Glycine transporter type 2 (GlyT2) inhibitor ameliorates bladder overactivity and nociceptive behavior in rats

Abstract

Background: Glycine is a major inhibitory neurotransmitter in the spinal cord, the concentration of which is regulated by two types of glycine transporters (GlyTs): GlyT1 and GlyT2. We hypothesized that the inhibition of GlyTs could ameliorate bladder overactivity and/or pain sensation in the lower urinary tract.

Objective: Investigate the effects of GlyT inhibitors on bladder overactivity and pain behavior in rats.

Design, setting, and participants: Cystometry was performed under urethane anesthesia in cyclophosphamide (CYP)-treated rats. In behavioral studies using conscious rats, nociceptive responses were induced by intravesical administration of resiniferatoxin (3μM). Selective GlyT1 or GlyT2 inhibitors were administered intrathecally to evaluate their effects.

Measurements: Cystometric parameters, nociceptive behaviors (licking and freezing), and messenger RNA (mRNA) levels of GlyTs and glycine receptor (GlyR) subunits in the dorsal spinal cord (L6-S1) were measured.

Results and limitations: During cystometry in CYP-treated rats, significant increases in intercontraction interval and micturition pressure threshold were elicited by ALX-1393, a selective GlyT2 inhibitor, but not by sarcosine, a GlyT1 inhibitor. These effects were completely reversed by strychnine, a GlyR antagonist. ALX-1393 also significantly suppressed nociceptive behaviors in a dose-dependent manner. In sham rats, GlyT2 mRNA was expressed at a much higher level (23-fold) in the dorsal spinal cord than GlyT1 mRNA. In CYP-treated rats, mRNA levels of GlyT2 and the GlyR α1 and β subunits were significantly reduced.

Conclusions: These results indicate that GlyT2 plays a major role in the clearance of extracellular glycine in the spinal cord and that GlyT2 inhibition leads to amelioration of CYP-induced bladder overactivity and pain behavior. GlyT2 may be a novel therapeutic target for the treatment of overactive bladder and/or bladder hypersensitive disorders such as bladder pain syndrome/interstitial cystitis.

Fig. 1

Photomicrographs of hematoxylin and eosin staining of the bladder wall in sham (saline) and cyclophosphamide (CYP)–treated rats: (a) sham rat bladder (×40); (b) sham rat bladder (×200); (c) CYP-treated rat bladder (×40); (d) CYP-treated rat bladder (×200). Figure 1b and 1d show the areas indicated by rectangles in Figure 1a and 1c, respectively, with higher magnification. Inflammatory cell infiltration in the suburothelial layer and tissue edema shown by an increase in the mucosal layer width are observed in the CYP-treated group (c and d). Scale bars: 100 µm.

Fig. 2

Typical traces showing the effect of intrathecal administration of (a) sarcosine (glycine transporter [GlyT] type 1 inhibitor) and (b) ALX-1393 (GlyT2 inhibitor) on continuous cystometrograms in sham rats. Arrows indicate the timing of drug administration. ALX-1393 showed some inhibitory effects on bladder activity (b), while sarcosine has no apparent effects (a). Vehicle 1: distilled water. Vehicle 2: 100% dimethyl sulfoxide. IVP = intravesical pressure; i.t. = intrathecal.

Fig. 3

Typical traces showing the effect of intrathecal administration of (a) sarcosine, (b) ALX-1393, and (c) the combined application of ALX-1393 and strychnine, a glycine receptor antagonist, on continuous cystometrograms in cyclophosphamide (CYP)–treated rats. Arrows indicate the timing of drug administration. ALX-1393 showed inhibitory effects on CYP-induced bladder overactivity (b), while sarcosine has no apparent effects (a). Strychnine antagonized the inhibitory effect of ALX-1393 (c). Vehicle 1: distilled water. Vehicle 2: 100% dimethyl sulfoxide. Vehicle 3: saline). IVP = intravesical pressure; i.t. = intrathecal.

