PKC activation reduces contractile force and BMS spontaneous activity at low concentrations and increases contractility force at higher concentrations
The biphasic effects of PKC activation by PDBu on urothelium intact isolated bladder strips in response to increasing concentrations of the drug is displayed in Figure 1. This phenomenon has been previously reported in urothelium denuded BSM strips isolated from the rabbit urinary bladder, and confirmed that the urothelium did not play a role in PKC-induced inhibition of bladder contractions [22]. Present data reveal that at low levels of PKC activation (20 nM), PDBu inhibited spontaneous contractions, and lowered the basal smooth muscle tone from 0.2 ± 0.014 g to 0.15 ± 0.011 g tension, a 25% decrease (Figure 1 B, N = 5, n = 7, p ≤ 0.05 to baseline). Further exposure to PDBu up to a maximal concentration of 640 nM led to a reversal of the initial reaction and an increased muscle tone up to 0.25 ± 0.017 g of tension, a 25% increase above the initial basal level (N = 5, n = 7, p ≤ 0.05 to baseline).
High level of PKC activation by PDBu enhances the maximal contractile force of BMS to EFS-induced nerve stimulation
Our preliminary studies indicated that PKC activation by PDBu had a concentration-dependent effect on EFS-induced nerve stimulation in isolated BMS. High (1 μM) concentration of PDBu amplified both the peak force (PF) response, and integral force (IF) of the bladder strips at low levels of EFS (0-4 Hz). We, therefore, followed the changes in BMS contractility at different concentrations of PDBu to determine the threshold for EFS-induced sensitivity in BMS. Our results (Figure 2) indicate that at a concentration of 100 nM and above, PDBu induced a significant increase in both PF, and IF in BMS from 0.5 to 4 Hz of EFS. At 4 Hz of EFS, the PF response in the presence of 100 nM PDBu was already 98 ± 11% of maximal force generation (measured at 32 Hz), and 117 ± 13% of maximum value for IF (Figure 2 B and C, N = 5, n = 8, p ≤ 0.05 for both parameters). By comparison to PDBu, the control bladder strip developed only 52 ± 6% of PF at 4 Hz, and 69 ± 8% of maximum value for IF. The greatest difference was seen at 0.5 Hz with 100 nM PDBu treatment. These data confirm the concentration-dependent effects of the PKC activator, PDBu on EFS-mediated contractility of isolated BMS.
PKC activator, PMA, enhances the contractile force response to EFS in BMS
In order to provide additional evidence for our abovementioned findings using PDBu, we used a similar PKC activator, PMA, to evaluate its effect on EFS-induced contractions. The data from this experimental set showed that PMA had significant effects on both PF and IF but required much higher concentrations to be effective. Figure 3 (A and B) present summarized data confirming that PMA (100 μM) stimulated an increase in PF and IF up to 4 Hz of EFS in comparison with control strips not treated with PMA. Similar to PDBu results, the greatest change was observed at lower frequencies (0.5-2Hz), where the PF increased by 228 ± 20.9%, 228 ± 21.5%, and 168 ± 16.2% (Figure 3 A), and the IF increased by 561 ± 52.3%, 219 ± 21.9%, and 196 ± 20.2%, respectively (Figure 3 B, N = 4, n = 6 in each group, p ≤ 0.05 to control values). Starting from 25 μM of PMA, and at 8-32 Hz of EFS stimulation, the drug caused an average increase in IF of 82 ± 7.9%, 48 ± 5.4%, and 80 ± 9.3% for 8, 16, and 32 Hz of EFS, respectively (Figure 3 B, N = 4, n = 6 in each group, p ≤ 0.05 to control values).
Inhibition of PKC with Bim-1 reduces the ability of BMS to maintain muscle force
Previous studies established that down-regulation of PKC in PBOO is associated with frequency of urination and a decreased volume of voided urine [19]. Additionally, Bim-1, a specific inhibitor of PKC, did not affect the PF at 32 Hz but reduced IF (ability to maintain muscle force) by ~50% in isolated BMS during in vitro studies when compared with control strips [22]. This observation suggested that Bim-1 induced a significant loss of smooth muscle ability to maintain force which is a requirement for physiological bladder emptying. We, therefore, evaluated BMS force generation over a range of EFS frequencies in the presence and absence of Bim-1, at different concentrations, to determine if this finding was dependent on the frequency of stimulation (Figure 4). Our data did not determine a significant difference between PF with and without Bim-1 over a range of applied frequencies (0.5-32 Hz, Figure 4 A) and concentrations of Bim-1 (4 nM- 32 nM). There was no substantial difference between IF values of strips with or without Bim-1 treatment at lower frequencies of EFS (0.5-2 Hz) either (Figure 4 B). However, EFS with frequencies above 2 Hz caused a significant reduction in IF of BMS subjected to Bim-1 (16 and 32 nM) in comparison with control group (Figure 4 B). The IF declined significantly at stimulation frequencies from 4 to 32 Hz at 16 and 32 nM of Bim-1. At a concentration of 16 nM Bim-1, the decline in IF from control values was 53 ± 5.1%, 55 ± 4.9%, 52 ± 5.2%, and 52 ± 4.8% at 4, 8, 16, and 32 Hz of EFS, respectively (N = 3, n = 5, p ≤ 0.05 to control group).
