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Oxytocin-Induced Ca²⁺ Responses in Human Myometrial Cells¹

^a Departments of Veterinary Anatomy & Public Health, Texas A&M University, College Station, Texas 77843-4458 ^b Department of Biochemistry and Molecular Biology, and Obstetrics, Gynecology and Reproductive Sciences, University of Texas Medical School at Houston, Houston, Texas 77225-0708 ^c Department of Obstetrics, Gynecology and Reproductive Medicine, School of Medicine, SUNY at Stony Brook, Stony Brook, New York 11794-8091

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Complex spatiotemporal changes in intracellular Ca²⁺ were monitored in an immortalized human myometrial cell line (PHM1-41) and first-passage human myometrial cells after oxytocin stimulation (1.0–1000 nM). Laser cytometry revealed intracellular Ca²⁺ oscillations in both culture systems starting at 1.0 nM, which were followed by repetitive Ca²⁺ transients by 10–15 min that lasted for at least 90 min. The amplitude of the initial Ca²⁺ spike was dose dependent, while the frequency of Ca²⁺ oscillations identified by Fast Fourier Transform (FFT) tended to increase with dose. Removal of oxytocin resulted in termination of oscillations. Analysis of the sources of the Ca²⁺ involved in oscillations indicated that the major contribution to oscillation frequencies of 6 mHz in cells was from the inositol 1,4,5-trisphosphate-sensitive pool, accounting for about 60% of the frequencies. Most of the remaining frequencies were attributable to extracellular Ca²⁺, which presumably comes from plasma membrane channels other than L-type channels. When oscillation frequencies exceeded 6 mHz, a significant contribution from a ryanodine-sensitive Ca²⁺ pool was detected. Eight-bromo-cAMP suppressed both the initial Ca²⁺ spike and the long-term oscillations. Prostaglandin E₁ and E₂ caused a significant increase in the frequency of oxytocin-induced Ca²⁺ oscillations. FFT analysis may be of considerable value for study of the mechanisms of rhythmic Ca²⁺ transients and their function in myometrial cells, as well as the mechanisms by which uterotonins and tocolytic agents impact myometrial Ca²⁺ regulation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The myometrium is capable of both spontaneous contractions and phasic contractions induced by a number of uterotonins including oxytocin [1–4]. The intracellular mechanisms responsible for the generation of phasic contractions are thought to rely on the production of repetitive intracellular Ca²⁺ transients activated primarily by the G protein-coupled formation of inositol 1,4,5-trisphosphate (IP₃) via phosphoinositide-specific phospholipase C (PI-PLC) and the subsequent release of Ca²⁺ from IP₃-sensitive intracellular stores. Elevation of cytosolic Ca²⁺ may stimulate influx of extracellular Ca²⁺ and activate Ca²⁺-induced Ca²⁺ release (CICR) from an IP₃-insensitive store through ryanodine receptors [5–7]. Subsequent cycles of Ca²⁺ release and uptake into the Ca²⁺ stores result in sustained oscillations of intracellular Ca²⁺ that are thought to precede the development of mechanical activity during smooth muscle cell contractions [8].

The agonist-induced periodic intracellular Ca²⁺ spikes that increase with increasing agonist concentration are thought to constitute a frequency-encoded signal with a high signal-to-noise ratio that limits prolonged exposure of cells to high intracellular Ca²⁺ concentrations [9]. These frequency-encoded signals may have other physiological roles, since a number of cellular targets have been identified that may be involved in the decoding of these Ca²⁺ signals [10–12]. In addition to stimulation of phasic myometrial contractions, it is likely that Ca²⁺ oscillations may regulate multiple physiological processes in myometrial cells.

While Ca²⁺ oscillations in rodent myometrial cells have been well characterized, relatively few studies have been performed on human myometrial cells (e.g., [4]). Recently, a human myometrial cell line was developed that retains a number of useful morphological, biochemical, and functional properties of human myometrial cells, including communication competence and the presence of oxytocin, relaxin, and estrogen receptors [13, 14]. In the present study, we have examined the utility of this cell line to study Ca²⁺ oscillations in human myometrial cells over an extended period of time (90 min).

