Dipyridamole

D. discoideum AX4 (wild type), pkaR⁻, and pkaC^OE strains are described in (49 (link)–52 (link)). D. purpureum DpAX1 is described in (53 (link)). Cells were grown in shaking suspension at 220 rpm at 22°C in HL5 medium supplemented with penicillin (50U/mL) and streptomycin (300μg/mL) (54 (link)). abcB3 shRNA expressing cells were maintained as above and supplemented with G418 (10μg/mL). For development on black nitrocellulose filters, cells were harvested at the logarithmic growth phase and washed twice with KK2 buffer (16.3mM KH₂PO₄, 3.7 mM K₂HPO₄, pH 6.2). Cells (5×10⁷) were deposited onto a 5 cm filter atop a paper pad saturated with 2 mL PDF buffer (9.2 mM K₂HPO₄, 13.2 mM KH₂PO₄, 20 mM KCl, 1.2 mM MgSO₄, pH 6.5) and incubated in a dark humid chamber at 22°C. Where indicated, vehicle control or 1 mM of the ABC inhibitors Indomethacin, Probenecid, Dipyridamole, or Sulfynpyrazone (Sigma-aldrich, MO, USA) was added to the PDF buffer. For developmental streaming assays, 1.56×10⁶ cells/cm² were deposited in a 12 well plate (BD Biosciences, CA, USA) under 666μL of PDF containing vehicle control or 50μM of one of the ABC transporter inhibitors. Cells were incubated at 22°C in a dark chamber and imaged at 16 hours.

ATP-binding cassette (ABC) transporters have been implicated in ATP release, including the multidrug resistance gene product, MDR1 (also known as P-glycoprotein, ABCB1 ^{59 (link)}), and the cystic fibrosis transmembrane conductance regulator CFTR (ABCC7) ^{58 (link)}. We tested for a contribution of ABC transporters to SDH by administering ABC transporter inhibitors dipyridamole (Sigma Chemical Co.), an inhibitor of MDR1/P-glycoprotein/BCRP ^{52 (link)}, and nicardipine (Santa Cruz Biotechnology, Paso Robles, CA), a dihydropyridine in different chemical class from dipyridamole, that is another potent inhibitor of MDR1/P-glycoprotein ^{52 (link)}, as well as breast cancer resistance protein ^{67 (link)}. We also tested the highly selective inhibitor of CFTR, CFTR_inh-172 (Santa Cruz Biotechnology), a thiazolidinone compound that acts directly on the CFTR ^{57 (link), 58 (link)}.