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Biology of Reproduction 63, 781-788 (2000)
© 2000 Society for the Study of Reproduction, Inc.

Regular article

Divergence in Murine Myometrium Spontaneous and Oxytocin-Stimulated Contractile Responses to Serine/Threonine Protein Phosphatase-1 Inhibition1

Gary D. Smith2,,a, Xiao-Tie Liua, and Mark Phillippeb

a Departments of Obstetrics and Gynecology, Physiology, and Urology; Reproductive Sciences Program, University of Michigan, Ann Arbor, Michigan 48109-0617 b Department of Obstetrics and Gynecology, The University of Chicago, Chicago, Illinois 60637

ABSTRACT

Reversible phosphorylation is essential in regulating uterine contractions. Identification, characterization, and functional understanding of myometrium protein phosphatase(s) are lacking. Okadaic acid (OA), which inhibits protein phosphatase-1 (PP1) and PP2A, has been shown to alter uterine contractions. Experiments were conducted to determine the 1) identity of the myometrial OA-sensitive PP, 2) influence of OA on spontaneous and oxytocin (OT)-stimulated myometrial contractions, and 3) expression of uterine PPs during sexual development. Western blot analysis indicated the presence of PP1{alpha} and PP2A in immature and mature mice. As determined by immunohistochemistry, gonadotropin-stimulated adult mouse uteri contain PP1{alpha} in longitudinal and circular myometrial layers and endometrial epithelium. Conversely, PP2A was localized to the endometrial stroma. Cumulative addition of OA (n = 9; 10, 100, 250, 500, 1000 nM) did not significantly alter spontaneous contractions of mouse uterine horns in comparison to vehicle-treated controls (n = 9). By the end of the test period OA- and vehicle-treated uteri displayed a comparable decline in uterine contractions to 79.2% and 63.7%, respectively, of basal contractile activity. Pretreatment of uterine tissue with OA (1 µM; n = 7) significantly reduced contractile response to increasing concentrations of OT (8, 16, 32, 64 nM) in comparison to vehicle pretreatment (dimethyl sulfoxide; n = 7). At the end of the OT-administration period, contractile activity was 160.4% and 67.3% of basal contractile activity for vehicle (no OA) and OA-pretreated groups, respectively. During the early prepubertal period PP1{alpha} was expressed in longitudinal myometrium and absent in circular myometrium; whereas, during the transition to sexual maturity PP1{alpha} was observed in both the longitudinal and circular myometrium. In summary, these studies have indicated 1) that PP1 is the primary myometrial OA-sensitive PP; 2) that inhibition of PP1 had no effect on spontaneous contractions, whereas it markedly inhibited OT-stimulated uterine contractions; and 3) that PP1 is differentially expressed in the circular and longitudinal myometrium in relation to sexual development.

parturition, phosphatases, puberty, signal transduction, uterus

INTRODUCTION

Phosphorylation and dephosphorylation of myosin light chains [1, 2] regulate contraction and relaxation of myometrial tissue. While the cascade of events leading to phosphorylation of myosin has been investigated extensively, characterization, regulation, and substrate identification of myometrial phosphatases are lacking. Intracellular phosphorylation events involved with myometrial contractions begin with an increase in intracellular Ca2+. This intracellular Ca2+ binds to calmodulin and the resulting complex stimulates activation of myosin light chain kinase (MLCK). Myosin light chain kinase catalyzes incorporation of a phosphate residue onto serine 19 of the 20-kDa light chain subunit of myosin [3]. Phosphorylation of myosin results in a conformational change in the myosin head and activation of myosin ATPase activity that ultimately causes force generation and muscle shortening.

