Cell experiment:

Individual CCD segments are dissected from rabbit and mouse kidney.The measurements of [Na+]i are used as a reflection of Na+ transport across the apical membrane. EnaC-specific activity is assessed by applying 10–6 M of the ENaC blocker Benzamil hydrochloride, a concentration that does not affect Na+:H+ exchange. Experiments are performed in the presence/absence of luminal Benzamil hydrochloride or the Na:H exchanger blocker HOE694 administers with/without the AT1 receptor blocker candesartan. Final DMSO concentrations are below 0.1% [2].

Animal experiment:

For the acute intracerebroventricular injection in anesthetized rats. A guide cannula is inserted into the right lateral cerebral ventricle, and Benzamil hydrochloride (0.1 nmol/kg, n=6; 1 nmol/kg, n=6; 10 nmol/kg,n=7) or vehicle (n=6) is injected into the right lateral ventricle through a cannula connected to a microsyringe 15 min before the start of intracerebroventricular infusion of hypertonic NaCl. Each injection consists of a volume of 10 μL deliver manually over a period of 30 s. To investigate the effects of Benzamil hydrochloride on the pressor response induced by intracerebroventricular injection of the pressor agent other than hypertonic NaCl, endothelin-1, which has a potent pressor action in the brain of rats, is intracerebroventricularly injected (1 nmol/10 μL) 15 min after the intracerebroventricular preinjection of Benzamil hydrochloride (10 nmol/kg, n=6) or the vehicle (n=6), and mean arterial pressure, heart rate, and abdominal sympathetic nerve firings are recorded for 20 min. Catheters are implanted into both the femoral artery and vein of 12-wk-old male rats anesthetized with urethan. Both catheters are filled with heparinized saline (50 U/mL). Fifteen minutes before the start of intracerebroventricular infusion of hypertonic NaCl, Benzamil hydrochloride (10 nmol/kg, n=6) or vehicle (10 μL, n=6) is injected into a tube connects to the femoral venous catheter by directly inserting the microsyringe, and 0.1 ml of isotonic saline solution is injected through the catheter to deliver the contents into the venous circulation[1].

Benzamil hydrochloride is a specific blocker of sodium channel (ENaC).
The specific ENaC blocker Benzamil hydrochloride adds to the luminal perfusate (10–6 M) significantly decreases baseline [Na+]i by 19.2 mM (n=5) and almost completely inhibits the [NaCl]L-dependent increasing in [Na+]i when coadministers with luminal AngII[2].
Intracerebroventricular injection of endothelin-1 (1 nmol) after intracerebroventricular preinjection of the vehicle of Benzamil hydrochloride markedly increases both mean arterial pressure and abdominal sympathetic nervous activity for 10 min. In SHRSP, intracerebroventricular infusion either of 1 or 10 nmol/kg/day Benzamil hydrochloride decreases systolic blood pressure (4th day: F=6.131, 1 nmol/kg/day, P<0.05, 10 nmol/kg/day, P<0.01; 5th day: F=6.842, 1 nmol/kg/day, P<0.05, 10 nmol/kg/day, P<0.01; 6th day: F=5.988, 1 nmol/kg/day, P<0.05, 10 nmol/kg/day, P<0.01; 7th day: F=7.935, 1 nmol/kg/day, P<0.01, 10 nmol/kg/day, P<0.01), urinary excretion of vasopressin (5th day: F=9.385, 1 nmol/kg/day, P<0.05, 10 nmol/kg/day, P<0.05; 6th day: F=8.783, 1 nmol/kg/day, P<0.01, 10 nmol/kg/day, P<0.01; 7th day: F=8.996, 1 nmol/kg/day, P<0.01, 10 nmol/kg/day, P< 0.01), and norepinephrine (3rd day: F=4.341, 1 nmol/kg/day, P<0.05, 10 nmol/kg/day, P<0.01; 4th day: F=9.865, 1 nmol/kg/day, P<0.01, 10 nmol/kg/day, P<0.01; 5th day: F=14.652, 1 nmol/kg/day, P<0.01, 10 nmol/kg/day, P<0.01; 6th day: F=13.376, 1 nmol/kg/day, P<0.01, 10 nmol/kg/day, P<0.01; 7th day: F=8.594, 1 nmol/kg/day, P<0.01, 10 nmol/kg/day, P<0.01) compare with vehicle[1].
Reference：
[1]. Nishimura M, et al. Benzamil blockade of brain Na+ channels averts Na(+)-induced hypertension in rats. Am J Physiol. 1998 Mar;274(3 Pt 2):R635-44.
[2]. Peti-Peterdi J, et al. Angiotensin II directly stimulates ENaC activity in the cortical collecting duct via AT(1) receptors. J Am Soc Nephrol. 2002 May;13(5):1131-5.