| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Female Reproductive Tract |
Center for Perinatal Biology, Departments of Physiology/Pharmacology and Pediatrics, Loma Linda University School of Medicine, Loma Linda, California 92350
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
environment, oxytocin, parturition, uterus
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Many factors contribute to the process of smooth muscle contraction (e.g., membrane receptors, second messengers, enzymes, ion channels), and theoretically, each may be responsible for the significant reduction of contractile activity in myometrium after long-term hypoxia (LTH). Our studies with the rat demonstrated a reduction in oxytocin-receptor density after LTH, which appeared to be the principal mechanism for reduced contractility [7, 8]. However, in these studies, the circular and longitudinal smooth muscle layers were not differentiated. Because the myometrium consists of two distinct layers, which have different physiological and pharmacological characteristics, differentiation of these layers may be important to discern the specific effects of chronic hypoxia on contractile function.
From an embryological perspective, the origins of longitudinal and circular smooth muscle are different. Circular muscle develops from the müllerian duct; longitudinal muscle is derived from the connective tissue between the circular layer and the peritoneum. Therefore, different electrophysiological and pharmacological characteristics of each muscle layer may be expected. Osa and Katase [11] showed differences in response to mechanical and electrical stimuli in circular versus longitudinal muscle of the pregnant rat uterus. Differential contractile responses to oxytocin [12–14], prostaglandin F2
[14], and catecholamines [15, 16], have also been reported. These data suggest a muscle layer-specific distribution of receptors in myometrial tissue. Our previous studies in the rat [7–9] and sheep [10], however, did not differentiate between the two smooth muscle layers.
The pregnant ewe has been an extensively utilized model of parturition [17, 18]. Because of its large size and robust nature, the fetus lends itself to endocrine and physiological studies of both fetal and maternal responses. However, only a few experiments have used this model for studies of in vitro contractility [19, 20]. Although the physiological and pharmacological characteristics of both layers from pregnant rat myometrium are well established, no similar data are available regarding sheep myometrium.
The present study was designed to test the hypothesis that chronic hypoxia during pregnancy suppresses myometrial contractility in the sheep. Specifically, we collected myometrium from pregnant ewes exposed to hypoxia for approximately the last 110 days of gestation, and we examined in vitro contractile responses of individual longitudinal and circular layers to oxytocin. We also evaluated changes in oxytocin-receptor protein by Western blot analysis.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Time-dated pregnant sheep of mixed Western breed (term = 147 days) were divided between two treatment groups: normoxic controls (n = 6), and LTH (n = 6). At 30 days of gestation, the ewes in the LTH group were transported to Barcroft Laboratory White Mountain Research Station (Bishop, CA; elevation, 3820 m; maternal partial pressure of oxygen [PO2], 59.1 ± 5.4 mm Hg [mean ± SEM]), where they were maintained until 137 days of gestation. The ewes were then transported to Loma Linda University Medical Center Animal Research Facility (elevation, 346 m); immediately after arrival, they were implanted with an arterial and tracheal catheter. The maternal PO2 for the LTH group was maintained at approximately 60 mm Hg by adjusting humidified nitrogen (N2) gas flow through a maternal tracheal catheter. The normoxic control ewes were maintained near sea level (
300 m) throughout gestation.
Between Days 137 and 142 of gestation, the animals were killed for collection of myometrial tissue from the middle third of the uterine horns. All procedures were approved by Animal Research Committee of Loma Linda University and followed the guidelines in the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Contractility Studies
Tissue preparation Myometrial tissue was immediately placed in cold, oxygenated Na+ Krebs buffer, and both endometrium and perimetrium were removed. Under a dissecting microscope, the longitudinal and circular layers were separated. The tissue was cut into strips (1 x 3 mm) and mounted in a standard organ bath filled with Na+ Krebs buffer (122.09 mM NaCl, 25.59 mM NaHCO3, 5.16 mM KCl, 2.49 mM MgSO4, 11.10 mM glucose, 1.60 mM CaCl2, and 0.114 mM ascorbic acid) bubbled with 95% O2:5% CO2 at pH 7.4 and 37°C. Tissues were equilibrated for a minimum of 1 h before experiments. Calibrated isometric force transducers (Radnoti Glass Technology, Monrovia, CA) coupled to on-line data acquisition software (Labview 2.2.1; National Instruments, Austin, TX) were used for measuring contractile tensions. The methodology is similar to that previously described in detail [7, 9].
