Regulation Of Cyclic AMP Research Articles

Bioinformatics and systems biology approaches to identify molecular targets and pathways shared between Schizophrenia and bipolar disorder

Schizophrenia is a heterogeneous spectrum disorder of unknown etiology that is going to be a public concern around the world. The molecular association between the pathophysiology of schizophrenia (SP) and bipolar disorder (BD) is still not clear enough. To address this gap, we developed an integrated bioinformatics approach to identify common molecular signatures and possible interactive pathways that are common in the molecular pathogenic process between SP and BD. We employed pluripotent stem cell (iPSC)-derived cortical interneuron transcriptomic data and post-mortem brain transcriptomic data from patients with SP and BD, respectively. Twenty common differentially expressed genes (DEGs) were identified between SP and BD. Positive regulation of cyclic AMP (cAMP)-mediated signaling and regulation of the metabolic process were found to be common biological pathways between these two disorders. Protein-protein interactions and transcriptional regulatory interactions identified with hub-bottleneck proteins (LY6G5B, DLGAP2, DXO, EGR3, BAG6, DL-GAP2, SKIV2L, CSNK2B and RXRB), regulatory transcription factors (FOXC1, GATA2, RELA and ELK4), and regulatory microRNAs (hsa-mir-34a-5p, hsa-mir23b-3p, hsa-mir-146a-5p, hsa-mir-27a-3p, and hsa-mir-1-3p) are as key molecular signatures between SP and BD. Protein-chemical and protein-drug interactions revealed valproic acid, antirheumatic agents, trichostatin A, aarsenic, and 4-(5-benzo (1,3) dioxol-5-yl-4-pyridin-2yl-1H-imidazole-2-yl) benzamide and isopropyl alcohol compounds could be used as a potential therapeutic target molecule against SP and BD. The research findings would be used in wet lab-based research to further clarify potential therapeutic targets for the treatment of SP and BP disorders.

Phosphodiesterase regulation of alcohol drinking in rodents

Alcohol use disorders are chronically relapsing conditions characterized by persistent drinking despite the negative impact on one's life. The difficulty of achieving and maintaining sobriety suggests that current treatments fail to fully address the underlying causes of alcohol use disorders. Identifying additional pathways controlling alcohol consumption may uncover novel targets for medication development to improve treatment options. One family of proteins recently implicated in the regulation of alcohol consumption is the cyclic nucleotide phosphodiesterases (PDEs). As an integral component in the regulation of the second messengers cyclic AMP and cyclic GMP, and thus their cognate signaling pathways, PDEs present intriguing targets for pharmacotherapies to combat alcohol use disorders. As activation of cAMP/cGMP-dependent signaling cascades can dampen alcohol intake, PDE inhibitors may provide a novel target for reducing excessive alcohol consumption, as has been proposed for PDE4 and PDE10A. This review highlights preclinical literature demonstrating the involvement of cyclic nucleotide-dependent signaling in neuronal and behavioral responses to alcohol, as well as detailing the capacity of various PDE inhibitors to modulate alcohol intake. Together these data provide a framework for evaluating the potential utility of PDE inhibitors as novel treatments for alcohol use disorders.

Neuronal NF1/RAS regulation of cyclic AMP requires atypical PKC activation.

Neurofibromatosis type 1 (NF1) is a common neurodevelopmental disorder in which affected individuals are prone to learning, attention and behavioral problems. Previous studies in mice and flies have yielded conflicting results regarding the specific effector pathways responsible for NF1 protein (neurofibromin) regulation of neuronal function, with both cyclic AMP (cAMP)- and RAS-dependent mechanisms described. Herein, we leverage a combination of induced pluripotent stem cell-derived NF1 patient neural progenitor cells and Nf1 genetically engineered mice to establish, for the first time, that neurofibromin regulation of cAMP requires RAS activation in human and mouse neurons. However, instead of involving RAS-mediated MEK/AKT signaling, RAS regulation of cAMP homeostasis operates through the activation of atypical protein kinase C zeta, leading to GRK2-driven Gαs inactivation. These findings reveal a novel mechanism by which RAS can regulate cAMP levels in the mammalian brain.

