Degradation Of Extracellular ATP Research Articles

Synergistic augmentation of ATP-induced interleukin-6 production by arsenite in HaCaT cells.

Chronic arsenic exposure causes cutaneous diseases such as hyperkeratosis and skin cancer. However, little information has been available regarding the molecular mechanisms underlying these symptoms. Because extracellular ATP and interleukin-6 (IL-6) are involved in pathological aspects of cutaneous diseases, we examined whether sodium arsenite (As(III)) affects ATP-induced IL-6 production in human epidermal keratinocyte HaCaT cells. The results showed that the addition of As(III) into the medium of HaCaT cells dose dependently increased the production of IL-6 induced by extracellular ATP, although As(III) alone had no effect on IL-6 production. To elucidate the mechanism of the synergistic effect of As(III) on IL-6 production by extracellular ATP, we next examined the phosphorylation of p38, ERK and epidermal growth factor receptor (EGFR), since we found that these signaling molecules were stimulated by exposure to extracellular ATP. The results indicated that ATP-induced phosphorylation of p38, ERK and EGFR was synergistically enhanced by co-exposure to As(III). To clarify the mechanisms underlying the enhanced phosphorylation of p38, ERK and EGFR by As(III), we explored two possible mechanisms: the inhibition of extracellular ATP degradation and the inhibition of protein tyrosine phosphatases (PTPs) activity by As(III). The degradation of extracellular ATP was not changed by As(III), whereas the activity of PTPs was significantly inhibited by As(III). Our results suggest that As(III) augments ATP-induced IL-6 production in HaCaT cells through enhanced phosphorylation of the EGFR and p38/ERK pathways, which is associated with the inhibition of PTPs activity.

Pannexin 1‐dependent ATP release from venous endothelium promotes acute vascular inflammation (669.8)

Tumor necrosis factor α (TNFα) activates endothelial cells (EC) promoting EC‐leukocyte interactions in the venous circulation which is mediated, in part, by purinergic signaling. However, it is currently not known if ECs release ATP to TNFα. Employing in vitro ATP release assays, we report that TNFα significantly increased ATP release from venous ECs (Human Umbilical and Saphenous) which was not observed in arterial ECs (Human Coronary and Aortic). ATP release was inhibited by WP9QY, a peptide antagonist of the TNF receptor 1 (TNFR1), indicating receptor activation in the initiation of this event. Pharmacological inhibition of Pannexin 1 (Panx1) channels in venous ECs with CBX or 10panx1 and siRNA knockdown of Panx1 significantly inhibited ATP release, whereas inhibition of connexin hemichannels, CALHM1 channels and exocytosis had no effect. This indicates that Panx1 channels are the conduit for ATP liberation. Additionally, we show that Src family kinases mediate Panx1 channel activation, as the Src inhibitor PP2 significantly reduced TNFα‐induced ATP release. Functionally, both Panx1 inhibition and degradation of extracellular ATP attenuated leukocyte‐venous EC interactions to TNFα stimulation. Utilizing ex vivo cannulated mesenteric venules, luminal perfusion of TNFα significantly increased ATP release into the perfusate, supporting a role for TNFα‐mediated ATP release in intact venules. Finally, we developed an EC‐specific, inducible Panx1 knockout mouse (VEcadCreERT2+/Panx1fl/fl) to determine the role of Panx1 in vascular inflammation in vivo. In conclusion, TNFα activates Panx1 channels in venous ECs inducing ATP release to promote leukocyte‐EC interactions.Grant Funding Source: Supported by NIH, AHA

Purinergic control of AMPK activation by ATP released through connexin 43 hemichannels: pivotal roles in hemichannel-mediated cell injury

Connexin hemichannels regulate many cell functions. However, the molecular mechanisms involved remain elusive. Hemichannel opening causes loss of ATP, we therefore speculated a potential role for AMPK in the biological actions of hemichannels. Activation of hemichannels by removal of extracellular Ca(2+) led to an efflux of ATP and a weak activation of AMPK. Unexpectedly, dysfunction of hemichannels markedly potentiated AMPK activation, which was reproduced by promotion of extracellular ATP degradation or inhibition of P2 purinoceptors but counteracted by exogenous ATP. Further analysis revealed that ATP induced a purinoceptor-dependent activation of Akt and mTOR. Suppression of Akt or mTOR augmented AMPK activation, whereas activation of Akt by transfection of cells with myristoylated Akt, a constitutively active form of Akt, abolished AMPK activation. In a pathological model of hemichannel opening triggered by Cd(2+), disclosure of hemichannels similarly enhanced AMPK activity, which protected cells from Cd(2+)-induced cell injury through suppression of mTOR. In summary, our data point to a channel-mediated mechanism for the regulation of AMPK through a purinergic signaling pathway. Furthermore, we define AMPK as a pivotal molecule that underlies the regulatory effects of hemichannels on cell survival.

