Three new potential cAMP affinity labels. Inactivation of human platelet low Km cAMP phosphodiesterase by 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate

Three new analogues of cAMP have been synthesized and characterized: 2-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate (2-BDB-TcAMP), 2-[(3-bromo-2-oxopropyl)thio]-adenosine 3',5'-cyclic monophosphate (2-BOP-tcAMP), and 8-[(4-bromo-2,3-dioxobutyl)thio]adenosine 3',5'-cyclic monophosphate (8-BDB-TcAMP). The bromoketo moiety has the ability to react with the nucleophilic side chains of several amino acids, while the dioxobutyl group can interact with arginine. These cAMP analogues were tested for their ability to inactivate the low Km (high affinity) cAMP phosphodiesterase from human platelets. The 2-BDB-TcAMP and 2-BOP-TcAMP were competitive inhibitors of cAMP hydrolysis by the phosphodiesterase with Ki values of 0.96 +/- 0.12 and 0.70 +/- 0.12 microM, respectively. However, 2-BDB-TcAMP and 2-BOP-TcAMP did not irreversibly inactivate the phosphodiesterase at pH values from 6.0 to 7.5 and at concentrations up to 10 mM. These results indicate that although the 2-substituted TcAMP analogues bind to the enzyme, there are no reactive amino acids in the vicinity of the 2-position of the cAMP binding site. In contrast, incubation of the platelet low Km cAMP phosphodiesterase with 8-BDB-TcAMP resulted in a time-dependent, irreversible inactivation of the enzyme with a second-order rate constant of 0.031 +/- 0.009 min-1 mM1. Addition of the substrates, cAMP and cGMP, and the product, AMP, to the reaction mixture resulted in marked decreases in the inactivation rate, suggesting that the inactivation was due to reaction at the active site of the phosphodiesterase.(ABSTRACT TRUNCATED AT 250 WORDS)     

The conversion of ATP to IMP by muscle surface enzymes

These experiments showed that an isolated muscle bundle could be used to study, simultaneously, ion transport and the activity of surface enzymes. Frog muscles were carefully dissected and incubated for four hours in Ringer's solution containing a tris buffer and 3 mM ATP; at various times samples of the medium were chromatographed and analysed spectrophotometrically for nucleotide content. Samples of the final medium were analysed for inorganic phoshpate and for ammonia. The results demonstrated the conversion of adenosine triphosphate to inosine monophosphate, via adenosine diphosphate and adenosine monophosphate. Under the conditions of the experiment IMP was detected on chromatograms within 20 minutes of incubation; at the end of four hours the media had concentrations of 2.3 mM IMP, 4.4 mM inorganic phosphate and 2.6 mM ammonia, showing a stoichiometric relation among the products formed. There was convincing evidence that the enzymes involved (ATPase, adenylate kinase and AMP deaminase) must be situated close to or on the muscle surface. No effect of ouabain (1 μM) on the activity of the ATPase, adenylate kinase or deaminase could be found in these experiments, but the drug inhibited Na and K recovery from a Na-loaded, K-depleted state.     

Long acting cAMP analogues enhance sulfate incorporation into matrix proteoglycans and suppress cell division of fetal rat chondrocytes in monolayer culture.

