Cellular Cyclic Nucleotide Levels Research Articles

Comparison of cAMP-specific phosphodiesterase mRNAs distribution in mouse and rat brain

There are eleven families of phosphodiesterases that regulate cellular levels of cyclic nucleotides by degradation of cAMP or cGMP. Knowledge of the expression sites of different PDE genes in brain is of special importance for studies on development of specific inhibitors considering that, for example, PDE4 inhibitor treatments exhibit profound anti-inflammatory effects. To address possible species differences we examined the expression of mRNAs coding for the cAMP specific PDE4 and PDE7 families since inhibitors have been used in clinic for schizophrenia, mood disorders, cognition and inflammatory diseases treatment. We have compared the expression of these PDEs in mouse brain by in situ hybridization histochemistry in comparison with rat brain and found that their neuroanatomical distribution differs in a few areas.

Cyclic Nucleotide Phosphodiesterase Assay Technology

Because of their critical role in modulating cellular cyclic nucleotide levels, phosphodiesterases (PDEs) are involved in many disease-related signaling pathways. The PDE family is large and diverse, with members having different tissue distribution, sub-cellular localizations, and substrate specificities. Because of these characteristics, the PDEs represent a broad group of potential drug targets. Described in the present unit are the assay development and validation procedures needed to establish a high-throughput screening system for these important enzymes. The assays provide a structured approach for determining the kinetic parameters of related enzyme families to facilitate the characterization of PDE inhibitors.

Prevention of Platelet Glycoprotein IIb/IIIa Activation by 3,4-Methylenedioxy-β-Nitrostyrene, A Novel Tyrosine Kinase Inhibitor

Binding fibrinogen to activated glycoprotein (GP)IIb/IIIa is the final common pathway of platelet aggregation and has become a successful target for antiplatelet therapy. In the present study, we found that a small chemical compound, 3,4-methyl-enedioxy-beta-nitrostyrene (MNS), exhibited potent and broad-spectrum inhibitory effects on human platelet aggregation caused by various stimulators. Moreover, addition of MNS to human platelets that had been aggregated by ADP caused a rapid disaggregation. We demonstrated that the antiaggregatory activity of MNS is due to inhibition of GPIIb/IIIa activation by measuring the binding amount of PAC-1 in platelets. In contrast, MNS is not a direct antagonist of GPIIb/IIIa, because MNS did not affect fibrinogen binding to fixed ADP-stimulated platelets. By investigating how MNS inhibits GPIIb/IIIa activation, we found that MNS potently inhibited the activity of tyrosine kinases (Src and Syk) and prevented protein tyrosine phosphorylation and cytoskeletal association of GPIIb/IIIa and talin, but it had no direct effects on protein kinase C, Ca2+ mobilization, Ca2+-dependent enzymes (myosin light chain kinase and calpain), and arachidonic acid metabolism, and it did not affect the cellular levels of cyclic nucleotides. Therefore, MNS represents a new class of tyrosine kinase inhibitor that potently prevents GPIIb/IIIa activation and platelet aggregation without directly affecting other signaling pathways required for platelet activation. Because MNS inhibits GPIIb/IIIa functions in a manner different from GPIIb/IIIa antagonists, this feature may provide a new strategy for treatment of platelet-dependent thrombosis.

Involvement of Cyclic GMP in Nitric-Oxide-induced Gastric Relaxation: Comparison of the Actions of Cyclic GMP and Cyclic AMP

