Endogenous ATP Research Articles

Development of a P2X1-purinoceptor mediated contractile response in the aged mouse prostate gland through slowing down of ATP breakdown

An age-related increase in prostatic smooth muscle tone is partly responsible for the lower urinary tract symptoms associated with benign prostatic hyperplasia (BPH). Changes in the effectors of prostatic smooth muscle contraction with age may play a role in the development of these symptoms. Using a mouse model of prostate contractility, this study investigated the effect of age on the different components of contractility in the prostate gland. The isometric force developed in response to electrical field stimulation or exogenously applied agonists by mouse prostates mounted in organ baths, was evaluated to determine the effect of age on contractile mechanisms. Changes with age in the rate of ATP breakdown and levels of the P2rx1 gene and P2X1-purinoceptor expression in mouse prostate were measured by a modified luciferin-luciferase assay, RT-PCR and western blot, respectively. Nerve mediated contractile responses containing a component elicited by P2X1-purinoceptors were observed in prostates taken from aged mice, but not in prostates taken from young adult mice. Furthermore, the potency of the endogenous purinoceptor agonist ATP was 50-fold greater in aged mice, whereas the potency of its stable analogue α,β-metATP was unchanged. An age-related decrease in ATP metabolism was also observed. With age, a purinergic contractile response to nerve stimulation develops in the mouse prostate gland due to a decrease in the rate of ATP breakdown. This may contribute to the increase in muscular tone observed in BPH and suggests that P2X1-purinoceptors are an additional target for the treatment of BPH.

IRFP is a sensitive marker for cell number and tumor growth in high-throughput systems

GFP and luciferase are used extensively as markers both in vitro and in vivo although both have limitations. The utility of GFP fluorescence is restricted by high background signal and poor tissue penetrance. Luciferase throughput is limited in vitro by the requirement for cell lysis, while in vivo, luciferase readout is complicated by the need for substrate injection and the dependence on endogenous ATP. Here we show that near-infrared fluorescent protein in combination with widely available near-infrared scanners overcomes these obstacles and allows for the accurate determination of cell number in vitro and tumor growth in vivo in a high-throughput manner and at negligible per-well costs. This system represents a significant advance in tracking cell proliferation in tissue culture as well as in animals, with widespread applications in cell biology.

P2X3 receptors induced inflammatory nociception modulated by TRPA1, 5-HT3 and 5-HT1A receptors

It has been described that endogenous ATP via activation of P2X3 and P2X2/3 receptors contributes to inflammatory nociception in different models, including the formalin injected in subcutaneous tissue of the rat's hind paw. In this study, we have evaluated whether TRPA1, 5-HT3 and 5-HT1A receptors, whose activation is essential to formalin-induced inflammatory nociception, are involved in the nociception induced by activation of P2X3 receptors on subcutaneous tissue of the rat's hind paw. We have also evaluated whether the activation of P2X3 receptors increases the susceptibility of primary afferent neurons to formalin action modulated by activation of TRPA1, 5-HT3 or 5-HT1A receptors. Nociceptive response intensity was measured by observing the rat's behavior and considering the number of times the animal reflexively raised its hind paw (flinches) in 60min. Local subcutaneous administration of the selective TRPA1, 5-HT3 or 5-HT1A receptor antagonists HC 030031, tropisetron and WAY 100,135, respectively, prevented the nociceptive responses induced by the administration in the same site of the non-selective P2X3 receptor agonist αβmeATP. Administration of the selective P2X3 and P2X2/3 receptor antagonist A-317491 or pretreatment with oligonucleotides antisense against P2X3 receptor prevented the formalin-induced behavioral nociceptive responses during the first and second phases. Also, the co-administration of a subthreshold dose of αβmeATP with a subthreshold dose of formalin induced nociceptive behavior, which was prevented by local administration of tropisetron, HC 030031 or WAY 100, 135. These findings have demonstrated that the activation of P2X3 receptors induces inflammatory nociception modulated by TRPA1, 5-HT3 and 5-HT1A receptors. Also, they suggest that inflammatory nociception is modulated by the release of endogenous ATP and P2X3 receptor activation, which in turn, increases primary afferent nociceptor susceptibility to the action of inflammatory mediators via interaction with TRPA1, 5-HT3 and 5-HT1A receptors in the peripheral tissue.

