ABSTRACT
We have reported previously that interferon-alpha (IFN-alpha) induces apoptosis that is counteracted by an epidermal growth factor (EGF) --> Ras --> extracellular signal-regulated kinase (ERK)-dependent survival response in human epidermoid cancer KB cells. We have studied the effects of the cytokine on the cAMP-dependent pathway in these cells. A decrease in the intracellular cAMP levels was recorded in KB cells treated with IFN-alpha, whereas forskolin induced an increase in the production of cAMP that was reduced in the presence of IFN-alpha, suggesting a reduction in the activity of adenylate cyclase (AC) induced by IFN-alpha. These effects were paralleled by significant change in the expression of some AC catalytic subunit(s) and by reduction in the activity of protein kinase A (PKA). 8-Br-cAMP completely antagonized the reduction of PKA activity induced by IFN-alpha, whereas PKA inhibitor KT5720 enhanced the reduction of the enzyme activity induced by IFN-alpha. We have found that IFN-alpha induced a decrease in cAMP response element binding protein (CREB) phosphorylation without changes in its total expression. The concomitant treatment with IFN-alpha and 8-Br-cAMP potentiated and KT5720 counteracted apoptosis induced by IFN-alpha alone. In conclusion, these data suggest that the decrease in AC/cAMP pathway activity is a survival response to the apoptosis induced by IFN-alpha. Therefore, this pathway could represent a target to enhance the antitumor activity of IFN-alpha.
Subject(s)
Adenylyl Cyclases/metabolism , Apoptosis/drug effects , Carcinoma, Squamous Cell/enzymology , Immunologic Factors/pharmacology , Interferon-alpha/pharmacology , Signal Transduction/drug effects , 8-Bromo Cyclic Adenosine Monophosphate/pharmacology , Carbazoles/pharmacology , Cell Line, Tumor , Cyclic AMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic AMP-Dependent Protein Kinases/metabolism , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Gene Expression Regulation, Enzymologic/drug effects , Humans , Indoles/pharmacology , Pyrroles/pharmacologyABSTRACT
The adenylate cyclase (AC)/cAMP/cAMP-dependent protein kinase pathway controls many biological phenomena. The molecular mechanisms by which cAMP induces alternative commitment towards differentiation or proliferation are not still completely known. The differentiation of myoblast cell lines into myocytes/myotubes represents a well-established model of skeletal muscle differentiation. We analyzed the AC/cAMP pathway during terminal differentiation of H9c2 myoblasts. When cultured in low-serum containing medium, H9c2 myoblasts exit the cell cycle and differentiate into myocytes/myotubes. A key step of this process is the expression of myogenin, an essential transcription factor for the terminal differentiation into myocytes. During this phenomenon we observed a decrease in both cAMP levels and AC activity, which suggests a functional negative role of cAMP on the differentiation process of H9c2 cells. 8-Br-cAMP and other cAMP-elevating agents, such as forskolin, IBMX, and isoproterenol, negatively affected skeletal muscle differentiation of H9c2 myoblasts. Both AC activity down-regulation and intracellular cAMP reduction were accompanied by significant variations in the levels of membrane proteins belonging to the AC system (AC catalytic subunit, G(alphai-1), G(alphas)). The functional relationship between intracellular cAMP content and protein levels of AC system is discussed.
Subject(s)
Adenylyl Cyclases/physiology , Cell Differentiation/physiology , Cyclic AMP/analysis , Myoblasts, Cardiac/physiology , Animals , Blotting, Western , Cell Line , Cyclic AMP/physiology , Fluorescent Antibody Technique , Intracellular Fluid/chemistry , Muscle, Skeletal/physiology , Myogenin/biosynthesis , RatsABSTRACT
The effect of nontoxic, low concentrations (10(-8) M) of retinoic acid (RA) for a relatively long time (28 days) on a Kirsten ras-virus transformed cell line (Ki-SVC1), derived from the rat seminal vesicle epithelium, was investigated. In these experimental conditions, the cell treatment with RA induced a decrease of the proliferation rate, apoptosis and a marked reduction of both anchorage-independent growth and tumorigenicity. These biological responses were either preceded or associated with important changes in adenylate cyclase/protein kinase C signaling pathways, the activation of important apoptosis-linked genes and a marked decrease of the v-Ki-ras p21 protein. The significance of these findings is discussed.
