Three hours after the last injection of NE inhibitor or PBS, the palatal gingival tissues were homogenized in Tris–HCl buffer using BioMasher (Nippi, Tokyo, Japan). These samples were centrifuged at 200 g for 5 min, and NE activity in the supernatant was determined by a method using the NE-specific substrate N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (Merck Millipore, Billerica, MA, USA), as previously described39 (link).
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