Alterations in detrusor contractility in rat model of bladder cancer

Abstract

Urinary incontinence of idiopathic nature is a common complication of bladder cancer, yet, the mechanisms underlying changes in bladder contractility associated with cancer are not known. Here by using tensiometry on detrusor smooth muscle (DSM) strips from normal rats and rats with bladder cancer induced by known urothelial carcinogen, N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN), we show that bladder cancer is associated with considerable changes in DSM contractility. These changes include: (1) decrease in the amplitude and frequency of spontaneous contractions, consistent with the decline of luminal pressures during filling, and detrusor underactivity; (2) diminution of parasympathetic DSM stimulation mainly at the expense of m-cholinergic excitatory transmission, suggestive of difficulty in bladder emptying and weakening of urine stream; (3) strengthening of TRPV1-dependent afferent limb of micturition reflex and TRPV1-mediated local contractility, promoting urge incontinence; (4) attenuation of stretch-dependent, TRPV4-mediated spontaneous contractility leading to overflow incontinence. These changes are consistent with the symptomatic of bladder dysfunction in bladder cancer patients. Considering that BBN-induced urothelial lesions in rodents largely resemble human urothelial lesions at least in their morphology, our studies establish for the first time underlying reasons for bladder dysfunction in bladder cancer.

Figure 1

Macroscopic appearance of the rat bladder in rat model of BBN-induced bladder cancer. (a) Representative images of incised and everted bladders of the rats exposed to BBN in drinking water for 16 weeks, showing the heterogeneity of bladder cancer lesions. (b) For comparison normal intact (left) and incised (right) rat bladder is presented. (c) Examples of detrusor smooth muscle (DSM) strips obtained from the bladders of rats with BBN-induced bladder cancer used for contraction measurements; note papillary-type outgrowths. (d) Histopathological evaluation of lesions identified by H&E stain in rat BBN-induced bladder cancer, from top to bottom: normal urothelium (control), papillary carcinomas showing various degree of dysplasia (cancer 1–3) and simple hyperplasia; right images represent magnified views of boxed parts on the left; calibration bars are in micrometers.

Figure 2

Bladder cancer reduces spontaneous contractions. (a) Representative recording of spontaneous contractions of DSM strip from control rat (top) with the part of the recoding presented at expanded amplitude and time scales (bottom right); A_SC and f_SC designate amplitude and frequency of spontaneous contractions, respectively, with f_SC = 1/T_SC, where T_SC is the period of spontaneous contractions; dashed and dotted lines correspond to the baseline and A_SC levels, respectively; bottom left panel presents amplitude histogram of the recording with two Gaussians fit to the histogram; the distance between Gaussians maximums corresponds to A_SC. (b) Typical power spectrum of spontaneous contractions used to estimate f_SC. (c,d) Bar graphs showing the decrease of the amplitude (c) and frequency (d) of spontaneous contractions in cancer (Cncr, dark gray, n = 9) vs. normal (Ctrl, light grey, n = 9) DSM strips; mean ± SD, “*” significant difference (P < 0.05).

Figure 3

Bladder cancer down-regulates DSM contractions evoked by electric field stimulation (EFS) mostly at the expense of m-cholinergic component. (a) Representative original recordings of EFS-contractions of control-DSM in response to EFS of increasing duration (from left to right 0.4, 1.6, 3.2, 4, 6, 8, 9 and 10 s) before (left) and after exposure to m-cholinergic inhibitor atropine (ATR, 1 µM, right). (b) Quantification (mean ± SD) of the dependence of EFS-contraction amplitude (A_EFS) in control DSM on EFS duration in the absence (filled symbols, n = 10) and in the presence of ATR (1 µM, open symbols, n = 10). (c,d) Same as in (a,b), respectively, but for cancer-DSM (mean ± SD, n = 10); note, the decrease of both overall A_EFS and the size of ATR-blockable component in cancer- vs. control-DSM; for (b,d) “*” significant difference (P < 0.05) between data points.

Figure 4

Bladder cancer up-regulates DSM contractility dependent on activation of heat and capsaicin-sensitive TRPV1-channel. (a,b) Representative original recordings of normal (control, a) and cancerous (b) DSM strips contractions in response to application of TRPV1 agonist capsaicin (CAP, 10 µM) on the background of atropine (ATR, 1 µM); upward spikes are EFS-contractions; note, development of stronger tension and stronger increase of EFS-contractions in cancer (b) vs. normal (a) DSM in response to CAP; in these as well as other original recordings from subsequent figures the first upward spike—contraction in response to KCl (60 mM) used for data normalization, dashed line—the level of baseline tension, thick solid lines—markers of drugs applications. (c,d) Quantification of the changes in tension (c) and amplitude of EFS-contractions (A_EFS, d) at indicated interventions in normal (Ctrl, light grey bars, mean ± SD, n = 8) and cancerous (Cncr, dark grey bars, mean ± SD, n = 10) DSM strips; “*” significant difference (P < 0.05) between corresponding values; note, development of stronger tension and A_EFS enhancement in response to CAP in cancer vs. control DSM.

Figure 5

TRPV4-dependent contractility is largely preserved in mucosa-striped normal-DSM and is reduced in cancer-DSM. (a–c) Representative recordings of contractions of control DSM strips (i.e. from normal rats) with intact (+ urothelium, a) and removed (-urothelium, b) urothelium, and cancer DSM strips with intact (+ urothelium, c) in response to the application of TRPV4 agonist GSK1016790A (1 µM). (d–f) Quantification of the changes in GSK1016790A-induced tension (d), amplitude (A_SC, e) and frequency (f_SC, f) of spontaneous contractions in normal (Ctrl) urothelium-preserved (+ uroth., light grey bars, mean ± SD, n = 10) and urothelium-devoid (-uroth., hatched light grey bars, mean ± SD, n = 10) DSM strips and in cancer-DSM strips (Cncr) with intact urothelium (+ uroth., dark grey bars, mean ± SD, n = 9); “*” significant difference (P < 0.05) between corresponding values.

Figure 6

Bladder cancer eliminates BK channel-dependent modulation of spontaneous contractions frequency. (a,b) Representative recordings of contractions of DSM strips from normal (control, a) and cancerous (b) bladder in response to application of BK-channel blocker TEA (3 mM); note, strong increase of spontaneous contractions frequency in response to TEA in control DSM (a) and lack thereof in cancer one (b). (c–e) Quantification of the changes in TEA-induced tension (c), amplitude (A_SC, d) and frequency (f_SC, e) of spontaneous contractions in control (Ctrl, light grey bars, mean ± SD, n = 10) and cancerous (Cncr, dark grey bars, mean ± SD, n = 8) DSM strips; “*” significant difference (P < 0.05) between corresponding values.