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Interleukin-1ß Induces Calcium Transients and Enhances Basal and Store Operated Calcium Entry in Human Myometrial Smooth Muscle¹

Parturition Research, Maternal and Fetal Research Unit,³ Department of Women's Health, Centre for Cardiovascular Biology and Medicine,⁴ King's College London, St. Thomas' Hospital Campus, London SE1 7EH, United Kingdom

	ABSTRACT

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We have previously reported increased protein expression of sarcoplasmic reticulum calcium ATPase (SERCA) 2b in myometrium from women in labor at term, but the stimulus for this change is unknown. Proinflammatory cytokines have been implicated in the cascade of events leading to preterm and term labor, and we hypothesize that interleukin (IL)-1ß may induce changes in key calcium homeostatic mechanisms and, in turn, augment myometrial contractility before labor. The aim of the present study was to investigate the long-term effects of IL-1ß on SERCA 2b protein expression, calcium mobilization from intracellular stores, and store-operated calcium entry. Myometrial biopsies were obtained, with consent, from women undergoing elective cesarean section at term. Primary-cultured human myometrial smooth muscle (HMSM) cells were exposed to IL-1ß (10 ng/ml) for 24 h or to culture medium alone (control). Cells were subsequently used in Western blot studies or loaded with fura-2 to assess calcium dynamics using fluorescent digital imaging. The present study clearly demonstrated that IL-1ß significantly increased SERCA 2b protein expression in HMSM cells. Cyclopiazonic acid-induced calcium transients were also augmented, predominantly by activation of lanthanum-sensitive, store-operated calcium entry. HMSM cell excitability was enhanced, as evidenced by increased basal calcium entry and the initiation of spontaneous calcium transients in 37% of IL-1ß-treated cells. IL-1ß modulation of calcium mobilization may be an important mechanism in the cascade of events preparing the pregnant uterus for labor.

calcium, cytokine, parturition, signal transduction, uterus

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
During human pregnancy, the smooth muscle of the uterus, the myometrium, is relatively quiescent until the onset of contractile activity in association with labor. The cascade of events precipitating human labor remains unclear, but it is proposed that the myometrium becomes primed to contract before the initiation of labor by activation of a "cassette of genes" encoding for a number of contraction-associated proteins [1], which include cyclooxygenase-2 [2] and oxytocin receptors [1]. The proposed stimuli for increased contraction-associated protein expression include placental hormones, stretch, and cytokines [3]. Increasingly, it appears that proinflammatory cytokines play a role in human preterm and term labor. Cytokines (interleukin [IL]-1ß, IL-8, and IL-6) are present in myometrial tissue [4–6], and raised concentrations of cytokines in cervicovaginal secretions are reported in women with preterm rupture of membranes and during term and preterm labor [7, 8]. In primates, inoculation of amniotic fluid with IL-1ß initiates uterine contractility [9], and in vitro IL-1ß and IL-6 increase cyclooxygenase-2 [10] and oxytocin-receptor [11] protein expression, respectively, in human myometrial smooth muscle (HMSM) cells.

Recently, we have demonstrated that calcium ATPases are gestationally regulated and that sarcoplasmic reticulum calcium ATPase (SERCA) 2b protein expression is increased in the myometrium in association with the onset of human labor [12]. Furthermore, that study suggested that SERCA 2b and store-operated Ca²⁺ (SOC) entry play a greater role in the regulation of contractile activity of myometrium from women in active labor compared to that from women at term who had not progressed to labor [12]. The precise role of SOC entry in human myometrium has not been defined, but in many types of mammalian smooth muscle, SOC entry is responsible for refilling sarcoplasmic reticulum (SR) Ca²⁺ stores and augments agonist-induced responses [13]. Studies of the hormonal regulation of SERCA and SOC entry are limited, although growth factors and serum are reported to modulate SERCA and SOC entry in vascular smooth muscle and endothelial cells [14–18].

The present study explores the hypothesis that proinflammatory cytokines may mediate an increase in human myometrial contractility during labor by modulation of Ca²⁺ mobilization. Specifically, the effect of prolonged exposure to IL-1ß on SERCA 2b protein expression, Ca²⁺ mobilization, and SOC entry in term pregnant human myometrium was studied.