Serine/threonine protein phosphatases (PPs) that hydrolyze and remove phosphate groups from phosphoproteins and thus antagonize protein kinase phosphorylation, have been implicated in the dephosphorylation of myosin and relaxation of smooth muscle. Classification of serine/threonine PPs is based on substrate specificity and sensitivity to a defined set of inhibitors and activators [4]. Protein phosphatase 1 (PP1) preferentially dephosphorylates the ß subunit of phosphorylase kinase, whereas PP2 dephosphorylates the {alpha} subunit of phosphorylase kinase to a greater extent than the ß subunit [5]. Type 1 PP is sensitive to the heat- and acid-stable inhibitors 1 and 2 (I1 and I2), whereas type 2 PPs are insensitive to I1 and I2 [6]. Type 2 PPs can be subclassified into PP2A, PP2B, and PP2C based upon cation requirements. Protein phosphatase-1 and PP2A do not require divalent cations for activity, whereas PP2B and PP2C require Ca2+/calmodulin and Mg+, respectively, for activity [7]. In addition, there are at least four isotypes of the catalytic subunit of PP1 that originate from three genes designated PP1C{alpha}, PP1C{delta}, and PP1C{gamma} [8]. Two spliced variants of PP1C{gamma}, PP1C{gamma}1, and PP1C{gamma}2 that differ by ~2 kDa due to the extended carboxyl terminus of PP1C{gamma}2 have been described [9–11].

An important discovery in phosphatase research was identification of the polyether monocarboxylic acid, okadaic acid (OA) that originates from dinoflagellates and is a specific inhibitor of PP1 and PP2A [4, 12]. Type 1 PP and PP2A activities are extremely sensitive to OA inhibition, displaying IC50 values of ~10.0 and ~0.1 nM, respectively. In comparison, PP2B is only slightly sensitive to OA inhibition (IC50 ~5 µM) and PP2C essentially is insensitive to OA [13, 14]. This distinguishing sensitivity of PP1 and PP2s to exogenous inhibitors is highly conserved in several systems including mammals, plants, and yeast [4, 7].

Aorta [15], gizzard [16–18], and bladder [19] are smooth muscle sources that have been investigated with respect to characterization of smooth muscle phosphatases. Presently, there is a lack of agreement in regard to whether PP1 and/or PP2A are the primary smooth muscle PP within these tissues. Likewise, OA has been shown to both inhibit and stimulate contractions of rat myometrial strips depending on concentrations used [20–23]. Low concentrations (100 nM) inhibit contractions and higher concentrations cause a biphasic, contractile/relaxation response [21]. Effects of OA on myometrial strips would indicate that PP1 and/or PP2A are involved; however, no reports exist characterizing the PP in the myometrium. Therefore the goals of these studies were to utilize immunological means to identify and characterize the PP in developing and adult mouse uteri and to investigate further the influence of PP inhibition on spontaneous and oxytocin (OT)-stimulated uterine contractions.

MATERIALS AND METHODS

Uterine Collection

Uterine tissue was obtained from CF1 female mice, utilizing a protocol approved by the Institutional Animal Care and Utilization Committee at the University of Chicago. Uteri were collected from mice on Days 11, 12, 13, 15, 16, 17, and 19 (Day 0 = day of birth) without hormonal stimulation (low estrogen milieu); adult mice primed with 10 IU pregnant mare serum gonadotropin (eCG; elevated estrogen milieu); or adult mice during proestrus/estrus (elevating/elevated estrogen milieu). Following cranial/cervical dislocation, uteri were surgically removed and rinsed in Earles buffered saline solution (EBSS; 117 mM NaCl, 1.8 mM CaCl2, 5.3 mM KCl, 0.8 mM MgSO4, 1.0 mM NaH2PO4, 26.2 mM NaHCO3, and 5.6 mM glucose) prior to use in subsequent experiments.

Western Blot Analysis

Uterine tissue isolated from prepubertal (Day 12 and Day 16) and adult eCG-stimulated mice were frozen in liquid nitrogen. Frozen uteri were thawed in cell lysis buffer (50 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.2% SDS, 360 mM 2-ß-mercaptoethanol, 0.6 mM PMSF, 1 µg/ml aprotinin and leupeptin), homogenized, and placed on ice for 15 min. Following centrifugation at 12 000 x g, 4°C for 10 min, a small portion of the supernatant was assessed for protein content [24]. An aliquot of SDS PAGE sample buffer was added (15.6 mM Tris-HCl [pH 6.8], 5.0% glycerol, 0.5% SDS, 360 mM 2-ß-mercaptoethanol, and bromophenol blue) to the remainder of the supernatant, and samples were boiled for 5 min, centrifuged, and supernatants were stored at -20°C until electrophoresis was performed.

