Blockage of the adenosine A2B receptor prevents cardiac fibroblasts overgrowth in rats with pulmonary arterial hypertension.

Sustained pressure overload and fibrosis of the right ventricle (RV) are the leading causes of mortality in pulmonary arterial hypertension (PAH). Although the role of adenosine in PAH has been attributed to the control of pulmonary vascular tone, cardiac reserve, and inflammatory processes, the involvement of the nucleoside in RV remodelling remains poorly understood. Conflicting results exist on targeting the low-affinity adenosine A2B receptor (A2BAR) for the treatment of PAH mostly because it displays dual roles in acute vs. chronic lung diseases. Herein, we investigated the role of the A2BAR in the viability/proliferation and collagen production by cardiac fibroblasts (CFs) isolated from RVs of rats with monocrotaline (MCT)-induced PAH. CFs from MCT-treated rats display higher cell viability/proliferation capacity and overexpress A2BAR compared to the cells from healthy littermates. The enzymatically stable adenosine analogue, 5'-N-ethylcarboxamidoadenosine (NECA, 1-30 µM), concentration-dependently increased growth, and type I collagen production by CFs originated from control and PAH rats, but its effects were more prominent in cells from rats with PAH. Blockage of the A2BAR with PSB603 (100 nM), but not of the A2AAR with SCH442416 (100 nM), attenuated the proliferative effect of NECA in CFs from PAH rats. The A2AAR agonist, CGS21680 (3 and 10 nM), was virtually devoid of effect. Overall, data suggest that adenosine signalling via A2BAR may contribute to RV overgrowth secondary to PAH. Therefore, blockage of the A2AAR may be a valuable therapeutic alternative to mitigate cardiac remodelling and prevent right heart failure in PAH patients.

2,4,5-Triaminopyrimidines as blue fluorescent probes for cell viability monitoring: synthesis, photophysical properties, and microscopy applications.

Monitoring cell viability is critical in cell biology, pathology, and drug discovery. Most cell viability assays are cell-destructive, time-consuming, expensive, and/or hazardous. Herein, we present a series of newly synthesized 2,4,5-triaminopyrimidine derivatives able to discriminate between live and dead cells. To our knowledge, these compounds are the first fluorescent nucleobase analogues (FNAs) with cell viability monitoring potential. These new fluorescent molecules are synthesized using highly efficient and cost-effective methods and feature unprecedented photophysical properties (longer absorption and emission wavelengths, environment-sensitive emission, and unprecedented brightness within FNAs). Using a live-dead Saccharomyces cerevisiae cell and theoretical assays, the fluorescent 2,4,5-triaminopyrimidine derivatives were found to specifically accumulate inside dead cells by interacting with dsDNA grooves, thus paving the way for the emergence of novel and safe fluorescent cell viability markers emitting in the blue region. As the majority of commercially available viability dyes emit in the green to red region of the visible spectrum, these novel markers might be useful to meet the needs of blue markers for co-staining combinations.

Adenosinergic System and BDNF Signaling Changes as a Cross-Sectional Feature of RTT: Characterization of Mecp2 Heterozygous Mouse Females.

Rett Syndrome is an X-linked neurodevelopmental disorder (RTT; OMIM#312750) associated to MECP2 mutations. MeCP2 dysfunction is seen as one cause for the deficiencies found in brain-derived neurotrophic factor (BDNF) signaling, since BDNF is one of the genes under MeCP2 jurisdiction. BDNF signaling is also dependent on the proper function of the adenosinergic system. Indeed, both BDNF signaling and the adenosinergic system are altered in Mecp2-null mice (Mecp2-/y), a representative model of severe manifestation of RTT. Considering that symptoms severity largely differs among RTT patients, we set out to investigate the BDNF and ADO signaling modifications in Mecp2 heterozygous female mice (Mecp2+/-) presenting a less severe phenotype. Symptomatic Mecp2+/- mice have lower BDNF levels in the cortex and hippocampus. This is accompanied by a loss of BDNF-induced facilitation of hippocampal long-term potentiation (LTP), which could be restored upon selective activation of adenosine A2A receptors (A2AR). While no differences were observed in the amount of adenosine in the cortex and hippocampus of Mecp2+/- mice compared with healthy littermates, the density of the A1R and A2AR subtype receptors was, respectively, upregulated and downregulated in the hippocampus. Data suggest that significant changes in BDNF and adenosine signaling pathways are present in an RTT model with a milder disease phenotype: Mecp2+/- female animals. These features strengthen the theory that boosting adenosinergic activity may be a valid therapeutic strategy for RTT patients, regardless of their genetic penetrance.

