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Regular Article |
a Department of Physiology and Pharmacology, James Cook University, Townsville, Queensland 4811, Australia
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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estradiol, female reproductive tract, mechanisms of hormone action, neuropeptides, uterus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tachykinins are predominantly localized in primary afferent sensory neurons and are released both centrally and locally. Thus, researchers have speculated that the release of sensory neurotransmitters in peripheral tissues is important for normal regulation and coordination of function [5]. Early studies of the distribution of sensory nerve fibers in the female rat reproductive tract suggested that calcitonin gene-related peptide was the most abundant neuropeptide, colocalizing with both SP and NKA in many of the sensory fibers [6]. This arrangement of sensory innervation of the female reproductive tract has been described in virtually all mammalian species studied to date [7]. Recently, the localization of NKB within the rat uterus [8] and in the rat and human uteroplacental unit [9] has also been reported, suggesting that peripheral release of NKB may play a role in regulating function within the uterus.
Expression of NK-Rs in the rat uterus has been investigated by reverse transcription polymerase chain reaction (RT-PCR) amplification of specific NK-R mRNAs [10–12] and by demonstration of uterine contractility in response to selective NK-R agonists [11–16]. These studies suggest that all three NK-Rs are expressed in the rat uterus. In a number of contractility studies, researchers have suggested that the dominant uterine contractile agonist is NKA preferentially activating NK2-Rs. SP activation of NK1-R also may play a minor role in mediating uterine contractility [13–15]. However, the presence and functional significance of NK3-R in the rat uterus was proposed as early as 1991 [17]. Recently we reported that activation of all three NK-Rs, using highly selective NK-R agonists, results in potent contractions in the rat uterus [16]. Further, we observed that the hormonal environment in the uterus significantly altered both the magnitude and nature of the contraction in response to NK-R activation. In uterine tissue obtained from estrogen-dominated rats (ovariectomized estrogen-treated rats and rats in estrus), the maximum contractile response to activation of all three NK-Rs was significantly reduced compared with the response in tissue obtained from non-estrogen-dominated animals (ovariectomized and diestrus). Furthermore, we observed that in non-estrogen-dominated uterine tissue activation of the NK3-R resulted in a sustained uterotonic response, whereas in estrogen-dominated tissue a monophasic contraction was elicited upon NK3-R activation. Other researchers investigating the expression of NK-Rs by RT-PCR have suggested that NK3-R is particularly strongly regulated by estrogen [10, 12].
In the present study, we furthered our investigations of the role of estrogen in the regulation of NK3-R-mediated uterine contractility.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All studies used tissue obtained from female Sprague-Dawley rats (150–200 g). Animals were housed in standard temperature-controlled conditions with a 14L:10D photoperiod and food (Commercial Rat Cubes; Norco, Lismore, NSW, Australia) and water available ad libitum. To obtain animals in different stages of the estrous cycle, vaginal smears were taken at the start of the light period, and animals were not used unless they had demonstrated at least two consecutive 4-day estrous cycles. In experiments using ovariectomized animals, surgery was performed 7–10 days prior to each experiment, and animals received an s.c. injection of 0.1 mg/kg 17ß-estradiol benzoate (Intervet, Castle Hill, NSW, Australia) prior to collection of estrogen-dominated tissue. For routine studies of the effect of estrogen, tissue was collected approximately 48 h after treatment with 17ß-estradiol benzoate. In one series of experiments, tissue was collected at four hourly intervals between 12 and 60 h after treatment with 17ß-estradiol benzoate. Animal studies were conducted in accordance with the Guide for Care and Use of Laboratory Animals (1996, National Academy of Science) as approved by the James Cook University Animal Ethics Review Committee.