Background: Smooth muscle relaxation induced by various agents that increase the cellular levels of cyclic nucleotides (cAMP and cGMP) is accompanied by a decrease in intracellular Ca2+ concentration. However, little is known about the differences between the inhibitory effects of cAMP and cGMP on the contraction of smooth muscle. Objective: To compare the effects and underlying mechanisms of cAMP and cGMP on the inhibition of gastric smooth muscle contraction, cyclic nucleotide promoting agents, as well as cell membrane permeable cyclic nucleotides were used. Methods: Isometric contraction was measured from circular muscle strips prepared from the fundus of cat stomach in a cylinder-shaped chamber filled with Krebs-Ringer solution (pH 7.4, temperature 36°C) bubbled with 5% CO2 in O2. The level of inositol phosphates (IPs) was measured. Results: Forskolin and sodium nitroprusside significantly inhibited acetylcholine (ACh)-induced gastric smooth muscle contraction and increased the cellular levels of cAMP and cGMP, respectively. Direct application of 8-Br-cAMP and 8-Br-cGMP also significantly inhibited ACh-induced contraction. Both verapamil and TMB-8 inhibited ACh-induced contraction. The combined inhibitory effect of verapamil and TMB-8 was significantly greater than the effect of either one, separately. Forskolin or sodium nitroprusside similarly augmented the effect of verapamil. However, the inhibitory effect of TMB-8 was augmented only by 8-Br-cGMP or sodium nitroprusside but not by 8-BrcAMP or forskolin. Forskolin and 8-Br-cAMP significantly inhibited the formation of inositol phosphates stimulated by ACh. Conclusions: cAMP inhibits the contraction mechanism associated with intracellular Ca2+ mobilization as well as extracellular Ca2+ influx, while cGMP inhibits contraction by inhibiting the mechanism associated with extracellular Ca2+ influx.

Nitric Oxide Activation of p38 Mitogen-activated Protein Kinase in 293T Fibroblasts Requires cGMP-dependent Protein Kinase

An increase in cellular levels of cyclic nucleotides activates serine/threonine-dependent kinases that lead to diverse physiological effects. Recently we reported the activation of the p38 mitogen-activated protein kinase (MAPK) pathway in neutrophils by a cGMP-dependent mechanism. In this study we demonstrated that exogenously supplied nitric oxide leads to activation of p38 MAPK in 293T fibroblasts. Phosphorylation of p38 corresponded with an increase in ATF-2-dependent gene expression. The effect of nitric oxide was mimicked by addition of 8-bromo-cGMP, indicating that activation of soluble guanylyl cyclase was involved. The importance of cGMP-dependent protein kinase in the activation of p38 MAPK by nitric oxide in 293T cells was assessed in a transfection based assay. Overexpression of cGMP-dependent protein kinase-1alpha caused phosphorylation of p38 in these cells and potentiated the effectiveness of cGMP. Overexpression of a catalytically inactive mutant form of this enzyme (T516A) blocked the ability of both nitric oxide and 8-bromo-cGMP to activate p38 as measured by both p38 phosphorylation and ATF-2 driven gene expression. Together, these data demonstrate that nitric oxide stimulates a novel pathway leading to activation of p38 MAPK that requires activation of cGMP-dependent protein kinase.

Aflatoxin B 1 is an inhibitor of cyclic nucleotide

Aflatoxin B 1 (AFB 1) action on cyclic nucleotide phosphodiesterase (PDE) activity has been tested on tissue extracts of various organs. In the presence of 100 μM AFB 1 a significant inhibition of cAMP and cGMP hydrolytic activity is observed in all tested tissue extracts. However, cGMP hydrolytic activity appears more sensitive to AFB 1 inhibition than cAMP hydrolytic activity and a considerably higher inhibition is observed in lung and spleen, than in liver, brain, kidney, and heart. When cGMP is used as substrate, the inhibitory response reaches 72% in lung and spleen extracts. We have also tested AFB 1 effects on lung and liver PDE activity peaks separated by DEAE-cellulose chromatography. These data confirm the poor sensitivity to the toxin of all PDE activities present in liver, while the lung peak (where PDE V in present) shows a higher sensitivity to AFB 1. In order to establish whether PDE V is in fact more sensitive to AFB 1, we have used mouse neuroblastoma cells, in which cGMP hydrolytic activity has been shown to be due to PDE V only. In this case, the calculated IC 50 is 24 μM and Dixon plot analysis shows a competitive inhibitory effect with a Ki of 16.7 μM. We have also used aflatoxin B 2 and M 2, and they proved to be much less effective than AFB 1: AFB 2 inhibits PDE V with an IC 50 of 117 μM, while AFM 2 does not show any effect. These results provide the first evidence of a competitive inhibition of AFB 1 on an enzymatic activity and suggest that an alteration of cellular cyclic nucleotide levels may play a role in the mechanism of aflatoxin action. All rights reserved.