Counteracting Colon Cancer by Inhibiting Mitochondrial Respiration and Glycolysis with a Selective PKCδ Activator.

Metabolic reprogramming is a central hub in tumor development and progression. Therefore, several efforts have been developed to find improved therapeutic approaches targeting cancer cell metabolism. Recently, we identified the 7α-acetoxy-6ß-benzoyloxy-12-O-benzoylroyleanone (Roy-Bz) as a PKCδ-selective activator with potent anti-proliferative activity in colon cancer by stimulating a PKCδ-dependent mitochondrial apoptotic pathway. Herein, we investigated whether the antitumor activity of Roy-Bz, in colon cancer, could be related to glucose metabolism interference. The results showed that Roy-Bz decreased the mitochondrial respiration in human colon HCT116 cancer cells, by reducing electron transfer chain complexes I/III. Consistently, this effect was associated with downregulation of the mitochondrial markers cytochrome c oxidase subunit 4 (COX4), voltage-dependent anion channel (VDAC) and mitochondrial import receptor subunit TOM20 homolog (TOM20), and upregulation of synthesis of cytochrome c oxidase 2 (SCO2). Roy-Bz also dropped glycolysis, decreasing the expression of critical glycolytic markers directly implicated in glucose metabolism such as glucose transporter 1 (GLUT1), hexokinase 2 (HK2) and monocarboxylate transporter 4 (MCT4), and increasing TP53-induced glycolysis and apoptosis regulator (TIGAR) protein levels. These results were further corroborated in tumor xenografts of colon cancer. Altogether, using a PKCδ-selective activator, this work evidenced a potential dual role of PKCδ in tumor cell metabolism, resulting from the inhibition of both mitochondrial respiration and glycolysis. Additionally, it reinforces the antitumor therapeutic potential of Roy-Bz in colon cancer by targeting glucose metabolism.

Nicotinic α7 receptor-induced adenosine release from perisynaptic Schwann cells controls acetylcholine spillover from motor endplates.

Acetylcholine (ACh) spillover from motor endplates occurs after neuronal firing bursts being potentiated by cholinesterase inhibitors (e.g., neostigmine). Nicotinic α7 receptors (α7nAChR) on perisynaptic Schwann cells (PSCs) can control ACh spillover by unknown mechanisms. We hypothesized that adenosine might be the gliotransmitter underlying PSCs-nerve terminal communication. Rat isolated hemidiaphragm preparations were used to measure (1) the outflow of [3 H]ACh, (2) real-time transmitter exocytosis by video-microscopy with the FM4-64 fluorescent dye, and (3) skeletal muscle contractions during high-frequency (50 Hz) nerve stimulation bursts in the presence of a selective α7nAChR agonist, PNU 282987, or upon inhibition of cholinesterase activity with neostigmine. To confirm our prediction that α7nAChR-mediated effects require direct activation of PSCs, we used fluorescence video-microscopy in the real-time mode to measure PNU 282987-induced [Ca2+ ]i transients from Fluo-4 NW loaded PSCs in non-stimulated preparations. The α7nAChR agonist, PNU 282987, decreased nerve-evoked diaphragm tetanic contractions. PNU 282987-induced inhibition was mimicked by neostigmine and results from the reduction of ACh exocytosis measured as decreases in [3 H]ACh release and FM4-64 fluorescent dye unloading. Methyllycaconitine blockage of α7nAChR and the fluoroacetate gliotoxin both prevented inhibition of nerve-evoked ACh release and PSCs [Ca2+ ]i transients triggered by PNU 282987 and neostigmine. Adenosine deamination, inhibition of the ENT1 nucleoside outflow, and blockage of A1 receptors prevented PNU 282987-induced inhibition of transmitter release. Data suggest that α7nAChR controls tetanic-induced ACh spillover from the neuromuscular synapse by promoting adenosine outflow from PSCs via ENT1 transporters and retrograde activation of presynaptic A1 inhibitory receptors.

Impairment of adenosinergic system in Rett syndrome: Novel therapeutic target to boost BDNF signalling.