Ectonucleotidase NTPDase3 is abundant in pancreatic β-cells and regulates glucose-induced insulin secretion

Extracellular ATP released from pancreatic β-cells acts as a potent insulinotropic agent through activation of P2 purinergic receptors. Ectonucleotidases, a family of membrane-bound nucleotide-metabolizing enzymes, regulate extracellular ATP levels by degrading ATP and related nucleotides. Ectonucleotidase activity affects the relative proportion of ATP and its metabolites, which in turn will impact the level of purinergic receptor stimulation exerted by extracellular ATP. Therefore, we investigated the expression and role of ectonucleotidases in pancreatic β-cells. Of the ectonucleotidases studied, only ENTPD3 (gene encoding the NTPDase3 enzyme) mRNA was detected at fairly abundant levels in human and mouse pancreatic islets as well as in insulin-secreting MIN6 cells. ARL67156, a selective ectonucleotidase inhibitor, blocked degradation of extracellular ATP that was added to MIN6 cells. The compound also decreased degradation of endogenous ATP released from cells. Measurements of insulin secretion in MIN6 cells as well as in mouse and human pancreatic islets demonstrated that ARL67156 potentiated glucose-dependent insulin secretion. Downregulation of NTPDase3 expression in MIN6 cells with the specific siRNA replicated the effects of ARL67156 on extracellular ATP hydrolysis and insulin secretion. Our results demonstrate that NTPDase3 is the major ectonucleotidase in pancreatic β-cells in multiple species and that it modulates insulin secretion by controlling activation of purinergic receptors.

Glial cells modulate the synaptic transmission of NTS neurons sending projections to ventral medulla of Wistar rats.

There is evidence that sympathoexcitatory and respiratory responses to chemoreflex activation involve ventrolateral medulla-projecting nucleus tractus solitarius (NTS) neurons (NTS-VLM neurons) and also that ATP modulates this neurotransmission. Here, we evaluated whether or not astrocytes is the source of endogenous ATP modulating the synaptic transmission in NTS-VLM neurons. Synaptic activities of putative astrocytes or NTS-VLM neurons were recorded using whole cell patch clamp. Tractus solitarius (TS) stimulation induced TS-evoked excitatory postsynaptic currents (TS-eEPSCs) in NTS-VLM neurons as well in NTS putative astrocytes, which were also identified by previous labeling. Fluoracetate (FAC), an inhibitor of glial metabolism, reduced TS-eEPSCs amplitude (−85.6 ± 16 vs. −39 ± 7.1 pA, n = 12) and sEPSCs frequency (2.8 ± 0.5 vs. 1.8 ± 0.46 Hz, n = 10) in recorded NTS-VLM neurons, indicating a gliomodulation of glutamatergic currents. To verify the involvement of endogenous ATP a purinergic antagonist was used, which reduced the TS-eEPSCs amplitude (−207 ± 50 vs. −149 ± 50 pA, n = 6), the sEPSCs frequency (1.19 ± 0.2 vs. 0.62 ± 0.11 Hz, n = 6), and increased the paired-pulse ratio (PPR) values (∼20%) in NTS-VLM neurons. Simultaneous perfusion of Pyridoxalphosphate-6-azophenyl-2′,5′-disulfonic acid (iso-PPADS) and FAC produced reduction in TS-eEPSCs similar to that observed with iso-PPADS or FAC alone, indicating that glial cells are the source of ATP released after TS stimulation. Extracellular ATP measurement showed that FAC reduced evoked and spontaneous ATP release. All together these data show that putative astrocytes are the source of endogenous ATP, which via activation of presynaptic P2X receptors, facilitates the evoked glutamate release and increases the synaptic transmission efficacy in the NTS-VLM neurons probably involved with the peripheral chemoreflex pathways.

Extracellular ATP Released by Osteoblasts Is A Key Local Inhibitor of Bone Mineralisation