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.

The P2Y13 receptor regulates extracellular ATP metabolism and the osteogenic response to mechanical loading

ATP release and subsequent activation of purinergic receptors has been suggested to be one of the key transduction pathways activated by mechanical stimulation of bone. The P2Y(13) receptor, recently found to be expressed by osteoblasts, has been suggested to provide a negative feedback pathway for ATP release in different cell types. Therefore, we hypothesized that the P2Y(13) receptor may contribute to the mediation of osteogenic responses to mechanical stimulation by regulating ATP metabolism by osteoblasts. To test this hypothesis, wild-type (WT) and P2Y(13) receptor knockout (P2Y(13)R-/-) mice were subject to non-invasive axial mechanical loading of the left tibiae to induce an osteogenic response. Micro-computed tomography analysis showed mechanical loading induced an osteogenic response in both strains of mice in terms of increased total bone volume and cortical bone volume, with the P2Y(13)R-/- mice having a significantly greater response. The extent of the increased osteogenic response was defined by dynamic histomorphometry data showing dramatically increased bone formation and mineral apposition rates in P2Y(13)R-/- mice compared with controls. In vitro, primary P2Y(13)R-/- osteoblasts had an accumulation of mechanically induced extracellular ATP and reduced levels of hydrolysis. In addition, P2Y(13)R-/- osteoblasts also had a reduction in their maximal alkaline phosphatase (ALP) activity, one of the main ecto-enzymes expressed by osteoblasts, which hydrolyzes extracellular ATP. In conclusion, deletion of the P2Y(13) receptor leads to an enhanced osteogenic response to mechanical loading in vivo, possibly because of the reduced extracellular ATP degradation by ALP. The augmented osteogenic response to mechanical stimulation, combined with suppressed bone remodeling activities and protection from OVX-induced bone loss after P2Y(13) receptor depletion as previously described, suggests a potential role for P2Y(13) receptor antagonist-based therapy, possibly in combination with mechanical loading, for the treatment of osteoporosis.

Contribution of intravascular versus interstitial purines and nitric oxide in the regulation of exercise hyperaemia in humans

The regulation of blood flow to skeletal muscle involves a complex interaction between several locally formed vasodilators that are produced both in the skeletal muscle interstitium and intravascularly. The gas nitric oxide (NO) and the purines ATP and adenosine, are potent vasodilators that are formed by multiple cell types and released into the skeletal muscle interstitium and in plasma in response to muscle contraction. Cellular sources of ATP and NO in plasma are erythrocytes and endothelial cells, whereas interstitial sources are skeletal muscle cells and endothelial cells. Adenosine originates primarily from extracellular degradation of ATP. During exercise the concentrations of ATP and adenosine increase markedly in the interstitium with smaller increases occurring in plasma, and thus the interstitial concentration during exercise is severalfold higher than in plasma. The concentration of NO metabolites (NOx) in interstitium and plasma does not change during exercise and is similar in the two compartments. Adenosine and NO have been shown to contribute to exercise hyperaemia whereas the role of ATP remains unclear due to lack of specific purinergic receptor blockers. The relative role of intravascular versus interstitial vasodilators is not known but evidence suggests that both compartments are important. In cardiovascular disease, a reduced capacity to form adenosine in the muscle interstitium may be a contributing factor in increased peripheral vascular resistance.