	MATERIALS AND METHODS

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Subjects

Written informed consent was obtained for the removal of myometrial biopsy specimens during elective cesarean section at term from women who had not progressed to labor (n = 25). None of the pregnant women included in the present study had underlying disease. The medical reason for cesarean section was breech presentation, placenta previa, or maternal request. Biopsy specimens were taken from the midline of the upper edge of a lower segment incision and placed immediately in cold physiological salt solution (PSS; 140 mM NaCl, 5.9 mM KCl, 1.2 mM NaH₂PO₄, 5 mM NaHCO₃, 1.4 mM MgCl₂, 1.8 mM CaCl₂, 11.5 mM glucose, and 10 mM Hepes titrated to pH 7.4 with NaOH) and used immediately for primary cell culture. The present study was approved by the Ethics Committee of St. Thomas' Hospital, London, United Kingdom (EC94/037), and was performed in accordance with the Declaration of Helsinki.

Isolation and Primary Culture of HMSM Cells

The HMSM cells were isolated as previously described [12]. Briefly, small segments of myometrium were incubated (37°C, 30–40 min) with 1 mg/ml of collagenase 1A and 1 mg/ml of collagenase XI. Isolated myometrial smooth muscle cells were resuspended in Dulbecco modified Eagle medium supplemented with 5% fetal calf serum (Invitrogen, Paisley, Renfrewshire, Scotland), 40 U/ml of penicillin, and 40 µg/ml of streptomycin. Myocytes were incubated (37°C, 95% air, 5% CO₂) and maintained as a primary culture until confluent (on average, 7 days). The culture medium was changed every 2–3 days. The purity of myocyte cultures was routinely assessed by immunocytochemistry using -actin and calponin monoclonal antibodies.

Treatment of HMSM Cells with IL-1ß

Confluent cells were serum deprived for 24 h (Dulbecco modified eagle medium plus 0.5% fetal calf serum). The medium was aspirated and replaced with Dulbecco modified Eagle medium plus 0.5% fetal calf serum supplemented with IL-1ß (10 ng/ml; R&D Systems, Abingdon, Oxon, U.K.) or Dulbecco modified Eagle medium plus 0.5% fetal calf serum (control) for a further 1, 4, or 24 h. The IL-1ß concentration used was within the range reported for cervicovaginal secretions of women in labor and women with preterm rupture of membranes [7, 8].

Western Blot Analysis

As previously described [12], primary-cultured HMSM cells (either IL-1ß-treated or respective time controls) were washed in phosphate-buffered saline, and then 250 µl of ice-cold lysis buffer (10 mM of Hepes-KOH [pH 7], 1 mM of dithiothreitol, 1% nonident-P40, and protease-inhibitor cocktail [COMPLETE tablets; Boehringer-Mannheim Biochemicals, Lewes, Sussex, U.K.]) were added. The protein content of the lysates was determined using a detergent-compatible protein ELISA-based assay kit with BSA as a standard (Bio-Rad Laboratories Ltd., Hemel Hempstead, Herts, U.K.).

Before electrophoresis, cell lysates (5 µg) were diluted 1:1 with SDS sample buffer containing 0.9% (v/v) 2-mercaptoethanol for 10 min at room temperature, and samples were separated by 7.5% SDS-PAGE gels. The proteins were transferred to polyvinylidene difluoride membranes [19]. All subsequent steps were carried as previously reported [12]. Transfer efficiency was assessed using a Ponceau red stain. Each membrane was blocked for 1 h with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween-20, and the membranes were incubated for 1 h with either polyclonal SERCA 2b antibody (gift from Dr. F. Wuytack, University of Leuven, Belgium; diluted 1:5000 in blocking buffer) or monoclonal -actin antibody (Sigma, Poole, Dorset, U.K.; diluted 1:100 000). Secondary-antibody incubation was carried out for 1 h with a horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibody (Amersham International Plc., Little Chalfont, Bucks, U.K.). Protein bands were visualized with enhanced chemiluminescence reagents according to the manufacturer's instructions (Amersham). Western blots were analyzed using the Kodak EDAS 120 system (Eastman Kodak, Rochester, NY). The signal-intensity values of SERCA 2b and -actin protein bands from IL-1ß-treated cells were expressed as a percentage of the respective untreated time controls. Negative controls were performed as described above but in the absence of primary antibody.

