Inhibitor Isobutylmethylxanthine Research Articles

CAMP controls cytosolic Ca2+ levels in Dictyostelium discoideum

BackgroundDifferentiating Dictyostelium discoideum amoebae respond upon cAMP-stimulation with an increase in the cytosolic free Ca2+ concentration ([Ca2+]i) that is composed of liberation of stored Ca2+ and extracellular Ca2+-influx. In this study we investigated whether intracellular cAMP is involved in the control of [Ca2+]i.ResultsWe analyzed Ca2+-fluxes in a mutant that is devoid of the main cAMP-phosphodiesterase (PDE) RegA and displays an altered cAMP metabolism. In suspensions of developing cells cAMP-activated influx of extracellular Ca2+ was reduced as compared to wild type. Yet, single cell [Ca2+]i-imaging of regA- amoebae revealed a cAMP-induced [Ca2+]i increase even in the absence of extracellular Ca2+. The cytosolic presence of the cAMP PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) induced elevated basal [Ca2+]i in both, mutant and wild type cells. Under this condition wild type cells displayed cAMP-activated [Ca2+]i-transients also in nominally Ca2+-free medium. In the mutant strain the amplitude of light scattering oscillations and of accompanying cAMP oscillations were strongly reduced to almost basal levels. In addition, chemotactic performance during challenge with a cAMP-filled glass capillary was altered by EGTA-incubation. Cells were more sensitive to EGTA treatment than wild type: already at 2 mM EGTA only small pseudopods were extended and chemotactic speed was reduced.ConclusionWe conclude that there is a link between the second messengers cAMP and Ca2+. cAMP-dependent protein kinase (PKA) could provide for this link as a membrane-permeable PKA-activator also increased basal [Ca2+]i of regA- cells. Intracellular cAMP levels control [Ca2+]i by regulating Ca2+-fluxes of stores which in turn affect Ca2+-influx, light scattering oscillations and chemotactic performance.

NSAIDs increase GM-CSF release by human synoviocytes: comparison with nitric oxide-donating derivatives

Non-steroidal anti-inflammatory drugs (NSAIDs) are used to treat the condition of rheumatoid arthritis, where levels of prostaglandin E 2 (PGE 2) and granulocyte macrophage-colony stimulating factor (GM-CSF) are elevated in the synovial fluid. NO-NSAIDs are a new class of cyclooxygenase (COX)-inhibitors developed by coupling a nitric oxide (NO)-donating moiety to conventional NSAIDs. We show that, in cytokine-treated synoviocytes (from non-rheumatic patients), NO-naproxen and NO-flurbiprofen like their parent compounds concentration-dependently reduce the levels of PGE 2 (an index of COX-2 activity), with a corresponding rise in the release of GM-CSF. Unlike acetylsalicylic acid (ASA), NO-ASA reduces the levels of PGE 2, without increasing GM-CSF release, although cell viability is reduced at the highest concentration (1 mM). The effects of NSAIDs and NO-NSAIDs on GM-CSF release were attributable to the PGE 2 mediated cyclic (c) AMP pathway because PGE 2 reversed the effects of COX blockade. Second, phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine (IBMX) and Ro-201724 (both of which elevate cAMP levels) decreased GM-CSF release, in the presence of PGE 2. Finally, neither sodium nitroprusside nor zaprinast (both of which elevate cGMP levels) affected GM-CSF or PGE 2 release. Our findings demonstrate that GM-CSF is regulated by NSAIDs and NO-NSAIDs via inhibition of COX and appears to be mediated via the cAMP pathway. NO-ASA is the exception, because it does not increase GM-CSF release, although at millimolar concentrations cell viability is reduced.

