Inhibits cAMP Synthesis Research Articles

Real-Time Determination of Intracellular cAMP Reveals Functional Coupling of Gs Protein to the Melatonin MT1 Receptor.

Melatonin is a neuroendocrine hormone that regulates the circadian rhythm and many other physiological processes. Its functions are primarily exerted through two subtypes of human melatonin receptors, termed melatonin type-1 (MT1) and type-2 (MT2) receptors. Both MT1 and MT2 receptors are generally classified as Gi-coupled receptors owing to their well-recognized ability to inhibit cAMP accumulation in cells. However, it remains an enigma as to why melatonin stimulates cAMP production in a number of cell types that express the MT1 receptor. To address if MT1 can dually couple to Gs and Gi proteins, we employed a highly sensitive luminescent biosensor (GloSensorTM) to monitor the real-time changes in the intracellular cAMP level in intact live HEK293 cells that express MT1 and/or MT2. Our results demonstrate that the activation of MT1, but not MT2, leads to a robust enhancement on the forskolin-stimulated cAMP formation. In contrast, the activation of either MT1 or MT2 inhibited cAMP synthesis driven by the activation of the Gs-coupled β2-adrenergic receptor, which is consistent with a typical Gi-mediated response. The co-expression of MT1 with Gs enabled melatonin itself to stimulate cAMP production, indicating a productive coupling between MT1 and Gs. The possible existence of a MT1-Gs complex was supported through molecular modeling as the predicted complex exhibited structural and thermodynamic characteristics that are comparable to that of MT1-Gi. Taken together, our data reveal that MT1, but not MT2, can dually couple to Gs and Gi proteins, thereby enabling the bi-directional regulation of adenylyl cyclase to differentially modulate cAMP levels in cells that express different complements of MT1, MT2, and G proteins.

Novel M2 -selective, Gi -biased agonists of muscarinic acetylcholine receptors.

More than 30% of currently marketed medications act via GPCRs. Thus, GPCRs represent one of the most important pharmacotherapeutic targets. In contrast to traditional agonists activating multiple signalling pathways, agonists activating a single signalling pathway represent a new generation of drugs with increased specificity and fewer adverse effects. We have synthesized novel agonists of muscarinic ACh receptors and tested their binding and function (on levels of cAMP and inositol phosphates) in CHO cells expressing individual subtypes of muscarinic receptors, primary cultures of rat aortic smooth muscle cells and suspensions of digested native tissues from rats. Binding of the novel compounds to M2 receptors was modelled in silico. Two of the tested new compounds (1-(thiophen-2-ylmethyl)-3,6-dihydro-2H-pyridinium and 1-methyl-1-(thiophen-2-ylmethyl)-3,6-dihydro-2H-pyridinium) only inhibited cAMP synthesis in CHO cells, primary cultures, and native tissues, with selectivity for M2 muscarinic receptors and displaying bias towards the Gi signalling pathway at all subtypes of muscarinic receptors. Molecular modelling revealed interactions with the orthosteric binding site in a way specific for a given agonist followed by agonist-specific changes in the conformation of the receptor. The identified compounds may serve as lead structures in the search for novel non-steroidal and non-opioid analgesics acting via M2 and M4 muscarinic receptors with reduced side effects associated with activation of the phospholipase C signalling pathway.

Caffeine inhibits PI3K and mTORC2 in Dictyostelium and differentially affects multiple other cAMP chemoattractant signaling effectors.

Caffeine is commonly used in Dictyostelium to inhibit the synthesis of the chemoattractant cAMP and, therefore, its secretion and the autocrine stimulation of cells, in order to prevent its interference with the study of chemoattractant-induced responses. However, the mechanism through which caffeine inhibits cAMP synthesis in Dictyostelium has not been characterized. Here, we report the effects of caffeine on the cAMP chemoattractant signaling network. We found that caffeine inhibits phosphatidylinositol 3-kinase (PI3K) and mechanistic target of rapamycin complex 2 (mTORC2). Both PI3K and mTORC2 are essential for the chemoattractant-stimulated cAMP production, thereby providing a mechanism for the caffeine-mediated inhibition of cAMP synthesis. Our results also reveal that caffeine treatment of cells leads to an increase in cAMP-induced RasG and Rap1 activation, and inhibition of the PKA, cGMP, MyoII, and ERK1 responses. Finally, we observed that caffeine has opposite effects on F-actin and ERK2 depending on the assay and Dictyostelium strain used, respectively. Altogether, our findings reveal that caffeine considerably affects the cAMP-induced chemotactic signaling pathways in Dictyostelium, most likely acting through multiple targets that include PI3K and mTORC2.

