Phosphatidylinositol 3-kinase activity is required for the expression of glial fibrillary acidic protein upon cAMP-dependent induction of differentiation in rat C6 glioma

Glial fibrillary acidic protein (GFAP) is an intermediate filament (IF) protein expressed upon maturation of astrocytes and upregulated during reactive astrogliosis. Its expression is modulated by several growth factors and hormones. Although an upregulation of intracellular cAMP is required for the induction of GFAP expression in astrocytes, little information is available on other downstream factors of the signal transduction pathways involved in the regulation of its expression. In this communication, we identified phosphatidylinositol 3-kinase (PI 3-K) as a necessary enzyme for GFAP expression in rat C6 glioma cells. Use of the specific PI 3-K inhibitors wortmannin and LY294002 and transfection of C6 cells with a dominant negative PI 3-K construct, resulting in a decrease of the enzymatic activity of PI 3-K, inhibited the cAMP-dependent expression of GFAP. Furthermore, confocal laser scanning microscopy demonstrated that inhibition of the PI 3-K activity by LY294002 or wortmannin concomitant with induction of differentiation changes the cellular distribution leading to a pericentrosomal localization of GFAP and an altered cell shape lacking process formation. We conclude that the expression and cellular distribution of GFAP is mediated through a PI 3-K-dependent mechanism.     

Improvement in cardiac function of diabetic rats by bosentan is not associated with changes in the activation of PKC isoforms

We previously demonstrated that chronic treatment with the mixed endothelin A and B (ET_A and ET_B) receptor blocker bosentan improved isolated working heart function in streptozotocin (STZ) diabetic rats. Endothelin-1 (ET-1) peptide levels, ET-1 mRNA and ET_A and ET_B receptor mRNA were all increased in diabetic hearts, but were unaffected by bosentan treatment, indicating that the beneficial effects of bosentan on heart appear to be on downstream effectors of ET-1 and ET receptors rather than the ET-1 system itself. Stimulation of ET-1 receptors leads to increased activation of protein kinase C (PKC), which is associated with PKC translocation from the cytosol to the membrane. Persistent activation of specific PKC isoforms has been proposed to contribute to diabetic cardiomyopathy. The purpose of this study was to determine whether chronic treatment with bosentan influences the activation of PKC isoforms in hearts from diabetic rats. Male Wistar rats were divided into four groups: control, bosentan-treated control, diabetic, and bosentan-treated diabetic. Diabetes was induced by the intravenous injection of 60 mg/kg streptozotocin. One week later, treatment with bosentan (100 mg/kg/day) by oral gavage was begun and continued for 10 weeks. The heart was then removed, homogenized, separated into soluble (cytosolic) and particulate (membrane) fractions and PKC isoform content in each fraction was determined by Western blotting. PKC α, β2, δ, ε and ζ were all detected in hearts from both control and diabetic rats. However, no change in the levels or distribution between the soluble and particulate fractions of any of these isoforms could be detected in chronic diabetic hearts compared to control, whether untreated or treated with bosentan. These observations indicate that bosentan does not improve cardiac performance in STZ diabetic rats by affecting the activation of PKC isoforms.