Rett syndrome (RTT; OMIM#312750) is mainly caused by mutations in the X-linked MECP2 gene (methyl-CpG-binding protein 2 gene; OMIM*300005), which leads to impairments in the brain-derived neurotrophic factor (BDNF) signalling. The boost of BDNF mediated effects would be a significant breakthrough but it has been hampered by the difficulty to administer BDNF to the central nervous system. Adenosine, an endogenous neuromodulator, may accomplish that role since through A2AR it potentiates BDNF synaptic actions in healthy animals. We thus characterized several hallmarks of the adenosinergic and BDNF signalling in RTT and explored whether A2AR activation could boost BDNF actions. For this study, the RTT animal model, the Mecp2 knockout (Mecp2-/y) (B6.129P2 (C)-Mecp2tm1.1Bird/J) mouse was used. Whenever possible, parallel data was also obtained from post-mortem brain samples from one RTT patient. Ex vivo extracellular recordings of field excitatory post-synaptic potentials in CA1 hippocampal area were performed to evaluate synaptic transmission and long-term potentiation (LTP). RT-PCR was used to assess mRNA levels and Western Blot or radioligand binding assays were performed to evaluate protein levels. Changes in cortical and hippocampal adenosine content were assessed by liquid chromatography with diode array detection (LC/DAD). Hippocampal ex vivo experiments revealed that the facilitatory actions of BDNF upon LTP is absent in Mecp2-/y mice and that TrkB full-length (TrkB-FL) receptor levels are significantly decreased. Extracts of the hippocampus and cortex of Mecp2-/y mice revealed less adenosine amount as well as less A2AR protein levels when compared to WT littermates, which may partially explain the deficits in adenosinergic tonus in these animals. Remarkably, the lack of BDNF effect on hippocampal LTP in Mecp2-/y mice was overcome by selective activation of A2AR with CGS21680. Overall, in Mecp2-/y mice there is an impairment on adenosinergic system and BDNF signalling. These findings set the stage for adenosine-based pharmacological therapeutic strategies for RTT, highlighting A2AR as a therapeutic target in this devastating pathology.

Resolving the Ionotropic P2X4 Receptor Mystery Points Towards a New Therapeutic Target for Cardiovascular Diseases.

Adenosine triphosphate (ATP) is a primordial versatile autacoid that changes its role from an intracellular energy saver to a signaling molecule once released to the extracellular milieu. Extracellular ATP and its adenosine metabolite are the main activators of the P2 and P1 purinoceptor families, respectively. Mounting evidence suggests that the ionotropic P2X4 receptor (P2X4R) plays pivotal roles in the regulation of the cardiovascular system, yet further therapeutic advances have been hampered by the lack of selective P2X4R agonists. In this review, we provide the state of the art of the P2X4R activity in the cardiovascular system. We also discuss the role of P2X4R activation in kidney and lungs vis a vis their interplay to control cardiovascular functions and dysfunctions, including putative adverse effects emerging from P2X4R activation. Gathering this information may prompt further development of selective P2X4R agonists and its translation to the clinical practice.

Tetanic Facilitation of Neuromuscular Transmission by Adenosine A2A and Muscarinic M1 Receptors is Dependent on the Uptake of Choline via High-Affinity Transporters.

BACKGROUND/AIMS: In this study, we evaluated the functional impact of facilitatory presynaptic adenosine A2A and muscarinic M1 receptors in the recovery of neuromuscular tetanic depression caused by the blockage of high-affinity choline transporter (HChT) by hemicholinium-3 (HC-3), a condition that mimics a myasthenia-like condition. METHODS: Rat diaphragm preparations were indirectly stimulated via the phrenic nerve trunk with 50-Hz frequency trains, each consisting of 500-750 supramaximal intensity pulses. The tension at the beginning (A) and at the end (B) of the tetanus was recorded and the ratio (R) B/A calculated. RESULTS: Activation of A2A and M1 receptors with CGS21680 (CGS; 2 nmol/L) and McN-A-343c (McN; 3 µmol/L) increased R values. Similar facilitatory effects were obtained with forskolin (FSK; 3 µmol/L) and phorbol 12-myristate 13-acetate (PMA; 10 µmol/L), which activate adenylate cyclase and protein kinase C respectively. HC-3 (4 µmol/L) decreased transmitter exocytosis measured by real-time videomicroscopy with the FM4-64 fluorescent dye and prevented the facilitation of neuromuscular transmission caused by CGS, McN, and FSK, with a minor effect on PMA. The acetylcholinesterase inhibitor, neostigmine (NEO; 0.5 µmol/L), also decreased transmitter exocytosis. The paradoxical neuromuscular tetanic fade caused by NEO (0.5 µmol/L) was also prevented by HC-3 (4 µmol/L) and might result from the rundown of the positive feedback mechanism operated by neuronal nicotinic receptors (blocked by hexamethonium, 120 µmol/L). CONCLUSION: Data suggest that the recovery of tetanic neuromuscular facilitation by adenosine A2A and M1 receptors is highly dependent on HChT activity and may be weakened in myasthenic patients when HChT is inoperative.