β-Adrenergic Regulation of Cyclic AMP and Ca Current at the T-Tubules and Surface Membrane in Rat Cardiomyocytes

β-Adrenoceptor (β-AR) signalling is severely impaired in heart failure (HF), and this is accompanied by a loss and disorganization of t-tubules. However, how the latter affects the former is unknown. Here, we examined how acute detubulation affects the β-AR response of L-type Ca2+ channel (LTCC) current (ICa,L) and membrane cAMP ([cAMP]m) in adult rat ventricular myocytes (ARVMs) and the contribution of phosphodiesterases PDE3 and PDE4 in this process. ARVMs were infected with an adenovirus encoding a mutant of the olfactory cyclic nucleotide-gated (CNG) channels α subunit to follow [cAMP]m dynamics by recording the associated cationic current. [cAMP]m was also monitored by fluorescent imaging using a FRET-based sensor encoding a plasma-membrane targeted cAMP probe (Epac2-camps). Osmotic shock treatment with 1.5M formamide during 15 min induced a loss of t-tubule network as verified by confocal imaging with di-8-ANEPPS staining. Detubulation of ARVMs reduced membrane capacitance by 40% and basal ICa,L density ∼3-fold. Short (15s) applications of isoprenaline (Iso 100nM) increased ICa,L ∼2.5-fold similarly in both conditions but had a 30% smaller effect on [cAMP]m in detubulated ARVMs. After Iso washout, ICa,L and [cAMP]m returned to basal with a ∼1.5-fold faster kinetic in detubulated ARVMs than in control, suggesting a faster cAMP degradation by PDEs. The contribution of PDE3 and PDE4 was thus tested using the inhibitors cilostamide (1µM) and Ro20-1724 (10µM), respectively. Both inhibitors had a more pronounced effect on Iso response of ICa,L and [cAMP]m in detubulated ARVMs. Thus, detubulation per se has a profound effect on the β-AR signalling cascade which may contribute to its impairment in HF. This is partly due to a change in the respective contributions of PDE3 and PDE4 to the hydrolysis of cAMP near the membrane and LTCCs.

Phosphodiesterase 4D single-nucleotide polymorphism 83 and cognitive dysfunction in carotid endarterectomy patients.

Phosphodiesterase 4D (PDE4D), through the regulation of cyclic AMP, modulates inflammation and other processes that affect atherosclerosis and stroke. A PDE4D polymorphism, single-nucleotide polymorphism (SNP) 83 (rs966221), is associated with ischemic stroke. The association of SNP 83 with postoperative cognitive dysfunction has never been investigated. To determine whether SNP 83 is associated with cognitive dysfunction 1 day and 1 month following carotid endarterectomy (CEA). Three hundred fourteen patients with high-grade carotid stenosis scheduled for CEA consented to participate in this single-center cohort study of cognitive dysfunction. Patients with the C/C genotype of SNP 83 exhibited significantly more cognitive dysfunction at 1 day (29.7%) than the C/T (15.8%, P = .008) and T/T (12.7%, P = .01) genotypes. In a multivariate logistic regression model, C/T and T/T genotypes were both associated with significantly decreased odds of cognitive dysfunction compared with the C/C genotype (odds ratio, 0.45 [0.24-0.83], P = .01 and odds ratio, 0.33 [0.12-0.77], P = .02). There were no significant associations at 1 month. The C/C genotype of SNP 83 is significantly associated with the highest incidence of cognitive dysfunction 1 day following CEA in comparison with the C/T and T/T genotypes. This PDE4D genotype may lead to accelerated cyclic AMP degradation and subsequently elevated inflammation 1 day after CEA. These observations, in conjunction with previous studies, suggest that elevated inflammatory states may be partially responsible for the development of cognitive dysfunction after CEA, but more investigation is required.

