Nitric oxide and the myometrium

Pharmacol. Thu. Vol. 70, No. 2, pp. 91-100, Copynght 0 1996 Elsevier Science Inc.

1996

ISSN 0163-7258/96 $32.00 PII SOl63-7258(96)00004-6

ELSEVIER

Associate

Nitric

Oxide

Editor:

F! Rubin

and the Myometrium Jane Norman

DEPARTMENT OF OBSTETRICS AND

GYNAECOLOGY, GLASGOWROYALINFIRMARY,UNlVERSITYOFGLASGOW,l0ALEXANDRAPARADE,GLASGOWG31

ZER, UK

ABSTRACT. Nitric oxide (NO) is a potent smooth muscle relaxant in blood vessels, the gastrointestinal tract and the respiratory system. Recent evidence has shown that NO has a relaxant (tocolytic) effect on myometrium. NO is produced within the female genital tract during pregnancy, and a reduction in NO synthesis may be involved in the initiation of parturition. Furthermore, the administration of NO donors may be useful in inhibiting uterine contractions in situations where such activity is unwanted, e.g., in preterm labour. NO is also produced in the myometrium in the nonpregnant state, and has potential roles in the facilitation of implantation and the prevention of dysmenorrhoea. This article aims to examine the evidence suggesting that NO has a physiological role in the maintenance of pregnancy and potential pharmacological use in the treatment of preterm labour. PHARMACOL. THER. 70(2): 91-100, 1996. KEY

Nitric

WORDS.

oxide, myometrium,

parturition,

preterm

labour,

uterine

contractions.

CONTENTS 91 92 92

.................... 1. INTRODUCTION ................ 2. THE MYOMETRIUM 2.1. PHYSIOLOGICALROLE ........... 2.2. PHYSIOLOGY OF MYOMETRIAL CONTRACTIONS ................ 2.3. MECHANISMOFACTION OFNITRIC OXIDEWITHINTHE MYOMETRIUM ................. ........ 3. NITRIC OXIDE BIOCHEMISTRY 3.1. NITRICOXIDE SYNTHESIS AND ACTIVITY ..................... 3.2. MEASUREMENTOFNITRICOXIDE 3.3. ESTABLISHED ROLES FOR NITRIC OXIDEINOTHERORGANS ........ OF NITRIC OXIDE BY 4. PRODUCTION MYOMETRIAL STRUCTURES .......... 4.1. NONPREGNANTSUBJECTS ........ 4.2. PREGNANCY .................. OF NONPREGNANT 5. FUNCTION MYOMETRIUM ..................... OFPREGNANT 6. FUNCTION MYOMETRIUM:THE GENERATION OF UTERINE CONTRACTIONS ............ 6.1. MEASUREMENTOFMYOMETRIAL CONTRACTION .................

92

92 93 93 93 93 93 93 94 94

94 94

6.2.

EFFECTOFNITRICOXIDEON MYOMETRIALCONTRAaIONS

. . . . . . . . . . . . . . . . . . . . 95 6.2.1. ANIMALDATA . . . . . . . . . . . 95 6.2.2. HUMAN DATA . . . . . . . . . . . 95 6.2.3. THE EFFECT OFNITRIC OXIDEONMYOMETRIAL CONTRACTIONSIS DEPENDENTON GESTATION ANDSTAGEOFLABOUR . . . . 96 6.3. EFFECTOFNITRICOXIDEON MYOMETRIALCONTRACTILITY INVIVO . . . . . . . . . . . . . . . . . . . . . 96 6.3.1. ANIMALDATA . . . . . . . . . . . 96 6.3.2. HUMANDATA . . . . . . . . . . . 96 7. PHYSIOLOGICALCONTROLOFTHE ONSET OF PARTURITION . . . . . . . . . . . 97 OF NITRIC OXIDE 8. REGULATION PRODUCTION BY STEROID HORMONES 97 9. EFFECT OF INHIBITORS AND DONORS OF NITRIC OXIDE DURING PREGNANCY . . . . . . . . . . . . . . . . . . . . . 98 . . . . . . . . . . . . . . . . . . . . . 98 10. CONCLUSION ACKNOWLEDGEMENTS ................. 98 REFERENCES ......................... 98 INVITRO

ABBREVIATIONS. bNOS, calcium-dependent, neurone NO synthase; eNOS, calcium-dependent, endothelial cell NO synthase; cGMP, cyclic GMP; GTN, glyceryl trinitrate; iNOS, calcium-independent, inducible NO synthase; Kcaz+, calcium-dependent potassium; L-NAME, NC-nitro-L-arginine methyl ester; L-NMMA, NC’-monomethyl-L-arginine; MLCK, myosin light chain kinase; NO, nitric oxide; NOS, NO synthase; SNP, sodium nitroprusside.

1.

INTRODUCT

The oxide

(NO),

a tiny

endothelium-derived and

in the physiology

ION

last 10 years have seen an explosion

has

many

metrium,

the female

originally

vasodilator.

functions,

to the formation Within

molecule from

of memory genital

myometrium,

of interest

NO smooth

described

tract,

as an

is widely

distributed

muscle

relaxation

and the killing

decidua

in nitric

of foreign

NO is produced and placenta.

cells.

by the endoIt is implicated

dilatation 1992),

both

volved

et al.,

in pathological

cluding 1993;

(Izumi intrauterine

labour, Molnar

and

(Telfer

to and during

and in the maintenance

pregnancy

term

of menstruation prior

of uterine

1993).

menorrhagia

et al.,

1994;

Diket

NO

arising

retardation,

vasoet al., during

may be in-

in the uterus, pre-eclampsia,

(Yallampalli et al.,

1995), (Myatt

quiescence

Furthermore,

conditions growth

et al.,

pregnancy

1994;

and

inpre-

Garfield,

Telfer

et al.,

J. Norman

92 1995).

