P2Y(2)R activation by nucleotides released from oxLDL-treated endothelial cells (ECs) mediates the interaction between ECs and immune cells through RAGE expression and reactive oxygen species production.

Lipoprotein oxidation, inflammation, and immune responses involving the vascular endothelium and immune cells contribute to the pathogenesis of atherosclerosis. In an atherosclerotic animal model, P2Y2 receptor (P2Y2R) upregulation and stimulation were previously shown to induce intimal hyperplasia and increased intimal monocyte infiltration. Thus, we investigated the role of P2Y2R in oxidized low-density lipoprotein (oxLDL)-mediated oxidative stress and the subsequent interaction between endothelial cells (ECs) and immune cells. The treatment of human ECs with oxLDL caused the rapid release of ATP (maximum after 5 min). ECs treated with oxLDL or the P2Y2R agonists ATP/UTP for 1h exhibited significant reactive oxygen species (ROS) production, but this effect was not observed in P2Y2R siRNA-transfected ECs. In addition, oxLDL and ATP/UTP both induced RAGE expression, which was P2Y2R dependent. Oxidized LDL- and ATP/UTP-mediated ROS production was diminished in RAGE siRNA-transfected ECs, suggesting that RAGE is an important mediator in P2Y2R-mediated ROS production. Treatment with oxLDL for 24h induced P2Y2R expression in the human monocyte cell line THP-1 and increased THP-1 cell migration toward ECs. The addition of apyrase, an enzyme that hydrolyzes nucleotides, or diphenyleneiodonium (DPI), a well-known inhibitor of NADPH oxidase, significantly inhibited the increase in cell migration caused by oxLDL. P2Y2R siRNA-transfected THP-1 cells did not migrate in response to oxLDL or ATP/UTP treatment, indicating a critical role for P2Y2R and nucleotide release in oxLDL-induced monocyte migration. Last, oxLDL and ATP/UTP effectively increased ICAM-1 and VCAM-1 expression and the subsequent binding of THP-1 cells to ECs, which was inhibited by pretreatment with DPI or by siRNA against P2Y2R or RAGE, suggesting that P2Y2R is an important mediator in oxLDL-mediated monocyte adhesion to ECs through the regulation of ROS-dependent adhesion molecule expression in ECs. Taken together, our findings suggest that P2Y2R could be a therapeutic target for the prevention of vascular disorders, including atherosclerosis.

Peripheral sensitisation of nociceptors via G-protein-dependent potentiation of mechanotransduction currents.

Mechanical stimuli impinging on the skin are converted into electrical signals by mechanically gated ion channels located at the peripheral nerve endings of dorsal root ganglion (DRG) neurons. Under inflammatory conditions sensory neurons are commonly sensitised to mechanical stimuli; a putative mechanism that may contribute to such sensitisation of sensory neurons is enhanced responsiveness of mechanotransduction ion channels. Here we show that the algogens UTP and ATP potentiate mechanosensitive RA currents in peptidergic nociceptive DRG neurons and reduce thresholds for mechanically induced action potential firing in these neurones. Pharmacological characterisation suggests that this effect is mediated by the Gq-coupled P2Y(2) nucleotide receptor. Moreover, using the in vitro skin nerve technique, we show that UTP also increases action potential firing rates in response to mechanical stimuli in a subpopulation of skin C-fibre nociceptors. Together our findings suggest that UTP sensitises a subpopulation of cutaneous C-fibre nociceptors via a previously undescribed G-protein-dependent potentiation of mechanically activated RA-type currents.

P2X2, P2X4 and P2Y1 receptors elevate intracellular Ca2+ in mouse embryonic stem cell-derived GABAergic neurons.

BACKGROUND AND PURPOSE: Neurons derived from mouse embryonic stem cells (mESCs) are a valuable resource for basic pharmacological research. With the exception of cardiomyocytes, there is relatively little understanding of the pharmacology of stem cell-derived differentiated cells. In this study we investigate P2 receptor agonist effects on GABAergic neurons derived from mESCs. EXPERIMENTAL APPROACH: mESCs were differentiated into GABAergic neurons in the presence of N2B27 culture medium. At day 24 of differentiation GABAergic neuronal responsiveness to purinergic agonists was investigated using calcium imaging and [3H]-GABA release studies. KEY RESULTS: Sub-populations of GABAergic neurons responded to some or all of the adenine and uracil nucleotides ATP, ADP, UTP and UDP (all 100 microM) with elevations of intracellular Ca2+ ([Ca2+]i). The number of neurons responding to ATP was reduced by suramin (100 microM), PPADS (10 microM) and MRS2179 (10 microM), but not by NF023 (10 microM). The response to ATP was modulated by extracellular Zn2+ and pH. Neurons also responded to ATP (100 microM) with the release of [3H]-GABA, an effect completely inhibited by tetrodotoxin (100 nM). Ap4A and 2-methylthioATP both elicited significant [3H]-GABA release. Reverse transcriptase PCR showed the presence of P2X1,2,3,4,5,6 and P2X7, and P2Y1,2 and P2Y6 receptors. mESCs expressed P2X2,5 and P2X7 and P2Y1,2 and P2Y6 receptors. CONCLUSIONS AND IMPLICATIONS: GABAergic neurons derived from stem cells elevate [Ca2+]i predominantly via the activation of P2X2, P2X4 and P2Y1 receptors. This study shows that mESCs generate good models of neuronal function for in vitro pharmacological investigation.