CD73-Deficient Mice Are Resistant to Carcinogenesis

CD73 is a cell surface 5'-nucleotidase that converts AMP to adenosine, an immune suppressive molecule. CD73 may promote immune escape in cancer by contributing to the degradation of extracellular ATP released by dying cancer cells in hypoxic tumors or following chemotherapy. However, whether CD73 exerts a critical oncogenic function during tumorigenesis is unknown. In this study, we used genetically deficient mice to investigate its contribution to autochthonous tumor formation. CD73 deficiency suppressed the development of 3-methylcholanthrene (MCA)-induced fibrosarcomas through a mechanism relying upon IFN-γ, natural killer (NK) cells, and CD8(+) T cells. Similarly, CD73 deficiency also suppressed prostate tumorigenesis in TRAMP transgenic mice. Importantly, treatment with an anti-CD73 monoclonal antibody effectively suppressed growth of established MCA-induced tumors or TRAMP-C1 prostate tumors and inhibited the development of TRAMP-C1 lung metastases. The therapeutic activity of anti-CD73 monoclonal antibody against primary tumors was dependent on CD8(+) T cells, whereas its antimetastatic activity was dependent on host CD73 expression independent of T cells or NK cells. Taken together, our findings indicate that CD73 is a critical factor in tumorigenesis and that anti-CD73 antibodies may offer a novel generalized strategy to blunt immune escape and treat cancer.

Highlights in basic autonomic neurosciences: Glia and neuromodulation

Adenosine is an important neuromodulator in the CNS. In response to insults, such as hypoxia, adenosine is released and exerts a neuroprotective effect by inhibiting excitatory synaptic transmission. Martin et al. investigated whether the hypoxia-induced excitatory synaptic depression might be mediated by adenosine release from astrocytes by using electrophysiological and calcium imaging techniques in hippocampal slices of transgenic mice with dominant negativesoluble N-ethylmaleimide-sensitive factor attachment protein receptor (dn-SNARE) and in pure astrocytic cultures,. The inhibition of glial cell metabolismwithfluorocitrate or theblockadeofA1 adenosine receptors with 8-cyclopentyltheophylline was effective in preventing the hypoxia-induced reduction in excitatory synaptic transmission. It was also shown that adenosine transporter inhibitors failed to modify hypoxiainduced effects, demonstrating that hypoxia-induced adenosine release was transporter independent. Moreover, adenosine release under hypoxic conditions was shown to be calcium independent. As under normoxic physiological conditions extracellular adenosine is mainly derived from extracellular catabolism of astrocytic ATP released by exocytosis, the presence of adenosine-mediated modulation of excitatory synaptic transmission during hypoxia in transgenic mice with impaired astrocytic ATP exocytosis (dn-SNARE) indicates that the adenosine mediating the hypoxic effects is not produced by extracellular ATP degradation. Thus, the authors conclude that under hypoxic conditions astrocytes regulate the excitatory synaptic transmission by direct release of adenosine, which acts on A1 receptors to reduce presynaptic transmitter release.

Extracellular ATP and the P2X7receptor in astrocyte-mediated motor neuron death: implications for amyotrophic lateral sclerosis

BackgroundDuring pathology of the nervous system, increased extracellular ATP acts both as a cytotoxic factor and pro-inflammatory mediator through P2X7 receptors. In animal models of amyotrophic lateral sclerosis (ALS), astrocytes expressing superoxide dismutase 1 (SOD1G93A) mutations display a neuroinflammatory phenotype and contribute to disease progression and motor neuron death. Here we studied the role of extracellular ATP acting through P2X7 receptors as an initiator of a neurotoxic phenotype that leads to astrocyte-mediated motor neuron death in non-transgenic and SOD1G93A astrocytes.MethodsWe evaluated motor neuron survival after co-culture with SOD1G93A or non-transgenic astrocytes pretreated with agents known to modulate ATP release or P2X7 receptor. We also characterized astrocyte proliferation and extracellular ATP degradation.ResultsRepeated stimulation by ATP or the P2X7-selective agonist BzATP caused astrocytes to become neurotoxic, inducing death of motor neurons. Involvement of P2X7 receptor was further confirmed by Brilliant blue G inhibition of ATP and BzATP effects. In SOD1G93A astrocyte cultures, pharmacological inhibition of P2X7 receptor or increased extracellular ATP degradation with the enzyme apyrase was sufficient to completely abolish their toxicity towards motor neurons. SOD1G93A astrocytes also displayed increased ATP-dependent proliferation and a basal increase in extracellular ATP degradation.ConclusionsHere we found that P2X7 receptor activation in spinal cord astrocytes initiated a neurotoxic phenotype that leads to motor neuron death. Remarkably, the neurotoxic phenotype of SOD1G93A astrocytes depended upon basal activation the P2X7 receptor. Thus, pharmacological inhibition of P2X7 receptor might reduce neuroinflammation in ALS through astrocytes.

