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Endocrinology Vol. 141, No. 7 2458-2464
Copyright © 2000 by The Endocrine Society

ARTICLES

Inhibition of Nitric Oxide Synthase Activity Diminishes the Acute Effects of Relaxin on Growth, But Not Softening, of the Cervix in the Rat1

O. D. Sherwood, L. M. Olson, S. Zhao and H. R. Little

Department of Molecular and Integrative Physiology (O.D.S., S.Z., H.R.L.) and College of Medicine (O.D.S.), University of Illinois at Urbana-Champaign, Urbana, Illinois 61801; and Department of Obstetrics and Gynecology, Washington University School of Medicine (L.M.O.), St. Louis, Missouri 63110

Address all correspondence and requests for reprints to: Dr. O. D. Sherwood, Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign, 524 Burrill Hall, 407 South Goodwin Avenue, Urbana, Illinois 61801. E-mail: od-sherw{at}uiuc.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Relaxin promotes growth and softening of the cervix during pregnancy in the rat. This study examined the hypothesis that nitric oxide (NO) mediates the effects of relaxin on the rat cervix. To test that hypothesis, N{omega}-nitro-L-arginine methyl ester (L-NAME) was used to inhibit NO synthase, the enzyme that converts arginine to NO and L-citrulline. Nonpregnant rats were ovariectomized when they were 78 days old (day 1 of treatment). At ovariectomy each animal was fitted with silicon tubing implants containing progesterone (P) and estrogen (E) in doses that provide blood levels similar to those during late pregnancy. Rats were assigned to three treatment groups. The control group OPE (n = 6 rats) received 0.5 ml L-NAME vehicle (PBS) sc at 6-h intervals from 0600 h on day 7 through 1200 h on day 8 and 0.5 ml relaxin vehicle (PBS) sc at 0600 and 1200 h on day 8. Group OPER (n = 6 rats) was treated in the same way as group OPE, except that 20 µg porcine relaxin were administered. Group OPERI (n = 7 rats) was treated in the same way as group OPER, except that L-NAME was administered at a dose of 100 mg/kg·6 h. Between 1400–1500 h on day 8, the cervices were removed and weighed. Cervical wet weight and extensibility were markedly greater (P < 0.01) in relaxin-treated group OPER rats than in group OPE controls. Treatment with L-NAME diminished relaxin’s effects on cervical wet weight, but not cervical extensibility. In conclusion, this study provides evidence that NO contributes to the acute effects of relaxin on the growth, but not the softening, of the rat cervix.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
RELAXIN is produced and secreted by the corpora lutea during the second half of pregnancy in the rat (1). One vital physiological role of relaxin throughout this period is to promote the progressive and marked growth and softening of the cervix that is required for rapid and safe delivery (2, 3, 4, 5). Studies at the light microscope level demonstrated that relaxin increases the accumulation of new epithelial and stromal cells (6, 7), reduces the organization and density of collagen fiber bundles (6), reduces the length of elastin fibers (6), and increases the cross-sectional area of arteries (6).

The molecular mechanisms that mediate the effects of relaxin in the cervix are poorly understood. The free radical molecule nitric oxide (NO) is known to play a key role in the regulation of mammalian physiology through its effects in cardiovascular, nervous, renal and other systems (8, 9, 10). NO is produced from one of the two guanidino moieties of the amino acid L-arginine in a reaction catalyzed by the oxidoreductase enzyme NO synthase (NOS) with L-citrulline as coproduct. There are three isoforms of NOS: the so-called constitutive isoforms, endothelial NOS (eNOS or NOS-III) and neuronal NOS (nNOS or NOS-I), and inducible NOS (iNOS or NOS-II). All three NOS isoforms are expressed in the rat cervix during pregnancy (11, 12). There are reasons to suspect that the effects of relaxin on the rat cervix are mediated at least in part through NO. When NO synthesis was inhibited with N{omega}-nitro-L-arginine methyl ester (L-NAME) throughout the last 6 days of rat pregnancy, the duration of delivery was prolonged (11). Additionally, when cervices were removed on day 20 of gestation and incubated overnight with L-NAME, they were less extensible than controls (11).

