Inhibitors Of Cyclic AMP Research Articles

Acute morphine alters GABAergic transmission in the central amygdala during naloxone-precipitated morphine withdrawal: role of cyclic AMP.

The central amygdala (CeA) plays an important role in opioid addiction. Therefore, we examined the effects of naloxone-precipitated morphine withdrawal (WD) on GABAergic transmission in rat CeA neurons using whole-cell recordings with naloxone in the bath. The basal frequency of miniature inhibitory postsynaptic currents (mIPSCs) increased in CeA neurons from WD compared to placebo rats. Acute morphine (10 μ M) had mixed effects (≥20% change from baseline) on mIPSCs in placebo and WD rats. In most CeA neurons (64%) from placebo rats, morphine significantly decreased mIPSC frequency and amplitude. In 32% of placebo neurons, morphine significantly increased mIPSC amplitudes but had no effect on mIPSC frequency. In WD rats, acute morphine significantly increased mIPSC frequency but had no effect on mIPSC amplitude in 41% of CeA neurons. In 45% of cells, acute morphine significantly decreased mIPSC frequency and amplitude. Pre-treatment with the cyclic AMP inhibitor (R)-adenosine, cyclic 3',5'-(hydrogenphosphorothioate) triethylammonium (RP), prevented acute morphine-induced potentiation of mIPSCs. Pre-treatment of slices with the Gi/o G-protein subunit inhibitor pertussis toxin (PTX) did not prevent the acute morphine-induced enhancement or inhibition of mIPSCs. PTX and RP decreased basal mIPSC frequencies and amplitudes only in WD rats. The results suggest that inhibition of GABAergic transmission in the CeA by acute morphine is mediated by PTX-insensitive mechanisms, although PTX-sensitive mechanisms cannot be ruled out for non-morphine responsive cells; by contrast, potentiation of GABAergic transmission is mediated by activated cAMP signaling that also mediates the increased basal GABAergic transmission in WD rats. Our data indicate that during the acute phase of WD, the CeA opioid and GABAergic systems undergo neuroadaptative changes conditioned by a previous chronic morphine exposure and dependence.

Feedback Control of Cyclooxygenase-2 Expression by Prostaglandin E2 in Rheumatoid Synoviocytes

Objective: The role of prostaglandin E2 (PGE2) in the etiopathogenesis of immune and inflammatory diseases has become the subject of recent debate. To determine the role of PGE2 in rheumatoid arthritis (RA), we tested the effect of exogenous PGE2 on the production of cyclooxygenase-2 (COX-2) by rheumatoid synoviocytes. Methods: Fibroblast-like synoviocytes (FLS) were prepared from the synovial tissues of RA patients, and cultured in the presence of PGE2. The COX-2 mRNA and protein expression levels were determined by RT-PCR and Western blot analysis, respectively. The PGE2 receptor subtypes in the FLS were analyzed by RT-PCR. Electrophoretic mobility shift assay (EMSA) was used to measure the NF-κB binding activity for COX-2 transcription. The in vivoeffect of PGE2 on the development of arthritis was also tested in collagen induced arthritis (CIA) animals. Results: PGE2 (10 to 10 M) dose-dependently inhibited the expression of COX-2 mRNA and the COX-2 protein stimulated with IL-1β, but not COX-1 mRNA. NS-398, a selective COX-2 inhibitor, displayed an additive effect on PGE2-induced COX-2 downregulation. The FLS predominantly expressed the PGE2 receptor (EP) 2 and EP4, which mediated the COX-2 suppression by PGE2. Treatment with anti-IL-10 monoclonal antibodies partially reversed the PGE2-induced suppression of COX-2 mRNA, suggesting that IL-10 may be involved in modulating COX-2 by PGE2. Experiments using an inducer and an inhibitor of cyclic AMP (cAMP) suggest that cAMP is the major intracellular signal that mediates the regulatory effect of PGE2 on COX-2 expression. EMSA revealed that PGE2 inhibited the binding of NF-κB in the COX-2 promoter via a cAMP dependent pathway. In addition, a subcutaneous injection of PGE2 twice daily for 2 weeks significantly reduced the incidence and severity of CIA as well as the production of IgG antibodies to type II collagen. Conclusion: Our data suggest that overproduced PGE2 in the RA joints may function as an autocrine regulator of its own synthesis by inhibiting COX-2 production and may, in part, play an anti-inflammatory role in the arthritic joints. (Immune Network 2003;3(3):201-210)

