Effect of N-2, 2'-O-dibutyryl cyclic AMP upon the interconvertible forms of cyclic AMP phosphodiesterase from human platelets.

The influence of dibutyryl cyclic AMP upon the equilibrium existing between three interconvertible forms - 2.5 S, 4.8 S and 7 S - of cyclic AMP phosphodiesterase from human platelets was investigated. It shifted the equilibrium towards the lighter form. It also exerted a protective effect against the thermo-inactivation of the enzyme. It is suggested that the analogue-induced equilibrium shift towards the dissociated high Km form of the phosphodiesterase might reflect a regulatory mechanism occurring also with natural cyclic nucleotides.     

Increase in hepatic tyrosine aminotransferase mRNA during enzyme induction by N6,O2'-dibutyryl cyclic AMP.

Tyrosine aminotransferase mRNA was quantitated by translation in a cell-free system derived from wheat germ followed by specific immunoprecipitation of the newly synthesized enzyme subunit. Hepatic poly(A)-containg RNA prepared from rats treated for 4 h with N6, O2'-dibutyryl cyclic AMP and theophylline was approximately 5.6 times more active in directing the synthesis of the tyrosine aminotransferase subunit relative to untreated controls. The overall template activity of the RNA prepared from control and cyclic AMP-treated animals was virtually identical, demonstrating that the cyclic nucleotide effect was specific for the tyrosine aminotransferase mRNA. At all times, after a single injection of dibutyryl cyclic AMP and theophylline, the increase in hepatic enzyme activity was accompanied by corresponding induction in the level of functional tyrosine aminotransferase mRNA. Other inducers of tyrosine aminotransferase, such as glucagon and hydrocortisone, also increased the level of tyrosine aminotransferase mRNA in proportion to their effect on enzyme activity. The RNA polymerase II inhibitor, alpha-amanitin, completely blocked the dibutyryl cyclic AMP-mediated increase in tyrosine aminotransferase mRNA activity. These studies demonstrate that, in intact animals, the induction of tyrosine aminotransferase activity by dibutyryl cyclic AMP can be completely accounted for by a corresponding increase in the level of functional mRNA coding for the enzyme.     

The effects of norepinephrine on the release of cyclic AMP by the isolated perfused heart of the guinea pig.

In the isolated perfused heart preparation of the guinea pig, continuous infusions of norepinephrine in doses of 0.06, 0.2 and 0.6 μg/min resulted in a dose-dependent increase in the heart rate, augmentation of the heart contractility measured as increments of dp/dt max. and an increase in the coronary flow. Simultaneous with these changes, a dose-dependent release of substantial quantities of cyclic AMP into the perfusate was observed, and there was a close moment to moment correlation between the increments of dp/dt max. and the increases in the amount of released cyclic AMP produced by norepinephrine. A linear correlation, though less marked than that described above, was also observed between the positive chronotropic and coronary vasodilatatory effects and the increases in the amount of released cyclic AMP, while there was no dynamic correlation between the tissue cyclic AMP level and the augmentation of the mechanical performances of the heart; Increases in the tissue cyclic AMP level were observed only with the highest dose and were transient and a significant decrease was noted with the lowest dose. The decrease was abolished by phentolamine. It is concluded that cyclic AMP exists in different ntracellular compartments in the heart, only one of which -- comprising a small fraction of the total cellular cyclic AMP, -- is functionally-relevant, and that much of this fraction can pass relatively freely across the cell membrane.     

Na+ channel blockade by cyclic AMP and other 6-aminopurines in neonatal rat heart