Length-tension study Length-tension curves for both smooth muscle layers were generated to determine optimal resting tension. The initial length of each strip was measured, and contractile response was assessed by a series of 120 mM K+ challenges after each length adjustment stretched to 400% of initial length. Maximum active tensions at each length were measured.
Oxytocin dose-response study Myometrial strips were gradually stretched to optimal resting tension and were initially stimulated with 120 mM K+. The tissues were then washed with Na+ Krebs buffer three times and allowed to re-equilibrate for 30 min. The same process was performed three times before adding oxytocin. After equilibration, strips were exposed to increasing half-log doses of oxytocin ranging from 10-12 to 10-6.5 M. The final stretched length of each strip was measured, and the wet weight was calculated on an analytic balance. The value of 1.05 g/cm3 was used for the density of wet tissue. The cross-sectional area (cm2) was calculated as weight/(density x length). Values for the cross-sectional area were used to normalize the tension generated by each strip (calculated as the integrated area under the contraction curves) as previously described [7].
Western blot analysis
Immediately after collection, myometrial tissue was placed in chilled, oxygenated Na+ Krebs buffer. The endometrium and perimetrium were removed, and the longitudinal and circular layers were separated under a dissecting microscope. Tissue samples were snap-frozen in liquid nitrogen and stored at -70°C until analyzed.
After thawing, the longitudinal and circular samples of myometrium were first cut into small pieces and then homogenized (twice for 15 sec) in chilled Tris-EDTA (TED) buffer (50 mM Tris [pH 7.4], 10 mM EDTA, 1 mM diethyldithiocarbamic acid [DEDTC], and 2 mM octyl glucoside). The samples were then centrifuged at 30 000 x g for 30 min at 4°C. The crude pellets containing cell membranes were sonicated (8-sec cycle, three cycles) in 500 µl of TED sonication buffer (20 mM Tris [pH 7.4], 50 mM EDTA, 0.1 mM DEDTC, and 45 mM octyl glucoside). The sonicates were centrifuged at 13 000 x g for 25 min at 4°C. The recovered supernatant was stored at -70°C until electrophoretic analyses were performed. The protein concentration was determined by the BCA Protein Assay (Pierce, Rockford, IL).
The proteins (10 µg/lane) were then separated on ready-made 7.5% Tris-HCl gel (Bio-Rad, Hercules, CA) and electrophoretically transferred to nitrocellulose membrane (Amersham, Little Chalfont, UK) using a Bio-Rad semidry transfer blot cell. The membranes for immunostaining were blocked with 5% nonfat dry milk (Bio-Rad) in wash buffer (10 mM Tris-HCl [pH 7.4], 100 mM NaCl, and 0.1% Tween-20) and then incubated overnight at 4°C. After blocking, the blots were washed three times with wash buffer (15 min each) at room temperature and then incubated overnight at 4°C with primary antibody for the oxytocin receptor (OTR primary Ab no. 3579, kindly provided by Dr. G. E. Hoffman, University of Maryland, Baltimore, MD) using 1:10 000 dilution in 5% nonfat dry milk in wash buffer. The primary antibody is a polyclonal rabbit anti-OTR antiserum generated using a synthetic dodecapeptide (WQNLRLKTAAAA) corresponding to the third intracellular loop of the rat OTR sequence, which tends to be the area of least homology between receptors of the AVP/OT family [21]. This antiserum has previously been validated for use in the sheep [22].
