Kt5823

Recordings were obtained, using a whole-cell patch clamp with an Axopatch 200B Patch Clamp Amplifier (Axon Instruments, Union City, CA, USA) at room temperature. The pCLAMP 9.0 software (Axon Instruments) was used for data acquisition and analysis of whole-cell currents. Activated currents were filtered at 2 kHz and digitized at 10 kHz. Recording patch pipettes were prepared from filament-containing borosilicate tubes (TW150F-4; World Precision Instruments, Sarasota, FL, USA), using a two-stage microelectrode puller (PC-10; Narishige, Tokyo, Japan), and were then fire polished on a microforge (MF-830; Narishige). When filled with pipette solution, the pipettes exhibited a resistance of 2–3 MΩ.
The bath solution to record K_V currents contained (in mM): 150 NaCl, 5.4 KCl, 1 CaCl₂, 1 MgCl₂, 10 glucose, and 5 HEPES (pH adjusted to 7.35 with NaOH). The pipette solution contained (in mM): 130 KCl, 1 CaCl₂, 2 MgCl₂, 10 HEPES, 10 EGTA, and 2 Mg-ATP (pH adjusted to 7.3 with KOH). TEA, 4-AP, SNAP, KT5823, ODQ, 8-Br-cGMP, KT5720, SQ22536, 8-Br-cAMP, and all other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Tetraethylammonium (TEA) was purchased from Santa Cruz Biotech, California, USA. Iberiotoxin was purchased from Alomone, Jerusalem, Israel. Glibenclamide was purchased from Research Biochemical International, Massachusetts, USA. Tetrodotoxin (TTX) was purchased from Tocris Bioscience, Bristol, UK. ω-conotoxin GVIA (CTX) was purchased from Bachem, Bubendorf, Switzerland. KT 5720, KT 5823, NG-Nitro-L-arginine (L-NNA), apamine, papaverine, and resveratrol were obtained from Sigma-Aldrich, Missouri, USA. Charybdotoxin was purchased from AnaSpec Inc., California, USA.

Tadalafil and vardenafil were obtained from Selleckchem (Houston, TX, USA).
KT5823, 8-Br(Bromo)-cGMP, Alizarin Red S, and SB216763 were from Sigma (St Louis,
MO, USA). Recombinant murine Wnt3a protein was purchased from R&D System
(Minneapolis, MN, USA). Antibodies against PKG1, PKG2, Lamin B, Dkk1, IgG,
β-actin, and protein A/G PLUS-Agarose were from Santa Cruz
Biotechnology (Santa Cruz, CA, USA). Antibodies against p-β-cat
(Ser33/37/Thr41), p-β-cat (Ser45), β-cat,
p-GSK3β (Ser9) and GSK3β were from Cell Signaling
(Danvers, MA, USA). Antibodies against AP and RunX2 were purchased from Abcam
(Cambridge, UK), and the antibody against Lef1 was from Proteintech (Chicago, IL,
USA). The IRDye 680 and 800 second antibodies were from LI-COR Bioscience
(Lincoln, Nebraska) and the Alexa 555-conjugated secondary antibodies were from
Life Technologies (Grand Island, NY, USA). Cyclic GMP Direct Immunoassay kits were
obtained from Abcam and TRAP staining kits were from Sigma.

KT-5823, SNAP, carboxy-PTIO and 1H-[1 (link),2 (link),4 (link)]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) were from Sigma (St. Louis, MO). LY-83583 and BAY-60-7550 were from Santa Cruz Biotechnology (Santa Cruz, CA). ODQ, LY-83583 and BAY-60-7550 were dissolved in dimethyl sulfoxide (DMSO). To ensure that the channel activity was not affected by DMSO, its final concentration in the bath was less than 0.1%.

Cells were washed twice with phosphate-buffered saline (PBS) and then incubated in Tyrode solution for 2 hours (h) before experiments. To examine the effect of NECA (Tocris, UK) on mitochondrial membrane potential, GSK-3β phosphorylation (at Ser9), and GRP94, cells were exposed to 800 μM H₂O₂ (Sigma, USA) for 20 minutes (min) to cause oxidant damage. A range concentrations of NECA (0.1–10 μM) were given 10 min before exposing to H₂O₂. In the study exploring roles of ERS and the cGMP/PKG signaling pathway in NECA-induced cardioprotection against oxidative damage, cells were exposed to 0.1 μM NECA/20 mM 2-DG (Sigma, USA)/0.1 μM KT5823 (Sigma, USA) for 20 min. The 2-DG and KT5823 were applied for 10 min before the cells were exposed to NECA.