The relationship between replication and the synthesis of matrix sulfated proteoglycans was investigated with fetal rat chondrocytes grown in monolayer culture. The effect of N6 O2' dibutyryl adenosine 3', 5' cyclic monophosphate (DBcAMP), adenosine 3', 5' cyclic monophosphate (cAMP), 8 Bromo adenosine 3', 5' cyclic monophosphate (8 Br-cAMP), sodium butyrate and hydroxyurea was examined. Between 0.05 and 0.5 mM DBcAMP, a dose related inhibition of cell division and stimulation of [35SO=/4] incorporation into matrix proteoglycans was demonstrated. At the higher concentrations of DBcAMP, cell division was completely inhibited and the enhancement of [35SO=/4] incorporation into matrix proteoglycans ranged between 40 and 120% (P less than 0.01). Utilizing 14C-glucosamine and photometric determination of proteoglycans with Alcian Blue, it was demonstrated that the increase in sulfate incorporation reflected enhanced accumulation of extracellular matrix. The effects of DBcAMP were mimicked by 8 Br-cAMP, suggesting they were mediated by the adenylyl cyclase system. cAMP (0.05-0.5 mM), sodium butyrate (0.1-0.5 mM) and hydroxyurea (0.5-5 mM) partially or fully inhibited cell division, but either failed or only slightly enhanced sulfate incorporation. The enhanced sulfated proteoglycan deposition promoted by DBcAMP began 8 to 12 hours after serum stimulation, its onset occurred prior to thymidine incorporation and the effect persisted for 28 hours. Determination of cell volume demonstrated an increase in size of DBcAMP treated chondrocytes between 8 to 12 hours, coincident with the onset of increased sulfate incorporation. These results are consistent with a model where matrix sulfated proteoglycan deposition by chondrocytes is mediated by intracellular cAMP levels and occurs in the G1 phase of the cell cycle.     

The induction of filopodia in the cellular slime mold Dictyostelium discoideum by cyclic adenosine monophosphate: mechanism of aggregation.

Dictyostelium discoideum vegetative amoebae grown axenically can be induced to extend microprojections, filopodia, in response to cyclic 3′,5′-adenosine monophosphate. Cyclic 3′-5′-guanosine monophosphate, adenosine monophosphate, or adenosine diphosphate at concentrations of 1.0 mm have no effect. After incubation for 15 min, 1.0 mM adenosine triphosphate will also cause filopodial formation. Treatment with 0.1 mM 2–4 dinitrophenol or 1.0 mM sodium azide does not prevent the induction by cyclic adenosine monophosphate. The induced cells can be more extensively agglutinated with Concanavalin A at 0.5 mg/ml than noninduced cells. A model is presented that describes a possible mechanism whereby cells may aggregate via the cyclic adenosine monophosphate induced filopodia.     

Effect of insulin on metabolism of cyclic adenosine 3',5'-monophosphate by plasma cell tumor (MPC-11).

Addition of insulin to cultured mouse plasma tumor cells (MPC-11) increases the entry of tritiated cyclic adenosine 3',5'-monophosphate (3H-cAMP). No increase of entry of N6-O2-dibutyryl adenosine 3',5' cyclic monophosphate (DBcAMP), 5' adenosine monophosphate (5' AMP) or adenosine was noted in the presence of insulin. The stimulation of cAMP transport by insulin was concentration dependent and inactivated insulin had no effect on nucleotide transport. Intracellular radioactivity after transport of cAMP was largely 5'AMP, while most of the extracellular radioactivity remained as cAMP after incubation.     

Cyclic AMP and serum arrest of the mitotic activity of human diploid fibroblasts.

Mitotic activity in confluent cultures of human diploid fibroblasts was arrested by the reduction of the serum concentration of the incubation medium to 0.5% or by the addition of 0.5 mM 6-N, 2'-O-dibutyryl-adenosine 3':5'-cyclic monophosphate (db cAMP). Under either of these conditions, cultures maintained a constant cell number for 14 days; cultures continuously exposed to medium containing 10% serum doubled their cell number during this 14-day period. The protein cotent per cell decreased by 20% when cells were maintained with 0.5% serum whereas that of cells exposed to db cAMP remained constant. Ultrastructural studies revealed that cells exposed to db cAMP exhibited a morphology typical of cells cultures with 10% serum alone, whereas cells incubated with 0.5% serum showed the ultrastructural changes in mitochondria, endoplasmic reticulum and Golgi complex previously identified with low-serum arrest. Cellular adenosine 3':5'-cyclic monophosphate (cAMP) levels remained constant during the 7-day growth period in which confluency was attained, as well as during the 14-day arrested period with 0.5% serum. These results indicated that the mitotic inhibition induced by reducing the serum concentration of the incubation medium was not mediated by increased intracellular levels of cAMP and differed from that induced by the addition of exogenous db cAMP.     