Endomyocardial Fibrosis: an Update After 70 Years.

PURPOSE OF REVIEW: This review aims at highlighting the need to better understand the pathogenesis and natural history of endomyocardial fibrosis when set against its changing endemicity and disease burden, improvements in diagnosis, and new options for clinical management. RECENT FINDINGS: Progress in imaging diagnostic techniques and availability of new targets for drug and surgical treatment of heart failure are contributing to earlier diagnosis and may lead to improvement in patient survival. Endomyocardial fibrosis was first described in Uganda by Davies more than 70 years ago (1948). Despite its poor prognosis, the etiology of this neglected tropical restrictive cardiomyopathy still remains enigmatic nowadays. Our review reflects on the journey of scientific discovery and construction of the current guiding concepts on this mysterious and fascinating condition, bringing to light the contemporary knowledge acquired over these years. Here we describe novel tools for diagnosis, give an overview of the improvement in clinical management, and finally, suggest research themes that can help improve patient outcomes focusing (whenever possible) on novel players coming into action.

Regulation of corneal noradrenaline release and topography of sympathetic innervation: Functional implications for adrenergic mechanisms in the human cornea.

Having established a main neuronal origin for noradrenaline (NA) in the cornea, we set out to study the physiologic determinants of its release and to correlate functional findings with sympathetic nerve density and overall topography. Whole corneas were obtained from 3 to 4 month-old rabbits and human donors. Study of prejunctional effects was carried out after incubation with radiolabelled NA (3H-NA). Corneas were superfused with warm aerated amine-free medium with cocaine and hydrocortisone to block subsequent neuronal and extraneuronal NA uptake. Samples were collected every 5 min. Four periods of transmural electrical stimulation were applied to assess evoked release of 3H-NA in the absence and in the presence of alpha-2 adrenoceptor antagonists. Catecholamines were extracted with alumina from the superfusate collected and quantified by high pressure liquid chromatography with electrochemical detection (HPLC-ED). Corneal nerve morphology was studied by immunofluorescence staining with monoclonal antibodies and subsequent confocal microscopy. Corneal lamellar sections were also produced (epithelium, stroma, endothelium) and endogenous NA and adrenaline (AD) were quantified by HPLC-ED. Results are means ±â€¯SEM. ANOVA and t-tests were used for statistical analysis. Ratios between enzymatic end products and their substrates were calculated. In both rabbit and human corneas, electrical stimulation increased the outflow of 3H-NA per minute and per shock. Addition of the alpha-2 adrenoceptor antagonist rauwolscine further increased the electrically-evoked overflow of 3H-NA in a concentration-dependent manner. Immunofluorescence revealed particular staining patterns for sensory and sympathetic fibres, epithelial cells and stromal keratocytes. In human corneal lamellar sections only NA was identified, particularly in the endothelium and epithelium. In the rabbit, concentration of NA was ten times that of AD. Electrically-evoked overflow reflects action potential-induced NA release by sympathetic nerves in the cornea and an alpha-2 adrenoceptor-mediated mechanism for its release is presented. Sympathetic innervation has similar functional relevance in both rabbit and human corneas.

ADP-Induced Ca2+ Signaling and Proliferation of Rat Ventricular Myofibroblasts Depend on Phospholipase C-Linked TRP Channels Activation Within Lipid Rafts.