Summary Elementary Na⁺ currents were recorded at 19°C in cell attached and inside-out patches from cultured neonatal rat cardiocytes in order to study the effect of cAMP and other 6-aminopurines.The treatment of the cardiocytes with db-cAMP (1×10^–3 mol/liter) led to a decline of reconstructed macroscopic peakI _Na to 62±7.6% of the initial control value. This reduction in NP₀ was mostly accompanied by a decrease in burst activity. Openstate kinetics were preserved even in DPI-modified, noninactivating Na⁺ channels. Since the stimulator of the adenylate cyclase, forskolin (1×10^–6 mol/liter), evoked a similar pattern of response, the NP₀ decrease can be considered as the functional correlate of Na⁺ channel phosphorylation brought about by cAMP-dependent protein kinase. As found in inside-out patches, cAMP (1×10^–3 mol/liter) remained effective under cell-free conditions and reduced reconstructed macroscopic peakI _NA to about 50% of the initial control value when the absence of Mg-ATP at the cytoplasmic membrane surface prevents phosphorylation reactions. A very similar response developed in the cytoplasmic presence of other 6-aminopurines including ATP (1×10³ mol/liter), adenosine (1×10^–4 mol/liter), adenine (1×10^–5 mol/liter) and hypoxanthine (1×10^–5 mol/liter). This susceptibility to adenine suggests that cardiac Na⁺ channelsin situ could sense intracellular fluctuations of adenine nucleotides, most likely of ATP.     

Effects of CuCl2 on the hydrolysis of cyclic GMP and cyclic AMP by the activator-dependent cyclic nucleotide phosphodiesterase from bovine heart.

CuCl2 non-competitively inhibited the hydrolysis of cyclic GMP and cyclic AMP by the activator-dependent phosphodiesterase from bovine heart in the presence of 5 mM Mg2+, 10 muM Ca2+ and phosphodiesterase activator with Ki values of approximately 2 muM for both substrates. CuCl2 inhibition was also non-competitive with Mg2+, Ca2+ and phosphodiesterase activator. Dialysis demonstrated that CuCl2 inhibition is reversible. Treatment of the enzyme with p-hydroxymercuribenzoate resulted in the loss of enzyme activity, suggesting the presence of sulfhydryl groups essential for enzyme activity. The inhibitory activity of CuCl2 was not additive with that of p-hydroxymercuribenzoate, therefore CuCl2 may inhibit enzyme activity by binding to one or more essential sulfhydryl groups. CuCl2 also inhibited the hydrolysis of cyclic AMP by the cyclic AMP-specific phosphodiesterase from bovine heart with an I50 value of 18 muM. Several effects of Cu2+ are discussed which have been noted in other studies and might be due, in part, to changes in cyclic nucleotide levels following alterations in phosphodiesterase activity.     

Protein phosphorylation in human peripheral lymphocytes - stimulation by phytohemagglutinin and N6 monobutyryl cyclic AMP.

Protein phosphorylation was studied in human peripheral lymphocytes. The cells were preincubated with

, exposed to phytohemagglutinin (PHA), N⁶ monobutyryl cAMP (MBcAMP) or 8 bromo cGMP (BcGMP), homogenized and analyzed by SDS-polyacrylamide gel electrophoresis. Both PHA and MBcAMP produced early increases in the [³²P]content of multiple proteins in the 30,000–100,000 molecular weight range. After further incubation with PHA there was a shift in the phosphorylation response to smaller molecular weight proteins. BcGMP (1 mM-10 pM) had no effect on protein phosphorylation. These results suggest a role for cAMP in the early action of PHA on human peripheral lymphocytes.     

Effects of CuCl2 on the hydrolysis of cyclic GMP and cyclic AMP by the activator-dependent cyclic nucleotide phosphodiesterase from bovine heart

CuCl₂ non-comepetitively inhibited the hydrolysis of cyclic GMP and cyclic AMP by the activator-dependent phosphodiesterase from bovine heart in the presence of 5 mM Mg²⁺, 10 μM Ca²⁺ and phosphodiesterase activator with K_i values of approximately 2 μM for both substrates. CuCl₂ inhibition was also non-competitive with Mg²⁺, Ca²⁺ and phosphodiesterase activator. Dialysis demonstrated that CuCl₂ inhibition in reversible. Treatment of the enzyme with p-hydroxymercuribenzoate resulted in the loss of enzyme activity, suggesting the presence of sulfhydryl groups essential for enzyme activity. The inhibitory activity of CuCl₂ was not additive with that p-hydroxymercuribenzoate, therefore CuCl₂ may inhibit enzyme activity by binding to one or more essential sulfhydryl groups. CuCl₂ also inhibited the hydrolysis of cyclic AMP by the cyclic AMP-specific phosphodiesterase from bovine heart with an I₅₀ value of 18 μM. Several effects of Cu²⁺ are discussed which have been noted in other studies and might be due, in part, to changes in cyclic nucleotide levels following alterations in phosphodiesterase activity.