Oxytocin is commonly used to induce or augment labor, but its mode of action is uncertain. To address the issue, isometric tension and the intracellular free Ca2+ concentration ([Ca2+]i) were simultaneously recorded from isolated strips of pregnant human myometrium loaded with fura 2. The changes in [Ca2+]iand tension during phasic contractions were indistinguishable in myometrium taken before or after the onset of labor, enabling samples to be pooled. Oxytocin (10 nM) had no effect on basal [Ca2+]ior tension, but it increased both the [Ca2+]iand the tension recorded during phasic contractions. Analysis of the [Ca2+]i-tension relationship revealed that during the falling (relaxation) phase of the contractile response, oxytocin increased the tension recorded at each [Ca2+]i. By manipulating extracellular Ca2+during phasic contractions, it was possible to ensure that the [Ca2+]isignals were similar in the presence and absence of oxytocin, yet oxytocin still improved the [Ca2+]i-tension relationship. We conclude that 10 nM oxytocin increases the [Ca2+]isensitivity of the contractile proteins only after a contraction has begun, possibly by causing inhibition of myosin light chain phosphatase.
- smooth muscle
- signal transduction
oxytocin increases the force and duration of myometrial contractions and is used clinically for induction and augmentation of labor. Whether endogenous oxytocin is involved in the onset and progression of normal human labor is controversial (27). Neither the maternal nor fetal plasma oxytocin concentrations have been conclusively demonstrated to increase during labor (27). However, the sensitivity of the uterus to oxytocin increases during pregnancy (2), and oxytocin antagonists reduce the frequency of spontaneous contractions (5).
Oxytocin interacts with a member of the family of receptors that couple to G proteins and thereby stimulates, via Gq/11 and possibly Gh, phospholipase C-mediated hydrolysis of polyphosphoinositides (10, 15, 17). The links between this or indeed other signalling pathways activated by oxytocin (14) and myometrial contractility are ill defined: they may involve regulation of both the intracellular free Ca2+ concentration ([Ca2+]i) and the Ca2+ sensitivity of the contractile machinery.
In common with other smooth muscles (23), myometrial contractions are associated with increases in [Ca2+]i(30). Ca2+ bound to calmodulin activates myosin light chain kinase, which then phosphorylates the regulatory myosin light chains, allowing them to rapidly bind to and detach from actin filaments and so generate tension. Although an increase in [Ca2+]iis a major control of smooth muscle contraction, hormones can also enhance contractile activity without directly increasing [Ca2+]i(29). Inhibition of myosin phosphatase causing a decrease in the rate of dephosphorylation of myosin light chain is a likely means of such Ca2+ sensitization (22, 23, 28). In permeabilized rat myometrium (8), oxytocin causes Ca2+ sensitization, but results from intact human tissue loaded with aequorin are contradictory (24). The effects of oxytocin on spontaneously active myometrium have not yet been examined under conditions that allow its influence on the [Ca2+]i-tension relationship to be defined.
In the present study, myometrium taken before or after the onset of labor was loaded with fura 2, and the effects of oxytocin on the [Ca2+]i-tension relationship were examined by simultaneous measurement of tension and [Ca2+]iduring phasic contractions.
Simultaneous measurements of isometric tension and [Ca2+]i.
Myometrial biopsies were obtained with informed written consent and local ethical committee approval (LREC DEC 89/56) at term caesarean section (37–42 wk gestation) either before or after the onset of labor. Indications for caesarean section included noncephalic presentation, previous caesarean section, failure to progress in labor, or fetal distress. Women had no significant medical conditions, and labor was defined as progressive (>2 cm) cervical dilation accompanied by regular uterine contractions.
Small strips of myometrium (2 × 2 × 15 mm) were dissected so that the longitudinal axis aligned with the direction of the muscle fibers. Strips were incubated for 15 h at 20°C in Krebs-Henseleit solution (KHS) containing 50 μM fura 2-acetoxymethyl ester (Molecular Probes, Leiden, The Netherlands) dissolved in anhydrous dimethyl sulfoxide (10%) and pluronic acid (0.5%) and then were washed in KHS (30 min). KHS, equilibrated with 5% CO2-95% O2, had the following composition (in mM): 118 NaCl, 4.7 KCl, 1.2 CaCl2, 1.2 MgSO4, 25 NaH2CO3, 1.2 K2PO4, and 11 glucose, pH 7.4. Strips were mounted (Fig.1 A) in a polymethylacrylate cuvette within a Perkin-Elmer LS50B spectrofluorimeter. One end of the muscle was attached by cotton to an isometric tension recording system comprising a Fort-10 transducer (World Precision Instruments, Aston, UK: bandwidth 0–10 g) and an EpiCompact amplifier and data acquisition system (Cambridge Research Systems, Cambridge, UK). A rapidly rotating filter wheel provided light of appropriate excitation wavelength (340 and 380 nm), and emitted light was collected through a long-pass barrier filter (510 nm). Autofluorescence (<10% of the fluorescence recorded from strips loaded with fura 2) was determined at the end of each experiment by addition of MnCl2 (2 mM) and ionomycin (5 μM) to quench the fura 2 fluorescence. [Ca2+]iwas determined from the ratio of the corrected fluorescence at 340 and 380 nm (R340/380) using a look-up table created from Ca2+standard solutions obtained from Molecular Probes (12). The fluorescence ratio (R340/380) and isometric tension were sampled simultaneously at 1 Hz (5 Hz for the high-resolution recordings in Fig. 3).
