Differential inhibition of cyclic AMP and cyclic GMP hydrolysis in rat renal cortex.

Adenosine 3′,5′-monophosphate (cyclic AMP) and guanosine 3′,5′-monophosphate (cyclic GMP) metabolism in rat renal cortex was examined. Athough the cyclic AMP and cyclic GMP phosphodiesterases are similarly distributed between the soluble and particulate fractions following differential centrifugation, their susceptibility to inhibition by theophylline, dl-4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724), and 1-methyl-3-isobutylxanthine (MIX) are quite different. Ro 20-1724 selectively inhibited both renal cortical-soluble and particulate cyclic AMP degradation, but had little effect on cyclic GMP hydrolysis. Theophylline and MIX effectively inhibited degradation of both cyclic nucleotides, with MIX the more potent inhibitor. Effects of these agents on the cyclic AMP and cyclic GMP content of cortical slices corresponded to their relative potency in broken cell preparations. Thus, in cortical slices, Ro 20-1724 (2 mm) had the least effect on basal (without agonist), carbamylcholine, and NaN₃-stimulated cyclic GMP accumulation, but markedly increased basal and (parathyroid hormone) PTH-mediated cyclic AMP accumulation, MIX (2 mm) which was as effective as Ro 20-1724 in potentiating basal and PTH-stimulated increases in cyclic AMP also mediated the greatest augmentation of basal, carbamylcholine, and NaN₃-stimulated accumulation of cyclic GMP. By contrast, theophylline (10 mm) which was only 12% as effective as Ro 20-1724 in increasing the total slice cyclic AMP content in the presence of PTH was much more effective than Ro 20-1724 in potentiating carbamylcholine and NaN₃-mediated increases in cyclic GMP. These results demonstrate selective inhibition of cyclic nucleotide phosphodiesterase activities in the rat renal cortex and support the possibility of multiple cyclic nucleotide phosphodiesterases in this tissue. Furthermore, both cyclic nucleotides appear to be rapidly degraded in the renal cortex.     

Regulation of adenosine 3':5'-monophosphate-dependent protein kinase in cerebral cortex

Alterations in the cyclic AMP-dependent protein kinase activity ratio in response to putative neurotransmitters and other cyclic AMP-elevating agents in intact cerebral cortical slices and Krebs-Ringer particulate preparations from cerebral cortex were examined. Both norepinephrine (30 microM) and forskolin (20 microM) produced a time-dependent increase in intracellular levels of cyclic AMP in cerebral cortical slices which was paralleled by an increase in both cyclic AMP and the protein kinase activity ratio. The increases were maximal at 5 min. and remained elevated for at least 15 min. Forskolin, norepinephrine, adenosine and isoproterenol produced a concentration-dependent increase in both cyclic AMP and the protein kinase activity ratio, however, the degree of increase observed was dissimilar. Thus, a 5-fold change in intracellular cyclic AMP resulted in only a 2-fold increase in the activity ratio. Of the agents examined, forskolin produced the most marked change in the activity ratio (from 0.23 to 0.78 at 100 microM) while isoproterenol at 100 microM produced only a 50% increase in the activity ratio. The half-time for the decline in forskolin elicited elevations of either the activity ratio or cyclic AMP was about 4-6 min. In the presence of the phosphodiesterase inhibitor, Ro 20-1724, both were significantly prolonged being 60-70% of the maximum observed immediately after forskolin stimulation, at 15 min. Potentiation of forskolin elicited increases in the activity ratio by Ro 20-1724 were also observed but the increase in the activity ratio was maximal at 7.5 min. while cyclic AMP accumulations continued to rise during the entire 15 min. incubation. Particulate preparations from cerebral cortex were found to contain a cyclic AMP-dependent protein kinase which could be activated 2 to 3-fold with either forskolin, norepinephrine, or adenosine. Unlike the intact brain slice the changes in protein kinase activity ratio and intracellular levels of cyclic AMP in cell-free particulate preparations were similar in both time and degree.     

Cyclic nucleotide hydrolysis in the thyroid gland. General properties and key role in the interrelations between concentrations of adenosine 3':5'-monophosphate and guanosine 3':5'-monophosphate.

