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Regular Article |
a Department of Physiology and Biophysics, University of Miami School of Medicine, Miami, Florida 33101
ABSTRACT
Calcitonin gene-related peptide (CGRP) inhibits myometrial contractile activity. However, the responsiveness of the mouse myometrium to CGRP is dependent on the hormonal and gestational stage. The inhibitory effect of CGRP in the myometrium is prominent during gestation and declines at parturition. The present study was undertaken to examine if nitric oxide (NO) production by nitric oxide synthase (NOS) isoforms mediates the inhibitory action of CGRP on uterine contractions as has been suggested earlier. Transgenic mice deficient in either of the three major NOS isoforms: endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS) were used. Isometric force measurements on myometrial strips obtained from NOS-deficient mice were carried out and the inhibitory capacity of CGRP was monitored. CGRP inhibited KCl-induced contractions of the myometrial strips obtained from eNOS(-/-), iNOS(-/-), and nNOS(-/-) mice with equal efficiency as in wild-type animals. Additionally, NOS protein expression in the mouse uterus during gestation and during the estrous cycle was examined by means of Western immunoblot analysis. No correlation between NOS expression and inhibitory activity of CGRP was evident. The results suggest that the inhibitory action of CGRP in the mouse uterus is independent of the activity of these NOS isoforms.
nitric oxide, signal transduction, uterus
INTRODUCTION
The smooth muscle of the uterus, the myometrium, undergoes large changes that are conducive of the diametrical functional roles of the uterus in the pregnant and the non pregnant state. During gestation, the uterus assumes a state of prolonged myometrial quiescence in contrast to the well-synchronized contractions during expulsion of the fetus. Several potential inhibitory factors of myometrial contractility—including calcitonin gene-related peptide (CGRP), relaxin, nitric oxide, and carbon monoxide [1–4]—have been identified.
CGRP is a neuropeptide with many diverse biological activities, including relaxation of myometrial contractility [1]. During pregnancy, the responsiveness of myometrium to CGRP is increased and a significant decline in CGRP responsiveness of the myometrium occurs at parturition [5]. Therefore, CGRP may be an important inhibitory factor that contributes to the maintenance of uterine quiescence during pregnancy. In the nonpregnant state, the responsiveness of myometrium to CGRP declines during estrus when the contractile activity of the myometrium may be essential for facilitation of sperm entry into the uterus [6].
The mechanisms by which CGRP induces relaxation of myometrial smooth muscle have not been clearly determined. A series of different mechanisms have been proposed in the literature: some could co-exist and some appear to be mutually exclusive. Proposed mechanisms include activation by CGRP of a calcium-activated potassium channel [7], CGRP-induced release of nitric oxide (NO) [3, 8], activation of adenylate cyclase, and generation of cAMP as a result of CGRP binding to its receptor [9–11]. In the myometrium, CGRP induces a 90-fold increase in cAMP concentrations [12].
Shew et al. [3] have reported that pharmacological inhibitors of nitric oxide synthase (NOS) can block the relaxing effect of CGRP but not of isoproterenol in the myometrium. Furthermore, they observed that NADPH-diaphorase, thought to be indicative of NOS, is localized in uterine nerve fibers. Based on these observations the authors concluded that the inhibitory effect of CGRP may depend on NO formation. NO-mediated CGRP-induced relaxation of vascular smooth muscle has also been reported [13, 14] but appears to be dependent on the presence of endothelium [15], which probably is the site of NO release. NO, however, may not be involved in potassium channel activation [16].