Several in vitro studies provide direct evidence that endogenous production of NO plays a role in mediating the effects of relaxin on several target cells and organs other than the cervix. Relaxin increased NO production by bovine aorta smooth muscle cells (13) and both rat and rabbit perfused hearts (14, 15). Also, it was recently reported that the inhibitory effect of relaxin on the contractility of the mouse uterus was blunted by the NOS inhibitor N{omega}-nitro-L-arginine (16).

In view of the evidence that 1) relaxin plays a major role in promoting cervical growth and softening in the rat, 2) inhibition of cervical NOS decreases cervical extensibility in rats, and 3) NOS plays a role in mediating the effects of relaxin in numerous target cells and organs other than the cervix, we hypothesized that the effects of relaxin on growth and/or softening of the rat cervix are mediated at least in part through NO. The present study examined that hypothesis by blocking NO synthesis in vivo in relaxin-treated rats.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Relaxin-induced growth and softening of the cervix does not normally occur in nonpregnant rats. Nevertheless, as in a recent study (17), nonpregnant rats were used for several reasons. First, it is possible to administer uniform doses of progesterone, estrogen, and relaxin and thereby limit variations in blood levels of these hormones. Second, it is possible to administer acutely a high dose of porcine relaxin that provides a marked increase in cervical wet weight and softening within 8 h of the initial relaxin injection (17). This short duration of relaxin treatment permits the use of an extremely high dose of NOS inhibitor, one that would probably be too toxic for a prolonged treatment period. Finally, by employing nonpregnant rats, it is possible to avoid the confounding effects that the high dose of NOS inhibitor might cause through detrimental effects on the fetuses and placentas. Prolonged blockade of NOS with L-NAME during late pregnancy produces signs similar to those of preeclampsia in rats. There is marked hypertension and growth retardation of the fetuses (18, 19, 20, 21). Also justifying the use of nonpregnant rats is the fact that the effects of relaxin on the rat cervix, uterus, and vagina were either discovered or have been demonstrated in nonpregnant rats (1, 22).

Nonpregnant female Sprague Dawley-derived rats were obtained at approximately 70 days of age from Harlan Sprague Dawley, Inc. (Indianapolis, IN). Rats were housed individually in a temperature (23–25 C)- and light-controlled room, with alternating 14 h of light (2100–1100 h) and 10 h of darkness. Rats were provided free access to water and Teklad 6% Mouse/Rat Diet (Harlan/Teklad, Madison, WI). The animal experimentation described in this report was approved by the University of Illinois at Urbana-Champaign laboratory animal care advisory committee.

Exp 1: surgical and hormonal treatment
Exp 1 was conducted to determine whether the administration of L-NAME inhibited both NOS activity and the acute stimulatory effect of relaxin on cervical wet weight. Nineteen 78-day-old rats were anesthetized with ether and bilaterally ovariectomized between 0900–1100 h on day 1 of treatment via a midline incision into the abdominal cavity (23). Rats were randomly assigned to one of three treatment groups: 1) ovariectomized (O) and treated with progesterone (P) and estrogen (E) (group OPE; n = 6); 2) ovariectomized and treated with progesterone, estrogen, and relaxin (R; group OPER; n = 6); 3) ovariectomized and treated with progesterone, estrogen, relaxin, and the nonselective NOS inhibitor (I) N{omega}-nitro-L-arginine methyl ester (L-NAME; group OPERI; n = 7; see Fig. 1Go).



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  		Figure 1. Diagram of the experimental design for Exp 1. See Materials and Methods for details.

 
Progesterone and estrogen were administered to all 19 animals throughout the 8-day treatment period following the procedure described by Burger and Sherwood (5). Because the effects of relaxin on the cervix are estrogen dependent (1), treatment with estrogen was necessary. Moreover, it was considered advantageous to use a procedure that provides serum levels of both progesterone and estrogen similar to those during the second half of rat pregnancy (5). In brief, progesterone capsules were constructed from 52-mm lengths of SILASTIC brand silicon tubing (Dow Corning Corp., Midland, MI; id, 1.5 mm; od, 1.9 mm). The tubing was sealed at one end with a 1-mm SILASTIC glue plug (Dow Corning Corp.), filled with 60 mg crystalline progesterone (Sigma, St. Louis, MO), and the open end was closed with a glue plug. Progesterone implants were rinsed twice in 100% ethanol and stored at room temperature. On the day before ovariectomy, progesterone implants were soaked in PBS (0.14 M NaCl and 0.01 M NaPO4, pH 7.0) at 25 C overnight. Immediately after ovariectomy on day 1, two capsules were inserted sc over each flank with a 10-gauge trochar (Innovative Research of America, Toledo, OH).