The blots were then washed three times with wash buffer (15 min each) and incubated for 75 min at room temperature with secondary antibody (Anti-Rabbit IgG, Heavy & Light Chain [Goat] Peroxidase Conjugate; Calbiochem, La Jolla, CA) using 1:5000 dilution in 5% nonfat dry milk in wash buffer. After incubation, membranes were washed three times with wash buffer (15 min each). The blots were subjected to enhanced chemiluminescence using Chemiglow Reagent (Alpha Innotech, San Leandro, CA) and visualized by using a powerful digital imaging system (The ChemiImager; Alpha Innotech). The images of immunoreactive staining were measured and analyzed by AlphaEase software (Alpha Innotech). The density of each band represents a sum of all the pixel values after subtraction of background. The molecular weight of the proteins was determined by running Multicolored Protein Marker NEL-312 (New Life Science Products, Boston, MA) in an adjacent lane.
Relative density was normalized by loading equal protein amounts. Furthermore, an oxytocin-receptor protein standard was run on each blot, and sample values were expressed as densitometric units relative to the standard (relative optical density). Immunoblots were repeated at least twice on each tissue with similar results. Samples from each animal were run in duplicate, and the mean of these duplicates represented the density of oxytocin-receptor protein for each animal. The values from each animal were then used to calculate the mean ± SEM for each treatment group.
Statistical Analysis
The maximum tension generated (Tmax) and the -log of concentration yielding 50% of maximum response (pD2) were calculated with logistic-curve fitting software (Igor; National Instruments, Austin, TX). Comparisons between the two experimental groups were analyzed by the Student t-test. Differences were considered to be significant at P < 0.05.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Figure 1 illustrates the length-tension relationship between myometrial strips from normoxic and hypoxic sheep. We observed no difference in the length-tension curves between control and LTH tissues in either longitudinal (Fig. 1A) or circular (Fig. 1B) myometrial smooth muscle layers. Chronic hypoxia did not change active tension produced by 120 mM K+ at optimal resting tension in either longitudinal muscle (1.18 ± 0.21 and 1.22 ± 0.11 g for control and hypoxia, respectively) or in circular muscle (1.61 ± 0.15 and 1.65 ± 0.11 g for control and LTH, respectively).
|
Oxytocin Dose-Response Study
The contractile responses of myometrial strips from both control and hypoxic animals to oxytocin are shown in Figure 2 and Table 1. The Tmax value of longitudinal strips did not differ between control and LTH animals (762.1 ± 65.9 vs. 737.3 ± 43.6 g · sec/cm2 for control and LTH, respectively), whereas circular muscle from LTH animals demonstrated a significant reduction in Tmax (1441.0 ± 72.6 vs. 1056.0 ± 47.5 g · sec/cm2 for control and LTH, respectively; P < 0.05). It is worth noting that in both the normoxic and LTH groups, maximum tension of the circular muscle was greater than that of the longitudinal muscle. The pD2 values of both layers in the LTH group were significantly higher than the values in the normoxic group (longitudinal muscle: 8.7 ± 0.3 vs. 9.8 ± 0.2 for control and LTH, respectively; circular muscle: 8.4 ± 0.1 vs. 9.5 ± 0.1 for control and LTH, respectively).
|
|
Western Blot Analysis
The relative expression of oxytocin-receptor protein was determined using Western blot analysis. Immunoblotting with a specific monoclonal antibody for the oxytocin receptor detected a single band, representing a protein with an approximate molecular weight of 62 kDa, which is consistent with the range demonstrated in another study [22]. Figure 3A shows a representative Western blot. Quantitative densitometry of the immunoblots revealed that the density of oxytocin-receptor protein in longitudinal muscle showed a significant increase in tissue collected from LTH sheep (0.56 ± 0.05 vs. 0.88 ± 0.05 relative optical density for control and LTH, respectively; P < 0.05) (Fig. 3B). In marked contrast, oxytocin-receptor protein was significantly lower (P < 0.05) in the circular layer of myometrium from the LTH group (0.53 ± 0.11 relative optical density) compared with control animals (0.82 ± 0.09 relative optical density) (Fig. 3C).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Oxytocin is one of the most potent uterotonic agents at term, and it seems to play a key role in the initiation and maintenance of labor. In the present study, LTH had a differential effect on the responses of longitudinal versus circular myometrial smooth muscle to oxytocin. The Tmax value of the circular muscle in the LTH group was significantly lower than the value for similar tissue from normoxic animals, suggesting that the maximal ability of circular muscle from hypoxic animals to contract in response to oxytocin is impaired. These data further confirm and expand our earlier observations in the LTH ewe [10]. In contrast, the Tmax values of the longitudinal layer were not significantly different between groups. Interestingly, the pD2 values of both layers in tissues from LTH animals were significantly higher than those values in normoxic animals (Table 1), indicating that LTH increased the sensitivity of both myometrial layers to oxytocin.
