Psychological stress plays an important role in the induction of frequent urination and exacerbation of bladder dysfunction including overactive bladder (OAB) and bladder pain syndrome/interstitial cystitis (BPS/IC).  Psychological stress-related information is conveyed to the brain, and then the brain recruits neuronal and neuroendocrine systems for adaptation to stressful conditions.  However, the brain pathophysiological mechanisms underlying psychological stress-induced effects on bladder function are still unclear.
  Previously, we reported that the sympatho-adrenomedullary (SA) system, a representative response to stressful conditions, is activated by centrally administered SIN-1 [1], a nitric oxide (NO) donor.  Peripheral NO has a local inhibitory effect on urination [2], while, in the
central nervous system, NO seems to have both inhibitory and facilitatory effects on micturition [3].  In this study, therefore, we investigated effects of centrally administered SIN-1 on micturition concerning with their dependence on the SA system in rats.

Urethane anesthetized (0.8 g/kg, ip) male Wistar rats (300-400 g) were used. 
(1) Catheters were inserted into the bladder from the dome and the femoral artery to perform continuous cystometry (CMG, 12 ml/h saline infusion) and to collect blood samples, respectively.  Three hours after the surgery, SIN-1 (100 or 250 µg/rat) or vehicle (10 µl saline/rat) was intracerebroventricularly (icv) administered.  Saline infusion into the bladder and evaluation of intercontraction intervals (ICI) and maximal voiding pressure (MVP) were started 60 min before the icv administration.  Plasma noradrenaline (NA) and adrenaline (Ad) levels were measured at just before and at 5 min after the icv administration.  In some experiments, acute bilateral adrenalectomy (ADX) was performed before catheters insertion.
(2) Catheters were inserted into the bladder from the dome and the femoral vein to perform continuous cystometry (CMG, 12 ml/h saline infusion) and to administer drugs intravenously (iv), respectively.  Three hours after the surgery, SIN-1 (250 µg/rat) or vehicle (200 µl saline/rat) was iv administered.  Continuous CMG was performed as described in (1).
(3) Three hours after the surgery of a bladder catheter insertion, single CMG (12 ml/h saline infusion) was performed.  After 4-5 times of single CMG, SIN-1 (250 µg/rat) or vehicle (10 µl saline/rat) was icv administered, then single CMG was continued for 60 min.
(4) Effects of pretreatment with carboxy-PTIO (PTIO, an NO scavenger, 750 µg in 5 µl saline/rat, icv) on the SIN-1 (250 µg/rat, icv)-induced responses were also evaluated.

(1) Centrally administered SIN-1 (100 or 250 µg/rat, icv) dose-dependently reduced ICI and elevated plasma Ad without altering MVP or plasma NA compared to the vehicle-treated group (Fig. 1A).  The SIN-1 (250 µg/rat, icv)-induced ICI reduction was not affected by ADX, which abolished the SIN-1-induced elevation of plasma Ad (Fig. 1B)
(2) Systemic administration of SIN-1 (250 µg/rat, iv) showed no significant effect on ICI or MVP compared to the vehicle-treated group (data not shown).
(3) Centrally administered SIN-1 (250 µg/rat, icv) significantly reduced single voided volume (Vv) and bladder capacity (BC) without affecting post-voiding residual urine volume (Rv) or voiding efficiency (VE) compared to the vehicle-treated group (Table 1).
(4) Pretreatment with PTIO (750 µg/rat, icv) significantly supressed the SIN-1 (250 µg/rat, icv)-induced reduction in ICI (Fig. 2) and elevation of plasma Ad (data not shown).  There were no significant effects on the treatment with PTIO alone (750 µg/rat, icv) without SIN-1 on ICI or MVP (data not shown).

(1) These results suggest that SIN-1 centrally stimulates bladder activity as shown by centrally, but not systemically, administered SIN-1-induced reduction in ICI.  The SIN-1-induced stimulation seems to be independent of the SIN-1-induced activation of central SA outflow because ADX, which abolished the SIN-1-induced elevation of plasma Ad, had no effect on the SIN-1-induced reduction in ICI.
(2) Because centrally administered SIN-1 reduced ICI, Vv and BC without altering Rv or VE, SIN-1 centrally induced frequent urination.  The SIN-1-induced ICI reduction was supressed by PTIO, indicating that SIN-1 induced frequent urination via NO production in the brain.  In line with our results, it is reported that centrally administered L-NAME, a non-selective inhibitor of NO synthases, prolonged ICI in urethane-anesthetized female rats [3].  Interestingly, in awake female rats, L-NAME centrally reduced ICI [3], indicating that roles of brain NO in regulation of micturition might be different in anesthetized (facilitatory role) and awake (inhibitory role) conditions.  On the other hand, in our anesthetized male rats, centrally administered PTIO by itself had no effect on ICI or MVP, indicating that endogenous NO in the brain do not seem to affect bladder function in the normal conditions.  Therefore, further studies are necessary to clarify brain NO-mediated regulation mechanisms for micturition in detail.

Brain NO centrally induces frequent urination, which is independent of the SA outflow modulation, at least in our anesthetized conditions.  Thus, the central nitrergic pathway that can directly regulate micturition might be a new target for alleviation of psychological stress-induced exacerbation of urinary bladder dysfunction such as OAB and BPS/IC.

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