Uncovering molecular elements of brain–body communication during development and treatment of neuropathic pain

Integral to neuropathic pain is a reciprocal interaction between tumor necrosis factor-α (TNF) production and the α 2-adrenergic receptor response, offering an attractive therapeutic target. The effects of varying levels of brain TNF on α 2-adrenergic regulation of cyclic AMP (cAMP) production in the hippocampus and sciatic nerve were investigated during the development and amitriptyline treatment of chronic pain. Increased levels of TNF during the development of chronic pain transform α 2-adrenergic inhibition of cAMP production in the brain to potentiation. While α 2-adrenergic receptors regulate TNF production, they also affect descending noradrenergic pathways. Increases in levels of TNF in the brain deeply impact peripheral inflammation through regulating α 2-adrenergic receptors, offering insight into brain–body interactions during neuropathic pain. Amitriptyline as an analgesic inhibits pain-induced increases in brain-associated TNF and transforms peripheral α 2-adrenergic receptors. The dynamic equilibrium between TNF levels and α 2-adrenergic functioning is uniquely altered during development and treatment of neuropathic pain. Proper manipulations of this interaction offer efficacious treatment of neuropathic pain.

Phosphodiesterase 4 regulation of cyclic AMP in pulmonary remodelling: Potential roles for isoform selective inhibitors

Phosphodiesterase 4 regulation of cyclic AMP in pulmonary remodelling: Potential roles for isoform selective inhibitors

Differentiation-induced alterations in cyclic AMP signaling in the Cath.a differentiated (CAD) neuronal cell line.

Regulation of intracellular cyclic AMP is critical to the modulation of many cellular activities, including cellular differentiation. Moreover, morphological differentiation has been linked to subsequent alterations in the cAMP signaling pathway in various cellular models. The current study was designed to explore the mechanism for the previously reported enhancement of adenylate cyclase activity in Cath.a differentiated cells following differentiation. Differentiation of Cath.a differentiated cells stably expressing the D2L dopamine receptor markedly potentiated both forskolin- and A2-adenosine receptor-stimulated cAMP accumulation. This enhancement was accompanied by a twofold increase in adenylate cyclase 6 (AC6) expression and a dramatic loss in the expression of AC9. The ability of Ca2+ to inhibit drug-stimulated cAMP accumulation was enhanced following differentiation, as was D2L dopamine receptor-mediated inhibition of Galphas-stimulated cAMP accumulation. Differentiation altered basal and drug-stimulated phosphorylation of the cAMP-response element-binding protein, which was independent of changes in protein kinase A expression. The current data suggest that differentiation of the neuronal cell model, Cath.a differentiated cells induces significant alterations in the expression and function of both the proximal and distal portions of the cAMP signaling pathway and may impact cellular operations dependent upon this pathway.

T cell receptor-independent CD4 signalling: CD4–MHC class II interactions regulate intracellular calcium and cyclic AMP

CD4 is a coreceptor on T helper (Th) cells that interacts with MHC class II molecules (MHCII). The mechanisms mediating the effects of CD4 on responses by T helper cells to stimulation of the antigen-specific T cell receptor (TCR) are still poorly understood. Here, we demonstrate T cell costimulation via CD4 signalling independent of T cell receptor-mediated signals. Incubation of T helper cells with peptide mimetics of the CD4-binding region on the MHC class II β2 domain caused intracellular calcium mobilization in the absence of antigen or other T cell receptor stimuli. Engagement of CD4 by peptide mimetics or wild-type MHC class II, but not by mutant MHC class II molecules incapable of engaging CD4, inhibited the T cell receptor-mediated increase in cyclic AMP (cAMP) concentrations in T helper cells. CD4-mediated signals activated cyclic AMP phosphodiesterases (PDEs) and inhibited adenylyl cyclase. Full activation and clonal expansion of antigen-stimulated T helper cells required the CD4-mediated regulation of cyclic AMP. Our results suggest a costimulatory mechanism of CD4 function that acts on the second messengers, calcium and cyclic AMP.

Regulation of intracellular cyclic AMP in skeletal muscle cells involves the efflux of cyclic nucleotide to the extracellular compartment.

