Inhibition of Spontaneous Activity by Mibefradil

In the first step of our investigation, we evaluated the inhibition of spontaneous uterine contraction by increasing doses (10 nM to 10 μM) of the t-type calcium channel inhibitor mibefradil. The dosing range was selected based on the observed uterine strip contractile response. 10 nM was the smallest drug concentration at which an effect was revealed. Beyond 10 µM, no further activity reduction was detected. Virgin (V) and proven breeder (PB) myometrial strips were suspended with a passive tension of 0.5 g in Krebs solution containing 2.5 mM CaCl2 (regular Krebs). Four parameters were quantified: area under the curve (AUC), amplitude, frequency, and duration. Figure 1.A displays sample traces of the experiment, while Fig. 1.B shows the relative summary curves (for both V and PB, rat n = 4). The curve parameters are reported in Table 1.A. Typically, area-under-the-curve data are reported normalized for the AUC recorded for a challenge with 40 mM KCl. Treatment with KCl induces massive depolarization of the plasma membrane and thus maximal contraction of the strips. The resulting AUC reflects the activation of all the muscle fibers of a particular strip. Normalization by the AUC in 40 mM KCl allows us to account for unintended variations between strips. However, we previously observed [1] that spontaneous contractility is considerably reduced by the washes that add and remove the KCl. Thus, for both the mibefradil and verapamil inhibition experiments, we chose to forgo the KCl challenge and sacrifice our ability to account for uneven sample characteristics to get a clearer estimate of the full inhibitory effect of these drugs on the two groups. The raw AUC data show that the spontaneous activity of V samples was higher than that of PB rats (P < 0.001, see Table 1), which is consistent with our previous observations [1]. The mibefradil-induced inhibition range (Max–Min) was larger in V than in PB strips (P < 0.001, see Table 1). Some, albeit limited, contractile activity was maintained even at the highest mibefradil concentration (10 µM). At this highest dose of inhibitor, no difference in AUC was detected between sample types. Also, the IC50 was similar. The amplitude of spontaneous contractions (Table 1.B.) and the range of the mibefradil-induced inhibition were higher in V samples than in PB samples. In 10 µM mibefradil, no differences in amplitude were recorded between the groups. The IC50 calculated for the amplitude curve was also similar in V than in PB.

Fig. 1

Dose-inhibition response to mibefradil. a. Sample traces recorded in uterine samples from virgin (top panel) and proven breeders (bottom panel) female rats. b. Dose inhibition response curves summarizing data obtained from experiments conducted on 4 virgin (V, black circles) and 4 proven breeder (PB, white circles) rats (6 uterine strips/rat). The top left panel shows the overall contractility decline under increasing doses of mibefradil, represented by the area under the curve measured over 5 min. For this experiment, no normalization was conducted. The top right panel shows amplitude changes. The bottom left panel represents mibefradil’s impact on contraction frequency. The bottom right panel shows changes in duration with increasing amounts of the T-type channel inhibitor

Table 1 Dose inhibition response to mibefradil and verapamil parameters

The frequency of spontaneous contractions was stronger in PB than in V (Table 1.C) and was consistently higher in PB strips during the entire sequence of mibefradil doses. Thus, the curve minimum was also significantly higher in PB samples. The range of inhibition was larger in V than in PB. This suggests that T-type channel inhibition might impair the formation of calcium-calmodulin complexes more in V strips in PB. The differences in IC50 did not reach significance.

In terms of durations, V and PB did not differ in the absence of inhibitors, but other curve parameters were significantly different (Table 1.D). In the presence of 10 µM mibefradil, the minimal duration recorded was higher in V than in PB (5.9 ± 0.4 vs 3 ± 0.1 s, P < 0.001). Thus, PB strips were more affected by the treatment than the V strips. 50% inhibition was achieved with a lower mibefradil concentration for PB than V.