Anomalous binding of MgADP to myosin of skeletal muscle

Binding of adenosine diphosphate to skeletal muscle myosin was studied using a range of concentrations from 0 to 2 mM. Up to 0.2 mM adenosine diphosphate two equivalent and independent nucleotide binding sites were detected, characterized by the single association constant of 5 x 10(4)M(-1). At greater adenosine diphosphate concentrations a decreasing binding capacity was noticed, bound nucleotide being essentially approximately 0.1 mol/mol at a 1-2mM adenosine diphosphate concentration. We tentatively propose that nucleotides act indirectly on myosin by promoting the perturbation of the solvent, which is supported by the fact that polyphosphates are known powerful kosmotropes.     

Increase of cyclic GMP induced in murine thymocytes by thymosin fraction 5

Using a radioimmunoassay (RIA) for the determination of adenosine 3'5' cyclic monophosphate (cAMP) and an acetylation-RIA procedure to measure guanosine 3'5' cyclic monophosphate (cGMP), we observed that cGMP levels, but not cAMP levels, were significantly elevated in murine thymocytes which had been incubated with preparations containing the thymic hormone, thymosin. Stimulation of intracellular cGMP levels was seen as early as 1 minute after incubation with thymosin fraction 5 and was maximal at approximately 10 minutes. Dose response studies indicated an optimum stimulation of cGMP with a thymosin concentration of 100 microg/ml. A control spleen fraction prepared by an identical procedure as fraction 5 did not affect the levels of either cyclic nucleotide.     

Hypoxanthine and adenosine in murine ovarian follicular fluid: concentrations and activity in maintaining oocyte meiotic arrest

The concentrations of hypoxanthine and adenosine in ovarian follicular fluid were estimated, using high-performance liquid chromatography, for three groups of mice: 1) pregnant mare's serum gonadotropin (PMSG)-primed mice; 2) PMSG-primed mice 2 h after injection with human chorionic gonadotropin (hCG); and 3) PMSG-primed mice 5 h after injection with hCG. The concentration of hypoxanthine in follicular fluid of Group 1 mice was 2-4 mM and of adenosine was 0.35-0.70 mM. There was no difference in the concentrations of these purines in the follicular fluid of Group 2 mice, in which maturation had been induced with hCG but the samples were taken just before germinal vesicle breakdown (GVBD). Therefore, a decrease in the concentrations of these purines does not appear to induce GVBD. A significant decrease in the concentrations of hypoxanthine and adenosine was observed in the follicular fluid of Group 3 mice in which GVBD had already occurred. This decrease was probably a result of an increase in follicular fluid volume. Adenosine had a significant, but transient, effect in maintaining both cumulus cell-enclosed and denuded oocytes in meiotic arrest; all oocytes had undergone GVBD by 100 min incubation in 1 mM adenosine. When GVBD was assessed after 3 h culture, concentrations up to 5 mM adenosine failed to maintain meiotic arrest. In contrast, hypoxanthine (2-5 mM) had a dose-dependent effect in maintaining both cumulus cell-enclosed and denuded oocytes in meiotic arrest that was sustained up to 24 h. Cumulus cell-enclosed oocytes were always more sensitive to hypoxanthine than were denuded oocytes. There was a strong synergistic effect of adenosine and hypoxanthine in maintaining meiotic arrest; 4 mM hypoxanthine and 0.75 mM adenosine maintained more than 95% of the oocytes in meiotic arrest for culture periods up to 24 h. This action was completely reversible by withdrawal of the purines. It is hypothesized that the synergistic effect of these purines may result both by promoting cyclic adenosine monophosphate synthesis (adenosine), and by preventing its hydrolysis (hypoxanthine).     

Influence of adenine nucleotides on the inhibition of photophosphorylation in spinach chloroplasts by N-ethylmaleimide.

