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Mepivacaine is an aminoamide local anesthetic that produces vasoconstriction in vivo and in vitro. The goals of this in vitro study were to determine whether mepivacaine-induced contraction involves calcium sensitization in isolated endothelium-denuded aortas, and to investigate the specific protein kinases involved. The effects of mepivacaine and potassium chloride on intracellular calcium concentrations ([Ca(2+)]i) and tension in the presence or absence of Y-27632 or GF 109203X were measured simultaneously using the acetoxymethyl ester of fura-2-loaded aortic strips. Cumulative mepivacaine concentration-response curves were generated in the presence or absence of the following inhibitors: Rho kinase inhibitor Y-27632, protein kinase C (PKC) inhibitor GF 109203X, extracellular signal-regulated kinase (ERK) inhibitor PD 98059, c-Jun NH2-terminal kinase (JNK) inhibitor SP600125, and p38 mitogen-activated protein kinase (MAPK) inhibitor SB 203580. Phosphorylation of PKC and MAPK, and membrane translocation of Rho kinase were detected in vascular smooth muscle cells by Western blotting. The slope of the mepivacaine-induced [Ca(2+)]i-tension curve was higher than that of the KCl-induced [Ca(2+)]i-tension curve. Pretreatment with Y-27632 or GF 109203X shifted the mepivacaine-induced [Ca(2+)]i-tension curve to the lower right. Pretreatment with Y-27632, GF 109203X, PD 98059, or SP600125 attenuated mepivacaine-induced contraction in a concentration-dependent manner. Y-27632 and GF 109203X attenuated mepivacaine-induced Rho kinase membrane translocation and PKC phosphorylation, respectively. PD 98059 and SP600125 attenuated mepivacaine-induced ERK and JNK phosphorylation, respectively. Taken together, these results indicate that mepivacaine-induced contraction involves increased calcium sensitization mediated by Rho kinase and PKC. Such contraction mainly involves activation of ERK- and JNK-mediated pathways., (© 2013 Published by Elsevier B.V.)

The goals of this study were to determine which lipid emulsion (Intralipid(®) and Lipofundin MCT/LCT(®)) is more effective in reversing high-dose levobupivacaine-induced reduced vasoconstriction in isolated rat aortas and to examine the associated cellular mechanisms with a particular focus on the endothelium. Two lipid emulsion concentration-response curves were generated using high-dose levobupivacaine-induced reduced vasoconstriction and vasodilation of isolated aortas pretreated with or without 60 mM KCl. Endothelial nitric oxide synthase (eNOS) and caveolin-1 phosphorylation were measured in rat aortic tissue treated with levobupivacaine in the presence or absence of lipid emulsion. Dichlorofluorescein oxidation, a measure of reactive oxygen species production, was measured in lipid emulsion-treated human umbilical vein endothelial cells. In levobupivacaine (0.3 mM)-induced reduced vasoconstriction of isolated aorta, the magnitude of the Intralipid(®)- and Lipofundin MCT/LCT(®)-mediated reversal was not significantly different. Lipid emulsion reversal of levobupivacaine-induced reduced vasoconstriction was greater in endothelium-intact aortas than in endothelium-denuded aortas. The two lipid emulsions similarly inhibited levobupivacaine-induced eNOS phosphorylation in aortic tissue. Pretreatment with both lipid emulsions increased dichlorofluorescein oxidation. Both Intralipid(®) and Lipofundin MCT/LCT(®) are equally effective for vascular tone recovery from high-dose levobupivacaine-induced reduced vasoconstriction. This reversal is mediated partially by decreasing nitric oxide bioavailability.

