Adenosine Monophosphate Formation Research Articles

Rare Heterozygous Loss-of-Function Variants in the Human GLP-1 Receptor Are Not Associated With Cardiometabolic Phenotypes.

Lost glucagon-like peptide 1 receptor (GLP-1R) function affects human physiology. This work aimed to identify coding nonsynonymous GLP1R variants in Danish individuals to link their in vitro phenotypes and clinical phenotypic associations. We sequenced GLP1R in 8642 Danish individuals with type 2 diabetes or normal glucose tolerance and examined the ability of nonsynonymous variants to bind GLP-1 and to signal in transfected cells via cyclic adenosine monophosphate (cAMP) formation and β-arrestin recruitment. We performed a cross-sectional study between the burden of loss-of-signaling (LoS) variants and cardiometabolic phenotypes in 2930 patients with type 2 diabetes and 5712 participants in a population-based cohort. Furthermore, we studied the association between cardiometabolic phenotypes and the burden of the LoS variants and 60 partly overlapping predicted loss-of-function (pLoF) GLP1R variants found in 330 566 unrelated White exome-sequenced participants in the UK Biobank cohort. We identified 36 nonsynonymous variants in GLP1R, of which 10 had a statistically significant loss in GLP-1-induced cAMP signaling compared to wild-type. However, no association was observed between the LoS variants and type 2 diabetes, although LoS variant carriers had a minor increased fasting plasma glucose level. Moreover, pLoF variants from the UK Biobank also did not reveal substantial cardiometabolic associations, despite a small effect on glycated hemoglobin A1c. Since no homozygous LoS nor pLoF variants were identified and heterozygous carriers had similar cardiometabolic phenotype as noncarriers, we conclude that GLP-1R may be of particular importance in human physiology, due to a potential evolutionary intolerance of harmful homozygous GLP1R variants.

5-HT7 Receptor Is Involved in Electroacupuncture Inhibition of Chronic Pain in the Spinal Cord.

Knee osteoarthritis (KOA) is a common and disabling condition characterized by attacks of pain around the joints, and it is a typical disease that develops chronic pain. Previous studies have proved that 5-HT1, 5-HT2, and 5-HT3 receptors in the spinal cord are involved in electroacupuncture (EA) analgesia. The 5-HT7 receptor plays antinociceptive role in the spinal cord. However, it is unclear whether the 5-HT7 receptor is involved in EA analgesia. The 5-HT7 receptor is a stimulatory G-protein (Gs)-coupled receptor that activates adenylyl cyclase (AC) to stimulate cyclic adenosine monophosphate (cAMP) formation, which in turn activates protein kinase A (PKA). In the present study, we found that EA significantly increased the tactile threshold and the expression of the 5-HT7 receptor in the dorsal spinal cord. Intrathecal injection of 5-HT7 receptor agonist AS-19 mimicked the analgesic effect of EA, while a selective 5-HT7 receptor antagonist reversed this effect. Moreover, intrathecal injection of AC and PKA antagonists prior to EA intervention prevented its anti-allodynic effect. In addition, GABAA receptor antagonist bicuculline administered (intrathecal, i.t.) prior to EA intervention blocked the EA effect on pain hypersensitivity. Our data suggest that the spinal 5-HT7 receptor activates GABAergic neurons through the Gs–cAMP–PKA pathway and participates in EA-mediated inhibition of chronic pain in a mouse model of KOA.

Chronic morphine induces cyclic adenosine monophosphate formation and hyperpolarization-activated cyclic nucleotide-gated channel expression in the spinal cord of mice

Chronic morphine exposure persistently activates Gαi/o protein-coupled receptors and enhances adenylyl cyclase (AC) activity, which can increase cyclic adenosine monophosphate (cAMP) production. Direct binding of cAMP to the cytoplasmic site on hyperpolarization-activated cyclic nucleotide-gated (HCN) channels increases the probability of channel opening. HCN channels play a prominent role in chronic pain the disease that shares some common mechanisms with opioid tolerance. This compensatory AC activation may be responsible for the induction of morphine-induced analgesic tolerance. We investigated spinal cAMP formation and expression of HCN2 in the spinal cord, and observed the effect of AC inhibition on the induction of morphine analgesic tolerance. We found that chronic morphine-induced antinociceptive tolerance increased spinal cAMP formation and the expression of spinal HCN2. Inhibition of spinal AC partially blocked chronic morphine-induced cAMP formation and prevented the induction of morphine-induced analgesic tolerance. Inhibition of HCN2 also showed a partial preventive effect on morphine-induced tolerance, hypothermia tolerance and also the right-shift of the dose-response curve. We conclude that repeated morphine treatment increases AC activity and cAMP formation, and also spinal HCN2 expression, blockade of AC or HCN2 can prevent the development of morphine-induced analgesic tolerance.