At the end of the dose-inhibition experiment, the uterine strips were treated with 50 nM oxytocin. This caused a reactivation of contractility that appeared 33% stronger in V than in PB (403 ± 37 vs 294 ± 19 g*s respectively, P < 0.001). This was somewhat unexpected since typically PB strips are more responsive to oxytocin (Fig. 5.A top panel, Table 3). Amplitude/frequency/duration analysis revealed that the main determinant of the stronger response of V was the event duration, which was significantly longer in V than in PB (9.0 ± 0.4 s vs 6.1 ± 0.1 s, P < 0.001). Hence, 50 nM oxytocin restored the duration to the pre-mibefradil levels in V but not PB samples. Frequency, which appeared to be the main player in the stronger response to oxytocin in the absence of mibefradil [1] increased in equal measure in the two groups here.

Inhibition of Spontaneous Activity by Verapamil

The inhibition of spontaneous V and PB uterine contractions by increasing doses (10 nM to 10 µM) of verapamil was then also evaluated. The dosing range once again was selected based on the observed strip response. As before, the strips were suspended with a passive tension of 0.5 g in Krebs solution containing 2.5 mM CaCl2 (regular Krebs), then treated with the L-type calcium channel inhibitor. As in the mibefradil experiments, to maximize the initial spontaneous activity of the uterine samples we did not conduct the challenge with 40 mM KCl. Figure 2.A shows sample traces of the experiment and Fig. 2.B the summary curves for AUC, amplitude, frequency, and duration (for both V and PB, rat n = 4). The curves’ parameters are listed in Table 1. Raw AUC showed once again a stronger spontaneous activity in V. Maximal concentrations of verapamil (10 µM) almost completely inhibited activity in both groups. The Max–Min Range was larger in V strips. IC50 was similar in V and PB (Table 1.A). The Amplitude curves follow the trend of the raw AUC curves, with significantly larger values for spontaneous values (= Max) and range in V samples (Table 1.B). The spontaneous frequencies once again were higher in PB. The inhibition achieved by 10 µM reduced frequency to levels close to zero in both groups. The inhibition range therefore appeared larger in PB than in P (Table 1.C.). No significant difference in IC50 was detected. The duration curves under verapamil were somewhat similar in the two groups. Spontaneous duration was similar in the two groups. Since high levels of verapamil abolished most contractions, the minimum duration was very low and essentially identical in both V and PB samples. Hence, the inhibition ranges did not differ significantly. However, the calculated IC50s resulted to be moderately different (Table 1.D), with PB strips event durations being inhibited more effectively than V strips (−7.21 ± 0.08 in PB vs −6.79 ± 0.07, P = 0.001).

Fig. 2

Dose-inhibition response to verapamil. a. Sample traces recorded in uterine samples from virgin (top panel) and proven breeders (bottom panel) female rats. b. Dose inhibition response curves summarizing data obtained from experiments conducted on 4 virgin (V, black circles) and 4 proven breeder (PB, white circles) rats (6 uterine strips/rat). The top left panel shows the overall contractility decline under increasing doses of verapamil, represented by the area under the curve measured over 5 min. For this experiment, no normalization was conducted. The top right panel shows amplitude changes. The bottom left panel represents verapamil’s impact on contraction frequency. The bottom right panel shows changes in duration with increasing amounts of the L-channel inhibitor

Thus, inhibition of spontaneous contractile activity by mibefradil and verapamil featured similar characteristics, with amplitude accounting for most of the AUC differences noted between V and PB in both cases.

As in the inhibition by mibefradil experiment, after the last dosage of verapamil, we added 50 nM oxytocin, which reactivated contractility. In the verapamil case, the oxytocin-induced activation was not as intense as in the mibefradil case. Treatment with oxytocin after 10 µM verapamil restored AUC only to 50% of V spontaneous levels (109 ± 8 vs the initial 221 ± 18 g*s) and 80% of PB spontaneous levels (134 ± 9 vs the initial 163 ± 12 g*s). Instead, treatment with oxytocin after 10 µM mibefradil caused AUC to double the spontaneous contractility levels in both V and PB samples (Fig. 5.A top panel and Table 3). This is not surprising, considering that, with 10 µM verapamil, the inhibition of contractility had been complete. Another difference from the mibefradil inhibition experiment is that PB responded twice as robustly as V strips to this single high dose of oxytocin (P < 0.001). This result is consistent with previous experiments with oxytocin and no VGCC inhibitors.