Mepivacaine is an aminoamide local anesthetic with an intermediate duration that intrinsically produces vasoconstriction both in vivo and in vitro. This study investigated the arachidonic acid metabolic pathways involved in mepivacaine-induced contraction, and elucidated the associated cellular mechanism with a particular focus on extracellular signal-regulated kinase (ERK) in endothelium-denuded rat aorta. Isolated rat thoracic aortic rings were suspended for isometric tension recording. Cumulative mepivacaine concentration-response curves were generated in the presence or absence of the following inhibitors: quinacrine dihydrochloride, nordihydroguaiaretic acid, phenidone, AA-861, indomethacin, NS-398, SC-560, fluconazole, PD 98059, and verapamil. Mepivacaine-induced ERK phosphorylation, 5-lipoxygenase (5-LOX) expression, and cyclooxygenase (COX)-2 expression in rat aortic smooth muscle cells were detected by Western blot analysis in the presence or absence of inhibitors. Mepivacaine produced tonic contraction in isolated endothelium-denuded rat aorta. Quinacrine dihydrochloride, nordihydroguaiaretic acid, phenidone, AA-861, NS-398, PD 98059, and verapamil attenuated mepivacaine-induced contraction in a concentration-dependent manner. However, fluconazole had no effect on mepivacaine-induced contraction. PD 98059, quinacrine dihydrochloride, nordihydroguaiaretic acid, AA-861, phenidone, and indomethacin attenuated mepivacaine-induced ERK phosphorylation. Mepivacaine upregulated 5-LOX and COX-2 expression. These results suggest that mepivacaine-induced contraction involves ERK activation, which is primarily mediated by the 5-LOX pathway and in part by the COX-2 pathway.

Background: The goal of this in vitro study was to investigate the effects of lipid emulsion (LE) on local anesthetic levobupivacaine-induced responses in isolated rat aorta and to determine whether the effect of LE is related to the lipid solubility of local anesthetics., Methods: Isolated rat aortic rings were suspended for isometric tension recording. The effects of LE were determined during levobupivacaine-, ropivacaine-, and mepivacaine-induced responses. Endothelial nitric oxide synthase and caveolin-1 phosphorylation was measured in human umbilical vein endothelial cells treated with levobupivacaine alone and with the addition of LE., Results: Levobupivacaine produced vasoconstriction at lower, and vasodilation at higher, concentrations, and both were significantly reversed by treatment with LE. Levobupivacaine and ropivacaine inhibited the high potassium chloride-mediated contraction, which was restored by LE. The magnitude of LE-mediated reversal was greater with levobupivacaine treatment than with ropivacaine, whereas this reversal was not observed in mepivacaine-induced responses. In LE-pretreated rings, low-dose levobupivacaine- and ropivacaine-induced contraction was attenuated, whereas low-dose mepivacaine-induced contraction was not significantly altered. Treatment with LE also inhibited the phosphorylation of endothelial nitric oxide synthase induced by levobupivacaine in human umbilical vein endothelial cells., Conclusions: These results indicate that reversal of levobupivacaine-induced vasodilation by LE is mediated mainly through the attenuation of levobupivacaine-mediated inhibition of L-type calcium channel-dependent contraction and, in part, by inhibition of levobupivacaine-induced nitric oxide release. LE-mediated reversal of responses induced by local anesthetics may be related to their lipid solubility.