(1) This report analyses the intracellular and extracellular accumulation of cyclic AMP in primary rat skeletal muscle cultures, after direct and receptor-dependent stimulation of adenylyl cyclase (AC). (2) Isoprenaline, calcitonin gene-related peptide (CGRP) and forskolin induced a transient increase in the intracellular cyclic AMP that peaked 5 min after onset stimulation. (3) Under stimulation with isoprenaline or CGRP, the intracellular cyclic AMP initial rise was followed by an exponential decline, reaching 46 and 52% of peak levels in 10 min, respectively. (4) Conversely, the forskolin-dependent accumulation of intracellular cyclic AMP decreased slowly and linearly, reaching 49% of the peak level in 30 min. (5) The loss of intracellular cyclic AMP from peak levels, induced by direct or receptor-induced activation of AC, was followed by an increase in the extracellular cyclic AMP. (6) This effect was independent on PDEs, since it was obtained in the presence of 3-isobutyl-1-methylxanthine (IBMX). (7) Besides, in isoprenaline treated cells, the beta-adrenoceptor antagonist propranolol reduced both intra- and extracellular accumulation of cyclic AMP, whereas the organic anion transporter inhibitor probenecid reduced exclusively the extracellular accumulation. (8) Together our data show that direct or receptor-dependent activation of skeletal muscle AC results in a transient increase in the intracellular cyclic AMP, despite the continuous presence of the stimulus. The temporal declining of intracellular cyclic AMP was not dependent on the cyclic AMP breakdown but associated to the efflux of cyclic nucleotide to the extracellular compartment, by an active transport since it was prevented by probenecid.

Regulation of cyclic AMP in rat pulmonary microvascular endothelial cells by rolipram-sensitive cyclic AMP phosphodiesterase (PDE4)

We report here studies on the regulation of the metabolism of adenosine 3′,5′-monophosphate (cAMP) in established and primary cultures of rat pulmonary microvascular endothelial cells (RPMVEC). Inhibition by rolipram, a selective inhibitor of cAMP phosphodiesterase (PDE) of the PDE4 gene family, was required to achieve maximal cAMP accumulation induced by direct or receptor-mediated adenylate cyclase activation when measured by [ 3 H ]-adenine prelabeling. Rolipram increased cAMP accumulation more effectively than did forskolin, isoproterenol, or adenosine derivatives alone, although extensive synergy was seen with combined agents. High-affinity PDE4 inhibitors, but not low-affinity or non-selective inhibitors, were effective inducers of cAMP accumulation in intact cells. The maximum effects (i.e. intrinsic activities) of these agents in the intact cell did not correlate with their in vitro PDE4 inhibitory affinities. RPMVEC were shown to express almost exclusively the PDE4 gene family isoforms A6 and B3. Guanosine 3′,5′-monophosphate hydrolysis, observed in other types of endothelial cells was not found in early or late passage RPMVEC. Reverse transcription-polymerase chain reaction identification of mRNAse supported these conclusions with the exception that PDE2 and PDE4D mRNA isoform transcripts were present. These studies also support the conclusion that the mechanism of rolipram reversal of rat lung ischemia-reperfusion-induced permeability involves PDE4 inhibition in the microvascular endothelial cells of the lung.

Regulation of cyclic AMP by extracellular ATP in cultured brain capillary endothelial cells

1 In primary unpassaged rat brain capillary endothelial cell cultures (RBECs), using reverse-transcriptase PCR with primers specific for P2Y receptor subtypes, we detected mRNA for P2Y2, P2Y4 and P2Y6, but not P2Y1 receptors. 2 None of the various nucleotides tested reduced forskolin elevated cyclic AMP levels in RBECs. ATP and ATPgammaS, as well as adenosine, enhanced cyclic AMP accumulation in the presence of forskolin. 3 Comparison of the concentration response curves to ATPgammaS with those for ATP and adenosine, at different incubation times, indicated that the response to purine nucleotides was not wholly dependent on conversion to adenosine. Adenosine deaminase abolished the response to adenosine but only reduced the response to ATP by about 50%. These results suggest the participation of a receptor responsive to nucleotides. 4 Isobutylmethylxanthine and 8-sulphophenyltheophylline prevented the cyclic AMP response, while neither 8-cyclopentyl-1, 3-dipropylxanthine nor SCH58261 were effective antagonists. 2-chloradenosine gave a robust response, but neither 2-chloro-N6-cyclopentyladenosine nor CGS 21680 were agonists. 5 These results show that adenosine and ATP can elevate the cyclic AMP levels of brain endothelial cells by acting on receptors which have a pharmacology apparently distinct from known P2Y and adenosine receptors.

Day/night differences in the stimulation of adenylate cyclase activity by calcium/calmodulin in chick pineal cell cultures: evidence for circadian regulation of cyclic AMP.