Guinea-pig lung adenylyl and guanylyl cyclase and PDE activities associated with airway hyper- and hypo-reactivity following LPS inhalation

The relationships between changes in in vivo airway reactivity and levels cyclicAMP and cyclicGMP were determined in guinea-pig lungs after exposure to inhaled lipopolysaccharide (LPS). After LPS (30 μg.ml −1, 1 h), guinea-pigs displayed in vivo airway hyperreactivity (AHR) at 1 h and hyporeactivity (AHOR) at 48 h, to inhaled (20 s) histamine (1 or 3 mM, respectively). Isoprenaline-stimulated cAMP or SNAP-stimulated cGMP were determined in the lungs isolated from guinea-pigs exposed to LPS inhalation to determine whether there was a relationship between AHR or AHOR and adenylyl/guanylyl cyclase and phosphodiesterase (PDE) activities. Assays were performed in the absence and presence of the non-selective PDE inhibitor, 3-isobutyl-1-methylxanthine (IBMX). Levels of cAMP and cGMP in its presence indicated adenylyl and guanylyl cyclase activities, respectively. The difference between cAMP and cGMP levels, in the absence and presence of IBMX, reflected relevant PDE activity. In vivo AHR was associated with increased PDE activity towards cAMP and cGMP (67 and 278%, respectively) and also increased adenylyl (47%) and guanylyl (210%) cyclase activities. In vivo AHOR at 48 h after LPS inhalation was also associated with raised cyclase activity ( p < 0.05), whereas relevant PDE activity declined by 79 and 68%, compared with 48 h after vehicle. Although net stimulated cGMP levels increased during AHR and AHOR and net stimulated cAMP increased during AHOR, our index of PDE activity increased during AHR and decreased during AHOR. These results therefore support the rationale for the use of PDE-inhibitors in the treatment of respiratory diseases associated with AHR.

Irsogladine, an anti-ulcer drug, suppresses superoxide production by inhibiting phosphodiesterase type 4 in human neutrophils

Neutrophil superoxide production is implicated in the pathogenesis of gastric mucosal damage induced by various ulcerative agents and Helicobacter pylori infection. We investigated here the effects of an anti-ulcer drug irsogladine [2, 4-diamino-6-(2, 5-dichlorophenyl)-s-triazine maleate] on cAMP formation in isolated human neutrophils. The cAMP level in human neutrophils was elevated by a phosphodiesterase (PDE) type 4 selective inhibitor rolipram, but not by any inhibitors of PDE1, PDE2 and PDE3. Irsogladine also increased cAMP formation in a concentration-dependent manner in neutrophils. A non-selective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) alone significantly increased cAMP level, whereas irsogladine was unable to further increase cAMP level in the presence of IBMX. Irsogladine inhibited concentration-dependently the superoxide (O2−) production induced by various stimuli including formyl-methionyl-leucyl-phenylalanine, opsonized zymosan, guanosine 5′-[gamma-thio] triphosphate, A23187 and phorbol 12-myristate 13-acetate. These effects of irsogladine were mimicked by rolipram, IBMX and dibutyryl cAMP. The inhibitory effects of irsogladine and rolipram on the O2− production were reversed by a protein kinase A inhibitor H-89. These results indicate that irsogladine inhibits the superoxide production in human neutrophils by the increase of cAMP content by PDE 4 inhibition, which in turn contributing to the anti-ulcer effects of irsogladine on gastric mucosal lesions associated with oxidative stress.

Atrazine is a competitive inhibitor of phosphodiesterase but does not affect the estrogen receptor

Atrazine (ATR), 2-chloro-4-ethylamino-6-isopropylamino- s-triazine, has been implicated in numerous studies to act as an endocrine disruptor, specifically by altering estradiol signaling via increased aromatase activity. Fluorescence polarization (FP) was used to show that the binding equilibria between estrogen receptor-α or estrogen receptor-β, and estradiol were not affected by ATR and its metabolites: ATR-desethyl (ADE), ATR-desisopropyl (ADI), ATR-desethyldesisopropyl (ADD) and terbuthylazine (TBZ). Therefore, ATR and its degradation products were studied to determine their ability to inhibit phosphodiesterase (PDE), the enzyme responsible for hydrolyzing the second messenger cAMP to 5′-AMP. Using FP, it was found that ATR inhibited PDE with an IC 50 value of 1.8 μM. This was lower than the known PDE inhibitor isobutyl methylxanthine (IBMX), which had an IC 50 value of 4.6 μM. The ATR degradation products ADE, ADI, ADD and TBZ were less effective than ATR at inhibiting PDE when assayed using FP. Classical competitive binding assays, using radiolabeled 14C-cAMP in conjunction with thin layer chromatography (TLC), were used to determine that ATR was a competitive inhibitor of PDE with an association constant of 85 μM.