Before an experiment, muscle strips were superfused for 1 h with KHS (∼1 ml/min) at 37°C under 2 g of tension and then were superfused continuously throughout the experiment. Strips that failed to spontaneously contract during the first hour were discarded. Exchanges of media were complete within 210 s, and contractions occurring within this time were excluded from subsequent analyses. Oxytocin acetate was obtained from Sigma (Poole, UK).
Analysis. Results were corrected for the small drift in basal tension and fluorescence ratio in some of these protracted experiments. Integrated areas under the curve (AUC) for [Ca2+]iand tension for each contraction were calculated using an Excel spreadsheet; AUC data therefore describe individual contractions and are independent of their frequency. To allow direct analysis of the [Ca2+]i-tension relationship, each measurement of the increase in [Ca2+]iwas plotted against the simultaneously recorded increase in isometric tension. Each of these [Ca2+]i-tension plots therefore had a rising phase (to the peak [Ca2+]i) and a falling phase (as [Ca2+]ireturned to baseline). To compare tissues from different patients, the maximal separation (S) between the rising and falling phases of these plots was used as an index of the extent to which oxytocin selectively affects the falling phase of the [Ca2+]i-tension relationship (see results).
Unless otherwise stated, only a single strip from each patient was included in the final analysis. Results are presented as means ± SE. Statistical analyses employed the Mann-WhitneyU-test and Student’st-test, withP < 0.05 taken as significant.
[Ca2+]iand tension in spontaneously active myometrium taken before or after the onset of labor.
Although myometrial strips are intrinsically fluorescent at the wavelengths used to record fura 2 fluorescence, our results demonstrate that it is possible to simultaneously measure tension and [Ca2+]i. First, the fluorescence ratio of strips that had not been loaded with fura 2 was unaffected by contractions (Fig.1 B). Second, only muscles in which contractions were accompanied by reciprocal changes in the fura 2 fluorescence recorded at the two excitation wavelengths (340 and 380 nm) were analyzed (Fig. 1 C). Third, in paired comparisons of myometrial strips (24 from 3 patients), fura 2 loading had no effect on spontaneous contractile activity: 11 of the 12 fura 2-loaded strips and 9 of the 12 control strips were spontaneously contractile; the peak tension (113 ± 39% of control,P > 0.05), frequency of contractions (109 ± 9%, P > 0.05), and AUC (130 ± 44%, P > 0.05) were all similar under the two conditions. Removal of extracellular Ca2+ or addition of nimodipine (100 nM) abolished both spontaneous contractions and the changes in fluorescence ratio (not shown), in keeping with previous reports (6).
Comparison of the changes in [Ca2+]iand tension recorded during spontaneous contractions (60 min) of muscle strips taken before or after the onset of labor (Fig.2) revealed no significant differences, nor were the effects of oxytocin different between the two samples (Table1). Therefore, in all subsequent experiments, biopsies taken from patients before and after the onset of labor were pooled.
To better resolve the temporal relationship between changes in [Ca2+]iand tension during spontaneous contractions, recordings were made at 5 Hz. The increase in [Ca2+]ipreceded the increase in tension (Fig. 3) by 436 ± 89 ms (n = 11) at the onset of a contraction and by 13 ± 3 s (n = 17) at its peak. These substantial delays, reflecting the cascade of biochemical events separating an increase in [Ca2+]ifrom contraction, are similar to those observed in other smooth muscles (23, 31).
Our results establish that spontaneous contractile activity is dependent on Ca2+ entry through L-type Ca2+ channels (19, 32), that tissue taken before or after the onset of labor behaves similarly (although loss of labor characteristics during fura 2 loading cannot be excluded), and that fura 2 can be used to measure [Ca2+]iin myometrium without interfering with its contractile activity.