Approximately 20 µg of mouse uterine total protein was added per lane and separated by one-dimensional SDS-PAGE [25]. Resolving gels were cast using 12% acrylamide; stacking gels contained 4% acrylamide. Gels were equilibrated in 25 mM Tris, 192 mM glycine, and 20% methanol for 15 min before protein electrophoretic transfer to polyvinylidene fluoride membrane (Bio-Rad Laboratories, Hercules, CA) in the same buffer. Blots were blocked with 5% nonfat dry milk in 20 mM Tris-HCl (pH 7.5), 0.1% Tween 20, and 137 mM NaCl (TTBS; ingredients from Sigma, St. Louis, MO) for 1 h and washed three times for 10 min in TTBS. Blots were incubated with either anti-PP1{alpha} (diluted 1:2000; Upstate Biotechnology, Lake Placid, NY) or anti-PP2A (diluted 1:2000; Upstate Biotechnology) antibodies in TTBS plus 1.0% nonfat milk overnight at 4°C. Blots were washed three times in TTBS, incubated with peroxidase-linked anti-rabbit IgG (Amersham Life Science, Arlington Heights, IL) for 1 h, washed twice in TTBS, then three times in TBS, followed by development with a chemiluminescence detection kit (Amersham Life Sciences) according to the manufacturer's instructions.

Immunohistochemistry

Uteri collected from prepubertal (Days 11–17), peripubertal (Day 19) and adult eCG-stimulated mice were fixed in 4% paraformaldehyde (prepared fresh daily), rinsed in PBS, and embedded in paraffin. Paraffin-embedded uteri were sectioned at 5-µm intervals and placed on superfrost-plus slides (Fisher Scientific, Itasca, IL), deparaffinized, placed in 100 mM glycine buffer (pH 3.65), and microwaved for 10 min for antigen retrieval [26]. To reduce background signal, samples were placed in Tris-buffered saline (TBS; 50 mM Tris, pH 7.6, 150 mM NaCl) containing 5.0% dimethyl sulfoxide (DMSO) and 0.2% Tween-20 for 10 min, rinsed in TBS, and incubated for 10 min in TBS containing 0.3% BSA, 1 mg/ml sodium azide, and 1.6% normal goat serum (Vector Laboratories, Burlingame, CA). Slides were rinsed and incubated overnight with anti-PP1{alpha} (diluted 1:500) or anti-PP2A (diluted 1:500) antibodies at 4°C in a humidified chamber. Slides were subsequently rinsed, washed (TBS + 0.05% Tween-20), rinsed again, and incubated with the appropriate biotinylated secondary antibody for 30 min at room temperature. After secondary antibody exposure, slides were quenched in 3.0% H2O2 in 90% methanol for 30 min, then rinsed and incubated for 30 min in avidin-biotin conjugated to peroxidase (Vector Laboratories). After several rinses, sections were exposed to 0.025% 3,3'-diaminobenzidine (DAB; Dojindo Labs-Wako Chemical, Richmond, VA), rinsed, counterstained with Mayers hematoxylin (Sigma), dehydrated by three changes of ethanol then three changes of xylene, and mounted. Positive controls consisted of mouse heart. Negative controls included 1) elimination of the primary antibody (not shown) and 2) nonimmune rabbit serum in place of the primary antibody.

