ω agatoxin iva

We tested several Ca_V antagonists to verify that high K⁺-stimulated MeAIB transport activity is dependent on presynaptic exocytotic Glu release. Verapamil and nifedipine were obtained from Sigma. All peptide antagonists (ω-conotoxin MVIIC, ω-conotoxin GVIA, ω-conotoxin SVIB, ω-agatoxin TK, ω-agatoxin IVA, and SNX-482) were from Alomone labs (Jerusalem, Israel). We performed concentration-response curves for Verapamil and nifedipine (25–0.78 μM), and for ω-conotoxin MVIIC (2.5–0.078 μM) and Cd²⁺ (100–3.125 μM). We also examined the % inhibition values for ω-agatoxin TK and ω-agatoxin IVA at 500 nM, and ω-conotoxin GVIA, ω-conotoxin SVIB, SNX-482 at 1 μM, or nickel ion (Ni²⁺) at 100 μM. The Ca_V inhibitors were added during a 5 min pretreatment in normal Krebs buffer and then during the high K⁺ (60 mM) exposure during 5 min. The cultures were then rinsed in N buffer and uptake of ¹⁴C-MeAIB (5 min) was then started in N buffer. Inhibition of spontaneous transport activity was also examined with a 5 min pretreatment followed by a 15 min period of ¹⁴C-MeAIB uptake with MVIIC (1.25–0.039 μM) or Cd²⁺ (50–1.56 μM) in Ca²⁺-containing (2.5 mM) Krebs buffer.
Following the uptake of ¹⁴C-MeAIB under spontaneous or high K⁺-stimulated transport conditions, the cultures were removed from the 37 °C incubator, placed on ice, and washed with ice-cold N buffer (2.5 ml). SDS (1%, 2 ml) was then added to solubilize the cells and the amount of radioactivity was determined by liquid scintillation counting using EcoScint (National Diagnostics).