Molecular targets for endogenous glial cell line-derived neurotrophic factor modulation in striatal parvalbumin interneurons

Administration of recombinant glial cell line-derived neurotrophic factor into the putamen has been tested in preclinical and clinical studies to evaluate its neuroprotective effects on the progressive dopaminergic neuronal degeneration that characterizes Parkinson’s disease. However, intracerebral glial cell line-derived neurotrophic factor infusion is a challenging therapeutic strategy, with numerous potential technical and medical limitations. Most of these limitations could be avoided if the production of endogenous glial cell line-derived neurotrophic factor could be increased. Glial cell line-derived neurotrophic factor is naturally produced in the striatum from where it exerts a trophic action on the nigrostriatal dopaminergic pathway. Most of striatal glial cell line-derived neurotrophic factor is synthesized by a subset of GABAergic interneurons characterized by the expression of parvalbumin. We sought to identify molecular targets specific to those neurons and which are putatively associated with glial cell line-derived neurotrophic factor synthesis. To this end, the transcriptomic differences between glial cell line-derived neurotrophic factor-positive parvalbumin neurons in the striatum and parvalbumin neurons located in the nearby cortex, which do not express glial cell line-derived neurotrophic factor, were analysed. Using mouse reporter models, we have defined the genomic signature of striatal parvalbumin interneurons obtained by fluorescence-activated cell sorting followed by microarray comparison. Short-listed genes were validated by additional histological and molecular analyses. These genes code for membrane receptors (Kit, Gpr83, Tacr1, Tacr3, Mc3r), cytosolic proteins (Pde3a, Crabp1, Rarres2, Moxd1) and a transcription factor (Lhx8). We also found the proto-oncogene cKit to be highly specific of parvalbumin interneurons in the non-human primate striatum, thus highlighting a conserved expression between species and suggesting that specific genes identified in mouse parvalbumin neurons could be putative targets in the human brain. Pharmacological stimulation of four G-protein-coupled receptors enriched in the striatal parvalbumin interneurons inhibited Gdnf expression presumably by decreasing cyclic adenosine monophosphate formation. Additional experiments with pharmacological modulators of adenylyl cyclase and protein kinase A indicated that this pathway is a relevant intracellular route to induce Gdnf gene activation. This preclinical study is an important step in the ongoing development of a specific pro-endo-glial cell line-derived neurotrophic factor pharmacological strategy to treat Parkinson’s disease.

Calcilytic NPS2143 promotes proliferation and inhibits apoptosis of spontaneously hypertensive rat vascular smooth muscle cells via activation of the renin-angiotensin system.

Vascular smooth muscle cell (VSMC) proliferation and apoptosis and the renin-angiotensin system (RAS) play critical roles in the development of essential hypertension. The activation of calcium-sensing receptor (CaSR), functionally expressed in VSMCs, inhibits cyclic adenosine monophosphate (cAMP) formation by elevating intracellular calcium ([Ca2+]i) and then suppressing renin release. The present study aimed to investigate the effects of NPS2143-mediated inhibition of CaSR on VSMC proliferation and apoptosis in spontaneously hypertensive rat (SHR) VSMCs and to assess whether these effects were mediated by alterations to RAS signaling. Primary VSMCs were isolated from the aortas of SHRs and Wistar-Kyoto rats. SHR VSMCs were treated with CaSR antagonist NPS2143 and cell proliferation and CaSR and RAS-related protein expression levels were measured to assess the effect. The results indicated that NPS2143 treatment promoted SHR VSMC proliferation, lower CaSR expression levels and higher RAS-related proteins levels when compared with control treatment. Additional measurement of the expression levels of proteins related to proliferation, remodeling, apoptosis and RAS related proteins, as well as cell viability, cell cycle, cell apoptosis ratio, [Ca2+]i, and the concentration of cAMP was performed after treatment with NPS2143, PLC inhibitor U73122, IP3 receptor antagonist 2-aminoethoxydiphenylborane (APB), adenylyl cyclase-V inhibitor MDL12330A, angiotensin converting enzyme inhibitor captopril, angiotensin I receptor (AT1R) inhibitor losartan, NPS2143 + U73122, NPS2143 + 2-APB, NPS2143 + MDL12330A, NPS2143 + captopril and NPS2143 + losartan. The results suggested that NPS2143 promoted cell proliferation, inhibited cell apoptosis, decreased [Ca2+]i and increased the expression of RAS compared with control treatments. NPS2143 + U73122 and NPS2143 + 2-APB enhanced the effects of NPS2143, while NPS2143 + MDL12330A, NPS2143 + captopril, NPS2143 + losartan attenuated the effected of NPS2143 in SHR VSMCs. Furthermore, the knockdown of AT1R by AT1R-short hairpin RNA also attenuated the effects of NPS2143 compared with NPS2143 alone. Collectively, these data indicated that NPS2143 promoted proliferation and inhibited apoptosis of VSMCs in SHRs, the effect of which was achieved by activation of RAS signaling.