These

findings

lead

to the

exciting

possibility

that

such conditions may be treated effectively by manipulation of NO synthesis. The role of NO in the uterine and fetoplacental circulation and the physiology and pathology of the female genital tract recently has been reviewed (Norman and Cameron, 1996; Poston et al., 1995). This review aims to describe the role of NO in myometrial physiology and pathology, and

i Ca2+

in particular, its contractile function. Clearly, the myometrium should not be considered in isolation from other tissues of the female genital tract, as signalling from other reproductive the myometrium.

2. 2.1.

THE

molecules

tissues may affect the function

\

calmodulin

MLCK activatnn

of :

MYOMETRIUM

Physiological Role

The major function of the myometrium is to contract and to expel the fetus at the end of pregnancy. To serve this purpose, the myometrium is composed largely of smooth muscle cells. However, blood and lymphatic vessels, fibroblasts, immune cells and connective

tissue are also present (Garfield

and Yallampalli, 1994). In contrast to many other smooth muscles, the myometrium spends much of its time in a “relaxed” state. Indeed, the maintenance of this state is essen-

FIGURE 1. Mechanism myosin light chain; SR,

of smooth muscle contraction. sarcoplasmic reticulum.

MLC,

tial if preterm labour and delivery are not to occur. However, during parturition and in the immediate puerperium, the myometrium has to be quickly converted to an efficiently contracting organ capable of expelling the fully grown fetus.

the binding of calcium to calmodulin. Calcium-calmodulin activates myosin light chain kinase (MLCK), which itself phosphorylates myosin. The phosphorylated myosin filaments bind to actin, and contraction occurs with the hydrolysis of ATI? Intracellular Cal+ can be increased by

2.2.

several mechanisms, and different agents are thought to operate via different routes. Firstly, voltage-gated calcium channels in the cell membrane may open in response either to

Physiology

of Myometrial

Contractions

The mechanism by which the myometrium contracts has been reviewed recently (Wray, 1993). Myometrial cells have

spontaneous

pacemaker activity or to hormonal

or neuro-

a negative membrane potential. When the magnitude of this potential is reduced beyond a certain threshold, an action potential may be stimulated. This action potential is then

nal stimulation. Secondly, calcium may be released into the cytoplasm from stores within the cell (e.g., the sarcoplasmic reticulum) in response either to inositol 1,4,5+risphosphate or to an increase in intracellular Ca2+ itself. Relaxation

converted into a contractile force. Gap junctions (intercellular channels that permit the passage of inorganic ions and small molecules) allow the orderly propagation of the action

occurs following both a fall in intracellular Ca2+ concentration, with inactivation of the calcium-calmodulin MLCK complex, and dephosphorylation of myosin light chains by

potential into other muscle cells, so that a wave of contraction can spread throughout the uterus (Garfield and Yallam-

a phosphatase.

palli, 1994). This imparts directional force and causes progressive cervical dilatation and delivery of the fetus. Myometrial contractile activity is controlled by myogenic,

2.3. Mechanism of Action of Nitric Oxide within the Myometrium

neurogenic and hormonal mechanisms. The intrinsic properties of rhe uterine smooth muscle are such that the myomerrium contracts spontaneously in the absence of any other input. This intrinsic myogenic activity is regulated by endocrine and paracrine activity, which suppresses contractile activity during pregnancy and stimulates it during parturition. Although the myometrium is innervated by postganglionic nerve fibres from the autonomic nervous system,

The smooth muscle relaxant effects of NO were described originally in blood vessels (Palmer et al., 1987). More recently, NO has been shown to cause bronchodilatation (Hogman

neurogenic mechanisms are thought to play little, if any, part in the control of human uterine activity. The mechanism by which smooth muscle cells contract is shown in Fig. 1. A rise in intracellular Ca2+ promotes

tial mechanisms by which NO may cause smooth muscle relaxation: the activation of guanylate cyclase, the stimulation of calcium-dependent potassium (Kc,:+) channels, and ADP ribosylation. The most important mechanism is

et al., 1993) and to inhibit contractions in the gastrointestinal tract (Desai et al., 1991) and the myometrium (Yallampalli et al., 1993a). Most information on the mechanism of action of NO comes from studies in vascular tissue. There are three poten-

Nitric Oxide and the Myometrium probably the activation

93

of soluble guanylate cyclase, which

catalyses the formation of cyclic GMP (cGMP) (Ignarro, 1992). This is supported by data showing that in myometrium, as in other smooth muscle, the relaxant NO or L-arginine are mimicked by 8-bromo-cGMP

actions of (a plasma-

in vascular tissue have shown that exposure to NO can downregulate NOS

activity (Buga et al., 1993; Bult et al., 1995).