Estimation of Intracellular Ca²⁺ Concentration

The method was modified from that previously described by Borin et al. [20]. Briefly, HMSM cells exposed to IL-1ß for 24 h, and respective control cells were loaded with the acetoxymethyl ester of the fluorescent Ca²⁺-indicator fura-2 (3 µM, 40 min at room temperature) and subsequently perfused with PSS for 30 min to allow for desterification of the fura-2 and removal of extracellular dye. Digital Ca²⁺ imaging was performed: Fura-2 fluorescence (emission wavelength, 510 nm) excited by 360 and 380 nm from HMSM cells and background fluorescence was imaged every 4–6 sec using a digital camera (Pentamax cCCD; Princeton Instruments, Trenton, NJ) controlled by Universal Imaging Metafluor computer software (Downington, PA). All cells within the field of view were simultaneously imaged and areas of interest within a cell, limited to the cytosol, monitored. The 360:380 ratios were calculated from background-subtracted images, with an increase in the ratio indicating a rise in intracellular Ca²⁺ concentration ([Ca²⁺]_cyt). The role of SERCA was examined by initiating Ca²⁺ release from intracellular stores using cyclopiazonic acid (CPA; 5 µM) in either PSS or in the absence of extracellular Ca²⁺ ([Ca²⁺]_o) plus 1 mM EGTA (separate experiments, Ca²⁺-free PSS applied 1 min before CPA addition). The peak change in fura-2 360:380 ratio (F_360/380) from baseline is reported. The effect of IL-1ß on SOC entry was also examined by replacing PSS with Ca²⁺-free PSS after 5-min exposure of cells to CPA. After 5 min in Ca²⁺-free PSS, Ca²⁺ was restored and the subsequent rate of [Ca²⁺]_cyt increase (F_360/380/sec) determined (using linear regression) as a measure of SOC entry. Experiments were repeated in the presence of the L-type Ca²⁺ channel-inhibitor diltiazem (20 µM). Experiments were also performed to determine the La³⁺-sensitivity (50 µM in PSS) of basal and SOC entry in IL-1ß-treated and control cells. SOC entry was further investigated by the application of extracellular Mn²⁺ (200 µM, MnCl₂ in nominally Ca²⁺-free PSS, no EGTA), a surrogate ion for Ca²⁺ that is not extruded by the plasma membrane calcium ATPase [21] or by Na/Ca exchange [22]. The initial rate of Mn²⁺ influx (quench of fura-2 360-nm fluorescence, [F₃₆₀/sec]) was measured. Modified PSS (sodium phosphate and sodium bicarbonate removed) was used for La³⁺ and Mn²⁺ experiments.

Whole-Cell Current Recording

Recordings were made using the tight-seal, whole-cell configuration of the patch-clamp technique [23]. Experiments were performed in single control and IL-1ß-treated HMSM cells using the standard patch-clamp technique with Axopatch 200A amplifier and pCLAMP-6 software (Axon Instruments, Union City, CA). The patch-pipette solution contained 135 mM CsCl, 2.5 mM MgCl₂, 10 mM EGTA, 10 mM Hepes, and 5 mM Na₂ATP. The bath solution contained 120 mM NaCl, 1 mM CsCl, 4 mM tetraethylammonium chloride, 1.2 mM MgCl₂, 10 mM BaCl₂, 10 mM Hepes, and 10 mM glucose. The L-type Ca²⁺-channel current was examined by holding cells at -50 mV and stepping to +20 mV, whereas the T-type Ca²⁺-channel current was examined by holding cells at -80 mV and stepping to -10 mV.

Statistical Analysis

Numerical data were analyzed in Microsoft Excel 97 (Redmond, WA), Microcal Origin 5.0 (Northampton, MA), Sigmaplot 2000 (Clecom, Birmingham, U.K.) and Graphpad Instat (San Diego, CA) and were expressed as the mean ± SEM. An ANOVA with Dunnett correction for multiple comparisons, Kruskal-Wallis nonparametric ANOVA with Dunn correction for multiple comparisons, or Student t-test were used as applicable. A value of P 0.05 was considered to be significant. When n refers to the number of cells, protocols were performed in cells originating from at least three subjects.

	RESULTS

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Effect of IL-1ß Treatment on SERCA 2b Protein Expression in HMSM Cells

Serum-deprived, primary-cultured HMSM cells expressed SERCA 2b protein (Fig. 1A). Treatment with IL-1ß significantly increased SERCA 2b protein expression at 24 h (% SERCA 2b expression of paired time control: 151.29% ± 22.64%) compared to 1 h (86.17% ± 5.03%) and 4 h (86.42% ± 13.93%, P < 0.05) (Fig. 1A). At the same time points, IL-1ß did not significantly alter smooth muscle actin protein expression (Fig. 1B).