A Computational Approach for Understanding the Interactions between Graphene Oxide and Nucleoside Diphosphate Kinase with Implications for Heart Failure.

During a heart failure, an increased content and activity of nucleoside diphosphate kinase (NDPK) in the sarcolemmal membrane is responsible for suppressing the formation of the second messenger cyclic adenosine monophosphate (cAMP)—a key component required for calcium ion homeostasis for the proper systolic and diastolic functions. Typically, this increased NDPK content lets the surplus NDPK react with a mutated G protein in the beta-adrenergic signal transduction pathway, thereby inhibiting cAMP synthesis. Thus, it is thus that inhibition of NDPK may cause a substantial increase in adenylate cyclase activity, which in turn may be a potential therapy for end-stage heart failure patients. However, there is little information available about the molecular events at the interface of NDPK and any prospective molecule that may potentially influence its reactive site (His118). Here we report a novel computational approach for understanding the interactions between graphene oxide (GO) and NDPK. Using molecular dynamics, it is found that GO interacts favorably with the His118 residue of NDPK to potentially prevent its binding with adenosine triphosphate (ATP), which otherwise would trigger the phosphorylation of the mutated G protein. Therefore, this will result in an increase in cAMP levels during heart failure.

Adenylyl Cyclase 5 Regulation by Gβγ Involves Isoform-Specific Use of Multiple Interaction Sites.

Adenylyl cyclase (AC) converts ATP into cyclic AMP (cAMP), an important second messenger in cell signaling. Heterotrimeric G proteins and other regulators are important for control of AC activity. Depending on the AC isoform, Gβγ subunits can either conditionally stimulate or inhibit cAMP synthesis. We previously showed that the Gαs-βγ heterotrimer binds to the N terminus (NT) of type 5 AC (AC5). We now show that Gβγ binds to the NT of a wide variety of AC isoforms. We hypothesized that Gβγ/AC5 interactions involving inactive heterotrimer and Gβγ stimulation of AC5 were separable events. Mutations of the Gβγ "hotspot" show that this site is necessary for AC5 stimulation but not for interactions with the first 198 aa of AC5NT, which is a G protein scaffolding site. This contrasts with AC6, where the Gβγ hotspot is required for both interactions with AC6NT and for stimulation of AC6. Additionally, the SIGK hotspot peptide disrupts Gβγ regulation of AC isoforms 1, 2, and 6, but not AC5. Gβγ also binds the C1/C2 catalytic domains of AC5 and AC6. Finally, cellular interactions with full-length AC5 depend on multiple sites on Gβγ. This suggests an isoform-specific mechanism in which bound Gβγ at the AC5NT is ideally situated for spatiotemporal control of AC5. We propose Gβγ regulation of AC involves multiple binding events, and the role of the AC NT for mechanisms of regulation by heterotrimeric G protein subunits is isoform-specific.

Cyclic AMP in oocytes controls meiotic prophase I and primordial folliculogenesis in the perinatal mouse ovary.

In mammalian ovaries, a fixed population of primordial follicles forms during the perinatal stage and the oocytes contained within are arrested at the dictyate stage of meiotic prophase I. In the current study, we provide evidence that the level of cyclic AMP (cAMP) in oocytes regulates oocyte meiotic prophase I and primordial folliculogenesis in the perinatal mouse ovary. Our results show that the early meiotic development of oocytes is closely correlated with increased levels of intra-oocyte cAMP. Inhibiting cAMP synthesis in fetal ovaries delayed oocyte meiotic progression and inhibited the disassembly and degradation of synaptonemal complex protein 1. In addition, inhibiting cAMP synthesis in in vitro cultured fetal ovaries prevented primordial follicle formation. Finally, using an in situ oocyte chromosome analysis approach, we found that the dictyate arrest of oocytes is essential for primordial follicle formation under physiological conditions. Taken together, these results suggest a role for cAMP in early meiotic development and primordial follicle formation in the mouse ovary.