Amplitude was increased by oxytocin to similar values (P = 0.5) in V and PB, thus it does not seem to play a role in this divergence. Duration in V strips grew more than in PB strips (7.2 s vs. 5.4 s, P < 0.001) after the oxytocin treatment. This aligned with the duration data obtained in the 10 µM mibefradil + 50 nM oxytocin study. However, event frequency was twice as high in PB than in V (P < 0.001) samples. Thus, frequency variations seem to drive the predominant response to oxytocin in PB. These findings match the data obtained in our earlier experiments with oxytocin alone [1]. The fact that frequency did not seem to play a lead role in the differential response to oxytocin in the mibefradil experiments was puzzling. Thus, to explore this aspect more thoroughly, we run dose responses to oxytocin in the presence of a fixed amount of VGCC inhibitor.

Response to Oxytocin in 1 \(\mu\)M Mibefradil

To test the hypothesis that T-type channels contribute to the differential response of V and PB uteri to oxytocin stimulation we preconditioned V and PB uterine strips with 1 μM mibefradil for 30 min. We were also interested in clarifying whether the effects of oxytocin on the frequency of PB strips are indeed suppressed by mibefradil as suggested by the previous experiment. In these experiments, all AUC values were normalized to the values obtained from a 40 mM KCl challenge. Figure 3.A displays sample traces, while Fig. 3.B the summary curves for AUC/AUC in 40 mM KCl, amplitude, frequency, and duration (for both V and PB, rat n = 4).

Fig. 3

Dose–response to oxytocin in 1 μM mibefradil. a. Sample traces recorded in uterine samples from virgin (top panel) and proven breeders (bottom panel) female rats. b. Dose–response curves summarizing data obtained from experiments on 5 virgin (V, black circles) and 5 proven breeder (PB, white circles) rats (4–6 uterine strips/rat). Top left panel: overall contractility dose response to oxytocin in the presence of mibefradil (area under the curve over 5 min normalized by the area under the curve over 5 min of the 40 mM KCl challenge. Top right panel: contraction amplitude. Bottom left panel: contraction frequency. Bottom right panel: contraction duration. In the presence of 1 μM mibefradil, spontaneous activity and response to oxytocin were reduced. Dose–response curves for AUC, amplitude, and frequency displayed a hormetic trend. The AUC minimum was smaller and the maximum higher in PB than in V. This is reflected in the frequency curves and resembles the findings in the absence of mibefradil [1]. The amplitude and duration were steadily higher in V than in PB samples throughout the experiment

Except for duration, all the curves obtained in this experiment display a hormetic, bell-shaped trend. For example, the AUC/AUC in 40 mM KCl curve peaked at 50 nM oxytocin, then descended. In the presence of 1 μM mibefradil and no oxytocin, PB strips contracted less robustly than V strips (AUC/AUC in 40 mM KCl 0.09 ± 0.03 vs. 0.17 ± 0.01 in V, P = 0.02). At the peak of the response to oxytocin (Max), contractility was slightly stronger in PB than in V strips, and so was the range (Max–Min). The range of the descendent branch of the dose–response curve was similar in the two groups. No difference was found in the V and PB EC50 for oxytocin in both the ascending and descending branches.

The amplitude curve peaked at 5 nM oxytocin. V samples maintained a greater amplitude than PB at all oxytocin concentrations. The range of the oxytocin response was also more marked in V in the ascending and descending branches of the curve. V strips amplitude increased by 42% vs 15% in PB strips (0.87 ± 0.03 vs 0.31 ± 0.04 g, P < 0.0001).

The hormetic trend of amplitudes was previously observed in dose–response to oxytocin in the absence of VGCC inhibitor preconditioning. Yet, in those experiments, the minima were twice as high as in this case and the curve peaked at 50 nM oxytocin. The EC50 of the ascending branch of amplitude was significantly smaller than the EC50 of the AUC ascending branch (P < 0.001), while the EC50 of the descending branch was consistent between AUC and amplitude.

