Cell experiment:

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℃. 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℃ for 1 hour in the presence of both dye and Probenecid, then moved to a Flexstation II-384 set for 32℃. 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].

Animal experiment:

Mice[1] In order to obtain a dose response curve, male C57 WT (n=39) mice 12-16 weeks of age are anesthetized with isoflurane while intravenous jugular access (IV) is obtained under a microscope. Subsequently, an echocardiographic study with both M-mode and B-mode is obtained in parasternal long axis (PSLAX) as described below. Either saline or different doses of Probenecid (increasing from 2 to 200mg/kg) are injected (bolus IV) for the initial contractility studies in WT mice.

Probenecid is an inhibitor of organic anion transport and MRP [1] [2]. Also, probenecid inhibited pannexin-1 channel with IC50 value of 150μM [3].

Multidrug resistance-associated proteins (MRPs) are ATP-binding cassette (ABC) transporters that transport various molecules across cellular membranes and are involved in multi-drug resistance.

Probenecid is an inhibitor of organic anion transport, MRP and pannexin-1 channel. In MRP-overexpressing HL60/AR and H69/AR tumor cell lines, probenecid reversed resistance to daunorubicin (DNR) and vincristine (VCR) in a concentration-dependent way [1]. In wild-type AML-2 cells, probenecid increased the MRP levels in a dose- and time-dependent way. In MRP-overexpressing AML cells, probenecid exhibited a significant chemosensitizing effect. These results suggested that probenecid functioned as an effective chemosensitizer of multidrug resistance (MDR) tumor cells but also an MRP activator [2].

In ischemia/reperfusion (I/R) injury rats, probenecid protected against CA1 neuronal death. Probenecid strengthened the upregulation of Hsp70 and inhibited the expression of calpain-1 and the released of cathepsin B. Also, probenecid inhibited the proliferation of astrocytes and microglia [4].

References:
[1]. Gollapudi S, Kim CH, Tran BN, et al. Probenecid reverses multidrug resistance in multidrug resistance-associated protein-overexpressing HL60/AR and H69/AR cells but not in P-glycoprotein-overexpressing HL60/Tax and P388/ADR cells. Cancer Chemother Pharmacol, 1997, 40(2): 150-158.
[2]. Kim HS, Min YD, Choi CH. Double-edged sword of chemosensitizer: increase of multidrug resistance protein (MRP) in leukemic cells by an MRP inhibitor probenecid. Biochem Biophys Res Commun, 2001, 283(1): 64-71.
[3]. Silverman W, Locovei S, Dahl G. Probenecid, a gout remedy, inhibits pannexin 1 channels. Am J Physiol Cell Physiol, 2008, 295(3): C761-767.
[4]. Wei R, Wang J, Xu Y, et al. Probenecid protects against cerebral ischemia/reperfusion injury by inhibiting lysosomal and inflammatory damage in rats. Neuroscience, 2015, 301: 168-177.