In order to better understand the regulation and physiological significance of frequency-encoded Ca²⁺ signals, we recently developed an experimental strategy employing Fast Fourier Transform (FFT) to study complex patterns of Ca²⁺ oscillations in a cell line (Clone 9) that responds to oxytocin and vasopressin [15]. Given the potential importance of frequency-encoded signals [9], FFT provided a useful approach to determining the frequencies of oscillations. This experimental approach was adapted to characterize stable, long-term Ca²⁺ oscillations induced in PHM1-41 and first-passage human myometrial cells. It also provides the opportunity to identify the relationship between the frequency of oscillations and the hormone concentration as well as the contributions of the extracellular and intracellular Ca²⁺ pools to the final frequency of Ca²⁺ oscillations.

	MATERIALS AND METHODS

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Materials

Dulbecco's Modified Eagle's medium with F-12 salts (DME-F12), Dulbecco's PBS, nifedipine, neomycin, thapsigargin, Bay K 8644, ryanodine, caffeine, oxytocin, vasopressin, prostaglandins E₁ and E₂ (PGE₁, PGE₂), EGTA, and all general chemical reagents were purchased from Sigma Chemical Co. (St. Louis, MO). Fetal bovine serum (FBS) was purchased from Equitech, Inc. (Ingram, TX). Tissue culture flasks were obtained from Corning (Oneonta, NY), and 2-well Lab-Tek Chambered Coverglass slides were purchased from Nunc, Inc. (Naperville, IL). Fluo 3-AM and 8-bromo-cAMP were purchased from Molecular Probes, Inc. (Eugene, OR).

Stock solution of 1.0 mM fluo 3-AM was prepared in dimethyl sulfoxide (DMSO) and diluted with medium to 3.0 µM (0.3% final DMSO concentration) for loading in cultured cells. Thapsigargin stock (1.0 mM) was prepared in DMSO and used at a concentration of 1.0 µM (0.1% DMSO). Nifedipine was prepared in ethanol (10 mM) and diluted in medium to 10 µM (0.1% ethanol); ryanodine was prepared in ethanol (10 mM) and diluted in medium to 5 µM for experiments. Bay K 8644 was prepared in ethanol and diluted to 1 µM. The 8-bromo-cAMP was prepared as a 10 mM stock, and neomycin and caffeine were prepared in 100 mM stocks in DME-F12. PGE₁ and PGE₂ stocks (1 and 50 mM, respectively) were prepared in ethanol and diluted to 50 µM–10 nM in DME-F12.

Cell Culture of Immortalized and Normal Human Myometrial Cells

The myometrial cell line, PHM1-41, was derived from term-pregnant human myometrium (patient not in labor) and immortalized using a vector expressing human papilloma virus E6 and E7 proteins [13]. Cells were cultured in DME-F12 plus 10% fetal calf serum and used between passages 15 and 23. A number of properties of the cell line have been described [13, 14, 16, 17].

Responses of PHM1-41 cells to oxytocin were compared with those of first-passage human myometrial cells. Use of human myometrial tissues was approved by the Stony Brook University Hospital's committee on research involving human subjects (no. 95-1224). Human myometrial tissue from premenopausal women was obtained from surplus pieces of surgical specimens after hysterectomies. The primary human myometrial cells, like PHM1-41 cells, are communication competent and exhibit comparable properties including connexin43 gap junctions and estrogen, progesterone, and oxytocin receptors [13, 14, 18–20]. Myometrial tissue was processed as described by Andersen et al. [18]. First-passage normal myometrial cells were analyzed 72 h after plating on Chambered Coverglass slides.

Analysis of Intracellular Ca²⁺ in PHM1-41 Cells

Oxytocin-induced changes in intracellular Ca²⁺ in myometrial cultures 72 h after plating were monitored with the Ca²⁺-sensitive fluorophore, fluo 3-AM [21], using a Meridian Ultima Confocal Microscope (Meridian Instruments, Okemos, MI). To minimize differences in fluo 3-AM loading from experiment to experiment, cells were seeded at the same density, all experiments were performed with the same fluo 3-AM stock, and each treatment was compared to a separate control. Cells were loaded with 3.0 µM fluo 3-AM for 1 h in serum- and phenol red-free medium at 37°C and then washed with serum- and phenol red-free medium. Cells were then placed on the stage of the confocal microscope, and an area of the chamber slide was selected for analysis. For image collection, scan parameters were adjusted for maximum detection of fluorescence with minimal cellular photobleaching. Fluorescence was generated in the cells by excitation at 488 nm, and fluorescence emission from scanned individual cells was collected (530 nm) by means of a photomultiplier tube. The basal fluo 3 fluorescence intensity was determined prior to addition of the hormones and/or pharmacologic agents.