Frequency also peaked at 5 nM oxytocin. However, no significant difference was detected between the two groups. This result was consistent with the dose-inhibition response to mibefradil + 50 nM oxytocin experiments, in which we noted similar frequency in the two samples. The EC50 of the frequency curves matched the EC50 of the AUC curves. The lack of difference in frequency is the most remarkable finding of this experiment, as frequency was previously identified as the driving factor for the differential response to oxytocin associated with parity [1].

The duration curves show a stronger response to oxytocin in V than in PB after pretreatment with 1 µM mibefradil. Event duration before the addition of oxytocin was similar in 1 µM mibefradil and untreated samples. Duration of V contractions increased by 42%, in PB by 25% (3.9 ± 0.4 vs 2.0 ± 0.2 s respectively, P < 0.0001). The maximal response elicited by oxytocin exceeded both the spontaneous duration and the duration in 10 µM mibefradil + 50 nM oxytocin.

Response to Oxytocin in 1 \(\mu\)M Verapamil

To evaluate the impact of L-type channel inhibition on the response to oxytocin, we preconditioned V and PB uterine strips with 1 μM verapamil for 30 min. Figure 4.A displays sample traces, Fig. 4.B the summary curves for AUC/AUC in 40 mM KCl, amplitude, frequency, and duration (for both V and PB, rat n = 4). The AUC/AUC in 40 mM KCl curves show a stronger contractile response from PB strips, with a trend closely resembling the dose–response to oxytocin in the absence of VGCC inhibitors from earlier experiments. A single sigmoidal model adequately captured the minor hormesis observed in the curve. This contrasts with the more pronounced hormetic effect of mibefradil preconditioning, which necessitates a more complex model for fitting.

Fig. 4

Dose–response to oxytocin in 1 μM Verapamil. a. Sample traces recorded in uterine samples from virgin (top panel) and proven breeders (bottom panel) female rats. b. Dose–response curves summarizing data obtained from experiments on 4 virgin (V, black circles) and 4 proven breeder (PB, white circles) rats (4–6 uterine strips/rat). Top left panel: overall contractility dose response to oxytocin in the presence of verapamil (area under the curve normalized by the area under the curve of the 40 mM KCl challenge. Top right panel: contraction amplitude. Bottom left panel: contraction frequency. Bottom right panel: contraction duration. In the presence of 1 μM verapamil, spontaneous activity and response to oxytocin were reduced. Amplitude and frequency but not AUC displayed a hormetic trend. The AUC minimum was smaller and the maximum higher in PB than in V. This is reflected in the frequency curves as with mibefradil and in the absence of VGCC blockers [1]. The amplitude and duration were steadily higher in V than in PB samples throughout the experiment

The maximal response to oxytocin for both groups was roughly half the one recorded without VGCC inhibitors [1] and comparable in intensity to the one recorded in 1 μM mibefradil. The initial treatment with verapamil greatly inhibited phasic contractions, so that no difference was detected in the two groups (P = 0.5). However, both maximal response and range were higher in PB. The calculated oxytocin EC50s did not differ.

The amplitude curve had a bell-shaped trend. In the ascending branch, the minimum was similar in the two groups, due to the strong inhibition of calcium entry by verapamil. However, the maximal amplitude and the range of amplitude increase elicited by oxytocin were higher in V strips (Table 2.B). V and PB strips gained 35% and 15% of their initial amplitude respectively (from 1.27 ± 0.04 to 2.72 ± 0.03 g in V, from 0.99 ± 0.02 to 1.14 ± 0.01 g). In the previously published oxytocin experiment with no VGCC inhibitors [1], the range of amplitude increase was higher in PB (+ 59%, from 2.2 ± 0.01 to 3.5 ± 0.1 g) than in V (+ 26%,from 3.12 ± 0.04 to 3.94 ± 0.03 in V,). In addition, with verapamil, the V amplitude reached its peak at 5 nM oxytocin, while the PB amplitude at 50 nM. This was reflected in the significantly different EC50 of the ascending branch. In the descending branches, V amplitude decreased by 20% from its maximum, PB amplitude by 13% (0.29 ± 0.05 g vs 0.13 ± 0.06, P = 0.02). The EC50s of the descending branches were comparable and similar to the EC50s of the amplitude curves in mibefradil pretreated samples.

