Role of 4-Hydroxylated Estradiol Reducing Ca2 + Uptake of Uterine

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and phenylephrine. (ROC activator) ... synthesized by the porcine ...... Synthesis of catechol estrone by porcine uterine arteries. Endo. Soc: Abst. 30. Van. Orden.

OF

BIOLOGY

36, 36 1-368

REPRODUCTION

(1987)

Role of 4-Hydroxylated Estradiol in Reducing Ca2 + Uptake of Uterine Smooth Muscle Cells through Potential-Sensitive Channels’ S. L. STICE,3

J. P. ROSAZZA,5

S. P. FORD,2’3 Department

of Animal

Iowa

D. E. VAN

ORDEN4

Science3

University

State

Ames,

and

Arterial

Iowa

50011

and of Obstetrics and Gynecology4

Departments and

Medical

Chemistry5

University Iowa City,

of Iowa Iowa 52240

ABSTRACT Entry of ionic Ca2 + into two major membrane channels. by membrane depolarization, csl-receptor-ligand interactions.

lations

of

suring

the

specific

Ca2

+

Ca2

inhibitors

+

used

through

PSCs,

a

4-hydoxylated

the

blockade +

of

and

Ca2

by agonists

no may

by known

1969).

This

effect

indicate

on ROCs. a refined

lag time, inhibited These

regardless of site by cycloheximide data

effects Recent

suggest

through evidence

Accepted ‘iournal Economics

Paper No. Experiment

and

and

2444; 2

11

that local from

begins of

after

a 20-

administration, (Killam et

estrogen

and is 1973).

a!.,

exerts

its

production of our laboratory

30-mm

a

vascular

mediator. and others

supported

J-11946 Station, in

Kildee

Hall,

request: Iowa

Dr. State

a,nrinone

mechanisms

of the Ames, party

by

Iowa Iowa;

Agriculture Projects

United

States

and 1994, Public

S. P. Ford, University,

Department Ames, IA

of

Animal

was

uterine

The

presence

system

for

choosing

of separate

an

muscle

Ca2

+

appropriate

excluding

the

inter-

was also determined

Ca2 uterine

as their

segments induced by Furthermore, it was

additive, it. It flow

by mea-

as well

two smooth of extracellular

arterial inhibited.

blood

controlling

studies,

By

for opening

increase

This was studied

towards uptake

ROCs.

of uterine be selectively

in pigs, +

decreased

pathways

blood

that Ca2

that

can

+

be

flow.

Gelbke porcine artery

et al., 1975) and are synthesized by the conceptus (Mondschein et al., 1985), uterine (Van Orden et al., 1983a), and endometrium

(S.

Ford,

P.

Orden et hyperemia Home

Iowa

State

al., 1983b), is unaffected

tion of cycloheximidt been reported that bound receptors for target cells (Schaeffer

2443 Health

Grant.

Reprint

sensitivities inhibits

through

and

pigs.

perfusion

University,

and

D.

E. Van

Orden, University of Iowa, unpublished observations). In addition, 4-hydroxylated estradiol (40H-E2) is equipotent with estradio!-17a (E2) in increasing UBF when injected into the uterine artery of gilts (Van

September 4, 1986. May 5, 1986.

Received

Service

in UBF

uptake

+

in

suggests that catechol estrogens (2- and 4-hydroxylated estrogens) may serve as this mediator. These metabolites of estrogen are found in the circulation when estrogens are high (Fishman and Dixon, 1967;

of estrogen results in an increase flow (UBF) in the pig, as has been mammalian species (Dickson et a!.,

increase

arteries in in vitro

different specifically

to

INTRODUCTION

The administration in uterine blood observed in other

uterine arteries

D-600

two separate

a compound

exhibited

Ca2

uptake

+

the

uptake and contractions (ROC activator) could

+

with

of

markedly D-600

inhibits Ca2

pathway

(40H-E2),

PSCs

cells

of uterine

showed amrinone.

specifically

or amrinone, and phenylephrine

Ca2

independently

muscle properties

parameters D-600 and

amrinone

common the

in smooth contractile

These study:

estradiol

through

activated

uptakes. in this

that of

uptake

and

of D-600 activator)

demonstrated

pretation

tone

while

concentration high-K (PSC

vascular smooth muscle cell for contraction is thought to be mediated by The first are designated as potential-sensitive channels (PSCs), which are opened and the second, as receptor-operated channels (ROCs), which are activated by This study was designed to determine the presence of these 2 distinct popu-

channels

entry

baseline

the

Science,

50011.

