| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Article |
a Departments of Obstetrics and Gynecology
b and Clinical Pharmacology, University Hospital, SE-221 85 Lund, Sweden
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Several recent studies show the presence of NOS in the uterus from animal species and the human [4–6]. However, there appear to be differences with respect to the predominant form, subcellular localization, and Ca2+ dependence in different species [4–6]. These may be due to real differences in species, hormonal status of the animal, or methodology employed or to a combination of these. In the nonpregnant sheep, there was considerably greater NOS activity in the particulate than in the soluble fraction, which was highly sensitive to Ca2+ [7]. In pregnant rat myometrium, the concentration of NOS per milligram of protein was roughly equal in cytosolic and particulate fractions, and this activity was generally insensitive to Ca2+ [5]. In the pregnant rabbit uterus, the NOS activity was primarily detected in the particulate fractions and was relatively insensitive to Ca2+ [4]. We have recently reported that in the nonpregnant rabbit uterus, cytosolic NOS was the predominant form and that NOS activity in both cytosolic and particulate fractions was highly Ca2+ dependent [8].
Although a regulation by ovarian steroids of uterine NOS would be expected, there is no consensus in the limited data that are available as to the nature of this regulation [4–7]. We have very recently shown a relatively high concentration of NOS activity in the nonpregnant rabbit uterus and importantly in the vagina [8]. The NOS concentration in the vagina was more than 3-fold higher than in the uterus. We also demonstrated for the first time that under well-controlled conditions, there is a clear down-regulation by estrogen of rabbit uterine and vaginal NOS [8]. Interestingly, the down-regulation was seen in cytosolic NOS, which was the predominant form, and not particulate NOS.
Although considerable evidence suggests that NO plays an important role in the regulation of myometrial activity, particularly in maintaining uterine quiescence, the role of NO in vaginal physiology is not known. Based on immunohistochemical studies, suggestions implying NO in the regulation of capillary permeability, vaginal blood flow, and vaginal smooth muscle dilation have been made [9, 10]. On the basis of the differences in type III NOS immunoreactivity in the vagina during the estrous cycle, it has been suggested that NO may stimulate vaginal secretion [11].
As both the uterus and the vagina are the prime targets, not only for estrogen but also for progesterone, and as both estrogen and progesterone are markedly elevated during pregnancy, in the present study we have examined the effect of estrogen or progesterone given alone and a combination of the two ovarian steroids on NOS activity obtained from the uterus and vagina of rabbits. Since we found considerable NOS activity in the vagina, 3- to 4-fold higher than in the uterus, experiments were done to see whether NO could be of relevance in the muscular activity in vaginal tissue. In addition, NOS-containing nerves were examined by immunohistochemistry.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Using Hypnorm, containing 0.315 mg/ml fentanyl citrate and 10 mg/ml fluanisone (Jansson Animal Health, Saunderton, UK), 5 ml/kg i.m., and 2 mg/kg i.m. midalozam (Dormicum; Roche, Nutley, NJ) anesthesia, mature female albino rabbits weighing 2.7–3.2 kg were bilaterally ovariectomized at least 2 wk before any further treatment. They were divided into four groups consisting of 6 animals each. While the control group received no further treatment, the rabbits in the estrogen (E2)-treated group were given an i.m. injection of 1 mg/kg polyestradiol phosphate (Estradurin; Pharmacia Upjohn, Kalamazoo, MI) dissolved in saline solution. E2+P4-treated rabbits, after 4 days of E2 treatment, received an injection daily for 3 consecutive days of 2 mg/kg P4 s.c. in oil. The rabbits were killed 7 days after the E2 injection. The P4-treated group received only P4 for 3 consecutive days as above and were killed on the fourth day. Data from a previous study have shown that a single injection of polyestradiol phosphate maintains steady and high levels of plasma estradiol in rabbits for at least 10 days [12].