Cyclic AMP and mitogen‐activated protein kinases are required for glutamate‐dependent cyclic AMP response element binding protein and Elk‐1 phosphorylation in the dorsal striatum in vivo

Dopaminergic and glutamatergic signalling cascades are integrated in striatal medium spiny neurones by cyclic AMP response-element binding protein and Elk-1 phosphorylation. Phosphorylated cyclic AMP response-element binding protein and phosphorylated Elk-1 contribute to c-fos expression by binding to the calcium and cyclic AMP response-element and the serum response element, respectively, in the c-fos promoter. The role of cyclic AMP and mitogen-activated protein kinase signalling cascades in glutamate-induced cyclic AMP response-element binding protein and Elk-1 phosphorylation and Fos expression was investigated using semiquantitative immunocytochemistry in vivo. Intracerebroventricular infusion of the sodium channel blocker, tetrodotoxin, decreased the glutamate-induced increase in phosphorylated cyclic AMP response-element binding protein, phosphorylated Elk-1, and Fos immunoreactivity. Intracerebroventricular infusion of the mitogen-activated and extracellular signal-regulated kinase inhibitor, PD98059, the p38 mitogen-activated protein kinase inhibitor, SB203580, or the cyclic AMP inhibitor, Rp-8-Br-cAMPS, decreased glutamate-induced phosphorylated cyclic AMP response-element binding protein, phosphorylated Elk-1, and Fos immunoreactivity. Simultaneous infusion of glutamate and Sp-8-Br-cAMPS, a cyclic AMP analogue, augmented induction of Fos immunoreactivity but not phosphorylated cyclic AMP response-element binding protein or phosphorylated Elk-1 immunoreactivity. These data indicate that cyclic AMP and mitogen-activated protein kinase signalling cascades are necessary for glutamate to induce cyclic AMP response-element binding protein and Elk-1 phosphorylation and Fos expression in the striatum. Furthermore, neuronal activity plays an important role in glutamate-induced signalling cascades in vivo.

Phosphodiesterase inhibitors, pentoxifylline and rolipram, increase bone mass mainly by promoting bone formation in normal mice

The administration of either Pentoxifylline (PTX), a methylxanthine derivative and an inhibitor of cyclic AMP (c-AMP) phosphodiesterases (PDEs), or Rolipram, an inhibitor specific to type-4 PDE (PDE4) in normal mice, significantly increased both cortical and cancellous bone mass. Vertebrae and tibiae from mice treated with PTX or Rolipram were analyzed by means of bone densitometry and histomorphometry. The results revealed that both PTX and Rolipram increased bone mass in normal mice mainly through the acceleration of bone formation. These findings suggest that both PTX and Rolipram can enhance physiological bone formation and thereby increase bone mass in normal mice. The possibility that these agents may be of value for the treatment of osteoporosis is discussed.

Internalization of voltage-dependent sodium channels in fetal rat brain neurons: a study of the regulation of endocytosis.