NO production is catalyzed from L-arginine by enzymatic activity of NOS [17]. Three major isoforms of NOS have been identified in a variety of tissues [18]: neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). Previous studies of the expression and cellular localization of NOS enzymes in the uterus have produced conflicting results, which may in part be due to the qualitative nature of the experimental techniques (immunohistochemistry), experimental design, or the particular animal model used in the study. NO has been implicated in events associated with maintenance and termination of pregnancy [19–22]. NOS isoforms have been identified alone or in combination in the uterus of many species, based on immunohistochemistry, Western blot analysis, or measurement of NOS enzymatic activity [19, 21, 23]. In rats and rabbits a decline in NOS activity at term has been reported [19, 24]. In human uterus, no significant change in either NOS mRNA levels or activity appears to be associated with labor and delivery, as reported by some investigators [25–27], while others report a decline in NOS expression associated with labor and delivery [28]. The NOS isoforms expressed in the uterus may be species and/or gestational stage dependent. Rabbit uterine NOS is reported to be primarily the inducible isoform [19], whereas both constitutive and inducible isoforms have been found in pregnant rat uterus [21].
The present study was undertaken to gain insight in the mechanism of CGRP relaxation and to determine the involvement of NO in the relaxant effect of CGRP on the myometrium, in the mouse uterus. Previous studies have relied on the use of inhibitors of NOS to investigate the role of second messenger NO in the relaxant effect of CGRP in the uterus. We chose to study the involvement of NOS isoforms as a potential mediator of CGRP action, by using knockout mice in which the gene for each of the three isoforms has been inactivated [29–31]. This approach has advantages over pharmacological inhibition of NOS activity because it allows the study of the role of each isoform independently of other NOS enzymes and does not contain the risk of nonspecific effects of an inhibitory drug on CGRP signaling.
To determine which specific NOS isoform, if any, plays a role in the inhibitory capacity of CGRP in the myometrium, we used uteri from mice deficient in each of three NOS isoforms. We performed isometric force measurements on myometrial strips obtained from NOS-deficient mice and wild-type controls to assess the inhibitory capacity of CGRP on KCl-induced contractions of the myometrial strips.
To test for compensatory changes we quantitated the expression levels of the isoforms in knockout mice. We also determined the expression of NOS isoforms (nNOS, eNOS, and iNOS) in the uterus of cycling nonpregnant mice and also gestating and parturient animals by Western immunoblot analysis.
MATERIALS AND METHODS
Isometric Force Measurements of the Myometrial Strips
Adult female wild-type, nNOS-, iNOS-, and eNOS-deficient (B6,129-Nos 1tm1Plh; C57BL/6-NOS 2tm1Lau, C57BL/6J-Nos 3tm1Unc) mice were obtained from Jackson Laboratories (Bar Harbor, ME). The stage of the estrous cycle was determined by daily monitoring of mice with vaginal smears. Strips of myometrium, 0.05–0.07 mm in diameter, were dissected from the longitudinal muscle layer of the uterus of cycling mice at metestrus and were placed in Krebs-bicarbonate solution (mM: NaCl, 119; NaHCO3, 25; MgSO4 1.2; KCl, 3.6; KH2PO4, 1.2; CaCl2, 2.5; glucose, 11; pH 7.4). Myometrial strips were placed in a 0.5-ml perfusion chamber, fixed at one end and connected to a force transducer at the other end. Changes in isometric tension were measured by a force-displacement transducer (Grass FT03E) and recorded on a Gould model TA240 recorder. The mounted strips were perfused continuously with gassed (95% O2, 5% CO2) Krebs-bicarbonate solution and equilibrated for 30 min before the experiment was started. With force recorded continuously, myometrial strips were induced to contract by perfusion with 25 mM KCl for 10 sec. The preparations were then washed and allowed to relax. Myometrial strips were next incubated with 0.1 µM CGRP for 1 min, and then incubated with 25 mM KCl for 10 sec and washed with Krebs buffer. Strips were induced to contract with 10-sec pulses of KCl per minute to obtain a time course of attenuation of contractile force by CGRP. Solution changes were made via a hand operated valve.
In other experiments, the inhibitory effect of CGRP in the presence of NOS inhibitor, NG-monomethyl-L-arginine (1 mM), was assessed. Myometrial strips were first induced to contract with 25 mM KCl, allowed to relax and next pre-incubated for 15 min with the NOS inhibitor. The above paradigm to determine the inhibitory effect of CGRP on KCl-induced contractions was then followed, in the continued presence of 1 mM NG-monomethyl-L-arginine (L-NMMA).