Uterine Contractions

The isolated uteri from mice in proestrus/estrus were divided into two individual uterine horns of similar size thus allowing administration of vehicle and treatment to each animal. For in vitro contraction studies uterine horns were connected to isometric contraction transducers (Harvard Bioscience, South Natick, MA) and placed in 50-ml muscle baths containing EBSS bubbled continuously with 95% oxygen/5% carbon dioxide, and maintained at 37°C. Contraction studies were performed after uterine horns were equilibrated with 2 g stretch tension for a 20-min period. Contractile activity at the end of the equilibration period was utilized for the calculation of the baseline uterine activity. Cumulative dose-response studies were performed with the addition of either OA, a PP1 and PP2A inhibitor (Calbiochem-Novabiochem Inc., La Jolla, CA) or vehicle (DMSO). Following cumulative addition of OA or vehicle, uterine horns were treated with OT (Sigma), KCl, or both in a sequential manner following a 1-h washout period. Addition experiments were conducted in which uterine horns were pretreated with 1 µM OA or vehicle followed by cumulative addition of OT.

Analog data recorded by the isometric contraction force transducer were computer digitized using an OMEGA PCL-718 A/D board (Omega Technologies, Stamford, CT), then collected, and stored using LABTECH Notebook (Laboratory Technologies, Wilmington, MA). The digitized contraction data were reported as the grams of tension generated or normalized for tissue cross section area, then analyzed to determine the area under the contraction curve for 5-min intervals using Sigma Plot (Jandel Scientific, Corte Madera, CA) and reported as the percentage of baseline contractile activity. These contraction studies were performed using seven to nine replicates as noted; each replicate represents studies performed using uterine tissue from a single animal. Statistical analysis was performed using Kruskal-Wallis one-way ANOVA on ranks and the Student-Newman-Keuls and Dunns test for multiple comparisons, when appropriate. Differences were considered to be significant statistically if P <= 0.05.

RESULTS

Identification of Uterine PP1 and PP2A

Western blot analysis of mouse uterine tissue collected during the prepubertal period (Days 12 and 16 of age) and from adults shows presence of both PP1{alpha} and PP2A (Fig. 1) at approximately 37 and 36 kDa, respectively. These uterine tissue samples contain numerous cell types in addition to endometrial and myometrial cells that could influence Western blot analysis observations; thus, we also identified the cell-specific expression and localization of PP1 and PP2A using immunohistochemistry (Fig. 2). Mouse heart was used as positive control tissue for both PP1{alpha} and PP2A (data not shown). Uteri collected from eCG-primed adult mice displayed negligible background staining when primary antibodies were replaced with nonimmune rabbit serum (NIRS). The enzyme PP1{alpha} was located in both longitudinal and circular layers of the myometrium, as well as in endometrial epithelium. In contrast to the uterine PP1{alpha} localization, PP2A was not observed to be expressed in the myometrium; however, it was highly expressed in the endometrial stroma and observed at lower levels in the endometrial epithelial cells.



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  		FIG. 1. Western blot analysis of PP1{alpha} and PP2A in immature (Day 12 and Day 16) and mature (adult) mouse uteri. Approximately 20 µg of total protein from mouse uteri was added per lane. Antibodies against PP1{alpha} and PP2A recognized proteins of 37 and 36 kDa, respectively



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  		FIG. 2. Representative localization of PP1{alpha} (B1–B3) and PP2A (C1–C3) by immunohistochemistry in uteri collected from PMSG-stimulated adult mice. Tissues were fixed, paraffin embedded, and exposed to microwave antigen retrieval prior to incubation with primary antibodies or NIRS (A1–A3) followed by biotinylated secondary antibody and colorimetric evaluation with avidin-biotin-peroxidase and DAB. Slides were counterstained with Meyers hematoxylin. Epi, Endometrial epithelium; Myo, myometrium; Str, endometrial stroma. Optical magnification: A1, B1, C1 = x100; A2, A3 = x400; B2, B3, C2, C3 = x1000

Okadaic Acid and Spontaneous Uterine Contractions

Addition of the PP1/PP2A inhibitor, OA, to muscle baths did not alter spontaneous uterine contractions significantly in comparison to vehicle treatment as seen in the representative contraction data presented in Figure 3. Cumulative addition of OA and vehicle, at 10-min intervals, resulted in a gradual decline in spontaneous contractile activity over time. Cumulative addition of OA at 10, 100, 250, 500, and 1000 nM yielded percentage of spontaneous baseline contraction activity (mean ± SEM) of 96 ± 5, 92 ± 7, 84 ± 8, 80 ± 9, and 79 ± 9, respectively. The OA effects were not significantly different from those produced by cumulative addition of vehicle, yielding percentages of baseline contraction activity (mean ± SEM) of 93 ± 6, 83 ± 6, 78 ± 11, 68 ± 8, and 64 ± 7 (Fig. 4).