Calcium-induced Calpain Mediates Apoptosis via Caspase-3 in a Mouse Photoreceptor Cell Line

The rd mouse, an accepted animal model for photoreceptor degeneration in retinitis pigmentosa, has a recessive mutation for the gene encoding the beta-subunit of the cGMP phosphodiesterase. This mutation results in high levels of cGMP, which leaves an increased number of the cGMP-gated channels in the open state, thus allowing intracellular calcium (Ca(2+)) to rise to toxic levels, and rapid photoreceptor degeneration follows. To delineate the events in rd photoreceptor degeneration, we demonstrated an increase in calpain and caspase-3 activity, hypothesizing that Ca(2+)-mediated apoptosis in photoreceptors is mediated by calpain, involving mitochondrial depolarization and caspase-3 activation. To examine this hypothesis further, a murine photoreceptor-derived cell line (661W) was treated with the Ca(2+) ionophore A23187, cGMP-gated channel agonist 8-bromo-cGMP, or phosphodiesterase inhibitor isobutylmethylxanthine to mimic the increased Ca(2+) influx seen in the rd photoreceptors. Ca(2+)-induced cell death in 661W cells was found to be mediated by calpain and caspase-3 and could be completely inhibited by the calpain inhibitor SJA6017, implicating both calpain and caspases in the apoptotic process. The apoptotic events correlated in an SJA6017-inhibitable manner with bid cleavage, mitochondrial depolarization, cytochrome c release, and caspase-3 and -9 activation. We concluded that Ca(2+) influx in the rd model of photoreceptor degeneration leads to the activation of the cysteine protease calpain, which executes apoptosis via modulation of caspase-3 activity.

Cholinergic–adrenergic antagonism in the induction of tachyarrhythmia by electrical stimulation in isolated rat atria

Atrial tachyarrhythmias (AT) are the most common cardiac rhythm disturbance. In the present study, we analyzed the cholinergic–adrenergic interaction in the in vitro induction of cholinergic-dependent tachyarrhythmia by high-frequency electric stimulation. Tachyarrhythmia was evoked in isolated rat right atria by trains of electric stimuli. Atrial response was expressed as the tachyarrhythmia induction index (ATI, i.e. the fraction of applied trains that resulted in arrhythmia induction). ATI was reversibly increased by 0.6 μM carbachol (CCh), which also decreased atrial spontaneous rate (ASR). In contrast, 10 nM isoproterenol (ISO), 100 μM tyramine and the phosphodiesterase inhibitor isobutyl-methylxanthine (IBMX, 100 μM) increased ASR and decreased ATI. Amiodarone (AMI, 10 μM) reduced ATI in the presence and absence of CCh. Further CCh addition restored ATI in atria treated with either IBMX or AMI, but not when both compounds were present. Increase in ATI by CCh in atria pretreated with IBMX plus ISO was significantly attenuated by 3 mM NaF. The antagonism between cholinergic muscarinic and β-adrenergic receptor stimulation (the former facilitating and the latter inhibiting tachyarrhythmia installation) possibly involves regulation of the phosphorylation status of adenosine cyclic 3′-5′-monophosphate (cAMP)-dependent protein kinase substrates. Additionally, cAMP-independent, AMI-sensitive mechanism stimulated by CCh (possibly muscarinic-dependent K + current activation) seems to contribute to AT facilitation.

Crystal structure of phosphodiesterase 9 shows orientation variation of inhibitor 3-isobutyl-1-methylxanthine binding.

Cyclic nucleotide phosphodiesterases (PDEs) are enzymes controlling cellular concentrations of the second messengers cAMP and cGMP. The crystal structure of the catalytic domain of PDE9A2, a member of a PDE family specifically hydrolyzing cGMP, has been determined at 2.23-A resolution. The PDE9A2 catalytic domain closely resembles the cAMP-specific PDE4D2 but is significantly different from the cGMP-specific PDE5A1, implying that each individual PDE family has its own characteristic substrate recognition mechanism. The different conformations of the H and M loops between PDE9A2 and PDE5A1 imply their less critical roles in nucleotide recognition. The nonselective inhibitor 3-isobutyl-1-methylxanthine (IBMX) binds to a similar subpocket in the active sites of PDE4, PDE5, and PDE9 and has a common pattern of the binding. However, significantly different orientations and interactions of IBMXs are observed among the three PDE families and also between two monomers of the PDE9A2 dimer. The kinetic properties of the PDE9A2 catalytic domain similar to those of full-length PDE9A imply that the N-terminal regulatory domain does not significantly alter the catalytic activity and the IBMX inhibition.