In chick pineal cell culture, stimulation of adenylate cyclase with the diterpene forskolin was greater during the subjective night than during the subjective day. This rhythm of cyclic AMP (cAMP) stimulation mimicked the rhythm of unstimulated cAMP measured previously during LD cycles from flow-through culture. Direct measurement of adenylate cyclase activity in permeabilized cells revealed an adenylate cyclase activity activated by Ca2+/calmodulin during the night but not during the day. However, this difference in adenylate cyclase activity at two times of the circadian cycle is apparent only when permeabilized cells were prewashed with buffer containing GTE When cAMP was measured from flow-through cultures maintained in continuous darkness to determine whether a circadian clock may regulate cAMP, a low-amplitude rhythm was measured. The circadian rhythm of cAMP was similar to the cAMP rhythm previously measured on LD cycles except that the rhythm in darkness had a lower amplitude. Similar to the suppression of melatonin, cAMP was suppressed by light presented during the middle of the night. LD differences in nocturnal cAMP levels were abolished with dipyridamole, an inhibitor of cyclic GMP (cGMP) phosphodiesterase. These results suggest that the rhythm of cAMP in chick pineal cells involves the stimulation of adenylate cyclase by Ca2+/calmodulin during the night and a GTP-dependent suppression of adenylate cyclase activity during the day. The photic suppression of cAMP at night involves the activation of a dipyridamole-sensitive, cGMP phosphodiesterase.

Regulation of cell cyclic AMP in medullary thick ascending limb of Henle in a rat model of chronic renal failure.

Chronic renal failure (CRF) is accompanied by adaptive changes in electrolyte reabsorption in the thick ascending limb of Henle of surviving nephrons. To study the cellular mechanism of this adaptation, we measured intracellular cAMP in micro-dissected medullary thick ascending limb (mTAL) segments in rats with CRF. mTAL exhibited in CRF an increase of basal cAMP from 25.6 +/- 10.0 in controls to 65.8 +/- 11.3 fmol mm-1 tubule in CRF (P < 0.05). Vasopressin and calcitonin stimulated mTAL adenylate-cyclase in a dose-dependent manner in controls but failed to stimulate in CRF. Likewise, maximal stimulation with 10(-3) M 3-isobutyl-1-methylxanthine (IBMX) plus 10(-5) M forskolin increased cAMP in controls to 63.0 +/- 16.0 but not in CRF, where maximal stimulated values remained at 63.1 +/- 18.8 fmol mm-1 tubule (P NS). Alpha2-adrenoreceptor activation with clonidine at concentrations ranging from 10(-8) to 10(-6) M diminished cAMP production by 37% in CRF (P < 0.05), whereas no differences were found in controls. Thus, the basal intracellular cAMP is increased in rat mTAL in CRF. The finding that neither forskolin nor vasopressin were able to further augment intracellular cAMP would suggest that stimulatory pathways of the adenylate-cyclase system are activated in the basal state. However, mTAL cells in CRF seem to retain the response of normal epithelium to inhibitory pathways such as the one mediated by alpha2-adrenoreceptors.

Role of G protein betagamma subunits in muscarinic receptor-induced stimulation and inhibition of adenylyl cyclase activity in rat olfactory bulb.

In the olfactory bulb, muscarinic receptors exert a bimodal control on cyclic AMP, enhancing basal and Gs-stimulated adenylyl cyclase activities and inhibiting the Ca2+/calmodulin- and forskolin-stimulated enzyme activities. In the present study, we investigated the involvement of G protein betagamma subunits by examining whether the muscarinic responses were reproduced by the addition of betagamma subunits of transducin (betagamma(t)) and blocked by putative betagamma scavengers. Membrane incubation with betagamma(t) caused a stimulation of basal adenylyl cyclase activity that was not additive with that produced by carbachol. Like carbachol, betagamma(t) potentiated the enzyme stimulations elicited by vasoactive intestinal peptide and corticotropin-releasing hormone. RT-PCR analysis revealed the expression of mRNAs encoding both type II and type IV adenylyl cyclase, two isoforms stimulated by betagamma synergistically with activated Gs. In addition, betagamma(t) inhibited the Ca2+/calmodulin- and forskolin-stimulated enzyme activities, and this effect was not additive with that elicited by carbachol. Membrane incubation with either one of two betagamma scavengers, the GDP-bound form of the alpha subunit of transducin and the QEHA fragment of type II adenylyl cyclase, reduced both the stimulatory and inhibitory effects of carbachol. These data provide evidence that in rat olfactory bulb the dual regulation of cyclic AMP by muscarinic receptors is mediated by betagamma subunits likely acting on distinct isoforms of adenylyl cyclase.