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  		FIG. 3. Representative in vitro contraction study demonstrating lack of effect of cumulative addition of PP1/PP2A inhibitor OA on spontaneous uterine contractions. Contractile activity in grams of tension generated in response to cumulative addition of vehicle (OA treatment equivalence of DMSO; A) or OA (B). Horizontal lines indicate the times when vehicle and OA additions occurred



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  		FIG. 4. Contractile activity (in percentage of baseline activity) of mouse uterine horns in response to cumulative addition of vehicle (n = 9) or OA (n = 9). Area under the contractile curve for a 5-min interval during the end of the 20-min equilibration period (baseline) and during each cumulative addition period for the vehicle or OA were utilized to calculate the contractile activity. Okadaic acid was added at 10-min intervals in a cumulative manner resulting in 10, 100, 250, 500, and 1000 nM concentrations. Equivalent volumes of vehicle (DMSO) alone were added in a similar manner. Data are reported as mean ± SEM

When OT was administered following the cumulative additions of vehicle, uterine tissue responded with an increase in contractile activity (Fig. 5A). In contrast, OT administered following the cumulative additions of OA (for a total of 1 µM) did not increase contractile activity (Fig. 5B). However, when KCl was administered after OA treatment, after an initial small peak, a small tonic contraction was observed (Fig. 5C). When the uterine tissue was subsequently washed and equilibrated in fresh EBSS buffer for 1 h (following the previous treatment with OA) and then treated with OT, the uterine tissue responded with increased phasic contractile activity (Fig. 5D). The addition of KCl after OT administration resulted in an initial large peak, followed by a slowly decreasing tonic contraction (Fig. 5D).



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  		FIG. 5. Representative in vitro contraction studies demonstrating the effects of OT and/or KCl treatment following the cumulative addition of vehicle or OA, with or without OA washout as noted. Contractile activity reported in grams of tension. A) Cumulative vehicle treatment for 70 min, followed by a 10-min treatment with OT (64 nM). B) Cumulative additions of OA (10 nM to 1 µM) for 70 min followed by a 10-min treatment with OT (64 nM). C) Cumulative additions of OA (10 nM to 1 µM) for 70 min followed by treatment with KCl (55 mM). D) Cumulative additions of OA (10 nM to 1 µM) for 70 min followed by washout and a 1-h equilibration with fresh EBSS, then treatments with OT (64 nM), followed by KCl (55 mM)

Okadaic Acid and OT-Stimulated Uterine Contractions

As seen in Figure 6A, cumulative-dose addition of OT following vehicle administration resulted in increased phasic contractile activity. However, when uterine tissue was pretreated with OA, cumulative additions of OT resulted in reduced uterine contractions (Fig. 6B). These differences in OT-stimulated uterine contractility, after pretreatment with vehicle versus OA, were statistically significant (Fig. 7). When expressed as a percentage of baseline contractile activity, there was no significant difference between vehicle (112 ± 8) and OA (88 ± 5) at initial treatment. Whereas, as observed in Figure 7, after pretreatment with vehicle (DMSO), cumulative administration of OT at 8, 16, 32, and 64 nM resulted in percentages of baseline contraction activity (mean ± SEM) of 128 ± 8, 128 ± 8, 145 ± 11, and 160 ± 19, respectively. While contractile activity increased with increasing doses of OT, these differences were not significant. Okadaic acid pretreatment, prior to cumulative administration of OT, resulted in decreasing percentages of baseline contraction activity (mean ± SEM) of 95 ± 9, 72 ± 12, 66 ± 18, and 67 ± 18, respectively.