Dexamethasone inhibits proliferation and stimulates ecto-5′-nucleotidase/CD73 activity in C6 rat glioma cell line

Malignant gliomas are the most common and devastating primary tumors of the adult central nervous system. Dexamethasone, a synthetic glucocorticoid, is commonly co-administered to control edema in the management of brain tumors during chemotherapy and radiotherapy. In the present study, the effect of dexamethasone on proliferation and ectonucleotidase activities in rat C6 glioma cell line was investigated. Dexamethasone concentrations ranging from 0.001 to 10 microM induced a time- and concentration-dependent inhibition of C6 rat glioma cell proliferation after 24, 48 and 72-h treatment. The tetrazolium reduction assay (MTT) indicated a reduction of in cell viability (44 +/- 7.6%) after 48-h treatment with 1 microM dexamethasone. Pretreatment with 10 microM of RU38486, an antagonist of glucocorticoid receptors, abolished the effect of 1 microM dexamethasone by 78 +/- 9.8% after 48 h of treatment, indicating that this action is mediated via the glucocorticoid receptor. Members of the E-NTPDase family and ecto-5'-nucleotidase/CD73 can modulate extracellular ATP degradation and adenosine formation, both of which have been described as proliferation factors. Treatment of C6 glioma cells for 48 h with 1 microM dexamethasone increased in 38 +/- 8.09% the AMP hydrolysis and in 3.7-fold the ecto-5'-nucleotidase/CD73 expression, suggesting an increase in adenosine formation and, therefore, a possible modulatory role in the elicitation of cell death responses. In addition, pretreatment with 5 microM GF 109203X, a protein kinase C (PKC) inhibitor, abolished the effect of dexamethasone on cell proliferation and on ecto-5'-NT activity, suggesting that dexamethasone could exert this action via PKC. The alterations in the catabolism of extracellular purines induced by dexamethasone treatment in glioma C6 cells could be related to its pharmacological effects.

NTPDase and 5′-nucleotidase Activities of Synaptosomes from Hippocampus of Rats Subjected to Hyperargininemia

ATP is an important excitatory neurotransmitter and adenosine acts as a neuromodulatory structure inhibiting neurotransmitters release in the central nervous system. Since the ecto-nucleotidase cascade that hydrolyzes ATP to adenosine is involved in the control of brain functions and previous studies realized in our laboratory have recently reported that acute administration of Arg decreases the NTPDase and 5'-nucleotidase activities of rat blood serum, in the present study we investigated the effect of arginine administration on NTPDase and 5'-nucleotidase activities by synaptosomes from hippocampus of rats. First, sixty-days-old rats were treated with a single or a triple intraperitoneal injection of arginine (0.8 g/Kg) or an equivalent volume of 0.9% saline solution (control) and were killed 1 h later. Second, rats received an intracerebroventricular injection of 1.5 mM arginine solution or saline (5 microL) and were killed 1 h later. We also tested the in vitro effect of arginine (0.1-1.5 mM) on nucleotide hydrolysis in synaptosomes from rat hippocampus. Results showed that intraperitoneal arginine administration did not alter nucleotide hydrolysis. On the other hand, arginine administered intracerebroventricularly reduced ATP (32%), ADP (30%) and AMP (21%) hydrolysis, respectively. In addition, arginine added to the incubation medium, provoked a decrease on ATP (19%), ADP (17%) and AMP (23%) hydrolysis, respectively. Furthermore, kinetic studies showed that the inhibitory effect of arginine was uncompetitive in relation to ATP, ADP and AMP. In conclusion, according to our results it seems reasonable to postulate that arginine alters the cascade involved in the extracellular degradation of ATP to adenosine.

Role of p38 map kinase in glycine-induced hepatocyte resistance to hypoxic injury

Glycine hepatoprotection is well known. However, the mechanisms involved are still poorly characterized. Glycine protection was investigated in isolated rat hepatocytes pretreated with 2 mmol/L glycine 15 min before incubation under hypoxic conditions. Glycine significantly reduced Na+ overload and hepatocyte death caused by hypoxia. Glycine protection required the activation of a signal pathway involving Src, Pyk2 and p38 MAP kinases. Glycine treatment also induced a 11% increase of hepatocyte volume and transient ATP release. The prevention of cell swelling by hepatocyte incubation in a hypertonic medium as well as the degradation of extracellular ATP with apyrase or the block P2 purinergic receptors with suramin reverted glycine-induced cytoprotection and inhibited Src, Pyk2 and p38 MAPK activation. Glycine down-modulated Na+/H+ exchanger (NHE) activity, without affecting the development of intracellular acidosis during hypoxia. Such an effect was reverted by inhibiting p38 MAPK that also abolished glycine protection against Na+ overload caused by hypoxia. Glycine-induced ATP release in response to a moderate hepatocyte swelling led to the autocrine stimulation of P2 receptors and to the activation of Src, Pyk2 and p38 MAPK that increased hepatocyte resistance to hypoxia by preventing Na+ influx through NHE.