Adenosine A 2 receptors modulate hippocampal synaptic transmission via a cyclic-AMP-dependent pathway

Blockade of adenosine A 2 receptors has been shown to significantly reduce the level of tetanus-induced long-term potentiation in area CA1 of rat hippocampus [Kessey K. et al. (1997) Brain Res. 756, 184–190; Sekino Y. et al. (1991) Biochem. biophys. Res. Commun. 181, 1010–1014]. In the present study, the effects of A 2 receptor activation and blockade on the modulation of normal synaptic transmission and tetanus-induced long-term potentiation were examined at the Schaffer–CA1 synapse in rat hippocampal slices. A 2 receptor activation reversibly enhanced synaptic transmission evoked by low-frequency test pulses as measured by the dendritic field excitatory postsynaptic potential. In the presence of A 1 receptor blockade, A 2 activation further enhanced the excitatory postsynaptic potential, while A 2 receptor blockade resulted in a reversible decrease of the excitatory postsynaptic potential. The A 2a receptor agonist, CGS21680, had no effect on the excitatory postsynaptic potential, suggesting that tonic activation of A 2b receptors contributes to synaptic transmission under normal physiological conditions. Furthermore, we investigated the contribution of A 2 receptors to the level of tetanus-induced long-term potentiation. Under control conditions, a single tetanus potentiated the excitatory postsynaptic potential by 63.5% relative to baseline 30 min post-tetanus. In contrast, tetanus-induced long-term potentiation during A 2 blockade was 21.3%. A 2 receptor activation increased the level of tetanus-induced long-term potentiation to 90.2%. Because A 2 receptors are known to stimulate cyclic-AMP accumulation, the possible involvement of cyclic-AMP was examined. Forskolin, a direct adenylate cyclase activator, and 8-bromo-cyclic-AMP, a membrane-permeable analog of cyclic-AMP, were able to reconstitute tetanus-induced long-term potentiation during A 2 receptor blockade; however, the inactive analog 1,9-dideoxy-forskolin had no effect, indicating that the effects of A 2 activation on synaptic transmission were mediated largely through the regulation of intracellular cyclic-AMP. Because A 1 receptors exert an opposing effect on synaptic transmission relative to A 2 receptors, these results suggest that the stoichiometry of A 1 versus A 2 receptor activation appears to play an important role in the modulation of normal synaptic transmission and long-term potentiation in the CA1 region of the hippocampus.

Mechanisms regulating adipocyte lipolysis.

Mechanisms regulating adipocyte lipolysis are reviewed in three stages. The first stage examines plasma membrane hormone receptors and G-proteins. The primary regulators of adipose tissue lipolysis, the catecholamines, bind to the alpha 2, beta 1, beta 2, and beta 3 adrenergic receptors. The alpha 2 receptor couples with Gi-proteins to inhibit cyclic AMP formation and lipolysis, while the beta receptors couple with Gs-proteins to stimulate cyclic AMP formation and lipolysis. The beta 1 receptor may mediate low level catecholamine stimulation, while the beta 3 receptor, which is activated by higher levels of catecholamines, may deliver a more sustained signal. The second stage examines the regulation of cyclic AMP, the intracellular messenger that activates protein kinase A. Adenylyl cyclase synthesizes cyclic AMP from ATP and is regulated by the G-proteins. Phosphodiesterase 3B hydrolyzes cyclic AMP to AMP and is activated and phosphorylated by both insulin and the catecholamines norepinephrine and epinephrine. The third stage focuses on the rate-limiting enzyme of lipolysis, hormone-sensitive lipase (HSL). This 82 to 88 kDa protein is regulated by reversible phosphorylation. Protein kinase A activates and phosphorylates the enzyme at 2 sites, and 3 phosphatases have been implicated in HSL dephosphorylation. The translocation of HSL from the cytosol to the lipid droplet in response to lipolytic stimulation may be facilitated by a family of lipid-associated droplets called perilipins that are heavily phosphorylated by protein kinase A and dephosphorylated by insulin. As the mechanisms regulating adipocyte lipolysis continue to be uncovered, we look forward to the challenges of integrating these findings with research at the in situ and in vivo levels.