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  		FIG. 6. Representative in vitro contraction study demonstrating the inhibitory effect of OA pretreatment on OT-stimulated contractions. Contractile activity reported in grams of tension generated in response to OT after pretreatment with vehicle (A) or OA (B). Horizontal lines indicate the times for the cumulative additions of OT



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  		FIG. 7. Bar graph demonstrating the contractile activity (reported in percentage of baseline activity) of mouse uterine horns in response to pretreatment with vehicle (DMSO; n = 7) or OA (n = 7) followed by cumulative addition of OT. Area under the contractile curve for a 5-min interval during the end of the 20-min equilibration period (baseline), following the addition of vehicle (DMSO) or OA (1 µM), and during each of the cumulative additions of OT were utilized to calculate the contractile activity. Vehicle or OA were added following the 20-min baseline period and were maintained in the bath during the remainder of the experiment. Oxytocin was added at 10-min intervals in a cumulative manner resulting in an 8, 16, 32, and 64 nM concentration. Data are reported in mean ± SEM. For each concentration of OT, bars with different letters are significantly different (P < 0.05)

Type 1{alpha} PP Expression During Sexual Development

The questions of when and where myometrial PP1 is first expressed, as well as if PP2A is expressed in the myometrium in relation to sexual development were assessed by immunohistochemical studies (Fig. 8). Substituting NIRS for the primary antibodies showed no DAB staining. Mouse heart was used as positive control tissue for both PP1{alpha} and PP2A. During the prepubertal period, Days 11 to 17, PP1{alpha} was highly expressed in the longitudinal layer of the myometrium and only slightly expressed in the circular myometrial layer. In the late peripubertal period (i.e., Day 19), PP1{alpha} was highly expressed in both the longitudinal and circular layers of the myometrium (similar to the tissue expression pattern observed in adult eCG-treated uteri). At no point during this period of sexual development was PP1{alpha} expression observed in the endometrial stroma. During this prepubertal period, PP2A expression was not observed in the myometrium; whereas, it was present in the endometrial stroma, particularly in the perilumenal region (data not shown).



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  		FIG. 8. Representative immunohistochemical studies demonstrating the localization of PP1{alpha} and PP2A in mouse heart (A1, B1, C1; control) and uteri during sexual development. Uteri were collected from mice during the prepubertal period on Days 11 (A2, B2, C2), 13 (A3, B3, C3), 15 (A4, B4, C4), 17 (A5, B5, C5), and the peripubertal period on Day 19 (A6, B6, C6). Tissues were fixed, paraffin embedded, and exposed to microwave antigen retrieval prior to incubation with primary antibodies or NIRS (A1–A6) followed by biotinylated secondary antibody and colorimetric evaluation with avidin-biotin-peroxidase and DAB. Slides were counterstained with Meyers hematoxylin. Optical magnification: A2–A6 = x100; remainder = x400. CL, Circular layer of the myometrium; LL, longitudinal layer of the myometrium

DISCUSSION

Early studies on smooth muscle PPs using chicken gizzards [16] and bovine aorta [15] indicated that PP2A was the predominant PP based on subunit composition. Turkey gizzard was found to contain four separate PPs termed smooth muscle protein phosphatases I–IV (SMP-I, SMP-II, SMP-III, SMP-IV [27–29]). The SMP-I and SMP-II were characterized as PP2A and PP2C, respectively [27], whereas SMP-III and SMP-IV were found to have PP1-like activity [28, 30]. More recent approaches to smooth muscle phosphatase characterization have emphasized identification of the PP that is physically associated with myosin (or actomyosin) with the assumption that the actual myosin light chain phosphatase is expected to interact with myosin heads. In these studies the myosin (actomyosin)-associated PP in gizzard and bladder preferentially dephosphorylates the ß subunit of phosphorylase kinase. Furthermore, it is inhibited by OA, I1, and I2 and thus appears to be PP1 [14, 17–19, 31]. Presently there are no reports characterizing the myometrial PP.

