Krebs solution contained the nitric oxide synthase inhibitor NW-nitro-L-arginine methyl Naringenin ester (LNAME, 10 5 M) to prevent the release of endogenous nitric oxide from any residual endothelium. In the preliminary study, L-NAME (10 5 M) had no effect on levobupivacaine-induced contraction in endothelium-denuded aortae (data not shown). 2.3. Study design The effect of protein kinase inhibitors on the cumulative levobupivacaine concentration (10 6 to 3×10 4 M)–response curves in endothelium-denuded aortae was assessed by comparing the contractile response in the presence or absence of the following protein kinase inhibitors: PKC inhibitor GF 109203X (10 6, 3×10 6, 10 5 M); Rho-kinase inhibitor Y-27632 (10 7, 3×10 7, 10 6 M); tyrosine kinase inhibitor genistein parthenolide (3×10 6, 10 5, 5×10 5 M); c-Jun NH2- terminal kinase (JNK) inhibitor SP600125 (10 6, 3×10 6, 10 5 M); extracellular signal-regulated kinase (ERK) inhibitor PD 98059 (3×10 6, 10 5, 3×10 5 M); and p38 MAPK inhibitor SB 203580 (10 6, 10 5, 3×10 5 M).

In all cases, the inhibitor was added to the organ bath 20 min before the addition of levobupivacaine. A subsequent concentration of levobupivacainewas added after the buy SNX-5422 previous concentration elicited a sustained and stable contraction. Inhibitor concentrations were chosen on the basis of concentrations previously shown to be effective in similar preparations (Abdel-Latif, 2001; Filipeanu et al., 1995; Lee et al., 2006, 2007; Rohra et al., 2004). 2.4. Cell culture Vascular smooth muscle cells (VSMCs) were isolated from the thoracic aortae of male rats by enzymatic dissociation and grown in Dulbecco’s modified Eagle’s medium supplemented with 10% heatinactivated fetal bovine serum, 2 mML-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (Ok et al., 2011). Cells were subcultured twice per week by harvesting with trypsin/ethylenediaminetetraacetic acid and seeding in 7.5×105/mm2 flasks. For experiments, cells between passage numbers 2 and 10 were seeded into dishes (107/100-mm dish), fed every other day, and used at confluence (6–7 days).

Cells were deprived of serum overnight prior to treatment. 2.5. Western blot analysis purchase Naringin Western blot analysis was performed according to the method described by Park et al. (2008). Membrane and cytosolic fractions were isolated from cells using a Mem-PER  Eukaryotic membrane protein extraction reagent kit (Pierce, Rockford, IL, USA) according to the manufacturer’s specifications. Cells were lysed in PRO-PREP protein extract solution to obtain total cell lysates, and the lysates were centrifuged at 100,000 ×g for 20 min at 4 °C. Protein concentration was determined using the Bradford method. For preparation of sample loading, equal volumes of 2 sodium dodecyl sulfate sample buffer (0.1 mol/l Tris–HCI, 20% glycerol, 4% sodium dodecyl sulfate, and 0.01% bromophenol blue) and supernatant fractions from the lysates were mixed. Proteins (60 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis for 90 min at 110 V.

The separated proteins were transferred to polyvinylidene difluoride membranes for 2 h at Gastrointestinal physiology 20 mA using SD Semi-dry Transfer Cells (Bio-Rad Laboratories, Hercules, CA, USA). After blocking the membranes using 5% nonfat milk in Tris-buffed saline (pH 7.0).