Cerebrovascular endothelial GPCR function was assessed using adenosine 5′-triphosphate disodium salt hydrate (ATP, 100 μmol/L) and acetylcholine chloride (ACh, 10 μmol/L) to activate endothelial purinergic and muscarinic GPCRs, respectively [12 (link), 13 (link), 28 (link)]. Effective concentrations of SKA-31 (10 μmol/L; Tocris Bioscience, Bristol, UK) and NS309 (1 μmol/L; Tocris) were used to directly evaluate function of SKCa/IKCa channels to produce hyperpolarization [13 (link), 29 (link)] and hyperpolarization-dependent [Ca2+]i increases [11 (link), 30 (link), 31 (link)]. Elevated level of extracellular KCl ([K+]o: 15 mmol/L) was used to stimulate endothelial KIR channels [13 (link)]. Finally, to examine mitochondrial Ca2+ content, a maximal but reversible concentration of FCCP (1 μmol/L) was applied [20 (link), 21 (link)]. DMSO solvent for working stocks of SKA-31, NS309, and FCCP was ≤0.1% which we have found to have no effect on endothelial Ca2+ and Vm on its own for ≤5 min applications used for respective drug treatments [32 (link)].
Resting [Ca2+]i and Vm were typically allowed ≥2 min to stabilize before application of a pharmacological agent, whereby each application was allowed sufficient time (≥3 min) to record peak [Ca2+]i and Vm responses. In between individual applications, the endothelial tube was washed with PSS to baseline conditions.
Resting [Ca2+]i and Vm were typically allowed ≥2 min to stabilize before application of a pharmacological agent, whereby each application was allowed sufficient time (≥3 min) to record peak [Ca2+]i and Vm responses. In between individual applications, the endothelial tube was washed with PSS to baseline conditions.