Tissue Preparation
Animals were anesthetized by brief exposure to CO2 and killed by cervical dislocation. The uterus was removed by careful dissection, and uterine strips were mounted in a 20-ml tissue bath containing De Jalon solution consisting of 154 mM NaCl, 5.6 mM KCl, 5.9 mM NaHCO3, 2.5 mM glucose, and 0.27 mM CaCl2 in an atmosphere of 95% O2/5% CO2 (BOC Gases, Townsville, QLD, Australia) at 32°C. This protocol was previously used to produce a stable myometrial preparation in which contractile effects of agonists could be clearly seen [16, 18]. The preparations were left to equilibrate for 60 min, during which time they were washed at 15-min intervals. Resting tension was maintained at 0.4–0.5 g throughout the experiment. In estrogen-dominated tissue, no spontaneous contractions were observed after the equilibration period. In non-estrogen-dominated tissue, spontaneous contractions were infrequent, irregular, and clearly distinguishable and did not exceed 5–10% of the response to acetylcholine. Isometric contractions were recorded using a Grass FT03 strain gauge transducer (Grass Instruments, Quincy, MA) and a MacLab data acquisition system (AD Instruments, Castle Hill, NSW, Australia).
Experimental Protocol
We previously determined that the minimal effective dose needed to induce the maximal contraction for each of the selective NK-R agonists is 10-7 M [16]. This dose of agonist was used throughout this study to enable evaluation of the influence of hormonal state on myometrial responses to NK-R stimulation and to enable investigation of the effect of sequential doses of the same agonist on the maximal contractile response to the agonist. The neurokinin agonists were chosen based on their NK-R type specificity [16]: [Sar9,Met(O2)11]substance P, [ß-Ala8]neurokinin A (4–10), and senktide (Research Biochemicals International, Castle Hill, NSW, Australia) for the NK1-R, NK2-R, and NK3-R, respectively [19, 20]. In one series of experiments, a second NK3-R-selective agonist, [MePhe7]neurokinin B (Research Biochemicals), was used to examine the effect of sequential additions of different NK3-R-selective agonists on NK3-R-mediated uterine contractions. In each experiment, 10-4 M acetylcholine (Sigma Chemical Co., Castle Hill, NSW, Australia) was applied to the tissue to check stability after the equilibration period and at the end of the experimental procedure to determine tissue viability. In all experiments, tissue responsiveness to this final dose of acetylcholine was always at least 90% of the initial response. In all experiments, uterine strips were exposed to agonist for 90 sec followed by rapid washing three times over a 90-sec interval and by a 5 min re-equilibration period before the next addition. In one series of experiments, uterine strips were pretreated with two consecutive 10-min applications of capsaicin (10-4 M; Sigma) or vehicle (30 µl ethanol). The time period between consecutive applications of capsaicin was 25 min, which included three rapid 90-sec washes and a 20-min incubation period. After pretreatment, tissue was washed rapidly (three times in 90 sec) and left to re-equilibrate for 20 min (washed every 5 min) prior to subsequent agonist additions as described above.
Reagents
Unless otherwise specified, all laboratory chemicals were of analytical research grade and were obtained from Ajax Chemicals (Auburn, NSW, Australia).
Data Analysis
The response to each agonist addition was evaluated as the maximum net increase in contractile force (measured in milligrams). The net maximum increase in contractile force is expressed in one of two ways, either as a percentage of the response to the first dose of agonist or as a percentage of the maximal net increase in contractile force in response to acetylcholine (10-4 M). The response of the myometrium to acetylcholine is independent of hormonal status [21]. Uterine contractile responses are routinely expressed as a percentage of the maximum response due to activation of muscarinic receptors by either acetylcholine or carbachol.
Statistical Analysis
Where appropriate, statistical analyses were performed by one-way ANOVA followed by comparison of groups by a Newman-Keuls multiple comparison test. Variation between group means (uteri obtained from 
4 animals) was designated as significantly different with a value of P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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As previously reported, a single dose of the NK3-R-selective agonist senktide promotes a strong contractile response in uterine tissue, approximately 60–100% of the maximal response to acetylcholine, dependent upon the hormonal environment. The magnitude of the maximal contractile response was significantly reduced in estrogen-dominated uterine tissue [16]. In the current series of experiments, we examined the contractile response to sequential activation of the NK3-R in estrogen-dominated and non-estrogen-dominated tissue.