Phosphodiesterase activities of horse (and dog) thyroid soluble fraction were compared with either cyclic AMP (adenosine 3':3'-monophosphate) or cyclic GMP (guanosine 3':5'-monophosphate) as substrate. Optimal activity for cyclic AMP hydrolysis was observed at pH 8, and at pH 7.6 for cyclic GMP. Increasing concentrations of ethyleneglycol bis(2-aminoethyl)-N,N'-tetraacetic acid inhibited both phosphodiesterase activities; in the presence of exogenous Ca2+, this effect was shifted to higher concentrations of the chelator. In a dialysed supernatant preparation, Ca2+ had no significant stimulatory effect, but both Mg2+ and Mn2+ increased cyclic nucleotides breakdown. Mn2+ promoted the hydrolysis of cyclic AMP more effectively than that of cyclic GMP. For both substrates, substrate velocity curves exhibited a two-slope pattern in a Hofstee plot. Cyclic GMP stimulated cyclic AMP hydrolysis, both nucleotides being at micromolar concentrations. Conversely, at no concentration had cyclic AMP any stimulatory effect on cyclic GMP hydrolysis. 1-Methyl-3-isobutylxanthine and theophylline blocked the activation by cyclic GMP of cyclic GMP of cyclic AMP hydrolysis, whereas Ro 20-1724 (4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone), a non-methylxanthine inhibitor of phosphodiesterases, did not alter this effect. In dog thyroid slices, carbamoylcholine, which promotes an accumulation of cyclic GMP, inhibits the thyrotropin-induced increase in cyclic AMP. This inhibitory effect of carbamoylcholine was blocked by theophylline and 1-methyl-3-isobutylxanthine, but not by Ro 20-1724. It is suggested that the cholinergic inhibitory effect on cyclic AMP accumulation is mediated by cyclic GMP, through a direct activation of phosphodiesterase activity.     

Serum-coated zymosan stimulates the synthesis of leukotriene B4 in human polymorphonuclear leukocytes. Inhibition by cyclic AMP

Addition of serum-treated zymosan particles to a suspension of human peripheral blood polymorphonuclear leukocytes led to the formation of leukotriene B₄. Prostaglandin I₂ and RO20-1724 (an inhibitor of cyclic 3′:5′-nucleotide phosphodiesterase) decreased the synthesis of this compound, indicating that cyclic AMP exerts an inhibitory effect on the formation of leukotriene B₄.     

Forskolin and phosphodiesterase inhibitors release adenosine but inhibit morphine-evoked release of adenosine from spinal cord synaptosomes.

The effects of forskolin, Ro 20-1724, rolipram, and 3-isobutyl-1-methylxanthine (IBMX) on morphine-evoked release of adenosine from dorsal spinal cord synaptosomes were evaluated to examine the potential involvement of cyclic AMP in this action of morphine. Ro 20-1724 (1-100 microM), rolipram (1-100 microM), and forskolin (1-10 microM) increased basal release of adenosine, and at 1 microM inhibited morphine-evoked release of adenosine. Release of adenosine by Ro 20-1724, rolipram, and forskolin was reduced 42-77% in the presence of alpha,beta-methylene ADP and GMP, which inhibits ecto-5'-nucleotidase activity by 81%, indicating that this adenosine originated predominantly as nucleotide(s). Significant amounts of adenosine also were released from the ventral spinal cord by these agents. Ro 20-1724 and rolipram did not significantly alter the uptake of adenosine into synaptosomes. Although Ro 20-1724 and rolipram had only limited effects on the extrasynaptosomal conversion of added cyclic AMP to adenosine, IBMX, a phosphodiesterase inhibitor with a broader spectrum of inhibitory activity for phosphodiesterase isoenzymes, significantly inhibited the conversion of cyclic AMP to adenosine and resulted in recovery of a substantial amount of cyclic AMP. As with the non-xanthine phosphodiesterase inhibitors, IBMX increased basal release of adenosine and reduced morphine-evoked release of adenosine. Adenosine released by IBMX was reduced 70% in the presence of alpha,beta-methylene ADP and GMP, and release from the ventral spinal cord was 61% of that from the dorsal spinal cord. Collectively, these results indicate that forskolin and phosphodiesterase inhibitors release nucleotide(s) which is (are) converted extrasynaptosomally to adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regional Difference in Responsiveness of Adenosine-Sensitive Cyclic AMP-Generating Systems in Chronic Epileptic Cerebral Cortex of the Rat

