cyclic adenosine 3,5-monophosphate analogues modulate ratp

BIOLOGY OF REPRODUCTION 40,435-447 (1989)

435

Cyclic Adenosine 3,5-Monophosphate Analogues Modulate Rat P$acental

Cell Growth and Differentiation1

MICHAEL J. SOARES,2’3 MAMATA DE,3 CATHERINE S. PINAL,3

and JOAN S. HUNT4

Departments of Physiology3 and Pathology4

Ralph L. Smith Mental Retardation Research Center

University of Kansas Medical Center

Kansas City, Kansas 66103

ABSTRACT

Cyclic adenosine 3’,S’-monophosphate (cAMP) has been implicated in the control of placental function. The

present investigation was designed to evaluate the actions of cAMP analogues on the control of rat placental

development. Two model systems were used to assess the actions of cAMP in the placenta: 1) a rat placental cell

line and 2) rat labyrinth placental explants. Elevation of intracellular cAMP via treatment with cAMP analogues,

3-isobutyl-J -methylxanthine, forskolin, or cholera toxin inhibited placental cell DNA synthesis whereas treat-

ment with an analogue to cyclic guanosine 3’,5’-monophosphate was without effect. The inhibitory actions

of dibutyryl cAMP on DNA synthesis were at least partially reversible and were not the result of metabolic

toxicity. Dibutyryl cAMP had dramatic effects on the organization and morphology of placental cells growing

in vitro and diminished the ability of the placental cells to grow following transplantation into allogeneic hosts.

Dijferentiatio n-associated characteristics of rat placental cells were also affected by cAMP, cAMP analogues

stimulated placental cell progesterone release and inhibited placental cell alkaline phosphatase activity. Di-

butyryl cAMP had effects on placental labyrinth explants similar to its effects on the placental cell line. Di-

butyryl cAMP inhibited explant outgrowth while stimulating explant release of progesterone. In summary,

cAMP effectively modulates the growth and differentiation of rat placental cells in vitro.

INTRODUCTION

Growth and differentiation of the rat chorioallan-

toic placenta are poorly understood processes,

undoubtedly involving a variety of regulatory factors

acting on a number of different cell types. The

specific intercellular modulators have not been iden-

tified; however, some progress has been made towards

identifying intracellular mediators. Recent investiga-

tions with the human placenta have implicated cyclic

adenosine 3’,5’-monophosphate (cAMP) as an intra-

cellular regulator of placental function. Analogues of

cAMP stimulate human placental cell production of

Accepted September 29, 1988.

Received August 2, 1988.

‘Supported by grants from the National Institutes of Child Health

and Human Development, HD-20676 and HD-22208, the Flossie WestMemorial Trust, and a Mental Retardation Research Center grant from

the National Institutes of Health. C.S.P. is supported by a fellowship

from the University of Kansas Medical Center Minority Access to

Research Careers Summer Training Program.2 Reprint requests.

steroid and peptide hormones (Hussa, 1980; Caritis

et al., 1983; Zeitler et al., 1983; Tonkowicz and

Poisner, 1985; Feinman et al., 1986; Harman et a!.,

1987; Petraglia et al., 1987a,b,c) and are involved in

modulating the deposition of extracellular matrix by

human placental cells (Queenan et aL, 1987; Ulloa-

Aquirre et al., 1987). These observations have been

derived from term human placentas, whereas the

actions of cAMP in the developing placenta are un-

certain. In mice, cAMP has been shown to be in-

volved in early mouse embryonic development. Both

blastocoel formation (Manejwala et al., 1986) and

embryo growth (Skreb and Hofman, 1977) are stimu-

lated by intracellular elevation of cAMP. However, a

role for cAMP in the development of the rat chorio-

allantoic placenta has not yet been demonstrated.

The present investigation was designed to assess the

actions of cAMP on rat placental cell growth and dif-

ferentiation. Two model systems were used to assess

the actions of cAMP in the placenta; a rat placental

cell line generated from midgestation placental pri-

436 SOARES ET AL.

mordia (Soares et al., 1987; Hunt et al., 1988; Hunt

and Soares, 1988) that responds to growth regulators

(De et al., 1988), and explants of midgestation rat

placental primordia (Soares and Glasser, 1987). The

results indicate that cAMP has a profound effect on

the morphogenesis of the rat chorioallantoic placenta.

Animals

MATERIALS AND METHODS

Holtzman rats were obtained from the Holtzman

Company (Madison, WI). The animals were housed in

an environmentally controlled facility with lights on

from 0600 to 2000 h and allowed free access to food

and water. Timed pregnancies were obtained by

housing female rats with male rats and examining

vaginal smears daily during the cohabitation. Success-

ful matings were confirmed by the presence of a

copulatory plug and/or the presence of sperma-

tozoa in the vaginal smear (designated as Day 0 of

gestation).