Effect of oxytocin on phasic contractions. Because the half-maximal effect of oxytocin on myometrial contractility occurs when its concentration is ∼10 nM (24, 26), this concentration was used in all subsequent experiments. Oxytocin had no significant effect on basal [Ca2+]ior tension (Fig. 4): in paired comparisons of measurements during the first (pretreatment) and second (±oxytocin) hour of phasic contractile activity, the basal [Ca2+]iand tension responses recorded from muscles stimulated with oxytocin were 98 ± 3 and 96 ± 3% (n = 11, P > 0.05 for each) of control responses, respectively. Control samples (n = 4) were not treated with oxytocin during the second hour to account for time-dependent changes in activity. Oxytocin did, however, increase the magnitude of both the tension and [Ca2+]irecorded during phasic contractions (Fig.5). Oxytocin had no significant effect on the frequency of contractions (7.7 ± 1.7 and 5.1 ± 0.8 h−1 before and after oxytocin, respectively, P > 0.05,n = 11), consistent with previous studies of myometrium in vitro (24). Because oxytocin more markedly increased tension than [Ca2+]i, the relationship between [Ca2+]iand tension was examined in detail.
Figure 6 shows the instantaneous relationship between [Ca2+]iand tension during phasic contractions in the presence and absence of oxytocin. In both cases, the relationships are strikingly asymmetric and comprise a rising phase during which tension is less sensitive to [Ca2+]ithan during the subsequent falling phase (Fig. 3), consistent with slow steps linking Ca2+ to contraction (31). Oxytocin had no effect on the rising phase of the [Ca2+]i-tension relationship but significantly affected the falling phase such that at each [Ca2+]ithere was a greater increase in tension (Fig. 6,B andD). To accommodate the variability between tissues from different patients (24) and yet allow quantitative analysis of the effects of oxytocin on the [Ca2+]i-tension relationship, we adopted the following analysis. The average half-maximal increase in [Ca2+]ifor all muscle strips was 39 nM, and the tension recorded at this [Ca2+]iwas therefore compared on the rising and falling phases of the [Ca2+]i-tension relationship. Comparison of these values from the last contraction before oxytocin addition with the first after its addition demonstrates that oxytocin significantly increases the tension only during the falling phase of the response (Table 2).
A simple means of examining changes in the [Ca2+]i-tension relationship is provided by quantifying the maximalS of the rising and falling phases of the response (see methods). To eliminate problems resulting from slow changes in muscle properties during our protracted recordings, [Ca2+]i-tension relationships were plotted for each contraction,S was calculated for each contraction during 2 h of activity, and S values were then expressed as percentages of the preceding contraction (Fig.7). This form of analysis was applied to control myometrial strips and those treated with oxytocin (10 nM) for the second hour of phasic activity. The results demonstrate that there is no change in the S of the [Ca2+]i-tension relationship between sequential control contractions but that, after oxytocin addition, the S abruptly increases and is thereafter maintained throughout the period of stimulation with oxytocin (Fig. 7).
Oxytocin increases the Ca2+ sensitivity of the contractile machinery.
The results are so far consistent with oxytocin selectively increasing the Ca2+ sensitivity of the contractile apparatus during the falling phase of the contractile response. However, oxytocin also significantly increased the duration of the increase in [Ca2+]i(and thereby the AUC), and this effect may have contributed to the change in the [Ca2+]i-tension relationship (Figs. 5 and 6 C and Table1). To resolve the issue, [Ca2+]itransients of similar duration were produced in the presence and absence of oxytocin by rapidly chelating extracellular Ca2+ using EGTA during the rising phase of a phasic contraction (Fig. 8). Under these conditions, the [Ca2+]iAUC was similar for control and oxytocin-treated muscle (107 ± 18% of control, n = 5,P > 0.05), and the rate at which [Ca2+]imonoexponentially returned to baseline was indistinguishable (time constant 18 ± 4 and 19 ± 4 s for control and oxytocin treated, respectively). Despite the similar Ca2+ signals, oxytocin still caused an increase in the tension recorded during a phasic contraction. In the presence of oxytocin, the peak and AUC tension were increased to 159 ± 13% (n = 5,P < 0.01) and 177 ± 31% (n = 5,P > 0.05) of their control values, and from the [Ca2+]i-tension relationship, oxytocin increased S to 203 ± 15% (n = 5,P < 0.05) of its pretreatment value (Fig. 8). These results establish that oxytocin increases the Ca2+ sensitivity of the contractile apparatus during phasic contractions independent of its ability to enhance the increase in [Ca2+]i.
In the only previous simultaneous measurements of [Ca2+]iand tension in human myometrium, the luminescent indicator aequorin was used (24). This indicator has several limitations: the membrane must be transiently permeabilized to allow loading, it is difficult to calibrate, and it is not amenable to the protracted recording required for multiple measurements from a single strip. Fura 2, which has been used in rat myometrium (18-20), overcomes these limitations, and we have established that it can be used to quantify [Ca2+]iin spontaneously active human myometrium without interfering with its contractile behavior (Fig. 1).