Cyclic AMP accumulation in brain slices incubated with adenosine or the adenosine analogue 2-chloroadenosine was examined in different areas of rat cerebral cortex following a unilateral injection of FeCl2 solution into the sensorimotor cortex to induce chronic epileptic activity. In the epileptic cortex, cyclic AMP accumulation in cortical slices was elicited three- to 11-fold by adenosine. The elicitation by adenosine of cyclic AMP accumulation was markedly inhibited by the adenosine antagonist 8-phenyltheophylline. In anterior cortical areas of rats in which the appearance of electrographic isolated spikes was dominant either ipsilateral or contralateral to the injection site 8 days or more after the injection, the adenosine-elicited accumulation of cyclic AMP was greater on the side of dominant spike activity than on the other. In anterior cortical areas of rats showing nearly equal spike activity on the two sides 19 days or more after the injection, the cyclic AMP accumulation was greater on the side ipsilateral to the injection site than on the other. In anterior and posterior cortical areas of rats showing spike-and-wave complexes and isolated spikes 1 month or more after the injection, the cyclic AMP accumulation was greater on the ipsilateral side than on the other. Similar regional differences in the adenosine-elicited accumulation of cyclic AMP were detected in the presence of the adenosine uptake inhibitor dipyridamole or the phosphodiesterase inhibitor DL-4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724). The cyclic AMP accumulation was elicited five- to 17-fold by 2-chloroadenosine, in which case the elicitation was markedly inhibited by 8-phenyltheophylline. Regional differences in the 2-chloroadenosine-elicited accumulation of cyclic AMP were similar to those with adenosine and were detected in the presence of Ro 20-1724 or adenosine deaminase. The regional differences which correlated with the electrographic discharge patterns were due mainly to persistent changes in cyclic AMP accumulation on the primary epileptic side. These results suggest that alterations in adenosine-sensitive cyclic AMP generation in the cortex are associated with the neurochemical process leading to chronic iron-induced epilepsy.     

Cyclic 3′,5′-AMP phosphodiesterase in Physarum polycephalum I. Chemotaxis toward inhibitors and cyclic nucleotides

The effect of several inhibitors of the enzyme cyclic 3′,5′-AMP phosphodiesterase as chemoattractants in Physarum polycephalum was examined. Of the compounds tested, 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Roche 20-1724/001) and 1-ethyl-4-(isopropylidinehydrazino)-1H-pyrazolo-(3,4-b)-pyridine-5-carboxylic acid ethyl ester, hydrochloride (Squibb 20009) were the most potent attractants. 3-Isobutyl-1-methyl xanthine, theophylline, and morin (a flavanoid) were moderate attractants and sometimes gave negative chemotaxis at high concentrations. Cyclic 3′,5′-AMP was an effective, but not potent attractant. A repellent effect following the positive chemotactic action was sometimes observed with cyclic 3′,5′-AMP at concentrations as high as 1 · 10^?2 M. Dibutyryl cyclic AMP appeared to be a somewhat more potent attractant than cyclic 3′,5′-AMP. The 8-thiomethyl and 8-bromoderivatives of cyclic AMP, which are poorly hydrolyzed by the phosphodiesterase, were not attractants in Physarum. Possible participation of cyclic 3′,5′-AMP in the directional movement in P. polycephalum is discussed.     

