Psychological stress plays an important role in the development of lower urinary tract symptoms. However, the pathophysiological mechanism of psychological stress-induced frequent urination is not well understood [1]. Psychological stress increases the levels of angiotensin II (Ang II), a stress-related neuropeptide in the brain [1]. Our previous report showed that involvement of brain γ-aminobutyric acid (GABA)A receptor for central Ang II-induced frequent urination in rats.  Moreover, central Ang II induces frequent urination by acting on the Ang II type 1 (AT1) receptor in the brain [1]. In this study, we further investigated the pathophysiological mechanism by which central Ang II induced frequent urination focusing on the GABAergic nervous system and AT1 receptor downstream signaling pathway.

Male Wistar rats (330-440 g) were anesthetized with urethane (1.0 g/kg, ip), and a catheter was then inserted into the bladder dome to perform continuous cystometry (12 ml/h saline infusion). Three hours after the surgery, each drug was centrally administered into the right ventricle.
Study 1: Vehicle 1 (sterile phosphate buffer saline/3 µL/rat), muscimol (GABAA receptor agonist, 100, 300 or 1,000 pmol/rat) or baclofen (a GABAB receptor agonist, 30, 100 or 300 pmol/rat) was intracerebroventricularly (icv) administered. Vehicle 1, muscimol (100, 300 pmol/rat) or baclofen (30 or 100 pmol/rat) was icv pre-treated 30 min before icv Ang II (30 pmol/3 µL/rat) administration. 
Study 2: Vehicle 2 [sterile 10% N,N-dimethylmethanamide (DMF)/3 µL/rat], telmisartan (an AT1 receptor antagonist, 3 or 10 nmol/rat), valsartan (an AT1 receptor antagonist, 10 nmol/rat), PD123319 (an Ang II type 2 receptor antagonist, 100 nmol/rat), U-73122 [a phospholipase C (PLC) inhibitor, 300 or 1000 pmol/rat] or chelerythrine chloride [a protein kinase C (PKC) inhibitor, 300 or 1,000 pmol/rat] was icv pre-treated 30 min before icv Ang II (30 pmol/3 µL/rat) administration.
Study 3: Vehicle 3 (sterile 10% DMF/5 µL/rat), apocynin [a nicotinamide adenine dinucleotide phosphate oxidase (NOX) inhibitor, 20 or 200 nmol/rat], or tempol (an anti-oxidant, 2 or 20 nmol/rat) was icv pre-treated 30 min before icv Ang II (30 pmol/rat) administration. 
The intercontraction interval (ICI) and maximum voiding pressure (MVP) were evaluated 20 min before and after the central Ang II administration (0 to 60 min) (n = 5-7).

A centrally administered highest dose of muscimol (1,000 pmol/rat) or baclofen (300 pmol/rat) significantly extended the ICI for 0-60 min, compared with Vehicle 1 treatment without altering the MVP (Figure 1A and 1B).  Centrally administered Ang II significantly shortened the ICI for 0-60 min after the injection compared to the value before central Ang II administration (-20-0 min) (data not shown). On the other hand, central pre-treatment with muscimol (300 pmol/rat) or baclofen (100 pmol/rat) alone which each dose of the drugs did not affect ICI, and significantly suppressed central Ang II-induced shortening of the ICI for 0-60 min after central Ang II administration, compared with Vehicle 1 pre-treatment (Figure 1C). Central pre-treatment with telmisartan, valsartan, U-73122 or chelerythrine chloride but not PD123319 suppressed central Ang II-induced shortening of the ICI in a dose-dependent manner compared with Vehicle 2 pre-treatment (Figure 2A and 2B). Moreover, central pre-treatment with apocynin or tempol significantly suppressed central Ang II-induced shortening of the ICI in a dose-dependent manner compared with Vehicle 3 pre-treatment (Figure 2C).

The GABAergic nervous system is known to modulate micturition [1]. Current data showed that a centrally administered highest dose of muscimol or baclofen extended the micturition interval. Thus, stimulating the GABAergic nervous system could inhibit micturition. Central pre-treatment with muscimol or baclofen suppressed central Ang II-induced shortening of the ICI. A previous study demonstrated that central Ang II-induced stimulation of AT1 receptors suppressed GABA release in the periaqueductal gray [2]. These data support a previous report [1] and suggested that central Ang II can induce frequent urination via inhibition of the GABAergic nervous system in rats. Moreover, Ang II is known to activate the canonical Gq protein/PLC/PKC signaling and NOX in various tissues through stimulation of the AT1 receptor [3]. Current data demonstrated that central pre-treatment with telmisartan, valsartan, U-73122, chelerythrine chloride, apocynin, or tempol in a dose-dependent manner inhibited central Ang II-induced shortening of the ICI. These data suggested that central AT1 receptor downstream signaling (PLC/PKC/NOX/super oxide anion) is involved in central Ang II-induced frequent urination.

Central Ang II induces frequent urination through the inhibition of the GABAergic nervous system and activation of AT1 receptor/PLC/PKC/NOX/superoxide anion pathways in rats. The central AT1 receptor might be a possible therapeutic target against psychological stress-induced frequent urination.

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