Nov 30, 1993 - guanosine monophosphate (cGMP) pathway in the rat uterus and that NO inhibits contractility during pregnancy but not during delivery.
0013-7227/94/1344-1971$03.00/0 Endocrinology Copyright 0 1994 by The Endocrine
Vol. 134, No. 4 Printed in U.S.A.
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STEROID HORMONES
MODULATE THE PRODUCTION AND cGMP IN THE RAT UTERUS
OF NITRIC
OXIDE
C!handrase.khar Yallampalli,DVM, F’h.D,Mary Byam-Smith,M.S..Sharon 0. Nelson, Ph.D.* and Robert E. Garfield, Ph.D. Departments of Obstetricsand Gynecology and Anesthesiology* The University of TexasMedical Branch Galveston Galveston,Texas ABSTRACT: deviously, we demonstrated the presence of an L-arginine-nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) pathway in the rat uterus and that NO inhibits contractility during pregnancy but not during delivery. In the present study, we investigated the possible role of sex steroid hormonesin the regulation of NO synthesisand cGME generation. NO, measured as nitrite production, and cGMP were determined in full thicknessuterine tissues from either pregnant rats on different gestational days or nonpregnant animals after treatment with steroid hormones. NO formation was low in tissues from nonpregnant rats, substantially increased during pregnancyand decreasedduring labor and immediately postpartum. The cGME content in the same tissues followed a similar trend. Uterine nitrite production and cGMF’ levels from animals treated with estradiol and estradiol + progesterone were significantly lower compared to those treated with vehicle or progesterone. Thesedata provide strong evidencethat the NO-cGMP system is upregulated during pregnancy to maintain uterine quiescenceand a rise in estrogen at term could inhibit this systemand thus initiate labor.
Uterine contractility is well known to be influenced by female sex steroid hormones (10,ll). During pregnancy in many species, including rat, the myomeuium is relatively quiescentwhen progesterone levels are elevated. At term, when progesteronelevels decline and estrogen levels rise, there is a sharp increasein myometrial activity that culminates with the expulsion of pups. The changes in the NO-cGMErelaxation pathway during pregnancy and parturition could be regulated by changes in steroid hormone levels. Indeed, some studies suggest that the production and actions of NO in vascular tissue are modulated by steroid hormones (12). However, there are no studies indicating what controls the change in synthesisor action of NO in the uterus. Therefore, these studieswere designed to determine if pregnancy and the sex steroid hormones modulate the NO-cGMP syntheticpathway in the uterus. Animals: Rats that were either non pregnant, pregnant on day 15 of gestation or prepubertal on day 16 of life were received from Harlan-Sprague-Dawley (Houston, TX). Pregnant animals were sacrificed on day 18 of gestation, at term during spontaneousdelivery (one to three pups delivered) and on day 1 postpartum. The prepubertal rats were weaned on day 20 and were injected S.C.daily for 3 days, with either Sug 17pestradiol, 1 mg progesterone, estradiol plus progesterone in 0.2ml of sesameoil, or vehicle only. Non pregnant animals were used on unspecified days of the estrus cycle. All animals were sacrificed in a CO* inhalation chamber.
Nitric oxide (NO) is now INTRODUCTION: recognized as the principal mediator of several functions including the relaxation of smooth musclesin a variety of tissues (l-5). Nitric oxide is generated from L-arginine by NO synthaseand multiple isoforms of this enzyme have been reported (6). The effects of NO are mediated via activation of soluble guanylate cyclase and subsequent production of cGMP (7,8). Recently we have reported the existence of an Larginine-NO-cGMP system in the rat uterus and showed that the system inhibits uterine contractility during gestation but not during delivery and immediately postpartum (9). Nitric oxide is generated in the uterus, and both substrate and donors of NO produce relaxation of the rat uterus. Furthermore, inhibitors of both NO synthaseand guanylate cyclase block the effects of L-arginine. However, the relaxation effects of the L-arginine-NO-cGMP system are substantially decreased during spontaneous &livery at term and during preterm delivery after antiprogesterone treatment. These studies provided convincing evidence that an L-arginine NO-cGMPrelaxation pathway could contribute to the maintenance of uterine quiescenceduring pregnancy and a change in this pathway could lead to the initiation of term or preterm labor. Date Received 11/30/93
Tissue incubations and nitrite assay: Uterine tissues, obtained from rats on different days of gestation or at the end of in vivo treatments,were rinsed thoroughly in minimum essential medium (MEM, Gibco, Grand Island, N.Y.). Full thickness tissue pieces were incubated in MEM in a COZ incubator with humidified chamber at 37°C for 24 hours. Following the incubations the medium was collected for quantitation of nitrites. Nitrates in the medium were reduced to nitrites by the acid treated cadmium method (13) and the total nitrites were measured by the Griess method (14). Briefly, Griess reagent was added to 1OOul aliquots of medium and optical densities were measuredat 550nm in a microtitre plate, using NaNOZ as a standard.