Effect of Isozyme-Selective Inhibitors of Phosphodiesterase on Histamine-Stimulated Cyclic AMP Accumulation in Guinea-Pig Hippocampus

Addition of histamine (0.1 mM) to guinea-pig hippocampal slices causes a 20- to 30-fold increase in the accumulation of cyclic AMP compared with basal levels. This accumulation represents a balance between cyclic AMP production by adenylate cyclase and cyclic AMP breakdown mediated by phosphodiesterase (PDE). However, brain tissues are known to contain several different PDE isozymes. To determine which are involved in this response to histamine, the effect of isozyme-specific PDE inhibitors on cyclic AMP accumulation was examined in the hippocampus. MB 22948 (0.1 mM), an inhibitor of PDEs I and II, had no significant effect on the response to either 1 microM or 0.1 mM histamine. SKF 94120 (0.1 mM), a PDE III inhibitor, was also without effect in the presence of 1 microM histamine, although with 0.1 mM histamine, it caused a weak (1.25-fold compared with control), but statistically significant, enhancement of cyclic AMP accumulation. However, both rolipram (0.1 mM), a PDE IV inhibitor, and 3-isobutyl-1-methylxanthine (0.1 or 1 mM), an inhibitor of all forms of PDE, significantly increased cyclic AMP accumulation (2.8- to 6.5-fold compared with controls), and the relative size of this effect decreased with increasing histamine concentration. It is concluded that PDE IV is the main PDE isozyme involved in cyclic AMP turnover in guinea-pig hippocampal slices responding to histamine.     

Morphone abstinence and quasi-abstinence effects after phosphodiesterase inhibitors and naloxone.

Naive or morphine-dependent rats received a single subcutaneous injection of a phosphodiesterase inhibitor; their behavioral responses were then recorded after a small subcutaneous dose of naloxone. In naive rats, the potent phosphidiesterase inhibitor, 3-isobutyl-1-methylxanthine (IBMX) produced acutely a state in which a small dose of naloxone (0.03 to 1.0 mg/kg subcutaneously) precipitated a quasi-morphine abstinence syndrome that was difficult to distinguish from the true abstinence syndrome, precipitated by the same dose of naloxone in rats made dependent on morphine. IBMX also intensified the true morphine abstinence syndrome. The potency with which IBMX, theophylline, caffeine and RO 20–1724 exerted these effects corresponded with their potency as inhibitors of cyclic-3′, 5′-AMP phosphodiesterase in rat brain homogenate. These and previous findings indicate that: (i) morphine-abstinence effects express increased activity of a central cyclic AMP mechanism; and (ii) naloxone can potently stimulate behavior in animals not treated with any opiate drug.     

Negative control of TSH action by iodide and acetylcholine: mechanism of action in intact thyroid cells.

Iodide, a substrate of thyroid metabolism, and acetylcholine depress cyclic AMP intracellular content and secretion in dog thyroid slices under TSH stimulation. A direct or indirect pseudocompetitive effect at the level of TSH receptor interaction has been rejected. Iodide and carbachol, both inhibited cyclic AMP accumulation in TSH stimulated dog thyroid slices but only the effect of carbachol was suppressed in the presence of isobutylmethylanthine. Ro 20-1724 did not relieve either inhibitory effect. Carbachol greatly enhanced cyclic AMP disposal in TSH prestimulated slices after the cut off of hormone action by a trypsin treatment. This effect was also suppressed by isobutylmethylxanthine but not by Ro 20-1724. No action of iodide could be evidenced on cyclic AMP disposal in similar slices, although a clear effect after the same time of iodide action was observed on cyclic AMP accumulation. Neither carbachol, nor iodide depresses ATP levels in these slices. The data suggest that carbachol exerts its action through an activation of cyclic AMP disappearance probably by an activation of cyclic AMP phosphodiesterase and that iodide, through an oxidized intermediate, experts its inhibitory effect at the level of cyclic AMP synthesis.     

Cyclic 3′,5′-nucleotide phosphodiesterase: A continuous titrimetric assay

A direct and continuous assay for cyclic 3′,5′-nucleotide phosphodiesterase has been developed. This method is based on the fact that the phosphate group of adenosine 3′,5′-phosphate has one titratable species whereas that of 5′-adenosine monophosphate has two. Hydrolysis of cyclic AMP to 5′-AMP by phosphodiesterase is accompanied by a stoichiometric generation of protons. The rate of addition of an alkaline solution to the reaction mixture to maintain a constant pH with a pH stat is thus stoichiometrically related to the rate of cyclic AMP hydrolysis. A reaction producing 10 mμmoles of H⁺ or more per minute in 1.5 ml of reaction mixture is accurately measured by this technique. Duplicates are usually within 5% of each other. Results obtained by the titrimetric method correlate well with those obtained by conventional methods. This technique has been successfully used to assay phosphodiesterase of bovine brain in the purified as well as the crude stage.     