Contribution of phosphodiesterase isozymes to the regulation of the L-type calcium current in human cardiac myocytes.

1. To determine the contribution of the various phosphodiesterase (PDE) isozymes to the regulation of the L-type calcium current (ICa(L)) in the human myocardium, we investigated the effect of selective and non-selective PDE inhibitors on ICa(L) in single human atrial cells by use of the whole-cell patch-clamp method. We repeated some experiments in rabbit atrial myocytes, to make a species comparison. 2. In human atrial cells, 100 microM pimobendan increased ICa(L) (evoked by depolarization to +10 mV from a holding potential of -40 mV) by 250.4 +/- 45.0% (n = 15), with the concentration for half-maximal stimulation (EC50) being 1.13 microM. ICa(L) was increased by 100 microM UD-CG 212 by 174.5 +/- 30.2% (n = 10) with an EC50 value of 1.78 microM in human atrial cells. These two agents inhibit PDE III selectively. 3. A selective PDE IV inhibitor, rolipram (1-100 microM), did not itself affect ICa(L) in human atrial cells. However, 100 microM rolipram significantly enhanced the effect of 100 microM UD-CG 212 on ICa(L) (increase with UD-CG 212 alone, 167.9 +/- 33.9, n = 5; increase with the two agents together, 270.0 +/- 52.2%; n = 5, P < 0.05). Rolipram also enhanced isoprenaline (5 nM)-stimulated ICa(L) by 52.9 +/- 9.3% (n = 5) in human atrial cells. 4. In rabbit atrial cells, ICa(L) at +10 mV was increased by 22.1 +/- 9.0% by UD-CG 212 (n = 10) and by 67.4 +/- 12.0% (n = 10) by pimobendan (each at 100 microM). These values were significantly lower than those obtained in human atrial cells (P < 0.0001). Rolipram (1-100 microM) did not itself affect ICa(L) in rabbit atrial cells. However, ICa(L) was increased by 215.7 +/- 65.2% (n = 10) by the combination of 100 microM UD-CG 212 and 100 microM rolipram. This value was almost 10 times larger than that obtained for the effect of 100 microM UD-CG 212 alone. 5. These results imply a species difference: in the human atrium, the PDE III isoform seems dominant, whereas PDE IV may be more important in the rabbit atrium for regulating ICa(L). However, PDE IV might contribute significantly to the regulation of intracellular cyclic AMP in human myocardium when PDE III is already inhibited or when the myocardium is under beta-adrenoceptor-mediated stimulation.

Diurnal variation of the adenylyl cyclase type 1 in the rat pineal gland.

Nocturnal melatonin production in the pineal gland is under the control of norepinephrine released from superior cervical ganglia afferents in a rhythmic manner, and of cyclic AMP. Cyclic AMP increases the expression of serotonin N-acetyltransferase and of inducible cAMP early repressor that undergo circadian oscillations crucial for the maintenance and regulation of the biological clock. In the present study, we demonstrate a circadian pattern of expression of the calcium/calmodulin activated adenylyl cyclase type 1 (AC1) mRNA in the rat pineal gland. In situ hybridization revealed that maximal AC1 mRNA expression occurred at midday (12:00-15:00), with a very low signal at night (0:00-3:00). We established that this rhythmic pattern was controlled by the noradrenergic innervation of the pineal gland and by the environmental light conditions. Finally, we observed a circadian responsiveness of the pineal AC activity to calcium/calmodulin, with a lag due to the processing of the protein. At midday, AC activity was inhibited by calcium (40%) either in the presence or absence of calmodulin, while at night the enzyme was markedly (3-fold) activated by the calcium-calmodulin complex. These findings suggest (i) the involvement of AC1 acting as the center of a gating mechanism, between cyclic AMP and calcium signals, important for the fine tuning of the pineal circadian rhythm; and (ii) a possible regulation of cyclic AMP on the expression of AC1 in the rat pineal gland.