Mediators of tubuloglomerular feedback regulation of glomerular filtration: ATP and adenosine

In the juxtaglomerular apparatus of the kidney the loop of Henle gets into close contact to its parent glomerulus. This anatomical link between the tubular system and the vasculature of the afferent and efferent arteriole enables specialized tubular cells, the macula densa (MD) cells, to establish an intra-nephron feedback loop designed to control preglomerular resistance and thereby single nephron glomerular filtration rate. This review focuses on the signalling mechanisms which link salt-sensing MD cells and the regulation of preglomerular resistance, a feedback loop known as tubuloglomerular feedback (TGF). Two purinergic molecules, ATP and adenosine, have emerged over the years as most likely candidates to serve as mediators of TGF. Data will be reviewed supporting a role of either ATP or adenosine as mediators of TGF. In addition, a concept will be discussed that integrates both ATP and adenosine into one signalling cascade that includes (i) release of ATP from MD cells upon increases in tubular salt concentration, (ii) extracellular degradation of ATP to form adenosine, and (iii) adenosine-mediated vasoconstriction of the afferent arteriole.

Evidence for the involvement of purinergic P2X7 receptors in outer retinal processing

Extracellular ATP mediates fast excitatory neurotransmission in many regions of the central nervous system through activation of P2X receptors. Although several P2X receptor subunits have been identified in the mammalian retina, little is known about the functional role of these receptors in retinal signalling. The purpose of the present study was to investigate whether purinergic P2X(7) receptors are involved in outer retinal processing by assessing receptor localization, degradation of extracellular ATP and the effect of functional activation of P2X(7) receptors on the electroretinogram (ERG). Using light and electron microscopy, we demonstrated that P2X(7) receptors are expressed postsynaptically on horizontal cell processes as well as presynaptically on photoreceptor synaptic terminals in both the rat and marmoset retina. Using an enzyme cytochemical method, we showed that ecto-ATPases are active in the outer plexiform layer of the rat retina, providing a mechanism by which purinergic synaptic transmission can be rapidly terminated. Finally, we evaluated the role of P2X(7) receptors in retinal function by assessing changes to the ERG response of rats after intravitreal delivery of the P2X(7) receptor agonist benzoyl benzoyl ATP (BzATP). Intravitreal injection of BzATP resulted in a sustained increase (up to 58%) in the amplitude of the photoreceptor-derived a-wave of the ERG. In contrast, BzATP caused a transient reduction in the rod- and cone-derived postreceptoral responses. These results provide three lines of evidence for the involvement of extracellular purines in outer retinal processing.

Degradation of extracellular ATP by the retinal pigment epithelium

Stimulation of ATP or adenosine receptors causes important physiological changes in retinal pigment epithelial (RPE) cells that may influence their relationship to the adjacent photoreceptors. While RPE cells have been shown to release ATP, the regulation of extracellular ATP levels and the production of dephosphorylated purines is not clear. This study examined the degradation of ATP by RPE cells and the physiological effects of the adenosine diphosphate (ADP) that result. ATP was readily broken down by both cultured human ARPE-19 cells and the apical membrane of fresh bovine RPE cells. The compounds ARL67156 and betagamma-mATP inhibited this degradation in both cell types. RT-PCR analysis of ARPE-19 cells found mRNA message for multiple extracellular degradative enzymes; ectonucleotide pyrophosphatase/phosphodiesterase eNPP1, eNPP2, and eNPP3; the ectoATPase ectonucleoside triphosphate diphosphohydrolase NTPDase2, NTPDase3, and some message for NTPDase1. Considerable levels of ADP bathed RPE cells, consistent with a role for NTPDase2. ADP and ATP increased levels of intracellular Ca(2+). Both responses were inhibited by thapsigargin and P2Y(1) receptor inhibitor MRS 2179. Message for both P2Y(1) and P2Y(12) receptors was detected in ARPE-19 cells. These results suggest that extracellular degradation of ATP in subretinal space can result in the production of ADP. This ADP can stimulate P2Y receptors and augment Ca(2+) signaling in the RPE.