Measurement

3.2.

of Nitric Oxide

permeable form of cGMP) (Izumi and Garfield, 1995; Izumi et al., 1993; Yallampalli et al., 1993b) and inhibited by

The short half-life of NO makes direct measurement difficult. Several approaches have been developed, therefore, as an

methylene

index of NO activity. One of the most widely used methods is the Griess reaction, which measures nitrite, the oxidation

blue (an inhibitor

of guanylate cyclase) (Yallam-

palli et al., 1993a,b). Despite this role for guanylate cyclase, the myometrium seems to be less sensitive to the relaxant

product

of NO (Green

et al., 1982). Briefly, nitrite reacts

effects of cGMP than vascular smooth muscle (Word et al., 1991). An alternative mechanism by which NO could cause

with the Griess reagent (1 part 0.1% naphthylethylenediamine hydrochloride and 1 part 1% sulfanilamide in 5% con-

smooth muscle relaxation is via activation of Kc,:* channels (Bolotina et al., 1994). Blockade of Kca:+ channels

centrated

causes a reduction in the cell membrane potential, an increase in intracellular Ca2+ and the stimulation of contractions

HJ’O,)

to

form

a purple

azo dye,

whose

absorbance at 546 nm can be detected by spectrophotometry and compared with a nitrite standard. NOS can be localised in vitro using NADPH diaphorase

et al., 1993). Lastly,

activity. NADPH diaphorase-positive enzymes produce a blue

NO causes ADP ribosylation in platelets (Brune and Lapetina, 1990), which ultimately may inhibit glycolysis (Kots

precipitate from nitroblue tetrazolium in the presence of NADPH, and in neuronal tissue, NADPH diaphorase activ-

ec al., 1992).

ity co-localises with NOS (Hope et al., 1991). This technique has been widely used to localise NOS in tissue sections.

in isolated myometrial

strips (Anwer

Recent evidence suggests that not all NADPH

3. NITRIC OXIDE BIOCHEMISTRY 3.1. Nitric Oxide Synthesis and Activity NO is derived from L-arginine by the action of NO synthase

diaphorase

activity is NOS, casting doubt on the validity of this technique (Tracey et al., 1993). An alternative approach is to localise the protein and mRNA for each of the three forms

(NOS) (Palmer et al., 1988). There are at least three forms of NOS. A constitutive calcium-dependent form is found

of NOS by immunocytochemistry

in endothelial cells (eNOS) and neurones (bNOS), whilst a calcium-independent, inducible form (iNOS) is present in macrophages, neutrophils and other cell types, including

tein may be achieved by Northern and Western blots, respectively. The total activity of NOS in a tissue can be quantified by measuring the conversion of L-arginine to L-citrulline.

vascular smooth muscles (Anggard, 1994; Moncada and Higgs, 1993). There is significant homology between the amino acid sequences of NOS from different species and between each of the different isoforms. Constitutive NOS releases small quantities of NO in response to stimuli such as acetylcholine, bradykinin, endothelin and shear stress. There is a dramatic amplification of inducible NOS activity in response

to factors

such as lipopolysaccharide

and y-

interferon, generating large quantities of NO. NO has a short half-life in uiwo.The majority is converted to nitrate (Yoshida et al., 1980), which is excreted in urine (Leaf et al., 1989). Oxidised

NO may also form nitrosate

molecules with sulphydryl-containing compounds (Stamler et cd., 1992), giving biologically active molecules that are more stable than NO itself. In the presence of superoxide anions (Om), NO reacts to form peroxynitrite (ONOO-), which, in turn, is oxidised to nitrate (NOi-). The effects of NO, therefore, are reduced in the presence of compounds that generate superoxide anions (such as xanthine and xanthine oxidase), and they are potentiated in the presence of superoxide dismutase, a widely located enzyme that inactivates superoxide anions. Analogues of L-arginine, such as NC-monomethyl-Larginine (L-NMMA) and NC-nitro-L-arginine methyl ester (L-NAME), attenuate the effect of NOS by competitive inhibition in viva (Furchgott et al., 1990). NOS may also be regulated by negative feedback, as studies

respectively. Quantification

3.3.

and in situ hybridisation,

of tissue NOS mRNA and pro-

Established Roles for

Nitric Oxide in Other Organs The role of NO in other organs has been reviewed extensively (Anggard, 1994; Moncada and Higgs, 1993). Whilst production of small amounts of NO is thought to be involved in physiological regulatory processes such as vasodilatation in the cardiovascular system and neurotransmission, production of large amounts of NO have been associated with pathologies such as circulatory shock and the neuronal damage that occurs in stroke. In the immune system, the production

of large amounts

of NO is involved in the killing

of foreign cells and as such, is useful to the host organism. A role for NO in tumour growth, angiogenesis sion has been demonstrated recentlv.

4.

PRODUCTION

BY MYOMETRIAL 4.1. Nonpregnant

OF NITRIC

and progres-

OXIDE

STRUCTURES Subjects

Prior to examining the effect of NO on myometrial function, it is appropriate to determine potential sources of NO, within the genital tract, both prior to and during pregnancy. Evidence from animal and human studies will be considered separately. There are no animal data on whether uterine tissue from

94

J. Norman

nonpregnant dence

individuals

can

that the nonpregnant

ucts of NO, and contains production

does

synthesising pregnant with

uterus

antibodies

raised

against

et al.,

et ul., 1995; Schmidt 1995). fibres

suggested

the

these

with

as mast

metrial

function. in the

Papka

(probably

iNOS+

cells

Prior of

However,

in organ

1995).

nonpregnant culture

NADPH

in human

diaphorase

myometrial

1995; Yoshida appeared positive

smooth

for

cells

together,

animal

and

ity is excessive,

Whether

that

prior

the

estrus

steroid

hormones.

neural

and

to pregnancy.