Drug Interaction of Aminophylline and Salbutamol Induced extreme Tachycardia in Chronic Obstructive Pulmonary Diseases -A Case Report

Aminophylline and salbutamol is a more often used drugs as bronchodilators in COPD and asthma worldwide, because it is inexpensive and widely available. Aminophylline (1, 3-dimethyl-7H-purine-2, 6-dione; ethane-1, 2-diamine) causes bronchodilatation, cardiac stimulation and vasodilatation by increasing cyclic adenosine monophosphate (cAMP) via the inhibition of phosphodiesterase (PDE). Salbutamol (4-[2-(tert-butylamino)-1 hydroxyethyl]-2-(hydroxymethyl) phenol) causes broncho-dilatation through beta-2 receptor stimulation thereby increasing cyclic adenosine monophosphate (cAMP) formation in bronchial muscle cell. Apart from bronchodilation activity salbutamol also produce vasodilatation, uterine relaxation and lesser cardiac stimulation. Patients receiving salbutamol and aminophylline should be monitored periodically for potential drug interactions, adverse effects, toxicity and other drug related problems. Symptoms of potential interaction between salbutamol and aminophylline is difficult to identify and may becomes increased risk for stroke, sudden cardiac arrest and death. Such potential drug interaction can be identified and minimized by performing therapeutic drug monitoring. We report a case of interaction of salbutamol and aminophylline induced extreme tachycardia in a 45 year old female patient of chronic obstructive pulmonary diseases (COPD) with acute exacerbations. Patient's ECG interpretation was abnormal with elevated vital signs (i.e. Heart rate). Both the sympathomimetic and the methylxanthine compounds frequently used in the management of COPD have been identified as being potentially arrhythmogenic. Although there is good evidence that medications from these two groups have additive bronchodilator effects, few studies have looked at a possible arrhythmogenic interaction between them in COPD. Hence closely monitoring the drug therapy prevent such type of potential interactions.

A New Highly Thyrotropin Receptor-Selective Small-Molecule Antagonist with Potential for the Treatment of Graves' Orbitopathy.

The thyrotropin receptor (TSHR) is the target for autoimmune thyroid stimulating antibodies (TSAb) triggering hyperthyroidism. Whereas elevated thyroid hormone synthesis by the thyroid in Graves' disease can be treated by antithyroid agents, for the pathogenic activation of TSHR in retro-orbital fibroblasts of the eye, leading to Graves' orbitopathy (GO), no causal TSHR directed therapy is available. Due to the therapeutic gap for severe GO, TSHR inhibitors were identified by high-throughput screening in Chinese hamster ovary cells expressing the TSHR. Stereo-selective synthesis of the screening hits led to the molecule S37, which contains seven chiral centers. Enantiomeric separation of the molecule S37 resulted in the enantiopure molecule S37a-a micro-molar antagonist of thyrotropin-induced cyclic adenosine monophosphate accumulation in HEK 293 cells expressing the TSHR. The unique rigid bent shape of molecule S37a may mediate the observed high TSHR selectivity. Most importantly, the closely related follitropin and lutropin receptors were not affected by this compound. S37a not only inhibits the TSHR activation by thyrotropin itself but also activation by monoclonal TSAb M22 (human), KSAb1 (murine), and the allosteric small-molecule agonist C2. Disease-related ex vivo studies in HEK 293 cells expressing the TSHR showed that S37a also inhibits cyclic adenosine monophosphate formation by oligoclonal TSAb, which are highly enriched in GO patients' sera. Initial in vivo pharmacokinetic studies revealed no toxicity of S37a and a remarkable 53% oral bioavailability in mice. In summary, a novel highly selective inhibitor for the TSHR is presented, which has promising potential for further development for the treatment of GO.

Calcimimetic R568 reduced the blood pressure and improved aortic remodeling in spontaneously hypertensive rats by inhibiting local renin-angiotensin system activity.