Cells

The cell line used in this study was derived from

chorioallantoic placentas of the Holtzman rat (Soares

et al., 1987) and is designated HRP. The cells were

maintained in RPMI-1 640 culture medium (Hazleton/

KC, Lenexa, KS) supplemented with 5-10% heat-

inactivated fetal bovine serum (FBS; Hazleton/KC),

50 MM 13-mercaptoethanol (BIORAD, Richmond,

CA), 1 mM sodium pyruvate (Sigma Chemical Com-

pany, St. Louis, MO), 100 units/ml of penicillin, and

100 pg/ml of streptomycin (Hazleton/KC).

cAMP and DNA Synthesis

HRP cells (2 X l0�) were plated in 15.5-mm-

diameter wells in culture medium supplemented with

5% FBS. The cells were allowed to attach to the

dishes overnight and the medium was replaced with

serum-free culture medium the following day. On the

third day of the assay, the medium was replaced with

fresh serum-free culture medium containing the test

substance. After 20 h of incubation, 1 pCi of 3H-

thymidine was added to the cultures. After a 4-h

incorporation period, the culture medium was re-

moved from the cells. The cells were washed twice

with phosphate-buffered saline (PBS, 10 mM sodium

phosphate, 150 mM sodium chloride, pH 7.2), twice

with 10% trichloroacetic acid (TCA), and once with

ethanol:ether (3:1). The cellular residues were solu-

bilized in 1 ml of 0.2 N sodium hydroxide. After a

15-mm incubation, 0.75 ml of the solubiized residue

was transferred to a scintillation vial and 100 i1 of

glacial acetic acid and 5 ml of counting cocktail

(Scinti-Verse, Fisher Scientific, St. Louis, MO) were

added to each vial. The radioactivity present in each

sample was then estimated with a Packard liquid

scintillation counter. The compounds examined in

these experimens included: N6 -2’ -O-dibutyryl cyclic

adenosine 3 ‘,5 ‘-monophosphate (dibutyryl cAMP),

N6 -2’ -0-diburyryl cyclic guanosine 3’, 5’-monophos-

phate (dibutyryl cGMP), 8-bromo cyclic adenosine

3’,5’-monophosphate (8 bromo cAMP), forskolin,

cholera toxin, pertussis toxin, and 3-isobutyl-1-

methylxanthine (MIX). All test reagents were ob-

tained from Sigma Chemical Company. The effect of

the test reagents on DNA synthesis were examined in

serum-containing medium (5% FBS), in serum-free

medium, and in serum-free medium supplemented

with rat transferrin (Pel-Freez, Rogers, AR; 2.5-5

j.zg/ml). We have previously demonstrated that trans-ferrin stimulates HRP cell DNA synthesis (De et aL,

1988).

In additional experiments, the time course of the

effects of dibutyryl cAMP were examined. The

experimental protocol used for these studies was

identical to that presented above, except for the dura-

tion of the cAMP treament (see Results section for

further information).

HRP cells (1 X 10�) also were plated in Lab-Tek

chamber slides (Miles Laboratories, Naperville, IL)

and used for autoradiographic analysis of DNA

synthesis. After overnight attachment in serum-

containing medium, the medium was replaced with

serum-free culture medium. After a 24-h incubation,

the medium was replaced with fresh, serum-free

culture medium containing 5 pg/ml of rat transferrin

or fresh, serum-free culture medium containing 5

pg/mi of rat transferrin + 1 mM dibutyryl cAMP.

The cells were incubated for 36 h; 3H-thymidine was

added at a concentration of 1 pCi/mi, and the sample

was incubated for 4 more hours. The slides were re-

moved from the Lab-Tek chambers, washed with PBS,

and dipped in photographic emulsion (Kodak,

Rochester, NY). The slides were developed after 3-5

days of exposure and counterstained with Toluidine

Blue. A coverslip was mounted on the slides, which

were then analyzed by light microscopy.

CYCLIC AMP AND PLACENTAL CELLS 437

Modulation of Placental Cell cAMP Accumulation

The accumulation of cAMP in medium conditioned

by HRP cells treated with forskolin, cholera toxin, or

pertussis toxin was measured with a radioimmuno-

assay (RIA) kit obtained from Biomedical Technolo-

gies, Inc. (Stoughton, MA). Culture medium condi-

tioned by the treated cells was measured without

extraction. Samples were acetylated and processed

according to the manufacturer’s recommendations.

The sensitivity of the assay was 0.005 pmoles per

tube and the assay shows limited cross-reactivity with

other related nucleotides (see technical information

provided by Biomedical Technologies, Inc.). All sam-

ples were measured in the same assay in which the

intraassay variation was less than 5.0%.

Effect of Dibutyryl cAMP

on Placental Cell Morphology

The pattern of HRP cell growth following ex-

posure to dibutyryl cAMP (1.0 mM) or to control

conditions was evaluated at 2-day intervals over an

8-day culture period. Cells were grown in medium

containing 5% FBS with or without the treatment.

