Guanylate cyclase and cyclic guanosine 3':5'-monophosphate phosphodiesterase activities and cyclic guanosine 3':5'-monophosphate levels in normal and transformed fibroblasts in culture.

To investigate the role of guanosine 3':5'-monophosphate (cyclic GMP) in cultured cells we have measured guanylate cyclase and cyclic GMP phosphodiesterase activities and cyclic GMP levels in normal and transformed fibroblastic cells. Guanylate cyclase activity is found almost exclusively in the particulate fraction of normal rat kidney (NRK) and BALB 3T3 cells. Enzyme activity is stimulated 3- to 10-fold by treatment with the detergent Lubrol PX. However, enhancement of guanylate cyclase by fibroblast growth factor could not be demonstrated under a variety of assay conditions. In both NRK and BALB 3T3 cells guanylate cyclase activity is low during logarithmic growth and increases as the cells crowd together and growth slows. Guanylate cyclase activity is undetectable in homogenates of NRK cells transformed by the Kirsten sarcoma virus (KNRK cells) either in the presence or absence of Lubrol PX. Guanylate cyclase activity is also greatly decreased in NRK cells transformed by Moloney, Schmidt-Ruppin, or Harvey viruses. BALB 3T3 cells transformed by RNA viruses (Kirsten, Harvey, or Moloney), by a DNA virus (SV40), by methylcholanthrene, or spontaneously, all have diminished but readily detectable guanylate cyclase activity. Cyclic GMP phosphodiesterase activity is found predominately in the soluble fraction of NRK cells. This activity increases slightly as NRK cells enter the stationary growth phase. Cyclic GMP phosphodiesterase activity is undetectable in two clones of KNRK cells under a variety of assay conditions, and is decreased relative to the level present in NRK cells in a third KNRK clone. However, both Moloney- and Schmidt-Ruppin-transformed NRK cells have a phosphodiesterase activity similar to that found in NRK cells. Boiled supernatant from both NRK and KNRK cells is observed to appreciably enhance the activity of activator-deficient phosphodiesterase from bovine heart. This result indicates that the absence of cyclic GMP phosphodiesterase activity in KNRK cells is not due to a loss of the phosphodiesterase activator. The intracellular concentration of cyclic GMP is found to be very low in transformed NRK cells when compared to levels measured in confluent NRK cells. The low levels of cyclic GMP in transformed NRK cells reflect the greatly decreased guanylate cyclase activity observed in these cells. These results do not appear to support the suggestion that cyclic GMP promotes the growth of fibroblastic cells.     

Influence of thyrotropin releasing hormone and dibutyryl cyclic adenosine 3',5'-monophosphate on ultrastructure of adenohypophysis in organ culture.

Ultrastructure of adenohypophysis in organ culture exposed to thyrotropin releasing hormone or dibutyryl cyclic adenosine 3',5'-monophosphate has been investigated. The features of increased secretory activity of glandular cells as well as the occurrence of a strongly osmiophilic substance in the intercellular spaces was observed in cultures exposed to both investigated factors.     

Self-phosphorylation of cyclic guanosine 3':5'-monophosphate-dependent protein kinase from bovine lung. Effect of cyclic adenosine 3':5'-monophosphate, cyclic guanosine 3':5'-monophosphate and histone.

Incubation of purified cyclic guanosine 3':5'-monophospate-dependent protein kinase with [gamma-32P]ATP and Mg2+ led to formation of one 32P-labeled protein, Mr = 75,000, which corresponded to the single protein band detected after polyacrylamide gel electrophoresis in sodium dodecyl sulfate. When electrophoresis was performed without detergent, the labeled protein coincided with the position of cGMP-dependent protein kinase activity. Phosphorylation was enhanced severalfold by either histone or cAMP and was inhibited by the addition of cGMP. Low concentrations of cGMP blocked the stimulatory effects of cAMP or histone (or both). Since neither cAMP-dependent protein kinase nor cGMP-dependent phosphoprotein phosphatase activities were detected in the purified enzyme, we concluded that the cGMP-dependent protein kinase is a substrate for its own phosphotransferase activity and that other protein substrates (histone) and cyclic nucleotides modulate the process of self-phosphorylation.     