Although labor is accompanied by profound changes in the physiology of the myometrium (3, 4, 9), our results suggest that the Ca2+ sensitivity of the contractile apparatus does not change in fura 2-loaded strips (Table1), consistent with results from skinned myometrium of the rat (7). We cannot, however, entirely eliminate the possibility that some differences between labor and nonlabor issues have been lost during the 15 h taken to load strips with fura 2.
Very high concentrations of oxytocin (μM) directly stimulate increases in [Ca2+]iand thereby tonic contractions (13). In our experiments, the characteristic phasic activity of normal myometrium (19, 30) was maintained by using a lower concentration (10 nM) of oxytocin, and the results pertain specifically to this concentration. Under these more physiological conditions, our results reveal two distinct modulatory effects of oxytocin on phasic activity.
First, oxytocin modestly increased the amplitude of the Ca2+ signal recorded during each spontaneous contraction (Table 1). We have not further addressed the mechanisms underlying this potentiation of the Ca2+ signals evoked by Ca2+ entry through L-type Ca2+ channels. The effect is not a consequence of oxytocin inhibiting Ca2+ removal from the cytosol, because after rapid removal of extracellular Ca2+, rates of [Ca2+]irecovery were unaffected by oxytocin (Fig. 8). In addition, it cannot simply reflect release of Ca2+stores by inositol 1,4,5-trisphosphate (IP3) because, even during several hours of exposure to oxytocin (Fig. 4), its effects were manifest only during spontaneous contractions; neither basal tension nor [Ca2+]iwas affected. IP3 receptors are stimulated by the concerted actions of IP3 and Ca2+ (25), and oxytocin may therefore have caused the formation of a subthreshold level of IP3 such that the increase in [Ca2+]iafter spontaneous opening of L-type Ca2+ channels would synergize with it to cause release of intracellular Ca2+ stores and so amplify the Ca2+ signal. Alternatively, oxytocin, possibly via protein kinase C (16), may have increased the sensitivity of the L-type Ca2+channels (11).
The second, and more striking, effect of this concentration of oxytocin was to selectively increase the tension evoked at each [Ca2+]iduring the falling phase of each phasic contraction (Table 2). The only previous simultaneous measurements of [Ca2+]iand tension in pregnant human myometrium concluded that oxytocin had no effect on the [Ca2+]i-tension relationship at either the peak of the response or during tonic contractions (24). In our experiments, the effect of oxytocin on the [Ca2+]i-tension relationship of spontaneously active myometrium was independent of its ability to prolong the Ca2+ signal (Fig. 8). How might oxytocin, without directly increasing [Ca2+]i, selectively increase the Ca2+sensitivity of the contractile apparatus during only the later stages of each phasic contraction?
Oxytocin stimulates the mitogen-activated protein kinase (MAPK) cascade (14), and in some smooth muscles MAPK has been shown to phosphorylate caldesmon (1), thereby increasing the [Ca2+]isensitivity of the contractile apparatus (21). Such a mechanism would not, however, readily explain why oxytocin increases the [Ca2+]isensitivity during only the falling phase of the response. A more likely mechanism would involve inhibition of myosin light chain phosphatase activity (23), the effect of which would be more pronounced after substantial phosphorylation of myosin light chains. Rho and its associated kinase (28), arachidonic acid, G proteins, and protein kinase C (23) have each been implicated in linking receptors to inhibition of myosin light chain phosphatase, but the links with the oxytocin receptor remain to be defined.
We conclude that oxytocin selectively increases the [Ca2+]isensitivity of the contractile apparatus during only the falling phase of a contraction, possibly by inhibition of myosin phosphatase. Selective manipulation of the mechanisms responsible for the [Ca2+]isensitization may ultimately provide additional means of controlling myometrial contractility for induction of labor or treatment of preterm labor.
We thank patients and staff of the Rosie Hospital, Cambridge, UK.
Address for reprint requests: S. Thornton, Dept. of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK.
This work was supported by The Sir Jules Thorn Charitable Trust. S. Thornton was a Medical Research Council Clinical Scientist.
Portions of this work were presented at the Physiological Society meeting in Cambridge, UK, December 15–17, 1997, and at the Society for Gynecologic Investigation in Atlanta, Georgia, March 11–14, 1998. The work has also been published as an abstract (J. Physiol. 506: 144P, 1998 andJ. Soc. Gynecol. Invest. 5: 185A, 1998).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.
- Copyright © 1999 the American Physiological Society