Uterine strips were sequentially exposed to four doses of agonist, each exposure separated by approximately 7 min during which time the tissue was washed. In tissue obtained from estrogen-treated ovariectomized animals, the contractile response to the second, third, and fourth dose of senktide was significantly reduced compared with the response to the first dose of senktide. The magnitude of the contractile response to the second dose of senktide was significantly reduced to 50.1% ± 6.0% (P < 0.001) of the response to the first dose (Fig. 1A). By the fourth exposure to senktide, the contractile response in tissue obtained from estrogen-treated ovariectomized animals had further been reduced to only 15.2% ± 1.8% (P < 0.001) of the response to the first dose (Fig. 1A). In contrast, in uterine tissue obtained from ovariectomized animals the sequential addition of senktide did not result in a reduction in the magnitude of the contractile response (Fig. 1A).
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The magnitude of contraction in response to consecutive doses of senktide in uterine tissue obtained from rats in estrus decreased in a manner similar to that of the responses observed in uterine tissue obtained from estrogen-treated ovariectomized rats. The magnitude of the contraction following the second dose of senktide in uterine tissue obtained from rats in estrus was significantly reduced to 69.9% ± 9.9% (P < 0.05) of the response to the first dose (Fig. 1B). Exposure to the fourth dose of senktide resulted in a further reduction in the contractile response to only 23.1% ± 7.6% (P < 0.001) of the response to the first dose (Fig. 1B). In contrast, sequential additions of senktide to uterine tissue obtained from diestrus rats had no significant effect on the contractile response compared with the initial response to the agonist (Fig. 1B).
Thus the contractile response in estrogen-dominated uterine tissue undergoes homologous desensitization in response to sequential activation of the NK3-R.
Desensitization of the Contractile Response to NK3-R Activation Does Not Affect the Contractile Response> to Activation of NK1-R or NK2-R in Estrogen-Dominated Uterine Tissue
We next determined whether the desensitization of NK3-R-induced contractility in the estrogen-dominated uterus was specific to the NK3-R or whether it reflected a more generalized loss in contractile responsiveness. We compared the level of NK1-R- and NK2-R-mediated uterine contractility before and after desensitization of the NK3-R-mediated response. Uterine tissue from estrogen-treated ovariectomized rats was challenged with the NK2-R-specific agonist, [ß-Ala8]neurokinin A (4–10), followed by the NK1-R-specific agonist, [Sar9,Met(O2)11]substance P. The tissue was then treated with four consecutive doses of the NK3-R-specific agonist, senktide, to induce desensitization of NK3-R-mediated contractility, followed by treatment with the NK2-R- and NK1-R-selective agonists, respectively. Desensitization of NK3-R-mediated contractility did not affect the magnitude of the contractile response to activation of NK1-R or NK2-R (Fig. 2). Furthermore, as we have previously reported [16] (data not shown), the order of addition of either the NK1-R- or NK2-R-selective agonists did not alter the magnitude of the contractile response to the first treatment with senktide, the NK3-R-selective agonist. Additionally the order of treatment with either the NK1-R- or NK2-R-selective agonists before and after the induction of NK3-R-mediated desensitization did not affect the magnitude of the contractile response to either NK1-R or NK2-R activation following desensitization (data not shown). At the completion of each experiment, tissue viability was determined by a final addition of acetylcholine (10-4 M). In all experiments, the sequential additions of senktide did not result in a decrease in tissue sensitivity to acetylcholine, which suggests that the desensitization is specific to the NK3-R and not due to a more generalized decrease in tissue contractility.
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Uterine Contractility in Response to NK3-R Activation Can Be Desensitized in Response to Different> NK3-R-Selective Agonists
The results described so far suggest that uterine contractility in response to NK3-R activation by the NK3-R-selective agonist senktide is subject to estrogen-dependent homologous desensitization. We next investigated whether the homologous desensitization is dependent upon the selective NK3-R agonist senktide.