Protein Extraction and Western Blot Analysis
Uterine tissue from cycling or timed-pregnant mice was homogenized in a Tris-based buffer containing 10 mM Tris-HCl, 1 mM EDTA, and 1% SDS plus proteinase inhibitors (100x: 50 mg/ml lima bean trypsin inhibitor, 2 mg/ml leupeptin, 16 mg/ml benzamidine, 2 mg/ml pepstatin A) and phenylmethylsulfonyl fluoride (100x: PMSF 10 mg/ml), and then centrifuged at 5000 x g for 15 min at 4oC to remove cellular debris, after which the supernatant (total lysate) was collected. Protein concentration of the total lysate was determined using a Pierce Micro-BCA kit (Pierce, Rockford, IL).
Protein samples containing 100 µg of protein were heated at 70°C for 10 min, after addition of an equal volume of a 2x loading buffer (0.125 mM Tris-Cl, 4% SDS, 20% w/v glycerol, 0.2 M DDT, 0.02% Bromophenol Blue, pH 6.8). The proteins were then resolved by electrophoresis on a 4–15% gradient Tris-HCl polyacrylamide gel (Bio-Rad, Hercules, CA) at a constant voltage of 140 volts with a running buffer containing 0.025 M Tris, 0.192 M glycine, 0.1% SDS, pH 8.3. The separated protein was electrotransferred to Immobilon-P transfer membranes (Millipore, Bedford, MA) at a constant voltage of 15 volts at room temperature, overnight. Following Millipore's Rapid Immunoblotting protocol (Millipore), membranes were immersed in methanol and allowed to dry prior to incubation with primary antibody. Blots were next incubated for 2 h with specific antibodies against either nNOS, iNOS, or eNOS. Commercial mouse monoclonal antibodies from Transduction Laboratories Inc. (Lexington, KY) against the C-terminal segments of human nNOS, mouse iNOS, and human eNOS were used. For nNos, a polyclonal rabbit antibody raised against the human protein was also employed. As positive controls the following sources were used: mouse brain homogenate for nNOS, mouse macrophage lysate from RAW 264.7 cell line for iNOS, and a lysate of a human endothelial cell line. Both cell line lysates were supplied with the antibodies. Unless stated otherwise, 10 µg protein of the lysates was loaded onto the gels. The blots were washed twice (10 min each) in 0.01 M Tris-buffered saline plus 0.05% Tween (TBS-T) and incubated with either a goat anti-mouse IgG antibody (1:2500) or with a goat anti-rabbit antibody (1:10000), conjugated to horseradish peroxidase (Transduction Laboratories, Inc.) for 30 min, and washed twice with TBS-T (10 min each). Immunoreactive protein in the preparations was detected by chemoluminescence using SuperSignal Western Blotting Detection System (Pierce).
RESULTS
Inhibitory Effect of CGRP on KCl-Induced Myometrial Contractions Using NOS-Deficient Mice
The inhibitory capacity of CGRP in the myometrium varies during the estrous cycle [6]. The maximal inhibition occurs during metestrus. In order to avoid cycle-related variability we determined the stage of the estrous cycle by daily vaginal smears; mice at metestrus were selected. Myometrial strips of mice deficient in each of the three nitric oxide synthase isoforms (nNOS[-/-], eNOS[-/-], iNOS[-/-], and wild-type animals) were used. The inhibitory effect of CGRP on myometrial contractility sets in slowly and is long lasting. Typically, the maximal inhibition requires 2–3 min to develop. In each case, the amplitude of the force of contraction corresponding to the maximally attenuated response was expressed as the percent of the amplitude of the control contraction (Fig. 1). Incubation with CGRP resulted in the inhibition of KCl-induced contractions of myometrial strips obtained from the uteri of mice, deficient in either nNOS, eNOS, or iNOS, which was not significantly different from the observed inhibition of contractions in wild-type animals (Fig. 1B).