In fetal rat brain neurons, activation of voltage-dependent Na+ channels induced their own internalization, probably triggered by an increase in intracellular Na+ level. To investigate the role of phosphorylation in internalization, neurons were exposed to either activators or inhibitors of cyclic AMP- and cyclic GMP-dependent protein kinases, protein kinase C, and tyrosine kinase. None of the tested compounds mimicked or inhibited the effect of Na+ channel activation. An increase in intracellular Ca2+ concentration induced either by thapsigargin, a Ca(2+)-ATPase blocker, or by A23187, a Ca2+ ionophore, was unable to provoke Na+ channel internalization. However, Ca2+ seems to be necessary because both neurotoxin- and amphotericin B-induced Na+ channel internalizations were partially inhibited by BAPTA-AM. The selective inhibitor of Ca2+/calmodulin-dependent protein kinase II, KN-62, caused a dose-dependent inhibition of neurotoxin-induced internalization due to a blockade of channel activity but did not prevent amphotericin B-induced internalization. The rate of increase in Na+ channel density at the neuronal cell surface was similar before and after channel internalization, suggesting that recycling of internalized Na+ channels back to the cell surface was almost negligible. Pretreatment of the cells with an acidotropic agent such as chloroquine prevented Na+ channel internalization, indicating that an acidic endosomal/lysosomal compartment is involved in Na+ channel internalization in neurons.

Role of protein kinase pathways in IL-1-induced chemoattractant expression by human mesangial cells

Human mesangial cells produce the monocyte-specific chemotactic factor monocyte chemoattractant protein-1 (MCP-1) in response to a variety of stimuli, including the pro-inflammatory cytokine interleukin-1 beta (IL-1). The intracellular signals responsible for mediating the effects of IL-1 on MCP-1 expression in human mesangial cells have not been defined. Evidence from other types of cells suggests that protein kinases are involved in MCP-1 gene regulation. We investigated the role of protein kinase pathways in mediating IL-1-induced MCP-1 expression. Activation of protein kinase C (PKC) by phorbol esters or diacyglycerol up-regulated mesangial MCP-1 message and bioactivity in a fashion similar to IL-1. However, while inhibition of PKC activity completely blocked phorbol-induced MCP-1 up-regulation, induction by IL-1 was not prevented. Inhibitors of cyclic AMP (cAMP)-dependent protein kinase (PKA) also failed to block IL-1-induced MCP-1 expression. Furthermore, increasing intracellular cAMP and activating PKA attenuated basal MCP-1 mRNA levels by 82% and blocked IL-1 induced MCP-1 expression by 88%. Finally, the role of protein tyrosine kinases was studied. The structurally distinct protein tyrosine kinase (PTK) inhibitors genistein, herbimycin A, and tyrphostin each caused a dose-dependent inhibition of the effects of IL-1 on mesangial MCP-1 activity. IL-1 treatment of mesangial cells resulted in the up-regulation of three tyrosine phosphoproteins with apparent molecular masses between 40 and 62 kD. These results suggest that the effects of IL-1 on MCP-1 expression are not mediated through PKC or cAMP-PKA, but may be transduced through PTKs.

Involvement of Calmodulin and Calmodulin-Dependent Myosin Light Chain Kinase in Blue Light-Dependent H+ Pumping by Guard Cell Protoplasts from Vicia faba L.

Signal transduction processes involved in blue light-dependent proton pumping were investigated using guard cell protoplasts from Vicia faba.N-[2-(Methylamino)ethyl]-5-isoquinolinesulfonamide, an inhibitor of cyclic AMP- and cyclic GMP-dependent protein kinases, had no effect. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H-7) and calphostin C, inhibitors of protein kinase C, produced slight inhibition of the blue light-dependent proton pumping. 1-[N, O-Bis(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl] -4-phenylpiperazine, a specific inhibitor of Ca(2+)/calmodulin (CaM)-dependent protein kinase II, did not inhibit the proton pumping, but 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine and 1-(5-chloro-naphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9), inhibitors of Ca(2+)/CaM-dependent myosin light chain kinase, strongly suppressed the proton pumping. A CaM antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), inhibited blue light-dependent proton pumping, whereas its less active structural analog, N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5), had little effect on the response. Other CaM antagonists, trifluoperazine, compound 48/80, prenylamine, and 3-(2-benzothiazolyl)-4,5-dimethoxy-N-[3-(4-phenyl-piperidinyl)- propylbenzenesulfonamide inhibited the proton pumping. In accord with these results, light-induced stomatal opening in the epidermis of Commelina benghalensis ssp. was inhibited by ML-9 and W-7, but not by H-7 and W-5. Thus, it is concluded that CaM and Ca(2+)/CaM-dependent myosin light chain kinase are the components of the signal transduction process in blue light-dependent proton pumping in guard cells.