Protein phosphatase 1 was observed to be highly expressed in the longitudinal and circular layers of the myometrium; whereas, PP2A was highly expressed only in the endometrial stroma. The line of demarcation between the myometrium and endometrium is recognizable with either PP1 or PP2A immunostaining. In addition to being highly expressed in myometrium, PP1 was also observed in the endometrial epithelium. The PP1 in endometrial epithelial cells might be involved with numerous cell functions ranging from control of cell proliferation [32], regulation of transcriptional activity [33], or controlling intracellular organelle trafficking [34] that might influence epithelial secretory activity. A role for endometrial PP1 in any of these cell functions remains to be elucidated and warrants further investigation.

Distribution of PP1c isoforms in different tissues has not been analyzed in detail; however, data exist in relation to isoform enrichment in specific tissues. For instance, PP1{gamma}1 is highly expressed in brain [35]; PP1{gamma}2 is enriched in testes [9, 36] and mammalian spermatozoa [37]. Type 1{delta} PP has been the primary isoform identified in both smooth and striated muscle [31, 38]. Type 1{delta} PP mRNA and protein have been reported in both gizzard and aorta smooth muscle [31] (Ito M, unpublished observation in Erdödi et al. [39]), yet no reports exist with respect to PP1{delta} isoform expression in the myometrium. In the screening of a gizzard cDNA library PP1{alpha} has been detected in this smooth muscle (Ito M, unpublished observation in Erdödi et al. [39]), and now we have reported PP1{alpha} protein expression in the myometrium. This observation of PP1{alpha} in the myometrium does not preclude the possible presence of PP1{delta}.

All past reports involving myometrial PPs have used OA to investigate the role of PPs in rat myometrial strips. Candenas and coworkers [20] first reported that OA could inhibit and stimulate uterine contractions at low (100 nM) and high (5 µM) concentrations, respectively. Okadaic acid-stimulated uterine contractions are partially inhibited when transmembrane Ca2+ movement was inhibited with Ca2+-free media plus EDTA [21, 22]. Administration of a cocktail of antagonists that block muscarinic, adrenergic, histaminergic, serotonergic, and opioid receptors were not found to alter significantly the stimulatory influence of 5–20 µM OA [22]. In our experience in other cell systems, functional effects of PP inhibition by OA have been recognized at doses between 50 nM and 1 µM [37, 40, 41]. With this in mind we designed the present study with OA doses lower than those previously used. Mouse uterine horn contractions were not altered significantly by increasing concentrations of OA (10 nM to 1 µM) in comparison to vehicle-treated control horns from the same animal. Following the cumulative addition of OA, uterine horns displayed a decreased responsiveness to OT- and KCl-stimulated contractions in comparison to vehicle-treated controls; however, if OA was removed by washout, the uterine horns displayed OT and KCl responsiveness.

To address this OA-dependent decreased OT responsiveness further, experiments were performed with a single administration of OA (1 µM) followed by cumulative addition of OT. Whereas the initial administration of OA had no significant influence on unstimulated uterine contractions, in relation to baseline and controls, this single OA treatment significantly inhibited the responsiveness of mouse uterine horns to OT-stimulated contractions. This OA-induced suppression of OT-stimulated contractions is similar to that recently described in the estrogen-primed rat myometrial strip system [42].

This divergence in spontaneous and OT-stimulated contractile responses to PP inhibition begins to provide insights into possible intracellular mechanisms by which PPs are involved with regulating myometrial contractions. There are at least three intracellular sites of action that could explain the observation that OA inhibition of myometrial PPs results in decreased responsiveness to OT. The first possibility is receptor desensitization. Regulation of receptor desensitization at the post-translational level can involve association with other proteins, internalization, or changes in subcellular location that collectively represent recycling of the receptor [43]. One of the primary proteins that interact with the agonist-bound receptor is ß-arrestin. Cytoplasmic ß-arrestin is constitutively phosphorylated, and dephosphorylation at Ser-412 is required for its function in receptor endocytosis [44]. Thus, inhibition of this Ser/Thr PP by OA might decrease dephosphorylation of ß-arrestin, blocking OT-receptor internalization, subsequent recycling of the receptor to the plasma membrane, and thus result in reduced responsiveness to OT-stimulated uterine contractions.

