Korean J Pain.  2014 Jul;27(3):229-238. 10.3344/kjp.2014.27.3.229.

Bupivacaine-induced Vasodilation Is Mediated by Decreased Calcium Sensitization in Isolated Endothelium-denuded Rat Aortas Precontracted with Phenylephrine

• Ok SH
• Bae SI
• Kwon SC
• Park JC
• Kim WC
• Park KE
• Shin IW
• Lee HK
• Chung YK
• Choi MJ
• Sohn JT

• KMID: 2278228
• DOI: http://doi.org/10.3344/kjp.2014.27.3.229

Abstract

BACKGROUND
A toxic dose of bupivacaine produces vasodilation in isolated aortas. The goal of this in vitro study was to investigate the cellular mechanism associated with bupivacaine-induced vasodilation in isolated endotheliumdenuded rat aortas precontracted with phenylephrine.
METHODS
Isolated endothelium-denuded rat aortas were suspended for isometric tension recordings. The effects of nifedipine, verapamil, iberiotoxin, 4-aminopyridine, barium chloride, and glibenclamide on bupivacaine concentration-response curves were assessed in endothelium-denuded aortas precontracted with phenylephrine. The effect of phenylephrine and KCl used for precontraction on bupivacaine-induced concentration-response curves was assessed. The effects of verapamil on phenylephrine concentration-response curves were assessed. The effects of bupivacaine on the intracellular calcium concentration ([Ca2+]i) and tension in aortas precontracted with phenylephrine were measured simultaneously with the acetoxymethyl ester of a fura-2-loaded aortic strip.
RESULTS
Pretreatment with potassium channel inhibitors had no effect on bupivacaine-induced relaxation in the endothelium-denuded aortas precontracted with phenylephrine, whereas verapamil or nifedipine attenuated bupivacaine-induced relaxation. The magnitude of the bupivacaine-induced relaxation was enhanced in the 100 mM KCl-induced precontracted aortas compared with the phenylephrine-induced precontracted aortas. Verapamil attenuated the phenylephrine-induced contraction. The magnitude of the bupivacaine-induced relaxation was higher than that of the bupivacaine-induced [Ca2+]i decrease in the aortas precontracted with phenylephrine.
CONCLUSIONS
Taken together, these results suggest that toxic-dose bupivacaine-induced vasodilation appears to be mediated by decreased calcium sensitization in endothelium-denuded aortas precontracted with phenylephrine. In addition, potassium channel inhibitors had no effect on bupivacaine-induced relaxation. Toxic-dose bupivacaine- induced vasodilation may be partially associated with the inhibitory effect of voltage-operated calcium channels.

Keyword

aorta; bupivacaine; calcium sensitization; phenylephrine; vasodilation; verapamil

MeSH Terms

4-Aminopyridine
Animals
Aorta*
Barium
Bupivacaine
Calcium Channels
Calcium*
Glyburide
Nifedipine
Phenylephrine*
Potassium Channels
Rats*
Relaxation
Vasodilation*
Verapamil
4-Aminopyridine
Barium
Bupivacaine
Calcium
Calcium Channels
Glyburide
Nifedipine
Phenylephrine
Potassium Channels
Verapamil

Figure

• 

  Fig. 1 The effects of iberiotoxin (10-7 M, A), 4-aminopyridine (2 × 10-3 M, B), glibenclamide (10-5 M, C), and barium chloride (3 × 10-5 M, D) on bupivacaine-induced concentration-response curves in isolated endothelium-denuded aortas precontracted with 10-7 M phenylephrine. Data are the means ± SD expressed as the percentage of the maximal contraction induced by phenylephrine (100% = 2.90 ± 0.97 g [n = 8] and 100% = 2.80 ± 0.66 g [n = 8] for endothelium-denuded rings with control and 10-7 M iberiotoxin, respectively, in A; 100% = 2.42 ± 0.53 g [n = 9] and 100% = 2.49 ± 0.55 g [n = 9] for endothelium-denuded rings with control and 2 × 10-3 M 4-aminopyridine, respectively, in B; 100% = 2.43 ± 0.67 g [n = 8] and 100% = 2.53 ± 0.34 g [n = 8] for endothelium-denuded rings with control and 10-5 M glibenclamide, respectively, in C; 100% = 3.20 ± 0.40 g [n = 6] and 100% = 3.09 ± 0.24 g [n = 6] for endothelium-denuded rings with control and 3 × 10-5 M barium chloride, respectively, in D). N indicates the number of isolated descending thoracic aortic rings.

