Fig. 7.
Activation of protein kinase B/Akt upstream of mammalian target of rapamycin in response to bupivacaine and lipid emulsion. (A) Western blots of cardiac lysates at different time points during recovery from bupivacaine toxicity with adjuvant IV lipid emulsion (ILE) for targets in the insulinergic pathway. Proteins blotted for include protein kinase B (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, insulin receptor substrate-1 (IRS1) phosphorylated at S612, and total glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as loading control. (B) Densitometry of cardiac lysates comparing relative phosphorylation level at baseline (n = 4) with the relative phosphorylation at the 5-min time point (after both bupivacaine and ILE) when animals are recovered and tissue concentrations are expected to be below sodium channel IC50 (less than 100 μM Bupi + ILE; n = 6; n = 3 for p70s6k); **P < 0.01, ***P < 0.001, **** P < 0.0001, Sidak post hoc test. (C) Regression across time of relative phosphorylation level of threonine 308 on Akt. Fit line: pT308-Akt = 0.16 ± 0.03 × time + 1.0 ± 0.2 R = 0.85. (D) Regression across time of relative phosphorylation level of threonine 421 on p70s6k. Fit line: pS421-p70s6k = 0.28 ± 0.06 × time − 0.55 ± 0.4 R = 0.89.