There

is, as yet, no evidence cells

tissue

Myometrial

presumably

muscle

NOS

under

can

both and

NO and impact

cycle,

smooth

staining

within

vascular

all may generate

not

antibody

ct al., 1995). No eNOS suggest

uterus,

but

vessels showed

studies

these system

on physiology

fibres

vessels,

cells.

human

during

rt al.,

nerve

muscle

the

varies

control

of

that isolated

generate

have

6.

can

generate

culture,

and

L-citrulline

large

that

the

uterus

(Yallampalli

were demonstrated

nancy

using

the

NADPH

that

of nitrate can

et ul.,

nerves al.,

amounts

shown convert

1993a,b).

diaphorase

tis-

nitrite

in

L-arginine

to

6.1.

Measurement

of Myometrial

The

variety

during

method

preg-

(Natuzzi

et

human

nitrite

in culture.

tween

pregnant

ability

to

(Buhimschi ising NOS

myometrium There

and

generate

can also generate

were no significant

nonpregnant nitrate/nitrite

differences

myometrium, or

in

nitrate/

cGMP

human

myometrium.

in

content

et al., 1995). No data have been published in pregnant

be-

either

However,

for

and for stimulation

difficult

to compare

tion

and Baird,

1985).

of this activity

has

in a meaningful

strips

generated

the

during

Alternatively,

of baseline

results

results

is to integrate

over the period either

to stimulate

of

can be expressed with a control

contractions,

can be expressed

e.g.,

as a percentage

activity. examining

the effect

as NO, the establishment cation

the

force and/or

approach

compared

the

be recorded.

maximum

lasts. Results

known

myometrium

transducer, can

contraction

the contraction

of the test agent

of the

to quantify

Another

for each

studied

on contractions

contraction used

of myo-

is normally

of agents end

it

is to be compared

to a tension

been

make

Standardisa-

sensitivity

function

is to calculate

of contractions. expended

studies.

if the

If one

each

have

of myometrial activity

of subjects

effect

attached

The simplest

as an effect KCI.

groups

of tissue.

methods

time for which

in different

way. Myometrial

in tjitro by examining

the force

quantification

is essential

from different

CONTRACTIONS

Contraction

of contractile

results

of methodology

mctrium

When

1993). Pregnant

of methods

activity

drug or with an agent

NOS-containing

in rat myometrium

the luteal menstrua-

MYOMETRIUM:

OF UTERINE

is fixed and the other

uterine

and

OF PREGNANT

GENERATION

frequency have

(Lumsden

aid

activ-

as dysmenor-

during during

plays a role in the control

FUNCTION

of isolated

NO.

Pregnancy of studies

clinically

in contractions

men-

would

if uterine

contractility

demonstrated

during

contractions

THE

Several

sues

been NO

obtained.

In the rat, a variety

is shed

the

yet to be investigated.

force

4.2.

increase

that

does not

localised

stained

a polyclonal

of myometrial

an

in order

However,

dur-

it is impor-

If conception

this may be manifest

can

et

contractions

in uterine

of the

is required

occurs,

endometrium

increase

function

for the endometrium.

contractility

of the shed endometrium.

and

(Telfer

smooth

cells of the immune

myometrial

expulsion

phase

(Buhimschi

blood

An

the main

is not extruded.

the decidualised

myometrium

vessels

blood

using

(Telfer

demon-

1995).

MYOMETRIUM

If fertilisation

myometrial

Inhibition

generate

cycle.

embryo

rhoea.

identified

has been

Myometrial

eNOS,

eNOS

in myometrial

Taken

activity and blood

myometrial

muscle.

staining bovine

was seen

cGMP

et al., 1995). The positively

to innervate

myometrial against

nerves

and

on myo-

has been

1993,

support

of myometrial

to inhibit

struation.

mice

stained

staining

myometrium

and contains

control

implanting occur,

of pregnancy,

is as a structural

tant

cells

throughout

the

onset

para-

nonpregnant

within

to the

myometrium

tion

human,

activity et al.,

OF NONPREGNANT

ing the menstrual

Rounded

of positively

FUNCTION

in

an iNOS-specific

and further

5.

have

studies

scattered

cycling

present

the density

1995).

against

number

diestrus-I,

of the

were

are autonomic raised

sites may impact

NOS

(Morris

and

et al., 1994; Con-

et ul., 1993a,b),

Myatt

of these

Minimal decidua

(Buttery

using and iNOS

Further

demonstrated

the syncytiotrophoblast

1995;

in either

strated

1992;

of the uterus,

of normally

during

fibres

et al.,

in both

of the placenta

Eis et al.,

1993;

production

NO

cells.