Previous studies have shown that exogenous ATP (>1µM) prevents bone formation in vitro by blocking mineralisation of the collagenous matrix. This effect is thought to be mediated via both P2 receptor-dependent pathways and a receptor-independent mechanism (hydrolysis of ATP to produce the mineralisation inhibitor pyrophosphate, PPi). Osteoblasts are also known to release ATP constitutively. To determine whether this endogenous ATP might exert significant biological effects, bone-forming primary rat osteoblasts were cultured with 0.5-2.5U/ml apyrase (which sequentially hydrolyses ATP to ADP to AMP + 2Pi). Addition of 0.5U/ml apyrase to osteoblast culture medium degraded extracellular ATP to <1% of control levels within 2 minutes; continuous exposure to apyrase maintained this inhibition for up to 14 days. Apyrase treatment for the first 72 hours of culture caused small decreases (≤25%) in osteoblast number, suggesting a role for endogenous ATP in stimulating cell proliferation. Continuous apyrase treatment for 14 days (≥0.5U/ml) increased mineralisation of bone nodules by up to 3-fold. Increases in bone mineralisation were also seen when osteoblasts were cultured with the ATP release inhibitors, NEM and brefeldin A, as well as with P2X1 and P2X7 receptor antagonists. Apyrase decreased alkaline phosphatase (TNAP) activity by up to 60%, whilst increasing the activity of the PPi-generating ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs) up to 2.7-fold. Both collagen production and adipocyte formation were unaffected. These data suggest that nucleotides released by osteoblasts in bone could act locally, via multiple mechanisms, to limit mineralisation.

P2X7 receptor‐induced death of motor neurons by a peroxynitrite/FAS‐dependent pathway

The P2X7 receptor/channel responds to extracellular ATP and is associated with neuronal death and neuroinflammation in spinal cord injury and amyotrophic lateral sclerosis. Whether activation of P2X7 directly causes motor neuron death is unknown. We found that cultured motor neurons isolated from embryonic rat spinal cord express P2X7 and underwent caspase-dependent apoptosis when exposed to exceptionally low concentrations of the P2X7 agonist 2'(3')-O-(4-Benzoylbenzoyl)-ATP. The P2X7 inhibitors BBG, oATP, and KN-62 prevented 2'(3')-O-(4-Benzoylbenzoyl)-ATP-induced motor neuron death. The endogenous P2X7 agonist ATP induced motor neuron death at low concentrations (1-100μM). High concentrations of ATP (1mM) paradoxically became protective due to degradation in the culture media to produce adenosine and activate adenosine receptors. P2X7-induced motor neuron death was dependent on neuronal nitric oxide synthase-mediated production of peroxynitrite, p38 activation, and autocrine FAS signaling. Taken together, our results indicate that motor neurons are highly sensitive to P2X7 activation, which triggers apoptosis by activation of the well-established peroxynitrite/FAS death pathway in motor neurons.

Astrocyte-derived ATP modulates depressive-like behaviors

Major depressive disorder (MDD) is a cause of disability that affects approximately 16% of the world's population; however, little is known regarding the underlying biology of this disorder. Animal studies, postmortem brain analyses and imaging studies of patients with depression have implicated glial dysfunction in MDD pathophysiology. However, the molecular mechanisms through which astrocytes modulate depressive behaviors are largely uncharacterized. Here, we identified ATP as a key factor involved in astrocytic modulation of depressive-like behavior in adult mice. We observed low ATP abundance in the brains of mice that were susceptible to chronic social defeat. Furthermore, we found that the administration of ATP induced a rapid antidepressant-like effect in these mice. Both a lack of inositol 1,4,5-trisphosphate receptor type 2 and transgenic blockage of vesicular gliotransmission induced deficiencies in astrocytic ATP release, causing depressive-like behaviors that could be rescued via the administration of ATP. Using transgenic mice that express a Gq G protein-coupled receptor only in astrocytes to enable selective activation of astrocytic Ca(2+) signaling, we found that stimulating endogenous ATP release from astrocytes induced antidepressant-like effects in mouse models of depression. Moreover, we found that P2X2 receptors in the medial prefrontal cortex mediated the antidepressant-like effects of ATP. These results highlight astrocytic ATP release as a biological mechanism of MDD.

Is It Time to Consider Photobiomodulation As a Drug Equivalent?