Tumor necrosis factor reduces cAMP production in rat microglia.

cAMP has been reported to exert a neuroprotective role in several in vivo and in vitro models of brain pathologies, mainly by regulating microglial activation and orienting these cells toward a neuroprotective phenotype. In order to elucidate the intracellular pathways regulated by tumor necrosis factor (TNF) in glial cells, I have studied the modulation of cAMP accumulation by TNF in microglia and astrocyte cultures obtained from the neonatal rat brain. Pre-treatment of microglia with TNF reduced in a dose- and time-dependent manner cAMP accumulation induced by forskolin (FSK), in the presence of the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine (IBMX). The TNF inhibitory action was 90% reverted by a neutralizing polyclonal anti-TNF antibody and was not prevented by a 16 h pre-treatment of microglial cultures with the Gi protein inhibitor pertussis toxin (PTx). These results suggest that TNF acts at a step of the cAMP transduction pathway other than receptors, G proteins, and phosphodiesterases. The target of TNF appeared to be adenylyl cyclase, whose ability to synthesize cAMP was markedly reduced (up to 50%) in membranes prepared from TNF-treated microglial cells, both in basal conditions and after stimulation with FSK. TNF induced a time-dependent degradation of IkappaB-alpha in microglial cells that was reverted by two inhibitors of nuclear factor kappaB activation, N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and N-CBZ-Leu-Leu-Leu-al (MG132). The same inhibitors also markedly prevented the reduction of FSK-evoked cAMP accumulation by TNF, suggesting the involvement of NFkappaB in the regulation of adenylyl cyclase by TNF in microglia. Conversely, cAMP accumulation in astrocytes was not affected by TNF. Based on these findings, it is proposed that the ability of TNF to inhibit cAMP synthesis in microglia may exacerbate its response and contribute to cell damage in neuroinflammation and neurodegeneration, possibly through enhanced release of proinflammatory and/or cytotoxic factors.

Cyclic adenosine monophosphate (cAMP) increases natriuretic peptide receptor C (NPR-C) expression in human aortic smooth muscle cells

Activation of the intracellular cAMP-signaling pathway by either forskolin or the cAMP-mimetic dibutyryl cAMP significantly increased transcript levels of NPR-C in primary cultures of human aortic smooth muscle cells. The time course of the increase was rapid, with significant differences from control occurring within 3 h of treatment and reaching ∼6 times control value after 24 h of exposure to 10 μM forskolin. Expression levels of the natriuretic peptide receptor B (NPR-B), but not the natriruetic peptide receptor A (NPR-A) were also increased by forskolin, rising to a level of ∼2 times control at 96 h. NPR-B transcript levels in the presence of dibutyryl cAMP were unaltered by the protein kinase A (PKA) inhibitor KT-5720, suggesting a PKA-independent pathway to NPR-B up-regulation. In contrast, KT-5720 reduced NPR-C transcript to a lower level that was not significantly different from control. Partial re-differentiation of AOSMC by culture in growth factor-reduced matrix (Matrigel) did not significantly change NPR-C transcript levels compared with cells grown on plastic, and the dibutyryl cAMP-induced increase in NPR-C (∼eight–nine-fold control value) was retained. The dibutyryl cAMP/forskolin effect on NPR-C transcript was not reproduced by the β2-selective adrenergic agonist isoproterenol (10 μM), but was replicated by incubation with the phosphodiesterase inhibitor isobutylmethylxanthine (0.5 mM). Up-regulated NPR-B and NPR-C transcript levels were reflected, respectively, in a two-fold increase in CNP-stimulated cGMP and an increase in 125 I -ANF binding competed by the NPR-C-specific natriuretic peptide, C-ANF(4–23) following a 4-day treatment with 0.125 mM dbcAMP. The present data suggest that elevation of cAMP in human vascular smooth muscle may potentiate the vasoactive effects of natriuretic peptides acting through the NPR-B and NPR-C receptors.