In the

al.,

nerve

(Papka

an antibody

majority

nitrite

nerves

et al., 1995). The

(Huang

was greatest

rad et al., NO

tissue

by co-localisation

and McNeill,

in the cervix.

also been

myometrium

the

Papka

whose uterus

1994).

and rat bNOS

and sensory

have

both

has been demonstrated the vascular

in the non-

porcine

of the body

that

staining

et ul.,

by immunoreactivity

some

was greatest

sympathetic) peptide

(Yallampalli

the

et cd., 1992; Shew et u2., 1993; Suburo

Although

myometrium

nerve

that

and

1994;

eviprod-

messenger

suggests

of the rat and mouse, diaphorase

(Grozdanovic

the

NO

However, oxidation

(the second

by NO),

produce

NO.

generates

nerve fibres have been demonstrated

NADPH

et al.,

cGMP

is increased

probably

generate

uterus

of the test agent

can he achieved ever, particularly

local-

lished

labour,

NOS

tissue

(Norman

simply

is essential. by stretching

in tissues stretch

of an inhibitory

of contractile

alone

obtained

activity In many

situations,

the muscle from

is insufficient

et uI., unpublished

agent

data).

such

prior to applistrip.

women

this How-

in estab-

to stimulate

the

Nitric Oxide and the Mvometrium 6.2.

95 by co-administration

Etiect of Nitric Oxide

on Myometrial Contractions In Vitro 6.2.1. Animal data. A wealth of evidence has accumulated

itor indomethacin.

of the prostaglandin

synthetase

inhib-

These findings may be explained by an

over the last few years indicating that NO is a powerful myometrial relaxant. The majority of experiments have used

increase in prostaglandin production induced by NO. It is known that NO stimulates cyclooxygenase, which is the enzyme that catalyses prostaglandin synthesis (Salvemini

myometrium removed from pregnant animals and set up in a tissue bath. NO gas (0.1 M) produced complete inhibition of spontaneous contractions of rat myometrium removed late in gestation prior to the onset of labour (Yallampalli er al., 1993a). Complete inhibition of spontaneous and

et al., 1993). Prostaglandins stimulate myometrial contractions. NO, therefore, has potentially opposing effects on uterine contractions: a direct inhibitory effect via the production of cGMP and an indirect stimulatory effect via an increase in prostaglandin synthesis. The net effect of NO in uivo may

carbachol-induced, but not KCI-induced, myometrial contractility was also effected by the application of the NO donor sodium nitroprusside (SNP) (0.1-5 mM) (Izumi and Garfield,

depend upon the physiological

1995; Yallampalli et ul., 1993a,b). Similarly, L-arginine (0.1-3 mM), the precursor

of NO,

inhibited spontaneous and carbachol-induced, but not KCIinduced, myometrial contractions (Izumi et al., 1993; Yallampalli et al., 1993a,b). This inhibition of contractility was itself abolished by inhibitors of NOS, such as 3 mM L-NAME (Yallampalli et ul., 1993a,b), or by inhibitors of guanylate cyclase, such as 0.1 mM methylene blue (Yallampalli et al., 1993a,b). Inhibitors of NOS, such as L-NAME (0.1 mM), had a small stimulatory effect on myometrial contractility when administered alone in vitro (Yallampalli et al., 1993a,b). 8-Bromo-cGMP, a plasma permeable analogue of cGMI’, inhibited spontaneous, oxytocin- and KCI-induced myometrial contractions in concentrations from lo-” M in vitro (Izumi and Garfield, 1995; Izumi et al., 1993; Yallampalli et ul., 1993b). There was a marked difference in sensitivity to 8-bromo-cGMI’, depending on the stimulating agent, with KCI-induced contractions being least sensitive to inhibition by 8-bromo-cGMI? There are fewer data on contractile activity in myometrium removed from nonpregnant animals. However, data that are available support a tocolytic (uterine relaxant) effect of NO. The NOS inhibitor

L-NMMA

inhibited

the spon-

taneous decline in the contractility of dissected uterine horns removed from rats pretreated with estrogen 24 hr previously

the enzyme required to convert L-arginine to NO. Evidence that NO is produced endogenously and inhibits spontaneous contractions of myometrial strips in viwo is provided by experiments showing that the application of inhibitors of NOS (e.g., L-NAME) stimulates activity. Conversely, one study has shown a stimulatory effect of NO on uterine contractions (Franchi er al., 1994). SNP (100 mM) was added to a tissue bath containing myometrium from nonpregnant estrogen-treated rats. Minimal contractile activity was observed prior to administration of the drug. Following the addition of SNP, a IO-min burst of uterine contractions was noted. These contractions were inhibited

at the time of

NO as a prophylactic

inhibitor

6.2.2.

There are no data showing an effect

Human

data.

of uterine

activity.

of NO gas on the contractions of myometrium obtained either from pregnant or nonpregnant women. Three studies have examined the effect of agents that release NO on contractility of isolated myometrial strips obtained from pregnant women undergoing Caesarean section. All studies showed inhibition of spontaneous and oxytocin-induced activity when amplitude or force of contractions was measured. However, NO-releasing appear to have more complex effects on frequency

agents of myo-

metrial contractions. Buhimschi et ~2. (1995) showed that diethylamine/NO (concentrations up to IO-4 M) caused a maximum reduction in “spontaneous” myometrial force to 20% of pretreatment in myometrium removed prior to the onset of labour, and to 40% of pretreatment in myometrium removed from women in active labour. In another study, glyceryl trinitrate (GTN) reduced the amplitude of spontaneous myometrial contractions by a maximum of 40%, when applied in concentrations of IO-4 M, and SNP reduced contractions

by a maximum

of 40% at 10mhM (Norman

et al.,

1995) (Fig. 2). Streptozotocin, which releases NO in response to UV radiation, increased the interval between contractions

(Franchi er al., 1994). Taken together, these experiments indicate that NO inhibits myometrial activity in e’itro in both pregnant and nonpregnant tissue. This effect is mediated by activation of guanylate cyclase and production of cGMI? Since the effects of NO are mimicked by L-arginine, the precursor of NO synthesis, it appears that the myometrium contains NOS-

conditions

administration; and it may be that when endogenous activity is minimal, NO will stimulate myometrial contractions. Clearly, this has implications for the clinical application of

70 60 5‘ cn ‘; .P .Z 8 .c $z

1

T

T

50403020-

GTN 10

SNP

1 01 -8

-7

-6

-5

-4

I -3

log [drug] M 2. Effect of GTN and SNP in vitro on amplitude of contractions in myometrial strips obtained from women undergoing Caesearean section at term prior to the onset of labour.