Uterine tissue from estrogen-treated ovariectomized rats was challenged with alternating doses of the NK3-R-selective agonists senktide or MePhe NKB, and the contractile response was standardized as a percentage of the response to acetylcholine (10-4 M). Regardless of the order of addition of NK3-R-selective agonist, the reduction of contractile response with each subsequent dose was similar. The maximal contractile response to both senktide and MePhe NKB was not significantly different, and concentration of agonist (10-7 M) needed to induce the maximal response was the same (data not shown). Figure 3 depicts the reduction in contractility observed when alternating doses of senktide and MePhe NKB were applied to uterine tissue. Tissue was first exposed to senktide, resulting in a contraction of 57.3% ± 2.6%. Subsequent exposure to MePhe NKB resulted in a contraction of 11.1% ± 3.6%, which was significantly weaker (P < 0.001) than the contraction resulting from the first exposure to senktide. Tissue was then exposed to senktide again (third exposure), resulting in a contraction of 1.2% ± 1.2%, which was significantly weaker than the response obtained with both the first exposure to senktide (P < 0.001) and the second exposure to MePhe NKB (P < 0.05). Following the fourth addition of NK3-R agonist, the magnitude of the contraction was not significantly different from the baseline value in all experimental replicates.
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These results suggest that desensitization is due to alteration in NK3-R functionality as it is not dependent on the type or nature of the NK3-R agonist. This hypothesis is further supported by the observations that the contractile response in uterine tissue obtained from estrogen-dominated animals in response to consecutive doses of MePhe NKB desensitized in a manner similar to the response to consecutive doses of senktide (data not shown).
Desensitization of NK3-R-Mediated Uterine Contractility Depends upon the Duration of Exposure to Estrogen
We next examined whether the desensitization to NK3-R-mediated contractility depended upon the duration of exposure of uterine tissue to an estrogen-dominated environment. In this series of experiments, ovariectomized rats were treated with estrogen, and uterine tissue was obtained at 4-h intervals between 12 and 60 h following estrogen treatment. The tissue was then stimulated with four sequential additions of senktide. Figure 4 displays the contractile response to the first and fourth treatments with senktide. This experiment clearly demonstrated that both estrogen-dependent homologous and heterologous desensitization of the NK3-R-mediated response is dependent upon the duration of exposure to estrogen.
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The contractile response to the first dose of senktide in uterine tissue obtained between 12 and 24 h after estrogen treatment did not differ and was between 95.8% ± 5.0% and 107.1% ± 1.2% of the response to acetylcholine. The contractile response to the first dose of senktide began to significantly weaken 28 h after estrogen treatment and continued to weaken until 60 h after estrogen treatment, when the response was barely above baseline. At 28 h after estrogen treatment, the magnitude of the contractile response to the first dose of senktide had significantly decreased to 77.6% ± 6.2% (P < 0.01) compared with the response between 12 and 24 h (Fig. 4). By 44 h after estrogen treatment, the response had decreased further to 51.5% ± 5.2% (P < 0.001), and by 60 h after estrogen treatment the response to senktide had dramatically decreased (P < 0.001) further to only 1.9% ± 0.3% (Fig. 4). Thus, estrogen-mediated heterologous desensitization of the NK3-R-mediated uterine contractile response is dependent upon the duration of exposure to estrogen.
Not only does estrogen mediate a time-dependent heterologous desensitization of the NK3-R-mediated uterine contractile response, the estrogen-dependent homologous desensitization of NK3-R-mediated uterine contractility (Fig. 1) also depends upon the duration of exposure to estrogen. Estrogen-dependent homologous desensitization of NK3-R-mediated contractility was first apparent 20 h after estrogen treatment, when the contractile response to the fourth dose of senktide had significantly decreased (P < 0.05) to 78.1% ± 8.0% compared with the response to the first dose of senktide of 95.8% ± 5.0% (Fig. 4). The estrogen-dependent homologous desensitization of NK3-R-mediated uterine contractility was observed between 20 and 56 h after estrogen treatment, with the most profound homologous desensitization observed between 40 and 56 h after estrogen treatment. For example, in uterine tissue obtained 44 h after estrogen treatment the contractile response of 51.5% ± 5.2% following the first dose of senktide was significantly reduced (P < 0.001) to 4.8% ± 0.7% following the fourth dose of senktide (Fig. 4).
