Korean J Pain.  2016 Oct;29(4):229-238. 10.3344/kjp.2016.29.4.229.

Lipid emulsion inhibits vasodilation induced by a toxic dose of bupivacaine by suppressing bupivacaine-induced PKC and CPI-17 dephosphorylation but has no effect on vasodilation induced by a toxic dose of mepivacaine

• Cho H
• Ok SH
• Kwon SC
• Lee SH
• Baik J
• Kang S
• Oh J
• Sohn JT

• KMID: 2354215
• DOI: http://doi.org/10.3344/kjp.2016.29.4.229

Abstract

BACKGROUND
The goal of this in vitro study was to investigate the effect of lipid emulsion on vasodilation caused by toxic doses of bupivacaine and mepivacaine during contraction induced by a protein kinase C (PKC) activator, phorbol 12,13-dibutyrate (PDBu), in an isolated endothelium-denuded rat aorta.
METHODS
The effects of lipid emulsion on the dose-response curves induced by bupivacaine or mepivacaine in an isolated aorta precontracted with PDBu were assessed. In addition, the effects of bupivacaine on the increased intracellular calcium concentration ([Ca²âº]áµ¢) and contraction induced by PDBu were investigated using fura-2 loaded aortic strips. Further, the effects of bupivacaine, the PKC inhibitor GF109203X and lipid emulsion, alone or in combination, on PDBu-induced PKC and phosphorylation-dependent inhibitory protein of myosin phosphatase (CPI-17) phosphorylation in rat aortic vascular smooth muscle cells (VSMCs) was examined by western blotting.
RESULTS
Lipid emulsion attenuated the vasodilation induced by bupivacaine, whereas it had no effect on that induced by mepivacaine. Lipid emulsion had no effect on PDBu-induced contraction. The magnitude of bupivacaine-induced vasodilation was higher than that of the bupivacaine-induced decrease in [Ca²âº]áµ¢. PDBu promoted PKC and CPI-17 phosphorylation in aortic VSMCs. Bupivacaine and GF109203X attenuated PDBu-induced PKC and CPI-17 phosphorylation, whereas lipid emulsion attenuated bupivacaine-mediated inhibition of PDBu-induced PKC and CPI-17 phosphorylation.
CONCLUSIONS
These results suggest that lipid emulsion attenuates the vasodilation induced by a toxic dose of bupivacaine via inhibition of bupivacaine-induced PKC and CPI-17 dephosphorylation. This lipid emulsion-mediated inhibition of vasodilation may be partly associated with the lipid solubility of local anesthetics.

Keyword

Bupivacaine; CPI-17; Lipid emulsion; PDBu; PKC; Vasodilation

MeSH Terms

Anesthetics, Local
Animals
Aorta
Blotting, Western
Bupivacaine*
Calcium
Fura-2
In Vitro Techniques
Mepivacaine*
Muscle, Smooth, Vascular
Myosin-Light-Chain Phosphatase
Phorbol 12,13-Dibutyrate
Phosphorylation
Protein Kinase C
Rats
Solubility
Vasodilation*
Anesthetics, Local
Bupivacaine
Calcium
Fura-2
Mepivacaine
Myosin-Light-Chain Phosphatase
Phorbol 12,13-Dibutyrate
Protein Kinase C

Figure

• 

  Fig. 1 (A) The effect of Intralipid® (lipid emulsion) on bupivacaine-induced vasodilation in an isolated endothelium-denuded rat aorta precontracted with 10-6 M phorbol 12,13-dibutyrate (PDBu). All values are presented as the mean ± SD (n = 6) and are expressed as a percentage of the maximal contraction induced by PDBu. N is the number of rats from which thoracic aortic rings were obtained. Lipid emulsion (0.35 and 0.8%): *P < 0.001 versus control. (B) The effect of lipid emulsion on mepivacaine-induced vasodilation in an isolated endothelium-denuded rat aorta precontracted with 10-6 M PDBu. All values are presented as the mean ± SD (n = 7) and are expressed as a percentage of the maximal contraction induced by PDBu. N is the number of thoracic aortic rings.