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Myometrial strips from uteri of wild-type animals were also used in isometric force measurements where NOS enzymes were pharmacologically inhibited, and the inhibitory capacity of CGRP was assessed. Previously, an apparent inhibition of CGRP effect on uterine contractions by an NOS inhibitor has been taken as evidence for the involvement of NOS in CGRP signaling. In the presence of 1 mM L-NMMA, a pharmacological inhibitor of NOS enzymes, the inhibitory capacity of CGRP was not significantly different from the control experiment (Fig. 1B).
Expression of NOS Isoforms in the Uterus During the Estrous Cycle and Gestation
If NO was involved in CGRP signaling one would expect a correlation between NOS expression and the inhibitory potency of CGRP, which varies during pregnancy and during the estrous cycle. To determine the expression of NOS isoforms in the uterus at the protein level, Western blot analysis was carried out using protein extracted from uteri of nonpregnant, gestating, and parturient mice. Since hormonal fluctuations in the nonpregnant state can influence the expression level of many proteins, cycling mice were monitored, the stage of the estrous cycle was determined, and uteri from each stage of the estrous cycle was used in order to test for any variations in the NOS protein expression during the estrous cycle.
Neuronal NOS Expression in the Uterus
Figure 2A is a representative Western blot showing the expression of nNOS protein in the uterus of wild-type mice during the estrous cycle (lanes 1–4), in the brain obtained from nNOS-deficient mice (lane 5), and the brain obtained from wild-type mice (lanes 6, 7, 8: dilution of 1/50, 1/10, and 1 compared to the uterus). A 155-kDa immunoreactive band corresponding to the nNOS was observed in lanes corresponding to brain tissue (positive control) as well as the uterine samples from nonpregnant mice. However, the nNOS protein expression in the uterus is less than 1/10 the amount expressed in the brain (Fig. 2A).
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Figure 2B is a representative Western blot using uterine protein samples obtained from gestating mice. During gestation, nNOS protein expression in the uterine protein samples obtained from pregnant mice was below the detection level by Western blot analysis using a monoclonal antibody (Fig. 2B).
Using a polyclonal rabbit antibody we were unable to detect expression of nNOS protein even in the nonpregnant uterus, although a 155-kDa immunoreactive band corresponding to nNOS was detected in the brain tissue used as a positive control. The polyclonal antibody however, cross-reacted with a protein of approximately 250 kDa in the uterus (data not shown).
Inducible NOS Expression in the Uterus
Previous studies by other investigators using immunohistochemical techniques had indicated the expression of iNOS protein in certain cell types present in the uterus, including macrophages, mast cells, uterine epithelial, and myometrial smooth muscle cells [32]. Other investigators have also reported iNOS expression in the uterus, an up-regulation of expression associated with pregnancy and down-regulation associated with labor and delivery, by Western blot analysis [28]. However, the support for these claims is unclear. The immunoreactive bands in the published figures, are not of the expected molecular mass (130 kDa) for iNOS and are different from the bands in the positive control lanes. The immunoreactive bands in the uterine samples may represent cross-reactivity of the antibody with unrelated cellular proteins. Consequently, it is unclear which protein was labeled in the immunohistochemical analysis.
Inducible NOS protein expression in the uterus during various stages of the estrous cycle was below the level of detection by Western blot analysis using 100 µg of protein (Fig. 3A). Furthermore, in the uterus of gestating animals at various stages of pregnancy or parturition, iNOS protein expression was not detected by Western blot analysis using a monoclonal antibody against iNOS (Fig. 3B). A 130-kDa immunoreactive band corresponding to iNOS protein was detected in the macrophage cell lysate used as positive control (Fig. 3).
