Naoki Yoshimura, Department of Urology, University of Pittsburgh School of Medicine, Suite 700 Kaufmann Medical Building, 3471 Fifth Avenue, Pittsburgh, PA 15213-3221, USA. e-mail: firstname.lastname@example.org
What's known on the subject? and What does the study add?
A novel family of G-protein-coupled receptors has been identified in rat dorsal root ganglia and named as sensory neuron-specific receptors (SNSRs) and these receptors are expressed exclusively in a subset of small-diameter primary afferent neurons involved in transmission of nociceptive information. However, it is not known whether SNSRs have a role in the control of the micturition reflex.
This study demonstrated that in urethane-anaesthetised rats activation of SNSRs can inhibit the micturition reflex via the pathways independent of capsaicin sensitive C-fibres.
• To investigate the effect of sensory neurone-specific receptors (SNSRs) activation on the micturition reflex in rats.
MATERIALS AND METHODS
• Continuous cystometrograms (CMGs, 0.04 mL/min) were performed in female Sprague-Dawley rats under urethane anaesthesia.
• After stable micturition cycles were established, a selective rat SNSR1 agonist, bovine adrenal medulla 8–22 (BAM8–22), was administered intravenously (i.v.) or intrathecally (i.t.) in normal rats or rats pretreated with capsaicin 4 days before the experiments.
• Micturition variables were recorded and compared before and after drug administration.
• Administration (i.v.) of BAM8–22 (3–100 µg/kg) significantly increased intercontraction intervals in a dose-dependent fashion, but did not affect residual urine or baseline pressure at any doses tested.
• Administration (i.t.) of BAM8–22 (0.01–0.3 µg) also increased intercontraction intervals in a dose-dependent fashion, but did not affect residual urine or baseline pressure at any doses tested.
• These inhibitory effects of i.v. (30 µg/kg) or i.t. (0.3 µg) administration of BAM8–22 still occurred after capsaicin pretreatment.
• These results indicate that in urethane-anaesthetized rats activation of SNSRs can inhibit the micturition reflex via pathways independent of capsaicin-sensitive C-fibres.
• Thus SNSRs could be a potential target for the treatment of bladder dysfunction, e.g. overactive bladder.
Previous studies have suggested that hyperexcitability of C-fibre bladder afferents, which are silent under normal conditions, is involved in the emergence of overactive bladder and bladder pain in various pathological conditions, e.g. spinal cord injury, BOO or interstitial cystitis . Thus, it has been postulated that targeting afferent hyperexcitability could be effective for treating detrusor overactivity and pain symptoms .
A family of G-protein-coupled receptors has been identified in rat dorsal root ganglia (DRG) and named as sensory neurone-specific receptors (SNSRs) . SNSRs are expressed exclusively in a subset of small-diameter sensory neurones in DRG and trigeminal ganglia . Based on several analyses, this family of receptors is comprised of four to six members in human (MrgX1–4 or SNSR1–6) and 32 receptors in mouse classified into three major subfamilies Mrg A, B, and C [3–6]. Initially, only one SNSR gene was identified in the rat . Recently, it has been shownthat more than one rat SNSR/Mrg subtype exists . These receptors have been subclassified in a similar scheme as described for human and mouse, rMrg A, B, C, and D. For the sake of simplicity; we refer to rSNSR/rMrgC as rSNSR1 in the present study, which corresponds to the first gene described in small DRG neurones . It appears that rSNSR1 is pronociceptive as intrathecal (i.t.) and intradermal administration of its agonists, bovine adrenal medulla 8–22 (BAM8–22) and (Tyr6)-γ2-MSH-6–12, enhance acute nociception . In contrast, it was reported that activation of rSNSR1 produces analgesia in a persistent pain model . However, to our knowledge, it is not known whether SNSRs are involved in the regulation of neural mechanisms controlling the micturition reflex. Therefore, we examined the effects of activation of rSNSR1 on the micturition reflex in urethane-anaesthetized rats.
