Aminoamide local anesthetics induce vasoconstriction in vivo and in vitro. The goals of this in vitro study were to investigate the potency of local anesthetic-induced vasoconstriction and to identify the physicochemical property (octanol/buffer partition coefficient, pKa, molecular weight, or potency) of local anesthetics that determines their potency in inducing isolated rat aortic ring contraction. Cumulative concentration-response curves to local anesthetics (levobupivacaine, ropivacaine, lidocaine, and mepivacaine) were obtained from isolated rat aorta. Regression analyses were performed to determine the relationship between the reported physicochemical properties of local anesthetics and the local anesthetic concentration that produced 50% (ED(50)) of the local anesthetic-induced maximum vasoconstriction. We determined the order of potency (ED(50)) of vasoconstriction among local anesthetics to be levobupivacaine > ropivacaine > lidocaine > mepivacaine. The relative importance of the independent variables that affect the vasoconstriction potency is octanol/buffer partition coefficient > potency > pKa > molecular weight. The ED(50) in endothelium-denuded aorta negatively correlated with the octanol/buffer partition coefficient of local anesthetics (r(2) = 0.9563; P < 0.001). The potency of the vasoconstriction in the endothelium-denuded aorta induced by local anesthetics is determined primarily by lipid solubility and, in part, by other physicochemical properties including potency and pKa.

The goals of this study were to examine the cellular signaling pathways associated with the phosphorylation of caldesmon, the phosphorylation-dependent inhibitory protein of myosin phosphatase (CPI-17), and the 20-kDa regulatory light chain of myosin (MLC20) induced by levobupivacaine in isolated rat aortas. The effects of genistein, tyrphostin 23, GF109203X, PD98059, Y-27632, 1-butanol, and ML-7 HCl on levobupivacaine-induced contraction were assessed. The effect of genistein on the simultaneous calcium-tension curves induced by levobupivacaine was examined. The effects of GF109203X, genistein, PD98059 and extracellular signal-regulated kinase (ERK) siRNA on levobupivacaine-induced caldesmon phosphorylation were investigated. The effect of genistein on the ERK and tyrosine phosphorylation induced by levobupivacaine was examined. The effect of GF109203X, PD98059, Y-27632, SP600125, and ML-7 HCl on the levobupivacaine-induced phosphorylation of CPI-17 and MLC20 were investigated. Genistein, tyrphostin 23, GF109203X, PD98059, Y-27632, ML-7 HCl, and 1-butanol attenuated levobupivacaine-induced contraction. Genistein caused a right downward shift of the calcium-tension curves induced by levobupivacaine. Genistein attenuated levobupivacaine-induced phosphorylation of protein tyrosine, ERK and caldesmon. PD98059, ERK siRNA and GF109203X attenuated levobupivacaine-induced caldesmon phosphorylation. GF109203X, Y-27632, SP600125, ML-7 HCl and PD98059 attenuated CPI-17 phosphorylation and MLC20 phosphorylation induced by levobupivacaine. These results suggest that levobupivacaine-induced caldesmon phosphorylation contributing to levobupivacaine-induced contraction is mediated by a pathway involving ERK, which is activated by tyrosine kinase or protein kinase C (PKC). The phosphorylation of CPI-17 and MLC20 induced by levobupivacaine is mediated by cellular signaling pathways involving PKC, Rho-kinase, and c-Jun NH2-terminal kinase or PKC, Rho-kinase, ERK, and myosin light chain kinase.

This study aimed to examine the effect of lipid emulsion (LE) on the vasoconstriction induced by dexmedetomidine (DMT) in the isolated rat aorta and elucidate the associated cellular mechanism. The effect of LE, NW-nitro-L-arginine methyl ester (L-NAME), and methyl-β-cyclodextrin (MβCD) on the DMT-induced contraction was examined. We investigated the effect of LE on the DMT-induced cyclic guanosine monophosphate (cGMP) formation and DMT concentration. The effect of DMT, LE, 4-Amino-3-(4-chlorophenyl)-1-(t-butyl)-1H-pyrazolo[3,4-d]pyrimidine,4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), and rauwolscine on the phosphorylation of endothelial nitric oxide synthase (eNOS), caveolin-1, and Src kinase was examined in the human umbilical vein endothelial cells. L-NAME, MβCD, and LE (1%, standardized mean difference (SMD): 2.517) increased the DMT-induced contraction in the endothelium-intact rat aorta. LE (1%) decreased the DMT (10-6 M) concentration (SMD: -6.795) and DMT-induced cGMP formation (SMD: -2.132). LE (1%) reversed the DMT-induced eNOS (Ser1177 and Thr496) phosphorylation. PP2 inhibited caveolin-1 and eNOS phosphorylation induced by DMT. DMT increased the Src kinase phosphorylation. Thus, LE (1%) enhanced the DMT-induced contraction by inhibition of NO synthesis, which may be caused by the decreased DMT concentration. DMT-induced NO synthesis may be caused by the increased eNOS (Ser1177) phosphorylation and decreased eNOS (Thr495) phosphorylation potentially mediated by Src kinase-induced caveolin-1 phosphorylation.