To determine the source of Ca²⁺ pools involved in oxytocin-induced Ca²⁺ oscillations and their corresponding input to the frequency of these oscillations, several pharmacologic agents were employed. Thapsigargin is an inhibitor of the microsomal Ca²⁺-ATPase pump that causes leakage of Ca²⁺ from intracellular IP₃-sensitive stores and prevents their refilling [22]. Nifedipine is primarily an inhibitor of L-type Ca²⁺ channels, although it may have inhibitory effects on receptor-operated Ca²⁺ channels [23, 24], and Bay K 8644 is an activator of these Ca²⁺ channels. The methylxanthine, caffeine (1.0–10 mM), can activate the Ca²⁺ store that is sensitive to the alkaloid inhibitor, ryanodine, an IP₃-independent Ca²⁺ release channel associated with the ryanodine receptor [25]. Neomycin is an inhibitor of PI-PLC, which suppresses IP₃ production [26].

To test each of the agents mentioned above, cells loaded with fluo 3 were placed on the Ultima stage, and basal fluorescence intensity was obtained from 5 image scans recorded from about 5–10 cells every 10 sec. After the fifth scan, cells were exposed to oxytocin, and image scans were acquired at the same sampling interval. Scanning continued until the cells established a uniform pattern of oscillations. At this time, a pharmacologic agent was added to the cells, and scanning was continued at the same sampling interval used with the hormone treatment. Control experiments were performed similarly with the addition of the corresponding solvent for each pharmacologic agent.

The fluorescence intensity of fluo 3 obtained from each cell was collected with a sampling frequency f_s (one scan every 10 sec) and analyzed using FFT [27] to identify the frequency or frequencies of Ca²⁺ oscillations if they exist. The FFT analysis involves the conversion of the experimental fluorescence intensity signal acquired in an interval of time into 1) one steady-state fluorescence intensity signal that represents the stable Ca²⁺ level within the cell at any given time during an experiment, and 2) the major frequencies of the oscillating Ca²⁺ signals resulting from the different Ca²⁺ pools involved, as well as their corresponding intensities. It is obvious that frequencies higher than the sampling frequency cannot be detected; however, preliminary experiments with video-rate sampling indicated that the 10-sec sampling interval was sufficient to monitor myometrial Ca²⁺ oscillations induced by even the highest pharmacologic oxytocin concentrations tested (1 µM).

Statistical Analysis

Data from 3 to 6 dishes per treatment from a minimum of 3 separate experiments were analyzed using the General Linear Models ANOVA procedure of SAS/STAT (Statistical Analysis Systems) and Duncan's New Multiple Range test [28] to determine the significance of difference between treatment groups.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Induction of Ca²⁺ Oscillations in PHM1-41 Cells and First-Passage Human Myometrial Cells

Recording of Ca²⁺ responses to oxytocin administration was performed in PHM1-41 cells and first-passage human myometrial cells. Under the culture conditions employed, spontaneous Ca²⁺ oscillations were not observed whereas spontaneous Ca²⁺ transients were only occasionally encountered. The behavior of first-passage human myometrial cells to oxytocin stimulation was indistinguishable from that of PHM1-41 cells in passages 15–23.

Analysis of data from individual PHM1-41 cells and first-passage myometrial cells over a relatively short time interval (5 min) indicated a dose-dependent increase in the amplitude of the initial Ca²⁺ transient upon addition of oxytocin (1–1000 nM) (Fig. 1). Intracellular Ca²⁺ levels in PHM1-41 cells returned to basal values within 60 sec of oxytocin treatment.

View larger version (26K): [in this window] [in a new window]   FIG. 1. The intracellular Ca2+ response of PHM1-41 cells during a 5-min interval after addition of graded concentrations of oxytocin (squares, 1 nM; inverted triangles, 10 nM; triangles, 100 nM; diamonds, 1000 nM). Data represent mean normalized intensity ± SEM of 20–30 cells per experiment.