Estrogen was administered in capsules constructed from 26-mm lengths of SILASTIC tubing (id, 1.6 mm; od, 3.2 mm; Dow Corning Corp.) that were filled with a 2-cm column of 17ß-estradiol (Sigma) dissolved at a concentration of 300 µg/ml in sesame oil (Sigma) and sealed with 3-mm SILASTIC glue plugs on each end. SILASTIC implants contained 12 µg 17ß-estradiol. As with the progesterone implants, estrogen implants were rinsed twice in 100% ethanol and incubated overnight in PBS before surgical insertion. A single estrogen implant was inserted sc over the spine through a skin incision that was located anterior to the skin incision used for the insertion of progesterone implants.

Rats were administered L-NAME (Sigma; 100 mg/kg BW 100 mg/kg/bh; group OPERI) or L-NAME vehicle (0.5 ml PBS; groups OPE and OPER) sc at 6-h intervals beginning at 0600 h on day 7 of treatment and ending at 1200 h on day 8 of treatment.

Highly purified porcine relaxin (24) (groups OPER and OPERI) or relaxin vehicle (0.5 ml PBS; group OPE) was administered sc at 0600 and 1200 h on day 8 of treatment. A dose of 20 µg porcine relaxin was used per injection because a previous study demonstrated that this dose stimulates marked cervical growth and softening within 8 h in nonpregnant rats (17).

Exp 1: influence of L-NAME on heart rate and systolic blood pressure
Between 0900–1200 h on days 6, 7, and 8 of treatment the rats were warmed to 30 C, and systolic blood pressure and heart rate were measured in the unanesthetized animals using tail-cuff plethysmography (25). Systolic blood pressure measurements were made with an IITC model 31 computerized blood pressure monitor (IITC, Inc./Life Sciences Instruments, Woodland Hills, CA) according to the manufacturer’s instructions. The average of three traces obtained without interfering movements on the part of the rat was recorded for each rat.

Exp 1: influence of L-NAME on urinary levels of nitrites/nitrates
On day 7 of treatment, animals were transferred to metabolic cages. To stimulate urination, 6 ml physiological saline were administered at 0600 h on day 8 when the collection of urine began. At 1400 h on day 8, the volume of urine collected was recorded, and 2-ml aliquots were frozen until levels of nitrites (nitrate plus nitrite) were determined. Urine samples were filtered through a 10,000 mol wt cut-off filter Ultrafree Microcentrifuge filter unit (Millipore Corp., Bedford, MA) at 8000 x g for 15 min at 25 C to remove hemoglobin. The amount of nitrite present in each 20-µl sample was determined after reduction of nitrates with nitrate reductase as described by Misko et al. (26). The amount of protein in each sample was determined with the bicinchoninic acid (BCA) method (27) using the BCA protein assay reagent kit (Pierce Chemical Co., Rockford, IL).

Exp 1: influence of relaxin and L-NAME on cervical wet weight and cervical NOS activity
Between 1400–1500 h on day 8, the animals were anesthetized with ether and killed by cervical dislocation. Their cervices were removed, trimmed of fat and connective tissue, weighed on an electronic analytical balance (H110, Sartorius Corp., Bohemia, NY), and frozen in liquid nitrogen. The cervices were maintained at -70 C until NOS activity levels were determined.

Individual cervices were crushed in a stainless steel chamber that was maintained at -195 C with liquid nitrogen. The pulverized cervical tissue was placed in 250 µl lysis buffer [50 mM sucrose, 25 mM HEPES buffer (pH 7.4), and 1 mM dithiothreitol] containing 10 µg/ml leupeptin, pepstatin A, chymostatin, aprotinin, antipain, soybean trypsin inhibitor, and 100 µg/ml phenylmethylsulfonylfluoride and sonicated three times for 10 sec each time. The mixture was centrifuged at 6000 rpm for 10 min at 4 C to collect the supernatant. A 20-µl portion of the supernatant was removed for determination of protein concentration using the BCA procedure (27).