Nucleotides released during heart injury affect myocardium electrophysiology and remodeling through P2 purinoceptors activation in cardiac myofibroblasts. ATP and UTP endorse [Ca2+ ]i accumulation and growth of DDR-2/α-SMA-expressing myofibroblasts from adult rat ventricles via P2Y4 and P2Y2 receptors activation, respectively. Ventricular myofibroblasts also express ADP-sensitive P2Y1 , P2Y12 , and P2Y13 receptors as demonstrated by immunofluorescence confocal microscopy and western blot analysis, but little information exists on ADP effects in these cells. ADP (0.003-3 mM) and its stable analogue, ADPßS (100 µM), caused fast [Ca2+ ]i transients originated from thapsigargin-sensitive internal stores, which partially declined to a plateau sustained by capacitative Ca2+ entry through transient receptor potential (TRP) channels inhibited by 2-APB (50 µM) and flufenamic acid (100 µM). Hydrophobic interactions between Gq/11 -coupled P2Y purinoceptors and TRP channels were suggested by prevention of the ADP-induced [Ca2+ ]i plateau following PIP2 depletion with LiCl (10 mM) and cholesterol removal from lipid rafts with methyl-ß-cyclodextrin (2 mM). ADP [Ca2+ ]i transients were insensitive to P2Y1 , P2Y12 , and P2Y13 receptor antagonists, MRS2179 (10µM), AR-C66096 (0.1 µM), and MRS2211 (10µM), respectively, but were attenuated by suramin and reactive blue-2 (100 µM) which also blocked P2Y4 receptors activation by UTP. Cardiac myofibroblasts growth and type I collagen production were favored upon activation of MRS2179-sensitive P2Y1 receptors with ADP or ADPßS (30 µM). In conclusion, ADP exerts a dual role on ventricular myofibroblasts: [Ca2+ ]i transients are mediated by fast-desensitizing P2Y4 receptors, whereas the pro-fibrotic effect of ADP involves the P2Y1 receptor activation. Data also show that ADP-induced capacitative Ca2+ influx depends on phospholipase C-linked TRP channels opening in lipid raft microdomains. J. Cell. Physiol. 232: 1511-1526, 2017. © 2016 Wiley Periodicals, Inc.

Inhibition of cholinergic neurotransmission by ß3-adrenoceptors depends on adenosine release and A1-receptor activation in human and rat urinary bladders.

The direct detrusor relaxant effect of ß3-adrenoceptor agonists as a primary mechanism to improve overactive bladder symptoms has been questioned. Among other targets, activation of ß3-adrenoceptors downmodulate nerve-evoked acetylcholine (ACh) release, but there is insufficient evidence for the presence of these receptors on bladder cholinergic nerve terminals. Our hypothesis is that adenosine formed from the catabolism of cyclic AMP in the detrusor may act as a retrograde messenger via prejunctional A1 receptors to explain inhibition of cholinergic activity by ß3-adrenoceptors. Isoprenaline (1 µM) decreased [3H]ACh release from stimulated (10 Hz, 200 pulses) human (-47 ± 5%) and rat (-38 ± 1%) detrusor strips. Mirabegron (0.1 µM, -53 ± 8%) and CL316,243 (1 µM, -37 ± 7%) mimicked isoprenaline (1 µM) inhibition, and their effects were prevented by blocking ß3-adrenoceptors with L748,337 (30 nM) and SR59230A (100 nM), respectively, in human and rat detrusor. Mirabegron and isoprenaline increased extracellular adenosine in the detrusor. Blockage of A1 receptors with 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 100 nM) or the equilibrative nucleoside transporters (ENT) with dipyridamole (0.5 µM) prevented mirabegron and isoprenaline inhibitory effects. Dipyridamole prevented isoprenaline-induced adenosine outflow from the rat detrusor, and this effect was mimicked by the ENT1 inhibitor, S-(4-nitrobenzyl)-6-thioinosine (NBTI, 30 µM). Cystometry recordings in anesthetized rats demonstrated that SR59230A, DPCPX, dipyridamole, and NBTI reversed the decrease in the voiding frequency caused by isoprenaline (0.1-1,000 nM). Data suggest that inhibition of cholinergic neurotransmission by ß3-adrenoceptors results from adenosine release via equilibrative nucleoside transporters and prejunctional A1-receptor stimulation in human and rat urinary bladder.

Neuromuscular paralysis by the basic phospholipase A2 subunit of crotoxin from Crotalus durissus terrificus snake venom needs its acid chaperone to concurrently inhibit acetylcholine release and produce muscle blockage.

BACKGROUND AND PURPOSE: Crotoxin (CTX), a heterodimeric phospholipase A2 (PLA2) neurotoxin from Crotalus durissus terrificus snake venom, promotes irreversible blockade of neuromuscular transmission. Indirect electrophysiological evidence suggests that CTX exerts a primary inhibitory action on transmitter exocytosis, yet contribution of a postsynaptic action of the toxin resulting from nicotinic receptor desensitization cannot be excluded. Here, we examined the blocking effect of CTX on nerve-evoked transmitter release measured directly using radioisotope neurochemistry and video microscopy with the FM4-64 fluorescent dye. EXPERIMENTAL APPROACH: Experiments were conducted using mice phrenic-diaphragm preparations. Real-time fluorescence video microscopy and liquid scintillation spectrometry techniques were used to detect transmitter exocytosis and nerve-evoked [3H]-acetylcholine ([3H]ACh) release, respectively. Nerve-evoked myographic recordings were also carried out for comparison purposes. KEY RESULTS: Both CTX (5µg/mL) and its basic PLA2 subunit (CB, 20µg/mL) had biphasic effects on nerve-evoked transmitter exocytosis characterized by a transient initial facilitation followed by a sustained decay. CTX and CB reduced nerve-evoked [3H]ACh release by 60% and 69%, respectively, but only the heterodimer, CTX, decreased the amplitude of nerve-evoked muscle twitches. CONCLUSION AND IMPLICATIONS: Data show that CTX exerts a presynaptic inhibitory action on ACh release that is highly dependent on its intrinsic PLA2 activity. Given the high safety margin of the neuromuscular transmission, one may argue that the presynaptic block caused by the toxin is not enough to produce muscle paralysis unless a concurrent postsynaptic inhibitory action is also exerted by the CTX heterodimer.