The question of whether photobiomodulation should be used as a drug equivalent arose in my mind after listening to presentations at the recent conference of the World Association for Laser Therapy (WALT)-2012 (Gold Cost City, Australia), and later at home when searching MEDLINE for the years 2009–2012. Photobiomodulation (earlier terms: low level laser therapy, LLLT, laser biostimulation) has been used in clinical practice for >40 years by now, and its action mechanisms on cellular and molecular levels have been studied for > 30 years. Enthusiastic medical specialists successfully used photobiomodulation in treating healing-resistant wounds and ulcers (e.g., chronic diabetic ulcers), in pain management, and in spinal cord and nervous system injuries when other methods had had limited success. However, photobiomodulation is still not a part of mainstream medicine. The goal of the present Editorial is to highlight some important recent developments in clinical applications and in studies of cellular and molecular mechanisms behind the clinical findings. One of the impressive and perspective challenges for photobiomodulation is its use in cases of Parkinson’s disease. Research in recent years evidenced that neuroprotective treatment with red and near infrared radiation (NIR) prevented mitochondrial dysfunction and dopamine loss in Parkinson’s disease patients. In another set of experiments, NIR normalized mitochondrial movement and axon transport, as well as stimulating respiration in cytoplasmic hybrid (‘‘cybrid’’) neurons. It is important to recall that reduced axonal transport contributes substantially to the degeneration of neuronal processes in Parkinson’s disease. Another development in recent years is the successful stimulation of stem cells with red and NIR radiation. One example is the treatment of myocardial infarction. The heart has been considered a post-mitotic organ lacking the capacity for self-renewal after injury. Surprisingly enough, human cardiac stem cells, in combination with bone marrow mesenchymal stem cells, were found to reduce infarct size and restore cardiac functions after myocardial infarction. This positive effect can even be increased by irradiation of stem cells. Mesenchymal stem cells were derived from bone marrow and adipose tissue, and stimulated by irradiation at k = 810 nm. Implantation of irradiated cells into the infarcted rat heart resulted in an *50% decrease in cardiac infarct size. An increase in proliferation rates and membrane potential was established after 532 nm irradiation of adipose tissue-derived stem cells. A recent review summarized data about enhancement of the proliferation of various cultured cell lines, including stem cells, as well as cell lines used for the production of viral vaccines and hybrid cell lines. The review underlined that photobiomodulation improves the proliferation of cells without causing any cytotoxic effects. One has to emphasize that laser therapy shares none of the risks associated with stem cell therapy, requires no anesthesia, and is painless. The optimal light parameters in this review were found to be as follows: doses were 0.5–4.0 J/ cm and wavelengths ranged from 600 to 700 nm. It is important to recall that, in this particular wavelength range, two peaks in absorption and action spectra connected with activation of cytochrome c oxidase (the primary photoacceptor for photobiomodulation effects) are situated. The peak at 620 nm belongs to reduced CuA, and that at 680 nm, to oxidized CuB atoms in cytochrome c oxidase molecule. The treatment of vitiligo (a depigmentary disorder) remains a challenge for clinical dermatologists. He-Ne laser irradiation was found to stimulate melanocyte proliferation. The expression of phosphorylated cyclic-adenosine monophosphate (AMP) response element-binding protein, an important regulator of melanocyte growth, was upregulated by He-Ne laser treatment. He-Ne laser irradiation imparted a growth stimulatory effect on functional melanocytes via mitochondria-related pathways. Irradiation with red light caused gene and noncoding RNA regulation for photoacceptor protection in the retina. This finding may open a new challenge for photobiomodulation. One of the major dose-limiting effects of chemotherapy drugs is oral mucositis of treated patients. Oral mucositis can affect up to 100% of patients undergoing high-dose chemotherapy and hematopoietic stem cell transplantation. Photobiomodulation can improve tissue repair and immune response in these patients. Photobiomodulation has been shown to improve functional outcome after surgical intervention to repair injured nerves. LED irradiation at 810 nm accelerated functional recovery and improved the quality of nerve regeneration after autograft repair of severely injured peripheral nerves. Forehead treatments with NIR reversed major depression and anxiety. Transcranial NIR laser therapy was investigated

Abstract 5506: In vivo activity of the ribonucleoside analogue 8-chloro-adenosine in breast cancer mouse models.