Fluid Shear-Induced ATP Secretion Mediates Prostaglandin Release in MC3T3-E1 Osteoblasts

ATP is rapidly released from osteoblasts in response to mechanical load. We examined the mechanisms involved in this release and established that shear-induced ATP release was mediated through vesicular fusion and was dependent on Ca2+ entry into the cell through L-type voltage-sensitive Ca2+ channels. Degradation of secreted ATP by apyrase prevented shear-induced PGE2 release. Fluid shear induces a rapid rise in intracellular calcium ([Ca2+]i) in osteoblasts that mediates many of the cellular responses associated with mechanotransduction in bone. A potential mechanism for this increase in [Ca2+]i is the activation of purinergic (P2) receptors resulting from shear-induced extracellular release of ATP. This study was designed to determine the effects of fluid shear on ATP release and the possible mechanisms associated with this release. MC3T3-E1 preosteoblasts were plated on type I collagen, allowed to proliferate to 90% confluency, and subjected to 12 dynes/cm2 laminar fluid flow using a parallel plate flow chamber. ATP release into the flow media was measured using a luciferin/luciferase assay. Inhibitors of channels, gap junctional intercellular communication (GJIC), and vesicular formation were added before shear and maintained in the flow medium for the duration of the experiment. Fluid shear produced a transient increase in ATP release compared with static MC3T3-E1 cells (59.8 +/- 15.7 versus 6.2 +/- 1.8 nM, respectively), peaking within 1 minute of onset. Inhibition of calcium entry through the L-type voltage-sensitive Ca2+ channel (L-VSCC) with nifedipine or verapamil significantly attenuated shear-induced ATP release. Channel inhibition had no effect on basal ATP release in static cells. Ca(2+)-dependent ATP release in response to shear seemed to result from vesicular release and not through gap hemichannels. Vesicle disruption with N-ethylmaleimide, brefeldin A, or monensin prevented increases in flow-induced ATP release, whereas inhibition of gap hemichannels with either 18alpha-glycyrrhetinic acid or 18beta-glycyrrhetinic acid did not. Degradation of extracellular ATP with apyrase prevented shear-induced increases in prostaglandin E2 (PGE2) release. These data suggest a time line of mechanotransduction wherein fluid shear activates L-VSCCs to promote Ca2+ entry that, in turn, stimulates vesicular ATP release. Furthermore, these data suggest that P2 receptor activation by secreted ATP mediates flow-induced prostaglandin release.

Degradation of extracellular nucleotides and their analogs in HeLa and HUVEC cell cultures.

The use of nucleotides and their analogs in the pharmacological studies of nucleotide receptors (P2 class) should be preceded by detailed studies on their degradation connected with ecto-enzymes of a given cell type. In the present studies we have analyzed stability of some phosphorothioate and phosphonate analogs of ATP and ADP in the HeLa epitheloid carcinoma and endothelial HUVEC cells cultures. Our studies have revealed that ecto-nucleotide pyrophosphatase (E-NPP) is one of the main enzymes involved in the extracellular degradation of ATP and other nucleotides in the HeLa cells. On the other hand, the ecto-ATPDase is responsible for the hydrolysis of extracellular nucleotides in human endothelial cell cultures, while the E-NPP-like enzymes of the HUVEC cells are not essential to this degradation. The concerted action of the aforementioned ecto-enzymes and nucleotide pyrophosphatase, 5'-nucleotidase and adenosine deaminase present in fetal bovine serum (FBS) supplied to the culture medium, results in partial or complete degradation of the phosphorothioate (ATPgammaS) and phosphonate analogs of adenosine nucleotides (alpha,beta-methylene-ATP and beta,gamma-methylene-ATP) in the cell cultures. Only ADPbetaS appears to be resistant to these enzymes. The influence of some nucleotides and their analogs on the proliferation of the HeLa cells in presence or absence of FBS is also discussed.

Degradation of extracellular nucleotides and their analogs in HeLa and HUVEC cell cultures.