The cells were plated in 15.5-mm-diameter wells

(2 X io� cells/well). At the termination of each

culture, the cell layers were stained with crystal

violet and photographed. Some cells were also grown

on Lab-Tek chamber slides similarly treated and then

were fixed in 10% phosphate-buffered formalin,

stained with hematoxylin and eosin, and photo-

graphed, or were fixed in 2% glutaraldehyde, post-

fixed with 1% osmium tetroxide, and prepared for

electron microscopic analysis as previously described

(Hunt et al., 1988). Sections were stained .with 35%

uranyl acetate and a lead citrate solution prior to

observation using a Zeiss transmission electron

microscope.


on Placental Cell Transplantability

HRP cells exposed to dibutyryl cAMP (0.5 mM)

and MIX (0.1 mM) or to control conditions were used

to test the growth response of placental cells follow-

ing transplantation. The cells were grown for 5 days

in medium containing 10% FBS with or without the

treatment. Control and treated HRP cells were in-

jected i.p. into male Holtzman rats (5 X 10� cells!

rat; n=6 for each treatment). Animals were monitored

daily for 4 wk, then were killed and autopsied. Pla-

cental cell growth following transplantation was

assessed as previously described (Soares et al., 1987).

Transplanted tissues were fixed in 10% phosphate-

buffered formalin (pH 7.2) and prepared either for

routine histological staining with hematoxylin and

eosin or for immunohistochemical staining for

laminin using an avidin-biotin immunoperoxidase kit

for rabbit immunoglobulin G (IgG) (Vectastain ABC,

Vector Laboratories, Burlingame, CA). A rabbit anti-

serum to rat laminin was used to determine the dis-

tribution of laminin in the placental cell transplants

(Soares et al., 1988).

Effect of Dibutyryl cAMP on Placental Cell

Progesterone and Placental Lactogen Release

HRP cells exposed to dibutyryl cAMP or to control

conditions were used to evaluate progesterone and

placental lactogen production. Cells (2 x 10�) were

plated in 15.5-mm-diameter wells in culture medium

supplemented with 5% FBS. Some of the wells were

exposed to dibutyryl cAMP (1 mM). On Days 2, 4, 6,

and 8 of culture, cells were incubated with 25-

hydroxycholesterol (5 pg/mi; Steraloids, Inc., Wilton,

NH) for 24 h; then, the conditioned medium was

collected and assayed for progesterone by RIA

(Soares et al., 1985) and placental lactogen by

radioreceptor assay (Shiu et al., 1973; Soares, 1987).

The measurement of placental lactogen utilizes a lac-

togen radioreceptor assay that does not discriminate

among molecules that specifically interact with lacto-

gen receptors (Shiu et al., 1973). Thus, both placental

lactogen-I and placental lactogen-Il would be active

in this assay. At the termination of the cultures, the

cell layers were stained with crystal violet to deter-

mine cell density (Glues et al., 1986). After removal

of the culture medium, crystal violet solution (300

p1/well; 5% formalin, 50% ethanol, 0.15 M NaCl,

0.5% crystal violet [Fisher Scientific]) was added,

incubated for 10 mm, centrifuged, decanted, and

rinsed with tap water. Culture dishes with the stained

cell layers were inverted and dried overnight; then,

the dye was eluted with 1 ml of ethylene glycol with

constant agitation for 15 mm. Solutions were trans-

ferred to cuvettes and monitored at 520 nm with a

spectrophotometer. A standard cuve of different cell

numbers was generated, processed as described above,

and used to determine cell densities in the cultures.

438 SOARES ET AL.

cAMP and Placental Cell Expression

of Alkaline Phosphatase

HRP cells were exposed to various concentrations

of dibutyryi cAMP, 8-bromo cAMP, or to control

conditions and then were evaluated for alkaline phos-

phatase expression. Cells (5 X 10�) were plated in

6.4-mm-diameter wells in culture medium supple-

meted with 5% FBS and with or without the cAMP

analogues. After a 48-h exposure to the treatments,

the culture medium was removed and replaced with

alkaline phosphatase substrate (200 p1; 100 mM tris

(hydroxymethyl) aminomethane [Tris] -HC1 [pH

9.5], 100 mM NaC1, 5 mM MgCl2, 8 mM disodium

p-nitrophenyl phosphate [Sigma]). The cells were

then incubated for 30 mm at room temperature. The

reaction was terminated by addition of 2 N NaOH

(50 p1). Absorbance was monitored with a Multiskan

microplate reader (Flow Laboratories, McLean, VA)

at 405 nm. Cell density was determined in duplicate

cultures by the crystal violet method described above.


on Placental Cell Protein Synthesis

HRP cells exposed to dibutyryl cAMP (1 mM) or

to control conditions were evaluated for their ability

to incorporate 35S-methionine into protein. Cells

(2 x 10�) were plated in 15.5-mm-diameter wells in

culture medium containing 5% FBS with or without

dibutyryl cAMP. After 44 h of culture, the medium

was replaced with serum-free and methionine-

deficient RPM! 1640 culture medium containing

L-35 C-methionine (10 pCi/well, ICN Radiochemicals,

Irvine, CA). The incorporation was terminated after

4 h. Medium was removed and the cell layers were

washed with PBS, 10% TCA twice, ethanol:ether

(3:1, vol/vol), and dried. Radioactive precipitates

were solubilized with 1 ml of 0.2 N NaOH, an aliquot

was transferred to scintillation vials, and 100 p1 of

acetic acid and 5 ml of counting cocktail were added.