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Endothelial NOS Expression in the Uterus
Western blot analysis demonstrated that eNOS is an abundant isoform expressed in the uterus of nonpregnant as well as pregnant mice. The monoclonal anti-eNOS antibody recognized a band of approximately 140 kDa in the uterus of nonpregnant mice (Fig. 4A) and also gestating mice at various stages of pregnancy (Fig. 4B). The level of expression of eNOS protein in the uterus of nonpregnant mice appears to vary during the estrous cycle.
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Loss of One NOS Isoform Is Not Compensated by an Increased Expression of Other Alternate NOS Isoforms
We examined the possibility that the loss of expression of one NOS isoform could be compensated by up-regulation of an alternate form in the knockout mice. We determined the expression level of each of the three NOS isoforms by Western blot analysis using protein extracts isolated from the uterus of mice lacking either of the three NOS isoforms {eNOS(-/-), iNOS(-/-), nNOS(-/-)} and wild-type animals (w/w). As shown in Figure 5, loss of one NOS isoform is not compensated by an increased expression of other forms. Neuronal NOS-deficient mice and iNOS-deficient mice expressed eNOS protein at levels comparable to the wild-type controls (Fig. 5A). Endothelial NOS-deficient and iNOS(-/-) mice expressed nNOS protein at levels comparable to wild-type controls (Fig. 5C). Inducible NOS was not detected in eNOS(-/-) nor nNOS(-/-) animals, as in wild-type controls (Fig. 5B).
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DISCUSSION
The present study indicates that NO is not an obligate mediator of the relaxing effect of CGRP in the myometrium. The inhibitory potency of CGRP on myometrial strips obtained from mice deficient in each of the three NOS isoforms was not significantly different from the CGRP effect on myometrial strips obtained from wild-type animals. Additionally, no correlation was observed between the expression levels of the NOS isoforms and the potency of CGRP to inhibit myometrial contractility which varies during the estrous cycle and during gestation and at parturition. These findings suggest that the inhibitory effect of CGRP on myometrial contractility is either completely independent of NO formation or that NOS activity constitutes only a minor component in the CGRP signaling pathway. Additionally, in the presence of L-NMMA, CGRP inhibition of KCl-induced contractions of the myometrium were not significantly different from CGRP effects in the absence of the inhibitor. This is in contrast to previous reports that in the presence of a NOS inhibitor, CGRP mediated attenuation of spontaneous contractions of the myometrium and of substance P induced contractions [3] was abolished. Hence the basis for the involvement of NO as a mediator of CGRP effects in the uterus is not clear-cut. This discrepancy might be explained by the fact that the CGRP effect requires several minutes to be maximal and may have been missed because of timing factors.
The relaxant effect of CGRP in the myometrium is well documented [1]. Our previous studies as well as reports by other investigators suggest a role for CGRP during pregnancy [5]. The relaxation of vascular smooth muscle by CGRP may be essential for the control of uterine blood flow and CGRP effect on myometrial smooth muscle may control uterine contractility during pregnancy. We have previously shown that the responsiveness of the myometrium to CGRP is influenced by the hormonal and the gestational state of the animal [6]. Increased sensitivity of the myometrium to CGRP during pregnancy inhibits uterine contractility, and at the onset of labor, a decrease in responsiveness releases the myometrium from the inhibitory effect of CGRP. Also, at estrus, a decrease in the myometrial sensitivity to CGRP allows for increased contractility of the uterus, which facilitates conception. Although there is an increase in CGRP responsiveness of the myometrium associated with gestation, we found no correlated change in the expression of NOS isoforms in the uterus. Indeed, nNOS and iNOS were not detected during gestation, whereas eNOS protein was expressed.