Involvement of mitochondrial proteins in calcium signaling and cell death induced by staurosporine in Neurospora crassa.

Staurosporine-induced cell death in Neurospora crassa includes a well defined sequence of alterations in cytosolic calcium levels, comprising extracellular Ca(2+) influx and mobilization of Ca(2+) from internal stores. Here, we show that cells undergoing respiratory stress due to the lack of certain components of the mitochondrial complex I (like the 51kDa and 14kDa subunits) or the Ca(2+)-binding alternative NADPH dehydrogenase NDE-1 are hypersensitive to staurosporine and incapable of setting up a proper intracellular Ca(2+) response. Cells expressing mutant forms of NUO51 that mimic human metabolic diseases also presented Ca(2+) signaling deficiencies. Accumulation of reactive oxygen species is increased in cells lacking NDE-1 and seems to be required for Ca(2+) oscillations in response to staurosporine. Measurement of the mitochondrial levels of Ca(2+) further supported the involvement of these organelles in staurosporine-induced Ca(2+) signaling. In summary, our data indicate that staurosporine-induced fungal cell death involves a sophisticated response linking Ca(2+) dynamics and bioenergetics.

Increased Urinary Adenosine Triphosphate in Patients With Bladder Outlet Obstruction Due to Benign Prostate Hyperplasia.

BACKGROUND: Diagnosis of bladder outflow obstruction (BOO) in patients with lower urinary tract (LUT) symptoms is challenging without using invasive urodynamic tests. Recently, we showed in vitro that urothelial strips from patients with benign prostatic hyperplasia (BPH) release more ATP than controls. Here, we tested whether urinary ATP can be used as a wall tension transducer non-invasive biomarker to detect BOO in patients with BPH. METHODS: 79 male patients with BOO and 22 asymptomatic controls were recruited prospectively. Patients were asked to complete the International Prostate Symptom Score (IPSS) questionnaire and to void at normal desire into a urinary flowmeter; the postvoid residual volume was determined by suprapubic ultrasonography. Urine samples from all individuals were examined for ATP, creatinine, and lactate dehydrogenase. RESULTS: BOO patients had significantly higher (P < 0.001) urinary ATP normalized by the voided volume (456 ± 36 nmol) than age-matched controls (209 ± 35 nmol). Urinary ATP amounts increased with the voided volume, but the slope of this rise was higher in BOO patients than in controls. A negative correlation was detected between urinary ATP and flow rate parameters, namely maximal flow rate (r = -0.310, P = 0.005), Siroky flow-volume normalization (r = -0.324, P = 0.004), and volume-normalized flow rate index (r = -0.320, P = 0.012). We found no correlation with LUT symptoms IPSS score. Areas under the receiver operator characteristics (ROC) curves were 0.91 (95%CI 0.86-0.96, P < 0.001) for ATP alone and 0.88 (95%CI 0.81-0.94, P < 0,001) when adjusted to urinary creatinine. CONCLUSIONS: Patients with BOO release higher amounts of ATP into the urine than the control group. The high area under the ROC curve suggests that urinary ATP can be a high-sensitive non-invasive biomarker of BOO, which may have a discriminative value of detrusor competence when comparing BPH patients with low urinary flow rates. Prostate 76:1353-1363, 2016. © 2016 Wiley Periodicals, Inc.

Activation of a TRP-like channel and intracellular Ca2+ dynamics during phospholipase-C-mediated cell death.