An equilibrium study of the cooperative binding of adenosine cyclic 3',5'-monophosphate and guanosine cyclic 3',5'-monophosphate to the adenosine cyclic 3',5'-monophosphate receptor protein from Escherichia coli

The binding of adenosine cyclic 3',5'-monophosphate (cAMP) and guanosine cyclic 3',5'-monophosphate (cGMP) to the adenosine cyclic 3',5'-monophosphate receptor protein (CRP) from Escherichia coli was investigated by equilibrium dialysis at pH 8.0 and 20 degrees C at different ionic strengths (0.05--0.60 M). Both cAMP and cGMP bind to CRP with a negative cooperativity that is progressively changed to positive as the ionic strength is increased. The binding data were analyzed with an interactive model for two identical sites and site/site interactions with the interaction free energy--RT ln alpha, and the intrinsic binding constant K and cooperativity parameter alpha were computed. Double-label experiments showed that cGMP is strictly competitive with cAMP, and its binding parameters K and alpha are not very different from that for cAMP. Since two binding sites exist for each of the cyclic nucleotides in dimeric CRP and no change in the quaternary structure of the protein is observed on binding the ligands, it is proposed that the cooperativity originates in ligand/ligand interactions. When bound to double-stranded deoxyribonucleic acid (dsDNA), CRP binds cAMP more efficiently, and the cooperativity is positive even in conditions of low ionic strength where it is negative for the free protein. By contrast, cGMP binding properties remained unperturbed in dsDNA-bound CRP. Neither the intrinsic binding constant K nor the cooperativity parameter alpha was found to be very sensitive to changes of pH between 6.0 and 8.0 at 0.2 M ionic strength and 20 degrees C. For these conditions, the intrinsic free energy and entropy of binding of cAMP are delta H degree = -1.7 kcal . mol-1 and delta S degree = 15.6 eu, respectively.     

Implantation in ovariectomized mice treated with dibutyryl adenosine 3',5'-monophosphate (dibutyryl cyclic AMP).

Experiments are described that demonstrate that uterine intraluminal injection of a 1-25 mM-solution of dibutyryl cyclic AMP (dcAMP) in phosphate buffered saline (PBS) induced implantation in ovariectomized pregnant mice. Pretreatment with progesterone was essential for this effect. When PBS was injected alone, it did not induce implantation in mice treated with progesterone. Bilateral adrenalectomy had no effect on the ability of dcAMP to substitute for oestradiol, showing that the effect was not due to dcAMP-induced oestrogen synthesis in the adrenal cortex. It is suggested that the dcAMP may act at the level of the uterus, the embryo, or both.     

18O-Labeling of guanosine monophosphate upon hydrolysis of cyclic guanosine 3':5'-monophosphate by phosphodiesterase

The hydrolysis of cGMP by phosphodiesterase was conducted in [18O]water to determine the site of bond cleavage and the stoichiometry of 18O incorporation into 5'-GMP. Three different forms of phosphodiesterase including a calmodulin-calcium-dependent enzyme in its basal and activated states were examined. The hydrolysis of cGMP catalyzed by each of the forms of phosphodiesterase proceeded with incorporation of 1 18O atom recoverable in the phosphate moiety of each molecule of 5'-GMP generated. No molecular species of phosphate deriving from the 5'-GMP generated containing two or three 18O were detectable. These results indicate that the phosphodiesterase-catalyzed hydrolysis of cGMP proceeds by nucleophilic substitution at phosphorus resulting in P-O bond cleavage. The stoichiometry of 18O incorporation indicates that the reaction proceeds without phosphate-water oxygen exchange when the hydrolytic reaction is catalyzed by diverse forms of phosphodiesterase in the basal or activated state. These considerations of the phosphodiesterase reaction help to establish the validity of monitoring the rate of enzyme-catalyzed hydrolysis of cGMP as a function of the rate of 18O-labeling of the phosphate of 5'-GMP when the reaction proceeds in a medium of predetermined 18O enrichment.     

Characterization and metabolism of cyclic guanosine 3'5'-monophosphate in Mycobacterium smegmatis

Cyclic guanosine 3'5'-monophosphate was isolated and purified from Mycobacterium smegmatis TMC 1515 using ion exchange chromatography. It was characterized by thin layer chromatography and radioimmunoassay. Guanylate cyclase and cGMP phosphodiesterase were detected in the cytosolic and particulate (37,000 X g pellet) fractions respectively. On the basis of our observations, cGMP appears to play a dual role (i) at the time of induction of cell proliferation and (ii) protects the bacteria against unfavourable surroundings during stationary phase of the growth. This is the first report demonstrating presence of cGMP, guanylate cyclase and cGMP phosphodiesterase in mycobacteria.     

Effects of hydralazine on guanosine cyclic 3', 5'-monophosphate levels in rat aorta

The mechanism of the vasodilator effect of hydralazine on isolated rat aorta was studied. Results demonstrated that the vasodilator effect of hydralazine was greater on intact aortas than on endothelium-denuded preparations, particularly at low concentrations of between 0.1 mM and 0.5 mM. In addition, hydralazine did not have any effect on cyclic GMP levels. We also found that methylene blue, an inhibitor of guanylate cyclase, completely abolished the vasorelaxant action of nitroglycerin but not that of hydralazine. These results indicate that the vasodilator effect of hydralazine was not due to elevating the cyclic GMP levels. On the other hand, hydralazine significantly inhibited both the contractions induced by norepinephrine and/or high-potassium. In conclusion, a part of the vasodilator effect of hydralazine seems to depend on the integrity of the vascular endothelium. However, this vasodilator effect was not associated with any elevation in cyclic GMP level. Thus, the direct vasodilator action of hydralazine may be related to its interference with the movement and/or translocation of calcium across the cell membrane.     