View larger version (38K): [in this window] [in a new window]   FIG. 1. Treatment with IL-1ß for 24 h enhances SERCA 2b protein expression in primary-cultured HMSM cells. A) Representative Western blot and summarized data (expressed as a percentage of paired time control [con]) of the effect of 1- to 24-h treatment with IL-1ß (10 ng/ml) on SERCA 2b protein expression (100 kDa) in serum-deprived HMSM cells. B) Representative Western blot and summarized data of the effect of 1- to 24-h treatment with IL-1ß (10 ng/ml) on -actin protein expression (42 kDa) in serum-deprived HMSM cells. Data are presented as the mean ± SEM (n = 6–8). *P < 0.05, 24-h treatment versus 1-h and 4-h treatments

Effect of 24h IL-1ß Treatment on Ca²⁺ Mobilization from SR Ca²⁺ Stores in HMSM Cells

Because increased SERCA 2b protein expression by IL-1ß might be expected to lead to an increase in the size of the releasable SR Ca²⁺ store, CPA-induced Ca²⁺ responses were assessed in IL-1ß-treated and control HMSM cells. Indeed, CPA induced a greater rise (89%) in [Ca²⁺]_cyt in cells treated with IL-1ß for 24 h (F_360/380: 0.469 ± 0.020, n = 71) compared to untreated cells (0.248 ± 0.017, n = 76, P < 0.001) (Fig. 2). However, the increase in peak [Ca²⁺]_cyt on addition of CPA reflects not only SR Ca²⁺ release but also the influence of Ca²⁺ entry and Ca²⁺ extrusion by the plasma membrane calcium ATPase and Na/Ca exchange. In an attempt to delineate the contribution of SR Ca²⁺ release from that of Ca²⁺ entry, further experiments were performed in Ca²⁺-free PSS (Fig. 2B). In the absence of [Ca²⁺]_o, the CPA-induced rise in [Ca²⁺]_cyt in IL-1ß-treated cells (0.270 ± 0.024, n = 60) was still significantly higher than that of the controls (0.146 ± 0.011, n = 44, P < 0.01). However, the peak CPA-induced response in IL-1ß-treated cells was reduced in Ca²⁺-free PSS, which strongly suggests that IL-1ß enhances Ca²⁺ entry as well as augments SR Ca²⁺ release (Fig. 2B). The potential involvement of Ca²⁺ entry via voltage-gated channels was assessed by addition of diltiazem and by direct measurement of L- and T-type Ca²⁺ currents. Neither control cells (n = 7) nor IL-1ß-treated cells (n = 9) demonstrated the presence of either L- or T-type Ca²⁺-channel currents (data not shown), and diltiazem did not affect the CPA-induced Ca²⁺ rise in IL-1ß-treated cells (n = 51), which remained significantly greater (95%) than in controls (n = 54, P < 0.00001). The increase in Ca²⁺ entry was therefore independent of altered activity of voltage-gated Ca²⁺ channels.

View larger version (27K): [in this window] [in a new window]   FIG. 2. Treatment with IL-1ß for 24 h increases CPA-induced Ca2+ entry in primary cultured HMSM cells. A) Representative traces to demonstrate CPA (5 µM)-induced Ca2+ mobilization in IL-1ß-treated (10 ng/ml, 24 h) versus serum-deprived control (Con) HMSM cells. B) Data are presented as the mean ± SEM to show the magnitude of CPA-induced Ca2+ mobilization in IL-1ß-treated (10 ng/ml, 24 h) versus control serum-deprived HMSM cells in the presence (PSS) and absence of [Ca2+]o (Ca2+-free PSS). ***P < 0.001, comparison between IL-1ß-treated and control cells in PSS; **P < 0.01, comparison between IL-1ß-treated and control cells in Ca2+-free PSS; P < 0.001, comparison between IL-1ß-treated cells in PSS and Ca2+-free PSS; P < 0.01 comparison in control cells in PSS versus Ca2+-free PSS (n = 44–76 cells)

Effect of IL-1ß on Basal and SOC Entry in HMSM Cells

Because a substantial portion of the CPA-induced Ca²⁺ mobilization in IL-1ß-treated cells appeared to result from an increase in SOC entry, this was further assessed by calculating the initial rate of Ca²⁺ entry following CPA-induced depletion of Ca²⁺ stores in a Ca²⁺-free medium. The readdition of [Ca²⁺]_o elicited a rapid rise in [Ca²⁺]_cyt in both treated and untreated cells. The slope of the initial rate of rise of [Ca²⁺]_cyt in IL-1ß-treated cells (F_360/380/sec: 0.0143 ± 0.0011, n = 72 cells) following readdition of extracellular Ca²⁺ was significantly steeper than in controls (0.0062 ± 0.0003, n = 74 cells, P < 0.00001) (Fig. 2A). This difference in the initial rate of rise remained in the presence of diltiazem (0.0132 ± 0.0011 with n = 52 IL-1ß-treated cells vs. 0.0024 ± 0.0002 with n = 54 control cells, P < 0.00001).