1-Isoamyl-3-isobutylxanthine: a remarkably potent agent for the potentiation of norepinephrine, histamine, and adenosine-elicited accumulations of cyclic AMP in brain slices.

1-Isoamyl-3-isobutylxanthine (EC₅₀ t 5 μM) potentiates by 2 to 6-fold the accumulations of cyclic AMP elicited in guinea pig cerebral cortical slices by norepinephrine, histamine, and adenosine. In addition, the xanthine derivative causes a 2 to 3-fold elevation of basal levels of cyclic AMP. 1-Isoamyl-3-isobutylxanthine has no effect on accumulations of cyclic AMP elicited by histamine or adenosine in the presence of a potent phosphodiesterase inhibitor, ZK 62771. The xanthine derivative retards the disappearance of cyclic AMP after a prior stimulation by adenosine. The results indicate that 1-isoamyl-3-isobutylxanthine is an extremely potent and effective inhibitor of phosphodiesterases involved in the regulation of cyclic AMP levels in guinea pig cerebral cortical slices. The 1-benzyl, 1-isoamyl, and 1-isobutyl derivatives of 3-isobutylxanthine potentiate the accumulation of cyclic AMP elicited by adenosine, while the 1-methyl derivative and 1-isoamyl-3-methylxanthine are inhibitory undoubtedly because of blockade of adenosine-receptors by these compounds. Xanthines with bulky 1- and 3- substituents appear to be relatively weak adenosine-antagonists and relatively specific and potent agents for inhibition of phosphodiesterases involved in cyclic AMP metabolism in brain tissue.     

High pressure liquid chromatography of cyclic nucleotide phosphodiesterase from purified human T-lymphocytes

The cyclic nucleotide phosphodiesterase (EC 3.1.4.17) in extracts of purified human peripheral blood T-lymphocytes was examined by ion exchange high pressure liquid chromatography. Four peaks of activity were isolated. The first peak of activity selectively hydrolyzed cyclic GMP. The following 3 peaks of activity (Ia, IIa and IIIa) were selective for cyclic AMP. The selective low Km cyclic AMP-phosphodiesterase inhibitor, Ro 20-1724 (d,1-1,4-[3-butoxy-4-methoxybenzyl]-2-imidazolidinone), did not inhibit the activity in Ia whereas it did inhibit the activity in IIa and IIIa (IC50 = 17 microM). The authors conclude that ion exchange high pressure liquid chromatography described in this communication is a useful method for the isolation of different forms of cyclic nucleotide phosphodiesterase activity from human T-lymphocytes.     

Potentiation of the 5-hydroxytryptamine-induced increases in myocardial contractility in Mercenaria mercenaria ventricle by forskolin

Forskolin (10(-5) M), an activator of adenylate cyclase in many mammalian cell types, potentiated the positive inotropic effects of 5-hydroxytryptamine (5-HT) on the bivalve, Mercenaria mercenaria, myocardium. Forskolin (10(-5) M) also significantly increased intracellular cyclic AMP concentrations when compared to 5-HT controls. Ro 20-1724 (10(-5) M), a putative inhibitor of cyclic AMP phosphodiesterase, had no effect on myocardial contractility or cyclic AMP concentration. A positive correlation between intracellular cyclic AMP concentration and the efficacy of 5-HT was obtained.     