Arteriolar vasodilatation in frog skeletal muscle in vivo: modification of second messenger systems

This study was concerned with the role of cyclic nucleotides in the post-junctional vasodilatation mechanism. Interventions with second messenger systems involving cyclic adenosine monophosphate (cyclic AMP) and cyclic guanosine monophosphate (cyclic GMP), allowed the role of these nucleotides in vascular smooth muscle to be evaluated in the autoperfused, transparent frog muscle, m. cutaneous pectoris. The microcirculation was observed by intravital microscopy, and arteriolar diameters were continuously recorded. Pre- and post-junctional effects were distinguished by comparing results in control frogs with those obtained in frogs that had been chemically sympathectomized with either 6-hydroxydopamine or tetrodotoxin. Arterioles that were pre-contracted with adrenaline dilated in response to topical application of forskolin or sodium nitroprusside, which are direct activators of intracellular adenylate cyclase and guanylate cyclase, respectively. Arterioles were also dilated by 3-isobutyl-1-methylxanthine (IBMX), which is a non-selective inhibitor of cyclic AMP- and cyclic GMP-phosphodiesterase, and by rolipram, which is a selective inhibitor of the calcium-independent cyclic AMP-phosphodiesterase. Dibutyryl-cyclic AMP and dibutyryl-cyclic GMP also caused vasodilatation. These results indicate that in vascular smooth muscle, intracellular mechanisms involving cyclic nucleotides (cyclic AMP and cyclic GMP) are important in vasodilatation. They may act in conjunction with pre-junctional inhibitory mechanisms on sympathetic nerves.

Inhibitory effects of AH 21–132 in guinea‐pig isolated ileum and taenia caeci

1. AH 21-132 is being investigated as a potential chemotherapeutic agent for bronchial asthma. The present experiments were designed to determine whether AH 21-132 shares the activity of theophylline as an antagonist at adenosine A1 receptors and to assess its potency as a relaxant in intestinal smooth muscle. 2. In the transmurally-stimulated guinea-pig ileum, theophylline (1 mM), but not AH 21-132 (1 and 10 microM), antagonized twitch depression induced by adenosine. Higher concentrations (100 microM and 1 mM) of AH 21-132 themselves had a depressant effect. Neither theophylline (1 mM) nor AH 21-132 (1 and 10 microM) antagonized twitch depression induced by noradrenaline. 3. AH 21-132 (100 microM and 1 mM) depressed maximum contractions of ileum induced by both acetylcholine (ACh) and histamine. 4. In ileum treated with hyoscine (1 microM), AH 21-132 (greater than 10 microM) caused a concentration-dependent depression of the log concentration-effect curve for potassium chloride. 5. Simultaneous extracellular electrophysiological and mechanical recording from taenia caeci showed that AH 21-132 (100 microM-1 mM) inhibited spontaneous tension waves and their associated bursts of electrical spike activity. 6. Intracellular electrophysiological recording from taenia caeci showed that the mechano-inhibitory effect of 1 mM AH 21-132 was accompanied by abolition of spontaneous spike activity. Following spike abolition, the membrane potential assumed a value very close to that observed during periods of electrical quiescence prior to drug exposure. 7. AH 21-132 inhibited the activity of cyclic AMP-dependent and cyclic GMP-dependent phosphodiesterases derived from homogenates of ileal smooth muscle. The effective concentration ranges were 0.1-1OOO microM and 1-1000 microM, respectively. Theophylline, too, inhibited these enzymes but in each case was less potent than AH 21-132. 8. It is concluded that AH 21-132 is devoid of antagonist activity at adenosine Al receptors which modulate ACh release from intramural cholinergic nerves in the ileum. At concentrations greater than IO microM, AH 21-132 has a relaxant effect on intestinal smooth muscle characterized by suppression of spontaneous action potentials but by minor change in resting membrane potential. AH 21-132 previously has been reported to depress the spontaneous tone of trachealis muscle with an EC50 value of less than lO microM and the present experiments therefore show that this agent is much less potent in inhibiting intestinal muscle. This potency difference cannot be attributed to a tissuerelated difference in the potency of AH 21-132 as an inhibitor of cyclic AMP- or cyclic GMPdependent phosphodiesterases.