Release of Endogenous Sensory Neuropeptides Can Mediate Desensitization of NK3-R-Mediated Uterine Contractility in Estrogen-Dominated Tissue
The experiments described so far suggest that exogenous administration of the selective NK3-R agonist senktide to estrogen-dominated uterine tissue promotes a contractile response that desensitizes with consecutive doses of agonist. In the next series of experiments, we investigated whether NK3-R-mediated uterine contractility could be desensitized by release of endogenous sensory neuropeptides. In these experiments, uterine tissue obtained from estrogen-treated ovariectomized rats was pretreated with two consecutive doses of capsaicin or vehicle or was not treated, followed by stimulation of NK3-R-mediated uterine contractions with four consecutive doses of senktide. In response to the first addition of capsaicin to the organ bath, there was a net increase in tension of approximately 40% of the maximal response to acetylcholine. The increase in tension in response to capsaicin developed more slowly than did the response to the selective NK-R agonists. The response to the second dose of capsaicin was similar but weaker (data not shown).
Regardless of the type of pretreatment, the contractile response to the four consecutive doses of senktide decreased with each consecutive dose (Fig. 5). The magnitude of the contractile response to the first addition of senktide was significantly reduced in capsaicin-pretreated tissue (22.8% ± 2.1%; n = 5) compared with the response to the first addition of senktide in tissue pretreated with vehicle (39.6% ± 3.6%; P < 0.01; n = 5) or not pretreated (51.7% ± 3.8%; P < 0.01; n = 6). This finding suggests that endogenous sensory neuropeptides released by the capsaicin pretreatment initiated desensitization of NK3-R-mediated contractions observed in response to the subsequent additions of senktide. The response is dependent upon the capsaicin pretreatment; pretreatment with vehicle had no significant effect on the magnitude of the response to the first dose of senktide compared with the response in tissue that was not pretreated.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Precise regulation of uterine contractility is central to a diverse range of reproductive events such as sperm transport, embryo positioning, and fetal expulsion. The classical neurotransmitters acetylcholine and norepinephrine mediate potent uterine contractile responses, but their actions alone are insufficient to explain the complexity of uterine contractile functions in response to neural stimulation. The uterus also receives rich sensory innervation. In the rat uterus, capsaicin-sensitive primary afferent sensory nerve fibers innervate vascular and nonvascular smooth muscle, with fibers branching and penetrating further into the uterus to innervate subepithelial layers [6, 22]. This innervation system has prompted some investigators to suggest that sensory neurotransmitters released in the uterus may be involved in regulating uterine contractility. Evidence is accumulating that the tachykinins, a group of sensory neurotransmitters, mediate potent uterine contractile responses and thus may play a significant role in the regulation of uterine contractility [11–17]. A significant proportion of these studies have been carried out in the estrogen-dominated rat uterus and have led investigators to propose that tachykinin-mediated contractility in the rat uterus is primarily mediated by NK2-Rs [13–15]. Results of our earlier studies also suggest that NK2-R activation causes strong uterine contractions in both the estrogen-dominated and non-estrogen-dominated rat uterus [16] and that activation of both NK1-Rs and NK3-Rs plays a significant role in mediating sensory neuropeptide control of uterine contractility. This hypothesis is supported by the identification of expression of NK1-R, NK2-R, and NK3-R mRNA in the rat uterus [10–12]. Perhaps of greater significance is the accumulating evidence suggesting that uterine contractility mediated particularly through the NK3-R may be regulated by estrogen. Investigators of NK3-R mRNA expression in the rat uterus have reported a dramatic reduction in NK3-R mRNA levels in the estrogen-dominated uterus [10, 12]. Results of functional studies also suggest that NK3-R-mediated uterine contractility is downregulated by estrogen [10, 12, 16].
The current study clearly illustrates an important regulatory role for estrogen in the control of NK3-R-mediated uterine contractility. The NK3-R-mediated uterine contractility was subject to estrogen-dependent heterologous and homologous desensitization. Further, the duration of exposure to estrogen was central in mediating the interplay between homologous and heterologous desensitization of NK3-R-mediated contractility. Uterine tissue obtained from ovariectomized rats between 12 and 20 h after estrogen treatment showed no evidence of reduction of response to initial exposure to the NK3-R-selective agonist senktide and no reduction in response upon repeated stimulation of the NK3-R. However, after 28 h of exposure to estrogen, the initial uterine contractile response to NK3-R activation was significantly reduced, and by 60 h after estrogen treatment contractility in response to NK3-R activation was barely distinguishable from baseline. In earlier studies in which little evidence of a role for NK3-Rs in mediating uterine contractility was reported, experiments were focused on tissue obtained from rats after at least 60 h of exposure to estrogen [13, 14]. The results of the current study suggests that exposure to estrogen of that duration would most likely have resulted in the complete heterologous desensitization of NK3-R-mediated contractility.