When intracellular Ca²⁺ was monitored over a longer time interval (90 min) after addition of oxytocin to either PHM1-41 or first-passage myometrial cells, repetitive Ca²⁺ transients were detected by 10–15 min and lasted for at least 90 min. Due to the consistency of responses in both PHM1-41 and first-passage myometrial cells, further analysis of these uniform Ca²⁺ transients was performed on PHM1-41 cells. Analysis of Ca²⁺ transients indicated that a time window between 2500 and 5000 sec was suitable for investigation of changes in the frequency of oxytocin-induced Ca²⁺ oscillations as well as the effects of a variety of pharmacologic agents on the frequency of these Ca²⁺ oscillations. Consequently, the interval from 2500 to 3800 sec was used to determine the reference frequency, and the interval from 3800 to 5000 sec was used to evaluate the effects of experimental treatments on this frequency (Fig. 2). Control experiments were performed to verify that the corresponding solvent for each treatment had no effect on the oxytocin-induced oscillations. FFT analysis of individual PHM1-41 cells exposed to 1–1000 nM oxytocin indicated that within the window of time from 2500 to 5000 sec, the frequency of periodic intracellular Ca²⁺ spikes tended to increase with increasing oxytocin concentration; however, the increases were not statistically significant with the exception of the 1000 nM concentration (Table 1).

View larger version (19K): [in this window] [in a new window]   FIG. 2. An example of Ca2+ oscillations induced in a single PHM1-41 cell by 100 nM oxytocin. The left panel is representative of the responses of both first-passage human myometrial cells and PHM1-41 cells during the first 40 min after administration of oxytocin. The right panel is representative of the continuation of this response during the next 40 min. Note that during the window of time between 2500 and 5000 sec, the Ca2+ oscillations are very uniform, providing an optimal interval for identifying the effects of experimental treatments on oxytocin-induced oscillations.

In PHM1-41 cells exposed to 100 nM oxytocin, the oscillation frequency of individual cells between 40 min and 1 h ranged from 3.0 ± 0.6 to 8.25 ± 0.8 mHz, and the oscillation frequency of each cell remained the same for the next 20 min if normal extracellular Ca²⁺ concentrations in the media were maintained (1.8 mM). During the same time intervals (2500–5000 sec), the steady-state level of intracellular Ca²⁺ about which cells oscillated did not change from the basal Ca²⁺ detected prior to initiation of oscillations with oxytocin.

PHM1-41 cells treated with 10 or 100 nM vasopressin exhibited Ca²⁺ oscillations virtually identical to those obtained using 10 and 100 nM oxytocin. Once the stable oscillations were established, removal of oxytocin (or vasopressin) from the medium resulted in termination of oscillations. It is noteworthy that addition of 10% FBS to PHM1-41 cells that were serum deprived for 3 h also induced Ca²⁺ oscillations with a frequency similar to that induced by 100 nM oxytocin. These oscillations were less uniform and lasted for up to 40 min. Addition of 10% FBS just prior to addition of 100 nM oxytocin doubled the oxytocin-induced oscillation frequency during the first 40 min. This frequency dropped to 6.19 ± 0.63 mHz during the subsequent 40 min. This frequency was still significantly increased compared to that induced by oxytocin alone (5.0 ± 0.58 mHz).

Contributions of Ca²⁺ Pools to the Oscillation Frequency in PHM1-41 Cells

The sources of the Ca²⁺ involved in oscillations were examined in cells challenged with 100 nM oxytocin. Addition of 1 mM EGTA to PHM1-41 cells 60 min after induction of oscillations with 100 nM oxytocin had no effect on the amplitude of the Ca²⁺ oscillations. However, addition of EGTA dropped the oscillation frequency from 5.0 ± 0.58 mHz to 1.41 ± 0.495 mHz (Fig. 3) even though the steady-state levels of intracellular Ca²⁺ changed less than 10% in relation to control values. Neither nifedipine (10 µM) nor Bay K 8644 (1 µM) had any significant effect when added to cells once the stable oscillations were established (data not shown).

View larger version (18K): [in this window] [in a new window]   FIG. 3. The effects of ryanodine, thapsigargin, and EGTA on ongoing Ca2+ oscillations in PHM1-41 cells induced by 100 nM oxytocin. Data represent the mean frequency of oscillations (mHz) ± SEM of 11–26 cells per treatment per experiment. Means identified with asterisk are significantly different from control at p < 0.05.

