Activation of muscarinic receptors in rat bladder sensory pathways alters reflex bladder activity

Abstract

Antimuscarinic drugs affect bladder sensory symptoms such as urgency and frequency, presumably by acting on muscarinic acetylcholine receptors (mAChRs) located in bladder sensory pathways including primary afferent nerves and urothelium. However, the expression and the function of these receptors are not well understood. This study investigated the role of mAChRs in bladder sensory pathways in vivo in urethane anesthetized rats. Intravesical administration of the mAChR agonist oxotremorine methiodide (OxoM) elicited concentration-dependent excitatory and inhibitory effects on the frequency of voiding. These effects were blocked by intravesical administration of the mAChR antagonist atropine methyl nitrate (5 microM) and were absent in rats pretreated with capsaicin to desensitize C-fiber afferent nerves. Low concentrations of OxoM (5 microM) decreased voiding frequency by approximately 30%, an effect blunted by inhibiting nitric oxide (NO) synthesis with L-NAME (N(omega)-nitro-L-arginine methyl ester hydrochloride; 5 mg/kg; i.v.). High concentrations of OxoM (40 microM) increased voiding frequency by approximately 45%, an effect blunted by blocking purinergic receptors with PPADS (0.1-1 mM; intravesically). mAChR agonists stimulated release of ATP from cultured urothelial cells. Intravenous administration of OxoM (0.01-5 microg/kg) did not mimic the intravesical effects on voiding frequency. These results suggest that activation of mAChRs located near the luminal surface of the bladder affects voiding functions via mechanisms involving ATP and NO release presumably from the urothelium, that in turn could act on bladder C-fiber afferent nerves to alter their firing properties. These findings suggest that the urothelial-afferent nerve interactions can influence reflex voiding function.

Figure 1.

Inhibitory and excitatory effects of intravesical administration of OxoM. A, Typical CMG recording illustrating the parameters used for quantifying the effects of drugs on voiding. B, Variation of ICI during the course of 8 h of continuous saline infusion (n = 7 rats). C, Representative CMG recordings illustrating the inhibitory and excitatory effects of intravesical OxoM in the same animal. The duration of 5 and 40 μm OxoM application was ∼35 and 45 min respectively, with a wash period of ∼108 min between applications. D, Changes in ICI after intravesical administration of different concentrations of OxoM. n = 9 rats for 1 and 5 μm OxoM and n = 9 rats for 20, 40, and 80 μm OxoM. Because 40 and 80 μm OxoM produce similar effects, for the following experiments, data are pooled together. Asterisks indicate significant changes (p < 0.05) from control, tested with paired t test. E, Intravesical administration of low (white bars; 5 μm; n = 12 rats) and high (black bars; 40 or 80 μm; n = 18 rats) concentrations of OxoM do not significantly change A, PTh, and BP. Statistical significance was tested with paired t test for each parameter, p > 0.05. In D and E, dotted lines represent control set to 100%.

Figure 2.

Effects of intravesical and intravenous administration of AMN. A, Changes in CMG parameters after intravesical administration of 1, 5, 50, and 100 μm AMN. Multiple concentrations were tested in each animal. Summary from n = 3, 7, 4, and 4 animals for 1, 5, 50, and 100 μm AMN, respectively. B, Changes in CMG parameters after intravenous administration of AMN (0.1–2 mg/kg) in the presence of intravesical AMN (100 μm). Control refers to parameters measured during intravesical AMN (100 μm). Data are summarized during two time intervals: the first 10 min after intravenous delivery of AMN (white bars) and the remaining of 10–30 min (<10 min, gray bars; summary from n = 4 rats). C, Effects of intravenous administration of AMN (0.1 mg/kg) in the presence of 100 μm intravesical AMN. In A and B, asterisks indicate significant changes (p < 0.05) from control, tested with paired t test, and dotted lines represent control set to 100%.

Figure 3.

Inhibitory effects of intravesical administration of OxoM. Ai, Low concentrations of OxoM (5 μm) increased the ICI (summary from n = 12 rats). Aii, The inhibitory effects on ICI (white bars) were partially reversible after ∼80 min wash (gray bars), and a second application produced excitation (black bar; summary from n = 5 rats). Aiii, The effects on ICI (white bars) were significantly reduced by intravesical administration of AMN (5 μm; black bars; summary from n = 10 rats; example in C). Asterisks indicate significant changes (p < 0.05) from control, tested with paired t test. Dotted lines represent control set to 100%. s, Significant difference; ns, no significant difference (tested with paired t test between the groups indicated). Bi, Time course of the inhibitory effects of OxoM (n = 12 rats). ICI number (#) 0 indicates when OxoM was applied. A negative ICI # refers to ICIs before drug application and a positive ICI # refers to ICIs after drug application. The thick line represents mean and dotted lines represent confidence interval (5, 95%). Bii, Similar data from control experiments in which saline was instilled instead of OxoM (n = 7 rats). C, Inhibitory effect of OxoM (5 μm) on ICI, which is reversed by intravesical administration of AMN (5 μm).

Figure 4.