Cyclic adenosine monophosphate (cAMP) increases natriuretic peptide receptor C (NPR-C) expression in human aortic smooth muscle cells

Activation of the intracellular cAMP-signaling pathway by either forskolin or the cAMP-mimetic dibutyryl cAMP significantly increased transcript levels of NPR-C in primary cultures of human aortic smooth muscle cells. The time course of the increase was rapid, with significant differences from control occurring within 3 h of treatment and reaching ∼6 times control value after 24 h of exposure to 10 μM forskolin. Expression levels of the natriuretic peptide receptor B (NPR-B), but not the natriruetic peptide receptor A (NPR-A) were also increased by forskolin, rising to a level of ∼2 times control at 96 h. NPR-B transcript levels in the presence of dibutyryl cAMP were unaltered by the protein kinase A (PKA) inhibitor KT-5720, suggesting a PKA-independent pathway to NPR-B up-regulation. In contrast, KT-5720 reduced NPR-C transcript to a lower level that was not significantly different from control. Partial re-differentiation of AOSMC by culture in growth factor-reduced matrix (Matrigel) did not significantly change NPR-C transcript levels compared with cells grown on plastic, and the dibutyryl cAMP-induced increase in NPR-C (∼eight–nine-fold control value) was retained. The dibutyryl cAMP/forskolin effect on NPR-C transcript was not reproduced by the β2-selective adrenergic agonist isoproterenol (10 μM), but was replicated by incubation with the phosphodiesterase inhibitor isobutylmethylxanthine (0.5 mM). Up-regulated NPR-B and NPR-C transcript levels were reflected, respectively, in a two-fold increase in CNP-stimulated cGMP and an increase in 125 I -ANF binding competed by the NPR-C-specific natriuretic peptide, C-ANF(4–23) following a 4-day treatment with 0.125 mM dbcAMP. The present data suggest that elevation of cAMP in human vascular smooth muscle may potentiate the vasoactive effects of natriuretic peptides acting through the NPR-B and NPR-C receptors.

Crystal Structures of Phosphodiesterases 4 and 5 in Complex with Inhibitor 3-Isobutyl-1-methylxanthine Suggest a Conformation Determinant of Inhibitor Selectivity

Cyclic nucleotide phosphodiesterases (PDEs) are a superfamily of enzymes controlling cellular concentrations of the second messengers cAMP and cGMP. Crystal structures of the catalytic domains of cGMP-specific PDE5A1 and cAMP-specific PDE4D2 in complex with the nonselective inhibitor 3-isobutyl-1-methylxanthine have been determined at medium resolution. The catalytic domain of PDE5A1 has the same topological folding as that of PDE4D2, but three regions show different tertiary structures, including residues 79-113, 208-224 (H-loop), and 341-364 (M-loop) in PDE4D2 or 535-566, 661-676, and 787-812 in PDE5A1, respectively. Because H- and M-loops are involved in binding of the selective inhibitors, the different conformations of the loops, thus the distinct shapes of the active sites, will be a determinant of inhibitor selectivity in PDEs. IBMX binds to a subpocket that comprises key residues Ile-336, Phe-340, Gln-369, and Phe-372 of PDE4D2 or Val-782, Phe-786, Gln-817, and Phe-820 of PDE5A1. This subpocket may be a common site for binding nonselective inhibitors of PDEs.

Effects of ghrelin on insulin and glucagon secretion: a study of isolated pancreatic islets and intact mice