MATERIALS AND METHODS
Adult female Sprague-Dawley rats weighing 248–267 g were used. All experiments were performed in accordance with institutional guidelines and approved by the University of Pittsburgh Institutional Animal Care and Use Committee.
BAM8–22 (Tocris Bioscience, Ellisville, MO, USA) was used. For i.v. and i.t. administration BAM8–22 was dissolved in saline (0.9% NaCl).
For i.v. administration of BAM8–22, rats were anaesthetized with isoflurane followed by urethane (1.2 g/kg s.c.; Sigma Chemical Co., St. Louis, MO, USA). Thereafter the abdomen was opened through a midline incision and a polyethylene catheter (PE-60; Clay-Adams, Parsippany, NJ, USA) connected to a pressure transducer and an amplifier was implanted into the bladder through the bladder dome. This catheter was used to record intravesical pressure during cystometry. The PowerLab (ADInstruments Pty, Ltd., Castle Hill, New South Wales, Australia) was used for data acquisition and manipulation. The catheter was also used to fill the bladder by continuous infusion of saline. The i.v. injections were made through a cannula (PE-10) inserted into the right jugular vein. After intravesical catheter insertion saline was continuously infused for ≈2 h at 0.04 mL/min to record cystometrograms during a control period. BAM8–22 (3, 10, 30 and 100 µg/kg, n= 8 per dose) was then administered i.v. and changes in bladder activity were monitored. In another group of rats BAM8–22 (30 µg/kg, n= 6) was administered i.v. after pretreatment with s.c. injection of capsaicin (125 mg/kg; Sigma Chemical Co.) 4 days before the experiments to determine whether the effect of BAM8–22 was mediated by capsaicin-sensitive C-fibre afferent pathways. The i.v. BAM8–22 was administered in a volume of 0.1 mL/100 g body weight. The intercontraction interval (ICI), maximum voiding pressure (MVP), pressure threshold (PT) and baseline pressure (BP) were measured before and after drug administration. The post-void residual urine volume (PVR) was also measured in a separate group of eight rats before BAM8–22 administration and at the end of two micturition cycles after drug administration. After constant voided volumes were collected bladder infusion was stopped and PVR was measured by withdrawing intravesical fluid through the catheter by gravity.
For i.t. administration of BAM8–22, a PE-10 i.t. catheter was implanted using isoflurane anaesthesia via an incision in the dura at the Th11 vertebra 3 days before the experiments. The catheter was directed caudally into the spinal subarachnoid space and positioned at the level of the L6–S1 spinal cord. The volume of fluid in the catheter was kept constant at 6 µL. Single doses of drugs were then administered in a volume of 2 µL, followed by a 7 µL flush with saline. At the end of experiments, a laminectomy was performed to verify the location of the catheter tip and the distribution of an injected dye (2 µL methylene blue flushed with 7 µL saline). The intravesical catheter (PE-60) was placed as described above, and cystometrograms were recorded for ≈2 h under urethane anaesthesia (1.2 g/kg,s.c.). Then BAM8–22 was administered i.t. (0.01, 0.03, 0.1 and 0.3 µg, n= 6 per dose). In another group of rats BAM8–22 (0.3 µg, n= 6) was administered i.t. after capsaicin pretreatment 4 days before the experiments. The ICI, MVP, PT and BP were measured before and after drug administration. The PVR before and after i.t. administration of BAM8–22 was also measured in a separate group of six rats as described above.
All data values are expressed as the mean (sem). In experiments with i.v. and i.t. administration of BAM8–22 the ICI, MVP, PT, BP values during 30 min before and after drug administration were averaged in each rat and then the averages in a group of rats were combined. One-way anova followed by Dunnett's multiple comparison test was used for the statistical analysis between the vehicle and drug-treated groups. The Student's paired t-test was used to compare cystometric variables before and after treatment, with P < 0.05 considered to indicate statistical significance.
