Isoflurane activates PKC and Ca(2+) -calmodulin-dependent protein kinase II via MAP kinase signaling in cultured vascular smooth muscle cells.

Abstract

Protein kinase C (PKC) and Ca(2+)-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 CaMKII. This study examined the role of the ERK1/2 signaling pathway in isoflurane activation of PKC and CaMKII using cultured vascular smooth muscle cells. 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 h 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 bisindolylmaleimide), or CaMKII (KN-93 and KN-62) at 10 microm. 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. 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 Go6976 reduced levels of phosphorylated ERK1/2. The membrane fraction of PKC epsilon but not of PKC alpha was increased by isoflurane. ERK1/2 signaling is downstream of PKC and CaMKII activated by isoflurane in vascular smooth muscle cells.