The incubation of spinach chloroplasts with 1 mM N-ethylmaleimide in light for 60 to 90 s results in a partial, irreversible inhibition of photophosphorylation. The inhibition was not overcome at infinite light intensity or at infinite concentrations of the phosphorylation substrates. Although the inhibition diminished with decreasing concentrations of adenosine diphosphate in the assay of phosphorylation, the inhibition of guanosine diphosphate phosphorylation was independent of the concentration of this nucleotide. Although adenosine di- or triphosphate (10 to 30 muM) alone partially prevented the development of the N-ethylmaleimide inhibition of phosphorylation, these nucleotides were more effective when either 1 mM inorganic phosphate or arsenate was also present. The light-dependent incorporation of N-ethylmaleimide into chloroplast-bound coupling factor 1 was affected by adenosine triphosphate and inorganic phosphate in a manner similar to the onset of N-ethylmaleimide inhibition. Since guanosine diphosphate did not protect phosphorylation from N-ethylmaleimide inhibition but is phosphorylated at rapid rates, it is apparent that coupling factor 1 in chloroplasts has multiple nucleotide recognition sites.     

Cyclic nucleotides and calcium in human lymphocytes induced to divide

Intracellular concentrations of cyclic adenosine 3'-5' monophosphate (cAMP) and cyclic guanosine 3'-5' monophosphate (cGMP) were measured in human lymphocytes induced to divide by the addition of lectins, 12-O-tetra-decanoylphorbol-13-acetate (TPA) and the calcium ionophore A 23187. cGMP levels rose within minutes without concomitant alterations in cAMP concentration. The cAMP and cGMP levels rose during the prereplicative and replicative phases respectively. Under calcium depleting conditions, both the fluctuations in cyclic nucleotide levels and the increase in [3H[ thymidine incorporation into DNA were abolished, suggesting a role for calcium ions in the regulation of lymphocyte proliferation.     

A RADIOISOTOPIC METHOD FOR MEASURING THE FORMATION OF ADENOSINE 3'',5''-CYCLIC MONOPHOSPHATE IN INCUBATED SLICES OF BRAIN

Abstract— A simple and sensitive method for measuring the effect of neurohormones and other chemical agents on the formation of adenosine 3',5'-cyclic monophosphate (cyclic 3',5'-AMP) in incubated slices of brain was developed. The principle of the method depends on pulse-labelling of adenosine-5'-triphosphate in slices of brain with [8-¹⁴C]adenine, followed by incubation in a medium containing the test substance, separation by thin-layer chromatography of the cyclic nucleotide formed in the slices, and radioassay. The purity of the cyclic nucleotide was confirmed by chromatography in a variety of systems and by hydrolysis with a specific, bovine-heart phosphodiesterase. The method was used to study the effect of histamine, norepinephrine, and adenosine on the accumulation of adenosine 3',5'-cyclic monophosphate in incubated slices of brain.     

Calcitonin stimulates adenylate cyclase activity, the accumulation of cyclic adenosine 3',5' monophosphate and the release of prostaglandin E2 in rat intestinal mucosa

Calcitonin stimulates intestinal fluid and electrolyte secretion by an unclear mechanism. The present results show that synthetic salmon calcitonin significantly stimulates adenylate cyclase activity in the membranes of rat intestinal mucosal cells. The effect of the hormone was in a dose-dependent manner for a dose range of 10(-9)-10(-7) M. The stimulated enzyme activity was followed by a progressive accumulation of cyclic adenosine 3',5' monophosphate and release of prostaglandin E2 in these cells during a 20 min experimental period of time. These results are discussed, and suggest that the formation of cyclic adenosine 3',5' monophosphate possibly mediates the action of calcitonin on intestinal cell functions.     