Hypertension is a cardiovascular disease that seriously affects human health. Activation of the calcium-sensing receptor (CaSR) inhibits cyclic adenosine monophosphate (cAMP) formation by increasing [Ca2+]i and subsequently inhibiting renin release. The renin-angiotensin system (RAS) plays an important role in the development of essential hypertension (EH). The purpose of this study was to determine the effects of NPSR568 (R568)-activated CaSR on blood pressure (BP), proliferation, and remodeling of vascular smooth muscle cells, and the activity of the RAS in spontaneously hypertensive rats (SHRs). In this study, we treated SHR and Wistar-Kyoto rats with R568 for 8 weeks. The tail-cuff method was used to assess rat BP weekly. Morphological changes in the thoracic aorta were evaluated with hematoxylin-eosin and Masson staining. Western blotting and immunohistochemistry were used to detect the expression of RAS-related proteins and proliferative remodeling proteins in the thoracic aorta. An enzyme-linked immunosorbent assay was used to detect the content of cAMP, the RAS, and the CaSR in plasma and the thoracic aorta. Finally, we found that treatment with R568 for 8 weeks reduced the BP and inhibited arterial vascular proliferation remodeling in SHRs. R568 administration significantly suppressed the activity of local RAS in the thoracic aortas of SHRs. Moreover, R568 treatment reversed the low expression of CaSR in SHRs. R568 may serve as an effective strategy against EH.

Abstract 447: The Role of Extracellular cAMP in the Pathogenesis of Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) is an uncommon, progressive, life-threatening and often fatal disease. Despite advances in PAH therapy, there is no cure for PAH, and new therapies need to be developed. Activation of G-protein-coupled receptors stimulates adenylyl cyclase, leading in vascular smooth muscle cells to an increase in intracellular cyclic adenosine monophosphate (cAMP) formation and reduced cell proliferation in vitro and in vivo . Yet, the last two decades of research have shown that there is more to the role of cAMP than ever expected from this molecule. Stimulated cells transport cAMP outside the cells, a process that is mediated by the multidrug resistance-associated proteins (MRPs). Herein, we investigated the role of extracellular cAMP in the lung and asked whether it may act on pulmonary vascular remodeling. By employing a fluorescence resonance energy transfer (FRET)-based sensor, we found that extracellular cAMP activates intracellular cAMP formation in human pulmonary artery smooth muscle cells (hPASMC) and endothelial cells (hPAEC). Extracellular cAMP, via binding of its metabolite adenosine to the type A2 receptor, reduced hPASMC and hPAEC proliferation and migration by controlling the PKA/CREB pathway. To test for a role of extracellular cAMP in the pulmonary vasculature, we used rat-monocrotaline and mouse-hypoxia as in vivo models of PAH. Rats treated with monocrotaline developed PAH with increased pulmonary artery pressures (PAP) and right ventricular (RV) hypertrophy. Extracellular cAMP infusion significantly prevented and reversed these structural changes. Extracellular-cAMP-treated rats displayed lower RV systolic pressure and Fulton index, as well as decreased PAP. In line with these observations, we found infused cAMP to potently repress RV hypertrophy, RV systolic pressure, perivascular lung fibrosis and pulmonary arteries remodeling in mice exposed to Sugen5416 and chronic hypoxia. Together, our results assign extracellular cAMP a potent regulatory role in pulmonary vascular cells, and suggest targeting the extracellular cAMP signaling pathway to limit pulmonary vascular remodeling and PAH.

Crystallization and preliminary X-ray diffraction study of recombinant adenine phosphoribosyltransferase from the thermophilic bacterium Thermus thermophilus strain HB27

Adenine phosphoribosyltransferase (APRT) belongs to the type I phosphoribosyltransferase family and catalyzes the formation of adenosine monophosphate via transfer of the 5-phosphoribosyl group from phosphoribosyl pyrophosphate to the nitrogen atom N9 of the adenine base. Proteins of this family are involved in a salvage pathway of nucleotide synthesis, thus providing purine base utilization and maintaining the optimal level of purine bases in the body. Adenine phosphoribosyltransferase from the extremely thermophilic Thermus thermophilus strain HB27 was produced using a highly efficient E. coli producer strain and was then purified by affinity and gel-filtration chromatography. This enzyme was successfully employed as a catalyst for the cascade biosynthesis of biologically important nucleotides. The screening of crystallization conditions for recombinant APRT from T. thermophilus HB27 was performed in order to determine the enzyme structure by X-ray diffraction. The crystallization conditions, which were found by the vapor-diffusion technique, were then optimized to apply the counter-diffusion technique. The crystals of the enzyme were grown by the capillary counter-diffusion method. The crystals belong to sp. gr. P1211 and have the following unitcell parameters: a = 69.86 A, b = 82.16 A, c = 91.39 A, α = γ = 90°, β = 102.58°. The X-ray diffraction data set suitable for the determination of the APRT structure at 2.6 A resolution was collected from the crystals at the SPring-8 synchrotron facility (Japan).