The samples were then counted in a liquid scintilla-

tion counter.

Effect of Dibutyryl cAMP on Midgestation

Placental Explant Outgrowth and


The labyrinth region of the midgestation (Day 11

or 12) chorioallantoic placenta grows well in vitro

(Soares et al., 1987). In vitro labyrinth growth in-

volves the migration of cells away from the explants

and proliferation of the migrating cells. The placental

labyrinth increases in size and becomes easier to dis-

sect as gestation progresses; however, we have pre-

viously shown that explants obtained from Day 15 of

gestation or later lose their ability for outgrowth. To

determine the day of gestation to most efficaciously

harvest placental tissue for examining growth re-

sponses, explants were dissected from the labyrinth

region of the chorionallantoic placenta on Days 12,

13, and 14 of gestation and placed in 15.5-mm-

diameter wells containing 1 ml of RPMI-1640 culture

medium supplemented with 10% FBS and other addi-

tives as previously described (Soares, 1987; Soares et

al., 1987). Culture medium was changed at 2-day

intervals and cultures were terminated after 8 days.

At the termination of the experiments, explants were

carefully removed and the amount of placental cell

outgrowth was determined by the crystal violet cell

proliferation assay described above. From these ex-

periments, Day 12 labyrinth tissue proved to possess

the greatest capacity for outgrowth (see Results) and

was selected for examining the effects of dibutyryl

CAMP on explant outgrowth and hormone produc-

tion. At the termination of the experiments (Days 4

and 8 of culture), placental cell outgrowth was

assessed as described above and conditioned media

were collected and stored frozen for later measure-

ment of progesterone by RIA (Soares et al., 1985)

and placental lactogen by radioreceptor assay (Shiu

et al., 1973;Soares, 1987).

Statistical Analysis

The data were analyzed by analyses of variance.

The source of variation from significant F-ratios was

determined with Newman-Keuls multiple comparison

test (Keppel, 1973).

RESU LTS

cAMP and Placental Cell DNA Synthesis

The addition of dibutyryl cAMP to rat placental

cell cultures resulted in a significant reduction in the

incorporation of 3H-thymidine into DNA (Fig. 1).

This diminishment of DNA synthesis was evident

with cells exposed to dibutyryl cAMP in the presence

of FBS and under serum-free conditions in the pres-

ence or absence of transferrin (Fig. 1). The minimal

effective dose for the inhibition of DNA synthesis in

the presence of FBS or transferrin was 0.05 mM

dibutyryl cAMP (p<O.Ol, Fig. 1). Inhibitory effects

of dibutyryl cAMP were first apparent within 4 h

I,

b

II

120

100

eo

60

40

20

(‘5

II

‘5

a ons 0.10 0.50 tOO

DIb� cycic MW (mM)

SF+T+�idyr�1 cAMP

12 48

0

(.5

I(p<O.O1, Fig. 2) and were maximal after 36 h of

exposure (Fig. 2). 8-Bromo cAMP and MIX also

showed inhibitory activities on HRP cell DNA syn-

thesis in a dose-dependent manner (Fig. 3). Minimal

effective concentrations for both of these compounds

was 0.05 mM (p<O.O1, Fig. 3). The cAMP analogues

were not found to have a significant effect on the

intracellular acid-soluble 3H-thymidine pool, indi-

cating that thymidine transport was not affected.

The inhibitory effect of cAMP analogues on HRP

cell DNA synthesis was also evident after autoradio-

graphic analysis of 3H-thymidine incorporation by

HRP cells cultured with and without dibutyryl cAMP

(Fig. 4).

Addition of dibutyryl cGMP had no significant

effects on HRP cell DNA synthesis at concentrations

ranging from 0.05 to 1.0 mM (control, 131.6 ± 6.2;

dibutyryl cAMP, 1 mM, 23.2 ± 0.08; dibutyryl cGMP

SF 061

Conc�WratI�t (mU)

0.1 LO

CYCLIC AMP AND PLACENTAL CELLS

Th� (�u�)

439

FIG. 1. Effects of dibutyryl cyclic adenosine 3’5’-monophosphate

(cAMP) on the incorporation of 3H-thymidine into DNA by Holtzman

rat placental cells (HRP cells). HRP cells were plated in culture mediumcontaining 5% fetal bovine serum. The medium was replaced with

serum-free medium after 24 h and replaced again with the respectivetreatments after an additional 24 h. After 20-h exposure to the treat-ments, 1 MCi of 3H-thymidine was added. The cells were harvested 4 h

later and the amount of 3H-thymidine incorporated into DNA wasdetermined by liquid scintillation counting. The effects of dibutyrylcAMP were examined in serum-free medium (.-.), serum-free mediumsupplemented with rat transferrin (2.5 Mg/mi. o-o), or in serum-

supplemented medium (5% FBS, U-u). Each point represents the mean

of 5-6 replicates, and the vertical bars represent the standard error of

the mean.