Abstract In contrast to deoxyribose or arabinose containing nucleoside analogs that are currently established for cancer therapeutics, 8-chloro-adenosine (8-Cl-Ado) possesses a ribose sugar. This unique nucleoside analog is RNA directed and is in a phase I clinical trial for hematological malignancies. Preclinically, it has been found to be cytotoxic to a number of malignant cell lines. Previously, we demonstrated in the breast cancer cell lines, MCF-7 and BT-474, 8-Cl-Ado accumulates as a triphosphate (8-Cl-ATP) reaching ∼125 and ∼400 μM, respectively, by 6 h of treatment with 10 μM 8-Cl-Ado [Breast Cancer Res Treat 121:355, 2010]. This accumulation was coupled with a parallel decrease in the endogenous cellular ATP pool levels. These changes in nucleotide levels lead to ∼50% inhibition of RNA synthesis. The net effect of these metabolic changes results in a 90% loss of clonogenic survival after a 3 d treatment with 10 μM 8-Cl-Ado. This loss in survival was associated with both apoptotic and autophagic cell death. Cellular pharmacology analyses on CD2F1 mice after i.v. administration of 50 and 100 mg/kg 8-Cl-Ado, revealed 8-Cl-ATP accumulated in the circulating peripheral blood mononuclear cells; within 1 hr of infusion the levels reached ∼350 and ∼1150 μM, respectively [Cancer Chemother Pharmacol 50:85, 2002] which was comparable to the levels seen in BT-474 cells treated with 10 μM for 6 and 72 hr, respectively. To test the in vivo antitumor activity of 8-Cl-Ado in breast cancer, orthotopic xenograft mouse models using both MCF-7 and BT-474 cell lines were evaluated. 8-Cl-Ado was administered i.p. 3 d/wk for 3 wk with a dose range of 25, 50 and 100 mg/kg/d once tumors reached ∼0.4 cm diameter. The MCF-7 xenograft mice showed dose dependent tumor growth inhibition evident after the second dose. In mice with the BT-474 tumors, the 100 mg/kg dose was highly effective as tumor sizes decreased after the first dose of treatment. Furthermore, in several of the mice there was an eventual loss of a palpable tumor. Likewise, the 100 mg/kg 8-Cl-Ado dose had a profound effect on the final tumor volumes in the BT-474 xenograft mice assessed during necropsy and completely eliminated any visible tumors in 9/21 (42.8%) mice assessed during necropsy. Importantly, none of these tested doses (25, 50 and 100 mg/kg/d) resulted in any untoward toxicity which was assessed by infusing various 8-Cl-Ado doses in CD1 mice. The results of the toxicology assessment showed that only repeated high i.v. doses (250 and 350 mg/kg/d for 5 d) showed a persistent nephrotoxicity 32 d after drug administration, while no other pathologic or hematologic toxicities were observed. In conclusion, our data indicates tumoricidal levels of 8-Cl-ATP are readily achieved in vivo without deleterious toxic effects and establishes 8-Cl-Ado in vivo efficacy against 2 breast cancer tumor models. Based on these findings, we plan to test 8-Cl-Ado in the clinic for patients with breast cancer. Citation Format: Christine M. Stellrecht, Shujun Shentu, Mary L. Ayres, Varsha Gandhi. In vivo activity of the ribonucleoside analogue 8-chloro-adenosine in breast cancer mouse models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5506. doi:10.1158/1538-7445.AM2013-5506

Purinergic modulation of smooth muscle intracellular Ca2+ concentration

Basal, steady‐state Ca2+ levels are determined by the balance between Ca2+ release/influx and uptake/extrusion mechanisms. Extrinsic factors can alter this balance to affect smooth muscle excitability. Extracellular ATP acts as an inhibitory neurotransmitter in gastrointestinal and vascular (portal vein) smooth muscle. This study examined the effect of extracellular ATP on the steady‐state intracellular Ca2+ concentration ([Ca2+]c). Extracellular ATP (1 mM; applied via picopritzer) reversibly decreased steady‐state [Ca2+]c (measured using fluo 4). To assess whether ATP functioned by altering influx or extrusion [Ca2+]c and inward currents were measured simultaneously in voltage‐clamped myocytes during a depolarizing pulse. ATP decreased the peak amplitude without altering the voltage‐dependence of the inward current. The P2 purinoceptor inhibitor, suramin, and the P2Y1 specific inhibitor, MRS2179, each prevented ATP inhibition of steady‐state [Ca2+]c and inward current. Interestingly, suramin and MRS2179 each also increased the magnitude of the inward current per se, indicating tonic ATP inhibition of Ca2+ channels by endogenous ATP. ATP did not alter Ca2+ uptake/extrusion mechanisms. ATP appears to influence Ca2+ influx mechanisms in smooth muscle via P2Y receptors to exert a negative regulation of excitability.

Charge Composition Features of Model Single-span Membrane Proteins That Determine Selection of YidC and SecYEG Translocase Pathways in Escherichia coli

We have investigated the features of single-span model membrane proteins based upon leader peptidase that determines whether the proteins insert by a YidC/Sec-independent, YidC-only, or YidC/Sec mechanism. We find that a protein with a highly hydrophobic transmembrane segment that inserts into the membrane by a YidC/Sec-independent mechanism becomes YidC-dependent if negatively charged residues are inserted into the translocated periplasmic domain or if the hydrophobicity of the transmembrane segment is reduced by substituting polar residues for nonpolar ones. This suggests that charged residues in the translocated domain and the hydrophobicity within the transmembrane segment are important determinants of the insertion pathway. Strikingly, the addition of a positively charged residue to either the translocated region or the transmembrane region can switch the insertion requirements such that insertion requires both YidC and SecYEG. To test conclusions from the model protein studies, we confirmed that a positively charged residue is a SecYEG determinant for the endogenous proteins ATP synthase subunits a and b and the TatC subunit of the Tat translocase. These findings provide deeper insights into how pathways are selected for the insertion of proteins into the Escherichia coli inner membrane.

