Adaptations of the beta-adrenoceptor-adenylyl cyclase system in rat skeletal muscle to endurance physical training.

beta-Adrenergic mechanisms may be important in the adaptation of skeletal muscle to endurance training. beta-Adrenergic signal transduction was examined in the gastrocnemius muscle of rats submitted to a progressive, 12-week treadmill running program and compared with sedentary controls. beta-Adrenoceptor density was significantly lower in exercised rats than in controls. The affinity constant for [125I]-(-) iodocyanopindolol binding was not different among the various groups. Adenosine cyclic monophosphate formation was significantly decreased in trained animals when isoproterenol plus guanosine triphosphate or forskolin plus Mn2+ were used to stimulate adenylyl cyclase. Immunoblot analyses revealed that the amount of the alpha-subunit of stimulatory guanine nucleotide-binding protein (Gs,alpha), both the small and the large isoforms, also decreased with physical exercise. Thus, the present report shows that endurance training results in alterations in beta-adrenergic receptor density, adenylyl cyclase activity and Gs protein level in rat gastrocnemius muscle.

Regulation of nerve growth factor secretion and mRNA expression by bacterial lipopolysaccharide in primary cultures of rat astrocytes.

The present work was undertaken to study the effect of bacterial lipopolysaccharide (LPS), a potent activator of the host inflammatory response, on the synthesis of nerve growth factor (NGF) by newborn rat brain astrocytes. Treatment of primary rat astroglial cells cultured in chemically defined medium with LPS resulted in a dose-dependent accumulation of NGF mRNA, and an increased release of NGF protein in the cell medium. NGF mRNA levels were maximal after 24 hr of stimulation (8-fold increase), whereas extracellular NGF peaked after 72 hours of treatment (17-fold increase). This dramatic increase of extracellular NGF was abrogated if cells were treated with actinomycin D or cycloheximide, a fact which implies that the accumulation of extracellular NGF by LPS-treated cells requires DNA transcription and RNA translation. Stimulation of NGF synthesis and secretion was: (i) unaffected by treatment with the protein kinase C inhibitor bisindolylmaleimide, and (ii) prevented by forskolin and 3-isobutyl-1-methylxanthine, two agents which increase cAMP levels. Inhibition of LPS effect was also obtained with apigenin, a proposed inhibitor of the mitogen-activated protein kinase pathway. Results thus show that LPS stimulates NGF synthesis by astroglial cells through a mechanism that is independent of protein kinase C (PKC), antagonized by cAMP-elevating agents, and probably mediated by the mitogen-activated protein kinase cascade. The data raise the possibility that LPS exerts stimulatory effects on NGF synthesis that are independent of those elicited by astrocyte-derived inflammatory lymphokines such as IL-1beta, TNF alpha or TGF beta1.

cAMP signalling mechanisms with aging in the Ceratitis capitata brain.

Aging has been associated with alterations in protein phosphorylation. This study was undertaken to examine eventual changes in cAMP-dependent protein kinase (PKA) activity and enzyme regulatory subunit levels from the dipterous Ceratitis capitata brain with postmaturational aging and senescence. PKA activity was determined in cytosolic and membrane fractions of the C. capitata brain during the adult stage of the insect lifespan. PKA activity markedly increased at the first stages of the life of the fly both in cytosol and in membranes. A lower peak of PKA activity was evident both in particulate and cytosolic fractions in the terminal phase of the life of the fly. Thus, PKA activity was significantly higher in the brain of mature flies when compared to the brain of aged flies. It is possible that increases in cAMP-dependent protein phosphorylation levels characterize the terminal aging process in the insect nervous tissue. On the other hand, levels of regulatory (R) subunit were also measured in membranes and cytosol by immunoblotting. Cytosolic regulatory subunit levels were more elevated near the terminal phase of life, whereas in membranes, regulatory subunit levels decrease in senescence in parallel with particulate PKA activity. The increased R subunit level in cytosol may reflect a cellular response mechanisms to down-regulate the kinase system in aged flies.

Addition of phosphatidylcholine-phospholipase C induces cellular redistribution and phosphorylation of protein kinase C zeta in C 6 glial cells.

Phosphatidylcholine breakdown has been shown to play a critical role in signal transduction involving generation of a number of second messengers [Exton, J.H., Biochim. Biophys. Acta, 1212 (1994) 26-42]. In the present report we demonstrate by immunofluorescence that short-treatment of C 6 glial cells with phosphatidylcholine-hydrolyzing phospholipase C (PC-PLC), changes the intracellular localization of protein kinase C (PKC) zeta from the cytoplasm to a perinuclear region. Western blot analysis also showed a redistribution of PKC zeta after incubation of cells with PC-PLC. To test whether these changes were accompanied by an activation of the enzyme, we measured the extent of phosphorylation of PKC zeta by immunoprecipitation from 32P-labelled cells. Short-treatment with PC-PLC resulted in enhanced phosphorylation of the higher Mr PKC zeta in C 6 glial cells.

Levels and activity of brain protein kinase C alpha and zeta during the aging of the medfly.

Brain protein kinase C (PKC) activity, as well as PKC alpha and PKC zeta levels detected by immunoblotting, were monitored during the lifespan of the Mediterranean fruit-fly Ceratitis capitata. PKC activity increased in the particulate fraction during the last stages of the life of C. capitata. Immunoblotting studies with an anti-PKC alpha antibody also demonstrated increased enzyme levels in the particulate fraction. Cytosolic levels of PKC zeta decreased in the terminal phase of the lifespan of the fly, whereas levels of membrane-bound PKC zeta increased at that stage. Results thus indicate that during C. capitata final phase of life a translocation of PKC alpha and PKC zeta to the particulate fraction occurs, and therefore both kinases could be involved in the terminal process of this fruit-fly.