Fig. 4

Effects of intrathecal administration of sarcosine and ALX-1393 on cystometric parameters in (a–d) sham and (e–h) cyclophosphamide (CYP)–treated rats. (a) Each bar represents the mean plus or minus standard error of the mean. ICI = intercontraction interval; PT = pressure threshold; MVP = maximum voiding pressure; BP = baseline pressure; V = vehicle; SCS = sarcosine (in micrograms, intrathecal); ALX = ALX-1393 (in micrograms, intrathecal); CYP = cyclophosphamide; N/A: not analyzed (ie, the analysis of the parameter was impossible because of the strong suppression of the micturition reflex); ANOVA = analysis of variance. * p < 0.05. ** p < 0.01 (compared to the vehicle group; one-way ANOVA followed by Bonferroni multiple comparison test); the number of animals (n) in each subgroup was shown in parentheses.

Fig. 5

Effects of intrathecal administration of ALX-1393 on (a) intercontraction interval and (b) pressure threshold in the presence or absence of strychnine in cyclophosphamide rats. Each bar represents the mean plus or minus the standard error of the mean. ICI = intercontraction interval; PT = pressure threshold; - = vehicle (dimethyl sulfoxide or saline); ANOVA = analysis of variance. * p < 0.05. ** p < 0.01 (compared among three groups; one-way ANOVA followed by Bonferroni multiple comparison test); the number of animals (n) in each subgroup was shown in parentheses.

Fig. 6

Changes in messenger RNA levels of (a) glycine transporters and (b) glycine receptor subunits in the dorsal L6–S1 spinal cord at the after cyclophosphamide treatment. Each bar represents the mean plus or minus the standard error of the mean from five different rats. S = sarcosine; CYP = cyclophosphamide; GlyT = glycine transporter; GlyR = glycine receptor; PCR = polymerase chain reaction; NCBI = National Center for Biotechnology Information. * p < 0.05. ** p < 0.01 (unpaired student t test); the real-time PCR was run by 40 cycles (denaturation at 95°C for 15 s; primer annealing at 55°C for 60 s; elongation at 72°C for 30 s); relative expression data were quantified using the 2-^ΔΔCT method, where CT is the cycle threshold. All target messenger RNA (mRNA) expression levels were normalized to that of the constitutive 18S ribosomal RNA. Primer sequences used for real-time PCR were as described in Table 2. All primers for PCR reaction were designed based on the NCBI database sequence of rat reference mRNA and checked for specificity with BLAST software from the NCBI Web site. PCR products were also validated by size determination after separation on 2% agarose gel.

Fig. 7

Effects of intrathecal administration of sarcosine on licking and freezing nociceptive behaviors in rats treated with intravesical resiniferatoxin (RTX; 3 µM). (a, b) The number of licking or freezing behaviors counted every minute. Intravesical application of RTX, which stimulates TRPV1 receptor-expressing C-fiber afferent nerves, increased the licking behavior, with a peak in the early phase (0–5 min) as described previously [14]. (c, d) The total number of licking or freezing behaviors for each 5-min period, showing that no significant effect of intrathecal administration of glycine transporter type 1 inhibitor was observed. V1 = vehicle for sarcosine; V2 = vehicle 10% ethanol, 10% tween 80, and 80% saline for RTX; RTX = resiniferatoxin (3 µM, intravesically); i.t. = intrathecal; SCS = sarcosine (250 µg, intrathecal). # p < 0.05. ## p < 0.01 (unpaired student t test).

Fig. 8

Effects of intrathecal administration of ALX-1393 on licking and freezing nociceptive behaviors in rats treated with intravesical resiniferatoxin (RTX; 3 µM). (a, b) The number of licking or freezing behaviors counted every minute. Intravesical application of RTX, which stimulates TRPV1 receptor-expressing C-fiber afferent nerves, increased the licking behavior, with a peak in the early phase (0–5 min) as described previously [14]. (c, d) The total number of licking or freezing behaviors for each 5-min period. The glycine transporter type 2 inhibitor significantly suppressed both nociceptive behaviors in a dose-dependent manner. Each point or the bar represents the mean plus or minus the standard error of the mean from five rats. V1 = vehicle for ALX-1393; V2 = vehicle for RTX; RTX = resiniferatoxin (3 µM, intravesically); ALX = ALX-1393 (in µg, intrathecal); V = vehicle. # p < 0.05. ## p < 0.01 (unpaired student t test). * p < 0.05. ** p < 0.01 (compared to the vehicle group).