Tissue Preparation
After the rabbits were killed, the uterus, vagina, and cerebellum were removed and placed in ice-cold saline solution. After excess fat and connective tissue had been trimmed, the tissues were washed with the saline solution. The uterine horn was opened, and a piece of the tissue from the middle of the horn (0.5 cm) was taken and processed for immunohistochemical study. The vagina was also opened by a longitudinal incision and divided into three longitudinal strips. One strip each was used for immunohistochemistry, NOS measurement, and recording of mechanical activity. The mechanical activity was recorded from three transversely excised regions of the vaginal strip: the introitus, the middle, and the proximal portion. The same three regions were excised separately for immunohistochemistry. Tissues designated for NOS determination were blotted dry, weighed, and immediately frozen on dry ice. Frozen tissues were stored at -80°C and used within 3 wk.
Measurement of NOS Activity
After thawing, the tissues were homogenized in approximately 10 vol buffer containing 320 mM sucrose, 10 mM Hepes buffer (pH 7.5), 0.1 mM EGTA, 1 mM dithiothreitol, 10 µg/ml trypsin inhibitor, 10 µg/ml leupeptin, and 2 µg/ml aprotinin with a Polytron (Kinematica GmbH, Lucerne, Switzerland) homogenizer for four periods of 15 sec (per gram tissue) with intermittent cooling pauses of 20 sec. The homogenate was centrifuged at 1000 x g for 10 min, and the pellet was discarded. The supernatant was filtered through one layer of gauze and centrifuged at 100 000 x g for 45 min, and the pellet and supernatant were saved as particulate and cytosolic fractions, respectively.
NOS activity was determined immediately after the preparation of subcellular fractions by measuring the formation of [14C]L-citrulline from [14C]L-arginine essentially according to the procedure previously described [8]. Incubation of cytosolic fraction (40–60 µg protein) was carried out with 100 µl of reaction mixture containing 50 mM K-phosphate (pH 7.5), 1.2 mM MgCl2, 0.24 mM CaCl2, 50 mM valine, 0.1 mM NADPH, 1 mM citrulline, 2 µM flavin adenine dinucleotide, 2 µM flavin mononucleotide, 10 µM tetrahydrobiopterin, 50 IU/ml calmodulin, 20 µM arginine, and 0.6–0.8 µM [14C]arginine (sp. act. 0.32 Ci/mmol). Samples were incubated for 20 min at 28°C. Reaction was terminated by removal of substrate and dilution by addition of 1.5 ml of 1:1 (v:v) prewashed resin AG50W-X8, in ice-cold water, and 5 ml of distilled water. After 10 min, 2 ml of the supernatant was removed and radioactivity was measured for quantification of [14C]citrulline by liquid scintillation spectrometry. [8]. In preliminary experiments, endogenous arginine was removed by passage of cytosol over a column (1 ml) of AG5OW-X-8 resin. No significant difference between these or untreated cytosol was observed.
Calculation of NOS Activity
Specific NOS activity in pmol/mg protein per minute is reported as the difference between the activity (total) and activity measured in a medium lacking CaCl2 and containing 1 mM NG-methyl-L-arginine methyl ester and 1 mM EGTA (nonspecific). Calcium-independent activity was the activity measured in the incubation mixture lacking CaCl2 and containing 1 mM EGTA. Protein concentration was determined by the method of Peterson [13].
Data are expressed as mean ± SEM. Comparisons were performed by one-way ANOVA, followed by Student's t-test with Bonferroni correction.