Sympathetic nerve-derived ATP regulates renal medullary vasa recta diameter via pericyte cells: a role for regulating medullary blood flow?

Pericyte cells are now known to be a novel locus of blood flow control, being able to regulate capillary diameter via their unique morphology and expression of contractile proteins. We have previously shown that exogenous ATP causes constriction of vasa recta via renal pericytes, acting at a variety of membrane bound P2 receptors on descending vasa recta (DVR), and therefore may be able to regulate medullary blood flow (MBF). Regulation of MBF is essential for appropriate urine concentration and providing essential oxygen and nutrients to this region of high, and variable, metabolic demand. Various sources of endogenous ATP have been proposed, including from epithelial, endothelial, and red blood cells in response to stimuli such as mechanical stimulation, local acidosis, hypoxia, and exposure to various hormones. Extensive sympathetic innervation of the nephron has previously been shown, however the innervation reported has focused around the proximal and distal tubules, and ascending loop of Henle. We hypothesize that sympathetic nerves are an additional source of ATP acting at renal pericytes and therefore regulate MBF. Using a rat live kidney slice model in combination with video imaging and confocal microscopy techniques we firstly show sympathetic nerves in close proximity to vasa recta pericytes in both the outer and inner medulla. Secondly, we demonstrate pharmacological stimulation of sympathetic nerves in situ (by tyramine) evokes pericyte-mediated vasoconstriction of vasa recta capillaries; inhibited by the application of the P2 receptor antagonist suramin. Lastly, tyramine-evoked vasoconstriction of vasa recta by pericytes is significantly less than ATP-evoked vasoconstriction. Sympathetic innervation may provide an additional level of functional regulation in the renal medulla that is highly localized. It now needs to be determined under which physiological/pathophysiological circumstances that sympathetic innervation of renal pericytes is important.

Enhancement of purinergic signalling by excessive endogenous ATP in resiniferatoxin (RTX) neuropathy

ATP is a ligand of P2X family purinoceptors, and exogenous ATP administration evokes pain behaviors. To date, there is a lack of systematic studies to address relationships between endogenous ATP and neuropathic pain. In this report, we took advantage of a mouse model of resiniferatoxin (RTX)-induced neuropathic pain to address the role of endogenous ATP in neuropathic pain. After RTX administration, endogenous ATP markedly increased in dorsal root ganglia (DRGs) (p < 0.01) and skin tissues (p < 0.001). The excessive endogenous ATP was removed by apyrase, an ATP hydrolyzing enzyme, administration via either a lumbar puncture route (p < 0.001) or an intraplantar injection (p < 0.001), which led to the normalization of neuropathic pain. In addition, intraplantar treatment with apyrase caused mechanical analgesia. Linear analyses showed that the densities of P2X3(+) neurons (r = -0.72, p < 0.0001) and P2X3(+) dermal nerves (r = -0.72, p < 0.0001) were inversely correlated with mechanical thresholds. Moreover, the contents of endogenous ATP in skin tissues were linearly correlated with P2X3(+) dermal nerves (r = 0.80, p < 0.0001) and mechanical thresholds (r = -0.80, p < 0.0001). In summary, this study demonstrated that enhanced purinergic signalling due to an increase in endogenous ATP after RTX-induced nerve injury contributed to the development of neuropathic pain. The data in this report provide a new therapeutic strategy for pain control by targeting the endogenous ligand of purinergic signalling.