Subject(s)
Antineoplastic Agents/pharmacology , Proto-Oncogene Proteins p21(ras)/metabolism , Tretinoin/pharmacology , Adenylyl Cyclases/metabolism , Animals , Apoptosis/drug effects , Cell Division/drug effects , Cell Line, Transformed , Cell Membrane/drug effects , Cell Membrane/metabolism , Cyclic AMP/analysis , Cytosol/drug effects , Cytosol/metabolism , Dose-Response Relationship, Drug , Down-Regulation , Hemangiosarcoma/pathology , Neoplasm Transplantation , Protein Kinase C/metabolism , Proto-Oncogene Proteins p21(ras)/analysis , Proto-Oncogene Proteins p21(ras)/genetics , RNA, Messenger/analysis , Rats , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/drug effectsABSTRACT
We have found that in the secretion of rat anterior prostate, a hydrolyzing activity on GTP is present with a high affinity for the substrate; ATP, GDP, and ADP are not substrates for enzymatic activity. At the same time we have shown that GTP is a negative modulator for the well-known type IV transglutaminase activity present in the prostatic secretion. The hydrolyzing activity on GTP appears to be due to two molecular species: a high-molecular-weight GTPase, having electrophoretical mobility higher than 100 kDa, and a low-molecular-weight GTPase, of about 30 kDa. The two enzymatic activities are associated in the prostatic secretion with the transglutaminase (type IV). We describe an experimental procedure to separate them.
Subject(s)
GTP Phosphohydrolases/metabolism , Prostate/metabolism , Transglutaminases/metabolism , Animals , Chromatography, Affinity , Chromatography, Gel , Electrophoresis, Polyacrylamide Gel , GTP Phosphohydrolases/chemistry , GTP Phosphohydrolases/isolation & purification , Guanosine Triphosphate/metabolism , Hydrolysis , Male , Molecular Weight , Prostate/enzymology , Rats , Rats, Wistar , Transglutaminases/isolation & purificationABSTRACT
Steroid sulphatase deficiency is a feature of recessive X-linked ichthyosis (RXLI) that causes the accumulation of sulphated steroids (SS) in various organs and cells. In a previous study, we detected elevated cholesterol sulphate (CS) and dehydroepiandrosterone sulphate (DHEAS) serum levels in a group of 15 RXLI patients selected in a narrow age range. In the present study both CS and DHEAS serum levels were qualitatively and quantitatively determined using gas-chromatographic analysis in a group of 33 RXLI patients ranging in age from 3 to 70 years. The levels of CS and DHEAS were significantly increased in all patients. Variations in SS were related both to patients' ages and clinical course of the disease; Serum SS levels start to increase in early infancy, peak at puberty, remain elevated in adults and decrease slightly in the elderly.
Subject(s)
Cholesterol Esters/blood , Dehydroepiandrosterone Sulfate/blood , Ichthyosis, X-Linked/blood , Adolescent , Adult , Age Factors , Aged , Child , Child, Preschool , Chromatography, Gas , Humans , Male , Middle AgedABSTRACT
Stably transfected Balb-C 3T3 fibroblasts (clone 5), overexpressing a catalytically active tissue transglutaminase, showed a basal adenylate cyclase activity lower than control cells (clone 1). Several modulators of the adenylate cyclase activity (forskolin, Mn2+ and pertussis toxin) showed the existence of a marked negative control on the adenylate cyclase activity present in clone 5 cells. Very interestingly, this same marked negative control was also found in a Balb-C 3T3 fibroblast clone stably transfected with a mutagenized human tissue transglutaminase (mut277 cys > ser) virtually devoid of transglutaminase catalytic activity (clone Ser). Conversely, a significant increase of the adenylate cyclase activity was observed in bovine aortic endothelial cells after the lowering of tissue transglutaminase expression levels by the transfection of an eukaryotic expression vector containing the gene for tissue transglutaminase in antisense orientation. All these findings suggest a possible role for type II tissue transglutaminase as a negative modulator of the adenylate cyclase activity in different cell types, beside its transglutaminase enzyme activity.
