ABSTRACT
Phosphodiesterase inhibition by papaverine is likely to play a minor role in the rat atrial inotropic response because non-significant changes in cyclic AMP were obtained. Isoprenaline however raised the nucleotide levels three-fold and up to seventeen-fold when papaverine was added in a similar preparation. A prominent effect of papaverine was to lengthen relaxation instead of shortening it as did isoprenaline. The results suggest different sites of action, although overlapping effects cannot be excluded.
Subject(s)
Cyclic AMP/metabolism , Myocardial Contraction/drug effects , Myocardium/metabolism , Papaverine/pharmacology , Animals , Electric Stimulation , Female , In Vitro Techniques , Isoproterenol/pharmacology , Rats , Stimulation, ChemicalABSTRACT
The existence of a GTP-binding protein of the Ns type in Trypanosoma cruzi was explored. Epimastigote membranes were labelled by cholera toxin in the presence of [adenine-14C]NAD+. After SDS/polyacrylamide-gel electrophoresis of extracted membrane proteins, a single labelled polypeptide band of apparent Mr approx. 45,000 was detected. Epimastigote cells were treated with N-ethylmaleimide and electrofused to lymphoma S49 cells lacking the Ns protein. Evidence indicates that in such electrofusion-generated cell hybrids a heterologous adenylate cyclase system was reconstituted with the Ns protein provided by T. cruzi epimastigotes.
Subject(s)
GTP-Binding Proteins/metabolism , Trypanosoma cruzi/metabolism , Adenylyl Cyclases/metabolism , Cell Fusion , Cell Membrane/drug effects , Cell Membrane/metabolism , Cholera Toxin/pharmacology , Electricity , Hybrid Cells/metabolism , Lymphoma/metabolism , Trypanosoma cruzi/drug effectsABSTRACT
Trypanosoma cruzi adenylate cyclase catalytic subunits may interact with regulatory factors from rat liver membranes, reconstituting heterologous systems which are catalytically active in assay mixtures containing MgATP. The systems show stimulatory responses to glucagon and guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) or fluoride. Reconstitution was obtained by three different methods: fusion of rat liver membranes (pretreated with N-ethylmaleimide) to T. cruzi membranes; interaction of detergent extracts of rat liver membranes with T. cruzi membranes; or interaction of purified preparations of T. cruzi adenylate cyclase and of liver membrane factors in phospholipid vesicles. The liver factors responsible for the guanine nucleotide effect were characterized as the NS protein. Data also indicate that reconstitution requires the presence of a membrane substrate.
Subject(s)
Adenylyl Cyclases/metabolism , Liver/metabolism , Trypanosoma cruzi/enzymology , Animals , Cell Membrane/drug effects , Cell Membrane/enzymology , Cholera Toxin/pharmacology , Glucagon/pharmacology , Liver/drug effects , NAD/pharmacology , Neurospora crassa/enzymology , Rats , Sodium Fluoride/pharmacologyABSTRACT
Adenylate cyclase activity associated with Trypanosoma cruzi sedimentable fractions was solubilized by treatment with the non-ionic detergent Lubrol PX and 0.5 M-(NH4)2SO4. The following hydrodynamic and molecular parameters were established for a partially purified enzyme-detergent complex: sedimentation coefficient 6.2 S; Stokes radius 5.65 nm; partial specific volume 0.83 ml/g; Mr 244 000; frictional ratio 1.33. A Mr of about 124 000 was calculated for the detergent-free protein from these parameters. The pI of this enzyme activity was 6.2. A monoclonal antibody to T. cruzi adenylate cyclase was obtained, which inhibited cyclase activities from several lower eukaryotic organisms. The T. cruzi adenylate cyclase was further purified by using this antibody in immunoaffinity chromatographic columns. Fractions obtained after this chromatography showed, on SDS/polyacrylamide-gel electrophoresis, a main polypeptide band with an apparent Mr of about 56 000, which specifically reacted with the monoclonal antibody.
Subject(s)
Adenylyl Cyclases/isolation & purification , Trypanosoma cruzi/enzymology , Adenylyl Cyclases/immunology , Animals , Antibodies, Monoclonal , Chromatography, Affinity , Clone Cells , Electrophoresis, Polyacrylamide Gel , Isoelectric Focusing , Molecular WeightABSTRACT
Right atria from rats were analyzed for chronotropic responses to phenylephrine in face of various drugs and procedures. Propranolol, 10(-8) M, produced a competitive antagonism against the agonist which concentration-effect curve was closely similar to that obtained from reserpinized animals. Prazosin, but not phentolamine (both 10(-6) M) showed inhibition of the phenylephrine-induced changes in heart rate, as judged by their -log EC50. Either of the alpha-adrenoceptor antagonists exhibited a greater steepness in the curve slope with respect to control. The simultaneous exposure of tissues to phentolamine and propranolol proved to effectively antagonize the chronotropic effect of the agonist. This held true for phentolamine assayed in atria from reserpine-pretreated rats. Previous incubation of tissues with papaverine, 10(-5) M, brought about supersensitivity to phenylephrine which was thoroughly inhibited by either phentolamine or propranolol. These results strongly suggest that beta-adrenoceptor stimulation of heart rate by phenylephrine takes place indirectly via norepinephrine release. There is also alpha 1-adrenoceptor stimulation (blocked by prazosin). Finally, it is hypothesized that supersensitivity develops by papaverine-enhanced Ca2+ influx, since numerous evidences are against a phosphodiesterase inhibition-dependent cAMP accumulation mechanism triggered by papaverine in the presence of phenylephrine.
Subject(s)
Heart Rate/drug effects , Papaverine/pharmacology , Phentolamine/pharmacology , Phenylephrine/antagonists & inhibitors , Prazosin/pharmacology , Quinazolines/pharmacology , Animals , Calcium/metabolism , Female , In Vitro Techniques , Myocardium/metabolism , RatsABSTRACT
Right atria from rats were analyzed for chronotropic responses to phenylephrine in face of various drugs and procedures. Propranolol, 10(-8) M, produced a competitive antagonism against the agonist which concentration-effect curve was closely similar to that obtained from reserpinized animals. Prazosin, but not phentolamine (both 10(-6) M) showed inhibition of the phenylephrine-induced changes in heart rate, as judged by their -log EC50. Either of the alpha-adrenoceptor antagonists exhibited a greater steepness in the curve slope with respect to control. The simultaneous exposure of tissues to phentolamine and propranolol proved to effectively antagonize the chronotropic effect of the agonist. This held true for phentolamine assayed in atria from reserpine-pretreated rats. Previous incubation of tissues with papaverine, 10(-5) M, brought about supersensitivity to phenylephrine which was thoroughly inhibited by either phentolamine or propranolol. These results strongly suggest that beta-adrenoceptor stimulation of heart rate by phenylephrine takes place indirectly via norepinephrine release. There is also alpha 1-adrenoceptor stimulation (blocked by prazosin). Finally, it is hypothesized that supersensitivity develops by papaverine-enhanced Ca2+ influx, since numerous evidences are against a phosphodiesterase inhibition-dependent cAMP accumulation mechanism triggered by papaverine in the presence of phenylephrine.