Background: Levobupivacaine is a long-acting local anesthetic with a clinical profile similar to that of racemic bupivacaine but with a greater margin of safety. Levobupivacaine produces dose-dependent vasoconstriction in vivo. Our goal in this in vitro study was to investigate the role of pathways involved in arachidonic acid metabolism in the levobupivacaine-induced contraction of isolated rat aorta and to determine which endothelium-derived vasodilators are involved in the modulation of levobupivacaine-induced contraction., Methods: Rat thoracic aortic rings were isolated and suspended for isometric tension recording. Cumulative levobupivacaine dose-response curves over a range of 10(-6) to 3 x 10(-4) M were constructed in 1) aortic rings with no drug pretreatment; 2) endothelium-denuded rings pretreated with quinacrine dihydrochloride (nonspecific phospholipase A(2) inhibitor: 2 x 10(-5), 4 x 10(-5) M), nordihydroguaiaretic acid (NDGA) (lipoxygenase inhibitor: 10(-5), 3 x 10(-5) M), indomethacin (nonspecific cyclooxygenase inhibitor: 10(-5) M), AA-861 (5-lipoxygenase inhibitor: 10(-5), 5 x 10(-5) M), fluconazole (cytochrome P450 epoxygenase inhibitor: 10(-5) M), verapamil (10(-5) M), or calcium-free solution; and 3) endothelium-intact rings pretreated with N(omega)-nitro-L-arginine methyl ester (L-NAME) (nitric oxide synthase inhibitor: 5 x 10(-5) M), indomethacin, or fluconazole. Levobupivacaine-induced contractile response at each concentration (10(-4), 3 x 10(-4) M) was assessed in endothelium-denuded rings. Dose-response curves for potassium chloride in endothelium-denuded rings were generated in the presence or absence of NDGA and AA-861. Intracellular Ca(2+) levels were monitored by Ca(2+) image analysis using Fluo-4 fluorescence in vascular smooth muscle cells treated with levobupivacaine alone or AA-861 plus levobupivacaine., Results: Levobupivacaine produced a tonic contraction in isolated rat aorta rings; this response was maximal at 10(-4) M levobupivacaine and gradually attenuated at 3 x 10(-4) M levobupivacaine. Levobupivacaine-induced contractions of endothelium-denuded rings were larger than those of endothelium-intact rings. Levobupivacaine-induced contraction of endothelium-denuded rings was attenuated by quinacrine dihydrochloride, NDGA, AA-861, verapamil, and calcium-free solution and, to a lesser extent, by indomethacin. L-NAME enhanced levobupivacaine-induced contraction of endothelium-intact rings and indomethacin slightly attenuated this contraction. NDGA and AA-861 attenuated the potassium chloride-induced contraction. AA-861 attenuated the levobupivacaine-induced intracellular calcium increase in vascular smooth muscle cells., Conclusions: Our data indicate that levobupivacaine-induced contraction of rat aortic smooth muscle is mediated mainly by activation of the lipoxygenase pathway and in part by activation of the cyclooxygenase pathway. In addition, activation of the lipoxygenase pathway seems to facilitate calcium influx via L-type calcium channels. Endothelial nitric oxide attenuates levobupivacaine-induced contraction.

Tramadol, a combination of R(-) and S(+) enantiomers, inhibits both the acetylcholine-mediated response of muscarinic receptors and the muscarine-induced accumulation of cyclic guanosine monophosphate. Our goals in this in vitro study were to investigate the effects of tramadol on endothelium-dependent relaxation induced by acetylcholine, to determine whether this effect of tramadol is stereoselective, and to elucidate the associated cellular mechanism in rat aorta. In endothelium-intact rings precontracted with phenylephrine with or without naloxone, dose-response curves for acetylcholine, histamine, and calcium ionophore A23187 were generated in the presence and absence of tramadol (racemic, R(-) and S(+)). Sodium nitroprusside dose-response curves were generated in the presence and absence of racemic tramadol. Racemic tramadol (5 x 10(-5) 10(-4) M) attenuated acetylcholine-induced relaxation in the rings with or without naloxone. R(-) tramadol, 5 x 10(-5) M, attenuated acetylcholine-induced relaxation, whereas S(+) tramadol, 5 x 10(-5) M, did not. Racemic tramadol (10(-4) M) had no effect on dose-response curves for calcium ionophore A23187 or sodium nitroprusside. Taken together, these results indicate that tramadol, at a supraclinical dose (5 x 10(-5) M), stereoselectively attenuates endothelium-dependent relaxation via an inhibitory effect at levels proximal to nitric oxide synthase activation on a pathway involving nonspecific endothelial receptor activation.

In this in vitro study we examined the effects of diazepam on a phenylephrine-induced contraction in rat aorta and determined the associated cellular mechanism focusing on the endothelium-derived vasodilators. The concentration-response curves for phenylephrine and potassium chloride were generated in the presence or absence of diazepam. Phenylephrine concentration-response curves were generated from the endothelium-intact rings pretreated independently with N(W)-nitro-L-arginine methyl ester, PK 11195, tetraethylammonium, and indomethacin in the presence or absence of diazepam. Diazepam (7 x 10(-7) M) attenuated the phenylephrine-induced contraction in the endothelium-intact rings, whereas a large dose (5 x 10(-6) M) of diazepam attenuated the phenylephrine-induced contraction in the aortic rings with or without the endothelium. A pretreatment with the N(W)-nitro-L-arginine methyl ester completely abolished the diazepam (7 x 10(-7) M)-induced attenuation of the phenylephrine concentration-response curve, as well as the diazepam (5 x 10(-6) M)-induced attenuation of the maximal contractile response to phenylephrine. The N(W)-nitro-L-arginine methyl ester (10(-4) M)-induced contraction was enhanced in the rings pretreated with diazepam (5 x 10(-6) M). These results indicate that a supraclinical concentration of diazepam attenuates phenylephrine-induced contraction by increasing endothelial nitric oxide activity and directly affecting vascular smooth muscle.