AIP inactivation leads to pituitary tumorigenesis through defective Gαi-cAMP signaling

The aryl hydrocarbon receptor interacting protein (AIP) is a tumor-suppressor gene underlying the pituitary adenoma predisposition. Thus far, the exact molecular mechanisms by which inactivated AIP exerts its tumor-promoting action have been unclear. To better understand the role of AIP in pituitary tumorigenesis, we performed gene expression microarray analysis to examine changes between Aip wild-type and knockout mouse embryonic fibroblast (MEF) cell lines. Transcriptional analyses implied that Aip deficiency causes a dysfunction in cyclic adenosine monophosphate (cAMP) signaling, as well as impairments in signaling cascades associated with developmental and immune-inflammatory responses. In vitro experiments showed that AIP deficiency increases intracellular cAMP concentrations in both MEF and murine pituitary adenoma cell lines. Based on knockdown of various G protein α subunits, we concluded that AIP deficiency leads to elevated cAMP concentrations through defective Gαi-2 and Gαi-3 proteins that normally inhibit cAMP synthesis. Furthermore, immunostaining of Gαi-2 revealed that AIP deficiency is associated with a clear reduction in Gαi-2 protein expression levels in human and mouse growth hormone (GH)-secreting pituitary adenomas, thus indicating defective Gαi signaling in these tumors. By contrast, all prolactin-secreting tumors showed prominent Gαi-2 protein levels, irrespective of Aip mutation status. We additionally observed reduced expression of phosphorylated extracellular signal-regulated kinases 1/2 and cAMP response element-binding protein levels in mouse and human AIP-deficient somatotropinomas. This study implies for the first time that a failure to inhibit cAMP synthesis through dysfunctional Gαi signaling underlies the development of GH-secreting pituitary adenomas in AIP mutation carriers.

Quantitative analysis of the dynamic signaling pathway involved in the cAMP mediated induction of l-carnitine biosynthesis in E. coli cultures

L-(-)-carnitine can be synthesized from waste bioprecursors in the form of crotonobetaine. Such biotransformation is carried out in E. coli by the enzymes encoded by operons regulated by the cAMP receptor proteins. Non-phosphorylated sugars, such as glycerol are used as energy and carbon source since glucose inhibits cAMP synthesis. Until now little attention has been paid to the regulatory signaling structure that operates during the transition from a glucose-consuming, non-l-carnitine producing steady state, to a glycerol-consuming l-carnitine producing steady state. In this work we aim to elucidate and quantify the underlying regulatory mechanisms operating in the abolition of the glucose inhibiting effect. For this purpose we make use of the systemic approach by integrating the available information and our own experimentally generated data to construct a mathematical model. The model is built using power-law representation and is used as a platform to make predictive simulations and to assess the consistency of the regulatory structure of the overall process. The model is subsequently checked for quality through stability and a special, dynamic sensitivity analysis. The results show that the model is able to deal with the observed system transient phase. The model is multi-hierarchical, comprising the metabolic, gene expression, signaling and bioreactor levels. It involves variables and parameters of a very different nature that develop in different time scales and orders of magnitude. Some of the most relevant conclusions obtained are: (i) the regulatory interactions among glucose, glycerol and cAMP metabolism are far stronger than those present in the l-carnitine transport, production and degradation processes; (ii) carnitine biosynthesis is very sensitive to the cAMP signaling system since it reacts at very low cAMP receptor concentrations, and (iii) ATP is a critical factor in the transient dynamics. All these model-derived observations have been experimentally confirmed by separate studies. As a whole, the model contributes to our general understanding of the transient regulation through the signal regulatory structure, thus enabling more accurate optimization strategies to be used.

Prostanoids in health and disease

The prostanoids are a family of lipid mediators generated by the action of cyclooxygenase on a 20-carbon unsaturated fatty acid, arachidonic acid. Prostanoids are generated widely in response to diverse stimuli and, acting in a paracrine or autocrine manner, play important roles in normal physiology and disease. This review summarizes the current knowledge on prostanoid generation and the roles of individual mediators, their biosynthetic pathways, and their receptors in health and disease.