361

and unlike E2, by simultaneous

its uterine administra-

(Ford et al., 1986). It has also there are specific membranecatecho! estrogen on estrogen et a!., 1980). Catechol estrogens

STICE

362

may The

reach the pig uterus

uterine possesses

artery via a system

is capable of carrying lumen to the adventitial (Magness

and

estrogens surface

Ford,

1982).

estrogens to the uterine rather than a vascular methoxylation

of

0-methyltransferase, (Ball and Several estrogens

is found

a!.

(1978) Ca2

+

marked increase in UBF no effects on the general researchers observed that estrogen

hyperemia

UBF in Redman

response (1984)

observed channel

mo;

13)

cells

further Since time

a a

increase

in

Walters and lag between

the administration of nifedipine and a reduction in blood pressure of pregnant women, we hypothesized a role of hydroxylated estrogens in reducing Ca2 + uptake by smooth muscle cells of the uterine arteries. It is known that uptake of extracellular Ca2 + is required

for

that drugs

vascular that

vascular

Further, membrane an exposure time contractions 30-mm time vasodilation subsequent Membrane

muscle occurs channels

contractions,

and

after exposure to (Bolton, 1979).

Ca2 + channel blockade requires of several mm to block Ca2 k-induced

(Meisheri et a!., lag associated

1981). with

Thus, the 20- to estrogen-induced

may result from its hydroxylation blockade of Ca2 + channels. channels mediating Ca2 + entry

been designated channels (PSCs) cell membrane concentration,

smooth

relaxation block these

as two major are activated

and have

types. Potential-sensitive by depolarization of the

after an elevation of extracellular K and Ca2 + influx through these channels

is selectively Meisheri et

inhibited a!., 1981).

by D-600 (Bolton, Receptor-operated

(ROCs) are phrine-induced

opened by activation

norepinephrineof al-adrenergic

or

1979; channels pheny!ereceptors,

and Ca2 + influx through these channels is selectively blocked by amrinone (Meisheri et a!., 1981). This study was therefore conducted to verify the presence of each type of calcium channel (ROC and PSC) in uterine possible interaction

arterial with

smooth 4OH-E2

muscle

and

gilts of similar age and weight (10-12 kg) that were exhibiting consecutive

the was

estrous

their

At artery

cycle.

designated

Contraction

at doses that had Further, these uteri were under

no

METHODS

cycles of normal duration to be killed during the

of

estrus

that hydroxylated of Ca2 + channels.

to nifedipine. observed a 20-mm

Yorkshire 140-160

estrous assigned

catecholblood

AND

General

that nifedipine, blocker, caused

in the rat circulation. rats whose

exhibited

uterine artery pathway the rapid

by in red

MATERIALS

catechol

via a lymphatic would avoid

Knuppen, 1980). lines of evidence suggest act through blockage

Sandahl et potential-sensitive

of

compounds

which

route. that

from the of the uterine Delivery

artery route those

a lymphatic of lymphatics

ET AL.

The

as Day

(18-22 days) were lutea! phase (LP; Day first

day

of behavioral

0.