FIG. 2. Time-course effects of dibutyryl cyclic adenosine 3’,5’-

monophosphate (cAMP) on the incorporation of 3H-thymidine into

DNA by Holtzman rat placental cells (HRP cells). HRP cells were platedin culture medium containing 5% fetal bovine serum. The medium was

replaced with serum-free medium after 24 h and replaced again with

serum-free medium containing transferrin (5 �ig/ml) in the presence(o--o) or absence (.-s) of dibutyl cAMP (1 mM) after an additional

24 h. The cells were harvested at 4, 8, 12, 24, 36, and 48 h. Each

point represents the mean of 5-6 replicates, and the vertical bars repre-

sent the standard error of the mean.

FIG. 3. Effects of 8-bromo cyclic adenosine 3’,5’-monophosphate

(cAMP) and 3-isobutyl-1-methylxanthine (MIX) on the incorporationof 3H-thymidine into DNA Holtzman rat placental cells (HRP cells).See Figure 1 and the text for details of the experimental design. Eachpoint represents the mean of 5-6 replicates, and the vertical bars

represent the standard error of the mean.

440 SOARES ET AL.

=, 0.05 mM, 134.7 ± 3.6; dibutyryl cGMP 0.1 mM,

129.4 ± 6.9; dibutyryl cGMP 0.5 mM, 129.3 ± 6.3;

dibutyryl cGMP, 1.0 mM, 121.6 ± 4.4; all values

expressed as cpm X i0� and are means of 5

replicates).

Forskolin and cholera toxin significantly inhibited

HRP cell DNA synthesis and stimulated the accumu-

lation of cAMP (p<0.01 for each comparison),

whereas pertussis toxin did not significantly influence

HRP cell DNA synthesis and cAMP accumulation

(Table 1).

The inhibitory actions of dibutyryl cAMP were

found to be at least partially reversible (Table 2).

Treatment of HRP cells for three consecutive days

with serum-free culture medium resulted in very low

levels of 3H-thymidine incorporation (Line 1). As was

noted in the time-course experiment, cells exposed to

serum-free conditions following exposure to trans-

ferrin continued to incorporate 3H-thymidine into

TABLE 1. Effects of forskolin, cholera toxin, and pertussis toxin on rat

placental cell DNA synthesis and accumulation of cyclic adenosine3’,5’-monophosphate (cAMP) (mean ± SEM).

H-Thymidine

Treatmentincorporation(cpm X 10�)

cAMP(pmoles/24 h)

Control 80.8 ± 9.2 1.53 ± 0.25Forskolin(100MM) 30.8 ± 1�4a 9�33 ± 0.74�

Cholera toxin (1 Mg/mI) 51.1 ± 34a 5.01 ± 014a

Pertussis toxin (200 ng/ml) 77.6 ± 5.4 2.64 ± 0.49

aValues are significantly different from values for the control treat-

ment, p<0.01.

DNA (Line 3), however, at a reduced level compared

to cells continuously exposed to transferrin (Lines

2, 4) (p<0.01). Dibutyryl cAMP significantly in-

hibited the stimulating effects of transferrin on HRP

cell 3H-thymidine incorporation (Line 5). Two con-

FIG. 4. Autoradiograrns of incorporation of 3H-thymidine into DNA by Holtzman rat placental cells (HRP cells). HRP cells were plated in the pres-ence of 5% fetal bovine serum (FBS); the medium was replaced with serum-free culture medium after 24 h, and replaced again with it) 5% FBS-

supplemented medium or B) 5% FBS-supplemented medium containing dibutyryl cyclic adenosine 3’,5’-monophosphate following an additional 24

h. The cells were incubated with the respective treatments for 36 h followed by a 4-h exposure to 3H-thymidine. The cells were dipped in photo-graphic emulsion, exposed for 3 days, developed, and counterstained. This figure depicts representative autoradiograms from 3 experiments. (X400.)

Day of culture


TABLE 2. Reversibility of the inhibitory effects of dibutyryl cyclic

adenosine 3’,5’-monophosphate (cAMP) on DNA synthesis by rat pla-cental cells (mean ± SEM).

Treatment

Day 3

Treatment

Day 4

H-Thymidine

incorporation(cpm X 10�)

1. Serum-free (SF)

2. SF + Transferrin (T)a,b

SF

. . .2.1 ± 0.2

115.4 ± 5.93. SF+T SF 74.3±2.7

4. SF + T SF + T 122.2 ± 6.3

5. SF+T

6. SF + T + cAMPb

SF+T+cAMPc

. . .23.2±4.9

21.9 ± 3.1

7. SF+T+cAMP8. SF+T+cAMP

SF+T+cAMPSF+T

10.1 ±0.7643+20d

aTransferrin was used at a concentration of 5 Mg/mI in all treatments

where it appeared.

bAssayed at the end of Day 3.

cDibutyryl cAMP was used at a concentration of 0.5 mM in all treat-

ments where it appeared.

dValues are significantly different from cells treated with dibutyryl

cAMP and harvested on Day 3 of the experiment (21.9 ± 3.1 vs. 64.3 ±

2.0, p<0.01).

