; Williams et al., 1978; Dubin et

al., 1982). The relationship between PG!2 production

and uterine contractility is not understood, since

PGI2 has variable effects on uterine contractions

(Omini et a!., 1979; Williams et al., 1979). Naproxen

treatment attenuated the in vitro release of PGI2,

PGF20., and PGE2. The greatest effect was seen in

PG!2 release. Inhibition of PG synthesis markedly

reduced the development of OT sensitivity in the

parturient uterus, as we had previously demonstrated

(Chan, 1983, 1987b).

The developments of UT receptors and gap junc-

tions were affected differently by the inhibition of

PG synthesis with naproxen. The control group and

the placebo-treated group showed a normal develop-

ment of uterine UT receptors, as originally described

by Soloff et al. (1979). There was an abrupt and

marked increase in uterine UT receptor concentra-

tions at term (Day 23). This was not seen in the

naproxen-treated group. Compared to the BMaX

of 290 ± 21 and 242 ± 23 fmol/mg membrane protein

of the Day 23 control and placebo-treated group,

respectively, the naproxen-treated group had a

BMax of only 79.7 ± 17.4 fmol/mg membrane protein.

Naproxen treatment, however, did not prevent but

only delayed the increase of UT receptors. Gestational

length was prolonged in the naproxen-treated rat.

It is worthy to note that luteinizing hormone-releasing

hormone-induced delay of parturition was also

associated with a low UT receptor concentration

(Bercu et al., 1980). Term pregnancy for the control

and the placebo-treated groups was 23 days, but the

naproxen-treated group had not delivered on the

morning of Day 24. The BMax for naproxen-treated

rats when measured on Day 24 was not significantly

different from those of term control or term placebo-

treated rats on Day 23. It cannot be excluded from

the findings presented here that the suppressive

effect of naproxen on UT receptor formation may be

mediated by mechanism(s) unrelated to its PG

synthesis inhibitory action. However, the reversal of

naproxen’s suppressive effect by PGF2a co-administra-

tion strongly supports a PG-mediated mechanism.

PGF2a co-administration not only reversed the ef-

fect of naproxen but, in fact, increased the UT

receptor concentration in the Day 23 pregnant uterus

compared to the uteri from control and placebo-

treated groups. One explanation for this could be

related to the fact that PGF2a-treated rats were

in an early stage of labor due to PGF2a stimulation

when killed on Day 23 for the experiment. It is

of interest to note that although PGI2 was the major

prostanoid produced by the parturient uterus and

was the most markedly inhibited by naproxen treat-

ment, PGF2a alone was sufficient to reverse the

naproxen’s suppressive effect on OT receptor forma-

tion. It has been shown that PGF2a-induced preterm

delivery was also associated with an increase in myo-

metnial OT receptor concentrations (Alexandrova and

Soloff, 1980b). It therefore appears that PGF2a

plays a functional role in the increase in uterine UT

receptor concentrations that normally occur at par-

turition.The development of gap junctions, unlike UT

receptor formation, was not delayed by naproxen

treatment. Gap junctions are normally absent in

1126 CHAN ET AL.

myometrial cells during early pregnancy but they

increase rapidly 1-2 days prior to term and reach

a maximum at term (Garfield et a!., 1978, 1980a).

It should be noted that Day 22 of these earlier studies

corresponds to Day 23 in the present study owing to

a difference in the procedure for timing the onset of

pregnancy. The animals in all three groups in this

study-the control, placebo-treated, and naproxen-

treated groups-developed the normal complement

of gap junctions on Day 23 (term). PGF2a co-

administration with naproxen likewise had no appar-

ent effect on normal gap junction development. Al-

though a normal amount of gap junction membrane

was present on Day 23 in naproxen-treated rats,

there appeared to be a delay in the onset of gap junc-

tion formation. The onset of gap junction formation

began on Day 22 in control rats and placebo-treated

rats, but none was apparent in naproxen-treated rats.

However, the difference between naproxen-treated

rats and placebo-treated rats was not statistically

significant. Inhibition of PG synthesis with naproxen

delayed delivery as well as disappearance of gap

junctions. Gap junction densities in undelivered rats

on Day 24 were higher than on Day 23, suggesting

that the synthesis of gap junctions was extended to

Day 24. There is evidence that the disappearance

of gap junctions may be triggered by events con-

nected with delivery rather than with ovarian hor-

mone levels (Berezin et a!., 1982). The results of the

present study are consistent with that suggestion and

further indicate that neither withdrawal of PGs nor

addition of PGF2a triggers gap junction degradation.

