In keeping with the toxicity studies, we found that adding VU625 (0.77 mM) to the vehicle, which delivers 690 pmol of VU625 per mosquito, did not significantly decrease the excretory capacity (583.329.52 nL/female) compared to the vehicle controls (Fig. were performed at room temperature. The compositions of the solutions used in these experiments are shown in Table 1. When present, VU625 was dissolved in solution or solution to a final concentration of 0.1, 1, 5, 15, or 50 M (0.05% DMSO). All solutions were delivered by gravity to a RC-3Z oocyte chamber (Warner Instruments, Hamden, CT) via polyethylene tubing at a flow rate of 2 ml/min. Solution changes were made with a Rheodyne Teflon 8-way Rotary valve (Model 5012, Rheodyne, Rohnert Park, CA). Table 1 Compositions (in mM) of solutions used in oocyte electrophysiology. and impaled with two conventional-glass microelectrodes backfilled with 3 M KCl (resistances of 0.5C1.5 M) to measure membrane potential (Vm) and whole-cell membrane current (Im), respectively. Current-voltage (ICV) relationships of oocytes were acquired as described previously [6]. In brief, the oocytes were subjected to a voltage-stepping protocol consisting of 20 mV actions from ?140 mV to +40 mV (100 ms each). After the conclusion of the voltage-stepping protocol, the clamp was turned off and a new solution was superfused through the chamber for 90 s before BMP2 acquiring another ICV relationship. All Vm and Im values were recorded by a Digidata 1440A Data Acquisition System (Molecular Devices) and the Clampex module of pCLAMP. The ICV plots were generated SL251188 using the Clampfit module of pCLAMP. To evaluate the inhibition of (i.e., 0.5 mM K+) were subtracted from those in 1) solution (i.e., 10 mM K+) to calculate the total inward current for an oocyte before exposure to VU625 (IA), and 2) solution with VU625 to calculate the inward current after exposure to the small molecule (IB). The percent inhibition of the inward current was calculated by subtracting IB from IA and then dividing by IA. For replaced solution and solution replaced solution mosquito colony used in the present study is identical to that described previously [6]. As before, only adult female mosquitoes 3C10 days post emergence were utilized for experiments. Mosquito toxicology experiments Adult female mosquitoes for injection were anesthetized on ice and impaled through the metapleuron using a pulled-glass capillary attached to a nanoliter injector (Nanoject II, Drummond Scientific Company, Broomall, PA). Each mosquito received a single hemolymph injection of 69 nL of SL251188 solution. The injection solution consisted of a potassium-rich phosphate buffered saline (K+-PBS), 15% DMSO, 1% -cyclodextrin, 0.1% Solutol, and a concentration of VU625 to deliver the doses indicated. In experiments where probenecid was used, water-soluble probenecid (Biotium, Hayward CA) was included in the injection solution at 50 mM, thereby providing a dose of 3.4 nmol per mosquito. The K+-PBS solution consisted of the following in mM: 92.2 NaCl, 47.5 KCl, 10 Na2HPO4, and 2 KH2PO4 (pH 7.5). A total of 10 mosquitoes were injected for a given treatment or dose, and then were placed into small cages within a rearing chamber (28C, 80% relative humidity, 1212 light:dark) and allowed free access to a solution of 10% sucrose. The mosquitoes were observed at 24 hr after injection. For each treatment, 3C7 replicates of 10 mosquitoes each were performed. Mosquito excretion experiments The excretory capacity of mosquitoes was measured as described [6]. In brief, after anesthetizing mosquitoes on ice, their hemolymph was injected as described above with 900 nL of a K+-PBS vehicle made up of 1.15% DMSO, 0.077% -cyclodextrin, and 0.008% Solutol, or the vehicle containing VU625 (0.77 mM) to deliver a dose of 690 pmol of VU625 per mosquito. In experiments where probenecid was used, the vehicle was supplemented with water- soluble probenecid (3.08 mM) to deliver a dose of 3.4 nmol of probenecid per mosquito. After injection, the mosquitoes were placed immediately in a graduated, packed-cell volume tube (MidSci, St. Louis, MO; 5 mosquitoes per tube) and held SL251188 at 28C. The volume of urine excreted at 60 min post injection was measured as described previously [6], and all mosquitoes were confirmed to.