‘Each value represents the mean of a minimum of 5 determinations.Placental cells were plated in 5% fetal bovine serum-containing medium

and incubated for 24 h; the medium was replaced with serum-freemedium, and the sample was incubated for a second 24 h and subse-quently exposed to the respective treatments described above.

secutive days of cAMP exposure were more effective

than a single 24-h exposure period (Lines 6, 7)

(p< 0.05). Replacing cAMP-containing medium with

Control

Dibutyryl

cAMP

medium supplemented with transferrin resulted in

significantly higher rates of 3H-thymidine incorpora-

tion (Line 8) compared with cells exposed to cAMP

(Line 7; p<0.01), demonstrating at least partial

reversibility of the cAMP inhibition.

Effect of Dibutyryl cAMP on Placental

Cell Growth Patterns and Morphology

Control HRP cells displayed a stacking or piling

phenomenon that was evident after 4 days of culture

and was extensive at 8 days of culture (Fig. 5). Cell

stacking was not evident in cultures incubated with

dibutyryl cAMP (Fig. 5). Cell-cell contacts were more

noticeable in dibutyryl cAMP-treated cultures than in

control cultures. Long processes extending from the

cells cultured with dibutyryl cAMP were noted (Fig.

6) and may give some further indication of the

differences in intercellular organization of the control

and cAMP-treated cells. Electron microscopic exami-

nation revealed that dibutyryl cAMP-treated cells

possessed a more rounded appearance and excep-

tionally dilated rough endoplasmic reticula (Fig. 7).

The cytoplasmic extensions visualized by light micro-

scopy were also observed by electron microscopy and

did not appear to contain any unusual cytoplasmic

constituents.

FIG. 5. Analysis of growth characteristics of control and dibutyryl cyclic adenosine 3’,5�.monophosphate (cAMP)-treated Holtzman rat placental

cells (HRP cells). The photographs depict representative 15.5-mm wells from Days 2, 4, 6, and 8 of culture. Note the absence of cell piling or stack-ing in the dibutyryl cAMP-treated cultures. (X 20.)

/

-I

B 4.- 4��’;�III’

442 SOARES ET AL.

FIG. 6. Histological analysis of control (A) and dibutyryl cyclic adenosine 3,5-monophosphate (cAMP) (8)-treated Hoitzman rat placental cells

(HRP cells). Cells were fixed in phosphate-buffered formalin and stained with hematoxylin and eosin. Note the location of the long processes ex-tending from the cAMP-treated cells. (X400.)


on Placental Cell Transplantability

Exposure of cells to dibutyryl cAMP and MIX

prior to i.p. transplantation reduced the ability of the

cells to grow in recipient animals. We have previously

shown that HRP cells transplanted to the peritoneum

grow as cystic structures suspended in the peritoneal

fluid and as solid growths adhered to various abdomi-

nal structures (Soares et al., 1987). Cells treated with

dibutyryl cAMP and MIX did not form cystic struc-

tues after i.p. transplantation (n=6) and showed very

poor growth as solid masses adhered to the mesen-

teries, whereas control cells showed extensive growth

of both types of transplants in 5 of 6 animals in-

jected. By light microscopy, the morphologies of the

solid masses generated from control and dibutyryl

cAMP-treated HRP cells were similar and indistin-

guishable from those reported earlier (Soares et al.,

1987). The extracellular matrix glycoprotein,

laminin, was widely distributed in solid masses

derived from both control and dibutyryl cAMP-

treated cells (data not shown). The abundance of

laminin in placental cell transplants has been pre-

viously reported (Soares et al., 1988).

Effect of Dibutyiyl cAMP on Placental

Cell Progesterone and Placental Lactogen Release

HRP cells exposed to dibutyryl cAMP produced

significantly more progesterone than did controls on

Days 6 and 8 of culture (Table 3). Placental lactogen

production by control or dibutyryl cAMP-treated

HRP cells was not detectable at any time during the

8-day culture period. Cell numbers were significantly

FIG. 7. Electron microscopic analysis of control (A and C) and

dibutyryl cyclic adenosine 3’,5’-monophosphate (cAMP) (B and D)-

treated Floltzman rat placental cells (HRP cells). Arrows denote the

location of the rough endoplasmic reticulum. (A and B, X 1928, C and

D, X 3850.)

C

.. %


A

-

.5

- -- 5.

#{149} -

�a- 8-bromo cAMP

�- Dibutyryl cAMP

Control .01 .1

444 SOARES ET AL.

TABLE 3. Effect of dibutyryl cyclic adenosine 3’,5’-monophosphate

(cAMP) on progesterone release by placental cells (mean ± SEM).