Calcium-dependent cyclic nucleotide phosphodiesterase from brain: comparison of adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate as substrates.

A Ca²⁺-dependent cyclic nucleotide phosphodiesterase has been partially purified from extracts of porcine brain by column chromatography on Sepharose 6 B containing covalently linked protamine residues, ammonium sulfate salt fractionation, and ECTEOLA-cellulose column chromatography. The resultant preparation contained a single form of cyclic nucleotide phosphodiesterase activity by the criteria of isoelectric focusing, gel filtration chromatography on Sephadex G-200, and electrophoretic migration on polyacrylamide gels. When fully activated by the addition of Ca²⁺ and microgram quantities of a purified Ca²⁺-binding protein (CDR), the phosphodiesterase hydrolyzed both adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP), with apparent K_m values of 180 and 8 μm, respectively. Approximately 15% of the total enzymic activity was present in the absence of added CDR and Ca²⁺. This activity exhibited apparent K_m values for the two nucleotides identical to those observed for the maximally activated enzyme. Competitive substrate kinetics and heat destabilization studies demonstrated that both cyclic nucleotides were hydrolyzed by the same phosphodiesterase. The purified enzyme was identical to a Ca²⁺-dependent phosphodiesterase present in crude extract by the criteria of gel filtration chromatography, polyacrylamide-gel electrophoresis, and kinetic behavior.Apparent K_m values of the Ca²⁺-dependent phosphodiesterase for cyclic AMP and cyclic GMP were lowered more than 20-fold as CDR quantities in the assay were increased to microgram amounts, whereas the respective maximal velocities remained constant. The apparent K_m for Mg²⁺ was lowered more than 50-fold as CDR was increased to microgram amounts. Half-maximal activation of the phosphodiesterase occurred with lower amounts of CDR as a function of either increasing degrees of substrate saturation or increasing concentrations of Mg²⁺. At low cyclic nucleotide substrate concentrations i.e., 2.5 μm, cyclic GMP was hydrolyzed at a fourfold greater velocity than cyclic AMP. At high substrate concentrations (millimolar range) cyclic AMP was hydrolyzed at a threefold greater rate than cyclic GMP.     

Conversion of guanosine 3', 5'-monophosphate to adenosine 3', 5'-monophosphate in frog myocardial tissue.

One of the transformation products of tritiated cyclic GMP in frog heart tissue homogenate was identified with cyclic AMP. The purified product met all criteria tested for: solubility in ZnCO₃, behavior on various ion-exchangers and sensitivity to cyclic nucleotide phosphodiesterase. Our findings may provide insight into the understanding of heart pacemaker activity.     

Inactivation of guanosine cyclic 3',5'-monophosphate dependent protein kinase from bovine lung by o-phthalaldehyde

Guanosine cyclic 3',5'-monophosphate (cGMP) dependent protein kinase is inactivated by o-phthalaldehyde. The loss of phosphotransferase activity following treatment with o-phthalaldehyde was rapid, and the second-order rate constant at 25 degrees C and pH 7.3 was 35 M-1 s-1. The inactivation reaction did not follow saturation kinetics. The cGMP-dependent protein kinase was protected from inactivation by its substrates, MgATP and Ser-peptide. Fluorescence excitation and emission spectroscopic data showed that an isoindole derivative was formed following the reaction between cGMP-dependent protein kinase and o-phthalaldehyde. Four moles of isoindole per mole of the cGMP-dependent protein kinase dimer was formed following complete inactivation by o-phthalaldehyde. In the absence of cGMP, the protein kinase lost only 50% of its cGMP binding activity while there was almost a complete loss of its phosphotransferase activity. Studies in the presence of 20 microM cGMP, however, showed that about 2 mol of isoindole groups per mole of the protein kinase dimer was formed following complete inactivation by o-phthalaldehyde. The second-order rate constant for inactivation of cGMP-dependent protein kinase by o-phthalaldehyde in the presence of 20 microM cGMP was 40 M-1 s-1. Fluorescence measurements of samples containing inactivated, iodoacetamide-modified, or 5'-[p-(fluorosulfonyl)benzoyl]adenosine-modified, cGMP-dependent protein kinase and o-phthalaldehyde showed that the intensity of fluorescence in each case was about 50% of that obtained from unmodified, active cGMP-dependent protein kinase and o-phthalaldehyde.(ABSTRACT TRUNCATED AT 250 WORDS)