The i.v. administered BAM8–22 inhibited the micturition reflex (Fig. 1a). BAM 8–22 at 3, 10, 30 and 100 µg/kg increased the ICI at doses of ≥10 µg/kg in a dose-dependent fashion to 100.2 (2.0)%, 118.1 (7.8)%, 141.1 (7.7)% and 170.4 (12.4)% of the control value, respectively (at 10, 30 and 100 µg/kg, P < 0.01; Fig. 2). This inhibitory effect was seen immediately after administration and returned to the pre-control level within 50 min (Fig. 1a). BAM8–22 also increased PT and MVP in a dose-dependent fashion, but did not affect BP or PVR at any doses tested (Table 1). Injection of vehicle (saline) had no effect on the ICI, PT, MVP, BP or PVR (Table 1). In rats with capsaicin pretreatment, there was still an inhibitory effect of BAM 8–22 at 30 µg/kg (n= 6; Fig. 3a).The i.v. administration of BAM8–22 in capsaicin pretreated rats increased the ICI with a similar efficacy asin the normal rats, and increased the PT and MVP significantly (Fig. 3a and Table 2). There was no significant change in BP or PVR (Table 2).
Table 1. Changes in cystometric variables after i.v. BAM 8–22 administration in normal rats
BAM 8–22, µg/kg
P < 0.01 (paired t-test);
P < 0.01 vs vehicle injection (Dunnett's multiple comparison test).
The i.t. administration of BAM 8–22 also inhibited the micturition reflex (Fig. 1b). BAM 8–22 at 0.01, 0.03, 0.1 and 0.3 µg increased the ICI at doses of ≥0.03 µg in a dose-dependent fashion to 100.6 (1.6)%, 124.0 (5.4)%, 144.3 (5.8)% and 157.4 (8.6)% of the control value, respectively (at 0.03, 0.1 and 0.3 µg, P < 0.01; Fig. 4). The inhibitory effect of BAM 8–22 decreased with time and returned to the pre-control level within 50 min (Fig. 1b). BAM 8–22 also dose-dependently increased PT and MVP (Table 3). However, there were no significant changes in BP or PVR (Table 3). The i.t. injection of vehicle (saline) had no effect on the ICI, PT, MVP, BP or PVR (Table 3). The inhibitory effect of i.t. administered BAM 8–22 (0.3 µg, n= 6) still occurred after capsaicin pretreatment (Fig. 3b). The i.t. administration of BAM8–22 in capsaicin pretreated rats increased the ICI with a similar efficacy as in normal rats, and increased the PT and MVP significantly (Fig. 3b and Table 4). There was no significant change in BP or PVR (Table 4).
Table 3. Changes in cystometric variables after i.v.BAM 8–22 administration in capsaicin-pretreated rats
BAM 8–22, µg/kg
P < 0.01,
P < 0.05 (paired t-test);
P < 0.01,
P < 0.05 vs vehicle injection (Dunnett's multiple comparison test).
The goal of the present study was to assess the effects of activation of rSNSR1 on the micturition reflex in urethane-anaesthetized rats. Our findings indicate that activation of rSNSR1 by BAM8–22 has an inhibitory effect on the micturition reflex, with i.v. or i.t. administration of BAM8–22 dose-dependently increasing the ICI and PT in normal rats.
BAM8–22 is a synthesized peptide with 15 amino acids. It differs from BAM22, an opioid peptide with 22 amino acids and one of the natural cleavage products of proenkephalin A , in that BAM8–22 does not contain the N-terminal YGGFM motif of BAM22 . It has also been shown that in vitro BAM8–22 does not interact directly with the opioid receptor . Furthermore, it has been reported that the effect of BAM8–22 on nociception is not mediated by opioid receptors, as this peptide displayed identical efficacy in inhibiting the nocifensive behaviours in the absence or presence of naloxone, a non-selective opioid receptor antagonist . In addition, to date endogenous BAM8–22 has not been found to exist although BAM22, an opioid peptide, is widely distributed in the CNS [11,12]. Therefore, it seems reasonable to assume that the inhibitory effects of BAM8–22 on normal bladder activity were mediated by activation of rSNSR1.