Role played by P2X and P2Y receptors in evoking the muscle chemoreflex.

The role played by purinergic 2Y receptors in evoking the muscle chemoreflex is not well defined. To shed light on this issue, we compared the pressor responses with popliteal arterial injection of UTP (1 mg/kg), a selective P2Y agonist, with those to popliteal arterial injection of ATP (1 mg/kg), a P2X and P2Y agonist, and to alpha,beta-methylene ATP (50 mug/kg), a selective P2X1 and P2X3 agonist, in decerebrate unanesthetized cats. We found that injection of ATP and alpha,beta-methylene ATP increased mean arterial pressure by 19 +/- 2 and 15 +/- 4 mmHg, whereas UTP had no affect on arterial pressure. In addition, the pressor responses to injection of ATP and alpha,beta-methylene ATP were abolished by section of the sciatic nerve, demonstrating that they were reflex in origin. We conclude that P2Y receptors on thin fiber muscle afferents play no role in evoking the muscle chemoreflex.

Purinergic substances promote murine keratinocyte proliferation and enhance impaired wound healing in mice.

As membrane-bound receptors for adenosine, purines, and pyrimidines, purinoceptors are expressed in nearly all cell types throughout the mammalian organism. Previous studies showed that purinoceptors are involved in the regulation of proliferation and differentiation of most target cells. The present study was performed to elucidate their role in keratinocyte proliferation and wound healing. The expression of the mRNA of several adenosine and P2Y receptors was shown in the immortalized murine keratinocyte cell line MSC-P5 and primary cultured keratinocytes of four different mouse strains. The nonselective adenosine receptor agonist 5'-(N-ethyl)-carboxamidoadenosine enhanced the growth of MSC-P5 cells in vitro via the A2B receptor. The proliferative stimulus of adenosine triphosphate and uridine triphosphate on this cell line was mediated by the P2Y2 receptor. The mitogenic effect of the purinergic substances was inhibited by simultaneous treatment with respective antagonists. Studies in a mouse model of dexamethasone-induced impaired wound healing showed the in vivo efficacy of the purinoceptor agonists. These studies confirm that pharmacological actions via purinoceptors offer an intriguing possibility in the treatment of impaired wound healing. Nevertheless, further investigations are needed to elucidate fully the role of purinergic mechanisms involved in wound healing.

Extracellular ATP stimulates NO production in rat thick ascending limb.

NO produced by NO synthase (NOS) 3 acts as an autacoid to regulate NaCl absorption in the thick ascending limb. ATP induces NO production by NOS 3 in endothelial cells. We hypothesized that extracellular ATP activates NOS in thick ascending limbs through P2 receptors. To test this, we measured intracellular NO production using the NO-selective fluorescent dye DAF-2 in suspensions of rat medullary thick ascending limbs. We found that ATP increased DAF-2 fluorescence in a concentration-dependent manner, reaching saturation at &200 micromol/L with an EC50 of 37 micromol/L. The increase was blunted by 74% by the nonselective NOS inhibitor L-omega-nitro-arginine-methyl-ester (2 mmol/L; 60+/-7 versus 16+/-6 arbitrary fluorescence units; P<0.02; n=5). In the presence of the P2 receptor antagonist suramin (300 micromol/L), ATP-induced NO production was reduced by 64% (101+/-11 versus 37+/-5 arbitrary fluorescence units; P<0.002; n=5). Blocking ATP hydrolysis with a 5'-ectonucleotidase inhibitor, ARL67156 (30 micromol/L) enhanced the response to ATP and shifted the EC(50) to 0.8 micromol/L. In the presence of ARL67156, the EC50 of the P2X-selective agonist beta,gamma-methylene-adenosine 5'-triphosphate was 4.8 micromol/L and the EC50 for the P2Y-selective agonist UTP was 40.4 micromol/L. The maximal responses for both agonists were similar. Taken together, these data indicate that ATP stimulates NO production in the thick ascending limb primarily through P2X receptor activation and that ATP hydrolysis may regulate NO production.

