Animal experiment:

The experiments are performed on male and female Sprague-Dawley rats that have been maintained in a heat controlled room maintained at 23° C with a 12h/12h light-dark schedule. A total of 116 adult rats are injected i.p. with Amiloride hydrochloride dihydrate (0.1, 0.5, and 2.0 mg/kg). After 15, 30, 45, or 120 min post-injection, the rats are anesthetized with 8% chloral hydrate, the chest is opened, and blood samples are obtained by cardiac puncture. The blood is centrifuged at 5000 RPM for 5 min, and the plasma (1.5 mL) separated and stored at -80° C[1].

Amiloride hydrochloride dihydrate is an inhibitor of both Epithelial sodium channel (ENaC) and urokinase-type plasminogen activator receptor (uTPA).

Amiloride hydrochloride dihydrate is a potent epithelial sodium channels (ENaCs) blocker. Amiloride is highly concentrated in the plasma 15 to 30 minutes following the injections. 2 mg/kg dose of Amiloride hydrochloride dihydrate has no effect on blood pressure, heart rate, mesenteric vascular resistance, or hindquarters vascular resistance as compare to the baseline measurements (n=7). Over a 2 hour period, Amiloride hydrochloride dihydrate elicits negligible responses in arterial pressure (-1±1 mmHg) and heart rate (-10±6 bpm/min) as compare to baseline levels. Results show an Amiloride hydrochloride dihydrate dose-related response pattern for the c-Fos activation in the area postrema (AP). Even at the lowest dose of Amiloride hydrochloride dihydrate (0.1 mg/kg), the number of c-Fos labeled neurons in the AP is statistically different from the control rats at a p<0.01 level[1].

References:
[1]. Miller RL, et al. Blockade of ENaCs by amiloride induces c-Fos activation of the area postrema. Brain Res. 2015 Mar 19;1601:40-51.
[2]. Xu LB, et al. Amiloride, a urokinase-type plasminogen activator receptor (uTPA) inhibitor, reduces proteinurea in podocytes. Genet Mol Res. 2015 Aug 14;14(3):9518-29.