The present study failed to delineate a clear func-

tion of PGs in myometrial gap junction develop-

ment. Previous studies with indomethacin, another

cyclooxygenase inhibitor, have shown that in vitro

treatment attenuated whereas in vivo treatment

enhanced the estrogen stimulation of gap junctions

(Garfield et a!., 1980a,b; MacKenzie and Garfield,

1985). It is probable that estrogen and progesterone

are the primary regulators of gap junction develop-

ment (Garfield et a!., 1978, 1980a; MacKenzie and

Garfield, 1985) and PGs modulate or mediate this

hormonal control. This same hormonal control mech-

anism has also been proposed for UT receptor forma-

tions (Roberts et a!., 1976; Alexandrova and Soloff,

1980a,b; Soloff, 1985). However, the different effects

of naproxen treatment on UT receptor and gap junc-

tion formations observed in this study indicate that

the two events are regulated independently at least

temporally. It should be noted that in the present

experiment, UT binding sites and gap junction

densities were determined in myometrial specimens

from the same uterus. This is the first demonstra-

tion of a dissociation or a different assembly time

for UT receptor and gap junction formations. The

correlation coefficient of the two parameters yielded

a low value of 0.551 and an r2 of 0.303. But, it should

be pointed out that we measured UT receptors in

membrane fractions prepared from whole uterine

tissue whereas gap junctions were measured in longi-

tudinal muscle fibers. It is possible that UT receptor

formation may differ in time course between longi-

tudinal and circular muscle and decidual tissues.

Therefore, gap junction densities in longitudinal

muscles may not correlate to total OT receptor

concentrations.

There is no evidence that gap junction formations

differ between the two muscle layers (Garfield et

a!., 1978, 1982). UT receptors in myometria and

endometria/decidua also appear to undergo similar

changes during parturition (Fuchs et a!., 1984;

Riemer et a!., 1986). However, in vitro contractile

studies have shown that longitudinal and circular

myometrial fibers respond differently to UT and

suggest that the two muscle layers have different UT

receptor populations (Crankshaw, 1987; Tuross et a!.,

1987). Although Crankshaw (1987) concluded that

only the response of the circular muscle was compati-

ble with the marked increase in UT binding site

number of the parturient uterus, Tuross et a!. (1987)

found that the responses of both the circular and

longitudinal muscles were compatible with the UT

receptor hypothesis. Furthermore, UT responsiveness

of the whole uterus has been shown to exhibit a high

degree of correlation with total UT receptor concen-

trations (Fuchs et a!., 1983; Riemer et al., 1986).

We are of the opinion that our data are highly sugges-

tive that the formations of myometrial UT receptors

and gap junctions proceed independently and can be

regulated separately.

There is now cumulating evidence implicating a

physiological role for PGs, UT receptors, and gap

junctions in the initiation of labor. Our observations

here are in accord with this view. It appears that

PGF2� stimulates OT receptor formation. The in-

creased OT receptor concentration may also lead to

enhanced uterine PG production (Roberts et a!.,

1976; Chan, 1977, 1983). Gap junctions provide the

necessary network for propagation of electrical activity

PG, OT RECEPTORS AND GAP JUNCTIONS 1127

and synchronized contractions (Garfield et a!., 1978,

1982; Cole et a!., 1985). Our finding that parturition

failed to occur in naproxen-treated rats on Day 23,

despite the presence of gap junctions in normal

numbers and densities, indicates that gap junction

formation alone in the absence of an increase in UT

receptors is not sufficient for initiation of labor. It is

possible that the gap junctions formed after naproxen

treatment may not be functional for some unknown

reason. There is evidence that gap junction perme-

ability may be subjected to physiological regulation

(Cole and Garfield, 1986). It appears that increases in

both UT receptor concentrations and gap junction

densities may be required for labor

ACKNOW LEDGMENTS

The authors wish to acknowledge the excellent technical assistance

of Ms. Pearl Chua-Eoan, Mrs. Lisette Soo-Kyung Kang, and Ms. Lore A.

von Hoffen.

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