Influence of naloxone on inhibitory pudendal-to-bladder reflex in cats

Abstract

To determine the involvement of opioid receptors in the inhibitory pudendal-to-bladder reflex, the effect of naloxone (0.01-1 mg/kg, i.v.), an opioid receptor antagonist, on the inhibition of bladder activity evoked by pudendal nerve stimulation was investigated in alpha-chloralose anesthetized cats. The inhibition of reflex isovolumetric bladder contractions induced by pudendal nerve stimulation (5-10 Hz) at intensity threshold (T) for producing complete inhibition was significantly suppressed by naloxone at a high dose (0.3 mg/kg). However, the inhibition elicited at higher intensities (1.5-3 T) was not changed. Naloxone (1 mg/kg) did not alter the frequency dependence of the inhibitory effect of pudendal stimulation. During cystometrograms (CMGs) pudendal nerve stimulation significantly increased bladder capacity to 155.1+/-24.5% and 163.4+/-10% of the control at stimulation intensities of 1 T and 1.5-3 T, respectively. After administration of naloxone (1 mg/kg), the bladder capacity during pudendal nerve stimulation at inhibition threshold (1 T) was not significantly different from control, but it was significantly increased at higher intensities (1.5-3 T). Naloxone alone markedly reduced bladder capacity to 43+/-11.1% of the control, and pudendal stimulation completely reversed this facilitatory effect. This study revealed that activation of opioid receptors contributes to or facilitates the inhibitory pudendal-to-bladder reflex. The reduction in bladder capacity after naloxone treatment also indicates that endogenous opioid peptides mediate a tonic inhibition of micturition. Understanding the neurotransmitter mechanisms involved in the inhibitory pudendal-to-bladder reflex could promote the development of new treatments for bladder overactivity and incontinence.

Fig. 1

Intensity and frequency dependent inhibitory effect of pudendal nerve stimulation on isovolumetric bladder contractions. A. At low intensities (1.2–1.8 V) of a 5 Hz stimulation, isovolumetric bladder contractions exhibited an initial enhancement of the contraction at the onset of the stimulation followed by a post-stimulus suppression of the contractions. However once the intensity reached the inhibition threshold (T = 2 V), the bladder was completely inhibited during the stimulation. B. In another experiment, 5–10 Hz stimulation at inhibition threshold (3 V) completely suppressed contractions and lowered baseline bladder pressure; whereas low (1–3 Hz) and high (20–40 Hz) frequency stimulation elicited a weak excitatory effect that was in some instances (20 Hz) followed by a period of inhibition. C. Summary of the frequency dependent inhibitory effect induced at stimulation intensity of 1–3 T. The measurement of area under curve during stimulation was normalized to the control measurement before stimulation (see inserted figure in C). Total 8 animals. Stimulation: 1–6 V in C; 0.1 ms pulse width in A–C. * indicates a statistically significant (P<0.05) difference from 100%. The thin lines under the pressure traces indicate the zero pressure. The thick black bars under the zero pressure lines indicate periods of stimulation.

Fig. 2

Changes in bladder activity recorded under isovolumetric conditions after different doses of naloxone (Nx). A. In one experiment a large increase in the amplitude of rhythmic contractions occurred at a dose of 0.01 mg/kg, but not at a higher dosage of 0.1 mg/kg. B. In another experiment, a transient increase in baseline bladder pressure occurred at dose of 0.1 mg/kg, but not at a higher dosage of 1 mg/kg. The thin lines under pressure traces indicate the zero pressure. A2 trace starts about 25 minutes after the end of A1 trace. B2 trace starts about 15 minutes after the end of B1 trace.

Fig. 3

Influence of naloxone (Nx) at different doses on isovolumetric bladder contractions recorded after the transient Nx response (see Fig.2) terminated. A. In one experiment, the baseline bladder pressure and contraction amplitude were not significantly changed. B. In another experiment, both baseline pressure and contraction amplitude were increased. The thin lines under pressure traces indicate the zero pressure.

Fig. 4

Influence of naloxone (Nx) on isovolumetric bladder contractions during the time period after the transient Nx response terminated (see Fig. 3). A. Normalized area under the bladder contraction curve. B. Normalized peak amplitude of bladder contraction. C. Normalized baseline bladder pressure. Data at different Nx doses were normalized to the measurements during the control periods as indicated in Fig. 3. Total 8 animals.

Fig. 5

Influence of naloxone (Nx) at different cumulative doses (0.01–1 mg/kg) on pudendal inhibition of isovolumetric bladder contractions. A. As the Nx dose increased, the minimal stimulation voltage required to completely inhibit bladder contractions also increased. B. The pudendal nerve evoked inhibitory effect was significantly weakened by Nx at threshold intensity (1 T). However, even after the highest dose of Nx (1 mg/kg) increasing the intensity of stimulation to 1.5–3 T still significantly inhibited bladder activity. Total 7 animals. Stimulation in B: 1–6V, 5–10 Hz. 0.1 ms pulse width in both A and B. * indicates a statistically significant (P < 0.05) difference from 100%. # indicates a statistically significant (P<0.05) difference between 1 T and 1.5–3 T groups. The thin lines under the pressure traces in A indicate the zero pressure. The black bars under the zero pressure lines indicate periods of stimulation.

Fig. 6

Frequency dependent inhibitory effect on isovolumetric bladder contractions after the maximal dose of naloxone (1 mg/kg). A. An example of pudendal nerve inhibitory effect at different frequencies. Black bars under the zero pressure baseline indicate stimulation durations. B. Summary of the frequency dependent inhibitory effect of pudendal nerve stimulation after naloxone treatment. Total 5 animals. Stimulation: intensity 4V for A; 3–6V for B; 0.1 ms pulse width for both A and B. * indicates a statistically significant (P < 0.05) difference from 100%.

Fig. 7

Effect of inhibitory pudendal nerve stimulation on bladder capacity before and after administration of 1 mg/kg naloxone (Nx). A. Repeated CMG recordings with 10 Hz stimulation. Black bars indicate stimulation durations. Th – micturition threshold pressure, A – micturition contraction amplitude, D – micturition contraction duration. B. Summary of the naloxone effect on bladder capacity with 5–10 Hz stimulation. Total 8 animals. T – threshold intensity for completely inhibiting bladder activity under isovolumetric conditions. Stimulation: 4 V (1 T) in A; but 1–6 V in B; 0.1 ms pulse width in both A and B. * indicates statistically significant (P < 0.05).

Fig. 8

Effect of inhibitory pudendal nerve stimulation at frequencies of 5–10 Hz on threshold pressure (A), amplitude (B), and duration (C) of the first micturition contraction during CMG before and after administration of 1 mg/kg naloxone (Nx). The definitions for each measured parameter in A–C were illustrated in Fig. 7A. Stimulation: 1–6 V, 0.1 ms pulse width. *indicates a statistically significant (P < 0.05) difference from 100%. Total 8 animals.