We combined in vitro and in vivo methods to investigate the effects of ghrelin, a novel gastric hormone, on insulin and glucagon release. Studies of isolated mouse islets showed that ghrelin concentrations in the physiological range (0.5–3 nmol l −1) had no effect on glucose-stimulated insulin release, while low ghrelin concentrations (1–100 pmol l −1) inhibited and high (0.1 and 1 μmol l −1) stimulated. The insulin response to glucose was enhanced in the presence of a high ghrelin concentration (100 nmol l −1). Glucagon release was stimulated by ghrelin (0.1 pmol l −1 to 1 μmol l −1); this effect was maintained in the presence of glucose (0–20 mmol l −1). In intact mice, basal plasma insulin was suppressed by 1 and 10 nmol kg −1 of ghrelin, 2 and 6 min after i.v. injection. Ghrelin (0.2–10 nmol kg −1 i.v.) suppressed also the glucose-stimulated insulin response and impaired the glucose tolerance (at a ghrelin dose of 3.3 nmol kg −1). Ghrelin (1 or 10 nmol kg −1 i.v.) inhibited the insulin response to the phospholipase C stimulating agent carbachol and enhanced the insulin response to the phosphodiesterase inhibitor isobutyl-methylxanthine (IBMX) but did not affect the response to the membrane-depolarizing amino acid l-arginine. These observations suggest that the inhibitory effect of ghrelin on glucose-induced insulin release is in part exerted on phospholipase C pathways (and not on Ca 2+entry), while the stimulatory effect of high doses of ghrelin depends on cyclic AMP. In contrast to the spectacular glucagon-releasing effect of ghrelin in vitro, ghrelin did not raise plasma glucagon. Carbachol, IBMX and l-arginine stimulated glucagon release. These responses were impaired by ghrelin, suggesting that it suppresses the various intracellular pathways (phospholipase C, cyclic AMP and Ca 2+), that are activated by the glucagon secretagogues. Together these observations highlight (but do not explain) the different effects of ghrelin on glucagon release in vitro and in vivo. The results show that ghrelin has powerful effects on islet cells, suggesting that endogenous ghrelin may contribute to the physiological control of insulin and glucagon release. However, the narrow “window” of circulating ghrelin concentrations makes this doubtful.

Evidence for a possible role for nitric oxide in the modulation of heart activity in Achatina fulica and Helix aspersa

The effects of nitric oxide (NO) donors, S-nitroso- N-acetylpenicillamine, S-nitroso- l-glutathione, sodium nitroprusside and sodium nitrite were investigated on the activity of the isolated hearts of Achatina fulica and Helix aspersa. NO donors inhibited heart activity in a concentration-dependent manner. The only exception was sodium nitroprusside, which excited H. aspersa heart. The inhibitory effects of these NO donors were reduced by the NO scavenger, methylene blue, the guanylyl cyclase inhibitor, 1H-(1,2,4) Oxadiazolo(4,3-a)quinoxalin-1-one (ODQ), and potentiated by 8-Br-cGMP and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX). Acetylcholine also inhibited the heart activity, and this inhibition was reduced by methylene blue and ODQ. Positive NADPH-diaphorase staining was located in the outer pericardial layer of the heart of A. fulica. The present results provide evidence that NO may modulate the activity of gastropod hearts, and this modulation may modify the inhibitory action of acetylcholine on heart activity.

Cyclic adenosine 3',5'-monophosphate-elevating agents inhibit transforming growth factor-beta-induced SMAD3/4-dependent transcription via a protein kinase A-dependent mechanism.

Transforming growth factor-beta (TGF-beta) plays complex roles in carcinogenesis, as it may exert both tumor suppressor and pro-oncogenic activities depending on the stage of the tumor. SMAD proteins transduce signals from the TGF-beta receptors to regulate the transcription of specific target genes. Crosstalks with other signaling pathways may contribute to the specificity of TGF-beta effects. In this report, we have investigated the effects of cyclic adenosine 3',5'-monophosphate (cAMP), a key second messenger in the cellular response to various hormones, on SMAD-dependent signaling in human HaCaT keratinocytes. Using either an artificial SMAD3/4-dependent reporter construct or the natural TGF-beta target, plasminogen activator inhibitor-1, we show that membrane-permeable dibutyryl cAMP, and other intracellular cAMP-elevating agents such as the phosphodiesterase inhibitor isobutyl-methylxanthine, the adenylate cyclase activator forskolin, or exogenous prostaglandin E2 (PGE2), interfere with TGF-beta-induced SMAD-specific gene transactivation. Inhibition of protein kinase A (PKA), the main downstream effector of cAMP, with H-89, suppressed cAMP-dependent repression of SMAD-driven gene expression. Inversely, coexpression of either an active PKA catalytic subunit or that of the cAMP response element (CRE)-binding protein (CREB) blocked SMAD-driven gene transactivation. cAMP-elevating agents did not inhibit nuclear translocation and DNA binding of SMAD3/4 complexes, but abolished the interactions of SMAD3 with the transcription coactivators CREB-binding protein (CBP) and p300 in a PKA-dependent manner. These results suggest that suppression of TGF-beta/SMAD signaling and resulting gene transactivation by cAMP-inducing agents occurs via PKA-dependent, CREB-mediated, disruption of SMAD-CBP/p300 complexes.