The contribution of the IP₃-sensitive pool to the oxytocin-induced Ca²⁺ oscillations was determined after the establishment of stable oscillations and subsequent treatment of cells with 1 µM thapsigargin to deplete and prevent replenishment of Ca²⁺ from intracellular IP₃-sensitive stores. FFT analysis indicated that thapsigargin decreased the frequency of oxytocin-induced oscillation from 5.0 ± 0.58 mHz to 1.74 ± 0.35 mHz (Fig. 3) during the 20-min treatment interval. The steady-state Ca²⁺ level increased 1.7-fold relative to the basal level of Ca²⁺ prior to addition of oxytocin.

To determine the contribution of the ryanodine-sensitive (IP₃-insensitive) pool to the frequency of oxytocin-induced Ca²⁺ oscillations, ryanodine (5 µM) or caffeine (1 mM) was added to PHM1-41 cells once the stable oscillations were established. FFT analysis revealed that ryanodine-treated cells exhibited steady-state intracellular Ca²⁺ levels similar to the basal Ca²⁺ levels and that the frequency of oscillations was not significantly changed (4.46 ± 0.50 mHz). However, when data from PHM1-41 cells treated with 100 nM oxytocin were separated into low ( 6 mHz)- and high-frequency oscillations ( 6 mHz), the effect of ryanodine treatment was discernibly different. Ryanodine did not significantly alter the low-frequency oscillations (p > 0.2), whereas the higher-frequency alterations were suppressed (p = 0.053). Treatment of cells with 1 mM caffeine did not cause a significant effect on the oxytocin-induced Ca²⁺ oscillations (5.15 ± 0.40 mHz).

Effects of Inhibition of PI-PLC on Stable Ca²⁺ Oscillations in PHM1-41 Cells

A dose-dependent decrease in the frequency but not the amplitude of Ca²⁺ oscillations induced by 100 nM oxytocin resulted from suppression of IP₃ production by neomycin, an inhibitor of PI-PLC. In the 1–10 mM range, the frequency of oscillations was decreased from 81% of control (oxytocin alone) to 37% of control (Fig. 4). However, the steady-state intracellular Ca²⁺ levels were similar to the basal Ca²⁺ level prior to addition of oxytocin for all neomycin concentrations. Neomycin in the absence of oxytocin had no effect on the basal Ca²⁺ levels.

View larger version (19K): [in this window] [in a new window]   FIG. 4. The effect of neomycin on ongoing Ca2+ oscillations in PHM1-41cells induced by 100 nM oxytocin. Data represent the mean frequency of oscillations (mHz) ± SEM of 12–30 cells per dose per experiment. Means identified with asterisk are significantly different from control at p < 0.05.

Effects of 8-Br-cAMP on Ca²⁺ Oscillations in PHM1-41 Cells

Addition of 1 mM 8-bromo-cAMP to PHM1-41 cells exhibiting stable oxytocin-induced oscillations resulted in no immediate changes in the oscillation frequency. However, pretreatment of PHM1-41 cells for 12 h with 500 µM 8-bromo-cAMP prior to oxytocin challenge resulted in suppression in the oscillation frequency by nearly 50% (i.e., from 5.0 ± 0.58 mHz to 2.6 ± 0.65 mHz) with no effect on the amplitude of oscillations.

Effects of PGE₁ and PGE₂ on Stable Ca²⁺ Oscillations in PHM1-41 Cells

Addition of 10 or 100 nM PGE₁ or PGE₂ to ongoing oxytocin-induced oscillations did not alter the frequency of oscillations; however, 1.0 µM PGE₁ and 50 µM PGE₂ significantly increased the frequency of oscillations compared to the control value (Table 2). There was no significant change in the steady-state intracellular Ca²⁺ levels compared to the control value.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The link between uterotonin-induced excitation and myometrial contraction involves an IP₃-induced rise in intracellular Ca²⁺ and the formation of a Ca²⁺-calmodulin complex that activates myosin light-chain kinase, followed by phosphorylation of myosin light chains, thereby permitting force-generating interactions of myosin with actin [1]. The generation of phasic contractions is thought to rely on the production of repetitive intracellular Ca²⁺ transients [2, 3] that precede the development of mechanical activity leading to contractions [8].

