G. Illiano

Increased adenylate cyclase activity in rat thyroid epithelial cells expressing viral ras genes

The activity of the adenylate cyclase catalytic subunit is higher in Harvey and Kirsten Murine Sarcoma Viruses-infected thyroid epithelial cells than in uninfected control cells either in the presence of Mg2+ alone or following stimulation by Mn2+ or forskolin. The higher activity is associated with an increased cAMP cellular content. The Gpp(NH)p and F- anion are more effective positive modulators in the control than in the virus infected cells: these results exclude therefore that the ras p21 proteins can act as the G-protein alpha-subunit and suggest that they negatively interfere with the G-protein modulation of the adenylate cyclase system.

Protein kinase C activities are increased in rat thyroid epithelial cells expressing V-ras genes

Both cytoplasmic and membrane-bound protein kinase C activities are increased in: Harvey-Sarcoma Virus, infected thyroid epithelial cells. The cytoplasmic kinase C increase is found in the chromatographic fraction eluted at lower salt concentration (100 mM NaCl-S100), while the more acidic protein fraction eluted at higher salt concentration (35 mM NaCl-S350) is virtually absent. Although the cytoplasmic S100 fraction from the control and ras-virus infected cells display a comparable PBt2 binding activity, they are different in the Ca+2-dependence and the TPA down regulation. In addition, the membranes from the control and ras-virus infected cells are different phosphate acceptors in place of the H1 histones.

ras oncogene-induced transformation of a rat seminal vesicle epithelial cell line produces a marked increase of adenylate cyclase and protein kinase C activities

Cells transformed by Kirsten murine sarcoma virus (Ki‐MSV) have basal adenylate cyclase activity (AC) higher than control cells and comparable level of forskolin‐stimulated AC activity. Moreover, a higher protein kinase C (PKC) activity was found to be present in the transformed cells. The molecular mechanism underlying the increase of AC activity was investigated. Our findings strongly suggest that this biochemical event is due to a marked decrease of the αi negative control of the enzyme, even though the αi of transformed cells appears to possess fully functional domains interacting with both the effector enzyme and the agonist‐activated receptor.

Relationship between the fluidity of the membrane lipids and the activity of the membrane-bound proteins: The effects of lidocaine on the adenylate cyclase activity of rat myocardium

The lidocaine effects on the basal adenylate cyclase activity of the studied membranes depend on the substrate concentration: the drug has an inhibiting effect on non-saturating ATP concentrations, but displays a stimulating effect on the saturating ones. In the presence of maximally stimulating NaF concentrations, lidocaine further stimulates the adenylate cyclase activity through all the ATP concentrations used. In the presence of ATP saturating concentrations, the adenylate cyclase activity stimulated by various Gpp(NH)p concentrations, is inhibited in the presence of lidocaine as well as the enzymic activity stimulated by several l-isoproterenol concentrations in the presence of a fixed Gpp(NH)p concentration.

Endotoxin inhibits the fluoride-stimulated adenylate cyclase activity of rat liver plasma membranes enriched with bile canaliculi

Escherichia coli endotoxin inhibited the fluoride-stimulated adenylate cyclase activity of liver plasma membranes enriched with bile canaliculi. Inhibition was of a mixed competitive and uncompetitive type. This effect, which may result from changes of membrane organization, may have relevance in the understanding of endotoxin-cell membrane interactions.

Inhibition of adenylate cyclase by transglutaminase-catalyzed reactions in pigeon erythrocyte ghosts

We report the occurrence in pigeon erythrocytes of a soluble Ca2+-dependent transglutaminase (TGase) activity. The effect of the erythrocyte ghost protein modifications, determined by TGase-catalyzed reactions, on adenylate cyclase, phospholipid methyltransferase I and II activities and on the lipidic matrix fluidity of the membrane was investigated by using a purified guinea pig liver TGase preparation. The results showed a significant inhibitory effect of such modifications both on the basal and on the variously stimulated (by NaF, Gpp(NH)p alone or in the presence of 1-isoproterenol) adenylate cyclase activity. By contrast, both the phospholipid methylation and the fluidity of the lipidic matrix of the membrane were unaffected by TGase-mediated reactions. These data suggest a new possible inhibitory mechanism of the cyclic AMP synthesis which might be triggered by the enhancement of the cytosolic Ca2+ concentration.

The effects of polyamines on the cyclic amp efflux and metabolism in E. coli b cells

1. 1. The effects of polyamines spermine, spermidine and putrescine have been investigated on the adenylate cyclase and phosphodiesterase activities bound to E. coli B-cell membranes. 2. 2. Putrescine, which is physiologically present in bacteria, affects the cAMP metabolism in a coordinate manner, depressing the adenylate cyclase and stimulating the phosphodiesterase activities. In vivo this is reflected by a decreased cAMP efflux toward the external medium. 3. 3. Spermidine stimulates the phosphodiesterase activities but has no effect on the adenylate cyclase; furthermore no effect was observed in vivo on the cAMP efflux. li[4. Spermine stimulates both the enzymic activities but with a prevailing effect on the adenylate cyclase. In vivo the effect is an increase in the cAMP efflux. 4. 5. In vivo the intracellular cAMP content is anyway not modified in presence of polyamines, in contrast with (and may be because of) the broad variation of the nucleotide efflux toward the external medium.

Protein Kinase C and G Proteins

In crude preparations of human platelet membranes we have shown a PKC-mediated phosphorylation both of ai and a G protein subunits. The experiments have been carried out in conditions of carefully controlled phosphatase activities.

The effects of lidocaine and procainamide on the sarcolemmal membrane high affinity cyclic AMP phosphodiesterase of rat myocardium.

The incubation of rat myocardial sarcolemmal membranes with 10(-4)M lidocaine or procainamide results in a decreased fluorescence polarization of the extrinsic probes (diphenylhexatriene and 12-anthroylstearate) and in the stimulation of the membrane-bound high affinity cyclic AMP phosphodiesterase activity (PDE). Lidocaine or procainamide do not contrast, but facilitate the aminophylline inhibitory action on the PDE activity, without modifying the basic molecular mechanism of the inhibitory action. The amplitudes of the effects described are inversely correlated to the temperature, being greater at the lower tested temperature (25 degrees greater than 30 degrees greater than 37 degrees C).

A stimulating effect of guanyl nucleotides on the rat-liver soluble cyclic GMP high-affinity phosphodiesterase activity.

The high affinity (low K m) cyclic GMP phosphodiesterase (PDE) is activated by GTP, while the cyclic AMP PDE is not. GTP and its hydrolysis‐resistant analogue, guanylylimidodiphosphate (GppNHp), display a half‐maximal stimulating effect at almost the same concentration (5 × 10−6M). The GTP stimulating effect is not observed when the socalled cyclic GMP low affinity (high K m) PDE is operative. GTP cooperates with the increase of the substrate concentration on removing the IBMX inhibitory effect. The isolation through a classical chromatographic procedure on a DEAE‐cellulose column, of a PDE fraction specific for cyclic GMP, results in the loss of the GTP stimulating effect.