Valproate activates phosphodiesterase-mediated cAMP degradation: relevance to C6 glioma G1 phase progression

Forskolin, a diterpene activator of adenylate cyclase, stimulates the production of cyclic adenosine monophosphate (cAMP) in a wide variety of cell types. In C6 glioma, used in this study, the anticonvulsant agent valproic acid (VPA) inhibited forskolin-stimulated cAMP accumulation in intact cells in a concentration-dependent manner. Kinetic studies indicated this valproate effect not to be mediated by direct inhibition of adenylate cyclase activity. The valproate-induced inhibition of cAMP accumulation was partially reversed by the phosphodiesterase (PDE) inhibitor isobutylmethyl xanthine (IBMX). Degradation of cAMP over time was more rapid in valproate-treated cells than in controls, and this effect was also reversed by IBMX. In synchronised C6 glioma, phosphodiesterase type IV (PDE4A1) expression was selectively upregulated during the G1 phase, in tandem with temporal biphasic peaks of cAMP. However, the expression of PDE4 isoforms was not affected by a 48-h exposure to valproate. These findings suggest inhibition of forskolin-stimulated cAMP levels in C6 glioma by valproate to be mediated by increased activation of PDE in the G1 phase. Since the degree of cell cycle arrest induced by valproate is intimately associated with its teratogenic potency, it appears that PDE-mediated inhibition of cAMP may contribute to the molecular mechanisms of valproate-induced teratogenicity.

Modulation of AMPA receptors by cAMP‐dependent protein kinase in PreBötzinger complex inspiratory neurons regulates respiratory rhythm in the rat

We hypothesize that phosphorylation of AMPA receptors or associated synaptic proteins modulates the excitability of respiratory neurons in the preBötzinger Complex (preBötC), affecting respiratory rhythm. Using neonatal rat medullary slices that spontaneously generate respiratory rhythm, we examined the role of the cAMP-PKA pathway (PKA: cAMP-dependent protein kinase) in modulating glutamatergic synaptic transmission, the excitability of inspiratory neurons in the preBötC and respiratory rhythm. Microinjection of forskolin, an activator of adenylate cyclase, into the preBötC with or without the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), decreased the period (increased the frequency) of respiratory-related rhythmic motor output in the hypoglossal nerve (XIIn) to 84 % (without IBMX) and to 72 % (with IBMX) of the pre-injection baseline. In the presence of MK-801, a non-competitive NMDA receptor antagonist, microinjection of forskolin plus IBMX decreased the period to 66 % of baseline levels. Microinjection of Rp-adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS), a PKA inhibitor, increased the period to 145 % of baseline levels. Concurrent microinjection of Rp-cAMPS and forskolin had no effect on the period. Bath application of 7beta-deacetyl-7beta-[gamma-(morpholino)butyryl]-forskolin hydrochloride (7Db-forskolin, a water-soluble derivative of forskolin): (1) decreased the period to 67 % of baseline levels without affecting the amplitude of integrated XIIn inspiratory discharge, (2) induced a tonic inward current of 29 pA and enhanced inspiratory drive current (the amplitude increased to 183 % and the integral increased to 184 % of baseline) in voltage-clamped (holding potential = -60 mV) preBötC inspiratory neurons and (3) increased the frequency to 195 % and amplitude to 118 % of spontaneous excitatory postsynaptic currents (sEPSCs) during expiratory periods. Dideoxy-forskolin did not have these effects. Intracellular perfusion with the catalytic subunit of PKA (cPKA) into preBötC inspiratory neurons progressively enhanced inspiratory drive currents and, in the presence of TTX, increased the inward currents induced by local ejection of AMPA; the latter currents were blocked by 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulphonamide (NBQX, an AMPA/kainate receptor antagonist). The effects of cPKA were blocked by co-application of PKA inhibitor (6-22) amide (PKI). These results suggest that phosphorylation of postsynaptic AMPA receptors through the cAMP-PKA pathway modulates both tonic and phasic excitatory amino acid synaptic transmission and excitability of inspiratory neurons in the preBötC and, therefore, regulates respiratory rhythm. Moreover, the basal level of endogenous PKA activity appears to be a determinant of resting respiratory frequency.