Table 2 Dose response to oxytocin parameter

Frequency curves were also hormetic. In both ascending and descending branches, all parameters except EC50 were higher in PB strips. PB and V frequencies both peaked at 5 nM consistently with the frequency curves of the dose–response to oxytocin in mibefradil. The ascending branches EC50 were similar between groups and consistent with the AUC curves EC50s. This trend is similar to that of the dose response to oxytocin in the absence of inhibitors [1].

In both groups, the event duration of spontaneous contractions was cut in half when 1 μM verapamil rather than 1 μM mibefradil was used (−51 ± 16% in V, −57 ± 14% in PB). Also, the maximal duration was shorter with verapamil than with mibefradil (−29 ± 11% in V, −31 ± 12% in PB). V strips preserved longer durations when treated with verapamil and showed a higher range of response to oxytocin than PB strips throughout the experiment.

Response to Oxytocin in 10 µM Verapamil

In another set of dose–response to oxytocin experiments, we preconditioned the uterine strips for 20 min with 10 µM verapamil. With this higher dosage of inhibitor, all phasic contractions were completely abolished. The application of increasing oxytocin concentrations resulted in a dose-dependent increase in AUC/AUC in 40 mM KCl, due to an increase in the strips’ basal tone since phasic contractions did not resume.

Under these extreme conditions, the response to oxytocin was still more intense in PB samples than in V samples (see summarizing histograms of Fig. 5 and corresponding data in Table 3). The AUC curves were both sigmoidal. The curve minimum was very similar (0.003 ± 0.001 in PB vs. 0.004 ± 0.002 in V). PB samples showed a higher oxytocin curve maximum and a wider overall response range compared to virgin V samples (Max: 0.106 ± 0.002 vs. 0.077 ± 0.002 in V, P = 10–12; Range: 0.103 ± 0.003 vs. 0.073 ± 0.003 in V, P = 6–6). Hence, AUC increased 3.4 folds in PB, 1.8 folds in V. The oxytocin EC50 was identical (−8.36 ± 0.08 for both). The maximal PB strip response to oxytocin in 10 µM verapamil was 88% weaker than the maximal response achieved in the absence of verapamil (0.106 ± 0.002 vs. 0.905 ± 0.02, according to our previously published data [1]) and 78% less robust than the maximal response to oxytocin in the presence of 1 µM verapamil. For V strips, the maximal response to oxytocin in 10 µM verapamil was 90% weaker than the maximal response in the absence of verapamil (0.077. ± 0.002 vs. 0.756 ± 0.001, as per Porta et al. [1]) and 80% less intense than the maximal response with 1 µM verapamil.

Fig. 5

Contractility Histograms. a. Top panel: Comparison of raw AUC data of untreated samples (control), in the presence of 10 μM of either mibefradil or verapamil alone and with the addition of 10–7.5 M oxytocin. These data originate from inhibition-response experiments. Bottom panel: Comparison of AUC/AUC in 40 mM data of untreated samples (control), in the presence of 1 μM of either mibefradil or verapamil alone and with the addition of 10–7.5 M oxytocin. These data originate from the dose–response to oxytocin in the presence of voltage-gated calcium channels blocker experiments. b. Reference histogram of AUC/AUC in 40 mM KCl in the presence and absence of oxytocin, according to data previously published by Porta et al. [1]

Table 3 Comparison of response to oxytocin with and without preconditioning with voltage-gated calcium channels inhibitors

These data are consistent with the results obtained from the inhibition-response experiment as well as with the results of the dose–response to oxytocin with 1 µM verapamil. Given the absence of phasic contractions, no data on amplitude, frequency, or duration are available for these experiments.