In this study, we identified an estrogen-dependent homologous desensitization of NK3-R-mediated contractility. The homologous desensitization is readily apparent between 20 and 56 h after estrogen treatment and is characterized by a reduction in the magnitude of the contractile response elicited by repeated stimulation of uterine tissue with the NK3-R-selective agonist senktide. The degree of homologous desensitization is quite dramatic; at 44 h after estrogen treatment the initial contractile response to senktide of 51.5% ± 5.2% was reduced to levels barely detectable above baseline following the fourth addition of senktide. To further characterize the estrogen-dependent homologous desensitization of NK3-R-mediated uterine contractility, we used the vanilloid receptor agonist capsaicin to induce release of sensory neuropeptides in estrogen-dominated uterine tissue exposed to capsaicin in the organ bath. Capsaicin activation of vanilloid receptors promotes the release of sensory neuropeptides, and repeated capsaicin treatment can deplete sensory neuropeptide content to a degree where the loss of sensory perception of pain occurs (reviewed in [23]). Capsaicin-mediated release of endogenous sensory neurotransmitters resulted in a decrease in contractile response to senktide in estrogen-dominated uterine tissue, further suggesting that this regulatory mechanism occurs in the intact animal. NKB has not been localized in sensory nerve fibers in the uterus. However, in a recent study [8] mRNA for the precursor of NKB (preprotachykinin-B) was demonstrated in the rat uterus, but the cellular location was not determined. Thus, we cannot propose that capsaicin treatment of isolated uteri activated the release of endogenous stores of NKB. Capsaicin treatment probably activated the release of large amounts of SP and NKA from uterine sensory nerves sufficient to activate all three NK-R subtypes. The most interesting observation from the experiment is that pretreatment with capsaicin only resulted in the desensitization of NK3-R-mediated contractility. These findings suggest that in estrogen-dominated tissue the extent of uterine contractility mediated by the NK3-R in response to release of tachykinins is tightly regulated.
We found no evidence for desensitization of NK2-R-mediated uterine contractility in any of the experiments. In some experiments, sequential additions of the NK1-R-selective agonist SarMet SP did appear to result in desensitization, but no consistent pattern in either estrogen-dominated or non-estrogen-dominated tissue could be established (data not shown). Other investigators have reported a similar inconsistency and/or complexity in hormonal regulation of NK1-R expression and functionality [12, 16, 17].
In further support of our hypothesis that uterine contractility in response to NK3-R activation is tightly regulated in the presence of estrogen, Candenas et al. [24] described the expression of and functional state of NK3-Rs during pregnancy in the rat. They found that uterine mRNA levels of the NK3-R reached a peak at Day 3 of pregnancy and then declined throughout the remainder of pregnancy, concomitant with rising estrogen levels. In those experiments, NK3-R-mediated contractility was significantly reduced in uterine strips obtained from the latter stages of pregnancy when estrogen levels were greatest. Candenas et al. also reported that uterine contraction in response to NK3-R activation was characterized by bell-shaped dose-response curves. These curves were generated by sequential additions of increasing concentrations of the NK3-R-selective agonist MePhe NKB. The results of our experiments suggest that these bell-shaped dose-response curves are a result of estrogen-dependent homologous desensitization of the NK3-R.
Despite the increasing accumulation of evidence suggesting that NK3-Rs are expressed in the uterus and functionally mediate uterine contractility, there have been few reports of production of the endogenous primary activator of the NK3-R, NKB, in the uterus. Page et al. [9] recently demonstrated that trophoblast cells in the placenta of rats and humans secrete NKB. This discovery has led to the suggestion that NKB secreted by trophoblasts targets the uterus, playing a central role in the remodeling of uterine blood flow during the establishment and maintenance of placentation [9, 25]. Pinto et al. [8] recently reported the expression of NKB mRNA in the rat uterus and found that this expression was strongly negatively regulated by estrogen. These findings and those of our current study strongly suggest that estrogen plays a central role in regulating the actions of NKB and NK3-R activation in the uterus.