Subject(s)
Adenylyl Cyclase Inhibitors , Transglutaminases/biosynthesis , 3T3 Cells , Animals , Blotting, Western , Cattle , Cell Membrane/enzymology , Cross-Linking Reagents , Endothelium, Vascular/enzymology , Gene Expression , Humans , Mice , Mice, Inbred BALB C , Mutagenesis , Polymerase Chain Reaction , Transfection , Transglutaminases/geneticsABSTRACT
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 alpha i negative control of the enzyme, even though the alpha i of transformed cells appears to possess fully functional domains interacting with both the effector enzyme and the agonist-activated receptor.
Subject(s)
Adenylyl Cyclases/metabolism , Cell Transformation, Viral/genetics , Genes, ras , Protein Kinase C/metabolism , Seminal Vesicles/enzymology , Adenosine Diphosphate Ribose/metabolism , Adenylate Cyclase Toxin , Animals , Colforsin/pharmacology , Enzyme Activation , Epithelial Cells , Epithelium/enzymology , Epithelium/metabolism , GTP-Binding Proteins/metabolism , Male , Rats , Seminal Vesicles/cytology , Seminal Vesicles/drug effects , Signal Transduction , Virulence Factors, Bordetella/pharmacologyABSTRACT
Effects of Ca2+ on adenosine 3',5'-cyclic monophosphate (cAMP)-mediated Cl- secretion were investigated in intact mucosa and isolated crypt cells of rabbit descending colon. Addition of 10 microM prostaglandin (PG)E2 or forskolin to tissues incubated in Ca(2+)-free medium increased the size of short-circuit current (Isc) and Cl- secretion as estimated by unidirectional 36Cl flux measurements (net flux = -2.31 +/- 0.24 vs. -1.22 +/- 0.10 mueq.h-1.cm-2, n = 4, P < 0.001). Addition of 10 microM PGE2 to tissues incubated in 1.2 mM Ca2+ Ringer induced a 7-fold increase in mean cAMP level, whereas it produced an 11-fold increase in tissues exposed to Ca(2+)-free medium. Membrane preparations from whole mucosa incubated in Ca(2+)-free medium displayed a cyclic nucleotide phosphodiesterase activity significantly lower than controls (18.76 +/- 0.54 vs. 31.20 +/- 0.39 pmol cAMP. mg protein-1.min-1, means +/- SE, n = 4, P < 0.001). Ca2+ removal also affected adenylate cyclase (AC) responsiveness to agonists; AC activity increased in controls by 54 and 226% after stimulation with 10 microM PGE2 and forskolin, respectively, but it increased more (77 and 325%, respectively) after incubation in Ca(2+)-free solutions.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Calcium/physiology , Chlorides/metabolism , Colon/metabolism , Cyclic AMP/metabolism , Intestinal Mucosa/metabolism , Adenylyl Cyclases/metabolism , Animals , Electrophysiology , Male , Osmolar Concentration , Phosphoric Diester Hydrolases/metabolism , RabbitsABSTRACT
Literature reports that patients affected by X-linked ichthyosis (XLI) have a reduction of sweat glands and a decrease of sweat production. The sweat physiology of 28 patients, 14 with XLI, 7 with lamellar ichthyosis, 7 with dominant ichthyosis and 28 control subjects were examined with sweat test, performed by pilocarpine iontophoresis. In the same patients we have performed skin biopsy to evaluate quantitative and qualitative reduction of sweat glands.
Subject(s)
Ichthyosis, X-Linked/physiopathology , Sweating , Adolescent , Adult , Biopsy , Child , Child, Preschool , Female , Humans , Ichthyosiform Erythroderma, Congenital/physiopathology , Ichthyosis Vulgaris/physiopathology , Iontophoresis , Male , Pilocarpine , Sweat Glands/pathologyABSTRACT
The metabolic basis of X-linked ichthyosis is a deficiency of steroid sulphatase, a microsomal enzyme which removes sulphate groups from sulphated steroids. We report on a carefully controlled group of 15 patients with recessive X-linked ichthyosis, selected in a narrow age range (22-33 years), in whom, through the use of gas chromatographic analysis and conventional radioimmunoassay, we have measured not only elevated serum cholesterol sulphate levels but also significantly elevated serum dehydroepiandrosterone sulphate levels. The latter finding has been controversial in previous reports. We believe that the radioimmunoassay procedure generally used should be held responsible for such controversy since it often gives rise to false positive and/or false negative values. Gas chromatography, although more exacting, appears to be far more reliable for the assessment of elevated serum dehydroepiandrosterone.