The Effect of Local Delivery of IBMX on the Neointimal Proliferation in Rat Carotid Balloon Injury Model

Background：Hyperplastic neointima is one of the major mechanisms of restenosis following balloon angioplasty in selected patients with symptomatic angina pectoris. Elevation of cellular cyclic nucleotide levels such as cAMP and cGMP are known to inhibit the proliferation of vascular smooth muscle cells. 3-isobutyl-1-methylxanthine (IBMX increases intracellular cAMP and cGMP by nonselective inhibition of phosphodiesterases (PDEs. We conducted this study under the hypothesis that local delivery of IBMX could inhibit neointimal hyperplasia after balloon injury of the rat carotid artery. Methods：Left common carotid artery of 10 week old male Sprague-Dawley rats were subjected to arterial injury by 2F Fogarty balloon catheter. After injury, animals were allocated to the control groups (control 1；injury control and control 2；pluronic gel plus DMSO control and IBMX group, which received pluronic polymer gel, DMSO and IBMX mixture periadventitially. After 3 weeks, the rats were killed by overdose of ketamine, and the injured left arteries were pressure-fixed with 10% formalin and subjected to histomorphological analysis. Results：Mean body weight of rats was not statistically different among study groups. The mean area of neointima (control group 1：0.28±0.05 mm 2 , control group 2：0.27±0. 08 mm 2 , IBMX group：0.18±0.08 mm 2 ；p<0.05 and the mean ratio of neointima to medial area[versus (control group 1：1.89±0.37, control group 2：1.95±0.41, IBMX group：1.41±0.47；p<0.05] were significantly less in IBMX group. The mean area of external elastic lamina was significantly larger in control group 1 than IBMX group (0.57±0.07 mm 2 versus 0.47±0.10 mm 2 and mean luminal area showed no significant difference among groups (control group1：0.14±0.07 mm 2 , control group 2：0.10±0.05 mm 2 , control group 3：0.16±0.06 mm 2 . Conclusion ：Peri-adventitial single administration of IBMX showed its effectiveness in reducing the neointimal proliferation in rat carotid balloon injury model. Furthermore we observed the positive

Cyclic nucleotide phosphodiesterase isozymes in rat mesangial cells

We characterized cyclic nucleotide phosphodiesterases isolated from rat mesangial cells and asserted their roles in regulating cellular cyclic nucleotide levels. Three peaks of phosphodiesterase activity were eluted by a linear sodium acetate gradient from a Q Sepharose column loaded with the mesangial cell extract. The first peak activity was stimulated by Ca 2+-calmodulin and inhibited by calmodulin-stimulated phosphodiesterase inhibitors but not by a selective cGMP specific phosphodiesterase V inhibitor. The second, minor activity peak was stimulated by cyclic GMP and inhibited by EHNA [erythro-9-(2-hydroxy-3-nonyl)-adenine], a selective inhibitor of cyclic GMP-stimulated phosphodiesterase II. The last peak activity was not inhibited by cyclic GMP but selectively inhibited by rolipram [4-(3-cyclopentyloxy-4-methoxyphenyl)-2-pyrrolidene] or Ro 20-1724 [4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone], inhibitors of cyclic AMP specific, cyclic GMP insensitive phosphodiesterase IV. Based on their order of chromatographic elution, kinetic properties and sensitivity to allosteric agents and inhibitors, the peak 1, 2 and 3 correspond to phosphodiesterase I, II and IV. The basal cyclic GMP level was raised more effectively by selective inhibitor of phosphodiesterase I than phosphodiesterase II. In contrast, the atrial natriuretic factor-induced cyclic GMP elevation was potentiated more effectively by selective inhibitors of phosphodiesterase II than phosphodiesterase I. The forskolin-induced cyclic AMP increase was greatly potentiated by selective phosphodiesterase IV inhibitors but not by other phosphodiesterase inhibitors. These data suggest that phosphodiesterase I and II are responsible for cyclic GMP hydrolysis whereas phosphodiesterase IV is mainly responsible for cyclic AMP hydrolysis. The relative importance of phosphodiesterase I and II varied with the intracellular cyclic GMP concentration.

