RESUMO
Abstract Type V adenylyl cyclase (AC) was stably over-expressed in HEK293 cells (293AC-V). Forskolin-stimulated cAMP accumulation in 293AC-V was 5 times as great as that in control cells. PMA, a protein kinase C (PKC) activator, enhanced cAMP accumulation in 293AC-V cells dose-and time-dependently and this enhancement was abolished by staurosporine. Insulin also enhanced cAMP accumulation in 293AC-V cells. Co-transfection of PKC-zeta, but not PKC-alpha, potentiated the effects of insulin. These data suggest that type V AC activity is regulated in cells by PKC isoenzymes through different extracellular stimuli.
Assuntos
Adenilil Ciclases/metabolismo , Isoenzimas/metabolismo , Rim/citologia , Proteína Quinase C/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Adenilil Ciclases/genética , Alcaloides/farmacologia , Células Cultivadas , AMP Cíclico/biossíntese , AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Insulina/farmacologia , Rim/embriologia , Dados de Sequência Molecular , Proteína Quinase C/antagonistas & inibidores , Estaurosporina , Fatores de Tempo , TransfecçãoRESUMO
It is now accepted that caveolin plays a key role in signal transduction by directly binding to and regulating the function of molecules involved in transmembrane signaling, such as ras, suggesting that the amount of caveolin within cells may be an important factor in determining the cellular signaling. We investigated the ontogenic changes in the protein amount of caveolin subtypes, as well as ras protein expression in various organs (the heart, lungs, and muscles) obtained from aging rats (neonates, young and old adults). Our results demonstrated that caveolin protein expression changed ontogenically in a subtype-dependent manner. In lungs, for example, caveolin-1 expression changed in an opposite manner to caveolin-3 expression, while in the heart caveolin-1 and -3 changed in parallel. Ras expression showed an ontogenic increase in lungs and a decrease in muscles, which were both parallel to caveolin-1 expression. Our results suggest that the regulation of transmembrane signaling by caveolin may differ among developmental stages and caveolin subtypes.
Assuntos
Envelhecimento/fisiologia , Caveolinas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Animais , Animais Recém-Nascidos , Western Blotting , Caveolina 1 , Caveolina 2 , Caveolina 3 , Centrifugação com Gradiente de Concentração , Pulmão/metabolismo , Músculos/metabolismo , Miocárdio/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , RatosRESUMO
An insect ovarian cell, Spodoptera frugiperda (Sf9), has been widely used to express recombinant proteins, including adenylyl cyclase, as a host cell in the baculovirus expression system. We report the presence and characterization of a soluble adenylyl cyclase (sAC) distinct from a membrane-bound form of adenylyl cyclase (mAC) that is also present in Sf9 cells. sAC was purified 3,500-fold to near homogeneity; a single band at 25 kDa on SDS-polyacrylamide gel electrophoresis correlated well with adenylyl cyclase catalytic activity. The purified enzyme had a catalytic activity of 0.1 micromol/min.mg and the Km of 0.55 mM for the substrate ATP. In contrast to mAC, sAC was heat-stable. Enzymatic activity of sAC was not stimulated by forskolin and was inhibited by salts at high concentrations. sAC utilized both manganese- and magnesium-ATP as substrate. Di- or triphosphate-containing nucleotides, such as GTP and GDP, as well as pyrophosphate, noncompetitively inhibited sAC. Our data suggest that the physical and biochemical characteristics of sAC are different from those of mAC in Sf9 cells as well as from those of other known forms of adenylyl cyclase in animal cells; sAC in Sf9 cells may constitute a new member of adenylyl cyclase found in animals.