Usefulness for the combination of G-protein- and β-arrestin-biased ligands of μ-opioid receptors: Prevention of antinociceptive tolerance.

Backgroundµ-Opioid receptor internalization is considered to be critically linked to antinociceptive tolerance. Although µ-opioid receptor agonists have been administered simultaneously with other drugs to control pain, little information is available regarding opioid–opioid interactions. Therefore, the present study was designed to further investigate the utility of a new G protein-biased ligand for µ-opioid receptors, TRV130, which has an antinociceptive effect without β-arrestin-dependent µ-opioid receptor internalization, and its combination with fentanyl using µ-opioid receptor-expressing cells and mice.ResultsIn the present study, we confirmed that fentanyl produced a profound increase in β-arrestin-2 recruitment accompanied by µ-opioid receptor internalization, whereas TRV130 did not induce either the recruitment of β-arrestin-2 or µ-opioid receptor internalization in µ-opioid receptor-expressing cells. Under these conditions, β-arrestin-2 recruitment accompanied by µ-opioid receptor internalization induced by fentanyl was abolished by TRV130, whereas TRV130 did not alter the reduction of cyclic adenosine monophosphate formation by fentanyl in µ-opioid receptor-expressing cells. In a behavioral assay, TRV130 exerted an antinociceptive effect in a hot-plate test in mice. In a combination test, the antinociceptive effect of TRV130 was synergistically increased by fentanyl. Fentanyl induced antihyperalgesia and development of its tolerance under a neuropathic pain-like state following sciatic nerve ligation. However, treatment of mice with an antinociceptive dose of TRV130 did not induce the rapid development of tolerance to its antihyperalgesic effect under a neuropathic pain-like state. Furthermore, the rapid development of tolerance to the antihyperalgesic effect induced by fentanyl plus TRV130 in mice with sciatic nerve ligation was not observed, unlike in the case of fentanyl alone.ConclusionsThese findings provide evidence that activation of the G protein-biased pathway through µ-opioid receptors can alter signaling in the β-arrestin-2 pathway linked to the stimulation of µ-opioid receptors. Furthermore, the combination of G protein-biased and β-arrestin-biased ligands of µ-opioid receptors exerts an ideal antinociceptive effect without the rapid development of antinociceptive tolerance.

Adenylyl Cyclase 6 Enhances NKCC2 Expression and Mediates Vasopressin-Induced Phosphorylation of NKCC2 and NCC

Arginine vasopressin (AVP) affects kidney function via vasopressin V2 receptors that are linked to activation of adenylyl cyclase (AC) and an increase in cyclic adenosine monophosphate formation. AVP/cyclic adenosine monophosphate enhance the phosphorylation of the Na-K-2Cl cotransporter (NKCC2) at serine residue 126 (pS126 NKCC2) and of the Na-Cl cotransporter (NCC) at threonine 58 (pT58 NCC). The isoform(s) of AC involved in these responses, however, were unknown. Phosphorylation of S126 NKCC2 and T58 NCC, induced by the V2 receptor agonist (1-desamino-8-D-arginine vasopressin) in wild-type mice, is lacking in knockout mice for AC isoform 6 (AC6). With regard to NKCC2 phosphorylation, the stimulatory effect of 1-desamino-8-D-AVP and the defect in AC6(-/-) mice seem to be restricted to the medullary portion of the thick ascending limb. AC6 is also a stimulator of total renal NKCC2 protein abundance in medullary and cortical thick ascending limb. Consequently, mice lacking AC6 have lower NKCC2 expression and a mild Bartter syndrome-like phenotype, including lower plasma concentrations of K+ and H+ and compensatory upregulation of NCC. Increased AC6-independent phosphorylation of NKCC2 at S126 might help to stabilize NKCC2 activity in the absence of AC6. Renal AC6 determines total NKCC2 expression and mediates vasopressin-induced NKCC2/NCC phosphorylation. These regulatory mechanisms, which are defective in AC knockout mice, are likely responsible for the observed mild Bartter syndrome.