Antagonists of the EP3 Receptor for Prostaglandin E2 Are Novel Antiplatelet Agents That Do Not Prolong Bleeding

Myocardial infarction and stroke are caused by blood clots forming over a ruptured or denuded atherosclerotic plaque (atherothrombosis). Production of prostaglandin E(2) (PGE(2)) by an inflamed plaque exacerbates atherothrombosis and may limit the effectiveness of current therapeutics. Platelets express multiple G-protein coupled receptors, including receptors for ADP and PGE(2). ADP can mobilize Ca(2+) and through the P(2)Y(12) receptor can inhibit cAMP production, causing platelet activation and aggregation. Clopidogrel (Plavix), a selective P(2)Y(12) antagonist, prevents platelets from clotting but thereby increases the risk of severe or fatal bleeding. The platelet EP(3) receptor for PGE(2), like the P(2)Y(12) receptor, also inhibits cAMP synthesis. However, unlike ADP, facilitation of platelet aggregation via the PGE(2)/EP(3) pathway is dependent on co-agonists that can mobilize Ca(2+). We used a ligand-based design strategy to develop peri-substituted bicylic acylsulfonamides as potent and selective EP(3) antagonists. We show that DG-041, a selective EP(3) antagonist, inhibits PGE(2) facilitation of platelet aggregation in vitro and ex vivo. PGE(2) can resensitize platelets to agonist even when the P(2)Y(12) receptor has been blocked by clopidogrel, and this can be inhibited by DG-041. Unlike clopidogrel, DG-041 does not affect bleeding time in rats, nor is bleeding time further increased when DG-041 is co-administered with clopidogrel. This indicates that EP(3) antagonists potentially have a superior safety profile compared to P(2)Y(12) antagonists and represent a novel class of antiplatelet agents.

Muscarinic M2Receptors Directly Activate Gq/11and GsG-Proteins

Muscarinic M(2) receptors preferentially couple with the G(i/o) class of G-proteins to inhibit cAMP synthesis. However, they can also stimulate net synthesis of cAMP and inositol phosphate (IP) accumulation. We investigated in intact Chinese hamster ovary (CHO) cells expressing human M(2) receptors (CHO-M(2) cells) whether direct interaction of M(2) receptors with G(s) and G(q/11) G-proteins is responsible for the latter effects. Suppression of the G(s)alpha subunit using RNA interference abolished stimulation of cAMP synthesis induced by 1 mM carbachol in both control and pertussis toxin-treated CHO-M(2) cells but had no effect on the inhibition of forskolin-stimulated cAMP synthesis. Carbachol stimulated accumulation of IP with an EC(50) of 79 microM. Removal of the G(q),G(11), or both alpha subunits reduced this response by 78, 54, and 92%, respectively, whereas suppression of the G(s)alpha subunit had no effect. Similar results obtained in CHO cells expressing M(1) receptors that preferentially couple with G(s) and G(q/11) G-proteins confirmed the efficiency of siRNA treatments. Stimulation of M(2) receptors in control and pertussis toxin-treated cells by a series of full agonists with respect to inhibition of adenylyl cyclase displayed different efficacies in stimulating IP accumulation. Carbachol, acetylcholine, and oxotremorine-M [N,N,N-trimethyl-4-(2-oxo-1-pyrolidinyl)-2-butyn-1-ammonium] behaved as full agonists, furmethide (N,N,N-trimethyl-2-furanmethammonium) and methylfurmethide [(5-methyl-2-furyl)methyltrimethylammonium] were partial agonists, and oxotremorine (1-[4-(1-pyrrolidinyl)-2-butynyl]-2-pyrrolidinone) had no effect. Our results provide direct evidence of M(2) receptor coupling with the alpha subunits of G(s) and G(q/11) G-proteins and demonstrate induction of multiple receptor conformational states dependent on both the concentration and the nature of the agonist used.

Dynamic Regulation of cAMP Synthesis through Anchored PKA-Adenylyl Cyclase V/VI Complexes

Spatiotemporal organization of cAMP signaling begins with the tight control of second messenger synthesis. In response to agonist stimulation of G protein-coupled receptors, membrane-associated adenylyl cyclases (ACs) generate cAMP that diffuses throughout the cell. The availability of cAMP activates various intracellular effectors, including protein kinase A (PKA). Specificity in PKA action is achieved by the localization of the enzyme near its substrates through association with A-kinase anchoring proteins (AKAPs). Here, we provide evidence for interactions between AKAP79/150 and ACV and ACVI. PKA anchoring facilitates the preferential phosphorylation of AC to inhibit cAMP synthesis. Real-time cellular imaging experiments show that PKA anchoring with the cAMP synthesis machinery ensures rapid termination of cAMP signaling upon activation of the kinase. This protein configuration permits the formation of a negative feedback loop that temporally regulates cAMP production.