Immunohistochemistry
The tissues were immersion fixed for 4 h in cold 4% formaldehyde in 0.1 M PBS and then rinsed in PBS containing 15% sucrose for 2–3 days. Both fixation and rinsing were performed at 4°C, after which the specimens were frozen in isopenthane at -40°C and stored at -70°C before sectioning. Tissue sections were cut at a thickness of 8–12 µm and preincubated with PBS containing 0.25% Triton X-100 for 2 h at room temperature. Incubation with primary antiserum was performed overnight in the presence of a sheep antiserum raised against nNOS (1:4000; Dr. P. Emson, Dept. of Neurobiology, Babraham Institute, Cambridge, UK). The primary antiserum was diluted in PBS containing 1% BSA and 0.25% Triton X-100. For the visualization of the immunoreactive products, the sections were rinsed in PBS and then incubated for 90 min with fluorescein isothiocyanate (FITC)-conjugated donkey anti-sheep IgG (1:80; Sigma Chemical Co., St. Louis, MO) diluted in PBS containing 1% BSA. All incubations with primary and secondary antisera were performed at room temperature in moisture chambers. The sections were finally rinsed, treated with PBS/glycerol with p-phenylenediamine to prevent fluorescence fading, and mounted. An Olympus (Tokyo, Japan) epifluorescence microscope equipped with appropriate filter settings for FITC immunofluorescence was used. In control experiments, no immunoreactivity could be detected in sections incubated in the absence of primary antiserum. As the possibility of cross reactions with antigens sharing similar sequences cannot be excluded, the structures demonstrated are referred to as nNOS-immunoreactive (IR).
Recording of Mechanical Activity
The vagina was dissected out from six control animals, and strips (1 x 1 x 5 mm) were prepared from the introitus, middle, and proximal areas. The strips were transferred to 5-ml tissue baths containing Krebs solution (119 mM NaCl, 4.6 mM KCl, 1.5 mM CaCl2, 1.2 mM MgCl2, 15 mM NaHCO3, 1.2 mM NaH2PO4, 11 mM glucose) maintained at 37°C by a thermoregulated circuit. The Krebs solution was continuously bubbled with a mixture of 95% O2 and 5% CO2, resulting in a pH of 7.4. The muscle strips were suspended between two L-shaped hooks by means of silk ligatures. One hook was connected to a movable unit allowing adjustment of passive tension, and the other to a Grass (Quincy, MA) FT03C force transducer. Isometric tension was recorded using a Grass polygraph (model 7D). After mounting, the strips were stretched to a passive tension of 4–6 mN and allowed to equilibrate for 45–60 min. Each experiment was started by exposing the preparations to a potassium (124 mM) Krebs solution (NaCl in the Krebs solution was substituted with KCl). After two reproducible potassium-induced contractions were obtained, the strips were exposed to noradrenaline (NA; 10-5 M) to induce contraction. After exposure of the contracted strip for approximately 15 min to 10-5 M zaprinast (inhibitor of cGMP-dependent phosphodiesterase V), the strips were subjected to electrical field stimulation (EFS) by means of two platinum electrodes placed on either side of the preparations resulting in transmural stimulation of nerves. The EFS was carried out using a Grass S48 or S88 stimulator delivering single square wave pulses (duration 0.5 ms) at various frequencies (2–40 Hz). The voltage giving maximal response was determined individually for each strip. Train duration was 5 sec, and the stimulation interval was 2 min. The polarity of the electrodes was shifted after each pulse by means of a polarity changing unit. The response to EFS was studied at various frequencies in the absence of or after 15-min exposure to NG-nitro-L-arginine (L-NOARG; 10-4 M).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
After E2 treatment, the uterine NOS activity was markedly down-regulated, as it was only approximately 25% of the control value (Fig. 1A). P4 treatment also decreased uterine NOS significantly, but not as markedly as E2 treatment. A combination of E2 and P4 treatment caused a further decrease compared to that with E2 alone, but the difference between the two was not significant.
In the vagina, NOS activity was nearly 4-fold higher than in the uterus (Fig. 1B). E2 treatment caused a significant reduction as the value was nearly halved compared with that for the control. P4+E2 treatment resulted in down-regulation similar to that with E2 alone. However, P4 treatment caused a considerable increase, 50% over the control value, in NOS activity (P < 0.01).
Figure 1C shows NOS activity in the cerebellum from each group. E2, P4, or E2+P4 treatment caused no significant change in cerebellar NOS.