Resting [Ca²⁺]_cyt in PSS was also higher in IL-1ß-treated HMSM cells (F_360/380: 1.214 ± 0.011, n = 233) compared to control cells (1.087 ± 0.008, n = 216, P < 0.00001) (data not shown). This suggests that basal Ca²⁺ entry may be enhanced and potentially contribute to the larger Ca²⁺ response observed on SR Ca²⁺-store depletion by CPA in IL-1ß-treated cells.

This possibility was examined further by assessing [Ca²⁺]_cyt in the presence and absence of [Ca²⁺]_o. Whereas basal Ca²⁺ entry was apparent in both control (F_360/380: PSS, 0.926 ± 0.009; Ca²⁺-free PSS, 0.900 ± 0.008; n = 44, P < 0.0001) and IL-1ß-treated cells (F_360/380: IL-1ß, 1.154 ± 0.032; Ca²⁺-free PSS, 1.062 ± 0.015; n = 60, P < 0.001). The effect of [Ca²⁺]_o removal on basal Ca²⁺ in IL-1ß-treated cells ( F_360/380, 0.092 ± 0.024, n = 60) was significantly greater than in controls (0.026 ± 0.004, n = 44, P < 0.05) (see Fig. 3). However, because basal Ca²⁺ entry in IL-1ß-treated cells was numerically much less than the change in Ca²⁺ evoked by CPA, the contribution to the augmented CPA response was probably small. Basal Ca²⁺ entry in control and IL-1ß-treated cells was insensitive to diltiazem (data not shown).

View larger version (34K): [in this window] [in a new window]   FIG. 3. Entry of [Ca2+]o-dependent- and La3+-sensitive Ca2+ in IL-1ß-treated and control (Con) primary-cultured HMSM cells. Data are presented as the mean ± SEM to show the effect of [Ca2+]o removal on basal Ca2+ entry in control (n = 35) versus IL-1ß-treated (10 ng/ml, 24 h, n = 43) HMSM cells and addition of La3+ (50 µM) on basal Ca2+ entry (control, n = 60; IL-1ß, n = 66) and CPA-induced Ca2+ entry in control (n = 61) and IL-1ß-treated cells (n = 45). N.S., Comparison between La3+-sensitive basal and CPA-induced Ca2+ entry in control cells. *P < 0.05, ***P < 0.001, comparison between control and IL-1ß treated cells; P < 0.05, comparison between La3+-sensitive basal and CPA-induced Ca2+ entry in IL-1ß-treated cells

La³⁺ Sensitivity of Basal Ca²⁺ Entry, Spontaneous Ca²⁺ Transients, and CPA-Induced Ca²⁺ Entry in Control and IL-1ß-Treated Cells

Basal Ca²⁺ entry was slightly reduced by 50 µM La³⁺ in control cells (F_360/380: PSS baseline, 1.108 ± 0.008; PSS + La³⁺, 1.070 ± 0.009; n = 60, P < 0.00001) (Fig. 4A). La³⁺ also significantly inhibited basal Ca²⁺ entry in IL-1ß-treated cells (F_380/360: PSS baseline, 1.210 ± 0.008; PSS + La³⁺, 1.127 ± 0.005; n = 66, P < 0.00001) (Fig. 4B); however, the La-sensitive portion of basal Ca²⁺ entry was substantially larger in the IL-1ß-treated cells ( F_360/380: 0.083 ± 0.006) compared to controls (0.037 ± 0.004, P < 0.001) (see Fig. 3).

View larger version (18K): [in this window] [in a new window]   FIG. 4. Entry of La3+-sensitive basal Ca2+ in IL-1ß-treated and control primary-cultured HMSM cells. A) Representative traces to demonstrate La3+ (50 µM) sensitivity of basal Ca2+ entry in three serum-deprived, control HMSM cells. Washout out of La3+ did not affect basal Ca2+. B) Representative traces to demonstrate the inhibitory effect of La3+ (50 µM) on basal Ca2+ entry and Ca2+ oscillations in four serum-deprived, IL-1ß-treated HMSM cells. On removal of La3+, basal Ca2+ responses were restored and oscillations enhanced

Of 53 cells treated with IL-1ß, 20 (37%) exhibited spontaneous Ca²⁺ transients (Fig. 4B), whereas no spontaneous transients were observed in 35 control cells (Fig. 4A). La³⁺ inhibited the generation of spontaneous Ca²⁺ transients, and on washout, a rebound increase in [Ca²⁺]_cyt was observed in all cells, which often resulted in the resumption (or initiation) of spontaneous Ca²⁺ transients (Fig. 4B). No effect of La³⁺ washout was observed in control cells (Fig. 4A).