Clearance receptor-binding atrial natriuretic peptides inhibit mitogenesis and proliferation of rat aortic smooth muscle cells

The current studies were designed to explore the effects of C-receptor-binding atrial natriuretic peptide analogues on serum-induced mitogenesis in cultured rat aortic smooth muscle cells. To this end, rANF99-126 and a series of truncated (rANF103-126, rANF103-125), ring-deleted (des[Gln116, Ser117, Gly118, Leu119, Gly120]rANF102-121-NH2 (c-ANF) and linear des(Cys105, Cys121)rANF104-126 peptide analogues were used. The latter two peptides have been reported to be selective for the ANF-C receptor. In cells subcultured between passage 3 to 19, rANF99-126, rANF103-126, and rANF103-125 concentration-dependently (0.1-1000 nM) inhibited serum-induced (3H) thymidine incorporation with maximal inhibition observed at 1 microM for each peptide (approximately 40, 31 and 56%) respectively. Furthermore, des[Cys105, Cys121]rANF104-126 inhibited serum-induced (3H)thymidine incorporation concentration-dependently without altering basal or elevated cellular cAMP or cGMP levels. Moreover, the reduction in thymidine incorporation was associated with inhibition of serum-induced clonal cell proliferation. In contrast, c-ANF failed to inhibit serum-induced mitogenesis, yet at a concentration of 100 nM it antagonized the antimitogenic effects of des[Cys105, Cys121]rANF104-126 or rANF99-126 without having any effect on basal or elevated cellular cyclic nucleotide levels. We conclude that the antimitogenic effect of atrial peptides is mediated through interaction with the ANF-C receptor and may be independent of changes in cellular cyclic nucleotide levels.

The role of mitochondria and sarcoplasmic reticulum calcium handling upon reoxygenation of hypoxic myocardium.

The mechanism of twitch prolongation of hypoxic myocardium after reoxygenation was studied before and after interventions that affect cellular cyclic nucleotide levels, subcellular calcium handling, or oxygen-derived free radical production/survival. Right ventricular ferret papillary muscles were subjected to two 20-minute periods of hypoxia, each followed by 1 hour of reoxygenation. The first sequence of hypoxia/reoxygenation was done without intervention. Before the second sequence, the pharmacological agent under study was added to the superfusate or the superfusate calcium concentration was increased from 2.5 to 8 mM. Time from peak to 80% decline in twitch tension was measured in the presence and absence of each intervention immediately before each period of hypoxia and after reoxygenation at maximal twitch prolongation. Interventions that affect Ca2+ flux across the sarcolemma (verapamil and 8 mM [Ca2+]o) or agents that affect oxygen free radical production/survival (dimethyl sulfoxide and allopurinol) did not affect twitch prolongation. Pharmacological agents that increase cyclic AMP levels (forskolin and milrinone) or those that inhibit mitochondrial activity (oligomycin B and ruthenium red) attenuated twitch prolongation. Pharmacological agents that decrease cyclic AMP levels (carbachol) or inhibit function of the sarcoplasmic reticulum (ryanodine) augmented twitch prolongation. The effect of mitochondrial inhibitors on intracellular calcium handling during hypoxia and reoxygenation was examined using muscles loaded with the bioluminescent calcium indicator aequorin. Mitochondrial inhibitors abbreviated the calcium transient and maximal twitch prolongation after hypoxia. We conclude that alterations in sarcoplasmic reticulum and mitochondria calcium handling contribute to the prolonged relaxation seen upon reoxygenation of hypoxic myocardium.

Cyclic nucleotide regulation of oosporogenesis by Lagenidium giganteum and related fungi

Incorporation of phosphodiesterase inhibitors into basal growth media limits the inducation of oosporogenesis by Lagenidium giganteum and related pythiaceous fungi. Cyclic AMP enhanced while cyclic GMP antagonized the effects of these compounds. Adenosine, guanosine, and their 5′-monophosphate derivatives acted primarily by reducing oospore maturation rather than induction. Asexual reproduction was generally promoted by cyclic AMP and phosphodiesterase inhibitors. It is suggested that cyclic nucleotides and sterols play a pivotal role in the induction of meiosis and that sterols and other factors affecting reproductive development by these fungi may act in part by regulating cellular cyclic nucleotide levels.