Administration of exogenous adenosine triphosphate to ischemic skeletal muscle induces an energy-sparing effect: Role of adenosine receptors

BackgroundIschemia–reperfusion injury is a devastating complication that occurs in allotransplantation and replantation of limbs. Over the years, several preservation strategies have been used to conserve the critical levels of intracellular adenosine triphosphate (ATP) during ischemia to sustain the ion gradients across the membranes and thus the tissue viability. The administration of exogenous ATP to ischemic tissues is known to provide beneficial effects during reperfusion, but it is unclear whether it provides protection during ischemia. The purpose of the present study was to determine the effect of ATP administration on high-energy phosphate levels in ischemic skeletal muscle and to examine the role of purinergic and adenosine receptors in mediating the response to exogenous ATP. MethodsThe extensor digitorum longus muscles of Fischer rats were subjected to ischemia and treated with different concentrations of ATP with or without purinergic and adenosine receptor blockers. Phosphorus-31 nuclear magnetic resonance spectroscopy was used to measure the rate of decay of ATP, phosphocreatine (PCr), and the formation of adenosine monophosphate and acidification. Phosphorylated compounds were analyzed using a simple model of energy metabolism, and the PCr half-life was used as an index of internal depletion of ATP to distinguish between intracellular and extracellular ATP. ResultsPCr decay was rapid in all muscle groups and was followed by gradual ATP decay. The half-life of PCr was significantly longer in the ATP-treated muscles than in the vehicle controls and was maximally prolonged by treating with slow hydrolyzing adenosine 5′-O-(3-thio)triphosphate. Purinoceptor (P2X) blockade with ATP treatment significantly increased the half-life of PCr, and adenosine receptor blockers blunted the response. Administration of adenosine to ischemic muscles significantly increased the half-life of PCr compared with that in the vehicle controls. ConclusionsExogenous ATP administration to ischemic skeletal muscles appears to spare intracellular energy by acting primarily through adenosine receptors.

Effect of paricalcitol and GcMAF on angiogenesis and human peripheral blood mononuclear cell proliferation and signaling

In addition to its role in calcium homeostasis and bone mineralization, vitamin D is involved in immune defence, cardiovascular function, inflammation and angiogenesis, and these pleiotropic effects are of interested in the treatment of chronic kidney disease. Here we investigated the effects of paricalcitol, a nonhypercalcemic vitamin D analogue, on human peripheral blood mononuclear cell proliferation and signaling, and on angiogenesis. These effects were compared with those of a known inhibitor of angiogenesis pertaining to the vitamin D axis, the vitamin D-binding protein-derived Gc-macrophage activating factor (GcMAF). Since the effects of vitamin D receptor agonists are associated with polymorphisms of the gene coding for the receptor, we measured the effects of both compounds on mononuclear cells harvested from subjects harboring different BsmI polymorphisms. Paricalcitol inhibited mononuclear cell viability with the bb genotype showing the highest effect. GcMAF, on the contrary, stimulated cell proliferation, with the bb genotype showing the highest stimulatory effect. Both compounds stimulated 3'-5'-cyclic adenosine monophosphate formation in mononuclear cells with the highest effect on the bb genotype. Paricalcitol and GcMAF inhibited the angiogenesis induced by proinflammatory prostaglandin E1. Polymorphisms of the vitamin D receptor gene, known to be associated with the highest responses to vitamin D receptor agonists, are also associated with the highest responses to GcMAF. These results highlight the role of the vitamin D axis in chronic kidney disease, an axis which includes vitamin D, its receptor and vitamin D-binding protein-derived GcMAF.

Functional characterization of novel loss-of-function mutations in the vasopressin type 2 receptor gene causing nephrogenic diabetes insipidus

X-linked nephrogenic diabetes insipidus (NDI) is a rare polyuric disorder caused by inactivating mutations in the arginine vasopressin receptor Type 2 (AVPR2) gene. NDI patients from six unrelated families were subjected to mutational analysis of the AVPR2 gene. In-depth in vitro characterization of novel AVPR2 mutants by a combination of functional and immunological techniques provided further insight into molecular mechanisms causing receptor dysfunction. Mutational analysis revealed four novel (A89P, G107R, Q174R, W208X) and three recurrent (V277A, R337X, ΔR247-G250) mutations within the AVPR2 gene. One family carried the missense mutation R337X and a 12-bp deletion (ΔR247-G250), corresponding to a fragment in the third intracellular loop (ICL3), which was not genetically linked to R337X. The functionally tested missense mutations A89P, G107R and Q174R led to reduced receptor cell surface expression in transfected COS-7 cells, most probably due to misfolding and intracellular retention, and consequently to reduction or loss of agonist-mediated cyclic adenosine monophosphate formation. Deletion of R247-G250 had no effect on receptor function in vitro. Comparison with other mammalian AVPR2 orthologs showed that this part of the ICL3 is structurally not conserved and, therefore, less relevant for receptor function. In contrast, all missense mutations (A89P, G107R, Q174R, V277A) affect receptor positions that were fully preserved during mammalian evolution. Our results provide valuable information about residues critical for AVPR2 folding, trafficking and function and proof that these mutations are responsible for causing NDI in the affected subjects.