Reply

A series of xanthine derivatives and papaverine have been studied to determine their abilities to alter tissue levels of cyclic AMP and cyclic GMP, inhibit phosphodiesterase activities, and cause relaxation of pig coronary arteries. The agents selected for this study exhibited a wide range of potencies to inhibit phosphodiesterase activities in the coronary artery supernatant fraction. In addition, some of these agents were up to 10 times more potent as inhibitors of cyclic GMP hydrolysis than of cyclic AMP hydrolysis, while others were 2-4 times more potent as inhibitors of cyclic AMP than of cyclic GMP hydrolysis. The rank order of potencies of these agents to cause relaxation of coronary artery strips was similar to the rank order of potencies to inhibit cyclic nucleotide phosphodiesterase activities. There were, however, some notable exceptions to the correlation between inhibition of cyclic nucleotide phosphodiesterase activities and relaxation. 1-Isoamyl-3-isobutylxanthine was a more potent relaxing agent than might be expected from its relatively low potency to inhibit cyclic nucleotide hydrolysis in tissue extracts. On the other hand, 1-methyl-3-isobutyl-7-(3-chlorobenzyl)-xanthine was one of the more potent inhibitors of cyclic nucleotide hydrolysis but was not as potent in causing relaxation as might have been expected. Exposure of the coronary artery strips to inhibitors caused increases in tissue levels of cyclic AMP and cyclic GMP and there was a statistically significant multiple linear regression of cyclic AMP and cyclic GMP levels on percent relaxation after 5 min of exposure to the agents. Cyclic AMP and cyclic GMP levels made approximately equal contributions to the regression of changes in percent relaxation, as determined by analysis of variance methods. While 1-isoamyl-3-isobutylxanthine did not fit the correlation between phosphodiesterase inhibition and potency to relax the arterial strips as well as the other agents, this agent also was found to cause unexpectedly large increases in cyclic AMP levels. Some agents caused relaxation accompanied by significant elevation of cyclic GMP levels and no significant change in cyclic AMP levels while other agents caused relaxation accompanied by significant increases in cyclic AMP but not cyclic GMP. These data offer some support for a hypothesis that both cyclic AMP and cyclic GMP are involved in the relaxation processes of pig coronary arteries.

Antiallergic activity profile in vitro of RHC 2963 and related compounds

RHC 2963 (7-methyl-pyrido (3′, 2′:4,5)-thieno (3,2-d)-1,2,3 triazine-4(3H)-one and 20 related compounds have been investigated for their antiallergic activities in 3 in vitro models of anaphylaxis and for their effects on cyclic nucleoticle phosphosphodiesterases (cNUC-PDE) from purified rat mast cells (RMC). Nine compounds were potent (I 50⩽80 μM) inhibitors of antigen-induced release of histamine (AIR) from RMC, 2 compounds inhibited anti-IgE-induced release of histamine from human basophils (I 50⩽60 μM) and one compound inhibited AIR from guinea pig lung slices (I 50 = 55 μM). RHC 2963 was 18 times more potent than disodium cromoglycate (DSCG) as inhibitor of AIR from RMC and had an activity profile identical to that of DSCG in the following respects: loss of inhibitory activity with increasing preincubation time, tachyphylactic properties and inability to inhibit non-immunologic release of histamine induced by compound 48/80. Neither RHC 2963 nor DSCG had any effect on anti-IgE-induced release of histamine from human basophils or IgG 1-mediated release of histamine from guinea pig lung. Twelve of the compounds in this chemical series were more potent than theophylline as inhibitors of cyclic AMP and/or cyclic GMP phosphodiesterase (PDE) from RMC. Paired regression analysis of the I 50 values for inhibition of AIR and cNUC-PDE from RMC revealed no statistically significant correlation between the inhibition of AIR and inhibition of cAMP- or cGMP-PDE. We conclude: (1) RHC 2963 and some of the related compounds are potent inhibitors of immunologic release of histamine from RMC with a mechanism of action similar to that of DSCG, and (2) inhibition of cAMP- or cGMP-PDE by these compounds is not the biochemical mechanism by which they inhibit AIR from RMC.