FIGURE

J. Norman

96 by 120%

and reduced

when

applied

1995).

the amplitude

in concentrations

Initial

contractile

heterogeneous

mixture

induced!’

of these

Each

donors

can reduce

metrial

contractions.

on which a 40%

with

The

useful

clinically

trations

M GTN,

studies

L-arginine, ous

the precursor

At a concentration taneous

contractions

(Lee

the time In both

of these

of NO,

arginine

Chang,

activity

may

of L-arginine

and L-NAME,

stimulate

1995;

et cd.,

some

Lee

of L-arginine (N-nitro-L-

of NO

and

myometrial

istered

SNP

Growing

evidence

metrium

to the inhibitory

ing the onset

of labour.

of this reduction only

suggests

that

determining

of NO.

The

Human

data.

published

over

could

in contributing

the potential

in the treatment

of preterm

from

to the onset

rats prior

3 mM L-arginine tility

completely

for 8 min,

was taken inhibited

had

trations

of 1000

similar

inhibitory

3 mM

induced

8-bromo-cGMP, inhibitory

removed times effect

completely

carbachol

higher when

were

myometrium

be involved,

since

no studies

decade

study

was followed

that

GTN

was removed

(which

deliver

over 24 hr) were applied intervals

Only

delivery

would

be undesirable,

to circumvent

of women

undergoing

this

problem

therapeutic

should

accurately

transducer.

in women

pregof this

studies

be quantified

may itself stimulate

attempted

results

in the treatment

use.

is unethical

women,

the

widespread

pressure

de-

was attributed

remaining

Although

may be effective

only

This

of preterm

one of the women

before

can

in the first

thereafter.

be awaited

in whom a

weeks

10 patches

of controlled

activity

in women

22-33

the results

such

to produce

patches

labour,

of the transducer

uterine

between

dose of 2 patches

In the

unaffected.

of

but concen-

NO

e.g., to assist dur-

and in this case, delivery

the use of an intrauterine

on contractions

have

that

or to correct

of GTN

at 24-hr

incompetence.

a transducer

NO

case reports

suggesting

of preterm Uterine

in which

for each of the 20 episodes

et

(Izumi

ges-

labour.

the uterus,

labour

of 13 women.

continued suggest

of a study

by the adminis-

uncontrolled

the effect

2 patches

and then

(1993) adminHowever,

animals

of the placenta,

hour,

nancy

but had no effect

required

must

in preterm

last

to a maximum

to cervical

when

in a concentration

Several the

to the abdomen

in a total

(Yallampalli

effect

in

(Lees rt al., 1994). D e p onit

prematurely,

labour

been

of preterm

labour

L-arginine

at mid-gestation,

a diagnosis

livered

contrac-

delivery

a change

et al., 1992; Peng er al., 1989). A more recent

taken

the tissue

during

removal

in vitro,

spontaneous

at mid-gestation,

at

than

rats as part

in these

have

has investigated

with

regimen

donors

2 min when

by 1 mM

was removed

a profound

of rat myometrium

only

effect

other

a total of 10 mg G-TN transdermally

not

myometrium was studied

in established

was removed

myometrium

ul., 1993). Similarly, 10 nM,

with

Furthermore,

contractions

myometrium when

animals

When

of NO

late preget ul., 1979;

a NO-specific

in pre-eclampsia.

be used to relax

(Bayhi

gestation

follow-

of parturition,

efficacy

of labour inhibited

compared

from

1993a).

et al.,

labour.

study

of the myo-

importance,

to our understanding

but also in determining

inversion

of the mechanism

is of crucial

during

For

activat-

is also seen at the level of cGMI?

to animals

been

is reduced

elucidation

in sensitivity

There

6.3.2. donors

of NO

platelet

observed

of pregnant

was not prolonged

as L-NMMA

1995).

effects

cause

1994).

to NO

activity

the role of NO

in e’itro (Buhim-

the sensitivity

If

mechanisms

to a group

ing manual

tions

1994).

cyclase

that

1995; Soloff

et al.,

in sensitivity

in sensitivity

was administered

The effect of nitric oxide on myometrial contracis dependent on gestation and stage of labour.