Dexmedetomidine is a highly selective α(2)-adrenoceptor agonist that is widely used for sedation and analgesia during the perioperative period. Intravenous administration of dexmedetomidine induces transient hypertension due to vasoconstriction via the activation of the α(2)-adrenoceptor on vascular smooth muscle. The goal of this in vitro study is to investigate the calcium-dependent mechanism underlying dexmedetomidine-induced contraction of isolated endothelium-denuded rat aorta.

Mepivacaine induces contraction or decreased blood flow both in vivo and in vitro. Vasoconstriction is associated with an increase in the intracellular calcium concentration ([Ca(2+)]i). However, the mechanism responsible for the mepivacaine-evoked [Ca(2+)]i increase remains to be determined. Therefore, the objective of this in vitro study was to examine the mechanism responsible for the mepivacaine-evoked [Ca(2+)]i increment in isolated rat aorta.

This study aims to examine the effect of linolenic acid on the vasodilation or vasoconstriction induced by acetylcholine and bupivacaine in isolated rat aortae and its underlying mechanism. The effect of linolenic acid on the vasodilation induced by acetylcholine, the calcium ionophore A23187, sodium nitroprusside, and 8-bromoguanosine 3',5'-cyclic monophosphate sodium salt (bromo-cyclic guanosine monophosphate [bromo-cGMP]) in endothelium-intact and endothelium-denuded aortae was examined. Linolenic acid inhibited vasodilation induced by acetylcholine, calcium ionophore A23187, and sodium nitroprusside. However, this fatty acid increased bromo-cGMP-induced vasodilation in endothelium-denuded aortae. Linolenic acid increased bupivacaine-induced contraction in endothelium-intact aortae, whereas it decreased bupivacaine-induced contraction in endothelium-intact aortae with Nω-nitro-l-arginine methyl ester and endothelium-denuded aortae. Linolenic acid inhibited acetylcholine- and bupivacaine-induced phosphorylation of endothelial nitric oxide synthase. Sodium nitroprusside increased cGMP in endothelium-denuded aortic strips, whereas bupivacaine decreased cGMP in endothelium-intact aortic strips. Linolenic acid decreased cGMP levels produced by bupivacaine and sodium nitroprusside. Together, these results suggest that linolenic acid inhibits acetylcholine-induced relaxation by inhibiting a step just prior to nitric oxide-induced cGMP formation. In addition, linolenic acid-mediated inhibition of vasodilation induced by a toxic concentration (3 × 10-4 M) of bupivacaine seems to be partially associated with inhibition of the nitric oxide-cGMP pathway.

Dexmedetomidine, a highly selective α-2 adrenoceptor agonist, produces vasoconstriction, which leads to transiently increased blood pressure. The goal of this study was to investigate specific protein kinases and the associated cellular signal pathways responsible for the increased calcium sensitization induced by dexmedetomidine in isolated rat aortas, with a particular focus on phosphorylation-dependent inhibitory protein of myosin phosphatase (CPI-17). The effect of Y-27632 and chelerythrine on the dexmedetomidine-induced intracellular calcium concentration ([Ca2+]i) and tension were assessed using fura-2-loaded aortic strips. The effects of rauwolscine, Y-27632, chelerythrine, and ML-7 hydrochloride on the dexmedetomidine-induced phosphorylation of CPI-17 or of the 20-kDa regulatory light chain of myosin (MLC20) were investigated in rat aortic vascular smooth muscle cells. The effects of rauwolscine, Y-27632, and chelerythrine on the membrane translocation of Rho-kinase and protein kinase C (PKC) phosphorylation induced by dexmedetomidine were assessed. Y-27632 and chelerythrine each reduced the slopes of the [Ca2+]i-tension curves of dexmedetomidine-induced contraction, and Y-27632 more strongly reduced these slopes than did chelerythrine. Rauwolscine, Y-27632, chelerythrine, and ML-7 hydrochloride attenuated the dexmedetomidine-induced phosphorylation of CPI-17 and MLC20. Taken together, these results suggest that dexmedetomidine-induced contraction involves calcium sensitization, which appears to be mediated by CPI-17 phosphorylation via Rho-kinase or PKC.

