Vinpocetine

Vinpocetine (VP, ethyl-apovincaminate) was applied as a model active substance purchased from Sigma-Aldrich Co., Ltd. (Budapest, Hungary). The Soluplus^® (SP, poly(vinyl caprolactam)–poly(vinyl acetate)–poly(ethylene glycol) graft co-polymer (PCL-PVAc-PEG)) was kindly gifted from BASF GmbH (Hannover, Germany), and Poloxamer 188 (P 188, poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol) (PEG-PPG-PEG)) was also acquired from Sigma-Aldrich Co., Ltd. D-trehalose dihydrate (D-TRE), chitosan (Chit, <100 kDa), sodium hyaluronate (Hya, low molecular weight, 20–40 kDa), hydroxypropyl methylcellulose (HPMC, average molecular weight: 10 kDa, viscosity of 80–120 cP, 2% in water (20 °C)); type III mucin and materials for the simulated nasal electrolyte solution (SNES) were also acquired from Sigma-Aldrich Co., Ltd. The composition of SNES was the following: 8.77 g/L of sodium chloride, 2.98 g/L of potassium chloride and 0.59 g/L of anhydrous calcium chloride in 1000 mL of purified water, adjusted to a pH of 5.6.

Resazurin sodium salt (cat. R7017), dimethyl sulfoxide (DMSO, cat. D8418) as well as cAMP and cGMP (sodium salts) were obtained from Sigma (Buchs, Switzerland). Radiochemicals were from Hartmann Analytic (Braunschweig, Germany). PDE inhibitors were from the following sources: isobutyl-methyl-xanthine (IBMX), 8-methoxymethyl-IBMX, vinpocetine, rolipram, dipyridamole, papaverine, BAY 73–6691, BRL 50481 were from Sigma; zardaverine, cilostamide were from BioMol Anawa Trading (Wangen, Switzerland); pentoxifylline was from Calbiochem (Merck Millipore, Schaffhausen, Switzerland); etatolate was from Tocris Bioscience (Lucerna-Chem, Lucerne, Switzerland); erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), milrinone, trequinsin, Ro 20–1724, dipyridamole, zaprinast were obtained from Fisher Scientific; BAY 60–7550 was from Biotrend Chemicals (Destin, FL, USA), sildenafil citrate was generously provided by Pfizer, Inc. The following compounds were synthesized using methods previously described: NEU28 (syn. PQ-10, CAS 927691-21-2) [23 (link)], NEU222 [24 (link)], PFE-PDE1 (patent EP911333A1) [24 (link)], PFE-PDE9 (patent WO2003037899A1) [24 (link)], tadalafil [25 (link)], piclamilast (patent WO9212961), roflumilast (patent WO9501338), NPD-001 (syn. CpdA, PPS54019) [26 (link),17 (link)] and VUF13525 (compound 20b in Orrling KM et al (2012) [18 (link)]).

Peripheral blood was withdrawn from antecubital vein of each donor and taken into EDTA vacuum tubes (15 IU/ml). Hematocrit was set to 40% for the experiments with autologous plasma. Blood samples were treated with the stimulators or inhibitors of the enzymes involved in the PKA pathway. Forskolin (10 μM) and SQ22536 (100 μM) were used for the stimulation and the inhibition of AC, respectively. H-89 (10 μM) and Pentoxifylline (10 μM) were used for the inhibition of PKA and PDEs, respectively. For the selective inhibition of PDEs, Vinpocetine (30 μM), Milrinone (20 μM), and Rolipram (10 μM) were used to block the activities of PDE1, PDE3, and PDE4, respectively. All chemical agents were purchased from Sigma-Aldrich Co (MO, United States) and incubated with blood samples at 37 C for 15 min, except Vinpocetine which was incubated for 30 min. A vehicle (DMSO or PBS) was prepared in the same volume (v/v) and studied under the same conditions as a control. After the incubation with the agents, whole blood samples were measured directly. All experiments were performed within 4 h after blood sampling.

CSE-siRNA and negative siRNA were purchased from GenePharma (Shanghai, China), and atelocollagen was purchased from KOKEN (Tokyo, Japan); CSE antibody was purchased from Santa Cruz (Delaware Ave, USA); NaHS, Acetylcholine(ACh), bradykinin, 9, 11-dideoxy-11α, 9α-epoxy-methanoprostaglandin F_2α (U46619), and Vinpocetine were purchased from Sigma Chemicals (St. Louis, USA); lactate dehydrogenase (LDH) and malondialdehyde (MDA) assay kits were purchased from Nanjing Jiancheng Biological Co (Nanjing, China). ChTx, Apamin, L-Cys, L-NG-nitroarginine methyl ester (L-NAME), and indomethacin (Indo) were purchased from sigma Chemicals (St. Louis, USA); Krebs solution (comprising the following (mM): NaCl 118, KCl 3.4, CaCl₂ 2.5, KH₂PO₄ 1.2, MgSO₄ 1.2, NaHCO₃ 25, and glucose 11.1) was aerated with a mixture of 95% O₂ and 5% CO₂ and oxygenated during the incubation period.

Cyclic hydrolysis both cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) levels in ADSCs were measured using cyclic AMP and GMP XP Assay kit (CST, Danvers, MA, USA). 7 × 10³ ADSCs were cultured with α-MEM and treated with 50 μM vinpocetine (Sigma-Aldrich). After 24 h, the cells were rinsed twice with PBS, and lysed with lysis buffer. 50 μl cell lysate with 50 μl of the HRP cAMP solution were added to the cAMP assay plate. Add 100 μl TMB substrate, incubate for 30 min, and absorbance were measured at 450 nm using a microplate reader (Bio-Rad Laboratories).

M199 medium (Invitrogen UK, 11150), taurine (Biochemica, A1141), creatine monohydrate (Sigma Aldrich, C3630), penicillin/streptomycin (Merck, A2212), carnitine hydrochloride (Sigma Aldrich, C9500) BSA (Sigma Aldrich, A6003), laminin (Sigma Aldrich L2020), isoproterenol hydrochloride (Sigma Aldrich, I6504), ICI118551 (Tocris UK, 0821), CGP 20712A (Tocris UK, 1024), SR 59230A hydrochloride (Tocris UK, 1511), L-NAME hydrochloride (Tocris UK, 0665), Vinpocetine (Sigma Aldrich, V6383), EHNA hydrochloride (Sigma Aldrich, E114), Cilostamide (Tocris UK, 0915), Tadalafil (Santa Cruz USA, sc-208412), IBMX (Santa Cruz sc-201188), self-made rabbit sGCα and β subunit antibodies (specificity tested in KO animals; Friebe et al., 2018 (link)), mouse α-actinin (Sigma Aldrich, A7732), mouse Caveolin-3 (BD Transduction Laboratories, 610421, specificity tested in KO animals; Woodman et al., 2002 (link)), secondary Alexa Fluor antibodies 488 nm, 514 nm, 568 nm and 633 nm (Life Technologies), BSA (Fisher Scientific UK, BPE9704), fluorescence mounting medium (Vectashield Germany, H-1000), MaTek glass-bottom dishes (MaTek USA, P35G-1.5–10 C), TAT-scram and TAT-Cav3 peptides (a gift from Dr. Sarah Calaghan from Leeds, England).