The effect of glucagon-induced adenosine 3' ,5' -monophosphate concentrations on bile acid synthesis in isolated rat liver cells

The effect of increased intracellular adenosine 3' ,5' -monophosphate (cAMP) concentrations on bile acid synthesis in isolated rat hepatocytes was investigated. When the cells were incubated in the presence of glucagon (0.2 microM) and theophylline (1 mM) the observed rise in the level of cAMP was accompanied by an increase in bile acid production. Hepatocyte cAMP concentrations after 1 h of incubation showed a highly significant positive linear correlation with the amounts of bile acid synthesised by the cells during this time. These results suggest that bile acid production is related to the concentration of cAMP in isolated hepatocytes and provide evidence for a role for the cyclic nucleotide in the regulation of bile acid synthesis.     

Cyclic AMP phosphodiesterase activity at the external surface of intact skeletal muscles and stimulation of the enzyme by insulin

Small intact frog skeletal muscles were exposed to radioactively labeled adenosine 3′,5′-cyclic monophosphate (cAMP) during incubation in frog Ringer's solution buffered with Tris (RT). The fate of the nucleotide was followed by measuring the products in the incubation media. Paper chromatography was used for the separation and identification of these products; the amounts were measured using liquid scintillation spectrometry. It was found that cAMP was degraded to AMP, which was then converted to IMP and, to some extent, inosine. The degradation of cAMP to AMP was markedly inhibited by theophylline (10 mM) suggesting the presence of cAMP phosphodiesterase activity at the muscle surface. Kinetic studies of enzyme activity in situ revealed two apparent K_m values: 0.33 μm and 55 μm. Insulin (0.3 unit/ml) increased the phosphodiesterase activity at concentrations of cAMP ranging from 2 to 17 μm. The possible roles of the surface phosphodiesterase were discussed.     

Development of steroidogenic activity in the ovary of the prepubertal hamster: II. Production of steroids from steroidal precursors and response in vitro to cyclic adenosine monophosphate and luteinizing hormone

In vitro exposure for 2 h to 250 ng/ml of pregnenolone led to increased production of progesterone and 17 alpha-hydroxyprogesterone (17 alpha-OHP) by hamster ovaries on Days 5, 10 and 15 of age. Similar incubations with 250 ng/ml progesterone or androstenedione caused significant increases in 17 alpha-OHP or testosterone, respectively. When testosterone was added in doses of 32.5, 250 and 500 ng/ml to ovaries on Days 5-30, as early as Day 5 the ovaries aromatized the androgen to estradiol. Day 30 ovaries were the most efficient in the conversion because antral follicles, the principal site for aromatization, were then present. In terms of progesterone production, 400 ng/ml of luteinizing hormone (LH) during 4 h of in vitro incubation stimulated ovaries on Days 5, 10 and 15. Cyclic adenosine 3':5' monophosphate (cAMP) at a dose of 1 mM and 5 mM stimulated progesterone production by Days 5 and 10 ovaries more efficiently than LH. However, Day 15 ovaries produced more progesterone in response to LH compared to cAMP. These experiments establish that the steroidogenic enzymes differentiate at a very early age in the hamster ovary, even before the appearance of gonadotropin receptors. The inability of the early postnatal ovary to produce steroids is apparently attributable to lack of precursors such as cholesterol or cholesterol side chain cleavage enzymes.     

Growth hormone inhibition of glucagon- and cAMP-induced lipolysis by chicken adipose tissue in vitro

The ability of growth hormone (GH) to inhibit the early (first hour) lipolytic response to glucagon and cAMP was investigated using chicken adipose tissue explants in vitro. In the first hour of incubation, GH inhibited glucagon, 8-bromo-3',5'-cyclic adenosine monophosphate (8-bromo-cAMP), and 1-isobutyl-3-methyl-xanthine (IBMX) induced glycerol release. The antilipolytic effect of GH was dose dependent, with inhibition of glucagon and 8-bromo-cAMP observed in the presence of as little as 100 ng/ml GH. In the fourth hour of incubation (late lipolytic response), GH (10, 100, or 1000 ng/ml) enhanced the lipolytic action of glucagon.     