Steroidogenic properties of phorbol ester and a Ca 2+ ionophore in bovine adrenocortical cell suspensions

When added independently to bovine adrenocortical fasciculata cell suspensions, 12 tetradecanoyl-phorbol-13 acetate (TPA) and the Ca 2+ ionophore A23187 activated net cortisol production in a time and dose-dependent manner during one hour incubation. When added together (each at 1 μM concentration), the drugs appeared synergistic and mimicked the steroidogenic effect of suboptimal concentration of angiotensin II or acetylcholine on these cells, with no detectable variation of cellular cyclic nucleotide levels. In addition, the drug mixture markedly enhanced the steroidogenic effect of acetylcholine. These observations suggest that Ca 2+-activated, phospholipid dependent protein kinase, which is present in adrenal cortex, might be considered as a possible target in the mechanism of action of steroidogenic agents such as angiotensin and acetylcholine, acting in adrenocortical cell through cyclic AMP independent processes.

Spinal cord metabolic changes in murine retrovirus-induced motor neuron disease

Decreased cerebrospinal fluid concentrations of cyclic nucleotides in human motor neuron disease and decreased spinal cord concentrations of cyclic nucleotides in murine (Wobbler) motor neuron disease suggest that an abnormality in cyclic nucleotide metabolism may play a role in motor neuron degeneration. Retroviruses cause decreased cellular levels of cyclic nucleotides in infected cells. We induced a motor neuron degeneration with a neurotropic retrovirus, but not with a non-neurotropic retrovirus. In paralyzed mice, mean cAMP was decreased 21% in posterior horn segments and 34% in anterior horn segments compared to controls. In these mice, mean glycogen content was increased 25% in posterior horn segments and 32% in anterior horn segments compared to controls. The proportion of spinal cord phosphorylase a decreased 24% in paralyzed mice compared to controls. The content of cGMP decreased 48% in the cerebellum and 25% in both anterior and posterior horn segments of the spinal cords of paralyzed mice compared to controls. White matter content of these chemicals did not decrease in the posterior column of affected animals. Spinal cord content of ATP increased 20–22% in all three compartments, but the spinal cord content of phosphocreatine increased dramatically in white matter (46%), posterior horn gray matter (69%), and anterior horn gray matter (103%) compared to controls. Changes in high-energy phosphate intermediate and cyclic nucleotide metabolites occurred only in topographical regions showing neuronal and astrocyte pathological changes, but did not occur in the cerebral cortex.

The fluorescence response of chlortetracycline-loaded human neutrophils is modulated by prostaglandin E 1, but not by cyclic nucleotides

Human neutrophils preloaded with chlortetracycline, commonly used as a probe of membrane-bound calcium, demonstrate a prompt decrease in fluorescence when exposed to surface stimuli such as the chemotactic peptide fMet—Leu—Phe. The fluorescence response was highly sensitive to preincubation with prostaglandin E 1. This effect was apparently not due to elevated levels of cAMP since exogenous dibutyryl-cAMP did not alter the chlortetracycline fluorescence response to fMet—Leu—Phe. This is one of the few instances of prostaglandin E 1 affecting neutrophils at physiologic concentrations, dissociated from changes in cellular cyclic nucleotide levels

The effect of serum and multiplication stimulating activity on L6 myoblast growth: The lack of correlation with cyclic nucleotide changes

The role of cAMP and cGMP in triggering proliferation of rat myoblasts was evaluated by: (1) measuring effects of mitogens on intracellular cyclic nucleotide levels, and (2) observing effects of agents which altered cyclic nucleotide levels on cell proliferation. Multiplication stimulating activity (MSA, 1 microgram/ml), a member of the somatomedin family, stimulated cell proliferation after 48 hr. It had little effect on cellular cyclic nucleotide levels, measured by radioimmunoassay. Horse serum (HS) and fetal bovine serum (FBS) stimulated cell proliferation approximately equally. Neither affected cAMP levels; FBS reduced cGMP to 33% of control values, but HS had no effect. Thus, there was no simple correlation between mitogenic action and cyclic nucleotide levels at any time from 5 min to 24 hr after addition of a purified mitogen or serum. Furthermore, agents which caused substantial changes in cyclic nucleotide levels had no effect on cell proliferation. Prostaglandin E1 (5 microM) elevated cAMP 440% without affecting cGMP levels or cell growth. A potent phosphodiesterase inhibitor, 1-methyl-3-isobutylxanthine (MIX, 0.1 mM), when added alone or in the presence of MSA, HS, or FBS, elevated cAMP 200% and cGMP 167%, but it had little effect on their mitogenic action. Lastly, a purified mitogen such as MSA, unlike serum, must be present for extended periods of time in order to stimulate cell proliferation. This makes it unlikely that a trigger mechanism functions in initiating cell division. We conclude that neither cAMP nor cGMP appear to be second messengers for the mitogenic action of MSA or serum on muscle cells.