Human keratinocytes express multiple P2Y-receptors: evidence for functional P2Y1, P2Y2, and P2Y4 receptors.

Extracellular nucleotides are agonists at the family of receptors known as the P2 receptors, and in keratinocytes the P2Y2 subtype is known to elevate the intracellular free calcium concentration (Cai) and stimulate proliferation. In this study, we have investigated the presence of other functional members of the P2Y subgroup in both normal human keratinocytes and the HaCaT cell line. Using reverse transcription polymerase chain reaction, the expression of mRNA for P2Y1, P2Y2, P2Y4, and P2Y6 receptors was demonstrated in HaCaT cells and differentiated and undifferentiated normal human keratinocytes. Cai was monitored in response to a panel of P2Y receptor agonists. To couple mobilized Cai to a downstream cellular response, cell proliferation was also addressed. In both cell types, adenosine 5'-triphosphate and uridine 5'-triphosphate induced Cai transients of approximately equal duration, magnitude, and shape, confirming the presence of functional P2Y2 receptors. In HaCaT cells, additional characteristic responses were observed in a subpopulation of cells; adenosine 5'-triphosphate failed to elevate Cai in some cells responding to uridine 5'-triphosphate, indicating the presence of P2Y4 receptors, whereas the P2Y1-specific agonist 2-methylthio-5'-adenosine diphosphate was, again, only effective in a small subpopulation. Uridine 5'-diphosphate was ineffective, indicating the absence of functional P2Y6 receptors. Adenosine 5'-triphosphate and uridine 5'-triphosphate equally promoted cell growth in normal human keratinocytes in comparison with the control. In HaCaT cells, adenosine 5'-triphosphate, uridine 5'-triphosphate, and adenosine 5'-diphosphate significantly increased proliferation in comparison to the controls, with a 30% higher response to uridine 5'-triphosphate than with adenosine 5'-triphosphate. These data demonstrate that multiple P2Y receptors (P2Y1, P2Y2, and P2Y4 subtypes) are differentially involved in the regulation of proliferation in human keratinocytes and therefore may be important in wound healing.

Improved sputum expectoration following a single dose of INS316 in patients with chronic bronchitis.

UNLABELLED: Uridine 5'-triphosphate (UTP) is a naturally occurring agonist for P2Y(2) receptors on the apical surface of ciliated respiratory epithelium. UTP stimulates salt and water transport and cilia beat frequency in human airway epithelium in vitro. Single, inhaled doses of UTP stimulate mucociliary clearance in conscious, intubated sheep and in patients with mild chronic bronchitis (smokers and former smokers), suggesting that UTP may be useful for obtaining deep-lung sputum specimens suitable for diagnostic purposes. STUDY OBJECTIVE: UTP is being developed for the cytologic diagnosis of lung cancer under the compound number INS316 Solution for Inhalation (Inspire Pharmaceuticals; Durham, NC). Its ability to improve the quality of expectorated sputum was tested in the current study. DESIGN: Placebo-controlled, double-blind, escalating two-period cross-over study. SETTING: Outpatient volunteers. PATIENTS OR PARTICIPANTS: Twenty-six patients with mild chronic bronchitis. INTERVENTION: Patients attempted to expectorate a specimen spontaneously, following a single inhaled dose of INS316 (10 to 180 mg), and following placebo. MEASUREMENTS AND RESULTS: Sputum weight, sputum cell content, spirometry, and oxyhemoglobin saturation. Only 28% of these patients were able to expectorate a macrophage-containing, deep-lung specimen spontaneously or following inhalation of placebo. In contrast, 85% of the patients were able to produce a specimen following inhalation of INS316. The average weight of the sputum expectorated was increased fourfold by placebo and 10-fold by INS316. A mild transient decrease in pulmonary function was observed following INS316 administration. CONCLUSION: A single dose of INS316 safely improves the ability of patients with mild chronic bronchitis to expectorate a deep-lung sputum specimen suitable for cytologic evaluation.

Analgesic effects of intrathecal administration of P2Y nucleotide receptor agonists UTP and UDP in normal and neuropathic pain model rats.