In the present study, time-resolved digital fluorescence imaging of Ca²⁺ dynamics in conjunction with FFT was used to characterize Ca²⁺ oscillations in PHM1-41 cells over a relatively long interval of time. These studies suggest that oxytocin stimulates uniform, relatively low frequency oscillations (i.e., ranging from approximately one oscillation every 5 min to one every 2 min) in PHM1-41 and first-passage human myometrial cells after an initial lag of about 10 min following the first Ca²⁺ transient. The basis for this initial lag is not known but may be attributable to initial feedback loops and the balance between Ca²⁺ release/entry and Ca²⁺ pumps that become stabilized following this interval. The uniform Ca²⁺ oscillations presumably result from activation of PI-PLC via G protein-linked receptors, which results in sustained generation of IP₃ and subsequent release of Ca²⁺ from IP₃-sensitive stores in the endoplasmic reticulum balanced by the actions of Ca²⁺ pumps that lower intracellular Ca²⁺.

While the initial oxytocin-induced Ca²⁺ transient was dose dependent, the slight increases in the frequency of oscillations with increasing oxytocin concentration were not significant with the exception of the 1000 nM concentration. Despite a large concentration range, oxytocin appears to induce a relatively narrow range of oscillation frequencies that may be regulated by the rate of IP₃ production, which governs the cycles of Ca²⁺ release and uptake into the Ca²⁺ stores. The response of PHM1-41 to vasopressin suggests the presence of vasopressin receptors in this cell line; however, vasopressin may simply be acting on the oxytocin receptor at 1000 nM levels.

The Ca²⁺ pool providing the major contribution to oxytocin-induced oscillation frequencies is the IP₃-sensitive pool, which is responsible for about 60% of the oscillation frequencies. Close to 40% of the remaining frequencies are due to extracellular Ca²⁺ that presumably enters through plasma membrane channels. It does not appear, however, that L-type Ca²⁺ channels contribute significantly to the stable oscillations, as neither nifedipine nor Bay K 8644 affects oscillations once they are established. Although the specific channels contributing to these Ca²⁺ oscillations have not yet been identified, there is recent evidence for a capacitative Ca²⁺ entry mechanism in PHM1-41 cells that may involve Ca²⁺ release-activated channels [29].

Human myometrial cells have been shown to express one or more members of a family of intracellular ryanodine receptors (RyR) that play a role in CICR [30]. However, the contribution of this IP₃-insensitive pool to the relatively low frequency stable oscillations stimulated by oxytocin in PHM1-41 cells was not statistically significant when the cells were oscillating at a frequency of 6 mHz; it was significant (p = 0.053), though, when the oscillation frequency exceeded 6 mHz. This suggests that the contribution of CICR may be small (up to 20% of total oscillation frequencies) but operational during high-frequency Ca²⁺ oscillations in PHM1-41 cells. Since FBS can up-regulate the 100 nM oxytocin-induced frequency of oscillation to 10 mHz, the CICR contribution to these oscillations is currently under investigation to address this possibility. The absence of a caffeine effect on stable oscillations in PHM1-41 cells may suggest that the caffeine-insensitive RyR₃ receptor [30, 31] is predominant in PHM1–41 cells rather than RyR₁ and RyR₂. Alternatively, the ryanodine-sensitive Ca²⁺ pool may be more important relative to the initial oxytocin-induced response.

The action of 8-bromo-cAMP on oxytocin-induced oscillations is consistent with the effect of relaxin and ß-agonists on myometrial contractions. Significant direct effects of 8-bromo-cAMP on stable oxytocin-induced Ca²⁺ oscillations in PHM1-41 cells were not detected, although the design of the experiment does not eliminate permeability issues related to the cAMP analogue. However, PHM1-41 cells treated for 12 h with 500 µM 8-bromo-cAMP induced suppression of the initial Ca²⁺ signaling events [14] as well as long-term oscillations. The suppression of Ca²⁺ responses by cAMP in PHM1-41 may involve a number of mechanisms. Uterine relaxants and cAMP have been shown to reduce free Ca²⁺ concentration [14] and phosphatidylinositide turnover in PHM1-41 cells [13] via inhibition of G_q/phospholipase C by protein kinase A [17]. These agents also affect intracellular Ca²⁺ concentrations by stimulating Ca²⁺-activated K⁺ channels in PHM1-41 cells via protein kinase A [16]. Studies are under way to determine how specific Ca²⁺ pools involved in the oscillations are affected by these mechanisms.