Levobupivacaine is a long-acting amide local anesthetic that intrinsically produces vasoconstriction both in vivo and in vitro. Levobupivacaine increases intracellular calcium concentrations ([Ca(2+)](i)) in vascular smooth muscle cells. The goals of this in vitro study were to investigate whether levobupivacaine-induced contraction is associated with increased Ca(2+) sensitivity and to identify the protein kinases involved in mediating contraction in response to levobupivacaine in isolated rat aortic smooth muscle. The effect of levobupivacaine and potassium chloride (KCl) on the [Ca(2+)](i) and tension was measured simultaneously with acetoxymethyl ester of fura-2-loaded aortic strips. Cumulative levobupivacaine concentration-response curves were generated in the presence or absence of the following antagonists: GF 109203X; Y-27632; genistein; SP600125; PD 98059; and SB 203580. Levobupivacaine-induced protein kinase C (PKC), extracellular signal-regulated kinase (ERK), and c-Jun NH(2)-terminal kinase (JNK) phosphorylation and Rho-kinase (ROCK-2) membrane translocation were detected in rat aortic vascular smooth muscle cells using Western blotting. The slope of the [Ca(2+)](i)-tension curve for levobupivacaine was higher than that for KCl. Y-27632, GF 109203X, and SP600125 attenuated levobupivacaine-induced contraction in a concentration-dependent manner. Genistein, PD 98059, and SB 203580 attenuated levobupivacaine-induced contraction. Pretreatment with GF 109203X and Y-27632 inhibited levobupivacaine-induced PKC phosphorylation and Rho-kinase (ROCK-2) membrane translocation, respectively. Pretreatment with SP600125 or PD 98059 attenuated the levobupivacaine-induced phosphorylation of JNK and ERK, respectively. These results indicate that levobupivacaine-induced contraction involving an increase in myofilament Ca(2+) sensitivity involves the primary activation of Rho-kinase-, PKC-, and JNK-mediated pathways of rat aortic smooth muscle.

Vasoconstriction mediated by the highly selective alpha-2 adrenoceptor agonist dexmedetomidine leads to transiently increased blood pressure and severe hypertension. The dexmedetomidine-induced contraction involves the protein kinase C (PKC)-mediated pathway. However, the main PKC isoform involved in the dexmedetomidine-induced contraction remains unknown. The goal of this in vitro study was to examine the specific PKC isoform that contributes to the dexmedetomidine-induced contraction in the isolated rat aorta. The endothelium-denuded rat aorta was suspended for isometric tension recording. Dexmedetomidine dose-response curves were generated in the presence or absence of the following inhibitors: the pan-PKC inhibitor, chelerythrine; the PKC-α and -β inhibitor, Go6976; the PKC-α inhibitor, safingol; the PKC-β inhibitor, ruboxistaurin; the PKC-δ inhibitor, rottlerin; the c-Jun NH2-terminal kinase (JNK) inhibitor, SP600125; and the myosin light chain kinase inhibitor, ML-7 hydrochloride. Western blot analysis was used to examine the effect of rottlerin on dexmedetomidine-induced PKC-δ expression and JNK phosphorylation in rat aortic vascular smooth muscle cells (VSMCs) and to investigate the effect of dexmedetomidine on PKC-δ expression in VSMCs transfected with PKC-δ small interfering RNA (siRNA) or control siRNA. Chelerythrine as well as SP600125 and ML-7 hydrochloride attenuated the dexmedetomidine-induced contraction. Go6976, safingol, and ruboxistaurin had no effect on the dexmedetomidine-induced contraction, whereas rottlerin inhibited the dexmedetomidine-induced contraction. Dexmedetomidine induced PKC-δ expression, whereas rottlerin and PKC-δ siRNA transfection inhibited dexmedetomidine-induced PKC-δ expression. Dexmedetomidine also induced JNK phosphorylation, which was inhibited by rottlerin. Taken together, these results suggest that the dexmedetomidine-induced contraction involves PKC-δ-dependent JNK phosphorylation in the isolated rat aorta.

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.