The second possible mechanism by which PP inhibition decreases OT responsiveness might involve the availability of MLCK. Phosphorylation of MLCK at Ser-815 and Ser-828 decreases the affinity of MLCK for Ca2+/calmodulin [3]. This phosphorylation of MLCK results in a 10-fold increase in the concentration of Ca2+/calmodulin necessary for half-maximal activation of MLCK [45]. Dephosphorylation of MLCK at these Ser residues increases the pool of MLCK available for activation and therefore increases the responsiveness of the system to uterotonin-stimulated Ca2+/calmodulin. Thus, one can envision that inhibition of a Ser-PP by OA would decrease dephosphorylation of MLCK (increase the phosphorylation of MLCK), increase the concentration of Ca2+/calmodulin necessary for half-maximal activation of MLCK, and thus desensitize the system to OT stimulation.

The final explanation is that myosin light chain dephosphorylation is important for resetting the system for subsequent stimulation [3]. If dephosphorylation of myosin is inhibited myosin light chains would lose responsiveness to MLCK, and consequently, further stimulation with OT would result in diminished contractile responsiveness. Any, or all, of these mechanisms might explain the observed effects of OA inhibition of myometrial PP(s) and contractile activity in response to OT stimulation. Whether specific PP-control pathways predominate during different physiological conditions or under specific stimulatory situations remains to be elucidated.

In an attempt to understand extracellular and intracellular mechanisms regulating myometrial contractions many investigators have focused on comparing specific factors (responsiveness to various uterotonic agonists, receptors, G-proteins, second messengers, etc.) between differing hormonal milieus and physiological states. One such comparison is between the prepubertal (low estrogen) and pubertal (high estrogen) states. While sexually immature uteri are capable of spontaneous contractions [46–48], they have been demonstrated to be less sensitive to OT stimulation in comparison to uteri from sexually mature animals [46]. In this study we have asked the question: might PP1 expression in the myometrium be a factor contributing to the change in contractility between the prepubertal and pubertal states? During the prepubertal period, PP1 expression is low in the circular myometrium, yet present in the longitudinal layer. There is a change in PP1 myometrial expression during the peripubertal period where PP1 begins to be highly expressed not only in the longitudinal layer, but also in the circular layer by Day 19. This expression pattern is similar to what we have observed for eCG-stimulated adult uteri. When one considers these expression data, the influence of OA inhibition and its effects on spontaneous and OT-stimulated contractions, and the lack of uterine OT sensitivity during the prepubertal period, we begin to see a scenario unfold whereby PP1 appears to be important for responsiveness of uterine tissue to OT. The observed change in PP1 expression during the pubertal transition may contribute to OT responsiveness during sexual development and requires future investigative attention.

In conclusion we have reported, for the first time, the direct identification of PP1 as the OA-sensitive PP within the myometrium. Type 2A PP was absent from the myometrium but present in the endometrial stroma. Type 1 PP was differentially expressed in the circular and longitudinal myometrium in relation to sexual development and may account for changes in responsiveness to uterotonic agonists during maturation. In addition, OA inhibition of myometrial PP1 had no effect on spontaneous, but inhibited OT-stimulated, contractions of mouse uterine horns.

ACKNOWLEDGMENTS

The authors thank Andrew Basa for technical assistance and Dr. Carrie Cosola-Smith for critical review of the manuscript.

FOOTNOTES

First decision: 7 January 2000.

1 A portion of this work was supported by NIH grant CA66132 (G.D.S.). Back

2 Correspondence: Gary D. Smith, 6428 Medical Sciences Building I, 1301 E. Catherine St., Ann Arbor, MI 48109-0617. FAX: 734 647 1006; smithgd{at}umich.edu Back

Accepted: April 17, 2000.

Received: December 7, 1999.

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