NOS activity was determined by measuring the ability of cervical supernatants to convert [14C]arginine to [14C]citrulline following the method of Conrad et al. (28) with modifications. Briefly, 100 µl of a 2 x reaction buffer containing 20 µM L-arginine, 4 µCi/ml [14C]arginine (SA, 240 mCi/mmol; American Radiolabeled Chemicals, St. Louis, MO), 1 mM NADPH, 2.5 mM CaCl2, 20 µg/ml calmodulin, 20 µM tetrahydropterin, 8 µM FAD, 2 mM magnesium acetate, and 25 mM HEPES were added to 100 µl cervical supernatant, and the mixture was incubated for 45–60 min in a shaking water bath at 37 C. The reaction was stopped by the addition of 0.9 ml stop buffer (100 mM HEPES and 10 mM EDTA, pH 5.5) at 4 C, and the [14C]citrulline was resolved over columns containing 1 ml of the cationic exchange resin AG 50W-X8 (100–200 mesh, sodium form, Bio-Rad Laboratories, Inc., Hercules, CA) in 100 mM HEPES and 10 mM EDTA, pH 4.3. The columns were washed with 1 ml stop buffer, the flow-through and wash were placed in 10 ml Bio-Safe II liquid scintillation buffer (Research Products International, Mount Prospect, IL), and the quantity of [14C]citrulline was determined with a liquid scintillation counter (model 2500, Hewlett-Packard Co., Roseville, CA). The specificity of the reaction was controlled by including a sample containing reaction buffer and lysis buffer, but no tissue extract. In addition, reaction of remaining cervical supernatant was conducted in the presence of 1 mM L-NAME. In every assay L-NAME completely inhibited NOS activity. The amount of [14C]citrulline produced by the cervical homogenates was determined as picomolar concentrations per µM protein. Each cervical sample (n = 6 or 7/group) was tested in duplicate.

Exp 2: surgical and hormonal treatment
Exp 2 was conducted for two reasons: first, to determine whether administration of L-NAME inhibited relaxin-induced cervical softening, and second, to determine whether administration of the NOS substrate L-arginine reversed the influence of L-NAME on the cervix in relaxin-treated rats.

Thirty-four 78-day-old rats were anesthetized with ether and bilaterally ovariectomized via a midline incision into the abdominal cavity (23). Rats were randomly assigned to one of four treatment groups. Three of the treatment groups were treated as described for groups OPE, OPER, and OPERI in Exp 1 (Fig. 1Go). A fourth group (OPERIA) was treated as described for group OPERI and also injected sc with L-arginine (A) at a dose of 200 mg/kg BW·6 h.

Exp 2: influence of relaxin and L-NAME on cervical softening
Between 1400–1500 h on day 8, the cervices were collected, weighed, and placed in Krebs-Ringer bicarbonate buffer, pH 7.5, at 4 C until their extensibilities were determined within 1 h of tissue collection. Cervical extensibility was determined as previously described (17, 29). In brief, each cervix was suspended between two metal hooks (1.3-mm diameter stainless steel), with the lower hook fixed in position, and the upper mobile hook connected to a Grass FT103 force displacement transducer (Grass Instruments, Quincy, MA). The cervix was placed in a 60-ml organ bath containing Krebs-Ringer bicarbonate buffer, pH 7.5, which was oxygenated with 95% O2-5% CO2. The temperature was maintained at 37 C by circulating water through the outer chamber of the organ bath. Transducers were calibrated in grams before use, and tension generated within the cervices was expressed in grams. Outputs from the transducers were recorded on a Macintosh computer with a MacLab/4 data acquisition system (AD Instruments, Mountain View, CA). For each cervix, the distance between the two hooks was gradually increased until approximately 5 g tension were recorded. This was designated the resting tension of the cervix. The distance between the two hooks was then increased by 1-mm increments at 15-min intervals until a total of 6-mm extension was applied. Tension that developed at each extension was determined. Linear regressions of grams of tension per mm extension were used to compare the effects of treatment on the tensile properties of the cervix.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Exp 1: influence of L-NAME on heart rate and systolic blood pressure
The administration of L-NAME to group OPERI on days 7 and 8 reduced heart rate (P < 0.05) to levels below those in groups OPE and OPER (Fig. 2AGo). Heart rate was reduced modestly (P < 0.05) in rats treated with relaxin alone on day 8. Systolic blood pressure was not significantly greater on days 7 and 8 in group OPERI than in groups OPE and OPER (Fig. 2BGo).



