Spadin

Tertiapin-Q (STT-170, Alomone Labs), CGP54626 (1088, Tocris), baclofen (0417, Tocris), linopirdine (1999, Tocris), XE991 (2000, Tocris), PK-THPP (5338, Tocris), spadin (5594, Tocris), and tolbutamide (T0891, dissolution with alcohol, Sigma) were used in whole-cell patch clamp mode. Kynurenic acid (K3375, Sigma), picrotoxin (1128, Tocris), and tetrodotoxin (T-550, Alomone Labs) were used to block synaptic currents. All solutions used in this study were made according to manufacturer’s specifications, and stock solutions of all drugs were dissolved in autoclaved de-ionized water unless specifically stated.

VIC_FB were stretched in culture, using a Flexercell FZ-4000T strain unit (Flexcell International Corporation, Burlington, USA). VIC were seeded at 5x10⁴ cells per well onto the centre of BioFlex 6-well culture plates, pre-coated with collagen type I and maintained in fibroblast medium not containing Streptomycin. Cells were allowed to adhere (without stretch application) for 2 days in an incubator at 37°C with 5% CO₂. Prior to being stretched, a visual check was made that cells had covered the deformable membrane in each well. Culture dish membranes were cyclically stretched equibiaxially by 15%, at 1 Hz for 24 hours. The amount of stretch applied reflects the level of strain experienced by cells under physiological conditions [4 (link), 5 (link)]. VIC_FB cultured on BioFlex plates as described above, but not stretched served as the control group. In a subset of experiments, cells were pre-incubated with one of the following: 100 μM Streptomycin (a blocker of MSC_NS [23 (link), 24 (link)]), 1 μM Spadin (TREK-1 blocker, [25 (link), 26 (link)]), 0.5 μM RN-9893 (TRPV4 blocker, [27 (link)]), or 1 μM GSK417651A (TRPC6/3 blocker, [28 (link), 29 (link)]). (Streptomycin was purchased from Sigma; GSK417651A was purchased from Tebu-Bio Ltd, Peterborough, UK; Spadin and RN-9893 were purchased from Tocris, Abingdon, UK).

Arachidonic acid (AA), BL-1249, CDC, cis-4,7,10,13,16,19-docosahexaenoic acid (DHA), and barium chloride (BaCl₂) were purchased from Sigma-Aldrich, spadin was bought from Tocris. All substances, except BaCl₂, were stored as stocks (1 mM–1 M) at −20°C and diluted to the required concentrations in standard bath solution immediately prior to experimentation.