Psychological stress plays an important role not only in the induction of frequent urination but also in exacerbation of bladder dysfunction such as overactive bladder (OAB) and bladder pain syndrome/interstitial cystitis (BPS/IC).  The brain integrates psychological stress-related information, thereby inducing physical and behavioural responses to psychological stress (stress responses) for adaptation to stressful conditions.  However, the brain pathophysiological mechanisms underlying psychological stress-induced effects on the micturition reflex are still unknown.
  In the brain, nitric oxide (NO) plays a role in neuromodulation.  In addition, several groups have reported the involvement of brain NO in responses to psychological stress [1], and another group previously reported that centrally administered SIN-1, an NO donor, induced activation of the sympatho-adrenomedullary (SA) system, a representative stress response, in rats [2].  In the lower urinary tract, peripheral NO is well known as a relaxation factor in the urethral smooth muscle, while roles of brain NO in regulation of the micturition reflex seem to be still controversial, both inhibitory and facilitatory roles are reported [3].  In this study, we investigated effects of centrally administered SIN-1 on the micturition reflex focusing on the dependence on the SA system and brain glutamatergic receptors in rats.

Urethane anesthetized (0.8 g/kg, ip) male Wistar rats (300-400 g) were used.
(1) Catheters were inserted into the bladder dome and the femoral artery to perform cystometry (12 ml/h saline infusion) and to collect blood samples, respectively.  Two hours after the surgery, continuous cystometry was started to evaluate intercontraction intervals (ICI) and maximal voiding pressure (MVP).  One hour after the start, SIN-1 (100 or 250 µg/rat) or vehicle (10 µl saline/rat) was intracerebroventricularly (icv) administered.  Plasma noradrenaline (NA) and adrenaline (Ad) levels were measured at just before and at 5 min after the icv administration.  We also confirmed effects of centrally pretreated carboxy-PTIO (PTIO, an NO scavenger, 750 µg in 5 µl saline/rat, icv) on the SIN-1 (250 µg/rat, icv)-induced responses.
(2) Three hours after the surgery of a bladder catheter insertion, single cystometry (12 ml/h saline infusion) was performed.  After 4-5 times of single cystometry, SIN-1 (250 µg/rat) or vehicle (10 µl saline/rat) was icv administered, then single cystometry was continued for 60 min.
(3) In some experiments, acute bilateral adrenalectomy (ADX) was performed before insertion of catheters described in (1).  After the ADX, hydrocortisone was administered (5 mg/kg, im) to maintain levels of glucocorticoid.  SIN-1 administration (250 µg/rat, icv), cystometry and collection of blood samples were performed as described in (1).
(4) Effects of central pretreatment with MK-801 [an antagonist of N-methyl-D-aspartate type glutamatergic receptors (NMDA receptors), 10 or 30 nmol in 5 µl saline/rat, icv] or DNQX [an antagonist of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate type glutamatergic receptors (AMPA receptors), 3 nmol in 5 µl saline/rat, icv] on the SIN-1 (250 µg/rat, icv)-induced responses were also investigated.

(1) Centrally administered SIN-1 dose-dependently reduced ICI and elevated plasma Ad without altering MVP or plasma NA compared to the vehicle-treated group (Fig. 1A).  These SIN-1-induced changes were significantly attenuated by central pretreatment with PTIO (data not shown).  
(2) Centrally administered SIN-1 significantly reduced single-voided volume (Vv) and bladder capacity (BC) without affecting post-voiding residual volume (Rv) or voiding efficiency (VE) compared to the vehicle-treated group (Table 1).
(3) The SIN-1-induced ICI reduction was not affected by ADX, which abolished the SIN-1-induced elevation of plasma Ad (Fig. 1B).
(4) Central pretreatment with MK-801 significantly attenuated the SIN-1-induced ICI reduction (Fig. 2), while DNQX showed no significant effect on the SIN-1-induced response (data not shown).

Our present data indicate that SIN-1 centrally induces facilitation of the micturition reflex as shown by centrally administered SIN-1-induced reduction in ICI, Vv and BC without altering Rv or VE.  The SIN-1-induced ICI reduction was supressed by PTIO, indicating that SIN-1 induced facilitation of the micturition reflex via NO production in the brain.  ADX, which abolished centrally administered SIN-1-induced elevation of plasma Ad, had no effect on the SIN-1-induced ICI reduction, therefore, brain NO-mediated facilitation of the micturition reflex seems to be independent of activation of the central SA outflow.  NO in the brain might induce facilitation of the micturition reflex through facilitation of sensory inputs to the micturition center because centrally administered SIN-1 showed no effect on urodynamic parameters of bladder efferent activity such as MVP, Rv or VE.  Since NO can stimulate glutamatergic NMDA and AMPA receptor-mediated signalling and centrally administered SIN-1 rapidly reduced ICI, we investigated roles of these ionotropic glutamatergic receptors in the SIN-1-induced response.  Centrally pretreated MK-801, but not DNQX, suppressed centrally administered ICI reduction, therefore, brain NO-mediated stimulation of brain NMDA receptor-mediated signalling might be involved in the SIN-1-induced facilitation of the micturition reflex.

Brain NO centrally induces facilitation of the micturition reflex through brain NMDA receptors, independently of the SA outflow modulation.  These findings would be useful for understanding the underlying mechanisms of psychological stress-related exacerbation of lower urinary tract symptoms in OAB and BPS/IC, for which brain NO could be a new therapeutic target.

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