On the Mechanism of Relaxation of Tracheal Muscle by Theophylline and Other Cyclic Nucleotide Phosphodiesterase Inhibitors

The mechamism of action of theophylline was studied by investigating the relationship between relaxant effect and inhibition of cyclic nucleotide phosphodiesterase (PDE) and by studying interactions with adenosine actions. Guinea pig tracheal smooth muscle cyclic AMP PDE had two apparent KmS': 0.4 and 70 microM for cyclic AMP. Theophylline and papaverine competetively inhibited the low Km form. Hydrolysis of 2.0 microM cyclic AMP and cyclic GMP was inhibited by several drugs. Some agents (e.g. ZK 62 711, ICI 63,197, Ro 20--1724, dipyridamol) were considerably more potent as inhibitors of cyclic AMP than of cyclic GMP hydrolysis, while other agents (M & B 22.948 and dilazep) selectively inhibited cyclic GMP breakdown, and some (theophylline, papaverine, IBMX and SQ 20,006) showed little selectivity. There was a weak but significant correlation between inhibition of cyclic AMP phosphodiesterase and relaxation of tracheal smooth muscle in vitro. There was also a correlation between the ratio of IC25 cyclic AMP/IC25 cyclic GMP and the smooth muscle relaxation, indicating that inhibition of cyclic AMP rather than cyclic GMP hydrolysis determined relaxation. However, there was a marked tachyphylaxis to the relaxant effect of the cyclic AMP selective PDE-inhibitors, while the nonselective methylxanthines did not show tachyphylaxis. The effect of theophylline was antagonized by low concentrations of adenosine, which by itself caused a weak tracheal contraction. The effect of PDI-inhibitors can be partly explained by decreased cyclic AMP breakdown but other mechanisms, such as antagonism of endogenous adenosine, may contribute to the observed relaxant action.

Studies with the Cyclic Adenosine Monophosphate Receptor and Stimulation of in Vitro Transcription of the Gal Operon

Abstract To elucidate further how cyclic adenosine 3' : 5'-monophosphate (cyclic AMP) and the cyclic AMP receptor are able to stimulate gal mRNA synthesis we have utilized an antiserum specific for cyclic AMP receptor and also cyclic GMP as inhibitors of cyclic AMP and cyclic AMP receptor action. When cyclic AMP, cyclic AMP receptor, DNA, and RNA polymerase are incubated together a rifampicin-resistant preinitiation complex is formed and, upon the addition of missing ribonucleoside triphosphates or Mg2+, gal mRNA synthesis occurs. If the preinitiation complex is formed in the absence of nucleoside triphosphates the antiserum is able to dissociate the complex. If the complex is formed in the presence of nucleoside triphosphates (no Mg2+), it is converted to a more stable form which is not reversed by the addition of antiserum. Cyclic GMP can prevent the formation of the preinitiation complex. However, we find that once the preinitiation complex has been formed cyclic GMP does not inhibit cyclic AMP receptor-cyclic AMP-dependent transcription, suggesting that RNA polymerase stabilizes cyclic AMP receptor-cyclic AMP binding to the gal promoter region.