8 br cgmp

For the LTP experiment, all the compounds were dissolved in ACSF to achieve the final concentration required. DEA/NO, ODQ, BAY41–2272, and 8-Br-cGMP were diluted in 0.1% DMSO; sildenafil and compound 7a in 0.05% DMSO. DEA/NO was stored for 24 h in alkaline solution (0.01 m NaOH) and diluted in ACSF immediately before use. Different treatments were interleaved on slices from the same mice. For the behavioral experiments, compound 7a and 8-pCPT-cGMP were dissolved in 2% DMSO and 2% Tween 80. Compound 7a was synthesized in six steps [35 (link)], while DEA/NO and BAY41–2272 were purchased from Enzo life Science (Farmingdale, NY, USA), 8-Br-cGMP from Biolog Life Science Institute (Bremen, Germany), ODQ from Cayman Chemical Company (Ann Arbor, MI, USA), sildenafil and 8-pCPT-cGMP from MilliporeSigma (St. Louis, MO, USA). For biochemical experiments compound 7a was dissolved in 4% DMSO and 2% Tween 80.

The cyclic nucleotides cGMP and cAMP were obtained from Sigma. All synthesized derivatives were monitored and analyzed by reversed phase HPLC and thin layer chromatography (TLC), visualization was effected by UV (254 nm) and confirmed by mass spectroscopy. The structures of all C8-modified cNMP derivatives are given in Table 1. Three of the analogs (2_G, 4_A/G) were previously studied in other channel types or isolated CNBDs^{29 (link),30 (link)}.
8-Br-cAMP and 8-Br-cGMP (Biolog Life Science Institute, Bremen) are appropriate compounds for the synthesis of the C8 substituted cAMP and cGMP derivatives, respectively. Following the synthesis route described by Brown et al.^{30 (link)} with minor modifications, the 8-Br-cNMP was first substituted to the thiol by thiourea. The desired thioether was then obtained with an appropriate linker derivative. The respective linker derivative comprised an N-terminal protected bromoalkylamine or bromoheteroalkylamine. The compounds described herein were obtained by deprotection of the terminal amino group and part of them was further acetylated. A detailed description of the synthesis is provided in Supplementary Methods.

The fibroblasts of wt‐ and cGKI‐KO‐mice were isolated and stained as described previously 5. The cells were pretreated with 8Br‐cGMP (Biolog, Bremen, Germany; 1 mm, 1 h, 37 °C) or vehicle followed by exposure to TGFβ (Biomol, Hamburg, Germany; 2 ng·mL⁻¹, 1 h, 37 °C) or vehicle. Nuclei were stained with DAPI (gift from Armin Kurtz, University Regensburg). P‐smad3 and P‐smad2 (Cell Signalling), respectively, were detected using an Alexa647‐conjugated anti‐rabbit secondary‐antibody (1 : 200; Invitrogen, Karlsruhe, Germany) for 2 h at room temperature. Coverslips were washed, mounted with glycerol and analysed using an Axiovert 200 microscope (Zeiss, Jena, Germany). To ensure a valid comparison, images were randomly selected from different fields. The intranuclear and extranuclear fluorescence‐intensity of three to six equal areas was measured respectively. Then, the mean value of intranuclear and extranuclear fluorescence intensity of P‐smad3 was determined respectively. For the quantification of the fluorescence intensity all values were related to values of untreated wt‐fibroblasts (control) using the metamorphic offline software.