Hexamethonium chloride

Sprague-Dawley rats (230∼270 g) and C57BL/6 mice (25∼30 g) were purchased from Sino-British SIPPR/BK Laboratory Animals (Shanghai, China). α7^-/- mice were generated and genotyped by PCR analysis as described previously (Liu et al., 2009 (link)). Animals were housed at 22°C under a 12/12 light schedule (on: 08:00), with free access to tap water and standard rat chow. All experimental procedures were in accordance with institutional animal care guidelines and approved by ethics committee of Second Military Medical University. Ani (Ani hydrochloride: C₁₇H₂₄NO₄) was purchased from Fu-Ma Chemical and Engineering Company (Hangzhou, China). Mecamylamine hydrochloride, methyllycaconitine (MLA) citrate, hexamethonium chloride, ACh chloride, nicotine, PNU282987 (PNU), static, ouabain octahydrate, potassium chloride, and antibody against α7nAChR were purchased from Sigma–Aldrich (St. Louis, MO, USA). HNMPA-(AM)3 and antibody against p-Na/K-ATPase were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). antibody against insulin receptor, Alexa-488-labeled and Cy3-labeled second antibodies, and DAPI were purchased from Abcam (Cambridge, MA, USA). LY 294002 and rapamycin were purchased from Merck Millipore (Darmstadt, Germany).

After 15 min recovery, slices were gently removed from the storage chamber using a cutoff plastic Pasteur pipette, and transferred into an Eppendorf tube. The milieu was removed and replaced by 200 µl pre-warmed and pre-carbogened Ringer’s saline. After 5 min, the supernatant was removed and stored at −20°C. This step referred to basal (B) secretion. Slices were next challenged for 5 min with a test (T) solution containing either 10 µM acetylcholine chloride (ACh, Sigma Aldrich, Saint-Quentin Fallavier, France), 10–100 µM nicotine (tartrate salt, Sigma Aldrich), 200 µM hexamethonium chloride (Sigma Aldrich), or saline as control. The slice supernatant (200 μl) was then collected and stored at −20°C before use. For each slice, results were expressed as the stimulation T/B ratio. Basal and stimulated amounts of E and NE were expressed in picomoles and in picomole per cubic meters of medullary tissue after normalization to the medulla volume.

During the development of these experimental protocols, the compounds employed in the present study (obtained from the sources indicated) were sodium pentobarbital (PISA Agropecuaria, Mexico City, Mexico); gallamine triethiodide, hexamethonium chloride, glibenclamide, rat α-CGRP, methoxamine hydrochloride and adenosine-5′-[β-thio]diphosphate trilithium salt (ADPβS) (Sigma Chemical Co., St. Louis, MO, USA); (1R*,2S*)-4-[2-Iodo-6-(methylamino)-9H-purin-9-yl]-2-(phosphonooxy) bicyclo [3.1.0]hexane-1-methanol dihydrogen phosphate ester tetra ammonium salt (MRS2500); 1-amino-9,10-dihydro-9,10-dioxo-4-[[4-(phenylamino)-3-sulfophenyl]amino]-2-anthracenesulfonic acid sodium salt (PSB0739) and 2-[(2-chloro-5-nitrophenyl)azo]-5-hydroxy-6-methyl-3-[(phosphonooxy)methyl]–4-pyridinecarboxaldehyde disodium salt (MRS2211) (TOCRIS, Avonmouth, Bristol, UK).
As previously reported (i) “gallamine, hexamethonium, α-CGRP and methoxamine were dissolved in physiological saline” [26 (link),27 (link),28 (link),29 (link),30 (link),54 (link),55 (link),57 (link)]; (ii) “ADPβS, MRS2500, PSB0739 and MRRS2211 were dissolved in bidistilled water” [36 (link)]; and (iii) “glibenclamide was dissolved in a vehicle combination of 33% PEG, 33% ethanol and 34% NaOH 0.2 M” [53 (link)]. None of these vehicles affected the baseline values of DBP or heart rate (not shown).