Excitatory effects of intravesical administration of OxoM. Ai, High concentrations of OxoM (40, 80 μm) reduced ICI (summary from n = 18 rats). Aii, The effects of OxoM (40, 80 μm) on ICI (white bars) were reversible after washout (gray bars) and repeatable (black bar); summary from n = 5 rats. Aiii, The effects of OxoM (80 μm) were blocked by intravesical administration of AMN (5 μm; summary from n = 5 rats). Dotted lines represent control set to 100%. Asterisks indicate significant changes (p < 0.05) from control, tested with paired t test. s, Significant differences tested with paired t test between the groups indicated in figure. B, Time dependence of the excitatory effects of intravesical OxoM. Examples from three rats. Bi illustrates the most common pattern (found in 78% of rats): initially the ICI becomes shorter (phase 1), then ICI lengthens (phase 2), but it is still shorter than control, without any changes in other CMG parameters. Numbers on top of voiding contractions represent the number of the ICI before (negative values) and after (positive values) OxoM application, which are used for quantification in C. Bii illustrates an example in which PTh increases slightly during phase 2. Biii illustrates an example in which BP increases during phase 2. Dotted lines indicate baseline pressure before OxoM application. Arrows in each panel indicate the time when OxoM infusion was started. C, Quantification of the time dependence of the excitatory effects of OxoM (40, 80 μm; summary from n = 18 animals). ICI number (#) 0 indicates when OxoM was applied. The line shows average of control data and dotted line represents linear fitting of the data during OxoM application.

Figure 5.

Effects of intravenous administration of OxoM on CMG parameters. A, Effects of increasing doses of intravenous OxoM on CMG parameters. The highest dose (5 μg/kg) was administered in the presence of intravesical AMN (5 μm for ∼50 min before intravenous administration of OxoM). Note that the effects of intravenous OxoM were not blocked by intravesical administration of AMN (5 μm). Bi and Bii show enlargements of the dotted areas in A illustrating an acute increase in the IVP after administration of OxoM (black circle). IVP was measured before (IVP₁) and after (IVP₂) intravenous OxoM delivery at the time points indicated by arrows. C, Quantification of the changes in CMG parameters produced by intravenous administration of OxoM in the absence (white bars) and in the presence (black bars) of intravesical AMN (5 μm; summary from n = 5 rats). Asterisks indicate statistically significant differences (paired t test p < 0.05) relative to control.

Figure 6.

Effects of intravesical and intravenous administration of OxoM in capsaicin pretreated rats. A, Comparison of CMG parameters between untreated and capsaicin pretreated rats, before OxoM application (summary from n = 21 untreated rats and n = 14 capsaicin pretreated rats). Asterisks indicate significant changes (p < 0.05) from control, tested with paired t test. B, CMG recordings from an untreated rat (Bi) and a capsaicin pretreated rat (Bii), illustrating the differences in ICI. Traces are on the same time scale for comparison. C, Effects of intravesical OxoM in a capsaicin pretreated rat. The arrow points to PTh. D, Intravesical administration of OxoM at increasing concentrations did not affect ICI, A, or BP, but increased PTh (summary from n = 5, 3, 9, 7, and 3, animals for OxoM 5, 20, 40, 80, and 160 μm, respectively). Dotted line represents control set to 100%. Asterisks indicate significant changes (p < 0.05) from control, tested with paired t test. E, Effects of intravenous OxoM in a capsaicin pretreated rat in the absence and in the presence of intravesical AMN (5 μm for >50 min before i.v. administration of OxoM). Note that the effects of intravenous OxoM were not blocked by intravesical administration of AMN.

Figure 7.

Inhibitory effects of intravesical administration of OxoM involve NO. Ai, Summary of the effects of intravenous administration of 5 mg/kg l-NAME (white bars; n = 9 rats) and 25 mg/kg l-NAME (black bars; n = 6 rats) on CMG parameters. Asterisks represent significant changes from control (paired t test between control and l-NAME p < 0.05). Significant (s; unpaired t test p < 0.05) or nonsignificant (ns; unpaired t test p > 0.05) differences in between 5 mg/kg l-NAME and 25 mg/kg l-NAME are shown. Aii, Effects of 5 mg/kg (i.v.) l-NAME on CMG parameters. Aiii, Effects of 25 mg/kg (i.v.) l-NAME on CMG parameters. Arrows indicate the time when l-NAME (i.v.) was injected. B, Time course of the effects of intravesical administration of low concentrations of OxoM (5 μm) in rats treated with l-NAME (n = 7 rats). Negative ICI values represent the ICI number (#) before OxoM application and positive ICI values represent ICI # after OxoM application. ICI # 0 was taken when OxoM was applied. C, The effects of intravesical administration of low concentrations of OxoM (5 μm) on CMG parameters after l-NAME, illustrating the absence of OxoM-induced inhibition. D, Time dependence of the effects of high concentrations of OxoM (40 μm) in rats treated with l-NAME (n = 6 rats). E, The effects of 40 μm OxoM on CMG parameters after l-NAME illustrating the presence of OxoM-induced excitation.

Figure 8.

Mechanisms of the excitatory effects of intravesical administration of OxoM. A, The excitatory effect of OxoM (40–80 μm) in the absence and in the presence of PPADS (0.1–1 mm) are illustrated for all rats (n = 13). Each empty round symbols represent data from a single rat and lines connect data from the same rat. Filled round symbols represent the average data from all rats before (left) and after (right) PPADS. Asterisk indicates significant differences (p = 0.038) between the effect of OxoM on ICI before and after PPADS. B, Time course of the excitatory effects of OxoM (40–80 μm) in the presence of PPADS (0.1–1 mm; intravesical) (n = 13 rats). Negative ICI values represent the ICI number (#) before OxoM application and positive ICI values represent ICI # after OxoM application. ICI # 0 was taken when OxoM was applied. C, Effects of OxoM in the absence (i) and in the presence (ii) of PPADS. D, In cultured urothelial cells, two consecutive applications of OxoS (10 μm) evoked ATP release of similar magnitude (summary from n = 5 coverslips). Baseline indicates baseline ATP levels and Ionomycin indicates ATP release in response to the calcium ionophore ionomycin (5 μm). E, OxoS-evoked ATP release was greatly reduced by AMN (20 μm; summary from n = 5 coverslips). In D and E, asterisks indicate significant differences tested with ANOVA between groups followed by Newman–Keuls post hoc test.