A rapid batch assay for cyclic AMP phosphodiesterase.

A simple new assay for cyclic AMP phosphodiesterase has been devised. The assay uses tritiated cyclic AMP as a substrate and measures the radioactivity present in the reaction product 5′-AMP. The samples are spotted on PEI-cellulose and then washed batchwise with 10–12 mm LiCl. Under these conditions, over 99,9% of the cyclic AMP is removed, while about 50% of the 5′-AMP product remains. The assay is very rapid and is sultable for hundreds of samples. The assay has been used for following cyclic AMP phosphodiesterase activity from Myxococcus xanthus on native polyacrylamide gels and for screening mutagenized colonies for enzyme activity.     

An improved protein binding assay for cyclic AMP

A simplified and inexpensive protein binding assay for cAMP has been developed for the rapid measurement of subpicomole quantities of the nucleotide. Conditions of assay were established in which interferences from salts and nucleotides present in biological samples were eliminated. Sodium chloride was demonstrated to reduce cAMP binding by denaturing the regulatory subunit of the protein kinase.     

A convenient erythrocyte membrane cyclic AMP binding assay.

Formaldehyde reacts with tryptophan in the presence of sulfuric acid and iron, nickel, or cobalt and gives a violet color with absorption maximum at 575 nm. Nanomoles of formaldehyde can be estimated spectrophotometrically by using this reaction which is also very specific. Other aminoacids, sugars and related compounds, higher aldehydes, and heavy metals do not react. 3-Substituted indoles give similar colors of less intensity. The tryptophan reaction can be used to measure formaldehyde formed from biochemical reactions using fractions of liver and other tissues.     

A simple ultrasensitive method for the assay of cyclic AMP and cyclic GMP in tissues.

A simple and rapid method for a highly sensitive radioimmunoassay of cyclic AMP and GMP is described. The method is based on the observation that the affinity of the cyclic nucleotide antibodies for the 2′-0-succinyl or acetyl derivatives is considerably greater than that for the nonacylated cyclic nucleotides. With the present method, 3–10 fmoles of cyclic AMP and cyclic GMP can be assayed using commercially available antisera against cyclic AMP or cyclic GMP. A reproducible conversion of the cyclic nucleotides in aqueous samples to the 2′-0-acylated derivative is brought about by a simple one-step addition of premixed reagents containing either succinic anhydride or acetic anhydride and triethylamine. The time required for succinylation or acetylation of 100 samples is less than 5 min using this technique. Tissue extracts after purification through anion exchange resins do not interfere with the acylation. After acylation, labeled antigen and antiserum are added and incubated at 4°C for 15 hr. The bound antingen is separated from the unbound antigen by a simple ethanol precipitation using bovine serum albumin to ensure complete precipitation. The specificity of the method has been validated by the following criteria: (1) treatment of the samples with purified phosphodiesterase results in complete loss of cyclic nucleotide immunoreactivity, (2) cyclic nucleotide content was a linear function of the tissue weight, and (3) in one instance using bovine rod outer segments, identical values of cyclic GMP were obtained with the present method and a purely physical method (high pressure liquid chromatography).     

The cardiac sarcoplasmic reticulum-glycogenolytic complex. A possible effector site for cyclic AMP.

Cardiac sarcoplasmic reticulum-glycogenolytic complex, isolated as a single peak on sucrose density gradient, may function as a "compartmented" effector site for cyclic AMP resulting in modulation of both glycogenolysis and calcium transport. The conversion of phosphorylase b to a is stimulated by ATP and inhibited by protein kinase inhibitor. Cyclic AMP alone stimulated neither phosphorylase b to a conversion nor calcium uptake. An inhibitor of adenylate cyclase depressed both calcium uptake and phosphorylase activation and both functions were subsequently stimulated by micromolar concentrations of cyclic AMP. Endogenous phosphorylation of sarcoplasmic reticulum was also inhibited by adenylate cyclase inhibitor and the inhibition was reversed by cyclic AMP. These results suggest that the sarcoplasmic reticulum of cardiac muscle is an internal effector site for cyclic AMP which may regulate both calcium and metabolism. It appears that cyclic AMP formation in vitro is not the rate-controlling step in the activation sequence.     

Enzymatic radioiodination of succinyl cyclic AMP tyrosine methyl ester by lactoperoxidase and radioimmunoassay for cyclic AMP.

A highly specific and simple radioimmunoassay for cyclic AMP with a sensitivity of 0.04 picomoles/tube has been developed according to the method of Steiner et al., using 125I-succinyl cyclic AMP tyrosine methyl ester as a tracer. The tracer with higher immunoactivities could be simply and constantly prepared by an enzymatic iodination procedure utilizing lactoperoxidase, radioactive iodide and hydrogen peroxide generated by glucose-glucose oxidase system, rather than by chloramine-T procedure.     