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  		Figure 2. Influence of L-NAME on heart rate (A) and systolic blood pressure (B) on days 6–8 of treatment. All values are means (±SE). Values with different superscripts differ (P < 0.05). The number of rats per group is indicated at the base of each bar.

 
Exp 1: influence of L-NAME on urine levels of nitrites/nitrates
Whereas there was no difference in the volume of urine collected among the three treatment groups on day 8 (data not shown), the concentration of urine nitrites/nitrates was markedly lower (P < 0.01) in group OPERI than in the other two treatment groups (Fig. 3AGo).



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  		Figure 3. Effects of L-NAME on urinary levels of nitrites/nitrates (A), wet weight of the cervix (B), and cervical NOS activity (C). All values are means (±SE). Values with different superscripts differ (P < 0.01). The number of rats per group is indicated at the base of each bar.

 
Exp 1: influence of L-NAME on cervical wet weight and cervical NOS activity
The mean wet weight of cervices in relaxin-treated OPER rats was approximately 80% greater (P < 0.01) than that in the relaxin-deficient OPE control rats (Fig. 3BGo). The mean cervical weight in rats treated with L-NAME plus relaxin (group OPERI rats) was lower (P < 0.01) than that in group OPER rats, but higher (P < 0.01) than that in group OPE controls.

Relaxin did not increase cervical NOS activity. Levels of NOS activity in group OPER did not differ from those in group OPE (Fig. 3CGo). The administration of L-NAME on days 7 and 8 reduced NOS activity. Cervical citrulline levels in group OPERI were below those in group OPE (P < 0.01).

Exp 2: influence of relaxin and L-NAME on cervical softening
Consistent with Exp 1, the mean wet weight of cervices in relaxin-treated OPER rats was far greater (P < 0.01) than that in relaxin-deficient OPE control rats (Fig. 4AGo). Also, the mean cervical weight in rats treated with L-NAME plus relaxin (group OPERI rats) was lower (P < 0.05) than that in group OPER rats, but higher (P < 0.01) than that in group OPE controls. The mean cervical weight in rats treated with L-arginine plus L-NAME (group OPERIA) did not differ from that in group OPERI.



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  		Figure 4. Influence of relaxin and L-NAME on the wet weight (A) and tension (B) of cervices at extension. All values are means (±SE). Values with different superscripts differ (P < 0.01). The number of rats per group is indicated at the base of each bar.

 
The mean tension with extension of cervices from group OPER rats was approximately half (P < 0.01) that in cervices from group OPE rats, and it did not differ from that in cervices from group OPERI and OPERIA rats (Fig. 4BGo).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This report provides evidence that NO contributes to relaxin’s acute effects on growth, but not softening, of the cervix in the rat. Because the rat cervix contains eNOS, iNOS, and nNOS (11, 12), the nonselective and irreversible NOS inhibitor L-NAME was employed. Steps were taken to administer L-NAME in such a way as to optimize the chances that it would effectively inhibit NOS activity. First, L-NAME administration began 1 day before relaxin administration to assure that cervical NOS had ample access to L-NAME before the cervix was stimulated with relaxin. Second, an extremely high dose of L-NAME was employed. The dose of 100 mg/kg·6 h is 2.5- to 5-fold greater than that which causes signs similar to preeclampsia and fetal growth retardation in pregnant rats (18, 19, 20, 21). The fact that relaxin promotes marked increases in both wet weight and softening of the cervix within 8 h of the initial relaxin injection enabled use of the high dose of L-NAME for a sufficiently brief duration to avoid unacceptable deleterious effects on the animal. The only apparent deleterious effect of L-NAME treatment was a slight, but significant (P < 0.05), reduction in body weight between days 6–8 of treatment (data not shown). Finally, L-NAME was administered at 6-h intervals to assure that NOS activity was inhibited throughout the 32-h period in which it was administered. Pfeiffer et al. (30) provided evidence that L-NAME is rapidly hydrolyzed in whole blood to the potent and long-lasting inhibitor N{omega}-nitro-L-arginine (L-NNA). A bolus iv injection of as little as 10 mg/kg L-NNA (1/10th the dose of L-NAME used in this study) increased mean arterial pressure for at least 5 h in sodium-depleted rats (31).