La³⁺ had a negligible influence on the CPA-induced Ca²⁺ plateau in control cells ( F_360/380: 0.037 ± 0.003, n = 61), with the response being of a similar magnitude to the small effect of La³⁺ on basal Ca²⁺ entry in the absence of CPA (Figs. 5A and 3). In comparison, La³⁺ significantly reduced the CPA-induced Ca²⁺ plateau in IL-1ß-treated cells ( F_360/380: 0.456 ± 0.053, n = 45 cells, P < 0.001) (Figs. 5B and 3). The La³⁺-sensitive component of the CPA-induced response was greater than that of basal Ca²⁺ entry (Fig. 3), suggesting that IL-1ß increases SOC entry.

View larger version (23K): [in this window] [in a new window]   FIG. 5. Effect of La3+ on CPA-induced Ca2+ responses in IL-1ß-treated and control HMSM cells. A) Representative traces to demonstrate that La3+ (50 µM) only slightly reduced the CPA-induced Ca2+ plateau in two control HMSM cells. Subsequent removal of [Ca2+]o (Ca2+-free), in the continued presence of La3+, reduced [Ca2+]cyt to just below baseline. Readdition of [Ca2+]o (PSS) increased [Ca2+]cyt to above basal levels. B) Representative traces to demonstrate the La3+ sensitivity of the CPA-induced Ca2+ plateau in IL-1ß-treated (10 ng/ml, 24 h) HMSM cells. La3+ reduced [Ca2+]cyt to basal levels, and removal of [Ca2+]o reduced this further. Readdition of [Ca2+]o increased [Ca2+]cyt to just above basal levels, but on washout of La3+, [Ca2+]cyt returned to a level similar to that of the CPA-induced plateau

Removal of [Ca²⁺]_o in both IL-1ß-treated and control cells, in the continued presence of CPA and La³⁺, further reduced [Ca²⁺]_cyt (Fig. 5). On readdition of [Ca²⁺]_o (in the continued presence of CPA and La³⁺), [Ca²⁺]_cyt increased. Washout of La³⁺ initiated a further rise in [Ca²⁺]_cyt in IL-1ß-treated, but not in control, cells (Fig. 5).

Basal and CPA-Induced Mn²⁺ Influx in IL-1ß and Control HMSM Cells

To confirm that the enhanced La³⁺-sensitive basal and CPA-induced Ca²⁺ responses were caused by increased Ca²⁺ influx as opposed to reduced Ca²⁺ extrusion, Mn²⁺ quench of fura-2 fluorescence at 360 nm was examined. In concordance with the lack of La³⁺-sensitive Ca²⁺ entry, Mn²⁺ quench of fura-2 fluorescence was not apparent in basal and CPA-treated control cells. The basal rate of Mn²⁺ entry was, however, significantly greater in IL-1ß cells (F₃₆₀/sec: 2-fold, n = 47) compared to controls (n = 54, P < 0.001) (Fig. 6A). The CPA-stimulated Mn²⁺ entry was also greater in IL-1ß-treated cells (F₃₆₀/sec: 5-fold, n = 60) compared to controls (n = 62, P < 0.001) (Fig. 6B).

View larger version (13K): [in this window] [in a new window]   FIG. 6. Basal and CPA-induced changes in Mn2+ influx in control (Con) and IL-1ß-treated primary cultured HMSM cells. A) Representative plot to show basal Mn2+ quench of fura-2 fluorescence (% change) in control versus IL-1ß (10 ng/ml, 360 nM)-treated HMSM cells. B) Representative plot to show CPA-activated Mn2+ quench of fura-2 fluorescence at 360 nM (% change) in control versus IL-1ß (10 ng/ml)-treated HMSM cells

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The present study has explored the hypothesis that the proinflammatory cytokine IL-1ß plays a role in the initiation of molecular events that underlie the functional preparation of the myometrium for labor. We have clearly demonstrated that prolonged exposure to IL-1ß increases SERCA 2b protein expression and augments CPA-induced Ca²⁺ mobilization from the SR and SOC entry in primary-cultured HMSM cells from pregnant women. Cell excitability was also enhanced, as evidenced by increased basal Ca²⁺ entry and the spontaneous initiation of Ca²⁺ transients in 37% of cells exposed to IL-1ß. This is, to our knowledge, the first demonstration of IL-1ß-induced stimulation of Ca²⁺ mobilization, SOC entry, and SERCA expression in any human smooth muscle.