• 

  Fig. 2 The effects of verapamil (10-6 and 10-5 M; A) and nifedipine (10-9, 10-8 and 10-7 M; B) on bupivacaine-induced concentration-response curves in isolated endothelium-denuded aortas precontracted with 3 × 10-6 M phenylephrine. Data are the means ± SD expressed as the percentage of the maximal contraction induced by phenylephrine (100% = 2.42 ± 0.61 g [n = 6], 100% = 2.02 ± 0.41 g [n = 5], and 100% = 1.85 ± 0.38 g [n = 5] for endothelium-denuded rings with control, 10-6 M verapamil, and 10-5 M verapamil, respectively, in A; 100% = 2.81 ± 0.65 g [n = 8], 100% = 3.06 ± 0.45 g [n = 7], 100% = 3.01 ± 0.41 g [n = 6], and 100% = 2.50 ± 0.77 g [n = 6] for endothelium-denuded rings with control, 10-9 M nifedipine, 10-8 nifedipine, and 10-7 M nifedipine, respectively, in B). N indicates the number of isolated descending thoracic aortic rings. *P < 0.001, †P < 0.05, and ‡P < 0.01 versus control. §P < 0.01 versus 10-9 M nifedipine.

• 

  Fig. 3 Bupivacaine concentration-response curve in isolated endothelium-denuded aortas precontracted with 3 × 10-6 M phenylephrine or 100 mM KCl. Data are the means ± SD expressed as the percentage of the maximal contraction induced by the contractile agonist (100% = 2.85 ± 0.59 [n = 8] and 100% = 2.68 ± 0.34 g [n = 8] for endothelium-denuded rings with 3 × 10-6 M phenylephrine and 100 mM KCl, respectively). N indicates the number of isolated descending thoracic aortic rings. *P < 0.001 and †P < 0.01 versus 3 × 10-6 M phenylephrine. ‡P < 0.01 and §P < 0.001 versus 5.36 × 10-7 M bupivacaine.

• 

  Fig. 4 The effects of verapamil (10-6 and 10-5 M) on phenylephrine concentration-response curves in isolated endothelium-denuded aortas. Data are the means ± SD expressed as the percentage of the maximal contraction induced by isotonic 60 mM KCl (100% = 2.33 ± 0.45 g [n = 5], 100% = 2.78 ± 0.08 g [n = 5], and 100% = 2.67 ± 0.48 g [n = 5] for endothelium-denuded rings with control, 10-6 M verapamil and 10-5 M verapamil, respectively). N indicates the number of isolated descending thoracic aortic rings. *P < 0.001 versus control. †P < 0.01, ‡P < 0.001, and §P < 0.05 versus 10-6 M verapamil.

• 

  Fig. 5 (A) Effect of bupivacaine on phenylephrine (3 × 10-6 M)-stimulated intracellular calcium concentration ([Ca2+]i) (upper trace) and muscle tension (lower trace) in endothelium-denuded rat thoracic aortas. The [Ca2+]i of fura-2-loaded aortic strips was detected using a fluorometer and expressed as the ratio F340/F380. 100% represents the phenylephrine (3 × 10-6 M)-induced increases in both [Ca2+]i and muscle tension before the cumulative addition of bupivacaine. When the [Ca2+]i and muscle tension induced by phenylephrine reached a steady state level, various bupivacaine contents (5.36 × 10-6, 1.61 × 10-5, 5.36 × 10-5, 2.52 × 10-4 and 9.51 × 10-4 M) were cumulatively added. (B) Concentration-inhibition curve for bupivacaine in phenylephrine (3 × 10-6 M)-stimulated endothelium-denuded rat thoracic aortas. Various bupivacaine contents were cumulatively applied during the sustained increases in both [Ca2+]i and tension induced by phenylephrine (3 × 10-6 M). 100% represents the phenylephrine (3 × 10-6 M)-induced increase in both [Ca2+]i and muscle tension before the cumulative addition of bupivacaine. Each point represents the mean of 5 experiments, and SD is shown by vertical bars. *P < 0.05 and †P < 0.001 versus phenylephrine (3 × 10-6 M). ‡P < 0.001 versus F340/F380.

• 

  Fig. 6 (A) Effect of bupivacaine on high KCl (100 mM)-stimulated intracellular calcium concentration ([Ca2+]i) (upper trace) and muscle tension (lower trace) in endothelium-denuded rat thoracic aortas. The [Ca2+]i of fura-2-loaded aortic strips was detected using a fluorometer and expressed as the ratio F340/F380. 100% represents the 100 mM KCl-induced increases in both [Ca2+]i and muscle tension before the cumulative addition of bupivacaine. When the [Ca2+]i and muscle tension induced by high KCl reached a steady state level, various bupivacaine contents (5.36 × 10-6, 1.61 × 10-5, 5.36 × 10-5, 2.52 × 10-4 and 9.51 × 10-4 M) were cumulatively added. (B) Concentration-inhibition curve for bupivacaine in high KCl (100 mM)-stimulated endothelium-denuded rat thoracic aortas. Various bupivacaine contents were cumulatively applied during the sustained increases in both [Ca2+]i and tension induced by high KCl (100 mM). 100% represents 100 mM KCl-induced increase in both [Ca2+]i and muscle tension before the cumulative addition of bupivacaine. Each point represents the mean of 5 experiments, and SD is shown by vertical bars. *P < 0.05, †P < 0.001 and ‡P < 0.01 versus 100 mM KCl.

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