Three lines of evidence indicate that L-NAME effectively inhibited NOS activity throughout the body. First, cervical NOS activity levels were lower in L-NAME-treated animals than in controls. Second, urinary levels of nitrites/nitrates in L-NAME-treated rats were approximately 30% those in controls, and this finding is consistent with previous reports (32, 33) that concluded that reduced urine nitrite/nitrate levels in L-NAME-treated rats reflects inhibition of NO synthesis in the renal circulation. Third, the observation that heart rate was reduced in L-NAME-treated rats is consistent with previous findings in both male (34, 35) and female (36) rats. There is evidence that L-NAME-induced bradycardia is attributable to inhibition of NOS activity in the central nervous system (34, 37, 38).

Because there is evidence that L-NAME inhibited NOS activity in the cervix and other organs, it seems reasonable to postulate that the attenuation of cervical wet weight that occurred when L-NAME was administered in conjunction with relaxin is attributable at least in part to a reduction in NOS activity. One hypothesis is that relaxin induces NOS biosynthesis. Consistent with that hypothesis is the recent report that relaxin increases the expression of eNOS in mouse uterine cells (16). However, in view of the fact that cervical NOS activity in the present study was not higher in relaxin-treated OPER rats than in OPE rats, it seems likely that relaxin’s effects on cervical wet weight are not dependent upon induction of NOS activity. An alternate hypothesis, which we favor, is that existing NOS activity plays a permissive role in enabling the acute relaxin-induced increase in cervical wet weight.

Whereas the present work does not provide clear insight into the isoform(s) of NOS that enables relaxin to induce maximal acute increases in cervical wet weight, we postulate that eNOS may be involved. The cervix has been demonstrated to contain eNOS in both nonpregnant (39) and pregnant (11, 12) rats. A key role of eNOS is to produce NO that diffuses into nearby vascular smooth muscle cells and dilates blood vessels through the activation of soluble guanylyl cyclase and production of cGMP (8, 9, 10). It is also known that relaxin promotes dilation of arteries in the cervix (6) and other target tissues (14, 15, 40, 41, 42, 43). Recently, it was reported that relaxin acts directly on vascular smooth muscle cells from bovine aortas in vitro to cause changes in cell shape that are consistent with cell relaxation and does so by activation of the L-arginine-NO pathway (13). Within the cervix, eNOS may enable relaxin to induce dilation of arteries and thereby contributes to the marked increase in cervical water content that occurs within a few hours after relaxin treatment (22).

Whereas the administration of L-NAME diminished relaxin’s effects on cervical wet weight, it had no apparent influence on cervical softening. This finding is of interest because it indicates that the acute effects of relaxin on cervical growth and softening either require different thresholds of NO and/or are mediated by different mechanisms. Numerous studies have demonstrated that relaxin-induced softening of the rat cervix is accompanied by a reduction in the density and organization of collagen fiber bundles in the extracellular matrix (1, 6, 44, 45), and it is generally accepted that this remodeling of collagen fibers contributes to cervical softening. Signal molecules that play key roles in relaxin-induced remodeling of the cervix have not been identified. We previously found that PGs do not mediate relaxin’s effects on cervical softening in the rat (17), and the present findings appear to rule out a key role for NO.

Cervical softening is a two-stage process in the rat. The first stage begins on about day 12 and increases progressively until the last day of pregnancy, when a second and more rapid stage occurs during the 3 h before birth (11, 46, 47). Whereas this study provides evidence that NO probably does not play a major role in the progressive and relaxin-dependent first stage of cervical softening that occurs over an approximately 10-day period, four lines of evidence indicate that NO may contribute to the second stage that occurs during the 3 h before delivery. First, cervical levels of iNOS (11) and iNOS messenger RNA (12) increase markedly during term labor in the rat when the second stage of cervical softening occurs. Second, extensibility of cervices removed on day 20 of pregnancy decreased significantly after overnight in vitro incubation with L-NAME (11). Third, the duration of delivery was prolonged about 2.4-fold in rats administered L-NAME sc via osmotic minipump from day 17 through delivery (11). Finally, the increased extensibility of the cervix observed 24 h after sc injection of the progesterone antagonist onapristone on day 18 of pregnancy was accompanied by increased iNOS protein and messenger RNA levels (11).