The NO system is thought to be involved in the regulation of multiple physiological processes in the uterus including myometrial relaxation, pregnancy maintenance, and cervical ripening [33]. In the literature however, there is a lack of general agreement regarding the particular NOS isoforms that mediate the relaxant effect of NO on the myometrium and the uterine cell types that express the enzymes. The discrepancy between reports as to particular isoform of NOS and the cell types expressing these isoforms may be due to specificity of the antibodies used or whether mRNA or protein was examined. Previous studies have relied on qualitative techniques such as in situ hybridization and immunohistochemistry, and indirect indicators of nNOS activity using NADPH-diaphorase immunoreactivity. These studies by in large have relied on immunohistochemistry and Western blot analysis using commercially available monoclonal and polyclonal antibodies. When uterine protein extracts have been used to detect NOS proteins, difficulty in visualizing expected size immunoreactive bands and/or presence of additional bands in Western blots have been reported; occasionally, similar observations were made in the present study. This indicates cross-reactivity of the antibody with other cellular proteins. Yet, bearing the risk of erroneous assignment of proteins, the same antibodies were used for immunohistochemistry to visualize and determine the cellular localization of NOS isoforms.
NO appears to be part of a redundant set of mechanisms that warrant myometrial quiescence during pregnancy. NOS-deficient mice have normal pregnancies and do not show a compensatory increased expression of other NOS isoforms. Thus, in the absence of the NO system, uterine quiescence can still be accomplished by other mechanisms including CGRP.
Based on Western immunoblot analysis, an abundant isoform of NOS in the mouse uterus is the eNOS. This result is in agreement with previous reports of eNOS expression in human myometrium as a prominent isoform [34–36]. The presence of eNOS in the uterus is most likely for homeostasis purposes and control of uterine blood flow. Furthermore, eNOS protein expression was greater at diestrus as compared to other stages of the estrous cycle, possibly indicating that eNOS expression in the mouse uterus may be influenced by hormonal changes associated with the estrous cycle. Hormonal regulation of NOS activity in rat uterus has been reported [37]. In order to directly asses the effects of ovarian hormones on NOS protein expression in the uterus, further studies are required in which protein extracts from uteri of ovariectomized, hormone-treated animals are used for Western analysis using specific antibodies raised against each isoform.
Inducible NOS expression was not detectable in the uterus of nonpregnant cycling mice, nor in the uterus of pregnant mice during gestation. This suggests that either iNOS is absent or is present in such low quantities, expressed only by certain cell types in the uterus, that it was below the detection threshold by Western analysis. This is in agreement with a previous report regarding lack of expression of iNOS protein in the human myometrium, in both the pregnant and in the nonpregnant state [38], where the authors conclude that uterine quiescence during pregnancy is not maintained by NOS activity. Similarly, the myometrium from pregnant rats is not sensitive to relaxation by cGMP, probably due to reduced expression of cGMP-dependent protein kinase [39]. On the other hand, expression of iNOS protein in uterine leukocytes during gestation in rats and mice has been reported [40]. Increased expression of iNOS during delivery and in response to hormonal treatment has also been reported [32, 41]. However, no classical estrogen response element (ERE) or progesterone response element (PRE) is present in the 5' promoter region of the iNOS gene. Other investigators have reported differences in localization of iNOS protein in the human myometrium when different antibodies were employed [26].
The uterine samples obtained from nonpregnant mice at various stages of the estrous cycle, contained low levels of nNOS protein as revealed by Western analysis using a monoclonal antibody. During gestation, however, nNOS protein expression in the uterus was below detection level. This observation is consistent with the reports that myometrial innervation is markedly reduced during pregnancy [42].
In summary, CGRP inhibits myometrial contractions in NOS-deficient mice as effectively as in the wild-type animals, CGRP inhibits myometrial contractions in the presence of a pharmacological inhibitor of NOS, and NOS-deficient mice have normal pregnancies. These observations suggest that the CGRP system in the uterus represents a mechanism that produces myometrial relaxation, independently of the NO system.
FOOTNOTES
First decision: 12 April 2000.
1 Supported by National Institute of Health grant GM48610. 
2 Correspondence: G. Dahl, Department of Physiology & Biophysics (R-430), University of Miami School of Medicine, 1600 NW 10th Ave., Miami, FL 33136. FAX: 305 243 5931; gdahl{at}miami.edu
Accepted: June 14, 2000.
Received: March 10, 2000.
REFERENCES
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