Effect of IL-1ß on SERCA 2b Protein Expression

The hypothesis that proinflammatory cytokines may contribute to altered myometrial SERCA expression at the time of labor [12] is supported by the observation that IL-1ß augmented SERCA 2b protein expression in HMSM cells at 24 h. An influence of proinflammatory cytokines on SERCA 2b expression per se has not been previously reported, although an endocrine/paracrine influence on SERCA 2 has been implied by previous observations [14, 16]. Platelet-derived growth factor (PDGF) enhances SERCA 2a protein expression in porcine aortic smooth muscle [16], and epidermal growth factor costimulates SERCA and plasma membrane calcium ATPase mRNA expression in rat aortic endothelial cells [14]. The pathway by which IL-1ß regulates SERCA 2b expression in HMSM cells remains to be elucidated, although Kuo et al. [14] have convincingly demonstrated that the "state of filling" of the SR Ca²⁺ store, which is partly dependent on SOC entry [24], is a pivotal factor in SERCA and plasma membrane calcium ATPase gene expression. Similarly, PDGF-stimulated SERCA 2a expression also appears to be modulated by Ca²⁺ entry [16]. It could be hypothesized, therefore, that IL-1ß regulates SERCA gene or protein expression indirectly through modulation of Ca²⁺ entry, and indeed, IL-1ß increases Ca²⁺ entry (and CPA-releasable SR Ca²⁺ store) in parallel with SERCA 2b protein expression.

Effect of IL-1ß on Basal Ca²⁺ and Cell Excitability

In the present study, IL-1ß profoundly altered Ca²⁺ dynamics in resting cells, resulting in enhanced basal Ca²⁺ entry in all cells and initiation of spontaneous Ca²⁺ transients in more than one third of cells. Both responses were sensitive to La³⁺ at concentrations reported to block store-operated and agonist-induced, nonstore-operated Ca²⁺ entry [25–29], and the effect of La³⁺ on basal Ca²⁺ entry was similar to that of [Ca²⁺]_o removal. This infers that generation of spontaneous Ca²⁺ transients is related to stimulation of basal Ca²⁺ entry and supports previous observations regarding the dependence of Ca²⁺ spikes in cultured myometrium [30] and other smooth muscle types [31, 32] on Ca²⁺ entry. In A7r5 cells (a vascular smooth muscle cell line), spontaneous Ca²⁺ oscillations appear to involve voltage-gated Ca²⁺ entry [33]; however, in the present study, L- and T-type Ca²⁺-channel activity was not detectable in the HMSM cells. In a few IL-1ß-treated cells, spontaneous activity was initially maintained in the presence of CPA but disappeared following extended exposure to this inhibitor or to CPA and La³⁺. Spontaneous transients did not reappear on washout of La³⁺ if CPA was present, which implies that the transients rely on a replete Ca²⁺ store and that store depletion drives basal Ca²⁺ entry. The large rebound rise in [Ca²⁺]_cyt generated after La³⁺ removal in IL-1ß-treated cells supports this, because prolonged depletion of the SR in the absence of functioning SOC entry would provide a stimulus for rapid Ca²⁺ influx following washout of La³⁺. However, it cannot be ruled out that the overshoot may, in part, be caused by nonspecific effects of prolonged exposure to La³⁺ on Ca²⁺ homeostasis.

An alternative explanation for our observations could be that IL-1ß primarily stimulates a constitutively active, noncapacitative (SR-independent) basal Ca²⁺ entry [29, 34–36], which initiates spontaneous Ca²⁺ transients either by triggering Ca²⁺-induced Ca²⁺ release and/or loading of the SR Ca²⁺ store, which, when full, sporadically releases Ca²⁺. This would explain why basal entry and resting [Ca²⁺]_cyt are enhanced in IL-1ß-treated cells but spontaneous Ca²⁺ transients are only observed (during the short 5-min observation period) in a smaller percentage of cells. Augmented basal entry could also stimulate SERCA 2b expression. The abolition of spontaneous transients by CPA-induced store depletion would still be consistent with this explanation.