The use of nucleotides and their analogs in the pharmacological studies of nucleotide receptors (P2 class) should be preceded by detailed studies on their degradation connected with ecto-enzymes of a given cell type. In the present studies we have analyzed stability of some phosphorothioate and phosphonate analogs of ATP and ADP in the HeLa epitheloid carcinoma and endothelial HUVEC cells cultures. Our studies have revealed that ecto-nucleotide pyrophosphatase (E-NPP) is one of the main enzymes involved in the extracellular degradation of ATP and other nucleotides in the HeLa cells. On the other hand, the ecto-ATPDase is responsible for the hydrolysis of extracellular nucleotides in human endothelial cell cultures, while the E-NPP-like enzymes of the HUVEC cells are not essential to this degradation. The concerted action of the aforementioned ecto-enzymes and nucleotide pyrophosphatase, 5'-nucleotidase and adenosine deaminase present in fetal bovine serum (FBS) supplied to the culture medium, results in partial or complete degradation of the phosphorothioate (ATPgammaS) and phosphonate analogs of adenosine nucleotides (alpha,beta-methylene-ATP and beta,gamma-methylene-ATP) in the cell cultures. Only ADPbetaS appears to be resistant to these enzymes. The influence of some nucleotides and their analogs on the proliferation of the HeLa cells in presence or absence of FBS is also discussed.

Third-generation beta-blockers stimulate nitric oxide release from endothelial cells through ATP efflux: a novel mechanism for antihypertensive action.

Nebivolol and carvedilol are third-generation beta-adrenoreceptor antagonists, which unlike classic beta-blockers, have additional endothelium-dependent vasodilating properties specifically related to microcirculation by a molecular mechanism that still remains unclear. We hypothesized that nebivolol and carvedilol stimulate NO release from microvascular endothelial cells by extracellular ATP, which is a well-established potent autocrine and paracrine signaling factor modulating a variety of cellular functions through the activation of P2-purinoceptors. Contraction and relaxation of renal glomerular vasculature were measured by determination of intracapillary volume with [3H]-inulin. Biologically active NO was measured with highly sensitive porphyrinic NO microsensors in a single glomerular endothelial cell (GEC). Extracellular ATP was measured by a luciferin-luciferase assay. Enzymatic degradation of extracellular ATP by apyrase and blockade of P2Y-purinoceptors by suramin or reactive blue 2 inhibited both beta-blocker-induced glomerular vasorelaxations and beta-blocker-stimulated NO release from GECs. Both beta-blocker-induced vasorelaxations were in the micromolar concentration range identical to that required for the beta-blocker stimulation of ATP and NO release from GECs. The maximum of NO release for nebivolol and carvedilol was very similar (188+/-14 and 226+/-17, respectively). Blockade of ATP release by a mechanosensitive ion channel blocker, Gd3+, inhibited the beta-blocker-dependent release of ATP and NO from GECs. These results demonstrate for the first time that nebivolol and carvedilol induce relaxation of renal glomerular microvasculature through ATP efflux with consequent stimulation of P2Y-purinoceptor-mediated NO release from GECs.

Purinergic regulation of glucose and glutamine synthesis in isolated rabbit kidney–cortex tubules

The effects of extracellular purinergic agonists and their breakdown products on glucose and glutamine synthesis in rabbit kidney–cortex tubules incubated with aspartate+glycerol or alanine+glycerol+octanoate were investigated. A rapid extracellular degradation of ATP was accompanied by an accumulation of AMP, inosine, and hypoxanthine. Extracellular ATP and its breakdown products accelerated glucose synthesis in renal tubules, while ammonium released from adenine-containing compounds enhanced glutamine synthesis and diminished the degree of gluconeogenesis stimulation. In contrast to AMP and inosine, ATP evoked calcium signals, while both ATP and inosine decreased intracellular cAMP content and accelerated the flux through fructose-1,6-bisphosphatase as concluded from changes in gluconeogenic intermediates. Since (i) the activity of partially purified renal fructose-1,6-bisphosphatase was increased upon protein phosphatase-1 treatment and decreased following treatment of previously dephosphorylated enzyme with protein kinase A catalytic subunit and (ii) both 8-bromoadenosine 3 ′,5 ′-cyclic monophosphate and 8-(4-chlorophenyltio)-cAMP inhibited renal glucose synthesis, it seems likely that in rabbit renal tubules ATP and inosine stimulate gluconeogenesis via cAMP decrease, which favors the appearance of a more active, dephosphorylated form of fructose-1,6-bisphosphatase, a key gluconeogenic enzyme.