6.2.3.

to agents

6.3. Effect of Nitric Oxide on Myometrial Contractility In Vivo 6.3.1. Animal data. Yallampalli and Garfield

tration

NO

of contractile

such

activity

or guanylate

tation

by NOS.

inhibition

Chang,

that the

endogenous

of NOS,

contractile

at

of labour.

in inhibiting

of labour,

myometrial

effect

obtained

suggests

NOS

Garfield,

potential)

induce

are increased (Kim

in myo-

in gap junc-

membrane

of oxytocin,

in

in sen-

changes

and Yallampalli,

effects

labour

this change

that

sensitivity

(Garfield

for the decrease

onset

by a reduction

that

in

to agents

reduce

and

myometrium

an increase

a decrease

endothelin

the difference

effect

myometrium,

inhibitors

and

inhibitors This

production

maintain

since

in ho,

a greater

to the onset

of NOS

as in animal

production

prior

respectively.

was via the

In human

with

the inhibitory

action

1993)

in myometrium

section

studies,

et al.,

and

accounts the

spon-

and oxytocin-induced

1995),

amplitude,

of action

contraction

schi

and

in vitro.

abolished

and

or during

Yallampalli

spontane-

contractility

(Izumi

and L-NAME),

mechanism

also inhibits

It is possible

the contractile

ing factor

in

the onset

to the inhibitory

followed

(including

relaxation

example,

concen-

to NO.

and

myometrial

in sensitivity

It is not clear whether

sensitivity

contractions

inhibitory

these

the

A reduction

following

in human

pregnancy,

labour

formation

increase

nancy

contractions

by the

large concen-

hypotension

L-arginine

of Caesarean

was reversed

tion

may be

and the use of such agents

of spontaneous

than

unpublished

donors

However,

of 0.3 mM,

a mixture

on frequency

NO

myometrial

myometrial

inhibited

et al.,

that

1993).

of NO

demonstrated

for an increase

during

et al.,

effect

1995).

during

is specific

metrium

caused

in duo.

or oxytocin-induced

reason of NO

sitivity

contractions,

induce

The effect

in frequency

to have a significant

by the profound

et al.,

a 30% decrease

agents.

may be limited

has been

(Buhimschi

is uncertain.

IO-4 M SNP

(Izumi

of labour

NO myo-

animals

to the tocolytic

labour,

(Norman

trations

would

that

of human

labouring

sensitivity

is dependent

suggest

as tocolytic

was a

“oxytocin-

from

on frequency whereas

are required

study

and

and force

is studied;

effect on myometrial

latter

has demonstrated

effect

in frequency

above

of NO

in this

studies

The

lo-4

increase

data).

activity

by 50%

M (Lee and Chang,

of “spontaneous”

the amplitude

NO donor

was noted

of contractions

of 10-j

in preterm since

with

Insertion

of

labour

the insertion

contractions.

We have

by using

the model

abortion

in the second

Nitric

Oxide

and

97

the Myometrium

decidua during the last 4 days of gestation

250 ii7 ‘”

q

N. saline

1993). cGMI’, the second messenger

T

(Sladek et al.,

whose production

is

simulated by NO, is elevated in rat uterus in pregnancy, but is markedly lower at the time of delivery (Yallampalli et al., 1994). There was markedly less NOS staining (using NADPH diaphorase) in myometrial tissues obtained during labour compared with during pregnancy (Natuzzi et al., 1993). There may be species differences in the role of NO. Changes

in NO appear to be less important

during guinea

pig parturition (Weiner et al., 1994a). There was a 200-fold amplification in cGMP content (measured by radioimmuno30

assay) from mid- to late-pregnancy.

d mln before

15-30min after

0-15min after

durmg

Time in relation to infusion

difference

FIGURE 3. Effect of GTN in viva on uterine activity of women at 12-16 weeks gestation. Each woman had been given 200 mg mifepristone 46 hr previously. N. saline, normal saline.

trimester.

The effect of GTN

on myometrial

myometrial

contractions.

units. Compared

with a placebo

mea-

infusion

towards the end of pregnancy

of

ONSET

are no data on such changes in human pregnancy, although if a fall in NO activity does occur at the end of pregnancy, it may contribute to the initiation of parturition. The effects of a fall in NO would be amplified by a reduction sitivity to the relaxant effects of NO.

CONTROL

in sen-

OF PARTURITION

Animal data suggest that NO may be involved in the timing of the onset of parturition (Table 1). NOS activity is high

8.

during

If NO is important

pregnancy

TABLE

and progressively

1. NO Within

decreases

the Uterus

in rabbit

in Nonpregnant

Nonpregnant

pregnancy

In labour

Blood vessels Nerves Cells of immune Endometrium

REGULATION

PRODUCTION

Subjects,

Potential source of NO within the uterus

Late

and in labour. NO may be

less important in other animals, and caution has to be taken in extrapolating from one species to another. As yet, there

(Fig. 3). The dose of GTN applied would be expected to generate plasma levels of GTN considerably in excess of those achieved by 2 patches, each releasing 10 mg GTN in 24 hr. 7. PHYSIOLOGICAL

of L-

Taken together, these experiments suggest that in some animal species at least, there is a decrease in NO production

normal saline, a 15-min infusion of GTN at a dose of 20 ,ug/min had no significant effect on uterine contractions

OF THE

as conversion

ble effect on NOS activity, again suggesting that changes in cGMP cannot be ascribed solely to changes in NO.

surements of uterine contractions were made in these women using an intrauterine pressure transducer and calculated in Montevideo

activity (measured

changes in NOS activity are responsible for the changes in cGMI? Furthermore, administration of L-NAME had no effect on myometrial cGMP content, despite a demonstra-

contractility

Accurate

in NOS

arginine to L-citrulline) was found between pregnant and nonpregnant animals, suggesting that factors other than

was quantified in these women (Norman et al., 1995). Each woman previously had been given mifepristone (200 mg), which stimulates

