Dpo 1

DPO-1 and TTX were from Tocris (Ellisville, MO), dendrotoxin-K, hongotoxin-1, margatoxin, were from Alomone Labs (Jerusalem, Israel), while the other substances were purchased from Sigma. The recording chamber was constantly perfused with aCSF (2–3 mL/min). The treatments were applied locally using a perfusion pencil system (tip diameter 100 µm, Automate Scientific) driven by gravity. Although we have not systematically constructed dose response curves, several concentrations were tested for each drug to assess its efficacy. After these trials we have chosen for each drug the concentration that produced the maximal effect. The doses used are similar with those reported in the literature.

PSS for vessel dissection, isolation and preparation contained (in mM) the following: KCl 6.0, NaCl 112, NaHCO₃ 1.18, MgSO₄ 1.18, KH₂PO₄ 1.18, CaCl₂ 1.8 and glucose 10. The pH of the PSS was adjusted to and maintained at 7.4 by continuous flow of normal air into the PSS. The amount of 60 mM K⁺-PSS (60K) was prepared by equimolar replacement of NaCl with KCl in the PSS. Empagliflozin was purchased from Ambeed Inc. (Arlington Heights, IL, USA) and used in the concentration range of 0.001–100 µM. Phenylephrine (PE) and 4-aminopyridine (4-AP) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used at a final concentration of 1 µM and 1 mM, respectively. Indomethacin (10 µM), DPO-1 (1 µM), Linopirdine (10 µM), Psora-4 (100 nM), Glibenclamide (10 µM), paxilline (10 µM), ODQ (10 µM), KT 5823 (1 µM), L-NNA (10 µM), SNP (10 µM) and acetylcholine (ACh, 1 µM)) were purchased from Tocris (Minneapolis, MN, USA). 4-AP, SNP and PE were dissolved in distilled water. Empagliflozin, DPO-1, Linopirdine, Psora-4, Indomethacin, Glibenclamide, paxilline, ODQ, KT5823, L-NNA and ACh were dissolved in dimethyl sulfoxide (DMSO). The final DMSO concentration (<0.1%) in the myograph chamber had no significant effect on arterial contractility.