Studies

death, a 3.5-cm supplying one

segment uterine

of the middle horn of each

uterine gilt was

excised immediately the mesometrium.

in front of its first bifurcation in Each arterial segment was placed

immediately oxygen,

a container dioxide)

into carbon

5%

(22#{176} C; composition

of oxygenated Krebs-Ringer

in millimoles

per

(95% solution

liter

=

sodium

chloride, 118.1; sodium bicarbonate, 25.0; potassium chloride, 4.7; calcium chloride’ 6 H2 0, 2.5; potassium dihydrophosphate, 1.2; magnesium sulfate’ 7 H2 0, 1.2; glucose, 11.1). At the laboratory, both arterial segments described arteries

were prepared for simultaneous perfusion as previously for ovine and bovine uterine (Ford et a!., 1976). Briefly, arterial segments

were

cannulated

tubing within

and 60

perfused continuously

at

mounted after

mm

each

end

with

polyethylene

in duplicate perfusion chambers death. Arterial segments were

intraluminally oxygenated

and

extraluminally Krebs-Ringer

with solution

(37#{176}C). An extra!uminal and intraluminal perfusion rate of 10 ml/min was maintained throughout the perfusion of each artery. Uterine arterial segments were allowed a 30-mm equilibration period, by which time all arteries had established a constant baseline perfusion flow and

pressure (BPP) against the intra-luminal were ready for the initiation of drug per-

fusions.

Drug

centrations Changes resistance

in the intra-luminal perfusion flow. in perfusion pressure arising from changes in to flow through the arterial segments were

measured recorded Hewlett Ca2

+

dosages

are

with Statham in millimeters Packard Uptake

Calcium previously

7700

reported

as the

pressure of mercury chart

final

transducers (mmHg)

con-

and by a

recorder.

Studies influx was measured reported by Meisheri

by using a technique et al. (1981). Mea-

ROLE surement

of unidirectional

study uptake

phenylephrine (influx). For

exposed

to (10

tissue

during

mm).

primarily

approach

amount

to

Ca2

+

us

to

from the of uterine

+

section

used

for Ca2 + placed into

for

entering

can

This

for

contraction

shown

from vascular 1981). Each

time

weighed,

the

on the

extracellular space. artery immediately

uptake

Brmefly, adjacent

studies

influx determinations. a container of oxygenated

been

to be

determine

or high-K

was

the to

utilized

Segments (95%

363

HYPEREMIA

Ca2 + Lynch,

to

experimental

selectively

UTERINE

Ca24 were the

be assumed

influx.

IN

out

a short

of

periods

of phenylephrine

of Ca2 segment

carried

solutions

short

allowed

influence

the

The

such

were

and high-K4-induced this, the arterial segments

Ca-containing

period due

fluxes

OF 4OH-E2

were 02, 5%

C02) physiological salt solution (PSS) at 22#{176} C, composition in millimoles per liter = sodium chloride, 140; potassium chloride, 4.5; D-glucose, 10; HEPES,

to

and

containing

inhibit

both

placed

0.5

efflux

smooth segment in

ml

of

a separate

Protoso!

PPO PA,

and and

0.3 250

International

g POPOP, ml Triton

Fisher X-100,

Corporation,

Mt.

dissolved in each liter of Fisher Scientific) was added for

radioactivity.

artery,

Additionally,

one

determine

0.5-cm

segments

binding

were

of

were

remaining

attached length

a 12 X 75 nonradioactive

was

culture PSS.

to clamp this length tube, thus suspending in the buffer solution. to

apply

muscle, These the

a constant

to

a 2.4-gram

used

to lower

After

each

tube containing A polyethylene

while

segment

4 ml stopper

of

the into

aerated was used

of silk against the side of the the arterial segment and weight The 2.4-gram weight was used tension

on

and resulted in a more by triplicate 0.5-cm segments tubes were kept aerated and

experiment.

weight,

a 60-mm

the

arterial

smooth

uniform uptake of from each animal. at 37#{176}C throughout equilibration

period,

the rings were exposed to control or experimental solutions containing E2, 4OH-E2, D-600, or amrinone for periods of 30 mm, followed by a 10-mm exposure to high-K4, pheny!ephrine, plus the inhibitor and/or protocol was designed to

At the end of the 10-mm were transferred to tubes Ca2 4-free 10 M maintained

D-600, additions

or amrinone of high-K4

incubation containing

buffer (PSS with CaCI2 omitted) lanthanum chloride (LaCl3) at 4#{176}C. At this concentration,

period, tissues 10 ml of a containing for 60 mm LaC13 has

the

of

was

the

of

segments

(n

relatively

consistent

for

all

uterine

was

used the

to

tissue.