PKC inhibitors reversed EFS-induced sensitivity of BMS upon high PKC stimulation, and decreased IF in bladder strips
In order to determine if increased sensitivity to EFS by high PDBu could be reversed by PKC inhibitors, we tested the effects of Bim-1 on muscle strips previously subjected to high concentration of PDBu. Figure 5 A (upper panel) shows the EFS-frequency response for the control strip without any treatment. Figure 5 A (lower panel) includes the effects of PDBu (100 nM) stimulation on EFS-dependent contractile responses alone, and after incubation with Bim-1 in the presence of PDBu. At the lowest frequency of EFS (0.5 Hz), PDBu increased the PF by 8-fold (N = 5, n = 6, p ≤ 0.05 to control); this effect was blocked by application of Bim-1 (Figure 5 B). At 0.5-4 Hz of EFS, the PF of PDBu-enhanced responses was reversed by addition of Bim-1, and the PF values were similar to the control group (Figure 5 B). PDBu had no significant effect on enhancement of PF at 8, 16 and 32 Hz of EFS, paralleling the absence of effect to Bim-1 stimulation at the same frequencies.
In order to provide further support for the effects of PKC inhibition on BMS contractility, we tested another PKC inhibitor, Ro318220 (Figure 5 C). Experiments with Ro318220 established that the drug significantly reduced IF of muscle strips, but required much higher concentrations in comparison with Bim-1 to be effective. Between 0.5 and 8 Hz of applied EFS, there was no significant difference in the level of IF at any concentration of Ro318220 (4.5-121.5 μM). However, at 16 and 32 Hz of EFS, Ro318220 caused a significant decrease in IF of the strips with the greatest effect observed at 40.5 μM and 121.5 μM (N = 5, n = 7, p ≤ 0.05 to control). At 40.5 and 121.5 μM, Ro318220 (at 16 Hz of EFS) decreased IF of BMS to 60 ± 7.2% and 58 ± 5.4% of control values (Figure 5 C, N = 4, n = 6, p ≤ 0.05).
Effects of extracellular calcium on the contractile responses of isolated BMS
Previous studies established that endogenous PKC activity is regulated by Ca2+-dependent and independent mechanisms [35,40,45,47]. Therefore, we recorded the contractile responses of BMS to EFS and PDBu in solutions with normal (1.8 mM) and very low (0.018 mM) calcium (Figure 6). A reduction in calcium concentration by 100-fold led to an 85% decrease in the response to EFS (32 Hz, Figure 6 A), and also caused a 50% decrease in the response to high concentration of PDBu (1.0 μM, Figure 6 B).
Effects of a high concentration of PDBu on BMS frequency-contractility response upon EFS
Our prior data (Figure 2) confirmed an increased sensitivity to EFS upon high level of stimulation of PKC activity, however, low levels of PKC activation had no significant effect on BMS response to EFS stimulation. In order to determine the stimulation frequency that produced half-maximal response (EFS50), we obtained a logarithmic frequency-response curve (0.1–100 Hz) in the presence of high PDBu. EFS50 values were determined for each strip using a sigmoidal curve fit of the data (SigmaPlot 11 Software, Systat Software Inc, San Jose, CA). Figure 7 A (upper panel) shows the contractile response of control strips whereas the lower panel presents the raw traces of BMS subjected to PDBu application (1 μM). Figure 7 B presents the summary data of contractile responses of BMS treated with and without PDBu. Application of PKC activator caused a significant leftward shift of dose response curve with EFS50 value of 2.14 ± 0.14 Hz in comparison with control value of 10.3 ± 1.2 Hz (Figure 7 B). This is indicative of a 5-fold increase in sensitivity of BMS strips to EFS incubated with a PKC activator (N = 5, n = 6, p ≤ 0.05 to control group).
Application of a PKC activator decreases BMS sensitivity to carbachol
It was previously shown that PDBu can inhibit spontaneous myogenic contractions in vitro at low levels of PKC activation. Our cystometric experiments also confirmed that the PKC inhibitors, Bim-1 and Ro318220, can induce frequency and NVC, and that PDBu can reverse Bim-1-induced frequency (please see below). We, therefore, tested the effects of different concentrations of PDBu on cholinergic sensitivity in DSM. As seen in Figure 8A, PDBu at 50 nM and 1 μM caused a significant rightward shift in the carbachol concentration response curve when compared to control group (Figure 8 B, N = 4 and n = 6 in each group, p ≤ 0.05). The EC50 values were 0.56 ± 0.049 nM, 4.2 ± 0.35 nM, and 4.7 ± 0.47 nM for control, PDBu (50 nM), and PDBu (1 μM) groups, respectively. This represented an approximate 8-fold decrease in sensitivity of BMS to carbachol after PDBu application. There was no effect on the maximum force produced with or without PDBu application.