Recent electrophysiological, behavioral, and biochemical studies revealed that ATP plays a role in facilitating spinal pain transmission via ionotropic P2X nucleotide receptors, although the involvement of metabotropic P2Y nucleotide receptors remains unclear. In the present study, we examined the effects of i.t. administration of P2Y receptor agonists UTP, UDP, and related compounds on nociception in normal rats and tactile allodynia in a neuropathic pain model. In the paw pressure test using normal rats, i.t. administration of UTP (30 and 100 nmol/rat) and UDP (30 and 100 nmol/rat), but not UMP (100 nmol/rat) or uridine (100 nmol/rat), significantly elevated the mechanical nociceptive thresholds, whereas ATP (30 and 100 nmol/rat) and alpha,beta-methylene-ATP (10 and 30 nmol/rat) lowered them. Similarly, in the tail-flick test, UTP (10, 30, and 100 nmol/rat) and UDP (100 nmol/rat) significantly prolonged the thermal nociceptive latency. In the von Frey filament test on normal rats, UTP (100 nmol/rat) and UDP (100 nmol/rat) produced no allodynia to the tactile stimulus, whereas ATP (100 nmol/rat) induced a significant and long-lasting tactile allodynia. In the neuropathic pain model, in which the sciatic nerves of rats were partially ligated, UTP (30 and 100 nmol/rat) and UDP (30 and 100 nmol/rat) produced significant antiallodynic effects. Furthermore, UTP (100 nmol/rat) and UDP (100 nmol/rat) caused no motor deficit in the inclined plane test. Taken together, these results suggest that the activation of UTP-sensitive P2Y(2) and/or P2Y(4) receptors and the UDP-sensitive P2Y(6) receptor, in contrast to P2X receptors, produces inhibitory effects on spinal pain transmission.

ATP and UTP at low concentrations strongly inhibit bone formation by osteoblasts: a novel role for the P2Y2 receptor in bone remodeling.

There is increasing evidence that extracellular nucleotides act on bone cells via multiple P2 receptors. The naturally-occurring ligand ATP is a potent agonist at all receptor subtypes, whereas ADP and UTP only act at specific receptor subtypes. We have reported that the formation and resorptive activity of rodent osteoclasts are stimulated powerfully by both extracellular ATP and its first degradation product, ADP, the latter acting at nanomolar concentrations, probably via the P2Y1 receptor subtype. In the present study, we investigated the actions of ATP, ADP, adenosine, and UTP on osteoblastic function. In 16-21 day cultures of primary rat calvarial osteoblasts, ADP and the selective P2Y1 agonist 2-methylthioADP were without effect on bone nodule formation at concentrations between 1 and 125 microM, as was adenosine. However, UTP, a P2Y2 and P2Y4 receptor agonist, known to be without effect on osteoclast function, strongly inhibited bone nodule formation at concentrations >or= 1 microM. ATP was inhibitory at >or= 10 microM. Rat osteoblasts express P2Y2, but not P2Y4 receptor mRNA, as determined by in situ hybridization. Thus, the low-dose effects of extracellular nucleotides on bone formation and bone resorption appear to be mediated via different P2Y receptor subtypes: ADP, signalling through the P2Y1 receptor on both osteoclasts and osteoblasts, is a powerful stimulator of osteoclast formation and activity, whereas UTP, signalling via the P2Y2 receptor on osteoblasts, blocks bone formation by osteoblasts. ATP, the 'universal' agonist, can simultaneously stimulate resorption and inhibit bone formation. These findings suggest that extracellular nucleotides could function locally as important negative modulators of bone metabolism, perhaps contributing to bone loss in a number of pathological states.

Ultrastructural analysis of nucleolar transcription in cells microinjected with 5-bromo-UTP.

In situ sites of nucleolar transcription in cells microinjected with 5-bromo-UTP (BrUTP) were visualized at an ultrastructural level. After injection the cells were maintained for 4-90 min at 37 degrees C, fixed, and embedded in LR White resin. Postembedding immunoelectron microscopic visualization with colloidal gold has been used for localizing both Br-labeled precursor incorporated into pre-rRNA and different nucleolar transcription or processing factors. This high resolution approach allowed us to identify significant signal as early as after 4-min incubation periods following BrUTP microinjection. It revealed the dense fibrillar component (DFC) as being the first nucleolar compartment labeled with anti-bromodeoxyuridine antibody. Moreover, RNA polymerase I, nucleolar transcription factor UBF, and fibrillarin were also detected almost exclusively in this same nucleolar compartment. From 30 min onward, following microinjection, Br-labeled rRNA occurred also in the granular component. The results indicate that the DFC is the site of pre-rRNA transcription and of initial steps of pre-rRNA processing. Moreover, it demonstrates that BrUTP microinjection followed by postembedding detection of Br-labeled RNA is a useful technique for high resolution studies of structure-function associations in the nucleolus.