An in vitro electrophysiological study showed that activation of SNSR4 produces inhibition, as application of BAM8–22 induced marked inhibition of synaptic responses (presynaptic inhibition) in hippocampal neurones expressing human SNSR4 . BAM8–22 also induced inhibition of high voltage-activated Ca2+ current in rat DRG and superior cervical ganglion neurones expressing human SNSR4 , which may lead to inhibition of excitatory neurotransmitter release at synapses formed between small-diameter DRG neurones and the spinal dorsal horn neurones . These events may underline the results obtained in the present study. If this is true, SNSRs could be categorized as the type of receptors that are expressed in the small-sized neurones in the DRG and function to negatively modulate excitability of the central terminals of primary afferents, similar to opioid , acetylcholine  and GABA receptors .
The present study also showed that the activation of rSNSR1 can inhibit the micturition reflex via pathways independent of capsaicin-sensitive C-fibre afferents. In the present study, the ICI and PT in capsaicin-pretreated rats were significantly increased compared with normal rats, as reported in previous studies using anaesthetized rats with afferent desensitization induced by pretreatment with capsaicin, a C-fibre neurotoxin . The inhibitory effects of i.v. or i.t. administration of BAM8–22 still occurred after capsaicin pretreatment. In a study using in situ hybridization analysis, rat SNSR mRNA was detected in a subset of small neurones within trigeminal ganglia and the DRG, but not in the sympathetic superior cervical ganglion or in the nodose ganglion, indicating SNSR is uniquely associated with sensory afferents in the rat . Moreover, in reverse transcriptase-PCR analyses of 25 different human tissues using oligonucleotides designed to conserved regions of SNSR 1–6, SNSR mRNA expression was detected exclusively in human DRG . Lembo et al.  used double-labelling studies combining in situ hybridization with immunohistochemical detection of neuronal markers such as substance P, calcitonin gene-related peptide, vanilloid receptor (TRPV1), isolectin B4 (IB4) on rat DRG tissue sections to identify the neuronal phenotype of SNSR-expressing cells. Most SNSR-positive neurones were found in the non-peptidergic, IB4-positive population. Also, a half of them were not co-localized with the TRPV1 capsaicin receptor. Therefore, it is possible that the effects of BAM8–22 are mediated by capsaicin-resistant C-fibres. The site of action may be afferent fibres in the bladder and/or the spinal cord although it is not known whether i.v. applied BAM8–22 can pass through the blood-brain barrier.
In the present study, i.t. or i.v. administration of BAM8–22 increased MVP in urethane-anesthaetized rats. There may be three possible mechanisms involved in the elevation of MVP. Firstly, BAM8–22 may increase urethral pressure during voiding by affecting the urethral reflex. Secondly, BAM8–22 may increase MVP because voiding has to be done for a larger volume after the increase in the ICI. Lastly, BAM8–22 may increase smooth muscle activity via efferent pathway stimulation. However, the first or second mechanism may be more reasonable as SNSRs are not expressed in smooth muscle or autonomic neurones, although further studies using isovolumetric cystometry or urethral perfusion pressure measurements are needed to clarify the mechanism inducing the BAM8–22-mediated increase in MVP.
Many receptors that can modulate micturition are also distributed in the neuronal pathways that regulate other functions, such as respiration and brain activity, etc. Targeting this type of receptor concurrently results in modulating the micturition reflex and producing unwanted effects. For example, opioids have been used clinically as effective analgesics for many pain conditions, but their use is limited by their considerable CNS-mediated side-effects. To date, it has been found that SNSRs are exclusively expressed in a subset of sensory neurones in the DRG and the trigeminal ganglion [3,8]. Thus, it is assumed that the sensory neurone-specific receptor could be an effective target for the pharmacological treatment of bladder dysfunction with less side-effects.
In conclusion, the results of the present study have shown that activation of SNSRs can inhibit the micturition reflex via suppression of afferent pathways independent of capsaicin-sensitive C-fibres. These findings raise the possibility that modulating SNSRs could be effective for treating bladder overactivity in various pathological conditions.
This study was supported by grants from NIH (DK057267, DK090919 and DK068557).