Fig. 5.
Blocking signaling exacerbates toxicity. (A) Simplified schematic representation of insulinergic signaling from insulin receptor substrate-1 (IRS1) to phosphoinositol-3-kinase (Pi3K) to protein kinase B (Akt) to p70 s6 kinase (p70s6k) with inhibition point by Wortmannin and LY294002 between Pi3K and Akt. (B) Western blots of cardiac lysates for 10-min time point for animals pretreated with Wortmannin before bupivacaine infusion. Proteins blotted for include Akt phosphorylated at S473; Akt phosphorylated at T308; total Akt, glycogen synthase kinase-3α (GSK-3α) and -3β (GSK-3β) phosphorylated at S21 and S9, respectively; p70 s6 kinase (p70s6k) phosphorylated at T421; ribosomal protein s6 (s6) phosphorylated at S235; and total glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as loading control. (C) Densitometry of cardiac lysates comparing protein phosphorylation in recovered animals treated with bupivacaine (n = 4, less than 100 μM bupivacaine) or bupivacaine and Wortmannin (n = 3, Wortmannin); **P < 0.01, ***P < 0.001, ****P < 0.0001, Sidak post hoc test. (D) Relative rate pressure product (RPP = mean arterial pressure × heart rate) at 10-min for animals recovering from bupivacaine toxicity either pretreated with Wortmannin (Wrtmn) or nothing (Bupi); *P < 0.05, Mann–Whitney U test. (E) Physiological parameters including maximal contraction (dp/dt), maximum relaxation (−dp/dt) and RPP from isolated Langendorff hearts at 2-min recovery point after treatment with 500 μM bupivacaine over 30 s. Hearts were either predosed with 50 μM LY294002 (LY294002) or nothing (bupivacaine). (F) Time to recovery of first beat for hearts from “E” after bupivacaine-induced asystole. (G) Time to asystole for hearts after bupivacaine treatment for hearts in “E.”