After the peak in cGMP

concentration, levels fell precipitously towards the end of gestation. However, in this animal model, no significant

During

OF NITRIC BY STEROID

OXIDE HORMONES

in the control

Late Pregnancy

Relative amount of NO production within the uterus

of myometrial

and During

Labour

Sensitivity of myometrium to tocolytic effects of NO

system

Low

Low

Blood vessels Nerves Cells of immune Placenta Decidua

system

High

High

Blood vessels Nerves Cells of immune Placenta Decidua

system

Low

Low

function,

J. Norman

98

it is no surprise that NO production is at least partially regulated by sex steroids. There is conflicting evidence on the effects of estrogen and progesterone. Much evidence suggests that NO production may be augmented by estrogens, and that induction by estrogen could account for some of the increase in NO production observed during pregnancy. In uie’o, administration of estradiol increased calciumdependent, but not calcium-independent, NOS activity in guinea-pig heart, kidney, skeletal muscle and cerebellum, with a maximum response after 10 days of treatment (Weiner et al., 1994b). In pregnancy,

the increase in NOS

activity

normally observed was prevented by the estrogen antagonist tamoxifen (Weiner et al., 1994~). In contrast, Yallampalli et ~1. (1994) have shown that estradiol or estradiol and progesterone treatment resulted in lower nitrite/cGMP content in rat uterus, compared with animals treated with vehicle

NOS inhibition (Molnar et al., 1994; Yallampalli and Garfield, 1993). Thus, NO may be essential for the maintenance of growth and of normal maternal blood pressure during pregnancy, and a reduction in NO synthesis may be implicated in intrauterine growth retardation and preeclampsia. However, these experiments do not support the theory that NO is involved in the prevention of preterm labour. A possible explanation is that some NOS activity was maintained within the genital tract of the treated rats. Alternatively, a reduction in NO may have been associated with a reduction in prostaglandin synthesis (Salvemini et ul., 1993), which would attenuate the stimulatory a reduction in NO on myometrial contractions.

10.

CONCLUSION

only. Further work indicates that the positive effect of estradiol

Data from both

may be limited to certain cell types, and that NOS in other cells may be inhibited by estradiol. In mice, iNOS+ stain-

nancy. NO normally

ing in mast cells and macrophages was low in ovariectomised mice, and was increased by the administration of estradiol or estradiol and progesterone, but not by progesterone alone (Huang etul., 1995). In contrast, epithelial cells showed positive staining for iNOS after the administration of progesterone

or estradiol and progesterone,

but not by

estradiol alone. Since the promoter region of the iNOS gene does not possess a classical estrogen, progesterone or glucocorticoid response gene (DiRosa et al., 1990), it seems likely that the effects of steroid hormones

on iNOS (and presum-

ably constitutive NOS) expression are mediated via other cytokines. This may explain why estradiol and progesterone have apparently different effects in different tissues and under different physiological conditions. A recent study has investigated the effect of estrogen on NOS activity in women (Ramsay et al., 1995). Thirty volunteers of reproductive age were given monthly depot injection of the gonadotrophin releasing hormone analogue decapeptyl. After 1 month, estradiol(2 mg daily) or placebo \vasadded to the treatment regimen. In the 3rd month, the treatment arms were reversed. Fasting plasma nitrate concentrations were significantly higher during the estradiol treatment phase, again suggesting that estradiol stimulated NO production. Nitrite concentrations did not differ between the t\vo groups.

9. EFFECT OF INHIBITORS AND DONORS OF NITRIC OXIDE DURING

PREGNANCY

Several groups have used animal models to investigate the effect of in e~iuo inhibition of NO synthesis. In each of three studies where L-NAME was administered during pregnancy, a significant reduction in fetal weight was observed (Diket er nl., 1994; Molnar et ul., 1994; Yallampalli and Garfield, 1993). In two studies, an increase in maternal blood pressure was observed (Molnar et ul., 1994; Yallampalli and Garfield, 1993). Gestational length did not appear to be affected by

effects of

animal and human

studies indicate that

NO is produced within the female genital tract during pregalthough

inhibits

uterine contractility

in one study, a stimulatory

in vitro,

effect was observed.

Preliminary human data suggest that NO may also inhibit myometrial contractions in ho. The mechanism of action of NO is probably the stimulation of guanylate cyclase, leading to the production of cGMP and a reduction in intracellular Ca?+. The physiological role of NO is probably to inhibit myometrial contractions synthesis initiation

during pregnancy. A reduction

at the time of parturition of labour. The inhibitory

in NO

may contribute to the effect of NO on myo-

metrial contractions would also be useful therapeutically in the treatment of preterm labour. Preterm labour is a major cause of perinatal death, and there are no agents currently available for treatment that improves fetal outcome. Further studies are required to determine the role of NO in myometrial physiology and pathology, and the conditions for which modification of NO may be useful therapeutically. NO has had an enormous pharmacological impact in cardiovascular and respiratory disease, and may have a similar impact in pathology

of the myometrium.

Acknowl&emem-I an grateful ro Professors William Martin and Ian Cameron for rhex wmmcnts on [his manuscrqx. Our work is supported bv grant\ from the Medical Research COUIICII, Wellbeing, Yorkhill NHS Trusr Research Support Group and Tenovus Scorland. foor which WC are vcrv grateful. 1 thank Linda Ward for excellenr rechnical help.

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