Enhancement of Neutrophil Chemotaxis and Alteration of Levels of Cellular Cyclic Nudeotides by Levamisole

Levamisole, an antihelminthic agent reported to enhance nonspecifically various parameters of the immune response, was examined for its effect on chemotaxis of human neutrophils and on levels of cellular cyclic nucleotides. This agent was found, in most instances, to enhance chemotactic responses of neutrophils to a bacterial chemotactic factor derived from Escherichia coli. At similar concentrations, levamisole produced increases in levels of guanosine 3':5'-cyclic phosphate in neutrophils. In contrast, a decrease in concentrations of adenosine 3':5'-cyclic phosphate was observed when neutrophils were incubated with levamisole. Neutrophil chemotaxis, with and without the addition of levamisole, was assessed in 10 patients with recurrent infections. The illnesses of these patients included Job's syndrome, Wiskott-Aldrich syndrome, eczema with an increased level of IgE and recurrent abscesses, chronic mucocutaneous candidiasis, and diabetes mellitus. Levamisole significantly enhanced chemotaxis of polymorphonuclear leukocytes from these patients. Levamisole appears to have a profound effect on chemotactic responses of neutrophils which probably results from alterations in cellular cyclic nucleotide levels. Levamisole may prove to be useful therapeutically in certain patients with defective neutrophil chemotaxis.

Papaverine: Its Effects on Cyclic Amp in Vitro and Psoriasis in Vivo

Psoriatic epidermis shows excessive cell proliferation, incomplete morphogenesis, and glycogen accumulation. These three features may result from coordinate misregulation by the previously reported abnormal lesional ratio of cyclic AMP/cyclic GMP. If so, it is possible that psoriasis might improve by topical application of an agent known to alter cellular levels of cyclic nucleotides. In the present study papaverine (3.0 x 10-4 m) produced a 210percent increase (.02 smaller than p smaller than .05) in epidermal cyclic AMP levels in vitro. Therefore, 1 percent papaverine cream (approximately 2.7 x 10-2m papaverine) was chosen to establish whether a cyclic AMP elevating agent could improve a soriatic lesion. A double-blind comparison of 1 percent papaverine cream versus cream alone was conducted on matched lesions in 45 patients. Of those patients showing differential improvement (i.e., only one lesion of the pair improved), 81 percent (p=0.011) of those showing a response in the center of the lesion and 77 percent (p=0.046) of those improving at the edge had applied papaverine. Although no attempts were made to develop a papaverine formulation with immediate therapeutic utility, the numerically significant papaverine results suggest future therapeutic developments.

Sequence of events mediating the effect of cholera toxin on rat thymocytes.

We have found that in rat thymocytes binding of [(125)I]choleragen is followed by cellular accumulation of cyclic 3',5'-AMP which, in turn, is followed by stimulation of amino acid transport. Binding of cholera toxin was complete by 30 min and remained constant for the subsequent 150 min. After stimulation by choleragen, cellular cyclic 3',5'-AMP became maximal by 30 min, after which it declined steadily so that by 90 min of incubation, cellular cyclic nucleotide levels were only 20% of those seen at 30 min. Stimulation of amino acid transport, although detectable by 15 min, did not become maximal until 120 min (by which time cellular cyclic 3',5'-AMP had decreased by more than 80%). We have also used this system to delineate the step at which various pharmacologic agents and hormones act to alter the sequence of events mediating the response of rat thymocytes to cholera toxin. The ability of cycloheximide to abolish choleragen-stimulated amino acid influx without reducing [(125)I]choleragen binding or cellular cyclic 3',5'-AMP suggests that cyclic nucleotide stimulation of amino acid transport includes a step involving protein synthesis.