Other investigators have reported that the inhibition of NOS with L-NAME or other inhibitors brings about an increase in blood pressure in rats (18, 19, 20, 21). We cannot account with certainty for the failure of L-NAME treatment to increase blood pressure. It may be attributable at least in part to the reproductive status of the female rats. Doses of L-NNA or L-NAME that had a marked effect on mean arterial pressure in pregnant rats had a more modest effect or no effect in nonpregnant rats (19, 35, 48). Our findings with ovariectomized virgin rats are consistent with a study conducted with nonpregnant Sprague Dawley rats (48) in which L-NAME had no significant effect on blood pressure.

The administration of the substrate L-arginine has been reported to reverse the effects of L-NAME or L-NNA on heart rate (35), blood pressure (20, 21, 35), and pup weight (20, 21). We do not know why the administration of L-arginine did not reverse the modest inhibitory effect of L-NAME on cervical wet weight. The dose of L-arginine employed provided an approximately 3-fold molar excess of L-arginine, and this is greater than the ratio of arginine to L-NAME reported to reverse the effects of L-NAME on blood pressure and fetal weight in pregnant rats (20, 21). It is possible that once L-NAME is metabolized to the potent and long-acting inhibitor L-NNA, L-NNA binds irreversibly to NOS and thereby prevents arginine from reversing the effects of NOS inhibition (10). It is also possible that a greater molar ratio of arginine to L-NAME than 3:1 is required to demonstrate that the inhibitory effects of L-NAME are attributable to reduced NOS activity. Finally, it is possible, if unlikely, that L-NAME attenuates relaxin’s effects on cervical wet weight by a mechanism other than the inhibition of NOS.

In conclusion, this report provides evidence that relaxin’s acute effects on growth, but not softening, of the cervix are dependent upon NO in the rat. Additionally, findings in this study support the view that cervical NOS activity is not increased by relaxin. Instead, the enzyme appears to act permissively to enable relaxin to increase cervical wet weight.


   Acknowledgments
 
The authors thank B. Sylavong and J. Hacker for supervision of animal care, the School of Life Sciences Artist Services for preparing the figures, and the College of Medicine Document Management Center for assistance with the preparation of the manuscript.


   Footnotes
 
1 This work was supported by USPHS NIH Grant HD-08700 (to O.D.S.) and a predoctoral fellowship from Reproductive Biology Training Grant USPHS 5-T32-HD07028 (to S.Z.). Back

Received December 30, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Sherwood OD 1994 Relaxin. In: Knobil E, Neill JD (eds) The Physiology of Reproduction, ed 2. Raven Press, New York, vol 1:861–1009
  2. Hwang J-J, Sherwood OD 1988 Monoclonal antibodies specific for rat relaxin. III. Passive immunization with monoclonal antibodies throughout the second half of pregnancy reduces cervical growth and extensibility in intact rats. Endocrinology 123:2486–2490[Abstract]
  3. Hwang J-J, Shanks RD, Sherwood OD 1989 Monoclonal antibodies specific for rat relaxin. IV. Passive immunization with monoclonal antibodies during the antepartum period reduces cervical growth and extensibility, disrupts birth, and reduces pup survival in intact rats. Endocrinology 125:260–266[Abstract]
  4. Lao Guico-Lamm M, Sherwood OD 1988 Monoclonal antibodies specific for rat relaxin. II. Passive immunization with monoclonal antibodies throughout the second half of pregnancy disrupts birth in intact rats. Endocrinology 123:2479–2485[Abstract]
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  6. Lee AB, Hwang J-J, Haab LM, Fields PA, Sherwood OD 1992 Monoclonal antibodies specific for rat relaxin. VI. Passive immunization with monoclonal antibodies throughout the second half of pregnancy disrupts histological changes associated with cervical softening at parturition in rats. Endocrinology 130:2386–2391[Abstract]
  7. Burger LL, Sherwood OD 1998 Relaxin increases the accumulation of new epithelial and stromal cells in the rat cervix during the second half of pregnancy. Endocrinology 139:3984–3995[Abstract/Free Full Text]
  8. Bredt DS, Snyder SH 1994 Nitric oxide: a physiological messenger molecule. Annu Rev Biochem 63:175–195[CrossRef][Medline]
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