Hypothesis / aims of study
Carbon monoxide (CO) is recognized as an endogenous gasotransmitter related to vasomodulation, neuromodulation, and cytoprotection [1]. CO is endogenously produced by heme oxygenase (HO)-induced heme degradation. In previous reports, intracerebroventricularly (icv) administered CORM-3 (CO donor) induced prolongation of intercontraction interval (ICI), and icv administered ZnPP (non-selective HO inhibitor) induced ICI shortening in rats [2]. In addition, the CORM-3-induced prolongation was attenuated by icv pretreated GABA receptor antagonists [2]. These findings suggest that brain CO can suppress the micturition reflex via the GABAergic nervous system. The system also mediates suppression of the reflex induced by activation of brain α7 nicotinic acetylcholine receptors (nAChRs) [3]. In this study, we investigated whether brain CO is involved in brain α7 nAChR-mediated suppression of the rat micturition reflex.
Study design, materials and methods
In urethane-anesthetized (0.8 g/kg, ip) male Wistar rats (270-420 g), a catheter was inserted into the bladder to perform cystometry. Two hours after the surgery, cystometry (saline instillation at 12 ml/h) was started to evaluate ICI. One hour after the start, CORM-3 (0.3 or 1 nmol/rat) or ZnPP (1 or 10 nmol/rat) was icv pretreated. Subsequently, PHA568487 (PHA, α7 nAChR agonist, 0.3 or 1 nmol/rat) was icv administered 30 min (ZnPP) or 60 min (CORM-3) after each pretreatment. When both CORM-3 and ZnPP were pretreated, CORM-3 (0.3 nmol/rat) was icv pretreated 30 min before ZnPP administration (10 nmol/rat, icv), and PHA (1 nmol/rat) was icv administered 30 min after ZnPP pretreatment. Evaluation of ICI was continued 1 h after the PHA administration.
Results
PHA at a lower dose (0.3 nmol/rat, icv) did not influence on ICI (Fig. A). In contrast, under icv pretreatment with CORM-3 (1 nmol/rat), PHA markedly prolonged ICI even at a lower dose (0.3 nmol/rat, icv) compared with that in the vehicle-pretreated group (Fig. A).
PHA at a higher dose (1 nmol/rat, icv) prolonged ICI (Fig. B). Icv pretreated ZnPP (10 nmol/rat) markedly attenuated the PHA-induced response (Fig. B).
Icv pretreated ZnPP (10 nmol/rat) significantly attenuated the PHA (1 nmol/rat, icv)-induced ICI prolongation, whereas the ZnPP-induced attenuation was significantly annulled under icv pretreatment with CORM-3 (0.3 nmol/rat) (Fig. C).
Interpretation of results
In this study, we used CORM-3 and PHA at an ineffective dose to induce ICI prolongation (CORM-3 at 1 nmol/rat; PHA at 0.3 nmol/rat) [2,3]. However, combination of both drugs induced ICI prolongation, indicating that CORM-3 potentiated the PHA-induced suppression of the micturition reflex. Therefore, brain CO might be involved in brain α7 nAChR-mediated suppression of the reflex.
Next, by using ZnPP at an ineffective dose to change ICI (10 nmol/rat) [2], we investigated roles of endogenous brain CO during PHA-induced ICI prolongation. In this experiment, icv pretreated ZnPP attenuated the PHA (1 nmol/rat, icv)-induced ICI prolongation. Owing to the lesser selectivity of ZnPP to inhibit HO, we confirmed whether the ZnPP-induced attenuation was mediated through ZnPP-induced inhibition of endogenous CO production. To validate this, we investigated effects of CORM-3-mediated central CO supplementation on the ZnPP-induced attenuation. In this experiment, we used ZnPP at an effective dose (10 nmol/rat) to attenuate the PHA-induced response and CORM-3 at an ineffective dose (0.3 nmol/rat) to potentiate the PHA-induced response. The ZnPP-induced attenuation of the PHA-induced ICI prolongation was negated by icv pretreated CORM-3, suggesting that the ZnPP-induced attenuation may be negated by the CORM-3-induced central supplementation of CO. Therefore, endogenous brain CO can be involved in brain α7 nAChR-mediated suppression of the reflex in rats.