The P2X7 Receptor Supports Both Life and Death in Fibrogenic Pancreatic Stellate Cells

The pancreatic stellate cells (PSCs) have complex roles in pancreas, including tissue repair and fibrosis. PSCs surround ATP releasing exocrine cells, but little is known about purinergic receptors and their function in PSCs. Our aim was to resolve whether PSCs express the multifunctional P2X7 receptor and elucidate how it regulates PSC viability. The number of PSCs isolated from wild type (WT) mice was 50% higher than those from the Pfizer P2X7 receptor knock out (KO) mice. The P2X7 receptor protein and mRNA of all known isoforms were expressed in WT PSCs, while KO PSCs only expressed truncated versions of the receptor. In culture, the proliferation rate of the KO PSCs was significantly lower. Inclusion of apyrase reduced the proliferation rate in both WT and KO PSCs, indicating importance of endogenous ATP. Exogenous ATP had a two-sided effect. Proliferation of both WT and KO cells was stimulated with ATP in a concentration-dependent manner with a maximum effect at 100 µM. At high ATP concentration (5 mM), WT PSCs, but not the KO PSCs died. The intracellular Ca2+ signals and proliferation rate induced by micromolar ATP concentrations were inhibited by the allosteric P2X7 receptor inhibitor az10606120. The P2X7 receptor-pore inhibitor A438079 partially prevented cell death induced by millimolar ATP concentrations. This study shows that ATP and P2X7 receptors are important regulators of PSC proliferation and death, and therefore might be potential targets for treatments of pancreatic fibrosis and cancer.

Involvement of ATP in noxious stimulus-evoked release of glutamate in rat medullary dorsal horn: A microdialysis study

Our electrophysiological studies have shown that both purinergic and glutamatergic receptors are involved in central sensitization of nociceptive neurons in the medullary dorsal horn (MDH). Here we assessed the effects of intrathecal administration of apyrase (a nucleotide degrading enzyme of endogenous adenosine 5-triphosphate [ATP]), a combination of apyrase and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, an adenosine A1 receptor antagonist), or 2,3-O-2,4,6-trinitrophenyl-adenosine triphosphate (TNP-ATP, a P2X1, P2X3, P2X2/3 receptor antagonist) on the release of glutamate in the rat MDH evoked by application of mustard oil (MO) to the molar tooth pulp. In vivo microdialysis was used to dialyse the MDH every 5min, and included 3 basal samples, 6 samples after drug treatment and 12 samples following application of MO. Tooth pulp application of MO induced a significant increase in glutamate release in the MDH. Superfusion of apyrase or TNP-ATP alone significantly reduced the MO-induced glutamate release in the MDH, as compared to vehicle. Furthermore, the suppressive effects of apyrase on glutamate release were reduced by combining it with DPCPX. This study demonstrates that application of an inflammatory irritant to the tooth pulp induces glutamate release in the rat MDH in vivo that may be reduced by processes involving endogenous ATP and adenosine.

ATP release and purinergic signaling: a common pathway for particle-mediated inflammasome activation

Deposition of uric acid crystals in joints causes the acute and chronic inflammatory disease known as gout and prolonged airway exposure to silica crystals leads to the development of silicosis, an irreversible fibrotic pulmonary disease. Aluminum salt (Alum) crystals are frequently used as vaccine adjuvant. The mechanisms by which crystals activate innate immunity through the Nlrp3 inflammasome are not well understood. Here, we show that uric acid, silica and Alum crystals trigger the extracellular delivery of endogenous ATP, which just precedes the secretion of mature interleukin-1β (IL-1β) by macrophages, both events depending on purinergic receptors and connexin/pannexin channels. Interestingly, not only ATP but also ADP and UTP are involved in IL-1β production upon these Nlrp3 inflammasome activators through multiple purinergic receptor signaling. These findings support a pivotal role for nucleotides as danger signals and provide a new molecular mechanism to explain how chemically and structurally diverse stimuli can activate the Nlrp3 inflammasome.

Enzyme-coupled assays for simultaneous detection of nanomolar ATP, ADP, AMP, adenosine, inosine and pyrophosphate concentrations in extracellular fluids

Purinergic signaling cascade includes the release of endogenous ATP and other agonists by chemical and mechanical stimuli, modulation of diverse cellular functions and subsequent ectoenzymatic inactivation. Basal release of extracellular purines and its physiological relevance remain controversial. Here we employed a combination of enzyme-coupled approaches for simultaneous bioluminescent (ATP, ADP, PPi) and fluorometric (AMP, adenosine, inosine, hypoxanthine) measurements of ATP and its metabolites without additional manipulations or derivatization of sampled biological fluids. By using these sensing techniques, extracellular purines were determined in various cells and tissues both at resting and pro-inflammatory conditions. The results obtained revealed the ability of endothelial, lymphoid and tumor cells to maintain extracellular ATP, ADP and adenosine at certain characteristic nanomolar levels. By quantifying the amounts of endogenously released and/or exogenously applied purines and their metabolites, these sensing techniques may be applied for evaluating purine-converting pathways on the cell surfaces and also for ex vivo analysis of purine homeostasis in the intact tissues. Furthermore, we provide novel insight into the mechanisms underlying tumorigenic effects of ATP by demonstrating the ability of metastatic prostate carcinoma PC3 and breast cancer MDA-MB-231 cells to maintain PPi, which derives from extracellular ATP in the course of nucleotide pyrophosphatase/phosphodiesterase reaction. Collectively, the results imply a complex pattern of nucleotide turnover where extracellular ATP, ADP and adenosine are maintained at steady-state levels via counterbalanced release and inactivation of ATP and other purines, and further suggest the importance of basal agonist release for continuous activation and/or desensitization of purinergic receptors.