IL-1ß Enhances SOC Entry

Several lines of evidence strongly indicate that SOC as well as basal Ca²⁺ entry is significantly enhanced by IL-1ß. The augmented CPA-induced Ca²⁺ response in IL-1ß-treated cells was predominantly dependent on [Ca²⁺]_o, and enhanced Ca²⁺ entry occurred in IL-1ß-treated cells on readdition of Ca²⁺ after a short period of time in Ca²⁺-free medium (in the continued presence of CPA). This entry was unlikely to be mediated via voltage-gated Ca²⁺ channels, because no evidence of L- or T-type Ca²⁺ currents was observed in these cells. The SOC entry pathway was also permeable to Mn²⁺, and the Mn²⁺ influx observed in IL-1ß-treated cells was significantly greater than in control cells. These data complement the observations that transforming growth factor ß enhances agonist-induced Ca²⁺ responses in human airway smooth muscle cells [37, 38]. La³⁺, [Ca²⁺]_o removal, or Mn²⁺ had little effect on basal or CPA-induced Ca²⁺ signals in control cells and may be associated with the growth-arrested state of the cells [17, 18]. The small La³⁺-insensitive component of CPA-induced Ca²⁺ entry in control cells and IL-1ß-treated cells (Fig. 4) may reflect the presence of another nonspecific cation-entry channel or reverse-mode Na/Ca exchange [39].

IL-1ß Mediated Effects on Gene Expression

The alterations in Ca²⁺ signaling evoked by 24-h treatment with IL-1ß may involve modulation of gene/protein expression, because preliminary studies (data not shown) indicate that acute application of IL-1ß does not influence Ca²⁺ mobilization. Altered gene expression is also implied from the time (24 h) that elapsed before a significant increase in SERCA 2b protein was demonstrable. Other genes of interest include members of the TrpC family (TrpC1, TrpC3, TrpC4, and TrpC6), which are putative components of SOC-entry channels [40, 41] present in our primary-cultured HMSM cells [42]. The regulation of these genes by pregnancy-related stimuli is the current focus of studies in our laboratory.

Some in vivo studies have proposed that IL-1ß responses are mediated by prostaglandins. Inoculation of IL-1ß into the amniotic fluid of nonhuman primates results in a delayed enhancement of myometrial contractility [9], which is inhibited by indomethacin. Additionally, IL-1ß enhances components of the prostaglandin-synthesis pathway, including cyclooxygenase-2 expression and prostaglandin F₂, in HMSM [10, 43]. In the present study, however, it is unlikely that paracrine factors such as prostaglandins were responsible for the initiation of Ca²⁺ transients, because only certain cells (within a given field of view) demonstrated spontaneous activity and because this activity did not propagate from cell to cell in the continually perfused preparation. However, we cannot rule out the possibility that prostaglandin synthesis during the 24-h treatment phase may have led directly, or indirectly, to permanent alterations in protein expression or Ca²⁺ signaling. Similarly, IL-1ß effects on Ca²⁺ mobilization could be modulated via stimulation of production of other cytokines (e.g., IL-8) [44].

Because no voltage-gated Ca²⁺-channel activity was demonstrable in the primary-cultured myometrial cells, we cannot discount a role for IL-1ß-induced modulation of these Ca²⁺ channels in human myometrium in vivo. However, the absence of the voltage-gated channels fortuitously allowed the study of basal and SOC pathways in isolation.

In summary, we have shown that long-term exposure to the proinflammatory cytokine IL-1ß can up-regulate SERCA 2b expression and enhance cell excitability as well as basal and SOC Ca²⁺ entry in primary-cultured HMSM cells. If the observed responses to IL-1ß in our myometrial cell model translate to myometrial tissue, then cytokine modulation of Ca²⁺ signaling may be an important mechanism controlling the cascade of events that prepare the quiescent uterine smooth muscle for the intense contractile activity during labor.

	ACKNOWLEDGMENTS

 
We are grateful to Dr. Frank Wuytack for his generous gift of the SERCA antibodies. Special thanks go to Ruth Brucker, Vasia Dekou, Jeannette Judah, and the Maternal and Fetal Research Unit "Myometrial Collection Team," the Labour Ward staff at St.Thomas' Hospital, and all the women who kindly participated in the present study.

	FOOTNOTES

 
¹ Supported by a grant from The Wellcome Trust (no. 061138) and Tommy's, the baby charity (reg. charity no. 1060508).

² Correspondence: R.M. Tribe, Parturition Research, Maternal and Fetal Research Unit, Department of Women's Health, Guy's, King's and St. Thomas' School of Medicine, St. Thomas' Hospital Campus, London SE1 7EH, U.K. FAX: 44 020 7620 1227; rachel.tribe{at}kcl.ac.uk

Received: 18 September 2002.

First decision: 18 October 2002.

Accepted: 9 December 2002.

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