Glucose synthesis by isolated guinea-pig hepatocytes. Effect of cyclic AMP and dibutyryl cyclic AMP.

In isolated guinea-pig hepatocytes, dibutyryl cyclic AMP stimulated gluconeogenesis from 2 mM galactose by 25 and 40% respectively. In the presence of 0.5 mM theophylline, cyclic AMP (0.1 mM) increased glucose synthesis from lactate and galactose by 26 and 34% respectively.     

Improvement on the competitive binding assay for the measurement of cyclic AMP by using ammonium sulphate precipitation.

The protein-binding assay developed by Brown, Albano, Ekins, Sgherzi & Tampion [(1971) Biochem. J. 121, 561-562] and Brown, Ekins & Albano [(1972) Adv. Cyclic Nucleotide Res. 2, 25-40] was modified by using precipitation with (NH4)2SO4 of the protein-cyclic AMP complex instead of adsorption of the free nucleotide on charcoal. The half-life of the protein-cyclic AMP complex obtained in the presence of charcoal was lower than that of the (NH4)2SO4-precipitated complex. In consequence, owing to the great stability of the precipitated protein-cyclic AMP complex, this method allows more accurate and reproducible determinations.     

Erythrophagocytosis by macrophages: suppression of heme oxygenase by cyclic AMP.

On incubation of peritoneal macrophages with antibody-coated radiolabeled erythrocytes, a reproducible fraction of the erythrocytes was phagocytized and heme oxygenase was induced. Addition of cyclic AMP, dbcyclic AMP, or theophylline to the incubation medium suppressed the substrate-mediated induction of heme oxygenase in a dose-related manner but did not impair the rate or extent of erythrophagocytosis. A similar effect was produced by epinephrine, norepinephrine, isoproterenol, and prostaglandins, which generate endogenous cyclic AMP by stimulating the adenyl cyclase system. Propanolol completely blocked the suppressive effect of epinephrine, while phentolamine was ineffective. In contrast to the cyclic adenosine nucleotide, cyclic GMP probably slightly enhanced the substrate-mediated induction of heme oxygenase and partly reversed the suppressive effect of cyclic AMP. Cyclic adenosine nucleotides, prostaglandin, and theophylline significantly reduced the incorporation of labeled uridine or leucine into RNA and protein of erythrophagocytic macrophages, but failed to impair the uptake of these precursors by the phagocytizing cells. These compounds also reduced the conversion of [1-¹⁴C] glucose to ¹⁴CO₂ by the incubated macrophages, whereas ¹⁴CO₂ formation was enhanced by epinephrine. None of these effects was reversible by addition of insulin or by glucose supplementation, which is in sharp contrast to the suppressive effect of glucocorticoids on heme oxygenase induction.     

Inhibition by cyclic AMP and dibutyryl cyclic AMP of transport of organic acids in kidney cortex.

1. Cyclic adenosine 3',5'-monophosphate and N-6-2'-O-dibutyryl cyclic adenosine 3',5'-monophosphate decrease the initial entry rate and the steady-state uptake of p-aminohippurate and uric acid by rabbit kidney cortex slices. 2. N-6-2'-O-Dibutyryl adenosine 3'-5'-monophosphate inhibits the tubular transport of p-aminohippurate competitively. 3. Isoproterenol, known to increase cyclic nucleotide concentration of the cortical tubules by activation of adenyl cyclase, decreases p-aminohippurate transport. Antidiuretic hormone which is known to stimulate only medullary adenyl cyclase has no effect on p-amino-hippurate uptake by cortical slices. 4. Theophylline, which inhibits cyclic nucleotide phosphodiesterase and, therefore, enhances the cellular accumulation of endogenous cyclic nucleotide, depresses p-aminohippurate transport.     

Iontophoresis of cyclic AMP.

The design, calibration, and operation of a source of controlled amounts of cyclic AMP (c-AMP) are described. Typically, 1.5 s pulses containing 10(10)-10(-12) molecules of c-AMP can be delivered to a region about 10 mum in diameter on an agar plate. The resulting concentration profiles are given as functions of distance and time. The diffusion coefficient of c-AMP in agar was measured to be 0.97 times 10(-5) cm2-s-1 at 21 degrees C.     

Isolation of cyclic AMP and cyclic GMP by thin-layer chromatography. Application to assay of adenylate cyclase, guanylate cyclase, and cyclic nucleotide phosphodiesterase

A procedure is described for isolation of cAMP and cGMP by thin-layer chromatography on polyethylenimine cellulose. Chromatographs are developed (descending) twice in the same direction with two different solvents. This procedure separates cAMP and cGMP from other radioactive metatolites of [³H] or [¹⁴C] ATP or GTP. Application of this isolation method to assay of adenylate cyclase, (EC 4.6.1.1), guanylate cyclase (EC 4.6.1.2), and cyclic nucleotide phosphodiesterase (EC 3.1.4.17) has proven convenient and provides results of unusual quality.     