Bim-1 does not affect carbachol concentration-response curve
Figure 9 A shows the representative raw data for control (upper trace), and Bim-1-treated (lower trace) muscle strips upon stimulation of muscarinic receptors with carbachol. The summary data in Figure 9 B reveal that Bim-1 had no significant effect on sensitivity to carbachol stimulation, nor on the PF generation in comparison with control (EC50 = 0.52 ± 0.06 μM for control group (N = 4, n = 7) vs EC50 = 0.54 ± 0.06 μM for Bim-1 group (N = 5, n = 8, p ≤ 0.05 to control).
Effects of PKC activation by PMA on urinary bladder function in vivo evaluated by cystometry
In addition to in vitro studies, we carried out similar experiments utilizing in vivo urodynamic approach in awake rats. We tested the effects of PMA, a PKC activator, on bladder function. Intravesical instillation of PMA (50 μM) caused a significant increase in frequency of micturition contractions, and decline in the voided volumes when compared to the control. Figure 10 A shows the representative cystometric traces from control group with a mean of 4 ± 0.5 voids in 2 hours (Figure 10 D). The voided volume recorded in control rats was 2.75 ± 0.23 ml, and decreased to 0.75 ± 0.43 ml with PMA (Figure 10 B and C, N = 4, p ≤ 0.05 to control). PMA also caused a significant increase in the voiding frequency to 8 ± 0.9 voids over the same time course (Figure 10 D).
Effects of PKC activation by PDBu on urinary bladder function in vivo evaluated by cystometry
In order to determine if low PDBu affected micturition contractions, we tested the effects of intravesical instillation of low (50 nM) and high (1 μM) concentrations of PDBu in in vivo studies. Figure 11 shows the cystometric recordings upon intravesical instillation of low (A, 50 nM) and high (B, 1 μM) concentrations of PDBu on urodynamic parameters of rat urinary bladder. The data revealed that low concentration of PDBu had no significant effect on frequency of micturition (Figure 11D), nor affected the voided volume (Figure 11C), and was similar to control (Figure 10). However, high concentration of PDBu caused a significant increase in the frequency of micturition contractions (Figure 11B and D) along with a commensurate decrease in the volume of voided urine from 2.7 ± 0.4 ml to 0.3 ± 0.048 ml per cycle (Figure 11C, p ≤ 0.05).
Intravesical instillation of Ro318220 decreases the micturition volume and increases the number of non-voiding contractions during cystometry
Urodynamic evaluation of bladder function was performed in the presence of a second PKC inhibitor, Ro318220. Figure 12 shows cystometry traces recorded in control rat without Ro318220 treatment (A) and after intravesical instillation of the drug (B). The data revealed that Ro318220 application caused a 3-fold decrease in voided volume (Figure 12 C), and an increase in NVC from 2 ± 0.5 to 6 ± 1.1 (Figure 12 D, N = 4, p ≤ 0.05 to control).
In vivo treatment with Bim-1 decreases the micturition volume and increases the number of non-voiding contractions during cystometry
This set of experiments was designed to determine whether or not Bim-1, a PKC inhibitor, promotes NVC in the urinary bladder under in vivo conditions. Cystometrograms recorded in awake rats reflect the changes in urodynamic parameters during intravesical instillation of either saline (Figure 13 A) or Bim-1 (Figure 13 B). The upper panels of the figures represent the intravesical pressure, whereas the lower panels show the volumes of voided urine. Intravesical instillation of Bim-1 (28 nM) decreased the voided volume of urine by 56 ± 4.8% (Figure 13 C, N = 4, p ≤ 0.05), and increased the number of non-voiding contractions by 3.5-fold (Figure 13 D, N = 4, p ≤ 0.05 to control). An increase in the number of NVC was consistent with the increased spontaneous activity seen in isolated muscle strips in response to Bim-1 application [22]. The reduced emptying, on the other hand, may be due to the inhibitory effect of Bim-1 on the IF or force maintenance in DSM as demonstrated in Figure 4, and as previously established in the rabbit bladder [22] .
Application of a low concentration of PDBu after intravesical application of Bim-1 rescues bladder function
In order to determine whether the effects of Bim-1 on bladder function can be reversed by subsequent application of the PKC activator, PDBu (50 nM) was infused following intravesical instillation of Bim-1 (Figure 14 A). The obtained results showed that a low concentration of PDBu inhibited Bim-1-induced non-voiding contractions in the urinary bladder, restored the duration of the micturition cycle, and increased the voided volume to control levels (Figure 14 C) when compared with the effects of Bim-1 (shown at the beginning of the tracing in Figure 14 A). Additional studies (Figure 14 B, and D) confirmed that these findings were not simply due to a washout effect of the Bim-1 during infusion of PDBu. In these experiments, we maintained the concentration of Bim-1 as in Figure 14 A, but had to increase the concentration of PDBu to 100 nM in order to see the same effect as in Figure 14 A. Since Bim-1 is a competitive inhibitor of PDBu, the higher concentration of PDBu was most likely able to overcome the effects of the initial Bim-1 infusion thus restoring a normal micturition cycle. These results confirm our in vitro findings and suggest that PKC down-regulation, e.g. as reported in PBOO [19], may contribute to frequency of micturition under pathophysiological conditions.