Probenecid (Benemid) is a benzoic acid derivative with antihyperuricemic property.

Probenecid acts as a competitive inhibitor of monoamine transport in the kidneys, liver, and eyes. It decreases the renal clearance of antibiotics, thereby enhancing the levels of antibiotics in the serum. As a competitive inhibitor of the organic anion transporter (OAT), Probenecid prevents OAT-mediated reuptake of uric acid from urine back into the serum, consequently promoting the excretion of uric acid by the kidneys.

In 1321N1 astrocytoma cells, Probenecid (2.5 mM) effectively inhibits the efflux of Fura-2 without altering basal calcium ion concentration or the calcium response to muscarine.

Probenecid is dissolved at 500 mM in 1 N NaOH and titrated to pH 7.0[1]. HEK-293T cells are transfected with hTAS2R expression constructs using Lipofectamine 2000 in poly-lysine coated, black 384-well plates with clear bottoms and incubated for 22 hours at 37°C. Growth media is removed and cells are washed twice with HBSS containing 20 mM HEPES, then loaded with a calcium indicator dye in HBSS containing 20 mM HEPES (Calcium 4 Assay kit) with or without 1 mM Probenecid. Cells are incubated at 37°C for 1 hour in the presence of both dye and Probenecid, then moved to a Flexstation II-384 set for 32°C. After a 15-minute temperature equilibration (without washout), indicated compounds are injected (at t=~25 seconds) and fluorescence is measured for 100 to 180 seconds, reading every 3 seconds. Data sets are analyzed and represented as % over baseline signal using Prism 5.0 software. For Schild plots, replicates of raw calcium flux values are expressed as % over baseline signal. The mean value at 36 seconds (corresponding to the maximum flux signal) for each concentration of TAS2R ligand in the presence of the indicated concentration of Probenecid is plotted against the log of ligand concentration. Data points are fit using non-linear regression in GraphPad Prism[1].