Hypothesis / aims of study
The heat-sensitive nonselective cation channel TRPM3 (Transient Receptor Potential channel Melastatin-3) is a nociceptor that plays a crucial role in the development of inflammatory hyperalgesia in the skin.(1) As many other TRP channels, it is also expressed in dorsal root ganglia innervating the bladder, but so far its function in bladder physiology was not known.(2) We aimed to elucidate the role of TRPM3 in normal bladder function and cystitis-induced bladder dysfunction by performing cystometry in wild type and TRPM3 knockout mice.
Study design, materials and methods
TRPM3 knockout mice were generated using homologous recombination in a C56/BL6J background.(1) In female C56/BL6J mice and TRPM3 knockout mice of 10-12 weeks old, a PE-50 tube was implanted in the bladder dome using the technique described earlier. (3) Immediately after implantation, animals were anesthetized using subcutaneous injection of urethane (1.2g/kg) and cystometry was carried out in both groups to assess normal bladder function at baseline. Normal saline (NaCl 0.9%) was infused into the bladder through the PE-50 tube at 0.04ml/min while simultaneously, bladder pressure was recorded using a Biopac pressure transducer. Basal pressure, threshold pressure, peak pressure, intercontractile interval, voided volume and residual volume were recorded during a baseline period of at least 30 minutes.
In a second series of experiments, C56/BL6J mice and TRPM3 knockout mice were injected intraperitoneal with cyclophosphamide (150 mg/kg) prior to cystometry. Twenty-four hours after cyclophosphamide injection, cystometry was carried out following the same protocol as described above.
Results
Under baseline conditions, genetic knockout of TRPM3 did not affect bladder function. In C56/BL6J mice, mean basal, threshold and peak pressure were 1.49 cm H2O (standard deviation 1.72) ; 8.86 cm H2O (standard deviation 2.44) and 40.33 cm H2O (standard deviation 3.39) respectively. The mean intercontractile interval was 256 seconds (standard deviation 103) and mean voided volume and residual volume were 63 microliter (standard deviation 24.8) and 50 microliter (standard deviation 35.2) respectively. In TRPM3 knockout mice, mean basal pressure (4.00 cm H2O; standard deviation 1.84), threshold pressure (9.17 cm H2O; standard deviation 2.92), peak pressure (43.22 cm H2O; standard deviation 6.09), intercontractile interval (169 seconds; standard deviation 52.7), voided volume (50 microliter; standard deviation 14.5) and residual volume (46 microliter; standard deviation 9.19) were not significantly different from those in C56/BL6J mice.
Induction of cyclophosphamide-induced cystitis had a different effect on TRPM3 knockout mice than on wild type B/6 mice. Cystitis-induced bladder dysfunction in wild type mice was evidenced by an increased basal (5.14 cm H2O, standard deviation 2.63) and threshold pressure (15.71 cm H2O, standard deviation 1.28), an effect that was absent in TRPM3 knockout mice where basal and threshold pressure remained stable at 3.06 cm H2O (standard deviation 1.31) and 9.96 cm H2O (standard deviation 2.24) respectively. Two-way anova for interaction between genotype and cyclophosphamide effect was significant for both threshold pressure (p = 0.00623) and basal pressure (p = 0.00602) with a power of 0.99 and 0.99. Secondly, there was a trend towards reduced intercontractile intervals (164 seconds, standard deviation 53.7) and voided volume (51 microliter, standard deviation 17.1) in wild type mice that was less pronounced in TRPM3 knockout mice with mean intercontractile intervals of 205 seconds (standard deviation 62.8) and mean voided volumes of 69 microliter (standard deviation 18.1). Two-way anova showed a significant interaction between genotype and the effect on intercontractile interval (p = 0.04203) but not on voided volume (p = 0.11275).
Interpretation of results
This is to our knowledge the first report showing a role for TRPM3 in bladder function.
Our results suggest that TRPM3 is not involved in normal bladder function in physiological circumstances. This is evidenced by a lack of difference in bladder function in TRPM3 knockout mice versus wild type mice.
In cyclophosphamide-induced cystitis however, typical cystometric changes that are seen in wild type mice are diminished in TRPM3 knockout mice. This suggests an important function of TRPM3 in inflammation-induced bladder dysfunction, similar to the role of TRPM3 in inflammatory hyperalgesia in the skin. Further research is needed to better understand the exact role of TRPM3 in the bladder.