Immunohistochemistry
Immunohistochemistry was done in tissues from at least four animals in each group. All along the vagina, a rich supply of nNOS-IR nerves was found in the smooth muscle (Fig. 2A). The nerve density appeared to be reduced in the E2-treated group and increased in the P4-treated group as compared with the control. However, no clearly quantifiable differences could be noted in the number of nNOS-IR nerves between any of the treatments. There appeared to be a somewhat higher density of nerves in the distal (introitus) and middle areas, which declined toward the proximal part. Some nNOS-IR nerve fibers were also seen below the epithelium (Fig. 2B). A few of these nerves appeared to penetrate through the basal membrane into the epithelium (Fig. 2C). In addition, considerable but scattered immunoactivity could be seen in structures within the epithelium, which could not be clearly characterized (Fig. 2, B and C). In the introitus region, relatively large ganglia with a high density of nNOS-IR nerve cell bodies could be found (Fig. 2D). In some cases, nerve cell bodies could also been seen in the middle region. These ganglia were, however, smaller than the ganglia in the introitus region.
|
In the uterus, nNOS-IR nerve fibers were mainly seen in the muscular layer. The density of the nNOS-IR nerves was considerably lower than in the vagina. No overt differences could be seen in the number of nNOS-IR nerves between any of the treatment groups.
Functional Studies of the Vagina
The response of the vaginal smooth muscle was studied in the three different isolated portions—distal (the introitus area), middle, and proximal—from the control animals. In the introitus, the muscle preparations responded with contraction after exposure to NA and with relaxation in response to EFS. The relaxant response was frequency dependent, with a maximum relaxation at about 12–20 Hz (Fig. 3A). The relaxation was attenuated in the presence of L-NOARG, and with high frequencies a contractile response could be seen after exposure to L-NOARG (Fig. 3B). In the middle part of the vagina, a similar pattern of responses was found, although the responses were generally weaker. Frequently the muscle had a spontaneous activity in this region, which sometimes made it difficult to interpret the response to EFS. In the proximal portion of the vagina, nearly all preparations showed spontaneous activity, and consequently a clear muscular response to NA and EFS was difficult to obtain.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Depending on the hormonal status, particularly E2 priming, organ, and species, P4 may act synergistically with E2 or have an anti-estrogenic action. The present data on P4-treated rabbits indicate that there are differences in the regulation by P4 of NOS depending on the tissue. In the uterus, P4 down-regulated NOS as was the case with E2, but the reduction was not as marked as with E2. A combination of E2 and P4 had some synergistic action as the down-regulation was slightly, although not significantly, augmented compared to that with E2 alone.
Although the effects of exogenously given P4 on uterine NOS have not been studied, from the large amount of data published by Chwalisz et al. [17] in pregnant rats, the authors have suggested that P4 up-regulates NO-cGMP and that its withdrawal before labor causes a down-regulation of NO production and as a result the initiation of labor. Although a sharp decline of plasma P4 also occurs in the rabbit as in the rat, but not in the human, before labor, the present data do not show any up-regulation of uterine NOS when P4 was given either alone or in combination with E2. During rabbit pregnancy, P4 is markedly elevated, as in rat pregnancy. It is not unlikely, however, that the level of P4 and duration of P4 dominance are different from those achieved by exogenous administration, which was the case in the present study. However, a marked down-regulation is difficult to accommodate even with this consideration. Alternatively, other factors associated with pregnancy, such as cytokines, may be contributory to the up-regulation of NO production during pregnancy in the rat as reported previously [5]. Massman et al. [18] recently showed that there was no significant change in NOS activity in sheep myometrium or endometrium with advancing gestation and labor. In this species the E2 levels increase during pregnancy and further increase near parturition while P4 levels decline, indicating little influence of ovarian hormones on uterine NOS.