of aerated

M LaC13 for 30 exposure to 0.5 of phenylephrine incubation

period,

ml of a Ca2 4-free at 4#{176}C for 60 mm,

determined

coefficient

multiple

each

in 4 ml

placed in 10 10 M LaC13

uptake

by running

end

were

Scintanalyzed, vial and counted

as

previously

variation, =

calculated

6) of a lutea!

uterine artery in a single assay, averaged an uptake of 237.6 ± 5.7 j.zmoles Ca/kg weight). Nonspecific binding of Ca, arterial

phase

6.9%, tissue which

segments

with (wet was in this

study, averaged 38.8 ± 2.6 i.imoles/kg tissue and may have resulted from binding to nonmuscular components of the arterial wall. Nonspecific binding, which averaged 18.1 ± 1.2% of specific uptake, was subtracted

from specific

Drugs

of and

At

described. The intraassay

obtain

and 0.5 .tCi/ml of Ca steroid. This experimental test the criteria of selective

inhibition by E2, 40H-E2, Ca2 + uptake induced by the phenylephrine.

high-K4.

IL,

Ca to

maintained

Ca

of silk

and

Pittsburgh, Products

segment

and

Two 20-cm lengths of 3-0 silk were passed through the lumen of each segment. Both ends of one length

Pro-

for

arterial

nonspecific

These

Toluene, to each

were again containing

determinations.

Research

Prospect,

tissues buffer

influx

vial

Scientific Research

0.5-cm

Ca24

of and dry,

scintillation

(NEN

Ca2 4-free buffer containing iO mm before and during the 10-mm j.zCi/ml of ‘ Ca in the presence

for

uptake

ducts, Boston, MA) and heated at 60#{176}Cfor 2 h to facilitate the breakdown of tissue. Ten milliliters of a Toluene Triton Scintillation cocktail (7.0 g

[N -2- hydroxyethylpiperazine - N’ -2 - ethanesulfonic acid], 5; calcium chloride, 1.5; magnesium chloride, 1.0). At the laboratory, the excess connective tissue was trimmed away, and each artery was cut into segments

and

muscle cells (Deth was then blotted

and

total

hydrochloride

into and

The 40H-E2 E2 by direct the phenolic purification

tography 1985).

each

artery

to

(Sigma,

St.

Louis,

(A. G. Knoll Pharmaceutical Laboratories, W. Germany), amrinone (Sterling

Winthrop, Rensselaer, New England Nuclear, study. from

for

Chemicals

Phenylephrine MO), D-600 Ludwigshafen,

‘ Ca influx

influx.

on

NY), and 45Ca (12.0 mCi/mg; Boston, MA) were used in this

used in the study was introduction of molecular

obtained oxygen

precursor by Aspergillus high performance liquid

alliaceus chroma-

as previously

described

(Williamson

et a!.,

STICE

364

Statistical For in uterine

perfusion

arterial

by split-plot design with

AL.

response

Analysis in vitro

ET

response

analysis perfusion

data,

treatment

differences

were

statistically

for a completely periods as the

ferences analyzed regression tionships

in Ca uptake of arteries by factorial design (Kirk, analyses were used to between inhibition of

uterine treatments.

arterial

segments



evaluated randomized subplot; dif-

from gilts were 1968). Simple determine relaCa uptake of

subjected

to

different

perfusion

sequence

In

Vitro

new

Experiment evaluate artermal

la.

The

the role responsiveness

segments perfusions.

from On

first

of

study

was

4OH-E2 in in vitro.

5 LP gilts were each experimental

period,

arteries received intra!uminal flow, 200

mM

luminal pressures

for

BPPs a bolus which

5 sec;

were

uterine arterial

to both

recorded

and

increase Ten mm a bolus

BPP,

the

vessel).

infusion,

mm

of

4OH-E2

the to

and phenylephrine activity of PSCs muscle membrane.

40H-E2 studies

used was to decrease

and

arteries (raninfused with vessel), and the 30 mm (control during

was used to estimate ROCs on the vascular The concentration of

which

using

studies

to

be

in the

middle

of their

doses from

with (p

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