ATP Serves as an Endogenous Inhibitor of UDP-Glucuronosyltransferase (UGT): A New Insight into the Latency of UGT

We have suggested that adenine-related compounds are allosteric inhibitors of UGT in rat liver microsomes (RLM) treated with detergent. To clarify whether the same occurs with a pore-forming peptide, alamethicin, the effects of adenine-related compounds on 4-metylumbelliferone (4-MU) glucuronidation were examined using RLM and human liver microsomes (HLM). ATP inhibited 4-MU glucuronidation when polyoxyethylene cetyl alcohol ether (Brij-58)-treated RLM were used (IC(50) = approximately 70 μM). However, alamethicin-treated RLM exhibited a lower susceptibility (IC(50) = approximately 460 μM) than Brij-58-treated RLM. A similar phenomenon was observed when pooled HLM were used. Then, the endogenous ATP content of RLM was determined in the presence and absence of alamethicin or detergent, and although no ATP remained in the microsomal pellets after Brij-58 treatment, more than half of the microsomal ATP remained even after treatment with alamethicin. Furthermore, the V(max) in the absence of an adenine-related compound was approximately three times higher in Brij-58-treated than in alamethicin-treated RLM. The difference in the inhibitory potency observed was due to the difference in remaining endogenous ATP and the accessibility of exogenous ATP to the luminal side of the endoplasmic reticulum (ER), where the active site of UDP-glucuronosyltransferase (UGT) is located. Gefitinib (Iressa), a protein tyrosine kinase inhibitor, markedly inhibited human UGT1A9 activity. It is interesting to note that AMP antagonized Gefitinib-provoked inhibition of UGT1A9, and ATP exhibited an additive inhibitory effect at a lower concentration. Therefore, Gefitinib inhibits UGT1A9 at the common ATP-binding site shared with ATP and AMP. Releasing adenine nucleotide from the ER is suggested to be one of the mechanisms that explain the "latency" of UGT.

Endogenous ATP and the Modulation of Sympathetic Vasoconstriction in Contracting Skeletal Muscle of Humans

Exogenous ATP administration in humans can blunt sympathetic vasoconstriction in a dose‐dependent manner, similar to what occurs within contracting skeletal muscle (sympatholysis). Here, we tested the hypothesis that endogenous ATP increases during exercise and is associated with the magnitude of sympatholysis in humans (N = 9). We measured deep venous plasma [ATP] and forearm blood flow (Doppler ultrasound), and calculated ATP effluent (FBF x [ATP]) at rest and during 5 and 15% MVC rhythmic handgrip exercise (EX), before and during superimposed lower body negative pressure (LBNP; −40 mmHg). LBNP‐induced vasoconstriction was similar during 5% EX vs rest (ΔFBF = −25 ± 2% vs −33 ± 5%), whereas this was blunted during 15% EX (−4 ± 2%). LBNP at rest did not alter [ATP] nor ATP effluent. During the 5% EX trial, [ATP] and ATP effluent were 44 ± 7, 70 ± 9, and 50 ± 10 nmol/L and 1 ± 1, 7 ± 1, and 4 ± 1 nmol/min at rest, EX, and EX + LBNP, respectively. During the 15% EX trial, [ATP] and ATP effluent were 47 ± 5, 79 ± 11, and 71 ± 8 nmol/L and 1 ± 1, 16 ± 3, and 14 ± 1 nmol/min at rest, EX, and EX + LBNP, respectively. We conclude that endogenous ATP release is increased during exercise and must remain elevated during sympathetic stimulation in order to blunt the vasoconstrictor response. Further, the contribution of intravascular ATP to vascular control may ultimately be dependent on an interaction between blood flow and [ATP].Supported by NIH HL102720