A possible role for cyclic AMP in the initiation of DNA synthesis by isoproterenol-activated parotid gland cells

An intraperitoneal injection of the β-adrenergic agonist dl-isoproterenol hydrochloride (100 mg/Kg body weight) into a rat caused an early, very large (400-fold) cyclic AMP surge (peaking at 10 minutes) in the parotid gland which was followed by a second, much smaller (two-fold) surge 12 to 16 hours later. DNA synthesis began about 16 to 20 hours after the isoproterenol injection and peaked between 24 and 28 hours. The maximum level of DNA-synthetic activity at 24 hours was correlated positively to the magnitude of the small cyclic AMP surge at 12 hours, but not to the size of the much larger cyclic AMP surge at 10 minutes. An intraperitoneal injection of dl-propranolol hydrochloride (59 mg/Kg body weight) at 8 hours after isoproterenol injection abolished the second cyclic AMP surge at 12 hours and markedly (65-75%) reduced the incorporation of [³H]-thymidine into DNA. Injection of dibutyryl cyclic AMP (6.3 mg/Kg body weight) and theophylline (25 mg/Kg body weight) at 8 hours prevented propranolol from inhibiting DNA synthesis. Propranolol appeared specifically to affect the cyclic AMP-dependent pre-DNA-synthetic step because it did not reduce [³H]-thymidine incorporation when injected after the second cyclic AMP surge had passed and DNA synthesis had just begun. Thus, the initial, large cyclic AMP surge following β-adrenergic stimulation may not be necessary for the initiation of prereplicative development, while the much smaller second surge may be needed for the initiation of DNA synthesis.     

Early cellular alterations induced by myocardial hypoxia: possible role of cyclic AMP (author's transl)

The ability of endogenous myocardial catecholamines to participate in the development of myocardial cellular alterations during a short period of severe hypoxia (30 min) was studied in isolated, Langendorff-perfused rat heart preparation, arrested by a high potassium concentration (16 mM) and perfused in the absence of exogenous substrate (Table I). Tyramine, which accelerated catecholamine depletion, also increased myocardial cell damage as assessed by a higher lactate dehydrogenase (LDH) release and a more marked reduction in cellular levels of high energy phosphates and glycogen (Table II). On the other hand, under conditions of beta-blockade (atenolol), hypoxia-induced tissular damage was reduced (Table II). These changes could be related to modifications in the cellular content of cyclic AMP (cAMP) since cAMP was consistently higher during the first 30 min of hypoxic perfusion than in control normoxic hearts (Table III) whereas cyclic GMP content remained unchanged. Moreover, interventions increasing cellular content of cAMP (theophylline, dibutyryl-cAMP) also increased hypoxic damage (Table IV), whereas N-methyl imidazole which reduced cellular content of cAMP lessened hypoxia-induced cellular alterations (Table IV). It is concluded that cellular lesions developing during the first 30 min of hypoxia in isolated arrested rat heart preparation perfused without exogenous substrate could be related to intracellular accumulation of cAMP occurring under the effect of endogenous catecholamine release.     

Activation of mammalian cyclic AMP phosphodiesterases by trypsin.

BHK fibroblasts contain two forms of cyclic AMP phosphodiesterase 3':5'-cyclic nucleotide 5'-nucleotidohydrolase EC 3.1.4.17) as analyzed by linear sucrose gradient fractionation; a 3.6-S form (peak I) and a 6.7-S form (peak II). Peak I is specific for cyclic AMP as substrate and displays Michaelis-Menten kinetics with an apparent Km of 2--3 micrometer. Peak II hydrolyzes cyclic GMP and displays anomalous kinetics for cyclic AMP hydrolysis. The activity of isolated peak II for cyclic AMP is increased by storage at 4 degrees C, treatment with trypsin, or treatment with rat brain and BHK fibroblast activator proteins. The activity of isolated peak I is unaffected by these conditions. Linear sucrose gradient fractionation demonstrates that activation of peak II by trypsin leads to the formation of a 3.6-S cyclic AMP-specific enzyme form, possibly peak I. In contrast to BHK fibroblasts (and most other mammalian tissues), rat uterus contains only one form of cyclic nucleotide phosphodiesterase on linear sucrose gradients, a 7-S form capable of hydrolyzing both cyclic AMP and cyclic GMP. Treatment of rat uterine supernatant with trypsin leads to the appearance of a 4-S, cyclic AMP-specific form with properties similar to that of BHK peak I. These data suggest that the kinetically complex, higher molecular weight cyclic nucleotide phosphodiesterases may consist of more than one catalytically active site and that multiple forms of the enzyme arise through dissociative mechanisms, possibly as a means of in vivo regulation.