While the prostaglandin E receptors in PHM1-41 cells have not yet been characterized, both PGE₁ and PGE₂ increase the frequency of ongoing oscillations induced by 100 nM oxytocin. Asbóth et al. [32] have provided pharmacologic evidence for several prostaglandin E receptor subtypes in cultured human myometrial cells (i.e., EP1, EP2, EP3A, and EP3D receptors). In primary human myometrial cells, PGE₂ was found to interact with 1) EP1 receptors that elevate intracellular Ca²⁺ independently from PLC, but involving a Gi protein and plasma membrane Ca²⁺ channels; 2) EP2 receptors that stimulate adenylyl cyclase; 3) EP3A receptors that inhibit adenylyl cyclase, and 4) EP3D receptors that stimulate PLC and also elevate intracellular Ca²⁺. The effect of PGE₁ and PGE₂ on PHM1-41 cells with ongoing oxytocin-induced oscillations would be consistent with effects of these prostaglandins operating primarily through EP1 and/or EP3D receptors.

The present studies have exploited an analytical method to identify, on a single-cell basis, signaling patterns that may regulate phasic myometrial cell contractions. This experimental approach is being developed to identify relationships between ion channel activity, cyclic depletion and refill of IP₃-sensitive and IP₃-insensitive stores, and oscillations in myometrial cells. Additionally, the application of FFT should prove useful to identify additional cellular targets involved in conversion of the frequency-encoded Ca²⁺ signal (i.e., frequency spectrum) into cellular functions in addition to phasic contractions. The frequency spectrum is significant in light of recent studies that have identified cellular targets capable of integrating or decoding frequency-encoded intracellular Ca²⁺ signals [11]. For example, the activity of CaM kinase II has recently been shown to be tightly linked to the frequency of Ca²⁺ oscillations and is adapted to discriminate between low- and high-frequency Ca²⁺ signals [12]. This enzyme can decode Ca²⁺ oscillations into graded levels of kinase activity based upon the degree of CaM kinase II phosphorylation [11, 12].

The Ca²⁺ frequency spectrum has also been shown to affect the activity of Ca²⁺-sensitive mitochondrial dehydrogenases in hepatocytes that are increased by intracellular Ca²⁺ oscillations and transmitted to mitochondria as mitochondrial Ca²⁺ oscillations. Oscillations in mitochondrial membrane potential appear to be synchronized with intracellular Ca²⁺ oscillations [15]. Thus, mitochondria appear to sense and integrate the frequency spectrum because of their intimate spatial association with the endoplasmic reticulum-associated Ca²⁺ release sites and their ability to retain accumulated Ca²⁺ for prolonged periods [10, 11].

Given the diversity of functions regulated by intracellular Ca²⁺, ranging from contraction and secretion to changes in gene expression and cell proliferation [1, 9, 33, 34], a better understanding of the coded Ca²⁺ signals and the specific contributions of the sources of Ca²⁺ that contribute to the signals should prove useful. The presently applied experimental approach involving FFT analysis of Ca²⁺ oscillations should be of considerable value for study of the underlying mechanisms of rhythmic Ca²⁺ transients and their functional significance in myometrial cells, as well as the mechanisms by which stimulatory and inhibitory uterotonins and tocolytic agents impact myometrial Ca²⁺ regulation. In addition, the actions of a number of xenobiotics including environmental estrogens, pesticides, heavy metals, and the like on contractile function in myometrial cells should be particularly amenable to this analysis.

	ACKNOWLEDGMENTS

 
The capable technical support of Clay Ainley and Emöke Rácz is gratefully acknowledged.

	FOOTNOTES

 
¹ Supported in part by Basic Research Grant No. 1-FY97–0426 from the March of Dimes Birth Defects Foundation and NIH P30-ES09106 ES (R.C.B.), NIH grant HD-09618 (B.M.S.), and NIH grant HD-30482 (J.A.).

² Correspondence. FAX: 409 847 8981; rburghardt{at}cvm.tamu.edu

Accepted: October 21, 1998.

Received: September 21, 1998.

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