Effects of isoproterenol on cyclic AMP and cyclic AMP-dependent protein kinase in developing chick myocardium

Embryonic chick (7–9 day) and newborn chick myocardia contain one major peak of cyclic AMP-dependent protein kinase activity as assessed by DEAE-cellulose chromatography. Evidence is presented that the cyclic AMP-dependent protein kinase activity ratios (activity in absence of cyclic AMP/activity in presence of added cyclic AMP) of homogenates prepared with low ionicf strength buffer reflect the endogenous activation state of the enzyme. The cyclic AMP content of newborn chick myocardium is lower than that of 7–9-day embryonic chick myocardium; the baseline cyclic AMP-dependent protein kinase activity is correspondingly reduced. Isoproterenol produces smaller elevations in cyclic AMP and in the cyclic AMP-dependent protein kinase activity ratio in newborn chick as compared to embryonic chick myocardium. Differences in the ability of isoproterenol to elevate cyclic AMP in the different preparations are not accompanined by appropriate changes in the adenylate cyclase or phosphodiesterase activities of the corresponding broken cell preparations. Studies with the phosphodiesterase inhibitor, Ro 20 1724 indicate that the changes in the ability of isoproterenol to elevate cyclic AMP in the developing chick myocardium are due to changes in the metabolism of the cyclic nucleotide by phosphodiesterase.     

Activation of cyclic AMP-dependent protein kinase and stimulation of protein phosphorylation in response to adenosine in C-1300 murine neuroblastoma

DEAE-cellulose chromatography of the 20,000g supernatant fraction of homogenates of C-1300 murine neuroblastoma (clone N2a) yields one major and two minor peaks of cyclic AMP-dependent protein kinase activity. Assessment of the endogenous activation state of the enzyme(s) reveals that the enzyme is fully activated by the treatment of whole cells with adenosine (10 μM) in the presence of the phosphodiesterase inhibitor Ro 20 1724 (0.7 mM). This treatment produces a large elevation in the cyclic AMP content of the cells. The treatment of whole cells with adenosine alone (1–100 μM) or Ro 20 1724 alone (0.1–0.7 mM) produces minimal elevations in cyclic AMP but nevertheless causes significant activations of cyclic AMP-dependent protein kinase. The autophosphorylation of whole homogenates of treated and untreated cells was studied using [γ-³²P] ATP, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Treatments which activate cyclic AMP-dependent protein kinase selectively stimulate the incorporation of ³²P into several proteins. This stimulation is most prominent in the 15,000-dalton protein band. The addition of cyclic AMP to phosphorylation reactions containing homogenate of untreated cells stimulates the phosphorylation of the same protein bands. These results indicate that adenosine may have regulatory functions through its effect on the cyclic AMP: cyclic AMP-dependent protein kinase system.     

Evidence for mediation of acetyl glyceryl ether phosphorylcholine stimulation of adenosine 3′,5′-(cyclic)monophosphate levels in human polymorphonuclear leukocytes by leukotriene B4

Acetyl glyceryl ether phosphorylcholine induces human neutrophil aggregation. Incubation of neutrophils with either prostaglandin I₂, or the cyclic AMP-dependent phosphodiesterase inhibitor, RO 20-1724 before the addition of PAF-acether attenuates subsequent aggregation. Paradoxically, a small elevation in cyclic AMP is observed coincident with the initiation of PAF-acether-stimulated aggregation. The elevation in cyclic AMP in response to PAF-acether is amplified by RO 20-1724, and the magnitude of the response is dependent upon the concentration of PAF-acether. The elevation in cyclic AMP is not due to prostaglandins, because indomethacin actually enhances the elevation in cyclic AMP induced by PAF-acether. The involvement of the neutrophil 5-lipoxygenase, and subsequent leukotriene B₄ synthesis, is suggested by the observation that 5-lipoxygenase inhibitors limit both the elevation in cyclic AMP induced by PAF-acether, and the indomethacin enhancement. This indirect evidence is supported by the fact that leukotriene B₄ itself elevates neutrophil cyclic AMP levels in intact cells, and stimulates the adenylate cyclase in broken cell preparations. Although the elevation in cyclic AMP induced by either PAF-acether or leukotriene B₄ is coincident with the onset of neutrophil aggregation, it is not obligatory for aggregation. The adenylate cyclase inhibitor 2′,5′-dideoxyadenosine blocks the PAF-acether-stimulated increase in cyclic AMP, and actually enhances aggregation. It is suggested that the increase in cyclic AMP observed after the addition of PAF-acether is due to concomitant leukotriene B₄ synthesis, and is not obligatory for neutrophil aggregation, but is actually part of a feed-back regulatory system through which PAF-acether and leukotriene B₄ can limit their own activity in neutrophils.     