These data suggested that LTH altered oxytocin-receptor expression. To investigate the hypothesis that LTH alters the density of oxytocin receptors in sheep myometrium, we performed Western blot analysis on longitudinal and circular myometrial samples. Immunoblotting with a specific monoclonal antibody for the oxytocin receptor detected a single band, representing a protein with an approximate molecular weight of 62 kDa, which is in the range previously reported by Wu et al. [22]. Additionally, those authors provided histological evidence for the role of oxytocin in the regulation of myometrial contractility by immunolocalization of the oxytocin receptor in myometrium. In their study, however, the myometrium was studied as a whole; it was not separated into distinct layers before analysis. The results of our study revealed that when circular and longitudinal layers were examined separately, LTH had opposing effects, with decreased expression in the circular layer and increased protein levels in the longitudinal layer.
These results provide additional evidence for differential regulation of contractile activity of smooth muscle layers after LTH exposure. Taken together with the contractility data, it is apparent that altered contractile activity in sheep myometrium is not solely the result of changes in the density of oxytocin-receptor proteins. Earlier studies from our laboratory using pregnant rats suggested that LTH-induced decreases in myometrial contractile responses may involve oxytocin-specific mechanisms, namely diminished oxytocin-binding sites [7]. However, it was not clear if the observed reduction in oxytocin-binding sites was primarily responsible for decreased contractile response. Because myometrial oxytocin receptors are coupled to the inositol-1,4,5-trisphosphate pathway [23], other potential mechanisms may play a role in the altered contractile response.
Changes in receptor density and sensitivity to oxytocin may only be partly responsible for observed differences in contractile responses between tissue from the control and LTH groups. Data from the present study support the hypothesis that steps in the contractile response process that occur downstream from the oxytocin receptor may also be altered by LTH. A number of studies have demonstrated that prolonged hypoxia alters receptor function in other smooth muscle types. The density of 1-adrenoreceptors was decreased in cerebral arteries from hypoxic sheep [24] as well as in the uterine artery, accompanied by reduced norepinephrine-binding affinity [25]. Li et al. [26] reported that chronic hypoxia downregulated 1-adrenoreceptors in rat cardiac myocytes and inhibited signaling mediated through this type of receptor. Moreover, ß-adrenergic receptors in rat heart were also downregulated after exposure to hypoxia [27], as were adenosine receptors. However, muscarinic-receptor affinity and density were increased [28]. Further studies from our laboratory demonstrated that LTH alters the inositol-1,4,5-triphosphate contraction coupling in the uterine artery from pregnant sheep [29]. These findings clearly indicate that in addition to changes in receptor number, changes in postreceptor G-protein coupling and inositol-1,4,5-triphosphate production by phospholipase C may be common pathways whereby LTH exerts effects.