The model organism Neurospora crassa undergoes programmed cell death when exposed to staurosporine. Here, we show that staurosporine causes defined changes in cytosolic free Ca(2+) ([Ca(2+)]c) dynamics and a distinct Ca(2+) signature that involves Ca(2+) influx from the external medium and internal Ca(2+) stores. We investigated the molecular basis of this Ca(2+) response by using [Ca(2+)]c measurements combined with pharmacological and genetic approaches. Phospholipase C was identified as a pivotal player during cell death, because modulation of the phospholipase C signaling pathway and deletion of PLC-2, which we show to be involved in hyphal development, results in an inability to trigger the characteristic staurosporine-induced Ca(2+) signature. Using Δcch-1, Δfig-1 and Δyvc-1 mutants and a range of inhibitors, we show that extracellular Ca(2+) entry does not occur through the hitherto described high- and low-affinity Ca(2+) uptake systems, but through the opening of plasma membrane channels with properties resembling the transient receptor potential (TRP) family. Partial blockage of the response to staurosporine after inhibition of a putative inositol-1,4,5-trisphosphate (IP3) receptor suggests that Ca(2+) release from internal stores following IP3 formation combines with the extracellular Ca(2+) influx.

Up-regulation of P2X7 receptor-mediated inhibition of GABA uptake by nerve terminals of the human epileptic neocortex.

OBJECTIVE: Thirty percent of patients with epilepsy are refractory to medication. The majority of these patients have mesial temporal lobe epilepsy (MTLE). This prompts for new pharmacologic targets, like ATP-mediated signaling pathways, since the extracellular levels of the nucleotide dramatically increase during in vitro epileptic seizures. In this study, we investigated whether sodium-dependent high-affinity γ-aminobutyric acid (GABA) and glutamate uptake by isolated nerve terminals of the human neocortex could be modulated by ATP acting via slow-desensitizing P2X7 receptor (P2X7R). METHODS: Modulation of [(3) H]GABA and [(14) C]glutamate uptake by ATP, through activation of P2X7R, was investigated in isolated nerve terminals of the neocortex of cadaveric controls and patients with drug-resistant epilepsy (non-MTLE or MTLE) submitted to surgery. Tissue density and distribution of P2X7R in the human neocortex was assessed by Western blot analysis and immunofluorescence confocal microscopy. RESULTS: The P2X7R agonist, 2'(3')-O-(4-benzoylbenzoyl)ATP (BzATP, 3-100 µm) decreased [(3) H]GABA and [(14) C]glutamate uptake by nerve terminals of the neocortex of controls and patients with epilepsy. The inhibitory effect of BzATP (100 µm) was prevented by the selective P2X7R antagonist, A-438079 (3 µm). Down-modulation of [(14) C]glutamate uptake by BzATP (100 µm) was roughly similar in controls and patients with epilepsy, but the P2X7R agonist inhibited more effectively [(3) H]GABA uptake in the epileptic tissue. Neocortical nerve terminals of patients with epilepsy express higher amounts of the P2X7R protein than control samples. SIGNIFICANCE: High-frequency cortical activity during epileptic seizures releases huge amounts of ATP, which by acting on low-affinity slowly desensitizing ionotropic P2X7R, leads to down-modulation of neuronal GABA and glutamate uptake. Increased P2X7R expression in neocortical nerve terminals of patients with epilepsy may, under high-frequency firing, endure GABA signaling and increase GABAergic rundown, thereby unbalancing glutamatergic neuroexcitation. This study highlights the relevance of the ATP-sensitive P2X7R as an important negative modulator of GABA and glutamate transport and prompts for novel antiepileptic therapeutic targets.

Adenosine A2A receptor and ecto-5'-nucleotidase/CD73 are upregulated in hippocampal astrocytes of human patients with mesial temporal lobe epilepsy (MTLE).

Refractoriness to existing medications of up to 80 % of the patients with mesial temporal lobe epilepsy (MTLE) prompts for finding new antiepileptic drug targets. The adenosine A2A receptor emerges as an interesting pharmacological target since its excitatory nature partially counteracts the dominant antiepileptic role of endogenous adenosine acting via inhibitory A1 receptors. Gain of function of the excitatory A2A receptor has been implicated in a significant number of brain pathologies commonly characterized by neuronal excitotoxicity. Here, we investigated changes in the expression and cellular localization of the A2A receptor and of the adenosine-generating enzyme, ecto-5'-nucleotidase/CD73, in the hippocampus of control individuals and MTLE human patients. Western blot analysis indicates that the A2A receptor is more abundant in the hippocampus of MTLE patients compared to control individuals. Immunoreactivity against the A2A receptor predominates in astrocytes staining positively for the glial fibrillary acidic protein (GFAP). No co-localization was observed between the A2A receptor and neuronal cell markers, like synaptotagmin 1/2 (nerve terminals) and neurofilament 200 (axon fibers). Hippocampal astrogliosis observed in MTLE patients was accompanied by a proportionate increase in A2A receptor and ecto-5'-nucleotidase/CD73 immunoreactivities. Given our data, we hypothesize that selective blockade of excessive activation of astrocytic A2A receptors and/or inhibition of surplus adenosine formation by membrane-bound ecto-5'-nucleotidase/CD73 may reduce neuronal excitability, thus providing a novel therapeutic target for drug-refractory seizures in MTLE patients.