Role of histamine H4 receptor in breast cancer cell proliferation

In order to better understand the role of histamine H4 (H4R) receptor in breast cancer, we studied the receptor expression pattern, associated signal transduction pathway and biological responses, in breast cancer cell lines with different malignant characteristics. A different pattern of protein expression was observed in MDA-MB-231 compared to MCF-7 cells determined by western blot, exhibiting the presence of a diverse range of molecular weight species of the H4R. H4R agonist reduced cyclic adenosine monophosphate (cAMP) formation induced by forskolin only in MCF-7 cells. In MDA-MB-231 cells, H4R agonists significantly decreased cell proliferation, augmented the Annexin-V and TdT-mediated UTP-biotin Nick End labelling (TUNEL) positive cells and produced a 2.5-fold increase in cell senescence. In MCF-7 cells, H4R agonists inhibited proliferation by 50%, increasing the exponential doubling time. This effect was associated to an augment in Annexin-V and TUNEL positive cells, and a 2-fold increase in cell senescence. We conclude that H4R is functionally expressed in human breast cancer cell lines, exhibiting a key role in histamine-mediated biological processes such as cell proliferation, senescence and apoptosis.

Gc protein-derived macrophage-activating factor (GcMAF) stimulates cAMP formation in human mononuclear cells and inhibits angiogenesis in chick embryo chorionallantoic membrane assay.

The effects of Gc protein-derived macrophage-activating factor (GcMAF) have been studied in cancer and other conditions where angiogenesis is deregulated. In this study, we demonstrate for the first time that the mitogenic response of human peripheral blood mononuclear cells (PBMCs) to GcMAF was associated with 3'-5'-cyclic adenosine monophosphate (cAMP) formation. The effect was dose dependent, and maximal stimulation was achieved using 0.1 ng/ml. Heparin inhibited the stimulatory effect of GcMAF on PBMCs. In addition, we demonstrate that GcMAF (1 ng/ml) inhibited prostaglandin E(1)- and human breast cancer cell-stimulated angiogenesis in chick embryo chorionallantoic membrane (CAM) assay. Finally, we tested different GcMAF preparations on CAM, and the assay proved to be a reliable, reproducible and inexpensive method to determine the relative potencies of different preparations and their stability; we observed that storage at room temperature for 15 days decreased GcMAF potency by about 50%. These data could prove useful for upcoming clinical trials on GcMAF.

Role of Transforming Growth Factor Beta in the Regulation of Thyroid Function and Growth

Transforming growth factor beta (TGF-beta) exists in nature as three isoforms. They exert their effects by binding to a type II receptor located at the cell membrane. The TGF-beta-type II receptor complex then recruits type I receptor, and this new complex stimulates the phosphorylation of Smads 2 and 3, which are subsequently transferred to the nucleus, where they regulate gene transcription. The thyroid gland expresses the TGF-beta1 gene mRNA and synthesizes the protein, which under physiologic conditions regulates thyroid growth and function. Different studies have demonstrated that TGF-beta1 inhibits cell proliferation and a number of functional parameters. These include cyclic adenosine monophosphate (AMP) formation, iodine uptake and organification, hormone secretion, and the expression of thyroglobulin, thyroid peroxidase, and Na(+)/I(-) symporter. The expression of the TGF-beta1 gene and protein may be stimulated by iodine under normal conditions. Since TGF-beta1 mimics some of the inhibitory actions of iodine, its participation in thyroid autoregulation has been proposed; however, this concept is still debated. In thyroid tumors, the inhibitory action of TGF-beta1 on cell proliferation is progressively lost as the tumor becomes more undifferentiated. The alterations in the signaling pathway of TGF-beta1 are not the same in tumors from different species. Even within the same species, such as the pig thyroid, the results may be different depending on whether monolayers or follicular suspensions are employed. The data suggest that it is not entirely possible to apply the results obtained in animal studies to normal or pathological human thyroid tissue. More studies are required to provide the information needed to develop treatments, based on targeting the signaling pathway of TGF-beta1, for undifferentiated thyroid cancer and other thyroid diseases.