• 

  Fig. 2 Effect of Intralipid® (lipid emulsion) on contraction induced by 10-6 M phorbol 12,13-dibutyrate (PDBu) in an isolated endothelium-denuded rat aorta. All values are presented as the mean ± SD (n = 5) and are expressed as a percentage of the maximal contraction induced by PDBu. N is the number of descending thoracic aortic rings.

• 

  Fig. 3 Effect of GF109203X on contraction induced by 10-6 M phorbol 12,13-dibutyrate (PDBu) in an isolated endothelium-denuded rat aorta. All values are presented as the mean ± SD (n = 6) and are expressed as a percentage of the maximal contraction induced by PDBu. N is the number of descending thoracic aortic rings. *P < 0.05, †P < 0.01 and #P < 0.001 versus control.

• 

  Fig. 4 (A) Effects of bupivacaine on the phorbol 12,13-dibutyrate (PDBu, 10-6 M)-stimulated intracellular calcium concentration ([Ca2+]i) (upper trace) and muscle tension (lower trace) in endothelium-denuded rat thoracic aortic strips. The [Ca2+]i of fura-2-loaded aortic strips was determined using a fluorometer and is expressed as the F340/F380 ratio. The value 100% represents the PDBu (10-6 M)-induced increases in both [Ca2+]i and muscle tension before the cumulative addition of bupivacaine. When [Ca2+]i and muscle tension induced by 10-6 M PDBu had reached steady-state levels, incremental concentrations of bupivacaine (10-6 to 10-3 M) were cumulatively added. WO: washout. (B) Cumulative concentration-response curve for bupivacaine in PDBu (10-6 M)-stimulated endothelium-denuded rat thoracic aortic strips. Incremental concentrations (10-6 to 10-3 M) of bupivacaine were cumulatively added during the sustained increases in [Ca2+]i and tension induced by PDBu (10-6 M). The value 100% represents the PDBu (10-6 M)-induced increases in both [Ca2+]i and muscle tension before the cumulative addition of bupivacaine. Each point represents the mean of five experiments, and the SD is indicated by the vertical bars. *P < 0.001 versus F340/F380.

• 

  Fig. 5 (A) The effects of bupivacaine (BPV), GF109203X, and combined treatment with Intralipid® (lipid emulsion, LE) and BPV on phorbol 12,13-dibutyrate (PDBu)-induced protein kinase C (PKC) phosphorylation in rat aortic vascular smooth muscle cells (VMSCs). VSMCs were treated with 10-6 M PDBu alone for 10 min, pretreated with 3 × 10-6 M GF109203X or 10-3 M BPV for 30 min, followed by treatment with 10-6 M PDBu for 10 min, or pretreated with LE (0.35 or 0.8%) for 1 h, followed by post-treatment with 10-3 M BPV for 30 min and a subsequent 10-min treatment with 10-6 M PDBu. PKC phosphorylation was examined by western blot analysis as described in the Materials and Methods section. The data are expressed as the mean ± SD (n = 4). N is the number of independent experiments. *P < 0.001 versus control. †P < 0.001 versus 10-6 M PDBu alone. ‡P < 0.001 versus combined treatment with 10-3 M BPV and 10-6 M PDBu. p-PKC: phosphorylated PKC. t-PKC, total PKC. (B) The effects of BPV, GF109203X, and combined treatment with LE and BPV on PDBu-induced phosphorylation-dependent inhibitory protein of myosin phosphatase (CPI-17) phosphorylation in rat aortic VMSCs. VSMCs were treated with 10-6 M PDBu alone for 20 min, pretreated with 3 × 10-6 M GF109203X or 10-3 M BPV for 30 min, followed by treatment with 10-6 M PDBu for 20 min, or pretreated with LE (0.35 or 0.8%) for 1 h, followed by post-treatment with 10-3 M BPV for 30 min and a subsequent 20-min treatment with 10-6 M PDBu. CPI-17 phosphorylation at Thr38 was examined by western blot analysis as described in the Materials and Methods section. The data are expressed as the mean ± SD (n = 6). N is the number of independent experiments. *P < 0.001 versus control. †P < 0.001 versus 10-6 M PDBu alone. ‡P < 0.001 versus combined treatment with 10-3 M BPV and 10-6 M PDBu. p-CPI-17: phosphorylated CPI-17, t-CPI-17: total CPI-17.

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