Determination of adenine and pyridine nucleotides in glucose-limited chemostat cultures of Penicillium simplicissimum by one-step ethanol extraction and ion-pairing liquid chromatography

Under specific conditions Penicillium simplicissimum excretes large amounts of organic acids, mainly citrate. As the energetic status of the hyphae might play a role in that respect, we developed a method for the determination of adenine (adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate) and pyridine (nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide (NADH)) nucleotides in hyphae of P. simplicissimum. An optimum separation of the five compounds in less than 15 min was possible on a C-8 column, utilizing 50 mM aqueous triethylamine-buffer (pH 6.5) and acetonitrile as mobile phase; detection was performed at 254 nm. With the exception of NADH, which could not be determined accurately due to stability problems, the method was sensitive (LOD < or = 0.7 ng on-column), repeatable (sigma(rel) < or = 4.4%), accurate (recovery rates between 97.9 and 104.9%), and precise (intraday variation < or = 9.4%, interday variation < or = 6.2 %). For an optimum extraction of the nucleotides the chemostat samples were directly placed into hot (90 degrees C) 50% ethanol, and shaken for 10 min, followed by evaporation of the solvent and a solid phase extraction cleanup of the redissolved aqueous samples. With this method the nucleotide concentrations in hyphae from a glucose-limited chemostat culture and the respective energy charge were determined. Additionally, the effect of the time lag between sampling and extraction and the effect of a glucose pulse on nucleotide concentrations were determined.     

Dynamics of hormone release from the perfused canine thyroid during cyclic AMP infusion.

In order to try to characterize the sequence of processes leading to hormone secretion from the stimulated thyroid, the effect of cyclic 3'5' adenosine monophosphate (cAMP) and related compounds were examined in 15 two-sided perfusions of canine thyroids isolated in situ. T4 and T3 concentrations in the effluent were measured radioimmunologically. cAMP 5 mM and TSH 100 muU per ml induced the same pattern of hormone release from the thyroid. After a latency period of 15--25 minutes a steep increase occurred in both T4 and T3 release. During the initial part of the stimulation the rise in T4 relase was somewhat slower than that of T3 release. The prolonged latency period before response earlier recorded in the same preparation during infusions of low concentrations of TSH was not observed during infusions of decreasing concentrations of cAMP (1, 0.8, 0.5 and 0.2 mM) or theophylline (5 and 1 mM). Either there was no response or the latency period was of the same length as that observed after a strong stimulus. These findings suggest that the latency period can be divided in two parts: () a variable, dose dependent satency period confined to the early part of the process sequence leading to secretion--i.e. before cAMP exerts its effect, and 2) an obligatory latency period related to the processes taking place after the formation of pseudopods. The duration of these late processes seems to be independent of the degree of stimulation.     

Blockade by cAMP of native sodium channels of adult rat skeletal muscle fibers

Although theskeletal muscle sodium channel is a good substrate for cAMP-dependentprotein kinase (PKA), no functional consequence was observed for thischannel expressed in heterologous systems. Therefore, we investigatedthe effect of 8-(4-chlorophenylthio)adenosine 3',5'-cyclicmonophosphate (CPT-cAMP), a membrane-permeable cAMP analog, on thenative sodium channels of freshly dissociated rat skeletal musclefibers by means of the cell-attached patch-clamp technique. Externallyapplied CPT-cAMP (0.5 mM) reduced peak ensemble average currents by~75% with no change in kinetics. Single-channel conductance andnormalized activation curves were unchanged by CPT-cAMP. In contrast,steady-state inactivation curves showed a reduction of the maximalavailable current and a negative shift of the half-inactivationpotential. Similar effects were observed with dibutyryl adenosine3',5'-cyclic monophosphate but not with cAMP, which doesnot easily permeate the cell membrane. Incubation of fibers for 1 hwith 10 µM H-89, a PKA inhibitor, did not prevent the effect ofCPT-cAMP. Finally, the -adrenoreceptor agonist isoproterenolmimicked CPT-cAMP when applied at 0.5 mM but had no effect at 0.1 mM.These results indicate that cAMP inhibits native skeletal muscle sodiumchannels by acting within the fiber, independently of PKA activation.