Treatmenta

Progesterone

(pg/b’ cells)

Day 4Control

Dibutyryl cAMP

91.2 ± 10.1

117.8 ± 11.7

Day 6

ControlDibutyryl cAMP

67.2 ± 10.7293.5 ± 45�5b

Day 8

Control

Dibutyryl cAMP

120.7 ± 12.2206.9 ± 12�3b

aprogesterone was not detectable on Day 2 of culture in samples

from either treatment.

bValues are significantly different from control values, p<0.01.

reduced in cultures exposed to the cAMP analogue

after 8 days of treatment (control: 6.37 ± 0.3 X 10�

cells/well vs. cAMP: 3.66 ± 0.09 X 1O� cells/well;

n=8 for each treatment, p<0.01).

cAMP and Placental Cell Alkaline

Phosphatase Expression

HRP cells showed a significant inhibition in their

expression of alkaline phosphatase when exposed to

either dibutyryl or 8-bromo cAMP (Fig. 8). The

1.1

�1.0

U’0�. 0.8

0.7C

0.3

0.2

0.1

0.0

Concentration (mM)

FIG. 8. Effect of 8-bromo and dibutyryl cyclic adenosine 3’,5’-

monophosphate (cAMP) on the expression of alkaline phosphatase byHoltzman rat placental cells (HRP). HRP cells were exposed to thetreatments for 48 h prior to alkaline phosphatase measurement. Eachbar or point represents the mean of 10 replicates and the vertical bars

represent the standard error of the mean.

inhibitory actions of the cAMP analogues were

concentration-dependent.


on Placental Cell Protein Synthesis

HRP cells exposed to dibutyryl cAMP or to control

conditions incorporated 35S-methionine into TCA-

precipitable protein at similar levels (control: 30.7 ±

0.8 X iO� cpm/well vs. dibutyryl cAMP 28.8 ± 2.2

X 10� cpm/well; the results represent 8 replicates for

each treatment). The ability of dibutyryl cAMP-

treated cells to incorporate 35S-methionine into TCA-

precipitable protein at levels comparable to controls

suggest an absence of toxicity in the growth sup-

pression induced by dibutyryl cAMP.

Effect of Dibutyryl cAMP on Midgestation

Placental Explant Outgrowth and


Placental tissue isolated from Day 12 of gestation

generated significantly more outgrowth than tissue

isolated from Day 13 or 14 of gestation. Absorbance

readings at 520 nm for the crystal violet-stained

placental explant outgrowths were as follows: Day

12, 0.81 ± 0.07; Day 13, 0.51 ± 0.03; and Day 14,

0.14 ± 0.01 (the results represent 8-10 replicates

per day of gestation). Explants of placental labyrinth,

isolated from Day 12 of gestation, showed impaired

outgrowth but enhanced progesterone release when

treated with dibutyryl cAMP (Table 4). Treatment

with the cAMP analogue did not significantly affect

placental lactogen release on Day 4 of culture but

significantly depressed placental lactogen output on

Day 8 of culture (Table 4).

DISCUSSION

The results of this investigation indicate that

modulation of intracellular cAMP levels significantly

affects the growth and differentiation of rat placental

cells.CAMP has been previously shown to have both

inhibitory and stimulatory actions on cell growth

(see Boynton and Whitfield, 1983; Gottesman andFleishmann, 1986). The direction of the action of

cAMP varies according to cell type and other experi-

mental conditions. Rat placental cell growth was

inhibited by cAMP analogues. Dibutyryl cAMP and

8-bromo cAMP each inhibited the incorporation of

3H-thymidine into rat placental cell DNA. Dibutyryl

cAMP also inhibited the outgrowth of cells from ex-


TABLE 4. Effect of dibutyryl cyclic adenosine 3,5’-monophosphate (cAMP) on midgestation placental explant outgrowth and progesterone and

placental lacrogen release (mean ± SEM).’

Placental

Treatment

Outgrowtha

(520 nm)

Progesterone

(ng/48 h)

lactogen

(ng/48 h)

Day 4

Control

Dibutyryl cAMPb

0.24 ± 0.02

0.12 ± 0�0jc

0.31 ± 0.03

1.28 ± 023c

85.7 ± 9.2

91.5 ± 13.5

Day 8

Control 0.79 ± 0.07 0.63 ± 0.07 234.9 ± 14.2

Dibutyryl cAMP 0.27 ± O.03’ 1.44 ± 0.21’ 133.5 ± 154C

aPlacental explant outgrowth was determined by removing explants from the cultures, staining the nuclei of the cellular outgrowths with crystal

violet, washing, drying, and eluting with ethylene glycol, and measuring the absorbance at 520 nm. See text for further details.

bDibutyryl cAMP was used at a concentration of 1 mM.

CVaIuts are significantly different from control values for the same day of culture. p<0.01.

