Downregulation of PDGFRα and SK channel in murine bladders from CYP-induced cystitis.
We compared the transcriptional expression of Pdgfra and Kcnn1-4 in detrusor muscles from CYP-treated and saline-treated mice. Pdgfra and Kcnn3 (SK3) were significantly downregulated (P<0.01 by unpaired t-test), but neither SK1-2 (Kcnn1 and Kcnn2) nor IK (Kcnn4) were changed significantly after CYP-treatment (n=4, Fig. 1A and B). Detrusor muscles from CYP-treated mice displayed a significant increase in Il6 and Tnf (inflammatory markers) (n= 4, Fig. 1C) suggesting the onset of bladder inflammation after CYP treatment. We further examined the transcriptional expression of Pdgfra and Kcnn3 in sorted PDGFRα+ cells from saline- and CYP-treated PDGFRα/eGFP mice (see Methods section). Pdgfra and Kcnn3 were significantly decreased in CYP-treated mice in comparison to mice treated with saline (n=4, P<0.01 in both genes, Fig. 1D). Transcriptional changes were also evaluated in detrusor smooth muscle cells (SMC). SMCs were isolated from saline- and CYP-treated smMHC/Cre/eGFP (see Methods section) mice, as previously described26. Main excitability-related genes in detrusor SMC, Kcnma1 (BK channels) and Cacna1c (L-type Ca2+ channels) were unchanged in CYP-treated SMC (n=4, Fig. 1E).
Levels of transcripts do not necessarily translate linearly into protein expression. Therefore, we also employed immunohistochemistry and Wes analysis to confirm parallel changes in protein expression. Immunohistochemistry revealed PDGFRα immune-positive cells were down-regulated in CYP-treated detrusor compared with age-matched saline-injected controls (Fig. 2A& B). Figure 2C shows a negative control image in which the primary antibody was omitted and the muscles were treated with only secondary antibody. Wes analysis showed in a more quantitative way that PDGFRα and SK3 were significantly downregulated in CYP-treated detrusor muscles, as compared to controls (n=4, Fig. 3)). These data are consistent with transcriptional data, and suggested the possibility that down-regulation of Pdgfrα and/or Kcnn3 transcripts could be involved in generation of DO.
The effect of SK channel activator on the generation of outward currents and membrane potentials in detrusor PDGFRα + cells from CYP-treated bladders.
We tested the effect of a SK channel activator (CyPPA, 10 µM) on freshly dispersed detrusor PDGFRα+ cells. The cells were dialyzed with K+-rich solution (see Methods). CyPPA hyperpolarized control detrusor PDGFRα+ cells from -31.4 ± 4.6 mV to -61.6 ± 2.9 mV under current clamp (I=0, red dot line, n= 5, Fig. 4A). In the same cells, CyPPA activated outward current at a holding potential of -40 mV in voltage-clamp (V-C) mode (Fig. 4A, blue dotted line). CyPPA-activated current amplitude averaged 39.3 ± 7.1 pA. Figure 4B shows currents evoked by ramp depolarization before (Fig. 4Ba, black trace) and in the presence of CyPPA (Fig. 4Bb, red trace). The resting membrane potentials (RMP) of CYP-treated detrusor PDGFRα+ cells were depolarized to -16 ± 1.4 mV (n=5, P<0.01, as compared to untreated control PDGFRα+ cells). CyPPA induced less hyperpolarization in CYP-treated detrusor PDGFRα+ cells (ΔmV 9.1 ± 2.0), as compared to untreated control PDGFRα+ cells (n=5, P<0.01, Fig. 4C) under current-clamp mode (I=0) and generated smaller outward currents under V-C mode (5.2 ± 0.9 pA; n= 5, P<0.01, Fig. 4D) at -40 mV. These data suggest that SK current density was significantly decreased in PDGFRα+ cells isolated from CYP-treated bladders, as compared with current density in control PDGFRα+ cells isolated from non-treated bladders.
The effect of SK channel blocker on detrusor muscle contractions in CYP-treated bladders
We examined the effect of an SK channel blocker to compare changes in functional expression of SK channels between saline-treated (control) and CYP-treated bladders using isometric force measurements. Detrusor muscle strips without submucosa exhibited spontaneous contractions. In saline-injected control, apamin (300nM, a selective blocker of SK channels) dramatically increased AUC from 62.9±7.9 mN*sec to 261.5±22.5 mN*sec during 5 min recordings (n=8, P<0.0001, Fig. 5A & C). These data are consistent with previous reports30–32. In CYP-treated detrusor muscle strips, spontaneous contractions were of high amplitude and irregular. AUC for these contractions was calculated since averaging the frequency and amplitude of these irregular contractions are not reliable measurements. AUC in CYP-treated muscles before and after apamin were 315.7±69.8 mN*sec and 341.3 ± 66.4 mN*sec (n=6, Fig. 5B & D), respectively. Thus, apamin had no significant effect on spontaneous contractile activity in CYP-treated muscles. We also calculated the apamin-sensitive contractions by normalized the effect of apamin from control AUC (before apamin). The sensitivity to apamin was significantly decreased in CYP-treated detrusor muscles (1.1±0.1 fold) compared to saline-injected detrusor muscle strips (4.6±0.6 fold, P<0.001, Fig. 5D).
The effect of SK channel blocker and agonist on CYP-treated bladders using ex vivo preparation.
We also examined the pressure-volume relationships of excised bladders in ex vivo preparations to investigate changes in functional expression of SK channel in control and CYP-treated bladders. Ex vivo pressure-volume measurements exclude extrinsic neural regulation during filling, so this technique highlights regulation via myogenic mechanisms. In in vivo cystometry, voiding contractions start around 20 cmH2O in murine bladder33. Thus, we analyzed the pressure amplitude and frequency up to 15 cmH2O (see Fig. 6A & B) to associate contractile activity (i.e. transient contractions or TCs) to the non-voiding contractions (NVCs) observed in in vivo cystometry. The passive pressure underlying was normalized to 0 cmH2O (see Fig. 6Aa & Bb). In control bladders, infusion of KRB solution (15 µl/min) induced a small increase in intravesical amplitude (1.3 ± 0.3 cmH2O) with the frequency of TCs equal to 17 ± 2 events at intravesical pressures up to 15 cmH2O. Addition of apamin (300 nM) into the bath increased TC frequency to 35 ± 3 events (P<0.001, Fig. 6A-C) and the amplitude of TCs to 3.8 ± 0.4 cmH2O (n=6, P<0.01, Fig. 6A, B & D). The SK chanel agonist, SKA-31, showed no significant effect on control bladders (data not shown). CYP-treated bladders displayed increased amplitude of TCs with high frequency before apamin treatment (Fig. 7A-C). SKA-31 and apamin treatment did not significantly affect the amplitude or frequency of TCs in CYP-treated bladders (n=6, Fig. 7B & C).