Hypothesis / aims of study
L-3,4-Dihydroxyphenylalanine (L-DOPA) has been recognized as a precursor of dopamine (DA). In contrast to this generally accepted idea, neurotransmitter roles of L-DOPA by itself have been also proposed [1]. L-DOPA receptor GPR143 is distributed in the central and peripheral nervous system [1], and previously, L-DOPA was reported to sensitize vasomotor response to sympathetic tone via activation of GPR143 [2]. However, roles of L-DOPA/GPR143 signaling in modulating autonomic nerves in the lower urinary tract tissues are unclear. In this study, to investigate roles of L-DOPA/GPR143 signaling in regulation of the rat micturition, we compared voiding behavior between wild-type (WT) and Gpr143 gene-deficient (Gpr143-/y) rats. We also investigated effects of L-DOPA on muscarinic receptor-mediated bladder contraction in both rats to clarify roles of L-DOPA/GPR143 signaling in regulation of parasympathetic tone in the bladder.
Study design, materials and methods
(1) In 9-week-old male Wistar (WT) and Gpr143-/y rats, voiding behavior studies were performed. All rats received food and water ad libitum from the time they were initially placed in metabolic cages. These rats were kept for 24 h for adaptation, and micturition frequency and total urine output were recorded for the next 24 h.
(2) Bladder dome and trigone (BL-D and BL-T) were prepared from nine-week-old male WT and Gpr143-/y rats sacrificed with an overdose of sodium pentobarbital (80 mg/kg, ip). From these tissues, 1 x 5 mm strips of BL-D and BL-T were prepared, and by using these strips, organ bath experiments were performed. Force induced by KCl (100 mM) was recorded as the control value for each experiment. Effects of pre-treatment with L-DOPA (10-8, 10-7 and 10-6 M) on carbachol (CCh, 10-8 to 3×10-4 M)-induced bladder strips contraction were investigated. Effects of pre-treatment with DA (10-6 M) on the CCh-induced contraction were also investigated by using strips prepared from the WT rat bladder.
Results
(1) In voiding behavior studies, between WT and Gpr143-/y rats, there was no significant difference in the urine output, the micturition frequency or the single-voided volume (Table 1).
(2) In both bladder strips from WT rats, L-DOPA alone showed no contraction or relaxation (data not shown). On the other hand, L-DOPA pre-treatment dose-dependently suppressed the CCh-induced contraction in strips of BL-D (Fig. 1A) and BL-T (data not shown) from WT rats. Values of maximum contraction and EC50 in BL-D strips from WT rats were shown in Table 2. EC50 values of the L-DOPA (10-7 M)-pre-treated group and of the L-DOPA (10-6 M)-pre-treated group were significantly higher than those of the control group (Table 2). In BL-T strips from WT rats, values (means ± SEM) of maximum contraction (%) and EC50 (M) were 207.7 ± 13.9 and 6.3 ± 0.6 (× 10-7) in the control group (n=16), 167.3 ± 6.3 and 9.0 ± 1.1 (× 10-7) in the L-DOPA (10-8 M)-pre-treated group (n=10), 169.2 ± 14.5 and 13.5 ± 1.9 (× 10-7) in the L-DOPA (10-7 M)-pre-treated group (n=12), and 171.3 ± 13.5 and 17.9 ± 3.6 (× 10-7) in the L-DOPA (10-6 M)-pre-treated group (n=14). EC50 values of the L-DOPA (10-6 M)-pre-treated group were significantly higher than those of the control group (P<0.05, statistical analysis was performed by ANOVA with Bonferroni post-hoc test).
In both bladder strips from Gpr143-/y rats, L-DOPA alone showed no contraction or relaxation (data not shown). On the other hand, L-DOPA pre-treatment (10-6 M) significantly suppressed the CCh-induced contraction in strips of BL-D (Fig. 1B) and BL-T (data not shown) from Gpr143-/y rats. Values of maximum contraction and EC50 in BL-D strips from Gpr143-/y rats were shown in Table 3. EC50 values of the L-DOPA (10-6 M)-pre-treated group were significantly higher than those of the control group (Table 3). In BL-T strips from Gpr143-/y rats, values of maximum contraction (%) and EC50 (M) were 243.6 ± 15.7 and 8.0 ± 0.6 (× 10-7) in the control group (n=12), and 235.3 ± 17.1 and 17.7 ± 2.0 (× 10-7) in the L-DOPA (10-6 M)-pre-treated group (n=12). EC50 values of the L-DOPA (10-6 M)-pre-treated group were significantly higher than those of the control group (P<0.05, statistical analysis was performed by ANOVA with Bonferroni post-hoc test).
In both bladder strips from WT rats, DA alone showed no contraction or relaxation (data not shown). Pre-treatment with DA showed no significant effect on the CCh-induced contraction in strips of BL-D (Fig. 2) and BL-T (data not shown) from WT rats. Values of maximum contraction and EC50 in BL-D strips from WT rats were shown in Table 4. In BL-T strips from WT rats, values of maximum contraction (%) and EC50 (M) were 139.8 ± 12.8 and 15.2 ± 6.8 (× 10-7) in the control group (n=10), and 137.6 ± 14.6 and 12.7 ± 1.4 (× 10-7) in the DA (10-6 M)-pre-treated group (n=10). There was no significant difference in both values between control and DA-pre-treated group in the BL-D and the BL-T strips.
Interpretation of results
There was no significant difference in voiding behavior between WT and Gpr143-/y rats. In organ bath experiments, L-DOPA alone showed no contractile or diastolic effects on the WT and Gpr143-/y rat bladder strips. These data suggest that roles of endogenous L-DOPA/GPR143 signaling in regulation of the micturition and the basal bladder contractility might be minor at least in normal conditions. On the other hand, in CCh-induced pre-contracted WT rat bladder strips, L-DOPA pre-treatment suppressed the bladder contractility. Interestingly, this L-DOPA-induced suppression was also observed in bladder strips from Gpr143-/y rats. These data indicate that L-DOPA can induce suppression of the muscarinic receptor-mediated bladder contraction independently of GPR143. Because the EC50 values, but not maximum contraction, for CCh were changed by L-DOPA, the L-DOPA-induced suppression might be induced via receptors other than GPR143. Thus, we investigated effects of DA, a metabolite of L-DOPA, on the bladder contractility in WT rats to clarify the involvement of DA receptors in the L-DOPA-induced suppression. DA alone showed no contractile or diastolic effects on the bladder strips, and unlike L-DOPA, DA pre-treatment showed no effect on the contractility in CCh-induced pre-contracted bladder strips. These results suggest that L-DOPA can induce suppression of the muscarinic receptor-mediated bladder contraction independently of DA receptors. Further studies are necessary to clarify which receptor can mediate the L-DOPA-induced suppression.