Bay k8644

Cells were patch-clamped in whole-cell mode using Cs⁺-filled pipettes as previously described^{35 (link)}. The following ion channel modulators were used: Tetraethylammonium chloride (TEA, T2265, Sigma-Aldrich) to block dominant BK channels, Nifedipine (N7634, Sigma-Aldrich) to inhibit Ca_V1.3 channels, Bay K 8644 (1544, Tocris) to activate Ca_V1.3 channels and Mibefradil (2198, Tocris) or NiCl₂ (Ni²⁺) to block Ca_V3.2 channels. Nifedipine and Bay K 8644 were prepared as stocks in DMSO and diluted so that the final vehicle concentration was < 0.1%. Cs⁺-pipette solution comprised (mM): 110 CsCl, 20 TEA-Cl; 5 MgCl₂; 1 EGTA; 0.1 Na₂GTP; 2.5 Na₂phosphocreatine; 4 Mg-ATP; 5 HEPES, 296.8 mOsm/L, pH 7.2. TEA-Ba²⁺ PSS comprised (mM): 115 NaCl, 10 TEA-Cl, 1 MgCl_2, 5.5 Glucose, 10 BaCl₂, 10 HEPES, 298.5 mOsm/L, pH 7.4. Divalent-free PSS comprised (mM): 129.8 NaCl, 5.4 KCl, 10 glucose, 2.9 sucrose, 4.2 NaHCO₃, 0.4 KH₂PO₄, 0.3 NaH₂PO₄, 5 EGTA, 10 HEPES, osmolarity 308.1 mOsm/L, pH 7.4.

This technique has been explained in detail elsewhere. Following the acquisition of a topographical image of the cell with SICM an area of interest is selected. The pipette is moved to a neutral area of the dish and clipped to give the patch-clamp resistance. It is then moved back to the original area of interest and the pipette is fused with the membrane patch and calcium channel recordings made as described in the references above. A sub-population of cells were treated with the non-specific calcium channel activator BAYK8644 (Tocris Biosciences, United States).

Verapamil hydrochloride (Tocris), Bay-K8644 (Tocris), caffeine (Sigma-Aldrich) and dbcAMP (Sigma-Aldrich) were dissolved in DMSO following the manufacturer’s instructions. Stock solutions were freshly prepared before experiments.

The following chemicals were used: phenylephrine hydrochloride (Sigma Chemical Co.), guanethidine (Tokyo Kasei, Tokyo, Japan), atenolol (Sigma Chemical Co.), nifedipine (Sigma Chemical Co.), BAY K 8644 (Tocris Bioscience, Bristol, UK), ryanodine (Wako Pure Chemical, Osaka, Japan), calyculin A (Wako Pure Chemical, Osaka, Japan).

Picrotoxin (50 μM), L-655 708 (11,12,13,13a-Tetrahydro-7-methoxy-9-oxo-9H-imidazo[1,5-a]pyrrolo[2,1 c][1,4]benzodiazepine-1-carboxylic acid, ethyl ester, 10 μM), SCS (Salicylidene salicylhydrazide, 1 μM), TPMPA ((1,2,5,6-Tetrahydropyridin-4-yl) methylphosphinic acid, 50 μM), etomidate (10 μM), allopregnanolone (100 nM), muscimol (5 μM), bumetanide (10 μM), nifedipine (10 μM), benidipine (10 μM), bay K 8644 (10 μM), bicuculline (50 μM), ATP (150 μM; all from Tocris Bioscience, Bristol, UK), SNAP ((S)-Nitroso-N-acetylpenicillamine, 50 μM), SC (semicarbazide 50 μM), GABA (5 μM), geneticin (10 μM; all from Sigma-Aldrich) and CPCPT (1-(3-Chlorophenethyl)−3-cyclopentylpyrimidine-2,4,6-(1H,3H,5H)-trione, 1 μM, Merck Millipore, Darmstadt, Germany) were used at the indicated concentrations, if not differently stated. All pharmacological treatments and live cell stainings were performed in CM at 37°C and 5% CO₂.

The drugs and solutions used in this work were as follows: dimethyl sulfoxide (DMSO) (Sigma-Aldrich, St. Louis, MO, USA), oleocanthal (PhytoLab), acetonitrile, hexane, electrochemiluminescence (ECL) immunoassay (Thermo), oxytocin (Sigma-Aldrich, St. Louis, MO, USA, O3251) (Sigma-Aldrich, St. Louis, MO, USA, O6379), carbachol (Sigma-Aldrich, St. Louis, MO, USA, C4382), PGF2α (Cayman, Ann Arbor, MI, USA, 16010), acetylcholine (Sigma-Aldrich, St. Louis, MO, USA, A2661), Bay K 8644 (TOCRIS, Taiwan, 1544), potassium chloride (KCl) (SHOWA, Saitama, Japan, 1630-5150), calcium chloride dehydrate (CaCl₂) (Wako, TX, USA, 038-00445), acetic acid (LAB-SCAN, Bangkok, Thailand, A8401E), β-Estradiol 3-benzoate (EB) (Sigma-Aldrich, St. Louis, MO, USA, E8515), ibuprofen (Sigma-Aldrich, St. Louis, MO, USA, I4883).