Cyclic AMP in Rat Ileum: Evidence for the Presence of an Extracellular Cyclic AMP-Adenosine Pathway

Extracellular adenosine plays a relevant role in regulating intestinal motility and preventing inflammatory processes. Adenosine 3',5'-cyclic monophosphate (cAMP) extruded from cells may be converted to adenosine monophosphate and then to adenosine by ecto-phosphodiesterase and CD73/ecto-5'nucleotidase, respectively, thus representing a source of adenosine. Our purpose was to assess the existence of a functional extracellular cAMP-adenosine pathway in intestinal tissue, obtaining evidence for CD73 expression and evaluating the effect of cAMP on ileum motility. The formation of cAMP metabolites in rat ileum strips incubated with exogenous cAMP or [(3)H]cAMP was monitored by high-performance liquid chromatography. CD73 was detected by immunoprecipitation and immunofluorescence. The functional activity of exogenous cAMP on ileum strips was recorded by measuring tension changes. In ileum strips, the generation of cAMP-derived adenosine monophosphate, adenosine, and inosine was time and concentration dependent and was blocked by phosphodiesterase or CD73 inhibitors in a manner consistent with exogenous cAMP being processed through the extracellular cAMP-adenosine pathway. Accordingly, [(3)H]cAMP uptake in ileum strips was negligible. Immunofluorescence revealed CD73 surface expression on intestinal smooth muscle cells and intact smooth muscle. Exogenous cAMP concentration-dependently increased ileum muscle tension partially inhibited by adenosine inactivation or receptor blockade. Forskolin-stimulated endogenous cAMP induced concentration-dependent ileum relaxations. A functioning extracellular cAMP-adenosine pathway featuring CD73 expression is present in rat ileum and affects intestinal motility. Extracellular cAMP may therefore act on intestinal muscle both directly by binding to specific smooth muscle cell membrane sites and indirectly through its degradation products.

Adenosine and its receptors

5′NT= : 5′-nucleotidase; AC= : adenylate cyclase; ACh= : acetylcholine; ADK= : adenosine kinase; AMP= : adenosine monophosphate; ATP= : adenosine triphosphate; CCL= : chemokine ligand; CGRP= : calcitonin gene related peptide; COX-2= : cyclooxygenase-2; EctoN= : ectonucleotidases; ENT= : equilibrative nucleoside transporters; ERK1,2= : extracellular signal regulated kinase 1,2; G= : guanine nucleotide binding protein; GABA= : γ-aminobutyric acid; GIRK= : G-protein gated inward rectifying potassium channels; IL= : interleukin; NGF= : nerve growth factor; NMDA= : N -methyl-d-aspartate; NO= : nitric oxide; PD= : Parkinson disease; PLA2= : phospholipase A2; PLC= : phospholipase C; PLD= : phospholipase D; SNc= : substantia nigra pars compacta; VLPO= : ventrolateral preoptic area. Adenosine is a ubiquitous chemical messenger that modulates cellular activity in both the CNS and peripheral organs.1–5 Adenosine levels in the brain extracellular space increase dramatically during metabolically stressful conditions, such as ischemia, seizures, or trauma. Adenosine, acting via different receptors, modulates excitability in the CNS2–4 and has a role in mechanisms of seizure susceptibility,5,6 sleep induction,7 basal ganglia function,8 pain perception,9 cerebral blood flow,10 and respiration.11 Adenosine receptors are inhibited by caffeine and other methylxanthines and provide a potential target for treatment of cerebral ischemia,12 seizures,13 pain,9 Parkinson disease (PD),8,14 and Huntington disease.15 There are several excellent reviews on recent experimental findings and insights into adenosine signaling in the CNS,1–6,8 and on adenosine as a potential therapeutic target in neurologic disorders.5–7,12–15 There is tight and dynamic regulation of adenosine levels in the nervous system.1–5 All cell types contribute to the accumulation of extracellular adenosine (figure). Extracellular concentrations of adenosine are determined by the interplay between the activity of intracellular and extracellular enzymes involved in adenosine metabolism and the transport of adenosine across the cell membrane. Metabolic stress triggers the dephosphorylation of adenosine triphosphate (ATP) and formation of adenosine monophosphate (AMP), which is converted to adenosine by 5′-nucleotidase. Adenosine is then released from cells by facilitated diffusion via nucleoside transporters, the best characterized being the equilibrative nucleoside transporters (ENT).16 Adenosine may also be produced by the extracellular metabolism of ATP via a cascade of ectonucleases.2–4 The primary route of adenosine metabolism is adenosine kinase (ADK), which is expressed primarily in astrocytes (figure). The concentration of extracellular adenosine increases dramatically in the setting of metabolic stress such as hypoxia, ischemia, trauma, or seizures.2–5,12 Transport from the astrocyte is the primary source of …