Carbon monoxide (CO), a well-known toxic gas, has a variety of physical functions related to vasomodulation, neuromodulation, and cytoprotection [1,2]. Regarding roles of CO in urinary function, there are a few reports showing the role of CO in relaxation of the urethral smooth muscle [3]. However, the detailed roles of CO in regulation of the micturition reflex are still unclear.
    CO is endogenously produced via degradation of heme to biliverdin by heme oxygenase-1 (HO-1) and HO-2 [1,2]. HO-1 is an inducible type, and is localized in the liver, spleen, brain, skin, and heart [1,2]. HO-2 is a constitutive type, and its localization is widely distributed, with particularly higher concentrations in the brain and testis [1,2].
    In this study, we investigated (1) effects of intravesically instilled CORM3, a CO donor, on the rat micturition reflex, (2) expression levels of HO-1 and HO-2 in the rat bladder, and (3) effects of CORM3 on contractility in the rat bladder smooth muscle.

(1) In urethane (0.8 g/kg, ip)-anesthetized male Wistar rats, a catheter was inserted into the bladder to instill reagents (4 ml/h) and to measure intravesical pressure. After detecting 4-7 stable micturition reflexes induced by saline instillation, CORM3 solution (10-8, 10-7 and 10-6 M, in turn) or vehicle (saline) was instilled.
(2) Bladder dome and trigone (BL-D and BL-T), liver and hypothalamus were prepared from male Wistar rats sacrificed with an overdose of sodium pentobarbital (80 mg/kg, ip). Expression levels of HO-1 and HO-2 in these tissues were investigated by quantitative real-time PCR. Liver and hypothalamus were used for positive controls.
(3) From bladder tissues prepared as described in (2), 1 x 5 mm strips of BL-D and BL-T were prepared. By using these strips, effects of CORM3 (1 x 10-8  to 3 x 10-5  M) were evaluated by organ bath experiments on pre-contracted bladder strips by carbachol (10-5  M).

(1) Intravesically instilled CORM3 (10-6 M) significantly prolonged intercontraction intervals (ICI) compared to the vehicle-treated group without affecting maximal voiding pressure (MVP) (Fig. 1).
(2) In the BL-D and BL-T, HO-2 mRNA was detected, while HO-1 mRNA levels were quite low (Fig. 2).
(3) In both bladder strips, CORM3 alone showed no contraction or relaxation (data not shown). In pre-contracted BL-D and BL-T strips by carbachol, CORM3 induced no relaxation compared with vehicle-pretreated strips (Table 1).

In this study, intravesically instilled CORM3 induced prolongation of ICI, suggesting that CORM3-derived CO in the bladder can suppress the rat micturition reflex. In the rat bladder, mainly a constitutive isozyme HO-2, but not an inducible isozyme HO-1, was detected, in line with a previous report using the pig bladder [3]. Therefore, endogenous CO produced by HO-2 in the bladder might suppressively regulate the rat micturition reflex. CO is reported to relax the vascular smooth muscle via a cGMP-mediated pathway [2], therefore, we predicted that the CORM3-induced suppression of the rat micturition reflex might be evoked by CO-mediated bladder smooth muscle relaxation. However, exogenous CORM3 showed no contractile or diastolic effects on the rat bladder strips, or no significant change on the rat MVP. It is reported that the pig bladder strips pre-contracted by carbachol (10-5 M) did not respond to exogenously applied CO (1.2 to 7.2 x 10-5 M) [3]. Thus, unlike the vascular smooth muscle, CO might show little effects on the bladder smooth muscle relaxation. CO is reported as a neuromodulator [1] and HO-2 immunoreactivity was detected in coarse nerve trunks within the smooth muscle of the pig bladder [3], suggesting a possibility that CO might regulate bladder contractility via modulation of peripheral nervous system in the bladder. Further studies are necessary to investigate mechanisms how CO in the bladder suppresses the rat micturition reflex focusing on CO-mediated neuromodulation.

Exogenously administered CO suppresses the rat micturition reflex independently of bladder smooth muscle relaxation. Thus, CO in the bladder might be a new therapeutic target for lower urinary tract dysfunctions such as overactive bladder.

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Continence 12S (2024) 101465
DOI: 10.1016/j.cont.2024.101465