Glucagon-like peptide 1 and fatty acids amplify pulsatile insulin secretion from perifused rat islets.

Glucose-induced insulin secretion from isolated, perifused rat islets is pulsatile with a period of about 5-10 min, similar to the insulin oscillations that are seen in healthy humans but which are impaired in Type II diabetes. We evaluated the pattern of enhancement by the potent incretin, glucagon-like peptide 1 (GLP-1). GLP-1 increased the amplitude of pulses and the magnitude of insulin secretion from the perifused islets, without affecting the average time interval between pulses. Forskolin and the phosphodiesterase inhibitor isobutylmethylxanthine had the same effect, suggesting that the effect was due to elevated cAMP levels. The possibility that cAMP might enhance the amplitude of pulses by reducing phosphofructo-2-kinase (PFK-2) activity was eliminated when the liver isoform of PFK-2 was shown to be absent from beta-cells. The possibility that cAMP enhanced pulsatile secretion, at least in part, by stimulating lipolysis was supported by the observations that added oleate had a similar effect on secretion, and that the incretin effect of GLP-1 was inhibited by the lipase inhibitor orlistat. These data show that the physiological incretin GLP-1 preserves and enhances normal pulsatile insulin secretion, which may be essential in proposed therapeutic uses of GLP-1 or its analogues.

CAMP-independent responses of olfactory neurons in Xenopus laevis tadpoles and their projection onto olfactory bulb neurons.

We report on responses of olfactory receptor neurons (ORNs) upon application of amino acids and forskolin using a novel slice preparation of the olfactory epithelium of Xenopus laevis tadpoles. Responses were measured using the patch-clamp technique. Both amino acids and forskolin proved to be potent stimuli. Interestingly, a number of ORNs that responded to amino acids did not respond to forskolin. This suggests that some amino acids activate transduction pathways other than the well-known cAMP-mediated one. The differential processing of cAMP-mediated stimuli on the one hand and amino acid stimuli on the other was further elucidated by calcium-imaging of olfactory bulb neurons using a novel nose-olfactory bulb preparation of Xenopus laevis tadpoles. The projection pattern of amino acid-sensitive ORNs to olfactory bulb neurons differed markedly from the projection pattern of forskolin-sensitive ORNs. Olfactory bulb neurons activated by amino acids were located laterally compared to those activated by forskolin, and only a small proportion responded to both stimuli. The ensemble of neurons activated by forskolin was also activated by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) and the membrane-permeant cAMP analogue 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (pCPT-cAMP). We therefore conclude that sensory transduction of a number of amino acids is cAMP independent, and amino acid- and cAMP-mediated responses are processed differentially at the level of the olfactory bulb.

Islet amyloid polypeptide inhibits glucagon release and exerts a dual action on insulin release from isolated islets

We have studied the influence of a wide concentration range of islet amyloid polypeptide (IAPP) on both glucagon and insulin release stimulated by various types of secretagogues. In an islet incubation medium devoid of glucose, the rate of glucagon release being high, we observed a marked suppressive action by low concentrations of IAPP, 10 −10 and 10 −8 M, on glucagon release. Similarly, glucagon release stimulated by l-arginine, the cholinergic agonist carbachol, or the phosphodiesterase inhibitor isobutylmethyl xanthine (IBMX), an activator of the cyclic AMP system, was inhibited by IAPP in the 10 −10 and 10 −8 M concentration range. Moreover, basal glucagon release at 7 and 10 mM glucose was suppressed by IAPP. In contrast, IAPP exerted a dual action on insulin release. Hence, low concentrations of IAPP brought about a modest increase of basal insulin secretion at 7 mM glucose and also of insulin release stimulated by carbachol. High concentrations of IAPP, however, inhibited insulin release stimulated by glucose (10 and 16.7 mM), IBMX, carbachol and l-arginine. In conclusion, our data suggest that IAPP has complex effects on islet hormone secretion serving as an inhibitor of glucagon release and having a dual action on insulin secretion exerting mainly a negative feedback on stimulated and a positive feedback on basal insulin release.