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of cGMP levels: Uterine tissuepieces were rinsed and equilibrated in Krebs solution, continuously gased with 5% COz - 95% O2 for one hour. The tissues were rapidly frozen in liquid nitrogen, placed in ice-cold 10% trichloroacetic acid and homogenized. The homogenate was centrifuged and the supematant was neutralized by the addition of CaCOr. The samples were clarified by low speed centrifugation and aliquots were assayedfor cGMP by radioimmunoassay (Biotrack cyclic GMB lZI Assay System with Amerlex TM-M Magnetic separation from Amersham, Arlington Heights, IL).
Determination
Statistical Analysis: Resultsate expressedas means+ SEM. Data were analyzed for statistical differences using oneway analysisof varianceand the differences were consideredsignificant if the P value was lessthan 0.05. w: In order to evaluate if the generation of NO and cGh4P were elevated during pregnancy and decreased during parturition, we measured the production of NO and cGMP by uterine tissuesfrom rats either nonpregnant or at different stages of gestation. Total nitrites produced by uterine tissues were low in nonpregnant rats (lOO%), substantially elevated during the mid stageof gestation (600%) and dramatically decreased at the time of spontaneous delivery (3%) and day 1 postpartum (10%) (Figure 1).
600
r-
*
b
NP
NONDEL
PP
DEL
Figure 2: Tissue cGMP content of full-thickness tissues from uterus obtained from non pregnant (NP); pregnant, on day 18 of gestation (NON DEL); spontaneously delivering (DEL) at term and day 1 postpartum (PP) rats. Values are means + SEM for tissues from 4 - 6 animals in each group. Bars with different letters at the top vary signtficantly.
To evaluate whether changes in uterine nitrite and cGMP are modulated by female sex steroid hormones, we measured the generation of nitrites and cGMP by the uterus from steroid hormone-treated rats. In order to reduce the variability commonly associatedwith the estruscycle, we utilized prepubertal rats. Total nitrites were significantly lower in uterine tissues from estradiol and estradiol + progesterone-treatedanimals compared to tissuesfrom animals receiving vehicle or progesteronealone (Figure 3). Nitric oxide production was substantially reduced in the presence of nitro-larginine methyl ester (data not shown) indicating the involvement of NO synthase.
a
NONDEL
DEL
PP
Figure 1: Total nitrites (nitrites + nitrates) produced by uterine tissues from nonpregnant (NP); pregnant on day 18 of gestation (NON DEL); spontaneously delivering at term (DEL) and day 1 postpartum (PP) rats. Values are means k SEA4 for tissues from 6 animals in each group. Bars with different letters at the top vary significantly.
The uterine content of cGMP in the same tissues followed a similar trend. cGMP levels were increased during pregnancy and decreasedduring parturition and postpartum (Figure 2).
b
b CONTROL
E
P
EAP
,
Figure 3: Total nitrites (nitrites + nitrates) produced by uterine tissues from prepubertal female rats treated with 5ug estradiol-178 (E), 1 mg progesterone (P), E + P or vehicle only (CONTROL). Values are means + SEA4 for tissues from S-6 animals in each group. Bars with dtflerent letters vary significantly.