In the present study, we confirmed that hypoxia decreased expression of oxytocin-receptor protein in circular muscle, and this effect seems to be consistent with results from contractility experiments (decreased Tmax response to oxytocin). However, the increased density of oxytocin-receptor protein in longitudinal muscle, without affecting maximal contractile response to this agonist, further supports the assertion that other mechanisms may contribute to the effect of LTH, at least in this muscle layer. Interestingly, in both the circular and longitudinal smooth muscle layers, we observed a significant increase in pD2 values, despite different contractile responses and opposite changes in the number of oxytocin receptors. This finding raises the important possibility that the binding affinity of oxytocin receptors might be altered after exposure to chronic hypoxia. Changes observed in the circular layer lead to the speculation that decreased oxytocin-receptor protein levels after hypoxia resulted in a compensatory response by increasing the coupling efficiency of these receptors (higher pD2 value). This mechanism may protect against any further decline of contractile activity in the circular muscle layer. Changes observed in longitudinal muscles seem to be more complex than those in the circular layer. Although both oxytocin-receptor protein expression and pD2 were enhanced, Tmax remained unchanged. It seems that LTH-induced alterations in the contractility response cascade downstream from the oxytocin receptor, and observed changes in oxytocin-receptor density and oxytocin-receptor coupling may also represent compensatory mechanisms that preserve contractile efficacy. For a better understanding of the cellular mechanisms involved in LTH regulation of myometrial contractility, the estimation of inositol-1,4,5-triphosphate generation as well as Ca2+ release will be important next steps in our research.
In the present study, we showed that LTH did not appear to affect the physical contractile characteristics of the smooth muscle layers of sheep myometrium. The degree of prestretch necessary to achieve maximum active tension was similar in tissue from control and LTH animals. Additionally, the percentage of initial length required to reach the maximum active tension in longitudinal muscle did not differ from that in circular muscle. Similarly, values of active tension elicited by 120 mM K+ at optimal resting tension were similar for control and LTH groups in both muscle layers. However, it is interesting to note that the active tension produced by the circular layer was higher compared with that produced by the longitudinal layer from both control and LTH animals. Differences in mechanical characteristics between the longitudinal and circular layers were also observed in rat myometrium [11]. These data suggest that the circular layer may play a more important role in the overall contractile process at term in the sheep.
The present study clearly demonstrated that LTH plays a regulatory role in myometrial function. Furthermore, the heterogeneity in responsiveness between circular and longitudinal myometrial smooth muscle to oxytocin and LTH emphasizes the importance of distinguishing between the two muscle layers in studies of uterine contractility.
|
FOOTNOTES |
|---|
2 Correspondence: Charles A. Ducsay, Center for Perinatal Biology, School of Medicine, Loma Linda University, Loma Linda, CA 92350. FAX: 909 558 4029; cducsay{at}som.llu.edu
Received: 22 April 2003.
First decision: 15 May 2003.
Accepted: 30 June 2003.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
on circular and longitudinal myometrium from the pregnant rat. Biol Reprod 1987 37:348-355[Abstract]- and ß-effects on the longitudinal and circular muscles of the pregnant rat myometrium. Jpn J Physiol 1976 26:403-416[Medline]. Biol Reprod 1998 58:73-781-adrenergic receptors and Ins(1,4,5)P3 responses in cerebral arteries. Am J Physiol 1997 272:R669-R6741-adrenergic receptors in the ovine uterine artery. Am J Physiol 1996 270:H1001-H10071-adrenergic receptor subtype mRNAs and inhibits 1-adrenergic receptor-stimulated cardiac hypertrophy and signaling. Circulation 1995 92:918-925This article has been cited by other articles:
|
|
 |
|
  B. Root, J. Abrassart, D. A. Myers, T. Monau, and C. A. Ducsay Expression and Distribution of Glucocorticoid Receptors in the Ovine Fetal Adrenal Cortex: Effect of Long-term Hypoxia Reproductive Sciences, May 1, 2008; 15(5): 517 - 528. [Abstract] [PDF]
|
|
|
|
 |
|
 B E Cross, H M O'Dea, and D J MacPhee Expression of small heat shock-related protein 20 (HSP20) in rat myometrium is markedly decreased during late pregnancy and labour Reproduction, April 1, 2007; 133(4): 807 - 817. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B G White, S J Williams, K Highmore, and D J MacPhee Small heat shock protein 27 (Hsp27) expression is highly induced in rat myometrium during late pregnancy and labour Reproduction, January 1, 2005; 129(1): 115 - 126. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T.K. Arakawa, M. Mlynarczyk, K.M. Kaushal, L. Zhang, and C.A. Ducsay Long-Term Hypoxia Alters Calcium Regulation in Near-Term Ovine Myometrium Biol Reprod, July 1, 2004; 71(1): 156 - 162. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