‘All values are means of 8 replicates.

plants of midgestation placental primordia. Treat-

ment with MIX, an inhibitor of phosphodiesterase

activity, or with forskolin, an activator of adenylate

cyclase, each resulted in a similar inhibition of rat

placental cell DNA synthesis. Bacterial toxins, such

as cholera and pertussis toxins, which are known to

alter the function of specific guanine nucleotide-

binding regulatory proteins (Casey and Gilman,

1988), had less dramatic effects on placental cell

cAMP generation and DNA synthesis. These observa-

tions are consistent with those recently reported for

the actions of bacterial toxins on the behavior of

human cytotrophoblast cells in vitro (Nulsen et al.,

1988) and support the notion that receptor-effector

coupling may be somewhat different in placental

cells. The effects of cAMP on human placental cell

DNA synthesis has yet to be determined. The limited

growth potential of term human placental cells has

made such investigations difficult.

Growth inhibition of the rat placental cells by

CAMP may have resulted from induction of differen-

tiation. The production of progesterone and placental

lactogen and the expression of alkaline phosphatase

are associated with rat trophoblast cell differentiation

(Sherman, 1983; Soares et al., 1985; Soares, 1987).

The elaboration of these indicators of trophoblast

cell differentiation during gestation is inversely

related to the growth potential of the rat chorioallan-

toic placenta and is different in the junctional and

labyrinth regions (Jolie, 1964; Peel and Bulmer,

1977; Soares, 1987). The junctional region, located

proximal to the uterine decidua, has a greater poten-

tial for hormone production and limited potential for

expression of alkaline phosphatase, whereas the

labyrinth region, located proximal to the developing

embryo, has a reduced potential for hormone produc-

tion and an enhanced capacity to express alkaline

phosphatase (Matt and MacDonald, 1985; Soares,

1987). Treatment of rat placental cells with dibutyryl

cAMP inhibited alkaline phosphatase expression and

stimulated progesterone biosynthesis, a phenotype

consistent with differentiation towards a junctional

zone type of placental cell. Increased progesterone

production was also observed in midgestation rat

placental primordia treated with dibutyryl cAMP. It

remains to be determined whether differentiation-

associated characteristics were induced in all cells or

were restricted to subpopulations of cells in the

placental cell line and placenta.

Treatment of rat placental cells with dibutyryl

cAMP had significant effects on in vitro growth

patterns, growth following transplantation, and

cellular morphology. Dibutyryl cAMP treatment

significantly reduced both the propensity of the rat

placental cells to pile in vitro and the growth poten-

tial of the placental cells in vivo. These observations

were associated with a cAMP-stimulated alteration in

cell-cell interactions and are consistent with cAMP

induction of rat placental cell differentiation. Pla-

cental cell growth in allogeneic hosts shares many

characteristics with tumor cell growth (Soares et al.,

1987). Previous reports on suppression of malignant

growth following induction of tumor cell differen-

tiation (see Sachs, 1986 and 1987, for reviews) are

446 SOAR ES ET AL.

in agreement with our findings on cAMP induction

of placental cell differentiation and reduced placen-

tal cell transplantability.

A note of caution regarding the interpretation of

studies utilizing dibutyryl cAMP is important. In

several experimental in vitro systems, butyrate, a

metabolite of dibutyryl cAMP, possesses the ability

to alter cellular function independent of elevating

intracellular cAMP (see Boynton and Whitfield, 1983,

for a discussion). Thus the effects we have observed

with dibutyryl cAMP may have been mediated by

cAMP and/or butyrate. Experimentation examining

the actions of 8-bromo cAMP and other elevators of

placental cell cAMP strengthens our contention that

intracellular cAMP elevation modulates placental cell

growth and differentiation (see Table 1 and Figs. 3

and 8).

The rat placenta has a hormonally responsive

adenylate cyclase enzyme system (Moore and Whit-

sett, 1982; Heller et al., 1986). Catecholamines stimu-

ulate the generation of cAMP in the rat placenta

(Moore and Whitsett, 1982; Heller et al., 1986).

Preliminary experiments examining the effects of

catecholamines on rat placental cell DNA synthesis

have been equivocal (unpublished observations).

Catecholamines may act on other trophoblast cell

types not represented in our cell line, on nontropho-

blast cells present in the placenta (mesenchymal cells,

endothelial cells, etc.), or possibly on our placental

cells but under different experimental conditions.

The physiological signals (intercellular or intracellu-

lar) responsible for stimulating rat placental cell

cAMP elevation and thus control of growth and

differentiation are yet to be identified.

In summary, the results of this investigation indi-

cate that cAMP may be an intracellular mediator in-

volved in inhibiting rat placental cell growth and

directing differentiation towards a placental cell

phenotype normally found in the junctional zone of

the chorioallantoic placenta.

ACKNOWLEDGMENTS

The authors gratefully acknowledge Linda Hicks for help in thepreparation of the manuscript, Kay von Bergen for assistance in the

histological preparation of the tissues, Douglas Larsen for excellent

technical assistance, Dr. Walter Morishige for the antiserum to proges-

terone, and Dr. A. Parlow and the National Institute of Arthritis,Diabetes and Digestive and Kidney Diseases for providing ovine pro-lactin that was used for radioiodination and as a standard in theplacental lactogen radioreceptor assay. We also acknowledge the useof the University of Kansas Medical Center Electron MicroscopyResearch Center.

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