Inhibition of bronchospasm and ozone-induced airway hyperresponsiveness in the guinea-pig by CDP840, a novel phosphodiesterase type 4 inhibitor.

1. The activity of CDP840, a novel, potent and selective cyclic nucleotide phosphodiesterase type 4 (PDE 4) inhibitor, was evaluated in guinea-pig models (in vitro and in vivo) of bronchospasm, ozone-induced airway hyperresponsiveness (AHR) and non-cholinergic bronchoconstriction. Comparisons were made with (i) other PDE 4 inhibitors: CT1731 (S-enantiomer of CDP840), rolipram, RP73401 and (ii) the clinically used agents salbutamol and theophylline. 2. CDP840 relaxed isolated trachea, under basal tone (EC50 4.5 +/- 1.1 microM) being 17 fold less potent than rolipram (EC50 0.26 +/- 0.13 microM) but attaining the same Emax (83 +/- 6% of the response to 300 microM papaverine). 3. CDP840 relaxed tracheae pre-contracted with carbachol (IC25 39 +/- 9 microM) and histamine (IC25 4 +/- 1 microM) producing monophasic curves. Stereoselectivity was not observed with CT1731 against either carbachol (IC25 33 +/- 11 microM) or histamine (IC25 17 +/- 10 microM). Aminophylline was 1.6 fold (carbachol) and 11 fold (histamine) less potent than CDP840. Rolipram and RP73401 produced tri-phasic relaxation curves but were of similar potency (at the IC25 level) to CDP840 against carbachol (rolipram 18 +/- 5 microM, RP73401 39 +/- 1 microM) whereas against histamine they were approximately 20 fold more potent (rolipram 0.2 +/- 0.1 microM, RP73401 0.2 +/- 0.1 microM). In producing > 30% (carbachol) and > 60% (histamine) relaxation these inhibitors had similar potency and were poor compared to salbutamol. 4. Pre-incubation with CDP840 (10 microM) did not antagonize histamine-induced contraction of isolated trachea; however, it did cause a slight potentiation of the subsequent relaxation to salbutamol (IC50 23 +/- 1 to 15 +/- 2 nM). 5. Pretreatment (1 h) with either CDP840 (1 mg kg-1, i.p. or 3 mg kg-1, i.v.) or rolipram (1 mg kg-1, i.p.) did not bronchodilate or antagonize bronchospasm due to inhaled histamine in anaesthetized, ventilated guinea-pigs. Salbutamol (1 mg kg-1, i.p.) did not bronchodilate but caused a parallel 7 fold rightward shift in the histamine dose-response curve. 6. Stimulation of the vagus nerve in the presence of atropine resulted in a frequency-related bronchoconstriction. CDP840 and rolipram (i.v.) inhibited the response being approximately equipotent (EC50 approximately 10 micrograms kg-1). Neither drug inhibited bronchospasm to inhaled substance P. 7. CDP840 (1-10 micrograms kg-1 i.p.) dose relatedly inhibited ozone-induced bronchoconstriction. CT1731 (1 mg kg-1), rolipram (1 mg kg-1), RP73401 (10 micrograms kg-1) and aminophylline (10 mg kg-1) had no effect. Ozone-induced AHR to inhaled histamine was inhibited by CDP840 in a dose-related manner, 10 micrograms kg-1 abolishing the AHR. This effect was stereoselective as CT1731 was approximately 30 fold less potent than CDP840. Rolipram was approximately 100 fold less potent and RP73401 and aminophylline had no effect. CDP840 was orally active being approximately 10 fold less potent compared to i.p. administration. 8. CDP840 is a poor spasmolytic and anti-spasmogenic agent in response to exogenous mediators; however, it potently inhibits vagally mediated non-cholinergic bronchoconstriction and ozone-induced AHR to histamine. It is possible that regulation of cyclic AMP by PDE 4 contributes to neuronal sensitivity in the airways. Furthermore, CDP840 may suppress AHR without being an overt bronchodilator. Such a profile of activity may have therapeutic benefit in airways diseases such as asthma.