The goals of this in vitro study were to investigate effects of etomidate on endothelium-dependent relaxation induced by acetylcholine in rat aorta, and to elucidate the associated cellular mechanism. In endothelium-intact rings precontracted with phenylephrine 10(-6) M, dose-response curves for acetylcholine (10(-9) to 10(-5) M) and calcium ionophore (10(-9) to 10(-6) M) were generated in the presence and absence of etomidate (5 x10(-6) 10(-5) M). In endothelium-intact or -denuded rings precontracted with phenylephrine 10(-6) M, sodium nitroprusside (10(-9) to 10(-6) M) dose-response curves were generated in the presence and absence of etomidate (10(-5)M). Etomidate (5 x10(-6), 10(-5)M) produced a significant rightward shift in the dose-response curves induced by acetylcholine (receptor-mediated endothelium-dependent agonist) and calcium ionophore A23187 (non receptor-mediated endothelium-dependent agonist). Etomidate (10(-5)M) had no effect on sodium nitroprusside (endothelium-independent nitric oxide donor)-induced vasorelaxant response in both endothelium-intact and -denuded rings. These results indicate that etomidate at clinically relevant concentrations attenuates endothelium-dependent relaxation induced by acetylcholine by an acting at a site distal to the endothelial muscarinic receptor, but proximal to guanylate cyclase activation of vascular smooth muscle in rat aorta.

Background: Previous study has shown that fentanyl attenuates acetylcholine-induced vasorelaxation. The goal of the current in vitro study was to identify the muscarinic receptor subtype that is mainly involved in the fentanyl-induced attenuation of endothelium-dependent relaxation elicited by acetylcholine., Methods: The effects of fentanyl and muscarinic receptor antagonists on the acetylcholine concentration-response curve were assessed in aortic vascular smooth muscle ring preparations precontracted with phenylephrine. In the rings pretreated independently with pirenzepine, 4-diphenylacetoxyl-N-methylpiperidine methiodide, and naloxone, acetylcholine concentration-response curves were generated in the presence and absence of fentanyl. The effect of fentanyl on the concentration-response curve for calcium ionophore A23187 was assessed., Results: Fentanyl (0.297 x 10 0.785 x 10 m) attenuated acetylcholine-induced vasorelaxation in ring preparations with or without 10 m naloxone. Pirenzepine (10 to 10 m) and 4-diphenylacetoxyl-N-methylpiperidine methiodide (10 to 10 m) produced a parallel rightward shift in the acetylcholine concentration-response curve. The concentrations (-log M) of pirenzepine and 4-diphenylacetoxyl-N-methylpiperidine methiodide necessary to displace the concentration-response curve of an acetylcholine by twofold were estimated to be 6.886 +/- 0.070 and 9.256 +/- 0.087, respectively. Methoctramine, 10 m, did not alter the acetylcholine concentration-response curve. Fentanyl, 0.785 x 10 m, attenuated acetylcholine-induced vasorelaxation in the rings pretreated with 10 m pirenzepine but had no effect on vasorelaxation in the rings pretreated with 10 m 4-diphenylacetoxyl-N-methylpiperidine methiodide. Fentanyl, 0.785 x 10 m, did not significantly alter calcium ionophore A23187-induced vasorelaxation., Conclusions: These results indicate that fentanyl attenuates acetylcholine-induced vasorelaxation via an inhibitory effect at a level proximal to nitric oxide synthase activation on the pathway involving endothelial M3 muscarinic receptor activation in rat aorta.