Subject(s)
Dehydroepiandrosterone/analogs & derivatives , Ichthyosis, X-Linked/blood , Acetylation , Adult , Aging/metabolism , Arylsulfatases/deficiency , Cholesterol/blood , Chromatography, Gas , Dehydroepiandrosterone/blood , Dehydroepiandrosterone Sulfate , Humans , Male , Radioimmunoassay , Steroids/blood , Steryl-SulfataseABSTRACT
The authors report on the occurrence of ocular abnormalities in X-linked ichthyosis (XLI) patients, in their carrier mothers and in healthy volunteers who served as controls. The diagnosis of XLI was based on: (1) demonstration of steroid sulfatase deficiency in cultured skin fibroblasts; (2) lack of hybridization of patient's deoxyribonucleic acid (DNA) with specific steroid sulfatase complementary DNA probe; (3) electrophoretic mobility of plasma lipoproteins. Cholesterol sulfate plasma levels were also determined. The incidence of corneal opacities was the same in XLI patients and in their carrier mothers (23.7 and 24.3%, respectively). Neither other corneal nor ophthalmological alterations were found. Moreover, in XLI patients the plasma levels of cholesterol sulfate were about twenty times higher than in controls. Our findings demonstrate that ocular changes do not seem to be an absolute criterion for a definite diagnosis of XLI and the fact that the pathogenesis of corneal opacities is not due to an accumulation of cholesterol sulfate, but rather that this compound probably induces physicochemical changes of the corneal tissue properties.
Subject(s)
Corneal Opacity/genetics , Genes, Recessive/genetics , Genetic Carrier Screening , Ichthyosis, X-Linked/genetics , Adolescent , Adult , Child , Child, Preschool , Cholesterol Esters/blood , Corneal Opacity/diagnosis , Humans , Ichthyosis, X-Linked/diagnosisSubject(s)
Adenylyl Cyclases/metabolism , Protein Kinase C/physiology , Signal Transduction/physiology , Alprostadil/pharmacology , Blood Platelets/ultrastructure , Calcium/pharmacology , Cell Membrane/metabolism , Colforsin/pharmacology , Enzyme Activation/drug effects , Humans , Phospholipids/pharmacology , Signal Transduction/drug effectsABSTRACT
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.
Subject(s)
Cell Transformation, Viral , Genes, ras , Harvey murine sarcoma virus , Protein Kinase C/metabolism , Sarcoma Viruses, Murine , Thyroid Gland/enzymology , Animals , Biological Transport , Calcium/pharmacology , Cell Membrane/enzymology , Chromatography , Cytoplasm/enzymology , Diglycerides/pharmacology , Enzyme Activation/drug effects , Epithelium/enzymology , Harvey murine sarcoma virus/genetics , Mutation , Phorbol 12,13-Dibutyrate/metabolism , Phosphatidylserines/pharmacology , Phosphorylation , Protein Kinase C/genetics , Rats , Sarcoma Viruses, Murine/genetics , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
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.
Subject(s)
Adenylyl Cyclases/analysis , Oncogenes , Thyroid Gland/enzymology , Animals , Colforsin/pharmacology , Cyclic AMP/analysis , Epithelium/enzymology , Guanylyl Imidodiphosphate/pharmacology , Kinetics , Magnesium/pharmacology , Manganese/pharmacology , Oncogene Protein p21(ras) , Oncogene Proteins, Viral/physiology , Rats , Rats, Inbred F344 , Sodium Fluoride/pharmacologyABSTRACT
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.
