Isoflurane activates PKC and Ca2+-calmodulin-dependent protein kinase II via MAP kinase signaling in cultured vascular smooth muscle cells

Background: Protein kinase C (PKC) and Ca2+-calmodulin-dependent protein kinase II (CaMKII) have been implicated in isoflurane-increased force in skinned femoral arterial strips. The extracellular signal-regulated kinases (ERK1/2) of mitogen-activated protein kinase have been shown to be target effectors of PKC and Ca-MKII. This study examined the role of the ERK1/2 signaling pathway in isoflurane activation of PKC and CaMKII using cultured vascular smooth muscle cells. Methods: Vascular smooth muscle cells were prepared by cell migration from isolated rabbit femoral arterial segments. Growth of passage of vascular smooth muscle cells (80-90% confluence, passage 5-10) was arrested for 48 It before experiments, during which time phorbol 1,3-diaceylester treatment was used to down-regulate PKC. Cells were treated for 30 min with one of the inhibitors of mitogen-activated protein kinase kinase (PD98059), PKC (Go6976 and bisindolymaleimide), or CaMKII (KN-93 and KN-62) at 10 muM. After administration of isoflurane, vascular smooth muscle cells were frozen rapidly, homogenized, and centrifuged. The homogenates were used for identification of phosphorylated ERK1/2 or for further centrifugation to separate the membrane from the cytosol for identification of PKC isoforms (alpha and epsilon) by Western blotting. Results: Isoflurane increased ERK1/2 phosphorylation in a dose-dependent manner and reached a plateau at 10 min. PD98059 or down-regulated PKC blocked the increase of phosphorylated ERK1/2 levels by isoflurane, and bisindolylmaleimide, KN-93, or KN-62, but not by Go69-6 reduced levels of phosphorylated ERK1/2. The membrane fraction of PKCepsilon but not of PKCalpha was increased by isoflurane. Conclusions: ERK1/2 signaling Is downstream of PKC and CaMKII activated by isoflurane in vascular smooth muscle cells.