Acute safety and effects on mucociliary clearance of aerosolized uridine 5'-triphosphate +/- amiloride in normal human adults.

Impaired mucociliary clearance contributes to the pathophysiology of several airways diseases including cystic fibrosis, asthma, and chronic bronchitis. Extracellular triphosphate nucleotides (adenosine 5'-triphosphate [ATP], uridine 5'-triphosphate [UTP]) activate several components of the mucociliary escalator, suggesting they may have potential as therapeutic agents for airways diseases. We conducted initial (Phase I) studies of acute safety and efficacy of aerosolized UTP alone and in combination with aerosolized amiloride, the sodium channel blocker, in normal human volunteers. Safety was assessed by measurement of pulmonary function. Neither UTP alone nor in combination with amiloride caused any clinically significant adverse effects on airway mechanics, (subdivisions of) lung volumes, or gas exchange. Acute efficacy of UTP and amiloride alone and in combination, was assessed by measuring changes in the clearance of inhaled radiolabeled particles. A 2.5-fold increase in mucociliary clearance was seen in response to UTP alone and in combination with amiloride. We conclude that aerosolized UTP +/- amiloride clearly enhances mucociliary clearance without acute adverse effects in normal adults, and may have therapeutic potential to enhance airways clearance in diseases characterized by retained airways secretions.

Effect of uridine 5'-triphosphate plus amiloride on mucociliary clearance in adult cystic fibrosis.

Cystic fibrosis (CF) is characterized by abnormal airway epithelial electrolyte transport leading to viscous airway secretions that are difficult to clear. By enhancing Cl- secretion onto and blocking Na+ absorption from the airway surface, treatment with aerosolized uridine 5'-triphosphate (UTP) plus amiloride may improve the rheology of airway secretions and enhance mucociliary clearance in patients with CF. After performing safety studies of aerosolized UTP/amiloride in adult patients with CF, we investigated the effects of inhaled vehicle and UTP/amiloride on mucociliary clearance of [99mTc] iron oxide particles from the airways of adult patients with CF (n = 14). We found no clinically significant adverse effects from inhalation of therapeutic doses of UTP/amiloride. Mean baseline peripheral clearance rates during the first 40 min of clearance measurements were significantly less in patients with CF than in normal subjects (mean +/- SE: 0.30 +/- 0.05 versus 0.54 +/- 0.07%/min, respectively; p = 0.01). Aerosolized UTP and amiloride in combination improved mucociliary clearance from the peripheral airways of the CF lungs to near normal values (0.51 +/- 0.09%/min; p = 0.04) during this period. These data support the concept for the use of UTP in combination with amiloride as therapy to improve clearance of secretions from the lungs of patients with CF.

Efecto del núcleo CMP forte en 46 pacientes con paraparesia espástica progresiva: estudio randomizado y ciego / Effect of CMP forte nucleus in 46 patients with progressive spastic paraparesis: randomized and blind study

Idiopatic or HTLV-1 associated progressive spastic paraparesis does not have a clear etiology or treatment. To assess the effects of a medication containing cytidinmonophosphate, uridintriphosphate and vitamin B 12 in the treatment of progressive spastic. Patients with the disease were randomly assigned to receive the Nucleus CMP forte (containing dysodic cytidinmonophosphate 5 mg,trisodic uridintriphosphate 3 mg and hydroxicobalamin 2 mg) tid or placebo during 6 months. Gait, spasticity, degree of neurogenic bladder and somatosensitive evoked potentials were assessed during treatment. Forty six patients aged 25 to 79 years old were studied, 24 were female and 29 HTLV-1 positive. Twenty two were treated with the drug and the rest with placebo. Gait and spasticity improved in 7 of 22 patients receiving the drug and 1 of 24 receiving placebo (p<0.05). Neurogenic bladder improved in 10 of 22 receiving the drug and 4 patients treated with the drug and in two of seven treated with placebo. The medication caused a modest improvement in patients with progressive spastic paraparesis and was free of side effects

[New aspects of therapy of mucoviscidosis (CF)]. / Neue Aspekte für die Therapie der Mukoviszidose (CF).

The authors survey of recent advances in CF research and their therapeutic implications: 1. the possibility of successful gene therapy by transfer of the normal gene to airway epithelial cells. 2. inhalations with the potassium-sparing diuretic amiloride that diminish the viscosity of the bronchial secretions, 3. application of adenosine or uridine triphosphate (ATP or UTP) to the apical surface of the respiratory epithelial cells which intervene with the function of ion channels, 4. enzymatic cleavage and liquidification of bronchial secretions by aerosolized human recombinante DNase. In addition, the possible advantages of (heart-) lung-transplantation are also discussed.