Activation of P2Y6 Receptors Facilitates Nonneuronal Adenosine Triphosphate and Acetylcholine Release from Urothelium with the Lamina Propria of Men with Bladder Outlet Obstruction.

PURPOSE: Deregulation of purinergic bladder signaling may contribute to persistent detrusor overactivity in patients with bladder outlet obstruction. Activation of uridine diphosphate sensitive P2Y6 receptors increases voiding frequency in rats indirectly by releasing adenosine triphosphate from the urothelium. To our knowledge this mechanism has never been tested in the human bladder. MATERIALS AND METHODS: We examined the role of the uridine diphosphate sensitive P2Y6 receptor on tetrodotoxin insensitive nonneuronal adenosine triphosphate and [(3)H]acetylcholine release from the human urothelium with the lamina propria of control organ donors and patients with benign prostatic hyperplasia. RESULTS: The adenosine triphosphate-to-[(3)H]acetylcholine ratio was fivefold higher in mucosal urothelium/lamina propria strips from benign prostatic hyperplasia patients than control men. The selective P2Y6 receptor agonist PSB0474 (100 nM) augmented by a similar amount adenosine triphosphate and [(3)H]acetylcholine release from mucosal urothelium/lamina propria strips from both groups of individuals. The facilitatory effect of PSB0474 was prevented by MRS2578 (50 nM) and by carbenoxolone (10 µM), which block P2Y6 receptor and pannexin-1 hemichannels, respectively. Blockade of P2X3 (and/or P2X2/3) receptors with A317491 (100 nM) also attenuated release facilitation by PSB0474 in control men but not in patients with benign prostatic hyperplasia. Immunolocalization studies showed that P2Y6, P2X2 and P2X3 receptors were present in choline acetyltransferase positive urothelial cells. In contrast to P2Y6 staining, choline acetyltransferase, P2X2 and P2X3 immunoreactivity decreased in the urothelium of benign prostatic hyperplasia patients. CONCLUSIONS: Activation of P2Y6 receptor amplifies mucosal adenosine triphosphate release underlying bladder overactivity in patients with benign prostatic hyperplasia. Therefore, we propose selective P2Y6 receptor blockade as a novel therapeutic strategy to control persistent storage symptoms in obstructed patients.

P2X7-induced zeiosis promotes osteogenic differentiation and mineralization of postmenopausal bone marrow-derived mesenchymal stem cells.

Polymorphisms of the P2X7 receptor have been associated with increased risk of fractures in postmenopausal women. Although both osteoblasts and osteoclasts express P2X7 receptors, their function in osteogenesis remains controversial. Here, we investigated the role of the P2X7 receptor on osteogenic differentiation and mineralization of bone marrow mesenchymal stem cell (BMSC) cultures from postmenopausal women (age 71±3 yr, n=18). We focused on the mechanisms related to intracellular [Ca(2+)]i oscillations and plasma membrane-dynamics. ATP, and the P2X7 agonist BzATP (100 µM), increased [Ca(2+)]i in parallel to the formation of membrane pores permeable to TO-PRO-3 dye uptake. ATP and BzATP elicited reversible membrane blebs (zeiosis) in 38 ± 1 and 70 ± 1% of the cells, respectively. P2X7-induced zeiosis was Ca(2+) independent, but involved phospholipase C, protein kinase C, and Rho-kinase activation. BzATP (100 µM) progressively increased the expression of Runx-2 and Osterix transcription factors by 452 and 226% (at d 21), respectively, alkaline phosphatase activity by 88% (at d 28), and mineralization by 329% (at d 43) of BMSC cultures in a Rho-kinase-dependent manner. In summary, reversible plasma membrane zeiosis involving cytoskeleton rearrangements due to activation of the P2X7-Rho-kinase axis promotes osteogenic differentiation and mineralization of BMSCs, thus providing new therapeutic targets for postmenopausal bone loss.