Characteristics of the renal C-type natriuretic peptide receptor in hypertrophied and developing rat kidney

This study investigates the effect of hypertrophy, using one kidney and one kidney/one clip rats, and development, comparing 3- and 12-week-old rats, on the expression of the 28-amino acid atrial natriuretic peptide (ANP(1-28)) binding sites in rat kidney. Here we report an increased B(max) value of glomerular binding sites for ANP(1-28) and C-type natriuretic peptide 1-22 (CNP(1-22)) in hypertrophied and developing kidney, without modifying their affinity, an effect that was prevented in the presence of the synthetic des[Gln(18), Ser(19), Gly(20), Leu(21), Gly(22)]ANP(4-23)-amide (C-ANF), suggesting that natriuretic peptide receptor (NPR)-C binding sites might be enhanced. The enhanced B(max) was only detected in the high affinity binding site for CNP(1-22), which has been identified as the 67 kDa NPR-C-like protein. A similar effect was observed in renal glomeruli from 3-week-old rats compared with 12-week-old rats. Our results indicate that ANP(1-28), CNP(1-22) and C-ANF inhibited cAMP synthesis stimulated by the physiological agonists histamine and 5-hydroxytryptamine or directly by forskolin. The inhibitory effect was found to be significantly greater in 1-kidney and 1-kidney/1-clip rats than in controls, and in 3-week-old rats compared with 12-week-old rats. Our observations suggest that this effect must be attributed to the 67 kDa NPR-C-like protein due to the enhanced B(max) values and the reported inhibitory role for this receptor on adenylyl cyclase activity. The enhanced inhibitory role of natriuretic peptides on cAMP synthesis in hypertrophied and developing kidney may influence glomerular function in the rat kidney and suggests a role for the 67 kDa NPR-C-like protein in growth.

Receptor subtypes for vasonatrin peptide in renal glomeruli and arteries

Vasonatrin peptide (VNP) is a synthetic new member of the natriuretic peptide family. VNP is a chimera of CNP and ANP, which possesses the 22-amino acid ringed structure of CNP and the COOH terminus of ANP. VNP shares properties with ANP and CNP but also shows functional characteristics distinct from those induced by the original natriuretic peptides. This study investigates VNP binding to specific sites in the kidney and femoral artery, in order to clarify the nature of the receptors through which VNP exerts its effects. Using autoradiographic techniques we have found that VNP binds to renal and arterial tissue sections. VNP binding was displaced by incubation in the presence of 1 μM ANP 1–28, CNP 1–22 and C-ANP, which suggests that VNP mostly binds to NPR-C. Cross-linking studies performed in rat glomerular membranes confirmed that VNP mainly binds to the 67 kDa-NPR-C-like protein and also to NPR-A. Consistent with this, our results indicate that VNP inhibits cAMP synthesis stimulated by the physiological agonist histamine in a concentration-dependent manner, without having any effect on basal cAMP production. Finally, we have found that VNP increases cGMP production in rat renal glomeruli, suggesting that this peptide functionally binds to NPR-A.

Agonist-induced polarized trafficking and surface expression of the adenosine 2b receptor in intestinal epithelial cells: role of SNARE proteins.

Adenosine, acting through the A2b receptor, induces vectorial chloride and IL-6 secretion in intestinal epithelia and may play an important role in intestinal inflammation. We have previously shown that apical or basolateral adenosine receptor stimulation results in the recruitment of the A2b receptor to the plasma membrane. In this study, we examined domain specificity of recruitment and the role of soluble N-ethylmaleimide (NEM) attachment receptor (SNARE) proteins in the agonist-mediated recruitment of the A2b receptor to the membrane. The colonic epithelial cell line T84 was used because it only expresses the A2b-subtype adenosine receptor. Cell fractionation, biotinylation, and electron microscopic studies showed that the A2b receptor is intracellular at rest and that apical or basolateral adenosine stimulation resulted in the recruitment of the receptor to the apical membrane. Upon agonist stimulation, the A2b receptor is enriched in the vesicle fraction containing vesicle-associated membrane protein (VAMP)-2. Furthermore, in cells stimulated with apical or basolateral adenosine, we demonstrate a complex consisting of VAMP-2, soluble NEM-sensitive factor attachment protein (SNAP)-23, and A2b receptor that is coimmunoprecipitated in cells stimulated with adenosine within 5 min and is no longer detected within 15 min. Inhibition of trafficking with NEM or nocodazole inhibits cAMP synthesis induced by apical or basolateral adenosine by 98 and 90%, respectively. cAMP synthesis induced by foskolin was not affected, suggesting that generalized signaling is not affected under these conditions. Collectively, our data suggest that 1) the A2b receptor is intracellular at rest; 2) apical or basolateral agonist stimulation induces recruitment of the A2b receptor to the apical membrane; 3) the SNARE proteins, VAMP-2 and SNAP-23, participate in the recruitment of the A2b receptor; and 4) the SNARE-mediated recruitment of the A2b receptor may be required for its signaling.