Assessment of selective inhibition of rat cerebral cortical calcium-independent and calcium-dependent phosphodiesterases in crude extracts using deoxycyclic AMP and potassium ions

The effects of various inhibitors on the activity of calcium-independent and calcium-dependent phosphodiesterases from rat cerebral cortex were examined. While the agents varied greatly in their relative potency, each was found to be approximately equipotent in inhibiting the calcium-dependent hydrolysis of either cyclic AMP or cyclic GMP. In contrast, the inhibitors displayed a marked substrate specificity for the calcium-independent enzyme with ratios of IC₅₀ values for inhibition of cyclic GMP hydrolysis when compared to cyclic AMP hydrolysis in decreasing order being: ZK 62711 (? 100) > Ro 20–1724 (?>25) papaverine (13) > 7-benzyl IBMX (4) > quercetin and kaempferol (2). The differential selectivity of the inhibitors for the two enzymes was most pronounced for ZK 62711 and Ro 20–1724 which were at least 25–100-times more potent in inhibiting the calcium-independent hydrolysis of cyclic AMP when compared to the calcium-dependent hydrolysis of cyclic AMP. In contrast, 7-benzyl IBMX, kaempferol and quercetin were 8–100-times more effective as inhibitors of cycluc GMP hydrolysis by the calcium-dependent phosphodiesterase while 7-benzyl IBMX and trimazosin displayed a similar enzyme selectivity using cyclic AMP as substrate. With the exception of papaverine, all agents were competitive inhibitors of the calcium-dependent phosphodiesterase. The type of inhibition observed with the calcium-independent enzyme was dependent on the substrate employed. The specificity of potassium ions in inhibiting the activity of the calcium-dependent phosphodiesterase and deoxycyclic AMP in inhibiting the calcium-independent enzyme was found to provide a convenient means to assess the effects of agents on these activities in crude extracts of cerebral cortex.     

Stimulation by Dibutyryl Cyclic AMP of Serotonin Synthesis and Tryptophan Transport in Brain Slices

N⁶,O²-′Dibutyryl cyclic AMP (dibutyryl cyclic AMP), a derivative of 3′,5′-adenosine monophosphate (cyclic AMP) resistant to phosphodiesterase inactivation, has been reported to stimulate serotonin and melatonin synthesis in the pineal gland in vitro^1–3. In brain adenyl cyclase and phosphodiesterase, which catalyse the formation and the inactivation of cyclic AMP, are found chiefly in the synaptosomal fraction of the tissue homogenates⁴, where vesicles containing monoamine are also present⁵. These factors prompted us to study the effects of cyclic AMP and its dibutyryl derivative on the synthesis of brain monoamines.     

Cyclic guanosine 3',5'-monophosphate and phosphodiesterase activity in mitogen-stimulated human lymphocytes.

The cyclic adenosine 3′,5′-monophosphate (cyclic AMP) phosphodiesterase from human leukemic lymphocytes differes from the normal cell enzyme in having a much higher activity and a loss of inhibition by cyclic guanosine 3′,5′-monophosphate (cyclic GMP). In an effort to determine the mechanism of these alterations, we have studied this enzyme in a model system, lectin-stimulated normal human lymphocytes. Following stimulation of cells with concanavalin A (con A) the enzyme activity gradually becomes altered, until it fully resembles the phosphodiesterase found in leukemic lymphocytes. The changes in the enzyme parallel cell proliferation as measured by increases in thymidine incorporation into DNA. The addition of a guanylate cyclase inhibitor preparation from the bitter melon prevents both the changes in the phosphodiesterase and the thymidine incorporation into DNA. This blockage can be partially reversed by addition of 8-bromo cyclic guanosine 3′,5′-monophosphate (8-bromo cyclic GMP) to the con A-stimulated normal lymphocytes. These results indicate a possible role of cyclic GMP in a growth related alteration of cyclic AMP phosphodiesterase.