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The changes in cGMP levels in steroid hormonetreated animals were similar to the changes in nitrite production (Figure 4). Uterine cGMP levels were significantly lower in estradiol-and estradiol + progesterone-treated animals compared to animals receiving vehicle or progesterone alone. a 30
t T
a
mz; ;
20 b
b
E” 2 15 c ;E’ 10 8 ”
5-
Ob
I CONTROL
E
P
E+P
Figure 4: Tissue cGMP content of Ihe uterus from prepubertal female rats treated with Sug estradiol-17p (E), I mg progesterone (P), E + P or vehicle only (CONTROL). Values are means + SEM for tissues from 5-6 animals in each group. Bars with different letters at the top vary significantly. B: These studies provide persuasive evidence to indicate that uterine NO production and cGMP generation are increased during pregnancy and decreased during parturition and postpartum. The synthesis of both NO and cGMP are substantially reduced by estradiol treatment. These studies clearly indicate that the NO-cGMP system is upregulated during gestation to maintain uterine quiescence and a fall in progesterone and a rise in estmdiol levels at term (10) could inhibit this system, thus facilitating labor and delivery. Our previous studies demonstrate the presence of an Larginine-NO-cGMP-relaxation system in the rat uterus (9). The major observations were that 1) the substrate and donors of NO caused relaxation, 2) inhibitors of NO synthase and soluble guanylate cyclase blocked the relaxation, 3) NO synthasc is localized to several cell types in the uterus, 4) NO and cGMP effects were decreased during delivery and 5) the cGMP-effector system is decreased during term and preterm delivery. Combined with the results from the previous study, the present findings provide strong evidence that the Larginine-NO-cGMP-effector pathway is increased during pregnancy to maintain uterine quiescence and decreased at term in response to steroid hormones to increase uterine contractility and initiate labor.
There are no other studies demonstrating hormonal control of the generation of NO and cGMP in the uterus during pregnancy. Our results showing that NO and cGMP generation by the uterus is substantially increased during pregnancy and decreased at term agrees well with other studies demonstrating changes in urine NO synthase activity (15,16) and a generalized increase in plasma and urinary levels of nitrites and cGMP (17). Changes in the levels of cGMP in the uterus reflect the generation of NO, indicating that in the rat uterus cGMP generation is primarily dependent upon NO production. The basal production of nitrites and cGMP per unit uterine tissue in adult rats (Figs 1 and 2) appeared to be lower than that in prepubertal rats (Figs 3 and 4). This may be due to the differences in the proportion of various uterine compartments contributing to the production of NO. However, compartmental changes could not account for the variation in NO and cGMP in tissues from pregnant and hormonally-treated rats because these conditions do not influence the size of any components to the extent needed to produce the observed effects. The underlying mechanism(s) controlling the increased NO and cGMP generation during pregnancy is not explained by our data. Although progesterone is well recognized as a major gestational hormone regulating uterine contractility, the precise events involved in the actions of this hormone during pregnancy are not well understood. We propose that progesterone upregulates NO synthesis in the uterus. This suggestion is supported by the present findings and by our previous studies demonstrating that the relaxation effects of Larginine in the uterus are increased during pregnancy and decreased at term (9). However, in the present study, the effects of progesterone on NO synthesis in the non pregnant uterus are not apparent. Estrogens stimulate progesterone receptors, and estrogens and progesterone generally have opposing actions. Progesterone alone was not effective in increasing NO and cGMP synthesis (Figs 3 and 4). However, estrogen priming prior to progesterone administration may be necessary for progesterone to be efficient and this may not be equivalent to treatment with estrogen and progesterone for the same time period (Figs 3 and were not systematically 4). These possibilities examined in the present study. It is possible that progesterone upregulates a specific isoform of NO synthasc in the uterus. Pregnancy-associated increases in NO synthesis could also be related to the effects of cytokines, because 1) cytokines modulate NO production in a variety of tissues (6.18) and 2) levels of cytokines in the uterus vary during pregnancy (19). In addition, NO generation in the uterus may be related to innervation. NO presence in uterine nerves has been demonstrated in tissues from non pregnant rats (20) and NO is thought to be involved in CGRP-induced relaxation (20). However, nerves in the uterus are thought to degenerate during late stages of pregnancy (21). Further studies are needed to examine the
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