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.

Water deprivation enhances the inhibitory effect of natriuretic peptides on cAMP synthesis in rat renal glomeruli

This study investigates the effect of water deprivation on the expression of atrial natiruretic peptide (ANP)(1-28) binding sites in rat kidney. Water deprivation increased the B(max) of glomerular binding sites for ANP(1-28) and C-type natriuretic peptide (CNP)(1-22) without modifying their affinity, an effect that was prevented in the presence of C-atrial natriuretic factor (C-ANF), suggesting that natriuretic peptide receptor-C (NPR-C) binding sites might be enhanced. Our results indicate that ANP(1-28), CNP(1-22), and C-ANF inhibit cAMP synthesis directly stimulated by forskolin or by the physiological agonists histamine and 5-hydroxytryptamine. The inhibitory effect was found to be significantly greater in water-deprived rats than in controls. Our observations suggest that this effect must be attributed to the 67-kDa NPR-C-like protein, because the 67- and 77-kDa NPR-C-like proteins show high and low affinities for CNP(1-22), respectively, and the enhanced inhibitory effect of CNP on cAMP generation in water-deprived rats was detected at subnanomolar concentrations. In addition, using affinity cross-linking studies we have observed that water deprivation increases the expression of the 67-kDa NPR-C-like protein, and HS-142, which binds to NPR-A and the 77-kDa NPR-C-like but not the 67-kDa protein, reduced ligand internalization without affecting cAMP inhibition by ANP(1-28). Finally, we have found that ligand binding to the 67-kDa NPR-C-like protein is reduced by GTPgammaS, suggesting that this receptor is associated with a G protein in renal glomeruli. The enhanced inhibitory role of natriuretic peptides on cAMP synthesis induced by water deprivation may influence glomerular function in the rat kidney.

Cyclic AMP signaling pathway modulates susceptibility of candida species and Saccharomyces cerevisiae to antifungal azoles and other sterol biosynthesis inhibitors.

Azoles are widely used antifungals; however, their efficacy is compromised by fungistatic activity and selection of resistant strains during treatment. Recent studies demonstrated roles for the protein kinase C and calcium signaling pathways in modulating azole activity. Here we explored a role for the signaling pathway mediated by cyclic AMP (cAMP), which is synthesized by the regulated action of adenylate cyclase (encoded by CDC35 in Candida albicans and CYR1 in Saccharomyces cerevisiae) and cyclase-associated protein (encoded by CAP1 and SRV2, respectively). Relative to wild-type strains, C. albicans and S. cerevisiae strains mutated in these genes were hypersusceptible to fluconazole (>4- to >16-fold-decreased 48-h MIC), itraconazole (>8- to >64-fold), or miconazole (16- to >64-fold). Similarly, they were hypersusceptible to terbinafine and fenpropimorph (2- to >16-fold), which, like azoles, inhibit sterol biosynthesis. Addition of cAMP to the medium at least partially reversed the hypersusceptibility of Ca-cdc35 and Sc-cyr1-2 mutants. An inhibitor of mammalian adenylate cyclase, MDL-12330A, was tested in combination with azoles; a synergistic effect was observed against azole-susceptible and -resistant strains of C. albicans and five of six non-C. albicans Candida species. Analysis of cAMP levels after glucose induction in the presence and absence of MDL-12330A confirmed that it acts by inhibiting cAMP synthesis in yeast. RNA analysis suggested that a defect in azole-dependent upregulation of the multidrug transporter gene CDR1 contributes to the hypersusceptibility of the Ca-cdc35 mutant. Our results implicate cAMP signaling in the yeast azole response; compounds similar to MDL-12330A may be useful adjuvants in azole therapy.