In humans, where P4 is greatly elevated during pregnancy and there is no decline before labor, no difference was found in NOS activity in the myometrium or the placenta or fetal membranes [19]. No changes in NOS mRNA expression were observed throughout gestation or with the onset of labor [20]. Furthermore, in a recent study in which some decline in plasma cGMP and NO3 could be seen before vaginal deliveries, no such change was observed in cesarean section deliveries, but plasma P4 levels in the two groups were not different [21]. Thus, our data in the rabbit, a species in which P4 domination during pregnancy and its withdrawal before parturition are known to occur, as in the rat—together with the data of the studies in human pregnancy [19–21]—do not support the argument for an up-regulation of NO-cGMP system by P4 during pregnancy but, rather, point to contribution by the fetus, placenta, or other factors. Using pregnant and pseudopregnant rabbits, we have previously reported data showing that factors associated with the fetus were highly important for the quiescence of myometrial activity during pregnancy [22, 23]. Both fetal membranes and placenta have been shown to produce a number of neurohormones, growth factors, cytokines, and prostaglandins [24].
We observed no significant change in NOS activity in the cerebellum in the present study after any hormonal treatment, which is in keeping with the view that this organ is not strictly a target for ovarian steroids [8]. Interestingly, in a previous study we showed that whereas E2 down-regulated NOS in the female rabbit lower urinary tract, there was no change in the upper urinary tract [25].
An interesting observation in the present study is the up-regulation by P4 of NOS activity in the vagina, indicating tissue-specific effects of P4; and this selectivity was observed among major target tissues for ovarian steroids like the uterus and vagina. We have previously presented data showing tissue-specific effects of P4 with respect to down-regulation of E2 receptors in the uterus and vagina [26]. Whether this effect can be observed in tissues from rabbits only or is a generalized phenomenon cannot be known until data from other species are available. The finding of a very substantial NOS activity in the vagina, which was nearly 4-fold higher than in the uterus, and its up-regulation by P4 deserve further investigation, particularly on the role of NOS in vaginal physiology. This is important in view of the present observation showing, for the first time, NO-dependent relaxation of vaginal smooth muscle, together with a rich supply of NOS-containing nerves observed by us and others [27, 28]. Nitrergic innervation may be important for the vasocongestion of the vagina and for the relaxation of the inner vaginal wall observed during sexual excitement [27].
Immunohistochemical studies have demonstrated the presence of NOS in the vagina from humans and other species [27, 28]. Compared to that in the mouse uterus where NOS activity was only sparse, the activity in the mouse vagina was abundant [28]. This is in agreement with the present immunohistochemical observations and biochemical data in the rabbit. Although a reduction in NOS was seen with E2 treatment in some of the specimens, it was not quantifiable with any reliability, because immunohistochemical data are only semiquantitative. Our biochemical data, however, clearly demonstrate a significant down-regulation by E2. An increase in NOS in estrous and proestrous phases in the rat, observed in immunocytochemical investigation reported previously [11], suggests again differences between species.
The results of the present study demonstrate a down-regulation by E2 and by P4 of NOS in the uterus; and while there was a down-regulation by E2 in the vagina also, P4 up-regulated NOS in this organ. None of the hormonal treatments had an effect on cerebellar NOS. A high NOS activity in the vagina and NO-mediated relaxation of vaginal smooth muscle suggest a physiological role and deserve further study, particularly on functional significance and possible differences among species.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 This work was supported by grants from the Royal Physiographic Society, the Crafoord and Anna-Lisa and Sven-Erik Lundgren Foundation. 
2 Correspondence. FAX: 46 46 157868; satish.batra{at}gyn.lu.se
Accepted: December 20, 1999.
Received: October 6, 1999.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  M. Basha, S. Chang, E. M. Smolock, R. S. Moreland, A. J. Wein, and S. Chacko Regional differences in myosin heavy chain isoform expression and maximal shortening velocity of the rat vaginal wall smooth muscle Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2006; 291(4): R1076 - R1084. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