A number of possible mechanisms could explain the desensitization of NK3-R-mediated contractility in the estrogen-dominated rat uterus. The observation that the desensitization is limited to NK3-R-mediated contractility and does not affect NK1-R- or NK2-R-mediated contractility suggests that the mechanism does not involve estrogen-dependent alteration of the physiological responsiveness of the tissue or the expression of a relaxant such as calcitonin gene-related peptide. Because the NK-Rs all signal through a common pathway, the mechanism of desensitization is unlikely to involve the depletion of key secondary messengers. Thus, the mechanism(s) of the desensitization of NK3-R-mediated contractility most likely involves an estrogen-dependent alteration in either the level of expression of NK3-Rs in the plasma membrane or the ligand-dependent activity of the receptor. The mechanism(s) also is likely to involve one or more of the following: reduction in receptor protein expression, internalization and sequestration of receptor following ligand activation, and phosphorylation of ligand-bound receptor resulting in the uncoupling of receptor-G protein interactions [26]. Results of recent studies suggest that a family of G protein-coupled receptor kinases (GRKs) and associated arrestin proteins play a significant role(s) in uncoupling G protein-coupled receptors from downstream signaling mediators such as the G proteins (reviewed in [27, 28]). These events have been associated with a number of examples of homologous desensitization. In recent reports, the role of GRKs 2/3 and arrestins 1/2 in the rapid desensitization of nerves in response to the NK1-R activation by substance P has been described [29, 30]. However, whether GRKs and arrestins are involved in the estrogen-dependent regulation of NK3-R-mediated uterine contractility remains to be determined.
In this study, we further characterized the role of estrogen in the regulation of functionality of the NK3-R in the rat uterus. Contractility in the rat uterus is initiated by activation of the NK3-R and is subject to both heterologous and homologous desensitization, with both of these processes depending upon the duration of exposure to estrogen. Ultimately, exposure to elevated estrogen levels results in the complete loss of uterine responsiveness to NK3-R activation. Thus, NK3-R desensitization following an elevation in estrogen levels may be important in the modulation of uterine contractility during implantation and pregnancy. The recent report that estrogen regulates expression of NKB in the rat uterus coupled with our current findings of the complexity of estrogen-regulated NK3-R functionality in the uterus strongly suggest that control of NK3-R contractility is important for normal uterine contractility. Our current studies are focusing on exploring the mechanism of estrogen-dependent homologous desensitization of NK3-R-mediated uterine contractility.
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FOOTNOTES |
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2 Correspondence: FAX: 61 07 4779 1526; gary.hamlin{at}jcu.edu.au
Received: 30 November 2001.
First decision: 19 December 2001.
Accepted: 7 June 2002.
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q/11, G-protein receptor kinase-2/3, and ß-arrestin-1/2. Mol Biol Cell 1998 9:2305-2324This article has been cited by other articles:
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  R. H. Lim and L. Kobzik Sexual Tension in the Airways: The Puzzling Duality of Estrogen in Asthma Am. J. Respir. Cell Mol. Biol., May 1, 2008; 38(5): 499 - 500. [Full Text] [PDF]
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 P. E. Losco, M. W. Leach, D. Sinha, P. Davis, T. J. Schmahai, A. Nomier, T. Kakkar, L. Reyderman, and M. E. Lynch Administration of an Antagonist of Neurokinin Receptors 1, 2, and 3 Results in Reproductive Tract Changes in Beagle Dogs, but Not Rats Toxicol Pathol, February 1, 2007; 35(2): 310 - 322. [Abstract] [Full Text] [PDF]
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 A. Klukovits, R. Gaspar, P. Santha, G. Jancso, and G. Falkay Role of Capsaicin-Sensitive Nerve Fibers in Uterine Contractility in the Rat Biol Reprod, January 1, 2004; 70(1): 184 - 190. [Abstract] [Full Text] [PDF]
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C. O. Pintado, F. M. Pinto, J. N. Pennefather, A. Hidalgo, A. Baamonde, T. Sanchez, and M. L. Candenas A Role for Tachykinins in Female Mouse and Rat Reproductive Function Biol Reprod, September 1, 2003; 69(3): 940 - 946. [Abstract] [Full Text] [PDF]
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