G protein diseases: newly recognized causes of metabolic encephalopathy

G proteins (guanine nucleotide-binding proteins) are trimeric proteins relaying signals from a large number of receptors to extremely complicated networks of intracellular effectors thereby leading to cellular action. They are composed of an a subunit that is loosely bound to a dimer consisting of a b subunit and a g subunit. Each subunit is encoded by several genes; there are at least 16 a, 6 b and 12 g genes. G protein trimers are defined by their a subunit: Gs contains as (stimulates cAMP synthesis), Gi contains ai (inhibits cAMP synthesis), etc. G protein activity is regulated through the binding and hydrolysis of guanosine triphosphate (GTP) by the a subunit. When a ligand (hormone, neurotransmitter or other) activates a receptor the abg-GDP complex releases GDP. This is followed by the binding of GTP to the a subunit and subsequently the dissociation of the a-GTP complex from bg. The a-GTP and bg complexes then activate different downstream signaling pathways. Signaling is terminated by the conversion of bound GTP to GDP and reassociation of a-GDP and bg (Fig. 1). Known G protein diseases are mainly defects in a subunit genes causing either a decreased transmission of signals (‘loss of function’) or an increased transmission of signals (‘gain of function’). They are essentially endocrinological/oncological diseases. Well-known inactivating mutations in the Gsa gene (MIM 139320; chromosome 20q13) are e.g. pseudohypoparathyroidism Ia and pseudopseudohypoparathyroidism. These are genomic mutations on the maternal and paternal allele, respectively (imprinted gene, see below). Examples of activating mutations in this gene are the classical McCuneAlbright syndrome as well as isolated adenomas of endocrine organs. These are somatic mutations, increasing cAMP, which stimulates cell proliferation. McCune-Albright syndrome is characterized by polyostotic fibrous dysplasia, hyperpigmented skin patches and autonomic hyperfunction of endocrine glands (causing acromegaly, Cushing syndrome a.o) (reviews in Refs. 1 and 2). Gsa is subject to genomic imprinting (at least in some tissues) and is part of the complex GNAS1 gene cluster with multiple promotors that are active depending on the parental origin of the allele, generating multiple alternative transcripts by splicing of different upstream exons to exon 2 of the ‘classical’ Gsa gene. One of these promotors is only active on the maternal allele and generates a transcript encoding NESP55, a chromogranin-like neurosecretory protein. Another promotor, only

Bis(31/31′){[Cys 31, Nva 34]NPY(27–36)-NH 2}: a neuropeptide Y (NPY) Y 5 receptor selective agonist with a latent stimulatory effect on food intake in rats

The actions of neuropeptide Y (NPY) are mediated by at least six G-protein coupled receptors denoted as Y 1, Y 2, Y 3, Y 4, Y 5, and y 6. Investigations using receptor selective ligands and receptor knock-out mice suggest that NPY effects on feeding are mediated by both Y 1 and Y 5 receptors. We have previously shown that Cys-dimers of NPY C-terminal peptides exhibit Y 1 selectivity relative to Y 2 receptors. Re-investigation of their selectivity with respect to the newly cloned receptors, has identified bis(31/31′) {[Cys 31, Nva 34]NPY(27–36)-NH 2} (BWX-46) as a Y 5 receptor selective agonist. BWX-46 selectively bound Y 5 receptors, and inhibited cAMP synthesis by Y 5 cells with potencies comparable to that of NPY. Moreover, BWX-46 (10 μM) exhibited no significant effect on the cAMP synthesis by Y 1, Y 2, and Y 4 cells. Thus, BWX-46 constitutes the lowest molecular weight Y 5 selective agonist reported to date. Intrahypothalamic (iht)-injection of 30 and 40 μg of BWX-46 stimulated the food intake by rats in a gradual manner, reaching maximal level 8 h after injection. This response was similar to that exhibited by other Y 5 selective agonists, but differed from that of NPY, which exhibited a rapid orexigenic stimulus within 1 h. It is suggested that the differences in the orexigenic stimuli of NPY and Y 5 agonists may be due to their differences in the signal transduction mechanisms.