Subject(s)
Adenylyl Cyclases/blood , Erythrocyte Membrane/enzymology , Transglutaminases/blood , Adenosine Triphosphate/blood , Adenylyl Cyclase Inhibitors , Animals , Calcium/pharmacology , Carbon Radioisotopes , Caseins/blood , Columbidae , Kinetics , Methyltransferases/blood , Phosphatidyl-N-Methylethanolamine N-Methyltransferase , Phosphatidylethanolamine N-Methyltransferase , Phosphorus Radioisotopes , Spermidine/bloodABSTRACT
Porins interact with macrophage membranes and inhibit their phagocyting activity. We have tested the porin effect on a biologically relevant membrane-bound enzymic activity, the adenylate cyclase system, which appears to be stimulated both in the presence of Mn2+ and Mg2+ or Mg2+ + Gpp(NH)p. Moreover, for mice macrophages incubated in the presence of porins, there is an increase in the intracellular cAMP content after 5 min of incubation, with a maximum after 15 min of incubation. The results shown suggest that the porin effects on the adenylate cyclase can represent the molecular basis of the porin-dependent inhibition of the macrophages phagocytosis. Our point of view, which proposes a cAMP role in inhibiting the phagocyting activity in macrophages, is supported also by the results of the experiments carried out in the presence of both dibutyryl-cAMP or aminophylline. The phagocyting activity is inhibited in all cases and independently of the bacteria to be phagocyted.
Subject(s)
Adenylyl Cyclases/metabolism , Bacterial Outer Membrane Proteins/pharmacology , Macrophages/physiology , Phagocytosis/drug effects , Aminophylline/pharmacology , Animals , Bacterial Outer Membrane Proteins/isolation & purification , Bucladesine/pharmacology , Cyclic AMP/metabolism , Guanylyl Imidodiphosphate/pharmacology , Ion Channels/physiology , Kinetics , Macrophages/enzymology , Magnesium/pharmacology , Mice , PorinsABSTRACT
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).
Subject(s)
3',5'-Cyclic-AMP Phosphodiesterases/metabolism , Lidocaine/pharmacology , Myocardium/enzymology , Procainamide/pharmacology , Sarcolemma/enzymology , Animals , Fluorescence Polarization , In Vitro Techniques , Intracellular Membranes/enzymology , Male , Membrane Fluidity/drug effects , Rats , Rats, Inbred StrainsABSTRACT
Glycosylated proteins, glycosylated albumin and HbA1 were studied in type I and type II diabetes. Glycosylated proteins were evaluated by a new method: aminophenylboronic acid affinity chromatography. A good correlation was found between total HbA1 and glycosylated proteins in both groups (r = 0.57, p less than 0.05; r = 0.67, p less than 0.01, respectively), but a positive correlation between stable HbA1 and glycosylated proteins was present only in maturity onset diabetics (r = 0.71, p less than 0.01). Glycosylated proteins correlated with glycosylated albumin only in type II diabetes (r = 0.67, p less than 0.01). We hypothesize that in maturity onset diabetes glycosylated proteins and HbA1 reflect a greater glycemic stability while in insulin-dependent diabetes the same proteins reflect a different periodic pattern of glycemic control. Moreover, our data suggest that aminophenylboronic acid affinity chromatography is a suitable tool for routine monitoring of metabolic control in maturity onset diabetes, while further investigations are needed to establish if this method is a useful index of altered glucose metabolism in type I diabetes.
Subject(s)
Blood Proteins/analysis , Diabetes Mellitus, Type 1/metabolism , Glycoproteins , Serum Albumin/analysis , Boronic Acids , Chromatography, Affinity/methods , Diabetes Mellitus/drug therapy , Glycated Hemoglobin/analysis , Glycation End Products, Advanced , Humans , Hypoglycemic Agents/therapeutic use , Glycated Serum Proteins , Glycated Serum AlbuminABSTRACT
The soluble guanylate cyclase activity of rat liver appears to be stimulated in VITRO by insulin at pMolar concentrations, while proinsulin, denaturated insulin or desoctapeptide insulin, are not able to stimulate the studied enzymic activity. Corresponding concentrations of other peptide hormones such as corticotropin (ACTH) or glucagon, either in the absence or in the presence of bacitracin, do not show any effect on the investigated enzymic system. Insulin stimulation of the soluble guanylate cyclase is characterized by a significant increase in the Vmax together with a decrease of the apparent Km. Insulin at low concentrations doesn't affect the cyclic GMP hydrolyzing activity; conversely higher concentrations of the hormone, while exerting a less marked effect on the guanylate cyclase activity, inhibit the cyclic GMP hydrolyzing activity.